Ofelia S. Ruiz

Angiotensin II Stimulation of Renal Epithelial Cell Na/HCO3 Cotransport Activity: A Central Role for Src Family Kinase/Classic MAPK Pathway Coupling

Angiotensin II (AII) plays an important role in renal proximal tubular acidification via the costimulation of basolateral Na/HCO3 cotransporter (NBC) and apical Na/H exchanger (NHE) activities. These effects are mediated by specific G protein-coupled AII receptors, but their corresponding downstream effectors are incompletely defined. Src family tyrosine kinases (SFKs) contribute to the regulation of both transport activities by a variety of stimuli and are coupled to classic mitogen-activated protein kinase (MAPK) pathway activation in this cell type. We therefore examined these signaling intermediates for involvement in AII-stimulated NBC activity in cultured proximal tubule cells. Subpressor concentrations of AII (0.1 nM) increased NBC activity within minutes, and this effect was abrogated by selective antagonism of AT1 angiotensin receptors, SFKs, or the classic MAPK pathway. AII directly activated Src, as well as the proximal (Raf) and distal (ERK) elements of the classic MAPK module, and the activation of Src was prevented by AT1 receptor antagonism. An associated increase in basolateral membrane NBC1 content is compatible with the involvement of this proximal tubule isoform in these changes. We conclude that AII stimulation of the AT1 receptor increases NBC activity via sequential activation of SFKs and the classic MAPK pathway. Similar requirements for SFK/MAPK coupling in both cholinergic and acidotic costimulation of NBC and NHE activities suggest a central role for these effectors in the coordinated regulation of epithelial transport by diverse stimuli.

Regulation of the renal Na-HCO3 cotransporter : IX. Modulation by insulin, epidermal growth factor and carbachol

To examine the role of tyrosine kinase (TK) on basolateral membrane (BLM) transport, we looked for the presence of TK activity in these membranes and showed that the synthetic substrate for TK, poly [Glu80 Na, Tyr20] caused a three-fold increase in tyrosine phosphorylation. This effect was completely blocked by the TK inhibitors, 2-hydroxy-5(2,5-dihydroxybenzyl) aminobenzoic acid (HAC), 1 microM, and methyl 2,5-dihydroxycinnamate (DHC), 5 microM. We then examined the effect of agents that cause TK stimulation on tyrosine kinase immunocontent and on the Na-HCO3 cotransporter activity in BLM and in primary cultures of the proximal tubule. We utilized the cholinergic agent, carbachol (10(-4) M), epidermal growth factor (EGF 10(-8) M), and insulin (10(-8) M), well known activators of TK. Carbachol, insulin, and EGF caused a significant increase in TK immunoreactive protein content which was blocked by HAC and DHC. In BLM, carbachol significantly stimulated HCO3-dependent 22Na uptake and this effect was totally prevented by the monoclonal antibody against TK. In cultured proximal tubule cells, carbachol, EGF and insulin at physiologic concentration caused a significant stimulation of the cotransporter activity and this effect was completely blocked by the TK inhibitor, HAC. Increasing the dose of insulin 100-fold did not cause further stimulation of the cotransporter indicating that insulin plays a permissive role on the cotransporter. These results demonstrate the presence of TK in renal proximal tubule cells and show that activation of this kinase by dissimilar agents enhance the activity of the Na-HCO3 cotransporter.

Glucocorticoids and the renal Na-H antiporter: role in respiratory acidosis.

We examined the role of glucocorticoids in the activity of the renal brush border Na-H antiporter under baseline conditions (5% CO2 gassing) and during respiratory acidosis (10% CO2 gassing) in cultured monolayers of a proximal tubule suspension (primary cultures of the proximal tubule). Primary cultures of the proximal tubule showed an adaptive increase in renal brush border Na-H antiporter activity in response to respiratory acidosis in presence but not in the absence of physiologic concentrations of hydrocortisone in the medium. The effect of hydrocortisone to increase the activity of the renal brush border Na-H antiporter in respiratory acidosis could also be elicited by dexamethasone. Deletion of hydrocortisone from the medium also impaired the baseline activity of the Na-H antiporter. The effect of hydrocortisone to increase the activity of the Na-H antiporter under baseline conditions and during respiratory acidosis was elicited by physiologic concentrations of the hormone and 100-fold increase in concentration did not further increase the activity of the Na-H antiporter. These results demonstrate that the presence of physiologic concentrations of glucocorticoids are necessary for the baseline activity of the renal brush border Na-H antiporter and its adaptive increase in response to respiratory acidosis.

Dual Effect of Cyclic GMP on Renal Brush Border Na-H Antiporter

Abstract. The Na-H antiporter of renal-brush border membranes is inhibited by cyclic AMP and stimulated by protein kinase C. The proximal tubule contains guanylate cyclase and is capable of cyclic GMP production. The effect of cGMP on renal Na-H antiporter activity was analyzed in phosphorylated brush border membranes by 22Na uptake in the presence or absence of 1 mM amiloride. 8-Bromo cyclic GMP (1 μM) increased the amiloride-sensitive 22Na uptake in control from 1.26 ± 0.13 to 1.54 ± 0.12 nmol/mg/protein/10 sec, P < 0.01, without altering the amiloride-insensitive component. In the absence of exogenous ATP, cGMP also stimulated the amiloride-sensitive 22Na uptake, which can be explained by the presence of endogenous ATP in concentrations of up to 50 μM in the membranes. In ATP-depleted membrane vesicles, however, cGMP inhibited the amiloride-sensitive 22Na uptake. These data indicate that cGMP acts on the Na-H antiporter by at least two different mechanisms, one of which is ATP dependent. It is likely that cGMP-dependent protein kinase mediates the stimulatory effects seen in the presence of ATP, and the inhibition seen in ATP-depleted membranes results from cGMP direct action on the Na-H antiporter.

Regulation of Renal Na-HCO3 Cotransporter: VIII. Mechanism of Stimulatory Effect of Respiratory Acidosis

Abstract. We examined the effect of respiratory acidosis on the Na-HCO3 cotransporter activity in primary cultures of the proximal tubule of the rabbit exposed to 10% CO2 for 5 min, 2, 4, 24 and 48 hr. Cells exposed to 10% CO2 showed a significant increase in Na-HCO3 cotransporter activity (expressed as % of control levels, 5 min: 142 ± 6, 2 hr: 144 ± 13, 4 hr: 145 ± 11, 24 hr: 150 ± 15, 48 hr: 162 ± 24). The increase in activity was reversible after 48 hr. The role of protein kinase C (PKC) on the stimulatory effect of respiratory acidosis on the cotransporter was examined in presence of PKC inhibitor calphostin C or in presence of PKC depletion. Both calphostin C and PKC depletion prevented the effect of 10% CO2 for 5 min or 4 hr to increase the activity of the cotransporter. 10% CO2 for 5 min or 4 hr increased total and particulate fraction PKC activity. To examine the role of phosphotyrosine kinase (PTK) on the increase in cotransporter activity we studied the effect of two different inhibitors, 2-hydroxy-5-(2,5-dihydroxylbenzyl) aminobenzoic acid (HAC) and methyl 2,5-dihydroxycinnamate (DHC) which inhibit phosphotyrosine kinase in basolateral membranes. Cells were pretreated either with vehicle or HAC or DHC and then exposed to 10% CO2 for 5 min or 4 hr. In cells treated with vehicle, 10% CO2 significantly increased cotransporter activity as compared to control cells exposed to 5% CO2. This stimulation by 10% CO2 was completely prevented by HAC or DHC at 5 min (5% CO2: 1.8 ± 0.2, 10% CO2: 2.6 ± 0.2, 10% CO2+ HAC: 1.6 ± 0.2, 10% CO2: +DHC: 2.0 ± 0.3 pH unit/min) and also at 4 hr. The protein synthesis inhibitors actinomycin D and cycloheximide appear to prevent the effect of 10% CO2 for 4 hr on the cotransporter. Our results show that early respiratory acidosis stimulates the Na-HCO3 cotransporter through PKC and PTK-dependent mechanisms and the late effect appears to be mediated through protein synthesis.

Renal cortical basolateral Na+/HCO3- cotransporter. I: Partial purification and reconstitution

The renal basolateral Na+/HCO3−cotransporter is the main system responsible for HCO3−transport from proximal tubule cells into the blood. The present study was aimed at purifying and functionally reconstituting the Na+/HCO3−cotransporter protein from rabbit renal cortex. Highly purified rabbit renal cortical basolateral membrane vesicles (hereafter designated as original basolateral membrane), enriched 12-fold in Na-K-ATPase, were solubilized in 2% octylglucoside, and then reconstituted in l-α-phosphatidylcholine (proteoliposomes). Na+/HCO3−cotransporter activity was assessed as the difference in 22Na uptake in the presence of HCO3−and gluconate. The activity of the Na+/HCO3−cotransporter was enhanced 18-fold in the solubilized protein reconstituted into proteoliposomes compared to the original basolateral membranes. The reconstituted solubilized purified protein exhibited kinetic properties similar to the cotransporter from original basolateral membranes. In addition, it was like the original cotransporter, inhibited by disulfonic stilbene SITS, and was eleetrogenic. The catalytic subunit of protein kinase A significantly inhibited Na+/HCO3−cotransporter activity in proteoliposomes. The octylglucoside-solubilized protein was further purified by hydroxylapatite column chromatography, and this resulted in an additional enhancement of Na+/HCO3−cotransporter activity of 80-fold over the original basolateral membranes. The fractions containing the highest activity were further processed by glycerol gradient centrifugation, resulting in a 124- to 300-fold increase in Na+/HCO3−cotransporter activity compared to the original basolateral membranes. SDS-PAGE analysis showed an enhancement of a protein doublet of 56 kD MW in the glycerol gradient fraction. Our results demonstrate that we have partially purified and reconstituted the renal Na+/HCO3−cotransporter and suggest that the 56 kD doublet protein may represent the Na+/HCO3−cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).

The role of phosphatidylinositol 3-kinase (PI3K) in CO2 stimulation of the Na+/HCO3- cotransporter (NBC).

The basolateral Na+/HCO3- cotransporter (NBC) is the major pathway for bicarbonate reabsorption in the renal proximal tubule cells. The cotransporter activity is enhanced by 10% CO2. Phosphatidylinositol 3-kinase (PI3K) has been shown to regulate the function and trafficking of cellular proteins by promoting their translocation to the plasma membrane. Therefore, we sought to examine the role of PI3K in CO2-mediated stimulation of NBC activity in OK cells. Our studies showed that wortmannin, a well-characterized PI3K inhibitor, had no effect on baseline NBC activity but prevented the stimulatory effect of 10% CO2. This effect was concentration-dependent and time-dependent. Another inhibitor of PI3K, LY294002, also prevented the CO2-mediated increase in NBC activity. CO2 stimulation of the cotransporter was paralleled by an increase in PI3K enzyme activity and this effect was blocked by wortmannin. Biotinylation studies also showed that 10% CO2 increased the immunoreactive NBC in the basolateral membranes and this was prevented by wortmannin. We previously showed that 10% CO2 stimulation of NBC activity involves the Src family kinase pathway. In the current studies, CO2 stimulation significantly increased Src phosphorylation and this effect was abrogated by wortmannin. In summary, CO2 stimulation of NBC is mediated at least in part by increased immunoreactive NBC protein in the basolateral membrane, a process which requires the interaction of PI3K with Src family kinase.

Regulation of the Renal Na-HCO3 Cotransporter X. Role of Nitric Oxide and Intracellular Calcium

Cholinergic agents increase the activity of the renal Na-HCO3 cotransporter and have been shown to stimulate the production of nitric oxide (NO) in other cells. To study the role of NO in mediating the effect of carbachol on Na-HCO3 cotransporter, we measured the activity of the cotransporter in rabbit proximal tubule cells treated with carbachol (10–4 M) or the NO inhibitor, L-NAME (10–3M), or carbachol+L-NAME. The activity of NaHCO3 cotransporter was measured by recovery of intracellular pH (pHi) in cells loaded with pH-sensitive dye, BCECF. In control cells, carbachol significantly increased Na-HCO3 cotransporter activity while L-NAME did not affect the activity of the cotransporter but completely blocked the enhancement induced by carbachol. Carbachol increased NO production by proximal tubule cells. We also studied the effect of the NO donor, SNAP (10–3M), on the cotransporter incubated for 1 h in cultured proximal tubule cells. SNAP caused a similar enhancement in the activity of the cotransporter sug- gesting that a different NO donor is capable of enhancing the activity of the cotransporter to the same extent as that observed with carbachol. Because the effect of NO is thought to involve cGMP, we examined the effect of 8-Br-cGMP (10–3 M) on the cotransporter. 8-Br-cGMP caused stimulation of the Na-HCO3 cotransporter activity although to a lesser degree than carbachol. We have previously shown that carbachol increases cytosolic calcium but the role of intracellular calcium (Cai) per se on the cotransporter has not been studied. We therefore studied the role of Cai on the activity of Na-HCO3 cotransporter in rabbit proximal tubule cells by utilizing the calcium ionophore, ionomycin, the microsomal Ca-ATPase inhibitor, thapsigargin, and the calcium chelator, BAPTA. Ionomycin, 5 µM, caused a significant stimulation of Na-HCO3 cotransporter which was prevented by BAPTA. The microsomal Ca-ATPase inhibitor, thapsigargin, also increased the cotransporter activity. As expected both ionomycin and thapsigargin caused a significant increase in Cai. Calyculin A, an inhibitor of protein phosphatase 2A prevented the stimulation of the cotransporter by calcium (in pH units/min: control 1.8±0.13; Ca 2.22±0.07; p<0.05; Ca+calyculin A 1.9±0.09, p<0.025) suggesting that calcium acting through kinases/phosphatases, plays a role in the phosphorylation of the cotransporter. These results demonstrate that NO and Cai modulate the activity of the cotransporter.

Renal cortical basolateral Na+/HCO 3 − cotransporter III. Evidence for a regulatory protein in the inhibitory effect of protein kinase A

The activity of the Na-H antiporter is inhibited by cyclic AMP-dependent protein kinase A (cAMP.PKA). The inhibitory effect of PKA on the Na-H antiporter is mediated through a regulatory protein that can be dissociated from the antiporter by limited protein digestion. PKA also inhibits the activity of the Na+/ HCO3−cotransporter. We investigated whether the activity of Na+/HCO3−cotransporter and the effect of PKA on this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubilized BLM reconstituted in liposomes (proteoliposomes), trypsin (100 μg) increased 22Na uptake in the presence of HCO3 but not in the presence of gluconate, indicating that trypsin does not alter diffusive 22Na uptake but directly stimulates the Na+/HCO3−cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CSU) of cAMP-PKA decreased the activity of the Na+/HCO3−cotransporter (expressed as nanomoles/mg protein/3s) from 23 ± 10 to 14 ± 6 (P < 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAMP-PKA on the activity of Na+/HCO3−cotransporter was blunted. To identify a fraction that was responsible for the inhibitory effect of the CSU on the Na+/HCO3−cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP-PKA on the Na+/HCO3−cotransporter activity was assayed in proteoliposomes digested with trypsin with the addition of a fraction containing the 42 kDa protein (fraction S+) or without the 42 kDa protein (fraction S−). With the addition of fraction S−, the CSU of cAMP-PKA failed to inhibit the Na+/HCO3−cotransporter activity (control 27 ± 6, CSU 27 ± 3) while the addition of fraction S+ restored the inhibitory effect of CSU (27 ± 6 to 3 ± 0.3 P < 0.01). The CSU of cAMP-PKA phosphorylated several proteins in solubilized protein including a 42 kDa protein. Fluorescein isothiocyanate (FITC) labels components of the Na+/HCO3−cotransporter including the 56 kDa and 42 kDa proteins. In trypsin-treated solubilized protein the 42 kDa protein was not identified with FITC labeling. The results demonstrate that the activity of the Na+/HCO3−cotransporter is regulated by protein(s) which mediates the inhibitory effect of PKA. Limited protein digestion can dissociate this protein from the cotransporter.

Renal Cortical Basolateral Na+/HCO− 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

Abstract. We have previously partially purified the basolateral Na+/HCO−3 cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na+/HCO−3 cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na+/HCO−3 cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na+/HCO−3 cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na+/HCO−3 cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO3 or Na2CO3) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na+/HCO−3 cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na+/HCO−3 cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na+/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues.