Involvement of different calcium channels in K+- and veratridine-induced increases of cytosolic calcium concentration in rat cerebral cortical synaptosomes

Intracellular calcium ion concentrations ([Ca²⁺]_i) in rat cerebral cortical synaptosomes were measured, using the calcium chelating fluorescence dye fura-2. The synaptosomes were depolarized by elevation of the extracellular K⁺ concentration or by addition of veratridine, which opens voltage-dependent Na⁺-channels and prevents their inactivation. Both enhancement of the concentration of extracellular K⁺ (up to 60 mM) and veratridine (1–100 μM) increased the [Ca²⁺]_i in a concentration-dependent manner. In the absence of extracellular Ca²⁺, the K⁺- and veratridine-induced increases in [Ca²⁺]_i were abolished, indicating that the increase in [Ca²⁺]_i was due to an influx of extracellular Ca²⁺. Tetrodotoxin (TTX), a blocker of the voltage-dependent Na⁺ channel, inhibited the veratridine-induced (10 μM) Ca²⁺ influx by more than 80%, while the K⁺-evoked (30 mM) increase of [Ca²⁺]_i was TTX-resistant. Both the K⁺- and the veratridine-induced Ca²⁺ influx were not reduced by nifedipine (1 μM), a blocker of L-type Ca²⁺ channels. Blockade of the voltage dependent N-type Ca²⁺ channels with ω-conotoxin GVIA (ω-CTx GVIA; 0.1 μM) and of the voltage-dependent P/Q-type channels with ω-agatoxin IVA (ω-AgaTx IVA; 0.2 μM) inhibited the K⁺-induced increase in [Ca²⁺]_i by about 30 and 55%, respectively; these effects were additive. ω-Conotoxin MVIIC (ω-CTx MVIIC) at a concentration of 0.2 μM, which may be assumed to block predominantly the Q-type Ca²⁺ channel, inhibited the K⁺-induced increase in [Ca²⁺]_i by 50%. The veratridine-induced increase in [Ca²⁺]_i was reduced by about 25% by ω-CTx GVIA (0.1 μM), but was resistant to ω-AgaTx IVA (0.2 μM) and ω-CTx MVIIC (0.2 μM). Mibefradil (former designation Ro 40-5967), a Ca²⁺ antagonist which blocks all types of voltage-dependent Ca²⁺ channels including the T and R channels, led to a concentration-dependent inhibition of the K⁺- and veratridine-induced increase in [Ca²⁺]_i (abolition at 10 μM mibefradil). Ifenprodil, another non-specific blocker of voltage-dependent Ca²⁺ channels, also inhibited the K⁺- and veratridine-induced increase in [Ca²⁺]_i in concentration-dependent manner and abolished it at 320 μM ifenprodil. In contrast, KB-R 7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; 1 and 3 μM), a highly potent and selective inhibitor of the Na⁺/Ca²⁺ exchanger (NCX1), failed to inhibit the K⁺- and veratridine-induced increase in [Ca²⁺]_i. It is concluded that the K⁺-induced increase in free cytosolic Ca²⁺ results from Ca²⁺ influx through voltage-dependent N- and, above all, Q-type Ca²⁺ channels. N-type Ca²⁺ channels also play a minor role in the veratridine-induced increase in [Ca²⁺]_i, but P/Q-type channels do not appear to be involved at all. The inhibition of the veratridine-induced, ω-CTx GVIA- and ω-AgaTx IVA-resistant increase in [Ca²⁺]_i by mibefradil and the failure of KB-R 7943 to inhibit this response are compatible with the suggestion that in rat cerebral cortical synaptosomes, Ca²⁺ influx via the R-type Ca²⁺ channel and/or another so far uncharacterized Ca²⁺ channel may substantially contribute to the veratridine-induced increase in [Ca²⁺]_i. Received: 7 March 1997 / Accepted: 9 September 1997