The nicotinic acetylcholine receptor of the bovine chromaffin cell, a new target for dihydropyridines

Abstract

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 Ca 2+ entry pathways was followed by measuring 45Ca 2+ or Mn 2+ uptake, or by the changes of [Ca 2+] i in fura-2-loaded chromaffin cells. Various dihydropyridine Ca 2+ channel blockers (nimodipine, PCA50938, nifedipine, nitrendipine, furnidipine) abolished the DMPP-mediated effects, but prevented only partially the activation by high [K +] 0 of 45Ca 2+ uptake. The IC 50 for DMPP-induced activation was around 1 μM. The L-type Ca 2+ channel activator Bay K 8644 potentiated the uptake of 45Ca + induced by K ++ depolarization at concentrations between 10 nM and 1 μM, but completely inhibited the uptake of 45Ca 2+ by DMPP (IC 50, 0.9 μM). 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 μM abolished the depolarization induced by DMPP, leaving the K +-evoked depolarization unaffected. Tetrodotoxin (2 μM) did not affect the DMPP- or high K +-mediated cell depolarization. Whole-cell inward current evoked by 100 μM DMPP (I DMPP) was measured in cells voltage-clamped at −80 mV. Nimodipine (10 μM) reduced I DMPP by 36%; Bay K 8644 (10 μM) inhibited I DMPP by 67%. DMPP-evoked catecholamine release from superfused chromaffin cells was reduced by over 90% with 10 μM 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 chromaffin cells contains a site for dihydropyridines whose occupation blocks Na + entry through the receptor ionophre. This limits the ensuing membrane depolarization, firing of action potentials, recruitment of Ca 2+ channels and entry of Ca 2+ in the cells, leading to a decrease in catecholamine secretion.