Damaging effects of the calcium paradox are reduced in isolated hearts from ethanol-dependent rats: paradoxic effects of dihydropyridine drugs.

Previous experiments showed that isolated hearts from ethanol-exposed rats show a marked increase in sensitivity to anoxic myocardial damage, and we suggested that this may be due to excess calcium entry through L-type voltage-operated calcium channels (L-VOCCs). To challenge this hypothesis, we investigated the effect of ethanol treatment ex vivo on a damaging stimulus, the "calcium paradox," which is associated with removal of calcium from the perfusate. Adult male Sprague-Dawley rats were exposed to intoxicating concentrations of ethanol for 6-10 days by inhalation. Isolated hearts from these animals were perfused with Krebs-Henseleit buffer by using a modified Langendorff technique, and the calcium paradox induced by a 10-min period of perfusion with calcium-free buffer, followed by reperfusion with calcium-containing buffer. Compared with controls, hearts from ethanol-exposed rats were significantly protected against myocardial damage, as shown by a marked reduction in release of intracellular proteins (lactate dehydrogenase, creatine phosphokinase, and myoglobin) during the reperfusion phase. These indices of myocardial damage were modified by the presence of the dihydropyridine (DHP) calcium channel antagonist nitrendipine (10(-6) M) and the DHP L-VOCC activator Bay K 8644 (10(-7) M) in the perfusate during the calcium paradox. Paradoxically, both drugs appeared to increase the damaging effects of calcium-free perfusion, with this effect being generally greater in the preparations from ethanol-exposed rats. As a result, the difference between these hearts and those from control rats was reduced, although a significant degree of protection against the calcium paradox remained. The results support the hypothesis that long-term exposure to ethanol in vivo produces marked alterations in the toxic effects of changes in myocardial calcium concentration. The increased sensitivity to DHP drugs of isolated hearts from ethanol-treated rats is consistent with previous experiments showing increased DHP radioligand-binding sites in these tissues.

Increased sensitivity to damaging effects of hypoxia and anoxia of isolated hearts from rats after prolonged exposure to ethanol: apparent protection by nitrendipine.

The studies described here investigate whether pathologic states that are thought to cause myocardial damage through excess calcium entry (i.e., hypoxia and anoxia) indeed cause greater damage in hearts from ethanol-exposed animals, and whether L-type voltage-operated calcium channels (L-VOCCs) are implicated. Adult male Sprague-Dawley rats were exposed to intoxicating concentrations of ethanol vapor for 6-10 days, and their isolated hearts compared with those of control animals in a Langendorff perfusion system. Hypoxia was induced by perfusion with Krebs-Henseleit buffer, which had not previously been bubbled with oxygen; anoxia was produced by perfusion with buffer bubbled with nitrogen. On reperfusion with oxygenated buffer, evidence of myocardial damage during the hypoxic/anoxic period was obtained by the release of intracellular proteins into the perfusate. After hypoxia, release of myoglobin (MYO) was significantly greater from hearts from ethanol-exposed rats than from controls; other indices of myocardial damage also were increased by hypoxia but did not differ significantly between treatment groups. After anoxic perfusion, release of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) as well as MYO were all markedly and significantly increased from ethanol-exposed hearts compared with those from control rats. The role of L-VOCCs in this damage was assessed with the calcium channel antagonist nitrendipine (10(-6) M) present in the perfusing buffer immediately before and during the anoxic stimulus. This completely reversed the situation so that preparations from ethanol-exposed rats now showed a reduced release of intracellular proteins compared with hearts from controls. Comparisons with absolute values from the previous experiments suggest that nitrendipine increased release of LDH and CPK from control hearts with little effect on these indices from ethanol-exposed hearts. However, in the case of anoxia-induced MYO release, nitrendipine markedly and significantly reduced this in hearts from ethanol-treated rats but had only a very small effect on the same parameter in controls. The results strongly suggest increased pathologic effects of hypoxia/anoxia in hearts from ethanol-exposed rats. This increased sensitivity may be at least partly a consequence of increased numbers of L-VOCCs in this tissue.

Effect of calcium, Bay K 8644, and reduced perfusion on basic indices of myocardial function in isolated hearts from rats after prolonged exposure to ethanol.

We previously reported findings consistent with a marked upregulation in functional L-type voltage-operated calcium channels (L-VOCCs) in hearts obtained from rats exposed over the long term to ethanol. These experiments were undertaken to establish whether detrimental effects on cardiac function were associated with excess calcium entry into the myocardium in these hearts. Isolated hearts from adult male Sprague-Dawley rats received intoxicating concentrations of ethanol for 6-10 days by inhalation, were perfused with Krebs-Henseleit buffer by a modified Langendorff technique, and several functional parameters were assessed continuously. In some experiments, the calcium concentration in the perfusate was first reduced from the physiologic range (1.2 mM) to 0.15 mM and then increased in a step-wise fashion to 4 mM. In other experiments, hearts were exposed to buffer containing concentrations of the L-VOCC activator, (+/-)Bay K 8644, increasing from 10(-9) to 10(-6) M. These perfusion protocols were repeated in hearts from treated animals subject to reduced coronary flow because of induction of partial left ventricular ischemia. There were some close similarities in the effects of these different stimuli. When the calcium concentration in the perfusate exceeded a physiologic level, there were signs of decreased function relative to controls in the hearts from ethanol-exposed rats. Thus R-wave amplitude and systolic pressure were lower, diastolic pressure also was reduced, but heart rate was elevated above that of controls. Similarly the presence of (+/-)Bay K 8644 in the perfusate caused a decrease in systolic and diastolic pressure and an increase in heart rate in hearts from ethanol-exposed rats. When cardiac perfusion was reduced in vitro by inflation of a balloon in the left ventricle, some of the effects of excess calcium and (+/-)Bay K 8644 were reproduced in control hearts. However, imposition of this "ischemic" stress did not appear to exacerbate the effects of prior exposure to ethanol. In general, in control hearts, indices of contractility were increased across the range of calcium concentration or by perfusing with (+/-)Bay K 8644. Hearts from ethanol-exposed rats, however, showed no further increase in these parameters once physiologic levels of calcium were exceeded, or showed inhibition of contractility in the presence of (+/-)Bay K 8644. The results are consistent with calcium entry through L-VOCCs in hearts from ethanol-exposed animals having detrimental effects on cardiac function once physiologic levels are exceeded. However, it is possible that these channels also may be involved in maintenance of cardiac function at hypocalcemic levels.

Effects of prolonged exposure to ethanol in vivo on functional parameters and sensitivity to nitrendipine in the isolated rat heart.

This study investigated the possibility that previously reported marked upregulation of binding sites for the dihydropyridine calcium channel antagonist nitrendipine in heart tissue during the development of ethanol dependence in the rat may represent functional L-type voltage-operated calcium channels (L-VOCCs). Isolated hearts obtained from adult Sprague-Dawley rats, which received intoxicating concentrations of ethanol for 6-10 days, by inhalation, were perfused with Krebs-Henseleit solution in the Langendorff mode. Basic measurements of cardiac function were compared with hearts from control rats not exposed to ethanol vapor. In another study, concentration-response curves were constructed for nitrendipine at concentrations in the range of 10(-10)-10(-6) M for hearts obtained from control and ethanol-exposed animals. Changes in measured cardiac parameters such as R-wave amplitude, heart rate, diastolic and systolic pressure, and (+)dP/dt(max) and coronary flow were recorded. All comparisons were made between preparations set to a similar left ventricular end-diastolic pressure. Under these conditions, there were no significant differences in R-wave amplitude, but isolated hearts from ethanol-dependent rats showed significantly greater indices of myocardial contraction than did controls. These included increased systolic and developed ventricular pressure and increased (+)dP/dt(max). Coronary flow also was significantly greater in hearts from ethanol-dependent rats compared with controls. Heart rate was higher in the alcohol-exposed group, but this difference did not achieve significance. When nitrendipine was added to the perfusate at concentrations between 10(-10) and 10(-6) M, hearts from ethanol-dependent animals showed a greater sensitivity to the effects of the drug on heart rate and systolic pressure. Effects on R-wave amplitude and (+)dP/dt(max) were less clear but also suggested a greater sensitivity to nitrendipine in hearts from ethanol-exposed rats. Effects on coronary flow were small and did not differ significantly between preparations from control and ethanol-dependent rats. The results suggest that the increase in Bmax of DHP binding previously observed in hearts from ethanol-dependent animals might represent an increase in L-VOCCs, which alters physiologic function, and pharmacologic responses in the isolated heart. These changes may represent the exposure of an adaptive mechanism designed to overcome the generally depressant effects of ethanol on cardiac function in vivo.