Pal L. Vaghy

Profile of the oppositely acting enantiomers of the dihydropyridine 202-791 in cardiac preparations: receptor binding, electrophysiological, and pharmacological studies.

Receptor binding, electrophysiological, and inotropic effects of the pure dihydropyridine enantiomers (+)S202-791 and (-)R202-791 were studied in cardiac preparations. The KI for (+)S202-791 binding correlated with the ED50s for an increase in contractile force and an increase in calcium current, the latter effect occurring at depolarized as well as resting holding potentials. The KI for (-)R202-791 binding was much lower than the IC50s for inhibition of calcium current measured at holding potentials of -80 or -90 mV and a negative inotropic effect, but correlated closely with the IC50 for inhibition of calcium current measured at -30 mV. Thus, (+)S202-791, is a voltage independent calcium channel activator and (-)R202-791 is a voltage dependent calcium channel inhibitor.

Receptor pharmacology of calcium entry blocking agents

The mechanism of action of calcium channel modulators, a class of drugs that includes 3 chemical groups--1,4-dihydropyridines, phenylalkylamines and benzothiazepines--has been extensively reviewed. The best known representatives of these 3 groups are nifedipine, verapamil and diltiazem, respectively. These drugs bind reversibly, stereospecifically and with high affinity to both the membrane-bound and the purified receptor complex. Non-dihydropyridines allosterically regulate dihydropyridine binding. This has been shown by using (-) [3H]202-791 and (+) [3H]PN200-110 as labeled ligands. The purified receptor complex that possesses binding sites for all 3 chemical groups is likely to be related to the voltage-dependent calcium channel. As the result of a drug-receptor interaction, voltage-dependent calcium channels are either activated or inactivated. The drugs that activate channels act by promoting long-lasting channel openings. The drugs that inhibit calcium channels, the calcium entry-blocking agents, act by preventing channel openings upon membrane depolarization. A complex pharmacologic, electrophysiologic, biochemical, immunologic and molecular genetic approach is required to determine the molecular mechanism of action of calcium channel modulators. Clinically, calcium entry-blocking agents are recommended for the treatment of angina pectoris, hypertension, posthemorrhagic cerebral vasospasm, supraventricular tachycardia, migraine and asthma and the protection of the ischemic myocardium.

Effects of dihydropyridine calcium channel modulators in the heart: Pharmacological and radioligand binding correlations

Bay k 8644 produced a concentration-dependent positive inotropic effect followed by a negative inotropic effect in isolated and intact cardiac preparations. Nimodipine in low concentrations produced slight positive inotropy and in higher concentrations, the usual negative inotropic action. Radioligand binding experiments revealed equilibrium dissociation constants that, taken together with the pharmacological data, suggest that dihydropyridines bind to receptor subtypes and have varying intrinsic activities.

Azidobutyryl clentiazem, a new photoactivatable diltiazem analog, labels benzothiazepine binding sites in the α1 subunit of the skeletal muscle calcium channel

[3H]Azidobutyryl clentiazem, a new photoactivatable diltiazem derivative, has a higher binding affinity than azidobutyryl diltiazem. It can be covalently incorporated into the α1 subunit of the skeletal muscle calcium channel by UV irradiation, which allows the benzothiazepine binding site to be determined. The photolabeled α1 subunit and its proteolytic fragments were analyzed with a panel of sequence‐directed antibodies. The results suggest that the linker region between segment S5 and S6 of domain IV is involved in benzothiazepine binding. This site is different from the dihydropyridine and verapamil binding sites.

Receptors for calcium antagonists

Calcium antagonists have been divided into 3 different subclasses represented by nifedipine, verapamil and diltiazem. These drugs have different pharmacologic effects and are not interchangeable. Previous studies suggested that all calcium antagonists bind to a 170 kd polypeptide (now called the alpha 2 subunit of the voltage-dependent calcium channel). The apparent molecular weight of this polypeptide characteristically decreased from 170 to 140 kd upon disulfide reduction as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recent studies demonstrated that calcium antagonists bind to a previously unrecognized 165 kd polypeptide (alpha 1 subunit) that does not change its electrophoretic mobility on disulfide reduction. Because of their similar molecular weights, the 2 polypeptides may overlap each other on polyacrylamide gels. The primary structure of both polypeptides clearly shows, however, that they are different from each other and only the alpha 1 subunit has the features expected of an ion channel.

Resolution of the identity of the Ca2+-antagonist receptor in skeletal muscle

Abstract The view on the identity of receptors for Ca 2+ -channel inhibitor drugs has changed during the last two years. Previous studies attributed all Ca 2+ -channel inhibitor drug receptors to a single 170 kDa polypeptide consisting of two (140 kDa and 30 kDa) disulfide-linked components. However, as reviewed here by Arnold Schwartz and colleagues, more recent studies show that 1,4-dihydropyridines and phenylalkylamines bind to another, previously unrecognized, approximately 165 kDa polypeptide which does not change its electrophoretic mobility upon disulfide reduction. The primary structure of the receptor exhibits homology with other voltage-dependent cation channels. This, and reconstitution of Ca 2+ -channel activity from purified receptor preparations: suggests that the Ca 2+ antagonist receptor, alone or in combination with other subunits, forms the L-type voltage-dependent Ca 2+ channel.

Protective effects of verapamil and diltiazem against inorganic phosphate induced impairment of oxidative phosphorylation of isolated heart mitochondria

Abstract The effects of verapamil and diltiazem on oxidative phosphorylation of isolated rabbit heart mitochondria were related to the experimental conditions employed. In an assay medium containing 250 mM sucrose, 1 mM pyruvate and 5 mM potassium phosphate buffer (pH 7) at 37° (sucrose medium), only a high concentration of verapamil (200–800 μM) or diltiazem (400–600 μM) affected mitochondria. State 4 respiration was stimulated, state 3 respiration was inhibited, and the ADP: O ratio was decreased by these drugs in sucrose medium. These effects resulted in a depression of the respiratory control index (RCI) and oxidative phosphorylation rate (OPR). On the other hand, in an assay medium containing 150 mM KCl, 1 mM pyruvate and 2 mM potassium phosphate buffer (pH 7) at 37° (KCl medium), the high rate of state 3 respiration and the normal value of the ADP: O ratio were not influenced significantly by diltiazem (400–800 μM) or verapamil (200–400 μM). These data indicate that neither verapamil nor diltiazem has an effect on the normal, functioning, isolated mitochondria in KCl medium. Elevation of inorganic phosphate (P 1 ) from 2 to 5 mM in the KCl medium induced a swelling of the mitochondria, inhibition of state 3 respiration, and a decrease in the ADP: O ratio, RCI and OPR. Under these conditions, a low concentration of verapamil (25–200 μM) or diltiazem (50–800 μM) inhibited the swelling effect of P i and at the same time prevented the P i -induced decrease in state 3 respiration, and the ADP: O ratio, RCI and OPR. In a medium containing 150 mM KCl, 1 mM pyruvate, 2 mM ADP and 10 μM palmitoyl-CoA, the addition of 5 mM P i induced swelling of mitochondria and a decreased rate of state 3 respiration. Under these conditions, even a low concentration of verapamil (6–200 μM) or diltiazem (25–400 μM) inhibited swelling and prevented the inhibition of state 3 respiration. It is concluded that low concentrations of verapamil and diltiazem had no effect on unswollen heart mitochondria. An increase in the free P i concentration induced swelling of mitochondria and resulted in an inhibition of oxidative phosphorylation, provided that the extramitochondrial potassium concentration was as high as that normally found in the cytosol. Under these conditions, a low concentration of verapamil and diltiazem was able to affect the mitochondrial membranes so as to prevent P i -induced swelling and the related inhibition of oxidative phosphorylation.

Phosphate induced swelling, inhibition and partial uncoupling of oxidative phosphorylation in heart mitochondria in the absence of external calcium and in the presence of EGTA

Summary Inorganic phosphate (Pi) induced swelling of heart mitochondria and resulted in an inhibition and a partial uncoupling of oxidative phosphorylation in an assay medium that contained 150 mM KCl and 1 mM pyruvate at 37°C. The swelling effect of Pi was partially inhibited and the uncoupling and inhibition of oxidative phosphorylation were completely prevented by ruthenium red (RR) but not by EGTA. The RR-insensitive swelling, on the other hand, was inhibited by rotenone, N-ethylmaleimide and 2,4-dinitrophenol but not by oligomycin. It is suggested that the Pi-induced swelling of mitochondria has two components. One component is insensitive to RR and related to the respiration-dependent uptake of potassium phosphate which by itself does not cause damage to oxidative phosphorylation. The other component of the swelling is RR- sensitive, may involve intramitochondrial calcium and results in inhibition and partial uncoupling of oxidative phosphorylation. Neither component, however, is inhibited by EGTA.

Isolation of Cardiac Muscle Mitochondria: An Update

In cardiac muscle cells, mitochondria occupy about 40% of the total volume (Page and McCallister, 1973) and provide about 90% of the ATP required under normal aerobic conditions (Neely and Morgan, 1974). In addition to being the major source of ATP, mitochondria contain distinct Ca2+ influx and efflux systems (Carafoli, 1979), the role of which in cell function is still incompletely understood. The structure and function of mitochondria are known to be damaged in such disease conditions as myocardial ischemia (Jennings and Ganote, 1974; Rouslin, 1983; Rouslin and Millard, 1980, 1981; Sordahl and Stewart, 1980; Wood et al., 1979; Nagao et al., 1980) and heart failure (Lindenmayer et al., 1968, 1970, 1971; Lochner et al., 1968; Meerson et al., 1964; Rouslin et al., 1979; Schwartz and Lee, 1962; Wollenberger et al., 1963). A major thrust of research in this area has been the elucidation of mechanisms and factors responsible for mitochondrial damage and the design of pharmacological interventions to prevent cellular injury. In most of these studies, it is necessary to study the structure and function of mitochondria in vitro after isolation and purification.

Molecular Characterization of the 1,4-Dihydropyridine Receptor in Skeletal Muscle

Calcium antagonists have been successfully used for the treatment of several cardiovascular disorders, such as coronary heart disease, supraventricular arrhythmias, and hypertension. The most selectively acting drugs belong to one of three chemical groups: 1,4-dihydropyridines (DHP), phenylalkylamines, and benzothiazepines. These drugs inhibit L-type Ca2+ channels [1-3] by binding at distinct but allosterically interacting sites [4-6]. The drug receptor is believed to comprise part of the L-type Ca2+ channel. Pharmacological, electrophysiological, and radioligand binding studies have provided evidence for the existence of pharmacologically relevant receptors in various tissues [4-6]. However, until very recently the identity of the DHP-binding polypeptide has been an enigma.