Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.2 (guanylate cyclase)
 8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitric oxide, which accounts for the biological activity of endothelium-derived relaxing factor (EDRF), is synthesized in endothelial cells from L-arginine by nitric oxide synthase (NOS). We report here the cloning and functional expression of a cDNA encoding human endothelial NOS. Oligonucleotides corresponding to amino acid sequences shared by cytochrome P450 reductase and the recently identified brain NOS were synthesized to amplify a partial cDNA encoding a bovine endothelial cell NOS-related protein. This partial cDNA was used to isolate a cDNA encoding a human vascular endothelial NOS. The translated human protein is 1294 amino acids long and shares 52% of its amino acid sequence with brain NOS. Using RNA blot hybridization, abundant endothelial NOS mRNA was detected in unstimulated human umbilical vein endothelial cells. To determine the functional activity of the endothelial protein, we ligated the cDNA into an expression vector and transfected it into NIH3T3 cells. Cells expressing this cDNA contained abundant NADPH diaphorase activity, a histochemical marker for NOS. In co-culture assays, nitric oxide production by transfected cells increased guanylate cyclase activity in reporter rat fetal lung fibroblasts. In addition, NOS-catalyzed conversion of arginine to citrulline in transfected cells was significantly increased by A23187, a calcium ionophore. Isolation of a cDNA encoding a calcium-regulated, constitutively expressed human endothelial NOS, capable of producing EDRF in blood vessels, will accelerate the characterization of the role of this enzyme in normal and abnormal endothelial regulation of vascular tone.
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PMID:Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. 138 4

We reported previously that the flavoprotein inhibitor diphenyleneiodonium sulfate (DPI) irreversibly inhibited the metabolic activation of glyceryl trinitrate (GTN) in isolated aorta, possibly through inhibition of vascular NADPH-cytochrome P450 reductase (CPR). We report that the content of CPR represents 0.03 to 0.1% of aortic microsomal protein and that DPI caused a concentration- and time-dependent inhibition of purified cDNA-expressed rat liver CPR and of aortic and hepatic microsomal NADPH-cytochrome c reductase activity. Purified CPR incubated with NADPH and GTN under anaerobic, but not aerobic conditions formed the GTN metabolites glyceryl-1,3-dinitrate (1,3-GDN) and glyceryl-1,2-dinitrate (1,2-GDN). GTN biotransformation by purified CPR and by aortic and hepatic microsomes was inhibited > 90% after treatment with DPI and NADPH. DPI treatment also inhibited the production of activators of guanylyl cyclase formed by hepatic microsomes. We also tested the effect of DPI on the hemodynamic-pharmacokinetic properties of GTN in conscious rats. Pretreatment with DPI (2 mg/kg) significantly inhibited the blood pressure lowering effect of GTN and inhibited the initial appearance of 1,2-GDN (1-5 min) and the clearance of 1,3-GDN. These data suggest that the rapid initial formation of 1,2-GDN is related to mechanism-based GTN biotransformation and to enzyme systems sensitive to DPI inhibition. We conclude that vascular CPR is a site of action for the inhibition by DPI of the metabolic activation of GTN, and that vascular CPR is a novel site of GTN biotransformation that should be considered when investigating the mechanism of GTN action in vascular tissue.
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PMID:Inhibition of NADPH-cytochrome P450 reductase and glyceryl trinitrate biotransformation by diphenyleneiodonium sulfate. 977 50