Multiple calcium channels are required for pituitary adenylate cyclase-activating polypeptide-induced catecholamine secretion from bovine cultured adrenal chromaffin cells

The effects of L-, N-, P- and Q-type calcium channel antagonists and (±)-BayK-8644 on catecholamine release induced by pituitary adenylate cyclase-activating polypeptide (PACAP-27) were investigated in bovine cultured adrenal chromaffin cells. PACAP-27 induced the release of 4-15% of the total cellular catecholamines over 7 min, with an EC₅₀ of 20 nM and the effect approaching maximum at 100 nM. Catecholamine release was fully dependent on the presence of extracellular calcium. The dihydropyridine nitrendipine which inhibits L-type calcium channels inhibited PACAP-27-induced secretion in a concentration dependent manner with an inhibition of 20-30% at 1 μM. In contrast, (±)-BayK-8644, which prolongs the opening of L-type calcium channels produced a concentration-dependent increase in PACAP-27-induced catecholamine release with 1 μM increasing release by 40-60%. Blockade of N-type calcium channels with ω-conotoxin GVIA reduced release by 5–15%. Block of P-type channels with low concentrations of ω-agatoxin IVA (≤ 30 nM) had no significant effect on release, while higher concentrations (100-300 nM) which block Q-type channels reduced release by up to 15%. ω-Conotoxin MVIIC, an antagonist of Q-type calcium channels and also of N- and P-type channels, inhibited release in a concentration-dependent manner with a near maximum effect of 30-50% produced by 300 nM. The combination of ω-conotoxin GVIA and ω-agatoxin IVA reduced release by 40-50%. Addition of ω-conotoxin MVIIC (300 nM) to the combination of ω-conotoxin GVIA (10 nM) and ω-agatoxin IVA (100 nM) did not inhibit catecholamine release more than with ω-conotoxin GVIA and ω-agatoxin IVA alone, indicating that 100 nM ω-agatoxin IVA was sufficient to block the Q-type calcium channels. When nitrendipine was used together with ω-conotoxin GVIA, ω-agatoxin IVA and ω-conotoxin MVIIC, catecholamine release induced by 20 nM or 100 nM PACAP-27 was reduced by 70–85%. Taken together these results suggest that influx of calcium through multiple different voltage-sensitive calcium channels mediate PACAP-27-induced catecholamine release from bovine chromaffin cells, and that L-, N- and Q-channels contribute to this response. Received: 11 March / Accepted: 20 June 1997