Effects of the putative P-type calcium channel blocker, R,R-(-)-daurisoline on neurotransmitter release.

ABSTRACT

The alkaloid and medicinal herb constituent, R,R-(-)-daurisoline, was originally reported to be a N-type Ca2+ channel blocker, but newer evidence indicates that it is a blocker of P-type Ca2+ channels. To clarify its specificity with respect to N- and P-channels, we compared its effects on the electrically induced release of endogenous glutamate, 3H-GABA and 3H-noradrenaline, from brain slices with those of omega-agatoxin IVA and omega-conotoxin GVIA. Like omega-agatoxin IVA (but with about 1000-fold lower potency), and unlike omega-conotoxin GVIA, R.R-(-)-daurisoline inhibited the release of 3H-GABA and glutamate, with IC50 values of 8 and 18 microM. However, inhibition particularly of 3H-GABA release was more complete than by omega-agatoxin IVA, indicating interaction with one or more additional voltage-sensitive Ca2+ channels, possibly the Q-type. Its potency to inhibit glutamate release elicited either electrically, by veratrine or by high concentrations of K+ was similar, in contrast to sodium channel blockers. The effects of R,R-(-)-daurisoline on the release of 3H-noradrenaline, 3H-dopamine and 3H-acetylcholine were in agreement with previous knowledge from experiments with omega-agatoxin IVA suggesting an involvement of P-channels. A weak inhibition of 3H-noradrenaline release at 10 microM, similar to that by omega-agatoxin IVA at 0.03 microM, was occluded by alpha 2-antagonistic properties and could be unmasked in presence of rauwolscine. At 10 microM, it also inhibited electrically evoked 3H-dopamine and 3H-5-hydroxytryptamine release and caused a marked spontaneous release of all three monoamines in a reserpine-like manner. Spontaneous and evoked release of 3H-acetylcholine was inhibited by about 25% at 10 microM. In radioligand binding studies, R,R-(-)-daurisoline interacted with alpha 1- and alpha 2-adrenoceptors, 5-HT2 and muscarinic cholinergic receptors with IC50 values close to 1 microM, and with mu opiate receptors even with 0.18 microM. Atropine reduced the weak inhibitory effect of R,R-(-)-daurisoline on 3H-acetylcholine release somewhat, suggesting that it was brought about by both P channel blockade and cholinergic agonist activity. The effect on 3H-GABA release was unaffected by naloxone, indicating that the interaction of R,R-(-)-daurisoline with mu opiate receptors is antagonistic. The pattern of effects on neurotransmitter release observed with R,R-(-)-daurisoline resembles that of omega-agatoxin IVA and supports previous electrophysiological data suggesting that the compound blocks P-type voltage-sensitive Ca2+ channels. However, the more complete blockade of amino acid release by R,R-(-)-daurisoline suggests interaction with additional Ca2+ channel subtypes. Although it does also possess other pharmacological properties, we think that the compound is suitable to test whether blockade of glutamate release via voltage-sensitive Ca2+ channels is a viable concept to obtain novel neuroprotective and/or anticonvulsant compounds.