Inhibitors caveolin-1 and protein kinase G show differential subcellular colocalization with Nitric oxide synthase.
BACKGROUND
Nitric oxide synthase (NOS) is negatively regulated by protein-protein interactions with caveolin-1 before extracellular activating signals release it for nitric oxide (NO) production. Smooth muscle protein kinase G (PKG) is a down-stream effector of NO signaling for relaxation of vascular smooth muscle cells (SMC). The PKG is also found in endothelial cells and it inhibits activated NOS within endothelial cells.


METHODS
We used confocal fluorescence microscopy to colocalize the inhibitors caveolin-1 and PKG with NOS in freshly isolated neonatal lamb endothelial cells in order to corroborate the speculation of their differential effects on NOS. The roles of caveolin-1 and PKG as regulators of NOS were investigated by examining their respective subcellular sites of colocalization with NOS using qualitative fluorescence immunohistochemistry and confocal microscopy.


RESULTS
Caveolin-1 was colocalized with NOS in the plasma membrane and Golgi. The PKG1-beta isoform was colocalized with serine116 phosphorylated NOS in the cytosol and in vesicular structures seen in the endoplasmic reticulum and in the nuclear region.


CONCLUSIONS
We conclude that unlike caveolin-1, a known pre-activation inhibitor of nascent NOS, PKG may be a post-activation inhibitor of NOS, possibly important for the recycling of the spent enzyme.
Department of Pharmacology, Lagos State University College of Medicine. 1 Oba Akinjobi Way, GRA, Ikeja, Lagos, P.M.B. 21266, Ikeja, Lagos, Nigeria. theresaadebola@yahoo.com
T J
TJ
Adebola
Raj
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Usha
D000818:Animals; D000831:Animals, Newborn; D051242:Caveolin 1 / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010766:Phosphorylation; D012756:Sheep
D051242:Caveolin 1; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D017869:Cyclic GMP-Dependent Protein Kinases