Hyposmolality stimulates Na+/H+ exchange and HCO3minus absorption in thick ascending limb via PI 3-kinase

David W. Good, John F. Di Mari, and Bruns A. Watts III

Deparments of Medicine and Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555


    ABSTRACT
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The signal transduction mechanisms that mediate osmotic regulation of Na+/H+ exchange are not understood. Recently we demonstrated that hyposmolality increases HCO3- absorption in the renal medullary thick ascending limb (MTAL) through stimulation of the apical membrane Na+/H+ exchanger NHE3. To investigate the mechanism of this stimulation, MTALs from rats were isolated and perfused in vitro with 25 mM HCO3--containing solutions. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (100 nM) and LY-294002 (20 µM) blocked completely the stimulation of HCO3- absorption by hyposmolality. In tissue strips dissected from the inner stripe of the outer medulla, the region of the kidney highly enriched in MTALs, hyposmolality increased PI 3-K activity 2.2-fold. Wortmannin blocked the hyposmolality-induced PI 3-K activation. Further studies examined the interaction between hyposmolality and vasopressin, which inhibits HCO3- absorption in the MTAL via cAMP and often is involved in the development of plasma hyposmolality in clinical disorders. Pretreatment with arginine vasopressin, forskolin, or 8-bromo-cAMP abolished hyposmotic stimulation of HCO3- absorption, due to an effect of cAMP to inhibit hyposmolality- induced activation of PI 3-K. In contrast to their effects to block stimulation by hyposmolality, PI 3-K inhibitors and vasopressin have no effect on inhibition of apical Na+/H+ exchange (NHE3) and HCO3- absorption by hyperosmolality. These results indicate that hyposmolality increases NHE3 activity and HCO3- absorption in the MTAL through activation of a PI 3-K-dependent pathway that is inhibited by vasopressin and cAMP. Hyposmotic stimulation and hyperosmotic inhibition of NHE3 are mediated through different signal transduction mechanisms.

signal transduction; adenosine 3',5'-cyclic monophosphate; phosphatidylinositol 3-kinase; hyperosmolality


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INTRODUCTION
METHODS
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DISCUSSION
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NA+/h+ exchangers are present in the plasma membrane of virtually all mammalian cells and participate in a variety of vital cell functions (20, 50, 57). At least five mammalian exchanger isoforms (NHE1-5) have been identified (38, 50). These isoforms differ in their tissue distribution, kinetic properties, and responses to physiological stimuli (38, 50, 57). NHE1 is expressed ubiquitously in nonpolar cells and on the basolateral membrane of epithelial cells, where it is involved in the regulation of cell volume and intracellular pH (20, 50, 57). NHE2, NHE3, and NHE4 exhibit a more restricted tissue distribution, with preferential localization in the kidney and gastrointestinal tract (38, 50, 57). NHE3 is expressed in the apical membrane of certain renal tubule and intestinal epithelial cells, where it mediates the transepithelial absorption of NaCl and NaHCO3 (2, 5, 15, 32, 38, 50, 54, 55). The activity of Na+/H+ exchangers is markedly influenced by changes in the extracellular osmolality. Hyperosmolality differentially regulates exchanger isoforms: it stimulates NHE1, NHE2, and NHE4 but inhibits NHE3 (7, 15, 22, 35, 44, 45, 52). Hyposmolality has been shown to inhibit NHE1 in several systems (19, 22, 30); however, the physiological responses of other exchanger isoforms to hyposmotic stress are poorly defined. In a study of epithelial isoforms expressed in Chinese hamster ovary (AP-1) cells, hyposmolality inhibited NHE2 but had no effect on NHE3 (22).

Transepithelial absorption of HCO3- by the medullary thick ascending limb (MTAL) of the mammalian kidney is mediated via apical membrane Na+/H+ exchange (14, 18, 54). Evidence from immunocytochemical, pharmacological, and functional studies indicates that this apical exchange activity is mediated by NHE3 (2, 5, 15, 18, 28, 52, 54). Recently we demonstrated that peritubular hyposmolality increases HCO3- absorption in the MTAL through stimulation of apical membrane Na+/H+ exchange (54). These studies provided the first evidence that NHE3 is regulated by hyposmotic stress. We also found that hyposmolality stimulates apical Na+/H+ exchange activity through an increase in maximal velocity (Vmax) (54), whereas hyperosmolality inhibits the apical exchanger by decreasing its apparent affinity for intracellular H+ (52). These different kinetic mechanisms suggest that the opposing effects of hyposmolality and hyperosmolality on NHE3 activity in the MTAL may be mediated through different signaling pathways. At present, however, the signaling mechanisms involved in osmotic regulation of Na+/H+ exchange activity are largely unknown.

Phosphatidylinositol 3-kinase (PI 3-K) phosphorylates the 3-position of the inositol ring of phosphatidylinositides, resulting in the production of lipid second messengers that regulate a variety of cellular processes, including proliferation and survival, glucose transport and metabolism, and cytoskeletal organization (40, 48). Recent studies indicate that PI 3-K is activated by hyposmotic cell swelling and plays a role in mediating swelling-induced stimulation of glycogen synthesis in hepatocytes and skeletal muscle cells (25, 29, 47). In addition, PI 3-K was found to play a role in the stimulation of apical membrane Na+/H+ exchange activity by epidermal growth factor in intestinal epithelial cells (23) and to increase plasma membrane NHE3 activity in transfected AP-1 cells (27). These findings suggest that PI 3-K could be a component of the signaling pathway that mediates hyposmotic stimulation of apical Na+/H+ exchange activity in the MTAL. At present, however, the role of PI 3-K in osmotic regulation of Na+/H+ exchange has not been defined. Also, whether PI 3-K activity is osmotically regulated in renal tubules is not known.

In a variety of clinical disorders, the development of plasma hyposmolality involves renal water retention mediated through elevated levels of vasopressin (4). We have demonstrated previously that arginine vasopressin (AVP) inhibits HCO3- absorption in the MTAL via cAMP (13), an effect opposite to the stimulation of HCO3- absorption by hyposmolality (54). These findings raise the possibility that AVP and hyposmolality could function in a negative feedback system, in which inhibition of HCO3- absorption by AVP is opposed by stimulation of HCO3- absorption by a decrease in osmolality. The feasibility of such a counterregulatory mechanism is supported by our previous observation that AVP and hyperosmolality regulate MTAL HCO3- absorption through independent pathways (15). However, despite the close association between hyposmolality and increased vasopressin levels in many clinical states, the interacting effects of hyposmolality and vasopressin in the regulation of ion transport and signaling pathways in renal tubules have not been investigated.

The aims of the present study were 1) to identify signaling pathways involved in the hyposmotic stimulation of apical membrane Na+/H+ exchange and HCO3- absorption in the rat MTAL and 2) to examine the interacting effects of hyposmolality and AVP in the regulation of HCO3- absorption. The results demonstrate that the effect of hyposmolality to increase HCO3- absorption through stimulation of apical membrane Na+/H+ exchange is mediated through activation of PI 3-K. Vasopressin blocks the hyposmotic stimulation of HCO3- absorption by increasing cAMP, which inhibits the hyposmolality-induced stimulation of PI 3-K activity. We also show that PI 3-K is not involved in the inhibition of HCO3- absorption by hyperosmolality, indicating that hyposmolality and hyperosmolality regulate apical Na+/H+ exchange (NHE3) activity in the MTAL through different signaling pathways.


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Tubule Perfusion and Measurement of Net HCO3- Absorption

MTAL from male Sprague-Dawley rats (60-90 g; Taconic, Germantown, NY) were isolated and perfused in vitro as previously described (13, 15, 54). In brief, the tubules were dissected from the inner stripe of the outer medulla, transferred to a bath chamber on the stage of an inverted microscope, and mounted on concentric glass micropipettes for perfusion at 37°C. In most experiments, the tubules were perfused and bathed in control solution that contained (in mM) 121 Na+, 4 K+, 97 Cl-, 25 HCO3-, 2.0 Ca2+, 1.5 Mg2+, 2.0 phosphate, 1.2 SO42-, 1.0 citrate, 2.0 lactate, 5.5 glucose, and 50 mannitol (osmolality = 295 mosmol/kgH2O). Hyposmotic solution was identical except for the removal of 50 mM mannitol (osmolality = 245 mosmol/kgH2O). In two experiments in Fig. 2, 25 mM NaCl replaced 50 mM mannitol in the control perfusate and bath (Na+ = 146 mM, Cl- = 122 mM, osmolality = 295 mosmol/kgH2O) and the hyposmotic solution was made by the removal of 25 mM NaCl. For experiments in Fig. 5, hyperosmotic solutions were prepared by the addition of 50 mM mannitol or 75 mM NaCl to the latter control solution (15). All solutions were equilibrated with 95% O2-5% CO2 (pH 7.45 at 37°C). Bath solutions also contained 0.2 g/100 ml fatty acid-free bovine albumin. Wortmannin (Sigma Chemical, St. Louis, MO) was prepared as a stock solution in dimethyl sulfoxide and LY-294002 (Sigma) as a stock solution in ethanol. These agents were diluted into bath solutions to final concentrations given in RESULTS; equal concentrations of vehicle were added to control solutions. Solutions containing other experimental agents were prepared as previously described (13, 15, 16). Tubules were dissected at 10°C in the control solution that contained 146 mM Na+ and 122 mM Cl- (see above). The length of the perfused tubule segments ranged from 0.48 to 0.70 mm.

The protocol for study of transepithelial HCO3- absorption was as described (13, 15, 54). The tubules were equilibrated for 20-30 min at 37°C in the initial perfusion and bath solutions, and the luminal flow rate (normalized per unit tubule length) was adjusted to 1.5-2.0 nl · min-1 · mm-1. Two or three 10-min tubule fluid samples were then collected for each period (initial, experimental, and recovery). The tubules were allowed to reequilibrate for 5-15 min after a change in the composition of the lumen and/or bath solutions. The absolute rate of HCO3- absorption (JHCO3-, pmol · min-1 · mm-1) was calculated from the luminal flow rate and the difference between total CO2 concentrations measured in perfused and collected fluids (13). An average HCO3- absorption rate was calculated for each period studied in a given tubule. When repeat measurements were made at the beginning and end of an experiment (initial and recovery periods), the values were averaged. Single tubule values are presented in the figures. Mean values ± SE (n = number of tubules) are reported in the text. In separate experiments, epithelial cell volume was determined from measurements of inner and outer tubule diameters as previously described (15, 52). The protocol and conditions for the cell volume experiments were virtually identical to those used in the HCO3- transport experiments.

Determination of PI 3-K Activity

Tissue preparation. The tissue preparation used to study PI 3-K activity has been described previously (3, 51). In brief, rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and both kidneys were removed and sliced in ice-cold control solution. The inner stripe of the outer medulla was cut from the slices and dissected into thin strips of tissue as described (3, 51). The strips were divided into four samples of equal amount and then incubated in vitro in the same solutions used for HCO3- transport experiments. The tissue samples were equilibrated at 37°C for 1 h in control solution and then either maintained in control solution or incubated in hyposmotic solution (50 mM mannitol removed) for an additional 15 min. Identical samples were run in individual experiments with either 100 nM wortmannin or 10-4 M 8-bromo-cAMP (8-BrcAMP) in control and hyposmotic solutions. These protocols were chosen to reproduce those used in the HCO3- transport experiments. The tissue samples were bubbled continuously with 95% O2-5% CO2 throughout the incubation for mixing and to maintain the oxygen tension and pH of the solutions. After incubation, the tissue samples were suspended immediately in ice-cold Triton lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 25 mM beta -glycerophosphate, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/ml leupeptin), homogenized using a Dounce pestle, and lysed for 2.5 h at 4°C on an orbital shaker. The cell lysates were then centrifuged at 3,600 g for 5 min, and the supernatants were isolated and assayed for protein concentration (Micro BCA kit, Pierce, Rockford, IL). The inner stripe of the outer medulla is highly enriched in MTALs, which comprise the majority of the protein mass and cellular volume of this region (3, 24). We demonstrated previously that changes in protein kinase activities measured in the inner stripe reproduce accurately changes measured in the MTAL (3, 51, 53).

Immunoprecipitation and PI 3-K assay. PI 3-K activity was determined in a lipid kinase assay with phosphatidylinositol as substrate, using previously described methods (12, 43). Equal amounts of protein (200 µg) from experimental samples were incubated for 2 h at 4°C with 10 µg of monoclonal antibody against the p85alpha subunit of PI 3-K (Santa Cruz Biotechnology, Santa Cruz, CA) and 20 µl of a protein A-agarose bead suspension (Santa Cruz). The immunoprecipitates were washed three times with lysis buffer and three times with 10 mM Tris · HCl, pH 7.4. To measure PI 3-K activity, the immune complexes were incubated for 10 min at 4°C in 10 µl of 1 mg/ml sonicated L-alpha -phosphatidylinositol (Avanti Polar Lipids, Alabaster, AL) in 30 mM HEPES, pH 7.4. Forty microliters of kinase buffer (30 mM HEPES, pH 7.4, 30 mM MgCl2, 200 µM adenosine, 50 µM ATP, and 20 µCi [gamma -32P]ATP) were then added, and the assays were carried out at room temperature for 15 min. The reactions were stopped with 100 µl of 1N HCl, and the lipids were extracted by the addition of 200 µl of chloroform-methanol (1:1). The organic phase was collected, and 20-µl samples were spotted onto silica-gel thin-layer chromatography (TLC) plates impregnated with 1% potassium oxalate. The TLC solvent was chloroform-methanol-water-ammonium hydroxide (18:14:3:1). The plates were dried, and phosphorylated substrate was detected by autoradiography. Autoradiograms were digitized, and substrate phosphorylation was quantified by densitometry (ImageQuant; Molecular Dynamics, Sunnyvale, CA). There was no detectable phosphorylation of substrate in negative control experiments in which sample protein was omitted from the assay. Equal amounts of PI 3-K in immunoprecipitates were verified in parallel samples for all protocols by immunoblotting with the same antibody used for immunoprecipitation.

Statistical Analysis

Results are presented as means ± SE. Differences between means were evaluated using the paired Student's t-test or ANOVA with Newman-Keuls multiple-range test, as appropriate. P < 0.05 was considered statistically significant.


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Hyposmolality Stimulates HCO3- Absorption

Previously we demonstrated that hyposmolality increases HCO3- absorption in the MTAL through stimulation of apical membrane Na+/H+ exchange activity (54). This finding was confirmed in three experiments in the present study (Fig. 1). Decreasing osmolality in the lumen and bath solutions by removal of 50 mM mannitol increased HCO3- absorption from 10.5 ± 0.8 to 14.3 ± 0.9 pmol · min-1 · mm-1 (P < 0.005). The stimulation was observed within 15 min after mannitol was removed and was reversible. A similar stimulation of HCO3- absorption is observed when osmolality is decreased by the removal of 25 mM NaCl (54).


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Fig. 1.   Hyposmolality stimulates HCO3- absorption in the medullary thick ascending limb (MTAL). Osmolality of the lumen and bath solutions was decreased from 295 (Control) to 245 mosmol/kgH2O (Hypo) by the removal of 50 mM mannitol. JHCO3-, absolute rate of HCO3- absorption. Data points are average values for single tubules. Lines connect paired measurements made in the same tubule. P value is for paired t-test. Mean values are given in RESULTS.

Role of PI 3-K in Hyposmotic Stimulation of HCO3- Absorption

Inhibitors of PI 3-K block stimulation of HCO3- absorption by hyposmolality. PI 3-K has been implicated in signal transduction by hyposmolality in several cell types (25, 29, 47). To determine whether PI 3-K is involved in hyposmotic stimulation of HCO3- absorption in the MTAL, we examined the effects of wortmannin and LY-294002, two selective inhibitors of PI 3-K activity (37, 49, 56). The results in Fig. 2 show that in tubules bathed with either 100 nM wortmannin or 20 µM LY-294002, decreasing osmolality by the removal of 50 mM mannitol or 25 mM NaCl had no effect on HCO3- absorption (11.7 ± 1.1, inhibitors, vs. 11.6 ± 1.1 pmol · min-1 · mm-1, inhibitors + hyposmolality, n = 6; P = not significant). These results suggest that PI 3-K plays an essential role in the stimulation of HCO3- absorption by hyposmolality.


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Fig. 2.   Inhibitors of phosphatidylinositol 3-kinase (PI 3-K) block hyposmotic stimulation of HCO3- absorption. MTAL were bathed with either 100 nM wortmannin () or 20 µM LY-294002 (open circle ) throughout the experiments. Hyposmolality was produced in lumen and bath by removal of 50 mM mannitol in 2 experiments with wortmannin and 2 experiments with LY-294002. In the other experiments, hyposmolality was produced by removal of 25 mM NaCl. JHCO3-, data points, lines, and P value are as in Fig. 1. Mean values are given in RESULTS.

Hyposmolality stimulates PI 3-K activity. To confirm that hyposmolality regulates PI 3-K activity in the MTAL, we examined PI 3-K in the inner stripe of the outer medulla, the region of the kidney highly enriched in MTALs (3, 24). Inner stripe tissue was incubated in control or hyposmotic (50 mM mannitol removed) solution for 15 min in the absence and presence of 100 nM wortmannin, and then PI 3-K activity was determined in an immune complex assay using phosphatidylinositol as substrate (see METHODS). As shown in Fig. 3, hyposmolality increased PI 3-K activity 2.2-fold (P < 0.05). This increase was blocked by pretreatment with wortmannin. In control solution, wortmannin decreased basal PI 3-K activity by 40% (cont vs. wort, Fig. 3). These results support the view that hyposmolality increases HCO3- absorption through stimulation of PI 3-K activity.


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Fig. 3.   Hyposmolality increases PI 3-K activity in inner stripe of outer medulla. A: inner stripe tissue was incubated for 1 h in control solution in the absence (Cont) and presence (Wort) of 100 nM wortmannin. The tissue was then either maintained in control solution or exposed to hyposmotic solution (50 mM mannitol removed) for 15 min in the absence (Hypo) or the continued presence (Wort + Hypo) of the inhibitor. Cell lysates were immunoprecipitated with antibody to the p85alpha subunit of PI 3-K, and PI 3-K activity was determined in a lipid kinase assay using phosphatidylinositol as substrate (see METHODS). The phosphorylated substrate (PI-3-P) was resolved by thin layer chromatography and detected by autoradiography. Autoradiogram shows 1 of 3 independent experiments. B: phosphorylated substrate was quantified by densitometry and PI 3-K activity presented as % of control activity measured in the absence of wortmannin (Cont). Data are means ± SE for 3 separate experiments. *P < 0.05 vs. Cont; #P < 0.05 vs. Hypo (ANOVA).

Inhibitors of PI 3-K decrease basal HCO3- absorption. The experiments in Fig. 3 show that the MTAL expresses constitutive PI 3-K activity that is inhibited by wortmannin. We therefore tested whether this constitutive activity influences the basal rate of HCO3- absorption. In tubules studied in control (isosmotic) solution, addition of wortmannin or LY-294002 to the bath decreased HCO3- absorption from 11.8 ± 0.8 to 10.0 ± 1.0 pmol · min-1 · mm-1 (n = 6; P < 0.001) (Fig. 4). This decrease was observed within 15 min after addition of the inhibitors and was stable for up to 60 min. Thus PI 3-K activity is a determinant of the basal rate of HCO3- absorption.


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Fig. 4.   Inhibitors of PI 3-K decrease basal HCO3- absorption. MTAL were studied in control solution, and then either 100 nM wortmannin () or 20 µM LY-294002 (open circle ) was added to the bath solution. JHCO3-, data points, lines, and P value are as in Fig. 1. Mean values are given in RESULTS.

Inhibitors of PI 3-K do not block inhibition of HCO3- absorption by hyperosmolality. In contrast to the stimulation by hyposmolality, hyperosmolality inhibits HCO3- absorption in the MTAL through inhibition of apical membrane Na+/H+ exchange (15, 52). The results in Fig. 5 show that in tubules bathed with wortmannin or LY-294002, increasing osmolality in the lumen and bath by the addition of 50 mM mannitol or 75 mM NaCl decreased HCO3- absorption by 46%, from 10.9 ± 1.0 to 5.8 ± 0.8 pmol · min-1 · mm-1 (n = 8; P < 0.001). This decrease is similar to that observed previously in the absence of the inhibitors (15, 51). Thus PI 3-K is not involved in the inhibition of HCO3- absorption by hyperosmolality.


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Fig. 5.   Inhibitors of PI 3-K do not block inhibition of HCO3- absorption by hyperosmolality. MTAL were bathed with 100 nM wortmannin () or 20 µM LY-294002 (open circle ) throughout the experiments. With each inhibitor, hyperosmolality was produced in lumen and bath by the addition of 50 mM mannitol in 2 experiments and by the addition of 75 mM NaCl in 2 experiments. JHCO3-, data points, lines, and P value are as in Fig. 1. Mean values are given in RESULTS.

Role of Protein Kinase C in Hyposmotic Stimulation of HCO3- Absorption

Protein kinase C (PKC) is a downstream target of PI 3-K products (48), and we have shown that PKC can mediate stimulation of HCO3- absorption in the MTAL under certain conditions (16, 17). The role of PKC in mediating the stimulation of HCO3- absorption by hyposmolality was examined using staurosporine and chelerythrine Cl, inhibitors that selectively abolish PKC-dependent regulation of HCO3- absorption in the MTAL (3, 16, 17). In tubules bathed with 10-7 M staurosporine or 10-7 M chelerythrine Cl, removal of 50 mM mannitol from the lumen and bath increased HCO3- absorption from 8.2 ± 0.6 to 12.2 ± 0.7 pmol · min-1 · mm-1 (n = 4; P < 0.005) (Fig. 6). Thus the stimulation of HCO3- absorption by hyposmolality does not involve PKC.


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Fig. 6.   Inhibitors of protein kinase C do not prevent hyposmotic stimulation of HCO3- absorption. MTAL were bathed with 10-7 M staurosporine () or 10-7 M chelerythrine Cl (open circle ) and then the lumen and bath solutions were made hyposmotic by removal of 50 mM mannitol. JHCO3-, data points, lines, and P value are as in Fig. 1. Mean values are given in RESULTS.

Interaction Between Hyposmolality and Vasopressin

AVP blocks stimulation of HCO3- absorption by hyposmolality. We showed previously that AVP inhibits HCO3- absorption in the MTAL (13); thus, in principle, the stimulation of HCO3- absorption by hyposmolality could counteract the inhibitory effect of AVP. To examine the interaction between hyposmolality and AVP in the regulation of HCO3- absorption, we tested the effect of hyposmolality in the presence of AVP. The results in Fig. 7A show that in tubules bathed with 10-10 M AVP, hyposmolality had no effect on HCO3- absorption (6.3 ± 0.5 pmol · min-1 · mm-1, AVP vs. 6.4 ± 0.5 pmol · min-1 · mm-1, AVP + hyposmolality, n = 4 ; P = not significant). Thus the stimulation of HCO3- absorption by hyposmolality is inhibited by AVP.


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Fig. 7.   Arginine vasopressin (AVP) blocks stimulation of HCO3- absorption by hyposmolality. MTAL were bathed with 10-10 M AVP in the absence (A) and presence (B) of lumen furosemide (10-4 M; Furos), and then hyposmolality was produced in lumen and bath by removal of 50 mM mannitol. JHCO3-, data points, lines, and P values are as in Fig. 1. Mean values are given in RESULTS.

In the MTAL, AVP increases apical salt entry via Na+-K+-2Cl- cotransport, resulting in the activation of volume regulatory mechanisms to minimize cell swelling (46). Thus one possible explanation for the effect of AVP to block hyposmotic stimulation of HCO3- absorption is that regulatory pathways responsive to cell swelling are already activated as the result of AVP-induced salt uptake, thereby precluding further activation by hyposmolality. To test this possibility, we examined the effect of hyposmolality in the presence of AVP in tubules perfused with furosemide to block Na+-K+-2Cl- cotransport-mediated salt uptake. The results in Fig. 7B show that in tubules studied with 10-10 M AVP in the bath and 10-4 M furosemide in the lumen, hyposmolality had no effect on HCO3- absorption (7.3 ± 0.8 pmol · min-1 · mm-1, AVP + furosemide, vs. 7.5 ± 0.7 pmol · min-1 · mm-1, AVP + furosemide + hyposmolality, n = 3; P = not significant). Thus the effect of AVP to block hyposmotic stimulation of HCO3- absorption occurs independent of effects on apical Na+-K+-2Cl- uptake and net NaCl absorption.

To examine further the possibility that AVP blocked hyposmotic stimulation of HCO3- absorption through an effect on cell volume, steady-state cell volume was determined under the same experimental conditions used in the HCO3- transport experiments (Figs. 1 and 7A). In the absence of AVP, removal of 50 mM mannitol from the lumen and bath increase cell volume from 0.30 ± 0.03 to 0.34 ± 0.03 nl/mm (n = 6; P < 0.001). In the presence of AVP, removal of 50 mM mannitol increased cell volume from 0.29 ± 0.04 to 0.33 ± 0.04 nl/mm (n = 4; P < 0.001). In both conditions, the hyposmolality-induced increase in cell volume was stable for up to 50 min, was reversible, and was the result of a decrease in the tubule inner (lumen) diameter. Thus the effect of AVP to block hyposmotic stimulation of HCO3- absorption is not mediated through an effect on cell volume. In similar experiments, wortmannin also had no effect on cell volume in control or hyposmotic solutions (data not shown).

cAMP blocks stimulation of HCO3- absorption by hyposmolality. AVP inhibits HCO3- absorption in the MTAL by increasing cAMP (13, 14). To determine whether cAMP mediates the effect of AVP to block stimulation by hyposmolality, tubules were bathed with forskolin or 8-BrcAMP, agents that induce maximal cAMP-dependent inhibition of HCO3- absorption (13). The results in Fig. 8 show that in the presence of either 10-6 M forskolin or 10-4 M 8-BrcAMP, hyposmolality had no effect on HCO3- absorption (8.0 ± 0.7, agent vs. 8.0 ± 0.7, agent + hyposmolality, n = 8; P = not significant). Thus agents that elevate cAMP prevent stimulation by hyposmolality. These results support the view that the effect of AVP to inhibit stimulation of HCO3- absorption by hyposmolality is mediated through cAMP.


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Fig. 8.   cAMP blocks stimulation of HCO3- absorption by hyposmolality. MTAL were bathed with 10-6 M forskolin () or 10-4 M 8-bromo-cAMP (8-BrcAMP; open circle ) and then hyposmolality was produced in lumen and bath by the removal of 50 mM mannitol. JHCO3-, data points, lines, and P value are as in Fig. 1. Mean values are given in RESULTS.

cAMP blocks stimulation of PI 3-K activity by hyposmolality. To investigate the mechanism by which AVP and cAMP block hyposmotic stimulation of HCO3- absorption, we examined the effect of cAMP on the PI 3-K signaling pathway. Inner stripe tissue was incubated in control or hyposmotic (50 mM mannitol removed) solution for 15 min in the absence and presence of 10-4 M 8-BrcAMP, and then PI 3-K activity was determined in an immune complex assay as described in METHODS. As shown in Fig. 9, 8-BrcAMP blocked completely the stimulation of PI 3-K activity by hyposmolality. 8-BrcAMP had no effect on basal PI 3-K activity (control vs. 8-BrcAMP, Fig. 9). These results support the conclusion that cAMP blocks hyposmotic stimulation of HCO3- absorption by inhibiting the hyposmolality-induced increase in PI 3-K activity.


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Fig. 9.   cAMP blocks stimulation of PI 3-K activity by hyposmolality. A: tissue from inner stripe of outer medulla was incubated for 1 h in control solution in the absence (Cont) and presence (8-BrcAMP) of 10-4 M 8-BrcAMP. The tissue was then maintained in control solution or exposed to hyposmotic solution (50 mM mannitol removed) for an additional 15 min in the absence (Hypo) or the continued presence (8-BrcAMP + Hypo) of 8-BrcAMP. PI 3-K activity was determined in a lipid kinase assay using phosphatidylinositol as substrate as described under METHODS. Phosphorylated substrate (PI-3-P) was resolved by thin layer chromatography and detected by autoradiography. Autoradiogram shows 1 of 4 independent experiments. B: phosphorylated substrate was quantified by densitometry and PI 3-K activity presented as % of control activity measured in the absence of 8-BrcAMP (Cont). Data are means ± SE of 4 separate experiments. *P < 0.05 vs. Cont; #P < 0.05 vs. Hypo (ANOVA).


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DISCUSSION
REFERENCES

The molecular mechanisms that mediate osmotic regulation of Na+/H+ exchange activity are not understood. Recently we demonstrated that hyposmolality increases HCO3- absorption in the MTAL through stimulation of the apical membrane Na+/H+ exchanger NHE3 (54). The present study demonstrates that this stimulation is mediated through activation of PI 3-K. We also show that the hyposmotic stimulation of HCO3- absorption is blocked by vasopressin and cAMP because of an effect of cAMP to inhibit hyposmolality-induced stimulation of PI 3-K activity. In contrast, inhibition of apical Na+/H+ exchange and HCO3- absorption by hyperosmolality does not involve PI 3-K and is not blocked by vasopressin (14, 15), indicating that hyposmolality and hyperosmolality regulate NHE3 activity in the MTAL through different signaling mechanisms. PI 3-K pathways are involved in the regulation of a number of cellular processes, including growth and survival, oncogenic transformation, vesicle trafficking, and glucose transport and metabolism (40, 48). Our studies establish a role for PI 3-K in the osmotic regulation of Na+/H+ exchange activity and identify an important interaction between the PI 3-K and cAMP signaling pathways in the control of renal epithelial transport.

Hyposmolality Stimulates NHE3 and HCO3- Absorption Through Activation of PI 3-K

Hyposmolality stimulates apical NHE3 in the MTAL, resulting in an increase in transepithelial HCO3- absorption (54). The stimulation of NHE3 occurs in the absence of a change in the driving force for the exchanger and is due to an increase in Vmax (54). The present study indicates that the hyposmotic stimulation of NHE3 is mediated through activation of PI 3-K. This conclusion is supported by several observations: 1) hyposmotic stimulation of HCO3- absorption was abolished by wortmannin and LY-294002, two structurally unrelated PI 3-K inhibitors with different mechanisms of action (37, 49, 56), 2) hyposmolality increased PI 3-K activity under conditions similar to those used in the HCO3- transport experiments, and 3) wortmannin blocked the hyposmolality-induced increase in PI 3-K activity. At the concentrations used in our study, wortmannin (100 nM) and LY-294002 (20 µM) are highly selective inhibitors of PI 3-K, with no significant action against a wide variety of protein serine-threonine kinases, protein tyrosine kinases, other lipid kinases, and ATPases (37, 49, 56). LY-294002 also does not inhibit phosphatidylinositol 4-kinase (49). The effects of the inhibitors to block hyposmotic stimulation of HCO3- absorption are not due to nonspecific metabolic or cytotoxic effects, because these compounds have no effect on the regulation of HCO3- absorption by hyperosmolality (Fig. 5) or AVP.1 In addition, we show that the effect of cAMP to block hyposmotic stimulation of HCO3- absorption is correlated directly with inhibition of PI 3-K activation (Figs. 8 and 9). Thus two different maneuvers (PI 3-K inhibitors and cAMP) that block hyposmotic stimulation of PI 3-K also block stimulation of HCO3- absorption. Taken together, these findings indicate that activation of PI 3-K is a critical component of the signaling pathway through which hyposmolality stimulates NHE3 and HCO3- absorption in the MTAL.

Recent studies indicate that NHE3 is present in intracellular vesicles and that its acute regulation involves the redistribution of transporters between the plasma membrane and one or more subapical vesicular compartments. In AP-1 cells expressing NHE3, NHE3 activity was localized both in the plasma membrane and in recycling endosomes (9). In human colon carcinoma (Caco-2) cells, inhibition of NHE3 activity by PKC was mediated in part by subcellular relocation of NHE3 from the apical membrane to subapical cytoplasmic compartments (21). Evidence for regulation of NHE3 through protein trafficking also has been presented in the renal proximal tubule. Immunocytochemical studies using NHE-specific antibodies showed that NHE3 was present in both the brush-border membrane and in subapical cytoplasmic vesicles in rat proximal tubule (5). Furthermore, the regulation of NHE3 activity in proximal tubule cells by a variety of factors, including parathyroid hormone (59), acute hypertension (58), metabolic acidosis (6), and endothelin (39), involves the redistribution of NHE3 protein between the apical membrane and intracellular vesicular compartments. Of particular relevance to the present study, PI 3-K has been shown to be involved in vesicular trafficking in both yeast and mammalian cells (40, 48) and recent studies implicate PI 3-K in the regulation of NHE3 recycling in transfected AP-1 cells (27). On the basis of these findings and the results of our current and previous (54) studies, we propose that hyposmolality stimulates HCO3- absorption in the MTAL via the following mechanism: hyposmotic cell swelling increases PI 3-K activity, which leads to the redistribution of NHE3 from subapical intracellular vesicles to the apical membrane. The recruitment of apical membrane transporters results in the increased apical NHE3 activity that mediates stimulation of HCO3- absorption. Our finding that hyposmolality increases NHE3 activity in the MTAL through an increase in Vmax is consistent with an increase in the number of membrane transporters through trafficking (54). In previous studies in which NHE3 was immunolocalized to the apical membrane of the MTAL (2, 5), an intracellular pool of NHE3 was not evident; however, there are reasons why NHE3 may not have been detected in subapical membrane compartments in the MTAL in these studies, including a lack of sufficient resolution and/or an inability of the antibodies to recognize NHE3 in the intracellular vesicles. Further studies are needed to define the contribution of PI 3-K-dependent trafficking to hyposmotic stimulation of NHE3 activity in the MTAL.

PKC has been identified as a downstream target of PI 3-K in a number of systems (40, 48) and recent studies indicate a role for PKC in regulating NHE3 trafficking in Caco-2 cells (21). Moreover, we found that PKC can mediate stimulation of HCO3- absorption in the MTAL under certain conditions (16, 17). These findings suggested that PKC may be a downstream mediator of the PI 3-K-dependent stimulation of NHE3 in the MTAL. We found, however, that inhibitors of PKC that abolish PKC-dependent stimulation of HCO3- absorption by other agonists (3, 16, 17) did not prevent stimulation by hyposmolality. Thus it is unlikely that PKC is involved in mediating the hyposmotic stimulation of NHE3. We also found that PI 3-K inhibitors (as well as AVP) blocked hyposmotic stimulation of HCO3- absorption but did not prevent cell swelling. This indicates that the mechanical stresses and/or structural changes associated with cell swelling are not in themselves sufficient to cause stimulation of NHE3 activity in the absence of a functioning PI 3-K signaling pathway. Our studies do not address whether cell swelling is necessary for PI 3-K activation. However, the observation in hepatocytes that cell swelling induced by either hyposmotic medium or increased amino acid uptake causes activation of PI 3-K (25) suggests that increased cell volume is the signal leading to kinase activation. Important goals for future work will be to define the mechanism(s) involved in hyposmotic activation of PI 3-K and the downstream effectors that mediate PI 3-K-dependent stimulation of NHE3 activity in the MTAL.

There is precedent for osmotic regulation of PI 3-K in other systems. In isolated hepatocytes and human intestine 407 cells, hyposmotic cell swelling induced a two- to threefold increase in PI 3-K activity (25, 47). Swelling-induced activation of PI 3-K has been shown to play a role in mediating increased glycogen synthesis in liver and skeletal muscle cells (25, 29) and in the regulation of Cl- channel activity and cell volume in intestine and hepatoma cells (11, 47). On the other hand, hyperosmotic stress decreased PI 3-K activity in a fibroblast cell line, leading to a decrease in the activity of the PI 3-K/Akt survival pathway and an increased susceptibility to agonist-induced apoptotic cell death (60). The results of the present study establish that PI 3-K is osmotically regulated in native renal tubule epithelial cells and demonstrate a role for this pathway in the control of MTAL ion transport. The identification of signaling pathways regulated in response to osmotic stress is of particular physiological relevance for cells of the renal medulla, which are routinely exposed to large and rapid changes in extracellular osmolality due to the normal function of the urinary concentrating mechanism (31). In this context, the role of PI 3-K as a component of signaling pathways that convey cell survival is noteworthy. Recent work suggests that osmotic stress and changes in cell volume modify cell growth and may be associated with the induction of programmed cell death in several cell types, including renal inner medullary collecting duct cells (8, 26, 41, 60). It is conceivable, therefore, that the effect of decreasing osmolality to stimulate PI 3-K activity in the MTAL may serve the dual function of regulating transepithelial ion transport and inducing cell survival signals that preserve the integrity of the MTAL epithelium in the constantly changing osmotic environment of the renal medulla. Further work is needed to explore this hypothesis.

Hyposmolality and Hyperosmolality Regulate NHE3 Activity Through Different Signaling Mechanisms

Hyperosmolality decreases HCO3- absorption in the MTAL through inhibition of apical membrane Na+/H+ exchange (NHE3) activity (15, 52). This inhibition is due predominantly to a decrease in the apparent affinity of the exchanger for intracellular H+ (52). In contrast, hyposmolality stimulates apical Na+/H+ exchange activity by increasing Vmax, with no effect on the apparent affinity for intracellular H+ (54). On the basis of these different kinetic mechanisms, we suggested that the contrary effects of hypo- and hyperosmolality on Na+/H+ exchange activity are not the simple result of opposite changes in a common regulatory mechanism but rather are mediated through the regulation of different signal transduction pathways. Two findings in the present study strongly support this view. First, inhibitors of PI 3-K abolished stimulation by hyposmolality but did not affect inhibition by hyperosmolality. Second, AVP and cAMP block stimulation by hyposmolality but have no effect on inhibition by hyperosmolality (14, 15). These findings indicate that hypo- and hyperosmolality regulate NHE3 activity in the MTAL through distinct signaling pathways that modify exchanger activity through different kinetic mechanisms: hyposmolality stimulates NHE3 by increasing Vmax via a PI 3-K-dependent pathway that is inhibited by AVP and cAMP; hyperosmolality inhibits NHE3 by decreasing its apparent affinity for H+ via a pathway that operates independently of both PI 3-K and cAMP. It is noteworthy that both hyposmotic stimulation and hyperosmotic inhibition of HCO3- absorption are blocked by inhibitors of tyrosine kinase pathways (15, 54). It is presently unknown whether hypo- and hyperosmolality regulate NHE3 through different tyrosine kinase pathways or through a common tyrosine kinase pathway that mediates both PI 3-K-dependent stimulation and PI 3-K-independent inhibition of NHE3 activity.

Role of PI 3-K in Basal HCO3- Absorption

Previous studies using cell lines transfected with NHE3 have reported differing results regarding the dependence of basal NHE3 activity on PI 3-K. In AP-1 cells, 100 nM wortmannin or 50 µM LY-294002 decreased basal NHE3 activity by 90% (27). In contrast, in Caco-2 cells, 100 nM wortmannin had no effect on basal NHE3 activity (23). The explanation for these differing results is unclear but may relate to differences in the basal rate of dynamic recycling of NHE3 in the two transfected cell lines (27). In the present study, we found that PI 3-K was constitutively active in the MTAL and that wortmannin and LY-294002 significantly decreased HCO3- absorption in control (isosmotic) solutions. These results indicate that the constitutive PI 3-K activity is a determinant of the basal rate of HCO3- absorption and support a role for PI 3-K in the control of apical NHE3 activity under isosmotic conditions. The decrease in HCO3- absorption induced by the PI 3-K inhibitors was relatively small (15%), however, suggesting that only a minor fraction of basal NHE3 activity is dependent on PI 3-K in native MTAL cells.

Vasopressin and cAMP Inhibit Stimulation by Hyposmolality

Decreases in plasma osmolality can be both the primary cause of, and the secondary consequence of, a change in the plasma vasopressin level (4, 31). Thus identification of the interacting effects of hyposmolality and vasopressin at the cellular level is important for understanding the pathophysiology of H2O balance. In the MTAL, vasopressin inhibits HCO3- absorption (13). We therefore tested the hypothesis that stimulation of HCO3- absorption by hyposmolality would antagonize the inhibition by AVP. Instead, we found that AVP blocks the hyposmotic stimulation. This suggests that a decrease in osmolality would be most effective at increasing HCO3- absorption in the MTAL when vasopressin levels are low. Such conditions usually are met during the normal regulation of H2O balance in vivo, where a decrease in plasma vasopressin results in decreased medullary interstitial osmolality and increased H2O excretion (4, 31). The effect of a decrease in osmolality to stimulate HCO3- absorption in the MTAL may be important physiologically for maintaining constant the HCO3- concentration and pH of the medullary interstitial fluid during changes in H2O balance (14, 54). In addition, as discussed previously, this effect may contribute to the urine-acidifying effects of diuretic drugs and to the increased urinary net acid excretion observed in response to hypotonic volume expansion (54). Moreover, the stimulation of NHE3 by hyposmolality in nephron segments such as the proximal tubule may play a role in other important pathophysiological processes, such as diuretic resistance and renal sodium retention in chronic edematous states (54). Finally, in a more general context, the effect of AVP to antagonize regulation by hyposmolality may be important for its role in volume and blood pressure regulation. In response to a decrease in the effective circulating volume, vasopressin secretion is markedly stimulated, resulting in renal H2O retention that aids in restoring blood pressure and the adequacy of the circulation. The vasopressin-dependent H2O retention that protects the circulation has the secondary consequence of causing plasma hyposmolality (4). If this secondary hyposmolality resulted in the production of intracellular signals that opposed vasopressin action, then the vital role of vasopressin in defense of the effective circulating volume would be compromised. On the basis of the findings of the present study, we suggest that the effect of AVP to inhibit hyposmolality-induced regulation may reflect an adaptation that permits vasopressin to carry out its volume regulatory function despite the development of secondary hyposmolality. As discussed below, this adaptive mechanism may involve an interaction at the cellular level between the vasopressin- and hyposmolality-induced signaling pathways.

Vasopressin inhibits the stimulation of HCO3- absorption by hyposmolality by increasing intracellular cAMP, which inhibits the hyposmolality-induced stimulation of PI 3-K activity. This conclusion is based on the following observations: 1) vasopressin increases cAMP production in the MTAL (34), 2) agents that increase cell cAMP reproduce the effect of vasopressin to block hyposmotic stimulation of HCO3- absorption, 3) stimulation of HCO3- absorption by hyposmolality requires the activation of PI 3-K, and 4) increased cAMP blocks the hyposmolality-induced stimulation of PI 3-K activity. These findings provide new evidence for an interaction between the cAMP and PI 3-K pathways in the control of renal epithelial transport and reveal a previously undescribed mechanism by which cAMP can influence Na+/H+ exchange activity, namely, through modulation of PI 3-K-dependent regulation. An effect of cAMP to inhibit agonist-induced activation of PI 3-K has been reported previously in a number of nonepithelial systems (1, 33, 36, 42). The mechanism of this interaction has not been defined. Of relevance to the present study, cAMP was found to inhibit insulin-induced stimulation of glucose transport in adipocytes by inhibiting PI 3-K-mediated translocation of the glucose transporter GLUT-4 to the plasma membrane (36). By analogy, it is plausible that cAMP may inhibit hyposmolality-induced stimulation of HCO3- absorption in the MTAL by inhibiting PI 3-K-mediated translocation of NHE3 to the apical membrane. The identification of the cAMP effect is important not only to understand the interactions between signal transduction pathways that control ion transport in the MTAL but also to provide insight into a new mechanism by which vasopressin and cAMP can influence other processes in renal cells that may be regulated through PI 3-K, such as growth and survival and responses to osmotic stress. Further work is needed to define the physiological roles of PI 3-K signaling pathways in the regulation of ion transporters and other cellular processes in the MTAL and to define the modulation of these processes by cAMP.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38217.


    FOOTNOTES

Address for reprint requests and other correspondence: D. W. Good, 4.200 John Sealy Annex 0562, Univ. of Texas Medical Branch, 301 Univ. Boulevard, Galveston, TX 77555.

1  In a recent study using A6 cells, a toad kidney-derived cell line, wortmannin blocked vasopressin stimulation of protein kinase A activity and Na+ transport (10). In contrast, in the MTAL we found no effect of wortmannin on AVP-induced inhibition of HCO3- absorption, a cAMP-mediated process (Good, unpublished results). Thus it is unlikely that PI 3-K is involved in mediating stimulation of cAMP production and activation of protein kinase A by AVP in this nephron segment.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 March 2000; accepted in final form 12 June 2000.


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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 279(5):C1443-C1454
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