Regulation of the Cellular Content of the Organic Osmolyte Taurine in Mammalian Cells

Change in the intracellular concentration of osmolytes or the extracellular tonicity results in a rapid transmembrane water flow in mammalian cells un...

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Neurochemical Research, Vol. 29, No. 1, January 2004 (© 2004), pp. 27–63

Regulation of the Cellular Content of the Organic Osmolyte Taurine in Mammalian Cells* Ian Henry Lambert1 (Accepted June 29, 2003)

Change in the intracellular concentration of osmolytes or the extracellular tonicity results in a rapid transmembrane water ﬂow in mammalian cells until intracellular and extracellular tonicities are equilibrated. Most cells respond to the osmotic cell swelling by activation of volume-sensitive flux pathways for ions and organic osmolytes to restore their original cell volume. Taurine is an important organic osmolyte in mammalian cells, and taurine release via a volume-sensitive taurine efﬂux pathway is increased and the active taurine uptake via the taurine speciﬁc taurine transporter TauT decreased following osmotic cell swelling. The cellular signaling cascades, the second messengers proﬁle, the activation of speciﬁc transporters, and the subsequent time course for the readjustment of the cellular content of osmolytes and volume vary from cell type to cell type. Using Ehrlich ascites tumor cells, NIH3T3 mouse ﬁbroblasts and HeLa cells as biological systems, it is revealed that phospholipase A2-mediated mobilization of arachidonic acid from phospholipids and subsequent oxidation of the fatty acid via lipoxygenase systems to potent eicosanoids are essential elements in the signaling cascade that is activated by cell swelling and leads to release of osmolytes. The cellular signaling cascade and the activity of the volume-sensitive taurine efﬂux pathway are modulated by elements of the cytoskeleton, protein tyrosine kinases/phosphatases, GTP-binding proteins, Ca2/calmodulin, and reactive oxygen species and nucleotides. Serine/threonine phosphorylation of the active taurine uptake system TauT or a putative regulator, as well as change in the membrane potential, are important elements in the regulation of TauT activity. A model describing the cellular sequence, which is activated by cell swelling and leads to activation of the volume-sensitive efﬂux pathway, is presented at the end of the review.

KEY WORDS: Regulatory volume decrease; iPLA2; cPLA2; leukotriene D4; reactive oxygen species; NADPH oxidase; protein tyrosine phosphatase; lysophosphatidyl choline.

which results in net movement of osmolytes and water across the plasma membrane and restoration of the cell volume. The volume regulatory response that follows cell swelling and cell shrinkage is designated regulatory volume decrease (RVD) and regulatory volume increase (RVI), respectively (1). In 1939 August Krogh reported that organic osmolytes contributed to the intracellular pool of osmotic compounds (2), and today it is recognized that net loss of compatible osmolytes, that is, free amino acids (taurine, alanine, glycine, proline, glutamate, glutamine, aspartate, -alanine), methylated compounds (glycerolphosphocholine, betaine), sugars, and polyols (sorbitol, myo-inositol), contributes signiﬁcantly to the

TAURINE: AN ORGANIC OSMOLYTE Mammalian cells have high water permeability, and most cells respond as almost perfect osmometers following exposure to hypotonic or hypertonic conditions. They subsequently activate a variety of plasma membrane–bound transporters for ions and compatible organic osmolytes,

* Special issue dedicated to Dr. Herminia Pasantes-Morales. 1 The August Krogh Institute, Biochemical Department, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark. Tel: 45-3532-1697; Fax: 45-3532-1567; E-mail: [email protected]

27 0364-3190/04/0100–0027/0 © 2004 Plenum Publishing Corporation

28 RVD process in vertebrates. Taurine, -aminoethanesulphonic acid (Fig. 1) is, with the exception of pancreatic -cells (3) and kidney epithelial cells (4), regarded as an important organic osmolyte in mammalian cells, and various cell lines, for example, astrocytes, neurones, pancreatic -cells, MDCK cells, and NIH3T3 cells, preferentially utilize taurine as an organic osmolyte following osmotic perturbation, even though the cellular concentration of other organic osmolytes is higher (3,5–7). Indeed, the high water solubility and poor ability of taurine to diffuse across cell membranes combined with its biochemical inertness and poor ability to chelate cations make the noncytotoxic taurine well suited as osmolyte for adjustment of the intra-

Lambert cellular osmotic pressure and cell volume after osmotic perturbation (1,8). The cellular to extracellular taurine concentration ratio has been estimated at 600 in Ehrlich ascites tumor cells (9), and it is a functional equilibrium between active uptake, passive release, and biosynthesis from cystein. Besides being an important organic osmolyte, taurine is also involved in a number of physiological processes (Fig. 1). This review deals with the regulation of the active taurine uptake system (TauT) and the volume-sensitive taurine leak pathway. Ehrlich ascites tumor cells, HeLa cells, and NIH3T3 mouse ﬁbroblasts are used in our laboratory as biological test systems and will be used in illustrating examples. Volume-sensitive ion transporting systems and

Fig. 1. Taurine synthesis and physiological effects of taurine. There are several pathways for taurine synthesis, but the pathway illustrated appears as the main pathway. Cysteine sulfonic acid decarboxylase, which converts cysteine sulfinic acid to hypotaurine, is the rate-limiting step in the taurine synthesis from methionine and cysteine. Taurine has a range of physiological roles. Bile salt formation: Taurine is an efficient conjugator for bile salts, as it remains ionized even at the high acidity that occurs at the upper intestine. Humans have the ability to switch from taurine conjugates to glycine conjugates when taurine availability is reduced. Cats are unable to make sufficient taurine and do not use glycine for conjugation and therefore require dietary taurine intake (312,313). Osmoregulation: See text for details. Membrane structure and function: Taurine binds to neutral phospholipids and the taurine–phospholipid interaction, that involves formation of ion pairs between the head groups, affects the membrane property, that is, architecture and fluidity. Taurine inhibits N-methylation of phospholipids, that is, conversion of phosphatidyl ethanol amine to phosphatidyl choline (8,314). Ca2 homeostasis: Ca2 binds to phosphatidyl inositol/phosphatidyl serine, and taurine increases the binding affinity of Ca2 to phospholipids but reduces the binding capacity, that is, Ca2 storage capacity. Taurine affects Ca2 uptake as well as release (314,315). Antioxidation: Taurine and to a greater extent hypotaurine have antioxidant activity (316). Ion channel function: Taurine affects Cl current and regulates the activity of anion channels (8,317). Modulation of neurotransmission: Taurine interacts with the GABA- and glycine-gated family of Cl channels (8,30,314).

Taurine Homeostasis their regulation have been described in a series of excellent reviews (1,10,11). Taurine is the end product of the metabolism of the sulphur-containing amino acids methionine and cysteine (Fig. 1), and although taurine has been reported to form the carboxylic end of some low-molecular-weight brain synaptic peptides (12) and to be incorporated in membrane lipids (taurolipids) in, for example, Tetrahymena (13), most of the taurine remains free as a zwitterion in the body ﬂuids. Most carnivores and omnivores are able to synthesize taurine. However, newborn mammals, including cats and certain monkeys, have little or no capacity for de novo taurine biosynthesis and therefore rely on a dietary supply of taurine (14). The total taurine pool in the body is controlled by an active taurine transporter located at the brush border of the renal proximal tubule and in the basolateral membrane of the distal nephron, and the expression of the transporter is upregulated or downregulated by the availability of taurine or the taurine precursors methionine and cysteine (15). Taurine deﬁciency leads to developmental retarda-

29 tion, neurological defects, inability of cells to grow and survive, and cardiac abnormalities (16,17).

ACTIVE TAURINE UPTAKE Taurine Uptake: TauT Taurine uptake in Ehrlich ascites cells is mediated by a high-afﬁnity, low-capacity, Na-, Cl- and pHdependent transport system, designated originally as the -system and now as TauT (Fig. 2) (18–21). TauT has been cloned, and it has turned out that TauT belongs to a larger family of Na- and Cl-dependent transporters that also includes transporters for serotonin, dopamine, noradrenalin, -aminobutyric acid (GABA), and creatine (22). TauT consists of 590–655 amino acids, the molecular weight is estimated at 65–74 kDa, and it has been suggested from the sequence and hydropathy plots that TauT has (i) 12 hydrophobic, membrane spanning domains with the N-terminal and C-terminal both being

Fig. 2. Taurine homeostasis in Ehrlich cells. Taurine is accumulated against a concentration gradient by the Na, Cl-dependent -amino acid transporter TauT, which is regulated by cAMP, protein kinase A (PKA), protein kinase C (PKC) and a calyculin A-sensitive phosphatase. State I-III represents TauT in different states of phosphorylation and increasing transport activity. Cell swelling involves cPLA2-mediated release of arachidonic acid from the nuclear membrane, oxidation of the fatty acid via the 5-LO system to LTC4/LTD4, which are subsequently released to the extracellular compartment. LTD4 binds to a cystein receptor (CysLT1) and triggers taurine release. The volume-sensitive taurine efflux pathway is inhibited by DIDS, MK196 and by membrane depolarization, but stimulated by arachidonic acid. Reduction in the cellular taurine content following hypotonic exposure is the result of an increased taurine release via the volume-sensitive taurine channel and a reduced taurine uptake via TauT (see text for details).

30 exposed to the cytosolic compartment; (ii) two potential N-glycosylation sites in the second extracellular loop; and (iii) several putative phosphorylation sites for protein kinase C (PKC), the cAMP-dependent protein kinase (PKA), and casein kinase II on the intracellular loops and the cytosolic C-terminal (23–25). It has also been indicated that the ﬁrst extracellular loop of TauT is involved in the interaction with Na and Cl (26). TauT: Speciﬁcity and Taurine Afﬁnity TauT in Ehrlich cells tolerates, in contrast to other amino acid transporting systems, a sulphonate or sulphinate group, and only -alanine and hypotaurine, which have an anionic site and a cationic site, separated by two methylene groups, are potent inhibitors of taurine inﬂux via TauT (20). These structural demands to the substrate agree with taurine transporters identiﬁed in, for example, rat brain synaptosomes (27), cultured rat glioma cells (28), neuroblast OMA cells (29), MDCK cells (24), and Bergmann glia (30). The afﬁnity of TauT for taurine varies from cell type to cell type, and the taurine concentration required for half maximal transport (Km) is below 15 M in, for example, neuroblastoma cells and LLC-PK1 and MDCK cells (29,31), and between 15 M and 60 M in, for example, Ehrlich cells, LoVo cells, cardiac myocytes, and nonmyocytes (19,32,33), or larger than 100 M in rat choroids plexus (34). It is noted that an additional saturable taurine transport system with low afﬁnity for taurine (Km ⬇ 0.3–1 mM) has been characterized in Ehrlich cells (19,35) and LoVo carcinoma cells (32). Taurine Uptake: Naⴙ and Clⴚ Requirement— Electrogenicity The initial taurine uptake in Ehrlich cells varies in a sigmoid manner as a function of the extracellular Na concentration, and no taurine is taken up in the absence of extracellular Na (19). Taking the energy in the electrochemical Na gradient into consideration (9) or ﬁtting the taurine uptake data to a Hill type equation have revealed that 2–3 Na ions are involved in the active uptake of 1 molecule of taurine in Ehrlich cells (19,21). The Na:taurine ratio has correspondingly been estimated at 3:1 in rabbit kidney and jejunum brush border (36,37) and at 2:1 in, for example, LLC-PK1, MDCK cells, cardiac myocytes, and nonmyocytes (31,33). It is emphasized that the Na:taurine ratio in most cases has been obtained by measuring the taurine inﬂux at various extracellular Na concentrations, which does not distinguish between the catalytic effect and the energetic

Lambert effect of the Na ions (38). Thus a ratio larger than 2 does not necessarily imply that two or more Na ions translocate together with one taurine molecule. An illustrating example is the pigeon erythrocyte, in which the Na:taurine ratio was estimated by the activation method at 2.4, whereas the ratio between the ﬂuxes was close to 1 (39). Reducing the extracellular Na concentration reduces the afﬁnity of the carrier for taurine but has no effect on the transport capacity in Ehrlich cells (19) or erythrocytes from the polychaete Glycera dibranchiata (40). This is in contrast to ﬂounder erythrocytes (41) and glia cells from bullfrogs (42), in which the afﬁnities and the capacity of the taurin inﬂux systems are reduced following reduction in the extracellular Na concentration. As Na ions increase the afﬁnity of the carrier for taurine, it is assumed that the Na ions bind to the taurine carrier, changing its tertiary structure, before binding of the taurine molecule takes place (43). The initial taurine uptake varies in a hyperbolic manner as a function of the extracellular Cl concentration and a 1:1 Cl to taurine coupling ratio has been suggested for the active taurine uptake in, for example, Ehrlich cells (20), rabbit kidney and jejunum brush border (36,37), and bovine brain capillary endothelial cells (44). The anion preference for taurine uptake is Cl SCN NO3 in Ehrlich cells (20), Cl Br W SCN I NO3 in rabbit kidney brush-border (36), and Cl Br SCN gluconate in MDCK cells (45). However, complete substitution of NO3 or SCN for Cl in Ehrlich cells reduced the initial taurine uptake by 75% and 20%, respectively, whereas the membrane potential in both cases depolarized by 20 mV (46). Thus the effect of anion substitution on taurine uptake seems not to be secondary to variation in the membrane potential. The observation that disulfonic stilbene compounds inhibit inorganic anion transport selectively in many tissues (47) and taurine transport in, for example, rat renal brush border (48) indicates that the translocation of taurine via TauT requires an intact binding site for anions. Concerning the mode of Cl action, Bogé et al. (49) suggested that Cl acts by increasing the accessibility of speciﬁc sites on the carrier to Na. Later Wolf et al. (50) demonstrated that Cl substitution reduced the Na:taurine ratio from 2:1 to 1:1, indicating that the role of Cl could be to facilitate binding of the second Na to TauT. A Cl gradient is required for taurine transport in rabbit kidney brush border, and it has been suggested that Cl stimulates taurine inﬂux primarily by facilitating the formation of the taurine:TauT complex (36). In this context it is noted that Cl alters the Na afﬁnity of the human Na, Cl-dependent GABA transporter

Taurine Homeostasis (GAT1) and the Na, glucose cotransporter (SLGT1), but Cl does not contribute to the net charge transport across the plasma membrane, most conceivably because Cl returns to the cis-side as part of the transport cycle (GAT1) or just binds to the carrier and affect the transport kinetics (SGLT1) (51). Taurine uptake in Ehrlich cells is stimulated at alkaline pH and following hyperpolarization of the membrane (52). Taurine uptake by brush-border membrane vesicles from rat jejunum (53), rabbit kidney (36), and rat kidney (48) is similarly stimulated by a negative potential on the trans side of the membrane. The isoelectric point pI of a taurine/-alanine transporter, cloned from a mouse brain cDNA library, has been estimated at 5.98 (54) and treating the taurine carrier as a protein with a single dissociable group, it is estimated that at alkaline extracellular pH we have simultaneously a carrier almost 100% on its anionic form and a highly negative membrane potential (46), which would increase the availability of unloaded carrier at the outside of the cell membrane and favor taurine uptake (52). However, taurine at a saturating concentration does not affect the membrane potential in Ehrlich cells (52), whereas it generates a current in Xenopus oocytes expressing a murine TauT (55) and in Bergmann glia (30). The current in the latter case is voltage dependent and strictly inwardly rectifying. Provided two to three Na ions and one Cl ion translocate to the cytosolic compartment together with one taurine, the apparent electroneutrality of the active taurine uptake in Ehrlich cells could indicate that cations (Na, K) return to the extracellular compartment via TauT as a part of the translocations cycle or that Na returns via the Na/K pump at an increased rate. It is noted that taurine, just like the inhibitory neurotransmitter GABA, increases the Cl permeability when added directly to neurons (8). However, the equilibrium potential for Cl in, for example, Ehrlich cells is more positive than the cell membrane potential (46) and as the cell membrane is not depolarized following addition of taurine, it is assumed that taurine does not increase the Cl conductance in the Ehrlich cells. In a recent report it was demonstrated that an antibody raised against the Nterminal part of the human TauT recognizes three protein bands from Ehrlich cells in the range of 50–70 kDa, as well as two protein in the 25–30 kDa region, and that stimulation with the PKC activator phorbol-myristateacetate (PMA) increased the intensity of the 70-kDa band (35). A 30-kDa protein has also been identiﬁed by Han et al. (56) using an antibody raised against TauT from rat. Furthermore, three translation products of TauT (107, 69, and 49 kDA) have been identiﬁed in the epididymis from mice (57). Whether the protein bands represent

31 TauT in conﬁgurations with different afﬁnities to taurine, Na and Cl and or different substrate coupling ratios has yet to be revealed.

Taurine Uptake: Regulation by Phosphorylation Taurine uptake is reduced following stimulation of PKC in a variety of cells, including Ehrlich cells (58), rat astrocytes (59), MDCK cells (56), and aortic endothelial cells from cow (60). Furthermore, microinjection of polyclonal antibodies, raised against an amino acid sequence that corresponds to a potential PKC phosphorylation site in the fourth intracellular segment, stimulates taurine inﬂux in Xenopus oocytes expressing TauT (56). It appears that Ser322 on TauT is the PKC target (61) and that PKC-mediated phosphorylation decreases the afﬁnity of TauT for taurine (56,59). Modulation of cellular cAMP stimulates Na-dependent taurine uptake in Ehrlich cells (58) and in rat heart (62), whereas lipid-permeable cAMP analogues reduce the taurine uptake in Xenopus oocytes expressing murine TauT (55). Calyculin A, which is a potent inhibitor of serin and threonine protein phosphatases (PP1, PP2A, PP3), inhibits taurine uptake and impairs the stimulating effect of cAMP on taurine uptake in Ehrlich cells (58). It has been proposed that TauT in Ehrlich cells exists in three conﬁgurations or states, that is, States I, II, and III, with low, normal and high transport activity, respectively (Fig. 2) (58). State I ↔ State II transition is governed by PKC-mediated phosphorylation and PP1, PP2, or PP3–mediated dephosphorylation, whereas State II : State III transition involves cAMP and PKA (58). Treatment of Ehrlich cells with calyculin A reduces the coupling ratio of Na:taurine, as well as the afﬁnity of TauT to Na and taurine (21). As Na is required for binding of taurine to TauT (19); it has been suggested that State II → State I involves PKC-mediated phosphorylation of TauT and subsequently a conformational change in TauT, which affects Na binding to the transporter (21).

Taurine Uptake: Regulation by Osmolarity Acute reduction in the extracellular osmolarity to 50% of the isotonic value reduces taurine uptake in Ehrlich cells by 70%, and it appears that the reduced uptake is the result of the reduction in the osmolarity rather than the results of a reduction in ionic concentration (9). The cell membrane depolarizes about 20 mV following cell swelling in media with half of the normal

32 osmolarity (46), which accounts for most of the reduction of the uptake (see 52). PKC activity is only slightly increased following hypotonic exposure (63), and, provided the reduced taurine uptake seen under hypotonic conditions reﬂects that a larger fraction of TauT is in State I, that is, in a state with reduced transport activity, it has to be assumed that cell swelling also reduces the activity of a calyculin A–sensitive phosphatase. Cells transferred to a hypertonic solution increase the transcription of genes coding for proteins involved in the active uptake and synthesis of compatible osmolytes. The synthesis of mRNA coding for TauT as well as taurine accumulation are increased in, for example, MDCK cells, astrocyte primary cultures, and Caco-2 cells, as part of the adaptation to hyperosmotic medium (24,64, 65). The accumulation of sorbitol and betaine is concomitantly increased, and a tonicity-responsive enhancer (TonE) has been identiﬁed at the 5 terminal of the genes coding for the sorbitol transporter (SMIT), the betaine transporter (BGT1), and aldose reductase, which is involved in the synthesis of sorbitol (66,67). It appears that a cytosolic transcription regulator TonEBP (TonE binding protein) becomes phosphorylated in a process that seems to involve tyrosine kinases and the mitogen-activated protein kinase (MAPK) p38, and that TonEBP translocates to the nucleus, where it binds to TonEs within a few hours after the hypertonic exposure (67–69). No tonicity-responsive element has yet been identiﬁed for the taurine transporter. However, exposing mammalian cells to a high concentration of extracellular taurine results in a reduction in the mRNA coding for TauT, the expression of TauT protein, as well as taurine uptake (65,70,71). The TauT gene is in itself considered as the target for adaptive regulation by dietary taurine availability (15,72). Two isoforms of mRNA encoding for TauT have been detected in LLCPK1 cells, that is, a 7.2-kb product, that is downregulated following taurine exposure and a 9.6-kb product, which is unaffected by taurine exposure (70). Han et al. (72) characterized the promoter region of the TauT gene from rat kidney and identiﬁed two estrogen receptor half sites, a TATA box, a TG22/(A-C)22, four DNA consensus binding sites for the transcription factors Sp1, an overlapping site for WT-1/EGR-1/Sp1, plus two consensus p53 half-sites (72). Sp1 is required for the basal promoter activation, whereas the TG repeat is critical for the full expression of the TauT gene (72). WT-1 and/or EGR1 are required for enhanced taurine transport activity (15). p53 binds directly to the TauT promoter and represses the transcription of the tauT gene (15). Taurine-deﬁcient kittens suffer from renal developmental abnormalities and p53-overexpressing mice

Lambert exhibit renal hypoplasia and renal insufﬁciency; thus it has been proposed that genes coding for TauT is a downstream target gene of p53 that couples p53 to renal development and apoptosis (15).

VOLUME-SENSITIVE TAURINE RELEASE Regulatory Volume Decrease: Swelling-Induced Taurine Release Taurine release from mammalian cell under isotonic conditions is low and resembles in the case of Ehrlich cells taurine uptake via TauT with respect to pH and membrane potential sensitivity (52). Taurine release via the Na-dependent taurine transporter has previously been demonstrated from cells during ischemia (73) and from Bergmann glia following depolarization of the membrane (30). Exposing cells to a hypotonic medium elicits an initial cell swelling, which reﬂects high water permeability and water equilibration, followed by a reduction in the cell volume toward the initial value (Fig. 3A), which reﬂects net loss of organic/inorganic osmolytes and cell water. The swelling-induce taurine efﬂux increases in a variety of cells more or less exponentially with the reduction in the extracellular medium, that is, with the increase in cell volume (9,74–76). The cellular taurine concentration in, for example, Ehrlich cells under isotonic (300 mOsm) conditions is estimated at 40–53 mM (9,21), and the taurine loss in Ehrlich cells accounts for about 10% of the total osmolyte loss within the initial minutes following cell swelling in the hypotonic medium (150 mOsm) (52). However, the swellinginduced Cl efﬂux pathway in Ehrlich cells inactivates within 10 min following hypotonic exposure (77), and the fraction of the osmolyte loss accounted for by taurine and other amino acids increases to 30% following 40-min hypotonic exposure (78). An increased catabolism of organic osmoeffectors contributes to the RVD response in some cell types (79), and it is estimated that oxidation accounts for about 30% of the reduction in the alanine content in Ehrlich cells (80), whereas the reduction in the cellular content of the other amino acids is accounted for by an increased efﬂux most probably via the swelling-induced taurine leak pathway (9). The cellular taurine concentration in NIH3T3 cells is estimated at 10 mM, and about 90% of the taurine is released from the NIH3T3 cell within the ﬁrst 15 min following exposure to the 50% hyposmotic solution (7). With respect to the swelling-induced release of organic osmolytes, focus has in the recent years been on (i) characterization of the cellular signaling cascades activated

Taurine Homeostasis

33

Fig. 3. Effect of PLA2 and 5-LO inhibitors on the rate of the regulatory volume decrease and the taurine efflux following hypotonic cell swelling. A, Ehrlich cells were at time zero exposed to hypotonic solution with half of the isotonic osmolarity and the cell volume followed electronically with time using a Coulter counter system. RO 31-4493 (1 M), RO 31-4639 (0.5 M), or ETH 615-139 (4 M) was present from the time of hypotonic exposure. B, Ehrlich cells, equilibrated with 14C-labeled taurine, were transferred to hypotonic solution with half the original osmolarity and the taurine release was followed within the initial 1.5 min. The rate constant for the initial taurine efflux (min1 g cell water/g cell dry wt) was estimated from slope of the efflux curve and given relative to the hypotonic control with no inhibitor added. RO 31-4639 (RO, 2.4 M), NDGA (50 M), and ETH 615-139 (10 M) were added at the time of hypotonic exposure. C, HeLa cells, grown to 80% confluence in 35-mm-diameter dishes and incubated with 14C-labeled taurine for 2 h, were washed with isosmotic solution to remove excess extracellular 14C-taurine. One milliliter of experimental solution was added to the dish after the final wash, left for 2 min, and transferred to a scintillation vial for estimation of 14C activity. This procedure was repeated every 2 min for 20–30 min with a reduction in osmolarity from 300 mOsm to 200 mOsm at time 6 or 8 min. RO 31-4639 (10 M), NDGA (50 M), ETYA (50 M), ETH 615-139 (10 M), or MK886 (1 M) was present during the whole efflux experiments. The 14C taurine activity remaining inside the cells at the end of the experiment was estimated by lysing the cells with NaOH, washing the dishes two times with distilled water and estimating the 14C activity in the NaOH lysate as well as in both water washouts. The total 14C activity in the cell system was estimated as the sum of activity in all the efflux samples plus the intracellular activity. The natural logarithm to the fraction of 14C activity remaining in the cells at a given time point was calculated and plotted versus time. The rate constant (min1) for the taurine efflux at each time point was estimated as the negative slope of the graph between the time point and the proceeding time point. The maximal rate constant (min1), obtained within 4–6 min following the hypotonic exposure, was estimated and given relative to the hypotonic control with no inhibitors added. D, NIH3T3 cells were loaded with 14C-labeled taurine and the maximal rate constant estimated as indicated for the HeLa cells. BEL (10 or 30 M) was added 30 min before and present during the efflux experiments. ETH 615-139 (10 M) was present during the efflux experiment. Maximal rate constants (min1), obtained by hypotonic cell swelling, are given relative to control value with no inhibitors added. All values in panel B, C, and D are given as mean values SEM. Data in panels A, B, C, and D are modified from references 318, 52, 83, and 76, respectively.

by cell swelling; (ii) evaluation of the role Ca2, eicosanoids, nucleotides, reactive oxygen species (ROS), and lysophospholipids as second messengers in the activation of volume-sensitive osmolyte transporting systems;

(iii) molecular identiﬁcation of the volume-sensitive efﬂux pathway for organic osmolytes; and (iv) identiﬁcation of the volume-sensing elements that elicit osmolyte release.

34 Swelling-Induced Activation of PLA2 It has turned out that activation of a phospholipase A2 (PLA2) and subsequent mobilization and oxidation of arachidonic acid via the 5-lipoxygenase (5-LO) to leukotrienes are essential events in the swelling-induced activation of the taurine-releasing pathways in, for example, Ehrlich cells (Fig. 2). PLA2-mediated hydrolysis of phospholipids to free fatty acids and lysophospholipids has also been demonstrated to be essential in the swellinginduced osmolyte release in human platelets (81), CHP100 neuroblastoma cells (82), and HeLa cells (83). The type of free fatty acid and lysophospholipid produced in a given physiological situation depends on the subcellular localization and the substrate speciﬁcity of the PLA2 activated. Based on functional criteria the PLA2 family has been divided into the cytosolic, Ca2-dependent PLA2 (cPLA2); the secretory, Ca2-dependent PLA2 (sPLA2); the cellular, Ca2-independent PLA2 (iPLA2); and the sn-2 PLA2, which has a remarkable speciﬁcity for short/oxidized groups in the sn-2 position of the phospholipids (84). Three isoforms of cPLA2 have been identiﬁed, that is, the cPLA2 (the classical, group IVA, 85 kDa), the cPLA2 (group IVB, 110 kDa), and the cPLA2 (group IVC, 60 kDa), and the homology between the isoforms is about 30% (84). All three isoforms have two catalytic domains, with a lipase consensus motif in the N-terminal catalytic domain (84). The cPLA2 and PLA2 have a Ca2-binding domain (CaLB) in the N-terminal portion that has a sequence homology with the C2 region of, for example, PKC. cPLA2 has a calveolin binding motif in the C-terminal, whereas cPLA2 is isoprenylated (84). cPLA2 is receptor regulated and activated by various agonists, including hormones, growth factors, cytokines, and endotoxins (84,85). The catalytic action of cPLA2 is Ca2 independent, but submicromolar Ca2 concentration is required for translocation of the enzyme from the cytosol to the Golgi complex, the endoplasmatic reticulum, and the perinuclear membrane and subsequent docking at the intermediate ﬁlament component, vimentin (84). cPLA2 contains several consensus sites for phosphorylation, and phosphorylation at Ser505 by members of the MAP kinase family (ERK, p38) is required to gain full catalytic activity (84,86). cPLA2 prefers phospholipids with choline as the polar head group and a polyunsaturated fatty acid in the sn-2 position (87). No preference for the fatty acid in the sn-1 position has been reported for cPLA2. Several types of low molecular mass sPLA2 have been identiﬁed (group I, II, III, V, X, XII), with sPLA2 (group I) being known for its function as a digestive enzyme (84). sPLA2 contains an N-terminal secretion signal peptide, and it has been suggested that sPLA2 that

Lambert belong to the heparin-binding type (group IIA, IID, V) interact with heparan sulphate proteoglycan, anchored to glycosyl phosphatidyl inositol (GPI) at the cell surface, and that they are sorted into caveolae, that is, the ﬂask-shaped invaginations of the plasma membrane, or internalized, causing mobilization of fatty acids and lysophospholipids at the caveolae or from intracellular domains (84). It is plausible that sPLA2 could be activated by changes in caveolae organization because of swelling-induced unfolding of the membrane and/or reorganization of the cytoskeleton. The nonheparin binding sPLA2, on the other hand, acts directly on the outer leaﬂet of the plasma membrane (84). The iPLA2 group consists of the -isoforms (group VIA, 85–88 kDa) and the -isoforms (group VIB, 90 kDa), but iPLA2 has apparently no demands for a speciﬁc fatty acid in the sn-2 position or polar head group (84). Activation of iPLA2 (VIA) activity requires oligomerization of four iPLA2 monomers, which involves ankyrin repeats (seven to eight per monomer) (86). iPLA2 (VIA) contains no ankyrin repeats but a co-terminal peroxisone localization signal (84). Caspase-3 cleaves iPLA2 at Asp183, but the truncated form, which lacks most of its ﬁrst ankyrin repeat, has an increased phospholipid turnover activity (88). iPLA2 (VIA) has a glycine-rich, nucleotide binding motif, and its catalytic activity appears to be protected by ATP and other nucleotides (86). Reactive oxygen species stimulate iPLA2 activity (89), and iPLA2 has been reported to be involved in eicosanoids synthesis (84). It has also been suggested that iPLA2 may potentiate the function of endogenous cPLA2 or that iPLA2 and cPLA2 in conjugation act on phospholipid microdomains rich in arachidonic acid (88). Various inhibitors have been used to identify the PLA2 type involved in swelling-induced cellular signaling. The RO 31-4493, RO 31-4639 compounds are designed to block pancreatic sPLA2 (90). AACOCF3 is a membranepermeable arachidonic acid analogue that presumably binds tightly to the active site of cPLA2 and reduces its activity (91), whereas bromoenol lactone (BEL) inhibits iPLA2 isoforms (84). The ability of Ehrlich cells to regulate the cell volume following osmotic exposure is impaired in the presence of RO 31-4493/RO 31-4639 (Fig. 3A) but only partly inhibited by AACOCF3 (92), indicating that several types of PLA2 are activated in Ehrlich cells by cell swelling. Tinel et al. (93) have similarly proposed that different types of PLA2 are involved in volume-sensitive signaling in rat inner medullar collecting duct cells (93). The cPLA2 is distributed uniformly throughout the Ehrlich cells (94) but predominantly in the nucleus in MDCK cells, HeLa cells, and bovine endothelial cells (95). In the case of Ehrlich cells

Taurine Homeostasis it has been demonstrated that the isoform, not the isoform, translocates to the nucleus within the ﬁrst minutes following hypotonic exposure and that the translocation process does not require MAP-kinase–mediated phosphorylation nor an intact F-actin cytoskeleton (94). Translocation of cPLA2 to the nuclear membrane has also been demonstrated following stimulation with Ca2 ionophores in, for example, rat alveolar cells (97) and Ehrlich cells (94). Using Ca2-mobilizing agonists it has been demonstrated that the differential membrane targeting mechanism of cPLA2, which involves the CaLB domain, is a function of the absolute amplitude of the intracellular Ca2 concentration ([Ca2]i) and the duration of the elevation, that is, cPLA2 translocate to the Golgi complex at a sustained [Ca2]i at 100–125 nM but to the endoplasmatic reticulum and the perinuclear membrane at 210–280 nM (98). Because no measurable Ca2 signaling is recorded in suspension of Ehrlich cells following hypotonic exposure (99) it is assumed that the prevailing cellular Ca2 concentration is sufﬁcient for translocation and binding of cPLA2 to the nuclear envelope in Ehrlich cells. The swelling-induced taurine efﬂux from Ehrlich cells (Fig. 3B) and HeLa cells (Fig. 3C) is reduced by RO 31-4639, whereas BEL inhibits the volume-sensitive taurine release from NIH3T3 cells (Fig. 3D) but has no effect on taurine release from HeLa cells (Lambert, unpublished data). AACOCF3 reduces the swellinginduced taurine efﬂux from the rat cerebral cortex (100) but has no effect on the volume-sensitive taurine efﬂux from NIH3T3 cells (76). Thus several types of PLA2 are involved in swelling-induced taurine efﬂux. Activation of sPLA2 by exogenous addition of melittin has been shown to induce taurine release from NIH3T3 cells under isotonic conditions in a process that involves the same cellular signaling system and taurine transporting system, as seen after hypotonic exposure (76). However, AACOCF3 reduces the melittin-/sPLA2–induced taurine efﬂux from NIH3T3 cells and, because sPLA2 activity apparently depends on active cPLA2 (84), it has been suggested that iPLA2, cPLA2, and sPLA2 all regulate taurine release in NIH3T3 cells (76). Arachidonic Acid: Direct Effects of a Fatty Acid Even though the phospholipids in mammalian cells constitute a large pool of arachidonic acid the concentration and availability of free arachidonic acid for synthesis of the biological potent eicosanoids are rather limited. However, arachidonic acid is rapidly mobilized following stimulation of phospholipases and diacylglycerol lipases with antigens, hormones, and Ca2-mobilizing agents (101).

35 Arachidonic acid is also released by osmotic exposure in, for example, Ehrlich cells (Fig. 4A) and the release occurs most likely at the nuclear envelope (94). Arachidonic acid, once released, is either reacylated into the membranes, assigned a role as a second messenger in it self or converted to (i) epoxides by cytochrom P450, (ii) prostaglandins, thromboxanes, and prostacyclines via the constitutive and inducible cyclooxygenases systems (COX1/COX2), or (iii) hydroxy fatty acids and leukotrienes by the lipoxygenase system. Patch-clamp studies of K have demonstrated that free fatty acids regulate the activity of ion channels and have raised the possibility that these compounds are endogenous channels regulators (102). The fatty acid– induced activation seems not to involve phosphorylation, GTP-dependent proteins, cyclic nucleotides, nor Ca2 but more likely either modulation of the ﬂuidity or curvature of the membrane, or a direct interaction of the fatty acid with the channel proteins or a closely associated component (102,103). Arachidonic acid is also reported to activate Cl and propionate transport via an unspeciﬁc anion transport pathway in Ehrlich cells suspended in isotonic medium (104), as well as a taurine efﬂux under isotonic conditions via a system that is sensitive to 4,4-diisothiocyanostilbene- 2,2-disulfonic acid (DIDS) (105). Organic osmolytes (sorbitol, betaine) are similarly demonstrated to be released under isotonic conditions from inner medulla collecting duct cells following addition of arachidonic acid (106). The swellingactivated K and Cl channels, on the other hand, are inhibited by unsaturated fatty acids in, for example, Ehrlich cells (104,107) and rat cerebellar astrocytes (108). In the case of Ehrlich cells it has been shown that arachidonic acid acts directly on the swellinginduced Cl channel (104). The effect of arachidonic acid on swelling-induced taurine efﬂux depends on the cell type, that is, the fatty acid stimulates taurine release from Ehrlich cells (52) but inhibits taurine release from HeLa cells (109) and 3T3NH cells (7). It is emphasized that the inhibitory and stimulatory effects of arachidonic acid on ion transporting systems all are obtained with fatty acids concentration in the micromolar range, indicating that their effect on osmolyte release under physiological conditions could be rather restricted. Lipoxygenase Products as Second Messengers Lipoxygenase activity has been demonstrated to be required for the RVD response in several cell types. Lipoxygenases catalyze the insertion of oxygen into arachidonic acid at positions 5 (5-LO), 8 (8-lipoxygenase, 8-LO), 12 (12-lipoxygenase, 12-LO), or 15 (15-lipoxygenase,

36

Lambert

Fig. 4. Eicosanoid metabolism and the effect of exogenous addition of LTD4 on the rate of the regulatory volume decrease following hypotonic exposure and on taurine release under isotonic conditions. A, Ehrlich cells, loaded with 3H-labeled arachidonic acid, were at time zero exposed to isotonic or hypotonic conditions and the release of the 3H-activity followed with time. Arachidonic acid release was linear under isotonic conditions for 8 min but biphasic under hypotonic conditions, that is, increased linearly within the initial 2 min and leveled within the subsequent 6 min. The rate of release, estimated from the slope of the synthesis curve in the time periods given in the figure, is given relative to the isotonic control value. B and C: The synthesis of LTC4/LTD4 and of PGE2 and PGF2 in Ehrlich cells was followed with time for 15 min under isotonic conditions and after exposure to hypotonic solution with half osmolarity by serially isolating cell-free medium (centrifugation) and estimating the leukotrienes/prostaglandins by radio immunoassay. The eicosanoid production (ng/g cell dry weight per min) was estimated, and values are given relative to the value from isotonic control cells. The radio immunoassay used for detection of leukotrienes did not discriminate between LTC4 and LTD4. D, Ehrlich cells were at time zero exposed to hypotonic solution with half of the isotonic osmolarity, and the cell volume followed electronically with time using a Coulter counter system. LTD4 (3 or 10 nM) was added at the time of maximal cell swelling, that is, at time 0.9 min. PGE2 (5 M) was present from the time of hypotonic exposure. The rate of volume recovery (fl/min) was calculated as the cell shrinkage within the first minute following addition of LTD4 or within 1 and 4 min in control cells and cells treated with PGE2. E, Ehrlich cells, equilibrated with 14C-labeled taurine, were transferred to isotonic solution with or without LTD4 (1, 5, 10 nM), and the initial taurine release followed within the initial 1.5 min. The rate constant for the initial taurine efflux (min1 g cell water/g cell dry wt) was estimated from slope of the efflux curve. All data in panels A–E are given as mean values SEM and modified from (92) (panel A), (124) (panels B and C), (127) (LTD4, panel D), (124) (PGE2, panel D), and (105) (panel E).

15-LO). The 5-LO is a single polypeptide chain with a molecular weight of about 72–80 kDa, and it has a large catalytic domain containing a single nonheme iron (110). The 5-LO catalyzes the ﬁrst two steps in the synthesis of leukotrienes, that is, oxidation of arachidonic acid into 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and the subsequent dehydration of the 5-HPETE to the unstable

leukotriene A4 (LTA4). A gluthation S-transferase conjugates gluthathione (-glutamylcysteinylglycine, GSH) with LTA4 to form LTC4. Sequential loss of the glutamic acid and the glycine residues from LTC4 leads to the formation of LTD4 and LTE4 (111). The 5-LO requires substrates with double bonds at carbons 5 and 8 for catalysis and arachidonic acid (5,8,11,14-eicosatetraenoic acid)

Taurine Homeostasis as well as EPA (5,8,11,14,17-eicosapentaenoic acid) are consequently good substrates (112). The 5-LO resides in the cytosol in blood PMNLs and inside the nucleus in alveolar macrophages and stimulation of cells leads to translocation of the 5-LO to the nuclear membrane, where it associates with the 5-lipoxygenase protein (FLAP) (110). FLAP, a 1-kDa protein with three transmembrane spanning regions and two hydrophilic loops, seems to be involved in either the activation of 5-LO or presentation of arachidonic acid to the 5-LO (113). The 5-LO/FLAP inhibitor MK886 binds to FLAP with high afﬁnity and prevents the membrane association of 5-LO and the subsequent oxidation of arachidonic acid (114). H2O2 and hydroperoxides activate the 5-LO by oxidation of its nonheme iron from Fe2 to Fe3 (113). Ca2 bind to the 5-LO (KD ⬃ 6 M, two Ca2 binds per 5-LO molecule) causing an increase in the hydrophobicity of the 5-LO and promotion of the 5-LO association with FLAP and the membrane (110). ATP is also required for 5-LO activity and it appears that the 5-LO has a single ATP binding site at low Ca2 concentrations but two ATP binding sites at high Ca2 concentrations (115). The MAP-kinases ERK1/2 and p38 (116), as well as tyrosine kinase activity (117), have been demonstrated to be involved in phosphorylation and activation of 5-LO. The 5-LO also contains six conserved histidine residues, an actin binding sequence, a nuclear localizing sequence, as well as a Src homology 3–binding domain (SH3) domain near the carboxyl terminus, and it is plausible that these motifs are involved in the translocation process (110,117). The RVD response in Ehrlich cells (Fig. 3A) and human ﬁbroblasts (118), as well as the volume-sensitive taurine efﬂux pathway in, for example, Ehrlich cells (Fig. 3B), HeLa cells (Fig. 3C), NIH3T3 ﬁbroblasts cells (Fig. 3D), C2C12 myotubes (119), and pig muscle (120), are blocked by addition of the 5-LO inhibitor ETH 615139. Swelling-induced taurine efﬂux from HeLa cells is furthermore reduced when the 5-LO is inhibited by addition of nordihydroguaiaretic acid (NDGA)—a potent antioxidant, eicosatetraynoic acid (ETYA)—which looks like arachidonic acid but contains triple bonds instead of double bonds and that acts as a substrate inhibitor or the 5-LO/FLAP inhibitor MK886 (Fig. 3C). Similarly, it has been demonstrated that ETYA and the 5-LO inhibitor ketoconazole inhibit release of sorbitol, betaine, and myoinositol from renal papillary epithelial cells (121). In human platelets, 12-LO oxidizes arachidonic acid following osmotic cell swelling (122). Thus lipoxygenase activity is required to activate the volume-sensitive release of organic osmolytes. The leukotrienes LTB4, LTC4, LTD4, and LTE4 are biologically very potent substances, that is LTB4 stim-

37 ulates chemokinesis, chemotaxis, adherence, aggregation of cells, and lysosomal degradation, whereas LTC4, LTD4, and LTE4 produce vaso-constriction and bronchoconstriction, increase the vascular permeability, and stimulate mucus secretion in the lungs (111). Ehrlich cells produce a variety of 5-LO products (123), and the leukotriene synthesis is increased (Fig. 4B), whereas the prostaglandin synthesis is reduced (Fig. 4C) following hypotonic exposure. It is emphasized that the prostaglandin synthesis in the Ehrlich cells is also increased by osmotic cell swelling in the presence of exogenous arachidonic acid, indicating that the 5-LO is favored under hypotonic conditions in the absence of exogenous substrate (124). Thus PLA2 and 5-LO apparently translocate to the same site on nuclear membrane in Ehrlich cells following cell swelling, and this intimate contact appears to be important for the subsequent RVD response. LTD4 has been assigned a key role as a second messenger in the swelling-induced activation of taurine efﬂux in Ehrlich cells. This assignment is based on the following observation: (i) arachidonic acid is released during the initial minutes following hypotonic exposure (Fig. 4A); (ii) the synthesis and release of LTC4/LTD4 is concomitantly increased (Fig. 4B), whereas the synthesis of PGE2 and PGF2 is reduced (Fig. 4C); (iii) an increase in the precursor availability enhances the rate of the volume regulatory response in hypotonic medium (125); (iv) exogenous addition of LTD4, at a concentration and under experimental conditions that do not provoke Ca2 mobilization, accelerates the RVD response following hypotonic exposure (Fig. 4D), whereas the LTD4 precursor LTC4 and the LTD4 metabolite LTE4 (124) and PGE2 (Fig. 4D) have no accelerating effect on the rate of the swelling-induced volume reduction; (v) LTD4 receptor antagonists reduce the rate of volume regulation (126,127); and (vi) LTD4 induces taurine efﬂux under isotonic conditions with an EC50 value less than 1 nM (Fig. 4E). Because 1 nM LTD4 causes no increase in cellular Ca2 and taurine release from Ehrlich cells is larger in the absence of extracellular Ca2 (52), it is assumed that the effect of LTD4 represents a Ca2-independent effect on the taurine-releasing system. LTD4 has also been assigned a role in the swelling-induced K loss from Ehrlich cells (128), whereas LTD4 is not involved in the concomitant activation of the volumesensitive Cl channel in Ehrlich cells (129). Leukotrienes also appear to be involved in release of inositol in glia cells (130), and LTD4 in particular plays a role in the RVD response in the rat colonic enterocytes (131) and the rat distal colon (132). The 5-HETE has been shown to potentiate swelling-induced taurine loss from HeLa cells (83), whereas the RVD response in swollen human

38 platelets involves the 12-lipoxygenase product hepoxilin A3 (81).

Role of Ca2ⴙ and Calmodulin A reduction in the extracellular osmolarity is in some cells accompanied by an increase in [Ca2]i, and even though the subsequent RVD response has been demonstrated to be Ca2 dependent in, for example, isolated crypts of mouse distal colon (132), astrocytes (133), and human cervical cancer cells (134). It has been suggested that the swelling-induced increase in [Ca2]i is an epiphenomenona, which is not related to RVD response (135,136). The swelling-induced increase in [Ca2]i often involves an increased Ca2 inﬂux from the extracellular compartment, and the inﬂux is in the case of the rat inner medullar collecting duct cells proceeded by Ca2 release from intracellular stores by a process that involves arachidonic acid metabolism (93). Other cell types, including human lymphocytes and Ehrlich cells, perform RVD response without any detectable increase in [Ca2]i (99,137). Swelling-induced taurine efﬂux from neonatal rat astrocytes (138) and human jurkat T-lymphocytes (139) requires extracellular Ca2 for activation, whereas the volume-sensitive taurine release from, for example, astrocytes (133), cerebellar granule neurons (140), and cells in the rat supraoptic nucleus (141), seems not to require Ca2 inﬂux for activation. In the case of Ehrlich cells (52) and Xenopus laevis erythrocytes (142) it has even been demonstrated that the swelling-induced taurine efflux is even improved in the absence of extracellular Ca2. Addition of a Ca2 ionophore at the time of maximal cell swelling in hypotonic medium accelerates the volume recovery in, for example, Ehrlich cells (124), and because the rate of the RVD response in many cell types is limited by the K conductance, the accelerating effect of Ca2 agonists on the RVD response is often taken to reﬂect an increase in [Ca2] and a subsequent activation of a Ca2-sensitive K conductance (1). Addition of Ca2 mobilizing agonists at the time of hypotonic exposure potentiates the swelling-induced taurine efﬂux from HeLa cells and accelerates the subsequent inactivation of the volume-sensitive taurine efﬂux pathway. It has been demonstrated that the potentiation of the swellinginduced taurine efﬂux in HeLa cells most likely reﬂects a Ca2 effect on Ca2-sensitive elements in the swellinginduced signaling cascade (PKC), whereas the Ca2 agonist effect on the inactivation of the volume-sensitive transport pathway reﬂects inactivation of the volumesensitive taurine channel as a result of an accelerated

Lambert restoration of the cell volume (Falktoft and Lambert, unpublished data). Ca2-mobilizing agonists have also been demonstrated to stimulate the volume-sensitive release of organic osmolytes from red blood cells (143) and airway epithelial cells (144). It is noted that LTD4 added at a high dose (100 nM) acts as a Ca2-mobilizing agonist in a variety of cells and that the LTD4induced increase in [Ca2]i in the case of Ehrlich cells is reduced in the absence of extracellular Ca2 (Fig. 5A) and represents a PLC- and inositol-1,4,5-triphosphate–mediated Ca2 release from the intracellular Ca2 stores, as well as Ca2 inﬂux from the extracellular compartment via a Ca2 channel that is neither voltage nor stretch activated (145). The LTD4-induced increase [Ca2]i is in the Ehrlich cells accompanied by net loss of KCl via Ca2-activated K and Cl channels, loss of cell water, and consequently cell shrinkage (107,124). It is emphasized that the effect of 100 nM LTD4 on taurine release under isotonic conditions is quantitatively similar to the effect of 1 nM LTD4 (Fig. 5B), and it is consequently assumed that the effect of LTD4 on taurine release in Ehrlich cells under isotonic conditions is Ca2 independent. The Ca2-mobilizing effect of LTD4 involves speciﬁc G-protein–coupled receptors (CysLT1 receptor) and it has been demonstrated in the case of human epithelial cells that the LTD4-induced Ca2 mobilization from intracellular stores involves a pertussis toxin–insensitive G-protein, whereas the LTD4-induced Ca2 inﬂux involves a pertussis toxin–sensitive, heterotrimeric G-protein (subtype Gi3) (146,147). Monomeric G-proteins (RhoA) are also demonstrated to be involved in LTD4-induced Ca2 signaling in epithelial cells (148) in a process that involves association of active RhoA with an Src kinase (c-Src), as well as a Gmediated targeting of RhoA and PLC (PLC1) to the plasma membrane (149). The swelling-induced taurine release from Ehrlich cells (Fig. 5C) and HeLa cells (Fig. 5D) (150) is inhibited in the presence of the calmodulin antagonists pimozide and W-7, whereas the taurine efﬂux from NIH3T3 cells is potentiated by W-7 (Fig. 5E). Inhibition of swelling-induced taurine release by calmodulin antagonist has also been demonstrated in human erythroleukemia cells (151) and cultured rat astrocytes (152). A linear correlation between the potency of a drug as a calmodulin antagonist and its inhibitory effect on the swelling-induced taurine efﬂux has previously been demonstrated in HeLa cells (150). cPLA2 activity is stimulated by calmodulin (84), whereas iPLA2 forms a catalytic inactive complex with calmodulin, and its activation requires dissociation from calmodulin (153). Thus Ca2 and calmodulin are involved in the regulation

Taurine Homeostasis

39

Fig. 5. Role of Ca2 and calmodulin in taurine release. A, Fura-2 loaded Ehrlich cells were preincubated in either Ca2-containing standard medium (1 mM Ca2) or in Ca2-free standard medium (2 mM EGTA) and the increase in cellular [Ca2]i was estimated on cells in suspension using a luminescence spectrometer, excitation wavelength of 340 nm and 380 nm, and measuring the emission at 510 nm. B, Ehrlich cells, equilibrated with 14C-labeled taurine, were transferred to isotonic solution in the absence or presence of LTD4 (1 nM/100 nM), ATP (10 M), or UTP (10 M). The initial taurine release was followed within the 1 min and the rate constant (min1 g cell water/g cell dry wt) for the initial taurine efflux estimated from slope of the efflux curve. C, Ehrlich cells, equilibrated with 14C-labeled taurine, were transferred to hypotonic solution with half of the isotonic osmolarity in the absence or presence of pimozide (12 M). The initial taurine release was followed within the 1 min and the rate constant (min1 g cell water/g cell dry wt) for the initial taurine efflux estimated from slope of the efflux curve. The rate constant from pimozide treated cells is given relative to the one for hypotonic control cells. D, Swelling-induced taurine release was followed from HeLa cells in hypotonic medium with two thirds of the original osmolarity in the absence or the presence of pimozide (10 M) or W-7 (50 M) and the maximal rate constant estimated as indicated under Fig. 3C. The rate constant from pimozide and W-7 is given relative to the hypotonic control cells. The W-7 data represents three sets of paired experiments. E, Swelling-induced taurine release was followed from NIH3T3 cells in hypotonic medium with two thirds of the original osmolarity in the absence or the presence of W-7 (50 M), and the maximal rate constant estimated as indicated under Fig. 3C. All values in panel A-E are given as mean values SEM. Data are modified from (127) (panel A), (105) (panels B), (52) (panel C), (83) (pimozide data, panel D) and (76) (panel E).

of the swelling-induced taurine release, and the opposing effect of calmodulin antagonists on the swellinginduced taurine efﬂux from, for example, HeLa cells and NIH3T3 cells most probably reﬂect that the PLA2 activated by cell swelling varies between the two cell lines. It has previously been demonstrated that the RVD response in Ehrlich cells is impaired in the presence of Ca2/calmodulin antagonists and that the inhibition is lifted by exogenous addition of LTD4 (126). It is noted that LTD4 binds to high-afﬁnity binding site in Ehrlich

cells and that LTD4 binding requires Ca2 (154). Furthermore, the accelerating effect of LTD4 on the RVD response in Ehrlich cells is improved and LTD4 binding to a high-afﬁnity receptor becomes irreversible in the presence of Ca2/calmodulin antagonists (126,154). Thus Ca2 and calmodulin appear to interfere at a step upstream to the LTD4 synthesis step, which could be not the PLA2 activation, and at a step that involves the LTD4-mediated signaling. The translocation and binding of cPLA2 and 5-LO to the nucleus requires Ca2

40 (see above) and could also represent a Ca2/calmodulin sensitive step. Other possibilities could be related to the components of the cytoskeleton, which are regulated by Ca2/calmodulin and myosin light chain kinase (96). On the other hand, the calmodulin-regulated step in the volume-sensitive signaling cascade in HeLa cells does not involve the calmodulin-dependent kinase II (CaMKII) (155). Furthermore, as the protein tyrosine phosphorylation pattern, induced by Ca2 exposure, differs from the pattern, induced by osmotic exposure, it also seems unlikely that a volume-sensitive tyrosine kinase is responsible for the Ca2-induced potentiation of the swelling-induced taurine loss (156,157).

Role of Endogenous and Exogenous ATP A number of electrophysiological studies have shown that intracellular ATP, or nonhydrolyzable analogues of ATP, are required for sustained activity of swelling-activated anion conductances (158). Ballatori et al. (159,160) reported a close correlation between intracellular ATP and swelling-activated taurine in the human hepatoma cell line, Hep G2, and they suggested that the inhibitory effect of a series of anion channel blockers could be secondary to a reduction in the cellular ATP concentration. ATP acts in some cell lines as an autocrine factor following osmotic cell swelling and elicits the subsequent ion loss (161,162) and modulates taurine loss (163). It is reported that swelling-induced ATP release is mediated by the volume-regulated anion channel (VRAC, 158), the volume-dependent, arachidonic acid sensitive large conductance anion channel (VDACL, 164) and is facilitated by CFTR expression (165). Furthermore, cibacron blue, which is a rather unspeciﬁc purinergic receptor antagonist, has been shown to inhibit the swelling-induced taurine release from HeLa cells (166). Extracellular application of ATP, in the millimolar concentration range, inhibits swelling-activated anion currents and osmolyte ﬂuxes in a number of cell types (167,168), whereas application of extracellular ATP in the micromolar concentration range to the human tracheal cell line, 9HTEo-, causes activation of taurine efﬂux under isotonic conditions and synergistically stimulates taurine efﬂux in hypotonic conditions in a Ca2dependent process (144). ATP binds to the purinergic receptors P1 and P2X/P2Y, and binding to P1 and P2Y receptors affects the intracellular concentration of cAMP, IP3, and Ca2 (169,170), whereas binding to P2X activates ligand-gated ion channels (171). It has been suggested that ATP, released by cell swelling,

Lambert binds to G-protein–coupled P2Y2 receptors and subsequently leads to a PLC-mediated Ca2 mobilization and activation of Ca2-sensitive K channels (see 162). It is noted that mechanical stress, by, for example, centrifugation, leads to release of ATP from Ehrlich cells and that ATP binds to P2Y1 and P2Y2 receptors, causing an increase in [Ca2]i, activation of Ca2 sensitive K- and Cl-currents and subsequent cell shrinkage (172,173). Activation of Ca2-sensitive channels has previously been demonstrated to accelerate the RVD response in osmotically swollen cells and to induce cell shrinkage in cells under isotonic conditions (127). ATP or UTP, when added in the micromolar concentration range, causes only a minor increase in the taurine efﬂux from Ehrlich cells (Fig. 5B) and HeLa cells (83) under isotonic conditions. However, because ATP at a similar concentration hyperpolarizes the plasma membrane in Ehrlich cells (173) and the taurine efﬂux is reduced under hyperpolarized conditions (52), it is assumed that the ATP effect on taurine release in Ehrlich cells is not secondary to a change in the membrane potential. ATP released to the extracellular compartment is rapidly degraded by ectonucleotidases, which constitute a heterogeneous group of enzymes with variation in tissue distribution and substrate speciﬁcity (174). However, even though exogenous addition of ATP or UTP in the micromolar range to HeLa cells at the time of hypotonic exposure potentiates the swelling-induced taurine efﬂux, inclusion of the ectoATPase apyrase to the hypotonic solution does not affect the swelling-induced taurine efﬂux from HeLa cells, indicating that ATP does not act as an autocrine factor in the swelling-induced activation of taurine transporting pathways in HeLa cells (83).

Reactive Oxygen Species and Tyrosine Phosphorylation Reactive oxygen species (ROS) have for many years been regarded as toxic by-products in the oxidative metabolism and to be involved in oxidative stress, lipid peroxidation, oxidative modiﬁcations of proteins, DNA damage, and apoptosis. However, in recent years it has turned out that intracellular signaling is regulated by the intracellular redox state and that ROS, besides being produced in phagocytic cells as part of the defence against bacteria, also act as intracellular signaling molecules, that is, ROS activate transcription factors and modulate growth stimulatory signaling cascades involving receptors coupled to tyrosine kinase activity or G-proteins in both phagocytic and nonphagocytic cells (see 175 and 176). More recently it has been demonstrated

Taurine Homeostasis that ROS are generated in NIH3T3 cells (76) and in skeletal muscle cells (120) following hypotonic exposure. The PLA2 products lysophosphatidylcholine (LPC) and arachidonic acid are known to generate ROS in a process that requires NAD(P)H-oxidase activity (177,178). Because of the swelling-induced ROS production in, for example, NIH3T3 cells is reduced in the presence of the iPLA2 inhibitor BEL (76), it has been suggest that the ROS-producing step following osmotic exposure is downstream to the PLA2 activation and most probably involves arachidonic acid and/or LPC (76). ROS include the oxygen radicals (superoxides, hydroxyl radicals, peroxyl radicals, alkoxyl radicals), as well as the nonradical oxygen species (hydrogen peroxide [H2O2], lipid hydroperoxide) (Fig. 6). Superoxides are produced from O2 (i) in the mitochondria as a result of leakage of electrons from the electron transferring systems, (ii) in the cytosol during oxidation of xanthine (hypoxanthine) by cytosolic xanthine oxidase, (iii) at the smooth endoplasmatic reticulum during oxidation of unsaturated fatty acids and xenobiotics by cytochrom P450, and (iv) at the plasma membrane by one-electron

41 reduction of molecular O2 by the NAD(P)H oxidase (Fig. 6). H2O2 is produced by dismutation of the superoxide and by a number of peroximal oxidases (urate and D-amino oxidase), whereas the hydroxyl radicals are generated from superoxides and H2O2 by either a HaberWeiss reaction or in the presence of iron ions by Fenton chemistry. H2O2 has the ability to diffuse across biological membranes and is a more stable but weaker oxidizing agent compared to superoxides. Nonenzymatic lipid peroxidation occurs when a free radical abstracts a hydrogen atom from methylene carbon of an unsaturated fatty acid and when the resulting carbon-centred radical reacts with molecular oxygen and forms a peroxy radical. This radical is in itself able to abstract hydrogen from an unsaturated fatty acid resulting in the formation of a lipid hydroperoxide and a carbon-centerd radical. Enzymatic lipid peroxidation involves the cyclooxygenases and the lipoxygenases. Cells possess an efﬁcient antioxidative system that limits the availability and action of free oxidizing radicals, this system includes enzymes (superoxide dismutase, catalase, GSHdependent peroxidase, phospholipid GSH peroxidase),

Fig. 6. Reactive oxygen species—their generation, elimination, and physiological effects. (See text for details [175,176,182,319,320]).

42 as well as dietary, water- and lipid-phase antioxidants (GSH, tocopherols (VitE), VitC, carotenes, VitA, phenolic compounds) (Fig. 6). It is noted that taurine and its precursor cysteine and hypotaurine have also been implicated in cellular defense against oxidative stress (8,179) and that taurine supplementation decreases lipid peroxidation (180). The mechanisms by which ROS exert their effect involve either alteration in the intracellular redox state, that is, a decrease in the ratio between the reduced and the oxidized state of the intracellular glutathione (GSH/GSSH), thioredoxin (TRXred/TRXox), and nicotinamide adenine dinucleotide phosphate (NAD(P)H/NADP), or oxidative modiﬁcation of proteins (175) (Fig. 6). Shifts toward a more oxidized condition are reported to cause inﬂammation and autoimmune and neurodegenerative diseases (181). GSH is an abundant tripeptide in mammalian cells and functions as an intracellular redox buffer, intracellular copper transporter/ buffer, cofactor for the GSH peroxidase, and substrate for the synthesis of, for example, leukotrienes. GSH is regenerated from oxidized GSH (GSSH) by GSH reductase and in enzymatic processes involving NAD(P)H. The concentration of the reduced form of gluthathione under nonstressed conditions is estimated to be about 10–100 fold higher than the concentration of the oxidized form (175,182). A massive extrusion of GSH together with cytochrome c from mitochondria following apoptotic stimuli has been reported, indicating that a decrease in cellular GSH could be associated with apoptosis (182). TRX, a small protein that contains an essential and a highly conserved -Cys-Gly-Pro-Cys- sequence in the active site, is a potent protein disulphide oxidoreductase and able to reduce ROS directly and refold oxidized proteins (182). Oxidized TRX (TRXox) is converted to reduced TRX (TRXred) by TRX reductase. ROS-induced protein modiﬁcations involve (i) oxidation of sulphydryl group in cysteine residues, (ii) formation of intramolecular disulﬁde linkage (conformational change) or intermolecular disulﬁde-linkage (dimerization), and (iii) dityrosine formation (cross-linkage) (Fig. 6). Oxidative stress has been associated with tyrosine phosphorylation (183), and it has been indicated that an increase in tyrosine phosphorylation prolongs the open time of the volume-sensitive taurine efﬂux pathway in NIH3T3 cells (184,185), whereas it shifts the osmosensitivity of the taurine efﬂux in supraoptic astrocytes (186). A tyrosine phosphorylation signaling step downstream to the initial unset of the RVD response seems also to be required for sustained activity of the volumeregulated anion channel (158). From Figs. 7A and 7B it is seen that the swelling-induced taurine release from NIH3T3 cells is rapidly inactivated and inhibited in the

Lambert presence of the selective NAD(P)H oxidase inhibitor diphenylene iodonium (DI) and the water-soluble antioxidant butylated hydroxytoluene (BHT), respectively. On the other hand, the maximal rate constant for the swelling-induced taurine efflux is significantly increased and the inactivation of the efﬂux delayed in the presence of H2O2 (Fig. 7C). The observation that the closing/inactivation of the swelling-induced taurine efﬂux is accelerated in the presence of DI and delayed in the presence of H2O2 could be taken to indicate that ROS most probably increase the open-probability of the volume-sensitive taurine efﬂux pathway in NIH3T3 cells. H2O2 has no immediate effect on taurine release when added to NIH3T3 cells under isotonic conditions, and H2O2 does not affect the osmosensitivity, that is, the degree of cell swelling required for activation of the volume-sensitive taurine efﬂux pathway (76). Furthermore, the effect of H2O2 on the volume-sensitive taurine efﬂux is unaffected by addition of the iPLA2 inhibitor BEL (76) but inhibited by the 5-LO inhibitor ETH 615-139 (76) and the anion channel blocker DIDS (Fig. 7C). Thus ROS seem to interfere with the volumesensitive PLA2/5-LO signaling cascade and/or the activity of the volume-sensitive taurine-releasing pathway in NIH3T3 cells at a step downstream to the swellinginduced activation of iPLA2. From Fig. 7D it is seen that vanadate, a phosphate analogue and competitive inhibitor of the protein tyrosine phosphatase PTP1B (183) in analogy with H2O2, increases the maximal rate constant and prolongs the open-time for the swellinginduced taurine efﬂux pathway in NIH3T3 cells and that the effect is abolished in the presence of DIDS. Vanadate also potentiates swelling-induced taurine efﬂux from rat supraoptic nucleus (186), cerebellar granule neurons (140), and HeLa cells (187). PTP1B contains an essential cystein residue in its catalytic site that forms a thiol phosphate complex during the dephosphorylation process, and H2O2 is reported to oxidize the sulphydryl group of cysteine to form disulphide bonds or to sulphinic acid (reversible), sulphenic acid, or sulphonic acid, causing loss of phosphatase activity (188). The potentiating effects of H2O2 and vanadate on swelling-induced taurine are synergistic (76) and because in vivo stimulation of rat-1 cells with exogenous H2O2 leads to oxidation of multiple protein tyrosine phosphatases (188), it has been suggested that the effect of ROS (H2O2) on the swelling-induced taurine efﬂux from NIH3T3 cells reﬂects oxidation and subsequent inhibition of protein tyrosine phosphatase (PTP1B) activity (76). A variety of protein serine/threonine kinases, for example, MAP kinase, S6 kinase, c-Jun NH2-terminal kinase, protein kinase B (Akt), and PKC, as well as

Taurine Homeostasis

43

Fig. 7. Effect of ROS, antioxidants, and vanadate on the swelling-induced taurine efflux from NIH3T3 mouse fibroblasts. NIH3T3 cells, equilibrated with 14C-taurine, were washed and the efflux followed with time in isotonic NaCl medium, with a shift in the osmolarity from 300 mOsm to 200 mOsm at the time indicated by the arrow. The rate constant (min1) for the taurine efflux was calculated and plotted versus time, as indicated in the legend to Fig. 3. DI (25 M, closed diamonds, panel A) was added to the loading medium 30 min before initiation of the efflux experiments and present throughout the experimental period. BHT (0.5 mM, closed squares, panel B), H2O2 (2 mM, open/closed squares, panel C), vanadate (50 M, open squares, panel D) and DIDS (closed symbols, panels C and D) were present throughout the efflux experiments. Data in panels A and B are reproduced (76). Data in panels C and D represent 4 sets of pained experiments. All values in panels A-D are given as mean values SEM.

protein tyrosine kinases, for example, epidermal growth factor receptor, insulin receptor, Src, and Lck, are regulated by redox in cells (189). Protein tyrosine kinase inhibitors block the RVD response in lymphocytes (190), as well as the swelling-induced taurine release from, for example, human intestine 407 cells (156), HeLa cells (187), NIH3T3 cells (Fig. 8A) and chicken retina (191). Furthermore, the potentiating effect of vanadate and H2O2 on the swelling-induced taurine release from NIH3T3 is impaired in the presence of the protein tyrosine kinase inhibitor genistein (76). Thus a yet unidentiﬁed protein tyrosine kinase seems to be activated by cell swelling, and the ROS-mediated effect on the volume-sensitive taurine efﬂux reﬂects a shift in pro-

tein tyrosine phosphorylation resulting from a concomitant inhibition of phosphatase (PTP1B) activity. In this context it should be noted that 5-LO is modulated by tyrosine kinases (117) and that mitogenic signaling following stimulation with platelet-derived or epidermal growth factors has been demonstrated to involve ROSmediated inactivation of PTP1B (175). Inhibitors of receptor protein tyrosine kinases have been demonstrated to partly suppress swelling-induce taurine release (76,192). However, addition of epidermal growth factor (EGF) has a minor potentiating or even inhibitory effect on swelling-induced taurine efﬂux from NIH3T3 cells and rat primary astrocytes (76,192), and it is assumed that the EGF receptor kinase is not the

44

Lambert

Fig. 8. Effect of PP2 and PMA on the swelling-induced taurine efflux from NIH3T3 cells. A and C, NIH3T3 cells, equilibrated with 14C-taurine, were washed and the efflux followed with time in isotonic NaCl medium, with a shift in the osmolarity from 300 mOsm to 200 mOsm at the time indicated by the arrow. The rate constant (min1) for the taurine efflux was calculated and plotted versus time as indicated in the legend to Fig. 3. PP2 (10 nM/50 nM) and genistein (100 M) were present throughout the experimental period. PMA (100 nM) was present only for 10 min before the initiation of the efflux experiment as indicated by the bar. B, Efflux experiments were carried out as indicated under panel A. PP2 (10 nM), DIDS (100 M), or arachidonic acid (10 M) was present throughout the whole experimental period. D, Efflux was carried out as indicated under panel C. DI (25 M) was added to the loading medium 30 min before initiation of the efflux experiments and present throughout the experimental period. PMA (100 nM) was present only for 10 min before the initiation of the efflux experiment. Data in panels A, C, and D are modified from references 76 and 185. Data in panel B represent three sets of paired observations. Values are given as mean values SEM.

main protein tyrosine kinase involved in the regulation of the volume-sensitive taurine efﬂux pathway. Protein tyrosine kinases belonging to the Src-family are involved in volume regulation, that is, p56Lck restores the osmotic action of the volume-regulated anion channel in lckdeﬁcient lymphocytes (190), and inhibition of p72Syk/ p56Lyn blocks swelling-induced taurine efﬂux in skate blood cells (193). Caveolin, which is the main constituent of caveolae, contains a scaffolding domain that is able to bind and inactivate c-Src (194), and it has been demonstrated that mutations that target c-Src to caveolae inhibit the volume-regulated anion channel

(195). The signiﬁcance of the caveolae and Src interaction in the regulation of volume-sensitive taurine transport pathways is unknown. Protein tyrosine phosphorylation occurs concomitant to activation of volume-sensitive ionic conductances (196) and taurine efﬂux (191) in a process that in some instances involves the focal adhesion kinase (FAK, p125FAK) (191,197). FAK is a nonreceptor tyrosine kinase involved in focal adhesion and integrin signaling, and FAK lacks the Src homology domains (SH2, SH3) but contains an integrin-binding domain, a kinase domain, two proline-rich sequences, a C-terminal FAT-domain,

Taurine Homeostasis which is required for targeting of FAK and downstreamsignaling, plus several tyrosine residues (198). Binding of FAK to 1-integrin elicits autophosphorylation of FAK at Tyr397, which creates a high-afﬁnity binding site for the p85 regulatory subunit of PI3K, as well as for members of the Src protein tyrosine kinase family (c-Src) (198). FAK-Src association leads to phosphorylation of Tyr576/Tyr577 and Tyr925 in the catalytic domain and the C-terminal of FAK, respectively (198,199). An elevated phosphorylation at Tyr576/Tyr577 is detected in vanadate-treated cells overexpressing chicken FAK, and the elevated tyrosine phosphorylation is essential for a concomitant increase in FAK kinase activity (200). ROS induce tyrosine phosphorylation of FAK (198) and activate Src in various cell types (201). Furthermore, H2O2 has been shown to induce a time-dependent phosphorylation of FAK in bovine pulmonary artery endothelial cells, which occurs after cytoskeletal reorganization (stress ﬁber appearance) and primarily through inhibition of protein tyrosine phosphatases speciﬁc to FAK (183,199). A phosphorylated Tyr925 residue in FAK acts as a potential docking site for the SH2-containing growth factor receptor-binding protein (Grb) and the guanine nucleotide exchange factor, Sos, and subsequently initiation of a Ras, Raf, and MAP kinase pathway (198). It is noted that translocation, phosphorylation, and activation of MAP-kinases, for example, ERK1/2, p38 and JNK1/2, often occur concomitantly to activation of volume-sensitive transporters (202,203). Tyrosine phosphorylation of p125FAK in platelets is attenuated by inhibitors of iPLA2 (204) and it is plausible that the inhibitory effect of the iPLA2 inhibitor BEL on the swelling-induced taurine efﬂux in NIH3T3 cells (Fig. 3D) reﬂects a role of p125FAK in the regulation of the swelling-induced taurine efﬂux pathway in NIH3T3 cells. However, addition of PP2, which prevents phosphorylation of FAK at Tyr577, potentiates the swelling-induced taurine efﬂux from NIH3T3 cells but does not delay the inactivation of the efﬂux (Fig. 8A). The potentiating effect of PP2 is impaired in the presence of arachidonic acid and DIDS (Fig. 8D), indicating that PP2 interferes with the activity of the volume-sensitive pathway for organic osmolytes and not with an alternative efﬂux pathway. Thus phosphorylation of FAK at the Tyr576 and Tyr577 residue seems to regulate the open probability of the volume-sensitive taurine efﬂux pathway in NIH3T3 cells. The monomeric GTP-binding protein RhoA has been reported to modulate the set point, that is, the degree of cell swelling required for activation of the volume-sensitive channels in NIH3T3 cells (184) and bovine endothelial cells (205). The maximal rate constant for the volume-sensitive taurine efﬂux is several-

45 fold higher in RhoA-transfected NIH3T3 cells (184), and as Rho stimulates FAK and as FAK once activated enhances the level of active Rho (206), it is conceivable that the increased taurine transport activity in Rho cells reﬂects an interaction between FAK and Rho. RhoA activates FAK and subsequent PI3K and the volume sensitive Cl current (197). However, PI3K is inhibited after cell swelling (207) and the swelling-induced taurine release from NIH3T3 cells is not affected by PI3K inhibitors, indicating that the Rho effect does not involve PI3K (184). Rho-associated kinases (ROK/ROCK) and most probably the myosin light-chain kinase (MLCK) have been reported to be involved in activation of Cl efﬂux in calf pulmonary artery endothelial cells (158). However, ROK/ROCK inhibition does not affect swelling-induced taurine efflux from NIH3T3 cells (184). Genistein is a weaker inhibitor of the swellinginduced taurine efﬂux in RhoA-transfected cells compared to the efﬂux in wild type cells, thus it has been suggested that RhoA represents a different regulatory mechanism, which is not affected by protein tyrosine kinase inhibition (184).

Swelling-Induced ROS Production: Role of NAD(P)H Oxidase The NAD(P)H oxidase is present in phagocytes, where it produces ROS in response to binding of various ligands (208) and functions, for example, in host defence against microorganisms and in nonphagocytes (ﬁbroblasts, condrocytes, epithelial and endothelial cells). The NAD(P)H oxidase in phagocytes consists of the cytosolic components p40phox, p47phox, p67phox, the small GTPase Rac2 (homologous to the ubiquitously expressed Rac1), plus the transmembrane, heterodimeric ﬂavocytochrom b558, which is composed of the glycoprotein gp91phox and p22phox (209). The cytosolic components translocate to the plasma membrane where they associate with b558 and form the catalytically active oxidase. p47phox and p67phox, as well as both subunits of b558, become phosphorylated in response to activation (210). It has been suggested that cPLA2 activity or the presence of arachidonic acid is required for activation of p47phox and p67phox in human monocytes (211). The NAD(P)H oxidase in nonphagocyte cells is also Rac regulated (208), and it appears that the p22phox, gp91phox (ﬁve homologous exists), p47phox, as well as p67phox components of the NAD(P)H oxidase in, for example, skeletal muscle cells are constitutively associated with the membrane of mitochondria, endoplasmatic reticulum, nucleus, and peroxisomes, and that the oxidase releases a signiﬁcant fraction of the superoxides to the

46 intracellular compartment (175,212). Phosphorylation of p22phox seems to involve a mechanism that is phospholipase D dependent and independent (213), whereas a phosphatidic acid (PA)–dependent protein kinase (209) and PKC (subtypes , II, , and ) (214) are reported to be involved in phosphorylation of p47phox and subsequently activation of the oxidase complex. From Fig. 8C and D it is seen that preexposure of NIH3T3 cells to the PKCactivating agent PMA leads to a two-fold increase of the rate constant for taurine efﬂux under a subsequent hypotonic shock and that the effect of PMA is impaired in the presence of the NAD(P)H oxidase inhibitor DI. Furthermore, expressing constitutively activated forms of Rac in ﬁbroblasts increases the intracellular ROS level (176) and accelerates the volume regulatory decrease following osmotic cell swelling (184). It has accordingly been suggested that ROS are generated by the NAD(P)H oxidase system in NIH3T3 cells upon cell swelling, and that the potentiating effect of PMA on the swelling-induced taurine release could reﬂect a ROS-mediated inhibition of PTP1B activity (185). It is noted that H2O2 at a low concentration induces tyrosine phosphorylation of PKC and enhances its enzymatic activity, whereas H2O2 at a high concentration impairs its activity (215,216), indicating a mutual PKC–ROS regulation, which is regulated by the actual ROS concentration.

Lysophospholipids and Release of Organic Osmolytes Lysophospholipids, produced by the action of PLA2, are natural components of lipoproteins, biological membranes, and serum, and they have within recent years been recognized as bioactive messengers (217). Lysophosphatidyl choline (LPC) is a bioactive molecule that at low concentrations acts as ligand for speciﬁc Gprotein–coupled receptors (218) and at high concentrations (25 M) affects the membrane ﬂuidity (219) and mediates a general permeabilization of the membrane (187,220,221). LPC is produced in Ehrlich cells following hypotonic exposure (92) and the free LPC concentration during in vivo conditions is estimated at 20 M in, for example, ischemic myocardium (222,223). The heparin-binding sPLA2 (group X) binds to phosphatidyl choline in the outer leaﬂet of the plasma membrane and is accordingly considered a generous supplier of exogenous LPC (84). Exogenous addition of a low concentration of LPC (5 M) releases taurine under isotonic conditions from Ehrlich cells (Fig. 9A), NIH3T3 cells (Fig. 9B), HeLa cells (Fig. 9D), C2C12 myotubes (119), and pig skeletal

Lambert muscle cells (120). The LPC-induced taurine release under isotonic conditions is transient and dose dependent (Fig. 9B, 187). Furthermore, the ability of a lysophospholipid to release taurine under isotonic conditions requires a saturated fatty acid in the sn-1 position and choline as the polar head group, that is, substitution of an unsaturated fatty acid for the saturated fatty acid or substitution of serine, inositol, ethanol, or an acidic group for choline results in lysophospholipids with reduced or no effect on taurine release in HeLa cells under isotonic conditions (187). LPC is cone shaped, that is, the cross-sectional area of the acyl chain is half the cross-sectional area of the polar head group, and LPC will, according to Lundbæk and Andersen (219), stabilize convex membrane surfaces and consequently affect the energetic costs of a membrane deformation and the conformation state of membrane proteins. Phosphatidyl choline has similar cross-sectional area in the hydrophilic and hydrophobic parts and organizes itself into a ﬂat structure. Thus activation of PLA2 not only releases a precursor for downstream signaling (arachidonic acid) but also leaves a lysophospholipid, which affects the physical state of the membrane. That the effect of LPC on taurine transport involves a change in the physical state of the plasma membrane is supported by the observation that addition of cholesterol, which affects membrane ﬂuidity, impairs the LPC effect on taurine release from pig muscle (120) and NIH3T3 ﬁbroblasts (Fig. 9E). LPC is also reported to activate speciﬁc K channels (TREK-1, TRAAK) in transiently transfected COS cells, and it appears that the activation of TREK requires a cytosolic factor and involves the carboxyl terminus of the K channel (103). ROS are produced following exogenous addition of LPC in NIH3T3 cells (Fig. 9C) and in muscle cells (120), and the LPC-induced taurine efﬂux is impaired in the presence of the antioxidants butulated hydroxy toluene (BHT) and VitE (Fig. 9B), indicating that LPC-induced taurine loss involves ROS production. In the case of HeLa cells it has furthermore been demonstrated that the effect of LPC on taurine release is modulated by tyrosine phosphorylation, Ca2/calmodulin, and the calmodulin-dependent kinase CaMKII (187). Unsaturated fatty acids generate ROS and lipidperoxidation, for example, in HeLa cells, and the ROS generation is blocked by calmodulin antagonists (224). Thus the calmodulin/CAMKII-regulated step in the LPC-induced signaling cascade seems to be upstream to the ROS production. It is emphasized that swelling-induced and LPC-induced taurine release from HeLa cells, NIH3T3 ﬁbroblasts, C2C12 myotubes, and pig muscle cells differ with respect to the sensitivity toward the 5-LO inhibitors

Taurine Homeostasis

47

Fig. 9. Effect of LPC on ROS production and taurine release from cells under isotonic conditions. A, Ehrlich cells, equilibrated with 14C-labeled taurine, were transferred to isotonic solution in the absence or presence of LPC (5 M). The initial taurine release was followed within the 1 min and the rate constant (min1 g cell water/g cell dry wt) for the initial taurine efflux estimated from slope of the efflux curve. B, NIH3T3 cells, equilibrated with 14C-taurine, were washed and the efflux followed with time in isotonic NaCl medium, with a shift at time 6 or 8 min to isotonic medium containing LPC (5 M, 10 M). BHT (0.5 mM) or VitE (100 g/ml) were present in the medium throughout the efflux experiment. Values represent 46 (5 M LPC), 6 (10 M LPC), 3 (BHT plus 5 M LPC), and 5 (VitE plus 5 M LPC) experiments. The effect of BHT and VitE was significant (P .05, paired Student’s t test). C, Cells, grown on coverslips (80% confluence) and loaded with carboxy-H2DCFDA, were washed and the ROS production estimated in isotonic NaCl medium in the absence or presence of LPC (10 M), using a luminescence spectrometer, excitation wavelength 490 nm and emission wavelength 515 nm. The traces are representative of three sets of experiments. D, HeLa cells, equilibrated with 14C-labeled taurine, were incubated for 2 h in the presence or absence of serum (serum starved). The cells were washed and the efflux followed with time in either isotonic NaCl medium, with addition of 5 M LPC at time 6 min or isotonic NaCl medium with a shift from 300 mOsm to 200 mOsm at time 6 or 8 min. The maximal values for the LPC- and swelling-induced rate constant (min1) were estimated and given as mean values. E, NIH3T3 cells, equilibrated with 14C-taurine, were washed and the efflux followed with time in either isotonic NaCl medium, with LPC (5 M) being included at time 6 or 8 min or in isotonic NaCl medium, with a shift in the osmolarity from 300 mOsm to 200 mOsm at time 6 or 8 min. Arachidonic acid (AA, 10 M) or cholesterol (CHO, 10 M) was present throughout the experimental period. The maximal rate constants were estimated and given relative to the respective control with no arachidonic acid or cholesterol added. The numbers of paired experiments are 5 (LPC arachidonic acid), 4 (LPC cholesterol), 3 (hypotonic, arachidonic acid), and 5 (hypotonic cholesterol). The effect of cholesterol on the LPC-induced taurine release and the effect of AA on the swelling-induced taurine release were significant (P 0.05, paired Student’s t test). Data in panels A and D are from (83) and (155), respectively. All values in panels A, B, D, E are mean values SEM.

(119,187), serum starvation (Fig. 9D), channel blockers (DIDS, MK196, arachidonic acid), and cholesterol (Fig. 9E; (83,119,120,187). Furthermore, the LPC concentration following hypotonic exposure does not reach a concentration that elicits taurine loss (187). Thus LPC is not assigned a role as an essential second messenger in the swelling-induced signaling cascade that leads to taurine release. However, as LPC and LPA both potentiate

swelling-induced taurine loss from, for example, HeLa cells (187), it seems reasonable to consider lysophospholipids (LPC, LPA) as modulators of the swelling-induced loss of organic osmolytes. Anoxia/ischemia, on the other hand, is associated with an increase in [Ca2]i, activation of various types of PLA2 (225), as well as generation of LPC plus ROS (226) and taurine release (73,120). It is therefore assumed that the LPC-sensitive signaling pathway

48 could be involved in ischemia-induced taurine efﬂux. The ROS production and the involvement of tyrosine phosphorylation most probably reﬂect a link between the signaling cascades activated by osmotic cell swelling and by exposure to LPC.

VOLUME-SENSITIVE TAURINE EFFLUX PATHWAY A volume-sensitive, Na-independent taurine efﬂux pathway different from the Na-dependent, highafﬁnity TauT has been demonstrated in rat astrocytes (227), cerebellar granule cells (228), MDCK-cells (229), HeLa cells (150), and NIH3T3 cells (7). Taurine is not concentrated in secretory granules but equally distributed in the cytoplasm and in the nucleus, as observed in rat pancreatic islet cells; therefore it is seems unlikely that taurine is excreted by exocytosis (3). Furthermore, swelling-induced taurine inﬂux in HeLa cells is nonsaturable (substrates tested up to 50 mM [109]) and the swelling-induced efﬂux and inﬂux exhibit a similar pharmacological proﬁle in bovine condrocytes (230) and HeLa cells (109), indicating that the volume-sensitive transporter is a diffusion pathway where the direction and net transport of osmolytes is determined by the electrochemical gradient across the plasma membrane. A volume-sensitive organic osmolyte anion channel, designated VSOAC, which is sensitive to a various pharmacological drugs, such as 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 1,9-dideoxyforskolin (DDF), NDGA, DIDS, and tamoxifen and by polyunsaturated fatty acids (arachidonic acid, linoleic acid), has been suggested to mediate the volume-sensitive, Cl current as well as the volume-sensitive efﬂux of organic osmolytes (231,232). The permeability sequence for the organic osmolytes in, for example, HeLa cells is taurine sorbitol choline thymidine sucrose and the minimum diameter of the pore is estimated at 8–9 Å in HeLa cells (109). It is noted that the terms volume-sensitive outwardly rectifying anion channel (VSOR, 233) and volume regulated anion channel (VRAC, 158) are most likely identical to VSOAC and that ICl,swell and ICl,vol refer to the wholecell Cl current evoked by cell swelling and estimated by patch clamp technique (234). VRAC is ubiquitously expressed and has been associated with cell proliferation, apoptosis, and regulation of cellular pH (158,233,235). VRAC is blocked by extracellular nucleotides in the millimolar range and its anion permeability sequence is SCN I NO3 Br Cl F(Eisenman series I, low ﬁeld strength) (158). The

Lambert ratio between the taurine and the Cl permeability is estimated at 0.2 in, for example, C6 glioma cells, skate hepatocytes, and rat inner modular collecting duct cells (236,237) and at 0.5 in MDCK cells (238). The molecular identity of the volume-sensitive, outwardly rectifying Cl channel (VSOAC/VRAC/VSOR) and the volume-sensitive taurine channel has not been revealed, but a series of proteins have been under consideration, that is, pICln (239), C1C-2 (240), C1C-3 (241), phospholemman (242), and the band 3 exchanger (AE1) (243). pICln is localized in the cytosol/nucleus, and translocation to the plasma membrane has been reported for some cells (244) but not in other cells (245,246). Furthermore, cell swelling does not activate the current generated by pICln, and its lack of pH sensitivity is in variance with the swelling-induced Cl current (245,247). Thus, even though pICln is permeable to taurine (248), it is now considered as an intracellular regulator of a volume-sensitive anion transporter. C1C-2 and C1C-3 belong to the C1C superfamily of Cl channels (235). C1C-2 is sensitive to cell swelling (235) and permeable to taurine (248). However, C1C-2 exhibits inward rectiﬁcation, it is activated by acidiﬁcation, and the anion selectivity of C1C2 is in variance with VSOAC (235). Furthermore, acinar cells from C1C-2 knockout mice still volume regulate (249), and astrocytes with no endogenous expression of C1C-2 still have a volume-sensitive Cl current and still regulate their volume following osmotic exposure (250). Thus C1C-2 is not considered a volume-sensitive anion transporter. The current generated by C1C-3 is constitutively active, outwardly rectifying, regulated by Ca2/calmodulin-dependent kinases plus PKC, and inhibited by DIDS, tamoxifen, and extracellular ATP (158,235). Expression of C1C-3 in NIH3T3 cells generates a Cl current that is enhanced by cell swelling (241,245), and expression of C1C-3 in oocytes has revealed a volume-sensitive taurine efﬂux (248). C1C-3 is present in HeLa cells (251), but knockout mice, lacking a functional C1C-3, still express ICl,vol and a normal volume regulatory response (252). Furthermore, C1C-3 seems to be localized to the cytosol in various cell types, indicating a regulatory role for C1C-3 in the swelling-induced release of anions (235). On the other hand, antisense–mediated downregulation of C1C3 in HeLa cells inhibits ICl,vol and impairs the volume regulatory response (251), indicating that C1C-3 could be the volume sensitive Cl channel or a volume-sensor in HeLa cells. Phospholemman, a small transmembrane peptide, exhibits anion and cation selectivity (253), and the transporter has a higher preference for taurine compared to Cl (254). The volume-sensitive taurine efﬂux is potentiated following overexpression of phospholemman

Taurine Homeostasis in, for example, HEK cells (255) and heart myocytes (242), but reduced following reduction in the expression in rat astrocytes (253). However, the large maximal conductance, the anion and cation selectivity, the inward rectiﬁcation, and the fact that the taurine permeability is higher than the Cl permeability does not agree with the data for the volume-sensitive Cl current (245,256). Phospholemman has not been excluded as being involved in volume-sensitive taurine release. That band 3 should be involved in swelling-induced taurine release followed from the observation that inhibitors of band 3 mediated anion exchange also inhibited swelling-induced release of anions and organic osmolytes. Trout erythrocytes have in contrast to mouse erythrocytes a swellinginduced osmolyte channel, and expression of band 3 (AE1 isoform) from the trout erythrocytes in Xenopus oocytes resulted in an increased anion conductance and taurine permeability, whereas expression of band 3 from the mouse had no effect (243). AE1 is also reported to be involved in volume-sensitive taurine release from rat erythrocytes (257). On the other hand, taurine efﬂux in Ehrlich cells is unaffected by substitution of gluconate for extracellular Cl, indicating that taurine does not leave the osmotically swollen Ehrlich cells in exchange for extracellular Cl via the anion exchanger (52). The hypothesis of a common osmosensitive transport pathway for organic osmolytes and anions is, however, questioned and data are now accumulating that separate, volume-sensitive efﬂux pathways exist for organic osmolytes and for Cl. The two efﬂux pathways can be discriminated by their differential regulation, sensitivity to expression of constitutive Rho, anion channels blockers, and kinase inhibitors and the time course for their activation/inactivation following hypotonic exposure. The swelling-induced taurine efflux from Ehrlich cells is reduced by membrane depolarization and exhibits a different pH proﬁle compared to the isotonic taurine efﬂux, indicating that the swelling-induced taurine efﬂux pathway is different from the taurine efﬂux systems working under isotonic conditions (9). The ICl,vol in Ehrlich cells is Ca2-independent, exhibits outward rectiﬁcation, and time- and voltage dependent activation at depolarized potentials, and the anion selectivity sequence is I Cl gluconate (258). Ehrlich cells also have a Ca2 activated Cl channel (ICl,Ca) that is outwardly rectifying, with an anion selectivity sequence I Cl gluconate, but ICl,Ca is in variance with ICl,vol activated by depolarization of the membrane (258). Arachidonic acid blocks the RVD response in Ehrlich cells (107), and from Fig. 10A it is seen that arachidonic acid also blocks cell shrinkage in osmotically swollen cells even under conditions in which a high

49 cation conductance has been ensured by addition of gramicidin. On the other hand, arachidonic acid has no effect on cell shrinkage induced under isotonic conditions by addition of the Ca2 ionophore A23187 (Fig. 10B). Thus arachidonic acid blocks ICl,vol but has no effect on ICl,Ca in Ehrlich cells. Arachidonic acid acts directly on the swelling-induced Cl efﬂux pathway (104), and the effect of arachidonic acid is dose dependent (Fig. 10A) and shared by other unsaturated fatty acids (107). The swelling-induced Cl efﬂux in Ehrlich cells is inhibited by MK196 (indacrinone), but relatively insensitive to DIDS, quinine, and DDF (105,259). That the volumesensitive Cl channel is more or less insensitive to DIDS has also been demonstrated in HeLa cells (260) and NIH3/3 cells (7). The swelling-induced taurine efﬂux from Ehrlich cells is stimulated by arachidonic acid (Fig. 10C), unaffected by addition of DDF, but inhibited by membrane depolarization, acidiﬁcation, and addition of the anion transport inhibitors DIDS and MK196 (52,105,259). Shennan and Thomson (261) observed a volume-sensitive taurine efflux in the absence of I efﬂux in rat mammary tissue, whereas Stegen et al. (248) demonstrated swelling-induced taurine release without chloride channel activity in Xenopus oocytes expressing pICln and ClC-3. Furthermore, Roman et al. (262) demonstrated an outwardly rectifying anion current and an increase in the I efﬂux (I used as marker for Cl) from human biliary cells following cell swelling without any increase in taurine efﬂux. The time course for the swelling-induced anion efﬂux from HeLa cells is transient, with a maximum at about 2 min after reduction in the osmolarity, whereas the taurine efﬂux is maximal after 4–6 min and remains elevated (Fig. 10D). Similar discrepancies between the time courses for the swelling-induced taurine and anion efﬂux have been demonstrated in cultured cerebellar astrocytes (263) and in NIH/3T3 cells (7). More recently it has been demonstrated that swelling-induced Cl and taurine loss from NIH/3T3 cells diverge with respect to their sensitivity to expression of constitutive Rho and Rho kinase inhibition (184). The protein tyrosine kinase p56Lck is activated by cell swelling and is involved in the activation of ICl,vol in jurkat lymphocytes and required for a normal volume regulatory response (190, 264). On the other hand, p56Lck seems not to be involved in the swelling-induced taurine efﬂux (139). Furthermore, the swelling-induced taurine efflux from HeLa cells is dramatically stimulated by low extracellular Cl, whereas the swelling-induced I, that is, the Cl efflux is unaffected by substitution of glutamate for Cl in the extracellular medium (265). Taken together it is assumed that the swelling-induced taurine and Cl

50

Lambert

Fig. 10. Comparison of the swelling-induced Cl and taurine efflux pathway. A, Ehrlich cells were at time zero transferred to hypotonic Nafree choline chloride medium with half of the isotonic osmolarity and the cell volume followed with time using a Coulter counter system. Arachidonic acid (5 M, 10 M) was added at the time of hypotonic exposure. Gramicidin (0.5 M) was added at the time of maximal cell swelling, that is, at the time of maximal cell swelling, to ensure high cation permeability. B, Ehrlich cells were at time zero transferred to isotonic Na-free choline chloride medium and the cell volume followed with time using a Coulter counter system. Gramicidin (0.5 M, squares) and arachidonic acid (10 M, closed squares) were added at time zero. A23187 (2 M) was added at the time indicated by the arrow, to activate Ca2-dependent Cl channels. C, Ehrlich cells, equilibrated with 14C-labeled taurine and 36Cl, were transferred to hypotonic Cl-free gluconate medium with half of the original osmolarity. The Cl efflux (cpm/g dry wt) was followed with time in the absence (con) or the presence of arachidonic acid (FAA, 200 M). The taurine efflux was followed with time in the absence or presence of arachidonic acid (25 M). The concentration was varied to compensate for differences in cell density, that is, the final cytocrit in Cl and taurine efflux experiment was 3% and 0.7%, respectively. The rate constants in the presence of inhibitors are given relative to the rate constants obtained in the absence of drugs. D, HeLa cells, equilibrated with 3H-labeled taurine and 125I were washed and the release of iodide (used as a tracer for Cl) and taurine was followed with time with a shift to hypotonicity as indicated by the arrow. The rate constants were calculated from the release activity and the sum of the activity remaining in the cells at the end of the experiments as indicated in the legend to Fig. 3C. E, HeLa cells, equilibrated with 14 C-labeled taurine were washed and transferred to isotonic KCl media with pH in the range 6.6 to 8.2, and the release of taurine was followed with time, with a shift to hypotonic KCl medium with two thirds of the original osmolarity but with similar pH. The maximal rate constants (min1) were calculated from the release activity and the sum of the activity remaining in the cells at the end of the experiments as indicated in the legend to Fig. 3C. The fraction of negatively charged taurine was calculated from the Henderson-Hasselbalch equation using pK1 1.5 and pK2 8.8. Data in panels A and B are modified from (107). Data in panel C are modified from (259). Data in panel D are reproduced from (265). Data in panel E represent three sets of experiments. Values in panels C–E are mean values SEM.

efﬂux from Ehrlich cells, NIH3T3 cells, HeLa cells, and jurkat lymphocytes occur via separate pathways. In this context it is noted that Mongin et al. (192) have demonstrated the presence of two volume-sensitive taurine efﬂux pathways in rat astrocytes, which are distinguished by their amino acid speciﬁcity and their regulation by tyrosine kinases.

Taurine is a zwitterionic amino acid (Fig. 1), and it is feasible that a fraction of the swelling-induced taurine efﬂux could be mediated by an anion channel under alkaline conditions. From Fig. 10E it is seen that the swelling-induced taurine efﬂux under hypotonic conditions increases as a function of extracellular alkalinisation. This is in accordance with the effect of pH on ICl,vol

Taurine Homeostasis and volume-sensitive taurine efﬂux described in astrocytes (266), BC3H1 myoblasts (267), and cultured endothelial cells from bovine pulmonary artery (268). It is noted that there seems to be a poor correlation between taurine efﬂux from HeLa cells and the fraction of taurine on its anionic form (Fig. 10E). Furthermore, the cellular acidiﬁcation in Ehrlich cells following hypotonic exposure (269) is far too small compared to the swelling-induced net loss of taurine (9) provided taurine left as an anion. Thus taurine seems to leave the Ehrlich cell as a zwitterion following osmotic cell swelling at physiological pH and via a volume-sensitive, potential-dependent taurine transporter. Guizouarn et al. (270) have accordingly demonstrated that taurine also leaves ﬁsh erythrocytes as an electroneutral molecule. Assuming that the Cl conductance in hypotonically swollen Ehrlich cells represents Cl ﬂux via a swelling-induced, arachidonic acid-sensitive Cl channel and via the swelling-induced taurine channel, it has been estimated that the volume-sensitive taurine channels contribute to the total Cl conductance by less than 5%.

SENSING A CHANGE IN THE CELL VOLUME Changes in the intracellular concentration of osmolytes (net uptake of solutes, metabolism, secretion of electrolytes) or the extracellular tonicity result in a rapid transmembrane water ﬂow in mammalian cells until intracellular and extracellular tonicities are equilibrated. Most swollen cells, with the exception of some cortical brain cells (271), respond to the osmotic cell swelling by activation of volume-sensitive ﬂux pathways for ions and organic osmolytes to restore their original cell volume (10). Translocation of enzymes (PLA2, 5-LO), shift in protein tyrosine phosphorylation pattern, as well as mobilization and release of various categories of second messengers (eicosanoids, nucleotides, Ca2, ROS) are important events in the activation and modulation of the volume-sensitive efﬂux pathway for taurine in Ehrlich cells, HeLa cells, and NIH3T3 cells. Osmotic cell swelling is inevitably accompanied by a change in (i) the intracellular ionic strength, macromolecular crowding, and activity of impermeant molecules, for example, enzymes involved in the activation/modulation of the RVD response (kinases, phosphatases) and (ii) the intracellular diffusion of cofactors and second messengers, that act as regulators of the volume-sensitive transporters (272,273). An increase in the ionic strength under conditions in which the cells are swollen, reduces the volume-sensitive taurine efflux from skate hepatocytes

51 (274) and skate red blood cells (275), whereas reduction in intracellular ionic concentration increases the activity of the volume-sensitive osmolytes channels for K, Cl and organic osmolytes in trout erythrocytes (276). Furthermore, Voets et al. (277) demonstrated that cell swelling did not activate the volume-sensitive outwardly rectifying current in pulmonary endothelial cells at constant ionic strength, whereas the current once activated remained under the control of the ionic strength and insensitive to change in cell volume. They consequently proposed that it is the decrease in the intracellular ionic strength and not an increase in cell volume that triggers activation of the volume sensitive Cl current (277). Ehrlich cells, exposed to hypotonic medium, regulate their cell volume back to a value (volume set point) that is higher than the initial value and presumably a function of extracellular osmolarity, and it has been suggested that the larger set point is associated with a lower intracellular ionic strength (278). It is recognized that even though transporters (Na/H exchanger, Na, K, 2Cl cotransporter) (279) and some protein kinases (280) are sensitive to changes in the cellular Cl concentration, no activation of protein tyrosine kinase by a reduction in ionic strength has yet been demonstrated. The cytoskeleton is a discrete ﬁlamentous network that provides the mechanical strength of the cytoplasm, and cell swelling has been demonstrated to cause reorganization of cortical F-actin (11). Swelling-induced rearrangement of cytoskeletal elements has been demonstrated to regulate the activation of the volume sensitive outwardly rectifying current in PC12 cells (281) and human intestine 407 cells (282), whereas disruption of the cytoskeleton had no effect on the channel in human endothelial cells (283). Evidence that the swelling-induced taurine efﬂux is modulated by components of the cytoskeleton has also been provided (284). However, as emphasized by Pedersen et al. (96), there is no clear correlation between the effects of cytochalasin on F-actin content, F-actin organization, and cell morphology, even though cell swelling and cell shrinkage have been demonstrated to be associated with a decrease and an increase in the F-actin content, respectively. Rho GTP-ases are key regulators of the actin cytoskeleton in various cells (285), and RhoA is known to regulate the formation of stress ﬁbers and focal adhesion (286). LTD4, which stimulates taurine release from Ehrlich cells under isotonic conditions (Fig. 4), also has an impact on the stress ﬁber production via activation of PKC (PKC) and RhoA, and on reorganization of the actin cytoskeleton in, for example, Int 407 cells and human bronchial smooth muscle cells (287,288). This could indicate that actin reorganization is not the initial event in the swelling-induced taurine release.

52 Integrins are adhesion receptors that via talin and vinculin link the extracellular matrix to the actin cytoskeleton (289). It has been indicated that a change in cell volume via modulation of integrin clustering triggers intracellular signal even in the absence of ligands (96). RhoA is essential in integrin signaling and has been reported to modulate swelling-induced currents in endothelial cells (290,291) and NIH3T3 cells (184). RhoA and Rac1 preferentially localize to caveolae (292), thus it becomes conceivable that cell swelling, that is, unfolding of the membrane and reorganization of the cytoskeleton induce a change in the caveolae organization (293) and subsequently activation of RhoA/integrin signaling. In this context it is noted that RhoA has been proposed as an initial element in the volume sensitive signaling sequence that regulates volume-sensitive anion channels, that is, the sequence RhoA, p125FAK → phosphatidyl inositol 3 kinase → phosphatidyl inositol biphosphate (197) and the sequence RhoA → Rho kinase → myosin light-chain phosphorylation (294) are proposed for modulation of volume-sensitive Cl channels. Several of the components, demonstrated to play a role in the regulation of taurine transport across the plasma membrane, for example, Src, FAK, PKC, and GTP binding proteins, have been reported to become physically associated at the focal adhesion complex following integrin activation (295). cPLA2 has a calveolin binding motif, the heparin binding sPLA2 is associated with caveolae (84) and components of complex receptor protein tyrosine kinase (RPTK) systems are known to cluster in caveolae (296). Because calveolin-1 restores the swellinginduced Cl current, that is, VRAC activity in calveolin1–deﬁcient cells, it has been proposed that the caveolae function as a potential endothelial mechanosensing unit for translation of mechanical stress into a biochemical response (297). Margalit et al. (298) demonstrated that mechanical stress activate the volume-sensitive cascade in human blood platelets, and they proposed that GTP-binding proteins and phospholipids interact as a response to the plasma membrane stretch occurring during cell swelling (81). In this context it is noted that direct stimulation of sPLA2 activity in NIH3T3 cells under isotonic conditions with the lipase activator melittin induces ROS production as well as taurine loss via a cellular signaling cascade and a taurine efﬂux pathway that exert a pharmacological proﬁle similar to the volume-sensitive signaling and efﬂux system (76). Thus stimulation of PLA2 activity is an essential upstream element in the swellinginduced signaling cascade leading to activation of osmolyte-releasing transport pathways. Kinnunen et al. have demonstrated that PLA2, incorporated in an artiﬁ-

Lambert cial unilamellar lipid vesicles, was directly activated by stretch and proposed that a shift in the lateral lipid packing in the plasma membrane could act as an osmosensor (299). Ingber (300) characterized the cell surface integrin receptors, the cytoskeletal ﬁlaments and the nuclear scaffolds as being “hard-wired” in the sense that a mechanical tug on the cell surface integrin results in an immediate change in the cytoskeleton and alteration in the molecular assemblies in the depth of the nucleus. Furthermore, intracellular perfusion with GTPS under conditions with unchanged volume has been demonstrated to lead to activation of VRAC in bovine endothelium (205). Thus cell swelling could conceivably affect the interaction and activity of PLA2 and G-proteins even though they are associated with the nuclear membrane. Stretch-activated channels respond to membrane stress by changing the open probability, and activation of nonselective, cation channels that could cause a localized increase in [Ca2]i have been demonstrated in a variety of cells (1). The transient receptor potential (TRP) superfamily consists of cation channels that respond to extracellular stimuli, that is, osmolarity, pH, and membrane stretch (301). OTRPC4 is a volume-sensitive, Ca2-selective member of the TRP family that has six transmembrane domains and several potential phosphorylations sites for PKC and PKA (302,303). However, TRPV4 is in contrast to ICl,vol GTPS insensitive and ATP independent, and its role in RVD has not been established (304). It is emphasized that cell swelling is not necessarily associated with membrane stretch because exposure of invaginated parts of the plasma membrane can account for a two- to four-fold increase in cell volume with no concomitant stretching of the membrane (233). Furthermore, cell swelling in hypotonic medium does not increase the membrane capacitance in astrocytes (305). Thus, even though the initial volume-sensitive mechanisms await identiﬁcation, it appears that unfolding of the plasma membrane, aggregation of elements (enzymes, channels, cofactors) at subcellular membrane microdomains or hot spots (caveolae, focal adhesion complex, nuclear envelope), and reorganization of the cytoskeleton are essential elements for downstream signaling following osmotic cell swelling.

MODEL FOR THE SWELLING-INDUCED REGULATION OF THE CELLULAR TAURINE CONTENT Based on the current knowledge the following model (Fig. 11) is proposed for the swelling-induced release of

Taurine Homeostasis

53

Fig. 11. Model for cellular signaling pathways involved in taurine release from mammalian cells. Osmotic cell swelling activates the volume sensitive organic osmolyte channel. The activation involves PLA2-mediated release of arachidonic acid, 5-LO–mediated oxidation of arachidonic acid, release of eicosanoids to the extracellular compartment, binding of the eicosanoid to a receptor (R), and activation of the volume-sensitive taurine efflux pathway. FLAP, the 5-LO activating protein, is assumed to present the arachidonic acid to 5-LO. ROS are generated by the NAD(P)H oxidase (NADPH 2 O2 → NADP 2 O2 2 H) and by the superoxide dismutase (2 O2 2 H → H2O2 2). ROS reduce the activity of protein tyrosine phosphatases, resulting in an increased open probability of the volume-sensitive taurine efflux pathway. PKC stimulates the NADPH oxidase and affects the eicosanoid receptor. The LPC-induced taurine leak pathway involves a more general permeabilization of the plasma membrane and ROS production. ROS comprise not only a potent amplification system via stimulation of iPLA2 and 5-LO, but also the cross-link between the two signaling pathways. PLA2 is essential for the activation of both pathways, but the downstream signaling coming into action depends on the type and subcellular localization of the PLA2 involved. The swelling-induced taurine efflux is inhibited by DIDS and serum-starvation but unaffected by cholesterol. The LPC-induced taurine efflux pathway is insensitive to DIDS and serum-starvation but inhibited by cholesterol. The model is modified from (185).

taurine in mammalian cells. Cell swelling leads to translocation and/or activation of PLA2 (cPLA2 in Ehrlich cells and iPLA2 in NIH3T3 cells) to a membrane, where it increases the availability of fatty acids (arachidonic acid) and lysophospholipids. Arachidonic acid acts as a Ca2mobilizing agent in some cells or as a precursor for the synthesis of highly potent eicosanoids in, for example, Ehrlich cells. The differential translocation of cPLA2 to the Golgi complex, the endoplasmatic reticulum, and the

nuclear envelope is reported to be a function of the absolute [Ca2]i (98), and because membranes of intracellular compartments exhibit different lipid composition, it is plausible that the products produced by PLA2 activation, that is, lysophospholipids/fatty acid and the subsequent cellular response, are determined by the actual [Ca2]i at the time of stimulation. The 5-LO translocates from the cytosolic or nuclear compartment to the nuclear envelope, where it associates with the

54 membrane-bound docking protein FLAP and subsequently oxidizes arachidonic acid into 5-HPETE. The latter is either reduced to 5-hydroxy eicosatetraenoic acid (5-HETE) or converted to the unstable LTA4. The 5-LO in Ehrlich cells is favored on the expense of COX1/COC2 in the absence of exogenous arachidonic acid, which is taken to indicate that the cPLA2 and 5-LO become intimately associated at the nuclear membrane upon cell swelling. The gluthation S-transferase conjugates gluthathione with LTA4 to form LTC4, and the subsequent loss of the glutamic acid residue from LTC4 leads to the formation of LTD4. The enzymes involved in the conversion of LTA4 to LTD4 are particulate enzymes located at the plasma membrane (306). It has been proposed that LTD4 is either released directly to the extracellular compartment (123) or alternatively that LTC4 is released to the extracellular space by a multiresistance-associated protein and subsequently converted to LTD4 (307). LTD4 binds to a leukotriene receptor (CysLT1) and promotes, in the case of the Ehrlich cells, activation of the volume-sensitive taurine efﬂux pathway as well as the K efﬂux pathway. Activation of the volume-sensitive Cl current in Ehrlich cells does not involve LTD4 (129). Trimeric G-proteins are involved in the volume-sensitive cascade at several steps, that is, at the activation of PLA2 as reported in, for example, human platelets and rat inner medullar collecting duct cells in primary culture (81,93) and in LTD4-mediated signaling, as reported in Ehrlich cells (126). Swelling-induced taurine efﬂux involves tyrosine phosphorylation by a yet unidentiﬁed protein tyrosine kinase and because the swelling-induced translocation of cPLA2 in Ehrlich cells is not affected following inhibition of tyrosine kinases (94), it is assumed that the tyrosine kinase step is downstream to the PLA2 translocation/activation step. In this context it is noted that LTD4 triggers a rapid Src-kinase–mediated phosphorylation of tyrosine residues on vinculin, which affects the vinculin–actin interaction at the focal adhesion (308). Arachidonic acid, LPC, and PKC-mediated phosphorylation lead to formation of ROS, presumably via activation of a NAD(P)H oxidase. ROS subsequently inhibit a protein tyrosine phosphatase and thereby increase the net protein tyrosine phosphorylation of proteins, which affect the open-probability of the volume-sensitive taurine channel. LPC also induces taurine loss from mammalian cells via a ROS-dependent mechanism that seems to involve controlled cellular signaling systems, as well as a more general permeabilization of the plasma membrane. The mechanisms responsible for LPC-induced ROS production have not yet been established. Stimulation of PKC and PKA modulates the activity of the

Lambert taurine efﬂux pathway in brain glial cells, but these kinases seem not to be required for swelling-induced activation (130). In the case of basophilic leukaemia cells it has been demonstrated that LTD4 activates a PKC (309) and that homologous desensitization of LTD4-induced Ca2 mobilization involves PKC (310). Exposing the cells to Ca2-mobilizing agents will inevitable lead to stimulation of Ca2-sensitive steps in the volume-sensitive signaling cascade (PLA2 translocation, 5-LO translocation and binding to FLAP, calmodulin modulation, PKC stimulation, binding of LTD4 to its receptor) and potentiation of the swellinginduced efﬂux. However, Ca2 mobilization will also activate Ca2-sensitive K and Cl efﬂux pathways, leading to an accelerated net loss of KCl and cell water and consequently an acceleration of volume restoration and inactivation of the volume-sensitive taurine efﬂux pathway. Taurine binds to neutral phospholipids and thereby affects membrane properties (architecture, ﬂuidity), and taurine reduces the Ca2 binding capacity of phospholipids (8). How taurine loss/removal from the cellular compartment during the RVD response affects Ca2 and ROS signaling is unknown. Lombardini (311) demonstrated that taurine depletion has a great effect on the phosphorylation of pyruvate dehydrogenase and histone H2B, and the latter could indicate a role for taurine in cell proliferation. Hypotonic cell swelling is also accompanied by a reduction in the active taurine uptake, which seems to be a result of the reduction in the extracellular Na and Cl concentration and the swelling-induced depolarization of the plasma membrane, and which has the physiological consequence that reuptake of taurine during the volume regulatory process is reduced. Little attention has been given to the mechanisms that shut off the volume-regulatory response following acute hypotonic exposure and what happens to the cellular signaling systems and the volume-sensitive transporters under prolonged hypotonic conditions. The swelling-induced Cl efﬂux in Ehrlich cells inactivates within a few minutes following hypotonic exposure (77). It is evident that the activity of the volume-sensitive taurine channel decreases and the activity of TauT increases as the cell volume and membrane potential approach the initial values. PLA2 and the 5-LO apparently translocate to microdomains on the nuclear envelope; thus a shutting down of the volume-sensitive taurine pathway could involve local restriction in the PLA2 substrate availability in the microdomain. This hypothesis is favored by the observation that an increase in the precursor pool improves the ability to perform a RVD response in Ehrlich cells (125). The observation that the RVD response can be elicited repetitively in HeLa cells (109)

Taurine Homeostasis could well imply that the microdomains or hot spots to which the PLA2 and 5-LO translocate upon osmotic exposure are located at random sites on the nuclear envelope to avoid substrate restriction. Signaling cascades that involve receptor-mediated steps can become less sensitive to the ligand as a result of either desensitization or downregulation of the receptor as has been demonstrated for the LTD4-induced Ca2 mobilization in basophilic leukemia cells (309,310). Whether longtime hypotonic exposure actually affects the expression of the enzymes in the swelling-induced signaling sequence or of the volume-sensitive transporters is currently under investigation.

ACKNOWLEDGMENT This work was supported by the Danish Research Council and Fonden af 1870.

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