Acidosis, cardiac stunning and its prevention by oxygen

When during aerobic perfusion of the 5 Hz paced rat Langendorff heart, under constant aortic pressure of 8.3 kPa, the pH of the medium is changed from 7.5 to 7.0 a short period of positive inotropy is followed by a dramatic loss of contractility. The hearts, rapidly frozen after 10 min pH 7.0 perfusion, show moderate loss of high-energy phosphates and accumulation of lactate and glycerol-3-phosphate, indicative of tissue anaerobiosis. This can be overcome by including fluorocarbon, an O2 vehicle, in the media. The transient positive inotropy is interpreted as H(+)-induced release of plasmalemma-bound Ca2+ into the cytosol. The accompanying morphologic alterations are as described in this issue by Vandeplassche and Borgers (1990) and by Verkleij et al. (1990).     

Cytoplasmic pH response to acid stress in individual cells of Escherichia coli and Bacillus subtilis observed by fluorescence ratio imaging microscopy

The ability of Escherichia coli and Bacillus subtilis to regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. The E. coli cytoplasmic pH fell to a value that varied among individual cells (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In an E. coli biofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells. B. subtilis cells were acid shifted from pH 7.5 to pH 6.0. In B. subtilis, unlike the case with E. coli, cytoplasmic pH showed no "overshoot" but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individual B. subtilis cells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.     

Activation of oxoglutarate dehydrogenase in the kidney in response to acute acidosis.

1. Activation by H+ and by Ca2+ of 2-oxoglutarate dehydrogenase extracted from mitochondria of normal or acidotic rat kidney is described. This effect, first shown for the enzyme from heart by McCormack & Denton [Biochem. J. (1979) 180, 533--544], is of a regulatory importance in kidney, in which organ, in contrast with heart, increased flux occurs during acute acidosis. 2. In renal-cortical tubules, 2-oxoglutarate concentration fell within 1 min of decreasing the pH and rose again 1--3 min after increasing the pH of the medium. The extent of the decrease in 2-oxoglutarate was directly related to the decrease in pH. A similar fall in the oxoglutarate concentration in the whole perfused kidney was noted within 5 min of inducing acidosis. 3. In tubules, the rates of gluconeogenesis and ammoniagenesis from 1 mM-glutamine were increased by 64 and 33% respectively on decreasing pH to 7.0, the increase in rates being proportional to the fall in pH between 7.4 and 7.0. 4. The increased rates of renal ammoniagenesis and gluconeogenesis seen in acute acidosis in vitro can be accounted for by the increased activity of 2-oxoglutarate dehydrogenase and the tissue concentrations of 2-oxoglutarate when calculated from the Km determined at normal and acidotic pH. 5. The decrease in 2-oxoglutarate concentration seen in acute acidosis implies a fall in intramitochondrial pH in kidney, and is the result of two phenomena, accelerated disposal via 2-oxoglutarate dehydrogenase and maintenance of near equilibrium of glutamate dehydrogenase.     

Excitation-contraction coupling in heart. XIX. Effect of hypoxia on calcium transport by subcellular particles in the isolated perfused rat heart.

To examine the role of changes in calcium transport by subcellular particles in the pathogenesis of contractile failure due to oxygen lack, both mitochondrial and microsomal fractions were obtained from the isolated hypoxic rat hearts and their calcium binding and uptake abilities were determined by the Millipore filtration technique. The contractile force decreased by about 40, 60 and 70% of the control within 5, 10 and 30 min respectively, of perfusing the heart with hypoxic medium containing glucose. In hearts perfused for 10 min with hypoxic medium containing glucose, calcium binding and uptake by the microsomal fraction decreased significantly. However, mitochondrial calcium binding, but not uptake, decreased significantly on perfusing the hearts with hypoxic medium containing glucose for 20 to 30 min when the microsomal calcium transport was markedly depressed. Reduction in contractile force, calcium binding and uptake by the microsomal fraction as well as calcium binding by mitochondria of hearts made hypoxic for 30 min recovered towards normal upon reperfusion with control medium for 15 min. On the other hand, omitting glucose from the hypoxic medium significantly decreased calcium binding by mitochondrial and microsomal fractions within 10 min of perfusion in comparison to the control and accelerated the effects of hypoxia upon contractile force and microsomal calcium uptake. In contrast to the hypoxic hearts, the mitochondrial calcium uptake decreased significantly and the magnitude of depression in the microsomal calcium binding was appreciably greater in hearts made to fail to a comparable degree upon perfusion with substrate-free medium. The observed defects in calcium transporting properties of microsomal and mitochondrial membranes appear secondary to the contactile failure in hypoxic hearts.     

Different effects of simple anoxic lactic acidosis and simulated in vivo anoxic acidosis on turtle heart.

We compared responses of turtle heart at 20 degrees C to an anoxic lactic acidosis solution (LA) containing 35 mM lactic acid in an otherwise normal turtle Ringers equilibrated with 3% CO2/97% N2 at pH 7.0) to a solution simulating in vivo anoxic acidosis (VA), with elevated concentrations of lactate, Ca2+, Mg2+, and K+, and decreased Cl-, equilibrated with 10.8% CO2/89.2% N2 at pH 7.0. We examined mechanical properties on cardiac muscle strips and determined intracellular pH (pHi) and high energy phosphates on perfused hearts using 31P-NMR. Maximum active force (Fmax) and the maximum rate of force development (dF/dtmax) of muscle strips were significantly higher during VA than during LA superfusion. An elevation of Ca2+ alone (to 6 mM) in LA significantly increased both Fmax and dF/dtmax but the effects diminished toward the end of the exposure; however, hypercapnic anoxic lactic acidosis (addition of 20 mM HCO3- to LA, equilibrated with 10.8% CO2/89.2% N2, pH 7.0) did not significantly affect Fmax or dF/dtmax. During VA perfusion, pHi (6.73 +/- 0.01) was significantly higher than that during LA perfusion (pHi 6.69 +/- 0.013), but the difference is probably too small to have physiological significance. ATP, creatine phosphate, and inorganic phosphate were not significantly different in the two anoxic solutions. We conclude that the reduction of cardiac mechanical function in vivo is minimized by the integrated effects of changes of ionic concentrations, but the observed changes in Ca2+ and pHi cannot fully explain the effect.     

Modification of intramuscular pH oscillations in the isolated perfused rat heart by different interventions.

Changes in the intramuscular pH oscillations were examined by the use of an antimony electrode upon perfusing the isolated rat heart under different experimental conditions. The pH oscillations were decreased upon perfusing the hearts with Na+- or Ca2+-free medium and increased upon perfusing with K+-free medium. Increasing the temperature of perfusion medium from 25 to 40 degrees C or omitting glucose from the perfusing medium decreased the magnitude of oscillations. On the other hand, complete interruption of the perfusion flow resulted in an increase in the amplitude of pH oscillation. An initial increase followed by a decrease in the pH oscillation was seen when hearts were perfused with medium containing lactic acid at pH 6.6. These results suggest that pH oscillations reflect fluctuations in myocardial metabolism.     

Subcellular calcium transport during contractile failure and recovery in heart perfused with Na-free medium

Perfusion of isolated rat hearts with Na-free medium resulted in an immediate increase in contractile force followed by a decline and complete loss of contractile force within 25 s. The recovery of the contractile force upon reperfusion was only partial if the duration of Na-free perfusions was 10 min or longer. Ca binding and uptake activities of mitochondria obtained from hearts perfused with Na-free medium did not change significantly. However, Ca binding and uptake activities of microsomes were depressed after 5 min of perfusion. The critical concentration of Na in the perfusion medium for inducing these changes was found to be less than 35 mM. The microsomal Ca-ATPase activity was decreased after 10 min of Na-free perfusion. Only partial recovery of microsomal Ca uptake was observed upon reperfusion of hearts preperfused with Na-free medium for 20 min or longer whereas Ca-ATPase activity in these hearts did not recover at all. These results suggest that the defect in the microsomal Ca transport may be secondary to the development of contractile failure and may partially be associated with the inability of Na-depleted hearts to recover fully their contractile force.     

Studies of acidosis in the ischaemic heart by phosphorus nuclear magnetic resonance.

1. Phosphorus-nuclear-magnetic-resonance measurements were made on perfused rat hearts at 37 degrees C. 2. With the improved sensitivity obtained by using a wide-bore 4.3 T superconducting magnet, spectra could be recorded in 1 min. 3. The concentrations of ATP, phosphocreatine and Pi and, from the position of the Pi resonance, the intracellular pH (pHi) were measured under a variety of conditions. 4. In a normal perfused heart pHi = 7.05 +/- 0.02 (mean +/- S.E.M. for seven hearts). 5. During global ischaemia pHi drops to 6.2 +/- 0.06 (mean +/- S.E.M.) in 13 min in a pseudoexponential decay with a rate constant of 0.25 min-1. 6. The relation between glycogen content and acidosis in ischaemia is studied in glycogen-depleted hearts. 7. Perfusion of hearts with a buffer containing 100 mM-Hepes before ischaemia gives a significant protective effect on the ischaemic myocardium. Intracellular pH and ATP and phosphocreatine concentrations decline more slowly under these conditions and metabolic recovery is observed on reperfusion after 30min of ischaemia at 37 degrees C. 8. The relation between acidosis and the export of protons is discussed and the significance of glycogenolysis in ischaemic acid production is evaluated.     

A review of 126 Caesarean sections by blood gas and the acid-base status of newborn calves

Blood gas and acid-base status was determined in 126 Caesarean-derived calves. The newborn calves were assigned by venous blood pH value at birth to three groups as follows: Group 1 (normal): pH above 7.2; Group 2 (slight acidosis): pH 7.2 to 7.0; and Group 3 (severe acidosis): pH below 7.0. Following Caesarean section births 80 (63.5%) calves had normal acid-base values, while 30 (23.8%) had a slight acidosis, and 16 (12.7%) had severe acidosis. The degree of hypoxia was similar in each group. Six calves (37.5%) in Group 3 died within 48 h of birth. The blood gas and acid-base status of Caesarean-derived. calves was not significantly influenced by any examined parameters with the exception of sex in Groups 1 and 2. The occurrence of meconium-stained calves was 9.1% (n = 11), and only two calves were slightly or severely acidotic immediately after birth.     

Protection of the ischemic diabetic heart by L-propionylcarnitine therapy

Diabetics suffer from an increased incidence of myocardial infarction and are less likely to survive an ischemic insult. Since L-propionylcarnitine (LPC) has been shown to protect against ischemic/reperfusion injury, we hypothesized that LPC may be of even greater benefit to the diabetic heart. Diabetes was induced by i.v. streptozotocin, 60 mg/kg; duration: 12 wks. The chronic effect of LPC was determined by daily i.p. injections (100 mg/kg) for 8 wks. The acute effects of LPC were determined by adding it to the perfusion medium (5 mM) of control and diabetic hearts. Initial cardiac contractile performance of isolated perfused working hearts was assessed by varying left atrial filling pressure. Hearts were then subjected to 90 min of low flow global ischemia followed by 30 min reperfusion. Chronic LPC treatment had no effect on initial cardiac performance in either control or diabetic hearts. Acute addition of LPC to the perfusion medium enhanced pump performance of control hearts, but had no effect in diabetic hearts. Both acute and chronic LPC significantly improved the ability of control and diabetic hearts to recover cardiac contractile performance after ischemia and reperfusion, however, chronic treatment was more effective in diabetic hearts.     

The activation of pyruvate dehydrogenase in the perfused rat heart by adrenaline and other inotropic agents.

Adrenaline resulted in a reversible 4-fold increase in the amount of pyruvate dehydrogenase in its active non-phosphorylated form in the perfused rat heart within 1 min. The increase was less in extent in hearts from starved or diabetic rats or in hearts from control rats oxidizing acetate, unless pyruvate was added to the perfusion medium. Increases could also be induced by other inotropic agents, supporting the hypothesis that increases in cytoplasmic Ca2+ can be relayed into mitochondria and influence oxidative metabolism.     

Myofibrillar Ca2+-stimulated Mg2+-ATPase from chronically ischemic canine heart

Functional properties of myofibrils from chronically ischemic canine myocardium were evaluated. Ischemia was produced by tight stenosis of left anterior descending artery (LAD), followed by 40 min acute ischemia with prior preconditioning. Animals of the first group were sacrificed after 8 weeks. In the second group, angioplasty of LAD was performed after 8 weeks of ischemia and animals were kept alive for other 4 weeks. Control animals were sham operated. Activity and kinetic parameters of myofibrillar Ca2+-stimulated Mg2+-ATPase were measured in myofibrils isolated from anterior and posterior parts of all hearts. We did not find any differences in maximal velocity (Vmax), half-maximal activation constant for calcium (K(Ca2+)50) and cooperativity coefficient (n(hill)) of myofibrils from different experimental groups as compared to controls, either at pH 7, pH 6.5 (acidosis) or pH 7.5 (alkalosis). K(Ca2+)50 increased in medium simulated acidosis (12.6-33.5 times) and n(hill) decreased significantly in all groups as compared with values obtained at pH 7. These results indicate that activity and Ca2+-sensitivity of myofibrillar Mg2+-ATPase remain unchanged despite deteriorated heart function 8 weeks after LAD obstruction. Experiments have confirmed that Ca2+-stimulated-ATPase from canine heart myofibrils responded to pH decrease by a decreased sensitivity to Ca2+ and a decreased cooperativity. However, sensitivity of the enzyme to the pH changes is unaltered by 8 weeks of chronic ischemia.     

Calcium exposure required for full expression of injury in the calcium paradox

Isolated hearts repleted with calcium after a short period of perfusion with a calcium-free medium undergo the injury of the calcium paradox and release cellular protein. In the present experiments, 15 min perfusion with a calcium containing HEPES solution subsequent to 10 min calcium-free perfusion resulted in the loss of 42.7 +/- 3.9 mg of protein/g heart. If hearts were repleted with calcium for 30 s only, then returned to calcium-free perfusion, resultant protein loss was the same. When calcium repletion was further decreased to 20 s, 23.9 +/- 1.3 mg/g of protein was lost. This critical period coincided with the onset of contracture but was prior to major ion movements. It is concluded that the transition to irreversible injury occurs within 30 s of calcium repletion and that subsequent removal of extra-cellular calcium does not alter the course of events leading to cell death.     

Properties of an L-glutamate-induced acid tolerance response which involves the functioning of extracellular induction components

Escherichia coli became more acid tolerant following incubation for 60 min in a medium containing L-glutamate at pH 7.0, 7.5 or 8.5. Several agents, including cAMP, NaCl, sucrose, SDS and DOC, prevented tolerance appearing if present with L-glutamate. Lesions in cysB, hns, fur, himA and relA, which frequently affect pH responses, failed to prevent L-glutamate-induced acid tolerance but a lesion in L-glutamate decarboxylase abolished the response. Induction of acid tolerance by L-glutamate was associated with the accumulation in the growth medium of a protein (or proteins) which was able to convert pH 7.0-grown cultures to acid tolerance, and the original L-glutamate-induced tolerance response was dependent on this component(s). Acid tolerance was also induced by L-aspartate at pH 7.0 and induction of such tolerance was dependent on an extracellular protein (or proteins). The L-glutamate and L-aspartate acid tolerance induction processes are further examples of a number of stress tolerance responses which differ from most inductions in that extracellular components, including extracellular sensors, are required.     

Effect of pH on lipolysis, cAMP and cAMP-dependent protein kinase activity in isolated rat fat cells

The effect of acidosis and alkalosis on lipolysis, cAMP production and cAMP-dependent protein kinase activity in isolated rat fat cells incubated in the presence of norepinephrine and norepinephrine plus theophylline has been investigated. The pH of the incubation medium was adjusted to 6.8, 7.4 and 7.8 respectively. Acidosis inhibited both norepinephrine- and norepinephrine plus theophylline-induced release of glycerol whereas alkalosis led to slight stimulation. Norepinephrine produced an increase in cAMP and cAMP-dependent protein kinase activity. However, comparison of both parameters in acidosis and alkalosis with those at pH 7.4 indicates that they were higher at pH 7.8 and lower at pH 6.8. Addition of theophylline in combination with norepinephrine increases cAMP production within 5 min, under acidosis to values similar to those obtained at pH 7.4 with norepinephrine. The same effect on protein kinase activity was obtained. In spite of this increment in cAMP and protein kinase activity produced by addition of norepinephrine plus theophylline, lipolysis remains inhibited by acidosis. Addition of theophylline at pH 7.4 and 7.8 induced a much higher cAMP production and cAMP-dependent protein kinase activity although at pH 7.8 there was a statistically significant increase in protein kinase activity at 10 min it did not induce a significant increase in lipolysis. This is discussed and possible mechanisms are suggested to explain the effect of acidosis and alkalosis on the lipolysis induced by norepinephrine in rat fat cells.     

pH值对沼液培养的普通小球藻生长及油含量积累的影响

以50％的沼液为普通小球藻的全营养培养基,考察培养基的起始pH值对小球藻生长及油脂含量的影响,普通小球藻对不同初始pH的沼液中氮、磷的去除情况。设定了2组实验,一组只调节初始接种培养液的pH,分别为6.0、6.5、7.0、7.5、8.0、8.5;另一组将培养液pH分别固定在6.0、6.5、7.0、7.5、8.0、8.5,pH用稀HCl和NaOH进行调节。研究发现在pH 6.5和pH 7.0的偏酸环境有利于小球藻生长,而pH在7.0~8.5的偏碱性条件下有利于小球藻油脂的积累,因此综合小球藻生长和油脂积累2个因素,得到最适合小球藻生长和油脂积累的pH为7.0。培养结束后沼液中氮磷的去除率分别达到了95％和97％,沼液中的总氮由原来的134.91 mg/L降至4.86 mg/L,总磷由10.19 mg/L降到0.32 mg/L。     

Origin of the cytoplasmic pH changes during anaerobic stress in higher plant cells. Carbon-13 and phosphorous-31 nuclear magnetic resonance studies

We tested the contribution of nucleoside triphosphate (NTP) hydrolysis, ethanol, and organic acid syntheses, and H(+)-pump ATPases activity in the acidosis of anoxic sycamore (Acer pseudoplatanus) plant cells. Culture cells were chosen to alter NTP pools and fermentation with specific nutrient media (phosphate [Pi]-deprived and adenine- or glycerol-supplied). In vivo (31)P- and (13)C-nuclear magnetic resonance (NMR) spectroscopy was utilized to noninvasively measure intracellular pHs, Pi, phosphomonoesters, nucleotides, lactate, and ethanol. Following the onset of anoxia, cytoplasmic (cyt) pH (7.5) decreased to 6.8 within 4 to 5 min, whereas vacuolar pH (5.7) and external pH (6.5) remained stable. The NTP pool simultaneously decreased from 210 to <20 nmol g(-1) cell wet weight, whereas nuceloside diphosphate, nucleoside monophosphate, and cyt pH increased correspondingly. The initial cytoplasmic acidification was at a minimum in Pi-deprived cells containing little NTP, and at a maximum in adenine-incubated cells showing the highest NTP concentration. Our data show that the release of H(+) ions accompanying the Pi-liberating hydrolysis of NTP was the principal cause of the initial cyt pH drop and that this cytoplasmic acidosis was not overcome by H(+) extrusion. After 15 min of anoxia, a partial cyt-pH recovery observed in cells supplied with Glc, but not with glycerol, was attributed to the H(+)-consuming ATP synthesis accompanying ethanolic fermentation. Following re-oxygenation, the cyt pH recovered its initial value (7.5) within 2 to 3 min, whereas external pH decreased abruptly. We suggest that the H(+)-pumping ATPase located in the plasma membrane was blocked in anoxia and quickly reactivated after re-oxygenation.     

Enhancement of PVA-degrading enzyme production by the application of pH control strategy

In batch culture for Poly(vinyl alcohol) (PVA)-degrading enzyme (PVAase) production by a mixed culture, higher pH (pH 7.5) was favorable for PVAase production at the prophase of cultivation, but lower pH (pH 7.0) was favorable at the anaphase. This situation was caused by the fact that the optimum pH for different key enzymes [PVA dehydrogenase (PVADH) and oxidized PVA hydrolase (OPH)] production is various. The activity and average specific production rate of PVADH reached the highest values at constant pH 7.5, whereas those of OPH appeared at pH 7.0. A two-stage pH control strategy was therefore developed and compared for its potential in improving PVAase production. By using this strategy, the maximal PVAase activity reached 2.05 U/ml, which increased by 15.2% and 24.2% over the fermentation at constant pH 7.5 and 7.0.     

The Role of Intracellular pH in Fertilization of Sand Dollar Eggs Analyzed by Microinjection Method

The change in intracellular pH (pH_i) upon fertilization and the effects of changing the pH_i by microinjection of pH buffers were investigated in the eggs of the sand dollar, Clypeaster japonicus. The pH_i was determined by the tint of a pH indicator, phenol red, microinjected into eggs. The pH_i ranged from 6.5 to 6.75 in unfertilized eggs and it rose by 0.4 to 0.5 unit within 3 min upon fertilization. The elevated pH_i ranging from 7.0 to 7.25 was maintained at least until the first cleavage. As reported in eggs of other species of sea urchin (1–4), development of fertilized eggs which had been transferred to Na-free sea water immediately after insemination was arrested and the pH_i did not rise remaining at the level of unfertilized eggs. Development was initiated in eggs arrested in Na-free sea water when the pH_i was elevated up to the level of fertilized eggs, i.e. 7.0 to 7.25, by microinjecting 1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-KOH buffer at pH 8.0. By microinjection of pH 7.5 buffer, some eggs started development though none of them underwent cleavage. By microinjection of pH 7.0 or pH 6.5 buffer, development was not initiated. The initiation of development depended on the pH value of microinjected pH buffer, and in consequence, on the final pH_i. The elongation of microvilli which had been arrested in eggs in Na-free sea water was also induced by microinjection of pH 8.0 or 7.5 buffer.     

Relationship between exogenous fuel availability and performance by teleost and elasmobranch hearts

Summary Performance by perfused isolated hearts of sea raven (Hemitripterus americanus) and skate (Raja erinecea), representatives of teleost and elasmobranch fishes, respectively, was monitored over a 30 min period under conditions of variable metabolic fuel availability. In both preparations initial cardiac output and hence fuel delivery to the myocardia were comparable to in vivo levels. Pressure development and hence overall work rate of the sea raven heart was also similar to in vivo levels.Fuel deprived sea raven hearts entered into a modest but significant contractile failure which could be prevented by the inclusion of 10 mM glucose or 1.0 mM palmitate in the perfusion medium. Addition of the glycolytic inhibitor iodoacetate to the medium resulted in rapid heart failure. Performance in the presence of iodoacetate could be improved by the inclusion of palmitate, lactate, or acetoacetate in the perfusion media but only high physiological levels of palmitate could completely alleviate the effect of iodoacetate.The inclusion of 1.0 mM palmitate in the perfusion medium of skate hearts resulted in a significant decrease in performance relative to fuel deprived hearts. Addition of iodoacetate to the medium resulted in rapid contractile failure. Hearts perfused with medium containing both iodoacetate and acetoacetate performed as well as fuel deprived hearts, indicating that this ketone body is an effective metabolic fuel.The performance data reported here are consistent with a previously established biochemical framework. The teleost heart has the capability of utilizing exogenous fatty acid as a metabolic fuel and this substrate may be able to support the contractile process independently. In contrast, fatty acid metabolism in the elasmobranch heart is poorly developed and appears to be more dependent upon the catabolism of blood borne ketone bodies.