Potassium depletion increases luminal Na+/H+ exchange and basolateral Na+:CO3=:HCO3- cotransport in rat renal cortex.

Most HCO3- reabsorption in proximal tubules occurs via electroneutral Na+/H+ exchange in brush border membranes (BBMS) and electrogenic Na+:CO3=:HCO3- cotransport in basolateral membranes (BLMS). Since potassium depletion (KD) increases HCO3- reabsorption in proximal tubules, we evaluated these transport systems using BBM and BLM vesicles, respectively, from control (C) and KD rats. Feeding rats a potassium deficient diet for 3-4 wk resulted in lower plasma [K+] (2.94 mEq/liter, KD vs. 4.47 C), and higher arterial pH (7.51 KD vs. 7.39 C). KD rats gained less weight than C but had higher renal cortical weight. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0, 10% CO2, 90% N2) into BLM vesicles was 44% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was DIDS sensitive, suggesting that Na+:CO3=:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.2 mM in KD vs. 7.6 mM in C and Vmax of 278 nmol/min/mg protein in KD vs. 177 nmol/min/mg protein in C. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0) into BBM vesicles was 34% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 6.2 mM in KD vs. 7.1 mM in C and Vmax of 209 nmol/min/mg protein in KD vs. 144 nmol/min/mg protein in C. Uptakes of Na(+)-dependent [3H]glucose into BBM and [14C]succinate into BLM vesicles were not different in KD and C groups, suggesting that the Na+/H+ exchanger and Na+:CO3=:HCO3- cotransporter activities were specifically altered in KD. We conclude that adaptive increases in basolateral Na+:CO3=:HCO3- cotransport and luminal Na+H+ exchange are likely responsible for increased HCO3- reabsorption in proximal tubules of KD animals.     

Effect of in vitro metabolic acidosis on luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport in rabbit kidney proximal tubules.

The aim of this study was to evaluate the role of the kidney in mediating the signals involved in adaptive changes in luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport systems in metabolic acidosis. Proximal tubular suspensions were prepared from rabbit kidney cortex and incubated in acidic (A) or control (C) media (pH 6.9 vs 7.4, 5% CO2) for 2 h. Brush border membrane (BBM) and basolateral membrane (BLM) vesicles were isolated from the tubular suspensions and studied for the activity of Na+/H+ exchange and Na+:HCO3- cotransport. Influx of 1 mM 22Na at 10 s (pH6 7.5, pH(i) 6.0) into BBM vesicles was 68% higher in group A compared to group C. The increment in Na+ influx in the group A was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.1 mM in C vs 9.2 in A and Vmax of 31 nmol/mg protein per min in group C vs 57 in A. Influx of 1 mM 22Na at 10 s (pH0 7.5, pH(i) 6.0, 20% CO2, 80% N2) into BLM vesicles was 83% higher in the group A compared to C. The HCO3-dependent increment in 22Na uptake in group A was 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid sensitive, suggesting that Na+:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 11.4 mM in C vs 13.6 in A and Vmax of 35 nmol/mg protein per min in C vs 64 in A. The presence of cyclohexamide during incubation in A medium had no effect on the increments in 22Na uptake in group A. We conclude that the adaptive increase in luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport systems in metabolic acidosis is acute and mediated via direct signal(s) at the level of renal tubule.     

Electroneutral K+/HCO3- cotransport in cells of medullary thick ascending limb of rat kidney.

The renal medullary thick ascending limb (MTAL) of the rat absorbs bicarbonate through luminal H+ secretion and basolateral HCO3- transport into the peritubular space. To characterize HCO3- transport, intracellular pH (pHi) was monitored by use of the pH-sensitive fluorescent probe (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein in fresh suspensions of rat MTAL tubules. When cells were preincubated in HCO3-/CO2-containing solutions and then abruptly diluted into HCO3-/CO2-free media, the pHi response was an initial alkalinization due to CO2 efflux, followed by an acidification (pHi recovery). The pHi recovery required intracellular HCO3-, was inhibited by 10(-4) M diisothiocyanostilbene-2-2'-disulphonic acid (DIDS), and was not dependent on Cl- or Na+. As assessed by use of the cell membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine, cell depolarization by abrupt Cl- removal from or addition of 2 mM barium into the external medium did not affect HCO3(-)-dependent pHi recovery, and the latter was not associated per se with any change in potential difference, which indicated that HCO3- transport was electroneutral. The HCO3(-)-dependent pHi recovery was inhibited by raising extracellular potassium concentration and by intracellular potassium depletion. Finally, as measured by use of a K(+)-selective extracellular electrode, a component of K+ efflux out of the cells was HCO3- dependent and DIDS sensitive. The results provide evidence for an electroneutral K+/HCO3- cotransport in rat MTAL cells.     

Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes. Evidence for Na+-HCO3- cotransport.

Using the pH-sensitive dye 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein and a continuously perfused subconfluent hepatocyte monolayer cell culture system, we studied rat hepatocyte intracellular pH (pHi) regulation in the presence (+HCO3-) and absence (-HCO3-) of bicarbonate. Baseline pHi was higher (7.28 +/- 09) in +HCO3- than in -HCO3- (7.16 +/- 0.14). Blocking Na+/H+ exchange with amiloride had no effect on pHi in +HCO3- but caused reversible 0.1-0.2-U acidification in -HCO3- or in +HCO3- after preincubation in the anion transport inhibitor 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). Acute Na+ replacement in +HCO3- alos caused acidification which was amiloride independent but DIDS inhibitible. The recovery of pHi from an intracellular acid load (maximum H+ efflux rate) was 50% higher in +HCO3- than in -HCO3-. Amiloride inhibited H+ effluxmax by 75% in -HCO3- but by only 27% in +HCO3-. The amiloride-independent pHi recovery in +HCO3- was inhibited 50-63% by DIDS and 79% by Na+ replacement but was unaffected by depletion of intracellular Cl-, suggesting that Cl-/HCO3- exchange is not involved. Depolarization of hepatocytes (raising external K+ from 5 to 25 mM) caused reversible 0.05-0.1-U alkalinization, which, however, was neither Na+ nor HCO3- dependent, nor DIDS inhibitible, findings consistent with electroneutral HCO3- transport. We conclude that Na+-HCO3- cotransport, in addition to Na+/H+ exchange, is an important regulator of pHi in rat hepatocytes.     

Stoichiometry of Na+-HCO-3 cotransport in basolateral membrane vesicles isolated from rabbit renal cortex.

The major pathway for HCO3- transport across the basolateral membrane of the proximal tubule cell is electrogenic Na+-HCO3- cotransport. In this study, we have determined the stoichiometry of the Na+-HCO3- cotransport system in basolateral membrane vesicles that were isolated from rabbit renal cortex by Percoll gradient centrifugation. When the membrane potential is approximated by the Nernst potential for K+, as in the presence of the K+ ionophore valinomycin, equilibrium thermodynamics predicts that the Na+-HCO3- cotransport system should come to equilibrium and mediate no net flux when (Na)i/(Na)o = [(HCO3)o/(HCO3)i]n[(K)o/(K)i]n-1, where n is the HCO3-:Na+ stoichiometry. Our experimental approach was to impose transmembrane Na+, HCO3-, and K+ gradients of varying magnitude and direction, and then to measure the net flux of Na+ over the subsequent 3-s period. In this way, we could determine the conditions for equilibrium of the transport system and thereby calculate n. The results of these experiments indicate that the value of n is greater than 2.6 and less than 3.5, consistent with a stoichiometry of 3 HCO3-:1 Na+, or a thermodynamically equivalent process. Based on reported intracellular potentials and ion activities, this value for the stoichiometry indicates that the inside-negative membrane potential is sufficient to drive HCO3- exit against the inward concentration gradients of HCO3- and Na+ that are present across the basolateral membrane of the intact proximal tubule cell under physiologic conditions.     

A pH modifier site regulates activity of the Na+:HCO3- cotransporter in basolateral membranes of kidney proximal tubules.

HCO3- exit across the basolateral membrane of the kidney proximal tubule cell is mediated via an electrogenic Na+:HCO3- cotransporter. In these experiments, we have studied the effect of internal pH on the activity of the Na+:HCO3- cotransport system in basolateral membrane vesicles isolated from rabbit renal cortex. Equilibrium thermodynamics predicts that in the presence of constant intravesicular concentration of Na+, an increasing concentration of HCO3- will be associated with an increasing driving force for Na+:HCO3- cotransport across the vesicles. Our experimental approach was to preequilibrate the membrane vesicles with 1 mM 22Na+ at pHi 6.8-8.0 and known concentrations of HCO3-. The vesicles were diluted 1:100 into Na(+)-free solution at pH 7.4 and the net flux of 22Na+ was assayed over 5 s. The results demonstrate that the net flux of Na+ was significantly higher at pHi 7.2 than pHi 8.0 despite much higher [HCO3-] at pHi 8.0. This suggests that an internal pH-sensitive site regulates the activity of the Na+:HCO3- cotransporter. This modifier site inhibits the cotransporter at alkaline pH despite significant base concentration and is maximally functional around physiologic pH. The combination of modifier sites on the luminal Na+/H+ exchanger and the basolateral Na+:HCO3- cotransporter should help maintain intracellular pH in a narrow range with changes in extracellular pH.     

Basolateral membrane Na+/H+ antiport, Na+/base cotransport, and Na+-independent Cl-/base exchange in the rabbit S3 proximal tubule.

The basolateral membrane Na+ and Cl(-)-dependent acid-base transport processes were studied in the isolated perfused rabbit S3 proximal straight tubule. Intracellular pH (pHi) was measured with 2'7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and a microfluorometer coupled to the tubule perfusion apparatus. Reduction of basolateral HCO3- from 25 to 5 mM caused pHi to decrease at a rate of 0.81 pH/min. Approximately 50% of this rate was Na+-dependent, 30% Cl(-)-dependent and 20% Na+ and Cl(-)-independent. Two basolateral Na+-dependent acid base transport pathways were detected: (a) an amiloride-sensitive Na+/H+ antiporter and (b) a stilbene-sensitive Na+/base cotransporter. No evidence was found for a Na+-dependent Cl-/base exchanger. The Cl(-)-dependent component of basolateral base efflux was mediated by a stilbene-sensitive Na+-independent Cl-/base exchange pathway. The results suggest that the acid base transport pathways of the basolateral membrane of the S3 proximal tubule differ from more proximal nephron segments.     

Microfilament-dependent activation of Na+/K+/2Cl- cotransport by cAMP in intestinal epithelial monolayers.

cAMP-mediated stimulation of Cl- secretion in the human intestinal cell line T84 is accompanied by significant remodeling of F-actin, and both the secretory and cytoskeletal responses may be largely ablated by previous cell loading with phalloidin derivatives, reagents that prevent dynamic reordering of microfilaments (1991. J. Clin. Invest. 87:1903-1909). In this study, we examined the effect of phalloidin loading on the cAMP-elicited activity of the individual membrane-associated transport proteins involved in electrogenic Cl- secretion. Efflux of 125I and 86Rb was used to assay forskolin-stimulated Cl- and K+ conductances, respectively, and no inhibitory effect of phalloidin could be detected. Na+/K(+)-ATPase pump activity, assessed as bumetanide-insensitive 86Rb uptake and the ability of monolayers to generate a Na+ absorptive current in response to apical addition of a Na+ ionophore, was not different between control and phalloidin-loaded monolayers. Forskolin was found to stimulate Na+/K+/2Cl- cotransport (bumetanide-sensitive 86Rb uptake) in time-dependent fashion. In the absence of any agonist, cotransporter activity was markedly decreased in phalloidin-loaded monolayers. Furthermore, under phalloidin-loaded conditions, the forskolin-elicited increase in bumetanide-sensitive 86Rb uptake was markedly attenuated. These findings suggest that cAMP-induced activity of Cl- channels, K+ channels, and the Na+/K(+)-ATPase are not influenced by F-actin stabilization. However, cAMP-induced activation of the Na+/K+/2Cl- cotransporter appears to be microfilament-dependent, and ablation of this event is likely to account for the inhibition of cAMP-elicited Cl- secretion seen in the phalloidin-loaded state. Such findings suggest that Na+/K+/2Cl- cotransporter is functionally linked to the cytoskeleton and is a regulated site of cAMP-elicited electrogenic Cl- secretion.     

Effects of acetazolamide on Na+-HCO-3 cotransport in basolateral membrane vesicles isolated from rabbit renal cortex.

We evaluated the effects of acetazolamide on Na+-HCO3- cotransport in basolateral membrane vesicles isolated from the rabbit renal cortex. Na+ uptake stimulated by an imposed inward HCO3- gradient was not significantly reduced by 1.2 mM acetazolamide, indicating that acetazolamide does not directly inhibit Na+-HCO3- cotransport. 4,4'-Diisothiocyanostilbene-2,2'-disulfonate (DIDS)-sensitive Na+-base cotransport was found to be absolutely CO2/HCO3--dependent. We therefore tested whether acetazolamide-sensitive availability of HCO3- at the basolateral membrane could be rate-limiting for Na+-base cotransport under some conditions. In the presence of a CO2/HCO3- buffer system but absence of an initial HCO3- gradient, Na+ influx was stimulated fivefold by an outward NH4+ gradient. This stimulation of Na+ influx by an outward NH4+ gradient was inhibited greater than 75% by 0.6 mM acetazolamide, suggesting that acetazolamide blocked the ability of the NH4+ gradient to generate an inward HCO3- gradient. In the presence of an inward HCO3- gradient, Na+ influx was inhibited greater than 70% by an inward NH4+ gradient. This inhibition of Na+ influx was reduced to only 35% by 0.6 mM acetazolamide, suggesting that acetazolamide blocked the ability of NH4+ to collapse the inward HCO3- gradient. Similarly, Na+ influx in the presence of an inward HCO3- gradient was inhibited greater than 80% by an outward acetate gradient, and this inhibition was reduced to only 50% by acetazolamide. Thus, acetazolamide caused either inhibition or stimulation of Na+ uptake depending on the conditions with respect to pH and HCO3- gradients. The indirect interaction of acetazolamide with the basolateral membrane Na+-HCO3- cotransport system may be an important mechanism underlying inhibition of proximal tubule acid secretion by this agent.     

Mineralocorticoids and acidosis regulate H+/HCO3- transport of intercalated cells.

The effects of acidosis and mineralocorticoids on cellular H+/HCO3- transport mechanisms were examined in intercalated cells of the outer stripe of outer medullary collecting duct (OMCDo) from rabbit. Intracellular pH (pHi) of intercalated cells was monitored by fluorescence ratio imaging using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). pHi recovered from an acid load at 2.8 +/- 0.5 x 10(-3) pHU/s in the absence of ambient Na+. This pHi recovery rate was similar in chronic acidosis induced by NH4Cl loading, but it was enhanced (+111%) by treatment with deoxycorticosterone acetate (DOCA). In a DOCA-treated group, luminal 10 microM SCH28080 and 0.1 mM omeprazole, H(+)-K(+)-ATPase inhibitors, did not change the pHi recovery rate, while luminal 0.5 mM N-ethylmaleimide blocked the rate by 68%. DOCA, but not acidosis, increased (approximately 40%) initial pHi response to bath HCO3- or Cl- reduction in Na(+)-free condition. After an acid load in the absence of Na+ and HCO3-, pHi response to basolateral Na+ addition was stimulated (+66%) by acidosis, but not by DOCA. Our results suggest that (a) mineralocorticoids stimulate H+/HCO3- transport mechanisms involved in transepithelial H+ secretion, i.e., a luminal NEM-sensitive H+ pump and basolateral Na(+)-independent Cl(-)-HCO3- exchange; and (b) acidosis enhances the activity of basolateral Na(+)-H+ exchange that may be responsible for pHi regulation.     

Specific inhibition of rat renal Na+/phosphate cotransport by picolinamide

Nicotinamide is both a precursor for NAD synthesis and an inhibitor of intracellular NAD hydrolysing enzymes. Overnight treatment of rats with nicotinamide causes dose-dependent inhibition of the Na+/phosphate cotransporter in the renal brush border membrane. Picolinamide is an isomer of nicotinamide that cannot be used for NAD synthesis. Picolinamide was used in the present study to explore the possibility that inhibition of Na+/phosphate cotransport may be related to inhibition of NAD hydrolyzing enzymes. Overnight treatment of rats with picolinamide, administered as a single injection (4 mmol/kg), inhibited Na+/phosphate cotransport by isolated renal brush border membrane vesicles. Like nicotinamide, the inhibition by picolinamide occurred in thyroparathyroidectomized rats, was specific for Na+/phosphate cotransport and was accompanied by a decrease in the apparent Vmax. In contrast to nicotinamide, there was only a small increase (1.5-fold) in renal cortical NAD content after picolinamide treatment. Direct incubation of isolated proximal tubules with thymidine, a potent inhibitor of NAD hydrolysis by intracellular enzymes, produced no change in Na+/phosphate cotransport. Thus, specific inhibition of renal Na+/phosphate cotrasport by picolinamide in vivo is unlikely due to inhibition of NAD hydrolyzing enzymes. It is suggested, based on data from in vitro studies, that a cyclic AMP-dependent mechanism may be involved.     

Mineralocorticoid modulation of apical and basolateral membrane H+/OH-/HCO3- transport processes in the rabbit inner stripe of outer medullary collecting duct.

To examine the mechanism by which mineralocorticoids regulate HCO3- absorption in the rabbit inner stripe of the outer medullary collecting duct, we microfluorometrically measured intracellular pH (pHi) in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) assaying the apical and basolateral membrane H+/OH-/HCO3- transport processes in three groups of animals: those receiving chronic in vivo DOCA treatment (5 mg/kg per d x 2 wk); those with surgical adrenalectomy (ADX, [chronic x 2 wk]) on glucocorticoid replacement; and controls. Baseline pHi was not different in the three groups. Cellular volume (vol/mm) was increased 38% in DOCA tubules versus controls, but unchanged in ADX tubules versus controls. Buffer capacities (BT) were not different in the three groups. Apical membrane H+ pump activity, assayed as the Na(+)-independent pHi recovery from an acid load (NH3/NH4+ prepulse) and expressed as JH (dpHi/dt.vol/mm.BT) was increased 76% in DOCA tubules versus controls, and decreased 56% in ADX tubules versus controls. Basolateral membrane Cl-/HCO3- exchange activity assayed as the pHi response to basolateral Cl- addition was increased 73% in DOCA tubules versus controls, and decreased 44% in ADX tubules versus controls. When examined as a function of varying [Cl-], the Vmax of Cl-/HCO3- exchange activity was significantly increased in DOCA tubules (control, 72.7 +/- 15.7 pmol.mm-1.min-1 vs DOCA, 132.3 +/- 22.5 pmol.mm-1.min-1, P less than 0.02), while the K1/2 for Cl- was unchanged. Basolateral membrane Na+/H+ antiporter activity assayed as the Na(+)-dependent pHi recovery from an acid load was not changed in chronic DOCA tubules versus controls. In conclusion, the apical membrane H+ pump and basolateral membrane Cl-/HCO3- exchanger of the rabbit OMCDi are regulated in parallel without chronic alterations in pHi under the conditions of mineralocorticoid excess and deficiency. The parallel changes in these transporters accounts for the alterations in OMCDi HCO3- absorption seen under these conditions.     

Na+/H+ and HCO3-/Cl- exchange in the control of intracellular pH in vivo in the spontaneously hypertensive rat.

1. We have previously shown that the cytosolic acid concentration changes in skeletal muscle during contraction in spontaneously hypertensive rats and normotensive Wistar-Kyoto rats in vivo. We have now found that this change was unaffected by 20% inhaled CO2 or by 4,4'-di-isothiocyanostilbene-2,2'-disulphonate. This is evidence that HCO3- exchange in vivo is not important in the control of cytosolic acid concentration during skeletal muscle contraction in either spontaneously hypertensive or Wistar-Kyoto rats. 2. We have also previously shown that the difference in cytosolic acid response during contraction between spontaneously hypertensive and Wistar-Kyoto rats is due to increased Na+/H+ antiporter activity in the spontaneously hypertensive rats. Our current findings suggest that this increase in Na+/H+ antiporter activity is more likely to be due to a change in the Km of the antiporter than to a change in the Vmax. We estimate that the Km of the antiporter changes in hypertension from pH 7.16 to 7.33. 3. We did not find any differences between adult spontaneously hypertensive and Wistar-Kyoto rats with regard to resting intracellular and extracellular pH and resting intracellular and extracellular HCO3- concentrations. In addition, we did not find any evidence of a difference in skeletal muscle HCO3-/Cl- exchange between adult spontaneously hypertensive and Wistar-Kyoto rats. 4. At rest, skeletal muscles of the spontaneously hypertensive and Wistar-Kyoto rats have the same lactate production, HCO3-/Cl- exchange and arterial partial pressure of CO2. In addition, we can also calculate that at a resting intracellular pH of 7.05 in the spontaneously hypertensive rats, the antiporter is 66% saturated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+/myo-inositol transport is regulated by basolateral tonicity in Madin-Darby canine kidney cells.

We investigated the effects of change in basolateral osmolality on Na(+)-dependent myo-inositol uptake in Madin-Darby canine kidney cells to test our hypothesis that the Na+/myo-inositol transporter (SMIT), an osmolyte transporter, is mainly regulated by osmolality on the basolateral surface. A significant osmotic gradient between both sides of the epithelium persisted at least 10 h after basolateral osmolality was increased. [3H]myo-inositol uptake increased in a basolateral osmolality-dependent manner. The magnitude of the increase is comparable to that for making both sides hypertonic. Apical hypertonicity also increased the uptake on the basal side, but the magnitude of the increase was significantly smaller than the basolateral or both sides hypertonicity. Betaine-gamma-amino-n-butyric acid transporter activity, measured by [3H]gamma-amino-n-butyric uptake, showed a pattern similar to SMIT activity in response to basolateral hypertonicity. The most plausible explanation for the polarized effect of hypertonicity is that the basal membrane is much more water permeable than the apical membrane. These results seem to be consistent with the localization and regulation of the SMIT in vivo.     

Chronic metabolic acidosis causes an adaptation in the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter in the rat proximal convoluted tubule.

The effect of chronic dietary acid on the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter was examined in the in vivo microperfused rat proximal tubule. Transporter activity was assayed with the epifluorescent measurement of cell pH using the intracellular, pH-sensitive fluorescent dye, (2'7')-bis(carboxyethyl)-(5,6)-carboxy-fluorescein (BCECF). BCECF was calibrated intracellularly, demonstrating similar pH-sensitivity of the dye in control and acidotic animals. In subsequent studies, lumen and peritubular capillaries were perfused to examine Na/H and Na(HCO3)3 transporter activity in the absence of contact with native fluid. The initial rate of change in cell pH (dpHi/dt) was 97, 50, and 44% faster in tubules from acidotic animals when peritubular [HCO3] was changed from 25 to 10 mM in the presence or absence of chloride, or peritubular [Na] was changed from 147 to 50 mM, respectively. dpHi/dt was 57% faster in tubules from acidotic animals when luminal [Na] was changed from 152 to 0 mM. Buffer capacities, measured using NH3/NH+4 addition, were similar in the two groups. The results demonstrate that chronic metabolic acidosis causes an adaptation in the intrinsic properties of both the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter.     

Electrogenic sodium/bicarbonate cotransport in rabbit renal cortical basolateral membrane vesicles.

The present studies examined the mechanism of bicarbonate transport across basolateral membrane vesicles prepared from rabbit renal cortex. Isotopic sodium uptake was stimulated by bicarbonate when compared with gluconate (2.5 nmol/mg protein per 5 s versus 1.4 nmol/mg protein per 5 s), and this process was inhibited by disulfonic stilbenes. Imposition of an interior-positive potassium diffusion potential further stimulated isotopic sodium uptake to 3.4 nmol/mg protein per 5 s, an effect that occurred only in the presence of bicarbonate and was blocked by disulfonic stilbenes. Kinetic analysis of the rate of bicarbonate-dependent sodium uptake as a function of sodium concentration revealed saturable stimulation with a Vmax of 2.7 nmol/mg protein per 2 s and a Km of 10.4 mM. The effect of bicarbonate concentration on bicarbonate-dependent sodium uptake was more complex. The present results demonstrate an electrogenic (negatively charged) sodium/bicarbonate cotransporter in basolateral membrane vesicles from the rabbit renal cortex. The electrogenicity implies a stoichiometry of at least two bicarbonate ions for each sodium ion.     

Regulation of rabbit medullary collecting duct cell pH by basolateral Na+/H+ and Cl-/base exchange.

The collecting duct of the inner stripe outer medulla (OMCDi) is a major site of distal nephron acidification. Using the pH sensitive fluorescent dye 2'-7'-bis(carboxyethyl)-5,6,-carboxyfluorescein (BCECF) and quantitative spectrofluorometry to measure intracellular pH in isolated perfused OMCDi, we have characterized basolateral transport processes responsible for regulation of intracellular pH. Experiments suggesting the existence of basolateral Cl-/base exchange were performed. In HCO3- containing buffers, bath Cl- replacement resulted in reversible alkalinization of the OMCDi from 7.22 +/- 0.05 to 7.57 +/- 0.12. Similarly 0.1 mM bath 4',4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) alkalinized the OMCDi from 7.14 +/- 0.09 to 7.34 +/- 0.09 and blocked further alkalinization by bath Cl- removal (delta = + 0.02 pH units). The concentration dependence kinetics of Cl-/base exchange revealed a K1/2 of 10 mM for external Cl- with a Vmax of 0.50 pH U/min. Experiments suggesting the existence of basolateral Na+/H+ exchange were also performed. Replacement of bath Na+ by tetramethylammonium resulted in reversible cell acidification (7.14 +/- 0.09 to 6.85 +/- 0.1). Tubules that were acidified by a brief exposure to NH4Cl displayed recovery of cell pH back to baseline at a rate that was highly dependent on bath Na+ concentration. Half maximal recovery rate was achieved at 7 mM bath Na+ and Vmax was 0.605 pH U/min. The Na+-dependent rate of cell pH recovery after acidification was blocked by 0.2 mM bath amiloride. These results suggest that intracellular pH in the OMCDi is regulated by parallel basolateral Na+/H+ exchange and Cl-/base exchange.     

Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange.

The cellular mechanisms responsible for abnormalities in spontaneously hypertensive rat (SHR) vascular smooth muscle cell (VSMC) growth and vasoreactivity are not defined. Because Na+/H+ exchange, which we have previously demonstrated in cultured VSMC, plays an essential role in mediating growth factor responses, we hypothesized that abnormalities in SHR growth regulation might be reflected in the activity of this transporter. To test this hypothesis, we studied DNA synthesis and Na+/H+ exchange (measured as the rate of amiloride-sensitive intracellular alkalinization or Na+ influx) in early subcultures (less than 6) of aortic VSMC from 12-wk-old SHR and Wistar Kyoto (WKY) animals. Serum-deprived SHR VSMC grew more rapidly in response to 10% serum with an increase in [3H]thymidine incorporation of 439% compared with 191% in WKY controls. Basal intracellular pH (pHi) values determined by fluorescent pH measurements were 7.37 +/- 0.04 and 7.27 +/- 0.03 (P less than 0.05) in early passage SHR and WKY, respectively. Acid recovery (initial pHi = 6.8) by SHR VSMC was faster than by WKY VSMC as measured by alkalinization (1.8 +/- 0.6 vs. 0.8 +/- 0.2 mmol H+/liter.min, P less than 0.05) or by amiloride-sensitive 22Na+ influx (14.5 +/- 1.2 vs. 4.0 +/- 0.5 nmol Na+/mg protein.min, P less than 0.05). In comparison to WKY cells early passage SHR VSMC exhibited 2.5-fold greater alkalinization and amiloride-sensitive 22Na+ influx in response to 100 nM angiotensin II. During serial passage, WKY cells acquired enhanced Na+/H+ exchange and growth rates so that by passage 6, these differences were no longer present. These findings in early cultures of SHR VSMC, removed from the in vivo neurohumoral milieu, suggest that increased Na+/H+ exchange in SHR may reflect alterations in Na+ homeostasis that might contribute to altered SHR VSMC function such as enhanced growth and vasoreactivity.     

Effects of Npt2 gene ablation and low-phosphate diet on renal Na+/phosphate cotransport and cotransporter gene expression

The renal Na(+)/phosphate (Pi) cotransporter Npt2 is expressed in the brush border membrane (BBM) of proximal tubular cells. We examined the effect of Npt2 gene knockout on age-dependent BBM Na(+)/Pi cotransport, expression of Na(+)/Pi cotransporter genes Npt1, Glvr-1, and Ram-1, and the adaptive response to chronic Pi deprivation. Na(+)/Pi cotransport declines with age in wild-type mice (Npt2(+/+)), but not in mice homozygous for the disrupted Npt2 allele (Npt2(-/-)). At all ages, Na(+)/Pi cotransport in Npt2(-/-) mice is approximately 15% of that in Npt2(+/+) littermates. Only Npt1 mRNA abundance increases with age in Npt2(+/+) mice, whereas Npt1, Glvr-1, and Ram-1 mRNAs show an age-dependent increase in Npt2(-/-) mice. Pi deprivation significantly increases Na(+)/Pi cotransport, Npt2 protein, and mRNA in Npt2(+/+) mice. In contrast, Pi-deprived Npt2(-/-) mice fail to show the adaptive increase in transport despite exhibiting a fall in serum Pi. We conclude that (a) Npt2 is a major determinant of BBM Na(+)/Pi cotransport; (b) the age-dependent increase in Npt1, Glvr-1, and Ram-1 mRNAs in Npt2(-/-) mice is insufficient to compensate for loss of Npt2; and (c) Npt2 is essential for the adaptive BBM Na(+)/Pi cotransport response to Pi deprivation.