Intracellular pH in vascular smooth muscle: regulation by sodium-hydrogen exchange and multiple sodium dependent HCO3- mechanisms

OBJECTIVES: The aim was to determine the mechanisms, particularly bicarbonate dependent mechanisms, of intracellular pH (pHi) recovery from various acidoses in vascular smooth muscle and to explore the ATP dependency of the respective mechanisms. METHODS: Experiments were conducted in rat aortic smooth muscle cells grown in primary culture and synchronised in a non-growing state by serum deprivation. pHi was measured in cells loaded with the pH sensitive fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF). Chloride efflux was studied by determination of the rate of efflux of 36Cl over 5 min. Cells were ATP depleted by substitution of glucose in the medium by 2-deoxyglucose. Acidoses were induced by CO2 influx and NH3 efflux techniques. RESULTS: In the absence of HCO3-, the 5-(N-ethyl-N-isopropyl) amiloride (EIPA) sensitive Na+/H+ exchange accounted for the recovery from intracellular acidosis. In the presence of HCO3- ions the response to respiratory acidosis (CO2 influx) was predominantly via activation of Na+/H+ exchange and an EIPA sensitive Na+ and HCO3- dependent mechanism. A 4-acetamido-4'-isothiocyanostilbene-2',2'-sulphonic acids (SITS) sensitive Na+ dependent Cl-/HCO3- mechanism which is also sensitive to EIPA makes a small contribution during severe intracellular acidosis. Under such conditions HCO3- dependent mechanisms contributed about 40% to the overall pHi regulating capacity of vascular smooth muscle cells. However, under conditions which deplete cellular ATP these pHi regulating mechanisms account for virtually all of theses cells' ability to regulate pHi. The inability of Na+/H+ exchange to participate in pHi recovery under these circumstances, reduces the ability of vascular smooth muscle cells to recover pHi by approximately 50-60%. Chloride efflux was approximately linear over 5 min and was increased by 36% in the presence of extracellular HCO3-. Efflux in the presence of HCO3- was inhibited similarly by both SITS and EIPA. CONCLUSIONS: At least three transporters contribute to recovery from acidosis in vascular smooth muscle: Na+/H+ exchange, an Na(+)-HCO3- cotransporter which is sensitive to EIPA, and an Na+ dependent HCO3-/Cl- exchange sensitive to both SITS and EIPA. The Na(+)-HCO3- cotransporter appears to be similar to that described in human vascular smooth muscle. When the Na+/H+ exchanger is attenuated by cellular ATP depletion, the alternative pathways, particularly the Na(+)-HCO3- cotransporter, ensure that substantial pHi regulatory capacity is maintained.     

Astrocytes and lentivirus infection in an experimental models of macaque infected with SIVmac251

OBJECTIVE: The aim was to examine the effects of lysophosphatidylcholine (LPC), an amphiphilic lipid metabolite in ischemic myocardium, on intracellular pH (pH(i)) regulatory systems in guinea pig papillary muscles. METHODS: In CO2/HCO(3-)-buffered Tyrode solution, pH(i), intracellular Na+ activity (aNai) and membrane potential of isolated guinea pig papillary muscles were measured using ion-selective microelectrode and conventional microelectrode. Standard ammonium prepulsing with 20 mM NH4Cl was used to produce an intracellular acid load, and effects of LPC on the pH(i) recovery from acidosis were evaluated in the absence and presence of a transport inhibitor. RESULTS: LPC acidified the resting pH(i) by 0.03 +/- 0.01 pH units (n = 15, p < 0.01) concomitantly with a slight decrease in resting membrane potential and an increase in aNai in quiescent preparations. The pH(i) recovery rate from an intracellular acid load was decreased to 83 +/- 4% of the control value by 30 microM LPC (n = 8, P < 0.05) but not by 30 microM phosphatidylcholine (PC). In the presence of 10 microM 5-(N,N-hexamethylene) amiloride (HMA), a Na(+)-H+ exchange inhibitor, LPC still slowed pH(i) recovery from an intracellular acid load to 77 +/- 4% of the control (n = 5, P < 0.05). However, LPC failed to alter the pH(i) recovery rate in the presence of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.5 mM), a Na(+)-HCO3- symport inhibitor. CONCLUSION: LPC impairs Na(+)-HCO3- symport but not Na(+)-H+ exchange, and LPC may potentiate its arrhythmogenic action by intensifying the intracellular acidosis in ischemic myocardium.     

Intracellular pH regulation in bovine aortic endothelial cells: evidence of both Na+/H+ exchange and Na+-dependent Cl-/HCO3- exchange

Regulation of intracellular pH (pHi) was studied in cultured bovine aortic endothelial cells, an important cell system for cardiovascular research. Suspended cells were acidified by the NH4Cl prepulse technique as well as by exposure to CO2/HCO3-. Subsequent rates of pHi recovery were monitored using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF). In HCO3(-)-free solutions, an EIPA-sensitive, Na+-dependent mechanism fully accounted for realkalinization, namely the Na+/H+ exchanger (NHE). In the presence of HCO3-, an additional acid efflux mechanism was found. This one was dependent on Na+ and intracellular Cl-, EIPA-insensitive but DIDS-sensitive, and therefore represented a Na+-dependent Cl-/HCO3- exchanger (NCBE). In summary, two acid-extruding mechanisms were identified in bovine aortic endothelial cells: NHE and NCBE.     

Na(+)-dependent and -independent Cl-/HCO-3 exchange mediate cellular HCO3- transport in cultured human intrahepatic bile duct cells

Biliary epithelial cells (cholangiocytes) modulate bile fluidity and alkalinity absorbing and/or secreting fluid and electrolytes, particularly HCO3- and Cl-. Mechanisms responsible for transepithelial H+/HCO3- secretion in human cholangiocytes are largely unknown. Human cholangiocytes isolated by enzymatic digestion and immunomagnetic purification from normal liver tissue obtained from reduced grafts used for pediatric liver transplantation were cultured in the presence of human hepatocyte growth factor. Maintenance of cholangiocyte phenotypic features was assessed using markers such as cytokeratin 19, gamma-glutamyltranspeptidase, vimentin, factor VIII-related antigen, desmin, epithelial membrane antigen (EMA), and human epithelial antigen (HEA) 125. Intracellular pH (pHi) transients were measured microfluorimetrically 2'7'-Bis(2-carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester (BCECF). In the absence of HCO3-, pHi recovery from an intracellular acid load (ammonia pre-pulse technique) was Na(+)-dependent and amiloride-inhibitable. No Na(+)-independent recovery was recorded even after stimulation with agents raising intracellular cyclic adenosine monophosphate (cAMP) concentrations. In the presence of HCO3-, recovery from an intracellular acid load required Na+, but was only partly inhibited by amiloride. In these conditions H+ extrusion was inhibited by 4,4-diisothiocyan atostilben-2,2-disulfonic acid (DIDS) and by intracellular Cl- depletion. Acute removal of extracellular Cl induced a pHi alkalinization that was inhibited by DIDS. pHi recovery from an intracellular alkaline load (isohydric CO2 changes) was Cl(-)-dependent and DIDS-inhibitable. Administration of agents raising intracellular cAMP concentrations increased both Na(+)-dependent and Na(+)-independent Cl-/HCO-3 exchange activity. Stimulation of Cl-/HCO3- exchange activity was not prevented by the Cl- channel inhibitor 5'-nitro-2(2)-phenylpropyl-amino-benzoate(NPPB). In conclusion, human cholangiocytes possess two acid extruders (Na+/H+exchanger and Na(+)-dependent Cl-/HCO3- exchange) and an acid loader (Cl-/HCO3- exchange), whereas no evidence was found for cAMP activated H(+)-ATPase. Bicarbonate influx is thus mainly mediated by Na-dependent Cl-/HCO3- exchange, whereas Na+:HCO-3 cotransport is not active in the physiological range of pHi. Stimulation of Na(+)-independent Cl-/HCO3- exchanger by cAMP does not require activation of Cl- conductances. These mechanisms may underlay hormone-regulated biliary HCO3- secretion in the human biliary tree.     

Changes in cytosol Ca(2+)-, Mg(2+)-, H(+)-, N(+)-, and K(+)-concentrations in cultivated hepatocytes in hypoxic conditions

AIM: It is assumed that disturbances of cellular ion homeostasis, especially an increase in the cytosolic Ca2+ concentration, are of decisive importance for hypoxic cell injury. The aim of this study is the determination of alterations in the cytosolic Ca2+, Mg2+, H+, Na+ and K+ concentration in cultured hepatocytes during hypoxia. METHODS: The alterations of ion homeostasis under hypoxic conditions were studied in primary cultures of isolated rat hepatocytes by using fluorescence microscopy. RESULTS: The measurements of cytosolic Ca2+ concentration showed no alterations during the first 3-4 h of hypoxia. About 1-2 h before cell injury became evident Ca2+ increased from 147 +/- 28 to 385 +/- 31 nM. Similarly the cytosolic Mg2+ concentration increased from 0.63 +/- 0.05 to 1.42 +/- 0.11 mM in a late stage of hypoxia. In contrast, the cytosolic Na+ concentration increased continuously from 16 +/- 2 mM at start to 76 +/- 9 mM after 5 h of hypoxic conditions. The cytosolic K+ concentration remained constant for 2 h (129 +/- 7 mM) but then decreased down to 31 +/- 18 mM. The intracellular H+ concentration increased slightly under hypoxic conditions, the pH decreased from 7.35 +/- 0.02 to 7.19 +/- 0.04. CONCLUSION: The results indicate that cytosolic Ca2+ plays only a minor role in the pathomechanism of hypoxic hepatocellular injury but suggest an important role of the cytosolic Na+ concentration in this process.     

Effects of sodium bicarbonate on striated muscle metabolism and intracellular pH during endotoxic shock

The effects of HCO3Na load on acid-base balance and muscle intracellular bioenergetics have been investigated using 31P-magnetic resonance spectroscopy in an experimental model of endotoxinic shock. Anesthetized, mechanically ventilated, and paralyzed rats (n = 16) were given an intravenous bolus of Escherichia coli lipopolysaccharide (15 mg/kg). When shock was established they were randomly assigned to receive either HCO3Na intravenously (2 mmol/kg in 2 min) or an equimolar saline injection. Lipopolysaccharide induced a significant decrease in the levels of mean arterial pressure (58 +/- 6 vs. 120 +/- 8 mmHg), arterial pH (7.20 +/- .03 vs. 7.35 +/- .01), intracellular pH (6.86 +/- .04 vs. 7.08 +/- .01), a marked hyperlactatemia (7 +/- 3 vs. 1.2 +/- .2 mmol/L) and a drop in the phosphocreatine-inorganic phosphate ratio. In the bicarbonate-loaded rats, mean arterial pressure further decreased whereas it remained unchanged in the saline group. Bicarbonate increased arterial pH and PaCO2 transiently. In the saline group, arterial pH decreased and PaCO2 remained stable. In both groups, intracellular pH and high energy phosphates had a similar evolution. In this model of septic shock, partial correction of arterial pH using HCO3Na did not reduce the metabolic cellular injury in skeletal muscle. Based on these results, HCO3Na may be of limited therapeutic value in severe septic metabolic acidosis.     

Glucose-induced changes in Na+/H+ antiport activity and gene expression in cultured vascular smooth muscle cells. Role of protein kinase C

Increased Na+/H+ antiport activity has been implicated in the pathogenesis of hypertension and vascular disease in diabetes mellitus. The independent effect of elevated extracellular glucose concentrations on Na+/H+ antiport activity in cultured rat vascular smooth muscle cells (VSMC) was thus examined. Amiloride-sensitive 22Na+ uptake by VSMC significantly increased twofold after 3 and 24 h of exposure to high glucose medium (20 mM) vs. control medium (5 mM). Direct glucose-induced Na+/H+ antiport activation was confirmed by measuring Na(+)-dependent intracellular pH recovery from intracellular acidosis. High glucose significantly increased protein kinase C (PKC) activity in VSMC and inhibition of PKC activation with H-7, staurosporine, or prior PKC downregulation prevented glucose-induced increases in Na+/H+ antiport activity in VSMC. Northern analysis of VSMC poly A+ RNA revealed that high glucose induced a threefold increase in Na+/H+ antiport (NHE-1) mRNA at 24 h. Inhibiting this increase in NHE-1 mRNA with actinomycin D prevented the sustained glucose-induced increase in Na+/H+ antiport activity. In conclusion, elevated glucose concentrations significantly influence vascular Na+/H+ antiport activity via glucose-induced PKC dependent mechanisms, thereby providing a biochemical basis for increased Na+/H+ antiport activity in the vascular tissues of patients with hypertension and diabetes mellitus.     

Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance

Osteoclasts resorb bone by secreting protons into an extracellular resorption zone through vacuolar-type proton pumps located in the ruffled border. The present study was undertaken to evaluate whether proton pumps also contribute to intracellular pH (pHi) regulation. Fluorescence imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit osteoclasts. The fluorescence of single osteoclasts cultured on glass coverslips and loaded with a pH-sensitive indicator was measured in nominally HCO(3-)-free solutions. When suspended in Na(+)-rich medium, the cells recovered from an acute acid load primarily by means of an amiloride-sensitive Na+/H+ antiporter. However, rapid recovery was also observed in Na(+)-free medium when K+ was used as the substitute. Bafilomycin-sensitive, vacuolar-type pumps were found to contribute marginally to pH regulation and no evidence was found for K+/H+ exchange. In contrast, pHi recovery in high K+ medium was largely attributed to a Zn(2+)-sensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configuration. Depolarizing pulses induced a slowly developing outward current and a concomitant cytosolic alkalinization. Determination of the reversal potential during ion substitution experiments indicated that the current was due to H+ (equivalent) translocation across the membrane. The H+ current was greatly stimulated by reducing pHi, consistent with a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data indicate the presence of a pH- and membrane potential-sensitive H+ conductance in the plasma membrane of osteoclasts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.     

Heterogeneity of intracellular pH and of mechanisms that regulate intracellular pH in populations of cultured cells

Cells within solid tumors are known to exist in a microenvironment that may be acidic and depend on membrane-based mechanisms (Na+/H+ antiport and Na+-dependent Cl-/HCO3- exchanger) that regulate intracellular pH (pHi). We have used the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl) 5 (and 6)-carboxyfluorescein and flow cytometry to study the distribution of pHi and the activity of these pHi-regulating mechanisms among populations of murine mammary sarcoma (EMT6), human breast cancer (MCF-7), and Chinese hamster ovary cells exposed to different levels of extracellular pH (pHe). Cells were exposed to Na+ buffer in the presence or absence of HCO3- and of 5-(N-ethyl-N-isopropyl)-amiloride (a potent inhibitor of the Na+/H+ antiport) to determine the relative importance of each exchanger in the regulation of pHi. Our results indicate that: (a) the distribution of pHi at any value of pHe is broader than can be accounted for by machine noise; (b) cells maintain levels of pHi that are higher than pHe under acidic conditions; (c) the distribution of pHi is narrower when the Na+-dependent Cl-/HCO3- exchanger is active; and (d) populations that are derived from selected cells with values of pHi at lower and higher ends of the pHi distribution generate pHi distributions that are similar to those of controls, suggesting a stochastic variation in the activity of membrane-based mechanisms that regulate pHi. Our data suggest that the Na+-dependent Cl-/HCO3- exchanger is the dominant mechanism for regulation of pHi under moderately acidic conditions such as may occur in the microenvironment of solid tumors.     

Effect of short-chain fatty acids on cell volume and intracellular pH in rat distal colon

Superfusion of isolated crypts from the rat colon with sodium-butyrate-containing solutions induced an increase in the crypt diameter indicating a swelling of the crypt cells. The response to butyrate (50 mmol l-1) was not uniform along the crypt axis, the most pronounced swelling being observed in the upper third of the crypt. The butyrate effect was concentration-dependent and was completely suppressed by amiloride, suggesting that it is caused by activation of the Na+/H+ exchanger. Acetate, propionate and isobutyrate had a similar action. In HEPES-buffered solution the butyrate-induced change in cell volume was monophasic, i. e. only a swelling took place, whereas in HCO3- buffer it was biphasic, i. e. swelling was followed by a regulatory volume decrease. This decrease was suppressed by K+ and Cl- channel blockers as well as inhibitors of leukotriene synthesis. Measurements of intracellular pH with the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) revealed that butyrate induced an acidification of the cell, which was stronger in HEPES than in HCO3- buffer. Estimation of Na+/H+ exchange activity, tested as recovery of intracellular pH from an acid load via an NH4Cl prepulse, revealed a much lower Na+/H+ exchange activity in the fundus region compared to the upper third of the crypt. The smaller volume response evoked by butyrate in the fundus region probably reflects the smaller Na+/H+ activity compared to the more differentiated cells near the opening of the crypt. It is concluded that cell swelling caused by short-chain fatty acids is a physiological stimulus for volume regulation. This response is restricted to the more differentiated cells.     

Expression cloning and characterization of a renal electrogenic Na+/HCO3- cotransporter

Bicarbonate transporters are the principal regulators of pH in animal cells, and play a vital role in acid-base movement in the stomach, pancreas, intestine, kidney, reproductive system and central nervous system. The functional family of HCO3- transporters includes Cl- -HCO3- exchangers, three Na+/HCO3- cotransporters, a K+/HCO3- cotransporter, and a Na+-driven Cl- -HCO3- exchanger. Molecular information is sparse on HCO3- transporters, apart from Cl- -HCO3- exchangers ('anion exchangers'), whose complementary DNAs were cloned several years ago. Attempts to clone other HCO3- transporters, based on binding of inhibitors, protein purification or homology with anion exchangers, have so far been unsuccessful. Here we monitor the intracellular pH and membrane voltage in Xenopus oocytes to follow the expression of the most electrogenic transporter known: the renal 1:3 electrogenic Na+/HCO3- cotransporter from the salamander Ambystoma tigrinum. We now report the successful cloning and characterization of a cDNA encoding a cation-coupled HCO3- transporter. The encoded protein is 1,035 amino acids long with several potential membrane-spanning domains. We show that when it is expressed in Xenopus oocytes, this protein is electrogenic, Na+ and HCO3- dependent, and blocked by the anion-transport inhibitor DIDS, and conclude that it is the renal electrogenic sodium bicarbonate cotransporter (NBC).     

The Na+/H+ antiporter potentiates growth and retinoic acid-induced differentiation of P19 embryonal carcinoma cells

The Na+/H+ exchanger is a ubiquitous plasma membrane protein that is responsible for pH regulation and is activated by growth factors. We examined the role of the Na+/H+ exchanger in cell growth and differentiation. Treatment of P19 cells with the Na+/H+ exchanger inhibitor Hoe 694 eliminated retinoic acid-induced differentiation in this cell line. We developed a P19 embryonal carcinoma cell line that was deficient in the Na+/H+ antiporter. Na+/H+ exchanger-deficient cells were reduced in the rate of cell growth and this effect was enhanced by the removal of added HCO3- and by reducing extracellular pH. The antiporter-deficient cells were also markedly deficient in their ability to differentiate to neuronal-like cells and recovered this ability when the Na+/H+ antiporter was reintroduced. The results show that the absence of Na+/H+ antiport as a pH regulatory mechanism can result in deficiencies in both cell growth and differentiation in embryonal carcinoma cells.     

NO3--induced pH changes in mammalian cells. Evidence for an NO3--H+ cotransporter

The effect of NO3- on intracellular pH (pHi) was assessed microfluorimetrically in mammalian cells in culture. In cells of human, hamster, and murine origin addition of extracellular NO3- induced an intracellular acidification. This acidification was eliminated when the cytosolic pH was clamped using ionophores or by perfusing the cytosol with highly buffered solutions using patch-pipettes, ruling out spectroscopic artifacts. The NO3-- induced pH change was not due to modulation of Na+/H+ exchange, since it was also observed in Na+/H+ antiport-deficient mutants. Though NO3- is known to inhibit vacuolar-type (V) H+-ATPases, this effect was not responsible for the acidification since it persisted in the presence of the potent V-ATPase inhibitor bafilomycin A1. NO3-/HCO3- exchange as the underlying mechanism was ruled out because acidification occurred despite nominal removal of HCO3-, despite inhibition of the anion exchanger with disulfonic stilbenes and in HEK 293 cells, which seemingly lack anion exchangers (Lee, B. S., R.B. Gunn, and R.R. Kopito. 1991. J. Biol. Chem. 266:11448- 11454). Accumulation of intracellular NO3-, measured by the Greiss method after reduction to NO2-, indicated that the anion is translocated into the cells along with the movement of acid equivalents. The simplest model to explain these observations is the cotransport of NO3- with H+ (or the equivalent counter-transport of NO3- for OH-). The transporter appears to be bi-directional, operating in the forward as well as reverse directions. A rough estimate of the fluxes of NO3- and acid equivalents suggests a one-to-one stoichiometry. Accordingly, the rate of transport was unaffected by sizable changes in transmembrane potential. The cytosolic acidification was a saturable function of the extracellular concentration of NO3- and was accentuated by acidification of the extracellular space. The putative NO3--H+ cotransport was inhibited markedly by ethacrynic acid and by alpha-cyano-4-hydroxycinnamate, but only marginally by 4, 4'-diisothiocyanostilbene-2,2' disulfonate or by p-chloromercuribenzene sulfonate. The transporter responsible for NO3--induced pH changes in mammalian cells may be related, though not identical, to the NO3--H+ cotransporter described in Arabidopsis and Aspergillus. The mammalian cotransporter may be important in eliminating the products of NO metabolism, particularly in cells that generate vast amounts of this messenger. By cotransporting NO3- with H+ the cells would additionally eliminate acid equivalents from activated cells that are metabolizing actively, without added energetic investment and with minimal disruption of the transmembrane potential, inasmuch as the cotransporter is likely electroneutral.     

Luminal acid increases apical cell membrane resistance in isolated Necturus antral mucosa

BACKGROUND & AIMS: The gastric mucosa must have efficient protective mechanisms to maintain physiological intracellular pH. The aim of this study was to investigate the effect of low luminal pH on apical membrane permeability. METHODS: Chambered Necturus antral mucosa was perfused with Ringer's/95% O2-5% CO2 at pH 7.25. The mucosal side was exposed to pH 4.0-2.0 with four microelectrodes placed in surface cells. Two-dimensional cable analysis was used to measure apical, basolateral, and shunt resistances. In some experiments, liquid sensor pH or Na(+)-selective microelectrodes were used. RESULTS: Luminal acidification hyperpolarized apical cell membrane potential and increased apical cell membrane resistance from 21.3 +/- 2.6 (pH 7.25) to 38.0 +/- 2.3 k omega.cm2 (pH 3.0; n = 8). The increase in apical cell membrane resistance was preceded by transient intracellular acidosis from 7.32 +/- 0.07 (pH 4.0) to 7.23 +/- 0.06 (pH 3.0; n = 6). Similar intracellular acidosis (provoked by NH4+ prepulse) failed to cause the effects observed with luminal acid. The increase in apical cell membrane resistance caused by luminal acid was eliminated when N-methyl-D-glucamine+, but not Na+, was substituted for all cations in the luminal solution. CONCLUSIONS: Luminal acidification (pH 3.0-2.0) closes apical amiloride-blockable Na+ channels. Protons are probably able to pass and even block these channels, but their effect in closing the channels does not occur intracellularly.     

Effect of SCFA on intracellular pH and intracellular pH regulation of guinea-pig caecal and colonic enterocytes and of HT29-19a monolayers

The response of the intracellular pH (pHi, measured with BCECF) of the caecal and distal colonic epithelium of guinea pig and of monolayers of HT29 clone 19a cells on the addition of short-chain fatty acids (SCFA) was assessed. Addition of SCFA to the luminal side of these cells had no major effect on pHi, independent of whether the apical Na+/H+ exchange or the apical K+/H+ ATPase was inhibited or not. Addition of SCFA to the serosal side, on the other hand, caused a marked decrease of pHi, followed by an effective regulation back to basal values, and after removal of the acid, the cells became alkalinized. Intracellular pH is mainly regulated by mechanisms in the basolateral membrane. The basolateral Na+/H+ exchanger and the Cl-/HCO3- exchanger were mainly responsible for pHi regulation. Inhibition studies are consistent with a NHE-1 type Na+/H+ exchanger in the basolateral membranes. The apical Na+/H+ exchanger of caecal enterocytes and in HT29 cells, and the apical K+/H+ ATPase in the apical membrane of the distal colon have no or little influence on pHi regulation. The comparison shows that the HT29-19a cell line is an adequate model for studying pHi phenomena of hind gut epithelial cells.     

Effects of HCO3- on cell composition of rabbit ciliary epithelium: a new model for aqueous humor secretion

PURPOSE: To determine whether the Na+-K+-2Cl- symport or the parallel Na+/H+ and Cl-/HCO3- antiports provide the dominant pathway for NaCl uptake into the ciliary epithelium. Both pathways are known to support NaCl entry from the stroma into the pigmented ciliary epithelial (PE) cells, after which Na+ and Cl- diffuse across the gap junctions into the nonpigmented ciliary epithelial (NPE) cells and are released into the aqueous humor. METHODS: Rabbit iris ciliary bodies were preincubated in HCO3-/CO2-containing or HCO3-/CO2-free solutions before quick freezing, cryosectioning, dehydration, and electron probe x-ray microanalysis. RESULTS: The NPE and the PE cells contained more K and Cl when incubated with bicarbonate. Inhibition of carbonic anhydrase with 0.5 mM acetazolamide had little effect in HCO3--free medium but prevented the increase in Cl in both cell types in HCO3-/CO2 solution. Inhibition of the Na+-K+-2Cl- symport with 10 to 500 microM bumetanide caused Cl loss from both cell types in HCO3--free solution, but bumetanide produced a paradoxical increase in Cl and Na in HCO3-/CO2 solution. Together, acetazolamide and bumetanide resulted in significant Cl loss in HCO3--free solution and prevented the gains of Cl and Na in HCO3-/CO2 solution. CONCLUSIONS: The present results indicate that the dominant entry pathway of NaCl from the stroma into the ciliary epithelial syncytium is through an acetazolamide-inhibitable Cl-/HCO3 and a parallel Na+/H+ antiport. The dominant release pathways into the aqueous humor appear to be a Na+-K+-2Cl-symport, which can be outwardly directed under physiological conditions, together with the Na+/K+-exchange pumps and Cl- channels.     

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.     

Uptake of 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate into isolated rat hepatocytes by three transport systems

Uptake of norcholansulfonate (3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate), an isogeometric analogue of cholate into isolated rat liver hepatocytes occurs only by saturable transport. In order to identify the transport systems involved, uptake of norcholansulfonate was studied using 7 beta-NBD-NCT ({N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl})-2'-aminoethanesulfonate) as a competing substrate. For transport of both bile salt derivatives, which mutually inhibit their mediated transport competitively, the existence of at least three transport systems must be assumed. Uptake studies using the cloned hepatic Na+/cholyltaurine cotransporting polypeptide stably expressed in CHO cells (Chinese hamster ovary cells) showed that both bile salt derivatives were transported and furnished the definite KT values of this single transport system and the ratio of the maximal uptake velocities. On the basis of these data, uptake of both bile salt derivatives into rat hepatocytes and their mutual competitive inhibition could be analyzed for three transport systems. The maximal flux rates J2 and the half-saturation constants KT2 in the presence of Na+ (143 mM) are for norcholansulfonate: J1(Na+ 143) = 1.0 +/- 0.2 nmol/(min . mg protein), KT1(Na+ 143) = 15 +/- 4 microM, J2(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT2(Na+ 143) = 15 +/- 2 microM, J3(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT3(Na+ 143) = 60 +/- 15 microM, and for 7 beta-NBD-NCT J1(Na+ 143) = 0.14 +/- 0.04 nmol/(min.mg protein), KT1(Na+ 143) = 3.1 +/- 0.5 microM, J2(Na+ 143) = 0.014 +/- 0.005 nmol/(min.mg protein), KT2(Na+ 143) = 21 +/- 2 microM, J3(Na+ 143) = 1.0 +/- 0.1 nmol/(min.mg protein), KT3(Na+ 143) = 190 +/- 25 microM. The kinetic parameters are in accordance with the assumptions that the cloned Na+/cholyltaurine cotransporting polypeptide represents transport system 2 and that the kinetically identified additional transport system 1 is either strictly or partially Na(+)-dependent.     

pH dependence of corneal oxygen consumption

PURPOSE: To determine whether corneal acidosis, which occurs during contact lens wear, alters corneal O2 consumption (QO2) and if so, whether increased ion transport activity could contribute to altered QO2 during acidosis. METHODS: PO2 was measured, using the phosphorescence quenching of Pd-meso-tetra-(4-carboxyphenyl) porphine, in an airtight chamber that held a trephined rabbit cornea. The rate of change in chamber PO2 was used as a measure of QO2. QO2 was measured at pH 7.5 and then at either pH 6.7, 7.1, or 7.3. Measurements of QO2 at pHs 7.5 and 6.7 were repeated in the presence of 0.5 mM amiloride and 0.5 mM ouabain. RESULTS: When pH was changed from 7.5 to 6.7, 7.1, or 7.3, O2 consumption increased by a factor of 1.80+/-0.11 (+/-SE), 1.65+/-0.12, and 1.44+/-0.06, respectively. Amiloride (0.5 mM) and ouabain (0.5 mM) inhibited 50% and 65%, respectively, of the increase in QO2 at pH 6.7. CONCLUSIONS: Corneal acidosis leads to increased QO2 in a dose-dependent manner. The increased QO2 is in part secondary to the activation of pH regulatory mechanisms, including Na+/H+ exchange, which then stimulates Na+/ K+-ATPase activity. These findings indicate that contact lens-induced acidosis can exacerbate corneal hypoxia and related complications.     

Proton secretion in the upper part of the descending limb of long-looped nephron from hamsters

The proton transport processes in the upper part of the descending limb of the long-looped nephron (LDLu) from hamsters were studied using a fluorescent dye, 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF) in microperfused single nephron preparations. Intracellular pH (pHi), as assessed by the measurement of the fluorescence of BCECF trapped in the cytoplasm, was 7.23 +/- 0.05 (n = 18) under nominally HCO3--free conditions. Ouabain, when added to the bath, decreased pHi by 0.22 units. After an NH4Cl prepulse, the initial proton extrusion rate was 1.23 +/- 0.26 (n = 9) pH units/min, and was retarded in the presence of 1 mM amiloride either in the bath or in the lumen. pHi failed to recover when Na+ was eliminated from ambient solutions. These observations suggest that Na+/H+ antiporters exist both in the apical and basolateral cell membranes. By measuring tubular fluid pH (pHt) under stopped flow conditions, we examined whether the hamster LDLu has the capacity to generate and maintain a transmural H+ gradient. After the tubular outflow was obstructed, the luminal fluid was rapidly acidified, reaching a steady-state pH of 6.84 +/- 0.09 (n = 7). The steady-state pH was influenced by bath pH. Tubular fluid acidification was not observed in the absence of Na+ and was prevented by ouabain. We conclude that the hamster LDLu has the capability to generate and maintain a transmural proton gradient by proton secretion via a luminal Na+/H+ antiporter which is secondarily driven by the Na+-K+ ATPase in the basolateral membrane.