Guppy, Leon J.

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 10−6M. 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.

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 (10−6M) 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.

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.

It is our contention that functional pharmacology, particularly in vivo, must play a greater role in drug discovery if the promises of reductionist techniques are to be brought to full fruition. Despite the advent of high-throughput screening, combinatorial chemistry and informatics, the number of new chemical entities (NCEs) coming on the market has fallen. While the advent of these methodologies makes it possible to move very quickly from molecular target to lead compound, the problem of demonstrating therapeutic utility remains. Has proof of concept (in vivo) become the bottleneck in drug discovery? This article focuses on this problem, and discusses what can be done about it. [ABSTRACT FROM AUTHOR]