INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE UBIQUITOUS AMINO ACID taurine (2-aminoethanesulfonic acid) constitutes a major fraction of the intracellular solutes in vertebrate species (14). Taurine is known to serve many important biological functions, the most important of which is its role in cell volume regulation. Changes in plasma osmolality are accompanied by corresponding changes in the content of intracellular taurine, which help to maintain cell volume (14). In general, taurine is poorly metabolized by vertebrate species (14, 15), and its renal excretion plays an important role in the regulation of body levels of taurine.

Taurine can be reabsorbed and/or secreted by the renal tubules of vertebrates (15). Mammalian kidneys physiologically exhibit only net reabsorption of taurine. On the other hand, net renal taurine secretion can occur in nonmammalian vertebrates such as marine fishes and ophidian reptiles (3, 22). Reptilian kidneys also have the distinct ability to both reabsorb and secrete taurine in vivo (1-4). The mechanisms for transepithelial reabsorption of taurine have been elucidated in mammals and reptiles, and they generally appear to be similar (5). In reptilian kidneys where bidirectional transport of taurine occurs, net transepithelial reabsorption of taurine occurs through luminal uptake via electrogenic, high-affinity, 3 Na⁺-2 Cl-1 taurine cotransporter coupled with passive mediated basolateral efflux (1, 2, 4). Additionally, the basolateral nonelectrogenic cotransporter (1 Na⁺-1 Cl-1 taurine), which normally transports taurine into the renal cells, could operate in reverse to facilitate taurine efflux in the reabsorptive process when intracellular taurine concentration is high (2).

Less is known about the mechanisms for transepithelial secretion of taurine that occurs in reptiles and marine fishes. Although taurine is secreted in vivo in reptiles, it has been difficult to characterize the taurine secretory mechanisms in these animals. This is due to the fact that the conditions under which taurine secretion occurs have not been defined (3). Therefore, to examine the mechanism of renal taurine secretion, we studied the process in a species known to exhibit only net secretion of the amino acid, a marine teleost, winter flounder (Pleuronectes americanus) (22).

More than 50% of the intracellular free amino acid pool in flounder tissues is taurine (14), which can be released into plasma during an environmental dilution as part of a volume regulatory response. Over a wide range of plasma taurine concentrations, from 0.02 to 0.26 mM, the flounder kidney exhibits a significant net tubular secretion of taurine (22). The ability of the flounder renal tubule to secrete taurine is critical for the maintenance of cell volume during hyposmotic stress as it allows the animal to respond more readily to changes in plasma levels of the amino acid. Studies of taurine secretion in isolated flounder renal tubules indicate that the secretion process involves a concentrating uptake of taurine across the basolateral membrane and a movement down its concentration gradient at the luminal membrane (13). With use of another in vitro system, the primary monolayer cultures of renal proximal tubule cells (PTCs) of the winter flounder mounted in Ussing chambers which, similar to the in vivo situation, exhibit net transepithelial secretion of taurine under open-circuited as well as short-circuited conditions, the taurine exit step from cell to lumen was found to be the critical step in the secretory process (18). This luminal pathway appears to be distinct from the Na⁺-taurine cotransporter; it is selective for taurine and inhibitable by bromcresol green and probenecid (18).

The purpose of the present study was to further characterize the mechanisms for transepithelial secretion of taurine using flounder PTCs. In particular, we were interested in the elucidation of the regulatory mechanisms of taurine transport in vertebrate kidneys that secrete taurine. Two factors have been found previously to influence renal excretion of taurine in both reptiles and marine fishes under in vivo conditions: 1) extracellular taurine concentration and 2) extracellular osmolality (3, 14, 22, and unpublished data). The use of renal cell culture systems in the present study permitted direct evaluation of the effects of these extracellular modulating factors on intact cells over short as well as long time courses. Our results indicate that taurine secretion by flounder renal proximal tubule cells could be modulated by acute changes in extracellular taurine concentration and osmolality. Indeed, the response of the flounder PTCs to 2-h exposure to hyposmotic medium mimics the in vivo response of the intact marine fish kidney to dilution of extracellular fluid.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Winter flounder (Pleuronectes americanus) was collected by otter trawl offshore of Niantic, CT, and generously supplied by the Northeast Utilities Environmental Laboratory. They were maintained, unfed, in artificial sea water at 10-12°C in the laboratory at the University of Connecticut.

Chemicals. The media for the isolation and maintenance of the primary renal cultures of the winter flounder were as described previously by Renfro and co-workers (7, 9). The radiolabeled [1,2-³H]taurine (32 Ci/mmol) or [2-³H]taurine (30 Ci/mmol) was obtained from Amersham and American Radiolabeled Chemicals, respectively. [1,2-³H]polyethylene glycol (PEG, mol wt 4,000, 250 µCi/ml) was obtained from Dupont-New England Nuclear. All other chemicals were purchased from Sigma Chemical and were of the highest purity available.

Preparation of primary renal monolayer cultures. Primary monolayer cultures of PTCs were prepared from kidney tissues of winter flounder. Renal cells were isolated, dispersed by cold trysinization, and plated to confluency on native rat tail collagen, according to the methods previously described by Renfro and co-workers (7, 9). Cultures were maintained in modified medium 199 and 10% fetal bovine serum (HyClone, Logan, UT) at 22°C in a humidified air incubator. After 4 days, the cultures on collagen gels were detached from the 35-mm plates, and the floating collagen rafts were allowed to contract over the next 7 days to 17-mm diameter when they were ready to be used. The PTCs were maintained without the amino acid taurine before flux experiments. The preparation yields monolayers of morphologically and functionally differentiated PTCs with transport characteristics similar to those of the intact flounder renal proximal tubules (7).

Transepithelial flux measurements. Transepithelial electrical properties and taurine fluxes were measured in 11- to 18-day-old monolayer cultures. The cultures, supported by 150-µm nylon mesh, were mounted in conventional Plexiglas Ussing chambers (aperture 0.332 cm²). Each hemichamber contained 1.6 ml of flounder Ringer solution [composition in mM, 150.0 NaCl, 4.0 KCl, 2.0 CaCl₂, 1.0 MgSO₄, 0.2 NaHCO₃, 25.0 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 0.5 glucose, 1.0 L-glutamine, 16.0 NaOH, varying concentrations of taurine (0.001-3.0 mM), pH 7.5 and osmolality 340 mosmol/kgH₂O] at 20 to 22°C, gassed with humidified 99% O₂-1% CO₂. Each hemichamber was independently stirred with a small magnetic bar (2 × 7 mm) turned by external water-driven turbines. Transepithelial taurine fluxes were measured under short-circuited conditions using two automatic dual voltage clamps (model DVC 1000; World Precision Instruments, Sarasota, FL) interfaced with a computer. Transepithelial electrical potential difference (PD) and resistance (R) across each monolayer culture were measured with 3 M KCl-2% agar bridges linked to external Ag-AgCl electrodes by triggering a computer-controlled 10-µA current pulse through the remote logic circuits of the clamps. The transepithelial PD, R, short-circuit current, and phloridzin-sensitive current (measured at the end of each flux determination as an indication of sodium-dependent glucose transport) were used to assess tissue viability and functional integrity.

Estimation of secretory and reabsorptive flux kinetics. For kinetic analysis, flux measurements were made when the concentration of taurine varied between 1 µM and 3 mM. This wide range of taurine concentrations encompasses the range of physiological concentrations of taurine found in the plasma of marine fish under isosmotic (~0.1-0.8 mM) and hyposmotic (~1.3-2.5 mM) conditions (14, 22). In addition, the use of a wide range of taurine concentrations in kinetic studies permits detection of multiple kinetic components of taurine transport (1, 10). The steady-state flux values at a particular taurine concentration were used in the analysis of kinetic characteristics of taurine unidirectional flux. Kinetic parameters for the observed unidirectional secretory and reabsorptive fluxes were estimated by a computer curve-fitting procedure, using an iterative, nonlinear method (Sigma Plot Scientific Graph System, Jandel Scientific), fitting the flux data to the Michaelis-Menten kinetic equation v = V_max[S]/(K_m + [S]), where v is steady-state unidirectional taurine flux (in nmol · cm² · h¹), [S] is the taurine concentration (in µM), V_max is the maximal rate of unidirectional taurine flux, and K_m is the concentration of taurine that yields one-half V_max. The nonlinear curve-fitting procedure yields a best fit with the least biasing of the nontransformed raw data, allowing detection of multiple saturable transport systems as well as estimation of the kinetic parameters for the taurine reabsorptive and secretory systems and their variances (10).

Exposure of flounder renal monolayer cultures to hyposmotic medium. The cultures were rinsed and placed in hyposmotic flounder Ringer solution containing 120 mM NaCl (osmolality = 287 ± 2 mosmol/kgH₂O) without taurine for 2 h at 22°C before beginning the flux measurements. The 2-h exposure period was chosen based on earlier in vivo observations that significant changes in renal function occur within 2-5 h on transfer of marine fish into dilute seawater (25). Unidirectional fluxes were measured in the same hyposmotic medium containing 0.5 mM taurine. Paired controls were treated similarly in isosmotic flounder Ringer solution.

Efflux studies. To determine apical and basolateral transmembrane taurine flux, cell-to-lumen and cell-to-peritubular bath fluxes of [³H]taurine from preloaded PTCs were examined. Cultures that had been in isosmotic or hyposmotic flounder Ringer solution for 2 h were mounted in Ussing chambers and loaded with taurine by addition of 0.5 mM taurine containing 2.5 µCi [³H]taurine in the respective osmolality in both sides of the hemichambers. After a 2-h incubation under open-circuit conditions at 22°C with constant stirring and aeration, the labeled medium was removed and each chamber was quickly rinsed twice with tracer-free Ringer solution containing cold taurine. The third rinse solution was left in the hemichambers for 15 min before removal. After the rinses, 1.6 ml of appropriate flounder Ringer solution containing 0.5 mM cold taurine were placed in each hemichamber, and the appearance of [³H]taurine was assessed in the medium on both sides of the epithelium. Duplicate 50-µl samples were taken, with volume replacement from each side at 5 min and at every 15 min thereafter over a total period of 1.5 h. The exit of [³H]taurine from the epithelium was measured as the accumulated disintegrations per minute in luminal and peritubular baths normalized to the mean specific activity of the preloading medium (averaged specific activity at the beginning and end of the loading period).

Estimation of taurine concentration in cultured cells. Intracellular concentrations of taurine in PTCs exposed to either isosmotic or hyposmotic flounder Ringer solution were estimated, using methods that were modified from the ones used in the earlier study of taurine secretion in cultured flounder renal epithelium (18). In the present study, the intracellular taurine radioactivity was extracted from individual culture without releasing the cells from the collagen gel. Paired experiments were carried out by simultaneously incubating individual cultures in 1.6 ml of either isosmotic or hyposmotic flounder Ringer solution (usually 3 cultures in each group), containing 0.5 mM taurine with 5 µCi [³H]taurine as tracer, for 3 h at 22°C. The 3-h period was chosen based on the prior observation that steady-state fluxes of [³H]taurine in the cultures mounted in Ussing chambers were usually attained by 2.5 h after the addition of the tracer. After 3 h, the PTCs were washed in three consecutive baths of respective Ringer solution containing no taurine and blotted on filter paper. The radioactivity accumulated intracellularly in the culture was extracted by vigorously mixing the culture with 0.5 ml of 0.1% Triton X-100 for 1 min followed by high-speed centrifugation. The extraction was repeated two more times, and the extracted fluid was pooled for determination of total radioactivity. The extracted collagen pellet was also analyzed for remaining radioactivity to assess the completeness of extraction. In all experiments, the amount of radioactivity remaining in the collagen pellet after extraction was <1% of that extracted into the Triton solution (0.79 ± 0.06%). The specific activity of [³H]taurine in the medium was determined at the beginning and end of the incubation period, averaged, and used to calculate intracellular taurine content of the particular culture (after equilibration the specific activities of extracellular and intracellular taurine were the same). The calculated intracellular taurine content for each culture was corrected for extracellular taurine trapped during the extraction. The amount of extracellular fluid trapped after extraction was determined by a parallel incubation of another set of cultures (n = 3) in isosmotic flounder Ringer containing 2.5 µCi of [1,2-³H]PEG.

Statistical analysis. Data are means ± SE (n = no. of different culture preparations), with the exception of the estimates of the kinetic parameters obtained from curve-fitting procedure, where data are estimates ± SD. Differences between means were analyzed for paired or unpaired data by Student's t-test or Wilcoxon's signed rank test as appropriate and were considered statistically significant at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Transepithelial taurine fluxes. Under short-circuited (PD clamped at zero) and zero transepithelial chemical gradient conditions (0.1 mM taurine on both peritubular and luminal sides representing physiological concentration in flounder plasma) (22), the PTCs exhibited net transepithelial secretion of taurine (Fig. 1). After equilibration the unidirectional taurine secretory flux (P to L) was ~1.5 times the corresponding unidirectional reabsorptive flux (L to P; 4.40 ± 0.27 vs. 2.92 ± 0.11 nmol · cm² · h¹), resulting in an average net transepithelial taurine secretory flux of 1.48 ± 0.22 nmol · cm² · h¹. Because there was no transepithelial electrochemical gradient for taurine, its net secretion is by definition an active process.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Time courses of transepithelial taurine fluxes in flounder renal proximal tubule primary monolayer cultures incubated in 0.1 mM taurine in Ussing chambers under short-circuited condition. Peritubular-to-lumen (P to L, , secretion), lumen-to-peritubular (L to P, , reabsorption, shown as negative to indicate flux in opposite direction), and net (difference in unidirectional fluxes, ) fluxes are as shown. Each point is the mean ± SE; n = 3 different culture preparations. The values for net taurine secretion at 1 h were significantly greater than zero (P < 0.01) in every preparation.

2

1

Kinetic characteristics of transepithelial taurine fluxes in flounder renal tubule monolayer cultures. The effects of increasing taurine concentration on the steady-state unidirectional reabsorptive and secretory fluxes were examined over a wide taurine concentration range of 1 µM to 3 mM (Fig. 2). With increasing taurine concentrations, both resultant increases in unidirectional reabsorptive and secretory taurine fluxes could be described by the Michaelis-Menten type kinetics (saturation kinetics) for systems with more than one kinetic component of transport. Analyses of the data in Fig. 2 suggest the presence of two independent saturable transport systems for taurine reabsorption and secretion in the flounder PTCs, each with distinct affinity and V_max, one operating at taurine concentrations in the micromolar range and the other operating in the millimolar range. Within both low- and high-taurine concentration ranges, unidirectional secretory and reabsorptive taurine fluxes could satisfactorily be fitted to the Michaelis-Menten equation with high degree of correlation (Table 1). At low concentrations (<500 µM), the taurine reabsorptive system showed higher affinity and lower capacity than the secretory system. At high concentrations (>500 µM), the taurine secretory system had higher affinity but the same capacity as the corresponding reabsorptive system.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Taurine unidirectional fluxes at steady state as a function of taurine concentration (1 µM to 3 mM). Simultaneously measured reabsorptive and secretory fluxes of [3H]taurine across flounder proximal tubule monolayer cultures are shown. Values at each taurine concentration are means ± SE of 3-4 simultaneous steady-state flux measurements using different culture preparations. In those cases where vertical bars are absent, SE is smaller than the graphical representations of the means (SE values < 0.5 nmol · cm2 · h1).

Effect of low medium osmolality on transepithelial fluxes of taurine across flounder renal tubule monolayer cultures. The monolayer cultures were incubated for 2 h in hyposmotic or isosmotic flounder Ringer solutions (osmolality 287 and 340 mosmol/kgH₂O, respectively) before initiation of flux measurements in the same solutions in the presence of 0.5 mM taurine. Exposure of the cultures to low osmolality over the 5-h period did not affect the culture viability and functional integrity as indicated by transepithelial PD and R [PD in hyposmotic medium 0.65 ± 0.08 mV (n = 11) vs. 0.60 ± 0.10 mV (n = 7) in isosmotic medium; R in hyposmotic medium 41 ± 0.4  · cm² (n = 11) vs. 41 ± 0.6  · cm² (n = 7) in isosmotic medium, n = number of cultures tested from 5 preparations]. More importantly, the reduction of NaCl in hyposmotic medium (from 150 to 120 mM) did not appear to affect the lumen-to-cell transmembrane gradient for Na⁺ in these cultures, as evidenced by the unaltered values for phloridzin-sensitive short-circuit current of the PTCs exposed to low osmolality (3.4 ± 0.8 µA · cm², n = 11, vs. isosmotic medium 3.5 ± 0.9 µA · cm², n = 7). Figure 3 illustrates the responses of the cultures to media of different osmolalities. Exposure of the cultures to hyposmotic medium (Fig. 3B) increased unidirectional secretory flux of taurine twofold while the reabsorptive flux remained unchanged, resulting in a threefold increase in net secretory flux of taurine (P < 0.05). The increase in net taurine secretory flux in response to hyposmotic medium was always observed regardless of seasonal differences in baseline taurine fluxes.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of 2-h exposure to hyposmotic medium on taurine secretion by flounder proximal tubule monolayer cultures. Time courses of secretory, reabsorptive, and net fluxes of taurine (0.5 mM) by cultures in isosmotic (340 mosmol/kgH2O, A) and hyposmotic (287 mosmol/kgH2O, B) media are shown. See Fig. 1 for description of symbols. Experimental cultures (B) were exposed to hyposmotic medium without taurine at 22°C for 2 h before initiation of flux measurements. Control cultures (A) were similarly preincubated in isosmotic medium without taurine for 2 h. Both groups were exposed to 0.5 mM taurine for about 45 min before the flux measurements. Values are means ± SE of 5 paired experiments, each using different culture preparations. The values for net taurine secretion for both groups at 1 h were significantly greater than zero (P < 0.01) in every preparation. * Significantly different from corresponding flux in paired control cultures in isosmotic medium at P < 0.05.

Cellular taurine content of flounder renal tubule monolayer cultures in isosmotic and hyposmotic media. After a 2-h incubation in hyposmotic medium, the water content of the cultured cells was only slightly elevated (540 ± 45 nl/mg wet wt, n = 4) compared with that of the culture mates in isosmotic medium (507 ± 42 nl/mg wet wt, n = 3). Intracellular content of taurine in PTCs exposed to hyposmotic solution was not significantly different from those in isosmotic medium (28.5 ± 1.8 nmol taurine/mg wet wt in cultures exposed to hyposmotic medium vs. 23.0 ± 4.6 nmol taurine/mg wet wt in cultures exposed to isosmotic medium, n = 6 for each). The intracellular concentration of taurine in hyposmotic cultures was 52.7 ± 3.3 mM compared with 48.7 ± 6.9 mM in isosmotic cultures.

Taurine fluxes across luminal and peritubular membranes of PTCs in isosmotic and hyposmotic media. To localize the stimulatory effect of hyposmotic medium on taurine secretory flux to a specific membrane of the renal epithelium, the cell-to-luminal bath and cell-to-peritubular bath fluxes of [³H]taurine from preloaded PTCs were examined. After steady state was attained in test medium, the total amounts of [³H]taurine exiting through luminal and peritubular surfaces were simultaneously measured in Ussing chambers over the next 1.5 h. Figure 4 shows time courses of taurine exit from PTCs through luminal (cell-to-luminal bath) and peritubular (cell-to-peritubular bath) surfaces of cells in hyposmotic medium. The effect of low osmolality on the individual membrane was evaluated by examining transmembrane taurine fluxes across each membrane in hyposmotic medium as percentage of the respective control fluxes in isosmotic medium. It can be seen that the enhanced taurine secretory flux observed after exposure of the cultures to hyposmotic medium was associated with an increase (>35%) in the amount of taurine exit across the luminal membrane and a decrease (~20%) in the cell-to-peritubular bath exit of taurine. Therefore, exposure to hyposmolality appeared to affect taurine transport at both poles of the flounder renal epithelium, increasing the luminal taurine efflux and decreasing the peritubular efflux.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Time courses of changes in taurine fluxes across the luminal (cell-to-luminal bath) and peritubular (cell-to-peritubular bath) membranes of flounder proximal tubule monolayer cultures in hyposmotic medium as percentage of respective fluxes in isosmotic medium. Cultures were preincubated in Ussing chambers for 2 h in flounder Ringer of respective osmolality containing 0.5 mM taurine and [3H]taurine and rinsed 3 times before the cumulative appearance of [3H]taurine in respective nonradioactive medium was measured and normalized to mean specific activity of preloading medium. Each bar is mean ± SE of 3 paired experiments. Error bars are not visible for SE values smaller than 0.5%. * Significantly different from isosmotic control (P < 0.05).

Effects of inhibitors on transepithelial taurine fluxes in PTCs exposed to hyposmotic medium. To determine whether the enhanced taurine secretory flux observed in cultures exposed to hyposmotic medium was related to a cellular regulatory volume response, the effect of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 0.1 mM) an inhibitor of band 3-mediated anion exchange and of the volume-activated osmolyte channel (16) was investigated. DIDS was applied to PTCs mounted in Ussing chambers in hyposmotic medium after steady-state taurine fluxes were achieved. In these experiments, only the effect of DIDS in the luminal bath was evaluated because the luminal transport carrier has been shown previously to be the rate-limiting step in taurine secretion (18). DIDS was ineffective in significantly altering hyposmolality-enhanced taurine fluxes (Table 2).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study confirms the earlier observation (18) that flounder PTCs perform net transepithelial secretion of taurine when mounted in Ussing chambers. The kinetic studies reported here indicate an overall system that is well suited for exhibiting net transepithelial secretion of taurine over a wide range of taurine concentrations. At low-taurine concentrations within the physiological range for flounders under isosmotic conditions (0.02-0.26 mM) (22), the high-affinity taurine reabsorptive system is saturated because of its low capacity while the higher capacity taurine secretory system dominates. At high-taurine concentrations similar to those observed physiologically in marine fishes under hyposmotic conditions (14, 22), the taurine secretory systems possess a twofold greater affinity for taurine than the reabsorptive counterpart, facilitating net taurine secretion. The presence of two saturable transepithelial transport systems for taurine with distinct affinities and capacities in flounder PTCs is in good agreement with similar findings of two saturable taurine transport systems on luminal and peritubular membranes in PTCs of mouse, rat, rabbit, and garter snake kidneys (see Table 1 in Ref. 1) as well as in two continuous renal epithelial cell lines (11).

In marine fishes that exhibit net renal secretion of taurine in vivo, plasma taurine concentration as well as the rates of tubular secretion of taurine are significantly elevated while the animals undergo environmental dilution (22). During acclimation to dilute seawater, the decreased plasma osmolality causes taurine to be released from the cells in the course of cellular volume regulation, raising plasma taurine concentrations (14, 22). The enhanced renal tubular secretion of taurine in a diluted environment helps the animal to excrete excess osmotically active material and obligated water more rapidly, facilitating the regulation of the extracellular fluid volume. The mechanism for the adaptive response of the renal taurine transport system during environmental dilution was investigated in the current study, in which the taurine concentration in Ussing chambers was raised from 0.1 mM in isosmotic medium to 0.5 mM in hyposmotic medium to mimic the conditions during in vivo dilution [plasma taurine concentration increases 3- to 10-fold in marine fish acclimated to dilute seawater (14, 22)]. Under these conditions, the flounder PTCs exhibited a twofold increase in taurine secretory flux with no change in the reabsorptive flux. It is clear from the kinetic characteristics of the renal taurine transport systems in the flounder monolayers (Table 1) that at 0.5 mM taurine concentration the high-affinity taurine reabsorptive system was already saturated in contrast to the taurine secretory system. Therefore, part of the enhanced unidirectional and net taurine secretion during dilution must be the effect of increased plasma taurine concentration on the unsaturated taurine secretory system. However, it is clear that an additional mechanism for enhanced taurine secretion must also play a role in the adaptive response to environmental dilution.

Although there was an enhanced net efflux of taurine out of the flounder renal PTCs in response to hyposmolality, this was not a regulatory cell volume decrease (RVD) response typically associated with short-term cell volume regulation. Generally, the time course for the enhanced taurine efflux during the RVD response of cells to reduction in osmolality is much shorter; during RVD, the rate of taurine efflux returns to normal parallel to the change in cell volume toward baseline, usually within an hour. In isolated proximal tubules of the freshwater teleost, Carassius auratus, exposed to hyposmotic medium of 100 mosmol/kgH₂O (8), the rate coefficient for taurine efflux peaked at 4 min and progressively declined toward the normal value by 20 min. An almost complete RVD was observed after 5 min in isolated perfused rabbit proximal convoluted tubules following a 40 mosmol/kgH₂O osmolality decrease (6). Short time courses for RVD were similarly observed in hypotonic-stressed renal cells of the distal nephron, Madin-Darby canine kidney cells (21), and rat inner medullary collecting duct cells (20). In the present study, when the flux measurements were carried out after a 2-h exposure to hyposmotic medium, the cell volume of the flounder PTCs (as indicated by the water content) was not different from that of the cultures in isosmotic medium.

Other lines of evidence further indicate that the response of the PTCs to hyposmotic medium was not a short-term RVD. Typical RVD responses of different renal cells include lowered cellular content of taurine in hyposmotic medium secondary to enhanced taurine efflux (8, 20, 21). In contrast, intracellular content of taurine in flounder PTCs exposed to hyposmotic solution was not significantly different from those in isosmotic medium. Furthermore, the inhibitor of the RVD response in other renal cells (20, 21), DIDS, failed to reduce the enhanced taurine secretory flux in flounder renal cells exposed to hyposmotic medium. Nevertheless, the observed enhanced taurine secretion in response to hyposmolality could still reflect the process of long-term cell volume regulation in which the flounder renal cells adapt to sustained alterations in extracellular osmolality. The response of the flounder PTCs to 2-h exposure to hyposmotic medium does mimic the in vivo increase in net taurine secretion shown in marine fish after 24-h acclimation to 70% sea water (22).

The hyposmolality-induced increase in taurine secretory flux was the result of changes in taurine transport on both luminal and peritubular membranes. There was a decrease in taurine efflux across the peritubular membrane and a concurrent increase in taurine efflux across the luminal membrane. The decreased peritubular taurine efflux could potentially help maintain the intracellular taurine concentration in hyposmotic cultures, facilitating increased secretion of taurine across the luminal membrane. In the kidney of the hypertaurinuric mouse, which normally exhibits net transepithelial taurine reabsorption, hypertaurinuria is caused by decreased taurine efflux across the renal basolateral membrane and resultant elevation of intracellular taurine concentration (17, 19). Taurine exit across the peritubular membrane of vertebrate renal cells can occur via a passive mediated process as well as via the taurine cotransporter operating in reverse (2, 6, 19). Hyposmolality probably did not affect (inhibit) the peritubular taurine cotransporter in the present study because it is the major taurine uptake system responsible for accumulating taurine inside fish renal cells (13, 22, 24), and intracellular taurine concentration did not change in hyposmotic medium. Furthermore, probenecid (1 mM), an inhibitor of the renal peritubular taurine cotransporter (2, 18), was found to further decrease the already reduced cell-to-peritubular bath movement of taurine from cultured flounder cells in hyposmotic medium by another 25% (cell-to-peritubular taurine efflux in hyposmotic medium with probenecid = 75.3 ± 1.2% of the rate in hyposmotic medium control, n = 3 preparations, data not shown). The additive nature of the inhibitory effects of hyposmolality and probenecid on peritubular taurine efflux possibly indicates actions through different pathways. This would suggest that exposure to hyposmolality probably decreased taurine efflux across the peritubular membrane of the cultured cells mostly by inhibiting the passive mediated taurine transport system. The decrease in the apparent permeability of the basolateral membrane to taurine in response to hyposmolality in flounder proximal tubule is unique. In other vertebrate renal cells of both proximal and distal in origin, including the proximal tubules of C. auratus, a freshwater teleost that also has the ability to secrete taurine (12), basolateral taurine permeability increases in hyposmotic medium (6, 8, 20, 23). It is not clear whether the different responses to hyposmolality reflect the difference in exposure time or the species differences.

The major site of action for hyposmolality to stimulate taurine secretory flux in flounder PTCs is at the luminal membrane where an actual increase in cell-to-lumen taurine efflux was observed. This conclusion was supported by the observation that the luminal addition of probenecid returned the enhanced cell-to-lumen taurine efflux in hyposmotic medium to the level observed in isosmotic medium. Luminal probenecid also inhibited both the enhanced transepithelial unidirectional and net taurine secretory fluxes in flounder PTCs in hyposmotic medium. The organic anions, probenecid and bromcresol green, have been shown previously to inhibit the luminal taurine transport system responsible for taurine secretion in flounder renal epithelium (18). In the present study, bromcresol green had no effect on the hyposmolality-stimulated taurine secretory flux. At present, not much is known about the characteristics of the luminal taurine efflux system in the flounder renal cells apart from the observations that it is specific for taurine (not other -amino acids like -alanine) and inhibitable by probenecid and bromcresol green (18). More information is needed before the pathway(s) underlying the stimulatory effect of hyposmolality on taurine secretion can be elucidated.

In conclusion, taurine secretory flux in flounder PTCs was modulated by changes in extracellular taurine concentration and osmolality. Similar to in vivo environmental dilution, the flounder renal epithelium enhanced taurine secretory flux in response to exposure to hyposmotic medium. This was accomplished by stimulation of the specific luminal taurine efflux pathway sensitive to probenecid and inhibition of the peritubular passive mediated taurine efflux system. The response of flounder PTCs to hyposmolality differs from the short-term regulatory volume decrease response observed in other types of renal cells and mimics the in vivo adaptive response of the intact marine fish kidney to dilution. The flounder renal epithelium can apparently maintain its cell volume while regulating the extracellular fluid volume through taurine secretion.