Ca(2+) entry into primary cultured pig coronary smooth muscle cells after previous store depletion by repetitive P2Y purinoceptor stimulation.

Store-operated Ca(2+) entry, stimulated by depletion of intracellular Ca(2+) pools, has not been fully elucidated in vascular smooth muscle cells of pig coronary arteries. Therefore, [Ca(2+)](i) was measured in cultured cells derived from extramural pig coronary arteries using the Fura-2/AM fluorometry. Divalent cation entry was visualized with the Fura-2 Mn(2+)-quenching technique. Ca(2+) stores were depleted either by repetitive stimulation of P2Y purinoceptors with ATP (10 micromol/L), or by the sarcoendoplasmic Ca(2+)-ATPase inhibitor 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ; 1 micromol/L) in Ca(2+)-free medium (EGTA 1 mmol/L). Addition of Ca(2+)(1 mmol/L) induced refilling of ATP-sensitive Ca(2+) stores and an increase in [Ca(2+)](i) in the presence of BHQ. Both could be significantly diminished by Ni(2+)(5 and 1mmol/L), La(3+)(10 micromol/L), Gd(3+)(10 micromol/L), and Mg(2+)(5.1 mmol/L). In contrast to the BHQ-mediated rise in [Ca(2+)](i), refilling of ATP-depleted stores was affected by neither flufenamate (0.1 mmol/L), nor by nitrendipine, nifedipine, and nisoldipine (each 1 micromol/L). The data suggest that after store depletion in pig coronary smooth muscle cells ATP and BHQ may converge on a common, Ni(2+)-, La(3+)-, Gd(3+)-, and Mg(2+)- sensitive Ca(2+) entry pathway, i.e. on a store-operated Ca(2+) entry. An additional contribution of the Na(+)/Ca(2+) exchanger cannot be excluded. Flufenamate-sensitive non-selective cation channels and dihydropyridine-sensitive L-type Ca(2+) channels are not involved in refilling of Ca(2+) stores after previous depletion by repetitive P2Y purinoceptor stimulation. The store-operated Ca(2+) entry in-between repetitive purinoceptor stimulation, i.e. in the absence of the agonist, may be responsible for the maintenance of agonist-induced rhythmic Ca(2+) responses.

Extracellular ATP increases [Ca2+]i in primarily cultured pig coronary smooth muscle cells via a P2Y purinoceptor subtype.

In primarily cultured pig coronary smooth muscle cells, extracellular adenosine triphosphate (ATP; 10(-9) to 10(-3) M) dose-dependently increases intracellular calcium ([Ca2+]i). The [Ca2+]i transients measured by fura-2 fluorescence consist of peak and plateau phases with [Ca2+]i values of 191.84 +/- 5.67 nM (n = 10) and 91.67 +/- 1.89 nM, respectively. In Ca(2+)-free solution, the peak phases persisted, but there was a loss of the plateau response, indicating an initial ATP-stimulated intracellular Ca2+ release and a subsequent transarcolemmal Ca2+ entry. Various agonists have been used to characterize the P2 purinoceptor subtype involved in the ATP-induced Ca2+ transients. The rank order of potency was uridine triphosphate (UTP) > ATP >> 2-meSATP > beta,gamma-meATP = alpha,beta-meATP = adenosine = 0. To examine the refilling of ATP-sensitive stores, four repetitive 60-s ATP responses were produced throughout with a 5-min recovery period in between. Now the ATP peaks gradually declined in Ca(2+)-free solution, indicating the emptying of the stores. If, however, Ca2+ entry was allowed in the "refilling period" (i.e., between the ATP pulses), the Ca2+ peaks could be maintained or restored, respectively. The data suggest that the ATP-dependent [Ca2+]i transients may be mediated via a UTP > ATP-activated P2Y purinoceptor subtype, mediating both an intracellular Ca2+ release and a transarcolemmal Ca2+ influx. The refilling of Ca2+ stores may occur through the unstimulated membrane after agonist stimulation. A putative pathway may be a "capacitative" Ca2+ entry induced on depletion of intracellular Ca2+ stores.

Voltage dependence of the pharmacological Mg2+ block of the Ca2+ entry into vascular smooth muscle cells.

We studied the voltage dependence of the inhibitory effects of the Ca2+ antagonist Mg2+, compared to nifedipine, on the transsarcolemmal Ca2+ uptake into primary monolayers of smooth muscle cells obtained from pig coronary arteries. The cellular Ca incorporation was analyzed by liquid scintillation. Depolarization was produced by elevation of K+0. The data suggest that depolarization known to improve the inhibition by organic Ca2+ antagonists of L-type voltage-gated Ca2+ channels attenuates the Ca2+ antagonism of Mg2+ at vascular smooth muscle cells.

Calcium antagonism in vascular smooth muscle cells.

Ca2+ ions play critical roles in physiological and pathological signal transduction in vascular smooth muscle cells (VSMC). The control of Ca2+ homeostasis is an important means for modulating excitability and response. A pathological increase of Ca(2+)-dependent vascular tone causes vasospasm and arterial hypertension. Moreover, Ca(2+)-mediated migration, proliferation, matrix production and necrotization of VSMC are important components of atherogenic plaque formation. At least two physiological Ca2+ antagonists are known that compete for Ca2+ binding sites at VSMC: H+ and Mg2+. Effective pharmacological control of Ca2+ homeostasis is exerted by organic Ca2+ antagonists. The prototypical compounds verapamil, nifedipine and diltiazem and their derivatives represent three separate structural categories of drugs that block transsarcolemmal Ca2+ influx by specific interaction at a set of binding sites associated with the alpha 1-subunit of the L-type, voltage-gated Ca2+ channel protein. Selectivity of action of the Ca2+ antagonists may arise from pharmacokinetics, class and subclass of the Ca2+ channel activated, state-dependent interactions or pathological alterations. In human therapy they are the drugs of choice in the treatment of arterial spasms and hypertension. The extent to which their antiatherogenic properties are related to Ca2+ channel antagonism at VSMC remains to be elucidated further.

Progression and regression by verapamil of vitamin D3-induced calcific medial degeneration in coronary arteries of rats.

Vitamin D3-induced mural calcification represents an animal model for investigating experimental calcium (Ca) overload and calcification of arterial walls. In this study, long-term progression of calcific degeneration in coronary arteries of rats after one intoxication with vitamin D3 was examined, as well as possible regression of preestablished mural Ca overload with the Ca antagonist verapamil. Sprague-Dawley rats were treated with one intramuscular (i.m.) overdose of vitamin D3 [300,000 IU/kg body weight (b.w.)]. Oral verapamil therapy (100 mg/kg/day b.w. for 24 weeks) was initiated 14 days after the vitamin D3 intoxication. Arteriosclerotic alterations were verified by microchemical analyses of tissue Ca and of cholesterol contents with atomic absorption spectroscopy (special graphite tube technique) and gas chromatography, respectively, and by standard histological techniques. Serum lipids were determined by sequential ultracentrifugation. Between week 3 and week 26 after the vitamin D3 injection, a progressive Ca incorporation from 448.8 +/- 110 to 1,310 +/- 166.3% of control values (i.e., coronary Ca content in 32-week-old untreated control rats = 100%) was observed, associated with calcific morphological lesions, and reactive intimal plaque formation. Verapamil prevented this progression and induced a regression of preestablished mural Ca overload. Therefore, the coronary Ca content after 24 weeks of verapamil treatment amounted to only 146.3 +/- 53.8% of controls. The data indicate that an initial calcific lesion of coronary arteries may serve as crystallization nucleus for advancing Ca overload and morphological alterations.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of calcium in arteriosclerosis--experimental evaluation of antiarteriosclerotic potencies of Ca antagonists. Study Group for Calcium Antagonism.

Chemical microanalyses of conventional human coronary artery plaques (stages I-III [WHO]) revealed the correlation between progressive mural Ca overload up to excessive degrees, and the severity of plaque formation, whereas only small amounts of cholesterol were found, even in complicated lesions. The pathogenetic role of Ca was tested in three types of experimental arteriosclerosis and atheromatosis, using Ca antagonists (verapamil, nitrendipine, diltiazem) as research tools: 1) The Ca type, in vitamin D3 plus nicotine-treated rats; 2) the cholesterol type, in cholesterol-fed New Zealand rabbits; 3) mixed types, in SHRs and NaCl-fed Dahl-S rats. Types (1) and (3) were demonstrated to be governed by a progressive arterial Ca uptake that could be established already in early lesions. The increased mural Ca supply promoted cellular necroses, migration, and proliferation, as well as calcification and degradation of elastic fibers. Ca antagonists prevented the increased Ca incorporation into arterial walls and inhibited the development of experimental arterioscleroses of types (1) and (3). Ca antagonists did not protect coronary arteries of cholesterol-fed rabbits (type [2]) from occlusive cholesterol accumulation. The data suggest an important pathogenetic role of Ca and pronounced antiarteriosclerotic potencies of Ca antagonists in Ca-dominated types of experimental arteriosclerosis. The significance of the present results for pathophysiology and therapy of conventional human arteriosclerosis remains to be clarified.

Experimental vasoprotection by calcium antagonists against calcium-mediated arteriosclerotic alterations.

According to chemical analyses, the development of conventional human coronary artery plaques from "fatty streaks" to "fibrous plaques" and "complicated lesions" is dominated by progressive mural calcium (Ca) incorporation. The atherogenic significance of Ca ions and arterial Ca overload was examined under the influence of nicotine, oxidatively modified low-density lipoproteins, spontaneous hypertension, and an elevated extracellular Ca concentration or vitamin D3. Experiments were carried out either in vitro on cultured medial cells of rats or in vivo on various types of experimental arteriosclerosis of rats. Suitable Ca antagonists (verapamil, diltiazem, nifedipine, or nitrendipine) prevented experimental Ca overload of arterial walls and transmembrane Ca uptake into cultured medial cells produced by risk factors. Thus they protected, in vivo and/or in vitro, against the atherogenic potential of Ca ions, i.e., migration, proliferation, matrix production and intracellular Ca overload of vascular smooth-muscle cells, as well as calcification of elastic fibers. The data indicate that various Ca-consuming processes demand a progressive uptake of Ca into arterial walls if Ca-dominated types of arteriosclerosis develop. Under experimental conditions, specific Ca antagonists inhibit Ca-mediated arteriosclerotic alterations by preventing progressive mural Ca incorporation.

Protective effects of various calcium antagonists against experimental arteriosclerosis.

Arterial walls altered by sclerotic processes accumulate lipids (particularly cholesterol) and calcium. Whereas the accumulation of lipids has long been incriminated as the major pathogenic factor involved in arteriosclerosis, concomitant arterial calcium overload has been considered of secondary importance. Using various animal models and specific calcium antagonists as experimental tools, we have shown the crucial role of excessive calcium uptake into arterial walls in the pathogenesis of arteriosclerotic lesions. Anticalcinotic vasoprotection with calcium antagonists has been demonstrated using light and electron microscopy, radiocalcium uptake experiments and calcium analyses with atomic absorption spectroscopy. The new 1,4-dihydropyridine calcium antagonist amlodipine has been shown to inhibit calcium accumulation in the internal elastic membrane of abdominal arteries of NaCl-loaded salt-sensitive Dahl-S rats, and consequently also exerts protective effects against arteriosclerotic lesions, shown particularly in the distal mesenteric artery branches. Formation of human coronary plaques is marked by a substantial local uptake of calcium, whereas there is a large overlap in the mural cholesterol content of healthy coronary arteries and plaques. Experimental findings in animals and with human tissue indicate that calcium antagonists such as amlodipine may provide a new approach to the prophylaxis of coronary artery lesions.

Calcium overload--an important cellular mechanism in hypertension and arteriosclerosis.

Arterial hypertension and arteriosclerosis are dramatic consequences of vascular calcium overload. Acute intracellular calcium overload of vascular smooth muscle cells produces hypercontractility. Hypertension develops if a general increase in systemic arteriolar tone leads to a rise in peripheral flow resistance. Moreover, progressive elevation of calcium destroys the structural integrity of the arterial and arteriolar walls. Thus, in various animals models, calcium overload initiates lesions of an arteriosclerotic character. Interestingly, conventional human coronary plaques also represent a calcium-dominated type of arteriosclerosis. With the advent of specific calcium antagonists, the pathogenic effects of calcium overload and its deleterious consequences have become, for the first time, accessible to therapeutic intervention. Accordingly, adequate treatment with calcium antagonists prevents calcium overload and can thereby protect arteries and arterioles from functional disturbances and structural damage. In spontaneously hypertensive rats, specific calcium antagonists of the verapamil, nifedipine and diltiazem type normalise blood pressure (BP) by reducing transmembrane calcium influx into vascular smooth muscle cells. However, in addition to controlling BP, these drugs also act as tissue protective agents. The long term effects of calcium antagonists such as verapamil in experimental hypertension include the prevention of severe arteriosclerosis, myocardial hypertrophy, and malignant nephrosclerosis. In humans, the antihypertensive efficacy of verapamil is well documented. Further clinical studies have yet to evaluate the antiarteriosclerotic and tissue protective potential of verapamil in humans.

Gallopamil: cardiovascular scope of action of a highly specific calcium antagonist.

According to Fleckenstein's original classification, gallopamil represents a prototype of highly specific calcium antagonists of Group A. Its large scope of cardiovascular effects is lastly based on one and the same mechanism of action: inhibition of transmembrane calcium influx into myocardial, cardiac pacemaker, and vascular smooth muscle cells. Thereby, in experimental studies that use electrophysiological, biochemical, isotopic, and histological techniques, gallopamil reduced calcium-dependent myocardial contractility and oxygen consumption; dampened nomotopic and ectopic cardiac pacemaker activity; exerted pronounced vasodilator effects; and protected myocardial cells from calcium overload-induced necrotization. The effects of gallopamil on myocardium, cardiac pacemakers, and vasculature are quantitatively comparable, surpassing those of verapamil by approximately one order of magnitude. Further clinical studies must demonstrate to what extent humans will benefit from the experimentally proven cardiovascular potential of gallopamil.

Differentiation between calcium- and cholesterol-dominated types of arteriosclerotic lesions: antiarteriosclerotic aspects of calcium antagonists.

In a series of animal studies we have clearly demonstrated since 1970 that calcium overload of the arterial wall is crucially involved in the pathogenesis of arteriosclerotic lesions. Following excessive calcium uptake, vascular stretch compliance and distensibility were lost together with structural integrity. Conversely, calcium antagonists that counteract abundant calcium uptake were found to prevent pathogenic mural calcium accumulation and damage. These effects were realized with light and electron microscopy as well as with measurements of calcium accumulation using atomic absorption spectrometry and radiocalcium. The experiments were carried out on rats exposed to various well-known risk factors such as nicotine, alloxan diabetes, high doses of vitamin D3, or dihydrotachysterol. Another particularly vasotoxic factor appeared to be hypertension, which develops in spontaneously hypertensive Okamoto rats (SHRs), in hypertensive NaCl-loaded salt-sensitive Dahl-S rats, and in rats with nephrogenic Goldblatt hypertension. Interestingly, aging arteries in animals and humans also exhibit, as a characteristic phenomenon, a steady increase in arterial calcium content. This natural age-dependent calcium accumulation is further enhanced in severe diabetics, heavy smokers, and hypertensive patients. However, the most excessive degree of toxic calcium overload, correlated with dramatic structural damage, occurred in human coronary artery plaques. Here, in "fatty streaks" (type I plaques according to World Health Organization classification), the mural calcium content exhibited a rise by 13 times above normal; in type II lesions (fibrous plaques), the increase in calcium was 24-fold; and in type III plaques, i.e., in "complicated lesions of stenosing character," calcium overload amounted to a value greater than 80 times above that found in healthy coronary segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium--a neglected key factor in arteriosclerosis. The pathogenic role of arterial calcium overload and its prevention by calcium antagonists.

Using specific calcium antagonists as experimental tools, both the physiological messenger and current carrying function of calcium ions as well as their pathogenetic potencies could be elucidated. Notably, excess intracellular calcium signalling and intra- and extracellular calcium overload turned out to be pathogenetic principles of general importance. In this context, progressive calcium overload of arteriosclerotic vascular walls and the antiarteriosclerotic effects of calcium antagonists, deserve particular interest. In fact, with the help of calcium antagonists, arterial calcium overload as decisive component of various types of experimental arteriosclerosis became accessible to a direct therapeutic intervention. According to their responsiveness to calcium antagonists, two pathophysiologically different types of experimental coronary plaques could be characterized: (1) The calcium type, i.e. coronary calcinosis of vitamin D3-intoxicated rats highly sensitive to calcium antagonist treatment, (2) the cholesterol type, represented by coronary atheromata of cholesterol-intoxicated rabbits; this primary coronary cholesterol accumulation could not be inhibited by calcium antagonists. The formation of conventional human coronary artery plaques is characterized from the very early lesion onwards by a progressive local uptake of calcium, finally leading to lethal consequences. Conversely, the analysis of the mural cholesterol does not allow to discriminate arteriosclerotic from normal coronary artery segments. Thereby, conventional human coronary plaques typically represent a calcium-dominated type of human arteriosclerosis and differ widely from plaques produced in cholesterol-fed rabbits. The results indicate the decisive pathophysiological role of calcium and calcium overload in both calcium-dominated types of experimental arteriosclerosis and conventional human coronary artery plaques. Moreover, the antiarteriosclerotic effects of calcium antagonists are demonstrated to be based--in various types of experimental arteriosclerosis--on the inhibition of intra- and extracellular calcium overload of arterial walls evoked by various risk factors (vitamin D3 intoxication, hypertension, nicotine, diabetes).

Excessive mural calcium overload--a predominant causal factor in the development of stenosing coronary plaques in humans.

Healthy human coronary artery walls contain, over their entire lifetime, more free and total cholesterol than calcium. However, as soon as arteriosclerotic alterations set in, the calcium content increases. Thus in coronary fatty streaks (arteriosclerotic plaques of WHO stage I), calcium was increased 13 times, in stage II plaques 25 times, and in fully developed stage III plaques 80 times above normal on average. The most dramatic calcium incrustation was found in coronary stage III plaques that had produced massive fatal coronary infarction. Here, the proportion of calcium salts (particularly hydroxyapatite) may amount to almost 50% of dry weight. Thus the most excessive accumulation of calcium seems to be correlated with the highest fatality. In contrast, there is no correlation between mural coronary free or total cholesterol content, and plaque severity. Accordingly, stenosing coronary stage III plaques contain less cholesterol than do fatty streaks. Moreover, in coronary stage III plaques the proportion of free cholesterol was 1.37%, and of total cholesterol only 2.34% of the whole mass, certainly not enough for directly causing coronary occlusion. Thus the calcium-rich plaques of human coronary arteries considerably differ from the well-known cholesterol-rich plaques (stage I and II) of human aortae. Our findings justify a new prophylactic approach with suitable calcium antagonists to interfere with deleterious calcium uptake in coronary plaque development.

Scope of vasodilatory effects of calcium antagonists.

Specific Ca antagonists of the verapamil, nifedipine, and diltiazem type have become the drugs of choice in the therapy of cardiac hyperkinetic disorders and of vascular hypertonicity (spasms). The specific mechanism of vasodilatation by Ca antagonists was substantiated by electrophysiological, radionuclear and mechanical measurements on rather different types of arterial vasculature, including the systemic resistance vessels, as well as on portal veins. The results indicate that the vasodilator efficacy of these Ca antagonists is mainly based on at least three components: (1) Suppression of the generation of Ca-carried membrane spike potentials; (2) decrease of direct transmembrane supply of activator Ca through potential- or receptor-operated membrane channels, and (3) interference with Ca-triggered intracellular Ca release in an indirect way, in that Ca antagonists block the influx of small amounts of trigger Ca or produce depletion of intracellular Ca stores. In any case, contractile activation practically ceases if transmembrane Ca supply is totally cut off upon addition of Ca antagonists to a Ca-deficient medium.