Effect Of Intracellular Acidification Research Articles

Intracellular acidification and damage of cellular membrane of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles

The effects of intracellular acidification and damage of cellular membrane on the inactivation of Saccharomyces pastorianus by a two-stage method of low-pressure carbon dioxide microbubbles (two-stage MBCO2) were investigated. Intra/extra cellular pH (pHin and pHex) and propidium iodide (PI) uptake of S. pastorianus cells treated with the two-stage MBCO2 were measured by fluorescence analysis, and were compared with the inactivation efficiency. The pHex and pHin of S. pastorianus cells treated with only mixing vessel at 10 °C of two-stage MBCO2 was lower than those that were untreated. However, the surviving number and the pHin of S. pastorianus cells treated with the two-stage MBCO2 containing the mixing vessel at 1.0 MPa and the heating coil of 40 °C decreased concomitant with the exposure time, and the increase of the PI uptake ratio was confirmed. In addition, when the pressure of the mixing vessel was 1.0 MPa, the surviving number and the pHin of S. pastorianus cells significantly decreased, and the PI uptake ratio increased by increasing the temperature to 45 and 50 °C with the heating coil of the two-stage MBCO2. These results suggested that the inactivation of S. pastorianus by two-stage MBCO2 might be induced by the damage to the cellular membrane at 45 and 50 °C, and by the lowering of the pHin at 40 °C.

Intracellular Acidification Is Associated with Changes in Free Cytosolic Calcium and Inhibition of Action Potentials in Rat Trigeminal Ganglion

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca(2+)-induced acidification and subsequent recovery of intracellular pH (pH(i)) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 μM) and capsaicin (1 μM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) with a following decrease in pH(i). The recovery of [Ca(2+)](i) to the prestimulus level was inhibited by LaCl(3) (1 mM) and o-vanadate (10 mM), a plasma membrane Ca(2+)/ATPase (PMCA) inhibitor. Removal of extracellular Ca(2+) also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na(+)/H(+) exchanger type 1 (NHE1), pancreatic Na(+)-HCO(3)(-) cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1-3 were detected by RT-PCR. pH(i) recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH(i). Application of the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (5 μM) or the pNBC1 inhibitor 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid (500 μM) delayed pH(i) recovery and decreased AP frequency. Simultaneous application of 5'-(N-ethyl-N-isopropyl) amiloride and 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca(2+)](i) in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH(i) recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.

Non-apoptotic concentrations of prodigiosin (H +/Cl − symporter) inhibit the acidification of lysosomes and induce cell cycle blockage in colon cancer cells

Prodigiosin (PG) is a bacterial red pigment with interesting immunosuppressive and apoptotic properties that have been partly attributed to its ability to uncouple V-ATPase through the promotion of the H +/Cl − symporter. In the present study, we investigate the effect of non-apoptotic concentrations of PG on the lysosomal-pH and on cell cycle progression in colon cancer cells. Lysosomal-pH was tested in DLD-1 cells using acridine orange vital staining. Orange granules, indicative of acidified lysosomes, decreased significantly in cells treated with 25 nM of PG for 1 / 2 h, and disappeared completely at 100 nM. This suggests that PG can induce lysosomal alkalinization without any apparent cytotoxic effect. Cell cycle progression was analysed in HT29 cells and we found that PG induces a blockage in the G1 phase. This blockage correlates with p21 WAF1/CIP1 induction, and it can be triggered either dependently or independently of p53. In conclusion, the reversible increase in lysosomal-pH and cytosol acidification induced by non-apoptotic concentrations of PG in colon cancer cells, suggests that the apoptotic process induced by PG can not be solely explained by changes in intracellular pH. The effect of intracellular acidification on cell cycle arrest must be analysed more exactly.

The effect of intracellular acidification on the relationship between cell volume and membrane potential in amphibian skeletal muscle.

The relationship between cell volume (V(c)) and membrane potential (E(m)) in Rana temporaria striated muscle fibres was investigated under different conditions of intracellular acidification. Confocal microscope xz-scanning monitored the changes in V(c), whilst conventional KCl and pH-sensitive microelectrodes measured E(m) and intracellular pH (pH(i)), respectively. Applications of Ringer solutions with added NH(4)Cl induced rapid reductions in V(c) that rapidly reversed upon their withdrawal. These could be directly attributed to the related alterations in extracellular tonicity. However: (1) a slower and persistent decrease in V(c) followed the NH(4)Cl withdrawal, leaving V(c) up to 10% below its resting value; (2) similar sustained decreases in resting V(c) were produced by the addition and subsequent withdrawal of extracellular solutions in which NaCl was isosmotically replaced with NH(4)Cl; (3) the same manoeuvres also produced a marked intracellular acidification, that depended upon the duration of the preceding exposure to NH(4)Cl, of up to 0.53 +/- 0.10 pH units; and (4) the corresponding reductions in V(c) similarly increased with this exposure time. These reductions in V(c) persisted and became more rapid with Cl(-) deprivation, thus excluding mechanisms involving either direct or indirect actions of pH(i) upon Cl(-)-dependent membrane transport. However they were abolished by the Na(+),K(+)-ATPase inhibitor ouabain. The E(m) changes that accompanied the addition and withdrawal of NH(4)(+) conformed to a Nernst equation modified to include realistic NH(4)(+) permeability terms, and thus the withdrawal of NH(4)(+) restored E(m) to close to control values despite a persistent change in V(c). Finally these E(m) changes persisted and assumed faster kinetics with Cl(-) deprivation. The relative changes in V(c), E(m) and pH(i) were compared to predictions from the recent model of Fraser and Huang published in 2004 that related steady-state values of V(c) and E(m) to the mean charge valency (z(x)) of intracellular membrane-impermeant anions, X(-)(i). By assuming accepted values of intracellular buffering capacity (beta(i)), intracellular acidification was shown to produce quantitatively predictable decreases in V(c). These findings thus provide experimental evidence that titration of the anionic z(x) by increased intracellular [H(+)] causes cellular volume decrease in the presence of normal Na(+),K(+)- ATPase activity, with Cl(-)-dependent membrane fluxes only influencing the kinetics of such changes.

Effects of intracellular acidification and varied temperature on force, stiffness, and speed of shortening in frog muscle fibers.

This study aimed to establish whether the temperature-dependent effect of acidification on maximum force observed in mammalian muscles also applies to frog muscle. Measurements of force, stiffness, and unloaded velocity of shortening in intact single muscle fibers from the anterior tibialis muscle of Rana temporaria were performed between 0 and 22 degrees C during fused tetani in H(2)CO(3)-CO(2)-buffered Ringer solution with pH adjusted to 7.0 and 6.3, respectively. The force-to-stiffness ratio increased as a rectilinear function of temperature between 0 and 20 degrees C at pH 7.0. Lowering the pH to 6.3 reduced the tetanic force by 13.5 +/- 1.2 and 11.5 +/- 1.4% at 2.8 and 20.5 degrees C, respectively, with only a minor reduction in fiber stiffness. The maximum speed of shortening was decreased by lowered pH by 12.9 +/- 1.5 and 7.8 +/- 1.1% at low and high temperature, respectively. Acidification increased the time to reach 70% of maximum force by 18.0% at approximately 2 degrees C; the same pH change performed at approximately 20 degrees C in the same fibers reduced the rise time by 24.1%. The same increase in the rate of rise of force at high temperature was also found at normal pH after the fibers were fatigued by frequent stimulation. It is concluded that, in frog muscle, the force-depressant effect of acidification does not vary significantly with temperature. By contrast, acidification affects the onset of activation in a manner that is critically dependent on temperature.

Dehydroascorbic acid uptake by coronary artery smooth muscle: effect of intracellular acidification

Dehydroascorbic acid (DHAA) enters cells via Na+-independent glucose transporters (GLUT) and is converted to ascorbate. However, we found that Na+ removal inhibited [14C]DHAA uptake by smooth-muscle cells cultured from pig coronary artery. The uptake was examined for 2–12min at 10–200μM DHAA in either the presence of 134mM Na+ or in its absence (N-methyl d-glucamine, choline or sucrose replaced Na+). This inhibition of DHAA uptake by Na+ removal was paradoxical because it was inhibited by 2-deoxyglucose and cytochalasin B, as expected of transport via the GLUT pathway. We tested the hypothesis that this paradox resulted from an inefficient intracellular reduction of [14C]DHAA into [14C]ascorbate upon intracellular acidosis caused by the Na+ removal. Consistent with this hypothesis: (i) the Na+/H+-exchange inhibitors ethylisopropyl amiloride and cariporide also decreased the uptake, (ii) Na+ removal and Na+/H+-exchange inhibitors lowered cytosolic pH, with the decrease being larger in 12min than in 2min, and (iii) less of the cellular 14C was present as ascorbate (determined by HPLC) in cells in Na+-free buffer than in those in Na+-containing buffer. This inability to obtain ascorbate from extracellular DHAA may be detrimental to the coronary artery under hypoxia-induced acidosis during ischaemia/reperfusion.

Dehydroascorbic acid uptake by coronary artery smooth muscle: effect of intracellular acidification.

Dehydroascorbic acid (DHAA) enters cells via Na(+)-independent glucose transporters (GLUT) and is converted to ascorbate. However, we found that Na(+) removal inhibited [(14)C]DHAA uptake by smooth-muscle cells cultured from pig coronary artery. The uptake was examined for 2-12 min at 10-200 microM DHAA in either the presence of 134 mM Na(+) or in its absence (N-methyl D-glucamine, choline or sucrose replaced Na(+)). This inhibition of DHAA uptake by Na(+) removal was paradoxical because it was inhibited by 2-deoxyglucose and cytochalasin B, as expected of transport via the GLUT pathway. We tested the hypothesis that this paradox resulted from an inefficient intracellular reduction of [(14)C]DHAA into [(14)C]ascorbate upon intracellular acidosis caused by the Na(+) removal. Consistent with this hypothesis: (i) the Na(+)/H(+)-exchange inhibitors ethylisopropyl amiloride and cariporide also decreased the uptake, (ii) Na(+) removal and Na(+)/H(+)-exchange inhibitors lowered cytosolic pH, with the decrease being larger in 12 min than in 2 min, and (iii) less of the cellular (14)C was present as ascorbate (determined by HPLC) in cells in Na(+)-free buffer than in those in Na(+)-containing buffer. This inability to obtain ascorbate from extracellular DHAA may be detrimental to the coronary artery under hypoxia-induced acidosis during ischaemia/reperfusion.

Modulation of ATP-sensitive K+ channels by internal acidification in insulin-secreting cells.

The effect of intracellular acidification (low pHi) on open probability of the ATP-sensitive K+ (KATP) channel was examined in insulin-secretion cells using an inside-out configuration of the patch-clamp technique. In an insulin-secreting cell line beta-TC3, KATP single-channel currents (IKATP) were readily recorded in the absence of internal ATP. ATP (50 microM and 0.5 mM) dramatically decreased the channel activity. A step decrease of intracellular pH (pHi) from 7.4 to 6.7 or 6.3 in the presence of ATP gradually increased the channel activity. In addition, low pHi in the presence of ATP could partially restore channel activity lost in a process called "rundown." Kinetic analysis revealed a change in channel gating at low pHi with ATP. The bursting durations of IKATP at pHi 6.3 in the presence of ATP were significantly longer than those at pHi 7.4 in the absence of ATP. These results suggest that the increased channel activity at low pHi might have resulted from a mechanism involving an alteration of channel conformation. We also observed an inhibitory effect of low pHi on channel activity. However, the inhibitory effect was much more apparent at pHi 5.7 and was only partially reversible. The activation effect of low pHi on IKATP in the presence of ATP was also observed in acutely isolated rat islet cells and in another insulin-secretion cell line RINm5F, although the effect was weaker and was variable among experiments. We conclude that, as in frog skeletal muscle and cardiac muscle, an increase in channel activity at low pHi is one of the mechanisms underlying proton modulation of IKATP in insulin-secreting cells.

Modulation of calcium homeostasis in cultured rat aortic endothelial cells by intracellular acidification

Acidosis produces vasodilation in a process that may involve the vascular endothelium. Because synthesis and release of endothelium-derived vasodilatory substances are linked to an increase in cytosolic calcium concentration ([Ca2+]i), we examined the effect of intracellular acidification on cultured rat aortic endothelial cells loaded either with the pH-sensitive probe carboxy-seminaphthorhodafluor-1 or the Ca(2+)-sensitive fluorescent probe indo 1. The basal cytosolic pH (pHi) of endothelial monolayers in a 5% CO2-HCO3- buffer was 7.27 +/- 0.02 and that in a bicarbonate-free solution was 7.22 +/- 0.03. Acidification was induced either by removal of NH4Cl (delta pHi = -0.10 +/- 0.02), changing from a bicarbonate-free to a 5% CO2-HCO3(-)-buffered solution at constant buffer pH (delta pHi = -0.18 +/- 0.03), or changing from a 5% to a 20% CO2-HCO3- solution (delta pHi = -0.27 +/- 0.07). Regardless of the method used, intracellular acidification increased [Ca2+]i as indexed by indo 1 fluorescence. The increase in [Ca2+]i induced by changing from a 5 to a 20% CO2-HCO3- solution was not significantly altered by removal of buffer Ca2+ either before or after depletion of bradykinin- and thapsigargin-sensitive intracellular Ca2+ stores. Thus intracellular acidification of vascular endothelial cells releases Ca2+ into the cytosol either from pH-sensitive intracellular buffer sites, mitochondria, or from bradykinin- and thapsigargin-insensitive intracellular stores. This Ca2+ mobilization may be linked to endothelial synthesis and release of vasodilatory substances during acidosis.

Effect of intracellular acidification on colonic NaCl absorption.

CO2 stimulates Na+ and Cl- absorption in rat distal colon. This is most likely due to intracellular generation of H+ and HCO3- and stimulation of apical Na(+)-H+ and Cl(-)-HCO3- exchangers. We examined whether intracellular acidification by means other than CO2 would also stimulate Na+ absorption. Stripped segments of distal colon from male Sprague-Dawley rats were studied under short-circuit conditions in Ussing chambers. Identically prepared tissues were used for intracellular pH (pHi) measurements with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. When the Ringer PCO2 was increased from 20 to 34 mmHg, pHi decreased from 7.50 +/- 0.04 to 7.35 +/- 0.04 and net Na absorption increased from 2.4 +/- 0.7 to 3.7 +/- 0.7 mu eq.cm-2 x h-1. A similar degree of intracellular acidification was obtained with 2.6 microM nigericin, but no stimulation of Na+ absorption was seen. When Ringer-HCO3- concentration was reduced from 39 to 11 mM at constant PCO2 = 35 mmHg, pHi decreased from 7.55 +/- 0.02 to 7.11 +/- 0.02 with no effect on net Na+ absorption. A similar reduction in pHi in a CO2-HCO3(-)-free, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Ringer also did not stimulate Na+ absorption. Methazolamide had no effect on steady-state pHi at any given PCO2 but caused marked reductions in net Na+ absorption (9.6 +/- 2.4 to 5.2 +/- 1.2 mu eq.cm-2 x h-1 at PCO2 = 70 mmHg). We conclude that Na+ absorption in rat distal colon is not stimulated by intracellular acidification per se but rather has an absolute requirement for CO2 and carbonic anhydrase activity.

Transient increase in Ca2+ influx in Saccharomyces cerevisiae in response to glucose: effects of intracellular acidification and cAMP levels

Influx of 45Ca2+ into Saccharomyces cerevisiae was measured under experimental conditions which enabled measurements of initial rate of transport across the plasma membrane, without interference by the vacuolar Ca2+ transport system. Addition of glucose or glycerol to the cells, after pre-incubation in glucose-free medium for 5 min, caused a rapid, transient increase in 45Ca2+ influx, reaching a peak at 3-5 min after addition of substrate. Ethanol, or glycerol added with antimycin A, had no effect on 45Ca2+ influx. We have shown previously that this increase is not mediated by an effect of the substrates on intracellular ATP levels. Changes in membrane potential accounted for only a part of the glucose-stimulated 45Ca2+ influx. The roles of intracellular acidification and changes in cellular cAMP in mediating the effects of glucose on 45Ca2+ influx were examined. After a short preincubation in glucose-free medium addition of glucose caused a decrease in the intracellular pH, [pH]i, which reached a minimum value after 3 min. A transient increase in the cellular cAMP level was also observed. Addition of glycerol also caused intracellular acidification, but ethanol or glycerol added with antimycin A had no effect on [pH]i. Artificial intracellular acidification induced by exposure to isobutyric acid or to CCCP caused a transient rise in Ca2+ influx but the extent of the increase was smaller than that caused by glucose, and the time-course was different. We conclude that intracellular acidification may be responsible for part of the glucose stimulation of Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Force during stretch and shortening of frog sartorius muscle: Effects of intracellular acidification due to increased carbon dioxide

The force-velocity relation of frog sartorius muscle was observed during slow stretch and during shortening in solutions with and without CO2 at extracellular pH (pHo) 6.9 and pHo 7.5 (5 degrees C). Less force was produced with CO2 than without CO2 during stretch, during shortening, and under isometric conditions. Compared with pHo 7.5, the effects were greater at pHo 6.9, where the concentration of CO2, a permeant acid, was greater and would cause a greater acidification of intracellular pH (pHi). The reduction of force caused by CO2 was smaller during stretch than during shortening or isometric contraction. This result indicates that the crossbridge states specific to stretch retain their ability to produce force better under acidic conditions than those characteristic of shortening and isometric conditions. This difference between stretch and shortening suggests that there may be compensating changes in the pattern of motor unit activity during fatigue in vivo.

Electrotonic synapses between Aplysia neurons in situ and in culture: aspects of regulation and measurements of permeability

Properties of electrotonic synapses between L14 neurons in the abdominal ganglion of the marine mollusc Aplysia californica were examined in situ and between unidentified buccal neurons maintained in tissue culture. In culture, depolarizing postsynaptic potentials in response to a train of action potentials showed apparent facilitation with increasing spike number, which was attributable to the low-pass filter properties of electrotonic transmission via gap junctions and to network properties. Gap junctional conductance (gj), calculated from current-clamp data or measured directly under voltage clamp, indicated no significant dependence of gj on transjunctional or inside-outside potential in situ or in culture. Octanol, a local anesthetic agent that reduces gj in many other systems, had no effect on gj between Aplysia neurons. The effect of intracellular acidification, a treatment that rapidly and reversibly uncouples a variety of cell types, reduced gj between Aplysia neurons but did not completely abolish it. The relationship between intracellular pH (pHi), measured with ion-sensitive microelectrodes, and gj was steeper in cultured neurons than in situ and was maximally reduced by 70-80%, as compared to 50% or less in situ at the lowest pHi values tested. The coupling coefficient (k) was reduced less by low pHi than was gj, which could be explained by a simultaneous increase in nonjunctional membrane resistance. Permeability properties of Aplysia electrotonic synapses to a variety of tracer molecules were also examined between identified L 14 neurons in situ and in dissociated buccal, abdominal, and bag neurons in culture. The fluorescent dyes Lucifer yellow, 6-carboxyfluorescein, and dichlorofluorescein (1.2-1.4 nm maximal diameters) did not spread detectably from an injected neuron to its electrically coupled neighbors, regardless of the strength of electrotonic coupling. However, the smaller tetraalkylammonium ions TMA and TEA (diameters 0.66 and 0.8 nm, concentrations measured with ion-selective electrodes), could be detected in neighboring cells within minutes. In culture, transfer of the tetraalkylammonium ions was slow and not easily detectable in cell pairs where gj was low (less than 20 nS). The permeability was as high as 10(-10) cm3/sec in situ and 10(-12) cm3/sec in culture, and values were roughly correlated with simultaneously measured values of gj. Electrotonic synapses in the nervous system of Aplysia, therefore, have a quantitatively different spectrum of sensitivities than has been found for gap junctions of other systems and appear to possess reduced permeability to tracer molecules.

単一心室筋細胞の電気活動に及ぼす細胞内酸性化の影響

単一心室筋細胞の電気活動に及ぼす細胞内酸性化の影響

Effects of intracellular acidification on membrane currents in ventricular cells of the guinea pig.

The membrane currents of single ventricular cells were measured under whole cell voltage clamp using a giga-sealed patch electrode, and the effects of intracellular acidification were examined by perfusing the electrode pipette with different pH solutions. The plateau of the action potential was shortened when the pH of the pipette solution was lowered from the control of 7.2 to 6, and finally to 5. The pH 6 pipette solution evoked a time-independent outward current at positive potentials and increased the slope conductance near the resting potential. These changes were suppressed by removal of both intra- and extracellular potassium ion, indicating that these currents were carried by potassium ions, but not by protons. Increasing the calcium concentration in the pipette from pCa 8 to pCa 6 induced a time-dependent outward current which had a reversal potential of about -13 mV. This result clearly differed from the changes induced by the acidic pipette solution, suggesting that the calcium-mediated conductance was not involved in the genesis of the acidic effects. The calcium current was not significantly affected by perfusion at pH 6, but was decreased by the more acidic (pH 5) solution. When the calcium current was recorded in sodium- and potassium-free external solution but with a cesium-rich internal solution, however, the calcium current was suppressed even with a weak acidic (pH 6.8) pipette solution. This effect was attributed not to an increased sensitivity of the calcium channel to protons, but to a more extensive intracellular acidification, which might have been caused by a depressed extrusion of proton via a sodium-hydrogen exchange mechanism on the surface membrane.

Stimulus-secretion coupling of glucose-induced insulin release. Effect of intracellular acidification upon calcium efflux from islet cells

When pancreatic islets were exposed to a medium of normal pH (7.4) equilibrated with a gaz mixture containing 30% (instead of 5%) CO 2, the intracellular pH of the islet cells, as judged by the apparent space of distribution of 14C-DMO, was decreased. The intracellular acidification was associated with a delayed decrease of [U- 14C] glucose oxidation, but no major change in glucose-stimulated proinsulin biosynthesis, 45calcium net uptake, or insulin release. The increase in p CO 2 provoked an immediate and sustained decrease in the fractional outflow rate of 45Ca from prelabeled and perfused islets. The latter decrease was most marked under conditions associated with stimulated 40Ca 45Ca exchange (i.e., at glucose 16.7 m M and normal Ca 2+ concentration), but was also present when a process of Na +Ca 2+ countertransport accounted for the major part of 45Ca efflux (e.g., in the absence of both glucose and extracellular Ca 2+). These findings are compatible with the view that the generation of H + derived from the metabolism of glucose in islet cells plays a role in the sugar-induced decrease in 45Ca fractional outflow rate.