Journal of Comparative s,,~176176176176176 Physiology B m..,.,

J Comp Physiol B (1985) 155:185-193

and EnvironPhysiology

9 Springer-Verlag 1985

Taurine transport by brush border membrane vesicles isolated from the flounder kidney Patricia A. King 1,a, Rolf Kinne 2,3,,, and Leon Goldstein l'a 1 Division of Biology and Medicine, Brown University, Providence, Rhode Island 02906, USA 2 Department of Physiology, Albert Einstein College of Medicine, Bronx, New York 10451, USA 3 Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672, USA Accepted May 30, 1984

Summary. Taurine transport was investigated in brush border membrane vesicles isolated from renal tubules of the winter flounder (Pseudopleuronectes americanus). Taurine uptake by the vesicles was greater in the presence of NaC1 as compared to uptake in KC1. The Na+-dependent taurine transport was electrogenic and demonstrated tracer replacement and inhibition by fl-alanine and HgCI2, indicating the presence of Na +-dependent, carrier-mediated taurine transport. In contrast to Na+-dependent taurine transport across the basolateral membrane, there was not a specific C1- dependency for transport in the brush border membrane. No evidence was obtained for Na+-inde pendent carrier-mediated taurine transport. The possible involvement of the brush border Na+-de pendent transport system in the net secretion of taurine from blood to tubular lumen in vivo (Schrock et al. 1982) is discussed.

Introduction

Taurine (2-aminoethane sulfonic acid) is one of the most commonly occurring amino acids in the urine of vertebrates, ranging from marine fishes (Schrock et al. 1982) to mammalian species including man (Gilbert et al. 1960; Jacobsen and Smith 1968). In mammals, the relatively high urinary concentration of taurine appears to result from a poor net renal reabsorption of this amino acid (Gilbert et al. 1960; Eisenbaeh et al. 1975; Dantzler and Silbernagl 1976). In contrast, in vivo investigations * Present address : Max-Planck-Institut ffir System Physiologic, Rheinland Damm 201, D-4600 Dortmund 1, FRG

of the renal handling of taurine in marine fish species have revealed a net secretion (Schrock et al. 1982). Net secretion of an amino acid is rarely observed (Schafer and Barfuss 1980) but in marine fish the renal secretion of taurine appears to be a general phenomenon with the important homeostatic function of regulating the plasma levels of this amino acid (King et al. 1980; Schrock et al. 1982). The mechanism of taurine secretion across the fish renal epithelium has been investigated in the winter flounder, Pseudopleuronectes americanus. The kidney of this species is primarily proximal tubule (Hickman and Trump 1969). Previously we examined taurine movement across the flounder renal basolateral membrane. Isolated tubule studies confirmed in vivo evidence of net taurine secretion by the flounder renal tubules and indicated taurine movement across the basolateral membrane as an uphill concentrating step dependent on the presence of Na + and CI-, and inhibited by other t-amino acids and y-amino butyric acid (King et al. 1982). Taurine movement across the apical membrane then occurs down a chemical concentration gradient from the cell into the lumen. The purpose of the current study was to investigate taurine movement across the luminal membrane of the flounder kidney by assaying taurine transport by brush border membrane vesicles. Membrane vesicles provide a means for studying transport across one membrane of an epithelium separate from the effects of cell metabolism and transport across the other membrane. Once isolated, brush border membranes form vesicles which, in the case of the rat renal brush border vesicles, have a diameter of 0.2 to 0.7 gm and are

186

P.A. King et al. : Taurine transport by flounder renal brush border membrane vesicles

8 5 % r i g h t - s i d e - o u t ( H a a s e et al. 1978). T h e m e m b r a n e in v i t r o t h e r e f o r e h a s t h e s a m e o r i e n t a t i o n a s in v i v o . I t is m o s t l i k e l y t h a t t h e f l o u n d e r r e n a l b r u s h b o r d e r vesicles h a v e s i m i l a r c h a r a c t e r i s t i c s of sidedness. I n t h e p r e s e n t s t u d y w e w e r e i n t e r e s t e d in i d e n tifying Na+-dependent and/or Na+-independent transport systems facilitating taurine movement across the flounder brush border membrane. For the rat kidney, which shows a net reabsorption (albeit poor) of this amino acid, taurine transport b y b r u s h b o r d e r m e m b r a n e vesicles h a s b e e n f o u n d to be a Na+-dependent process that displays an o v e r s h o o t in t h e p r e s e n c e o f a s o d i u m g r a d i e n t (100 m M ) w i t h n o e v i d e n c e o f a N a + - i n d e p e n d e n t c a r r i e r m e d i a t e d t r a n s p o r t ( R o z e n et al. 1979). I t w a s t h e r e f o r e o f i n t e r e s t t o d e t e r m i n e w h e t h e r diff e r e n c e s in a p i c a l m e m b r a n e t a u r i n e t r a n s p o r t syst e m s in m a m m a l i a n a n d m a r i n e fish k i d n e y s a c c o u n t f o r t h e n e t r e a b s o r p t i o n o f t a u r i n e in t h e f o r m e r a n d its n e t s e c r e t i o n in t h e l a t t e r .

Materials and methods Animals. Winter flounder, Pseudopleuronectes americanus, of mixed sex were caught by dragnet off Mount Desert Island, Maine. The animals were held in running seawater at 12-17 ~ until use, usually 2-3 days following capture. During this time, the fish were not fed. Preparation of brush border membrane vesicles. The protocol for preparation of the renal brush border membrane vesicles was modified from the procedures of Eveloff et al. (1979) for the preparation of flounder renal brush border membrane vesicles, and Booth and Kenny (1974) for the isolation of brush border membranes by calcium precipitation. All procedures were carried out at 0-4 ~ Twelve to fifteen flounders were killed by transection of the spinal cord, and the kidneys rapidly removed and placed in 20-40 ml ice cold buffer (135 mM NaC1, 2.5 mM KC1, 1.5 mM CaCI2, 1.0 mM MgCI2, 15.0 mM Tris, 0.5 mM NaHzPO 4, pH 8.5). The renal tissue was cut into small pieces with scissors until the minced tissue offered little resistance to being drawn into a 50 ml syringe. The hematopoeitic tissue was separated from the tubules by suction through the syringe (20 times) followed by centrifugation at 50 g for 1 min. The supernatant was removed, the tubule pellet resuspended in 20 ml of the buffer and the mixture centrifuged at 50 g for 1 min. The tubules were washed in this manner 3 times in order to completely remove the red blood cells. The final tubule pellet was taken up in approximately 100 ml ice cold homogenization buffer (10 mM mannitol, 2 mM Tris-HC1, and 2 mM CaCI2, pH 7.1) and homogenized at full speed for 1 rain in a precooled Waring blender. CaC12 was then added to the homogenate to give a final concentration of 30 raM, and the mixture was allowed to sit on ice for 15 min. At the end of this time, the suspension was centrifuged at 1,500g for 12 min. The supernatant was removed and recentrifuged at 15,000g for 20 rain. The resulting pellet was recovered in 25 ml of homogenization buffer, homogenized in a teflon-glass homogenizer to resuspend the membranes and then treated again with CaC12 (final concentration of CaC12 30 mM). After 15 rain on ice, the mixture was centrifuged at 2,400 g for 12 min and the supernatant was

then recentrifuged at 20,000 g for 12 min. The final pellet was taken as the brush border membranes and resuspended in vesicle buffer (200 mM mannitol, 20 mM Tris-HEPES, 2raM CaC12, pH 8.2) by gentle homogenization with a 100 gl Eppendoff pipette. The final protein concentration was approximately 10 mg/ml. The vesicles were stored at - 8 0 ~ until use (up to 5 days). We found that storage over this period did not effect the viability of the vesicles as measured by the rate of D-glucose uptake.

Uptake measurements. Taurine uptake was measured by a rapid filtration method (Hopfer et al. 1973; Eveloff et al. 1979). The vesicles were quickly thawed (37 ~ before use and the transport studies were initiated by adding 20 gl of the vesicle preparation to 130 gl of incubation medium that was 0.1 mM in taurine and contained 10 gCi 3H-taurine. The total composition of the incubation media and manipulations of the vesicles are described in the figure and table legends. At the indicated time points, transport was stopped by rapidly diluting 20 gl of the reaction medium into I ml of ice cold stop solution (240 mM mannitol, 100 mM NaC1, 2 mM CaC12, 20 mM Tris-HEPES, pH 8.2). The total volume was quickly pipetted onto a millipore filter (HA 0.45 gin) and the filter was then washed with an additional 3 ml of the stop solution. The filter and adhering membranes were combined with liquid scintillation fluid and counted by standard procedures. In some experiments glucose or alanine uptake was also measured. In these studies the incubation medium was 0.1 mM glucose or alanine and included 10 gCi 3H-glucose or 5 gCi 14C-alanine. All experiments were carried out at 15 ~ incubation solutions and vesicle buffer were filtered (Millex-GS, 0.22 gin) in order to avoid bacterial contamination. Enzymes. The brush border membranes, as well as the whole kidney homogenate and discarded fractions of the preparation, were analyzed for Na+-K+-ATPase and alkaline phosphatase activities (Heidrich et al. 1972). All assays were performed on previously frozen preparations, and activity was measured at 37 ~ Proteins were measured after precipitation with 10% TCA by the method of Lowry etal. (1951) using bovine serum albumin as a standard. Statistical analysis. Data are expressed as the means of the number of experiments indicated by n. Standard errors are included in the tables. Data were analyzed using one-way analysis of variance and the Student-Neuman-Keuls test; for comparison of only two means the Student t-test for group or paired data was employed. Significant differences in uptake are indicated by P values. Chemicals. Radiolabeled chemicals, [2-3H(N)]-taurine (22.7 Ci/ mmol), D-16-3H(N)]-glucose (25 Ci/mmol), and L-[t*C(U)]-alanine (150 mCi/mmol), were obtained from New England Nuclear. All other chemicals were purchased from Sigma Chemical Co. or Fisher Scientific and were of the highest purity available.

Results Enzymatic characterization o f the brush border membranes Alkaline phosphatase and Na+-K+-ATPase were u s e d as m a r k e r e n z y m e s f o r t h e b r u s h b o r d e r a n d basolateral membranes, respectively. This enzymatic characterization has previously been used to

P.A. King et al. : Taurine transport by flounder renal brush border membrane vesicles identify flounder renal membranes (Eveloff et al. 1979; Renfro and Pritchard 1982) and assumes that the distribution of these enzymes in the flounder kidney is similar to that in the rat renal cell (Kinne et al. 1976). Alkaline phosphatase activity in the vesicles averaged 1 0 . 5 + 2 . 2 g m o l e s substrate released/rag protein h (mean_+ SE, n = 8) and was enriched more than l l.5-fold compared to activity in the whole homogenate. Brush border N a + - K + - A T P a s e activity was 3.0_+1,3 ~tmoles substrate released/rag protein.h; the ratio of vesicle/homogenate activity for this enzyme was 0.37 and indicates little basolateral contamination of the final brush border membrane preparation. The total recovery of enzyme activity during the preparation of the membranes as compared to activity in the original homogenate was 97.6_+4.9% (mean_+SE, n = 8 ) for alkaline phosphatase and 83.3__+ 14.5% for Na+-K+-ATPase.

Functional characterization of the brush border vesicles Previous studies of flounder renal brush border membrane vesicles demonstrated that the uptake of o-glucose and L-alanine occur by Na+-depen dent transport systems (Eveloff et al. 1980). The functional viability of vesicles prepared in the present study was therefore tested by following the uptake of these two solutes. In the presence of a 75 m M inward-directed NaC1 gradient, the rate of glucose uptake was characterized by a large overshoot (Fig. 1) and was significantly greater than uptake in an equal KC1 gradient (not shown). Alanine uptake also displayed a slight overshoot under the NaC1 gradient conditions (Fig. 1). These data are in accord with the studies of Eveloff et al. (1980) and verify the functional integrity of the vesicle preparation. In comparison to o-glucose and L-alanine, the rates of taurine transport in the presence of the NaC1 gradient were much lower and did not display an overshoot. The equilibrium values for o-glucose, L-alanine, and taurine uptake by the brush border vesicles were similar (Fig. 1) and indicate that the 3 solutes enter the same intravesicular space, in the range of 1.5-2.0 gl/mg protein.

187

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Fig. 1. Uptake of D-glucose, L-alanine and taurine into flounder renal brush border membrane vesicles. Vesicleswere prepared in 200 mM mannitol, 20 mM Tris-HEPES, pH 8.2, and 2 mM CaC12. The incubation medium contained 75 mM NaC1; 2 mM CaC12; 20raM Tris-HEPES, pHS.2, 0.1 mM [3H]-glucose, [14C]-alanine, or [3H]-taurine, and 50 mM mannitol. Values are the means of 2 experiments

ity. The incubation osmolarity was modified by addition of sucrose, an impermeant solute, and taurine uptake was compared under incubation conditions of 300, 333,400, and 600 mOsm. Taurine uptake at equilibrium decreased linearly as the osmolarity of the incubation medium was increased. These data indicate that taurine is taken up into an osmotically reactive space and provide further evidence that taurine uptake reflects transport into the vesicles.

Osmotic dependence of taurine uptake by renal brush border membrane vesicles

Sodium and chloride dependence of taurine uptake

Uptake of taurine in the brush border membrane vesicles was tested as a function of intravesicular volume on the assumption that intravesicular volume varies inversely with the incubation osmolar-

The rate of taurine uptake by flounder renal brush border membrane vesicles displayed a sodium dependence (Fig. 2). At 15 s, uptake in a 75 m M NaC1 gradient was approximately 2 times that ob-

188

P.A. King et al. : Taurine transport by flounder renal brush border membrane vesicles

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100

90

90

80

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60

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Fig. 2. Uptake of taurine into flounder renal brush border membrane vesicles. Uptake is expressed as % of equilibrium uptake. Values are means+SE (n=6). Vesicles were prepared as in Fig. 1. Incubation media contained 20 mM Tris-HEPES, pH 8.2, 50 mM mannitol, 2 mM CaCI2, 0.1 mM [3H]-taurine, and 75 mM NaC1 (e); 75 mM KC1 (A); 75 mM NaCI+ 10 mM taurine (o); or 75 mM KCI+I0mM taurine (zx). Data were analyzed by one-way analysis of variance and the Student-Newman-Keuls test. Uptake in NaCI was significantly greater than uptake in KC1 at all time points (P<0.01 at 15 s, P<0.05 at 1, 2, 3 min). The addition of 10 mM taurine significantly lowered the uptake of 3H-taurine in NaC1 at 0.25 rain (P<0.01) and 1 min (P<0.05). There was no difference in 3H-taurine uptake between vesicles incubated in KC1 or KCl+10 mM taurine

served in an equal KC1 gradient and t r a n s p o r t rem a i n e d significantly higher for up to 3 min o f t r a n s p o r t ( P < 0 . 0 5 ) . The effect o f tracer replacem e n t (10 m M n o n l a b e l e d taurine) on N a + - d e p e n dent a n d N a + - i n d e p e n d e n t taurine t r a n s p o r t was also tested. The addition o f 10 m M n o n l a b e l e d taurine significantly decreased the rate o f labeled taurine u p t a k e in a NaC1 gradient (Fig. 2). After one m i n u t e o f t r a n s p o r t , taurine was a c c u m u l a t e d to 4 4 % o f the equilibrium in c o n t r o l vesicles a n d only 32% in vesicles u n d e r tracer replacement conditions ( P < 0 . 0 5 ) . There was n o effect o f tracer

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Fig. 3. Uptake of taurine into flounder renal brush border membrane vesicles under conditions of no salt gradient. Uptake is expressed as % of equilibrium uptake. Values are means ___SE (n= 4). Vesicles were prepared as in Fig. 1 and then preequilibrated in 20 mM Tris-HEPES, pH 8.2, 2 mM CaC12, and 75 mM NaC1 or 75 mM KC1 at 4 ~ for 1 h. The composition of the incubation medium was the same as the preincubation but included 0.1 mM [3H]-taurine. Significant differences between uptake in NaC1 and KC1 were analyzed by the Student t-test for group data and indicated by P values

replacement o n taurine u p t a k e by vesicles incubated in KC1 medium. T a u r i n e t r a n s p o r t and N a + - d e p e n d e n c e were then investigated u n d e r conditions o f no salt gradients, with vesicles pre-equilibrated with NaC1 or KC1. A N a + - d e p e n d e n c e for taurine u p t a k e by b r u s h b o r d e r m e m b r a n e vesicles was d e m o n s t r a t e d even in the absence o f these gradients (Fig. 3). After one minute o f incubation, taurine u p t a k e by vesicles in NaC1 was 1.6 times u p t a k e by vesicles t r a n s p o r t i n g in KC1 m e d i u m (P < 0.01). In a n o t h e r set o f experiments ( n = 4 ) , the c o n c e n t r a t i o n o f N a § was varied by substitution with K § such that N a § levels were 75, 50, a n d 20 r a M ; the vesicles were pre-equilibrated to eliminate salt gradients. As calculated by u p t a k e m e a s u r e m e n t s at 15 s

189

P.A. King et al. : Taurine transport by flounder renal brush border membrane vesicles Table 1. Effect of CI

on Na+-dependent flux of taurine into flounder renal brush border membrane vesicles

Incubation conditions

% Equilibrium uptake 0.25 rain

1.0 rain

2.0 min

3.0 min

75 mM N a S C N + 100 mM mannitol

24.6•

39.9•

49.6•

57.6•

75 mM NaC1 + 100 mM mannitol

17.1•

34.0•

44.6~1.7

56.2•

75 m M NaSCN + 50 mM KC1

23.7•

40.1•

50.5•

59.8•

Values are means _+SE (n = 8). The vesicles were prepared as in Fig. 1. In addition to the conditions indicated in the table, each incubation included 20 m M Tris-HEPES, pH 8.2, 2 m M CaC12 and 0.1 m M [3H]-taurine. Data were analyzed by one-way analysis of variance and the Student-Newman-Keuls test. Uptake in a NaC1 medium was significantly lower than uptake in NaSCN + mannitol or uptake in NaSCN + KC1 at 0.25 and 1.0 min, of transport, * ( P < 0.01) and ** ( P < 0.05)

these experiments revealed an apparent K m of 10.5 mM Na + for Na +-dependent taurine transport by the brush border membrane vesicles. Taurine transport across the brush border membrane did not display a specific C1- dependence. As shown in Table 1, taurine uptake by the vesicles was greater in a 75 mM NaSCN gradient than in an equal NaC1 gradient and taurine flux in the presence of NaSCN was not accelerated by the addition of C1- (50 mM KC1) to the incubation medium. These data indicate that the Na+-depen dent transport of taurine does not require C1- and also demonstrate an electrogenicity of the transport system (see Effects of membrane potential). The absence of a C1- dependence in this transport system differs from the Na+-dependent taurine flux across the basolateral membrane which shows a specific C1- requirement (King et al. 1982) and indicates that different systems for taurine transport operate at the two membranes.

Further evidence for a Na+-dependent taurine transport system Previous investigations of taurine transport in the flounder (King et al. 1982), dogfish (Schrock et al. 1982) and mammalian renal tubules (Chesney et al. 1976; Dantzler and Silbernagl 1976; Chesney and Jax 1977; Rozen et al. 1979) have shown that the carrier for Na+-dependent transport is shared by other ‚ acids. We therefore tested the effect of I mM fl-alanine on the uptake of taurine (0.1 raM). fl-Alanine significantly decreased Na +dependent taurine flux (75 mM NaC1 gradient) into the flounder brush border vesicles. At 15 s, I rain, and 2 min of transport, taurine uptake in a NaC1 medium averaged 21.8 • 2.8 %, 49.2 • 5.1% and 66.2 • 3.2% equilibrium (n = 4, mean _+SE), respectively. This was significantly greater than uptake in the presence of 1 mM fl-alanine: 17.2 • 1.8%, 43.5 • 5.0%, and 53.5 • 5.8% equilib-

rium, respectively (n=4, P<0.05 by paired data analysis). There was no effect offl-alanine on Na +independent uptake. The effect of HgClz, an inhibitor of facilitated transport systems, was also examined. For Na +dependent transport (75 mM NaC1 gradient), control taurine uptake averaged 93.0_+ 3.9 pmobs/mg protein at one minute of incubation and was significantly reduced by the presence of 0.1 mM HgC12 in the medium, 73.2 _+11.2 pmoles/mg protein rain (mean• n = 6 , P<0.05). There was no effect of HgC12 on Na+-independent transport (75 mM KC1 gradient). At one minute of transport, taurine uptake was 36.2+3.3pmoles/mg protein rain under control conditions and 36.3 + 11.1 pmoles/ mg protein min in the presence of HgC12 (mean_+ SE, n = 4).

Effect of membrane potential on Na+-dependent taurine transport The influence of electrical potential on Na +-dependent taurine transport was tested first by examining the effect of an inside positive membrane potential on taurine flux. The inside positive charge was generated by an inward-directed potassium diffusion potential in the presence of valinomycin. Under the potential-generating conditions, the rate of taurine uptake by the vesicles was significantly decreased (Table 2), indicating that Na+-depen dent taurine transport across the renal brush border is positive electrogenic. The effect of membrane potential on Na+-dependent D-glucose transport was also measured. In the presence of the potassium diffusion potential, the rate of glucose flux into the brush border vesicles was decreased as compared to control glucose transport rates (Table 2). Since the mechanism for Na +-dependent D-glucose transport by the flounder renal brush border membrane vesicles has previously been identified as positive electrogenic (Eveloff

190

P.A. King et al. : Taurine transport by flounder renal brush border membrane vesicles

Table 2. The effect of membrane potential on sodium dependent flux of taurine and glucose into flounder renal brush border vesicles % Equilibrium uptake 0.25 min

1.0 min

2.0 rain

3.0 rain

12.2• 7.9•

29.1• 19.8•

35.6• 29.6•

44.3• 33.8•

P<0.01

P<0.01

NS

P<0.05

68.5 25.5

78.0 57.5

74.0 65.6

80.0 78.8

24.6• 19.5•

39.9• 37.0•

49.6• 45.8•

57.6• 51.6•

P<0.02

NS

NS

P<0.05

A Potassium diffusion potential Taurine (4) :

Glucose (2):

control plus valinomycin

control plus valinomycin

B Anion Replacement Taurine (8) :

NaSCN Na +-gluconate

The values are the means'_ SE; n is shown in parentheses. The membranes were prepared as in Fig. 1. A Effects of potassium diffusion potential. The incubation medium contained 75 mM NaC1, 50 m M K+-gluconate, 20 m M Tris-HEPES, pH 8.2, 2 mM CaC12, and 0.1 m M [aHl-taurine or 0.1 m M [3H]-glucose. Valinomycin (dissolved in ethanol) was added to give a final concentration of 90 ~tM. The final incubating concentration of ethanol was less than 1.0% of the reaction volume; the incubation medium of the controls included an equal amount of ethanol only. B Effects of anion replacement. The incubation medium contained 75 mM N a S C N or Na+-gluconate, 20 m M Tris-HEPES, pH 8.2, 2 m M CaCI2, 0.1 mM [3H]-taurine, and 100 m M mannitol. Significant differences between means were tested by the Student t-test for group data and are indicated by P values. NS = not significantly different

et al. 1979) these experiments serve to confirm that an inside positive potential was generated under our experimental conditions. In a second set of experiments, the role of membrane potential in taurine transport was studied using anion substitution. If the Na § uptake of taurine by the brush border vesicles is electrogenic, transport will require an accompanying anion and the presence of more permeant anionic species should accelerate the rate of taurine uptake. Taurine uptake was studied in 75 mM Na § gradient conditions with thiocyanate, a more permeant anion, or gluconate, a less permeant anion, substituted for chloride. The rate of taurine transport was greater in the NaSCN medium than in the Na§ medium (Table 2) again indicating that Na+-dependent taurine transport is electrogenic.

Evaluation of possible basolateral membrane contamination In comparison to the Na +-dependent fluxes of glucose, the rate of taurine transport into the flounder renal brush border membrane vesicles is relatively low (Fig. 1). Because previous investigations (King et al. 1982) have identified a high rate of Na-dependent taurine transport across the flounder renal basolateral membrane, we felt it necessary to con-

sider the possibility that the low rate of taurine transport measured in the current study resulted from basolateral contamination of the brush border membrane preparation. As noted earlier, enzymatic analysis indicated minimal contamination of the brush border membranes; the vesicle fraction showed a low enrichment of Na +-K + ATPase activity (0.37) as compared to the whole homogenate. Secondly, among the vesicle preparations the rate of taurine uptake was constant over the range of basolateral contamination observed, Na+-K§ enrichment values of 0 to 0.7. In addition, we investigated the electrogenicity of Na § taurine transport under conditions in which an inside positive membrane potential was generated by sodium-glucose cotransport, a Na § conductive pathway. Since the system for Na § cotransport is not present in the basolateral membrane, a glucose-induced potential would only occur in brush border membrane vesicles. Transport was assayed under conditions of a 75 mm NaC1 gradient (as in Fig. 1) and uptake was measured after 1 rain of incubation. Control uptake averaged 61.8 _+1.0 pmoles taurine/mg protein.min (mean-q- SE, n = 6). In comparison, the presence of 0.1 mM glucose in the medium significantly reduced the uptake of taurine to 52.8__ 1.5 pmoles/mg protein- min (mean +_SE, n : 6, P < 0.05). This effect was prevented by the addition

P.A. King et al. : Taurine transport by flounder renal brush border membrane vesicles

191

of 0.1 m M phlorizin, known to inhibit Na+-glu cose transport (67.5_+4./pmoles/mg protein min, mean_+ SE, n = 6). These data, therefore, corroborate the enzyme enrichment values and provide further evidence identifying the vesicles as brush border membranes. Finally, unlike Na +-dependent transport across the basolateral membrane, Na +dependent flux of taurine into the brush border membrane vesicles did not exhibit a specific requirement for chloride. These results also indicate that taurine uptake by the vesicles is not a consequence of basolateral contamination and at the same time suggest different systems for Na+-de pendent movement operating in the apical versus basolateral membranes.

p H on the Na +-independent transport of taurine by the renal brush border membrane vesicles. Transport was assayed in the direction of uptake since these measurements were more sensitive in assessing taurine movement. The incubation p H was varied from 7.5 to 8.5 with the intravesicular p H at 8.2; at p H 7.5 94% of taurine is in the zwitterion form, while at 8.5, 37% is in this form. There was no difference in Na+-independent taurine uptake into the vesicles when measured at pH's 7.5, 8.0 and 8.5; at 15 s of transport, taurine uptake averaged 16.8 _ 1.7, 13.5 _ 1.0, 14.0 _ 0.7 pmoles/ mg protein, respectively ( m e a n _ SE, n = 4). These data indicate that the two species have similar rates of simple diffusion across the membrane.

Taurine effIux studies

Discussion

No evidence for Na +-independent facilitated taurine transport in the brush border membrane could be demonstrated by following the uptake of taurine into the brush border membrane vesicles. It is possible, however, that Na +-independent transport carriers, which would operate in vivo to facilitate exit of taurine from the cell, are asymmetric and therefore undetectable by uptake measurements. As a result, we performed a number of efflux studies that examined Na +-independent transport under conditions of no salt gradients (vesicles pre-equilibrated with the incubating salt concentration, 0.1 m M taurine, and 3H-taurine) and tested effects of tracer replacement and HgC12. The results of these experiments did not show an effect of either of these treatments, therefore providing no evidence for the presence of a Na +-independent carrier mediated efflux across the apical membrane. Incubation at cold temperature (0-4 ~ reduced rate of taurine efflux from the vesicles under conditions of KC1 or NaC1 incubations. This may have resulted from a change in the fluidity of the membranes at these low temperatures. At physiological p H for the flounder (pH 7.8), taurine exists predominantly as the zwitterion, the pK, of the sulfonic group being 1.5 and that of the amino group 8.7. Since the p K a of the amino group is relatively close to physiological pH, slight differences in the latter would have a discernable affect on the equilibrium between the zwitterion and anionic form of taurine, and if one of these species was more permeable than the other, transport could be affected. For instance, if the zwitterion form of taurine were more permeable than the anion, a cellular p H lower than the tubule lumen p H would facilitate a net efflux of taurine from the renal cell. Accordingly we tested the effect of

The results of the present study indicate the presence of a carrier facilitating Na+-dependent electrogenic taurine transport in the flounder renal brush border membrane. Taurine uptake was accelerated in the presence of a Na § gradient and the Na § dependence was observed even in the absence of a salt gradient suggesting that sodium facilitates the interaction of taurine with the carrier. The Na+-dependent taurine transport displayed tracer replacement as well as inhibition by HgC12, both characteristic of transport systems. In addition, Na § transport was inhibited by [~-amino acids (/]-alanine) as has been demonstrated for previously described taurine transport systems in the rat kidney (Dantzler and Silbernagl 1976; Chesney and Jax 1977). The characteristics identified for taurine transport in the flounder brush border membrane vesicles are like those that have been described for mammalian renal brush border membranes and suggest the presence of similar taurine transport systems in the fish and mammalian renal brush border. Rozen et al. (1979) found that the rate of taurine accumulation in rat renal brush border vesicles is sodium dependent, electrogenic, and specific for fl-amino acids. In the studies of the rat brush border membrane, however, taurine transport shows a concentrative effect (overshoot) in the presence of a sodium gradient; no overshoot was observed under similar incubation conditions for the flounder renal brush border vesicles (Fig. 1, 2). The absence of an overshoot for taurine uptake is indicative of a slower rate of transport and suggests a lower number of transport sites or a slower rate of turnover for the carrier system in the fish renal brush border. The current study provided no evidence for

192

P.A. King et al. : Taurine transport by flounder renal brush border membrane vesicles

LUMEN

TAURINE

Na* 145ram

2

PLASMA

CELL

/'~////

TAURINE ~,~" 0.1-0.2 mM

55ram Na+ \ x' ~"-,'",x

70mM

-70mY

/

Na§ 145mM

~ ":~,

CI-

TAURINE OI mM

-70mY

Fig. 4. Sodium and taurine gradients in the flounder kidney and a hypothetical scheme for the secretion of taurine across the flounder renal epithelium. A complete explanation is given in the Discussion. The dashed lines indicate simple diffusion Na +-independent taurine transport by the flounder brush border membrane, a carrier mechanism which would operate in vivo to facilitate efflux of taurine from the renal cell. Taurine transport in KC1 media did not demonstrate characteristics of carrier mediated systems i.e. tracer replacement, inhibition by HgC12, or competition with #-alanine. What process then accounts for the secretion of taurine by the flounder renal tubule? Although Na+-dependent solute transport would usually operate in the direction of cellular uptake, it is possible that the net gradients for Na § influx and taurine efflux promote a Na+-dependent exit of taurine from the cell into the lumen. A diagram depicting the Na § and taurine gradients across the apical membrane of the flounder renal tubule is shown in Fig. 4. We have estimated the cellular potential difference (PD) as - 7 0 mV, equal to the K § equilibrium potential as calculated from the K + concentration in teased tubules (Kleinzeller and D u b y a k 1977) corrected for extracellular and lumen space. This value is similar to the cellular P D for the mammalian proximal renal tubule. The transepithelial voltage is minimal, approximately 2 m V lumen negative (Beyenbach et al. 1980). Luminal N a § in the flounder renal tubule has been measured as 145 m M (Beyenbach 1982) and cellular Na + as 70 m M (from Kleinzeller and D u b y a k 1977, as measured in teased tubules and corrected for extracellular and lumen space). This gradient together with a cellular PD of - 7 0 mV provides a driving force of 87 mV for Na+-dependent taurine influx (calculated for conditions at 15 ~ The taurine concentrations, on the other hand, are 55 m M for the flounder renal cell (from King et al. 1982, corrected for extracellu-

lar and lumen space) and 0.1 m M in the tubular fluid (plasma concentration which would equal the taurine concentration in the glomerular filtrate) with the cell to lumen taurine gradient generating a driving force of 156 mV for the effiux of taurine into the tubular lumen. The resulting net force for Na+-dependent taurine movement across the apical membrane is 69 mV driving net effiux from the cell. This value most likely approximates a maximal driving force for net efflux. The cellular Na + concentration used here is derived from whole tissue measurements; cellular activites may in fact be much lower. In addition, a lumen taurine concentration of 0.1 m M may be an underestimate as glomerular filtration and tubular secretion contribute equally to the total excretion of taurine and lumenal taurine concentration could therefore be higher than the plasma level. Both of these factors would promote a lower driving force for net efflux. However, if the driving forces are recalculated using a cellular Na + concentration of 30 raM, and a luminal taurine concentration of 0.2 raM, the net force for taurine movement still results in efflux (30 mV) with driving forces of 109 mV for influx and 139 mV for efflux. Thus, for the flounder renal tubule, in vivo conditions indicate that Na+-de pendent taurine transport across the brush border membrane could operate in the direction of net effiux of the amino acid from the cell into the tubular lumen. For the transepithelial secretion of taurine, transport across the basolateral membrane must result in net uptake of taurine by the cell and transport across the apical membrane in net efflux. The fish renal epithelium is characterized by a taurine transport system at the basolateral membrane dependent on both Na + and CI-, and a carrier mediated system on the apical membrane dependent only on Na +. Although the gradient for taurine is the same at both membranes, the requirement for C1- and possibly the stoichiometry for the coupling of N a + and taurine differ in the two systems, promoting influx at one membrane and effiux at the other. A stoichiometry of I Na+/1 taurine could result in a net effiux of taurine across the luminal membrane while at the contraluminal membrane a stoichiometry of 2 N a + / l taurine would provide a driving force for net influx (20 mV or 62 mV using 70 m M or 30 raM, respectively, as the intracellular Na + concentration). Previously, we have found that the flounder renal tubules avidly accumulate taurine across the basolateral membrane; tissue/medium ratios for taurine uptake by teased renal tubules averaged 15 and cell/bath values for taurine transport by single iso-

P.A. King et al. : Taurine transport by flounder renai brush border membrane vesicles

lated renal tubules averaged 135 (King et al. 1982). Transport across this membrane therefore results in the very high cellular concentration of taurine (55 raM). This in turn provides a large gradient for efflux from the cell which is responsible for establishing the driving force for net efflux of taurine via a Na+-dependent carrier system at the brush border membrane. The difference between net reabsorption of taurine in the mammalian kidney and net secretion in the fish may result from the ability of the latter to accumulate taurine across the basolateral membrane to such a high cellular concentration. Chesney and Jax (1977) measured the tissue/medium ratio for taurine uptake by rat kidney cortex slices under similar conditions to those in our renal tubule studies (1 h incubation, 0.1 mM taurine in Ringer solution) but at 37 ~ rather than 15 ~ Taurine was accumulated to only half the extent in the rat kidney slices (tissue/ medium taurine ratio = 7) as it was in the flounder renal tubules (tissue/medium taurine ratio = 15). Thus, in the mammalian kidney, the cell/lumen taurine gradient may never be large enough to promote net efflux by the Na+-dependent transport system at the apical membrane. In addition to Na+-dependent transport, simple diffusion of taurine from the cell into the lumen may also contribute to the total efflux of the amino acid across the brush border membrane. As previously noted, taurine uptake by vesicles in KC1 medium did not demonstrate properties of carriermediated transport and therefore appears to represent simple diffusion of the amino acid. As a zwitteflon, however, taurine would be expected to have a low permeability to lipid membranes (Klein et al. 1971) and efflux by simple diffusion, like Na +dependent efflux, would be dependent on a large cell to lumen taurine gradient. Acknowledgements. The authors wish to thank P. Newsholme for valuable technical assistance, and V. Burrage and K. Cotter for help in the preparation and typing of the manuscript. The research was supported by NSF grants PCM 81-09706 (LG) and DEB-7826821 (Mount Desert Island Biological Laboratory) and the Whitehall Foundation (LG).

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ine in vivo and in vitro in hypertaurinic mice. J Clin Invest 57:183-193 Chesney RW, Jax DK (1977) Heterogeneity of the fl-aminopreferring transport system in rat kidney cortex. Differential influence of gtutathion oxidation. Biochem Biophys Acta 466 : 84-96 Dantzler WH, Silbernagl S (1976) Renal tubular reabsorption of taurine, )~-aminobutyric acid (GABA) and fl-alanine. Studies by continuous microperfusion. Pfliigers Arch 367:123-128 Eisenbach GM, Weige M, StoRe H (1975) Amino acid reabsorption in the rat nephron. Free flow micropuncture study. Pfltigers Arch 357:63-76 Eveloff J, Kinne R, Kinter WB (1979) ~-Aminohippuric acid transport into brush border vesicles isolated from flounder kidney. Am J Physiol 237:F291-F298 Eveloff J, Field M, Kinne R, Murer H (1980) Sodium-cotransport systems in intestine and kidney of the winter flounder. J Comp Physiol 135:175-182 Gilbert JB, Ku Y, Rogers LL, Williams RJ (1960) The increase in urinary taurine after intraperitoneal administration of amino acids to the mouse. J Biol Chem 235:1055-1060 Haase W, Schafer A, Murer H, Kinne R (1978) Studies on the orientation of brush-border membrane vesicles. Biochem J 172:5~62 Heidrich HG, Kinne R, Kinne-Saffran E, Hannig K (1972) The polarity of the proximal tubule cell in rat kidney. Different surface charges for the brush border microvilli and plasma membranes. J Cell Biol 54:232-245 Hickman CP, Trump BF (1969) The Kidney. In: Hoar WS, Randall DJ (eds) Fish physiology, vol I. New York, Academic Press, pp 91-239 Hopfer U, Nelson K, Perrotto J, Isselbacher KJ (1973) Glucose transport in isolated brush border membranes from rat small intestine. J Biol Chem 248 : 25-32 Jacobsen JG, Smith LG (1968) Biochemistry and physiology of taurine and taurine derivatives. Physiol Rev 48:424-511 King PA, Beyenbach KW, Goldstein L (1982) Taurine transport by isolated flounder renal tubules. J Exp Zool 223:103 114 King PA, Cha CJ, Goldstein L (1980) Amino acid metabolism and cell volume regulation in the little skate, Raja erinacea. I. Oxidation. J Exp Zool 212:69-77 Kinne R, Maier R, Eveloff J, Kinter WG (/976) Preparation and enzymatic properties of brush border and basal-lateral membranes from flounder kidney tubules. Bull Mt Desert Isl Biol Lab 16:69-72 Klein RA, Moore MJ, Smith MW (1971) Selective diffusion of neutral amino acids across lipid bilayers. Biochem Biophys Acta 233:420-433 Kleinzeller A, Dubyak GR (1977) Renal sugar transport in the winter flounder IV. Effect of Ca +z on sugar transport in teased renal tubules. J Cell Physiol 93:11-16 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275 Renfro LJ, Pritchard JB (1982) H+-dependent sulfate secretion in the marine teleost renal tubule. Am J Physiol 243 : F150F159 Rozen R, Tenenhouse HS, Scrivcr CR (1979) Taurine transport in renal brush-border membrane vesicles. Biochem J 180:245-248 Schafer JA, Barfuss DW (1980) Membrane mechanisms for transepithelial amino acid absorption and secretion. Am J Physiol 238 : F335-F346 Schrock H, Forster RP, Goldstein L (1982) Renal handling of taurine in marine fishes. Am J Physiol 242: R64-R69