Separation of two pathways for calcium entry into chromaffin cells

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Abstract
1 The effects of various drugs on 45Ca + 40Ca 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 40Ca, with 45Ca as a radiotracer, unstimulated cells took up an average of 0.13 fmol/cell 40Ca and 772 c.p.m./106 cells of 45Ca (n = 76). Upon stimulation with DMPP (100μm 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 μm, respectively for DMPP and K). A similar picture emerged with Cd ions, though Cd exhibited an IC50 against K (1.86 μm) slightly lower than the IC50 against DMPP (8.14 μm). 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 μm 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 μm). At 10 μm, 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.