Morphine prevents the mitochondrial permeability transition pore opening through NO/cGMP/PKG/Zn /GSK-3 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 /GSK-3 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 and glycogen synthase kinase 3 (GSK-3 ). 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 , nitric oxide (NO), and mitochondrial membrane potential ( m), respectively. Fluorescence images were obtained with confocal microscopy. Cardiomyocytes treated with morphine for 10 min showed a significant increase in Newport Green DCF fluorescence intensity, an effect that was reversed by the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME), indicating that morphine mobilizes Zn via NO. Morphine rapidly produced NO. ODQ and NS2028, the inhibitors of guanylyl cyclase, prevented Zn release by morphine, implying that cGMP is involved in the action of morphine. The effect of morphine on Zn release was also abolished by KT5823, a specific inhibitor of protein kinase G (PKG). Morphine prevented oxidant-induced loss of m, indicating that morphine can modulate the mPTP opening. The effect of morphine on the mPTP was reversed by KT5823 and the Zn 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 mutant, suggesting that morphine may prevent the mPTP opening by inactivating GSK3 . In support, morphine significantly enhanced phosphorylation of GSK-3 at Ser, and this was blocked by TPEN. GSK-3 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 through the NO/cGMP/PKG signaling pathway and prevents the mPTP opening by inactivating GSK-3 through Zn .