Morphine prevents the mitochondrial permeability transition pore opening through NO/cGMP/PKG/[Zn.sup.2+]/GSK-3[beta] signal pathway in cardiomyocytes

Xi J, Tian W, Zhang L, Jin Y, Xu Z. Morphine prevents the mitochondrial permeability transition pore opening through NO/ cGMP/PKG/[Zn.sup.2+]/GSK-3[beta] signal pathway in cardiomyocytes. Am J Physiol Heart Circ Physiol 298: H601-H607, 2010. First published December 4, 2009; doi:10.1152/ajpheart.00453.2009.--The aim of this study was to test whether morphine prevents the mitochondrial permeability transition pore (mPTP) opening through [Zn.sup.2+] and glycogen synthase kinase 3[beta] (GSK-3[beta]). Fluorescence dyes including Newport Green Dichlorofluorescein (DCF), 4-amino-5-methylamino2',7'-difluorofluorescein (DAF-FM), and tetramethylrhodamine ethyl ester (TMRE) were used to image free [Zn.sup.2+], nitric oxide (NO), and mitochondrial membrane potential ([DELTA] [PHI] m), respectively. Fluorescence images were obtained with confocal microscopy. Cardiomyocytes treated with morphine for 10 rain showed a significant increase in Newport Green DCF fluorescence intensity, an effect that was reversed by the NO synthase inhibitor [N.sup.G]-nitro-L-arginine methyl ester (L-NAME), indicating that morphine mobilizes [Zn.sup.2+] via NO. Morphine rapidly produced NO. ODQ and NS2028, the inhibitors of guanylyl cyclase, prevented [Zn.sup.2+] release by morphine, implying that cGMP is involved in the action of morphine. The effect of morphine on [Zn.sup.2+] release was also abolished by KT5823, a specific inhibitor of protein kinase G (PKG). Morphine prevented oxidant-induced loss of [DELTA][PHI]m, indicating that morphine can modulate the mPTP opening. The effect of morphine on the mPTP was reversed by KT5823 and the [Zn.sup.2+] chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). The action of morphine on the mPTP was lost in cells transfected with the constitutively active GSK-3[beta] mutant, suggesting that morphine may prevent the mPTP opening by inactivating GSK3[beta]. In support, morphine significantly enhanced phosphorylation of GSK-3[beta] at [Ser.sup.9], and this was blocked by TPEN. GSK-3[beta] small interfering RNA prevented the pore opening in the control cardiomyocytes but failed to enhance the effect of morphine on the mPTP opening. In conclusion, morphine mobilizes intracellular [Zn.sup.2+] through the NO/cGMP/PKG signaling pathway and prevents the mPTP opening by inactivating GSK-3[beta] through [Zn.sup.2+]. nitric oxide; protein kinase G; guanosine 3',5'-cyclic monophosphate; glycogen synthase kinase 3[beta] doi: 10.1152/ajpheart.00453.2009