Distinct effects of ω-toxins and various groups of Ca 2+-entry inhibitors on nicotinic acetylcholine receptor and Ca 2+ channels of chromaffin cells

Abstract

The effects of ω-toxins and various Ca 2+ antagonist subtypes on the 45Ca 2+ entry into bovine adrenal medullary chromaffin cells stimulated via nicotinic acetylcholine receptors or via direct depolarization with K +, have been compared. The conditions selected to stimulate the 45Ca 2+ entry consisted of a 60-s period of exposure of cells to 100 μM of the nicotinic acetylcholine receptor agonist dimethylphenylpiperazinium or to 70 mM K +. The N-type voltage-dependent Ca 2+ channel blockers ω-conotoxin GVIA and MVIIA (1 μM) inhibited 45Ca 2+ entry stimulated by dimethylphenylpiperazinium or K + by around 25–30%. The P-type Ca 2+ channel blocker ω-agatoxin IVA (10 nM) did not affect the dimethylphenylpiperazinium nor the K + responses; 1 μM (Q-channel blockade) inhibited both responses by around 50%. The N/P/Q-type Ca 2+ channel blocker ω-conotoxin MVIIC (1 μM) inhibited the K + evoked 45Ca 2+ entry by 70%, while dimethylphenylpiperazinium was blocked by 50% ( P<0.001). The L-type Ca 2+ channel blockers nifedipine, furnidipine, diltiazem or verapamil (3 μM each) inhibited much more the dimethylphenylpiperazinium than the K + response. The dimethylphenylpiperazinium signal was blocked 71, 88, 89, and 53%, respectively, by nifedipine, furnidipine, diltiazem and verapamil, and the K + response by 38, 29, 22, and 10%. Combined ω-conotoxin MVIIC (1 μM) and furnidipine (3 μM) blocked 100% of the K + evoked 45Ca 2+ entry. However, combined ω-conotoxin GVIA (1 μM), and furnidipine left unblocked 50% of the K + response. The `wide spectrum' Ca 2+ channel antagonists flunarizine or dotarizine (3 μM each) blocked the dimethylphenylpiperazinium and the K + responses to a similar extent (50%); cinnarizine (3 μM) inhibited more the dimethylphenylpiperazinium (82%) than the K + response (21%). At 3 μM, the highly lipophilic β-adrenoceptor antagonist (±)-propranolol, reduced by 68% the dimethylphenylpiperazinium signal and by 23% the K + signal. Other high lipophilic β-adrenoceptor antagonists such as metoprolol and labetalol, reduced little the dimethylphenylpiperazinium and the K + responses. The highly lipophilic agent penfluridol blocked the dimethylphenylpiperazinium response by 30% and the K + response by 50%. One of the least lipophilic compounds tested, (+)-lubeluzole, blocked by 40% the dimethylphenylpiperazinium and the K + responses. These data are compatible with the idea that the various ω-toxin peptides used to separate pharmacologically the different voltage-dependent Ca 2+ channels expressed by neurones, do not block the neuronal nicotinic acetylcholine receptor ion channel. In contrast the L-type Ca 2+ channel blockers do block the nicotinic acetylcholine receptor ionophore. Lipophilicity of the compounds is not a requirement for Ca 2+ channel or nicotinic acetylcholine receptor blockade.