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 mu M) 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 mu M) 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 mu>M), a blocker of L-type Ca2+ channels. Blockade of the voltage dependent N-type Ca2+ channels with omega-conotoxin GVIA (omega-CTx GVIA; 0.1 mu M) and of the voltage-dependent P/Q-type channels with omega-agatoxin IVA (omega-AgaTx IVA; 0.2 mu M) inhibited the K+-induced increase in [Ca2+](i) by about 30 and 55%, respectively; these effects were additive. omega-Conotoxin MVIIC (omega-CTx MVIIC) at a concentration of 0.2 mu M, 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 omega-CTx GVIA (0.1 mu M), but was resistant to omega-AgaTx TVA (0.2 mu M) and omega-CTx MVIIC (0.2 mu M). 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 mu M 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 mu M ifenprodil. In contrast, KB-R 7943 (2-[2-[4(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; 1 and 3 mu M), 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, omega-CTx GVIA-and omega-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).