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(+)‐PN200‐l 10 and Ouabain Binding Sites in Purified Bovine Adrenomedullary Plasma Membranes and Chromaffin Cells
Author(s) -
Castillo Carlos J. F.,
Fonteríz Rosalba I.,
López Manuela G.,
Rosenheck Kurt,
García Antonio G.
Publication year - 1989
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1989.tb08536.x
Subject(s) - chemistry , membrane , dissociation constant , binding site , receptor , endocrinology , biochemistry , biology
Bovine adrenal medulla plasma membranes were purified by a differential centrifugation procedure using sucrose and Urografin discontinuous density gradients; the membranes were enriched 10‐ 12‐fold in acetylcholinesterase activity and [3H]ouabain binding sites. Specific (+)‐[3H]PN200‐1 10 binding to these membranes amounted to 90% of total binding and was saturabie and of high affinity (K D = 41 pM; B max = 119 fmol/mg of protein) with a Hill coefficient close to I, a result suggesting the presence of a single, homogeneous population of dihydropyridine receptors. The association and dissociation rate constants were, respectively, 7.5 × 10′ M‐′ rnin‐l and 0.023 min‐I. Unlabeled (+)‐PN200‐110 displaced (+)‐[3H]PN200‐ 1 10 binding with a potency 100‐fold higher than (‐)‐PN200‐1 10 (ICsO, 0.5 and 45 nM, respectively). Although the two enantiomers of BAY K 8644 completely displaced (+)‐[3H]PN200‐1 10 binding, they exhibited no stereoselectivity (ICsO, 69 and 83 nM, respectively). Whereas (i‐)‐nitrendipine very potently displaced (+)‐[3H]PN200‐l 10 binding (ICsO = 1.3 nM), verapamil and cinnarizine displaced the binding by only 30 and 40% at 1 FM, and diltiazem increased it by 20% at 10 pM. [3H]Ouabain bound to plasma membranes with a KD of 34 nM and a B max of 9.75 pmol/mg of protein, a figure 80‐fold higher than the B max for (+)‐PN200‐ 1 10. t3H]Ouabain also bound to intact chromaffin cells with a B max of 244 fmol/106 cells. This figure, together with the plasma membrane ouabain/(+)‐PNZOO‐ I I0 ratio of 80, allows the calculation of 1,884 (+)‐PN200‐110 binding sites/cell. Assuming a 1: 1 stoichiometry, this means that intact chromaffin cells contain ‐ 1.5 L‐type, dihydropyridine‐associated, voltage‐dependent Ca2+ channels/pm2 of surface area.