Separation of two pathways for calcium entry into chromaffin cells

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./10(6) cells of 45Ca (n = 76). Upon stimulation with DMPP (100 microM for 60 s) or K (59 mM for 60 s), 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 microM, respectively for DMPP and K). A similar picture emerged with Cd ions, though Cd exhibited an IC50 against K (1.86 microM) slightly lower than the IC50 against DMPP (8.14 microM). 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 microM 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 microM). At 10 microM, 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.