Inhibition of GTP-dependent vesicle trafficking impairs internalization of plasmalemmal eNOS and cellular nitric oxide production.
The Ca2+ mobilizing peptide, bradykinin (BK), stimulates endothelial nitric oxide synthase (eNOS)-derived cellular nitric oxide (NO) production in association with altering the subcellular distribution of the enzyme. In the present study we examine the influence of cellular GTPases, particularly the large GTPase dynamin, on BK-mediated eNOS localization and cellular NO production. BK stimulation of ECV cells, which were stably transfected with eNOS-GFP (eNOS-GFP ECV304), increased NO production. This was associated with the mobilization of eNOS-GFP protein into Triton X-100-insoluble fractions of cell lysates, and an internalization of plasmalemmal eNOS-GFP in live and fixed ECV 304 cells. Incubation of digitonin-permeabilized ECV304 cells with the non-hydrolyzed GTP analog, GTP-gamma-S, abrogated the BK-mediated internalization of eNOS-GFP as assessed by confocal microscopy. Conversely, inhibition of clathrin-dependent endocytosis, via overexpression of AP 180 or pretreatment of cells with chlorpromazine, did not influence BK-mediated eNOS redistribution. Furthermore, specific inhibition of dynamin-2 GTPase function by overexpression of a dominant negative construct, K44A, prevented the BK-mediated enrichment of eNOS-GFP within low buoyant density, caveolin-enriched fractions of eNOS-GFP ECV304 cell lysates. Dynamin-2 K44A overexpression also markedly impaired BK-dependent, L-NAME-inhibited NO production as did incubation of permeabilized cells with GTP-gamma-s. These studies demonstrate that disruption of dynamin- and GTP-dependent, but clathrin-independent, vesicle trafficking pathways impairs BK-dependent cellular NO production, via inhibition of the internalization of eNOS-containing plasmalemmal vesicles.
Chatterjee
Suvro
S
GI Research Unit, Department of Physiology, and Tumor Biology Program, Mayo Clinic, Rochester, MN 55905, USA.
Cao
Sheng
S
Peterson
Timothy E
TE
Simari
Robert D
RD
Shah
Vijay
V
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism; D018492:Dopamine Antagonists / Q000494:pharmacology; D034285:Dynamin II / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D034041:Monomeric Clathrin Assembly Proteins / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D017830:Octoxynol / Q000494:pharmacology; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions; D022161:Transport Vesicles / Q000187:drug effects / Q000502:physiology*
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D002966:Clathrin; D018492:Dopamine Antagonists; D008164:Luminescent Proteins; D034041:Monomeric Clathrin Assembly Proteins; D011993:Recombinant Fusion Proteins; C439023:clathrin assembly protein AP180; D049452:Green Fluorescent Proteins; D009569:Nitric Oxide; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D017830:Octoxynol; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D034285:Dynamin II; D001920:Bradykinin; D002746:Chlorpromazine