Effect of the ion composition of the calcium-free medium and cardiomyocyte damage during "calcium paradox" in rats]

The findings reveal that the degree of myocardial damage in the "calcium paradox" does not depend on the contracture strength, that the contracture attenuation due to a decreased concentration in the Ca-free medium is not equal to the cardiocytes protection as compared with other means. Mg2+ and the studied means of myocardial protection seem to play a major role in assessment of the "calcium paradox" development and explain the difference in results of the hyposodium medium effects in 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.     

Some mechanisms of adenosine protective effect in the "calcium paradox"

A possibility of preventing the "calcium paradox" with the aid of adenosine was studied as well as some mechanisms of adenosine effect upon the heart in case of the "calcium paradox". Adenosine was found to suppress release of amino acids from the heart in perfusion with calcium-free medium, to efficiently prevent disorders in the energy-dependent functions of mitochondrion and myoglobin release from the heart in reperfusion with Ca2+ -containing solution. Adenosine was also found to increase 2-10-fold lactate release from the heart. Adenosine seems to be able to activate glycolysis. Iodine acetate was shown to completely suppress the adenosine ability to decrease amino acid release from the heart perfused with calcium-free medium. Under conditions of iodine acetate blocking of glycolysis was found to possess no protective properties against cytolysis in the "calcium paradox". The heart mitochondria isolated in the end of the experiment revealed low values of free or phosphorylating respiration and complete dissociation of oxidation. Also a protective effect of adenosine in inhibition of Na+, K+ -ATPhase with Strophantinum, was studied.     

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.     

Significance of extracellular concentration of sodium ions in the protective effect during the "calcium paradox"]

Increasing of extracellular sodium concentration up to 200 mM diminishes heart damage under "calcium paradox". Phosphocreatine (10(-4) M) potentiates the effect of high sodium perfusion media; in this case myoglobin release from the myocardium is minimal (5-9% of control). An the same time, ATP and phosphocreatine concentrations and oxidation to phosphorylation coupling in mitochondria remain at a sufficiently high level. Elevation of osmotic pressure by the effect of 120 mM sucrose enhances heart damage under "calcium paradox" both in the presence and absence of phosphocreatine. The protective effects of superhigh (200 mM) sodium concentrations and phosphocreatine are completely reversed by strophanthin or decreasing K+ concentration down to 0.5 mM.     

In vitro fertilization in inbred BALB/c mice II: effects of lactate, osmolarity and calcium on in vitro capacitation

To elucidate requirements for in vitro sperm capacitation in inbred BALB/c mice, osmolarity, calcium and lactate were optimized using modified simplex optimization medium (mKSOM). Modified human tubal fluid (mHTF), a capacitation-supporting medium, was used as a control. In the first series of experiments, the effects of calcium and osmolarity were studied in the presence of lactate. Although preincubation with >or=5 mM CaCl2 improved fertilization after insemination significantly, it was still significantly lower than incubation with mHTF. To obtain fertilization at the equivalent levels to that of mHTF, isotonic osmolarity (305 mOsmol) was required. Trehalose, an osmotic reagent, could substitute for NaCl partially. In the second series of experiments, the effects of lactate were examined using a concentration of 5 mM calcium and isotonic osmolarity. Preincubation with 75%), as well as the percentages of B (capacitated) pattern sperm (>or=40%) in chlortetracycline (CTC) staining, as compared with incubation in mHTF (46% and 28%, respectively; p<0.05). In the third series of experiments, the effects of osmolarity and calcium in the absence of lactate were examined. An increase in osmolarity during sperm preincubation increased both fertilization and B-pattern sperm significantly in a dose-dependent manner. Trehalose, sucrose and choline chloride could substitute for NaCl. An increase in CaCl2 concentration during preincubation had no effect on fertilization, but this increase reduced the percentages of B-pattern sperm. In vitro capacitation of inbred BALB/c mice is sensitive to lactate and osmolarity, but that sensitivity for calcium varies depending on the presence or absence of lactate.     

Effects of ouabain on calcium paradox in rat hearts

The effects of ouabain (10(-7) to 10(-5) M) on the interrelationship between cell-cell contacts, resting tension, and creatine phosphokinase (CK) leakage owing to myocardial cell injury during Ca2+ paradox were studied in isolated perfused rat heart preparations. After perfusing for 15 min with Ca2+ -containing medium, hearts were perfused for 5 min with Ca2+ -free medium followed by a reperfusion with Ca2+ -containing medium for 5 min. This resulted in a transient increase in resting tension and a substantial release of CK into the perfusate during the calcium reperfusion period. These changes were accompanied by extensive structural damage in the myocardial cell, including formation of contraction bands, swelling of the mitochondria, and cell-cell separation. Inclusion of 10(-5) M ouabain for 5 min in the Ca2+ -containing perfusion medium prior to the start of Ca2+ -free perfusion resulted in a higher and sustained resting tension that was accompanied by a reduced loss of CK from the heart during Ca2+ reperfusion. In a histological examination of these ouabain exposed hearts, most of the structural changes owing to calcium paradox were apparent, but the cell-cell contacts were maintained. The results are consistent with the hypothesis that the loss of cell-cell contacts in the intercalated disc during the occurrence of Ca2+ paradox may be the cause of the delayed decline in the resting tension and is only partially responsible for the loss of CK. These differences in myocardial changes during Ca2+ paradox with or without ouabain may be due to the retention of calcium at certain crucial sites under the influence of ouabain.     

Alterations in cardiac lysosomal hydrolases following induction of the calcium paradox

Although perfusion of the heart with calcium-free medium for a brief period followed by reperfusion with calcium-containing medium results in marked structural derangements (calcium paradox), the mechanisms for this cell damage are far from clear. Since activation of lysosomal enzymes has been associated with pathological damage, it was the purpose of this study to examine alterations in the activities of several lysosomal enzymes in rat hearts subjected to calcium paradox. No significant changes in the activities of beta-acetylglucosaminidase, beta-galactosidase, alpha-mannosidase, or acid phosphatase were seen in the homogenates of hearts exposed to the calcium paradox. However, there were dramatic alterations in the lysosomal enzyme activities in the sedimentable and nonsedimentable fractions during calcium paradox. The lysosomal enzyme activities were also detected in the perfusate collected during reperfusion with calcium-containing medium. These changes occurred during the reperfusion period since no alterations were apparent after calcium-free perfusion and were dependent upon the time of reperfusion with medium containing Ca2+ as well as the time of perfusion with Ca2+ -free medium before inducing Ca2+ paradox. These data indicate that alterations in lysosomal enzymes owing to reinstitution of calcium in Ca2+-deprived hearts may occur as a part of cardiac damage and general cellular disintegration.     

In vitro fertilization in inbred BALB/c mice I: isotonic osmolarity and increased calcium-enhanced sperm penetration through the zona pellucida and male pronuclear formation

To optimize IVF conditions for BALB/c mice, which are known to have poor in vitro fertilizability, the requirements for sperm-ova interaction were studied by use of modified simplex optimization medium (mKSOM) as a basic medium. Modified human tubal fluid (mHTF) was used for sperm preincubation and acted as a positive control. When the two media were compared, neither capacitation nor fertilization was supported in mKSOM. Increasing the calcium concentration in mKSOM to 5 mM or more during sperm: ova coincubation improved zona penetration but not male pronuclear (MPN) formation to the same level as those cells incubated in mHTF. When medium osmolarity was varied from 230-305 mOsmol by NaCl at 5 mM CaCl2, MPN formation improved at 280 mOsmol or higher osmolarity to the same level as that found when using mHTF. When NaCl equivalent to 25-75 mOsmol was substituted with trehalose, no significant reduction in fertilization was observed. Substitution of NaCl equivalent to 75 mOsmol with other osmotic reagents (sucrose, choline chloride and sorbitol) resulted in similar levels of fertilization as found with mHTF, except for sorbitol, which reduced fertilization significantly caused by its detrimental effect on sperm viability. At isotonic osmolarity (305 mOsmol), maximum fertilization was observed at 5 mM CaCl2; lower or higher concentrations of CaCl2 resulted in reduced fertilization. Calcium and osmolarity, therefore, are important for sperm : ova interaction in BALB/c mice and the increases in calcium to 5 mM and osmolarity to 305 mOsmol are optimal for BALB/c sperm to penetrate through the zona and to form MPN.     

Calcium regulates the mode of exocytosis induced by hypotonic shock in isolated neuronal presynaptic endings

A decrease in the osmolarity of incubation medium is accompanied by calcium influx in neuronal presynaptic endings. We studied the influence of Ca2+ on exocytosis induced by hypotonic shock using the hydrophilic fluorescent dye acridine orange and the hydrophobic fluorescent dye FM2-10. It was shown using acridine orange that lowering of osmolarity to 230 mOsm/l induces exocytosis both in calcium-containing and calcium-free medium. By contrast, we were able to demonstrate calcium-dependence of exocytosis using styryl dye FM2-10. Lowering of osmolarity leads to increase of [3H]D-aspartate and [3H]GABA release in calcium-free medium. Addition of calcium inhibits hypotonic-induced neurotransmitter release. Decreasing of NaCl concentration to 92 mM in isotonic medium is able to induce d-aspartate and GABA release. Thus, our data suggest that hypotonic swelling induces calcium-independent exocytosis possibly by a "kiss and run" mechanism. Calcium influx mediated by stretch channels is able to provoke full fusion between plasma membrane and synaptic vesicles. [3H]D-aspartate and [3H]GABA released by hypotonic shock is determined by sodium lowering rather than by osmolarity decreasing itself.     

Protective effects of manganese, cobalt, nickel, and barium against a calcium paradox in the isolated frog heart

The effect of inorganic slow channel blockers on the calcium paradox in the frog heart was examined. Addition of the divalent cations of manganese, cobalt, nickel, or barium during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that in the control paradox in the absence of divalent cations, there is an efflux of calcium from myocardial cells during calcium depletion and a massive influx of calcium during the following reperfusion, leading to a calcium overload. Divalent cations protected frog myocardial cells, when present in the calcium-free perfusion medium, by reducing both calcium efflux during calcium depletion and the massive calcium influx during reperfusion. The effectiveness of the added divalent cations showed a strong dependence upon their ionic radius. The most potent inhibitors of the calcium paradox in the frog heart were the divalent cations having an ionic radius closer to the ionic radius of calcium. These results are discussed in terms of the possible mechanism involved in the protective effect of manganese, cobalt, nickel, and barium.     

Taurine's possible protective role in age-dependent response to calcium paradox

When hearts were reperfused with Ca++ after a short period of Ca++-free perfusion, irreversible loss of electrical and mechanical activity was observed. This phenomenon, first described by Zimmerman and Hulsmann, was termed the "calcium paradox". Chizzonite and Zak recently reported that rat hearts exhibited an age-dependent response in a calcium paradox model. The taurine (2-aminoethanesulfonic acid) content of hearts in the newborn animal is high, and decreases rapidly during the first few days of life. The present experiments were performed to test whether the myocardial taurine content was closely linked to an age-dependent response in the calcium paradox model, using post-hatched chicks. The mechanical dysfunction of the heart was much more severe in 9-day-old post-hatched chicks than in 2-day-old chicks when the hearts were subjected to the calcium paradox. Myocardial taurine content was lower in the 9-day-old chicks than in the 2-day-old chicks. The age-related response to the calcium paradox was partially protected by oral pretreatment with taurine, and there was a small increase in myocardial taurine level. It is proposed that myocardial taurine is one factor in the protection against the calcium paradox phenomenon.     

Effects of changes in osmolarity on isolated human airways

The effects of hypo- and hyperosmolarity on the function of isolated human airways were studied. Changes in osmolarity induced an increasing bronchoconstriction that was proportional to the magnitude of the change in osmolarity. Hypertonicity-induced airway narrowing resulted when buffer was made hypertonic with sodium chloride or mannitol but not with urea. The airways showed no tachyphylaxis to repetitive exposure to hypo- and hypertonic buffer of 200 and 600 mosM, respectively. The bronchoconstriction was not secondary to stimulation of H1 or leukotriene C4/D4 receptors or the release of prostaglandins in the preparation. The bronchoconstriction in hypotonic buffer was totally dependent on extracellular calcium, whereas in hypertonic buffer the bronchoconstriction seemed partially dependent on intracellular calcium release. Isoprenaline prevented the bronchoconstriction in hyper- or hypotonic buffer of 450 and 250 mosM but not in buffer of 600 and 150 mosM. It is concluded that hypo- and hypertonic buffers lead to bronchoconstriction via different mechanisms, which relate to influx of extracellular calcium in hyposmolar buffer and probably to release of calcium from intracellular stores in hypertonic buffer. In strongly hypertonic buffer, part of the bronchoconstriction may be due to osmotic shrinkage. The relevance of our data for the mechanism of bronchoconstriction after inhalation of hypo- or hypertonic saline depends on whether changes in osmolarity around the airway smooth muscle occur in asthmatics but not in normal subjects, and this has not yet been established.     

Possible mechanisms involved in the development of the calcium paradox

Reperfusion of an isolated heart with calcium-containing solution after a short period of calcium-free perfusion may result in excessive influx of calcium into the cells and irreversible cell damage (calcium paradox). This paper describes the possible routes of calcium entry that occurs during the phase of calcium repletion, and the possible mechanisms involved in the development of the calcium paradox damage. The routes of calcium entry include the glycocalyx, the slow channels, the Na+-Ca2+ exchange mechanism, passive diffusion, and abnormal sites of calcium entry. In addition to an increased influx of calcium, a loss in the ability of the sarcolemma to remove calcium from the cells may contribute to the net gain of tissue calcium. The calcium paradox damage itself, which follows the massive influx of calcium into the myocardial cells, may be a result of calcium-triggered energy dependent reactions and a concomitant acidification of the cytoplasm. Mechanical factors may also be involved in the development of the calcium paradox.     

Ultrastructural alterations of the mitochondrial ATPase in the calcium paradox as revealed by negative staining

We have used a simple negative staining technique to study the structural alterations of mitochondria from biopsies of hearts subjected to the calcium paradox and treatment with diltiazem, a calcium channel blocker. A significant (P less than 0.05) decrease in the number of spheres on the mitochondrial membranes occurs during the calcium paradox (58.0 +/0 4.1/micrometer vs. control 80.5 +/- 6.5). Treatment with diltiazem prevented the loss of spheres from mitochondrial membranes during the calcium paradox (75.5 +20 5.0 micrometer). We found that this negative staining technique can be used for quick assessment of the condition of mitochondria in biopsies from normal and pathological organs.     

Effect of osmolarity on potassium transport in isolated cerebral microvessels

Potassium transport in microvessels isolated from rat brain by a technique involving density gradient centrifugation was studied in HEPES buffer solutions of varying osmolarity from 200 to 420 mosmols, containing different concentration of sodium chloride, choline chloride, or sodium nitrate. The flux of 86Rb (as a tracer for K) into and out of the endothelial cells was estimated. Potassium influx was very sensitive to the osmolarity of the medium. Ouabain-insensitive K-component was reduced in hypotonic medium and was increased in medium made hypertonic with sodium chloride or mannitol. Choline chloride replacement caused a large reduction in K influx. Potassium influx was significant decrease when nitrate is substituted for chloride ion in isotonic and hypertonic media, whereas a slight decrease was found in hypotonic medium. The decrease of K influx in the ion-replacement medium is due to a decrement of the ouabain-insensitive component. Potassium efflux was unchanged in hypotonic medium but was somewhat reduced in hypertonic medium. The marked effect of medium osmolarity on K fluxes suggests that these fluxes may be responsible for the volume regulatory K movements. The possible mechanism of changes of K flux under anisotonic media is also discussed.     

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.     

Changes in blood osmolarity,electrolytes, and metabolites among adult rats treated with a neurotoxic dose of MSG1

Adult rats were given large doses of MSG (4 g/kg) or isosmolar amounts of sodium chloride or L-alanine intraperitoneally or by forced intubation. Blood or plasma samples from these rats where assayed for osmolarity, hematocrit, pH, and concentrations of protein, sodium, potassium, chloride, calcium, magnesium, and urea nitrogen. Intraperitoneal MSG produced characteristic hypothalamic lesions; MSG by gavage failed to do so. Intraperitoneal MSG also caused major increases in plasma osmolarity, hemoconcentration, hypovolemia, alkalosis, hypernatremia, and uremia; plasma levels of chloride and potassium fell significantly. Administration of MSG by gavage caused much smaller changes in plasma osmolarity and sodium, and no significant changes in hematocrit, plasma protein or plasma urea nitrogen. Administration of sodium chloride or L-alanine (agents not known to produce the characteristics MSG brain lesions) caused some, but not all, of the metabolic changes seen after MSG. These observations suppot the hypothesis that the ability of large, concentrated doses of MSG to produce brain lesions in susceptible species involves a two-step process, i.e., initial damage to the blood-brain barrier for glutamate, followed by entry of the circulating amino acid into the extracellular space of the brain.     

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 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.