Intracellular pH homeostasis plays a role in the NaCl tolerance of Debaryomyces hansenii strains
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Debaryomyces hansenii
Homeostasis
Osmotic shock
Intracellular pH
Strain (injury)
Osmoregulation
Calcium ionophores such as ionomycin and A23187 are often used to determine the role of intracellular Ca++ in cellular processes. Ionomycin but not Ca+(+)-mobilizing agonists increases basal intracellular pH in hepatocytes. To explain this difference in effects of agents that increase intracellular Ca++ concentration, the mechanism of ionomycin-induced increases in basal intracellular pH in isolated rat hepatocytes was studied. Changes in intracellular pH and intracellular Ca++ concentration were measured with the fluorescent probes BCECF (2',7'-bis-2-[carboxyethyl ester]-5[6]carboxyfluorescein) and quin-2, respectively. Ionomycin produced dose-dependent increases in intracellular pH and intracellular Ca++ concentration, with the increase in intracellular Ca++ concentration preceded by the increase in intracellular pH. Ionomycin-induced increases in intracellular pH were not affected by 1 mmol/L amiloride, 100 mumol/L diisothiocyanostilbene disulfonate or removal of extracellular Na+, indicating that the effect is not mediated by Na+/H+ exchange, Cl-/HCO3- exchange or Na+/HCO3- cotransport. Ionomycin failed to increase intracellular pH or intracellular Ca++ concentration in the absence of extracellular Ca++, and both intracellular pH and intracellular Ca++ concentration increased promptly when extracellular Ca++ was reintroduced. Ionomycin-induced increases in intracellular Ca++ concentration but not intracellular pH were smaller in hepatocytes loaded with the Ca++ buffering agent MAPTA. Thapsigargin increased intracellular Ca++ concentration but failed to increase intracellular pH. Thus the effect of ionomycin is independent of the effect of ionomycin on intracellular Ca++ concentration and dependent on extracellular intracellular Ca++ concentration. Experimental conditions that produce cell depolarization did not increase basal intracellular pH but lowered ionomycin-induced increases in intracellular pH by 25% without affecting increases in intracellular Ca++ concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Ionomycin
Intracellular pH
Calcium in biology
Thapsigargin
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We investigated whether aging changed H+ homeostasis in hippocampal slices bathed in HEPES buffer. Intracellular pH in hippocampal slices from rats aged 26-27 months (7.06 +/- 0.02) was significantly lower compared with that in slices from rats aged 6-7 months (7.16 +/- 0.04). Age did not influence extracellular ph. Age-related reductions in intracellular pH may reflect altered pH regulation that potentially affects brain function and could contribute to the increased vulnerability of the aged brain to metabolic stress.
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HEPES
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A series of studies has indicated that the frequency of morphological transformation induced by chemical carcinogens in early passage Syrian hamster embryo (SHE) cells is significantly higher when these cells are cultured in medium of reduced bicarbonate concentration and pH (6.70) compared with cells cultured in medium of higher pH. It has also been shown that intercellular gap junctional communication is decreased in these cells when they are cultured at pH 6.70 compared with medium of higher pH. The purpose of the studies reported here was to characterize the effect of changing extracellular pH on intracellular pH in SHE cells. The frequency of morphological transformation induced by benzo(a)pyrene was established at various extracellular pHs and compared with intracellular pH values. Cells cultured in medium of pH ranging from 6.70 to 7.35 were loaded with the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, and either the steady-state intracellular pH values or the kinetics of change in intracellular pH following refeeding of the cultures with medium of pH ranging from pH 6.70 to pH 7.35 was monitored via image analysis techniques. Results from these studies indicate that, at culture medium pH above 6.95, SHE cells were relatively insensitive to changes in extracellular pH, maintaining an intracellular pH of 7.30 to 7.35 in medium containing 0% serum or pH 7.05 to 7.10 in medium containing 20% fetal bovine serum. At extracellular pHs below 6.95, intracellular pH decreased and, in the presence of serum, equilibrated with extracellular pH. The decrease in intracellular pH was closely associated with an increase in benzo(a)pyrene-induced morphological transformation frequency observed in parallel studies. These results indicate that SHE cells have active intracellular pH regulatory activities and suggest that intracellular acidification plays a role in the increased frequency of transformation observed in SHE cells cultured under acidic conditions.
Intracellular pH
pH indicator
Fetal bovine serum
Bicarbonate
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The effect of variable extracellular pH on intracellular pH, cell energy status, and thermal sensitivity was evaluated in CHO cells over the extracellular pH range of 6.0 to 8.6. Extracellular pH was adjusted with either lactic acid, HCl, or NaOH. Regardless of the method of pH adjustment, the results obtained were similar. The relationship between extracellular and intracellular pH was dependent upon the pH range examined. Intracellular pH was relatively resistant to a change in extracellular pH over the pHe range of 6.8 to 7.8 (i.e., delta pHi congruent to delta pHe X 0.33). Above and below this range, delta pHi congruent to delta pHe X 1.08 or X 0.76, respectively. Cellular survival after a 30-min heat treatment at 44 degrees C remained constant over the extracellular pH range of 7.0 to 8.4, but varied substantially over a similar intracellular pH range. The cellular concentration of the high energy phosphate reservoir, phosphocreatine, decreased with decreasing pH. However, the cellular concentrations of ATP, ADP, and AMP remained constant over the entire pH range examined. It is concluded that increased thermal sensitivity resulting from a change in extracellular pH is not due to cellular energy depletion. Furthermore, intracellular pH is a more accurate indicator of thermal sensitivity than is extracellular pH.
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The hydrogen ion concentration, usually expressed as pH, is highly regulated in both the intracellular and extracellular environments(1 2). Although the body has developed multiple systems to maintain pH homeostasis, changes in the pH of the extracellular and intracellular environments can occur under pathophysiological conditions. It has also become apparent in recent years that transient changes in intracellular pH (pHi) are part of the vascular smooth muscle cell (VSMC) response to physiologic stimuli.
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Nonproliferating rat hepatocytes in primary monolayer culture were used for determining liver cell intracellular pH and the degree of intracellular pH homeostasis. The dimethyloxazolidinedione weak acid distribution method was adapted for use in monolayer culture. Intracellular pH of cultured hepatocytes in bicarbonate:CO2 medium was relatively constant at 6.85-7.05 over the external pH range of 7.0-8.0. Below an external pH of 7.0, intracellular pH fell below 6.8. Varying PCO2 between 15 and 40 mmHg did not alter the extracellular versus intracellular pH curve. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid medium, in the absence of bicarbonate, intracellular pH homeostasis was less well defended. In this setting, the intracellular versus extracellular pH relationship curve could be described by a straight line with slope of 0.59 +/- 0.04. The system responded to the addition of the protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone with an increase in the transmembrane pH gradient. Addition of nigericin in 5 mM K+ medium resulted in intracellular acidification to pH 5.5 +/- 0.2. Metabolism of 20 mM added fructose resulted in intracellular acidification. Incubation in sodium-free media at extracellular pH of 7.6 reduced intracellular pH to 6.67 +/- 0.02 compared with an intracellular pH of 6.99 +/- 0.04 in cultures exposed to medium sodium concentrations of 20-80 meq/liter.
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Protonophore
Nigericin
Intracellular Fluid
Bicarbonate
HEPES
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Debaryomyces hansenii
Homeostasis
Osmotic shock
Intracellular pH
Strain (injury)
Osmoregulation
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Abstract The highly NaCl‐tolerant yeast Debaryomyces hansenii produces and obtains high levels of intracellular glycerol as a compatible solute when grown at high NaCl concentrations. The effect of high NaCl concentrations (4%, 8% and 12% w/v) on the glycerol production and the levels of intra‐ and extracellular glycerol was determined for two D. hansenii strains with different NaCl tolerance and compared to one strain of the moderately NaCl‐tolerant yeast Saccharomyces cerevisiae . Initially, high NaCl tolerance seems to be determined by enhanced glycerol production, due to an increased expression of DhGPD1 and DhGPP2 (AL436338) in D. hansenii and GPD1 and GPP2 in S. cerevisiae ; however, the ability to obtain high levels of intracellular glycerol seems to be more important. The two D. hansenii strains had higher levels of intracellular glycerol than the S. cerevisiae strain and were able to obtain high levels of intracellular glycerol, even at very high NaCl concentrations, indicating the presence of, for example, a type of closing channel, as previously described for other yeast species. Copyright © 2005 John Wiley & Sons, Ltd.
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Chapter 1: pH Homeostasis in Human Red Blood Cells The intracellular pH of normal and homozygous sickle cell red blood cells was measured in cell suspensions in plasma by NMR. Freshly drawn, metabolically active red cells maintain a transmembrane pH gradient that differs significantly from the expected Donnan equilibrium proton distribution. In the physiologically important extracellular pH range of 7.12 to 7.57 the intracellular pH is maintained within the narrow range of 7.20 to 7.37. Outside of this range, the intracellular pH is linearly dependent on the extracellular pH. Thus, red cells maintain an intracellular pH that is closer to 7.3 over the entire extracellular pH range than is expected from the Donnan equilibrium ion distribution. The ligation state of cellular hemoglobin shifted the position of the intra- vs extra-cellular pH relationship, but did not alter the ability of the cells to regulate intracellular pH. Deoxygenation of normal red blood cells resulted in an intracellular pH increase of 0.05 ± 0.02 compared to oxygenated cells over the extracellular pH range 7.00 to 7.80. Metabolically depleted cells are unable to maintain a non-Donnan equilibrium proton distribution. The regulation of intracellular pH was regained by restoring cellular ATP levels. Sickle cell blood demonstrated the same ability to regulate intracellular pH as was observed in normal blood. Deoxygenation of sickle cell blood also resulted in a net increase in intracellular pH. However, gelation of the sample prevented accurate intracellular pH measurements of completely deoxygenated sickle cell samples. Chapter 2: Measurements of Cell Volume by Nuclear Magnetic Resonance The Mean Hemoglobin Concentration (MHC) of red blood cells was measured non-invasively and non-destructively by NMR. The difference between intracellular and extracellular proton relaxation rates provides the basis for the determination of the MHC in red blood cells. T1 relaxation times were measured at a proton frequency of 200 MHz. The T1 relaxation time for water protons in serum is 2.20 ± 0.20 seconds. The T1 relaxation time of water protons in red blood cell pellets is 0.64 ± 0.15 seconds. In red blood cell lysate, the T1 relaxation time is 0.77 ± 0.11 seconds. The observed water T1 relaxation data from red blood cell samples under various conditions were fit to the complete equation for the time-dependent decay of magnetization for a two-compartment system including chemical exchange. The MHC for each sample was calculated from the hematocrit and the intracellular water fraction as determined by NMR. MHC values obtained in this manner ranged from 25% to 29% by volume for normal red blood cells in serum, in agreement with published values. The use of proton NMR to determine MHC values directly and non-destructively provides a method to evaluate the effect of various agents on the MHC in viable cells and has wide applicability to the study of antisickling agents in intact cells. The ability to monitor cell volume and to follow the effect of agents known to affect ion transport (valinomycin, nystatin, amiloride, etc.) on cell volume has enormous experimental potential. Chapter 3: 31P NMR Studies of the Binding Site of anti-Phosphorylcholine Antibodies The binding of the phosphorylcholine (PC) analogue, 2-(trimethylphosphonio)-ethylphosphate (phosphorylphosphocholine, PPC) to the PC binding myeloma proteins TEPC-15, McPC 603, and MOPC 167 was studied by 31P NMR. Binding of PPC to each of the proteins results in an observed phosphate chemical shift that is identical to the shift observed when PC is bound. Thus, the specific binding interactions of the phosphate subsite of the proteins with PC are maintained when PPC is bound. The chemical shift and titration behavior of the phosphonium resonance of PPC was studied as a probe of the choline subsite of these proteins. PPC bound to TEPC-15 or MOPC 167 exhibits a +0.1 ppm upfield shift from the free hapten. In contrast, PPC binding to McPC 603 results in a +2.7 ppm upfield shift that titrates with a pKa of 3.6. The shape of the titration curve indicates that the ionizations of 2 protons of equal pKa are responsible for the observed titration. Three acidic residues provide the major contribution to the choline subsite in both TEPC-15 and MOPC 167. The amino acid substitution ASP99H → Asn in the third hypervariable region of McPC 603 destroys the spatial symmetry of the choline subsite in McPC 603. The symmetry of the charge distribution of the choline subsite that is lost by this substitution is restored at low pH by titrating the negative charges of Glu35H and Glu59H.
Intracellular pH
Deoxygenation
Homeostasis
Intracellular Fluid
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The motile unicellular halotolerant alga Dunaliella salina, which can grow in media containing 0.08～~5.0 mol/L NaCl (Fig. 1), osmoregulated mostly by accumulating intracellularly a single compatible solute, glycerol (Fig. 3). With the increase of the extracellular NaCl concentrations from 0.50～4.0 mol/L, the amount of the intracellular glycerol rose markedly from 6.20～51.5 pg/cell (Fig. 3) and the intracellular Na~+ and K~+ contents had a small range of variation, around 30 and 35 mmol/L (Fig. 2) respectively. During a hypertonic treatment from 2.0 mol/L to 3.0 mol/L NaCl solution, the alga could recover their original volumes by raising their intracellular glycerol content in about 1h (Figs. 5,6). At the same time, the intracellular ATP content decreased (Fig. 8) and the H~+ secretion from the alga cells increased (Fig. 7). All of these changes could be fully inhi bited by 20 μmol / L Na_3VO_4, a specific inhibitor of the DM H~+-ATPase (Figs. 7,8).Thus it is suggested that the activity of the PM H~+-ATPase is stimulated by hypertonic stress and plays a central role in the ajustment of the alga against the stress. In a 2.0 mol/L NaCl medium, the increase of the intracellular Na~+ content and the decreases of the intracellular K~+ and ATP contents resulting from the treatment with 3.0 mmol/L KCN also show the important role played by the Phi H~+-ATPase of the alga against extracellular osmotic pressure (Fig. 9). The mechanism by which the PM H~+-APTase participates in the activition of glycerol synthesis in the alga is discussed.
Dunaliella salina
Osmoregulation
Dunaliella
Osmotic shock
Intracellular pH
Osmotic concentration
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