Pharmacological characterisation of the calcium channels coupled to the plateau phase of KCl-induced intracellular free Ca 2+ elevation in chicken and rat synaptosomes

Abstract

The effect of various blockers of voltage operated calcium channels (VOCCs) was studied on the non-inactivating, plateau phase of KCl-induced intracellular free Ca 2+ ([Ca 2+] i) elevation in rat cortical and chicken forebrain synaptosomes. In chicken synaptosomes, ω-CgTx GVIA (0.1 nM to 1 μM) and ω-CgTx MVIIA (0.1 nM to 1 μM), both selective blockers of N-type Ca 2+ channels, produced a concentration-dependent inhibition of the plateau phase of [Ca 2+] i elevation. ω-CgTx GVIA (IC 50 value 28 nM) was more potent than ω-CgTx MVIIA (IC 50 value 78 nM), but at submaximal concentrations, took longer to reach its maximum effect (20 min for ω-CgTx GVIA; 10 min for ω-CgTx MVIIA). At 1 μM, the highest concentration tested, each toxin blocked > 85% of [Ca 2+] i elevation. The effect of ω-CgTx GVIA on the extent and time-course of inhibition of [Ca 2+] i elevation was maintained in a Na +-free, choline substituted, medium. ω-Aga IVA (300 nM), a selective blocker of P-type calcium channels, inhibited 28 ± 5% of [Ca 2+] i elevation. The effect of a combination of submaximal inhibitory concentrations of ω-CgTx GVIA (100 nM) and ω-Aga IVA (300 nM) was less than additive. In rat synaptosomes, ω-CgTx GVIA (1 μM) and ω-CgTx MVIIA (1 μM), blocked only 18 ± 5% and 17 ± 4% of the plateau phase of free Ca 2+ elevation, respectively. ω-Aga IVA produced a concentration-dependent inhibition of [Ca 2+] i elevation in this preparation. Threshold inhibition was observed at 1 nM, and maximum inhibition (64 ± 8%) at 1 μM. The effect of a combination of ω-Aga IVA (300 nM) and ω-CgTx GVIA (1 μM) was fully additive. L-type calcium channel blockers, nifedipine, verapamil and diltiazem blocked only a minor component of the plateau phase of [Ca 2+] i elevation in both rat and chicken synaptosomes whereas flunarizine and fluspirilene, two atypical Ca 2+ channel blockers, each blocked the majority (> 85%) of [Ca 2+] i elevation in both species. Our data suggest that: (a) ω-CgTx-sensitive N-type Ca 2+ channels are the predominant component of the plateau phase of KCl-induced [Ca 2+] i elevation in chicken synaptosomes; (b) ω-Aga IVA-sensitive P-type Ca 2+ channels are the predominant component of this phase of [Ca 2+] i elevation in rat synaptosomes; and (c) flunarizine and fluspirilene block ω-CgTx-sensitive, N-type Ca 2+ channels in chicken synaptosomes and ω-Aga IVA sensitive, P-type Ca 2+ channels in rat synaptosomes.