Identification of two calcium channel receptor sites for [3H]nitrendipine in mammalian cardiac and smooth muscle membrane (original) (raw)
Related papers
The EMBO Journal, 1991
The dihydropyridine binding site of the rabbit skeletal muscle calcium channel a, subunit was identified using tritiated azidopine and nitrendipine as ligands. The purified receptor complex was incubated either with azidopine or nitrendipine at an cal subunit to ligand ratio of 1:1. The samples were then irradiated by a 200 W UV lamp. The ligands were only incorporated into the a, subunit, which was isolated by size exclusion chromatography and digested either by trypsin (azidopine) or endoproteinase Asp-N (nitrendipine). Each digest contained two radioactive peptides, which were isolated and sequenced. The azidopine peptides were identical with amino acids 13-18 (minor peak) and 1428-1437 (major peak) of the primary sequence of the skeletal muscle a, subunit. The nitrendipine peptides were identical with amino acids 1390-1399 (major peak) and 1410-1420 (minor peak). The sequence from amino acids 1390 to 1437 is identical in the a, subunits of skeletal, cardiac and smooth muscle and follows directly repeat IVS6. These results indicate that dihydropyridines bind to an area that is located at the putative cytosolic domain of the calcium channel.
Binding of [3H]nimodipine to cardiac and smooth muscle membranes
European Journal of Pharmacology, 1982
Specific binding of [3H]nimodipine to membranes from rat ventricle and guinea pig ileal longitudinal smooth muscle was studied. Dissociation constants were 0.24 and 0.12 nM, and the maximal number of binding sites were 0.4 and 0.75 pmol/mg protein for cardiac and smooth muscle, respectively. The values obtained for both types of muscle were similar to those obtained for [3H]nitrendipine binding, as were the potencies of a series of dihydropyridines for competing with [3H]nimodipine. These results support the hypothesis that the binding site characterized is that mediating the pharmacological effects of these compounds. Calcium antagonists Nifedipine Nitrendipine Receptor binding Calcium channel blocker
Circulation research, 1987
The interaction of 1,4-dihydropyridine derivatives with their receptors on voltage-dependent calcium channels in cardiac membranes was studied to determine if there are basic differences in the binding properties of ligands that cause inhibition or activation of calcium channels. The binding characteristics of 6 pure stereoisomers, (-) and (+)202-791, (-) and (+)Bay k 8644, (-) and (+)PN 200-110, as well as racemic Bay k 8644 and nitrendipine, were compared. Competition studies using the cold ligands and 3 different radiolabelled dihydropyridines, (+)[ J H]PN 200-110, (±)['H]nitrendipine, and (±)['H]Bay k 8644, showed that, for each combination tested, the labelled dihydropyridine could be displaced by the cold dihydropyridine. The binding reactions were markedly affected by temperature. The K d values for most compounds were significantly higher (5-19 times) at 0° than at 37° C. In contrast, the affinity of (+)PN 200-110 was similar at 0° and 37° C, but slightly higher at 25° C. A thermodynamic analysis indicated that the binding of the two pure isomers that are Ca 2+-channel activators ("agonists"), (-)Bay k 8644 and (+)202-791, was driven entirely by enthalpy and was associated with an unfavorable decrease in entropy. This was in marked contrast to the binding of the inhibitors ("antagonists"). The binding of (+)PN 200-110 and nitrendipine at low temperatures was driven largely or entirely by entropy. Other antagonist-binding reactions were driven mainly by enthalpy but were associated with favorable increases in entropy. The affinity of the three radiolabelled ligands for the dihydropyridine receptor differed 100 times and appeared to be due to large differences in dissociation rate constants for each of the ligands. The rates of dissociation of (+)[ 3 H]PN 200-110 and (±)[ 3 H]nitrendipine, but not of (±)[ 3 H]Bay k 8644, were significantly slowed by diltiazem, a calcium-channel inhibitor that binds to another receptor on the calcium channel. The results show that there were marked differences in the binding of the various dihydropyridines and suggest that the energetics of binding of Ca 2+-channel activators and inhibitors may be fundamentally different. (Circulation Research 1987;61:379-388) V oltage-dependent calcium channels are present in many cells. In certain cells, such as cardiac cells, these channels represent the major pathway through which Ca 2+ enters the cells. The opening and closing of calcium channels can be modulated by a wide variety of drugs' 2 that bind to receptors believed to be located on the channels themselves. 3 " 7 The major classes of these drugs are 1) the phenylalkylamines, such as verapamil and D600; 2) benzothiazepines, such as diltiazem; and 3) the 1,4-dihydropyridine derivatives, such as (+)PN 200-110, nifedipine, nimodipine, nitrendipine, and Bay k 8644. The dihydropyridine derivatives are interesting
Journal of Molecular and Cellular Cardiology, 1995
We have investigated the mechanism underlying the modulation of the cardiac L-type Ca 2؉ current by protein kinase C (PKC). Using the patch-clamp technique, we found that PKC activation by 4-␣-phorbol 12-myristate 13-acetate (PMA) or rac-1-oleyl-2-acetylglycerol (OAG) caused a substantial reduction in Ba 2؉ current through Ca v1.2 channels composed of ␣11.2, 1b, and ␣2␦1 subunits expressed in tsA-201 cells. In contrast, Ba 2؉ current through a cloned brain isoform of the Ca v1.2 channel (rbC-II) was unaffected by PKC activation. Two potential sites of PKC phosphorylation are present at positions 27 and 31 in the cardiac form of Ca v1.2, but not in the brain form. Deletion of N-terminal residues 2-46 prevented PKC inhibition. Conversion of the threonines at positions 27 and 31 to alanine also abolished the PKC sensitivity of Ca v1.2. Mutant Cav1.2 channels in which the threonines were converted singly to alanines were also insensitive to PKC modulation, suggesting that phosphorylation of both residues is required for PKC-dependent modulation. Consistent with this, mutating each of the threonines individually to aspartate in separate mutants restored the PKC sensitivity of Ca v1.2, indicating that a change in net charge by phosphorylation of both sites is responsible for inhibition. Our results define the molecular basis for inhibition of cardiac Ca v1.2 channels by the PKC pathway.
Naunyn-Schmiedeberg's Archives of Pharmacology, 1992
The ability of calcium antagonists and antiarrhythmic agents to potentiate the negative inotropic effects of calcium antagonists was investigated in guinea-pig left atria. The poteny of nitrendipine was enhanced by several amphiphilic agents by one order of magnitude or more (by pretreatment with quinidine or bepridil). The effect of preincubation with bepridil was investigated for a larger number of dihydropyridines. Only some of them were potentiated like nitrendipine. There was no potentiation between any two members of the same chemical group, i.e. between two dihydropyridines or two catamphiphilic calcium antagonists.
Drug Development Research, 2003
The selectivity of action of the clinically available calcium channel antagonists depends on a number of factors: 1) Mode of calcium mobilizationFintracellular and extracellular sources. 2) Class and subclass of calcium channel involved. 3) Pharmacokinetic considerations, including distribution and interactions with cell membranes. 4) State-dependent interactionsFvoltage-and frequency-dependent interactions. 5) Pathologic state of tissue and channel regulation. These factors will be illustrated with particular reference to the 1,4-dihydropyridine series of ligands. The Ca 2+ channel antagonists may be placed in several classes according to their rates of onset and offset of action, and the overall cardiovascular activity of an agent can profoundly alter its hemodynamic response. Additionally, third-generation agents such as lacidipine and lercenadipine partition extensively into plasma membranes according to membrane composition. Calcium mobilization through the L-type channel is the dominant target of the available antagonists: hence, systems that rely dominantly or exclusively on other mobilization processes are insensitive. Renal afferent and efferent arterioles that regulate glomerular flow and resistance are sensitive to the powerful vasoconstrictive effects of angiotensin II; however, only the processes in the afferent arteriole are sensitive to the Ca 2+ channel antagonists. An important and subtle process for conferring tissue selectivity of action is derived from statedependent interactions between channel and drug, whereby, according to the modulated-receptor hypothesis, drugs may exhibit preferential affinity for or access to different states of the channelFresting, open, or inactivated. Transitions between these states are determined by membrane potential and biochemical factors, including channel phosphorylation. The 1,4-dihydropyridine series exhibit significant and structure-dependent voltage-dependent interactions, which reflect a preferential interaction with open and/or inactivated channel states. The differential pharmacology of the L-type Ca 2+ channel is a further determinant of tissue selectivity. The cardiovascular system expresses three different a 1 subunitsFCa V 1.2 A-C Fand although a detailed pharmacological comparison is not yet available, there is sufficient evidence to indicate important differences. Thus, the 1,4-dihydropyridine nisoldipine interacts differentially with recombinant channels derived from cardiac and vascular smooth muscle. A number of disease states, both clinical and experimental, have been associated with changes in Ca 2+ channel expression and function, although the causal relationship is often not established. Thus, in cardiac hypertrophy and failure the majority of studies reveal a reduction in L-type channel number or function.