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Damaging Effects of the Calcium Paradox are Reduced in Isolated Hearts from Ethanol-Dependent Rats: Paradoxic Effects of Dihydropyridine Drugs

Guppy, Leon J.; Littleton, John M.*

Journal of Cardiovascular Pharmacology: December 1999 - Volume 34 - Issue 6 - p 765-771

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 (10−6M) and the DHP L-VOCC activator Bay K 8644 (10−7M) 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.

Pharmacology Section, Department of Basic Medical Sciences, Faculty of Medical Sciences, University of The West Indies, Mona, Kingston, Jamaica, West Indies; and *University of Kentucky, Tobacco and Health Research Institute, Lexington, Kentucky, U.S.A.

Received October 22, 1998; revision accepted May 6, 1999.

Address correspondence and reprint requests to Dr. L. J. Guppy at Pharmacology Section, Department of Basic Medical Sciences, Faculty of Medical Sciences, University of The West Indies, Mona, Kingston 7, Jamaica, West Indies. E-mail:

Dr. Guppy was formerly at the Department of Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada.

Previous experiments from this laboratory showed that prolonged exposure of rats to ethanol in vivo causes a very marked upregulation of [3H]dihydropyridine ([3H]DHP) calcium channel antagonist binding sites in heart and other tissues (1). These sites appear to represent functional L-type voltage-operated calcium channels (L-VOCCs) (2,3), and we suggested (4) that this change, leading to excess calcium entry through L-VOCCs, underlies the increase in susceptibility of these hearts to damaging effects of reperfusion after anoxia (the "oxygen paradox") described by ourselves and others (4-6). However, this suggestion relies heavily on effects of the DHP calcium channel antagonist nitrendipine, which appeared to be much more protective against one index of anoxia-induced damage (myoglobin release) in hearts from ethanol-exposed rats than in controls (4). Another possible explanation for our results is that ethanol exposure in vivo simply makes the myocardium more susceptible to damage produced by many different kinds of oxidative stress in vitro, rather than potentiating just those involving excess calcium entry. For this reason, we decided to investigate another form of myocardial damage in which cellular calcium metabolism is implicated, although in a rather different way, the "calcium paradox" (7).

In the calcium paradox, perfusion of the isolated heart with calcium-free buffer is thought to cause covert cellular damage by removal of calcium ions from the cellular matrix and from the cytosol (7,8). However, this damage is not apparent until the isolated heart is reperfused with buffer containing calcium; at this time, calcium ions entering the myocytes cause overt damage, which can be observed by efflux of the intracellular proteins myoglobin (MYO), lactate dehydrogenase (LDH), and creatine phosphokinase (CPK) into the perfusing buffer (6-8). The damage caused during the calcium paradox might therefore be influenced by an increase in functional L-VOCCs on the myocardium (as in hearts from ethanol-exposed rats) in two ways in vitro: during the calcium-free phase, increased efflux of calcium might occur, or increased influx of calcium through increased L-VOCCs might occur during reperfusion with calcium-containing buffer. Indeed it was recently reported that calcium-free perfusion is associated with the appearance of increased numbers of L-VOCCs on the myocardium, and that these upregulated channels then become a major route for the cytotoxic calcium entry occurring during reperfusion (9). On this basis, an increased number of L-VOCCs, as produced by ethanol exposure in vivo, should markedly potentiate the damaging effects of the calcium paradox by increasing calcium flux in vitro. This effect should be inhibited by the presence in the perfusing buffer of a calcium channel antagonist (e.g., nitrendipine) and mimicked in control preparations by the presence of a calcium channel "agonist" (e.g., Bay K 8644).

This simple model emphasizes the similarities between the oxygen paradox and the calcium paradox (see 7), but the situations are not identical, with regard to either the mechanisms of calcium entry (8) or the effects of calcium channel antagonists (10,11). Thus, although damage assessed by release of intracellular components during reperfusion undoubtedly reflects calcium entry, there is evidence that the route of this entry is less likely to be through L-VOCCs than it is during periods of hypoxia/anoxia and reperfusion (8). On this basis, increased numbers of L-VOCCs on the myocardial membrane in hearts from ethanol-treated rats may be largely irrelevant to the acutely damaging effects of the calcium paradox in isolated hearts in vitro. Additionally, there is another important factor to be considered: in the ethanol-treated rats, increased numbers of L-VOCCs have been present on the myocardium in vivo for several days before the exposure to the calcium paradox challenge in vitro (4). Increased calcium flux into the myocardium has almost certainly been occurring during this period, and this is likely to alter the consequences of imposition of the calcium paradox in the isolated heart. Thus, it has been reported that this type of calcium "preconditioning" can elicit marked protection against the calcium paradox in rat heart (12). If so, this aspect of ethanol treatment in vivo should induce a protective effect on the isolated heart against the calcium paradox. A very similar proposal has been made to explain the effects of short-term ethanol exposure in vitro on the calcium paradox in isolated rat heart (13). Under these conditions, ethanol increases cytosolic calcium by an unknown mechanism, and it is suggested that this limits the damaging effects of subsequent perfusion with calcium-free buffer, thus limiting the cellular damage caused by this stimulus (13).

The experiments described can thus have diametrically opposed outcomes, depending on the relative importance of the increased numbers of L-VOCCs on hearts from ethanol-exposed rats in vitro and in vivo. If in vitro effects are important, the consequences of ethanol exposure are predicted to be an increase in damage associated with the calcium paradox. This should be prevented by the presence of nitrendipine in the perfusing buffer, and mimicked in controls by the presence of Bay K 8644. If the effects of L-VOCC upregulation in vivo are more important, the prediction is that ethanol treatment should be protective against the calcium paradox in vitro, and the effects of the DHP drugs in vitro should be minimal. These hypotheses are not mutually exclusive, and the experimental design, in which interactions between treatments in vivo and drug exposures in vitro are investigated, should help elucidate this possibility.

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Ethanol administration to rats

Male Sprague-Dawley rats (300-500 g) were randomly divided into an ethanol-treatment group, exposed to ethanol vapor for 6-10 days, as previously described (14), and a control group, exposed to identical conditions but with ethanol vapor absent. The ethanol-treatment group inhaled ethanol vapor from ∼10 mg/L in the first 2 days to ∼22 mg/L by the end of the treatment period. Under these conditions, a physical syndrome of withdrawal was seen on removal of animals from ethanol (15).

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Isolated heart preparations

At the end of the treatment period, rats were anesthetized with ether bubbled into a large bell jar. After ∼5 min, the inferior vena cava was isolated, and 0.2 ml of 1,000 U/ml heparin was injected. Approximately 30-60 s later, the thoracic cavity was opened, and the heart was rapidly removed and placed in Krebs-Henseleit (K-H) buffer at 4°C, and the heart mounted for retrograde perfusion at 30°C via the aorta, according to a modification of the method of Langendorff (16). The Langendorff apparatus used in these studies consisted of eight water-jacketed reservoirs, connected to water-jacketed extension tubes, all arranged in series. All reservoirs that were in use at a particular time were maintained at 100 cm H2O above the heart. Different concentrations of drugs could be accessed instantly to perfuse the heart by simultaneously closing of one chamber and turning on perfusion from a drug-containing reservoir. K-H solution used in these functional studies was composed of the following salts (in mM) dissolved in distilled water: NaCl (119), NaHCO3 (25), KCl (4.6), KH2PO4 (1.2), MgSO4 (1.2), CaCl2 (1.2), and glucose (11). All K-H buffer solutions were bubbled continuously with 95% O2/5% CO2 throughout the entire experiment. The pH was maintained at 7.35 ± 0.03. After equilibration with K-H for 15-20 min, a fluid-filled balloon hand-made from Cling Film (LinPac Plastics Co., Sedgefield, U.K.) was inserted into the left ventricle and attached to a Statham pressure transducer connected to a low-level DC preamplifier of a Grass polygraph model 7P1 (Grass Instruments, Quincy, MA, U.S.A.). In addition to ventricular pressure, the first derivative of the pressure, (+)dP/dtmax, was obtained by means of a linear-differentiating circuit, the 7P20 polygraph differentiator (Grass Instruments). Initial end-diastolic pressure was set at 10-15 mm Hg and was identical for each pair of hearts (control vs. ethanol exposed). A three-lead platinum electrode configuration was used to record the ECG from the Langendorff heart preparation. The heart was then enclosed in a heated water-jacketed chamber throughout the experimental period. Several myocardial parameters were recorded. These included R-wave amplitude; heart rate; diastolic, systolic, and developed pressure; (+) dP/dtmax; and coronary flow.

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Perfusion protocol for the calcium paradox

The method of Nayler et al. (17) was used for the perfusion sequence in all calcium-paradox experiments, with slight modifications, to induce a mild Ca2+ paradox. The perfusion sequence was as follows. Hearts were excised and perfused with normal K-H buffer for 20 min in the Langendorff mode, followed by 10 min Ca2+-free K-H perfusion, and then 10 min of reperfusion with normal K-H, all at 37°C. Perfusion of hearts with DHPs was according to this basic procedure, except that hearts were initially perfused with normal K-H for 15 min, followed by 5 min of perfusion with normal K-H + drug, followed by 10 min Ca2+-free K-H + drug perfusion, and finally reperfusion with normal K-H + drug for 10 min. The drugs used in this study were nitrendipine at a concentration of 10−6M and Bay K 8644 at a concentration of 10−7M. These concentrations of nitrendipine and Bay K 8644 were chosen because preliminary experiments on isolated perfused hearts at these concentrations showed a substantial effect on measured myocardial parameters.

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Collection of perfusate

During the perfusion sequence, collections of perfusate were taken at 5, 10, 20 min, during the normal K-H perfusion period, 0, 2, and 10 min of Ca2+-free perfusion, and 0, 1, 2, 3, 4, 5, 6, 8, and 10 min of reperfusion with normal K-H. The perfusate was then examined biochemically for the presence of intracellular components, such as LDH. An ultraviolet (UV) spectroscopic method of assaying for LDH with pyruvate and reduced nicotinamide adenine dinucleotide (NADH) was used (18). The LDH activity was determined by the rate of oxidation of NADH. The change in extinction was measured at 340 nm. The assay was performed at 25°C and pH 7.5. All solutions were prepared fresh in distilled water, and assays were performed very soon after collection. This was to prevent loss of activity of LDH, which occurs on storage of this enzyme at various temperatures. The method for determination of CPK activity is based on the forward reaction by using creatine as the substrate (19). Measurements were carried out at 25°C and pH 9.0 at a wavelength of 360 nm. The rate of removal of NADH is measured as the extinction change at 360 nm and is proportional to the CPK activity. The assay for MYO (17) was performed at 25°C. Samples were read at 412 nm. The standard was myoglobin from rabbit skeletal muscle (Sigma Chemical Co., Poole, Dorset, U.K.) at a concentration of 400 mg/ml. The basis for this assay was that MYO is an intracellular protein (nonenzymatic) and can be lost from the cell only if the integrity of the cell membrane is compromised. It can be considered a good marker for the presence of myocardial cell damage. Volume activity is in terms of units (U), defined in reference 18 as the amount of product in micromoles produced from substrate in 1 min. This defines the amount of enzyme activity in our assay. We standardized it for heart weight.

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Animals and chemicals

Sprague-Dawley rats were obtained from Tucks Ltd., Rayleigh, Essex, U.K. Nitrendipine was a kind gift from Bayer, Newbury, Berks, U.K. All other chemicals were obtained from Sigma Chemical Co., Poole, Dorset, U.K. The investigation reported in this study conforms with the Guide for Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No. 85-23, revised 1985).

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Statistical analysis

A Student's t test was used to analyze two sample means, with a level of significance chosen at p < 0.05, 0.01, or 0.001. Curves of the biochemical markers of cardiac necrosis were analyzed by analysis of variance (ANOVA). All values are given as mean ± SEM.

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Comparison between in vivo treatment groups

The institution of calcium-free perfusion was associated with an almost immediate reduction in the size of the R-wave and an overall diminution of the ECG, although electrical activity continued throughout this phase. Heart rate, as determined from the ECG, increased slightly in both groups. None of these changes was significantly different between controls and hearts from ethanol-treated rats. In contrast to these relatively minor effects on electrical activity, mechanical activity ceased during calcium-free perfusion due to contracture; thus diastolic pressure increased and systolic pressure decreased, with developed pressure being completely abolished. Perfusion rate (coronary flow) decreased markedly in both groups of preparations. All these pathophysiologic changes were maintained during reperfusion with calcium-containing buffer, with the contracture becoming even more severe, and the electrical activity of the isolated hearts ceasing. There were no significant differences between hearts from different treatment groups in any of these parameters.

Release of the intracellular protein MYO and the activities of intracellular enzymes LDH and CPK in the perfusate were very low in the calcium-free perfusion phase, but increased rapidly during the reperfusion with calcium-containing buffer (Fig. 1A-C). The release of these intracellular components was markedly and significantly lower in hearts from animals that had received alcohol in vivo at almost all time points for all indices (Fig. 1A-C).

FIG. 1

FIG. 1

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Effects of perfusion with Bay K 8644

Within 1-2 min of beginning perfusion with buffer containing 0.1 μM Bay K 8644, both groups of preparations showed an increase in developed pressure and contractility. Alterations in ECG also were seen during this period, including ST elevation. Calcium-free perfusion in the presence of 0.1 μM Bay K 8644 was associated with a loss of electrical activity (with the exception of occasional p waves in some preparations-these were commonly larger in preparations from ethanol-treated rats and were sometimes accompanied by electrical activity resembling ventricular fibrillation). These were distinct differences from the effects of calcium-free perfusion in the absence of drug, but their random nature made quantitation difficult. All residual electrical activity was abolished on reperfusion with calcium-containing buffer. Alterations in contractility and coronary flow during calcium-free perfusion and reperfusion were similar to those observed in drug-free conditions, with no clear differences emerging between treatment groups.

Release of intracellular components was once again minimal during the calcium-free phase but increased markedly during reperfusion. In comparison with drug-free conditions, these indices of myocardial damage were all higher when the perfusing buffer contained Bay K 8644 (Figs. 2 and 4A-C). Enhancement by Bay K 8644 of LDH release was greater in the preparations from ethanol-treated animals, and this effect removed any protective effect of the ethanol treatment on this index during the calcium paradox (Figs. 2A and 4A). Essentially similar results were obtained for CPK release, except that some protection was still observed in the alcohol-treatment group (Figs. 2B and 4B). MYO release differed only in that potentiation of this index of myocardial damage by Bay K 8644 was similar for control and preparations from ethanol-treated rats, so that in vivo treatment retained a protective effect in the latter group (Figs. 2C and 4C).

FIG. 2

FIG. 2

FIG. 4

FIG. 4

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Effects of perfusion with nitrendipine

The high concentration of nitrendipine used in these studies (1 μM) caused pronounced atrioventricular (A-V) block (2:1, 3:1, and 4:1) in some hearts, particularly those from ethanol-treated rats. Heart rate decreased in both groups, and some hearts stopped beating even before calcium-free perfusion began. Similarly systolic pressure and contractility both decreased after perfusion with nitrendipine, and end-diastolic pressure increased. In general these effects of nitrendipine were greater in hearts from ethanol-treated rats than from controls. These differences were documented previously (3), and no attempt was made to quantify them here. Calcium-free perfusion in the presence of nitrendipine abolished the heartbeat in all hearts, with a reduction in coronary flow and the development of contracture during both this phase and reperfusion, as observed in the same periods with drug-free perfusion. There were no significant differences between treatment groups during these phases of calcium-free perfusion and reperfusion.

All indices of myocardial damage followed the same temporal pattern during the imposition of the calcium paradox in the presence of nitrendipine as during its absence. Thus there was very little release of LDH, CPK, or MYO during calcium-free perfusion, but these proteins were released immediately on reperfusion (Fig. 3A-C). The presence of nitrendipine increased the release of all intracellular proteins during the reperfusion phase compared with release in drug-free buffer (Fig. 4A-C). In the case of LDH, cumulative release from control preparations was substantially higher than that with ethanol-treated rats (Fig. 4A). However, the increase in LDH release when compared with the drug-free situation showed a greater percentage loss (250%) from hearts obtained from ethanol-dependent animals. For CPK and MYO release, potentiation of damage was greater in the ethanol-treatment group, resulting in almost identical patterns of release in these as in control hearts (Figs. 3B and C and 4B and C). In general, loss of intracellular components appears to be potentiated to a higher degree by nitrendipine and Bay K 8644 in preparations from ethanol-treated rats compared with the drug-free conditions.

FIG. 3

FIG. 3

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The experiments were designed to assess the effect of long-term ethanol exposure in vivo on the damaging effects of the calcium paradox on the isolated rat heart in vitro. The results show quite unequivocally that, as assessed by the release of intracellular proteins, previous exposure to ethanol in vivo results in protection against the calcium paradox in vitro. This result is in direct contrast to our previous findings on the effects of hypoxia/anoxia, which were potentiated in isolated hearts from the ethanol-treatment group (3). Other workers previously showed that short-term ethanol exposure in vitro is also capable of protecting the isolated rat heart against the calcium paradox (13) and suggested that this is because ethanol increases intracellular calcium concentrations. We believe that a similar mechanism may be responsible here. Thus the increased numbers of functional L-VOCCs found on myocardium during prolonged exposure to ethanol in vivo may increase cytosolic calcium concentrations, and this may then limit the damaging effects of perfusion with calcium-free buffer in the isolated heart. A very similar suggestion comes from recent work in which calcium preconditioning was shown to protect specifically against the damage associated with the calcium paradox (12).

This interpretation suggests that the contribution of calcium entry through increased numbers of L-VOCCs in vitro is minimal to the damaging effects of the calcium paradox. In an attempt to test this hypothesis, we also investigated the effects of perfusion with an activator (Bay K 8644) and an antagonist (nitrendipine) of L-VOCCs on the myocardial damage produced in control hearts and those from ethanol-treated rats. Perfusion with Bay K 8644 produced the predicted result, in that the damage associated with reperfusion was significantly increased by the presence of the drug. This suggests that calcium entry through L-VOCCs is involved in the damage caused by the calcium paradox, as suggested by several other workers (7,8), based on effects of calcium channel antagonists (10,11). The effect of Bay K 8644 in enhancing loss of LDH and CPK in our experiments was significantly greater in preparations from ethanol-exposed rats. An exception is noted in terms of its effect on the loss of MYO. This result is also as predicted, because these hearts have markedly increased numbers of [3H]DHP-binding sites, which retain their normal high affinity for Bay K 8644 (1). The results obtained with Bay K 8644 therefore suggest that an ethanol-induced upregulation of L-VOCCs on the myocardium can affect the response to the calcium paradox in two ways, a protective preconditioning effect in vivo and a damaging increase in calcium entry in vitro. Unfortunately the results obtained with nitrendipine, which might have elucidated this complex relation further, are difficult to interpret because this antagonist also tended to increase the indices of damage observed. Once again the detrimental effects were greater in the preparations from ethanol-treated animals, perhaps because the number of [3H]nitrendipine binding sites is increased in these (1). There are several potential reasons for this damaging effect of nitrendipine (see later).

From the preceding account, the effects of the DHP drugs against the calcium paradox in our experiments can themselves be regarded as paradoxic because both the "antagonist" nitrendipine and the "agonist" Bay K 8644 produced similar (detrimental) effects. However, it must be remembered that these drugs have complex effects on the heart (20) and are not antagonists and agonists in the usual meaning of the words; rather they are "modulators" capable of modifying calcium flux in very different ways depending on membrane polarization, calcium gradients, and the presence of activated G proteins (21). In addition, the concentrations of the drugs chosen for these experiments are high and are at the borderline between agonist/antagonist actions (this is particularly true for Bay K 8644, in which the racemic mixture was used (22). These high concentrations were based on effects of the DHPs observed in previous experiments (2,3) and in preliminary unpublished experiments on the calcium paradox, in which lower concentrations of nitrendipine and Bay K 8644 were found to be without significant effect. Thus it is possible that both drugs are increasing or decreasing calcium flux through L-VOCCs under the specific conditions of the calcium paradox in these experiments. Based on previously published work on the relation between L-VOCCs and the damage associated with the calcium paradox (9,11), the most likely explanation is that both DHPs increase calcium entry through these channels during the reperfusion phase, when external calcium concentrations are high. This would be compatible with our previous finding (4) that the presence of this concentration of nitrendipine enhanced some indices of myocardial damage during reperfusion after anoxia. Both detrimental effects of the DHP calcium channel antagonist might be relevant to reported detrimental effects of short-term high doses of nifedipine on the anoxic myocardium in humans (17). More direct measurements of calcium flux and cytosolic calcium concentration over a range of DHP concentrations would be necessary to test this hypothesis.

Unlike the imposition of anoxia (sometimes described as the "oxygen paradox"; see 20), the calcium paradox is essentially an experimental paradigm and has no direct pathophysiologic counterpart (7,20,21). The "protection" produced by in vivo ethanol exposure from damage produced in vitro here probably therefore has no relevance to the reported "beneficial" effects of alcohol clinically [e.g., against coronary thrombosis; 22). Nevertheless, there are other clinically relevant situations in which prior alcohol administration has cardioprotective effects, and that may be through a mechanism similar to that in the calcium paradox. Thus, long-term alcohol exposure protects the heart against septic shock (23), and the mechanism proposed to explain this protection, hyperexcitation in alcohol withdrawal, is very similar to that which we propose here for protection against the calcium paradox (24). Thus, in our hypothesis, an adaptive increase in L-VOCCs on the myocardial membrane in response to ethanol exposure is exposed by removal of the drug during perfusion in alcohol-free buffer in vitro (withdrawal) and confers protective effects against the calcium paradox.

The diametrically opposed effects of prior ethanol exposure on myocardial damage induced by anoxia (potentiation) and calcium-free perfusion (protection) suggest strongly that similarities between the two challenges are only superficial (7,20). We suggest that although both effects of ethanol might be explained by the increase in L-VOCCs that we previously described on the myocardium, the role of this increase in vivo and in vitro may be different. In anoxic damage, the increase in L-VOCCs increases the short-term calcium influx in vitro that is thought to cause myocardial damage in this paradigm. In the calcium paradox, prolonged prior calcium entry in vivo (and perhaps in vitro) through L-VOCCs reduces the impact of calcium flux during the calcium-free perfusion and reperfusion that leads to the damage associated with the calcium paradox. This hypothesis suggests that the calcium preconditioning effect that can protect against the calcium paradox is not effective against the oxygen paradox of anoxia-induced damage (12). If this is the case, then fundamental differences between the mechanisms for myocardial damage between the two paradigms must exist, and further work on this differential effect of ethanol is justified.

In conclusion, prior long-term exposure to ethanol in vivo protects the isolated rat heart from the damaging effects of the calcium paradox in vitro. This result is diametrically opposed to the effects of similar treatment on the damage caused by hypoxia/anoxia, which is potentiated by ethanol treatment in vivo. Both results may be explained by the marked increase in L-VOCCs previously found in the hearts of ethanol-treated rats but this is uncertain. To some extent, this conclusion is supported by the effects of perfusion with nitrendipine and Bay K 8644 in these paradigms, but these DHP drugs appear to have complex effects that cannot be interpreted solely in terms of inhibition or activation of L-VOCCs, and other explanations cannot therefore be excluded.

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Ethanol; Dependence; Heart; Calcium paradox; Calcium channels; Nitrendipine; Bay K 8644; Toxicity

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