Alkaline pHi Research Articles

Microfluorimetric imaging study of the mechanism of activation of the Na+/H+ antiport by muscarinic agonist in rat mandibular acinar cells.

The mechanism of regulation of intracellular pH (pHi) in dispersed acini from the rat mandibular salivary gland has been studied with a microfluorimetric imaging method and the pH probe 2',7'-bis(2-carboxyethyl)-5(and -6)-carboxyfluorescein. The pHi in the TRIS/HEPES-buffered standard solution was 7.29 +/- 0.01. Addition of 1 mumol/l acetylcholine (ACh) or ionomycin caused a sustained increase in the pHi. These agents decreased pHi in the absence of external Na+ or in the presence of amiloride. The rate of pHi recovery from an acid load after NH+4 prepulse was a linear function of pHi and increased as pHi became more acidic. Addition of ACh shifted the relationship towards a more alkaline pHi range. The increase in pHi induced by ACh or ionomycin was not inhibited by the protein kinase C inhibitors staurosporine (10 nM) and 1-(5-isoquinolinesulfonyl)-1-methylpiperazine (50 mumol/l). Addition of 0.1-1 mumol/l phorbol 12-myristate 13-acetate (TPA) had little effect on pHi within 10 min; however, exposure to TPA for 120 min resulted in a significant rise in pHi. In Ca(2+)-free solution with 50 mumol/l 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the ACh-induced rise in both pHi and cytosolic Ca2+ concentration was suppressed. ACh and ionomycin caused an increment of amiloride-sensitive acid output into the extracellular fluid, while 20 mumol/l 1-oleoyl-2-acetylglycerol had little effect on it. It was concluded that (a) stimulation with ACh activated the Na+/H+ antiport in the plasma membrane, (b) ACh also stimulated the intracellular acid production but acid extrusion by the Na+/H+ antiport prevented the cell from intracellular acidification, and (c) the major route of signal transduction for the ACh-induced activation of the Na+/H+ antiport was independent of protein kinase C but was dependent on the rise in cytosolic Ca2+ concentration. The implication of the cytosolic acidification and cell volume change in pHi regulation is discussed.

Regulation of Cl/HCO3 exchange in gastric parietal cells.

Microspectrofluorimetry of the fluorescent indicators 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein and 6-methoxy-N-(3-sulfopropyl)-quinolinium was used to measure intracellular pH (pHi), intracellular Cl (Cli), and transmembrane fluxes of HCO3 and Cl in single parietal cells (PC) in isolated rabbit gastric glands incubated in HCO3/CO2-buffered solutions. Steady-state pHi was 7.2 in both resting (50 microM cimetidine) and stimulated (100 microM histamine) PCs. Transmembrane anion (HCO3 or Cl) flux rates during Cl removal from or readdition to the perfusate were the same in resting and stimulated PCs. These rates increased at alkaline pHi, though this pHi dependence was small in the physiological range. Maximum velocity (Vmax) for Cl influx or HCO3 efflux was 80-110 mM/min at pHi 7.6-7.8, and the Km for extracellular concentrations of Cl (Clo) was 25 mM; in the physiological range (pHi 7.1-7.3), Vmax for anion fluxes was approximately 50 mM/min. Steady-state Cli in the unstimulated PC was 62 +/- 5 mM, but on histamine stimulation, Cli decreased rapidly to 25 mM and then increased back to a steady-state level of 44 mM. HCO3 fluxes due to Cl removal or readdition were completely blocked by 0.5 mM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS), but Cl fluxes were only inhibited by 80%. H2DIDS did not inhibit the decrease in Cli that occurred with histamine treatment. Diphenylamine carboxylate (0.5 mM) inhibited Cl flux by only 50% and caused no additional inhibition of Cl flux when used in conjunction with H2DIDS. Transmembrane anion fluxes during solution Cl removal or readdition occurred 80% through the anion exchanger at the basal membrane and 20% through other pathway(s), presumably the Cl channel in the apical membrane. We conclude that the increase in transport activity via the Cl/HCO3 exchanger that occurs during histamine-induced increases in HCl secretion is due mostly to the decrease in Cli. In the resting cell with Cli = 62 mM, Clo = 120 mM, pHi = 7.2, and extracellular pH = 7.4, the anion exchanger is poised near its thermodynamic equilibrium. During histamine stimulation Cli drops from 62 mM to 44 mM, the thermodynamic equilibrium of the anion exchanger at the basolateral membrane is disturbed, and the anion exchanger then exchanges cellular HCO3 for extracellular Cl. Cli serves a crucial regulatory role in stimulus-secretion coupling in the PC.

Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line

A new transient expression system has been developed to investigate the function of anion exchangers in vivo. Human 293 cells were cotransfected with AE2 or AE3 cDNA together with a plasmid encoding a cell surface marker protein. Staining of the cells with antibody directed against a cell surface epitope present in the marker protein permitted the detection of cells expressing functional anion exchangers. Intracellular pH (pHi) recording in individual transfectants loaded with the fluorescent pHi indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, was used to determine the flux of HCO3- as a measure of Cl-/HCO3- exchange activity. Cells expressing either anion exchanger displayed significantly enhanced Cl-/HCO3- exchange activity compared with controls expressing only the marker. Transfection with either anion exchanger or with control plasmid resulted in altered intrinsic buffering capacity profiles compared with untransfected controls. Expression of either AE2 or AE3 did not result in changes in resting pHi. The activities of both AE2 and AE3 were stimulated at alkaline pHi, suggesting that an internal protonation site in AE2 and AE3 may regulate their activities. Both exchangers were inhibited reversibly and irreversibly by the anion 4,4'-diisothiocyanostilbene-2,2'-disulfonate with IC50 values of 142 and 0.43 microM for AE2 and AE3, respectively. These data indicate that structural differences in these highly conserved anion exchangers give rise to differences in affinities at the external anion binding site.

Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat.

1. The dual-emission pH-sensitive fluoroprobe carboxy-SNARF-1 (carboxy-seminaptharhodofluor) was used to measure pHi in type I cells enzymically dispersed from the neonatal rat carotid body. 2. Steady-state pHi in cells bathed in a HEPES-buffered Tyrode solution (pH 7.4) was found to be remarkably alkaline (pHi = 7.77) whereas cells bathed in a CO2-HCO3(-)-buffered Tyrode solution (pH 7.4) had a more 'normal' pHi (pHi = 7.28). These observations were further substantiated by using an independent nullpoint test method to determine pHi. 3. Intracellular intrinsic buffering (beta, determined by acidifying the cell using an NH4Cl pre-pulse) was in the range 7-20 mM per pH unit and appeared to be dependent upon pHi with beta increasing as pHi decreased. 4. In cells bathed in a HEPES-buffered Tyrode solution, pHi recovery from an induced intracellular acid load (10 mM-NH4Cl pre-pulse) was inhibited by the Na(+)-H+ exchange inhibitor ethyl isopropyl amiloride (EIPA; 150 microM) or substitution of Nao+ with N-methyl-D-glucamine (NMG). Both EIPA and Nao+ removal also caused a rapid intracellular acidification, which in the case of Nao+ removal, was readily reversible. The rate of this acidification was similar for both Nao+ removal and EIPA addition. 5. Transferring cells from a HEPES-buffered Tyrode solution to one buffered with 5% CO2-HCO3- resulted in an intracellular acidification which was partially, or wholly, sustained. The rate of acidification upon transfer to CO2-HCO3- was considerably slowed by the membrane permeant carbonic anhydrase inhibitor, acetazolamide, thus indicating the presence of the enzyme in these cells. 6. In CO2-HCO3(-)-buffered Tyrode solution, pHi recovery from an intracellular acidosis (NH4+ pre-pulse) was only partially inhibited by EIPA or amiloride whereas Nao+ removal completely inhibited the recovery. The stilbene DIDS (4,4-diisothiocyanatostilbenedisulphonic acid, 200 microM) also partially inhibited pHi recovery following an induced intracellular acidosis. Furthermore, the pre-treatment with 200 microM-DIDS of a pre-acidified cell in Na(+)-free Tyrode solution completely inhibited pHi recovery when Nao+ was reintroduced together with concomitant addition of 150 microM-EIPA. We conclude, that in the presence of CO2-HCO3-, a Na(+)- and HCO3-dependent (DIDS inhibitable) mechanism aids acid extrusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Nonsteroidal Anti-Inflammatory Analgesic Drugs on Chloride/Bicarbonate Antiports

Several nonsteroidal anti-inflammatory drugs (NSAIDs) were tested for their action on the Cl−/HCO3 − exchange by antiports and on the regulation of cytosolic pH (pHi) in Vero cells. The Na+-independent Cl−/HCO3 − antiport restores cytosolic pH (pHi) to normal after alkalinisation of the cytosol, 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 neutral. Preincubation with presumably anti-inflammatory concentrations of aspirin (acetylsalicylic acid), salicyclic acid, indomethacin or piroxicam at pH < 7 strongly increased the activity of this antiport, producing a decrease in pHi. Higher doses of the drugs competitively inhibited the Na+-independent Cl−/HCO3 − antiport. The Na+-coupled Cl−/HCO3 − antiport, which is most active when the pHi is < 7 and which normalises pHi after acidification, was inhibited after preincubation with these drugs, both at therapeutic and at higher concentrations. Preincubation with the drugs reduced pHi by 0.05 to 0.4 pH units compared with that in cells incubated in the absence of drugs.

Human primary brain tumour metabolism in vivo: a phosphorus magnetic resonance spectroscopy study

Magnetic resonance spectroscopy was used to study intracellular pH and compounds which contain phosphorus in normal human brain and primary brain tumours non-invasively. In normal subjects (n = 7) intracellular pH (pHi) of the brain was 7.03 +/- 0.02 (mean +/- s.e.m.). The pHi did not vary between superficial (2 cm, majority grey matter) and deep brain (5 cm, majority white matter). The relative concentrations of phosphocreatine (PCr) and phosphomonoesters (PME) to ATP were also constant with depth. The relative concentration of phosphodiesters (PDE) increased from superficial to deep in normal brain. The astrocytomas (n = 7, grade II-IV) were significantly more alkaline (pHi = 7.08 +/- 0.03), and contained more PCr and PME, with respect to ATP, than normal brain at similar depth. The meningiomas (n = 4) were also more alkaline (pHi = 7.19 +/- 0.02) with a raised PME level but reduced PCr. The reduction in meningioma PCr may be due to the significant necrosis (greater than 20%) seen in the surgical biopsies. No significant necrosis was seen in the gliomas. Previous in vitro studies suggest that increased PME may be due to accumulation of phosphoethanolamine (PE), a phospholipid precursor. These results suggest that human primary brain tumours characteristically are more alkaline with increased PME than normal brain.

Acidic intracellular pH shift during Caenorhabditis elegans larval development.

During recovery from the developmentally arrested, nonfeeding dauer stage of the nematode Caenorhabditis elegans, metabolic activation is accompanied by a decrease in intracellular pH (pHi). Phosphorus-31 nuclear magnetic resonance (31P NMR) analyses of perchloric acid extracts show that inorganic phosphate predominates in dauer larvae, whereas ATP and other high-energy metabolites are abundant within 6 hr after dauer larvae have been placed in food to initiate development. Although metabolic activation has been associated with an alkaline pHi shift in other organisms, in vivo 31P NMR analysis of recovering dauer larvae shows a pHi decrease from approximately 7.3 to approximately 6.3 within 3 hr after the animals encounter food. This shift occurs before feeding begins, and it coincides with, or soon follows, the developmental commitment to recover from the dauer stage, suggesting that control of pHi may be important in the regulation of larval development in nematodes.

Cell membrane potential: a signal to control intracellular pH and transepithelial hydrogen ion secretion in frog kidney.

The dependence of intracellular pH (pHi) and transepithelial H+ secretion on the cell membrane potential (Vm) was tested applying pH-sensitive and conventional microelectrodes in giant cells fused from single epithelial cells of the diluting segment and in intact tubules of the frog kidney. An increase of extracellular K+ concentration from 3 to 15 mmol/l decreased Vm from -49 +/- 4 to -29 +/- 1 mV while pHi increased from 7.44 +/- 0.04 to 7.61 +/- 0.06. Addition of 1 mmol/l Ba2+ depolarized Vm from -45 +/- 3 to -32 +/- 2 mV, paralleled by an increase of pHi from 7.46 +/- 0.04 to 7.58 +/- 0.03. Application of 0.05 mmol/l furosemide hyperpolarized Vm from -48 +/- 3 to -53 +/- 3 mV and decreased pHi from 7.47 +/- 0.05 to 7.42 +/- 0.05. In the intact diluting segment of the isolated-perfused frog kidney an increase of peritubular K+ concentration from 3 to 15 mmol/l increased the luminal pH from 7.23 +/- 0.08 to 7.41 +/- 0.08. Addition of Ba2+ to the peritubular perfusate also increased luminal pH from 7.35 +/- 0.07 to 7.46 +/- 0.07. Addition of furosemide decreased luminal pH from 7.32 +/- 0.03 to 7.24 +/- 0.05. We conclude: cell depolarization reduces the driving force for the rheogenic HCO3- exit step across the basolateral cell membrane. HCO3- accumulates in the cytoplasm and pHi increases. An alkaline pHi inactivates the luminal Na+/H+ exchanger. This diminishes transepithelial H+ secretion. Cell hyperpolarization leads to the opposite phenomenon. Thus, pHi serves as signal transducer between cell voltage and Na+/H+ exchange.

Induction of SOS functions by alkaline intracellular pH in Escherichia coli.

Alkalinization of intracellular pH (pHi) causes an increase in UV resistance in wild-type and pH-sensitive mutant (DZ3) cells of Escherichia coli. Utilizing cells transformed with a plasmid (pA7) which bears the uvrA promoter fused to galK galactokinase structural gene, it was shown that alkaline pHi leads to an increase in the specific activity of galactokinase. This effect was not displayed in a mutant bearing a recA-insensitive lexA gene, nor in cells harboring a plasmid (pA8) in which the galK is fused to a lexA-insensitive uvrA promoter. Hence, the effects of pHi on cells functions may involve the lexA product of the SOS system.

Depletion of cellular ATP inhibits Na+/H+ antiport in cultured human cells. Modulation of the regulatory effect of intracellular protons on the antiporter activity.

The metabolic energy dependency of Na+/H+ exchange activity was investigated in cultured human A431 carcinoma cells and foreskin fibroblasts. Following the activation of Na+/H+ exchange which results from loading cells with Na+ or Li+, depletion of cellular ATP by incubation with metabolic inhibitors causes an inhibition of Na+/H+ exchange activity. This inhibition is reversible and correlates with the extent of the reduction in the ATP pool. On the other hand, reduction of the intracellular pH (pHi) to approximately 6.0-6.2 results in a similar effective activation of Na+/H+ exchange in both control and ATP-depleted cells. Na+/H+ exchange activity in ATP-depleted cells that either have or have not been loaded with Na+ shows a steep dependency on pHi, being essentially abolished above pHi of 6.4-6.5, whereas control cells show a considerable activity also at more alkaline pHi values. Thus, Na+/H+ exchange activity in ATP-depleted cells shows an increased dependency on intracellular protons. These findings suggest that although metabolic energy is not required as a driving force for Na+/H+ antiport, depletion of metabolic energy substrates inhibits the antiporter activity due to a modulation of an intracellular proton-dependent regulatory mechanism.

Fluorescence identifies an alkaline cell in turtle urinary bladder.

Intracellular pH (pHi) of turtle bladder mucosal cells was studied by the trapped fluorescent indicator technique. Bladders efficiently accumulated and converted 4-methylumbelliferyl acetate to its pH-sensitive derivative 4-methylumbelliferone (4MU). Excited at the pH-indifferent wavelength 334 nm, bladders fluoresced a uniform blue. Using pH-sensitive 365-nm excitation, 10-20% of the mucosal cells fluoresced distinctly brighter, suggesting a more alkaline pHi. Using the 365/334 ratio to quantitate pHi, this difference averaged 0.1 pH units. Bright cells were more distinct after SITS or acetazolamide but disappeared after digitonin permeabilization, dinitrophenol, or treatment with propionate, DMO, and NH4Cl. Essentially the same population of bright cells was identified by carboxyfluorescein diacetate. The brighter cell corresponded exactly to a population of cells with distinctive acridine orange staining and bright costaining with the potential-sensing probes Di-O-C5, Di-S-C3, and 4-Di-5-Asp. Two extremes of bright cell shape were seen: an elongate cell, prevalent under conditions stimulating H+ secretion, and a more compact cell, when acidification was inhibited. These observations support the hypothesis that acidification represents H+ secretion via the luminal membrane and that a primary role of carbonic anhydrase in this process is to support the exit of base from the cell. The more alkaline cells appear to be the carbonic anhydrase-rich cells. These cells are chemically isolated from the surrounding granular cells and change their morphology in response to changes in acidification. These special properties indicate a unique role for the carbonic anhydrase cell in H+ secretion.

The Na+/H+ antiport is activated by serum and phorbol esters in proliferating myoblasts but not in differentiated myotubes. Properties of the activation process.

The properties of the Na+/H+ exchange system have been studied with 22Na+ uptake techniques at two stages of muscle development: proliferating myoblasts and differentiated myotubes. The characteristics of the interactions of the exchanger with external H+, with external Na+, and with amiloride or its more potent analogs are the same at both stages of development. Differences between the two stages of development concern: (i) the internal pH (pHi) dependence of the Na+/H+ exchanger, and (ii) the activation of the Na+/H+ exchanger by serum and phorbol ester which is observed in myoblasts but not in myotubes. Properties of the Na+/H+ exchanger in myoblasts after serum activation seem to be identical to those observed in myotubes with or without serum as if myotube formation stabilized a fully activated state of the exchanger. The activation of the myoblast Na+/H+ exchange system by serum is due to a shift of the pHi dependence towards alkaline pHi values and to an increase in the maximal activity of the Na+/H+ exchange system at acidic pH. Phorbol esters which are well-known activators of protein kinase C can only partially mimic the effects of serum on the Na+/H+ exchanger: they produce a shift of the pH dependence, but they do not increase the maximal activity at acidic pH.

Pace-maker current changes during intracellular pH transients in sheep cardiac Purkinje fibres.

Intracellular acidosis, at constant extracellular pH, hyperpolarizes the resting potential and reduces the diastolic depolarization rate of cardiac Purkinje fibres. With alkaline pHi, the fibre depolarizes and spontaneous firing is observed. Intracellular pH transients induced either by superfusion with Tyrode buffered with 5% CO2/23 mM HCO3- or 16% CO2/61 mM HCO3-, or with solutions containing weak undissociated acids, transiently shifted the half-maximum activation potential E0.5 of the pace-maker current. Similar transients were observed when NH4Cl was added and subsequently withdrawn from the solution. Simultaneous pHi measurements demonstrate a close relation between the time course of the pHi and E0.5 variations. Acid pHi shifts E0.5 to more negative and alkaline pHi's to less negative potentials. These pace-maker current activation voltage shifts are interpreted as the direct consequence of fixed charges titration at the inside of the sarcolemma. Other effects, like the slowing-down and reduction of the pace-maker current by acid pHi, presumably result from other interactions of protons with the pace-maker channel.

Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH.

There is now good evidence that cytoplasmic pH (pHi) may have an important role in the metabolic activation of quiescent cells. In particular, growth stimulation of mammalian fibroblasts leads to a rapid increase in pHi (refs 3-6), due to activation of a Na+/H+ exchanger in the plasma membrane, and this alkalinization is necessary for the initiation of DNA synthesis. However, the mechanism by which mitogens activate the Na+/H+ exchanger to raise pHi is not known, although an increase in cytoplasmic free Ca2+ ([Ca2+]i) has been postulated as the primary trigger. We now present data suggesting that the Na+/H+ exchanger is set in motion through protein kinase C, a phospholipid- and Ca2+-dependent enzyme normally activated by diacylglycerol produced from inositol phospholipids in response to external stimuli. Using newly developed pH microelectrodes and fluorimetric techniques, we show that a tumour promoting phorbol ester and synthetic diacylglycerol, both potent activators of kinase C (refs 12-15), mimic the action of mitogens in rapidly elevating pHi in different cell types. Furthermore, we demonstrate that, contrary to previous views, an early rise in [Ca2+]i is not essential for the activation of Na+/H+ exchange and the resultant increase in pHi. Finally, we suggest that an alkaline pHi shift, mediated by Na+/H+ exchange, may be a common signal in the action of those hormones which elicit the breakdown of inositol phospholipids.

Activation of Na+/H+ exchange by epidermal growth factor elevates intracellular pH in A431 cells.

Epidermal growth factor (EGF) increases Na+ uptake in several cell types through an electroneutral, amiloride-sensitive pathway putatively identified as Na+/H+ countertransport. We have previously shown (Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983) J. Biol. Chem. 257, 4883-4889) that EGF rapidly activates amiloride-sensitive net Na+ influx in the A431 human epidermoid carcinoma cell line. We also described the presence of transmembrane, amiloride-sensitive Na+/H+ exchange in A431 cells using a new fluorescence technique for the measurement of intracellular pH (pHi) based on the incorporation of fluorescein-dextran into the cell cytoplasm. The low pKa of fluorescein (approximately 6.4) prevented the direct assessment of the inferred, EGF-induced cytoplasmic alkalinization, mediated by stimulated Na+/H+ exchange. In this paper, 4',5'-dimethylfluorescein (pKa 6.75) was coupled to dextran, allowing increased pH sensitivity of the fluorescence assay in the physiological range. Using this improved assay, basic features of pHi regulation in A431 cells are documented, including the role of Na+/H+ exchange and Na+-linked C1-/HCO3-exchange in acid extrusion. We directly demonstrate a rapid elevation of pHi by addition of EGF as well as by serum in A431 cells. The pHi increase is half-maximal at 5-10 ng/ml of EGF, is dependent on external Na+, independent of external Ca2+, and inhibited by millimolar amiloride. EGF and serum also enhance Na+/H+ exchange-mediated cytosolic acidification when the transmembrane Na+ concentration gradient favors Na+ efflux from the cells. An alkaline pHi shift, caused by activation of Na+/H+ exchange, may be an important primary event in the mechanism of EGF action.