Springer-Verlag 1998

Cell Tissue Res (1998) 291:337±349

REGULAR ARTICLE

Helmut Bartels ´ Ian C. Potter ´ Katja Pirlich Jon Mallatt

Categorization of the mitochondria-rich cells in the gill epithelium of the freshwater phases in the life cycle of lampreys

Received: 30 June 1997 / Accepted: 5 August 1997

Abstract The distribution and ultrastructure of the mitochondria-rich (MR) cells in the gills of larval (ammocoetes) and adult lampreys (Petromyzon marinus and Geotria australis) have been studied. One type of MR cell, which is found only in ammocoetes, occurs in groups on and between gill lamellae. Freeze-fracture replicas show that the apical membrane of this ammocoete MR cell contains globular particles. The second type of MR cell, which is present in both ammocoetes and adults in freshwater, is located between lamellae and at the base of the filament. This cell usually occurs singly and is typically intercalated between ammocoete MR cells in larval lampreys and between pavement cells and pavement and chloride cells in adult lampreys. It contains rod-shaped particles in either the apical membrane (subtype A) or, far less frequently, the lateral membrane (subtype B) and in membranes of cytoplasmic vesicles and tubules. These features characterize this intercalated MR cell as a member of a group of MR cells that are also found in urinary epithelia of tetrapods and the amphibian epidermis, where they are involved in H+ and HCO3- secretion. Because this type of MR cell disappears when the young adult lamprey enters This paper is dedicated to Prof. Enrico Reale on the occasion of his 70th birthday Financial support was provided by the Australian Research Grants Committee

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H. Bartels ( ) Anatomische Anstalt, Universität München, Pettenkoferstrasse 11, D-80336 München, Germany Tel.: +49-89-51604850, Fax: +49-89-51604857, e-mail: [email protected] H. Bartels ´ K. Pirlich Abteilung für Zellbiologie und Elektronenmikroskopie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany I. C. Potter School of Environmental and Life Sciences, Murdoch University, Murdoch, 6150, Western Australia J. Mallat Department of Zoology, Washington State University, Pullman, Washington, 91664-4236, USA

the sea and reappears immediately after the fully grown adult re-enters freshwater on its spawning run, it is presumably essential for osmoregulation in freshwater. On the basis of electrophysiological studies on frog skin, it is proposed that the subtype A of the branchial intercalated MR cell of lampreys provides the driving force for the Na+ uptake by active H+ secretion. By analogy with urinary epithelia, the subtype B cells may exchange Cl- for HCO3-. Key words Mitochondria-rich cell ´ Gill epithelium ´ Freshwater osmoregulation ´ Ion uptake ´ Lampreys, Geotria australis, Petromyzon marinus (Agnatha)

Introduction The larval phase in the life cycle of all species of lampreys is spent in freshwater and typically lasts for at least three years (Potter 1980). The microphagous larva (ammocoete) eventually undergoes a radical metamorphosis into a young adult, which, in anadromous species, migrates downstream to the sea, where it feeds on the blood and/or muscle tissue of fish (Potter and Hilliard 1987). After a period of rapid growth, the adult re-enters rivers and migrates upstream to shallow areas, where spawning and then death occur (Hardisty and Potter 1971). The ammocoete is unable to osmoregulate in water where the osmolality exceeds that of its serum (Morris 1972; Beamish 1980). Although lampreys have developed the capacity to osmoregulate in full-strength seawater by the end of metamorphosis (Potter and Huggins 1973; Potter and Beamish 1977; Potter et al. 1980), this ability is progressively lost during the upstream spawning migration that follows the completion of the marine trophic phase (Morris 1956, 1972). The mechanisms used by lampreys for osmoregulating in marine (hypertonic) environments are considered to be similar to those employed by marine teleosts. These involve drinking sea water, resorbing monovalent ions through the intestine

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and secreting the resultant excess Na+ and Cl- through the activity of chloride cells in the gill epithelium (Smith 1930; Morris 1972; Zadunaisky 1984; Karnaky 1986; Bartels and Potter 1991). In lampreys, the chloride cells develop during metamorphosis and disappear during the upstream spawning migration (Morris 1957; Peek and Youson 1979). The gill epithelia of lampreys and teleosts also play a crucial role in osmoregulation in freshwater. This occurs through the uptake of Na+ and Cl- from the environment in exchange for H+ and HCO3-, respectively (Krogh 1939; Morris 1972). In upstream-migrating adults of the lamprey Lampetra fluviatilis, the gill epithelium contains a mitochondria-rich (MR) cell that differs morphologically from the chloride cell found during the marine phase (Morris 1957; Morris and Pickering 1976; Nakao 1977; Bartels and Welsch 1986). The rod-shaped particles revealed in the plasma membranes of the MR cell by freeze fracture have led to the identification of this cell as a member of a group of MR cells that are present in various epithelia involved in ion transport and acid-base regulation (Bartels and Welsch 1986). However, in contrast to the situation in upstream-migrating lampreys, the gill epithelium of ammocoetes has been described as containing two types of MR cells that differ in their distribution, arrangement and morphology (Youson and Freeman 1976; Mallatt and Ridgway 1984). The use of freeze-fracture replicas during the present study indicates that one of the two types of MR cells in the gills of ammocoetes is identical to the MR cell found in upstream-migrating lampreys. This study also shows that this type of MR cell is present during all freshwater stages of the lamprey life cycle and that neither this type, nor the other type of MR cell, occurs during the period spent in seawater. The ultrastructural characteristics of this MR cell are considered in the context of models developed for other epithelia engaged in osmoregulation in freshwater in order to propose mechanisms whereby Na+ and Cl- are taken up by the gills of lampreys in freshwater. The different cell types in the lamprey gill epithelium are also assigned names that are consistent both with their location and structure and with those of comparable cells in other ion-transporting epithelia.

Fully grown adults of G. australis that were about to start their upstream migration were netted at night, soon after they had passed over the sandbar at the mouth of the Donnelly River, during periods of heavy freshwater discharge. Since the exchange of seawater across the sandbar is at best limited at these times, there is essentially no estuary to the Donnelly River. Thus, these migrating lampreys had moved suddenly from full-strength seawater to freshwater. Four of these adults were immediately placed in fresh (river) water, whereas another three were placed in full-strength seawater. These two groups of fully grown adult lampreys were immediately taken back to the laboratory where, for one week, they were kept in renewed fresh river water and seawater, respectively, and then killed under anaesthesia with benzocaine. Electron microscopy. The third and fourth gills of all animals used in the present study were fixed in a solution of 2.5% glutaraldehyde in 0.1 M Na-cacodylate-HCl buffer, pH 7.4. Small pieces of gill tissue, containing two to four filaments, were then removed and processed for thin-section and freeze-fracture electron microscopy. The material used for thin-section electron microscopy was postfixed either in 2% OsO4 in 0.1 M cacodylate buffer or in OsO4-ferrocyanide solution (Karnovsky 1971), dehydrated in ethanol and embedded in either Agar-Araldite or Epon 812. Thin sections were stained with uranyl acetate and lead citrate. The tissue samples for freeze-fracture electron microscopy were cryoprotected in 30% glycerol in Ringer solution, mounted on specimen holders, frozen in the liquid supernatant of melting Freon 22 and stored in liquid nitrogen. Replicas were prepared by using a BAM 360 Balzers freeze-fracture device, equipped with a QSG 201 quartz crystal thin-film monitor and an EVM 052 electron-beam gun (Balzers, Liechtenstein) at 2”10-6 Torr and ±100C. The replicas were cleaned in commercial bleach, chromic acid and distilled water and collected on Formvar-coated copper grids. Thin sections and replicas were examined in a Siemens Elmiskop IA and a Philips CM 10 electron microscope at 80 kV. Some of the gill tissues of ammocoetes and young adults of G. australis, which had been fixed in glutaraldehyde as described above, were used for scanning electron microscopy. These tissue samples were thoroughly rinsed in the same buffer that was used with the fixative and made conductive by using the thiocarbohydrazide method (Malick et al. 1975). They were dehydrated in a graded series of acetone, critical-point dried in a CPD 020 device (Balzers), mounted on metal stubs by means of conducting carbon paint (Leit-C, Neubauer, Münster, Germany) and examined in a JEOL SSM-35CF and a Philips 505 Scanning Electron Microscope.

Results Ammocoetes

The lamellae of the gills of ammocoetes are covered by a two-layered squamous epithelium, which, at the base of the lamellae, gradually changes into the cuboidal Materials and methods multi-layered epithelium that characterizes the interlamellar region of the filament (Fig. 1a). The different cell Animals. Ammocoetes of Petromyzon marinus and ammocoetes and types at the gill surface, i.e. the two types of MR cells downstream migrating young adults of Geotria australis were caught in the Black Mallard River, Michigan, and the Donnelly Riv- and the pavement cell, can be identified in freeze-fracer in south-western Australia, respectively. These animals were ture replicas on the basis of their cytological charactermaintained in laboratory aquaria under conditions approximating istics, such as the presence and size of cytoplasmic vestheir natural environment. After three weeks, six ammocoetes of both P. marinus and G. australis were decapitated under anaesthesia icles and granules, the frequency, size and location of with 0.25 ml/l phenoxyethanol or a 0.01% solution of benzocaine. mitochondria, and the structure of the apical surface Twelve young adults of G. australis were acclimated to full- (Figs. 2±6). strength seawater by transferring them first to 1/3 seawater for 4 days The most abundant type of MR cell, termed the ªmitoand then to 2/3 seawater for a further 4 days. After they had been in chondria-rich cellº by Morris and Pickering (1975), ªmifull-strength seawater (35½) for one week, these animals and 12 downstream migrants that had been maintained in river water tochondria-rich platelet cellº by Youson and Freeman (1976) and ªion-uptake cellº by Mallatt and Ridgway were killed under anaesthesia in benzocaine.

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Fig. 1a Survey electron micrograph of two successive interlamellar regions and the bases of gill lamellae (L) in larval G. australis. Intercalated MR cells (arrowheads) are located singly between ammocoete MR cells with ªdarkº mitochondria. b Scanning electron micrograph showing four intercalated MR cells (arrowheads) between ammocoete MR cells at the base of a lamella; G.australis. a ”2100, b ”2200 Fig. 2 Ammocoete MR cell (MR) next to a pavement cell (P) on a gill lamella of G. australis. Note the differences in the size of the secretory granules and in the electron density of the mitochondrial matrices. ”20 700

(1984) and Mallatt et al. (1995), is arranged in large groups over most of the surface of the lamellae and in the narrow region between the lamellae (Figs. 2, 3). These MR cells contain numerous mitochondria, which occupy about one third of the cellular volume (Mallatt et al. 1995) and are characterized by an electron-dense matrix (Fig. 2). Some of these MR cells possess small ovoid and round granules that are up to 0.3 mm in height and 0.1 mm in width (Fig. 2). These granules, which are located between the apical membrane and mitochondria (Fig. 2), probably contain mucus. The surface of the api-

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cal membrane is moderately enlarged in area by the presence of short microplicae and/or microvilli (Figs. 1b, 2, 3a). In freeze-fracture replicas, the apical membrane of these MR cells has the typical appearance of vertebrate cell membranes (Fig. 3). There are numerous globular particles (diameter 8±9 nm) on the protoplasmic face (P-face; Fig. 3b) and few particles but many small pits on the exoplasmic face (E-face; Fig. 3c). Because this type of gill MR cell occurs only in larval lampreys, we will subsequently refer to it as the ammocoete MR cell. This term also avoids implying a function, for which there is no experimental evidence as yet. The second and less abundant type of MR cell in ammocoetes has been termed the ªchloride cellº by Youson and Freeman (1976) and Mallatt and Ridgway (1984). These cells are restricted to the interlamellar region of the filament and to the base of the lamellae, where they occur singly or in pairs, intercalated between the ammocoete MR cells (Figs. 1, 4). Unlike the ammocoete MR cell described above, the matrix of the mitochondria of these cells has the same density as the cytoplasm (Fig. 4a). Furthermore, these cells lack mucous secretory granules. Instead, their cytoplasm contains numerous small vesicles and membranous tubules, which are interspersed between mitochondria and frequently occur directly below the apical membrane (Fig. 4a). In a few cases, a vesicle can be seen fusing with the apical membrane. The membrane of a tubule has never been observed to be continuous with the lateral cell membrane, contrasting with the situation typically found in the chloride cells of adult lampreys. The surface area of the apical membrane is enlarged by an elaborate system of slender branching microplicae (Fig. 4a, c). Apical crypts, similar to those in the chloride cells of marine teleosts (Karnaky 1986), have not been observed. Although such crypts sometimes appear to be present, these arise because the section has been cut at a slightly oblique angle through the system of microplicae at the apical cell pole. A coat of studs, projecting about 12 nm from the membrane, is present on the cytoplasmic side of the apical membrane (Fig. 4b). In freeze-fracture replicas (Fig. 5), the apical membrane of this second type of MR cell contained rodshaped particles and globular particles on its P-face (Fig. 5b) and rod-shaped pits on its E-face (Fig. 5c). The rod-shaped particles measured 16±18 or 24±27 nm Fig. 3a±c Freeze fracture of the ammocoete MR cells of P. marinus. a Survey micrograph showing small secretory granules (arrowheads) and large mitochondria (M) immediately underneath the apical membrane (A). b P-face (P) of the apical membrane (framed area in a), showing numerous globular particles. c The E-face (E) of the apical membrane is almost devoid of particles. Arrows in a, c Zonulae occludentes of the ammocoete MR cell. In this and the following freeze-fracture micrographs, the direction of platinum/carbon shadowing is approximately from the bottom to the top. a ”32 000, b, c ”80 000

in length and 8±9 nm in width and appeared to consist of two or three globular subunits (Bartels and Welsch 1984; Kohn et al. 1997). The density of the rod-shaped particles on the P-face of the apical membrane was related to the extent to which this membrane was amplified. In other words, the rod-shaped particles were most densely packed in those apical membranes where the microplicae were greatest in number and largest in size (cf. Figs. 5b, 8a). Rod-shaped particles and pits, similar to those in the apical membrane, were regularly present in the membranes of the cytoplasmic vesicles and tubules (Fig. 5d). In a few MR cells, these particles were also detected in the lateral membrane (shown in an adult in Fig. 8b) These MR cells share many ultrastructural characteristics, such as the presence of abundant mitochondria, cytoplasmic vesicles and tubules, microplicae that are often well-developed, a coat of studs on the cytoplasmic side of the apical membrane and rod-shaped intramembranous particles (Brown 1989; Brown and Breton 1996), with intercalated MR cells in the renal collecting duct and various other ion-transporting epithelia. For this reason, we subsequently refer to these MR cells in the gills of lampreys as intercalated MR cells. The pavement cells, called ªmitochondria-poor cellsº by Youson and Freeman (1976), ªmucous-platelet cellsº by Mallatt and Ridgway (1984) and ªmucous-pavement cellsº by Mallatt et al. (1995), are located towards the tip of the lamella, in a position apical to the ammocoete MR cells. They are characterized by abundant ovoid mucous secretory granules, which are 0.4 mm in height and 0.15 mm in width and are thus larger than those in ammocoete MR cells (Fig. 2). These granules are located immediately below the apical cell membrane, whose area is moderately increased by the presence of low microplicae or short microvilli. The mitochondria, which are few in number, have a less electron-dense matrix than those in the ammocoete MR cell (Fig. 2). In freeze-fracture replicas of the gills of larval P. marinus, the apical membrane of these latter cells is characterized by numerous large particles (diameter 10±13 nm) on the E-face (Fig. 6), whereas the P-face contains only a few small particles, thereby resembling the apical membrane of pavement cells in adult lampreys (Bartels 1989). These cells are identical to the only type of cell found on the surface of the gill lamellae of adult lampreys (Morris 1957; Youson and Freeman 1976; Bartels 1989). In adult lampreys, these cells have been termed ªpavement cellsº by Bartels (1989), as they are analogous to the pavement cells of the gill lamellae of teleost fishes (Laurent 1984). This term is now extended to these corresponding cells in ammocoetes (see also Mallatt et al. 1995). Adult lampreys The intercalated MR cell was the only one of the two types of MR cells described above for ammocoetes that

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Fig. 4a±c Intercalated MR cell (type A) of larval G. australis. a The area of the apical surface is enlarged by long slender microplicae and the apical cytoplasm contains numerous membranous vesicles and tubules. b A coat of studs (arrowheads) projects from the cyto-

plasmic surface of the apical membrane. c Scanning electron micrograph showing the elaborate system of microplicae at the apical surface. a ”12 700, b ”66 700, c ”6000

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Fig. 5a±d Freeze fracture of the intercalated MR cells (type A) of larval P. marinus. a Survey micrograph showing numerous membranous vesicles and tubules and mitochondria (M) in the cytoplasm. b P-face of the apical membrane (framed area in a), showing numer-

ous rod-shaped particles. c E-face of the apical membrane (E) showing rod-shaped pits and P-face (P) showing rod-shaped particles. d Rod-shaped particles and pits (arrowheads) in the membranes of cytoplasmic vesicles and tubules. a ”40 000, b±d ”80 000

344 Fig. 6 Freeze fracture of the apical membrane of a pavement cell of larval P. marinus. The Eface (E) is characterized by numerous large particles. ”66 700 Fig. 7 Scanning electron micrograph showing the gill epithelium at the base of the filament of a downstream migrating young adult G. australis kept in freshwater. Intercalated MR cells (stars) are singly located between pavement cells. Arrowheads Chloride cells arranged in rows. ”2800

was found in the gill epithelium of adult lampreys. This cell type was only found in young adult lampreys prior to their entry into seawater (Figs. 7, 8b) and in fully grown adults after they had embarked on their upstream migration (Fig. 8a). The intercalated MR cells were no longer present in young adults that had been acclimated to full-strength seawater for one week (Bartels et al. 1996). They were also absent from those adults that had been caught soon after they had re-entered the Donnelly River on their upstream migration and that were then held for one week in full-strength seawater. However, these cells were present in the corresponding group of upstream migrants that were examined after being maintained in river water for one week (Fig. 8a).

The intercalated MR cells of adult lampreys occur in the interlamellar region and at the base of the filament (Fig. 7) and are thus located in the same position as in ammocoetes (see above). However, the intercalated MR cells of ammocoetes are located beween ammocoete MR cells, whereas those of downstream migrants and early upstream migrants are situated between pavement cells and between pavement and chloride cells. As in ammocoetes, the vast majority of the intercalated MR cells in adult lampreys contain rodshaped particles in the apical membrane (Fig. 8a), rather than in the lateral membrane (Fig. 8b) and in the membranes of cytoplasmic vesicles and tubules. These MR cells are identical to the type 3 cells described by Nakao (1977) in upstream migrating Lampetra japonica.

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Fig. 8a, b Freeze fracture of the intercalated MR cells in adult lampreys. a Type A cell in a fully grown adult at the beginning of the upstream migration (one week in freshwater). A Apical membrane with low microplicae and globular and rod-shaped particles, L lateral membrane with only globular particles, ZO zonula occludens. b Type B cell in a young adult prior to entry into seawater. Small fragments of the lateral membrane (arrowheads) with rod-shaped particles are exposed. Stars Intercellular spaces between the intercalated MR cell and a chloride cell (CC). a ”50 000, b ”60 000

Discussion Ammocoete MR cell In contrast to the intercalated MR cell, the ammocoete MR cell disappears early in metamorphosis (Peek and Youson 1979) and never reappears during adult life. Since this cell type contains numerous mitochondria and shows a positive histochemical reaction for carbonic anhydrase (Conley and Mallatt 1988), it has been assumed to be involved in transporting ions, such as Na+, Cl-, H+ or HCO3(Morris and Pickering 1975; Youson and Freeman 1976; Mallatt and Ridgway 1984). However, there is as yet no experimental evidence that this is indeed the function of these cells. Since the ammocoete MR cell is not present in downstream- and upstream-migrating adults, which are faced with the same problems of osmoregulation in

freshwater as the ammocoete, the uptake of Na+ and Clmust be achieved in adult freshwater stages, either by the intercalated MR cell on its own or in conjunction with pavement cells, the only other cell type that is present throughout both freshwater periods in adult life (see below). It appears relevant, however, that the ammocoete MR cell is present only when the lamprey is feeding in freshwater. It is thus possible that this cell is responsible for the secretion of ion(s) or waste products that are related to the ingestion of food. The diet of ammocoetes comprises predominantly detritus and diatoms (Moore and Mallatt 1980) and thus fundamentally differs from that of adult lampreys, which ingest the tissues and body fluids of host fishes (Bahr 1952; Potter and Hilliard 1987). Intercalated MR cell Intercalated MR cells are present in the gill epithelium throughout the larval life and metamorphosis of lampreys but disappear soon after the young adult lamprey has entered seawater. This cell reappears when the fully grown adult re-enters rivers at the start of the spawning run. The timing of the presence, loss and reappearance of the intercalated MR cell provides strong circumstantial evidence that this cell plays a crucial role in osmoregulation

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throughout all freshwater phases in the life cycle of anadromous species of lampreys. The numerous rod-shaped particles present in membranes of the intercalated MR cells of the gill epithelium of larval and adult lampreys ally this cell type with a group of MR cells that are present in various ion-transporting epithelia. These cells include the MR cell in the toad and turtle urinary bladders, the flask cell in the amphibian renal collecting duct and epidermis, and the intercalated cell in the mammalian renal collecting duct, all of which are rich in cytosolic carbonic anhydrase and contain a vacuolar type proton pump (H+V-ATPase) in their plasma membrane and in cytoplasmic vesicular membranes (Brown and Breton 1996). Immunocytochemical studies of MR cells in the rat collecting duct, with antibodies against the H+V-ATPase, and rapid-freeze deepetch electron microscopy of MR cells in the toad urinary bladder epithelium have shown that the studs on the cytoplasmic surface of the membranes of MR cells represent the peripheral cytoplasmic V1 subunit of this proton pump (Brown et al. 1987). Since the density of rodshaped particles in the apical membrane is correlated with the amount of H+ flux across that membrane (Stetson and Steinmetz 1986), it has been proposed that these particles are either the transmembranous portion of the proton pump or an intimate associate of that pump, which is located in the same membrane domain (Brown et al. 1987). This hypothesis has been supported (1) by a quantitative study of MR cells in the turtle urinary bladder epithelium, demonstrating a one-to-one relationship between a submembranous stud and each of the globular subunits of an intramembranous rod-shaped particle (Kohn et al. 1997) and (2) the observation that all of the membranes that contain rod-shaped particles and have thus far been examined by freeze-fracture replicas express H+V-ATPase activity (Brown and Breton 1996). There are two subtypes of intercalated MR cells, termed A cells and B cells. These are involved in H+ and HCO3- secretion, respectively, and are distinguished in the renal collecting duct on the basis of the location of the H+V-ATPase, rod-shaped intramembranous particles and a bicarbonate exchanger. In the proton-secreting A cells, the H+V-ATPase and rod-shaped particles are located in the apical membrane and the membranes of cytoplasmic vesicles, whereas the basolateral membrane contains a Cl-/HCO3- exchanger, which is identified as an alternatively spliced kidney form of the band 3 protein AE1 (Brown and Breton 1996). The bicarbonate-secreting B cells were orginally identified by the location of the H+VATPase and rod-shaped particles in the basolateral membrane, whereas the Cl-/HCO3- exchanger is in the apical membrane and, although functionally detectable, is nonimmunoreactive for AE-1 and is still unidentified at the protein level. More recent studies have shown, however, that the B cells lacking AE-1 vary in the subcellular immunolocalization of H+V-ATPase, which has been detected in either the apical or basolateral membrane, in both membrane domains or diffusely in the cytoplasm (Brown and Breton 1996).

Since the vast majority of the intercalated MR cells that we have found in the gill epithelium of larval and adult lampreys contain numerous rod-shaped particles in their apical membranes, most of these cells probably belong to the subtype A of the MR cell and are thus regarded as responsible for secreting protons through the gill surface. Accordingly, the few MR cells that possess rod-shaped particles in the basolateral membrane could represent the bicarbonate-secreting subtype B. In view of the various immunocytochemical localizations of the H+V-ATPase in AE-1-negative type B-cells (see above), the possibility cannot be excluded that some of the lamprey MR cells, which possess rod-shaped particles in the apical membrane, belong to subtype B. Further immunocytochemical and functional studies are thus needed to differentiate more clearly between the two subtypes of intercalated MR cells in the lamprey gill epithelium. Models of ion uptake in freshwater Two models have been proposed for the site of ªNa+ for H+º exchange during osmoregulation in freshwater. Both are based mainly on work on the epithelium of the frog skin, which contains intercalated MR cells and granular cells, and imply that H+ is actively secreted by a proton pump, whereas Na+ uptake occurs through amiloride-sensitive Na+ channels (Kirschner 1983; Harvey and Ehrenfeld 1988; Larsen 1988, Nagel and Dörge 1996; Ehrenfeld and Klein 1997). Although both models invoke MR cells as being responsible for H+ secretion (see above), there is disagreement as to the participation of these two cell types in the uptake of Na+. The hypothesis that Na+ is taken up by the MR cell itself is based on indirect evidence from electrophysiological studies carried out under natural physiological conditions, i.e. dilute external Na+ concentrations and an open-circuit condition, whereas the view that it is taken up by adjacent granular cells was originally based on results obtained under ªUssing conditionsº, i.e. where short-circuited skin is bathed on both sides with Ringer solution (Harvey and Ehrenfeld 1988; Ehrenfeld et al. 1989; Ehrenfeld and Klein 1997). However, when re-examining the possible participation of MR cells in Na+ uptake by frog skin under ªnatural conditionsº, Nagel and Dörge (1996) have found that Na+ transport across MR cells appears to be negligible and thus conclude that it occurs through the granular cells. Studies on the transport of Na+ and H+ in other tight epithelia, such as those of the toad and turtle urinary bladders, which are used as models for the collecting duct of the mammalian kidney, are consistent with the model derived employing Ussing conditions on the frog skin (Durham and Nagel 1986; Lang 1988). These urinary epithelia also contain two cell types that are analogous to the MR cells and granular cells in the frog epidermis (Wade et al. 1975; Wade 1976; Rick et al. 1978; Durham and Nagel 1986; Brown and Breton 1996). The pavement cell, which is the only cell other than the intercalated MR cell that is found in the superficial

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layer of the gill epithelium of adult lampreys throughout their upstream migration is also considered to be analogous to the granular cell in the toad bladder and frog skin (Bartels 1989; Bartels and Potter 1993). We propose that the subtype A of the intercalated MR cells in the gill epithelium of lampreys in freshwater provides the driving force for the uptake of Na+ by active H+ secretion, whereas the pavement cell must be considered as the primary candidate for the cell type that takes up Na+, at least in adult lampreys. Since the intercalated MR cells of larval lampreys are surrounded by ammocoete MR cells, rather than by pavement cells as in adults, the possibility that the ammocoete MR cells contribute to the uptake of Na+, as originally proposed by Morris and Pickering (1975), Youson and Freeman (1976) and Mallatt and Ridgway (1984), cannot be excluded. The alternative model proposed by Harvey and Ehrenfeld (1988) for the frog skin, which implies that Na+ is taken up by the intercalated MR cell itself, would have the advantage of providing a mechanism for Na+ uptake in lampreys that can be applied equally to ammocoetes and adults. It has been proposed that MR cells are also the principal site of Cl- transport across the amphibian skin (Katz et al. 1985; Foskett and Ussing 1986) and that, in urinary epithelia, Cl- is taken up in exchange for secreted HCO3- via the subtype B intercalated MR cell (Stetson and Steinmetz 1985; Schuster 1993). However, in contrast to the situation found in the mammalian collecting duct and turtle bladder, a subtype of MR cells that might primarily be responsible for the uptake of Cl- has not been identified in the amphibian epidermis, where the electrogenic proton pump, a Cl-/HCO3- exchange transport system and a voltage-gated Cl channel have been colocalized in the apical membrane of the intercalated MR cells (Larsen et al. 1992; Brown and Breton 1996). The question arises as to whether the few MR cells of subtype B that we have identified morphologically in the gills of larval and adult lampreys are sufficient to balance Cl- losses under normal conditions or whether those cells identified as subtype A in the present study also contribute to the uptake of Cl-. Further studies are thus needed to measure Na+ and Cl- transport across the lamprey gill epithelium and to determine the role of intercalated MR cells under defined experimental conditions. Comparison between the gill epithelia of lampreys and teleosts in freshwater The intercalated MR cells differ ultrastructurally from the chloride cells, which in both lampreys and teleosts are essential for osmoregulation in a hypertonic environment. They lack a tubular system, which represents a vast amplification of the basolateral cell membrane in chloride cells, but do possess intramembrane rod-shaped particles, which are absent from the membranes of chloride cells (Nakao 1974, 1977; Sardet et al. 1979; Pisam and Rambourg 1991; Bartels et al. 1993). Collation of the results of this and previous studies on lamprey gill epithelia show

that the onset of the marine phase is accompanied by the rapid disappearance of the intercalated MR cells and the increased exposure to the environment of the chloride cells (Bartels et al. 1993, 1996), a cell type that develops during the immediately preceding period of metamorphosis (Peek and Youson 1979). In contrast, entry into freshwater on the spawning run is accompanied by the rapid reappearance of intercalated MR cells and a gradual loss of chloride cells (Morris 1957). The above changes in the gill epithelium are presumably responsible for the ability of young adult lampreys to survive direct transfer from freshwater to seawater (Potter and Huggins 1973; Potter and Beamish 1977; Potter et al. 1980) and for fully grown adults to pass rapidly from the sea into rivers. As is the case in seawater, the basic physiological mechanisms by which lampreys and teleosts osmoregulate in freshwater are considered to be the same (Morris 1972; Hardisty et al. 1989). However, the cellular composition of the gill epithelia of these two groups differ in freshwater. Thus, for example, chloride cells are not developed by anadromous lampreys until they are about to enter the sea and are lost after re-entry into fresh water (Morris 1957; Peek and Youson 1979), whereas such cells are present throughout the life cycle of all teleosts, irrespective of whether the environment is freshwater or seawater (Laurent 1984; Hwang and Hirano 1985; King et al. 1989; Pisam and Rambourg 1991; Li et al. 1995; Perry 1997). However, unlike the situation in lampreys, there are two types of chloride cells in teleosts, one of which is responsible for Cl- and Na+ secretion in marine environments. The latter occurs in groups and is linked to adjacent chloride cells by shallow (leaky) occluding junctions (Karnaky 1986); it is thus similar to that in adult lampreys in seawater (Bartels and Potter 1991; Bartels et al. 1996). The other type of chloride cell is found only in teleosts in freshwater and occurs singly between pavement cells as do the intercalated MR cell in lampreys (Pisam and Rambourg 1991; Perry 1997). However, unlike the intercalated MR cell of lampreys, the freshwater chloride cell of teleosts contains a tubular system that is similar to, but less elaborate than, that of marine chloride cells and it does not possess rod-shaped particles in its plasma and/or cytoplasmic membranes. Although the term ªmitochondriarich cellº has also been applied to the freshwater chloride cell of teleosts (Pisam and Rambourg 1991), cells with the characteristics of the intercalated MR cell of lampreys have not been found in the gills of teleosts. The role in the exchange of ªNa+ for H+º in freshwater that we have ascribed to subtype A of the intercalated MR cell in lampreys, in concert with the pavement or ammocoete MR cells, is apparently played entirely by the pavement cell in teleosts. This conclusion is based on (1) the observation that, when teleosts are exposed to acidosis, there is an increase in both the surface area of their pavement cells and the uptake of Na+ through the gills (Goss et al. 1992) and (2) the immunocytochemical demonstration that the pavement cells contain H+-ATPase (Sullivan et al. 1995). Experimental studies also indicate that the chloride cells of teleosts in freshwater are responsible for exchang-

348

ing Cl- for HCO3- (Perry 1997), a role that could be played by the intercalated MR cell in lampreys, possibly its subtype B. Acknowledgements The authors thank Drs. R.W. Hilliard and G.Power for collecting and acclimating Geotria australis, Dr. A. Moldenhauer for providing Fig. 7, Mrs. U. Fazekas and Mr. H. Heidrich for excellent technical assistance, and Mrs. S. Herzmann for expert assistance with photography.

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