Calcium Channel Antagonist Binding Sites Research Articles

Comparative potencies of calcium channel antagonists and antischizophrenic drugs on central and peripheral calcium channel binding sites

Dihydropyridines are potent agents on [3H]nitrendipine binding sites in heart and brain membranes. Like the phenylalkylamines, they are slightly more active on heart than on brain [3H]nitrendipine binding sites. On the other hand, the diphenylalkylamines, the diphenylpiperazines and the antischizophrenic drugs of the diphenylbutylpiperdine type are more potent on brain [3H]nitrendipine binding sites. The findings suggest tissue heterogeneity of [3H]nitrendipine binding sites and the possible development of diphenylbutylpiperidine-diphenylbutylpiperazine analogues that could selectively act on brain calcium channel antagonist binding sites.

The effects of halothane on voltage-dependent calcium channels in isolated Langendorff-perfused rat heart.

Halothane has been previously shown in vitro to decrease both the inward calcium current in isolated cells and the density of calcium antagonist binding sites in cardiac sarcolemmal membranes prepared from several species, including humans, presumably contributing to the negative inotropic effects seen with volatile anesthetics. In this study we examined whether halothane produced similar changes in calcium channel antagonist binding characteristics ex vivo in an intact perfused heart by using isradipine, a dihydropyridine calcium channel blocker that binds specifically to the alpha 1 subunit of the L-type voltage-dependent calcium channel. The rat hearts were perfused by the Langendorff method in the presence of halothane and unlabeled isradipine. After the hearts were homogenized and prepared into membranes, a radioligand binding assay was performed and binding curves obtained. Data were analyzed by nonlinear regression analysis of a one-site binding equation and were evaluated by a paired t test. Halothane protected or inhibited the binding of unlabeled isradipine to calcium channels in a dose-dependent manner such that as the halothane is removed during the membrane preparation process, previously obscured sites were then available for specific binding of the radioligand. The sites that were protected by halothane had a lower affinity for [3H]-isradipine than controls. In both isolated membranes and the intact heart, halothane changes the availability of calcium channel antagonist binding sites, indicating a change in conformation of the voltage-dependent calcium channel in the presence of anesthetic. This change may result from a direct effect on the protein or from an indirect effect mediated through the membrane lipid bilayer. It also is demonstrated that halothane "protected" channels are probably a modified class of channels compared to those in control tissues as exemplified by the much lower affinity that the protected channels have for [3H]-isradipine. We conclude that a major mechanism by which halothane depresses contractility is mediated through the voltage-dependent calcium channel, and this process results from a conformational change in the channel.

Altered sarcolemmal calcium channel density and Ca 2+-pump ATPase activity in tachycardia heart failure

Whether sarcolemmal (SL) calcium handling is altered in endstage heart failure produced by chronic rapid pacing is not known. To investigate this we paced 7 rabbits at a rate of 400 beats/min for 35 ± 11 days. 6 animals served as non-paced controls. Purified left ventricular SL membranes were then prepared and tested for [ 3H]-nitrendipine binding and (Ca 2++Mg 2+)-dependent ATPase (Ca 2+-pump) activity. Results show a 50% reduction in calcium channel antagonist binding sites with B max values reduced from 450 ± 40 to 230 ± 8 fmoles/mg protein in response to chronic rapid pacing ( P < 0.01). This change was accompanied by a modest decrease in K d from 0.29 ± 0.09 to 0.22 ± 0.03 nM (not significant). V max values for the SL Ca 2+-pump ATPase were decreased from 387 to 164 nmoles/mg protein/min ( P < 0.01) with K Ca 2+ values reduced from 0.91 to 0.28 μM Ca 2+ ( P < 0.05) in response to tachycardia induced failure as compared to controls. ATPase activity in both groups was very sensitive to 25 μM calmidazolium and 5 μM vanadate. Results from this study indicate that both a reduction in SL calcium channel density and decrease in SL Ca 2+-pump ATPase activity are evident in tachycardia heart failure. We conclude that sarcolemmal calcium handling is altered in heart failure induced by chronic rapid pacing and that such changes may contribute to systolic dysfunction associated with this model of heart failure.

The interaction of BBR 2160 with calcium channel antagonist binding sites

The interaction of BBR 2160 with calcium channel antagonist binding sites

Genetic up-regulation of calcium channels in a cellular model of ethanol dependence

Bovine adrenal chromaffin cells were maintained in culture medium containing ethanol (200mM) for 6 days. Cultures maintained in ethanol were viable and were morphologically similar to controls. There was a greater than 100% increase in the number of [ 3H]dihydropyridine calcium channel antagonist binding sites on the cell membranes from ethanol-treated cultures, which could be prevented by concomitant exposure to cycloheximide (5 μg·ml −1) and the mRNA synthesis inhibitor, anisomycin (10 μg·ml −1) implicating de novo synthesis of protein and genetic regulation, respectively. Release of catecholamines, induced by 18mM K +, from cultures grown in ethanol was enhanced. The increased release of catecholamines was inhibited by nM concentrations of dihydropyridine calcium channel antagonists, implying that an increase in the number of dihydropyridine-sensitive calcium channels accounts for this functional alteration.

Reduction of calcium channel antagonist binding sites by oxygen free radicals in rat heart

In view of the importance of Ca 2+-channels in controlling the entry of Ca 2+ into the myocardium, this study was undertaken to examine the effects of oxygen free radicals on the binding of Ca 2+-channel antagonists in rat heart by employing [ 3H]-nitrendipine as a ligand. Isolated heart membranes were incubated with xanthine + xanthine oxidase (a superoxide anion radicals generating system), hydrogen peroxide (an activated species of oxygen), or hydrogen peroxide + Fe 2+ (a hydroxyl radicals generating system). The assay of the [ 3H]-nitrendipine binding activity revealed that the maximal number of binding sites ( B max) were reduced in a time-dependent manner by superoxide radicals without any changes in the binding constant ( K d); a significant reduction of B max was seen after incubating membranes with xanthine + xanthine oxidase for a 10-min-period. Superoxide dismutase showed a protective effect on the superoxide radicals induced reduction in B max. Both hydrogen peroxide and hydroxyl radicals also depressed the B max for [ 3H]-nitrendipine binding without any significant change in K d; catalase and mannitol showed protective effects on hydrogen peroxide or hydroxyl radicals induced depression in B max, respectively. These results indicate that oxygen free radicals may reduce the number of Ca 2+-channels in the cell membrane and this change may contribute towards decreasing the voltage-dependent Ca 2+ influx in the cardiac cell.

Distribution of the ω-conotoxin receptor in rat brain. An autoradiographic mapping

The distribution of [ 125I]ω-conotoxin GVIA binding sites, the putative voltage-sensitive calcium channels, was studied by an autoradiographic method in the rat brain. The toxin binding sites were distributed throughout the brain in a highly heterogeneous manner. The highest density of the binding sites was observed in the cerebral cortex, hippocampus, amygdaloid complex, substantia nigra, caudate putamen, superior colliculus, nucleus of the solitary tract, and the dorsal horn of the cervical spine. The glomerular layer of the olfactory bulb, molecular layer of the cerebellar cortex, and posterior lobe of the hypophysis showed intermediate density but the density was higher than in the surrounding areas. The globus pallidus, thalamic areas, inferior olive, and pontine nuclei showed low density, while no binding sites were observed in the white matter tract regions such as the internal and external capsule, corpus callosum, fimbria of the hippocampus, fornix, stria medullaris of the thalamus, and fasciculus retroflexus. This distribution of ω-conotoxin binding sites indicates that the toxin binding sites are localized in those areas of the brain enriched in synaptic connections. This distribution pattern resembles that reported for voltage-sensitive sodium channels but it differs from that of the binding sites of dihydropyridines and verapamil. These results suggest that ω-conotoxin recognizes different molecules from organic calcium channel antagonist binding sites and that ω-conotoxin-sensitive voltage-sensitive calcium channels are concentrated in the synaptic zones and play a key role in the excitation-secretion coupling of neurotransmitters.

Dihydropyridine Binding Sites in Aerobically Perfused, Ischemic, and Reperfused Rat Hearts

The time course of normothermic (37 degrees C) and hypothermic (22 degrees C) global ischemia, and of post-ischemic reperfusion on the affinity (KD), selectivity, and density (Bmax) of the high-affinity 1,4 dihydropyridine (DHP) calcium channel antagonist binding sites were studied in isolated rat heart membranes, using (+)[3H]PN 200-110. Short periods (10 or 20 min) of ischemia at either 37 degrees C or 22 degrees C altered neither the KD nor Bmax of these binding sites as compared with aerobic controls. By contrast, longer periods of normothermic ischemia (30 or 60 min) caused a reduction (p less than 0.05) in Bmax (36% after 30 min and 32.7% after 60 min) without change of KD. This reduction in Bmax was reversed by 15-min reperfusion at 37 degrees C after 30 but not 60-min ischemia. There was no effect of ischemia (up to 60 min) on the density of DHP binding sites under hypothermic conditions. Irrespective of the experimental conditions the selectivity of the binding sites was maintained, with (+)PN 200-100 greater than (-)Bay K8644 greater than (-)PN 200-110 = (+)Bay K8644 much greater than (-)D600 in displacing (+)[3H]PN 200-110, and D-cis diltiazem stimulating the binding. These results show that the ischemia-induced change in Bmax is specific, reversible, and time- and temperature-dependent.

Characterization of calcium channel antagonist binding sites labeled by [ 3H]nitrendipine in porcine coronary artery and aorta

The receptor sites for dihydropyridine calcium channel antagonists in porcine stem coronary artery were characterized with [ 3H]nitrendipine (NTD), and were compared with those in the thoracic aorta. Specific [ 3H]NTD binding in the coronary artery and aorta was saturable, reversible and of high affinity. These sites showed a pharmacological specificity and stereoselectivity characteristic of the receptor sites for dihydropyridine calcium channel antagonists. The pharmacological potencies of nitrendipine, nifedipine, nisoldipine, (+)-PN 200-110 and (−)-PN 200-110 in the isolated porcine coronary artery correlated well with their potencies in competing for [ 3H]NTD binding sites. The Hill coefficients for the competition curves for these antagonists in the porcine coronary artery and aorta were close to one. Verapamil partially inhibited specific [ 3H]NTD binding in both tissues. The [ 3H]NTD binding was increased by Mg 2+ and Ca 2+ but was markedly reduced by EDTA. Thus, the present study has shown that [ 3H]NTD selectively labels the pharmacologically relevant dihydropyridine receptors in the porcine coronary artery and that there is no significant difference between the binding characteristics of the receptor sites in the coronary artery and aorta of pigs.

Solubilization of Calcium Channel Antagonist Binding Sites from Rat Brain

Calcium antagonist binding sites were solubilized from rat brain membranes using the detergent 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate (CHAPS). CHAPS-solubilized [3H]nitrendipine binding sites are saturable over a range of 0.05-4 nM and Scatchard analysis reveals a single, high-affinity (KD = 0.49 +/- 0.10 nM), low-capacity (Bmax = 56 +/- 4 fmol/mg of protein) binding site. Reversible ligand competition experiments using solubilized binding sites demonstrated appropriate pharmacologic specificity, with dihydropyridines (nifedipine = nitrendipine greater than Bay K 8644) completely displacing binding, verapamil partially displacing binding, and diltiazem enhancing binding, as previously described in membrane preparations. Lyophilized Crotalus atrox venom was purified by ion exchange chromatography followed by gel filtration to a single peptide band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This fraction of molecular weight 60,000 competitively inhibits [3H]nitrendipine binding to both membrane and soluble preparations with an IC50 of 5 micrograms/ml. This polypeptide should serve as a useful ligand for future efforts in purifying the dihydropyridine calcium channel binding site in brain.

Calcium Channel Receptor Binding Studies for Diltiazem and Its Major Metabolites

Pharmacologically distinct but allosterically interacting calcium channel antagonist binding sites have recently been identified using radiolabeled dihydropyridine derivatives (e.g., [3H]nitrendipine) and the benzothiazepine [3H]diltiazem. Whereas the functional significance of the dihydropyridine calcium channel antagonist receptor is well documented, it remains to be established whether drug interactions with the recognition site for [3H]diltiazem within the slow calcium channel or the allosteric interaction of the diltiazem binding site with the dihydropyridine receptor are of physiological significance. In a study of structure-activity relationships, we therefore examined the effects of diltiazem and five of its analogs on the binding of [3H]diltiazem and [3H]nitrendipine to the rat cerebral cortex. In parallel, we studied the effects of these drugs on the spontaneous myogenic contractions of the rat portal vein, a functional test of calcium antagonism. The diltiazem analogs used in this study correspond to its major metabolites in humans, i.e., N-desmethyl-(MA, desacetyl-(M1), N-desmethyl, desacetyl-(M2), O-desmethyl, desacetyl (M4), N-desmethyl, O-desmethyl, desacetyl-diltiazem (M6). Unlabeled diltiazem inhibited [3H]diltiazem binding at 37 degrees C with a pIC50 [-log IC50 (M)] of 6.87. pIC50 values for M1, MA, M2, M4, and M6 were 6.72, 6.49, 6.03, 5.51, and 5.33, respectively. pIC50 values for these drugs on [3H]diltiazem binding were significantly correlated (p less than 0.01) with their pEC50 values for enhancement of [3H]nitrendipine binding to cerebral cortical membranes at 37 degrees C. Maximal enhancement of [3H]nitrendipine binding by diltiazem, M1, MA, M2, M4, and M6 was 73, 50, 9.7, 11, 12, and 52%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic, benzodiazepine, gaba, chloride channel and other binding sites in frontal cortex in hepatic coma in man

1. 1. Alterations in several neurotransmitter systems in brain have been implicated in the pathophysiology of hepatic coma (HC). Studies on human autopsy material are few. We investigated H-quinuclidlnylbenzllate (QNB), 3H-spiperone, 3H-imipramine, 3H-PN-200-110, 3naloxone, 3H-Flunitrazepam, 3H-muscimol, 35S-t-butylbicyclophosphothionate and 3H-cyclohexyladenosine binding sites In frontal cortex from seven patients with HC and five controls. 2. 2. The density of 3H-QNB binding sites was significantly decreased and the affinity slightly increased in HC. The functional significance of these selective changes in muscarinic receptor binding sites Is unclear. Further studies evaluating cholinergic function In HC are Indicated. 3. 3. Acute studies in animals point to an Increase In GABA and BZ binding sites in HC. The present results show that the BZ/GABA-receptor-chloride-Ionophore complex is unchanged in HC in man. 4. 4. Serotonergic (5HT-2), adenosine (A-l), imipramlne (5HT uptake sites), opiate (naloxone) and calcium channel antagonist binding sites are unchanged in HC.

3HNitrendipine binding sites are decreased in the substantia nigra and striatum of the brain from patients with Parkinson's disease

We examined the degree of binding of the calcium antagonist, [ 3Hnitrendipine ([ 3HNDP), in the prefrontal cortex, caudate nucleus, putamen, pallidum and substantia nigra obtained at autopsy from patients with Parkinson's disease. The specific bindings of [ 3HNDP were significantly reduced in the caudate nucleus, putamen and substantia nigra, as compared to the relevant controls. Scatchard analyses revealed that these reductions resulted from decreased in the apparent maximum number of binding sites (B max). The affinity constants ( K d ) remained unchanged. Thus, it is highly likely that calcium channel antagonist binding sites on nigral dopamine (DA) neurons may be lost in the degenerative process of Parkinson's disease.

Enhancement of brain calcium antagonist binding by phencyclidine and related compounds.

The abilities of compounds structurally or pharmacologically related to phencyclidine to increase the apparent affinity of the [3H]dihydropyridine calcium channel antagonist [3H]nitrendipine were examined in lysed synaptosomal membrane preparations of rat brain. The p-bromo analog of phencyclidine (1-(1-(4-bromophenyl)cyclohexyl)piperidine) was the most efficacious compound tested in enhancing the apparent affinity of [3H]nitrendipine. The efficacy of this compound was approximately two-fold greater than PCP. The stereoisomers of PCMP (1-(1-phenylcyclohexyl-3-methylpiperidine) were also more efficacious than phencyclidine, although only a small degree of stereoselectivity was observed. Levoxadrol, dexoxadrol and the enantiomers of ketamine did not potentiate [3H]nitrendipine binding. The enantiomers of SKF 10047 (n-allylormetazocine), dextrorphan, levorphanol and the ion channel toxins histrionicotoxin and pumiliotoxin-B also increased the apparent affinity of [3H]nitrendipine, while several local anesthetics and mu-opiate receptor ligands were without effect. These studies suggest that the ability of phencyclidine and structurally related compounds to increase the apparent affinity of [3H]nitrendipine is not mediated through an interaction with phencyclidine receptors, but may represent a unique site for allosteric modulation of neuronal dihydropyridine calcium channel antagonist binding sites.

The current status of the dihydropyridine calcium channel antagonist binding sites in the brain

The current status of the dihydropyridine calcium channel antagonist binding sites in the brain

Comparative autoradiographic distribution of calcium channel antagonist binding sites for 1,4-dihydropyridine and phenylalkylamine in rat, guinea pig and human brain

1. The in vitro autoradiographic distribution of calcium channel antagonist binding sites for 1,4-dihydropyridine and phenylalkylamine has been investigated in rat, guinea pig and human brain. 2. 1,4-dihydropyridine([ 3H] (+) PN200-110) and phenylalkylamine ([ 3H] (−) D-888) binding sites are identically distributed in the brain of the three mammalian species studied. 3. High densities of calcium antagonist binding sites are present in brain areas enriched in synaptic contacts such as the hippocampus, cortex and striatum. Low to moderate densities of sites are found in other regions such as the thalamus, hypothalamus and brain stem. 4. These data demonstrate the existence of specific calcium antagonist binding sites in mammalian brain including man. These sites are discretely distributed with highest concentrations present in the hippocampus and cortex. Moreover, the similar distribution of binding sites for [ 3H](+)PN200-110 and [ 3H](−) D-188 suggests that 1,4-dihydropyridine and phenylalkylamine bind to the same receptor site complex in mammalian brain.

Calcium channel antagonist binding sites labeled by 3H-nimodipine in human brain.

In vitro binding of 3H-nimodipine to human brain membranes is demonstrated in this study. This binding was specific and saturable, and had an apparent affinity constant (KD) of 0.27 nM. The maximal number of binding sites for 3H-nimodipine was 5.8 pmoles/gm wet weight of human frontal cortex. The binding was shown to be dependent on calcium, with half-maximal stimulation obtained at 3 X 10(-5) M CaCl2. Other 1,4-dihyropyridine calcium antagonists were shown to be competitive antagonists of 3H-nimodipine binding. In contrast, the calcium antagonists, verapamil and diltiazem, had complex interactions with 3H-nimodipine binding. These results represent the first identification of 3H-calcium antagonist binding sites in human brain, and they confirm that various calcium antagonist drugs may differ with respect to both their potency and their molecular site of action.