Regulation of intracellular pH in pyramidal neurones from the rat hippocampus by Na(+)-dependent Cl(-)-HCO3- exchange.

1. We have measured intracellular pH (pHi) in freshly isolated pyramidal neurones from the CA1 region of the rat hippocampus using the fluorescent indicator 2',7'-bis(carboxy-ethyl)-5-(and-6)-carboxyfluorescein (BCECF). 2. The neurones selected by our isolation procedure, when studied in the nominal absence of CO2-HCO3-, had a mean steady-state pHi of 6.81 +/- 0.02 (n = 163). The recovery of pHi from acid loads was very slow. The rate of recovery from acid loads was reduced by Na+ removal, but only very slightly inhibited by 1 mM amiloride. 3. The addition of 5% CO2-25 mM HCO3- caused steady-state pHi to increase from 6.74 +/- 0.05 to 7.03 +/- 0.03 (n = 28). In the presence of 5% CO2-25 mM HCO3-, the rate of pHi recovery from acid loads was much faster than in its absence. 4. The HCO(3-)-induced alkalinization was reversible, and did not occur in the absence of extracellular Na+ or in the presence of DIDS (4,4'-diisothiocyanatostilbene- 2,2'-disulphonic acid). 5. In the absence of external Cl-, successive exposures to CO2-HCO3- elicited alkalinizations that were progressively reduced in rate and amplitude. This effect, presumably due to gradual depletion of internal Cl-, was rapidly reversed by returning Cl- to the external medium. 6. We conclude that the major acid-extrusion mechanism in pyramidal CA1 neurones is the Na(+)-dependent Cl(-)-HCO3- exchanger. The Na(+)-dependent mechanism that operates in the nominal absence of HCO3- is far less active.     

Contribution of Na+/H+ exchange to pH regulation in pulmonary artery smooth muscle cells.

In blood vessels in the systemic circulation, the plasmalemmal Na+/H+ exchanger has been implicated in a variety of cellular functions, including the regulation of intracellular pH (pHi) and cell volume, and the response to smooth muscle mitogens. The role of this transport system in pulmonary vascular smooth muscle has not been explored. The present study examined the characteristics of Na+/H+ exchange in cultured guinea pig pulmonary artery smooth muscle cells. These cells were subjected to an acid load, and the recovery from acid loading was monitored using the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In the absence of HCO3-, pHi recovery from acid loading was dependent on external Na+ and was inhibited by the Na+/H+ exchange inhibitor dimethylamiloride (DMA) (recovery rate was reduced from 54.4 +/- 5.5 to 12.8 +/- 2.0 mmol H+/liter.min). This exchanger was also active in the presence of HCO3-; DMA reduced resting pHi and slowed the rate of recovery from acid loading in HCO3- buffers. However, in the presence of HCO3-, other transport systems, presumably HCO3-/Cl- exchange, also contribute to the regulation of pHi. In HCO3- buffers, the rate of recovery from acid load averaged 40.8 +/- 1.8 mmol H+/liter.min. Addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of HCO3-/Cl- exchange, slowed this recovery to 25.5 +/- 1.6 mmol H+/liter.min. A combination of DIDS and DMA further slowed the recovery to 19.7 +/- 1.5 mmol H+/liter.min. These findings indicate that the Na+/H+ exchanger plays a significant role in the regulation of pHi in pulmonary artery smooth muscle cells, even in HCO(3-)-containing buffers.     

Evidence of chloride/bicarbonate exchange mediating bicarbonate efflux from S3 segments of rabbit renal proximal tubule

The mechanism of HCO3- exit from rabbit renal proximal tubule S3 segments was investigated. Isolated tubules were perfused luminally and peritubularly with test solutions and cell pH (pHi), cell Cl- activity ([Cl-]i) and cell Na+ activity ([Na+]i) were measured with ion-selective microelectrodes. From the response of pHi and [Cl-]i to changes in bath Cl- or HCO3- concentrations a Cl-/HCO3- exchanger was identified in the basolateral cell membrane. It was reversibly inhibited by millimolar concentrations of the disulfonic stilbene SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid). Cell potential measurements and preliminary determinations of initial ion flux rates suggested a stoichiometry of Cl- to HCO3- flux near 1.0. The transport rate appeared to saturate already at low bath Cl- concentrations (approximately 30 mmol/l), but it was independent of bath pH in the range of 7.4-6.4. Cl-/HCO3- exchange was not directly coupled to Na+ flux although in approximately half of the experiments long-term incubation in Na(+)-free solutions indirectly inhibited the exchanger. Sudden application of SITS under control conditions revealed that the exchanger normally facilitates the exit of HCO3- from cell to interstitium at the expense of Cl- uptake into the cell. How Cl- ions recirculate towards the peritubular surface is presently not known.     

Na(+)-H+ exchange is not important for pancreatic HCO3- secretion in the pig.

Pancreatic inter- and intralobular duct cells extrude H(+)-ions to interstitial fluid when they secrete HCO3- to pancreatic juice. This study assesses the potential importance of Na(+)-H(+)-ion exchange for H(+)-ion extrusion and secretion of HCO3-, using the Na(+)-H+ exchange blockers amiloride and hexamethylene-amiloride. Intracellular pH (pHi) in inter- and intralobular pancreatic duct epithelium was measured using BCECF fluorescence. H(+)-ion efflux was measured using a NH4Cl prepulse, acid-loading technique. In HCO3(-)-free media, pHi recovery following acid loading was blocked by amiloride (10(-4) M) and hexamethylene-amiloride (10(-6) M), demonstrating amiloride- and hexamethylene-amiloride-sensitive Na(+)-H+ exchange. However, 5 x 10(-6) M hexamethylene-amiloride did not reduce secretin-dependent pancreatic HCO3- secretion in vivo. Maximal H(+)-efflux through Na(+)-H+ exchange was 1.5 +/- 0.2 mumol min-1 ml cell volume-1, i.e. less than 1% of estimated net H(+)-ion efflux during HCO3- secretion. Conclusion: amiloride- and hexamethylene amiloride sensitive Na(+)-H+ exchange is not important for secretin-dependent pancreatic HCO3- secretion in the pig. Other mechanisms for H+ extrusion dominate.     

Dexamethasone lowers cytosolic pH in macrophages by altering alkalinizing pH-regulatory mechanisms

The effect of dexamethasone on cytosolic pH (pHc) in resident mouse peritoneal macrophages was investigated using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein tetra-acetoxymethyl ester (BCECF-AM). Dexamethasone was found to significantly lower pHc and this reduction of pHc evolved gradually with time, was near maximal at 10 nM dexamethasone, and could be prevented by the glucocorticoid receptor antagonist RU-38486. The lower pHc of dexamethasone-treated cells was neither due to a reduction of cellular buffer capacity nor to an altered regulation of pHc by Na+/H+-exchange or by acidifying Na+-independent Cl-/HCO3- exchange, as assessed by studies of pH recovery after acute acid and alkali loads, respectively. Instead, an impaired pHc recovery by both the H+-ATPase and the alkalinizing Na+-dependent Cl-/HCO3- exchange was observed. This impairment was most likely not caused by an altered expression or localization of the 39-kDa subunit of the proton pump. Dexamethasone treatment caused a reduction of pHc also in a HCO3--containing solution, suggesting that acid extrusion by both the H+-ATPase and Na+-dependent Cl-/HCO3- exchange is important for maintenance and regulation of macrophage resting pHc. The lowering of macrophage pHc might be one mechanism whereby glucocorticoids exert their anti-inflammatory effects.     

The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones

1. Intracellular pH (pH(i)), Cl(-) and Na(+) levels were recorded in snail neurones using ion-sensitive micro-electrodes, and the mechanism of the pH(i) recovery from internal acidification investigated.2. Reducing the external HCO(3) (-) concentration greatly inhibited the rate of pH(i) recovery from HCl injection.3. Reducing external Cl(-) did not inhibit pH(i) recovery, but reducing internal Cl(-), by exposing the cell to sulphate Ringer, inhibited pH(i) recovery from CO(2) application.4. During pH(i) recovery from CO(2) application the internal Cl(-) concentration decreased. The measured fall in internal Cl(-) concentration averaged about 25% of the calculated increase in internal HCO(3) (-).5. Removal of external Na inhibited the pH(i) recovery from either CO(2) application or HCl injection.6. During the pH(i) recovery from acidification there was an increase in the internal Na(+) concentration ([Na(+)](i)). The increase was larger than that occurring when the Na pump was inhibited by K-free Ringer.7. The increase in [Na(+)](i) that occurred during pH(i) recovery from an injection of HCl was about half of that produced by a similar injection of NaCl.8. The inhibitory effects of Na-free Ringer and of the anion exchange inhibitor SITS on pH(i) recovery after HCl injection were not additive.9. It is concluded that the pH(i) regulating system involves tightly linked Cl(-)-HCO(3) (-) and Na(+)-H(+) exchange, with Na entry down its concentration gradient probably providing the energy to drive the movement inwards of HCO(3) (-) and the movement outward of Cl(-) and H(+) ions.     

Secretin stimulates {\rm HCO}_{\rm 3}^{\rm - } and acetate efflux but not Na+/{\rm HCO}_{\rm 3}^{\rm - } uptake in rat pancreatic ducts

Pancreatic ducts secrete HCO3(-), but transport mechanisms are unresolved and possibly vary between species. Our aim was to study the intracellular pH (pHi) regulation and thus H+/HCO3- transport in rat pancreatic ducts. Of particular interest was the Na+/HCO3(-) cotransporter, thought to be important in HCO3(-) -transporting epithelia. pHi was measured with BCECF in freshly isolated intralobular ducts. A reduction in extracellular Na+ concentration or application of HOE 694 (1 microM) decreased pHi by 0.1 to 0.6 pH units, demonstrating Na+/H+ exchanger activity. A reduction in extracellular Cl- concentration or addition of H2DIDS (10 microM) increased pHi by 0.1 to 0.5 pH units, demonstrating Cl-/ HCO(3)- (OH ) exchanger activity. In experimental acidosis, extracellular HCO3(-)/CO2 buffer did not increase the rate of pHi recovery, indicating that provision of HCO3(-) by the Na+/HCO3(-) cotransporter was not apparent. Most importantly, Na+/HCO3(-) cotransport was not stimulated by secretin (1 nM). In contrast, in experimental alkalosis the pHi recovery was increased in HCO3(-)/CO2 buffer, possibly due to Na+/HCO3(-) cotransport in the efflux mode. Secretin (1 nM) and carbachol (1 microM) stimulated HCO3(-) efflux, which can account for the observed HCO3(-) concentrations in rat pancreatic juice. Acetate and HCO3(-) buffers were handled similarly, indicating similar transport mechanisms in pancreatic ducts.     

Intracellular pH changes in human aortic smooth muscle cells in response to fluid shear stress.

The smooth muscle cell (SMC) layers of human arteries may be exposed to blood flow after endothelium denudation, for example, following balloon angioplasty treatment. These SMCs are also constantly subjected to pressure driven transmural fluid flow. Flow-induced shear stress can alter SMC growth and metabolism. Signal transduction mechanisms involved in these flow effects on SMCs are still poorly understood. In this work, the hypothesis that shear stress alters the intracellular pH (pHi) of SMC is examined. When exposed to venous and arterial levels of shear stress, human aortic smooth muscle cells (hASMC) undergo alkalinization. The alkalinization plateau persisted even after 20 min of cell exposure to flow. Addition of amiloride (10 micromoles) or its 5-(N-ethyl-N-isopropyl) analog (EIPA, 10 micromoles), both Na+/H+ exchanger inhibitors, attenuated intracellular alkalinization, suggesting the involvement of the Na+/H+ exchanger in this response. The same concentrations of these inhibitors did not show an effect on pHi of hASMCs in static culture. 4-Acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS, 1 mM), a Cl-/HCO3- exchange inhibitor, affected the pHi of hASMCs both in static and flow conditions. Our results suggest that flow may perturb the Na+/H+ exchanger leading to an alkalinization of hASMCs, a different response from the flow-induced acidification seen with endothelial cells at the same levels of shear stress. Understanding the flow-induced signal transduction pathways in the vascular cells is of great importance in the tissue engineering of vascular grafts. In the case of SMCs, the involvement of pHi changes in nitric oxide production and proliferation regulation highlights further the significance of such studies.     

Sodium- and bicarbonate-independent regulation of intracellular pH in culture mouse astrocytes

The intracellular pH (pHi) of cultured mouse astrocytes was measured with double-barrelled pH-sensitive microelectrodes. In bicarbonate-buffered saline pHi was 7.05 and in HEPES-buffered saline 6.68. In both solutions H+ was not in electrochemical equilibrium; pHi was 0.7-1 pH unit more alkaline than expected from passive H+ distribution. Cells were acidified by applying NH4+ and the subsequent regulation of pHi was studied in bicarbonate-free saline. The mean rate of pHi recovery was 0.2 pH units min-1 which was not changed by amiloride or by removal of external Na+. Thus, the cells recovered from an acid load independently of Na(+)-H+ exchange, Na(+)-HCO3- cotransport or any other bicarbonate- or Na(+)-dependent mechanism.     

Intracellular pH regulation in U937 human monocytes: roles of V-ATPase and Na+/H+ exchange

The role of plasmalemmal V-type H+ translocating ATPase (V-ATPase) in regulation of intracellular pH (pHi) is unclear in monocytes. This study examined the plasmalemmal V-ATPase and Na+/H+ exchanger (NHE) in U937 human monocytes. The fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein was used to monitor baseline pHi and the kinetics of pHi recovery from cytosolic acid-loads (NH4Cl prepulse). Bafilomycin A1 and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were used to delineate the activities of the H+-pump and NHE, respectively. Baseline pHi was approximately 7.13 at an extracellular pH (pHo) of 7.4 and fell progressively at lower pHo values. EIPA had no effect on baseline pHi at pHo 7.4, but caused a sustained decrement in pHi at pHo 6.0-7.0. Bafilomycin A1 had biphasic effects on baseline pHi at pHo 6.5-7.4; pHi declined approximately 0.1 units over the course of several minutes and then recovered. At pHo 6.0, bafilomycin A1 caused a sustained decrement in baseline pHi. Recovery from the bafilomycin-induced acidosis at pHo 6.5-7.4 was prevented by EIPA. Similarly, pHi recovery from NH4Cl prepulse acid-loads (pHo 7.4) was sensitive to both EIPA and bafilomycin A1. During this recovery process, Na+/H+ exchange (EIPA-sensitive component of apparent H+ efflux) was the predominant mechanism for H+ extrusion at acid-loaded pHi values < 7.0. At acid-loaded pHi values > or = 7.0, the V-ATPase (bafilomycin-sensitive component) and NHE contributed almost equally to H+ extrusion. The data provide the first evidence that plasmalemmal V-ATPase participates in pHi regulation in U937 cells. The H+-pump and NHE interacted to set baseline pHi and for pHi recovery following cytosolic acid-loading of the monocytes.     

pH regulation in isolated in vitro perfused rat colonic crypts

We investigated disorders and regulation of cytosolic pH (pHi) in isolated perfused crypts from rat distal colon using the pH-sensitive dye BCECF. This preparation allows distinct examination of either luminal or basolateral transport. The effects of luminal weak organic acids and bases on pHi were examined. The physiological concentrations of both luminal CO2/HCO3- and acetic acid/acetate acidified pHi significantly, but less than when applied from the basolateral side. Corresponding changes (luminal versus basolateral) in pHi were -0.17+/-0.04 versus -0.39+/-0.04, (n=8) and -0.15+/-0.02 versus -0.41+/-0.04, (n=8), respectively. Basolateral versus luminal application of NH3/NH4+ led to a more marked change in pHi, namely 0.35+/-0.03 versus 0.008+/-0.007 pH units, (n=19). The luminal perfusion of NH3/NH4+ was controlled by applying fura-2 acid to the luminal side and at the same time recording fura-2-specific fluorescence. Hence, the influence of luminal acid/base on colonic pHi homeostasis was limited. To examine pHi regulation, we investigated the recovery from an intracellular acid load using the NH3/NH4+ pulse method. Recovery was completely dependent on basolateral Na+, indicating that luminal acid/base transport does not play a major role in pHi homeostasis. The basolateral transporters involved in pHi recovery are probably the EIPA- and HOE694-inhibitable (IC50=0.2 and 2 micromol/l, respectively) Na+/H+ exchanger NHE1 and the DIDS-inhibitable Na+-dependent HCO3- importer.     

Na+/H+ exchange in glial cells of Necturus optic nerve

Single and double-barreled pH-sensitive electrodes were used to study intracellular pH (pHi) regulation in glial cells of Necturus optic nerve in the nominal absence of HCO3-/CO2. After the cells were acidified by the addition and withdrawal of NH4+, the pHi recovered toward the original steady-state pHi. The recovery from acidification was Na+-dependent and inhibited by 1 mM amiloride. These results suggest the existence in intact vertebrate glial cells of a Na+/H+ exchanger which functions in acid extrusion.     

Intracellular pH recovery and lactate efflux in mouse soleus muscles stimulated in vitro: the involvement of sodium/proton exchange and a lactate carrier

The intracellular pH recovery after stimulation of mouse soleus muscles in vitro was studied by means of intracellular pH-sensitive microelectrodes. The lactate efflux and the total lactate content were measured by means of an enzymic method. During electrical stimulation for 2 min in a CO2/HCO3- -buffered Ringer's solution, pHi decreased by 0.5 units. The rate of pHi-recovery was independent of external bicarbonate, but dependent on the buffer concentration. The rate of intracellular pH recovery was reduced by the lactate transport inhibitors PCMBS and cinnamate, whereas the inhibitors of inorganic anion-exchange SITS and DIDS had no effect. The Na+/H+ exchange inhibitor amiloride reduced the rate of pHi recovery. The pHi recovery was faster than the lactate efflux, which could be accounted for by an Na+/H+ exchange. A number of inhibitor compounds were used in order to discriminate between the three possible lactate efflux pathways: the monocarboxylate carrier mechanism, the inorganic anion exchange, and the molecular (non-ionic) diffusion of lactic acid. The lactate efflux was partly inhibited by cinnamate, PCMBS and phloretin, but was unaffected by DIDS and tetrathionate. These experiments demonstrate the existence of a lactate carrier in mammalian skeletal muscles. The lactate carrier is responsible for more than half of the lactate efflux after muscle activity. Both the pHi recovery studies and the lactate efflux measurements showed that, under the given conditions, the inorganic anion-exchange mechanism is not essentially involved in the recovery processes after muscle activity.     

{\text{HCO}}^{{\text{ - }}}_{{\text{3}}}-dependent pHi recovery and overacidification induced by {\text{NH}}^{ + }_{4} pulse in rat lung alveolar type II cells: {\text{HCO}}^{ - }_{3} -dependent NH3 excretion from lungs?

Intracellular pH (pHi) after the NH+4 pulse addition and its removal were measured in isolated alveolar type II cells (ATII cells) using BCECF fluorescence. In the absence of HCO(-3), the NH+4 pulse addition increased pHi (alkali jump) and its removal decreased pH(i) (acid jump) to the control level (no overacidification). This pHi change was induced by reaction 1 (NH3 + H+ <--> NH+4). However, in the presence of HCO(-3), the NH+4 pulse removal decreased pHi (acid jump) with overacidification. The extent of overacidification was decreased by acetazolamide (a carbonic anhydrase inhibitor), bumetanide (an inhibitor of Na+/K+/2Cl(-) cotransporter [NKCC]), and NPPB (an inhibitor of Cl(-) channel). The NH+4 pulse addition led to the accumulation of NH+4 in ATII cells via reaction 1 and NKCC, and the NH+4 pulse removal induced reaction 2 (NH+4 + HCO(-3) --> NH3 + H+ HCO(-3)) in addition to the reversal of reaction 1. Thus, NH+4 that entered via NKCC reacts with HCO(-3) (reaction 2) to produce H+, which induces overacidification in the acid jump. After the overacidification, the pH(i) recovery consisted of a rapid recovery (first phase) followed by a slow recovery (second phase). The first phase was inhibited by NPPB, glybenclamide, amiloride, and an Na+-free solution, and the second phase was inhibited by DIDS, MIA, and an Na+-free solution. Both phases were accelerated by a high extracellular HCO(-3) concentration. These observations indicate that the first phase was induced by HCO(-3) entry via Cl(-) channels coupled with Na+ channels activities, and that the second phase was induced by H+ extrusion via Na+/H+ exchanger and by HCO(-3) entry via HCO(-3) cotransporter. Thus, in ATII cells, HCO(-3) entry via Cl(-) channels is essential for recovering pHi after overacidification during the acid jump and for removing NH+4 that entered via NKCC from ATII cells, suggesting HCO(-3)-dependent NH3 excretion from lungs.     

Intracellular pH in isolated rat renal papillary thin limbs of Henle's loop

Intracellular pH (pHi) was measured in isolated, nonperfused and perfused rat papillary thin limbs of Henle's loops in N-2-hydroxyethylpiperazine-N'-2-ethansulfonic acid (HEPES)- or HEPES/bicarbonate-buffered medium at pH 7.4 using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Resting pHi was about 6.7 in descending thin limbs (DTL) and about 6.9 in ascending thin limbs (ATL), even with a medium pH of 7.4. These values appeared to reflect the acid pH of the blood in the neighboring vasa recta found in vivo. The resting pHi did not differ whether or not the medium contained bicarbonate although the total buffering capacity of the tubule cells was increased in the presence of bicarbonate. In nonperfused DTL and ATL, pHi was further acidified following an NH4Cl pulse. The rate of recovery of pHi from this level to the resting pHi was reduced by Na+ removal from the bath in both DTL and ATL and by the addition of ethylisopropylamiloride (EIPA) to the bath in the presence of Na+ in DTL. The rate of recovery was not affected by Cl- removal from the bath or K+ (75 mM) or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) addition to the bath in either DTL or ATL. These results suggest that the common, amiloride-sensitive, basolateral Na+/H+ exchanger plays a role in the regulation of pHi in rat papillary DTL but that a different basolateral Na+/H+ exchanger or a luminal Na+/H+ exchanger is important in rat papillary ATL.     

Secretin stimulates bile ductules to secrete both H+ and HCO3(-)-ions.

Secretin-dependent ductular HCO3- secretion into bile may involve secretion of H+ to interstitial fluid and HCO3- to bile by the ductular epithelium. To determine whether secretin causes bile ductules to secrete H+, we have examined the effect of secretin on the elimination of an intracellular acid load from bile ductular epithelium during pharmacological blockade of Na(+)-H+ exchange and in the absence of HCO3-. Microdissected bile ductules from pigs were suspended in HCO3- free HEPES buffer and loaded with acid using an NH4Cl prepulse technique. Intracellular pH was measured using dual-wavelength excitation of BCECF fluorescence. Na(+)-H+ exchange was defined as a Na(+)-dependent and amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive efflux of H(+)-ions following acid loading. We found that secretin stimulated ductular H+ secretion independent of Na(+)-H+ exchange. Blockade of Na(+)-H+ exchange by hexamethylene-amiloride did not affect secretin-dependent ductular HCO3- choleresis in vivo. We conclude that secretin stimulates bile ductules to secrete H(+)-ions to interstitial fluid as well as HCO3- ions to bile by a mechanism independent of Na(+)-H+ exchange.     

Intracellular pH in mammalian stages of Trypanosoma cruzi is K+-dependent and regulated by H+-ATPases

Regulation of intracellular pH (pHi) was investigated in Trypanosoma cruzi amastigotes and trypomastigotes using 2',7'-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF). pHi was determined to be 7.33 +/- 0.08 and 7.35 +/- 0.07 in amastigotes and trypomastigotes, respectively, and there were no significant differences in the regulation of pH, between the two stages. Steady-state pHi, recovery of pHi from acidification, and H+-efflux were all decreased markedly by the H+-ATPase inhibitors N,N'-dicyclohexylcarbodi-imide (DCCD), diethylstilbestrol (DES) and N-ethylmaleimide (NEM) supporting a significant role for a plasma membrane H+-ATPase in the regulation of pHi. pHi was maintained at neutrality over a range of external pH (pHe) from 5-8 in parasites suspended in a buffer containing Na+ and K+ (standard buffer) but was acidified at low pHe in the absence of these cations (choline buffer). The pHi of trypomastigotes decreased significantly when they transformed into amastigotes. The rate of recovery of pHi by acidified parasites was similar in Na+-free buffer and standard buffer but was slower in the absence of K+ (K+-free or choline buffer) and parasites suspended in choline buffer were acidic by 0.25 pH units as compared with controls. Ba2+ and Cs+ decreased the pHi of parasites suspended in standard but not choline buffer suggesting the presence of an inward directed K+ channel. The pHi of amastigotes and trypomastigotes suspended in Cl(-)-free buffer was decreased by 0.13 and 0.2 pH units, respectively, supporting the presence of a chloride conductive channel. No evidence of pH regulation via a Na+/H+ or Cl-/HCO3- exchanger was found. These results are consistent with the presence of a plasma membrane H+-ATPase that regulates pHi and is supported by K+ and Cl- channels.     

Intracellular pH regulation in human preimplantation embryos

We report here that intracellular pH (pH(i)) in cleavage-stage human embryos (2-8-cell) is regulated by at least two mechanisms: the HCO(3)(-)/Cl(-) exchanger (relieves alkalosis) and the Na(+)/H(+) antiporter (relieves acidosis). The mean pH(i) of cleavage-stage embryos was 7.12 +/- 0.008 (n = 199) with little variation between different stages. Embryos demonstrated robust recovery from alkalosis that was appropriately Cl(-)-dependent, indicating the presence of the HCO(3)(-)/Cl(-) exchanger. This was further confirmed by measuring the rate of intracellular alkalinization upon Cl(-) removal, which was markedly inhibited by the anion transport inhibitor, 4,4'-diisocyanatostilbene-2,2'-disulphonic acid, disodium salt. The set-point of the HCO(3)(-)/Cl(-) exchanger was between pH(i) 7.2 and 7.3. Embryos also exhibited Na(+)-dependent recovery from intracellular acidosis. Na(+)/H(+) antiporter activity appeared to regulate recovery up to about pH(i) 6.8; this recovery was HCO(3)(-)-independent and amiloride-sensitive, with a pH(i) set-point of approximately 6.8-6.9. A second system that was both Na(+)- and HCO(3)(-)-dependent appeared to mediate further recovery from acidosis up to about pH(i) 7.1. Thus, pH(i) of early human preimplantation embryos appears to be regulated by opposing mechanisms (HCO(3)(-)/Cl(-) exchanger, Na(+)/H(+) antiporter, and possibly a third acid-alleviating transporter that was both Na(+)- and HCO(3)(-)-dependent) resulting in the maintenance of pH(i) within a narrow range.     

Intracellular pH transients in rat diaphragm muscle measured with DMO.

Changes of the intracellular pH of rat diaphragm muscle were monitored at 30-min intervals with the weak acid DMO (5,5-dimethyl-2,4-oxazolidinedione). Transferring the muscle from a CO2-containing to a CO2-free solution caused intracellular pH (pHi) to rise by an average of 0.18 during the first 30 min and then to level off at a slightly lower value over the next 60-90 min. Transferring the muscle from a CO2-free to a CO2-containing solution caused pHi to fall by 0.18 during the first 30 min and then to recover by 0.05 over the next 90 min. Subsequent return to the CO2-free solution caused pHi to overshoot the control value by 0.10. Both the recovery and the overshoot can be accounted for by an acid-extruding pump. Intracellular acid loading with 118 mM DMO similarly caused pHi to fall initially, to recover slowly during the acid loading, and then to overshoot the control pHi on removal of the acid load. In the absence of HCO3-/CO2, acid extrusion was reduced by about a fifth. SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) had no effect. The absence of either Na+ or Cl- from HCO3-/CO2- free solution reduced acid extrusion by about a half.