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.     

Regulation of intracellular pH by Na+/H+ exchange in human lymphocytes

Spectrofluorimetry and the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF) was used to measure the intracellular pH (pHi) of suspended human lymphocytes. A linear relationship exists between pHi and the fluorescent spinal-ratio (I490nm/I435nm, emission 526 nm) between pH 6.5 and pH 7.8. At the end of each experiment the ratio was calibrated using the high [K+] nigericin technique. All solutions were HEPES buffered. The pHi in resting cells was 7.27 +/- 0.02 (n = 37) at 25 degrees C. Na+ free solution caused a pHi decrease to 6.81 +/- 0.08. The ammonium prepulse technique (25 mM NH4Cl) dropped the pHi to pH 6.80. A rapid recovery of the pHi after this acidification was observed in NaCl Ringer solution. Na+ free solution completely blocked the recovery. 1 mM amiloride led to a partial block of recovery. pHi was restored to the basal value after readdition of Na+. We conclude that in HEPES buffered solutions human lymphocytes recover pHi via a mechanism dependent on extracellular Na+ and largely accomplished by an amiloride inhibitory Na+/H+ exchanger.     

Intracellular pH regulation in rabbit renal medullary collecting duct cells. Role of chloride-bicarbonate exchange.

The renal medullary collecting duct (MCD) secretes protons into its lumen and HCO3 into its basolateral space. Basolateral HCO3 transport is thought to occur via Cl/HCO3 exchange. To further characterize this Cl/HCO3 exchange process, intracellular pH (pHi) regulation was monitored in freshly prepared rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Cells were preincubated in bicarbonate-containing solutions and then abruptly diluted into bicarbonate-free media. The MCD cell pHi response to abrupt removal of CO2/HCO3 included an initial alkalinization due to rapid CO2 efflux, followed by an acidification due to HCO3 efflux and a gradual recovery to the resting pHi of 7.24 +/- 0.06 partly due to the action of a plasma membrane H+-ATPase. The initial alkalinization required a CO2/HCO3 gradient and did not occur in the presence of acetazolamide. The acidification phase required intracellular HCO3 and extracellular Cl, which was consistent with a Cl/HCO3 exchange. MCD HCO3 efflux exhibited saturable kinetics for extracellular Cl, with a Michaelis constant (Km) of 29.9 +/- 7.7 mM. HCO3 efflux also exhibited preference for halides over NO3, SCN, and gluconate, and striking sensitivity to disulfonic stilbene and acetazolamide inhibition, with an apparent K1 of 5 X 10(-7) M for DIDS. The final pHi recovery required intracellular ATP, which indicated that Cl/HCO3 and H+-ATPase activities are present in the same cells in these suspensions. The results provide direct evidence for MCD Cl/HCO3 exchange and describe some of the properties of this transport process.     

Mechanism of apical and basolateral Na(+)-independent Cl-/base exchange in the rabbit superficial proximal straight tubule.

The present study was undertaken to determine the magnitude and mechanism of base transport via the apical and basolateral Na(+)-independent Cl-/base exchangers in rabbit isolated perfused superficial S2 proximal tubules. The results demonstrate that there is an apical Na(+)-independent Cl-/base exchanger on both membranes. HCO3- fails to stimulate apical Cl-/base exchange in contrast to the basolateral exchanger. Inhibition of endogenous HCO3- production does not alter the rate of apical Cl-/base exchange in Hepes-buffered solutions. Both exchangers are inhibited by H2DIDS and furosemide; however, the basolateral anion exchanger is more sensitive to these inhibitors. The results indicate that the apical and basolateral Cl-/base exchangers differ in their transport properties and are able to transport base equivalents in the absence of formate. The formate concentration in rabbit arterial serum is approximately 6 microM and in vitro tubule formate production is < 0.6 pmol/min per mm. Formate in the micromolar range stimulates Jv in a dose-dependent manner in the absence of a transepithelial Na+ and Cl- gradient and without a measurable effect on Cl(-)-induced equivalent base flux. Apical formic acid recycling cannot be an important component of any cell model, which accounts for formic acid stimulation of transcellular NaCl transport in the rabbit superficial S2 proximal tubule. We propose that transcellular NaCl transport in this nephron segment is mediated by an apical Na+/H+ exchanger in parallel with a Cl-/OH- exchanger and that the secreted H+ and OH- ions form H2O in the tubule lumen.     

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)     

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.     

Basolateral membrane sodium-independent Cl-/HCO3- exchanger in rat inner medullary collecting duct cell.

Previous studies have shown that the middle third of the rat inner medullary collecting duct (IMCD-2) secretes protons despite the absence of intercalated cells, the cell thought to secrete protons in other portions of the collecting duct. A new cell, the IMCD cell, is the predominant cell in IMCD-2. The mechanism responsible for base exit in the IMCD cell was characterized by measuring cell pH of isolated perfused tubules with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Reduction of bath HCO3- caused a significant and reversible decrease in cell pH, whereas a similar change in luminal HCO3- had a significantly smaller effect, indicating that the HCO3-/H+ permeability of the basolateral membrane is much larger than the apical membrane. The rate of cell acidification induced by reduction in bath HCO3-, a measure of basolateral HCO3- transport, was significantly decreased in the absence of bath and lumen Cl. Decreases in bath Cl caused a significant and reversible increase in cell pH, which was not changed significantly by complete removal of Na from perfusate and bath, but was significantly inhibited by basolateral 4',5'-diisothiocyanostilbene-2,2'-disulfonic acid. A chemical voltage clamp did not inhibit the rate of cell alkalinization after bath Cl removal, indicating that Cl-/HCO3- exchange is not via parallel Cl and HCO3- conductances. Cell pH was measured in single cells by low-light-level imaging to show that most cells contain the chloride-dependent HCO3- pathway. We conclude that the rat IMCD cell possesses a basolateral Na-independent CL-/HCO3- exchanger which may serve as the base exit step for transepithelial proton secretion.     

Anion dependence of rabbit medullary collecting duct acidification.

Rabbit medullary collecting duct (MCD) acidification has been demonstrated to occur by means of a sodium-independent, aldosterone-stimulated mechanism. We have examined the anionic dependence of this process by means of the isolated perfused tubule technique. Total replacement of perfusate chloride with gluconate enhanced tubular bicarbonate reabsorption (JHCO3), from a basal rate of 10.7 +/- 1.0 pmol X mm-1 X min-1 to a rate of 15.01 +/- 1.0 pmol X mm-1 X min-1. Removal of bath chloride, with and without removal of perfusate chloride completely abolished acidification. Bath, but not luminal 4-acetamido-4' isothiocyano-2,2'-disulfonic stilbene provoked a marked decrease in JHCO3 from 10.1 +/- 1.2 pmol X mm-1 X min-1 to 2.3 +/- 0.3 pmol X mm-1 X min-1. Measurement of chloride reabsorptive rate (JCl) revealed colinearity between JHCO3 (9.18 +/- 0.9 pmol X mm-1 X min-1) and JCl (9.75 +/- 1.18 pmol X mm-1 X min-1). We propose a model of mammalian distal nephron acidification in which (a) cellular base exit is effected by means of a basolateral membrane Cl-base exchanger and (b) net electroneutrality of electrogenic proton secretion is maintained by the parallel movement of an anionic species, functionally chloride.     

Feedback inhibition of cyclic adenosine monophosphate-stimulated Na+ transport in the rabbit cortical collecting duct via Na(+)-dependent basolateral Ca++ entry.

Arginine vasopressin (AVP) transiently stimulates Na+ transport in the rabbit cortical collecting duct (CCD). However, the sustained effect of both AVP and its putative second messenger, cyclic adenosine monophosphate (cAMP), on Na+ transport in the rabbit CCD is inhibitory. Because maneuvers that increase [Ca++]i inhibit Na+ transport, the effects of AVP and cell-permeable cAMP analogues, on [Ca++]i were investigated in fura-2-loaded in vitro microperfused rabbit CCDs. Low-dose AVP (23-230 pM) selectively stimulated Ca++ influx, whereas 23 nM AVP additionally released calcium from intracellular stores. 8-chlorophenylthio-cAMP (8CPTcAMP) and 8-bromo-cAMP (8-Br-cAMP) also increased CCD [Ca++]i. The 8CPTcAMP-stimulated [Ca++]i increase was totally dependent on basolateral [Ca++]. In the absence of cAMP, peritubular Na+ removal produced a marked increase in [Ca++]i, which was also dependent on bath [Ca++], suggesting the existence of basolateral Na+/Ca++ exchange. Luminal Na+ removal in the absence of cAMP did not alter CCD [Ca++]i, but it completely blocked the cAMP-stimulated [Ca++]i increase. Thus the cAMP-dependent Ca++ increase is totally dependent on both luminal Na+ and basolateral Ca++, suggesting the [Ca++]i increase is secondary to cAMP effects on luminal Na+ entry and its coupling to basolateral Na+/Ca++ exchange. 8CPTcAMP inhibits lumen-to-bath 22Na flux [JNa(l-b)] in CCDs bathed in a normal Ca++ bath (2.4 mM). However, when bath Ca++ was lowered to 100 nM, a maneuver that also blocks the 8CPTcAMP [Ca++]i increase, 8CPTcAMP stimulated, rather than inhibited JNa(l-b). These results suggest that cAMP formation initially stimulates CCD Na+ transport, and that increased apical Na+ entry secondarily activates basolateral Ca++ entry. The cAMP-dependent [Ca++]i increase leads to inhibition Na+ transport in the rabbit CCD.     

Effect of autologous serum on human leucocyte Na+/H+ exchange and intracellular pH

1. Leucocyte Na+/H+ exchange and intracellular pH were investigated in physiological buffer containing bicarbonate. 2. The amiloride analogue 5-(N,N-hexamethylene) amiloride (1 x 10(-5) mol/l), an inhibitor of Na+/H+ exchange, had no significant effect on resting leucocyte pH, Na+ influx or Na+ content. 3. Ammonium chloride washout induced a profound intracellular acidosis, stimulating Na+/H+ exchange. This led to a 236% increase in Na+ influx. Eighty-eight per cent of this increase was inhibited by 5-(N,N-hexamethylene) amiloride. This demonstrates that 5-(N,N-hexamethylene) amiloride is an effective inhibitor of Na+/H+ exchange in human leucocytes. 4. The recovery from intracellular acidosis was shown to be dependent entirely on the presence of extracellular Na+. The Ki for inhibition by 5-(N,N-hexamethylene) amiloride of the recovery was 0.5 mumol/l. 5. Incubation with serum increased Na+ influx and Na+ content: the maximal effect was reached at 20% dilution. Serum (10%, v/v) increased influx by 40%, and 20% (v/v) serum by 102%, over resting levels. Only 43% of the serum-induced increase in Na+ influx was inhibited by 5-(N,N-hexamethylene) amiloride. This represented one-fifth of total Na+ influx. 6. Leucocyte intracellular pH increased on incubation with serum. This alkalinization was inhibited using 5-(N,N-hexamethylene) amiloride. 7. Studies of Na+/H+ exchange in leucocytes in physiological and pathological states are more likely to reflect the state in vivo if carried out in the presence of autologous serum.     

Enhancement of electrogenic Na+ transport across rat inner medullary collecting duct by glucocorticoid and by mineralocorticoid hormones.

We have investigated the effect of steroid hormones on Na+ transport by rat renal inner medullary collecting duct (IMCD) cells. These cells, grown on permeable supports in primary culture, grow to confluence and develop a transmonolayer voltage oriented such that the apical surface is negative with respect to the basal surface. The results of these experiments demonstrate that this voltage is predominantly (or exclusively) the result of electrogenic Na+ absorption. Na+ transport can be stimulated two- to fourfold by exposure to either dexamethasone or aldosterone (100 nM). Experiments using specific antagonists of the glucocorticoid and mineralocorticoid receptors indicate that activation of either receptor stimulates electrogenic Na+ transport; electroneutral Na+ transport is undetectable. Two other features of the IMCD emerge from these studies. (a) These cells appear to have the capacity to metabolize the naturally occurring glucocorticoid hormone corticosterone. (b) The capacity for K+ secretion is minimal and steroid hormones do not induce or stimulate conductive K+ secretion as they do in the cortical collecting duct.     

Regulation of the rabbit ileal brush-border Na+/H+ exchanger by an ATP-requiring Ca++/calmodulin-mediated process.

Brush-border vesicles purified from rabbit ileal villus cells were used to evaluate how Ca++/calmodulin (CaM) regulates the neutral linked NaCl absorptive process, part of which is a Na+/H+ exchanger. After freezing and thawing to allow incorporation of macromolecules into the vesicles, the effect of Ca++/CaM on brush-border Na+ uptake with an acid inside pH gradient, and on Na+/H+ exchange was determined. Freezing and thawing vesicles with 0.85 microM free Ca++ plus 5 microM exogenous CaM failed to alter Na+/H+ exchange as did the addition of exogenous ATP plus an ATP regenerating system, which was sufficient to elevate intravesicular ATP to 47 microM from a basal level of 0.4 microM. However, the combination of Ca++/CaM plus ATP inhibited Na+ uptake in the presence of an acid inside pH gradient and inhibited Na+/H+ exchange, while Na+ uptake in the absence of a pH gradient was not altered. This effect required a hydrolyzable form of ATP, and did not occur when the nonhydrolyzable ATP analogue, AMP-PNP, replaced ATP. Under the identical intravesicular conditions used for the transport studies, Ca++ (0.85 microM) plus exogenous CaM (5 microM), in the presence of magnesium plus ATP, increased phosphorylation of five brush-border peptides. These data are consistent with Ca++/CaM acting via phosphorylation to regulate the ileal brush-border Na+/H+ exchanger.     

Hormonal regulation of proton secretion in rabbit medullary collecting duct.

With the exception of aldosterone, little is known about the hormonal regulation of distal nephron acidification. These experiments investigated the effects of prostaglandin E2, indomethacin, lysyl-bradykinin, 8-bromo-cyclic AMP, and forskolin on proton secretion in the major acidifying segment of the distal nephron, the medullary collecting duct from inner stripe of outer medulla. Using in vitro microperfusion and microcalorimetry, net bicarbonate reabsorption (proton secretion) was measured in rabbit medullary collecting ducts before, during, and after exposure to each test substance. PGE2 reduced proton secretion 12.2%, while the following substances stimulated proton secretion: indomethacin 14.2%; 8-bromo-cyclic AMP 34.5%; forskolin 39%. Lysyl-bradykinin was without effect. These studies demonstrate that distal nephron acidification, in addition to being stimulated by aldosterone, is significantly inhibited by the hormone PGE2. The stimulation of proton secretion by cAMP suggests that other hormones known to activate adenylate cyclase may also influence distal nephron acidification.     

Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal tubule.

The apical transport processes responsible for proton secretion were studied in the isolated perfused rabbit S3 proximal tubule. Intracellular pH (pHi) was measured with the pH dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady state pHi in S3 tubules in nominally HCO3(-)-free solutions was 7.08 +/- 0.03. Removal of Na+ (lumen) caused a decrease in pHi of 0.34 +/- 0.06 pH/min. The decrease in pHi was inhibited 62% by 1 mM amiloride (lumen) and was unaffected by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (lumen) and Cl- removal (lumen, bath). After a brief exposure to 20 mM NH4Cl, pHi fell by approximately 0.7 and recovered at a rate of 0.89 +/- 0.15 pH/min in the nominal absence of Na+, HCO3-, organic anions, and SO4(2-) (lumen, bath). 1 mM N,N'-dicyclohexylcarbodiimide (lumen), 1 mM N-ethylmaleimide (lumen), 0.5 mM colchicine (bath), and 0.5 mM iodoacetic acid (lumen, bath) inhibited the Na+-independent pHi recovery rate by 73%, 55%, 77%, and 86%, respectively, whereas 1 mM KCN (lumen, bath) did not inhibit pHi recovery. Reduction of intracellular, but not extracellular chloride, also decreased the Na+-independent pHi recovery rate. In conclusion, the S3 proximal tubule has an apical Na+/H+ antiporter with a Michaelis constant for Na+ of 29 mM and a maximum velocity of 0.47 pH/min. S3 tubules also possess a plasma membrane H+-ATPase that can regulate pHi, has a requirement for intracellular chloride, and utilizes ATP derived primarily from glycolysis.     

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.     

Intracellular pH regulation and proton transport by rabbit renal medullary collecting duct cells. Role of plasma membrane proton adenosine triphosphatase.

Proton secretion in the renal medullary collecting duct is thought to occur via a luminal proton-ATPase. In order to determine what mechanism(s) participate in proton transport across medullary collecting duct (MCD) cells membranes, intracellular pH (pHi) regulation and proton extrusion rates were measured in freshly prepared suspensions of rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Proton extrusion rates were measured using a pH stat. The resting pHi of MCD cells was 7.19 +/- 0.05 (SE) in a nonbicarbonate medium of pH 7.30. When cells were acidified by exposure to acetate salts or by abrupt withdrawal of ammonium chloride, they exhibited pHi recovery to the resting pHi over a 5-min time-course. Depletion of greater than 95% of cellular ATP content by poisoning with KCN in the absence of glucose inhibited pHi recovery. ATP depletion inhibited proton extrusion from MCD cells. Treatment with N-ethylmaleimide also inhibited pHi recovery. In addition, cellular ATP content was dependent on transmembrane pH gradients, suggesting that proton extrusion stimulated ATP hydrolysis. Neither removal of extracellular sodium nor addition of amiloride inhibited pHi recovery. These results provide direct evidence that a plasma membrane proton-ATPase, but not a Na+/H+ exchanger, plays a role in proton transport and pHi regulation in rabbit MCD.     

Metabolic acidosis stimulates H+ secretion in the rabbit outer medullary collecting duct (inner stripe) of the kidney.

The outer medullary collecting duct (OMCD) absorbs HCO3- at high rates, but it is not clear if it responds to metabolic acidosis to increase H+ secretion. We measured net HCO3- transport in isolated perfused OMCDs taken from deep in the inner stripes of kidneys from control and acidotic (NH4Cl-fed for 3 d) rabbits. We used specific inhibitors to characterize the mechanisms of HCO3- transport: 10 microM Sch 28080 or luminal K+ removal to inhibit P-type H+,K+-ATPase activity, and 5-10 nM bafilomycin A1 or 1-10 nM concanamycin A to inhibit H+-ATPase activity. The results were comparable using either of each pair of inhibitors, and allowed us to show in control rabbits that 65% of net HCO3- absorption depended on H+-ATPase (H flux), and 35% depended on H+,K+-ATPase (H,K flux). Tubules from acidotic rabbits showed higher rates of HCO3- absorption (16.8+/-0.3 vs. 12.8+/-0.2 pmol/min per mm, P < 0.01). There was no difference in the H,K flux (5.9+/-0.2 vs. 5.8+/-0.2 pmol/min per mm), whereas there was a 61% higher H flux in segments from acidotic rabbits (11.3+/-0.2 vs. 7.0+/-0.2 pmol/min per mm, P < 0.01). Transport was then measured in other OMCDs before and after incubation for 1 h at pH 6.8, followed by 2 h at pH 7.4 (in vitro metabolic acidosis). Acid incubation in vitro stimulated HCO3- absorption (12.3+/-0.3 to 16.2+/-0.3 pmol/min per mm, P < 0.01), while incubation at pH 7.4 for 3 h did not change basal rate (11.8+/-0.4 to 11.7+/-0.4 pmol/min per mm). After acid incubation the H,K flux did not change, (4.7+/-0.4 to 4.6+/-0.4 pmol/min per mm), however, there was a 60% increase in H flux (6.6+/-0.3 to 10.8+/-0.3 pmol/min per mm, P < 0.01). In OMCDs from acidotic animals, and in OMCDs incubated in acid in vitro, there was a higher basal rate and a further increase in HCO3- absorption (16.7+/-0.4 to 21.3+/-0.3 pmol/min per mm, P < 0.01) because of increased H flux (11.5+/-0.3 to 15.7+/-0.2 pmol/min per mm, P < 0.01) without any change in H,K flux (5.4+/-0.3 to 5.6+/-0.3 pmol/min per mm). These data indicate that HCO3- absorption (H+ secretion) in OMCD is stimulated by metabolic acidosis in vivo and in vitro by an increase in H+-ATPase-sensitive HCO3- absorption. The mechanism of adaptation may involve increased synthesis and exocytosis to the apical membrane of proton pumps. This adaptation helps maintain homeostasis during metabolic acidosis.