title: Non-anticoagulant heparin increases endothelial nitric oxide synthase activity: role of inhibitory guanine nucleotide proteins.
abstract: Heparin, which is widely used clinically, has recently been shown to have specific properties affecting the vascular endothelium. We hypothesized that heparin stimulates endothelial nitric oxide synthase (eNOS) activity by a mechanism independent of its anticoagulant properties and dependent on an inhibitory guanine nucleotide regulatory protein (Gi). We determined the effect of both heparin and N-acetyl heparin (Non-Hep), a heparin derivative without anticoagulant properties, on eNOS activity in cultured bovine aortic endothelial cells and on endothelium-dependent relaxation in isolated vascular rings. The eNOS activity was determined by measuring both citrulline and nitric oxide (NO) metabolite formation. Heparin and Non-Hep dose-dependently increased basal eNOS activity (ED50 1.0 microgram/ml or 0.15 U/ml), an effect that was significantly inhibited by pertussis toxin (100 ng/ml), a Gi-protein inhibitor. Agonist-stimulated (acetylcholine, 10 microM) eNOS activity was potentiated following pre-treatment with both heparin and Non-Hep and reversed by pertussis toxin. Heparin and Non-Hep induced a dose-dependent relaxation in preconstricted thoracic aortic rings, an effect that was significantly inhibited by pertussis toxin, endothelial inactivation (following treatment with sodium deoxycholate) and NG-nitro-L-arginine-methyl ester (L-NAME). We conclude that heparin and non-anticoagulant heparin induce endothelium-dependent relaxation following activation of eNOS by a mechanism involving a Gi-protein. Administration of heparin derivatives without anticoagulant properties may have therapeutic implications for the preservation of eNOS in conditions characterized by endothelial dysfunction.
authors.affiliation: Department of Physiology and Biophysics, Georgetown University Medical Center, Washington DC 20007, USA.
authors.forename: P C
authors.initials: PC
authors.lastname: Kouretas
authors.forename: R L
authors.initials: RL
authors.lastname: Hannan
authors.forename: N K
authors.initials: NK
authors.lastname: Kapur
authors.forename: R
authors.initials: R
authors.lastname: Hendrickson
authors.forename: E M
authors.initials: EM
authors.lastname: Redmond
authors.forename: A K
authors.initials: AK
authors.lastname: Myers
authors.forename: Y D
authors.initials: YD
authors.lastname: Kim
authors.forename: P A
authors.initials: PA
authors.lastname: Cahill
authors.forename: J V
authors.initials: JV
authors.lastname: Sitzmann
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic / Q000502:physiology; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D002956:Citrulline / Q000494:pharmacology; D003840:Deoxycholic Acid / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000502:physiology*; D006493:Heparin / Q000031:analogs & derivatives / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D037342:Pertussis Toxin; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology