©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hyperosmolality Inhibits Bicarbonate Absorption in Rat Medullary Thick Ascending Limb via a Protein-tyrosine Kinase-dependent Pathway (*)

David W. Good (§) , With the technical assistance of Thampi George

From the (1) Departments of Internal Medicine and Physiology & Biophysics, University of Texas Medical Branch, Galveston, Texas 77555

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the rat medullary thick ascending limb (MTAL), hyperosmolality inhibits transepithelial HCO absorption (JHCO) by inhibiting apical membrane Na/Hexchange. To examine signaling mechanisms involved in this regulatory response, MTALs were isolated and perfused in vitro with 25 mM HCO solutions (290 mosmol/kg HO). Osmolality was increased in lumen and bath solutions by addition of 300 mM mannitol or 75 mM NaCl. Addition of mannitol reduced JHCO by 60% and addition of NaCl reduced JHCO by 50%. With the protein tyrosine kinase (PTK) inhibitor genistein (7 µM) or herbimycin A (1 µM) in the bath, addition of mannitol reduced JHCO only by 11% and addition of NaCl reduced JHCO only by 15%. Staurosporine (10 M) or forskolin (10 M) in the bath had no effect on inhibition of JHCO by hypertonic NaCl. Genistein had no effect on inhibition of JHCO by vasopressin (a cyclic AMP-dependent process) or stimulation of JHCO by prostaglandin E(a protein kinase C-dependent process). Under isosmotic conditions, addition of genistein or herbimycin A to the bath increased JHCO by 30% through stimulation of apical membrane Na/Hexchange. Addition of the tyrosine phosphatase inhibitor molybdate (50 µM) to the bath reproduced the inhibition of JHCO observed with hyperosmolality. These data indicate that 1) the effect of hyperosmolality to inhibit MTAL HCO absorption through inhibition of apical membrane Na/Hexchange is mediated via a PTK-dependent pathway that functions independent of regulation by cyclic AMP and protein kinase C, and 2) a constitutive PTK activity inhibits apical membrane Na/Hexchange and HCO absorption under isosmotic conditions. Our results suggest that tyrosine phosphorylation is a critical step in inhibition of the apical Na/Hexchanger isoform NHE-3 by hyperosmolality.


INTRODUCTION

In many cell types, Na/Hexchange is a primary transport pathway responsible for cell volume regulation in hyperosmotic conditions (1, 2, 3, 4) . In response to osmotic shrinkage, parallel activation of Na/Hand Cl/HCO exchangers results in net uptake of NaCl and water, thus returning cell volume toward its original value (5, 6, 7, 8, 9, 10, 11) . The stimulation of Na/Hexchange by hyperosmolality is the result of an increase in the sensitivity of the exchanger to internal H(1, 5, 8) but does not appear to require direct phosphorylation of the exchanger (11) . The intracellular signaling mechanisms involved in osmotic regulation of Na/Hexchange are largely unknown.

In the rat kidney, the medullary thick ascending limb (MTAL)() of the loop of Henle reabsorbs a sizable fraction of the HCO filtered at the glomerulus (12, 13) . The Hsecretion required for this HCO absorption is mediated virtually completely by apical membrane Na/Hexchange (13, 14, 15) . Thus, the rate of transepithelial HCO absorption serves as a measure of apical Na/Hexchange activity under steady-state transporting conditions. In recent studies, we demonstrated that apical membrane Na/Hexchange in the rat MTAL exhibits a unique functional response to hyperosmolality. In contrast to the activation of Na/Hexchange observed in other cell types, hyperosmolality markedly inhibited both apical membrane Na/Hexchange and net HCO absorption in MTAL segments (15, 16) . The inhibition of apical Na/Hexchange could not be explained by an increase in intracellular Naactivity or intracellular pH (pH) and was the result of a decrease in the sensitivity of the exchanger to internal H, reflected by an acid shift in the pHdependence curve (15) . The inhibition by hyperosmolality also was associated with a marked increase in the pHsensitivity of the exchanger over the physiologic pHrange, suggesting that this regulatory response may be a specialized adaptation that enables the MTAL to regulate pHor luminal acidification in the hyperosmotic environment of the renal medulla (15) . However, the signal transduction mechanisms by which hyperosmolality inhibits apical membrane Na/Hexchange have not been identified.

The present study was designed to examine intracellular signaling mechanisms involved in hyperosmotic regulation of HCO absorption and apical membrane Na/Hexchange in the isolated, perfused MTAL of the rat. The results indicate that the inhibition of HCO absorption by hyperosmolality is mediated via a protein tyrosine kinase (PTK)-dependent pathway and that regulation via this pathway occurs independent of regulation by cyclic AMP and protein kinase C. In addition, a constitutive PTK activity inhibits apical membrane Na/Hexchange and HCO absorption under isosmotic conditions.


EXPERIMENTAL PROCEDURES

Materials

Stock solutions of genistein (20 mM), herbimycin A (1.8 mM), staurosporine (0.2 mM), and 5-( N-ethyl- N-isopropyl)-amiloride (EIPA, 50 mM) were prepared in MeSO. Stock solutions (1 mg/ml) of prostaglandin E(PGE) and forskolin were prepared in ethanol. Arginine vasopressin (AVP) was prepared as a 4 10 M stock solution in water. The agents were diluted into bath and perfusion solutions to final concentrations given under ``Results.'' Equivalent concentrations of ethanol or MeSO were added to control solutions. All agents were purchased from Sigma, except genistein and EIPA (Research Biochemicals International, Natick, MA) and herbimycin A (Life Technologies, Inc.).

Tubule Perfusion

Medullary thick ascending limbs from male Sprague-Dawley rats (50-80 g, Taconic, Germantown, NY) were isolated and perfused in vitro as described previously (12, 16, 17) . Single tubules were dissected from the inner stripe of the outer medulla at 10 °C in control bath solution (see below), transferred to a bath chamber on the stage of an inverted stereomicroscope, and mounted on concentric glass pipettes for microperfusion (12, 16) . Tubule fluid emerging from the distal end of the tubules was collected for timed intervals into calibrated constriction pipettes for calculation of tubule fluid flow rates and for analysis of total COconcentrations. The luminal perfusion rate (normalized per unit of tubule length) averaged 1.5-1.7 nl/min/mm. In all experiments, the luminal perfusion solution contained (in mM): 146 Na, 4 K, 122 Cl, 25 HCO, 2.0 Ca, 1.5 Mg, 2.0 phosphate, 1.2 SO, 1.0 citrate, 2.0 lactate, and 5.5 glucose. The bath solution was identical except for addition of 0.2% fatty acid free bovine albumin (Sigma). Osmolality of the solutions was 290 mosmol/kg HO. Hyperosmotic solutions were prepared by addition of 300 mM mannitol (final osmolality = 590 mosmol/kg HO) or 75 mM NaCl (final osmolality = 425 mosmol/kg HO). Other experimental agents were added to perfusion or bath solutions as described under ``Results.'' All solutions were equilibrated with 95% O, 5% COand were pH 7.45-7.47 at 37 °C. The length of the perfused tubule segments ranged from 0.49 to 0.70 mm and averaged 0.60 ± 0.01 mm.

The tubule perfusion protocol was as described previously (16, 17) . In brief, after mounting on pipettes, the tubules were equilibrated for 20-40 min at 37 °C in the initial perfusion and bath solutions. Two to four 10-min tubule fluid samples were then collected for determination of the HCO transport rate (initial period). The perfusion and/or bath solutions were then changed to one of the experimental solutions (increase in osmolality, addition of inhibitor, etc.), and the tubule was allowed to re-equilibrate for 5-20 min. Two to four additional tubule fluid samples were then collected (experimental period). Finally, the initial solutions were returned to the perfusate and bath and the control measurements repeated (recovery period). In separate experiments, measurements of inner and outer tubule diameters were obtained for calculation of epithelial cell volume (15, 18, 19) . The protocol and conditions for the cell volume experiments were virtually identical to those used for HCO transport experiments.

Analysis

Total carbon dioxide concentrations in perfusion and bath solutions and in collected tubule fluid were measured by microcalorimetry as described previously (12) . Transepithelial voltage was measured between calomel cells using 140 mM NaCl-agar bridges (12, 16) . Because net fluid transport is absent in MTALs studied in symmetric isosmotic or hyperosmotic solutions (12, 16) , absolute rates of HCO absorption (JHCO, picomoles/min/mm)() were calculated as JHCO = V ([TCO] [TCO]])/ L, where V is fluid collection rate (nanoliters/min), [TCO] is total carbon dioxide concentration (mM) in perfused (o) and collected (c) fluid, and L is perfused tubule length (mm). A mean HCO absorption rate was calculated for each period studied in a given tubule. When control measurements were made at the beginning and end of an experiment, the values were averaged. Single tubule values are presented in the figures. Results in tables and text are means ± S.E. Differences between means were evaluated using Student's t test for paired data, with p < 0.05 considered statistically significant.


RESULTS

Effects of Genistein and Herbimycin A on Inhibition by Mannitol

The effects on HCO absorption of increasing osmolality with mannitol are shown in and Fig. 1. Adding 300 mM mannitol to the perfusion and bath solutions decreased HCO absorption by 60%, from 9.9 to 4.2 pmol/min/mm ( (A); Fig. 1 A). The inhibition by mannitol was reversible and was similar to that observed previously in the presence of vasopressin (16) . In contrast, in tubules bathed with the PTK inhibitor genistein (7 µM) or herbimycin A (1 µM), adding 300 mM mannitol to the perfusate and bath decreased HCO absorption only by 11%, from 11.7 to 10.4 pmol/min/mm ( (B); Fig. 1 B). Addition of mannitol decreased the transepithelial voltage in the absence or presence of inhibitor ().


Figure 1: Effects of adding mannitol ( Mann, 300 mM) to perfusate and bath on HCO absorption in the absence ( A) or presence ( B) of protein tyrosine kinase inhibitors. In B, genistein (7 µM) or herbimycin A (1 µM) was present in the bath throughout the experiments. Control and genistein solutions were 290 mosmol/kg HO; solutions containing mannitol were 590 mosmol/kg HO. Filled and open circles are mean values for single tubules. Lines connect paired measurements made in the same tubule. P values are for paired t test. Mean values are in Table I (A and B).



Effects of Genistein on Inhibition by NaCl

To determine whether the effect of genistein was specific for mannitol, we examined the effects of increasing osmolality with NaCl, the solute primarily responsible for the hyperosmolality of the renal outer medulla in vivo. Adding 75 mM NaCl to the perfusion and bath solutions decreased HCO absorption by 50%, from 14.0 to 7.1 pmol/min/mm ( (C); Fig. 2A). A similar inhibition was observed previously with addition of 150 mM NaCl (16) . In contrast, in tubules bathed with 7 µM genistein, adding 75 mM NaCl to the perfusate and bath decreased HCO absorption only by 15%, from 17.8 to 15.5 pmol/min/mm ( (D); Fig. 2 B). Thus, the effect of genistein to block hyperosmotic inhibition was independent of the solute used to produce hyperosmolality.


Figure 2: Effects of adding NaCl (75 mM) to perfusate and bath on HCO absorption in the absence ( A) or presence ( B) of genistein. In B, genistein ( Gen, 7 µM) was present in the bath throughout the experiments. Control and genistein solutions were 290 mosmol/kg HO; solutions containing added NaCl were 425 mosmol/kg HO. Filled circles, lines, and P values as in Fig. 1. Mean values are in Table I (C and D).



To determine whether the influence of genistein on hyperosmotic inhibition of HCO absorption was related to an effect on cell volume, steady-state cell volume was determined in MTALs studied under the same experimental conditions used in the preceding HCO transport experiments. In the absence of genistein, the initial control volume was 0.30 ± 0.03 nl/mm ( n = 3). Addition of 75 mM NaCl to the perfusate and bath decreased cell volume to 0.22 ± 0.02 nl/mm ( p < 0.001). The cells returned to their original volume (0.29 ± 0.03 nl/mm) following 75 mM NaCl removal.() In tubules bathed with 7 µM genistein, the initial cell volume was 0.29 ± 0.02 nl/mm, addition of 75 mM NaCl decreased cell volume to 0.22 ± 0.02 nl/mm ( p < 0.001), and cell volume recovered to 0.29 ± 0.02 nl/mm following 75 mM NaCl removal ( n = 3). Thus, genistein had no effect on cell volume under isosmotic or hyperosmotic conditions. In both the absence and presence of genistein, cell volume was stable in hyperosmotic NaCl for up to 30 min and the decrease in cell volume was the result of an increase in the tubule inner (luminal) diameter.

Effects of Staurosporine and Forskolin on Inhibition by NaCl

HCO absorption in the rat MTAL is inhibited by cyclic AMP and, under certain conditions, stimulated by activation of protein kinase C (13, 17, 20) . To determine whether these signaling pathways are involved in inhibition of HCO absorption by hyperosmolality, the effects of increasing NaCl concentration were examined in the presence of staurosporine, a protein kinase C inhibitor, or forskolin, a direct activator of adenylyl cyclase. At the concentrations used, forskolin mediates maximal cyclic AMP-dependent inhibition of HCO absorption (17) , and staurosporine eliminates completely protein kinase C-dependent regulation of HCO transport (20) . The results are shown in Fig. 3. In tubules bathed with 10 M staurosporine, addition of 75 mM NaCl to the perfusate and bath decreased HCO absorption by 53%, from 12.7 ± 1.8 to 6.0 ± 1.6 pmol/min/mm ( n = 3; Fig. 3A). In tubules bathed with 10 M forskolin, addition of 75 mM NaCl decreased HCO absorption by 75%, from 10.1 ± 0.4 to 2.5 ± 0.9 pmol/min/mm ( n = 3; Fig. 3B). In both cases, the inhibition by NaCl was reversible. Thus, the effect of hyperosmotic NaCl to inhibit HCO absorption was unimpaired by pretreatment with staurosporine or forskolin.


Figure 3: Effects of adding NaCl (75 mM) to perfusate and bath on HCO absorption in the presence of staurosporine ( A) or forskolin ( B). Either staurosporine ( Stauro, 10 M) or forskolin ( Forsk, 10 M) was present in the bath throughout the experiments. Staurosporine and forskolin solutions were 290 mosmol/kg HO; solutions containing added NaCl were 425 mosmol/kg HO. Filled circles, lines, and p values as in Fig. 1. Mean values are in the text.



Effects of Vasopressin and Prostaglandin Ein the Presence of Genistein

Previously, we demonstrated that AVP inhibits HCO absorption by 40-50% via cyclic AMP (17) and that this inhibition is reversed by PGEthrough activation of protein kinase C (20, 21) . To determine whether regulation of HCO absorption by AVP or PGEalso may involve PTK, the effects of AVP and PGEwere studied in the presence of genistein (Fig. 4). With 7 µM genistein in the bath, adding 10 M AVP to the bath decreased HCO absorption by 45%, from 14.9 ± 1.6 to 8.2 ± 1.1 pmol/min/mm ( p < 0.01; Fig. 4A). With both AVP and genistein in the bath, adding 10 M PGEto the bath increased HCO absorption from 8.3 ± 0.2 to 12.4 ± 0.4 pmol/min/mm ( p < 0.001; Fig. 4B). The effects of AVP and PGEwere reversible and are similar to results obtained previously in MTALs studied in the absence of genistein (17, 21) . Thus, at the same concentration that nearly abolished inhibition by hyperosmolality, genistein did not prevent AVP inhibition or PGEstimulation of HCO absorption. Effects of Genistein on Basal HCOAbsorption-The effects of PTK inhibitors on HCO absorption under control (isosmotic) conditions are shown in and Fig. 5. Addition of 7 µM genistein or 1 µM herbimycin A to the bath increased HCO absorption by 30%, from 13.3 to 17.2 pmol/min/mm ( (A)). The stimulation of HCO absorption was reversible (Fig. 5 A). Thus, a constitutive PTK activity appears to play a role in determining the base-line HCO absorption rate.


Figure 4: Effects of arginine vasopressin ( AVP, 10 M in the bath) and prostaglandin E ( PGE, 10 M in the bath) on HCO absorption in the presence of genistein. Genistein ( Gen, 7 µM) was present in the bath throughout the experiments. In PGEexperiments ( B), the bath also contained 10 M AVP. All solutions were 290 mosmol/kg HO. Filled circles, lines, and p values as in Fig. 1. Mean values are in the text.




Figure 5: Effects of genistein on basal HCO absorption. In A, either genistein (7 µM) or herbimycin A (1 µM) was added to control bath solution. In B, ethylisopropyl amiloride ( EIPA, 50 µM) was added to the luminal perfusate in tubules bathed with 7 µM genistein. All solutions were 290 mosmol/kg HO. Filled circles, lines, and p values as in Fig. 1. Mean values are in Table II (A and B).



Further experiments were performed to assess whether the increase in HCO absorption with genistein was mediated by an increase in apical membrane Na/Hexchange. The contribution of apical Na/Hexchange to HCO absorption was assessed by luminal addition of EIPA, a potent Na/Hexchange inhibitor. With 7 µM genistein in the bath solution, addition of 50 µM EIPA to the luminal perfusate decreased HCO absorption by 80%, from 16.3 to 3.6 pmol/min/mm ( (B); Fig. 5 B). In the absence of genistein, luminal EIPA decreased HCO absorption from 11.8 ± 0.4 to 2.8 ± 0.2 pmol/min/mm ( n = 3; p < 0.001).() Thus, the increase in HCO absorption observed with genistein was inhibitable by luminal EIPA.

Effects of Molybdate

To assess further the role of tyrosine phosphorylation in the regulation of HCO absorption, we examined the effect of molybdate, a potent protein tyrosine phosphatase inhibitor (22, 23) . In tubules perfused and bathed in isosmotic solution, addition of 50 µM sodium molybdate to the bath decreased HCO absorption by 48%, from 13.6 ± 1.1 to 7.1 ± 1.6 pmol/min/mm ( n = 4; p < 0.025). The inhibition by molybdate was fully reversible and occurred in the absence of a change in transepithelial voltage.() Thus, the effect of hyperosmolality to inhibit HCO absorption could be reproduced with an agent that inhibits tyrosine phosphatases. In three additional experiments, addition of molybdate to the bath had no effect on HCO absorption in tubules perfused and bathed with hypertonic NaCl (5.4 ± 0.4 pmol/min/mm, NaCl versus 5.3 ± 0.1 pmol/min/mm, NaCl + molybdate; p = NS). Thus, the inhibitory effects of hyperosmolality and molybdate were not additive, suggesting that the two factors may act through a similar mechanism of action. These results provide further support for a role for tyrosine phosphorylation in the inhibition of HCO absorption by hyperosmolality.


DISCUSSION

Activation of Na/Hexchange by hyperosmolality is an important mechanism for maintenance of cell volume in many cell types (1, 2, 3, 4) . In contrast, we have shown recently that hyperosmolality inhibits apical membrane Na/Hexchange in the MTAL of the rat (15) . Inhibition of the apical Na/Hexchanger accounts functionally for the effect of hyperosmolality to inhibit transepithelial HCO absorption (15, 16) . The results of the present study show that these inhibitory effects of hyperosmolality are mediated via a PTK-dependent signaling pathway. Furthermore, regulation of HCO absorption by hyperosmolality via the PTK pathway occurs independent of regulation by cyclic AMP and protein kinase C. Tyrosine phosphorylation has been suggested previously to be an important step in swelling-induced activation of ion channels in a human intestinal cell line (26) . Our data are the first to suggest that tyrosine phosphorylation also may play an essential role in osmotic regulation of Na/Hexchange.

Evidence supporting the conclusions of this study was obtained primarily from experiments examining the effects of PTK inhibitors. Several findings support the view that these agents influenced MTAL HCO absorption via their actions on PTK activity. First, virtually identical results were obtained with genistein and herbimycin A, two chemically unrelated PTK inhibitors with different mechanisms of action (27, 28) . Second, at the concentrations studied, both genistein and herbimycin A are selective PTK inhibitors, with no significant activity against a variety of other protein kinases, phosphatases, or phosphodiesterase.()(27, 28, 29, 30, 31, 32) . The apparent specificity of these agents was supported in the present study by the observation that genistein, at the same concentration that nearly eliminated inhibition by hyperosmolality, had no effect on regulation of HCO absorption by AVP (a cyclic AMP-dependent process) or PGE(a protein kinase C-dependent process). Third, both genistein and herbimycin A reversibly stimulated HCO absorption under isosmotic conditions. This finding, along with the lack of effect of genistein on regulation by AVP and PGE, suggests that the effect of these agents to inhibit hyperosmotic regulation was not the result of a toxic or nonspecific metabolic effect on the MTAL cells. Taken together, these results support the notion that genistein and herbimycin A prevent the inhibition of HCO absorption by hyperosmolality via their targeted actions to inhibit PTK activity. Role of PTK in Inhibition of HCOAbsorption by Hyperosmolality-In the rat, the osmolality of the renal medulla can vary from 290 mosmol/kg HO to more than 1500 mosmol/kg HO in response to changes in systemic HO balance. Thus, the osmolalities achieved in the present study with NaCl (425 mosmol/kg HO) and mannitol (590 mosmol/kg HO) represent reasonable estimates of the values expected to surround the MTAL in vivo. Previously, we demonstrated that hyperosmolality produced with a variety of solutes markedly inhibited MTAL HCO absorption, an effect that may be important physiologically for limiting delivery of HCO to the medullary interstitial fluid during antidiuresis (13, 16) . Results of the present study indicate that hyperosmotic inhibition of HCO absorption was eliminated nearly completely by inhibitors of PTK. These agents prevented inhibition of HCO absorption by both physiologic (NaCl) and nonphysiologic (mannitol) osmotic agents, indicating that the increase in osmolality rather than addition of a particular solute was the signal that initiates activation of the PTK regulatory pathway. Protein tyrosine phosphorylation thus appears to be a critical element in the inhibition of HCO absorption by hyperosmolality. Further support for this conclusion was obtained from the observation that the inhibitory effect of hyperosmolality could be reproduced with molybdate, a potent tyrosine phosphatase inhibitor (22, 23) . In addition, the lack of additivity of the effects of hyperosmolality and molybdate suggests that these factors inhibit HCO absorption through a common signaling mechanism, presumably an increase in tyrosine phosphorylation.

Apical membrane Na/Hexchange mediates virtually all of MTAL HCO absorption (13, 14, 15) . Furthermore, hyperosmolality inhibits HCO absorption through inhibition of apical membrane Na/Hexchange (15) . The effect of the PTK inhibitors to block hyperosmotic inhibition of HCO absorption is thus most likely the result of their preventing inhibition of apical Na/Hexchange. An alternative possibility is that exposure to PTK inhibitors may unmask a second Hsecretory pathway that is stimulated by hyperosmolality, thereby offsetting hyperosmotic inhibition of the apical Na/Hexchanger to maintain HCO absorption. This possibility seems unlikely, however, since stimulation of HCO absorption by genistein was inhibited by luminal EIPA. This indicates that PTK regulation of HCO absorption is mediated through regulation of apical membrane Na/Hexchange and that the apical exchanger mediates HCO absorption both in the absence and presence of PTK inhibitors. Our results are thus most consistent with the conclusion that hyperosmolality acts via a PTK-dependent signaling pathway to inhibit apical membrane Na/Hexchange, thereby inhibiting transepithelial HCO absorption. Hyperosmolality inhibits the apical Na/Hexchanger by decreasing its sensitivity to internal H, manifested as an acid shift in the pHdependence curve (15) . The present results suggest that this osmotic-induced acid shift requires PTK. Further work is needed to test this directly and to identify the molecular mechanisms involved in PTK-dependent regulation of the Na/Hexchanger.

At least four unique mammalian isoforms of Na/Hexchange (NHE-1 through NHE-4) have been identified (33) . Recent preliminary studies suggest that NHE-3 may be the Na/Hexchanger isoform in the apical membrane of the MTAL (34, 35) , as reported previously for the renal proximal tubule (36) and intestine (37) . Based on our demonstration that hyperosmolality directly inhibits apical Na/Hexchange in the rat MTAL (15) , we infer that hyperosmolality inhibits NHE-3 in this nephron segment. This view is supported by recent studies in transfected cell lines (38) and cultured renal epithelial cells (39) which reported that hyperosmolality inhibits NHE-3 but stimulates NHE-1 and NHE-2. The present study provides the first evidence that the effect of hyperosmolality to inhibit NHE-3 may depend critically on tyrosine phosphorylation.

Although our results suggest an important role for tyrosine phosphorylation in the inhibition of HCO absorption, they do not establish the nature of the link between PTK activity and hyperosmolality. An increase in tyrosine phosphorylation in response to hyperosmolality could result from stimulation of tyrosine kinase activity, inhibition of protein tyrosine phosphatase activity, or both. Alternatively, the role of PTK may be permissive, such that tyrosine phosphorylation is essential for intact functioning of the regulatory mechanism but is not a process regulated directly by hyperosmolality. In previous studies in cultured intestinal cells, hypotonicity induced a rapid increase in tyrosine phosphorylation that was potentiated by pretreatment with a tyrosine phosphatase inhibitor, suggesting a primary role for activation of tyrosine kinase (26) . Analyses of signal transduction mechanisms involved in osmotic activation of KCl cotransport in red blood cells and Na/Hexchange in lymphocytes also suggest a primary role for regulation via a protein kinase (4, 40, 41) . Recent studies in yeast and in mammalian cell lines have demonstrated that several members of the mitogen-activated protein (MAP) kinase, and MAP kinase kinase families are activated by osmotic stress (42, 43, 44, 45) . Furthermore, the activation of MAP kinases involves tyrosine phosphorylation (42, 44, 45) . These findings support the notion that hyperosmotic regulation occurs via activation of protein kinases and suggest that MAP kinases may be a component of a PTK-dependent signaling pathway that mediates hyperosmotic inhibition of apical membrane Na/Hexchange in the rat MTAL.

The role of protein phosphorylation in activation of Na/Hexchange during cell volume regulation is poorly understood. Stimulation of Na/Hexchange by hyperosmolality does not appear to require direct phosphorylation of the exchanger (at least for the NHE-1 isoform) (11) ; however, phosphotransferase reactions involving other proteins appear to be involved in the activation process (4, 6, 41, 46) . These reactions have not been identified, although protein kinases A and C do not appear to be involved (4, 10) . In view of the present results, it will be of interest to determine whether activation of Na/Hexchange may be a PTK-dependent process.

Genistein inhibited hyperosmotic regulation of HCO absorption but had no effect on basal (isosmotic) cell volume or on the extent of cell shrinkage in response to hypertonic NaCl. Thus, the PTK inhibitors did not influence HCO absorption through effects on cell volume, and cell shrinkage, by itself, was not sufficient to elicit the inhibition of HCO absorption. This latter result supports our previous conclusion (15) that an increase in cell Naactivity secondary to cell shrinkage appears to play little, if any, role in the hyperosmotic inhibition of apical membrane Na/Hexchange. Our experiments do not address the question of whether a decrease in cell volume may be necessary for activation of the PTK signaling pathway.

Regulation via the PTK-dependent Pathway Occurs Independent of Regulation by Cyclic AMP and Protein Kinase C

AVP inhibits MTAL HCO absorption by 40-50% by increasing intracellular cyclic AMP (17) . Cyclic AMP does not appear to be involved in inhibition of HCO absorption by hyperosmolality, since 1) inhibition by hyperosmolality is additive to the maximal inhibition that can be achieved with AVP or exogenous addition of 8-bromo-cyclic AMP (16, 17) , and 2) stimulation of cyclic AMP production with forskolin has no effect on hyperosmotic inhibition of HCO absorption (Fig. 3 B). We have also shown that the inhibition of HCO absorption by AVP is reversed by PGEthrough activation of protein kinase C (20, 21) . Evidence against the involvement of protein kinase C in hyperosmotic regulation includes 1) hyperosmolality has no effect on stimulation of HCO absorption by PGE(21) , and 2) staurosporine, at a concentration that eliminates completely protein kinase C-dependent regulation by PGE(20) , has no effect on inhibition by hyperosmolality (Fig. 3 A). A further dissociation of the signaling pathways was obtained from experiments demonstrating that genistein had no effect on either AVP inhibition or PGEstimulation of HCO absorption (Fig. 4). Thus, a genistein-sensitive PTK does not appear to be involved in regulation of HCO absorption by AVP and PGE. Taken together, these results indicate that hyperosmolality inhibits apical membrane Na/Hexchange and HCO absorption via a PTK-dependent pathway that does not involve cyclic AMP or protein kinase C and that operates independent of regulation by AVP and PGE. These findings are in contrast to results suggesting that AVP or cyclic AMP is necessary for hyperosmotic activation of basolateral Na/Hexchange in the mouse MTAL (19) but are consistent with evidence in other cell types that protein kinases A and C are not involved in hyperosmotic stimulation of Na/Hexchange (4, 10) . Role of PTK in Basal HCOAbsorption-Genistein or herbimycin A stimulated HCO absorption under isosmotic conditions in the absence of a change in cell volume. As discussed above, this stimulation is inhibited by luminal EIPA, indicating that genistein increased HCO absorption through stimulation of apical membrane Na/Hexchange. These data indicate that apical membrane Na/Hexchange is under basal control by PTK, such that a constitutive PTK activity inhibits apical Na/Hexchange and HCO absorption under isosmotic conditions in the absence of added agonist. Although it is reasonable to assume that this is the same PTK pathway that is involved in hyperosmotic inhibition of HCO absorption, this remains to be proven. Nonetheless, our results suggest that tyrosine phosphorylation is an important determinant of apical membrane Na/Hexchange activity and HCO absorption in the MTAL under both isosmotic and hyperosmotic conditions.

In summary, the effect of hyperosmolality to inhibit MTAL HCO absorption through inhibition of apical membrane Na/Hexchange is mediated via a PTK-dependent pathway. Regulation via this pathway occurs independent of regulation by cyclic AMP and protein kinase C. A constitutive PTK activity also controls basal HCO absorption and apical Na/Hexchange activity under isosmotic conditions. These data support an important role for protein tyrosine kinase in osmotic regulation of Na/Hexchange and suggest that tyrosine phosphorylation is an essential step in inhibition of NHE-3 by hyperosmolality in the MTAL.

  
Table: Effects of genistein on inhibition of HCOabsorption by hyperosmolality

Values are means ± S.E. Mannitol (300 mM) or NaCl (75 mM) was added to perfusate and bath in the absence or presence of bath genistein (7 µM). Numbers in parentheses are numbers of tubules. V, fluid flow rate; [TCO], total carbon dioxide concentration in collected tubule fluid; JTCO, absolute rate of total COabsorption; V, transepithelial voltage, oriented lumen positive with respect to bath. [TCO] was 25.4 ± 0.1 mM in perfusion fluid and 25.4 ± 0.1 mM in bath.


  
Table: Effects of genistein on basal HCOabsorption

Values are means ± S.E. In series A, genistein (7 µM) was added to control bath solution; in series B, ethylisopropyl amiloride (EIPA, 50 µM) was added to luminal perfusate in the presence of bath genistein. Numbers in parentheses, V, [TCO], JTCO, and Vas in Table I. [TCO] was 25.5 ± 0.1 mM in perfusion fluid and 25.1 ± 0.1 mM in bath.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK38217. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 4.200 John Sealy Hospital E-62, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0562.

The abbreviations used are: MTAL, medullary thick ascending limb; PTK, protein tyrosine kinase; EIPA, ethylisopropyl amiloride; PGE, prostaglandin E; AVP, arginine vasopressin; MAP, mitogen-activated protein.

In HCO transport experiments, the fall in total COconcentration along the tubule lumen is due to a decrease in the concentration of HCO because, at physiologic pH, HCO accounts for 95% of the total COof the perfusate (the remaining 5% is comprised primarily of dissolved COand carbonic acid). Thus, total COabsorption rates (JTCO) are virtually equal to HCO absorption rates (JHCO), and the terms are used interchangeably throughout the paper.

The decrease in cell volume in hypertonic NaCl is near that expected if the MTAL cells behaved as perfect osmometers, suggesting minimal cell volume regulation under the conditions of these experiments. Similar results have been reported for the mouse MTAL, in which a regulatory volume increase was not observed in hyperosmotic solutions unless vasopressin was present in the bath solution (19). In the MTAL of the rat, hyperosmolality inhibits HCO absorption in the absence or presence of vasopressin (Ref. 16 and present study), suggesting, by analogy with the mouse data, that hyperosmolality inhibits HCO absorption in the absence or presence of cell volume regulation.

The residual HCO absorption observed in the presence of EIPA is abolished by removal of luminal Naand is the result of incomplete inhibition of the apical membrane Na/Hexchanger by EIPA at physiological Naconcentrations (13-15).

Phenylarsine oxide, another agent that inhibits tyrosine phosphatases (23-25), also inhibited MTAL HCO absorption when added to the bath at low concentrations (0.02-0.05 µM). However, in contrast to results obtained with molybdate, the inhibition by phenylarsine oxide was virtually complete, irreversible, and associated with a sharp decrease in the transepithelial voltage, presumably reflecting nonspecific metabolic effects as observed with this agent in other cell types (25).

The ICfor inhibition of tyrosine kinase activity ranges from 1 to 100 µM for genistein and from 0.1 to 10 µM for herbimycin A, depending on the particular PTK or physiological process being studied, the tissue preparation, and the conditions of the assay ( e.g. in vitro versus in vivo) (27-32). In the present study, a low concentration of genistein (7 µM) or herbimycin A (1 µM) was sufficient to reduce hyperosmotic inhibition of HCO absorption by 80%. Whether higher concentrations of the inhibitors would have abolished the inhibition was not tested.


ACKNOWLEDGEMENTS

We thank L. Reuss and M. Jennings for critical reading of the manuscript.


REFERENCES
  1. Grinstein, S., and Rothstein, A. (1986) J. Membr. Biol. 90, 1-12 [Medline] [Order article via Infotrieve]
  2. Hoffman, E. K., and Simonsen, O. (1989) Physiol. Rev. 69, 315-382 [Free Full Text]
  3. Chamberlin, M., and Strange, K. (1989) Am. J. Physiol. 257, C159-C173
  4. Grinstein, S., Furuya, W., and Bianchini, L. (1992) News Physiol. Sci. 7, 232-237 [Abstract/Free Full Text]
  5. Grinstein, S., Rothstein, A., and Cohen, S. (1985) J. Gen. Physiol. 85, 765-787 [Abstract]
  6. Grinstein, S., Cohen, S., Goetz, J. D., and Rothstein, A. (1985) J. Cell Biol. 101, 269-276 [Abstract]
  7. Hebert, S. (1986) Am. J. Physiol. 250, C920-C931
  8. Green, J., Yamaguchi, D. T., Kleeman, C. R., and Muallem, S. (1988) J. Biol. Chem. 263, 5012-5015 [Abstract/Free Full Text]
  9. Parker, J. C., McManus, T. J., Starke, L. C., and Gitelman, H. J. (1990) J. Gen. Physiol. 96, 1141-1152 [Abstract]
  10. Davis, B. A., Hogan, E. M., and Boron, W. F. (1992) Am. J. Physiol. 262, C533-C536
  11. Grinstein, S., Woodside, M., Sardet, C., Pouyssegur, J., and Rotin, D. (1992) J. Biol. Chem. 267, 23823-23828 [Abstract/Free Full Text]
  12. Good, D. W., Knepper, M. A., and Burg, M. B. (1984) Am. J. Physiol. 247, F35-F44
  13. Good, D. W. (1993) Semin. Nephrol. 13, 225-235 [Medline] [Order article via Infotrieve]
  14. Watts, B. A., III, and Good, D. W. (1991) J. Am. Soc. Nephrol. 2, 715
  15. Watts, B. A., III, and Good, D. W. (1994) J. Biol. Chem. 269, 20250-20255 [Abstract/Free Full Text]
  16. Good, D. W. (1992) J. Clin. Invest. 89, 184-190 [Medline] [Order article via Infotrieve]
  17. Good, D. W. (1990) J. Clin. Invest. 85, 1006-1013 [Medline] [Order article via Infotrieve]
  18. Kirk, K. L., DiBona, D. R., and Schafer, J. A. (1984) J. Membr. Biol. 79, 53-64 [Medline] [Order article via Infotrieve]
  19. Hebert, S. C. (1986) Am. J. Physiol 250, C907-C919
  20. Good, D. W., and George, T. (1994) J. Am. Soc. Nephrol. 5, 679
  21. Good, D. W., George, T. (1992) J. Am. Soc. Nephrol. 3, 777
  22. Tonks, N. K., Dilz, C. D., and Fischer, E. H. (1988) J. Biol. Chem. 263, 6731-6737 [Abstract/Free Full Text]
  23. Walton, K. M., and Dixon, J. E. (1993) Annu. Rev. Biochem. 62, 101-120 [CrossRef][Medline] [Order article via Infotrieve]
  24. Garcia-Morales, P., Minami, Y., Luong, E., Klausner, R. D., and Samelson, L. E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9255-9259 [Abstract]
  25. Maher, P. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11177-11181 [Abstract]
  26. Tilly, B. C., van den Berghe, N., Tertoolen, L. G. J., Edixhoven, M. J., and de Jonge, H. R. (1993) J. Biol. Chem. 268, 19919-19922 [Abstract/Free Full Text]
  27. Akiyama, T., and Ogawara, H. (1991) Methods Enzymol. 201, 362-370 [Medline] [Order article via Infotrieve]
  28. Uehara, Y., and Fukazawa, H. (1991) Methods Enzymol. 201, 370-379 [Medline] [Order article via Infotrieve]
  29. Lane, P. J. L., Ledbetter, J. A., McConnell, F. M., Draves, K., Deans, J., Schieven, G. L., and Clark, E. A. (1991) J. Immunol. 146, 715-722 [Abstract/Free Full Text]
  30. Yao, X.-R., and Scott, D. W. (1993) Cell. Immunol. 149, 364-375 [CrossRef][Medline] [Order article via Infotrieve]
  31. Tsunoda, Y., Modlin, I. M., and Goldenring, J. R. (1993) Am. J. Physiol. 264, G351 -G356
  32. Knox, K. A., and Gordon, J. (1994) Cell. Immunol. 155, 62-76 [CrossRef][Medline] [Order article via Infotrieve]
  33. Tse, M., Levine, S., Yun, C., Brant, S., Pouyssegur, J., and Donowitz, M. (1993) J. Am. Soc. Nephrol. 4, 969-975 [Abstract]
  34. Borensztein, P., Laghmani, K., Froissart, M., Philippe, M., Bichara, M., and Paillard, M. (1994) J. Am. Soc. Nephrol. 5, 248
  35. Sun, A. M., Nillni, E. A., Huo, T. L., Dworkin, L. D., Bookstein, C., Rao, M. C., Chang, E. B., and Lytton, J. (1994) J. Am. Soc. Nephrol. 5, 263
  36. Biemesderfer, D., Pizzonia, J., Abu-Alfa, A., Exner, M., Reilly, R., Igarashi, P., and Aronson, P. S. (1993) Am. J. Physiol. 265, F736-F742
  37. Bookstein, C., DePaoli, A. M., Xie, Y., Niu, P., Musch, M. W., Rao, M. C., and Chang, E. B. (1994) J. Clin. Invest. 93, 106-113 [Medline] [Order article via Infotrieve]
  38. Kapus, A., Grinstein, S., Wasan, S., Kandasamy, R., and Orlowski, J. (1994) J. Biol. Chem. 269, 23544-23552 [Abstract/Free Full Text]
  39. Soleimani, M., Bookstein, C., McAteer, J. A., Hattabaugh, Y. J., Bizal, G. L., Musch, M. W., Villereal, M., Rao, M. C., Howard, R. L., and Chang, E. B. (1994) J. Biol. Chem. 269, 15613-15618 [Abstract/Free Full Text]
  40. Jennings, M. L., and Al-Rohil, N. (1990) J. Gen. Physiol. 95, 1021-1040 [Abstract]
  41. Bianchini, L., Woodside, M., Sardet, C., Pouyssegur, J., Takai, A., and Grinstein, S. (1991) J. Biol. Chem. 266, 15406-15413 [Abstract/Free Full Text]
  42. Brewster, J. L., deValoir, T., Dwyer, N. D., Winter, E., and Gustin, M. C. (1993) Science 259, 1760-1763 [Medline] [Order article via Infotrieve]
  43. Itoh, T., Yamauchi, A., Miyai, A., Yokoyama, K., Kamada, T., Ueda, N., and Fujiwara, Y. (1994) J. Clin. Invest. 93, 2387-2392 [Medline] [Order article via Infotrieve]
  44. Galcheva-Gargova, Z., Derijard, B., Wu, I.-H., and Davis, R. J. (1994) Science 265, 806-808 [Medline] [Order article via Infotrieve]
  45. Han, J., Lee, J.-D., Bibbs, L., and Ulevitch, R. J. (1994) Science 265, 808-811 [Medline] [Order article via Infotrieve]
  46. Grinstein, S., Goetz-Smith, J. D., Stewart, D., Beresford, B., and Mellors, A. (1986) J. Biol. Chem. 261, 8009-8016 [Abstract/Free Full Text]

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