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The Ca2+ antagonists are a novel group of drugs useful in management of a variety of cardiac disorders. They differ from one another in terms of their chemistry, tissue specificity and selectivity. As a group, however, they share the common property of slowing Ca2+ entry through voltage-activated, ion-selective channels. Some of them exhibit other properties, including that of interfering with Na+ transport. At least one of them, diltiazem, has an intracellular action. Specific high and low affinity binding sites have been identified for two of the major groups of Ca2+-antagonists, with the binding sites for verapamil and its derivatives being distinct from those which can be occupied by the dihydropyridines. The number (Bmax) and affinity (KD) of these binding sites changes under certain pathological conditions-including a reduction in ischaemia and in spontaneous hypertension, an increase in the latter, at present, only demonstrated for the dihydropyridine binding sites. The sensitivity of a particular tissue to these drugs will depend upon a number of factors including the number of binding sites that are present, the contribution made by the Ca2+ entering through the voltage-activated channels to the functioning of the tissue, and properties which are peculiar to a particular type of Ca2+ antagonist, for example, whether, as in the case of verapamil, they exhibit use-dependence. [ABSTRACT FROM AUTHOR]

A variety of animal models have been used to determine whether calcium channel blockers exert an inhibitory effect on atherosclerotic lesion formation. These models include the cholesterol-fed rabbit, in which the lesions resemble the fatty-streak stage of atherosclerotic lesion development in humans. Diet-induced atherosclerosis in monkeys is also used and, in this case, the lesions resemble those found in humans, both in pathology and distribution. Other models involve mechanical injury superimposed on cholesterol feeding. Cellular and subcellular preparations are being used to investigate the mechanisms involved in the antiatherosclerotic activity of the calcium channel blockers. The ability of calcium channel blockers to slow atherosclerotic lesion formation is a class effect that is independent of their blood pressure-lowering effect, and occurs without any significant change in the plasma lipid profile. It is accompanied by a reduction in vessel wall cholesterol and calcium and is maintained over prolonged periods of treatment. The mechanisms that may be involved include inhibition of smooth muscle cell proliferation and migration, slowed platelet aggregation, restructuring of cholesterol-enriched cell membranes, enhanced gene expression for low-density lipoprotein receptor protein, inhibition of growth factor release, slowed calcium uptake, and restoration of endothelium-dependent relaxation.

Recent advances in the field of calcium antagonism include the development of long-acting calcium antagonists, some of which have a slow onset of action. Some of these newly developed calcium antagonists are dihydropyridine derivatives. Others, including monatepil (AJ-2615), are chemically unrelated to either the dihydropyridine (nifedipine), phenylalkylamine (verapamil), or benzothiazepine (diltiazem) prototypes of the group. Irrespective of their chemistry, however, vascular selectivity is a prominent feature of the more recently developed calcium antagonists. This, coupled with their long duration and slow onset of action, properties that facilitate continuous protection rather than the intermittent protection of short-acting calcium antagonists, makes them suitable for use as blood pressure-lowering agents that provide "vascular protection"--as indicated by their ability to slow experimentally induced atherosclerotic lesion formation. Long-acting vasculoprotective calcium antagonists can be used to treat other disorders, including angina pectoris. Their efficacy as antiatherosclerotic agents, proven in experimental models, needs to be confirmed by clinical trials on natural atherosclerosis.

Atherosclerosis is a multifactorial disease which culminates in the ruptured plaque seen at autopsy. Hypercholesterolaemia, subintimal accumulation of lipid, monocyte adhesion followed by penetration across the endothelium, the conversion of monocytes to macrophages and smooth muscle cell proliferation and migration are some of the events involved in the early stages of lesion formation. Late events include the formation of excess ground substance and collagen, and the formation of the fibrotic cap. Young lesions tend to be more fragile than "old" calcified lesions, and it is these young lesions which rupture, haemorrhage and provide anchor points for platelets. Therapeutic interventions aimed at controlling lesion formation include those which reduce risk factors, including hypertension as well as those which interfere with the cascade of events involved in lesion formation. Agents which lower plasma cholesterol provide one approach. Another approach is to use calcium antagonists which not only lower blood pressure, but also directly interfere with some of the metabolic events involved in lesion formation.

Hypertension is a complex disease, the treatment of which should not only lower systolic and diastolic blood pressure but also attenuate the secondary consequences of the disease. These include vascular injury (including atherosclerosis), stroke, left ventricular hypertrophy, and renal damage. To establish whether the long-acting, vascular-selective calcium antagonist amlodipine attenuates some of these secondary consequences of hypertension, 5-week-old stroke-prone hypertensive and 8-week-old spontaneously hypertensive rats were treated (orally) with 5 mg/kg/day and 10 mg/kg/day amlodipine, respectively, for 30 weeks. The treatment resulted in a significant lowering of systolic blood pressure, accompanied by reduced cardiac hypertrophy and prolonged survival. Evidence for a protective effect of amlodipine on the vasculature was obtained by treating cholesterol-fed rabbits with 1-5 mg/kg/body weight/day. This resulted in a reduction in vascular Ca2+ overloading and a reduced incidence of sudanophilic lesion formation. Protection against ischemia-induced changes in the myocardium included a reduction in the ischemia-induced externalization of endothelin-1 binding sites.

Calcium-dependent processes involved in atherosclerotic lesion formation include platelet aggregation, monocyte adhesion, release of growth factors, cell proliferation and migration, protein and collagen secretion and synthesis, and endothelial necrosis. In addition, the calcium (Ca2+) component of smooth muscle cell contraction contributes to one of the main risk factors, hypertension. The ability of the calcium channel blockers (CCBs) to interrupt the sequence of events that culminates in the formation of atherosclerotic lesions has been demonstrated in animal models and clinical trials. These studies have involved the short-acting CCBs. The results of this animal study show that manidipine, a new long-acting CCB, produces a dose-dependent reduction in atherosclerotic lesion formation without reducing plasma cholesterol.

Calcium antagonists are widely used in the management of a variety of cardiovascular disorders, including angina pectoris, myocardial infarction, atherosclerosis and hypertension. At the cellular level these drugs act primarily by limiting calcium ion (Ca++) entry through voltage-sensitive, Ca(++)-selective channels. This effect contributes to the antiatherogenic properties of calcium antagonists and is primarily responsible for their ability to reduce systolic and diastolic blood pressure, and to protect the myocardium. The cardioprotective effect of calcium antagonists is a complex phenomenon that needs to be considered in terms of the immediate consequences of Ca(++)-channel blockade and subsequent reduction in cytosolic Ca++, together with the long term benefits. These long term benefits include protection of the vasculature, an attenuation of hypertension-induced hypertrophy and improved left ventricular diastolic function. However, irrespective of whether it is the short or long term effects of calcium antagonists that are being considered, it is their ability to lower cytosolic Ca++ that is primarily responsible for their cardio- and vascular-protective effects.

Atherosclerosis is a complex and multifactorial disease, the endpoint of which is the formation of a calcified plaque. Intermediate events include intimal injury, smooth muscle cell proliferation and migration, macrophage infiltration, lipid accumulation and excess formation of ground substance. To determine whether the newly developed, long-acting calcium antagonist, amlodipine, slows the development of atherosclerotic lesions under experimental conditions, young New Zealand white rabbits were fed on a diet of 2% cholesterol plus 1% peanut oil for up to 12 weeks. Half the rabbits received 1 or 5 mg amlodipine/kg body weight/day. Amlodipine caused a significant and dose-dependent reduction in lesion formation in the thoracic aorta. At the same time thoracic aorta Ca2+ and cholesterol content were maintained at near normal levels, despite the raised plasma cholesterol levels. The protective effect of amlodipine persisted throughout a treatment period of 12 weeks, indicating the absence of tachyphylaxis.

The binding characteristics of [125I]endothelin-1 (ET-1) to cardiac membranes isolated from rat hearts subjected to Ca(2+)-free perfusion or the Ca2+ paradox were examined. The effect of treatment with 2, 3 butanedione monoxime (BDM), which inhibits the tissue damage associated with the calcium paradox, was also investigated. Membranes from rat hearts perfused under control conditions bound [125I]ET-1 to a single population of sites with a Bmax of 107.7 +/- 3.7 fmol/mg protein and an affinity (KD) of 153 +/- 12 pM. Ten minutes of Ca(2+)-free perfusion resulted in a significant (P < 0.01) increase in Bmax to 167.5 +/- 8.3 fmol/mg protein without change in KD. Ca2+ repletion following Ca(2+)-free perfusion tended to increase further the Bmax (180.6 +/- 10.4 fmol/mg protein) without change in KD. Treatment with BDM attenuated but did not prevent the rise in Bmax following Ca(2+)-free perfusion. Following Ca2+ repletion, however, Bmax returned to control levels in the BDM treated group. These changes were not associated with changes in the ability of ET-1 and ET-3 to inhibit [125I]ET-1 binding. The results demonstrate that Ca(2+)-free perfusion is associated with an increase in the binding site density of [125I]ET-1 which is maintained or further increased upon Ca2+ repletion. If, however, the tissue damage associated with the Ca2+ paradox is prevented with BDM, Ca2+ repletion is associated with a reversal of the increase due to Ca(2+)-free perfusion.

Endothelin-1 (ET-1) may be implicated in the pathophysiology of myocardial ischaemia. To determine whether the long-acting calcium antagonist amlodipine attenuates the ischaemia- and reperfusion-induced increase in cardiac ET-1 binding sites, hearts from rats pretreated with amlodipine (0.25 or 0.5 mg/kg) 2 or 5 h before they were killed were made ischaemic for 20 or 40 min, reperfused, and subfractionated. Twenty- and 40-min ischaemia caused a time-dependent increase in ET-1 binding site density (Bmax) identified with [125I]ET-1. Amlodipine pretreatment attenuated this increase in a time- and dose-dependent manner. 0.25 and 0.5 mg/kg amlodipine also suppressed the reperfusion-induced increase in [125I]ET-1 binding site density, even when the 0.5-mg/kg pretreatment series reperfusion was administered after 40-min ischaemia.

To establish whether the density, affinity, or selectivity of endothelin-1 (ET-1) binding sites in cardiac ventricular membranes varies with age, membranes were harvested from 5- to 7-day-, 20-day-, and 8- to 9-week-old Sprague Dawley rats and labeled with [125I]ET-1. Selectivity was established by using cold ET-1, ET-2, ET-3, big ET-1, and (+)PN200-110 to inhibit specific binding of [125I]ET-1. Over the age span studied, selectivity and affinity of the [125I]ET-1 binding sites was unchanged, but density (Bmax) decreased from 209.7 +/- 18.4 at 5-7 days to 154.0 +/- 8.9 (p less than 0.02) at 20 days, and to 89.7 +/- 5.2 (p less than 0.01) fmol/mg protein at 8-9 weeks. These age-dependent differences in Bmax were not accompanied by a change in membrane yield and occurred at a time when the specific binding of (+)[3H]PN200-110 increased.

1. The postulate that the composition of solutions used to reperfuse ischaemic hearts may modulate their ability to synthesize high-energy compounds was tested in isolated rat hearts subjected to 30 min normothermic ischaemia and then reperfused with either Krebs'-Henseleit buffer (K-H) for 20 min (control reperfusion, CR), or a 'myocardial protective solution' (MPS) for 5 min, followed by 15 min K-H (modified reperfusion, MR). The 'myocardial protective solution' was designed to protect against damage caused by sodium and calcium accumulation and by free radicals. Metabolic precursors were also included to promote and support adenosine triphosphate (ATP) resynthesis during reperfusion under both aerobic and hypoxic conditions. 2. 31P nuclear magnetic resonance (NMR) was used to measure tissue ATP and creatine phosphate (CP), and atomic absorption spectrometry was used to measure Ca++. Early during CR, ATP recovered to 28% of the pre-ischaemic value, but fell to 5.5% with continued perfusion. Similarly, CP recovered to 45.5% of the pre-ischaemic level during early CR but fell to 25.5% with continued perfusion. 3. Better maintenance of ATP was seen during MR with oxygenated MPS (O2-MR), the final ATP remaining at 16.9% of the pre-ischaemic level. During O2-MR, CP recovered to 43.55 of the pre-ischaemic level but was not maintained and fell to a final level of 29.5%. 4. During MR with O2-free MPS (non-O2-MR), there was no reperfusion-associated fall in ATP or CP, with the levels maintained at 26.6% and 34.55, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The widespread distribution of L-type Ca2+ channels in the cardiovascular system makes that system a natural 'target' for drugs which inhibit L-type Ca2+ channel activity. Now that tissue-dependent differences in the chemical composition of the calcium antagonist binding sites have been recognized it may be possible to develop drugs with enhanced tissue selectivity. The search for new compounds should not be restricted to improvements in tissue selectivity, however. Some of the ancillary properties of the L-type Ca2+ channel inhibitors--including their ability to protect against lipid peroxidation--should not be lost because these ancillary properties may contribute significantly to their usefulness as therapeutic agents.

Oxy and hydroxy radicals produced during postischemic reperfusion may contribute to the mechanisms responsible for the sustained contractile dysfunction and ultrastructural injury that occur under these conditions. At the molecular level, the consequent peroxidation of membrane-located lipids (including membranes that delineate the sarcoplasmic reticulum, the mitochondria, and the myocytes) probably contributes to the associated loss of Ca2+ homeostasis. Protection against oxy and hydroxy radical-induced injury can be approached in several ways. Oxy and hydroxy radical formation can be limited, or the radicals "trapped." Alternatively, agents that protect membranes against lipid peroxidation-induced injury can be used. To determine whether the calcium antagonist nisoldipine has such a protective effect, isolated hearts were exposed to 0.9 mM H2O2 for short periods of time, and the functional recovery on removal of the H2O2 was used to assess the protective effect of 5 x 10(-9) M nisoldipine. In addition, further evidence of protection was obtained by exposing hearts to an oxy radical-generating system in the presence and absence of 10(-8) M nisoldipine and using the inhibitory effect of nisoldipine on the oxy radical-induced externalization of the endothelin-1 ETA binding sites to quantify protection.

Calcium antagonists are useful for the management of patients with ischaemic heart disease, particularly when used prophylactically. At the cellular level, these drugs act primarily by limiting calcium ion (Ca++) entry through the voltage-sensitive Ca(++)-selective channels, an effect that contributes markedly to their 'energy sparing' properties. However, the long term use of these drugs has additional advantages, particularly with respect to their ability to slow Ca(++)-dependent processes involved in the formation of atherogenic lesions, partially antagonise the effects of the raised levels of circulating endothelin-1 encountered during ischaemia-induced heart failure and hypertension, and trap and immobilise oxyradicals. Prolonged episodes of ischaemia result in an irreversible loss of homeostasis with respect to Ca++. However, the increase in myocardial cytosolic Ca++ caused by relatively short periods of ischaemia is small, reversible, and markedly attenuated by the prophylactic use of calcium antagonists. In the isolated, perfused rat heart, verapamil pretreatment produces statistically significant inhibition of the increase in cytosolic Ca++ during 20-minute global ischaemia. This stereospecific effect is associated with a decrease in the rise in total tissue Ca++ during reperfusion and amelioration of the adenosine triphosphate depletion caused by ischaemia. In general, discussion relating to the molecular basis of the use of calcium antagonists in the management of patients with ischaemic heart disease needs to take into account the duration of the ischaemic event, the workload on the myocardium, the need for prophylactic therapy, and the presence of exacerbating factors such as atherosclerosis and tobacco smoking. The early rise in cytosolic Ca++, the source of which remains uncertain, appears to be an important focus for anti-ischaemic drug therapy.

Calcium is a ubiquitous cation involved in a wide variety of physiological processes. Normally, cytosolic calcium is maintained within narrow limits but under certain conditions the levels rise--either because of excess calcium entry, internal release, or failure of the extrusion mechanisms. Such conditions include hypertension and myocardial ischemia. Calcium ions are also involved in the formation of atherosclerotic lesions. Hypertension, ischemic heart disease, and atherosclerosis are all amenable to calcium antagonist therapy. The efficacy of this class of drugs in the management of such a wide spectrum of disorders is in accord with the central role played by calcium in the etiology of these disorders. To some extent, however, the disorders are interrelated, with hypertension being a major risk factors for ischemic heart disease and atherosclerosis.

Oxidant stress has been implicated in reoxygenation damage following hypoxia and can lead to loss of membrane integrity and cell death. In this study the effects of oxidant stress, induced by tert-butyl hydroperoxide (tBHP), on cell conformation and intracellular free calcium ([Ca2+]i) of cardiac myocytes isolated from rat ventricles were examined. Incubation in the presence of 1 mM tBHP lead to a rise in [Ca2+]i, hypercontracture and loss of membrane integrity (as judged by trypan blue staining and loss of fluorescence of fura-2 loaded cells). Incubation in calcium-free medium or medium containing 2,3 butanedione-monoxime (BDM), which decreases myofibrillar calcium sensitivity, delayed but did not prevent the cell shape changes and loss of membrane integrity. In the presence of BDM, hypercontracture occurred at a higher [Ca2+]i than in control cells, indicating a possible role for [Ca2+]i in the generation of hypercontracture in this model. Treatment with calcium antagonists (10(-6) or 10(-7) M nisoldipine or 10(-6) M amlodipine) did not afford any protection against tBHP. ATP depletion did not accelerate loss of membrane integrity. Pretreatment of cells with the iron chelator, desferrioxamine mesylate greatly attenuated the effect of tBPH, delaying the rise in [Ca2+]i, cell shape changes and loss of membrane integrity. It appears, therefore, that tBHP-induced changes are mediated by the iron dependent generation of butyl alkoxyl radicals. The evidence suggests that tBHP-induced contracture is [Ca2+]i dependent rather than ATP dependent. Calcium modifies, but is not essential for the action of tBHP on isolated myocytes. During reoxygenation of hypoxic hearts calcium overload and free radical generation may act synergistically resulting in the characteristic changes associated with this condition, including loss of sarcolemmal integrity.

The specific binding of [125I]ET-1 to rat cardiac membrane fragments was inhibited by [D-Arg1,D-Phe, D-Try7,9,Leu11] substance P [substance P(D)], a potent bombesin antagonist. This inhibitory effect required high concentrations (greater than 3X10(-6)M) of substance P(D) and was accompanied by a steep increase in non-specific binding, and not a decrease in total binding. Such results indicate that substance P(D) does not competitively inhibit the specific binding of [125I]ET-1 to rat cardiac membrane fragments.

Amlodipine is a 'second generation', long-acting calcium antagonist. To characterise the binding properties of this drug saturation binding studies were undertaken using (-)[3H]amlodipine and rat cardiac membrane fragments. (-)[3H]Amlodipine bound to a single population of high affinity binding sites with a KD of 1.64 +/- 0.17 nM, a Bmax of 0.45 +/- 0.08 pmol/mg protein and a Hill coefficient approaching unity. Binding was slow and required up to 5 hours to reach asymptote during incubation at 25 degrees C. The specific binding was totally inhibited by (-)amlodipine and (-)D600 and partially inhibited by (+)PN200-110, Bay K8644, (+)D600 and d-cis diltiazem. These results indicate that (-)[3H]amlodipine interacts strongly with the phenylalkylamine as well as the dihydropyridine binding sites. It also interacts with the benzothiazepine binding sites. The inhibition of (-)[3H]amlodipine binding by D600 is stereospecific, (-) greater than (+)D600. Bound (-)[3H]amlodipine dissociated slowly from its binding sites, less than 40% dissociation occurring during 5 hours of incubation (k-1 = 1.53 x 10(-3) min-1). These results indicate that the binding profile of amlodipine differs from that of other dihydropyridine-based Ca2+ antagonists. In addition they explain its slow onset of action, and slowed recovery on withdrawal.

Acetylcholine elicited an increase in developed tension on isolated porcine coronary arterial rings at concentrations exceeding 1.1 x 10(-7) M. However, at concentrations greater than 3.3 x 10(-6) M, desensitization occurred with repeated exposures to acetylcholine. When rings were first exposed to 3.3 x 10(-6) M, then washed and subsequently exposed to 3.3 x 10(-5) M, tension was either unchanged or significantly reduced, giving rise to two different population effects. Although atropine completely antagonized the effect of acetylcholine, pretreatment with methysergide (5-HT2), mepyramine (H1) and prazosin (alpha 1) did not attenuate acetylcholine-induced desensitization, indicating that the phenomenon is unrelated to either serotonin, H1- or alpha-adrenergic receptor activation. Diltiazem and nifedipine significantly reduced or completely inhibited contractions produced by K+ depolarization, but not those produced by acetylcholine. Moreover, ryanodine did not alter the tension developed by repeated exposures to acetylcholine. Our results suggest that acetylcholine activates specific receptor-operated channels which are less sensitive to calcium antagonists than K(+)-activated voltage-dependent channels. Moreover, acetylcholine-induced contractions in this model do not appear to be the result of calcium release from the sarcoplasmic reticulum since ryanodine failed to attenuate contractions.

Abnormalities in regulatory mechanisms for calcium handling play a key role in cell death and tissue necrosis. In the cardiovascular system this applies to the vasculature and the myocardium alike. In the aged population, where hypertension is a known risk factor, manifestations of vascular injury include atherogenesis and stroke. The newly developed dihydropyridine-based calcium antagonist amlodipine was used in investigations to determine whether calcium antagonists with sustained activity, in addition to lowering blood pressure, slow the development of atherogenesis in rabbits receiving high cholesterol diets, or reduce mortality in stroke-prone hypertensive rats. To establish whether this drug protects the vasculature against excessive atheroma formation in the presence of high cholesterol intake, rabbits were given 2% cholesterol in addition to their normal food intake and either 0, 1, or 5 mg/kg/day amlodipine orally for either 8 or 12 weeks. One day after the conclusion of the treatment protocol, the thoracic aorta was excised, assayed for calcium or cholesterol concentrations, and stained to identify sudanophilic-positive lesions. Amlodipine caused a time- and dose-dependent reduction in lesion formation, calcium overload, and cholesterol level. In the second series of experiments, amlodipine (5 mg/kg/day) was added to the diets of stroke-prone hypertensive rats. Treatment was initiated at age 5 weeks and continued for 30 weeks. During the treatment period, systolic blood pressure was reduced in the amlodipine-treated rats (166 +/- 9 mm Hg) versus those treated with placebo (248 +/- 12 mm Hg) (p less than 0.001). A significant reduction in mortality was observed in the amlodipine-treated rats (p less than 0.001), with 93% surviving versus only 26% in the placebo group at the end of the 30-week treatment period. Concomitantly, cardiac hypertrophy was attenuated in the treated group compared with the placebo group (heart-to-body weight ratios of 4.5 +/- 0.01 vs 5.8 +/- 0.6, respectively [p less than 0.01]). These results extend the evidence that calcium antagonists provide vascular protection in animal models. This finding may become increasingly important in the management of an aging hypertensive population.

Amlodipine is a newly developed long-acting dihydropyridine-based calcium antagonist. To characterize the binding properties of this compound, saturation binding studies were undertaken, using (-)[3H]amlodipine and rat cardiac membrane fragments. (-)[3H]Amlodipine bound to a single population of high-affinity binding sites with a KD of 1.68 +/- 0.12 nM, a Bmax of 0.34 +/- 0.08 pmol/mg protein, and a Hill coefficient approaching unity. Binding required up to 5 h to reach asymptote, and was pH- and temperature-sensitive. The specific binding was totally inhibited by (-) amlodipine and (-) D600 (IC50 values of 9.20 +/- 5.56 and 6.58 +/- 6.57 nM, respectively) and only partially inhibited by (+) PN 200-110, (-) Bay K 8644, (+) D600, and d-cis diltiazem (IC50 values of 60 +/- 10, 160 +/- 20, 250 +/- 40, and 200 +/- 30 nM, respectively). These results indicate that in addition to its ability to bind to the dihydropyridine and benzothiazepine recognition sites in rat cardiac membrane fragments, (-)[3H]amlodipine also binds strongly to the recognition sites for the phenylalkylamine-based calcium antagonists. The results also show that the inhibition of (-)[3H]amlodipine binding by D600 is stereospecific with (-) greater than (+)D600. Dissociation of bound (-)[3H]amlodipine was slowed under acidotic (pH 6.0) and accelerated under alkalotic (pH 10.0) conditions.

The tissue-protective effect of the calcium antagonists is a complex phenomenon that needs to be considered at the cellular as well as the organ level, and with respect to both the vasculature and myocardium. As far as the vasculature and the myocardium are concerned, hypertension is a major risk factor. The dihydropyridine-based calcium antagonists are protective under these circumstances, not only because of their blood pressure-lowering effect but also because of their ability to slow plaque formation and possibly to provide some protection against oxyradical-induced injury. With regard to the myocardium, tissue protection in the presence of hypertension involves not only a reduction in hypertrophy, but also a reduction in ischemia-induced injury and the incidence of damage due to lipid peroxidation.

The use of calcium antagonists as cardioprotective agents is based on the assumption that uncontrolled Ca2+ gain is a key factor in causing cell death and tissue necrosis. This uncontrolled gain in Ca2+ is the ultimate expression of a series of metabolic events triggered by inadequate perfusion. One of the early events is a rise in cytosolic Ca2+ (Cai2+). Using 1,2-bis(e-amino-5-fluorophenoxy)ethan-N1N1N11N11tetraacetic acid and nuclear magnetic resonance spectroscopy to monitor this early rise in Cai2+, it is possible to show that, in isolated perfused rat hearts, Cai2+ increases (p less than 0.01) within the initial 10 min of ischemia and that the increase progresses with time. Possible causes of this early rise in Cai2+ include activation of the endothelin-1 receptors with the subsequent inositol triphosphate-induced activation of sarcoplasmic (SR) Ca2+ release, enhanced Ca(2+)-induced Ca2+ release from the SR reticulum, displacement of bound Ca2+ by the accumulating protons and entry of Ca2+ in exchange for Na+, or through the voltage-sensitive Ca2+ channels. Using the d and l isomers of verapamil it is possible to show that verapamil slows the early rise in Cai2+, the l isomer being more effective (p less than 0.01) than the d isomer. This, in addition to its established energy-sparing effect, may contribute to the effectiveness of verapamil as a cardioprotective agent when used prophylactically.

A sudden, severe and sustained reduction in coronary blood flow triggers progressive damage to the myocardium, starting with a loss of adenosine triphosphate, creatine phosphate, potassium ions and active tension-generating capacity, and proceeding until the myocytes are swollen, acidotic, incapable of maintaining ionic homeostasis, depleted of the purine precursors needed for energy (as adenosine triphosphate) repletion, and showing signs of structural disorganisation. In addition, the cells become electrically unstable and unable to relax. The early development of raised end-diastolic resting tension probably reflects the early rise in cytosolic Ca++ which occurs in part because of the generation of free radicals. The calcium antagonists can protect against the ischaemia-reperfusion-induced injury, provided they are used prophylactically, and adequate plasma levels are attained. The basis of this protective effect is complex and includes an energy-sparing effect, a slowed loss of adenosine precursors, a slowed rise in cytosolic Ca++, and sometimes, a slowed release of endogenous noradrenaline (norepinephrine). In the case of verapamil, evidence of its protective effect includes reduced creatine kinase release, reduction in infarct size, improved recovery of contractility, maintenance of energy-rich phosphates, preservation of ultrastructure, and maintenance of mitochondrial function. Prophylactic therapy with calcium antagonists has additional advantages in that these agents slow the development of atherogenic plaques, protect the vascular endothelial cells and, in some instances, protect against lipid peroxidation caused by free radicals.

Amlodipine is a long-acting dihydropyridine calcium antagonist with vascular selectivity. Although structurally related to nifedipine, amlodipine differs in several important respects, including its slow rate of onset and slow recovery. These effects probably reflect the relatively slow rate of association and dissociation of amlodipine with its binding site. The interaction of amlodipine with the calcium antagonist binding site associated with the slow Ca2+ channels differs from that of other dihydropyridines in that it involves the binding domains for the phenylalkylamine- and benzothiazepine-based antagonists, as well as for the dihydropyridines. The prolonged duration of action of amlodipine makes it suitable for use in conditions where calcium channel blockade is required on a 24-h basis. To determine whether amlodipine has a vascular protective effect, amlodipine was given orally to either cholesterol-fed rabbits or stroke-prone hypertensive rats. In the cholesterol-fed rabbits amlodipine (1 or 5 mg/kg/day) produced a significant, dose-dependent reduction in the incidence of atheromatous lesions in the thoracic aorta over an 8-week period. In stroke-prone rats the administration of amlodipine at a dose of 5 mg/kg/day reduced the incidence of mortality over a 30-week treatment period. In spontaneously hypertensive rats amlodipine (5 mg/kg/day) caused a fall in systolic blood pressure, accompanied by a significant (P less than 0.01) reduction in cardiac hypertrophy. When administered intravenously (0.25 mg/kg) 5 h before hearts were excised and made globally ischaemic for short periods (the 'stunned' heart) amlodipine pretreatment improved functional recovery associated with reperfusion.

Although calcium antagonists were originally developed for use in the management of patients with angina pectoris, they are now used in the management of other cardiovascular disorders, including hypertension. More recently, the calcium antagonists have been under investigation for their potential protective role in atherosclerosis. Coupled with these new possibilities for therapeutic use are the development of new, long-acting, tissue-specific calcium antagonists. Amlodipine belongs to this group, and although it is a dihydropyridine-based calcium antagonist, its pharmacologic profile differs from that of other dihydropyridine-based calcium antagonists. Differences include: different pH optimum for receptor binding, different rates of association and dissociation, and differences in allosteric interaction with the diltiazem and verapamil binding sites. Amlodipine, when given orally to rabbits receiving a high-cholesterol diet, reduces atheroma formation. Evidence of its ability to protect the vasculature is provided by its ability to significantly increase (p less than 0.001) survival in stroke-prone hypertensive rats.

Experimental studies using animal models designed to mimic the effect of ischemia and postischemic reperfusion have provided data indicating that the calcium antagonists might be cardioprotective. The laboratory studies have indicated consistently, however, that the timing of such drug administration is of critical importance. In the case of nifedipine (a dihydropyridine-based calcium antagonist), the laboratory studies have shown that when used prophylactically it has a protective effect during short (as in the "stunned heart") and long episodes (greater than 30 minutes) of ischemia. This protection has been quantitated in a variety of ways, including preservation of left ventricular function. A complete understanding of whether and how calcium antagonists can be used to protect the myocardium requires further detailed knowledge of not only of the voltage-activated calcium channel and its binding sites, but also of the sequence of events triggered by ischemia and reperfusion. Recent data from our laboratories indicate that cardiac membranes contain specific binding sites for the novel, endothelial-derived vasoconstrictor peptide, ET, and that the density of these sites increases during ischemia. ET promotes Ca2+ influx through the voltage-sensitive channels by a mechanism that does not involve a direct interaction with the dihydropyridine (DHP)-sensitive binding sites. Nevertheless, the ET-induced Ca2+ influx is attenuated by the dihydropyridine-based calcium channel blockers.

Cardiac contractile activity is usually controlled by intracellular Ca2+, but it can also be modified by oxidizing agents. Incubation of guinea pig heart myofibrils with diamide (3 mM, 1 h) increased basal (no Ca2+) ATPase activity by 580% and abolished Ca2+ dependence. The effect was proportional to diamide concentration (0.01-1 mM) and duration of preincubation (up to 2 h). Dithiothreitol (5 mM, 1 h) reversed most of the basal ATPase activation and restored Ca2+ sensitivity. Other sulfhydryl reagents produced a similar effect but also produced inhibition of total ATPase. In intact cell preparations, diamide produced a slow tonic contraction, consistent with myofibril activation. In the perfused rat heart, 1 mM diamide slowly increased diastolic ventricular pressure; this increase was partially reversed by dithioerythritol. In isolated rat heart myocytes, 1 mM diamide produced a slow tonic contraction, increased contractility in response to stimulation. Cardiocytes superfused for 1 h with buffer containing EGTA to deplete Ca2+ did not contract in response to stimulation but showed a slow tonic contraction with diamide. This contraction could be slowly and only partially reversed by dithioerythritol. Response to stimulation was restored by addition of Ca2+. The results show that diamide can produce contraction in viable cells. This contraction does not require extracellular Ca2+ and is unlikely to involve intracellular Ca2+. The direct activation of myofibrillar ATPase may contribute to the increased myocardial stiffness seen in ischemia and to ischemic contracture.

The effect of aerobic perfusion, of up to 90 min global ischemia, and of reperfusion on 125I-labeled porcine endothelin (125I-ET-1) binding site density (Bmax), affinity (KD), and selectivity was investigated using cardiac membranes harvested from adult Sprague-Dawley rats. Membranes from nonperfused hearts bound 125I-ET-1 with a Bmax of 117.0 +/- 8.8 fmol/mg protein, a KD of 0.076 +/- 0.005 nM, and a Hill coefficient approaching unity. Neither aerobic perfusion nor 10 min of normothermic ischemia and subsequent reperfusion altered these binding parameters. The extension of the ischemic episode increased Bmax (23, 32, 62, and 60% increase after 20, 30, 60, and 90 min ischemia, respectively). Reperfusion further increased Bmax (P less than 0.01 at 15 min reperfusion after 20 and 30 min ischemia, and after 30 min reperfusion following 60 min ischemia) without changing selectivity. Affinity was reduced after 60 (P less than 0.05) and 90 (P less than 0.01) min ischemia. Ischemia at 22 degrees C had no effect on the density or affinity of these binding sites.