Co-incident signalling between µ-opioid and M₃-muscarinic receptors at the level of Ca²⁺ release from intracellular stores: Lack of evidence for InsP₃ receptor sensitisation

Activation of G(i)/G(o)-coupled opioid receptors increases [Ca2+](i) (intracellular free-Ca2+ concentration), but only if there is concomitant G(q)-coupled receptor activation. This G(i)/G(o)-coupled receptor-mediated [Ca2+](i) increase does not appear to result from further production of InsP(3) [Ins(1,4,5)P-3] in SH-SY5Y cells. In the present study, fast-scanning confocal microscopy revealed that activation of mu-opioid receptors alone by I muM DAMGO ([D-Ala, NMe-Phe, Gly-ol]-enkephalin) did not stimulate the InsP(3)-dependent elementary Ca2+-signalling events (Ca2+ puffs), whereas DAMGO did evoke Ca2+ puffs when applied during concomitant activation Of M-3 muscarinic receptors with 1 muM carbachol. We next determined whether mu-opioid receptor activation might increase [Ca2+](i) by sensitizing the InSP3 receptor to InsP(3). DAMGO did not potentiate the amplitude of the [Ca2+](i) increase evoked by flash photolysis of the caged InsP(3) receptor agonist, caged 2,3-isopropylidene-InsP(3), whereas the InsP(3) receptor sensitizing agent, thimerosal (10 muM), did potentiate this response. DAMGO also did not prolong the rate of decay of the increase in [Ca2+](i) evoked by flash photolysis of caged 2,3-isopropylidene-InsP(3). Furthermore, DAMGO did not increase [Ca2+](i) in the presence of the cell-membrane-permeable InsP(3) receptor agonist, InsP(3) hexakis(butyryloxymethyl) ester. Therefore it appears that mu-opioid receptors do not increase [Ca2+](i) through either InsP(3) receptor sensitization, enhancing the releasable pool of Ca2+ or inhibition of Ca2+ removal from the cytoplasm.