Influence Of Intracellular pH Research Articles

The Antimicrobial Activity of Human Defensins at Physiological Non-Permeabilizing Concentrations Is Caused by the Inhibition of the Plasma Membrane H+-ATPases.

Human defensins are cysteine-rich peptides (Cys-rich peptides) of the innate immune system. Defensins contain an ancestral structural motif (i.e., γ-core motif) associated with the antimicrobial activity of natural Cys-rich peptides. In this study, low concentrations of human α- and β-defensins showed microbicidal activity that was not associated with cell membrane permeabilization. The cell death pathway was similar to that previously described for human lactoferrin, also an immunoprotein containing a γ-core motif. The common features were (1) cell death not related to plasma membrane (PM) disruption, (2) the inhibition of microbicidal activity via extracellular potassium, (3) the influence of cellular respiration on microbicidal activity, and (4) the influence of intracellular pH on bactericidal activity. In addition, in yeast, we also observed (1) partial K+-efflux mediated via Tok1p K+-channels, (2) the essential role of mitochondrial ATP synthase in cell death, (3) the increment of intracellular ATP, (4) plasma membrane depolarization, and (5) the inhibition of external acidification mediated via PM Pma1p H+-ATPase. Similar features were also observed with BM2, an antifungal peptide that inhibits Pma1p H+-ATPase, showing that the above coincident characteristics were a consequence of PM H+-ATPase inhibition. These findings suggest, for the first time, that human defensins inhibit PM H+-ATPases at physiological concentrations, and that the subsequent cytosolic acidification is responsible for the in vitro microbicidal activity. This mechanism of action is shared with human lactoferrin and probably other antimicrobial peptides containing γ-core motifs.

Nutrient-stimulated insulin secretion in mouse islets is critically dependent on intracellular pH.

BackgroundMany mechanistic steps underlying nutrient-stimulated insulin secretion (NSIS) are poorly understood. The influence of intracellular pH (pHi) on insulin secretion is widely documented, and can be used as an investigative tool. This study demonstrates previously unknown effects of pHi-alteration on insulin secretion in mouse islets, which may be utilized to correct defects in insulin secretion.MethodsDifferent components of insulin secretion in mouse islets were monitored in the presence and absence of forced changes in pHi. The parameters measured included time-dependent potentiation of insulin secretion by glucose, and direct insulin secretion by different mitochondrial and non-mitochondrial secretagogues. Islet pHi was altered using amiloride, removal of medium Cl-, and changing medium pH. Resulting changes in islet pHi were monitored by confocal microscopy using a pH-sensitive fluorescent indicator. To investigate the underlying mechanisms of the effects of pHi-alteration, cellular NAD(P)H levels were measured using two-photon excitation microscopy (TPEM). Data were analyzed using Student's t test.ResultsTime-dependent potentiation, a function normally absent in mouse islets, can be unmasked by a forced decrease in pHi. The optimal range of pHi for NSIS is 6.4–6.8. Bringing islet pHi to this range enhances insulin secretion by all mitochondrial fuels tested, reverses the inhibition of glucose-stimulated insulin secretion (GSIS) by mitochondrial inhibitors, and is associated with increased levels of cellular NAD(P)H.ConclusionsPharmacological alteration of pHi is a potential means to correct the secretory defect in non-insulin dependent diabetes mellitus (NIDDM), since forcing islet pHi to the optimal range enhances NSIS and induces secretory functions that are normally absent.

Bicarbonate promotes dye coupling in the epithelium and endothelium of the rabbit cornea

Purpose. Examine the mechanism of bicarbonate maintenance of cell-to-cell coupling in rabbit corneal epithelium and endothelium. Methods. Carboxyfluorescein was microinjected into rabbit corneal epithelial and endothelial cells. Adjacent cells were observed for fluorescence. Bathing solutions were buffered with bicarbonate, HEPES, phosphate, or acetate-citrate. The influence of intracellular pH and transmembrane voltage (V m) were examined. Results. Bicarbonate was the only buffer to increase dye coupling. Substitution of bicarbonate structural analogs bisulfite and carbamate in a HEPES-buffered solution increased dye coupling in both cell types. Intracellular pH and V m alterations in corneal epithelial cells bathed in HEPES vs. bicarbonate buffered media had no significant effects on dye coupling. Conclusions. Bicarbonate increases intercellular communication in the corneal epithelium and endothelium. This effect appears to result from an interaction of the bicarbonate molecule (or one of its structural analogs) with either gap junction proteins or an intermediary. We also demonstrate the presence of Cx43 in the rabbit corneal endothelium.

The effect of intracellular pH on cytosolic Ca2+ in HT29 cells.

The influence of intracellular pH (pHi) on intracellular Ca2+ activity ([Ca2+]i) in HT29 cells was examined microspectrofluorometrically. pHi was changed by replacing phosphate buffer by the diffusible buffers CO2/HCO3- or NH3/NH4+ (pH 7.4). CO2/HCO3- buffers at 2,5 or 10% acidified pHi by 0.1, 0.32 and 0.38 pH units, respectively, and increased [Ca2+]i by 8-15 nmol/l. This effect was independent of the extracellular Ca2+ activity and the filling state of thapsigargin-sensitive Ca2+ stores. Removing the CO2/HCO3- buffer alkalinized pHi by 0.14 (2%), 0.27 (5%), and 0.38 (10%) units and enhanced [Ca2+]i to a peak value of 20, 65, and 143 nmol/l, respectively. Experiments carried out with Ca2+-free solution and with thapsigargin showed that the [Ca2+]i transient was due to release from intracellular pools and stimulated Ca2+ entry. NH3/NH4+ (20 mmol/l) induced a transient intracellular alkalinization by 0.6 pHunits and increased [Ca2+]i to a peak (Delta [Ca2+]i = 164 nmol/l). The peak [Ca2+]i increase was not influenced by removal of external Ca2+, but the decline to basal [Ca2+]i was faster. Neither the phospholipase C inhibitor U73122 nor the inositol 1,4,5-trisphosphate (InsP3) antagonist theophylline had any influence on the NH3/NH4+-stimulated [Ca2+]i increase, whereas carbachol-induced [Ca2+]i transients were reduced by more than 80% and 30%, respectively. InsP3 measurements showed no change of InsP3 during exposure to NH3/NH4+, whereas carbachol enhanced the InsP3 concentration, and this effect was abolished by U73122. The pHi influence on "capacitative" Ca2+ influx was also examined. An acid pHi attenuated, and an alkaline pHi enhanced, carbachol- and thapsigargin-induced [Ca2+]i influx. We conclude that: (1) an alkaline pHi releases Ca2+ from InsP3-dependent intracellular stores; (2) the store release is InsP3 independent and occurs via an as yet unknown mechanism; (3) the store release stimulates capacitative Ca2+ influx; (4) the capacitative Ca2+ influx activated by InsP3 agonists is decreased by acidic and enhanced by alkaline pHi. The effects of pHi on [Ca2+]i should be of relevance under many physiological conditions.

A modulation of glutamate-induced phosphoinositide breakdown by intracellular pH changes

The influence of intracellular pH (pHi) changes on the formation of inositol phosphate metabolites (IPs) produced by glutamatergic stimulation was studied in 8-day-old rat brain synaptoneurosomes. For this purpose pHi was measured using 2′,7′-bis-(2-carboxyl)-5,6-carboxyfluorescein (BCECF) fluorimetric assay in parallel with the basal and receptor-mediated formations of inositol monophosphate (IP1) and inositol bisphosphate (IP2). We found that glutamate (1mM), which induces a transient acidification (ΔpH=−0.05), produces an identical accumulation of IP1 and IP2. K+ (30mM), which provokes an alkalinization of the internal medium (ΔpH=+0.22), mainly leads to the formation of IP1 metabolites. Paired combinations of glutamate with 1, 5 and 10mM NH4+ finally result in an alkalinization of the intrasynaptoneurosomal medium. These combinations produce a strong decrease of the IP2 level concomitant with an increase of the IP1 formation, compared to the levels of IP1 and IP2 evoked by glutamate alone. The total amount of IPs (IP1+IP2) produced by these combinations is not different from that obtained with glutamate alone. Paired combinations of carbachol with NH4+ produce an identical alkalinization to that produced by NH4+ alone. These combinations produce an increased IP1 accumulation, while the IP2 formation is slightly decreased. When the internal medium is acidified by dimishing the external concentration of Na+, the ratio IP1/IP2 produced after metabotropic glutamate receptor (mGluR) activation is shifted to lower values, while it is not affected for the muscarinic stimulation. These data suggest that the mGluR-associated pathway in synaptoneurosomes is sensitive to pHi shifts, while the muscarinic receptor-associated pathway is less altered when pHi is manipulated. It may be proposed that pH-sensitive inositol phosphate dephosphorylating systems, i.e. phosphatases, are associated with mGluRs in this preparation.

1043 Influence of intracellular pH on the apoptosis caused by hyperthermia

1043 Influence of intracellular pH on the apoptosis caused by hyperthermia

Effects of intracellular sodium and hydrogen ion on the sodium activated potassium channel in isolated patches from guinea pig ventricular myocytes

The Na+ activated K+ channel (IK(Na)) may be activated during ischaemia when the intracellular Na+ concentration is raised. As ischaemia is also associated with intracellular acidification, the influence of intracellular pH on this K+ channel was investigated. The effects of intracellular Na+ and H+ on the IK(Na) channel were investigated in isolated patches from guinea pig ventricular myocytes by the patch clamp technique. Increase of intracellular Na+ increased the open probability of the channel. Intracellular acidification had no effect on the single channel conductance, but significantly decreased the open probability of the channel in the pH range 7.5-6.5. The effect of intracellular pH on open probability was about the same in a wide range of intracellular Na+ concentrations. The lower open probability induced by acidification seemed to be caused by prolonged closed times. Because the effects of increased intracellular Na+ and decreased pH on open probability are opposite, it is suggested that the IK(Na) channel might be important under conditions of raised intracellular Na+ with relatively unchanged intracellular pH. Consequently, it is hypothesised that this channel may be involved in adaptation of action potential duration at high heart rate and in action potential shortening in the border zone of regional ischaemic areas.

The influence of intracellular pH on contraction, relaxation and [Ca2+]i in intact single fibres from mouse muscle.

1. The effects of intracellular pH (pHi) on myoplasmic free calcium concentration ([Ca2+]i) and contractile performance were studied in intact single fibres dissected from mouse skeletal muscle. Indo-1 was used to measure [Ca2+]i and pHi was altered by changing perfusate CO2. 2. Tetanic tension was decreased at acidic pHi and increased at alkaline pHi whereas the rate of mechanical relaxation was showed at both acidic and alkaline pHi. Resting and tetanic [Ca2+]i were increased at acidic pHi and decreased at alkaline pHi while the final rate of decline of [Ca2+]i after a tetanus was markedly slowed at acid pHi but only marginally accelerated at alkaline pHi. 3. Steady-state [Ca2+]i-tension curves were constructed from measurements of tetani at different stimulus frequencies. The curves at acid pHi showed a reduced maximum Ca(2+)-activated tension and a reduced Ca2+ sensitivity, and curves at alkaline pHi showed the opposite changes. 4. Two methods were devised to determine the contribution of [Ca2+]i to the rate of relaxation. In one method the instantaneous tension was plotted as a function of instantaneous [Ca2+]i throughout a tetanus and compared with the steady-state [Ca2+]i-tension relation. In a second method the [Ca2+]i signal during a tetanus was converted to a Ca(2+)-derived tension record by means of the steady-state [Ca2+]i-tension relation and this Ca(2+)-derived tension was then compared to the true tension. 5. The sarcoplasmic reticulum (SR) pump function was analysed by plotting -d[Ca2+]i/dt against [Ca2+]i during the final slow decline of [Ca2+]i after a tetanus. This analysis shows that the Ca2+ uptake by the SR is a third- or fourth-power function of [Ca2+]i and that acidosis substantially slows the rate of SR Ca2+ pumping. 6. In conclusion, the slowing of relaxation at acidic pHi could be attributed to a slowing of cross-bridge detachment rather than the observed slowing of the rate of uptake of Ca2+. Conversely the slowing of relaxation in alkaline pHi could be attributed to the increase of Ca2+ sensitivity of the myofibrillar proteins.

Influence of intracellular pH on light emission from a luxA/B derivative of Lactococcus lactis subsp. diacetylactis.

High levels of constitutive aldehyde-dependent light emission were obtained from nongrowing cells of Lactococcus lactis subsp. diacetylactis F712 transformed with IuxA/B when they were suspended in buffered solutions. Inductions of light emission was time-dependent and was not due to growth, synthesis of luciferase or stimulation of metabolism by fermentable carbohydrate. The major factor controlling light emission in such cells appears to be the intracellular pH value. Experiments with ionophores indicated that a transmembrane pH gradient was not essential for light emission.

Influence of intracellular pH on the membrane potential of cultured carotid body glomus cells

The influence of acidity on the resting potential (Em) of cultured rat glomus cells was studied within a narrow range (7.32-7.54) of extracellular pH (pH0). Under these conditions, the intracellular pH (pHi) ranged from 6.3 to 7.21. Resting potentials were most negative when pHi was lowest, and least negative when pHi was highest. These effects were determined by the equilibrium potential of H+ ions (EH) since Em linearly followed EH. However, EH was little affected by pH0. Therefore, intracellular, and not extracellular, acidity was most important in contributing to the Em of glomus cells.

Influence of intracellular pH on reduction of energy metabolism during hypoxia in the lugworm Arenicola marina

AbstractThe influence of intracellular pH (pHi) on the energy metabolism during hypoxia was studied in the lugworm Arenicola marina using in vivo 31P nuclear magnetic resonance (NMR) spectroscopy and biochemical analysis of the metabolic end products during hypoxia. By addition of phenylphosphonic acid (PPA) to the incubation medium to increase the extracellular H+ buffering capacity, different degrees of intracellular acidosis during hypoxia could be attained.At low extracellular PPA concentrations pHi dropped within 12 h of hypoxia from 7.3 to 6.7. High extracellular PPA concentrations (50 mM) kept pH1 stable at 7.2. Glycogenolysis was enhanced during hypoxia at high pHi, as shown by the measured amounts of accumulated end products. ATP production at high pHi was 31 μmol/g h vs. 15 at low pHi‐ The mobilisation of the phosphagen phosphotaurocyamine (PTC) was, in contrast, decreased at high pHi: the ratio PTC/β‐ATP dropped from 4.8 (normoxia) to 2.8 at high vs. 1.3 at low pHi. The reduction of the metabolic rate during hypoxia hence depends on intracellular acidosis: the rate of the Embden‐Meyerhof‐Parnas pathway at high pHi was 4.5 μmol/g h vs. 1.9 at low pHi.

Influence of intracellular pH on mitochondrial calcium during ischaemia of the isolated rat heart

Under physiological conditions cardiac mitochondria seem to play a minor role in maintaining intracellular Ca2+ homoeostasis. However, under conditions of cellular Ca2+ overload, mitochondria may accumulate large amounts of Ca2+. Using transmission and analytical electron microscopy, we investigated, in globally ischaemic rat heart preparations, the influence of intracellular pH on the development of Ca2+-containing intramitochondrial inclusions. We confirmed that under these experimental conditions Ca2+ was a major element of mitochondrial inclusions. The size of these inclusions increased with external Ca2+ concentration. An intracellular alkalinization, produced by addition of 20 mM NH4Cl to the perfusate prior to ischaemia, inhibited the formation of such inclusions. On the other hand, a pre-ischaemic intracellular acidification, produced by the addition and subsequent withdrawal of the 20 mM NH4Cl, increased the number of inclusions present at the end of an ischaemic episode. The presence of amiloride (10(-3) M), prior to and during ischaemia, increased the number of inclusions. These data suggest that cytoplasmic pH may be an important factor in mitochondrial Ca2+ accumulation in pathological conditions.

Ungulate cardiac purkinje fibres: the influence of intracellular pH on the electrical cell-to-cell coupling.

1. Passive electrical properties of sheep cardiac Purkinje fibres were assessed by performing linear cable analysis. In a separate set of experiments pH(i) was monitored using recessed-tip pH-sensitive micro-electrodes.2. In Tris-buffered Tyrode solution (nominally CO(2)-free), the pH(i) was 7.27, in bicarbonate-buffered solution equilibrated with 6% CO(2), the mean pH(i) was 7.02.3. Application of 15 mM-NH(4)Cl produced a rapid intracellular alkalinization (0.19 pH units), followed by a slower acidification. Removal of NH(4)Cl gave rise to a slow and transient intracellular acidification (0.5 pH units).4. The biphasic and transient shift in pH(i), induced by the NH(4)Cl treatment, was accompanied by a change of the inside longitudinal resistance per unit fibre length, r(i), displaying a similar time course. The increase in pH(i) produced a maximum decrease in r(i) of 16.4%, while the decrease in pH(i) yielded a maximum increase in r(i) of 30.4%.5. Changing from bicarbonate-buffered Tyrode solution equilibrated with 6% CO(2) to Tris-buffered Tyrode solution led to an increase in pH(i) (0.26 pH units). A subsequent change to bicarbonate-buffered Tyrode solution equilibrated with 15% CO(2) produced a decrease in pH(i) (0.48 pH units). Both changes were sustained.6. This CO(2) protocol gave rise to corresponding changes in r(i); the intracellular alkalosis was associated with a decrease in r(i) (21.2%), and the intracellular acidosis was accompanied by an increase in r(i) (30%).7. Based on recent findings showing an interaction between pH(i) and pCa(i) (Hess & Weingart, 1980), it is concluded that the changes in r(i) are directly caused by protons and not indirectly via secondary changes of the [Ca(2+)](i).8. The pH(i)-dependent changes in r(i) are likely to reflect alterations of the nexal resistance, r(n), because the cytoplasmic resistance, r(c), has the inverse sensitivity to pH(i).9. Unlike pCa(i), pH(i) would seem to be able to modify the cell-to-cell coupling by increasing or decreasing r(i) over a rather narrow range, without ever producing electrical uncoupling.10. Because of basic differences in the action of calcium and protons on the cell-to-cell coupling (magnitude of the effect, operative concentration range), it is tempting to conclude that there is more than one kind of binding site which controls the nexal conductance.

The effects of hydrogen ions on the kinetics of calcium transport by rat kidney mitochondria

The influence of pH on the kinetics of the initial rate of calcium uptake by isolated kidney mitochondria was studied using the ruthenium red-ethylene glycol bis(β-aminoethyl ether) N,N-tetraacetic acid quench method (K. Reed and F. Bygrave, 1975, Eur. J. Biochem. 55, 497–504). In the absence of phosphate, the K m is increased 50% and the V is decreased 57% when the pH is decreased from pH 7.4 to 7.0. Conversely, when the pH is increased to 7.8, the K m is decreased 25% while the V is not affected. The presence of 0.1 or 0.4 m m phosphate in the incubation medium abolishes the change in K m at a low pH while the V remains depressed by 36 and 25%, respectively. The presence of phosphate does not affect the decrease in the K m seen with an increased medium pH. Mitochondria incubated in steady-state conditions with a medium free calcium of 0.7 μ m also show significant changes in calcium exchange and distribution with pH. Two kinetic calcium pools are found in isolated mitochondria. Decreasing pH from 7.4 to 7.1 decreases mitochondria total calcium 32%, decreases the rapidly exchanging pool 28%, and depresses both the mitochondrial membrane and an intramitochondrial calcium exchange by 54 and 22%, respectively. Raising the pH to 7.7 increases both exchangeable pools (63 and 46%), and increases the mitochondrial membrane calcium exchange 44%. These results are consistent with previous studies on the influence of intracellular pH on calcium metabolism of kidney cells in which the mitochondrial pool was markedly affected by pH ( R. Studer, and A. Borle, 1979, J. Membrane Biol. 48, 325–341 ). Alterations in cellular pH may modify mitochondrial calcium transport and cellular calcium metabolism and thus affect cell functions which are calcium dependent.