CHARACTERIZATION OF CALCIUM CHANNEL RECEPTORS IN THE HEART AND BRAIN (DIHYDROPYRIDINE, NIFEDIPINE, NITRENDIPINE).
AuthorLEE, HOWARD RUSSELL.
KeywordsCalcium in the body.
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PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractRecently, [³H]nitrendipine ([³H]NTD), a substituted 1,4-dihydropyridine calcium channel antagonist, has been used to label in vitro dihydropyridine calcium channel receptor sites in cardiac and smooth muscle, in the brain, and in other peripheral tissues. We have demonstrated the in vivo labeling of [³H]NTD to brain and peripheral tissues. This specific binding is decreased in a dose dependent manner by pretreatment with increasing doses of nifedipine. Thin layer chromatography demonstrates that the bound tritium species is predominantly the unmetabolized, parent [³H]NTD for the cerebral cortex and the left ventricle, whereas in the liver, the main species are metabolites of NTD. The regional distribution of [³H]NTD specific binding parallels that of in vitro studies. Notable exceptions include the aorta, skeletal muscle, and the ileum. The complex hemodynamic effects of NTD may explain differences noted in these in vivo studies as compared to in vitro studies. A highly sensitive assay for dihydropyridine receptors in cerebral cortical and cardiac homogenates using [³H](+)PN 200-110 ([³H]PN), is described. [³H]PN shows 10-12 fold higher affinity for its binding site than [³H]NTD at physiologic temperatures, and exhibits lower nonspecific binding. D-cis diltiazem and verapamil interact with [³H]PN in an allosteric manner. D-cis diltiazem and verapamil decrease and increase, respectively, the K(d) of the [³H]PN-receptor complex without significantly altering the B(max) in both cerebral cortex and heart. The [³H]PN assay is a distinct improvement over the [³H]NTD assay, and should be a valuable tool in the further study of the dihydropyridine receptor binding site. A radioreceptor assay was developed to measure the free (unbound) and total NTD levels in serum. Standard curves are reproducible and linear from 4 x 10⁻¹¹ to 4 x 10⁻⁹ M NTD. The limits of sensitivity of the assay are 0.1-0.2 picomoles/ml. Using equilibrium dialysis, NTD was found to be 93-98 per cent protein bound in human serum. There was no concentration dependency for protein binding from 1 to 100 ng/ml. A pharmacokinetic study in hypertensive patients revealed a peak total NTD serum level at 90-100 minutes. Preliminary studies suggest a possible dose-dependent accumulation of NTD during chronic drug therapy. The in vitro binding of [³H]NTD was studied in deoxycorticosterone-NaCl hypertensive rats and matched control rats. The hypertensive rats showed a significant decrease in brainstem [³H]NTD receptor density as compared to controls after 6 weeks of treatment. There were no receptor alterations in other selected brain regions and the heart. In light of the role of the brainstem in the development of various forms of experimental hypertension, this study provides evidence of selective alterations in the [³H]NTD sites which may be involved in the development of hypertension. Also, this study provides evidence that dihydropyridine binding sites may be under dynamic regulation.
Degree ProgramPharmacology and Toxicology