Peter J. Bailey

The effect of extracellular calcium concentration and Ca-antagonist drugs on enzyme release and lactate production by anoxic heart cell cultures

Abstract Tissue cultures of neonate rat heart cells were subjected to anoxia for 16 h in the presence of 5 m m glucose and increasing concentrations of calcium in the incubation medium. When the calcium content of the culture medium was increased, release of intracellular enzyme was observed. Increased extracellular calcium concentration appeared to have no effect on normoxic tissue preparations. The time course of this anoxia-related change was examined. Increasing the calcium concentration of the medium caused an acceleration of the anoxia-induced rate of enzyme leakage when compared with anoxic cultures exposed to a low medium calicum concentration. The calcium-induced increase in anoxic enzyme release could be partially reversed by various concentrations of the calcium antagonist drugs verapamil and nifedipine. Verapamil and nifedipine both appear to reduce the extent to which an elevated medium calcium concentration is able to reduce myocyte ATP content under anoxic conditions. These drugs appear to be without effect on ATP content of anoxic cells exposed to a low calcium concentration on the medium. Calcium-induced increases in enzyme leakage cannot be modified by the provision of high medium glucose concentrations, and hypertonic solutions of mannitol exacerbate damage to myocytes under anoxic and hypercalcaemic conditions. These data suggest that high extracellular concentrations of calcium exacerbate damage to heart cells subjected to anoxia although the mechanism underlying this exacerbation is uncertain.

The relationship between glycolysis, fatty acid metabolism and membrane integrity in neonatal myocytes

Abstract The glycolytic rate of cultured neonatal rat cardiac cells was manipulated by varying the concentration of glucose, l -lactate and 2-deoxyglucose supplementing the normal growth medium under anoxic and normoxic conditions. The leakage of lactate dehydrogenase from the cells was found to show good inverse correlation with glycolytic rate under both normoxic and anoxic incubation conditions. Enzyme release correlated inversely with intracellular ATP concentrations in anoxia but not in normoxia. Treatment of cultures with iodoacetate caused enzyme leakage but interference with mitochondrial energy production using cyanide or 2,4-dinitrophenol was without effect on enzyme release. Lactate inhibited fatty acid oxidation in a concentration-dependent manner, but 2-deoxyglucose was without effect. Treatment of cultures with lactate or 2-deoxyglucose caused an increase and a decrease in intracellular fatty acid accumulation, respectively. Elevation of the medium glucose concentration promoted free fatty acid accumulation. Although lactate and 2-deoxyglucose influence fatty acid metabolism by the cardiac cell in different ways they both cause enzyme release, an effect which correlates well with their inhibition of glycolysis even under normoxic conditions.

Interrelationship between cellular metabolic status and susceptibility of heart cells to attack by phospholipase

Abstract Neonatal heart cell cultures incubated in the presence of phospholipase C released enzyme into the extracellular fluid. In the presence of varied medium glucose concentration and varied concentrations of l -lactate or 2-deoxyglucose it was observed that the extent to which this phenomenon occurred was related to both the ATP content of the cultures and their glycolytic activity. The susceptibility of the cells to attack by phospholipase C was markedly enhanced by the metabolic inhibitor iodoacetate but to a much lesser extent by an inhibitor or an uncoupler of mitochondrial function. The data suggest that cellular energy status may determine the resistance of the cell membrane to attack by phospholipases perhaps by causing some organizational rearrangement. The data also lend some support to the idea that ATP derived from glycolytic sources may be preferentially used for membrane maintenance.

The effects of cyanide and iodoacetate intoxication and ischaemia on enzyme release from the perfused rat heart.

Isolated rat hearts perfused in the presence of iodoacetate show inhibition of glycolysis and release enzymes into the perfusate. Hearts perfused with cyanide, a mitochondrial inhibitor, show acceleration of glycolysis and no enzyme release. The adenine nucleotide content of the iodoacetate, but not the cyanide-perfused hearts was reduced. These results indicate that the membranes were permeable in the former treatment group. The adenylate energy charge and the ATP content of both the cyanide and iodoacetate treatment groups were similar but, as the extent of enzyme release was quite different, it appears that the energy state of the cell was not the prime factor controlling membrane integrity. Isolated perfused hearts were rendered ischaemic by placing a one-way ball valve in the aortic outflow tract. ATP concentration declined, as did ADP after an initial rise of short duration. AMP concentrations rose as the time of ischaemia increased. At the time at which enzyme release was first determined, the intracellular total adenine nucleotide content began to decline, suggesting that the membrane had become permeable to both small and large molecules. Glycolysis was stimulated by the hypoxia induced in the preparation and then this increase became inhibited. The point at which this inhibition was observed was also the point at which membrane permeability was evident. Taken together, the data from these experiments suggest that the energy derived from the activity of the glycolytic pathway may be important to the heart for maintenance of membrane function, particularly in ischaemia.

The effect of β-adrenergic blocking drugs on the intrinsic beating rate of cultured rat myocytes

Cultured myocytes from neonate rats were subject to increasing doses of dl-, d- or l-propranolol or dl-atenolol, and the intrinsic heart rates were measured. Both propranolol isomers and racemate stopped beating activity at very high concentrations (10−3 m) while atenolol did not. The results are interpreted to indicate that constant β-adrenoceptor stimulation is not an obligatory requirement for maintenance of spontaneous beating in this system as has been suggested.

Mechanism of stimulation of cardiac myocyte beating rate by 5-hydroxytryptamine

Abstract The beating rate of cardiac myocyte cultures prepared from neonate rat ventricles increases in response to increasing concentrations of 5-hydroxytryptamine (5HT). Several methyl and methoxy derivatives of 5HT show stimulant properties to varying degrees, and the 5HT stimulated response is attenuated in the presence of various antagonists of 5HT and also by L-propranolol although DL-atenolol is without effect. The α-adrenergic blockers phentolamine and thymoxamine are able to antagonize the action of 5HT but only at high concentrations. The chronotropic activity of 5HT is not accompanied by increases in intracellular cyclic AMP (cAMP). The data are consistent with the idea that, in rat cardiac tissue, 5HT stimulates a specific receptor at a post synaptic site and does not owe its stimulatory properties solely to presynaptic stimulation of catecholamine release.

Cultured neonate rat myocytes as a model for the study of myocardial ischaemic necrosis

The preparation of cultures of neonate rat heart muscle cells is described. These cultures, when subjected to anoxia, show enzyme release that can be directly related to the uptake of a vital dye such as trypan blue. Enzyme release is a valid method of estimating cell necrosis in this model. The survival of anoxic cultures is closely associated with glycolytic activity. Glycolysis rate falls and enzyme release increases as the medium glucose concentration is reduced. If glycolysis is inhibited by either 2‐deoxyglucose or L‐lactate, enzyme release under anoxic conditions is enhanced. Enzyme release correlates inversely with glycolytic activity and the intracellular ATP content of the cultures. Addition of ATP to anoxic cultures partially ameliorates the effect of the anoxia on enzyme release. Elevation of the calcium content of the culture medium exacerbates the damage caused to cardiac myocytes by anoxic insult. This effect can be obtunded by calcium‐antagonist drugs such as verapamil or nifedipine and can be explained in terms of a reduction in utilization of intracellular ATP by the anoxic myocytes. These observations indicate that cultured myocytes may represent a useful model of hypoxic injury against which novel pharmacological agents, that may reduce hypoxic or ischaemic injury in vivo, could be evaluated.

Mechanisms of stimulation of rat cardiac muscle by 5-hydroxytryptamine

Abstract Administration of 5-hydroxytryptamine to isolated perfused working rat heart preparations caused an increase in contractile activity. The increase in activity due to low concentrations of 5-hydroxytryptamine (10 −5 M) could be blocked by either methysergide or propanolol but not by atenolol. At high concentrations of 5-hydroxytryptamine (10 −3 M) no increase in tissue cyclic AMP was found but a significant rise in the value of the cyclic AMP-dependent protein kinase activation ratio was observed, suggesting a slight increase in intracellular cyclic AMP had occurred. It was also observed that while low concentrations of 5-hydroxytryptamine (up to 10 −5 M) did not provoke catecholamine release from the intra-cardiac stores higher concentrations (10 −4 M) did. It is suggested that low concentrations of 5-hydroxytryptamine have a direct action on cardiac muscle but that at high concentrations a direct action cannot be separated from an action by which catecholamines are released from intra-cardiac stores.

The effects of incubation conditions on enzyme release from anoxic heart cell cultures

Abstract Cultured myocytes from neonate rats were subjected to varying concentrations of glucose and serum under normoxic and anoxic conditions. Cellular metabolism was measured in terms of glucose utilization and lactate production. Leakage of cytoplasmic lactate dehydrogenase (LDH) into the medium was measured to give an indication of cell injury. The effect of variations in temperature on these parameters was evaluated. Myocytes cultured under anoxic conditions for 16 h utilized more glucose than normoxic cells with a concomitant increase in lactate production. When the initial glucose concentration in the medium was below 3 m m , anoxic myocytes released large amounts of LDH. Also, at low initial glucose concentrations there was a surge of anoxic LDH release between 12 and 15 h of incubation. The addition of exogeneous lactate to anoxic cultures had no influence on enzyme leakage. However, reduced enzyme leakage was observed when medium lactate was increased under normoxic conditions. An increased concentration of serum in the medium stimulated glucose metabolism in both normoxic and anoxic myocytes. Despite this enhancement of glycolysis, anoxic cells released significant amounts of LDH into the medium. LDH release by anoxic myocytes is minimal in medium containing 1 to 2% v/v serum. Maximum glucose metabolism and lactate production at 5 m m glucose occurred between 37 to 40°C. The relation between LDH release and temperature was not marked. Significant release of LDH did occur in anoxic cultures incubated between 37°C and 40°C when compared with normoxic cultures at the same temperature. At 4°C, in the absence of glucose metabolism, LDH release occurred from both normoxic and anoxic cells. These data demonstrate that significant cellular enzyme release occurs during anoxia: (a) below a threshold glucose concentration and (b) in proportion to the serum content of the medium.

The effects of endogenous catecholamine and pure α- and β-adrenoceptor stimulation on myocardial glycogenolysis, lactate production and distribution

Abstract The stimulation of glycogenolysis and lactate production and distribution by endogenous catecholamines released from the perfused rat heart by high K + solutions in the presence and absence of an α- or β-adrenergic blocker has been assessed. In addition, the effects of pure α- (noradrenaline + atenolol) and pure β (isoprenaline + thymoxamine) adrenoceptor stimulation have been examined. The results suggest that while glycogenolysis is under the control of the β-adrenoceptor, lactate accumulation in the tissue and perfusate is probably under both α- and β-adrenoceptor control. The distribution of lactate between intracellular and extracellular compartments appear to be governed by β-adrenoceptors in the coronary vessels or possibly by β-receptor induced activation of a permease on the cell membrane of the myocardial cell. Of these two suggestions the former seems the most likely.