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We studied cardioprotective as well as Akt and extracellular signal-activated kinase (ERK) activating effect of a Ca(2+) antagonist and a beta-adrenergic receptor blocker during ischemia-reperfusion, and compared these properties of the substances with that of a poly(ADP-ribose) polymerase (PARP) inhibitor used as a positive control throughout the experiments. Langendorff-perfused isolated rat hearts were subjected to 25 min global ischemia followed by 45 min reperfusion, and recovery of energy metabolism as well as functional cardiac parameters were monitored. Although to varying extents, all substances improved recovery of creatine phosphate, ATP, intracellular pH, and reutilization of inorganic phosphate. These favorable changes were accompanied by improved recovery of heart function parameters and reduced infarct size. In addition and again to varying extents, all studied substances decreased oxidative damage (lipid peroxidation and protein oxidation), and activated Akt, glycogen synthase kinase (GSK)-3beta, and ERK1/2. Correlation between cardioprotective and kinase activating effectivity of the compounds proved to be statistically significant. Physiological significance of these kinase activations was established by demonstrating that inhibition of Akt by LY294002 and ERK1/2 by PD98059 compromised the cardioprotective effect of all the substances studied. In conclusion, we demonstrated for the first time that activation of phosphatidylinositol-3-kinase (PI-3K)-Akt and ERK2 pathways significantly contributed to cardioprotective effects of a Ca(2+) antagonist and a beta-adrenergic receptor blocker. Furthermore, we found a strong correlation between cardioprotective and kinase-activating potencies of the substances studied (Verapamil, Metoprolol and two PARP inhibitors), which indicated the potentiality of these kinases as drug-targets in the therapy of ischemic heart disease.

At the threshold concentration (1-10 pmol l(-1)), the neuropeptide hormones proctolin (PR) and the FLRFamide-like peptide (FLP) F(2) cause an increase in amplitude of electrically evoked contractions (each contraction is a brief tetanus) of lobster heart ostial muscle. At higher concentrations each peptide also induces an increase in tonus (contracture). The PR-induced contracture and augmentation of tetani are proportional to increases in [Ca2+]i. The rate of onset and recovery of peptide-induced effects on both tetani and contracture appeared to reduced by Ca2+ storage by the sarcoplasmic reticulum (SR). Enhanced tetani following a contracture may be due to enhanced voltage-gated Ca2+ current and sarcoplasmic reticular (SR) Ca2+ loading. The SR Ca2+ loading appears to be specific for PR and F2, since glutamic-acid-induced contractures are not followed by increased tetani. The prolonged elevation of [Ca2+]i during contracture causes a right-ward shift in the force-pCa curve indicating a decrease in myofibrillar sensitivity to Ca2+. Blocking voltage-gated Ca2+ channels with Cd2+, nifedipine or verapamil, while reducing tetani, does not prevent peptide-induced contracture and enhanced tetani. Opening SR Ca2+ channels and depleting SR Ca2+ with either caffeine or ryanodine blocked tetani but permitted accelerated peptide-induced contractures. We conclude that PR and F2 at low concentration enhance voltage-dependent Ca2+ induced Ca2+ release from the SR, while higher hormone levels directly gate Ca2+ entry across the sarcolemma.

A substantial number of drugs act either directly or indirectly on the heart, but surprisingly, little is known about drug oxidation in the heart. We therefore investigated the metabolism of the calcium antagonist verapamil in microsomal fractions isolated from the left and right ventricle of heart muscle and in primary cultures of cardiomyocytes of adult rats. Metabolism of verapamil proceeded predominantly with microsomal fractions isolated from the right ventricle of rat heart, and in liquid chromatographic-tandem mass spectrometry (LC-MS/MS) and LC-MS(3) experiments four metabolites (M1-M4) could be identified. Furthermore, the intermediate biotransformation products M5 to M8 could additionally be identified in cultures of primary cardiomyocytes, thus providing new insight into the mechanisms of the N-dealkylation and O-demethylation pathway of verapamil. We show metabolism of verapamil to be predominant in the right ventricle of the heart, and the data reported herein may explain metabolic inactivation and/or adverse drug reactions of certain cardiovascular drugs on the basis of tissue specific metabolism.

Background: Cytochrome P450 mono-oxygenases bring about metabolism of many drugs, including verapamil, but no information is available on the metabolism of such drugs in the human heart., Methods: We investigated the gene expression of major human cytochrome P450 mono-oxygenases in the various regions of explanted hearts from six patients with dilated cardiomyopathy and one with transposition of the arterial trunk. For comparison we also studied samples of liver and lung from three patients and two samples of normal heart. The biological significance of tissue-specific cytochrome mono-oxygenase gene expression was further investigated by study of the microsomal metabolism of verapamil in ventricular tissue., Findings: mRNA for the cytochromes 1A1, 2B6/7, 2C8-19, 2D6, and 4B1 was predominantly expressed in the right ventricle; the unilateral expression of the 2D6 gene in right-venticular tissue is important because of its key role in the metabolism of beta-blockers. A strong correlation between tissue-specific gene expression and enzyme activity was found; microsomal metabolism of verapamil was confined to the right ventricle. By contrast, cytochrome P450 3A genes (which are the major drug-metabolising enzymes in the liver and the lung) were not expressed in any of the human heart tissues, and mRNA transcripts for epoxide hydrolase, cytochrome P450 2E1, and flavin-containing mono-oxygenases 2 and 5 were detected in all regions of the human heart and the great vessels., Interpretation: These findings show that expression of genes for cytochrome P450 mono-oxygenases and verapamil metabolism are found predominantly in the right side of the heart. This finding may explain lack of efficacy of certain cardioselective drugs.

Mutations in motif IVS5 and IVS6 of the human cardiac calcium channel were made using homologous residues from the rat brain sodium channel 2a. [3H]PN200-110 and allosteric binding assays revealed that the dihydropyridine and benzothiazepine receptor sites maintained normal coupling in the chimeric mutant channels. Whole cell voltage clamp recording from Xenopus oocytes showed a dramatically slowed inactivation and a complete loss of use-dependent block for mutations in the cytoplasmic connecting link to IVS5 (HHT-5371) and in IVS5 transmembrane segment (HHT-5411) with both diltiazem and verapamil. However, the use-dependent block by isradipine was retained by these two mutants. For mutants HHT-5411 and HHT-5371, the residual current appeared associated with a loss of voltage dependence in the rate of inactivation indicating a destabilization of the inactivated state. Furthermore, both HHT-5371 and -5411 recovered from inactivation significantly faster after drug block than that of the wild type channel. Our data demonstrate that accelerated recovery of HHT-5371 and HHT-5411 decreased accumulation of these channels in inactivation during pulse trains and suggest a close link between inactivation gating of the channel and use-dependent block by phenylalkylamines and benzothiazepines and provide evidence of a role for the transmembrane and cytoplasmic regions of IVS5 in the use-dependent block by diltiazem and verapamil.

Elementary K+ currents were recorded at 19 degrees C in inside-out patches from cultured neonatal rat cardiocytes to elucidate the block phenomenology in cardiac ATP-sensitive K+ channels when inhibitory drug molecules, such as the sulfonylurea glibenclamide, the phenylalkylamine verapamil or sulfonamide derivatives (HE 93 and sotalol), are interacting in an attempt to stress the hypothesis of multiple channel-associated drug targets. Similar to their adult relatives, neonatal cardiac K(ATP) channels are characterized by very individual open state kinetics, even in cytoplasmically well-controlled, cell-free conditions; at -7 mV, tau open(1) ranged from 0.7 to 4.9 msec in more than 200 patches and tau open(2) from 10 to 64 msec--an argument for a heterogeneous channel population. Nevertheless, a common response to drugs was observed. Glibenclamide and the other inhibitory molecules caused long-lasting interruptions of channel activity, after cytoplasmic application, as if drug occupancy trapped cardiac K(ATP) channels in a very stable, nonconducting configuration. The resultant NPo depression was strongest with glibenclamide (apparent IC50 13 nmol/liter) and much weaker with verapamil (apparent IC50 9 mumol/liter), HE 93 (apparent IC50 29 mumol/liter) and sotalol (apparent IC50 43 mumol/liter) and may have resulted from the occupancy of a single site with drug-specific affinity or of two sites, the high affinity glibenclamide target and a distinct nonglibenclamide, low affinity target. Changes in open state kinetics, particularly in the transition between the O1 state and the O2 state, are other manifestations of drug occupancy of the channel. Any inhibitory drug molecule reduced the likelihood of attaining the O2 state, consistent with a critical reduction of the forward rate constant governing the O1-O2 transition. But only HE 93 (10 mumol/liter) associated (with an apparent association rate constant of 2.3 x 10(6) mol-1 sec-1) to shorten significantly tau open(2) to 60.6 +/- 6% of the pre-drug value, not the expected result when the entrance in and the exit from the O2 state would be drug-unspecifically influenced. Sotalol found yet another and definitely distinctly located binding site to interfere with K+ permeation; both enantiomers associated with a rate close to 5 x 10(5) mol-1 sec-1 with the open pore thereby flicker-blocking cardiac K(ATP) channels. Clearly, these channels accommodate more than one drug-binding domain.

Two models of heart failure were used in rat experiments to study the effect of isoptin on tolerance to the toxic effect of k-strophanthin. Calcium antagonist-induced changes in the cardiotonic and chronotropic effects of the cardiac glycoside and myocardial oxygen consumption were examined in isolated frog atria with a movable-electrode electron tube and polarography. Isoptin was shown to substantially enhance tolerance to the toxic effect of k-strophanthin and slightly decrease positive inotropic effects of the cardiotonic, by slowing down its development rate. The agent potentiated the negative chronotropic action and lowered myocardial oxygen consumption.

Chronically administered verapamil can accumulate in cardiac tissues and may depress cardiac function despite low plasma concentration. To examine this, two groups of rabbits were injected intraperitoneally twice a day with either 1 mg/kg verapamil or saline for 28 days. Fourteen hours after the last dose, right atrial tissue and right ventricular papillary muscle were isolated. Plasma and myocardial tissue concentrations of verapamil were determined. Plasma samples contained no detectable amounts of verapamil, but the myocardium contained 2.1 +/- 0.6 (mean +/- SE) ng of verapamil per gram of wet tissue. Spontaneous action potentials recorded from the sinoatrial node in the verapamil-treated group showed decreases in the phase 0 and phase 4 depolarization rates, a slower pacemaker rate, and an attenuated response to epinephrine compared with the saline-treated group. Papillary muscle in the verapamil-treated group exhibited a significantly lower force of contraction and a depressed contractile response to epinephrine compared with the control group. There were no differences in rate or contractile responses the day after single injection with 2 mg/kg verapamil or saline twice a day. Myocardial verapamil concentration increased with the duration of treatment and with the doses given; myocardial verapamil concentration was 3.0 +/- 0.6 ng/g wet tissue in the group treated for 28 days with 2 mg/kg verapamil. Our study shows that chronic treatment of rabbits with verapamil decreases cardiac chronotropic and inotropic responses to adrenergic stimuli despite no detectable verapamil in plasma, and that accumulation of verapamil in the myocardium is probably associated with these effects.

We have studied the effects of 60 min global ischaemia and 30 min of subsequent reperfusion on the binding of [3H]-(+)-PN 200-110 and [3H]-(-)-devapamil (desmethoxyverapamil or D888) in rat heart membranes. The hearts were perfused in the Langendorff-mode and pretreated with 1 mumol/l verapamil, 30 nmol/l and 1 mumol/l nifedipine. After 60 min of global ischaemia in the absence of drugs, we found a reduction of [3H]-(+)-PN 200-110 binding sites, without changes in the equilibrium dissociation binding constant (Kd). After the subsequent reperfusion maximum specific binding (Bmax) was further reduced, whereas the Kd remained constant. [3H]-devapamil binding sites were influenced to a lower extend and showed only a decrease in Bmax at reperfusion. Pretreatment with 1 mumol/l verapamil completely prevented the changes which were observed for [3H]-(+)-PN 200-110. Pretreatment with a low, vasodilating concentration (30 nmol/l) of nifedipine displayed selective protection against the extra reduction in Bmax which was observed during reperfusion. It is concluded that calcium antagonists show protection against the ischaemia-induced loss of dihydropyridine binding sites in relation to their negative inotropic, energy-saving activity. Furthermore, nifedipine at low, vasodilating but not negative inotropic concentrations protects against further reperfusion-induced injury, which protection may be related to an improved flow during reperfusion.

We explored the effect of age and of hypertrophy on Ca2+ antagonist binding site density (Bmax), affinity (Kd), and selectivity in cardiac membranes harvested from the hearts of young adult (9-week-old) and older (25-week-old) Sprague Dawley (SD) rats, Wistar Kyoto rats (WKY), and spontaneously hypertensive rats (SHR). Radioligand binding studies with (+)[3H]PN200-110 failed to show a significant difference between the Bmax obtained for the cardiac membranes of 9-week-old SD, WKY, or SHR. Similarly, at 25 weeks, the Bmax values were the same for each group, but in each group the Bmax tended to increase with age. The Kd and selectivity were unchanged. For (-)[3H]D888 binding, the Kd values changed with age, but there was no hypertension or hypertrophy-linked increase in Bmax. In the SD and SHR series, but not in the WKY, there was a tendency for the Bmax to increase with age. We interpreted these results to mean that age may contribute to the different Kd and Bmax values described for cardiac membranes from 25-week-old WKY and SHR.

The binding properties of the calcium channel antagonist, [3H]nitrendipine, were investigated in homogenates of the rat cerebral cortex, heart and ileum. The specific component of [3H]nitrendipine binding was consistent with mass-action behavior and was characterized by a high affinity dissociation constant in the range of 0.1-0.3 nM. A variety of other calcium channel antagonists inhibited the binding of [3H]nitrendipine with Ki's that agree generally with the ability of these drugs to block contractions of cardiac and smooth muscle. The inhibition of [3H]nitrendipine binding by other dihydropyridines was consistent with competitive antagonism whereas the inhibition caused by verapamil and D600 resembled negative heterotropic cooperativity. Consistent with this latter postulate was the observation that the kinetics of [3H]nitrendipine binding are altered by verapamil, with both the association rate and the dissociation rate being increased. La+3 and several divalent cations caused an inhibition of [3H]nitrendipine with the rank order of potency being Cd+2 greater than La+3 greater than Ni+2 greater than Co+2 = Mn+2 greater than Mg+2 = Ba+2 greater than Ca+2.

Drug receptors for the Ca-antagonists nitrendipine and (-)desmethoxyverapamil (D-888) were studied in porcine cardiac sarcolemma. The dihydropyridine (DHP) derivative [3H]nitrendipine binds to a single population of high affinity sites (KD = 0.44 +/- 0.10 nM, Bmax = 2.43 +/- 0.19 pmol/mg; (n = 8), whereas the phenylalkylamine derivative [3H](-)D-888 interacts with high and low affinity sites of KD = 5.1 +/- 1.4 nM and KD = 438 +/- 86 nM (n = 6) in the same membrane preparations. The antagonists tested differ also with respect to their pharmacological efficacy on cultured intact heart myocytes. The EC50 value of the negative chronotropic effect exerted by D-888 corresponds to its high affinity binding component. Nitrendipine exhibit a 200-fold lower pharmacological efficacy, as would be expected from binding data.

1 The effect of (+/-)-, (+)- and (-)-verapamil on the Ca2+-binding, Ca2+-transporting activity, and Ca2+-dependent adenosine triphosphatase (ATPase) activity of isolated cardiac sarcolemmal preparations was studied. Enzymatic treatment was used to establish the nature of the sites facilitating [14C]-(+/-)-verapamil binding. 2 (+/-)-Verapamil 1 microM inhibited the passive binding of 45Ca2+. The (+/-)- and (-)-isomers were equiactive. 3 (+/-)-Verapamil 1 microM inhibited the ATP-dependent transport of 45Ca2+ and the associated activation of the Ca2+-sensitive ATPase. The activity resided in the (-)-isomer. 4 Lineweaver-Burk plots for the initial rates of ATP-dependent transport showed that the inhibition induced by the (-)-isomer was accompanied by a reduced Km and Vmax. 5 Enzymatic removal of N-acetyl neuraminic acid and galactose residues increased [14C]-(+/-)-verapamil binding; removal of N-acetylglucosamine and treatment with phospholipase C and trypsin decreased the binding. 6 These results have been interpreted to mean that (-)-verapamil interferes with the ATP-dependent Ca2+-transporting properties of the sarcolemma, and that this effect is accompanied by an altered activity of the intrinsic Ca2+-sensitive ATPase. N-acetylneuramic acid and galactose residues do not provide binding sites for verapamil at the cell surface.

Verapamil (Vp) has been found to augment the creatine kinase (CK) and lactate dehydrogenase (LDH) leakage from isolated mouse heart incubated in vitro at 25 degrees C. This leakage was concentration-dependent, and in the second hour followed the Henri-Michaelis-Menten kinetics model. Least-squares estimates obtained with this model for the Km values of verapamil were 0.38 +/- 0.16 mM (CK leakage) and 0.51 +/- 0.21 mM (LDH leakage). Calcium ion inhibited this effect of verapamil, completely abolishing it when the [Ca2+]:[Vp] greater than or equal to 2. At lower [Ca2+], the effect of verapamil also followed hyperbolic kinetics; the Km of verapamil was increased, but the asymptomatic leakage rate at high [Vp] was not changed significantly. These features indicate that the calcium ion is a competitive inhibitor of verapamil-augmented enzyme leakage. These observations suggest that there may exist a common calcium ion and verapamil binding site, or two allosterically related binding sites, which represent a prime determinant of myocardial leakage. The concept of a vulnerable site that binds calcium ions and verapamil competitively and initiates or vitiates processes that influence myocardial enzyme leakage is attractive because it suggests new approaches to the problem of myocardial preservation. As verapamil is thought not to enter the cell, the site of the competitive inhibition may be the sarcolemmal coat.

The effect of the verapamil upon the stability of the membranes of lysosomes isolated from rabbit hearts was studied by means of the acid phosphatase activity, the mapping enzyme of the cytoplasmic organelles. A dose of 2.5 mg of verapamil was injected endovenously in each animal. After homogenization of a sample taken from the lower end of the heart, the lysosomes were isolated by fractionated centrifugation and refrigerated at 2 degrees C. The lysosomal activity of non treated animals was 43.1 +/- 8.9x10(-1)mU/mg. The activity of lysosomes isolated from verapamil treated animals without previous treatment "in vitro" with digitonin was 8.7 +/- 1.6x10(-1)mU/mg and the activity of those with previous treatment of digitonin was 9.1 +/- 2.1x10(-1)mU/mg. The activity of lysosomes isolated from the control group and treated "in vitro" by digitonin was 193.9 +/- 40.5x10(-10)mU/mg. Consequently, the verapamil proved to have a strong capacity of protection of the lysosome membranes when "in vivo".

Electrophysiological and pharmacological studies have shown that peripheral-type benzodiazepine receptors modulate voltage-sensitive calcium channels in the heart. We have compared these binding sites with binding sites for [3H]dihydropyridines, which are believed to label such channels. Although no direct or allosteric interaction could be demonstrated between the two sites, their subcellular distribution--sarcolemma and ryanodine-sensitive sarcoplasmic reticulum--was parallel. Size determination of the two sites suggests that the receptors for these two classes of compounds are separate molecules packaged in the same membrane compartment.

The [3H]-verapamil binding activity of rat cardiac sarcolemmal fragments was studied, using membranes harvested from non-perfused, aerobically-perfused and ischaemic hearts. Glass-fibre filters were found to contain specific, high affinity--(KD 38 +/- 3.1 nM) [3H]-verapamil binding sites--making them unsuitable for use in [3H]-verapamil binding studies. Incubation of membranes from non-perfused hearts in a medium containing 150 mM NaCl, 1 mM CaCl2 and 50 mM Tris revealed two populations of [3H]-verapamil binding sites. When centrifugation instead of filtration was used to separate bound and free [3H]-verapamil, high affinity sites with a KD of 0.57 +/- 0.19 microM and a Bmax of 38 +/- 5.2 pmol mg-1 protein, and low affinity sites with a KD of 78 +/- 27.5 microM and a Bmax of 2.9 +/- 1.3 nmol mg-1 protein were detected. However, only low affinity binding sites could be detected in membranes which had been incubated in a cation-free medium containing 50 mM Tris. [3H]-verapamil binding to the low and high affinity sites was saturable, reversible, stereospecific and displaceable by D600 greater than diltiazem greater than Ca2+ but not by nifedipine, nitrendipine, nisoldipine or prazosin. The two populations of binding sites survived aerobic perfusion and 60 min ischaemia at 37 degrees C. Ischaemia reduced the Bmax and KD but selectivity was maintained.

Plasma, heart, and extracardiac tissue verapamil concentrations were measured after sustained intravenous infusions in 11 dogs to determine the differential tissue accumulation of verapamil. A steady state verapamil concentration of 327 +/- 50 ng/ml decreased the mean arterial blood pressure from 104 +/- 9 to 90 +/- 6 mm Hg (p = 0.08) and the P-R interval increased from 118 +/- 4 to 176 +/- 13 ms (p less than 0.001) with second-degree atrioventricular block developing in 6 animals. Verapamil accumulated in organs in the following order: Lung much greater than kidney greater than spleen greater than ventricular myocardium = liver greater than atrial myocardium greater than cerebral cortex greater than fat = skeletal muscle. Levels in the ventricular free wall were consistently greater than atrial levels, but no difference was observed between left versus right-sided cardiac chambers. In summary, affinity of different organs for verapamil is highly variable and organ-specific; furthermore, differential intracardiac chamber accumulation occurs.

Voltage-dependent Ca channels were studied in living, tissue cultured rat heart cells using patch clamp analysis of single channels and radioligand binding studies at different membrane potentials. Ca channel activating and blocking dihydropyridines (DHP) show cooperative interaction and, therefore, bind to at least two different binding sites on the channel protein. Cooperative interactions between activating and blocking DHP, between DHP and verapamil and between DHP and diltiazem are all voltage dependent. The type of interaction in polarized cells and hence, the possible effects in vivo cannot be predicted from studies in cell homogenates.

The effects of two excitation-contraction uncoupling agents, manganese (Mn) and verapamil, are compared in heart and fast-twitch skeletal muscle. Particular attention is given to the effect of the agents on the first contraction following a period of quiescence when the agents are administered during the quiescent period. Mn significantly diminishes dP/dt of the first beat in heart muscle, whereas verapamil has no significant effect. Neither Mn nor verapamil has a significant effect on the first postquiescent contraction in skeletal muscle, though verapamil produces a diminution of dP/dt in subsequent contractions in both tissues. A comparison of the effect of the agents on 45Ca exchange in heart cells in tissue culture indicates that Mn induces a rapid displacement of a rapidly exchangeable component of heart-cell calcium with subsequent inhibiton of influx. Verapamil, by contrast, produces no rapid displacement but only an inhibiton of influx. The functional and Ca-exchange effect of the two agents are compatible with a model which places most of the contractile-dependent Ca at the cellular surface in heart muscle and at deeper, intracellular sites in fast-twitch skeletal muscle.

The Syrian cardiomyopathic hamster has a hereditary disease in which a progressive myocardial necrosis mimics human forms of cardiac hypertrophy. Lesions are associated with calcium overload and can be prevented with the calcium antagonist verapamil. Numbers of receptor binding sites for calcium antagonists in heart, brain, skeletal muscle, and smooth muscle were markedly increased in cardiomyopathic hamsters. The uptake of calcium-45 into brain synaptosomes was also increased in cardiomyopathic hamsters. The increase in calcium antagonist receptors and related voltage-sensitive calcium channels may be involved in the pathogenesis of this cardiomyopathy.

The disposition of verapamil was studied in anesthetized open-chested dogs following administration of intravenous doses of 0.5 mg/kg. The plasma and myocardial verapamil concentration-time data were fit to a three-compartment model to describe the disposition kinetics. The distribution equilibrium between myocardium and plasma was achieved rapidly and the concentration of verapamil decayed in parallel in these two tissues. The partition coefficient which describes the time averaged myocardial/plasma concentration ratio was 6.21 +/- 2.38. Examination of the relationship between the plasma and myocardial concentrations and the time course of the effect of verapamil, as defined as PR interval prolongation, revealed a hysteresis effect in some dogs. Despite this hysteresis, there was a linear relationship between plasma and myocardial concentrations of verapamil and the degree of prolongation of the PR interval. The results of this study indicate that the concentration in the plasma is in equilibrium with the myocardium and changes in plasma concentration are indicative of parallel changes in myocardial levels. The effect of verapamil on the atrioventricular node is related to the concentration in the myocardium and plasma, but there is substantial interanimal variability in the sensitivity to verapamil.

Stereospecific saturable and reversible binding of d-cis-diltiazem has been demonstrated in cardiac sarcolemmal membrane vesicles. Analysis of binding by either equilibrium or kinetic techniques indicates the presence of a single class of benzothiazepine receptors which bind diltiazem with a KD of 80 nM at 25 degrees C. Benzothiazepine receptors copurify with other sarcolemmal marker activities and exist in a complex with distinct receptors for dihydropyridine and aralkylamine Ca2+ entry blockers in a 1:1:1 stoichiometry. Ligand binding to one receptor of this complex influences binding reactions at the other two sites in a manner that depends on ambient temperature. Binding of either dihydropyridine agonists or antagonists causes partial inhibition of diltiazem binding at 25 degrees C (Bmax effect), while most dihydropyridine antagonists stimulate and agonists inhibit diltiazem binding at 37 degrees C (both are KD effects). This temperature-dependent change in receptor coupling was confirmed by Scatchard analyses and study of diltiazem dissociation kinetics. Verapamil, interacting at the aralkylamine receptor, inhibits diltiazem binding equivalently at 25 and 37 degrees C (KD effects). In addition, both classes of dihydropyridines inhibit verapamil binding in a temperature-independent fashion, as does diltiazem (all are KD effects). Allosteric coupling between benzothiazepine and dihydropyridine receptors is manifested in cardiac muscle since the negative inotropic potency of diltiazem is increased by nitrendipine and decreased by 4-(O-trifluromethy(phenyl)-2,6-dimethyl-5-nitro-1,4-dihydropyridin e-3- carboxylic acid, methyl ester. These results suggest a model in which the Ca2+ entry blocker receptor complex undergoes a change between 25 and 37 degrees C so that at the latter temperature all sites are directly coupled. Allosteric coupling may have important consequences in vivo since it can be detected in functional assays of Ca2+ channel activity.

Vogel et al. (1979; J. Pharmacol. Exp. Ther. 210, 378) reported that one calcium antagonist, bepridil, exerted an effect internally as well as its effect on blocking Ca2+ entry in cardiac muscle. Therefore, the uptakes of tritiated nifedipine, diltiazem, bepridil, and verapamil by cat ileal smooth muscle, chick embryonic ventricular muscle, and rabbit papillary muscle were investigated. It was found that the uptakes of verapamil and bepridil by the muscles were much higher than those of nifedipine and diltiazem. The uptake of bepridil was substantially greater than that of verapamil; thus, the order of uptake was: bepridil greater than verapamil much greater than nifedipine greater than diltiazem. The cardiac muscles accumulated at least 2-fold greater amount of calcium antagonists than the smooth muscle. The amount of a given calcium antagonist accumulated by a muscle was not a function of the ability of that calcium antagonist to inhibit Ca2+ uptake into the muscle, since nifedipine and diltiazem were more potent in depressing Ca2+ uptake, but had the smallest uptakes. The calcium antagonists were more effective in depressing Ca2+ uptake into smooth muscle than into cardiac muscle. Calculation indicates that internal drug concentration at steady state for both cardiac and smooth muscles was either equal to (diltiazem) or much higher than the drug concentration in the medium (bepridil and verapamil). It is concluded that bepridil and verapamil enter and accumulate in the muscle cells, whereas nifedipine and diltiazem permeate more slowly into the muscles. The ability of all four drugs to enter the muscle cells confers the possibility that these calcium antagonists may exert secondary actions on internal sites of the muscle, such as the sarcoplasmic reticulum.

Drug receptors associated with Ca2+-channels in isolated chick heart membranes were found to exist in high and low affinity states. When assays were conducted in the presence of EDTA most of the receptors detected with the dihydropyridines (+)[3H]PN 200-110 or [3H]nitrendipine appeared to be in the lower affinity state. Inclusion of either Mg2+ or Ca2+ in the binding reactions resulted in the disappearance of the lower affinity state and the conversion of the receptors to a single high affinity state. Similar results were obtained with the phenylalkylamine derivative [3H]desmethoxyverapamil (D888). The results suggest that both the dihydropyridine and phenylalkylamine receptors on the cardiac Ca2+-channel can exist in interconvertible high and low affinity states in vitro, and that the proportion of receptors in each affinity state can be altered by the absence or presence of divalent cations.

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding of the dihydropyridine calcium antagonist [3H]nitrendipine was studied in cardiac myocytes incubated in normal and high potassium buffer so that we might examine the voltage dependence of dihydropyridine binding. Hearts were obtained from adult male Wistar rats, and isolated calcium tolerant myocytes were dissociated by enzymatic dispersion. Cells in 5.6 mM extracellular potassium showed specific binding of [3H]nitrendipine with KD 587 +/- 50 (mean +/- SE) pM and maximum receptor density, Bmax, 10.8 +/- 1.3 fmol/mg wet weight. Cells depolarized in 50 mM potassium showed no change in KD, 661 +/- 77 pM, but approximate doubling of Bmax 25.6 +/- 3.7 fmol/mg wet weight. Binding equilibrium was reached within 5 minutes at 37 degrees C, and the KD determined by kinetic analysis was in good agreement with KD determined by saturation experiments. Unlabeled nitrendipine and nifedipine completely inhibited [3H]nitrendipine binding with slope factors less than one, whereas verapamil and diltiazem only partially inhibited binding with slope factors substantially less than one. As a function of increasing extracellular potassium concentration from 2.4-54.1 mM, the number of nitrendipine-binding sites increased gradually 115%. Aconitine also produced a 58% increase in binding sites over a concentration range of 1-30 micrograms/ml. The potassium-induced changes in number of binding sites occurred rapidly and were rapidly reversible with changes in extracellular potassium concentration. There was no change in the number of [3H]nitrendipine-binding sites as a function of potassium or aconitine concentration in dead or digitonin-treated myocytes. We conclude that nitrendipine receptor density in viable myocytes is voltage dependent, but we detect no change in KD as a function of voltage.

The plasma levels and myocardial content of verapamil and its metabolites norverapamil, N-dealkylverapamil and N-dealkylnorverapamil were determined in 15 patients with valvular [3] or ischaemic [12] heart disease. The mean myocardial plasma concentration ratio (M/P) was 7.05 for verapamil, 11.45 for norverapamil, 8.93 for N-dealkylverapamil, and 11.33 for N-dealkylnorverapamil, with great interpatient variability. The highest M/P ratios of verapamil were generally found in patients with the lowest plasma levels, suggesting that saturable tissue uptake may occur.

Binding properties of the calcium channel activating dihydropyridine (DHP), H-BAY K 8644, and the inhibiting 3H-verapamil were demonstrated in monolayer cultures of beating cardiac cells. 3H-BAY K 8644 specific binding was dependent on the presence of extracellular calcium, the affinity was modulated by Ca2+, but Hill coefficients remained unaffected. BAY K 8644 stimulated myocardial contractility in resting and beating myocytes. In contrast to beta-adrenoceptor agonists, however, cellular levels of cyclic AMP and cyclic GMP in cultured myocytes remained unchanged by the compound. Dihydropyridine derivatives of both the calcium channel activating BAY K 8644 as well as the Ca2+ entry blocking DHPs of the nifedipine or nimodipine type yielded very low affinity to other receptors measured in brain and heart membranes. 3H-BAY K 8644 binding sites proved to be highly specific for various potently displacing DHP derivatives and discriminated between optical isomers (stereoselectivity) with inhibition constants (Ki) in the nanomolar range. The heterogeneous shapes of the competition curves also imply interactions of these compounds with different (sub-)sites of the DHP receptor that represents one locus of interaction in regulating transmembranal Ca2+ currents. The other specific site of action for the potent diphenylalkylamines, clearly different to the DHP receptor was characterized with 3H-verapamil. The equilibrium dissociation constant, Kd in cultured myocytes ranged between 16-25 nM, and binding capacity, Bmax amounted to about 1.85 pmol/mg of protein. The different mode of competitions indicates the involvement of more than one 3H-verapamil binding site. The interrelation of the structurally heterogeneous channel modulators with the differently radiolabelled receptor (sub-)sites located in or near by the calcium channel may represent new approaches in investigating the nature of action of these potent compounds.

The calcium channel blocker [3H]verapamil showed specific binding of high affinity (equilibrium dissociation constant, KD, of 4.25 nM) and low density (maximal number of binding sites, Bmax, 50 fmol/mg protein) in frog heart membranes. Nitrendipine, a new calcium channel blocker, had a potency comparable to verapamil in the inhibition of [3H]verapamil binding. Concentrations of Ca2+ in the medium exceeding 3 mM decreased [3H]verapamil binding. These findings are consistent with the concept that high-affinity binding sites for calcium channel blockers are associated with sarcolemmal calcium channels.

Tritiated 1,4-dihydropyridines (nimodipine, nitrendipine, nifedipine, PN 200-110) and [3H]D-cis-diltiazem as well as [3H]verapamil were employed to directly identify calcium channels in membranes for excitable tissues. The channels, when probed with 1,4-dihydropyridines, exhibit the following properties: 1,4-Dihydropyridine calcium channel blockers bind in a temperature-dependent, reversible manner and with high affinity (dissociation constants 0.2-2 nM at 37 degrees C) to a finite number of sites. For chiral 1,4-dihydropyridines, the binding is stereoselective. Hill slopes of approximately 1.0 are observed. In brain, heart, and solubilized skeletal-muscle membranes, an absolute requirement for certain divalent cations exists in order to bind the ligands with high affinity. Cooperativity (negative and positive) between Me2+ and 1,4-dihydropyridine binding sites is observed. 1,4-Dihydropyridine binding sites are down-regulated in a complex manner by the optically pure enantiomers of D-600 and verapamil. These channel blockers induce, to a different extent, a low-affinity state of the 1,4-dihydropyridine binding site. It is postulated that this allosteric site, at which these blockers act, is closely coupled to the 1,4-dihydropyridine binding site and that a spectrum of compounds exists that differ in their affinity as well as their intrinsic activity to induce the down-regulation. The 1,4-dihydropyridine binding sites are up-regulated by D-cis-diltiazem and KB-944. The up-regulation is temperature-dependent and induces a high-affinity state for 1,4-dihydropyridine channel blockers, accompanied by distinct alterations of the kinetics as well as the pharmacological profile of the 1,4-dihydropyridine binding sites. Complex interactions exist between the channel blockers that induce up-regulation and those that induce down-regulation of the binding. For a given radiolabeled 1,4-dihydropyridine, a tissue-specific (but not species-specific) equilibrium binding dissociation constant is observed. Thus, all heart (human, rat, guinea pig, frog, bovine) have the same KD (0.25 nM at 37 degrees C) for, [3H]nimodipine. The same is observed for brain (KD = 0.5 nM) and for skeletal muscle (KD = 1-2 nM). Three subtypes of channels can be distinguished on the basis of the KD and the tissue-specific up-regulation by D-cis-diltiazem. Subtype-selective drugs exist; e.g., AQA 39 is an inhibitor of [3H]nimodipine binding at skeletal-muscle calcium channels, but not at brain channels. Despite their different pharmacological and kinetic profiles, calcium channels in skeletal muscle and brain have the same molecular size (Mr) when probed by radiation inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

The distribution of the bovine cardiac binding sites for the organic calcium-channel blockers was studied. Crude microsomal membranes were separated into three fractions, which contained mainly membranes derived from sarcolemma, 'junctional' sarcoplasmic reticulum containing transversal tubuli, and free sarcoplasmic reticulum. The high-affinity binding site for the dihydropyridines, determined in the presence of nitrobenzylthioinosine, was enriched 12-fold and 17-fold in sarcolemma and junctional sarcoplasmic reticulum. The binding sites for the phenylalkylamines, determined with [3H]verapamil or [3H](-)desmethoxyverapamil, were enriched 1.5-3.4-fold in sarcolemma and junctional sarcoplasmic reticulum but 6-10-fold in free sarcoplasmic reticulum. The phenylalkylamine-binding site, present in free sarcoplasmic reticulum, was partially destroyed by chymotrypsin or phospholipase A2 and C treatment. Specific binding was proportional to the concentration of the added membrane protein. The binding of (-)desmethoxyverapamil was half-maximally inhibited by 6.5 mM calcium chloride and was optimal in the presence of 5 mM EGTA. In three out of five preparations (-)desmethoxyverapamil bound to a single site with an apparent Kd value of 191 +/- 42.8 nM and a density of 34.5 +/- 7.7 pmol/mg protein. In two out of five preparations an additional high-affinity site (Kd approximately 0.67 nM) was detected. The low-affinity site bound other phenylalkylamines, but stereospecific binding of phenylalkylamines was not observed. Binding of phenylalkylamines to the low-affinity site was inhibited by some but not all calmodulin 'antagonists'. Furthermore dihydropyridines did not affect the binding of (--)desmethoxyverapamil suggesting that the low-affinity site differs considerably from the high-affinity sarcolemmal site. These results suggest that free sarcoplasmic reticulum contains a binding site for phenylalkylamines at a relative high density, which is not related to the high-affinity site present in the voltage-dependent calcium channel.

Specific, saturable, and reversible binding of verapamil has been demonstrated in crude cardiac sarcolemmal membranes. These receptors possess a Kd of approximately 50 nM for verapamil as determined by either equilibrium binding studies, competition binding analysis, or kinetic analysis of on and off rates and display an average density of 1.25 pmol/mg of protein. Specificity of binding is indicated by several criteria. Competition studies with the verapamil analog D-600 indicate that (-)D-600 is 200-fold more potent than the (+)-isomer in displacing bound verapamil. Likewise, several other aryl alkyl amine Ca2+ entry blockers effectively displace bound ligand. In addition, dihydropyridines and diltiazem promote partial (25-35%) displacement of bound verapamil with Ki values similar to the Kd values for their respective receptors. Characterization of nitrendipine binding in this preparation indicates an average density of 0.3 pmol of receptors/mg of protein suggesting that the verapamil:nitrendipine binding site ratio is approximately 4:1. Binding characteristics of verapamil and nitrendipine receptors in highly purified sarcolemmal vesicles are similar to those in the crude preparation except that the ratio of verapamil:nitrendipine sites approaches 1 and nitrendipine and diltiazem promote almost complete displacement of bound verapamil. Fractionation studies of crude sarcolemmal membranes indicate that excess verapamil receptors, insensitive to the action of dihydropyridines or diltiazem, are located in a high-density, nonmitochondrial, non-sarcolemmal membrane fraction. Thus, verapamil receptors exist in two locations in cardiac tissue but only in the sarcolemmal membrane are these receptors coupled to the dihydropyridine receptor.

We have studied allosteric effects of the Ca channel blockers d-(cis)-diltiazem, (+/-)-verapamil, and (S)- and (R)-devapamil on the specific binding of the 1,4-dihydropyridine derivative (+)-[3H]PN 200-110 to intact tissue cultured rat heart cells. In polarized cells (membrane potential, -38 +/- 4 mV) d-(cis)-diltiazem (5 microM) increased the affinity of the radiolabel 2-4-fold causing 100-187% enhancement of binding at (+)-PN 200-110 concentrations below 0.5 nM. (+/-)-Verapamil (0.1-3 microM) had a similar, although smaller, effect on (+)-PN 200-110 binding. The two enantiomers of devapamil were without effect. In depolarized cells (membrane potential, 0 mV) d-(cis)-diltiazem had a small and the phenylalkylamines a strong inhibitory effect on (+)-PN 200-110 binding, mainly due to a reduction of binding affinity. At 50% receptor occupation by the radioligand, (R)-devapamil, (+/-)-verapamil, and (S)-devapamil displaced 40, 55, and 75%, respectively, of specifically bound radiolabel. Half-maximal effects were reached with 50, 20, and 4.5 nM, respectively, of the three compounds. Compared with nominally Ca-free medium (containing 3-5 microM Ca), addition of 1.25 mM CaCl2 caused an increase in the maximal binding capacity for (+)-PN 200-110 in both polarized and depolarized cells. However, Ca had only marginal effects on the allosteric interactions between (+)-PN 200-110, d-(cis)-diltiazem, and verapamil. We conclude from our results that positive cooperative interactions between Ca channel blockers prevail under conditions in which the voltage-dependent Ca channel can fluctuate between closed, open, and inactivated states. Negative cooperativity is usually observed under conditions in which all channels are inactivated (depolarized cells, fragmented membranes). Therefore, it is impossible to predict the type and the extent of allosteric interactions in vivo from studies in cell homogenates.