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.     

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.     

Rat hepatocytes exhibit basolateral Na+/HCO3- cotransport.

Primary cultures and plasma membrane vesicles were used to characterize Na+ and HCO3- transport by rat hepatocytes. Na+ uptake into hepatocytes was stimulated approximately 10-fold by 25 mM extracellular HCO3-.HCO3--stimulated Na+ uptake was saturable, abolished by 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS), and unaffected by amiloride or Cl- removal. Neither propionate nor acetate reproduced this effect of HCO3-. 22Na efflux from preloaded hepatocytes was similarly increased approximately 10-fold by an in greater than out HCO3- concentration gradient. 22Na efflux was also increased by valinomycin and an in greater than out K+ concentration gradient in the presence but not absence of HCO3-. Intracellular pH (pHi) measured with the pH-sensitive fluorochrome 2',7'-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (BCECF) decreased at a rate of 0.227 (+/- 0.074 SEM) pH units/min when extracellular HCO3- concentration was lowered from 25 to 5 mM at constant PCO2. This intracellular acidification rate was decreased 50-60% in the absence of Na+ or presence of SITS, and was unaffected by amiloride or Cl- removal. Membrane hyperpolarization produced by valinomycin and an in greater than out K+ concentration gradient caused pHi to fall; the rate of fall was decreased 50-70% by Na+ removal or SITS, but not amiloride. An inside positive K+ diffusion potential and a simultaneous out greater than in HCO3- gradient produced a transient 4,4'-diisothiocyano-2,2' disulfonic acid stilbene (DIDS) sensitive, amiloride-insensitive 22Na accumulation in basolateral but not canalicular membrane vesicles. Rat hepatocytes thus exhibit electrogenic basolateral Na+/HCO3- cotransport.     

Intracellular pH regulation in isolated rat bile duct epithelial cells.

To evaluate ion transport mechanisms in bile duct epithelium (BDE), BDE cells were isolated from bile duct-ligated rats. After short-term culture pHi was measured with a single cell microfluorimetric set-up using the fluorescent pHi indicator BCECF, and calibrated with nigericin in high K+ concentration buffer. Major contaminants were identified using vital markers. In HCO3(-)-free media, baseline pHi (7.03 +/- 0.12) decreased by 0.45 +/- 0.18 pH units after Na+ removal and by 0.12 +/- .04 after amiloride administration (1 mM). After acid loading (20 mM NH4Cl) pHi recovery was inhibited by both Na+ removal and amiloride (JH+ = 0.74 +/- 1.1, and JH+ = 2.28 +/- 0.8, respectively, vs. 5.47 +/- 1.97 and 5.97 +/- 1.76 mM/min, in controls, respectively). In HCO3- containing media baseline pHi was higher (7.16 +/- 0.1, n = 36, P less than 0.05) and was decreased by Na+ substitution but not by amiloride. Na+ removal inhibited pHi recovery after an intracellular acid load (0.27 +/- 0.26, vs. 7.7 +/- 4.1 mM/min, in controls), whereas amiloride reduced JH+ only by 27%. pH recovery was inhibited by DIDS (0.5-1 mM), but not by Cl- depletion. Finally, acute Cl- removal increased pHi by 0.18 pH units in the absence but not presence of DIDS. These data indicate that BDE cells possess mechanisms for Na+/H+ exchange, Na+:HCO3- symport and Cl-/HCO3 exchange. Therefore BDE may be capable of transepithelial H+/HCO3- transport.     

Effect of secretion on intracellular pH regulation in isolated rat bile duct epithelial cells.

The effects of secretin on ion transport mechanisms involved in regulation of intracellular pH (pHi) and HCO3- excretion were characterized in bile duct epithelial (BDE) cells isolated from normal rat liver. pHi was measured with 2,7-bis(carboxy-ethyl)-5(6)-carboxy-fluorescein-acetomethylester (BCECF-AM) using a microfluorimetric method. Basal pHi of BDE was 7.04 +/- 0.06 in Hepes and 7.16 +/- 0.10 in KRB and was unaffected by secretin (50-200 nM). Recovery rates from an acid load in Hepes or in KRB media (with and without amiloride) were also not altered by secretin, indicating that Na+/H+ exchange and Na+/HCO3- cotransport were not affected by this hormone. After acute Cl- removal, pHi rose 0.24 +/- 0.08 pHU at a maximal rate of 0.125 +/- 0.06 pHU/min (H+ flux rates = 6.02 +/- 3.27 mM/min) and recovered after Cl- readmission (0.188 +/- 0.08 pHU/min; H+ flux rates = 11.82 +/- 5.34 mM/min). Pretreatment with 1 mM DIDS inhibited the effects of Cl- removal, while valinomycin, which induces cell depolarization, enhanced these effects, probably by stimulating electrogenic HCO3- influx. Secretin significantly increased both the maximal rate of alkalinization after Cl- removal (P < 0.012) and of pHi recovery after Cl- readmission (P < 0.025), indicating stimulation of Cl-/HCO3- exchange activity. These findings were reproduced with N6,2'-O-Dibutyryladenosine-3',5'-cyclic monophosphate (DBcAMP). The Cl- channel blocker 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB, 10 microM) significantly decreased the effects of secretin and DBcAMP on the pHi changes promoted by acute Cl- removal/readmission. These findings establish that secretin stimulates the activity of the Cl-/HCO3- exchanger in BDE cells, probably by activating Cl- channels via the intracellular messenger cAMP. This in turn depolarizes the cell, stimulating electrogenic Na+/HCO3- symport. The cell depolarization induced by Cl- channel activation should enhance HCO3- entrance through electrogenic Na+/HCO3- symport, which in turn stimulates the Cl-/HCO3- exchange. These mechanisms could account for secretin stimulated bicarbonate secretion in bile.     

Na+-dependent recovery of intracellular pH from acid loading in mouse colonic crypt cells

The membrane transport mechanism for regulating the intracellular pH value (pHi) was investigated in mouse distal colon crypt cells. pHi was measured by microfluorometry in an isolated crypt fragment loaded with the pH-sensitive fluoroprobe, 2',7'-bis-(2-carboxyethyl)-5-(6) carboxyfluorescein. The pHi recovery process after acid loading induced by a 40 mM NH4Cl prepulse was almost totally dependent on Na+ in both the presence and absence of CO2/HCO3- in the perfusion solution. In the CO2/HCO3(-)-free, HEPES-buffered solution, amiloride partially inhibited the pHi recovery rate from acid loading with an ED50 value of 15 microM and maximum inhibition of 83%. In a CO2/HCO3- solution, amiloride inhibited the pHi recovery rate with an ED50 value of 18 microM, which was similar to that in the HEPES-buffered solution, while the rate of pHi recovery remaining in the presence of the maximum effective concentration of amiloride was significantly larger than that in the HEPES-buffered solution. The Na+-dependent pHi recovery from the acid loading was significantly less (by 18%) in the presence of forskolin. These results suggest that the pHi recovery from acid loading was mediated by 1) amiloride-sensitive Na+/H+ exchanger, 2) the amiloride-insensitive Na+/H+ exchanger, and 3) the Na+- and HCO3(-)-dependent acid extruder. The pHi recovery could be inhibited by cAMP.     

Regulation of intracellular pH in the rabbit cortical collecting tubule.

The cortical collecting tubule (CCT) is an important nephron segment for Na+, K+, water and acid-base transport. Differential loading characteristics of the pH sensitive dye 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein (BCECF) and basolateral Cl- removal were used to identify and study intracellular pH (pHi) regulation in each of three cell types involved in this transport. Both principal cells and beta-intercalated cells were found to have a basolateral Na+/H+ exchanger based on the Na+ and amiloride sensitivity of pHi recovery from acid loads. Intercalated cells demonstrated abrupt pHi changes with basolateral Cl- removal. alpha-intercalated cells alkalinized; beta-intercalated cells acidified. In the beta-intercalated cells, luminal Cl- removal blocked changes in pHi in response to changes in luminal HCO3- or peritubular Cl-, providing direct evidence for a luminal Cl-/HCO3- exchanger. In principal cells, brief removal of either peritubular or luminal Cl- resulted in no change in pHi; however, return of peritubular Cl- after prolonged removal resulted in a rapid fall in pHi consistent with a basolateral Cl-/HCO3- exchanger, which may be relatively inactive under baseline conditions. Therefore, Cl-/HCO3- exchange is present in all three cell types but varies in location and activity.     

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.     

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.     

Effect of glucagon on intracellular pH regulation in isolated rat hepatocyte couplets.

To elucidate mechanisms of glucagon-induced bicarbonate-rich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pHi) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pHi, significantly stimulates the Cl-/HCO3- exchange activity. The effect of glucagon was associated with a sevenfold increase in cAMP levels in rat hepatocytes. The activity of the Cl-/HCO3- exchanger was also stimulated by DBcAMP + forskolin. The effect of glucagon on the Cl-/HCO3- exchange was individually blocked by two specific and selective inhibitors of protein kinase A, Rp-cAMPs (10 microM) and H-89 (30 microM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl- channel blocker, NPPB (10 microM), showed no effect on either the basal or the glucagon-stimulated Cl-/HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 microM), completely blocked the glucagon stimulation of the Cl-/HCO3- exchange; however, this effect was achieved through a significant inhibition of the glucagon-stimulated cAMP accumulation in rat hepatocytes. Colchicine pretreatment inhibited the basal as well as the glucagon-stimulated Cl-/HCO3- exchange activity. The Na+/H+ exchanger was unaffected by glucagon either at basal pHi or at acid pHi values. In contrast, glucagon, at basal pHi, stimulated the Na(+)-HCO3- symport. The main findings of this study indicate that glucagon, through the cAMP-dependent protein kinase A pathway, stimulates the activity of the Cl-/HCO3- exchanger in isolated rat hepatocyte couplets, a mechanism which could account for the in vivo induced bicarbonate-rich choleresis.     

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.     

Evidence for carrier-mediated chloride/bicarbonate exchange in canalicular rat liver plasma membrane vesicles.

To determine whether anion exchangers might play a role in hepatic bile formation, we looked for the presence of Cl-:OH- and Cl-:HCO3- exchange in highly purified canalicular (c) and basolateral (bl) rat liver plasma membrane (LPM) vesicles. In cLPM vesicles, a pH gradient (7.7 in/6.0 out) stimulated 36Cl- uptake twofold above values obtained during pH-equilibrated conditions (7.7 in = out). When 50 mM HCO3- was also present inside the vesicles, the same pH gradient (7.7 in/6.0 out) resulted in Cl- uptake to levels fourfold above pH- and HCO3--equilibrated controls and two- to threefold above Cl- equilibrium (overshoot). Initial rates of both pH and HCO3- gradient-stimulated Cl- uptake were completely inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). A valinomycin-induced K+ diffusion potential (inside positive) also stimulated Cl- uptake in cLPM, but this conductive Cl- pathway was insensitive to DIDS. The DIDS-sensitive, pH and HCO3- gradient-stimulated Cl- uptake demonstrated: saturation with Cl- (Km approximately 6.3 mM; Vmax approximately 51 nmol X mg-1 X min-1); partial inhibition by bumetanide (26%), furosemide (33%), probenecid (37%), and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (49%); cis-inhibition by chloride and nitrate but not by sulfate and various organic anions, and independence from the membrane potential. These data demonstrate the presence of an electroneutral Cl-:OH- and Cl-:HCO3- exchanger in rat liver canalicular membranes that favors Cl-:HCO3- exchange. In contrast, no evidence was found for the presence of a Cl-:HCO3- (OH-) exchange system in blLPM vesicles. Furthermore, neither blLPM nor cLPM vesicles exhibited Na+-stimulatable Cl- uptake, indicating the absence of a NaCl co-transport system in either LPM subfraction. These findings are consistent with a functional role for a Cl-:HCO3- (OH-) exchanger in canalicular bile formation.     

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.     

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.     

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 anti-inflammatory analgesic drugs on the regulation of cytosolic pH by anion antiport

Four nonsteroidal anti-inflammatory analgesic drugs acetylsalicylic acid (aspirin), salicylic acid, indomethacin and piroxicam were tested for their action on chloride/bicarbonate exchange by anion antiport and on the regulation of cytosolic pH (pHi) in Vero cells. The Na+-independent Cl-/HCO3- exchange regulates pHi back to normal after alkalinization and is therefore in a state of high activity at alkaline pHi, whereas it is in a state of low activity when pHi is below neutrality. Preincubation with the drugs at low pH strongly increased the activity of the Na+-independent Cl-/HCO3- exchange, whereas at high pH the antiport was inhibited. The Na+-dependent Cl-/HCO3- exchange, which is most active at low pHi and normalize pHi after acidification, was inhibited after preincubation with the drugs. In cells that had been incubated with the drugs, pHi was 0.1 to 0.4 pH units lower than in cells that had been incubated in the absence of drugs. The prostaglandins E2, F2 and I2 had little effect on anion antiport, and did not counteract the effects of the anti-inflammatory drugs on Cl-/HCO3- exchange, indicating that these actions of the drugs are not due to the inhibition of prostaglandin synthesis. The relevance of our findings to the clinical effects of these drugs is discussed.     

Beta-adrenergic upregulation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells.

We used the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6')-carboxyfluorescein to monitor the recovery of the intracellular pH (pHi) of rat parotid acini from an NH4(+)-induced alkaline load. This recovery was markedly inhibited by the loop diuretic bumetanide and by Cl- removal, indicating that it is largely due to NH4+ entry via the basolateral Na(+)-K(+)-2Cl- cotransporter. The rate of recovery of pHi was enhanced threefold by pretreatment (37.5 s) with isoproterenol (K1/2 = 21.5 nM) or norepinephrine (in the presence of phentolamine), and blocked by the beta 1-specific antagonist atenolol, indicating an upregulation of cotransport activity by beta 1-adrenergic stimulation. The effect of isoproterenol was prevented by protein kinase inhibitors and mimicked by cAMP analogues, and by maneuvers known to increase cytosolic cAMP levels in these cells, consistent with the involvement of protein kinase A. Physiologically, such an upregulation of the acinar Na(+)-K(+)-2Cl- cotransporter would lead to an increase in acinar chloride uptake across the basolateral membrane, and consequently, an increase in overall chloride and fluid secretion. Prevention of this upregulation by beta-blockers and possibly by other commonly used clinical agents may account for the dry mouth and dry eyes experienced by some patients taking these medications.     

Intracellular pH regulation in human SW-620 colon cancer cells

Intracellular pH (pHi) regulation is essential for basic functioning of the cell and activation of pHi regulatory mechanisms appears to be involved in the initial stage of cell division. Little is known about pHi regulation in human colonic carcinoma cells. We investigated SW-620 (CCL 227) cells, a cell-line derived from a human colonic adenocarcinoma. pHi changes were recorded by computer-assisted spectrofluorimetric monitoring of the pH-sensitive, fluorescent dye BCECF (2',7'-bis(carboxyethyl)- 5(6)carboxyfluorescein). Resting pHi in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffered solution was 7.53 +/- 0.01. Intracellular acidification after an ammonium prepulse produced a pHi decline of 0.5 units and pHi returned to normal value in NaCl Ringer's. Both 1 mM amiloride and Na-free solution completely inhibited recovery for 8 minutes. This inhibition was reversible in NaCl Ringer's. Na-free solution led to a pHi decrease to 7.39 +/- 0.04 after 16 min, pHi was also lowered by 8 minute incubation of cells with 1 mM amiloride (7.40 +/- 0.02). In HCO3/CO2-buffered solution resting pHi was 7.42 +/- 0.01 (n = 35). Recovery from an acute acid load, induced by NH4 prepulse or switching from HEPES- to bicarbonate-buffered solution, was Na dependent, Cl independent, reversible and only partially blocked by 1 mM amiloride - pHi slowly recovered from 6.83 +/- 0.03 to 7.00 +/- 0.06 in 8 minutes. In the presence of amiloride and 200 microns H2DIDS (dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) pHi recovery was completely inhibited for 8 minutes. In Na-free solution pHi decreased from 7.44 +/- 0.04 to 7.29 +/- 0.03 within 8 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular pH during "chemical hypoxia" in cultured rat hepatocytes. Protection by intracellular acidosis against the onset of cell death

The relationships between extracellular pH (pHo), intracellular pH (pHi), and loss of cell viability were evaluated in cultured rat hepatocytes after ATP depletion by metabolic inhibition with KCN and iodoacetate (chemical hypoxia). pHi was measured in single cells by ratio imaging of 2',7'-biscarboxy-ethyl-5,6-carboxyfluorescein (BCECF) fluorescence using multiparameter digitized video microscopy. During chemical hypoxia at pHo of 7.4, pHi decreased from 7.36 to 6.33 within 10 min. pHi remained at 6.1-6.5 for 30-40 min (plateau phase). Thereafter, pHi began to rise and cell death ensued within minutes, as evidenced by nuclear staining with propidium iodide and coincident leakage of BCECF from the cytoplasm. An acidic pHo produced a slightly greater drop in pHi, prolonged the plateau phase of intracellular acidosis, and delayed the onset of cell death. Inhibition of Na+/H+ exchange also prolonged the plateau phase and delayed cell death. In contrast, monensin or substitution of gluconate for Cl- in buffer containing HCO3- abolished the pH gradient across the plasma membrane and shortened cell survival. The results indicate that intracellular acidosis after ATP depletion delays the onset of cell death, whereas reduction of the degree of acidosis accelerates cell killing. We conclude that intracellular acidosis protects against hepatocellular death from ATP depletion, a phenomenon that may represent a protective adaptation against hypoxic and ischemic stress.