Review of preclinical data of calcium channel blockers and atherosclerosis.

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.

New therapeutic trends in calcium antagonism. Are they meaningful?

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.

Therapeutic approaches to the control of coronary 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.

End-organ involvement and calcium antagonist therapy: animal studies.

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.

The antiatherosclerotic effect of the calcium antagonists and their implications in hypertension.

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.

Pharmacological aspects of calcium antagonism. Short term and long term benefits.

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.

The effects of Ca(2+)-free perfusion and the calcium paradox on [125I] endothelin-1 binding to rat cardiac membranes.

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.

The antiatherogenic effects of amlodipine: promise of preclinical data.

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.

Effect of amlodipine pretreatment on ischaemia-reperfusion-induced increase in cardiac endothelin-1 binding site density.

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.

Effect of age on endothelin-1 binding sites in rat cardiac ventricular membranes.

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.

Relationship between ATP resynthesis and calcium accumulation in the reperfused rat heart.

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)

Calcium channels and their involvement in cardiovascular disease.

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.

The role of oxygen radicals during reperfusion.

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, calcium antagonism, atherosclerosis, and ischemia.

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.

The molecular basis for the use of calcium antagonists in ischaemic heart disease.

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.

The role of calcium in the toxic effects of tert-butyl hydroperoxide on adult rat cardiac myocytes.

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 inhibitory effect of [D-Arg1,D-Phe,D-Try7,9,Leu11] substance P on endothelin-1 binding sites in rat cardiac membranes.

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.

The unique binding properties of amlodipine: a long-acting calcium antagonist.

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.

Concentration-dependent desensitization of isolated porcine coronary arterial segments to acetylcholine.

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.