The effect of acid--base changes on skeletal muscle twitch tension.

The effects of acid--base alterations produced by changing bicarbonate (metabolic type), carbon dioxide tension (respiratory type), or both bicarbonate and carbon dioxide tension (compensated type) on skeletal muscle twitch tension, intracellular pH, and intracellular potassium were studied in vitro. Hemidiaphragm muscles from normal rats and rats fed a potassium-deficient diet were used. Decreasing the extracellular pH by decreasing bicarbonate or increasing CO2 in the bathing fluid produced a decrease in intracellular pH, intracellular K+, and muscle twitch tension. However, at a constant extracellular pH, an increase in CO2 (compensated by an increase in bicarbonate) produced an increase in intracellular K+ and twitch tension in spite of a decrease in intracellular pH. The effect on twitch tension of the hemidiaphragms showed a rapid onset, was reversible, persisted until the buffer composition was changed, and was independent of synaptic transmission. It is concluded that the twitch tension of the skeletal muscle decrease with a decrease in intracellular K+. The muscle tension also decreases with an increase in the ratio of intracellular and extracellular H+ concentration. However, there is no consistent relationship between muscle tension and extracellular or intracellular pH. The muscle tension of the diaphragms taken from K+-deficient rats is more sensitive to variations in CO2, PH, and bicarbonate concentration of the medium than that of the control rat diaphragms.     

The effects of pH on the sodium-withdrawal contractures evoked from frog atrial trabeculae

Contractures can be evoked from frog atrial trabeculae by raising extracellular pH in Na-free fluid after the spontaneous relaxatin of the tension evoked by removing extracellular Na+. These alkalinity contractures are unaffected by local anaesthetics but are absent following a brief perfusion with Ca-free fluid but are partially inhibited by local anaesthetics. Perfusion by solution containing a high Pco2 fails to induce contractures. The relationship between [Na]0 and tension is altered by changing extracellular pH; acidic fluids below pH 6.0 reduce tension and alkaline fluids increase tension over the whole range. NH4Cl has the same effect as alkaline fluids while CO2-containing fluid has no effect. These results can be interpreted if the effects of variation of extracellular pH, on Na-withdrawal contractures, are mediated by changes in intracellular pH which in turn affect the Ca-sensitivity of the contractile proteins.     

Effects of changes of intracellular pH on contraction in sheep cardiac Purkinje fibers

Intracellular pH (pHi) was measured with a pH-sensitive microelectrode in voltage-clamped sheep cardiac Purkinje fibers while tension was simultaneously measured. All solutions were nominally CO2/HCO3 free and were buffered with Tris. The addition of NH4Cl (5-20 mM) produced an initial intracellular alkalosis that was associated with an increase of twitch tension. At the same time, a component of voltage-dependent tonic tension developed. Prolonged exposure (greater than 5 min) to NH4Cl resulted in a slow recovery of pHi accompanied by a decrease of tension. Removal of NH4Cl produced a transient acidosis that was accompanied by a fall of force. In some experiments, there was then a transient recovery of force. If extracellular pH (pHo) was decreased, then pHi decreased slowly. Tension also fell slowly. An increase of pHo produced a corresponding increase of both force and pHi. The application of strophanthidin (10 microM) increased force and produced an intracellular acidosis. The addition of NH4Cl, to remove this acidosis partially, produced a significant increase of force. The above results show that contraction is sensitive to changes of intracellular but not extracellular pH. This pH dependence will therefore modify the contractile response to inotropic maneuvers that also affect pHi.     

CO2 induced acute respiratory acidosis and brain tissue intracellular pH: a 31P NMR study in swine

High concentration carbon dioxide (CO(2)) is used to promote pre-slaughter anaesthesia in swine and poultry, as well as short-lasting surgical anaesthesia and euthanasia in laboratory animals. Questions related to animal welfare have been raised, as CO(2) anaesthesia does not set in momentarily. Carbon dioxide promotes anaesthesia by lowering the intracellular pH in the brain cells, but the dynamics of the changes in response to a high concentration of CO(2) is not known. Based on (31)P NMR spectroscopy, we describe CO(2)-induced changes in intracellular pH in the brains of five pigs inhaling 90% CO(2) in ambient air for a period of 60 s, and compare the results to changes in arterial blood pH, P(CO2), O(2) saturation and HCO(3)(-) concentration. The intracellular pH paralleled the arterial pH and P(CO2) during inhalation of CO(2); and it is suggested that the acute reaction to CO(2) inhalation mainly reflects respiratory acidosis, and not metabolic regulation as for example transmembrane fluxes of H(+)/HCO(3)(-). The intracellular pH decreased to approximately 6.7 within the 60 s inhalation period, and the situation was metabolically reversible after the end of CO(2) inhalation. The fast decrease in intracellular pH supports the conclusion that high concentration CO(2) leads to anaesthesia soon after the start of inhalation.     

Influence of hypercapnia on certain aspects of heat exchanges at room temperature in the rabbit

Heat production as well as heat loss from the surface of the ear and airways during and after hypercapnia induced by one hour inhalation of 10% mixture was studied in conscious rabbits. The arterial CO2 tension increased by about 45 mm Hg and pH fell by about 0.3. These changes were associated with decreasing of heat production while heat loss from the ears and airways increased significantly. Consequently the rectal temperature fell meanly by about 0.9 degrees C. The data show that fall of body temperature in the rabbits exposed on 10% CO2 at room temperature is due both to heat production changes and heat loss changes.     

pH(i) responses to osmotic cell shrinkage in the presence of open-system buffers.

Changes in plasma volume in vivo cause rapid changes in extracellular pH by altering the plasma bicarbonate concentration at a constant Pco(2) (Garella S, Chang BS, and Kahn SI. Kidney Int 8: 279, 1975). Few studies have examined the possibility that changes in cell volume produce comparable changes in intracellular pH (pH(i)). In the present study, alveolar macrophages were exposed to hyperosmotic medium in the absence or presence of the open-system buffers CO(2)-HCO(3)(-), propionic acid-propionate, or NH(3)-NH(4)(+). In the absence of open-system buffers, exposure to twice-normal osmolarity (2T) produced a slow cellular alkalinization [change in pH(i) (DeltapH(i)) approximately 0.38; exponential time constant (tau) approximately 120 s]. In the presence of 5% CO(2), 2T caused a biphasic pH(i) response: a rapid increase (DeltapH(i) approximately 0.10, tau approximately 15 s) followed by a slower pH(i) increase. Identical rapid pH(i) increases were produced by 2T in the presence of propionic acid (20 mM). Conversely, 2T caused a rapid pH(i) decrease (DeltapH(i) approximately -0.21, tau approximately 10 s) in the presence of NH(3) (20 mM). Thus osmotic cell shrinkage caused rapid pH(i) changes of opposite direction in the presence of a weak acid buffer (contraction alkalosis with CO(2) or propionic acid) vs. a weak base buffer (contraction acidosis with NH(3)). Graded DeltapH(i) were produced by varying extracellular osmolarity in the presence of open-system buffers; osmolarity increases of as little as 5-10% produced significant DeltapH(i). The rapid pH(i) responses to 2T were insensitive to inhibitors of membrane H(+) transport (ethylisopropylamiloride and bafilomycin A(1)). The results are consistent with shrinkage-induced disequilibria in the total cellular buffer system (i.e., intrinsic buffers plus added weak acid-base buffer).     

Effect of decreased pH on force and phosphocreatine in mammalian skeletal muscle

Phosphocreatine (PCr) and intracellular pH changes were monitored by 31P-NMR spectroscopy in isolated, arterially perfused cat biceps and soleus muscles, while the pH of the CO2-bicarbonate buffered perfusate was decreased from 7.1-7.4 to 6.4-6.7 by increasing the CO2 in the equilibrating gas from 5 to up to 70%. In biceps (fast twitch) muscles, intracellular pH decreased from 7.0 to 6.6 (30% CO2, 30 degrees C), peak tetanic force decreased by 8%, but the rise and relaxation times of tetanic were not significantly changed. In soleus muscles, intracellular pH decreased from 7.0 to 6.6 (30% CO2, 30 degrees C), peak tetanic force was unchanged, but the rise and relaxation times of tetani were increased by 27 and 112%, respectively. In both muscles greater decreases in tetanic force were observed during repetitive or ischemic stimulation, which resulted in intracellular pH similar to that produced by hypercapnia. Contrary to previous reports, there was no significant decrease in PCr level in either muscle type with decreased intracellular pH. In the soleus at 30 degrees C there was a significant increase in PCr level with decreased pH.     

Vasoconstriction: a novel activity for low density lipoprotein

Low density lipoprotein plays an important role in the pathogenesis of atherosclerosis. Cumulative addition of 1-30 micrograms/ml of LDL from normolipidemic subjects produced a dose-dependent increase in contractile tension of thoracic aortic rings from rats. The maximal LDL-induced contractile response was approximately 30% of that induced by 1 microM norepinephrine. Similar concentrations of LDL induced a dose-dependent transient increase of the concentration of intracellular free calcium, and a biphasic change of the intracellular pH in cultured rat vascular smooth muscle cells. We conclude that low density lipoprotein occurring for example in the extravascular fluid can mediate vasoconstriction by changes in cytosolic calcium and intracellular pH.     

Intracellular pH of stimulated thymocytes measured with a new fluorescent indicator

A new fluorescent intracellular pH indicator is described ("quene 1") which is related to the tetracarboxylate Ca2+ indicator based on the quinoline fluorophor ("quin 2"). Quene 1 has excitation and emission maxima at 390 and 530 nm, respectively, and shows a 30-fold increase in fluorescence between pH 5 and 9 with a pK alpha of 7.3. The fluorescence is insensitive to Ca2+ and Mg2+ at free concentrations up to 10(-4) M and to the proportions of Na+ and K+ at total concentrations of Na+ and K+ from 100 to 200 mM. The indicator is loaded into thymocytes using the tetraacetoxymethyl ester derivative which is hydrolyzed in the cells to give the tetracarboxylate anion. Intracellular pH can be measured at intracellular quene 1 concentrations of approximately 0.1 mM and quene 1 does not perturb glycolysis or the ATP level in resting cells at concentrations up to 0.8 mM. The intracellular pH of mouse thymocytes indicated by quene 1 is 7.15 +/- 0.04 and it is insensitive to the concentration of Ca2+ or Mg2+ in the extracellular medium. The intracellular pH decreased when the pH of the medium was lowered by addition of HCl, but was insensitive to NaOH at extracellular pH values up to 8.0. Rapid transient changes in intracellular pH are induced by NH4Cl, NaCO2CH3, or HCO3-/CO2. The thymocytes showed no early changes (within 30 min) in intracellular pH in response to mitogenic concentrations of lectins or 4 beta-phorbol-12-myristate-13-acetate.     

Kinetics of the Bohr effect of menhaden hemoglobin, Brevoortia tyrannus.

The kinetics of proton release on ligation of menhaden hemoglobin was studied by flash photolysis over a range of pH. In contrast to all previous kinetic work with human hemoglobin, a nonlinear relationship between proton release and CO binding was found. Proton uptake was also observed in the course of O₂ replacement by CO at low pH. It follows that at least part of the proton release is associated with quaternary rather than tertiary conformational changes i.e. this result is consistent with a two-state model in which L is a function of pH.     

Is the change in intracellular pH during fatigue large enough to be the main cause of fatigue?

The intracellular pH of frog sartorius muscles exposed to an extracellular pH 8.0 (25 mM HCO3-, 1% CO2) was 6.9-7.1. Following a fatiguing stimulation period (one tetanic contraction per second for 3 min), the intracellular pH was 6.5-6.7. When similar experiments were repeated with frog sartorius muscles exposed to pH 6.4 (2mM HCO3-, 1% CO2), the intracellular pH was 6.8-6.9 at rest and 6.3-6.4 following fatigue. So, in both experiments the intracellular pH decreased by 0.4-0.5 pH unit during fatigue. When the CO2 concentration of the bathing solution was increased from 1 to 30%, the intracellular pH of resting muscles decreased from 7.0 to 6.2-6.3. Although the effect of CO2 on the intracellular pH was greater than the fatigue effect, the decrease in tetanic force with CO2 was less than 40%, while during fatigue the tetanic force decreased by at least 70%. Therefore in frog sartorius muscle the decrease in tetanic force during fatigue exceeds the decrease that is expected from just a change in intracellular pH.     

The effect of acid-base balance on fatigue of skeletal muscle

H+ ions are generated rapidly when muscles are maximally activated. This results in an intracellular proton load. Typical proton loads in active muscles reach a level of 20-25 mumol X g-1, resulting in a fall in intracellular pH of 0.3-0.5 units in mammalian muscle and 0.6-0.8 units in frog muscle. In isolated frog muscles stimulated to fatigue a proton load of this magnitude is developed, and at the same time maximum isometric force is suppressed by 70-80%. Proton loss is slowed when external pH is kept low. This is paralleled by a slow recovery of contractile tension and seems to support the idea that suppression results from intracellular acidosis. Nonfatigued muscles subjected to similar intracellular proton loads by high CO2 levels show a suppression of maximal tension by only about 30%. This indicates that only a part of the suppression during fatigue is normally due to the direct effect of intracellular acidosis. Further evidence for a component of fatigue that is not due to intracellular acidosis is provided by the fact that some muscle preparations (rat diaphragm) can be fatigued with very little lactate accumulation and very low proton loads. Even under these conditions, a low external pH (6.2) can slow recovery of tension development 10-fold compared with normal pH (7.4). We must conclude that there are at least two components to fatigue. One, due to a direct effect of intracellular acidosis, acting directly on the myofibrils, accounts for a part of the suppression of contractile force. A second, which in many cases may be the major component, is not dependent on intracellular acidosis. This component seems to be due to a change of state in one or more of the steps of the excitation-contraction coupling process. Reversal of this state is sensitive to external pH which suggests that this component is accessible from the outside of the cell.     

Relationships between extracellular pH, intracellular pH, and gene expression in Dictyostelium discoideum

A variety of studies have shown that differentiation of Dictyostelium discoideum amoebae in the presence of cAMP is strongly influenced by extracellular pH and various other treatments thought to act by modifying intracellular pH. Thus conditions expected to lower intracellular pH markedly enhance stalk cell formation, while treatments with the opposite effect favor spores. To directly test the idea that intracellular pH is a cell-type-specific messenger in Dictyostelium, we have measured intracellular pH in cells exposed to either low extracellular pH plus weak acid or high extracellular pH plus weak base using 31P nuclear magnetic resonance (NMR). Our results show that there is no significant difference in intracellular pH (cytosolic or mitochondrial) between pH conditions which strongly promote either stalk cell or spore formation, respectively. We have also examined the effects of external pH on the expression of various cell-type-specific markers, particularly mRNAs. Some mRNAs, such as those of the prestalk II (PL1 and 2H6) and prespore II (D19, 2H3) categories, are strongly regulated by external pH in a manner consistent with their cell-type specificity during normal development. Other markers such as mRNAs D14 (prestalk I), D18 (prespore I), 10C3 (common), or the enzyme UDP-galactose polysaccharide transferase are regulated only weakly or not at all by external pH. In sum, our results show that modulation of phenotype by extracellular pH in cell monolayers incubated with cAMP does not precisely mimic the regulation of stalk and spore pathways during normal development and that this phenotypic regulation by extracellular pH does not involve changes in intracellular pH.     

Alteration of sensitivity and time scale in invertebrate photoreceptors exposed to anoxia, dinitrophenol, and carbon dioxide

The effects of anoxia, 2,4-dinitrophenol (DNP), and carbon dioxide (CO2) on the late receptor potential of Balanus lateral ocelli, Limulus ventral eyes, and the retinular cells of Linulus lateral eyes have been studied. Either anoxia, DNP, or exposure to 100% CO2 causes a depolarization of 5-30 mV and a gradual reduction and eventually abolition of the late receptor potential and an increase in the latency and time to peak of the response. This lengthening of the time scale is in contrast to the response obtained in photoreceptors that have been light-adapted or injected with calcium. In that case a loss in sensitivity is associated with a decrease in latency and time to peak. Because of these observed differences, the effects of metabolic inhibition cannot be attributed merely to a loss in regulation of intracellular free calcium. Rather, because alteration of intracellular pH (pHi) by using either (NH4)2SO4 or CO2 produced changes in the photoresponse similar to those caused by metabolic inhibition, it is suggested that changes in pHi during metabolic inhibition can account in part for the lengthening of the time scale. In addition to the changes in pHi and internal Ca++ concentration due to metabolic inhibition, the possible role of other consequences of metabolism in the transduction mechanism is also discussed.     

Generation of intracellular pH gradients in single cardiac myocytes with a microperfusion system

This study describes the use of a microperfusion system to create rapid, large regional changes in intracellular pH (pH(i)) within single ventricular myocytes. The spatial distribution of pH(i) in single myocytes was measured with seminaphthorhodafluor-1 fluorescence using confocal imaging. Changes in pH(i) were induced by local external application of NH(4)Cl, CO(2), or sodium propionate. Local application was achieved by simultaneously directing two parallel square microstreams, each 275 microm wide, over a single myocyte oriented perpendicular to the direction of flow. One stream contained the control solution, and the other contained a weak acid or base. End-to-end, stable pH(i) gradients as large as 1 pH unit were readily created with this technique. This result indicates that pH within a single cardiac cell may not always be spatially uniform, particularly when weak acid or base gradients are present, which can occur, for example, in regional myocardial ischemia. The microperfusion method should be useful for studying the effects of localized acidosis on myocyte function, estimating intracellular ion diffusion rates, and, possibly, inducing regional changes in other important intracellular ions.     

Intracellular pH regulates basolateral K+ and Cl- conductances in colonic epithelial cells by modulating Ca2+ activation.

The role of intracellular pH as a modulator of basolateral K+ and Cl- conductances in epithelial cells was studied using digitonin-permeabilized colonic cell layers so that cytosolic pH could be clamped at specific values, while basolateral K+ and Cl- conductances were activated by stepwise increases in intracellular free Ca2+. Increasing the intracellular pH from 6.6 to 8.0 enhanced the sensitivity of both ionic conductances to intracellular Ca2+, but changing extracellular pH had no effect. Maximal K+ and Cl- currents activated by Ca2+ were not affected by changes in intracellular pH, suggesting that protons do not alter the conduction properties of the channels. Hill analysis of the Ca2+ activation process revealed that raising the cytosolic pH from 6.6 to 8.0 reduced the K1/2 for Ca2+ activation. In the absence of Ca2+, changes in intracellular pH did not have a significant effect on the basolateral K+ and Cl- conductances. These results are consistent with the notion that changes in cytosolic pH can modulate basolateral conductances by modifying the action of calcium, perhaps by acting at or near the activation site to provide a mechanism of variable "gain control."     

Induction of meiotic maturation in Xenopus oocytes by 12-O-tetradecanoylphorbol 13-acetate

Fully grown Xenopus oocytes are physiologically arrested at the G2/prophase border of the first meiotic division. Addition in vitro of progesterone or insulin causes release of the G2/prophase block and stimulates meiotic cell division of the oocyte, leading to maturation of the oocyte into an unfertilized egg. The possibility that the products of polyphosphoinositide breakdown, diacylglycerol and inositol-1,4,5-trisphosphate (IP3-, are involved in oocyte maturation was investigated. Microinjection of IP3 into oocytes just prior to addition of progesterone or insulin accelerated the rate of germinal vesicle breakdown (GVBD) by up to 25%. Half-maximal acceleration occurred at an intracellular IP3 concentration of 1 microM. Treatment of oocytes with the diacylglycerol analog and tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced GVBD in the absence of hormone. Half-maximal induction of GVBD occurred with 150 nM TPA and was blocked by pretreatment of oocytes with 10 nM cholera toxin. Microinjection of highly purified protein kinase C from rat brain into oocytes did not induce maturation but markedly accelerated the rate of insulin-induced oocyte maturation. However, injection of the enzyme had no effect on progesterone action. In oocytes with a basal intracellular pH below 7.6, TPA increased intracellular pH, but GVBD occurred with TPA in Na-substituted medium. Neomycin, a putative inhibitor of polyphosphoinositide breakdown, reversibly inhibited insulin- but not progesterone-induced maturation. Half-maximal inhibition occurred at 1.6 mM neomycin. These results indicate that protein kinase C is capable of regulating oocyte maturation in Xenopus.     

Changes in intracellular pH of Physarum plasmodium during the cell cycle and in response to starvation

The intracellular pH of Physarum plasmodia was monitored under conditions of growth and during starvation by means of recessed tip pH microelectrodes. There is a cycle of intracellular pH that corresponds to the period of the cell cycle, with a low point at mid-interphase of pH 7.0 and a peak of pH 7.5 just at mitosis. Experiments in which the intracellular pH is artificially lowered suggest that there is a critical period 1 h before mitosis in which the pH must be high (>7.2), but that mitosis itself can proceed at lower values. During the process of differentiation induced by starvation the intracellular pH drops to very low values (6.6 by 15 h) and refeeding can quickly reverse this condition and restore the pH cycle and nuclear division. Additionally, artificially lowering the intracellular pH will give rise to morphology which resembles the first stages of starvation-induced differentiation.     

Stimulation of Na:H exchange by insulin

In frog skeletal muscle, the increase of intracellular pH (pHi) induced by insulin is correlated with an increase in intracellular Na+ when the sodium pump is inhibited by ouabain. Reversing the Na+ free energy gradient by substituting either Mg2+ or choline for extracellular Na+ converts the effect of insulin to a decrease in pHi, indicating that the action of insulin upon pHi is determined by the Na+ free energy gradient. Moreover, estimates of the Na+ free energy gradient indicate that both the direction and magnitude satisfy the hypothesis that this is the source of energy for the observed changes in pHi. Both the increase in intracellular pH induced by insulin and the associated increase in intracellular Na+ produced by this hormone in the presence of ouabain are blocked by amiloride. This drug also blocks the decrease in pHi by insulin when Mg2+ is substituted for Na+ in the Ringer. In Ringer containing Na+, the increase in pHi by insulin occurs when both metabolic and atmospheric sources of CO2 are eliminated by using a 100% N2 atmosphere. Thus, the mechanism stimulated by insulin is not a Na+-CO3(2-) cotransport system, but is either an Na:H exchange or a Na+-OH- cotransport system which can be inhibited by amiloride. The suggestion is advanced that the Na:H exchange mechanism is part of the membrane transduction system for insulin.     

The effect of temperature on the contractile response of cardiac muscle from two frog species, Rana temporaria and Xenopus laevis, at two different calcium concentrations-II. At pH 7.0 and pH 6.6

1. Mechanical parameters were recorded from paced ventricular cardiac muscle strips of two amphibian species. Rana temporaria and xenopus laevis. 2. The strips contracted at pH 6.6 and 7.0 with either 1.6 or 3.0 mM Ca2+ while the temperature was changed from 5 to 30 degree C. 3. In the hearts of R. temporaria, but not in those of X. laevis, in increased Ca2+ concentration at low pH could compensate for the effect of lowered pH. 4. It is concluded that during high CO2 tension and/or low pH the myocardium of X. laevis was more temperature dependent, less Ca2+ dependent in the Ca2+ range examined and less sensitive to the pH and/or the CO2 tension during physiological temperature and Ca2+ concentration, than the cardiac muscle of R. temporaria.