EFFECT OF SUBTYPE-SPECIFIC CA2-ANTAGONISTS AND CA2+-FREE MEDIA ON THEFIELD STIMULATION-EVOKED RELEASE OF ATP AND [H-3] ACETYLCHOLINE FROM RAT HABENULA SLICES()

The involvement of different subtypes of voltage-sensitive Ca2+ channe ls in the initiation of field stimulation-induced endogenous adenosine triphosphate (ATP) and [H-3]acetylcholine ([H-3]ACh) release was inve stigated in the superfused rat habenula slices. ATP, measured by the l uciferin-luciferase assay, and [H-3]ACh were released simultaneously f rom the tissue in response to low frequency electrical stimulation (2 Hz, 2.5 msec, 360 shocks). The N-type Ca2+ channel blocker omega-conot oxin GVIA (omega-CgTX, 0.01-1 mu M) reduced the stimulation-evoked rel ease of ATP and [3H]ACh in a dose-dependent manner. Similarly, the P-t ype Ca2+ channel antagonist omega-agatoxin IVA (omega-Aga IVA) (0.05 m u M) and the inorganic Ca2+ channel blocker Cd2+ (0.2 mM) inhibited th e outflow of both transmitters, while Ni2+ (0.1 mM) was without signif icant effect. A high correlation was observed between the percent inhi bition of ATP release and percent inhibition of ACh release caused by the different Ca2+ antagonists. Long-term perfusion (i.e., 90 min) wit h Ca2+ free solution inhibited the evoked-release of ATP and [H-3]ACh. In contrast, perfusion of slices with the same media for a shorter ti me (i.e., 20 min) did not reduce the release of [H-3]ACh and ATP but e ven increased the evoked-release of ATP about fourfold. The breakdown of extracellular ATP was not blocked under low [Ca2+](0) condition, me asured by the creatine phosphokinase assay and HPLC-UV technique. Appl ication of extra- or intracellular Ca2+ chelators, and dipyridamole (2 mu M), the nucleoside transporter inhibitor, did not reduce the exces s release of ATP after short-term perfusion with Ca2+-free media. Tetr odotoxin (TTX, 1 mu M), while inhibiting the majority of ATP release u nder normal conditions, was also unable to reduce release under low [C a2+](0) conditions. In summary, we showed that both N- and P-type Ca2 channels are involved in the initiation of electrical stimulation-evo ked release of ATP and [H-3]ACh in the rat habenula under normal extra cellular calcium concentration. Under low [Ca2+](0) conditions an addi tional release of ATP occurs, which is not associated with action pote ntial propagation.