Dysfunction of Golgi tethers, SNAREs, and SNAPs in monocrotaline-induced pulmonary hypertension.
Monocrotaline (MCT)-induced pulmonary hypertension (PH) in the rat is a widely used experimental model. We have previously shown that MCT pyrrole (MCTP) produces loss of caveolin-1 (cav-1) and endothelial nitric oxide synthase from plasma membrane raft microdomains in pulmonary arterial endothelial cells (PAEC) with the trapping of these proteins in the Golgi organelle (the Golgi blockade hypothesis). In the present study, we investigated the mechanisms underlying this intracellular trafficking block in experiments in cell culture and in the MCT-treated rat. In cell culture, PAEC showed trapping of cav-1 in Golgi membranes as early as 6 h after exposure to MCTP. Phenotypic megalocytosis and a reduction in anterograde trafficking (assayed in terms of the secretion of horseradish peroxidase derived from exogenously transfected expression constructs) were evident within 12 h after MCTP. Cell fractionation and immunofluorescence techniques revealed the marked accumulation of diverse Golgi tethers, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), and soluble NSF attachment proteins (SNAPs), which mediate membrane fusion during vesicular trafficking (GM130, p115, giantin, golgin 84, clathrin heavy chain, syntaxin-4, -6, Vti1a, Vti1b, GS15, GS27, GS28, SNAP23, and alpha-SNAP) in the enlarged/circumnuclear Golgi in MCTP-treated PAEC and A549 lung epithelial cells. Moreover, NSF, an ATPase required for the "disassembly" of SNARE complexes subsequent to membrane fusion, was increasingly sequestered in non-Golgi membranes. Immunofluorescence studies of lung tissue from MCT-treated rats confirmed enlargement of perinuclear Golgi elements in lung arterial endothelial and parenchymal cells as early as 4 days after MCT. Thus MCT-induced PH represents a disease state characterized by dysfunction of Golgi tethers, SNAREs, and SNAPs and of intracellular vesicular trafficking.
Sehgal
Pravin B
PB
Depatment of Cell Biology and Anatomy, New York Medical College, Valhalla, NY 10595, USA. pravin_sehgal@nymc.edu
Mukhopadhyay
Somshuvra
S
Xu
Fang
F
Patel
Kirit
K
Shah
Mehul
M
D000818:Animals; D002460:Cell Line; D022162:Cytoplasmic Vesicles / Q000378:metabolism / Q000473:pathology; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology; D005455:Fluorescent Antibody Technique; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D006976:Hypertension, Pulmonary / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology*; D008297:Male; D008561:Membrane Fusion / Q000502:physiology; D016686:Monocrotaline / Q000633:toxicity; D010641:Phenotype; D021381:Protein Transport / Q000502:physiology; D011651:Pulmonary Artery / Q000378:metabolism / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020545:Respiratory Mucosa / Q000378:metabolism / Q000473:pathology; D050600:SNARE Proteins / Q000378:metabolism*; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism*
D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D016686:Monocrotaline; D006026:Glycoside Hydrolases