Effect of subtype-specific Ca(2+)-antagonists and Ca(2+)-free media on the field stimulation-evoked release of ATP and [3H]acetylcholine from rat habenula slices.

The involvement of different subtypes of voltage-sensitive (Ca2+ channels in the initiation of field stimulation-induced endogenous adenosine triphosphate (ATP) and [3H]acetylcholine ([3H]ACh) release was investigated in the superfused rat habenula slices. ATP, measured by the luciferin-luciferase assay, and [3H]ACH were released simultaneously from the tissue in response to low frequency electrical stimulation (2 Hz, 2.5 msec, 360 shocks). The N-type Ca(2+)-channel blocker omega-conotoxin GVIA (omega-CgTX, 0.01-1 microM) reduced the stimulation-evoked release of ATP and [3H]ACh in a dose-dependent manner. Similarly, the P-type Ca2+ channel antagonist omega-agatoxin IVA (omega-Aga IVA) (0.05 microM) and the inorganic Ca(2+)-channel blocker Ca2+ (0.2 mM) inhibited the outflow of both transmitters, while Ni2+ (0.1 mM) was without significant effect. A high correlation was observed between the percent inhibition of ATP release and percent inhibition of ACh release caused by the different Ca2+ antagonists. Long-term perfusion (i.e., 90 min) with Ca(2+)-free solution inhibited the evoked-release of ATP and [3H]ACh. In contrast, perfusion of slices with the same media for a shorter time (i.e., 20 min) did not reduce the release of [3H]ACh and ATP but even increased the evoked-release of ATP about fourfold. The breakdown of extracellular ATP was not blocked under low [Ca2+]0 condition, measured by the creatine phosphokinase assay and HPLC-UV technique. Application of extra- or intracellular Ca2+ chelators, and dipyridamole (2 microM), the nucleoside transporter inhibitor, did not reduce the excess release of ATP after short-term perfusion with Ca(2+)-free media. Tetrodotoxin (TTX, 1 microM), while inhibiting the majority of ATP release under normal conditions, was also unable to reduce release under low [Ca2+]0 conditions. In summary, we showed that both N- and P-type Ca2+ channels are involved in the initiation of electrical stimulation-evoked release of ATP and [3H]ACh in the rat habenula under normal extracellular calcium concentration. Under low [CA2+]0 conditions an additional release of ATP occurs, which is not associated with action potential propagation.