SEPARATION OF 2 PATHWAYS FOR CALCIUM ENTRY INTO CHROMAFFIN CELLS

1 The effects of various drugs on Ca-45 + Ca-40 uptake into cultured bovine adrenal chromaffin cells evoked by 1,1-dimethyl-4-phenylpiperazinium (DMPP) or high K, were studied. In the presence of 1 mM external Ca-40, with Ca-45 as a radiotracer, unstimulated cells took up an average of 0.13 fmol/cell Ca-40 and 772 c.p.m./10(6) cells of Ca-45 (n = 76). Upon stimulation with DMPP (100-mu-m for 60s) or K (59 mM for 60s), Ca uptake increased to 0.92 and 1 fmol/cell, respectively. 2 Flunarizine behaved as a potent blocker of both DMPP- and K-evoked Ca uptake (IC50 of 1.76 and 1.49-mu-M, respectively for DMPP and K). A similar picture emerged with Cd ions, though Cd exhibited an IC50 against K (1.86-mu-M) slightly lower than the IC50 against DMPP (8.14-mu-M). 3 Clear cut differences were observed with amiloride, guanethidine, nimodipine and nisoldipine which behaved as selective blockers of DMPP-mediated Ca uptake responses: IC50 values to block DMPP effects were 290, 27, 1.1 and 1.63-mu-M respectively for amiloride, guanethidine, nimodipine and nisoldipine. Amiloride blocked K-evoked Ca uptake by only 35% and guanethidine did not affect it. Nisoldipine inhibited K-evoked Ca uptake only partially at low concentrations (about 30%); a second blocking component was observed at the highest concentration used (10-mu-M). At 10-mu-M, nimodipine blocked K-evoked Ca uptake by 50%. 4 Thus, it seems that the nicotinic receptor mediated Ca uptake pathway can be pharmacologically separated from the K-activated pathway. The results are compatible with the hypothesis that in cultured bovine adrenal chromaffin cells, stimulation of nicotinic receptors recruits a single type of Ca channel which is sensitive to flunarizine, Cd, amiloride, guanethidine, nimodipine and nisoldipine. The results also suggest that K depolarization might be recruiting in addition to this channel, another Ca channel which is highly sensitive to Cd and flunarizine, resistant to nisoldipine, nimodipine and amiloride, and insensitive to guanethidine.