DENSITY OF APAMIN-SENSITIVE CA2-DEPENDENT K+ CHANNELS IN BOVINE CHROMAFFIN CELLS - RELEVANCE TO SECRETION()

Three objectives were defined when planning this study: (i) to identif y binding sites for [I-125]apamin in intact bovine adrenal medulla chr omaffin cells and to estimate their density and selectivity; (ii) to d etermine whether apamin modified the release of catecholamines evoked by brief pulses of dimethylphenylpiperazinium (DMPP, 1 or 5 mu M for 1 0 sec), histamine (10 mu M for 10 sec) or high K+ (20, 35 or 70 mM for 10 sec) applied to superfused cells; and (iii) to test whether apamin affected the profiles of the changes in cytosolic Ca2+ concentrations [Ca2+](i) obtained in suspensions of cells loaded with fura-2 and sti mulated with DMPP or histamine. At equilibrium, increasing concentrati ons of [I-125]apamin gave a saturation curve whose Scatchard transform ation produced a K-d of 132 pM and a B-max of 0.72 fmol/10(6) cells. Q uinine, tetraethylammonium, charybdotoxin or glibenclamide (blockers o f various subtypes of K+ channels) did not inhibit [I-125]apamin bindi ng. Binding was blocked by apamin and by d-tubocurarine, two blockers of small-conductance Ca2+-activated K+ channels (SK channels). The num ber of binding sites for [I-125]apamin amounted to approx. 900 per sin gle chromaffin cell, 0.72 sites per mu m(2) surface area. Apamin (1 mu M) enhanced the secretory response to histamine (10 mu M), DMPP (1 or 5 mu M) and high K+ (20 or 35 mM) by 2-3-fold. The response to 70 mM K+, however, was unaffected. Apamin also enhanced the peak [Ca2+](i) i ncrease produced by DMPP or histamine by approx. 30%. Overall, these r esults strongly support the hypothesis that under physiological condit ions, SK channels control some of the electrical activity of chromaffi n cells and indirectly, the opening of voltage-dependent Ca2+ channels , the access of Ca2+ to the secretory machinery and the rate of catech olamine release to the circulation from the intact adrenal gland.