Distinct effects of omega-toxins and various groups of Ca2+-entry inhibitors on nicotinic acetylcholine receptor and Ca2+ channels of chromaffin cells

The effects of omega-toxins and various Ca2+ antagonist subtypes on the Ca-45(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 Ca-45(2+) entry consisted of a 60-s period of exposure of cells to 100 mu M of the nicotinic acetylcholine receptor agonist dimethylphenylpiperazinium or to 70 mM K+. The N-type voltage-dependent Ca2+ channel blockers omega-conotoxin GVIA and MVIIA (1 mu M) inhibited Ca-45(2+) entry stimulated by dimethylphenylpiperazinium or K+ by around 25-30%. The P-type Ca2+ channel blocker omega-agatoxin IVA(10 nM) did not affect the dimethylphenylpiperazinium nor the K+ responses; 1 mu M (Q-channel blockade) inhibited both responses by around 50%. The N/P/Q-type Ca2+ channel blocker omega-conotoxin MVIIC (1 mu M) inhibited the K+ evoked Ca-45(2+) entry by 70%, while dimethylphenylpiperazinium was blocked by 50% (P <0.001). The L-type Ca2+ channel blockers nifedipine, furnidipine,diltiazem or verapamil (3 mu M 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 omega-conotoxin MVIIC (1 mu M) and furnidipine (3 mu M) blocked 100% of the K+ evoked Ca-45(2+) entry. However, combined omega-conotoxin GVIA (1 mu M), and furnidipine left unblocked 50% of the K+ response. The 'wide spectrum' Ca2+ channel antagonists flunarizine or dotarizine (3 mu M each) blocked the dimethylphenylpiperazinium and the K+ responses to a similar extent (50%); cinnarizine (3 mu M) inhibited more the dimethylphenylpiperazinium (82%) than the K+ response (21%). At 3 mu M, the highly lipophilic beta-adrenoceptor antagonist(+/-)-propranolol, reduced by 68% the dimethylphenylpiperazinium signal and by 23% the K+ signal. Other high lipophilic beta-adrenoceptor antagonists such as metoprolol and labetalol, reduced little the dimethylphenylpiperaziniun and the K+ responses. The highly lipophilic agent perfluridol 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 omega-toxin peptides used to separate pharmacologically the different voltage-dependent Ca2+ channels expressed by neurones, do not block the neuronal nicotinic acetylcholine receptor ion channel. In contrast the L-type Ca2+ channel blockers do block the nicotinic acetylcholine receptor ionophore. Lipophilicity of the compounds is not a requirement for Ca2+ channel or nicotinic acetylcholine receptor blockade.