9 research outputs found

    Molecular and functional properties of P2X receptors—recent progress and persisting challenges

    Full text link

    Co-incident signalling between the Gi/Go-coupled d-opoid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells

    No full text
    In SH-SY5Y cells, activation of delta -opioid receptors with [D-Pen(2,5)]-enkephalin (DPDPE; 1 muM) did not alter the intracellular free Ca2+ concentration [Ca2+](i). However, when DPDPE was applied during concomitant Gq-coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine-M, it produced an elevation of [Ca2+](i). The DPDPE-evoked increase in [Ca2+](i) was abolished when the carbachol-sensitive intracellular Ca2+ store was emptied. There was a marked difference between the concentration-response relationship for the elevation of [Ca2+](i) by carbachol (EC50 13 muM, Hill slope 1) and the concentration-response relationship for carbachol's permissive action in revealing the delta -opioid receptor-mediated elevation of [Ca2+] (EC50 0.7 mM; Hill slope 1.8). Sequestration of free G protein beta gamma dimers by transient transfection of cells with a beta gamma binding protein (residues 495-689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of delta opioid receptor activation to elevate [Ca2+](i). However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicating that delta -opioid receptor activation did not stimulate phospholipase C. Furthermore, delta -opioid receptor activation did not result in the reversal of muscarinic receptor desensitization, membrane hyperpolarization or stimulation of sphingosine kinase. There was no coincident signalling between the delta -opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca2+](i) in SH-SY5Y cells by a PLC-independent mechanism. In SH-SY5Y cells the coincident signalling between the endogenously expressed delta -opioid and m3 muscarinic receptors appears to occur in the receptor activation-Ca2+ release signalling pathway at a step after the activation of phospholipase C.</p

    Co-incident signalling between µ-opioid and M<sub>3</sub>-muscarinic receptors at the level of Ca<sup>2+</sup> release from intracellular stores: Lack of evidence for InsP<sub>3</sub> receptor sensitisation

    No full text
    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.</p
    corecore