Prevention of myocardial reperfusion injury by increasing artificial trans-membrane sodium gradient in "calcium paradox" and post-ischemic reperfusion

An effect of the high sodium gradient during "calcium paradox" and postischemic reperfusion has been studied. A decrease of Na/Ca exchange by high sodium gradient (200 mM NaCl in the perfusion solution) resulted in the reduction of myoglobin release from the heart during "calcium paradox". High sodium concentration solution (200 mM) increased protective effect of ATP during "calcium paradox". Exogenous phosphocreatine (100 mumol/mol) increased myoglobin release from the heart. During perfusion of the heart by high sodium concentration, phosphocreatine efficiently decreased myoglobin release from the heart during "calcium paradox". Exogenous ATP (as Na-pump activator) and high Na+ concentration solution (180 mM) prevented the LDH release from the myocardium, decreased ATP hydrolysis, inhibited Ca influx, maintained total adenine nucleotides, phosphate potential, energy charge of the cardiomyocytes.     

Metabolism of extracellular phosphocreatine during changes in the ionic composition of the medium in the perfused rat heart]

Perfusion of isolated rat hearts with a phosphocreatine (10(-4) M) containing solution to which strophanthin or KCl had been added up to a concentration of 27 mM as well as Ca2+ depletion decreased phosphocreatine concentration in the perfusate with a simultaneous increase in creatine and phosphocreatine concentrations in the myocardium. Neither high extracellular concentrations of Na+ (200 mM), nor phosphocreatine increased creatine and phosphocreatine levels in the myocardium. The effect of high sodium perfusion media was completely reversed by strophanthin. Phosphocreatine decreased the lactate content in the perfusate. Strophanthin or potassium chloride enhanced the effect of phosphocreatine on the lactate release. Conversely, creatine augmented the lactate content in the perfusate. A high specificity of the phosphocreatine effect on the myocardium independently of the ionic composition of the perfusate was postulated. A mechanism of protective effects of phosphocreatine and high sodium perfusion media on "calcium paradox" is proposed.     

Dependence of myocardial contracture on energy resources during the calcium paradox

Perfusion of the rat isolated hearts with calcium-free and calcium containing solution revealed a complex and deep myocardial damage called the calcium paradox. The reperfusion of the rat heart with calcium rich media resulted in myoglobin loss from the heart, significant decreasing of ATP and phosphocreatine level, complete uncoupling of respiration and phosphorylation in mitochondria, occurrence of myocardial contracture. Decreasing of sodium level to 30 mM--80 mM in calcium free media exacerbates the heart damage due to the calcium paradox with absence of contracture. Addition of phosphocreatine (1 mM, 5 mM, 10 mM) evoked some restoration of ATP contents in the tissue with appearance of significant contracture. Phosphocreatine exacerbated the loss of myoglobin from the heart subjected to the calcium paradox. A discrepancy between myocardial contracture and degree of cellular damage has been observed during the calcium paradox.     

The prevention of the development of myocardial contracture in the "calcium paradox" by action on Na-Ca metabolism]

The effect of artificial high sodium gradient on the rate of the myocardium contracture development during "calcium paradox" was studied in the experiments on the isolated heart of Langendorf-perfused rats. It is stated that artificial creation of a high sodium gradient decreases the rate of the myocardium contracture development. Exogenous nucleotides, activators of Na, K-ATPase, and their precursors intensified the protective action of the hypersodium medium. Phosphocreatine (100 mmol/l) had no protective effect during the "calcium paradox". However, under conditions of the high sodium gradient phosphocreatine efficiently prevented development of the contracture during the "calcium paradox". It is important to note that under analogous conditions creation of high osmosity of the solution adding 12 mmol/l of saccharose does not protect the heart from development of the myocardium contracture.     

Dependence of myocardium injury on extracellular K+ concentration during calcium paradox

It is well-known that the first stage of the calcium paradox involves decreasing of Na+ gradient. The decreased sodium gradient is a cause of activation of the Na(+)-Ca+ exchange and formation of cardiac injury during the calcium repletion. Potassium ions are natural extracellular activators of Na(+)-pump. It has been shown that heart perfusion by Ca(2+)-free medium evoked extrusion from cells of hydrophilic amino acids whose transport-depends on sodium gradient. The heart reperdusion with Ca(2+)-containing agent leads to myofibrillar contracture and extensive myoglobin release. The simultaneous events are: elevation in tissue water contents, decreasing of intracellular concentration of adeninnucleotides, uncoupling of oxidation and phosphorylation in mitochondria. The decreasing of K+ level to 0.5 mM exacerbates myocardial damage during the calcium paradox, despite absence of myocardial contracture. The elevation of K+ (to 10 mM or 20 mM) attenuated the calcium paradox development in the heart. The elevated K+ concentration protected isolated heart from extensive myoglobin release, development of myocardial contracture. The high K+ concentrations alleviate mitochondrial damage and elevate contents of adeninnucleotide in the tissue. The positive effect of the elevated K+ concentration can be completely blocked by strophanthine, the selective Na+, K(+)-pumb blocker.     

Oxidative damage to the myocardium: the protective action of exogenous phosphocreatine

Oxidative damage of the isolated perfused rat heart was caused by addition of 90 microM H2O into Krebs-Henseleit solution. After 20 min of H2O2 addition an elevation of diastolic pressure (irreversible contracture) was observed followed by decrease of developed tension and heart work. Addition of phosphocreatine (10 mM) at constant total sodium concentration prevented the development of contracture and diminished the decrease of cardiac work. This protective effect is probably related to the elevation of structural order of phospholipids by phosphocreatine.     

Dependence of myocardial damage on the anion composition and osmotic pressure in the extracellular fluid during "calcium paradox" in rats]

The data obtained reveal that elevation of extracellular osmolarity with sucrose during reintroduction of Ca-containing medium after 10 minutes of Ca2+ removal prevents loss of haemoglobin in a concentration-dependent mode. Reducing the extracellular osmolarity of the reperfusion medium by means of decreasing the concentration of sodium chloride and calcium chloride exacerbates the loss of haemoglobin from the cardiomyocytes. There is a close correlation between the water contents in tissues and the loss of haemoglobin during the "calcium paradox". The findings suggest dependence of the heart damage during the "calcium paradox" on anionic composition of extracellular space and activity of anionic transporters.     

Protective effect of adenosine against a calcium paradox in the isolated frog heart.

The effect of adenosine on the calcium paradox in the isolated frog heart was studied. Addition of adenosine during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein and creatine kinase release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that adenosine protected frog myocardial cells by reducing the massive calcium influx during reperfusion possibly through an action on calcium channels. Adenosine exerted its action in a dose-dependent manner; a concentration of 10 microM adenosine provided maximum protection of myocardial cells against the calcium paradox damage. Higher concentrations of adenosine produced side effects on both electrical and mechanical activity. These results are discussed in terms of the possible mechanism involved in the protective effect of adenosine.     

On the solubility of calcium deoxycholate: kinetics of precipitation and the effect of conjugated bile salts and lecithin

In view of the low solubility of calcium deoxycholate and the possible induction of cholesterol precipitation in the gallbladder by calcium insoluble salts, we find it of interest to study the precipitation of calcium deoxycholate and its dependence on other bile components. The findings of these studies were as follows: (i) Precipitation of calcium deoxycholate from mixtures of calcium chloride and monomeric deoxycholate (at concentrations below the critical micelle concentration (CMC] is very slow even at relatively high CaCl2 concentrations (more than 20 days at 50 mM CaCl2). (ii) At higher deoxycholic acid (DOC) concentrations, precipitation of micellar DOC is faster and requires much lower calcium chloride concentrations. For any given calcium concentration, the rate of precipitation is maximal at an optimal DOC concentration. In solutions containing 150 mM NaCl, the maximal rate of precipitation occurs at about 10 mM DOC, almost independent of Ca2+ concentration. At lower ionic strength (10 mM NaCl), the optimal DOC concentration is 30 mM. These observations suggest that the most important factors in determining the rate of Ca(DOC)2 precipitation are (a) the ratio between calcium ions bound to the surface of a DOC micelle, and the [DOC] (the Ca2+/DOC binding ratio) and (b) the concentration of DOC micelles. (iii) In the presence of conjugated deoxycholates, the crystallization of calcium deoxycholate is inhibited. Phosphatidylcholine has a similar, although smaller, inhibitory effect. Upon precipitation of calcium deoxycholate from a mixed micellar system containing sodium deoxycholate, phosphatidylcholine and cholesterol, the latter two components spontaneously form vesicles. The anti-nucleating effect of PC and conjugated bile salts is explained in terms of "poisoning" of the crystallization process. In view of the latter results we conclude that under normal conditions calcium deoxycholate is not likely to precipitate in the gallbladder.     

Creatine kinase of heart mitochondria. Control of oxidative phosphorylation by the extramitochondrial concentrations of creatine and phosphocreatine

Defining how extramitochondrial high-energy phosphate acceptors influence the rates of heart oxidative phosphorylation is essential for understanding the control of myocardial respiration. When the production of phosphocreatine is coupled to electron transport via mitochondrial creatine kinase, the net reaction can be expressed by the balanced equation: creatine + Pi----phosphocreatine + H2O. This suggests that rates of oxygen consumption could be regulated by changes in [creatine], [Pi], or [phosphocreatine], alone or in combination. The effects of altering these metabolites upon mitochondrial rates of respiration were examined in vitro. Rat heart mitochondria were incubated in succinate-containing oxygraph medium (pH 7.2, 37 degrees C) supplemented with five combinations of creatine (1.0-20 mM), phosphocreatine (0-25 mM), and Pi (0.25-5.0 mM). In all cases, the mitochondrial creatine kinase reaction was initiated by additions of 0.5 mM ATP. To emphasize the duality of control, the results are presented as three-dimensional stereoscopic projections. Under physiological conditions, with 5.0 mM creatine, increases in Pi or decreases in phosphocreatine had little influence upon mitochondrial respiration. When phosphocreatine was held constant (15 mM), changes in [creatine] modestly stimulated respiratory rates, whereas Pi again showed little effect. With 1.0 mM Pi, respiration clearly became dependent upon changes in [creatine] and [phosphocreatine]. Initially, respiratory rates increased as a function of [creatine]. However, at [phosphocreatine] values below 10 mM, product "deinhibition" was observed, and respiratory rates rapidly increased to 80% State 3. With 2.0 mM Pi or higher, respiration could be regulated from State 4 to 100% State 3. Overall, the data show how increasing [creatine] and decreasing [phosphocreatine] influence the rates of oxidative phosphorylation when mediated by mitochondrial creatine kinase. Thus, these changes may become secondary cytoplasmic signals regulating heart oxygen consumption.     

Glucose and octanoate utilization by isolated adult rat heart cells

Rat heart muscle cells continue to beat in the isolated state apparently independent of any innervation. The oxidation of ¹⁴C-glucose to ¹⁴CO₂ was linear for at least 60 minutes of incubation. The rate of glucose oxidation rose rapidly up to a medium glucose concentration of 2.5 mM and then plateaued. Lactate production reached a maximum at 5 mM glucose. Glucose uptake was linearly related to the concentration up to 40 mM. The addition of octanoate reduced, but did not eliminate, glucose oxidation. Octanoate utilization increased with increasing concentration and reached a maximum at 2 mM. The oxidation of octanoate was linearly related to the time of incubation for at least 90 minutes. The presence of glucose, at a concentration of 1.25 mM or higher, increase the oxidation of octanoate by the heart cells. The metabolic parameters measured with the isolated heart cells gave values comparable to those obtained with the perfused rat heart. Decreasing or increasing the concentration of sodium, potassium or magnesium did not effect the oxidation of either glucose or octanoate with the exception that when sodium was increased above 200 mM, a significant increase in glucose oxidation was observed. In contrast, the addition of calcium to a calcium free medium increased glucose oxidation, reaching a maximum at 0.2 mM calcium. The oxidation of octanoate reached a maximum at 0.2 mM and then decreased significantly with increasing calcium concentration. The metabolic activity appears to be independent of the concentration of sodium, potassium or magnesium. In contrast, the isolated heart cell is very sensitive to a change in calcium concentration.     

Effect of papaverine on the tonus and contraction of depolarized taenia coli musculature in guinea pigs

Experiments were performed on the isolated strips of guinea pig taenia coli. The smooth muscle was depolarized in a solution with high potassium concentration (120 mM KCl). The effect of papaverine (in concentration of from 10(-5) to 3.10(-5) g/ml) on the tonus and the contractile off-response originating after the ending of longlasting strong polarizing current was investigated. It was found that: 1) papaverine abolished the concentrations induced by drugs (histamine, acetylcholine, bradykinin); 2) papaverine reduced the tonus of depolarized muscle and eliminated its increase under the effect of a rise of the external calcium concentration; 3) papaverine had no effect on the amplitude and the ascending phase of the contractile off-response; 4) papaverine accelerated the discending phase of the contractile off-response. The data obtained suggest: 1) there are chemoexcitable calcium channels in the cellular membrane which are blocked by papaverin; 2) there are calcium "leakage" channels in the cellular membrane responsible for the tone maintenance which are blocked by papaverine; 3) papaverine has a negligible effect on the electroexcitable calcium channels.     

Sodium and ouabain induce proliferation of rat thymic lymphocytes via calcium- and magnesium- dependent reactions

When the concentrations of either calcium or of magnesium in the culture medium were increased from the normal 0.6 and 1.0 mM to 1.8 and 2.5 mM respectively mitotic activity of rat thymic lymphocytes increased. Very high (10(-4)M) ouabain concentrations abolished these mitogenic actions whilst lower (10(-7) and 10(-11)M) concentrations had no effect. However in the normal medium these lower concentrations of ouabain were themselves mitogenic. The stimulatory effect of 10(-7)M ouabain was calcium-dependent and oestradiol-blockable and that of 10(-11)M magnesium-dependent and testosterone-blockable. A 10 mM increment in extracellular sodium concentration also stimulated mitosis in these cells in a calcium-dependent manner whilst a 20 mM increment required the presence of magnesium to exert its mitogenic effect. However, when similar osmotic increments were provided by potassium and lithium salts, or sucrose no mitotic stimulation was provoked. Subtle interactions between sodium and the divalent cations are clearly involved in events which lead to mitosis and the steroids oestradiol and testosterone can somehow block these effects.     

Relation of Ca2+ efflux from the heart to the concentration of Ca-channel blockers

Dependence of calcium ion efflux from the heart ventricles on the concentration of verapamil, fenigidine and nicardipine was studied in frogs, using Ca-sensitive electrodes. The effect of sodium and potassium ions was also investigated. It was established that dependence of calcium ion efflux (delta Ca2+) on the concentration of Ca-antagonists (B) may be expressed by the following formula: (Formula: see text). With cellular membrane depolarization 50 mM KCl, none of the blockers (10(-5) M) caused Ca2+ efflux from ventricular cells. Analogous phenomenon was noted in low-sodium solution (60 mM).     

The effect of calcium on the antioxidant systems in the halophyte <Emphasis Type="Italic">Cakile maritima</Emphasis> under salt stress

The purpose of the current investigation was to study the effect of Ca²⁺ (0, 3.5 and 20 mM concentrations) on the antioxidant systems in the halophyte Cakile maritima under NaCl stress (0, 100, 200 and 400 mM NaCl). Plants treated with both moderate calcium (3.5 mM) and salt levels (100 mM) showed the maximum growth, and the addition of 20 mM calcium to the nutrient media did not significantly reduce the growth under the moderate salt treatment. The absence of calcium associated with high salt concentration induced a strong reduction of biomass production. The tolerance of C. maritima at moderate salinity and calcium was related with the lowest values of the parameters indicative of oxidative stress (malondialdehyde, electrolyte leakage and hydrogen peroxide concentration). This was accompanied with a higher peroxidase, superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities. In contrast, in the absence of calcium, those enzymes showed the lowest activities under all salt treatments. As a whole, it can be noticed that salt tolerance was improved by moderate calcium concentration; however, the absence of calcium has a drastic effect on C. maritima.     

Strophanthidin inotropy in cardiac Purkinje fibers under conditions that vary cellular sodium or calcium

The role of sodium and calcium on strophanthidin inotropy was studied in canine cardiac Purkinje fibers perfused in vitro under conditions that vary cellular sodium and calcium. With high concentrations of strophanthidin (greater than or equal to 10(-7) M), force increases more in the presence of low [Ca]0 or high [Na]0 and less in the presence of a low sodium-calcium concentration solution than in Tyrode solution. In a solution with a low concentration of sodium-calcium containing strophanthidin, restoring [Na]0 to normal decreases and then re-increases force: when [Na]0 is decreased again, the force transiently overshoots. These effects of strophanthidin are exaggerated by metabolic inhibitors. In a low [Ca] solution, low concentrations of strophanthidin (3 X 10(-8) or 5 X 10(-8) M) re-increase force a little or not at all. On recovery, the transient force increase is not exaggerated by low strophanthidin and is absent after manganese exposure. The inotropy of low concentrations of strophanthidin is potentiated by norepinephrine, high [Ca]0 (4 mM), or by lowering [Na]0. Thus, the present results suggest that the inotropic action of high strophanthidin concentrations depends primarily on sodium and secondarily on calcium, and that the inotropic action of low concentrations of strophanthidin involves a modification of the cell response to calcium.     

Three correlations between extracellular calcium concentration and myocardial cell injury induced by drugs

Primary cultures of newborn rat myocardial cells were treated in various extracellular calcium concentrations (0, 1.35, 2.7, 4.05, and 5.4 mM) with three different drugs; namely, ouabain, sulmazole, and chlorpromazine. Lactate dehydrogenase (LDH) release was used as an indicator of damage. The results showed 10(-3) M ouabain caused apparent damage of the cells and the damage was increased by an increased extracellular calcium concentration. Sulmazole (10(-3)M) caused damage of the cells in the absence of calcium; but it did not cause damage of the cells in the presence of calcium; it protected the cells from damage caused by high calcium concentrations (4.05 and 5.4 mM). Chlorpromazine (1.6 X 10(-4)M) caused severe damage of the cells. The various calcium concentrations had no influence on the degree of the damage. Correlation coefficients showed that correlations between the calcium concentrations and the cell damage caused by ouabain, sulmazole and chlorpromazine were positive correlation, negative correlation, and no correlation, respectively. It is suggested that influx of extracellular calcium is not a final common pathway of drug-induced myocardial cell injury, although it plays an important role in cell injury.     

In vivo 23Na nuclear magnetic resonance study of maintenance of a sodium gradient in the ruminal bacterium Fibrobacter succinogenes S85

Sodium gradients (DeltapNa) were measured in resting cells of Fibrobacter succinogenes by in vivo 23Na nuclear magnetic resonance using Tm(DOTP)5- [thulium(III) 1,4,7,10-tetraazacyclododecane-N',N",N"'-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a DeltapNa of -55 to -40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na+ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).     

Positive inotropic effect of Murraya koenigii (Linn.) Spreng extract on an isolated perfused frog heart

Ethanolic extract of fresh leaves of M. koenigii (MKEE) showed a dose dependent positive inotropic effect on isolated frog heart. The responses to MKEE (62.5-1000 microg) were not affected in either way by theophylline, imidazole, propranolol and sildenafil. The change in potassium and sodium concentration did not alter MKEE-induced positive inotropic effect. Lignocaine did not alter the responses to MKEE significantly. Responses to MKEE were significantly inhibited when calcium concentration was reduced to half (from 1.58 to 0.79 mM) and were significantly potentiated when calcium concentration was doubled (from 1.58 to 3.16 mM). Verapamil was found to inhibit the responses significantly. The results suggest that M. koenigii induced positive inotropic effect possibly by increasing availability of calcium from extra cellular sites.     

Calcium mediates the light-induced decrease in maintained K+ current in Limulus ventral photoreceptors

In addition to increasing the conductance to sodium, light reduces the maintained voltage-dependent potassium current (iK) in Limulus ventral photoreceptors. We have investigated the mechanism underlying this long-lasting decrease in ik. Intracellular injection of calcium produced a similar reduction of the voltage-dependent outward current. This reduction was not due to an activation of the voltage-dependent inward current (iin) because calcium injection reduced the outward current even under conditions where iin was blocked with Ni2+, and because calcium injection produced a decrease in conductance, as measured from the slope of the instantaneous i-V curve. The effect of light on ik could be blocked by injection of the calcium buffer EGTA (pCa 7.1) to an intracellular concentration of 50-70 mM. Even larger injections of the pH buffer MOPS (100-200 mM) did not reduce the effect of light on ik. These experiments show that intracellular free calcium (Cai2+) can reduce ik. Furthermore, since Cai2+ is known to increase in light, our results are consistent with the hypothesis that calcium is the internal transmitter for the light-induced decrease in ik.