Pronounced Acidification Research Articles

Paradoxical Effect of Bicarbonate on Cytoplasmic pH

The effect of an abrupt rise in bicarbonate concentration on cytoplasmic pH was studied in human platelets suspended in a Tyrode's buffer. Addition of bicarbonate raised extracellular pH but simultaneously caused pronounced cytoplasmic acidification. This effect may be due to combination of bicarbonate with hydrogen ions in extracellular fluid to form carbonic acid, which is converted by carbonic anhydrase to water and carbon dioxide. Bicarbonate ions do not diffuse rapidly across cell membranes, whereas carbon dioxide is highly diffusible and can combine with water in the cytoplasm, forming carbonic acid and reducing the intracellular pH. In accord with this explanation cytoplasmic acidification by bicarbonate was antagonised by acetazolamide (an inhibitor of carbonic anhydrase). Cytoplasmic acidification could contribute to adverse effects of intravenous sodium bicarbonate in patients with severe acidaemia. These findings add weight to the body of opinion that such treatment is both illogical and dangerous.

Lipoxygenase inhibitors suppress intracellular calcium rise induced by ionomycin in rat thymocytes

The lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA) and 16S-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE) have been found to suppress the rise in free cytoplasmic Ca 2+ concentration ([Ca 2+] i) induced by the Ca 2+ ionophores ionomycin and A23187 in rat thymocytes. Bromophenacyl bromide (BPB), a phospholipase A 2 (PLA 2) inhibitor, produced a much weaker inhibitory effect, and indomethacin, a cyclo-oxygenase inhibitor, practically did not influence the [Ca 2+] i response to ionomycin. These findings implicate the involvement of LO product(s) in the [Ca 2+] i rise triggered by the Ca 2+ ionophores. The contribution of the NDGA-sensitive component to the ionomycin-induced [Ca 2+] i rise was significant in the ionomycin concentration range of 0.1 nM to 0.1 μM, whereas at higher doses of the ionophore it gradually diminished. By contrast, the [Ca 2+] i rise induced by exogenous arachidonic acid (AA) or melittin, a PLA 2 activator, was not suppressed but potentiated by NDGA. Ionomycin and exogenous AA also elicited opposite changes in thymocyte cytoplasmic pH (pH i): the former elevated the pH i while the latter induced a pronounced acidification of the cytoplasm. This difference in the pH i responses may account for the different sensitivity of ionomycin- and AA-elicited [Ca 2+] i signal to LO inhibitors.

Role of Na(+)-H+ and Cl(-)-HCO3- antiports in the regulation of cytosolic pH near neutrality

In Vero cells, Na(+)-H+ antiport as well as Na(+)-coupled and Na(+)-independent Cl(-)-HCO3- antiport are involved in regulation of cytosolic pH (pHi) after large (unphysiological) deviations from neutrality. In this paper we have studied to which extent each of the three antiports is involved in regulation of pHi after small deviations from neutrality expected to occur under physiological conditions. At physiological extracellular pH (pHo), inhibition of Na(+)-H+ exchange by amiloride did not alter pHi. At neutral and alkaline pHo, pHi was found to be lower in the presence of HCO3- than in its absence, whereas at acidic pHo, pHi was higher in the presence of HCO3- than in its nominal absence. Above pHi 6.5, the activity of the Na(+)-coupled Cl(-)-HCO3- antiport was higher than the Na(+)-H+ antiport. After a small reduction of pHi, the recovery of steady-state pHi was entirely dependent on Na(+)-coupled Cl(-)-HCO3- antiport, whereas after more pronounced acidification, also Na(+)-H+ exchange contributed to the acid extrusion. The Na(+)-independent Cl(-)-HCO3- antiport, which acts as an acidifying mechanism, was strongly activated at pHi greater than 7.1. The results indicate that at physiological pHo the steady-state pHi is largely determined by the activity of the two Cl(-)-HCO3- antiports, and they suggest that Na(+)-H+ exchange does not influence the resting pHi under these conditions.

The effects of metabolic inhibition on uterine metabolism and intracellular pH in the rat.

1. Uterine metabolism was studied in pregnant and non-pregnant rats by measuring high energy phosphates and intracellular pH (with 31-phosphorus nuclear magnetic resonance (31P NMR) spectroscopy) and lactic acid efflux. Isolated, superfused uteri were investigated during control conditions (30 degrees C) and in the presence of metabolic inhibitors and high [K+]. 2. In control conditions the ratio of phosphocreatine and ATP concentrations ([PCr]/[ATP]) was higher in the pregnant (0.88 +/- 0.09) than the non-pregnant uterus (0.52 +/- 0.04). When oxidative phosphorylation was inhibited by cyanide, there was a significant (P less than 0.01) decrease in [PCr] and [ATP] and an increase in the concentration of inorganic phosphate ([Pi]). These changes were greater in the non-pregnant than the pregnant uterus. 3. There was no difference in the mean value of resting pHi found in pregnant and non-pregnant uterus (7.19 +/- 0.04 and 7.17 +/- 0.03, external pH 7.4, n = 10 and 12 respectively). There was a significant intracellular acidification in both pregnant (0.31 +/- 0.04 pH unit) and non-pregnant (0.27 +/- 0.02 pH unit) uterine tissue in the presence of cyanide. These effects of cyanide on metabolites and pHi were fully reversed upon return to control solutions. 4. When both aerobic and anaerobic glycolysis were blocked by iodoacetate, there was a rapid disappearance of high energy phosphates from the 31P NMR spectrum and a large increase in the phosphomonoester spectral region, where sugar phosphate intermediates of glycolysis resonate. These changes were seen in both pregnant and non-pregnant uteri and were irreversible. 5. Lactate production was detected, in the presence of oxygen, in both pregnant and non-pregnant preparations (0.43 +/- 0.07 and 0.25 +/- 0.09 mumols g-1 min-1, respectively). In both preparations the rate of lactate production was markedly increased in the presence of cyanide. The increase was much more marked in non-pregnant (approximately 10-fold) than pregnant (approximately 5-fold) uteri, resulting in a very similar rate of lactate efflux in cyanide. 6. When lactate efflux in non-pregnant uteri was blocked by alpha-cyano-4-hydroxycinnamate, there was a significant acidification (0.21 +/- 0.04 pH unit, n = 6). The addition of cyanide produced a more pronounced acidification (0.34 +/- 0.04 pH unit) than that seen with either cyanide or alpha-cyano-4-hydroxycinnamate alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Soil effects on water chemistry in three adjacent upland streams at Glendye in northeast Scotland

The soils and water chemistry of streams were studied in three superficially similar adjacent upland catchments in northeast Scotland. High stream baseflow total organic carbon concentrations were shown to be associated with stream water pH values between 5 and 6 and drainage predominantly from peat. Streams with pH values of 6.8–7.0 and low TOC concentrations at base flow were associated with inputs of water from mineral soils, and were more susceptible to pronounced acidification during storms as the hydrological pathway changed. Differences in relative contributions of water draining mineral soils in the three catchments are reflected also by changes in water chemistry along transects of the streams, and in different relationships between aluminium and stream water organic matter content. The results indicate that soil physical and chemical parameters must be incorporated into water chemistry models to be used for predictive purposes.

Charge and acidity compensation during proton-sugar symport in Chlorella: The H+-ATPase does not fully compensate for the sugar-coupled proton influx

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H(+)-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H(+) and K(+) fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H(+) symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H(+) by the hexoseproton symport and charge compensation by K(+) loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H(+)-ATPase caused a slow-down of the K(+) efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H(+) uptake and K(+) loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H(+) fluxes. The quantitative participation of the several phases of H(+) and K(+) flow depended on the pH of the medium, the ambient Ca(2+) concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H(+)-ATPase never fully compensated for H(+) uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K(+) efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.

The role of HCO3- and Na+/H+ exchange in the response of rat parotid acinar cells to muscarinic stimulation.

The intracellular pH (pHi) of a rat parotid acinar preparation was monitored using the pH-sensitive fluorescent dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Under resting (unstimulated) conditions both Na+/H+ exchange and CO2/HCO3- buffering contribute to the regulation of pHi. Muscarinic stimulation (carbachol) of the acini produced a gradual rise in pHi (approximately 0.1 unit by 10 min) possibly due to activation of the Na+/H+ exchanger. When the exchanger was blocked by amiloride or sodium removal, carbachol induced a dramatic (atropine inhibitable) decrease in pHi (approximately 0.4 pH unit with t1/2 approximately 0.5 min at 1 mM carbachol). The rate of this acidification was reduced by removal of exogenous HCO3- and by the carbonic anhydrase inhibitor methazolamide. Also, acini stimulated with carbachol in Cl- -free solutions showed a more pronounced acidification than in the corresponding Cl- -replete media. Taken together, these data indicate that the carbachol-induced acidification of rat parotid acinar cells unmasked by inhibition of the Na+/H+ exchanger is due to a rapid loss of intracellular HCO3-. Carbachol induced acidification was inhibited by the Cl- channel blocker diphenylamine 2-carboxylate but not by 4-acetomido-4'-isothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of Cl-/HCO3- exchange. In addition, this acidification could not be sustained in Ca2+-free media and was totally blocked by chelation of intracellular Ca2+. Interpreted in terms of HCO3- loss, these results closely parallel the pattern of carbachol-induced Cl- release from this same preparation and indicate that HCO3- is secreted in response to muscarinic stimulation via the same or a very similar exit pathway, presumably an apical anion channel. Under normal physiological conditions the intracellular acidification resulting from HCO3- secretion is buffered by the Na+/H+ exchanger.

Protonation of the sarcoplasmic reticulum Ca-ATPase during ATP hydrolysis.

pH changes of the reaction medium (pH 6.35) following addition of MgATP were determined at 4 degrees C with sarcoplasmic reticulum vesicles in the presence of 0.1 mM CaCl2 and 5 mM MgCl2. With intact vesicles, a pronounced acidification following a slight alkalinization was induced by MgATP, indicating H+ ejection from vesicles. This agrees with previous observations by several investigators that suggested H+ counter-transport coupled to Ca2+ uptake. When vesicles were made leaky, a markedly enhanced alkalinization occurred after addition of MgATP, and then the pH returned to the level before the MgATP addition. The decay of this alkalinization coincided with the disappearance of phosphoenzyme (EP) which was formed from MgATP and the Ca-ATPase of sarcoplasmic reticulum. The CaCl2 concentration dependence of the alkalinization agreed well with that of EP formation. Furthermore, the alkalinization was prevented by modification of a specific SH group of the enzyme essential for EP decomposition. These findings give evidence that the alkalinization with leaky vesicles represents protonation of the enzyme during ATP hydrolysis. The enhanced alkalinization with leaky vesicles suggests protonation occurring inside vesicles. When CaATP in place of MgATP was added in the absence of MgCl2, neither the H+ ejection from intact vesicles nor the enhanced alkalinization with leaky vesicles occurred. Under these conditions, CaATP did not efficiently support active Ca2+ uptake. These results are compatible with the view that the observed protonation could represent an initial event occurring inside vesicles in the H+ countertransport coupled to Ca2+ uptake.

The intracellular pH of frog skeletal muscle: its regulation in hypertonic solutions.

Intracellular pH (pHi) was followed with micro-electrodes in frog semitendinosus muscle, superfused at 22 degrees C with hypertonic solutions (external pH, pHo, 7.35) containing 2.5, 15 or 50 mM-K. Tonicity was doubled by addition of 250 mM-mannitol or, in a few cases, 125 mM-extra NaCl. Tripling of tonicity was accomplished by adding 500 mM-mannitol. Because of the ability of hypertonicity to minimize contracture, the course of pHi could be followed from the start of depolarization. The pHi of fibres after about 40 min in Ringer solution (2.5 mM-K, HEPES buffer) of twice normal tonicity was 7.40 +/- 0.04 (S.E. of mean) (n = 17), about 0.2 higher than at normal tonicity. The membrane potential, Vm, was -87.7 +/- 1.3 mV. When the muscle was depolarized in 50 mM-K to about -30 mV, the pHi rapidly fell by 0.3-0.5 unit (n = 9), and then promptly returned. This recovery was followed by a much slower and progressive rise to above control. Removing Na from the medium did not affect the degree of acidification, but the pHi recovered at a slightly slower rate, did not reach control value and showed no progressive rise. A less pronounced transient acidification was also observed when the muscle was depolarized in 15 mM-K to about -60 mV. When contracture was prevented either by 1-2 mM-tetracaine under isotonic conditions or by raising tonicity 3-fold, 50 mM-K produced no transient acidification. When the pHi of resting fibres in Ringer solution (2.5 mM-K) of twice normal tonicity was reduced by 5% CO2 from 7.40 to 7.12 +/- 0.07 (n = 3), it recovered at a slow rate (0.06 +/- 0.03 delta pHi h-1). Depolarization by 15 or 50 mM-K enhanced recovery rate 4-6-fold. These solutions of twice normal tonicity, as compared to those of normal tonicity, shifted the curve relating pHi recovery rate and membrane potential along the potential axis in the direction of hyperpolarization. This shift may be due to increased ionic shielding of fixed negative charges at the inner membrane surface. At twice normal tonicity, the very slow pHi recovery of resting fibres from CO2-induced acidification, as well as the more rapid recovery in depolarized fibres, could be abolished by 1 mM-amiloride or by removing Na. The application of amiloride during pHi recovery in 50 mM-K was not associated with an observable change in Vm. SITS had no significant effect on recovery.(ABSTRACT TRUNCATED AT 400 WORDS)

Pyrite oxidation in acid sulphate soils: The role of miroorganisms

A study has been made of microbial processes in the oxidation of pyrite in aicd sulphate soil material. Such soils are formed during aeration of marine muds rich in pyrite (FeS2). Bacteria of the type ofThiobacillus ferrooxidans are mainly responsible for the oxidation of pyrite, causing a pronounced acidification of the soil. However, becauseThiobacillus ferrooxidans functions optimally at pH values bellow 4.0, its activity cannot explain the initial pH drop from approximately neutral to about 4. This was shown to be a non-biological process, in which bacteria play an insignificant part. AlthoughThiobacillus thioparus andThiobacillus thiooxidans were isolated from the acidifying soil, they did not stimulate oxidation of FeS2, but utilized reduced sulphur compounds, which are formed during the non-biological oxidation of FeS2. Ethylene-oxide-sterilized and dry-sterilized soil inoculated with pure cultures of mixtures of various thiobacilli or with freshly sampled acid sulphate soil soil did not acidify faster than sterile blanks.Thiobacillus thiooxians. Thiobacillus thioparus. Thiobacillus intermedius andThiobacillus perometabolis increased from about 104 to 105 cells/ml in media with FeS2 as energy source. However, FeS2 oxidation in the inoculated media was not faster than in sterile blanks. Attempts to isolate microorganisms other thanThiobacillus ferrooxidans, like metallogenium orLeptospirillum ferrooxidans, which might also be involved in the oxidation of FeS2 were not successful. Addition of CaCO3 to the soil prevented acidification but did not stop non-biological oxidation of FeS2.

Pyrrole hydrogen ionization of imidazole derivatives in metal ion complexes and carbonic anhydrase.

Zinc ion-induced pyrrole hydrogen ionization from a histidyl residue has recently been suggested as the source of the activity-related deprotonation in carbonic anhydrase (EC 4.2.1.1; carbonate hydro-lyase) occurring at pH 7-8. The results offered to support the hypothesis are shown to be deficient. Examination of the literature reveals no precedent for such a pronounced acidification from pK(a) [unk] 14.2 in imidazole compounds by zinc ion. It is suggested that Zn(II)-induced pyrrole ionizations in imidazoles occur with pK(a) about 13.