Acid-induced responses in hamster chorda tympani and intracellular pH tracking by taste receptor cells

HCl- and NaCl-induced hamster chorda tympani nerve responseswere recorded during voltage clamp of the lingual receptive field. Voltage perturbations did not influence responses to HCl. In contrast, responses to NaCl were decreased by submucosal-positive and increased by submucosal-negative voltage clamp. Responses to HCl were insensitive to the Na⁺ channel blockers,amiloride and benzamil, and to methylisobutylamiloride (MIA), anNa⁺/H⁺exchange blocker. Responses to NaCl were unaffected by MIA but weresuppressed by benzamil. Microfluorometric and imaging techniques wereused to monitor the relationship between external pH(pH_o) and the intracellular pH(pH_i) of fungiform papilla tastereceptor cells (TRCs) following2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein loading.TRC pH_i responded rapidly andmonotonically to changes in pH_o.This response was unaffected byNa⁺ removal or the presence ofamiloride, benzamil, or MIA. The neural records and the data fromisolated TRCs suggest that the principal transduction pathway for acidtaste in hamster is similar to that in rat. This may involve themonitoring of changes in TRC pH_i mediated through amiloride-insensitiveH⁺ transport across TRC membranes.This is an example of cell monitoring of environmental pH through pHtracking, i.e., a linear change inpH_i in response to a change inpH_o, as has been proposed for carotid bodies. In taste, the H⁺transport sites may be concentrated on the basolateral membranes ofTRCs and, therefore, are responsive to an attenuatedH⁺ concentration from diffusion ofacids across the tight junctions.

     

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pH_i) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO₃^–) or 7.24 ± 0.03 (with HCO₃^–). Ion substitution and inhibitor experiments were performed to determine whether common H⁺-transporting species were operating to maintain basal pH_i. Removal of extracellular Na⁺ or Cl^– or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H₂DIDS) had no effect. Acidification with the K⁺/H⁺ exchanger nigericin reduced pH_i to 6.25 ± 0.15 (without HCO₃^–) or 6.53 ± 0.10 (with HCO₃^–). In the presence of extracellular Na⁺, recovery to basal pH_i was prompt and occurred at similar rates in the absence and presence of HCO₃^–. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pH_i after acidification. Recovery was inhibited by removal of Na⁺ or addition of amiloride, whereas removal of Cl^– and addition of H₂DIDS were ineffective. Addition of the Na⁺/H⁺ exchanger monensin to cells that had returned to basal pH_i elicited a further increase in pH_i to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH per minute as pH_i approached the basal level, despite the continued presence of a driving force for H⁺ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pH_i is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na⁺/H⁺ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load. sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein     

Glucose activates H(+)-ATPase in kidney epithelial cells

The vacuolar H⁺-ATPase (V-ATPase) acidifies compartments of the vacuolar system of eukaryotic cells. In renal epithelial cells, it resides on the plasma membrane and is essential for bicarbonate transport and acid-base homeostasis. The factors that regulate the H⁺-ATPase remain largely unknown. The present study examines the effect of glucose on H⁺-ATPase activity in the pig kidney epithelial cell line LLC-PK₁. Cellular pH was measured by performing ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. Intracellular acidification was induced with NH₃/NH₄⁺ prepulse, and rates of intracellular pH (pH_i) recovery (after in situ calibration) were determined by the slopes of linear regression lines during the first 3 min of recovery. The solutions contained 1 µM ethylisopropylamiloride and were K⁺ free to eliminate Na⁺/H⁺ exchange and H⁺-K⁺-ATPase activity. After NH₃/NH₄⁺-induced acidification, LLC-PK₁ cells had a significant pH_i recovery rate that was inhibited entirely by 100 nM of the V-ATPase inhibitor concanamycin A. Acute removal of glucose from medium markedly reduced V-ATPase-dependent pH_i recovery activity. Readdition of glucose induced concentration-dependent reactivation of V-ATPase pH_i recovery activity within 2 min. Glucose replacement produced no significant change in cell ATP or ADP content. H⁺-ATPase activity was completely inhibited by the glycolytic inhibitor 2-deoxy-D-glucose (20 mM) but only partially inhibited by the mitochondrial electron transport inhibitor antimycin A (20 µM). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (500 nM) abolished glucose activation of V-ATPase, and activity was restored after wortmannin removal. Glucose activates V-ATPase activity in kidney epithelial cells through the glycolytic pathway by a signaling pathway that requires PI3K activity. These findings represent an entirely new physiological effect of glucose, linking it to cellular proton secretion and vacuolar acidification. proton secretion; glycolysis; intracellular pH; concanamycin A     

Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells

Using thepH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF),we examined the effect of hyperosmolar solutions, which presumablycaused cell shrinkage, on intracellular pH(pH_i) regulation in mesangialcells (single cells or populations) cultured from the rat kidney. Thecalibration of BCECF is identical in shrunken and unshrunken mesangialcells if the extracellular K⁺concentration ([K⁺])is adjusted to match the predicted intracellular[K⁺]. ForpH_i values between ~6.7 and~7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH₂O) is threefold larger than in unshrunken cells (~300mosmol/kgH₂O). In the nominalabsence ofCO₂/HCO₃,exposing cell populations to a HEPES-buffered solution supplementedwith ~300 mM mannitol (600 mosmol/kgH₂O) causes steady-statepH_i to increase by ~0.4. The pH_i increase is due to activationofNa⁺/H⁺exchange because, in single cells, it is blocked in the absence ofexternal Na⁺ or in the presence of50 µM ethylisopropylamiloride (EIPA). Preincubating cells in aCl-free solution for atleast 14 min inhibits the shrinkage-induced pH_i increase by 80%. Wecalculated the pH_i dependence oftheNa⁺/H⁺exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) thepH_i dependence of the totalacid-extrusion rate and 2) thepH_i dependence of theEIPA-insensitive acid-loading rate. Shrinkage alkali shifts thepH_i dependence ofNa⁺/H⁺exchange by ~0.7 pH units.     

Regulation of cardiac AMP-specific 5'-nucleotidase during ischemia mediates ATP resynthesis on reflow

The ability toresynthesize ATP during recovery from ischemia is limited tothe size of endogenous pool of adenine nucleotides. CytosolicAMP-specific 5'-nucleotidase (5'-NT) plays a key role inATP degradation and hence the capacity for ATP resynthesis. We havesuggested (J. Clin. Invest. 93:40-49, 1994) that intracellular acidosis [intracellular pH(pH_i)] is a potentinhibitor of 5'-NT under in vivo conditions. To test thishypothesis further, we used the hyperthyroid rat heart because we couldalter pH_i during ischemiaand determine the consequences of lowerpH_i on AMPaccumulation (by chemical assay) and ATP resynthesis (by³¹P nuclear magnetic resonancespectroscopy) during reperfusion. Global no-flow ischemiacaused pH_i to decrease from 7.1 under well-oxygenated control perfusion to 6.7. We found thatdecreasing pH_i further from pH 6.7 to 6.4 leads to increased accumulation (30%) of AMP duringischemia and to a 2.5-fold increase in ATP resynthesis duringreperfusion. Analysis of all known substrates, products, activators,and inhibitors of the 5'-NT suggests that 5'-NT isactivated primarily by Mg²⁺ andADP and is inhibited by H⁺. Thusthese observations provide evidence for a salutary effect ofintracellular acidosis on preserving the AMP pool due to inhibition of5'-NT and suggest a novel role ofH⁺ in protecting ischemic tissue.

     

Tastants evoke cAMP signal in taste buds that is independent of calcium signaling

We previously showed that rat taste buds express several adenylyl cyclases (ACs) of which only AC8 is known to be stimulated by Ca²⁺. Here we demonstrate by direct measurements of cAMP levels that AC activity in taste buds is stimulated by treatments that elevate intracellular Ca²⁺. Specifically, 5 µM thapsigargin or 3 µM A-23187 (calcium ionophore), both of which increase intracellular Ca²⁺ concentration ([Ca²⁺]_i), lead to a significant elevation of cAMP levels. This calcium stimulation of AC activity requires extracellular Ca²⁺, suggesting that it is dependent on Ca²⁺ entry rather than release from stores. With immunofluorescence microscopy, we show that the calcium-stimulated AC8 is principally expressed in taste cells that also express phospholipase C₂ (i.e., cells that elevate [Ca²⁺]_i in response to sweet, bitter, or umami stimuli). Taste transduction for sucrose is known to result in an elevation of both cAMP and calcium in taste buds. Thus we tested whether the cAMP increase in response to sucrose is a downstream consequence of calcium elevation. Even under conditions of depletion of stored and extracellular calcium, the cAMP response to sucrose stimulation persists in taste cells. The cAMP signal in response to monosodium glutamate stimulation is similarly unperturbed by calcium depletion. Our results suggest that tastant-evoked cAMP signals are not simply a secondary consequence of calcium modulation. Instead, cAMP and released Ca²⁺ may represent independent second messenger signals downstream of taste receptors. calcium-sensitive adenylyl cyclase; capacitative entry; cross talk; taste transduction     

Incubation of OKP cells in low-K+ media increases NHE3 activity after early decrease in intracellular pH

Chronichypokalemia increases the activity of proximal tubule apical membraneNa⁺/H⁺antiporter NHE3. The present study examined the effect ofthe incubation of OKP cells (an opossum kidney, clone P cell line) incontrol medium {K⁺ concn([K⁺]) = 5.4 mM} or low-K⁺ medium([K⁺] = 2.7 mM) onNHE3. The activity of an ethylisopropyl amiloride-resistant Na⁺/H⁺antiporter, whose characteristics were consistent with those ofNHE3, was increased inlow-K⁺ cells beginning at 8 h.NHE3 mRNA and NHE3 protein abundance were increased 2.2-fold and 62%,respectively, at 24 h but not at 8 h. After incubation inlow-K⁺ medium, intracellular pH(pH_i) decreased by 0.27 pH units(maximum at 27 min) and then recovered to the control level.Intracellular acidosis induced by 5 mM sodium propionate increasedNa⁺/H⁺antiporter activity at 8 and 24 h. Herbimycin A, a tyrosine kinase inhibitor, blocked low-K⁺- andsodium propionate-induced activation of theNa⁺/H⁺antiporter at 8 and 24 h. Our results demonstrate thatlow-K⁺ medium causes an earlydecrease in pH_i, which leads to anincrease in NHE3 activity via a tyrosine kinase pathway.     

Oxidative stress decreases pHi and Na(+)/H(+) exchange and increases excitability of solitary complex neurons from rat brain slices

Putative chemoreceptors in the solitary complex (SC) are sensitive to hypercapnia and oxidative stress. We tested the hypothesis that oxidative stress stimulates SC neurons by a mechanism independent of intracellular pH (pH_i). pH_i was measured by using ratiometric fluorescence imaging microscopy, utilizing either the pH-sensitive fluorescent dye BCECF or, during whole cell recordings, pyranine in SC neurons in brain stem slices from rat pups. Oxidative stress decreased pH_i in 270 of 436 (62%) SC neurons tested. Chloramine-T (CT), N-chlorosuccinimide (NCS), dihydroxyfumaric acid, and H₂O₂ decreased pH_i by 0.19 ± 0.007, 0.20 ± 0.015, 0.15 ± 0.013, and 0.08 ± 0.002 pH unit, respectively. Hypercapnia decreased pH_i by 0.26 ± 0.006 pH unit (n = 95). The combination of hypercapnia and CT or NCS had an additive effect on pH_i, causing a 0.42 ± 0.03 (n = 21) pH unit acidification. CT slowed pH_i recovery mediated by Na⁺/H⁺ exchange (NHE) from NH₄Cl-induced acidification by 53% (n = 20) in -buffered medium and by 58% (n = 10) in HEPES-buffered medium. CT increased firing rate in 14 of 16 SC neurons, and there was no difference in the firing rate response to CT with or without a corresponding change in pH_i. These results indicate that oxidative stress 1) decreases pH_i in some SC neurons, 2) together with hypercapnia has an additive effect on pH_i, 3) partially inhibits NHE, and 4) directly affects excitability of CO₂/H⁺-chemosensitive SC neurons independently of pH_i changes. These findings suggest that oxidative stress acidifies SC neurons in part by inhibiting NHE, and this acidification may contribute ultimately to respiratory control dysfunction. hyperoxic hyperventilation; O₂ toxicity; pH regulation; brain stem; reactive oxygen species     

The role of intracellular pH in cell growth arrest induced by ATP

In this study, we investigated ionic mechanisms involved in growth arrest induced by extracellular ATP in androgen-independent prostate cancer cells. Extracellular ATP reversibly induced a rapid and sustained intracellular pH (pH_i) decrease from 7.41 to 7.11. Inhibition of Ca²⁺ influx, lowering extracellular Ca²⁺, and buffering cytoplasmic Ca²⁺ inhibited ATP-induced acidification, thereby demonstrating that acidification is a consequence of Ca²⁺ entry. We show that ATP induced reuptake of Ca²⁺ by the mitochondria and a transient depolarization of the inner mitochondrial membrane. ATP-induced acidification was reduced after the dissipation of the mitochondrial proton gradient by rotenone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, after inhibition of Ca²⁺ uptake into the mitochondria by ruthenium red, and after inhibition of the F₀F₁-ATPase with oligomycin. ATP-induced acidification was not induced by either stimulation of the Cl^–/HCO₃^– exchanger or inhibition of the Na⁺/H⁺ exchanger. In addition, intracellular acidification, induced by an ammonium prepulse method, reduced the amount of releasable Ca²⁺ from the endoplasmic reticulum, assessed by measuring change in cytosolic Ca²⁺ induced by thapsigargin or ATP in a Ca²⁺-free medium. This latter finding reveals cross talk between pH_i and Ca²⁺ homeostasis in which the Ca²⁺-induced intracellular acidification can in turn regulate the amount of Ca²⁺ that can be released from the endoplasmic reticulum. Furthermore, pH_i decrease was capable of reducing cell growth. Taken together, our results suggest that ATP-induced acidification in DU-145 cells results from specific effect of mitochondrial function and is one of the major mechanisms leading to growth arrest induced by ATP. prostate; cancer; acidification     

Role of Na(+)/H(+) exchanger during O(2) deprivation in mouse CA1 neurons

To determine the role ofmembrane transporters in intracellular pH (pH_i) regulationunder conditions of low microenvironmental O₂, we monitoredpH_i in isolated single CA1 neurons using the fluorescentindicator carboxyseminaphthorhodafluor-1 and confocal microscopy. Aftertotal O₂ deprivation or anoxia (PO₂ 0 Torr), a large increase in pH_i was seen in CA1neurons in HEPES buffer, but a drop in pH_i, albeit small,was observed in the presence of HCO. Ionicsubstitution and pharmacological experiments showed that the largeanoxia-induced pH_i increase in HEPES buffer was totallyNa⁺ dependent and was blocked by HOE-694, stronglysuggesting the activation of the Na⁺/H⁺exchanger (NHE). Also, this pH_i increase in HEPES bufferwas significantly smaller in Na⁺/H⁺ exchangerisoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons,demonstrating that NHE1 is responsible for part of the pH_iincrease following anoxia. Both chelerythrine and H-89 partly blocked,and H-7 totally eliminated, this anoxia-induced pH_iincrease in the absence of HCO. We conclude that1) O₂ deprivation activatesNa⁺/H⁺ exchange by enhancing protein kinaseactivity and 2) membrane proteins, such as NHE, activelyparticipate in regulating pH_i during low-O₂states in neurons.

     

Effects of osmolarity on taste receptor cell size and function

Osmotic effects onsalt taste were studied by recording from the rat chorda tympani (CT)nerve and by measuring changes in cell volume of isolated rat fungiformtaste receptor cells (TRCs). Mannitol, cellobiose, urea, or DMSO didnot induce CT responses. However, the steady-state CT responses to 150 mM NaCl were significantly increased when the stimulus solutions alsocontained 300 mM mannitol or cellobiose, but not 600 mM urea or DMSO.The enhanced CT responses to NaCl were reversed when the saccharideswere removed and were completely blocked by addition of 100 µMamiloride to the stimulus solution. Exposure of TRCs to hyperosmoticsolutions of mannitol or cellobiose induced a rapid and sustaineddecrease in cell volume that was completely reversible, whereasexposure to hypertonic urea or DMSO did not induce sustained reductionsin cell volume. These data suggest that the osmolyte-induced increasein the CT response to NaCl involves a sustained decrease in TRC volumeand the activation of amiloride-sensitive apicalNa⁺ channels.

     

Inhibition of NHE-1 Na+/H+ exchanger by natriuretic peptides in ocular nonpigmented ciliary epithelium

The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) display hypotensive effects in the mammalian eye by lowering the intraocular pressure (IOP), a function that is mediated by the bilayer ocular ciliary epithelium (CE), in conjunction with the trabecular meshwork. ANP regulates Na⁺/H⁺ exchanger (NHE) activity, and inhibitors of NHE have been shown to lower IOP. We examined whether NPs influence the NHE activity of the CE, which is comprised of pigmented (PE) and nonpigmented (NPE) epithelial cells, by directly recording the rate of intracellular pH (pH_i) recovery from its inner NPE cell layer. NPs inhibited, in a dose-dependent manner (1–100 nM), the rate of pH_i recovery with the order of potency CNP > ANP > BNP, indicative that this inhibition is mediated by the presence of NPR type B receptors. 8-Bromo-cGMP (8-BrcGMP), a nonhydrolyzable analog of cGMP, mimicked NPs in inhibiting the rate of Na⁺-dependent pH_i recovery. In contrast, ethylisopropyl amiloride (EIPA, 100 nM) or amiloride (10 µM) completely abolished the pH_i recovery by NHE. 18-Glycyrrhetinic acid (18-GA), a gap junction blocker, attenuated the inhibitory effect of CNP on the rate of pH_i recovery, suggesting that NHE activity in both cell layers of the CE is coregulated. This interpretation was supported, in part, by the coexpression of NHE-1 isoform mRNA in both NPE and PE cells. The mechanism by which the inhibitory effect of NPs on NHE-1 activity might influence the net solute movement or fluid transport by the bilayer CE remains to be determined. Na⁺/H⁺ exchanger type 1; intracellular pH; aqueous humor     

Regulation of the Cytoplasmic pH of Chara corallina in the Absence of External Ca2+: Its Significance in Relation to the Activity and Control of the H+ Pump

Effects of removal of external Ca²⁺ on the cytoplasmic pH (pH_c)of Chara corallina have been measured with the weak acid 5,5-dimethyl-oxazolidine-2,4-dione(DMO) as a function of external pH (pH₀) and of the externalconcentration of K⁺. Removal of Ca²⁺ always decreased pH_c whenpH₀ was below about 6.0; the decrease was about 0.2–0.4units at pH₀ 5.0, increasing to about 0.5 units at pH₀ 4.3.When pH₀ was 6.0 or higher the removal of Ca²⁺ had little orno effect on pH_c. This situation was not altered by changingthe concentration of K⁺, though in some experiments at pH₀ 5.0–5.2there was a slight decrease in pH₀ (about 0.2 units) when K⁺was increased from 0.2 to 2.0 mol m^–3, an effect apparentlyreversed when K⁺ was higher (5.0 or 10.0 mol m^–3). Theresults suggest that H⁺ transport continues in the absence ofexternal Ca²⁺, despite previous suggestions to the contrary,and that the H⁺ pump does not necessarily run near thermodynamicequilibrium with its chemical driving reaction. They indicate,rather, that the H⁺ pump is under kinetic control and providefurther evidence for the inadequacy of present models for theoperation of the H⁺ pump in charophyte cells, especially inrelation to its proposed role in regulating pH_c. Key words: Chara corallina, Cytoplasmic pH, Calcium     

The role of sodium and potassium transport pathways in taste transduction: an overview

Recent studies have shown that taste sensations are mediatedby a multiplicity of transduction mechanisms. The taste of saltis produced in part by the entry of Na⁺ through channels inthe apical taste cell membrane. Na⁺ transport also mediatessweet perception in some species. The taste of KCI requiresentry of K⁺ through apical potassium channels. The productionof second messengers such as cAMP by taste stimuli or tastemodifiers can depolarize taste cells by inducing an enzymaticcascade that alters K⁺ permeability.     

Changes in intracellular Ca2+ and pH in response to thapsigargin in human glioblastoma cells and normal astrocytes

Despite extensive work in the field of glioblastoma research no significant increase in survival rates for this devastating disease has been achieved. It is known that disturbance of intracellular Ca²⁺ ([Ca²⁺]_i) and intracellular pH (pH_i) regulation could be involved in tumor formation. The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) is a major regulator of [Ca²⁺]_i. We have investigated the effect of inhibition of SERCA by thapsigargin (TG) on [Ca²⁺]_i and pH_i in human primary glioblastoma multiforme (GBM) cells and GBM cell lines, compared with normal human astrocytes, using the fluorescent indicators fura-2 and BCECF, respectively. Basal [Ca²⁺]_i was higher in SK-MG-1 and U87 MG but not in human primary GBM cells compared with normal astrocytes. However, in tumor cells, TG evoked a much larger and faster [Ca²⁺]_i increase than in normal astrocytes. This increase was prevented in nominally Ca²⁺-free buffer and by 2-APB, an inhibitor of store-operated Ca²⁺ channels. In addition, TG-activated Ca²⁺ influx, which was sensitive to 2-APB, was higher in all tumor cell lines and primary GBM cells compared with normal astrocytes. The pH_i was also elevated in tumor cells compared with normal astrocytes. TG caused acidification of both normal and all GBM cells, but in the tumor cells, this acidification was followed by an amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive recovery, indicating involvement of a Na⁺/H⁺ exchanger. In summary, inhibition of SERCA function revealed a significant divergence in intracellular Ca²⁺ homeostasis and pH regulation in tumor cells compared with normal human astrocytes. fura-2; BCECF; store-operated calcium channels     

Modulation of rat chorda tympani NaCl responses and intracellular Na+ activity in polarized taste receptor cells by pH

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH (pH(i)) and Na(+) activity ([Na(+)](i)) in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the lingual surface with NaCl solutions adjusted to pHs ranging between 2.0 and 10.3 increased the magnitude of NaCl CT responses linearly with increasing external pH (pH(o)). At pH 7.0, the epithelial sodium channel (ENaC) blocker, benzamil, decreased NaCl CT responses and inhibited further changes in CT responses induced by varying pH(o) to 2.0 or 10.3. At constant pH(o), buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HCO(3)(-)/CO(2) inhibited NaCl CT responses relative to CT responses obtained with NaCl solutions buffered with HEPES. The carbonic anhydrase blockers, MK-507 and MK-417, attenuated the inhibition of NaCl CT responses in HCO(3)(-)/CO(2) buffer, suggesting a regulatory role for pH(i). In polarized TRCs step changes in apical pH(o) from 10.3 to 2.0 induced a linear decrease in pH(i) that remained within the physiological range (slope = 0.035; r(2) = 0.98). At constant pH(o), perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO(3)(-)/CO(2) decreased resting TRC pH(i), and MK-507 or MK-417 attenuated the decrease in pH(i) in TRCs perfused with HCO(3)(-)/CO(2) buffer. In parallel experiments, TRC [Na(+)](i) decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical Na(+), and (d) acid loading the cells with NH(4)Cl or sodium acetate at constant pH(o). Diethylpyrocarbonate and Zn(2+), modification reagents for histidine residues in proteins, attenuated the CO(2)-induced inhibition of NaCl CT responses and the pH(i)-induced inhibition of apical Na(+) influx in TRCs. We conclude that TRC pH(i) regulates Na(+)-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures.     

Proton Transport and pH Control in Sinapis alba Root Hairs: A Study carried out with Double-Barrelled pH Micro-electrodes

Continuous measurements of cytoplasmic pH (pH_c) in Sinapis roothairs have been carried out with double-barrelled pH-micro-electrodesin order to gain information on translocation of protons acrossthe plasmalemma and cytoplasmic pH control. (i) The cytoplasmicpH of Sinapis (7–33 ? 0–12, standard conditions)changes no more than 0.1 pH_c, per pH_o-unit, regardless of whethercyanide is present or not. (ii) Weak acids rapidly acidify pH_cand hyperpolarize, while weak bases alkalize pH_c and depolarizethe cells, (iii) 1.0 mol M,³ NaCN acidifies the cytoplasm by0.4 to 0.7 pH-units, but alkalizes the vacuole. (iv) 20 mmolm^–3 CCCP has no significant effect on pH_c, if added atpH 9.6 or 7.2, but acidifies pH_c by 1.3 units at pH 4.3. Inthe presence of CCCP, cyanide acidifies the cytoplasm, (v) Chloridetransiently acidifies pH_c, while K⁺, Na⁺, and have no significant effects, (vi) Cytoplasmic buffer capacityforms a bell-shaped curve versus pH_c with an optimum of about50 mol m^–3 H⁺pH_c-unit. The modes of proton re-entry and the effects of active and passiveproton transport on cellular pH control are critically discussed.It is suggested that the proton leak, consisting of H⁺-cotransport(e.g. H⁺/Cl^–) rather than H⁺-uniport, is no threat topH_c. The proton export pump, although itself reacting to changesin pH_c, influences pH_c only to a minor extent. It is concludedthat buffer capacity and membrane transport play moderate rolesin pH_c control in Sinapis, while the interlocked H⁺-producingand -consuming reactions of cellular metabolism are the mainregulating factors. This makes pH control in Sinapis quite differentfrom bacterial and animal cells. Key words: Cytoplasmic pH, double-barrelled pH micro-electrode, pH control, proton transport, Sinapis     

Light-Induced H+ Accumulation in the Vacuole of Nitellopsis obtusa

The effects of light on the pH in the vacuole and the electricpotential difference across the plasmalemma and the tonoplastof Nitellopsis obtusa were investigated by means of conventionaland H⁺-specific glass or antimony microelectrodes. Illuminationis found to bring about a decrease in the pH of the vacuolarsap by 0.1–0.5 units concomitant with a depolarizationof the cell. The light-induced changes of the potential differenceand the vacuolar pH depend in different ways on the pH of theexternal medium (pH_o). At pH_o 9.0 cells exhibit great light-inducedpotential changes (up to 100 mV), but only small pH changesof the vacuolar sap. At neutral or slightly acidic pH_o valuesthe amplitude of the light-induced pH changes in the vacuoleincreases up to 0.3–0.5 pH units, but the amplitudes ofthe potential changes at the plasmalemma are relatively small.At pH_o 9.0 a transient acidification of the medium is observedupon illumination whereas at lower pH values light-induced alkalinizationwas only seen. Transfer of the cells from pH_o 9.0 to pH_o 7.5results in a cell hyperpolarization by 60–80 mV and adecrease of the vacuolar pH by 0.4–0.5 units under lightconditions but has no significant effect on the potential andthe vacuolar pH in the darkness. It is proposed that mechanismsof active H⁺ extrusion from the cytoplasm are located both inthe plasmalemma and the tonoplast. The observed acidificationin the vacuole appears to be determined by a light-induced increaseof the concentration of H⁺ in the cytoplasm. The H⁺ conductionof the plasmalemma seems to increase on illumination. The patternof the light-induced H⁺ fluxes across the tonoplast and theplasmalemma depends crucially on the extent of the light-inducedchanges in the H⁺ conductance and on the electrochemical gradientfor H⁺ at the plasmalemma.     

Differences in regulation of pH(i) in large (>/=10 nuclei) and small (</=5 nuclei) osteoclasts

Osteoclasts aremultinucleated cells that resorb bone by extrusion of protons andproteolytic enzymes. They display marked heterogeneity in cell size,shape, and resorptive activity. Because high resorptive activity invivo is associated with an increase in the average size of osteoclastsin areas of greater resorption and because of the importance of protonextrusion in resorption, we investigated whether the activity of thebafilomycin A₁-sensitive vacuolar-typeH⁺-ATPase (V-ATPase) and amiloride-sensitiveNa⁺/H⁺ exchanger differed between large andsmall osteoclasts. Osteoclasts were obtained from newborn rabbit bones,cultured on glass coverslips, and loaded with the pH-sensitiveindicator2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF).Intracellular pH (pH_i) was recorded in single osteoclasts by monitoring fluorescence. Large (10 nuclei) and small (5 nuclei) osteoclasts differed in that large osteoclasts had a higher basal pH_i, their pH_i was decreased by bafilomycinA₁ addition or removal of extracellular Na⁺,and the realkalinization upon readdition of Na⁺ wasbafilomycin A₁ sensitive. After acid loading, asubpopulation of large osteoclasts (40%) recovered by V-ATPaseactivity alone, whereas all small osteoclasts recovered byNa⁺/H⁺ exchanger activity. Interestingly, in60% of the large osteoclasts, pH_i recovery was mediated byboth the Na⁺/H⁺ exchanger and V-ATPaseactivity. Our results show a striking difference betweenpH_i regulatory mechanisms of large and small osteoclaststhat we hypothesize may be associated with differences in the potentialresorptive activity of these cells.

     

Human cytomegalovirus infection enhances osmotic stimulation of Na+/H+ exchange in human fibroblasts

Infection withhuman cytomegalovirus (HCMV) causes an enlargement (cytomegaly) ofhuman fibroblasts (MRC-5). As a first step toward determining whethersolute uptake, mediated in part by Na⁺/H⁺exchange, is responsible for the development of cytomegaly, we studiedthe effects of HCMV infection on intracellular pH(pH_i) regulation (nominalCO₂/concn = 0) by comparing cytomegalic cells with mock-infected cells.Seventy-two hours after HCMV infection of MRC-5 cells we observed thefollowing changes relative to mock-infected cells: restingpH_i is 0.1-0.2 pH unit morealkaline; the intrinsic buffering power of the cytoplasm was reduced by~40-50%; acid-loadingH⁺-equivalent fluxes were reduced;and there were alterations of Na⁺/H⁺exchanger (NHE) properties, including an alkaline shift of the pH_i dependence of activity, areduction of the apparent affinity for extracellularNa⁺, and an increase of theapparent maximum velocity and a large increase in stimulation by ahyperosmotic challenge. These results indicate that HCMV infectionexerts a profound effect on functional properties of the NHE, onacid-loading mechanisms, and on intrinsic cellular buffering power.These effects are consistent with a role for the NHE in the developmentof cytomegaly.