Depletion of the ATPase NSF from Golgi membranes with hypo-S-nitrosylation of vasorelevant proteins in endothelial cells exposed to monocrotaline pyrrole.
Investigations of regulated S-nitrosylation and denitrosylation of vasorelevant proteins are a newly emergent area in vascular biology. We previously showed that monocrotaline pyrrole (MCTP)-induced megalocytosis of pulmonary arterial endothelial cells (PAECs), which underlies the development of pulmonary arterial hypertension, was associated with a Golgi blockade characterized by the trapping of diverse vesicle tethers, soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs), and soluble NSF-attachment proteins (SNAPs) in the Golgi; reduced trafficking of caveolin-1 (cav-1) and endotheial nitric oxide (NO) synthase (eNOS) from the Golgi to the plasma membrane; and decreased caveolar NO. We have investigated whether NSF, the ATPase involved in all SNARE disassembly, might be the upstream target of MCTP and whether MCTP might regulate NSF by S-nitrosylation. Immunofluorescence microscopy and Golgi purification techniques revealed the discordant decrease of NSF by approximately 50% in Golgi membranes after MCTP despite increases in alpha-SNAP, cav-1, eNOS, and syntaxin-6. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide failed to affect the initiation or progression of MCTP megalocytosis despite a reduction of 4,5-diaminofluorescein diacetate fluorescence and inhibition of S-nitrosylation of eNOS as assayed using the biotin-switch method. Moreover, the latter assay not only revealed constitutive S-nitrosylation of NSF, eNOS, cav-1, and clathrin heavy chain (CHC) in PAECs but also a dramatic 70-95% decrease in the S-nitrosylation of NSF, eNOS, cav-1, and CHC after MCTP. These data point to depletion of NSF from Golgi membranes as a mechanism for Golgi blockade after MCTP and to denitrosylation of vasorelevant proteins as critical to the development of endothelial cell megalocytosis.
Mukhopadhyay
Somshuvra
S
Department of Cell Biology & Anatomy, New York Medical College, Valhalla, NY 10595, USA.
Lee
Jason
J
Sehgal
Pravin B
PB
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D051242:Caveolin 1 / Q000378:metabolism; D002458:Cell Fractionation; D002478:Cells, Cultured; D033922:Clathrin Heavy Chains / Q000378:metabolism; D015536:Down-Regulation; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D016166:Free Radical Scavengers / Q000494:pharmacology; D006056:Golgi Apparatus / Q000187:drug effects* / Q000201:enzymology; D016686:Monocrotaline / Q000031:analogs & derivatives* / Q000494:pharmacology; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D021381:Protein Transport; D050600:SNARE Proteins / Q000378:metabolism; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism
D051242:Caveolin 1; D016166:Free Radical Scavengers; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033922:Clathrin Heavy Chains; C014114:monocrotaline pyrrole; D009569:Nitric Oxide; D016686:Monocrotaline; D052250:Nitric Oxide Synthase Type III; D052066:N-Ethylmaleimide-Sensitive Proteins
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