THE NICOTINIC ACETYLCHOLINE-RECEPTOR OF THE BOVINE CHROMAFFIN CELL, A NEW TARGET FOR DIHYDROPYRIDINES

The effects of 1,4-dihydropyridine derivatives on divalent cation transients and catecholamine release stimulated by either high K+ or the nicotinic receptor agonist dimethyl-phenyl-piperazinium (DMPP) have been compared in bovine adrenal chromaffin cells. The activation of Ca2+ entry pathways was followed by measuring Ca-45(2+) or Mn2+ uptake, or by the changes of [Ca2+]i in fura-2-loaded chromaffin cells, Various dihydropyridine Ca2+ channel blockers (nimodipine, PCA50938, nifedipine, nitrendipine, furnidipine) abolished the DMPP-mediated effects, but prevented only partially the activation by high [K+]0 of Ca-45(2+) uptake. The IC50 for DMPP-induced activation was around 1 muM. The L-type Ca2+ channel activator Bay K 8644 potentiated the uptake of Ca-45(2+) induced by K+ depolarization at concentrations between 10 nM and 1 muM, but completely inhibited the uptake of Ca-45(2+) by DMPP (IC50, 0.9 muM). Both high [K+]0 and DMPP produced membrane depolarization as measured using bis-oxonol. The DMPP-evoked, but not the K+-evoked membrane depolarization was prevented by Na+ removal, suggesting that the depolarization was due to Na+ entry through the acetylcholine receptor ionophore. Nimodipine at 10 muM abolished the depolarization induced by DMPP, leaving the K+-evoked depolarization unaffected. Tetrodotoxin (2 muM) did not affect the DMPP- or high K+-mediated cell depolarization. Whole-cell inward current evoked by 100 muM DMPP (I(DMPP)) was measured in cells voltage-clamped at -80 mV. Nimodipine (10 muM) reduced I(DMPP) by 36%; Bay K 8644 (10 muM) inhibited I(DMPP) by 67%. DMPP-evoked catecholamine release from superfused chromaffin cells was reduced by over 90% with 10 muM nimodipine; in contrast, K+-evoked release was decreased by 20%. The results suggest that the nicotinic acetylcholine receptor of bovine chromaffin cells contains a site for dihydropyridines whose occupation blocks Na+ entry through the receptor ionophore. This limits the ensuing membrane depolarization, firing of action potentials, recruitment of Ca2+ channels and entry of Ca2+ in the cells, leading to a decrease in catecholamine secretion.