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

The effects of ω-toxins and various Ca²⁺ antagonist subtypes on the ⁴⁵Ca²⁺ 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 ⁴⁵Ca²⁺ 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 Ca²⁺ channel blockers ω-conotoxin GVIA and MVIIA (1 μM) inhibited ⁴⁵Ca²⁺ entry stimulated by dimethylphenylpiperazinium or K⁺ by around 25–30%. The P-type Ca²⁺ 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 Ca²⁺ channel blocker ω-conotoxin MVIIC (1 μM) inhibited the K⁺ evoked ⁴⁵Ca²⁺ entry by 70%, while dimethylphenylpiperazinium was blocked by 50% (P<0.001). The L-type Ca²⁺ 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 ⁴⁵Ca²⁺ entry. However, combined ω-conotoxin GVIA (1 μM), and furnidipine left unblocked 50% of the K⁺ response. The ‘wide spectrum' Ca²⁺ 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 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.