Morphine prevents the mitochondrial permeability transition pore opening through NO/cGMP/PKG/Zn2+/GSK-3beta signal pathway in cardiomyocytes.
The aim of this study was to test whether morphine prevents the mitochondrial permeability transition pore (mPTP) opening through Zn(2+) and glycogen synthase kinase 3beta (GSK-3beta). Fluorescence dyes including Newport Green Dichlorofluorescein (DCF), 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), and tetramethylrhodamine ethyl ester (TMRE) were used to image free Zn(2+), nitric oxide (NO), and mitochondrial membrane potential (DeltaPsi(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 (G)-nitro-L-arginine methyl ester (L-NAME), indicating that morphine mobilizes Zn(2+) via NO. Morphine rapidly produced NO. ODQ and NS2028, the inhibitors of guanylyl cyclase, prevented Zn(2+) release by morphine, implying that cGMP is involved in the action of morphine. The effect of morphine on Zn(2+) release was also abolished by KT5823, a specific inhibitor of protein kinase G (PKG). Morphine prevented oxidant-induced loss of DeltaPsi(m), indicating that morphine can modulate the mPTP opening. The effect of morphine on the mPTP was reversed by KT5823 and the Zn(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-3beta mutant, suggesting that morphine may prevent the mPTP opening by inactivating GSK-3beta. In support, morphine significantly enhanced phosphorylation of GSK-3beta at Ser(9), and this was blocked by TPEN. GSK-3beta 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(2+) through the NO/cGMP/PKG signaling pathway and prevents the mPTP opening by inactivating GSK-3beta through Zn(2+).
Xi
Jinkun
J
Department of Anesthesiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Tian
Wei
W
Zhang
Lei
L
Jin
Yulan
Y
Xu
Zhelong
Z
D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005029:Ethylenediamines / Q000494:pharmacology; D005456:Fluorescent Dyes; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D008297:Male; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects* / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D023421:Models, Animal; D009020:Morphine / Q000494:pharmacology*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015032:Zinc / Q000378:metabolism*
D000701:Analgesics, Opioid; D002227:Carbazoles; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; C073601:KT 5823; D009569:Nitric Oxide; D009020:Morphine; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D017869:Cyclic GMP-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D006152:Cyclic GMP; D015032:Zinc; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine