Intracellular pH regulation in hepatocytes isolated from three teleost species.

The mechanisms of intracellular pH (pH(i)) regulation were studied in hepatocytes isolated from three species of teleost: rainbow trout (Oncorhynchus mykiss), black bullhead (Ameiurus melas) and American eel (Anguilla rostrata). Intracellular pH was monitored over time using the pH-sensitive fluorescent dye BCECF in response to acid loading under control conditions and in different experimental media containing either low Na(+) or Cl(-) concentrations, the Na(+)-H(+) exchanger blocker amiloride or the blocker of the V-type H(+)-ATPase, bafilomycin A(1). In trout and bullhead hepatocytes, recovery to an intracellular acid load occurred principally by way of a Na(+)-dependent amiloride-sensitive Na(+)-H(+) exchanger. In eel hepatocytes, the Na(+)-H(+) exchanger did not contribute to recovery to an acid load though evidence suggests that it is present on the cell membrane and participates in the maintenance of steady-state pH(i). The V-type H(+)-ATPase did not participate in recovery to an acid load in any species. A Cl(-)-HCO(3)(-) exchanger may play a role in recovery to an acid load in eel hepatocytes by switching off and retaining base that would normally be tonically extruded. Thus, it is clear that hepatocytes isolated from the three species are capable of regulating pH(i), principally by way of a Na(+)-H(+) exchanger and a Cl(-)-HCO(3)(-) exchanger, but do not exploit identical mechanisms for pH(i) recovery. J. Exp. Zool. 284:361-367, 1999.     

Regulation of pH in individual pancreatic beta-cells as evaluated by fluorescence ratio microscopy

Pancreatic beta-cells are known to maintain intracellular pH (pHi) at a value well above that predicted from the electrochemical gradient. The mechanisms for the active extrusion of protons were examined by continuously monitoring pHi in individual beta-cells from ob/ob mice using the fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In a medium nominally devoid of bicarbonate, the steady-state pHi was 6.82 +/- 0.02 and the intracellular buffering capacity was equivalent to 79 +/- 3 mM/pH unit. pHi remained unaffected after raising the glucose concentration from 3 to 20 mM, it was lowered when depolarizing the beta-cells with tolbutamide and it increased in the presence of carbachol. After removal of Na+ there was a significant drop of pHi and blockage of the pHi recovery following acid loading with the NH4+ prepulse technique. Whereas addition of amiloride had a similar, but less pronounced effect, omission of Cl- resulted in moderate alkalinisation. After switching to a medium containing bicarbonate, minor acidification was followed by adjustment of pHi to a steady state higher than the initial one. The results indicate that the acid load arising from glucose metabolism in the beta-cells is effectively buffered and the protons extruded both by Na+-H+ and Cl- -HCO3- exchangers.     

Intracellular pH-regulatory mechanisms in pancreatic acinar cells. I. Characterization of H+ and HCO3- transporters

Rat pancreatic acini loaded with the pH sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to characterize intracellular pH (pHi) regulatory mechanisms in these cells. The acini were attached to cover slips and continuously perfused. In 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered solutions recovery from acid load (H+ efflux) required extracellular Na+ (Na+out) and was blocked by amiloride. Likewise, H+ influx initiated by removal of Na+out was blocked by amiloride. Hence, in HEPES-buffered medium the major operative pHi regulatory mechanism is a Na+/H+ exchange. In HCO3(-)-buffered medium, amiloride only partially blocked recovery from acid load and acidification due to Na+out removal. The remaining fraction required Na+out, was inhibited by H2-4,4'-diisothiocyanostilbene-2,2'-disulfunic acid (H2DIDS) and was independent of C1-. Hence, a transporter with characteristics of a Na(+)-HCO3- cotransport exists in pancreatic acini. Measurement of pHi changes due to Na(+)-HCO3- cotransport, suggests that the transporter contributes to HCO3- efflux under physiological conditions. Changing the Cl- gradient across the plasma membrane of acini maintained in HCO3(-)-buffered solutions reveals the presence of an H2DIDS-sensitive, Na(+)-independent, Cl(-)-dependent, HCO3- transporter with characteristics of a Cl-/HCO3- exchanger. In pancreatic acini the exchanger transports HCO3- but not OH- and under physiological conditions functions to remove HCO3- from the cytosol. In summary, only the Na+/H+ exchanger is functional in HEPES-buffered medium to maintain pHi at 7.28 +/- 0.03. In the presence of 25 mM HCO3- at pHo of 7.4, all the transporters operate simultaneously to maintain a steady-state pHi of 7.13 +/- 0.04.     

Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium

BACKGROUND: The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+)/HCO(3)(-) cotransporter in the pH regulation in rat epididymis. METHOD/PRINCIPAL FINDINGS: Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH) solution, the intracellular pH (pHi) recovery from NH(4)Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+)/H(+) exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO(3)(-) buffered would cause another pHi recovery. The pHi recovery in HCO(3)(-) buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), the inhibitor of HCO(3)(-) transporter or by removal of extracellular Na(+). The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH. CONCLUSIONS: The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.     

Direct measurement of intracellular pH in identified glial cells and neurones of the leech central nervous system

Neutral carrier pH-sensitive double-barrelled microelectrodes were used to investigate intracellular pH (pHi) in leech neuropile glial cells and in Retzius neurones. The mean pHi of the glial cells was 6.87 +/- 0.13 (+/- SD, n = 27) in HEPES-buffered saline (pHo 7.4) and 7.18 +/- 0.19 (n = 13) in solutions buffered with 2% CO2- 11 mM HCO3-. The distribution of H+ ions in both the glia and neurones was found not to be in electrochemical equilibrium. To investigate pHi regulation, the pHi was decreased by exposure to CO2 or by adding and then removing NH4Cl. Acidification by any method was followed by a recovery to normal pHi values within minutes. The pHi recovery from acidification in neuropile glial cells in HEPES-buffered saline and CO2-HCO3- buffered saline was, however, blocked by removing external Na. In HCO3(-)-free solutions the diuretic amiloride (2 mM) reduced the rate of pHi recovery. In the presence of HCO3-, the rate of acid efflux was stimulated; the stilbene 4-acetamido-4'-isothiocyanatostilbene-2,3'-disulfonic acid (SITS; 0.5 mM) slowed pHi recovery. In HEPES buffered and CO2-HCO3- buffered solutions pHi regulation in neurones was inhibited by removing external Na. In HCO3(-)-free solutions amiloride reduced the rate of pHi recovery considerably. In the presence of HCO3-, SITS or amiloride slowed but did not completely block pHi recovery. We conclude that leech glial cells and neurones have two mechanisms of pHi regulation, one being Na+-H+ exchange and the other Na+ and HCO3- dependent.     

Regulation of intracellular pH in sheep cardiac Purkinje fibre: interactions among Na+, H+, and Ca2+1

Experiments were performed on sheep cardiac Purkinje fibres using pH- and sodium-selective microelectrodes, while simultaneously measuring tension, to determine if the fall in intracellular pH (pHi) following a rise in intracellular Na+ activity (aiNa) is caused by inhibition or reversal of acid extrusion on Na+-H+ exchange. A rise in aiNa was induced either by using the cardioactive steroid strophanthidin to inhibit the sarcolemmal Na+-K+ pump or by increasing the frequency of stimulation (0-4 Hz). Both of these manoeuvres led to an increase in aiNa and a decrease in pHi. Following exposure to strophanthidin, amiloride (an inhibitor of sarcolemmal Na+-H+ exchange) produced a decrease in both pHi and aiNa. These effects of amiloride increased with decreasing pHi, indicating that acid extrusion on Na+-H+ exchange is stimulated by the fall in pHi. The changes in intracellular Na+ and H+ caused by amiloride were quantitatively consistent with an electroneutral stoichiometry. The fall in pHi during strophanthidin exposure is therefore not caused by inhibition or reversal of acid extrusion Na+-H+ exchange. It is likely that the fall in pHi during a rate increase is also independent of Na+-H+ exchange. This is because (i) it has been shown previously to occur in the presence of amiloride and (ii) the calcium antagonist D600 completely abolished the stimulation-dependent fall in pHi. It is concluded that the intracellular acidosis following inhibition of the sarcolemmal Na+-K+ pump or following an increase in the rate of stimulation is secondary to a rise in intracellular calcium.     

Na+-independent Cl(-)-HCO-3- exchange in Madin-Darby canine kidney cells. Role in intracellular pH regulation

The role of plasma membrane Cl(-)-HCO-3-exchange in regulating intracellular pH (pHi) was examined in Madin-Darby canine kidney cell monolayers. In cells bathed in 25 mM HCO-3, pH 7.4, steady state pHi was 7.10 +/- 0.03 (n = 14) measured with the fluorescent pH probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Following acute alkaline loading, pHi recovered exponentially in approximately 4 min. The recovery rate was significantly decreased by Cl- or HCO-3 removal and in the presence of 50 microM 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS). Na+ removal or 10(-3) M amiloride did not inhibit the pHi recovery rate after an acute alkaline load. Following acute intracellular acidification, the pHi recovery rate was significantly inhibited by 10(-3) M amiloride but was not altered by Cl- removal or 50 microM DIDS. At an extracellular pH (pHo) of 7.4, pHi remained unchanged when the cells were bathed in either Cl- free media, HCO-3 free media, or in the presence of 50 microM DIDS. As pHo was increased to 8.0, steady state pHi was significantly greater than control in Cl(-)-free media and in the presence of 50 microM DIDS. It is concluded that Madin-Darby canine kidney cells possess a Na+-independent Cl(-)-HCO-3 exchanger with a Km for external Cl- of approximately 6 mM. The exchanger plays an important role in pHi regulation following an elevation of pHi above approximately 7.1. Recovery of pHi following intracellular acidification is mediated by the Na+/H+ antiporter and not the anion exchanger.     

Na+ effects on intracellular pH of isolated, perfused rabbit gastric mucosal surface cells.

The effects of extracellular Na+ on intracellular pH were studied by perfusing BCECF loaded gastric mucosal surface cells adherent to glass coverslips held in a spectrophotofluorometer. Removal of Na+ from a NaCl Ringer perfusate (pH 7.4) resulted in progressive intracellular acidification, which was partially blocked by amiloride. An H+ conductance did not appear to be present. Acidification induced either by Na+ removal or by a NH4 prepulse was reversed by extracellular Na+, but this effect was not completely prevented by amiloride. Amiloride significantly, but not completely, inhibited Na22 uptake by gastric mucosal surface cells. The data suggest that extracellular Na+ maintains intracellular pH of gastric mucosal surface cells through amiloride-sensitive and -insensitive pathways. In the absence of extracellular Na+, cellular acidification seemed to be partially due to Na+/H+ exchange.     

Cl--dependent and Cl--independent Na+/ HCO3- acid extrusion in cultured rat cerebellar astrocytes

It is still uncertain whether the Na+-dependent Cl--HCO3- exchanger (NCBE) is expressed in mammalian astrocytes. Using fluorescent indicators to monitor the intracellular pH (pHi) and intracellular Na+ or Cl- levels, the NCBE in cultured rat cerebellar astrocytes was examined in detail. In nominally bicarbonate-free (Hepes-buffered) medium, a marked pHi recovery from internal acid load was seen which could be blocked completely by 30 microM HOE 694, a specific Na+-H+ exchanger isoform 1(NHE-1) inhibitor, at a pHi above 6.9. These conditions were therefore used to block NHE activity in CO2/HCO3-buffered media when the NCBE was being studied at pHi above 6.9. After internal acid loading in completely Cl--free bicarbonate-buffered medium (containing HOE 694), the rates of pHi recovery and transient Na+ influx were considerably slowed, and the Cl--dependent acid extrusion was both Na+- and 4,4-diisothiocyano-stilbene-disulphonic acid (DIDS)-sensitive. Moreover, a HCO3-dependent Cl- efflux during internal acid injection was seen. These results suggest that the NCBE is present in astrocytes. Following repetitive internal acid loading by addition of 5% CO2 to internal Cl- depleted cells, a similar rate of pHi recovery was consistently seen, suggesting Cl--independent pHi regulation also occurred in astrocytes. Moreover, this pHi recovery was completely blocked in the absence of sodium or on addition of DIDS, confirming that the Na+-HCO3 cotransporter (NBC) is present. Thus, the present study provides evidence that both the NCBE and NBC play important roles in acid extrusion in cultured mammalian astrocytes.     

Intracellular pH regulation in ventral horn neurones cultured from embryonic rat spinal cord

Intracellular pH was measured with the pH-sensitive fluorescent probe BCECF in spinal cord neurones cultured from rat embryos. At an external pH of 7.3, the average steady-state pHi was 7.18 +/- 0.03 (SEM, n = 97) and 7.02 +/- 0.01 (n = 221) in HEPES-buffered and in bicarbonate-buffered medium, respectively. In both external media, pHi was strongly dependent on external pH (pHe). In HEPES-buffered medium, pHi recovery following an acid load induced by transient application of ammonium required external Na+ and was inhibited by amiloride, indicating the presence of a Na+/H+ exchange. Na(+)- and HCO3(-)-dependent, DIDS-sensitive alkalinizing mechanisms also contributed to pHi regulation in CO2/bicarbonate-buffered medium. The presence of an electrogenic Na(+)-HCO3- cotransporter was confirmed by the alkalinizing effect of KCl application. The fact that pHi is lower in CO2/bicarbonate- than in HEPES-buffered medium and the alkalinization observed upon suppression of external Cl- suggest that the acidifying Cl-/HCO3- transporter plays an important role in defining pHi.     

Extracellular ATP stimulates an amiloride-sensitive sodium influx in human lymphocytes

Extracellular ATP has been shown to increase the Na+ permeability of human lymphocytes by 3 to 12-fold. The kinetics of this ATP-induced response were studied by measuring 22Na+ influx into chronic lymphocytic leukemic lymphocytes incubated in low-sodium media without divalent cations. ATP-stimulated uptake of 22Na-ions was linear over 4 min incubation and this influx component showed a sigmoid dependence on ATP concentration. Hill analysis yielded a K1/2 of 160 microM and a n value of 2.5. The nucleotide ATP-gamma-S (1-2 mM) gave 30% of the permeability increase produced by ATP, but UTP (2 mM) and dTTP (2 mM) had no effect on 22Na influx. The amiloride analogs 5-(N-ethyl-N-isopropyl) amiloride and 5-(N,N-hexamethylene) amiloride, which are potent inhibitors of Na(+)-H+ countertransport, abolished 72-95% of the ATP-stimulated 22Na+ influx. However, the involvement of Na(+)-H+ countertransport in the ATP-stimulated Na+ influx was excluded by three lines of evidence. Sodium influx was stimulated 7-fold by extracellular ATP but only 2.4-fold by hypertonic conditions which are known to activate Na(+)-H+ countertransport. Addition of ATP to lymphocytes produced no change in intracellular pH when these cells were suspended in isotonic NaCl media. Finally ATP caused a membrane depolarization of lymphocytes which is inconsistent with stimulation of electroneutral Na(+)-H+ exchange. These data suggest that ATP acts cooperatively to induce the formation of membrane channels which allow increased Na+ influx by a pathway which is partially inhibited by amiloride and its analogs.     

Basolateral regulation of pHi in proximal tubules of avian loopless and long-looped nephrons in bicarbonate.

In isolated, nonperfused chicken proximal tubules from both loopless reptilian-type and long-looped mammalian-type nephrons, resting intracellular pH (pHi), measured with pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), was approximately 7.1 under control HCO3- conditions [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)/5 mM HCO3(-)-buffered medium with pH 7.4 at 37 degrees C] and was reduced to approximately 6.8 in response to NH4Cl pulse. The rate of recovery of pHi (dpHi/dt) from this level to the resting level in proximal tubules from both nephron types was (1) significantly reduced by the removal of Na+ or both Na+ and Cl- from the bath, and (2) unaffected by the removal of Cl- from the bath or the presence of a high K+ concentration or Ba2+ in the bath. In proximal tubules from long-looped mammalian-type, but not loopless reptilian-type, nephrons, dpHi/dt was significantly reduced by the addition of either 5-(N-ethyl-N-isopropyl) amiloride (EIPA) or 4,4'-diisothiocyanostilbene-2,2'disulfonate (DIDS) to the bath. These data suggest that a Na+/H+ exchanger and most likely a Na(+)-dependent Cl-/HCO3- exchanger are involved in basolateral regulation of pHi in mammalian-type nephrons whereas none of the commonly identified basolateral acid-base transporters appear to be involved in regulation of pHi in reptilian-type nephrons.     

Interaction of external H+ with the Na+-H+ exchanger in renal microvillus membrane vesicles

We examined the effects of external H+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. The initial rate of Na+ influx into vesicles with internal pH 6.0 was optimal at external pH 8.5 and was progressively inhibited as external pH was reduced to 6.0. A plot of 1/V versus [H+]o was linear and yielded apparent KH = 35 nM (apparent pK 7.5). In vesicles with internal pH 6.0 studied at external pH 7.5 or 6.6, apparent KNa was 13 or 54 mM, Ki for inhibition of Na+ influx by external Li+ was 1.2 or 5.2 mM, Ki for inhibition by external NH4+ was 11 or 50 mM, and Ki for inhibition by external amiloride was 7 or 25 microM, respectively. These findings were consistent with competition between each cation and H+ at a site with apparent pK 7.3-7.5. Lastly, stimulation of 22Na efflux by external Na+ (i.e. Na+-Na+ exchange) was inhibited as external pH was reduced from 7.5 to 6.0, also consistent with competition between external H+ and external Na+. Thus, in contrast with internal H+, which interacts at both transport and activator sites, external H+ interacts with the renal microvillus membrane Na+-H+ exchanger at a single site, namely the external transport site, where H+, Na+, Li+, NH4+, and amiloride all compete for binding.     

HCO3-secretion by bullfrog duodenum: dependence on nutrient Na+ during secretory stimulation

HCO3(-) secretion across in vitro duodenal mucosa of Rana catesbeiana was investigated under baseline conditions and during secretory stimulation. Baseline secretion was abolished by removal of CO2-HCO3(-)and reduced approximately 60% by removal of nutrient Na+, but was not sensitive to changes in Cl- or K+. Baseline secretion was not directly altered by exposure to 10(-3) M amiloride or 10(-3) M H2DIDS (dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the nutrient solution and only mildly reduced by acetazolamide. Following removal and restoration of Na+, recovery of secretion was impaired by exposure to acetazolamide (5 x 10(-4) M) or H2DIDS (5 x 10(-4) M) in the nutrient solution. Secretion stimulated by glucagon (10(-6) M) or 16,16-dimethyl prostaglandin E2 (10 microg.mL(-1)) was markedly attenuated by removal of Na+ or by exposure to H2DIDS, but secretion was not altered by acetazolamide (5 x 10(-4) M) or nutrient amiloride (1 mM). Thus, the HCO3(-) that is secreted under nonstimulated conditions derives partly from basolateral Na(+)-dependent uptake and partly from cellular CO2 hydration. Secretagogue-stimulated secretion by duodenal surface epithelium depends on stilbene-sensitive Na+(HCO3(-))n uptake across the basolateral membrane.     

Effects of nutrient and non-nutrient stimuli on cytosolic pH in cultured insulinoma (HIT-T15) cells

Intracellular pH (pHi) was measured in the insulin-secreting HIT-T15 cell line using the pH-sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF). It was observed that the addition of a weak acid (e.g., acetate or propionate) caused a rapid decrease in pHi, followed by a slower recovery to the resting pH value. Conversely the addition of N4Cl caused an increase in pHi followed by recovery. The addition of amiloride caused a fall in pHi; however, in this case no recovery to basal pH levels was observed. Subsequent addition of a weak acid caused a further fall in pHi with no recovery. The addition of glucose caused a transient acidification followed by alkalinization. When glucose was added to cells which had been pretreated with amiloride, the initial acidification was not followed by recovery or alkalinization. Addition of glyceraldehyde, alpha-ketoisocaproate, lactate or pyruvate to HIT cells also resulted in intracellular acidification followed by recovery. Similarly, depolarisation of HIT cells by treatment with high K+ or with Ba2+ was associated with a pronounced fall in pHi, followed by a gradual recovery. Insulin secretion from HIT cells was stimulated by glucose, glyceraldehyde, alpha-ketoisocaproate, lactate, pyruvate and KCl, whilst amiloride and weak acids exerted only modest effects in the absence of glucose, but amiloride in particular markedly potentiated glucose-induced insulin release. Thus, HIT cells appear to have an amiloride-sensitive mechanism for the extrusion of protons, probably Na+-H+ exchange. Whilst intracellular acidification appears to potentiate secretory responses to nutrient stimuli, it seems unlikely that the activation of HIT cells by these nutrients occurs as a result of intracellular acidification. The mechanisms by which various nutrient and non-nutrient stimuli might exert distinct effects on pHi are discussed.     

The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain

Cytosolic pH (pHi) was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxy-methyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound. pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 microM BCECF were estimated to have a pHi of 6.94 +/- 0.02 (mean +/- standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 +/- 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H+ with gramicidin and nigericin, pHi was estimated to be 7.19 +/- 0.03 (n = 12). With the latter method, pHi = 6.95 +/- 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pHi in synaptosomes was approximately 7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4Cl and then diluted into NH4Cl-free medium, pHi immediately acidified to a level of approximately 6.6. After the acidification, pHi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be approximately 50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (KI = 3 microM), and by abolition of the Nao/Nai gradient. pHi and its recovery after acidification were not affected by incubation in an HCO3-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of Cl with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H+ antiporter is the primary regulator of pHi in mammalian brain nerve terminals.     

Interactions between Na+ channels and Na+-HCO3- cotransporters in the freshwater fish gill MR cell: a model for transepithelial Na+ uptake

Isolated mitochondria-rich (MR) cells from the rainbow trout gill epithelium were subjected to intracellular pH (pH(i)) imaging with the pH-sensitive dye BCECF-AM. MR cells were categorized into two distinct functional subtypes based on their ability to recover pH(i) from an NH(4)Cl-induced acidification in the absence of Na(+). An apparent link between resting pH(i) and Na(+)-independent pH(i) recovery was made. We observed a unique pH(i) acidification event that was induced by extracellular Na(+) addition. This further classified the mixed MR cell population into two functional subtypes: the majority of cells (77%) demonstrated the Na(+)-induced pH(i) acidification, whereas the minority (23%) demonstrated an alkalinization of pH(i) under the same circumstances. The focus of this study was placed on the Na(+)-induced acidification and pharmacological analysis via the use of amiloride and phenamil, which revealed that Na(+) uptake was responsible for the intracellular acidification. Further experiments revealed that pH(i) acidification could be abolished when Na(+) was allowed entry into the cell, but the activity of an electrogenic Na(+)-HCO(3)(-) cotransporter (NBC) was inhibited by DIDS. The electrogenic NBC activity was supported by a DIDS-sensitive, Na(+)-induced membrane potential depolarization as observed via imaging of the voltage-sensitive dye bis-oxonol. We also demonstrated NBC immunoreactivity via Western blotting and immunohistochemistry in gill tissue. We propose a model for transepithelial Na(+) uptake occurring via an apical Na(+) channel linked to a basolateral, electrogenic NBC in one subpopulation of MR cells.     

Intracellular pH-regulatory mechanisms in pancreatic acinar cells. II. Regulation of H+ and HCO3- transporters by Ca2(+)-mobilizing agonists

Pancreatic acini loaded with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to examine the effect of Ca2(+)-mobilizing agonists on the activity of acid-base transporters in these cells. In the accompanying article (Muallen, S., and Loessberg, P. A. (1990) J. Biol. Chem. 265, 12813-12819) we showed that in 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES)-buffered medium the main pHi regulatory mechanism is the Na+/H+ exchanger, a while in HCO3(-)-buffered medium pHi is determined by the combined activities of a Na+/H+ exchanger, a Na(+)-HCO3- cotransporter and a Cl-/HCO3- exchanger. In this study we found that stimulation of acini with Ca2(+)-mobilizing agonists in HEPES or HCO3(-)-buffered media is followed by an initial acidification which is independent of any identified plasma membrane-located acid-base transporting mechanism, and thus may represent intracellularly produced acid. In HEPES-buffered medium there was a subsequent large alkalinization to pHi above that in resting cells, which could be attributed to the Na+/H+ exchanger. Measurements of the rate of recovery from acid load indicated that the Na+/H+ exchanger was stimulated by the agonists. In HCO3(-)-buffered medium the alkalinization observed after the initial acidification was greatly attenuated. Examination of the activity of each acid-base transporting mechanism in stimulated acini showed that in HCO3(-)-buffered medium: (a) recovery from acid load in the presence of H2-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS) (Na+/H+ exchange) was stimulated similar to that found in HEPES-buffered medium; (b) recovery from acid load in the presence of amiloride and acidification due to removal of external Na+ in the presence of amiloride (HCO3- influx and efflux, respectively, by Na(+)-HCO3- cotransport) were inhibited; and (c) HCO3- influx and efflux due to Cl-/HCO3- exchange, which was measured by changing the Cl- or HCO3- gradients across the plasma membrane, were stimulated. Furthermore, the rate of Cl-/HCO3- exchange in stimulated acini was higher than the sum of H+ efflux due to Na+/H+ exchange and HCO3- influx due to Na(+)-HCO3- cotransport. Use of H2DIDS showed that the latter accounted for the attenuated changes in pHi in HCO3(-)-buffered medium, as much as treating the acini with H2DIDS resulted in similar agonist-mediated pHi changes in HEPES- and HCO3(-)-buffered media. The effect of agonists on the various acid-base transporting mechanisms is discussed in terms of their possible role in transcellular NaCl transport, cell volume regulation, and cell proliferation in pancreatic acini.     

Hypertonicity-induced intracellular pH changes in rat mast cells

In a non-isotonic environment, cells can shrink or swell and return to their normal shape by activating ion transport pathways. Changes in intracellular pH (pHi) after osmotic stress have been identified in several cells. In order to study the mechanisms that regulate cytosolic pH of rat mast cells in a hypertonic medium, we used the pH sensitive dye, BCECF. Under these hypertonic conditions, pHi undergoes an alkalinization following an initial acidification. The alkalinization is mediated by a Na+/H+ exchanger, since it is inhibited by amiloride and lack of extracellular sodium. Under these conditions, the alkalinization is increased with the PKC activators, TPA and OAG, and partially blocked with trifluoperazine, an unspecific protein kinase C (PKC) and Ca2+ calmodulin-dependent protein kinases (Ca2+/CaM K) inhibitor. There is also an anion exchanger, blocked with DIDS but not activated by PKC, that participates in the observed alkalinization. However, Na+/H+ exchanger is the main mechanism involved in the alkalinization of pHi of mast cells in a hyperosmotic environment.