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PubMed1983

Acidification of endocytic vesicles by an ATP-dependent proton pump.

One of the early events in the pathway of receptor-mediated endocytosis is the acidification of the newly formed endocytic vesicle. To examine the mechanism of acidification, we used fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) as a probe for endocytic vesicle pH. Changes in pH were determined from the change in fluorescein fluorescence at 490-nm excitation as measured with a microscope spectrofluorometer. After endocytosis of F-alpha 2M, mouse fibroblast cells were permeabilized by brief exposure to the detergent digitonin. Treatment with the ionophore monensin or the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused a rapid increase in the pH of the endocytic vesicle. Upon removal of the ionophore, the endocytic vesicle rapidly acidified only when MgATP or MgGTP was added. Neither ADP nor the nonhydrolyzable analog, adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) could support acidification. The ATP-dependent acidification did not require a specific cation or anion in the external media. Acidification was insensitive to vanadate and amiloride but was inhibited by Zn2+ and the anion transport inhibitor diisothiocyanostilbene disulfonic acid (DIDS). We also examined the acidification of lysosomes with the permeabilized cell system, using fluorescein isothiocyanate dextran as probe. DIDS inhibited the ATP-dependent reacidification of lysosomes, although at a lower concentration than that for inhibition of endocytic vesicle reacidification. These results demonstrate that endocytic vesicles contain an ATP-dependent acidification mechanism that shares similar characteristics with the previously described lysosomal proton pump.

Adenosine Triphosphatases↗

PubMed1998

Regulation of acetylcholine release by intracellular acidification of developing motoneurons in Xenopus cell cultures.

1. The effects of intracellular pH changes on the acetylcholine (ACh) release and cytoplasmic Ca2+ concentration at developing neuromuscular synapses were studied in Xenopus nerve-muscle co-cultures. 2. Spontaneous and evoked ACh release of motoneurons was monitored by using whole-cell voltage-clamped myocytes. Intracellular alkalinization with 15 mM NH4Cl slightly reduced the frequency of spontaneous synaptic currents (SSCs). However, cytosolic acidification following withdrawal of extracellular NH4Cl caused a marked and transient increase in spontaneous ACh release. 3. Another method of cytosolic acidification was used in which NaCl in Ringer solution was replaced with weak organic acids. The increase in spontaneous ACh release paralleled the level of intracellular acidification resulting from addition of these organic acids. Acetate and propionate but not isethionate, methylsulphate and glucuronate, caused an increase in intracellular pH and a marked increase in spontaneous ACh release. 4. Impulse-evoked ACh release was slightly augmented by intracellular alkalinization and inhibited by cytosolic acidification. 5. Cytosolic acidification was accompanied by an elevation in the cytoplasmic Ca2+ concentration ([Ca2+]i), resulting from both external Ca2+ influx and intracellular Ca2+ mobilization. In contrast, the increase in [Ca2+]i induced by high K+ was inhibited by cytosolic acidification. 6. We conclude that cytosolic acidification regulates spontaneous and evoked ACh release differentially in Xenopus motoneurons, increasing spontaneous ACh release but inhibiting evoked ACh release.

Acetylcholine↗

PubMed1975

Acidification in the rat proximal jejunum.

1. Production of hydrogen ions by the rat proximal jejunum was investigated using the everted sac technique. 2. Acidification occurred in the absence of glucose, increasing on addition of glucose to reach a maximal value. An apparent Km of 1-78 mM was derived for the glucose-dependent process. 3. Acidification in the presence of glucose was inhibited by 10 mM-2:4-dinitrophenol, 10 mM phlorrhizin, 10 mM aminophylline and anaerobiosis. 4. Histamine, ethylenediamine tetraacetic acid (EDTA), ouabain and acetazolamide, compounds known to alter acid production in gastric mucosa had no effect on jejunal acidification. 5. Galactose and 3-O-methylglucose failed to increase acidification; in contrast, mannose and fructose did lead to increases, indicating metabolic origin of the hydrogen ions. 6. Serosal and mucosal lactate production were measured and the calculated percentage of hydrogen ions possibly derived from this source was shown to account for only a small proportion of acidification. 7. The greatest increase in acidification with minimal simultaneous production of lactate occurred with ATP which was shown not to enter intestinal tissues. 8. A hypothesis for acidification, that of the break-down at the mucosal surface of ATP from intracellular metabolic sources, is proposed and its relevance to the postulated microclimate is discussed.

Acetazolamide↗

PubMed1994

Vagovagal inhibition of motilin-induced phase III contractions by antral acidification in dog stomach.

Gastric acidification at pH 1.0 strongly inhibits the spontaneously occurring and motilin-induced phase III contractions in canine and human stomach. In this study, we examined inhibition by gastric acidification in dogs following gastrojejunostomy, truncal vagotomy, and antrectomy. As a result, gastric acidification with 0.1 N HCl solution at pH 1.0 for 30 min at a rate of 1.0 ml/min significantly inhibited motilin-induced phase III activity to 23.5 +/- 5.9% of the control in the normal intact dogs and to 17.2 +/- 3.4% in the gastrojejunostomized dogs. In the antrectomized dogs, gastric acidification did not significantly inhibit the action of motilin (81.7 +/- 10%), but, in the vagotomized dogs, gastric acidification inhibited the action of motilin to 72.0 +/- 4.9%; the inhibition was much weaker than in the intact and gastrojejunostomized dogs but was significant. The duodenal acidification had no effect at all on the action of motilin (94.6 +/- 12.5%) in the gastrojejunostomized dogs. These findings strongly suggest the existence of a vagovagal reflex in the inhibition of motilin-induced phase III contractions by gastric antral acidification, although the involvement of sympathetic regulation cannot be completely ruled out.

Animals↗

PubMedDate not supplied

Cisplatinum-induced lesion of proximal tubule acidification in the rat.

Rats were given a 4- to 6-mg/kg body weight intraperitoneal injection of the antitumor drug, Cisplatin, 5-7 days prior to experiments to study tubule acidification by clearance and stationary microperfusion techniques. Cisplatin reduced the glomerular filtration rate markedly and caused a moderate degree of metabolic acidosis, but urine acidification (pH) was well maintained. Proximal tubule stationary pH and bicarbonate concentrations, as measured by pH microelectrodes, were significantly increased. The defect of proximal H+ secretion is reflected by increased acidification half-times (from 4.44 to 10.2 s) and reduced bicarbonate reabsorption to 37% of control values. H-ion back flux, measured during tubule and capillary perfusions with Ringer's bicarbonate- and CO2-free phosphate solutions, was reduced to 68% of control values. The apparent H-ion permeability was lowered from 0.79 to 0.54 cm/s. These results indicate that proximal acidification is reduced by impairment of H+ transport and not by increased transepithelial H+ shunting. Blunted acidification is compatible with a reduction in the number of Na/H exchangers in the proximal brush border and/or a decrease in the apical sodium gradient, the driving force for proximal H-ion secretion. Cortical distal tubule acidification, measured by double-barreled ion-exchange resin/PD microelectrodes, was not significantly affected by Cisplatin. This accounts for the observation that, in spite of the impaired proximal acidification, urine pH is kept within the normal range.

Acid-Base Equilibrium↗

PubMed2000

[Disorders of urinary acidification in patients with monoclonal gammapathies].

BACKGROUND: In patients with monoclonal gammapathy (MG), renal impairment becomes the frequent complication. At the same time, its exact pathogenetic mechanism remains to be identified. We tried to detect urinary acidification defect that may become the first sign of the kidney parenchymal injury. The goal of our work was to estimate the incidence of the acidification impairment, the contribution of proximal and distal types of renal tubular acidosis and the dependency of the defect on the type of light chain paraprotein. METHODS AND RESULTS: In the group of 21 patients aged 38 to 82 years (14 females and 7 males) with monoclonal gammapathy accompanied with creatinemia lower than 180 micromol/l, the short acidification test was performed (calcium chloride was administrated and the urinary acidity was monitored). Renal tubular acidosis was diagnosed in those patients whose urinary acidity after the CaCl2 administration did not reach pH 5.25 or less. The control group consisted of 19 volunteers (6 males and 13 females) with normal renal functions. Results of the tests in MG patients and in control persons were compared using the unpaired Student's t-test. In 11 patients (52.4%) the urinary acidification defect of the incomplete type was detected. Eight of these patients (38.1%) had the distal and three (14.3%) had the proximal type of impairment. Eight patients with the acidification defect had the paraproteinuria kappa and four of them had that of lambda type. In persons without the acidification impairment kappa chains were found in 5 persons, lambda chains in 4 of them. CONCLUSIONS: In our group of MG patients acidification defect occurred in more than half of them. Contrary to the assumed higher nephrotoxicity of the lambda chains, the disturbance was more frequently found in patients with paraproteinemia of the kappa type.

Acidosis, Renal Tubular↗

PubMed1989

Acidification and ion permeabilities of highly purified rat liver endosomes.

While it is well established that acidic pH in endosomes plays a critical role in mediating the orderly traffic of receptors and ligands during endocytosis, little is known about the bioenergetics or regulation of endosome acidification. Using highly enriched fractions of rat liver endosomes prepared by free flow electrophoresis and sucrose density gradient centrifugation, we have analyzed the mechanism of ATP-dependent acidification and ion permeability properties of the endosomal membrane. This procedure permitted the isolation of endosome fractions which were up to 200-fold enriched as indicated by the increased specific activity of ATP-dependent proton transport. Acidification was monitored using hepatocyte and total liver endosomes selectively labeled with pH-sensitive markers of receptor-mediated endocytosis (fluorescein isothiocyanate asialoorosomucoid) or fluid-phase endocytosis (fluorescein isothiocyanate-dextran). In addition, changes in membrane potential accompanying ATP-dependent acidification were directly measured using the voltage-sensitive fluorescent dye Di-S-C3(5). Our results indicate that ATP-dependent acidification of liver endosomes is electrogenic, with proton transport being accompanied by the generation of an interior-positive membrane potential opposing further acidification. The membrane potential can be dissipated by the influx of permeant external anions or efflux of internal alkali cations. Replacement externally of permeable anions with less permeable anions (e.g. replacing Cl- with gluconate) diminished acidification, as did replacement internally of a more permeant cation K+ with less permeant species (such as Na+ or tetramethylammonium). ATP-dependent H+ transport was not coupled to any specific anion or cation, however. The endosomal membrane was found to be extremely permeable to protons, with protons able to leak out almost as fast as they are pumped in. Thus, the internal pH of endosomes is likely to reflect a dynamic equilibrium of protons regulated by the intrinsic ion permeabilities of the endosomal membrane, in addition to the activity of an ATP-driven proton pump.

Animals↗

PubMed1993

Ethinyl estradiol decreases acidification of rat liver endocytic vesicles.

Treatment with ethinyl estradiol is known to impair bile formation, bile acid transport and Na,K-ATPase activity, to alter receptor-mediated endocytosis and transcytosis of IgA and asialoorosomucoid and to affect membrane lipid composition and fluidity. Because appropriate sorting and trafficking of asialoorosomucoid requires adequate acidification of endocytic vesicles by a lipid-sensitive electrogenic proton pump, we examined the effects of 5 days of treatment with ethinyl estradiol (5 mg/kg body wt, subcutaneously) on acidification of early endosomes prepared from male rat livers. Littermate control animals received equal volumes of the solvent propylene glycol. Pretreatment with ethinyl estradiol reduced ATP-dependent initial rates of endosome acidification by 11% to 25% when measured in potassium medium containing 0 to 140 mmol/L chloride; these differences were significant at four of six chloride concentrations tested. The proton pumps of ethinyl estradiol and propylene glycol endosomes exhibited similar Michaelis-Menten constants for MgATP (Michaelis-Menten constant of 63 and 66 mumol/L in the absence of chloride and 101 and 126 mumol/L in the presence of chloride, respectively). Acidification of ethinyl estradiol and propylene glycol endosomes changed in the same manner when various cations or anions were substituted for potassium gluconate, although the effects of ethinyl estradiol were less marked in the absence of K+. Kinetics of inhibition for ethinyl estradiol and propylene glycol endosomes were similar for the proton pump inhibitors N-ethylmaleimide (50% inhibitory concentrations of 13.5 and 18.1 mumol/L), dicyclohexylcarbodiimide (50% inhibitory concentrations of 206 and 216 mumol/L) and bafilomycin A (50% inhibitory concentrations of 11 and 6 nmol/L). Although initial rates of acidification were slower in ethinyl estradiol endosomes, ATP-dependent steady-state vesicle interior pH was the same as that of propylene glycol endosomes over a range of chloride concentrations; this appeared to be due mainly to a trend toward decreased proton leak rates in ethinyl estradiol endosomes. Overall, ethinyl estradiol treatment modestly decreased initial rates of acidification and vesicle proton leakage, perhaps because of changes in endosome lipid composition; differences in the number, density or activation state of proton pumps; or differences in endosome geometry. Because the decrease in acidification rates was small, the effects of estrogen on the efficiency of uncoupling of endocytosed ligands such as asialoorosomucoid from their receptors in early endosomes; thus the rates of sorting and distribution of ligands remain unclear.

Adenosine Triphosphate↗

PubMed1995

Intracellular acidification and Ca2+ transients in cultured rat cerebellar astrocytes evoked by glutamate agonists and noradrenaline.

The effect of different neurotransmitters on the intracellular pH (pHi) and intracellular calcium (Ca2+i) was studied in cultured astrocytes from neonatal rat cerebellum, using the fluorescent dyes 2,7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) and Fura-2. Application of glutamate or kainate (100 microM) in a HEPES-buffered, CO2/HCO3(-) -free saline induced a decrease in pHi and an increase in Ca2+i. Amplitude and time course of the pHi and Ca2+i transients were different. Glutamate and kainate evoked a mean acidification of 0.22 +/- 0.05 (n = 29) and 0.20 +/- 0.09 (n = 12) pH units, respectively. The changes in pHi and Ca2+i induced by kainate, but not by glutamate, were inhibited by 6-cyano-7-dinitroquinozalin-2,3-dion (CNQX; 50 microM). In order to elucidate the mechanism of the agonist-induced acidification, whether the pHi changes were secondary to the Ca2+ rises was tested. In the absence of extracellular Ca2+, the kainate-induced Ca2+i transient was suppressed, while the intracellular acidification was only reduced by 13%. Removal of extracellular Ca2+ reduced the glutamate-induced pHi change by 8%, while the second component of the Ca2+i transient was abolished. Application of trans-( +/- )-1-amino-(1S,3R)-cyclopentadicarboxylic acid (t-ACPD, 100 microM), a metabotropic glutamate receptor agonist, and of noradrenaline (20 microM) evoked a Ca2+i increase, but no change of pHi. D-aspartate, which has a low affinity to glutamate receptors, but is known to be transported by the glutamate uptake system in some astrocytes, evoked an intracellular acidification, similar to that induced by glutamate, but no Ca2+i transient. The results suggest that the kainate-induced acidification is only partly due to the concomitant Ca2+i rise, while the glutamate/aspartate-induced acidification is mainly due to the activation of the glutamate uptake system.

Animals↗

PubMed2006

Role of the plasma membrane calcium adenosine triphosphatase on domoate-induced intracellular acidification in primary cultures of cerebelar granule cells.

Changes in intracellular pH (pH(i)) and cytosolic calcium concentration ([Ca(2+)](c)) caused by the glutamate agonist domoate (DOM) were studied in single cultured mouse cerebellar granule cells (CGC) by using the fluorescent probes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and simultaneous evaluation of cytosolic calcium concentration with the fluorescent dye Fura-2 acetoxymethyl ester (Fura-2 AM). DOM caused a concentration-dependent increase in [Ca(2+)](c) and a concentration-dependent intracellular acidification of CGC. DOM-induced intracellular acidification was completely abolished by the use of Ca(2+)-free medium, suggesting that it was due mostly to an influx of extracellular calcium. The pH(i) decrease caused by DOM was also completely blocked in the presence of the AMPA/kainate receptor antagonist CNQX, indicating that the DOM-induced intracellular acidification was caused by DOM activation of the AMPA/kainate subtype of glutamate receptors. Different mechanisms that could be involved in DOM-induced pH(i) decrease, such as displacement of H(+) by Ca(2+) from a common intracellular binding site, DOM-induced alteration of pH(i) regulation mechanisms, and a possible acidification caused by DOM-induced increase of mitochondrial Ca(2+) uptake, were excluded. DOM-induced intracellular acidification was completely prevented by inhibitors of the plasma membrane calcium adenosine triphosphatase (ATPase) (PMCA), including orthovanadate, lanthanum extracellular pH of 8.5, and the specific PMCA inhibitor caloxin 2A1. Our results therefore indicate that PMCA is involved in DOM-induced intracellular acidification in primary cultures of CGC. Simultaneous recording of [Ca(2+)](c) and pH(i) indicates that the increase in intracellular calcium evoked by DOM will activate the calcium extrusion mechanisms through the calcium pump, which, in turn, will decrease intracellular pH by countertransport of H(+) ions.

Acid-Base Equilibrium↗

PubMed1999

Intracellular acidification during apoptosis can occur in the absence of a nucleus.

During apoptosis, DNA fragmentation and intracellular acidification occur concurrently. Previous results have shown that intracellular acidification is not required for DNA fragmentation, while the alternative, that acidification is a consequence of DNA fragmentation was analyzed here. To obviate the requirement of any nuclear function in acidification, apoptosis was induced by staurosporine in cytoplasts made from the breast tumor cell line MDA-MB-468. Both cells and cytoplasts demonstrated externalization of phosphatidylserine that was prevented by the pan-caspase inhibitor zVAD-fluoromethylketone or by expression of Bcl-2. Intracellular acidification was observed in both cells and cytoplasts and this was also inhibited by both zVAD-fluoromethylketone and Bcl-2. These results show that intracellular acidification and DNA fragmentation are independent consequences of caspase action during apoptosis.

Acids↗

PubMed1990

Secondary hyperaldosteronism stimulates acidification in rat distal colon.

Recent studies from this laboratory have determined that colonic K+ absorption is altered by the PCO2 and by secondary hyperaldosteronism. Partial inhibition by vanadate and mucosal ouabain suggested the operation of an H+/K+ exchange pump. To determine the mechanism of acidification in rat distal colon, we measured in vitro acidification using the pH-stat technique by voltage-clamped segments of colonic epithelium in controls and in the presence of secondary hyperaldosteronism, induced by a sodium-deficient diet. Chronic stimulation with aldosterone resulted in increased mucosal acidification in vitro for at least 2 h. This effect could not be accounted for by lactate production and was not altered by elimination of the aldosterone-induced increase in voltage and short-circuit current with 10 microM amiloride. Studies with inhibitors and ion substitution revealed that mucosal acidification resulted from both Na-dependent and Na-independent mechanisms. Na-dependent acidification was inhibited by ATPase inhibitors and was mediated in part by a luminal Na+/H+ exchanger in the presence of secondary hyperaldosteronism. Na-independent acidification was mediated by a pathway dependent on luminal K+ that was inhibited by vanadate and mucosal ouabain, consistent with the operation of an H+/K+ exchange pump.

Adenosine Triphosphatases↗

PubMed1991

Cl-, Na+, and H+ fluxes during the acidification of rabbit reticulocyte endocytic vesicles.

The ionic fluxes associated with the ATP-dependent acidification of endocytic vesicles were studied in a preparation isolated from rabbit reticulocytes enriched for transferrin-transferrin receptor complexes. No vesicle acidification was observed in the absence of intra- and extravesicular ions (sucrose(in)/sucrose(out), while maximal acidification was observed with NaCl(in)/KCl(out).K+(in) was a poor substitute for Na+(in), and Cl-(out) could be replaced by other anions with the following efficacy of acidification: Cl- greater than Br- greater than I- greater than PO4(3-) greater than gluconate greater than SO4(2-). Flux studies using 36Cl- and 22Na+ showed that the vesicles had a permeability for Cl- and Na+, and that ATP-dependent H+ pumping was accompanied by a net influx of Cl- and a net efflux of Na+ provided that there was a Na+ concentration gradient. After 3 mins, the time necessary to maximal acidification, the electrical charge generated by the entrance of H+ was countered to about 45% by the Cl- influx and to about 42% by the Na+ efflux. These studies demonstrated that both Cl- and Na+ fluxes are necessary for optimal endocytic vesicle acidification.

Adenosine Triphosphatases↗

PubMed1981

Acidification and sodium entry in frog skin epithelium.

Acidification of the external medium by isolated frog skin epithelium (Rana catesbeiana, Rana temporaria, and Caudiververa caudiververa) and its relationship to Na+ uptake was studied. Acidification was measured by the pH-stat technique under short-circuit or open-circuit conditions. The results of this study demonstrate that (a) acidification by these species of in vitro frog skins is not directly coupled to Na+ or anion transport; (b) acidification can be inhibited by the diuretic drug amiloride, but only at high external Na+ concentrations; (c) acidification rate in these species of frog skin is controlled in part by the metabolic production of CO2; and (d) the positive correlation between net Na+ absorption and net acidification observed in whole animal studies could not be replicated in the in vitro skin preparation, even when the frogs were first chronically stressed by salt depletion, a physiological state comparable to that used in the in vivo experiments.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1991

Mechanisms of acid injury to rabbit esophageal epithelium. Role of basolateral cell membrane acidification.

Because mucosal HCl traverses the paracellular pathway before significant damage develops within esophageal epithelium, the effects of mucosal and serosal acidification were compared on Ussing-chambered rabbit esophageal epithelia. Notably serosal, but not mucosal, acidification was associated with cell necrosis, and the latter was accompanied by abolition of potential difference and short-circuit current. The reason for this difference was explored by exposing tissues serosally to ouabain, chloride-free solution, 4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid (SITS), or amiloride. The results show that serosal acidification, but not ouabainization, is associated with cell necrosis and that cell necrosis induced by serosal acidification can be blocked by SITS and chloride-free solution, but not by amiloride. These findings are compatible with the hypothesis that serosal acidification is more damaging than mucosal acidification because of the greater rate with which hydrogen ions can traverse the basolateral membrane; also, the route for more rapid entry appears to involve a SITS-sensitive, chloride-dependent mechanism (e.g., Cl/HCO3 antiport).

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1986

Cytoplasmic pH regulation in activated human neutrophils: effects of adenosine and pertussis toxin on Na+/H+ exchange and metabolic acidification.

When stimulated, neutrophils undergo a complex change in cytoplasmic pH (pHi): an incipient acidification, followed by an alkalinization which is due to activation of Na+/H+ exchange. When the latter is inhibited by amiloride or by removal of extracellular Na+, the actual magnitude of the initial acidification can be fully appreciated. The acidification is thought to be of metabolic origin, but the precise origin of the H+ (equivalents) remains undefined. We used adenosine, a modulator of neutrophil responsiveness, to identify the source of metabolic acid in cells stimulated by either formylmethionylleucylphenylalanine (fMet-Leu-Phe) or 12-O-tetradecanoylphorbol 13-acetate (TPA). Pretreatment of the cells with adenosine inhibited the fMet-Leu-Phe-induced respiratory burst, but secretion of specific and azurophilic granules, as well as aggregation were unaffected. In fMet-Leu-Phe-treated cells, adenosine reduced the acidification recorded in Na+-free media, but had no effect on the activation of the Na+/H+ antiport. Adenosine had little or no effect on the TPA-induced responses, including the pHi changes. The respiratory burst, as well as the cytoplasmic acidification were also inhibited in parallel by pretreating the cells with 'islet-activating protein' from Bordetella pertussis. It was concluded that activation of the NADPH-oxidase and/or the associated stimulation of the hexose monophosphate shunt play a major role in the metabolic acidification of stimulated neutrophils.

Adenosine↗

PubMed2005

Vinyl acetate induces intracellular acidification in mouse oral buccal epithelial cells.

Vinyl acetate exposure in drinking water has been associated with tumor formation in the upper gastrointestinal tract of rats and mice. One potential mechanism for inducing carcinogenesis involves acidification of the intracellular environment due to the metabolism of vinyl acetate to acetic acid. Prolonged intracellular acidification is thought to produce cytotoxic and/or mitogenic responses that are the sentinel pharmacodynamic steps toward cancer. To determine whether exposure to vinyl acetate affects the intracellular pH of intact oral cavity tissue, isolated mouse oral buccal epithelium was loaded with the pH-sensitive dye BCECF, and then exposed to vinyl acetate concentrations ranging from 10 to 1000 microM for up to 4 min. Extracellular vinyl acetate exposure induced a progressive intracellular acidification that was reversible upon removal of the vinyl acetate. The rate of the acidification was concentration-dependent and increased exponentially within the concentration range tested. The magnitude of the vinyl acetate-induced acidification was inhibited by pretreatment with the carboxylesterase inhibitor bis(p-nitrophenyl)phosphate. These results are consistent with the hypothesis that vinyl acetate contributes to the generation and progression of oral cavity tumors via a process of intracellular acidification. Such a process has been proposed to have practical dose-response thresholds below which the intracellular environment can be maintained within homeostatic bounds and the contribution of exposure to carcinogenic risk is negligible.

Acids↗

PubMed1997

Inhibition by xipamide of amiloride-induced acidification in cultured rat cardiocytes.

The diuretic drug xipamide improves myocardial relaxation in hypertensive patients with left ventricular hypertrophy, but its mechanism of action is unknown. Here, xipamide was tested in cultured rat heart myogenic H9c2 cells and newborn cardiomyocytes for its effects on cell acidification (and Ca2+ mobilization). In H9c2 cells, blocking Na+/H+ exchange with amiloride (2 mM) provoked cell acidification with rate = 0.82 +/- 0.17 pH units/h (n = 6). Xipamide 1 microM maximally inhibited 50 +/- 7% (n = 9) of cell acidification. The action of xipamide required the presence of HCO3- and was antagonized by the HCO3(-)-transport blocker DIDS (4,4'-diisothiocyanostilbene-2.2'-disulfonic acid). Conversely, the carbonic anhydrase (EC 4.2.1.1) inhibitor acetazolamide failed to prevent xipamide action. Finally, xipamide was without significant effect on the Ca2+ signals induced by endothelin-1, vasopressin or the Ca2+ ionophore ionomycin. In newborn rat cardiomyocytes, xipamide reduced amiloride-induced cell acidification at similar concentrations as in H9c2 cardiocytes, but with a slightly higher extent of maximal inhibition (70-80%). In conclusion, xipamide reduced amiloride-dependent cell acidification in the rat heart myogenic H9c2 cell line and in newborn rat cultured cardiomyocytes. This action of xipamide seems to be related to a complex interaction with DIDS-sensitive HCO3- movements. Prevention of cell acidification by xipamide could be involved in the beneficial effects of this compound in myocardial relaxation and left ventricle filling in hypertensive patients with left ventricular hypertrophy.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗