title: Neutral sphingomyelinase, NADPH oxidase and reactive oxygen species. Role in acute hypoxic pulmonary vasoconstriction.
abstract: The molecular mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are not yet properly understood. Mitochondrial electron transport chain (ETC) and NADPH oxidase have been proposed as possible oxygen sensors, with derived reactive oxygen species (ROS) playing key roles in coupling the sensor(s) to the contractile machinery. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C ζ (PKCζ) participate in the signalling cascade of HPV. Herein, we studied the significance of nSMase in controlling ROS production rate in rat pulmonary artery (PA) smooth muscle cells and thereby HPV in rat PA. ROS production (analyzed by dichlorofluorescein and dihydroethidium fluorescence) was increased by hypoxia in endothelium-denuded PA segments and their inhibition prevented hypoxia-induced voltage-gated potassium channel (K(V) ) inhibition and pulmonary vasoconstriction. Consistently, H(2) O(2) , or its analogue t-BHP, decreased K(V) currents and induced a contractile response, mimicking the effects of hypoxia. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia-induced ROS production, K(V) channel inhibition and vasoconstriction. Hypoxia induced p47(phox) phosphorylation and its interaction with caveolin-1. Inhibition of nSMase (GW4869) or PKCζ prevented p47(phox) phosphorylation and ROS production. The increase in ceramide induced by hypoxia (analyzed by immunocytochemistry) was inhibited by rotenone. Exogenous ceramide increased ROS production in a PKCζ sensitive manner. We propose an integrated signalling pathway for HPV which includes nSMase-PKCζ-NADPH oxidase as a necessary step required for ROS production and vasoconstriction.
authors.affiliation: Department of Pharmacology, School of Medicine, University Complutense of Madrid, Madrid, Spain.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Frazziano
authors.forename: Laura
authors.initials: L
authors.lastname: Moreno
authors.forename: Javier
authors.initials: J
authors.lastname: Moral-Sanz
authors.forename: Carmen
authors.initials: C
authors.lastname: Menendez
authors.forename: Lucía
authors.initials: L
authors.lastname: Escolano
authors.forename: Constancio
authors.initials: C
authors.lastname: Gonzalez
authors.forename: Eduardo
authors.initials: E
authors.lastname: Villamor
authors.forename: Jose Luis
authors.initials: JL
authors.lastname: Alvarez-Sala
authors.forename: Angel L
authors.initials: AL
authors.lastname: Cogolludo
authors.forename: Francisco
authors.initials: F
authors.lastname: Perez-Vizcaino
mesh_terms: D000208:Acute Disease; D000818:Animals; D002518:Ceramides / Q000037:antagonists & inhibitors / Q000378:metabolism; D004579:Electron Transport / Q000187:drug effects / Q000502:physiology; D000860:Hypoxia / Q000201:enzymology / Q000378:metabolism* / Q000503:physiopathology*; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D019255:NADPH Oxidases / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D024642:Potassium Channels, Voltage-Gated / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism; D011651:Pulmonary Artery / Q000201:enzymology / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014475:Uncoupling Agents / Q000494:pharmacology; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology*; D000099221:Protein Kinase C zeta
chemical_list: D002518:Ceramides; D019255:NADPH Oxidases; D024642:Potassium Channels, Voltage-Gated; D011493:Protein Kinase C; D017382:Reactive Oxygen Species; D012402:Rotenone; D013108:Sphingomyelin Phosphodiesterase; D014475:Uncoupling Agents; D000099221:Protein Kinase C zeta; C060505:neutrophil cytosolic factor 1