Superoxide from NADPH oxidase upregulates type 5 phosphodiesterase in human vascular smooth muscle cells: inhibition with iloprost and NONOate

• Published in: British journal of pharmacology Vol. 155; no. 6; pp. 847 - 856
• Main Authors: Muzaffar, S, Shukla, N, Bond, M, Sala‐Newby, G B, Newby, A C, Angelini, G D, Jeremy, J Y
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-11-2008; Nature Publishing Group
• Subjects: Biological and medical sciences; Cardiovascular system; Cells, Cultured; Cyclic Nucleotide Phosphodiesterases, Type 5 - metabolism; Humans; Iloprost - pharmacology; Medical sciences; Muscle, Smooth, Vascular - drug effects; Muscle, Smooth, Vascular - enzymology; Muscle, Smooth, Vascular - metabolism; NADPH oxidase; NADPH Oxidases - metabolism; nitric oxide; Nitric Oxide Donors - pharmacology; Nitroso Compounds - pharmacology; Pharmacology. Drug treatments; Phosphodiesterase 5 Inhibitors; Research Papers; Rho‐kinase; Saphenous Vein - cytology; superoxide; Superoxides - metabolism; Superoxides - pharmacology; type 5 PDE; Up-Regulation; vascular smooth muscle cell; Vasodilator Agents - pharmacology; Vasodilator agents. Cerebral vasodilators; Human; Iloprost; Vasodilator agent; Prostaglandin I2; Enzyme; 3',5'-Cyclic-GMP phosphodiesterase; Arachidonic acid derivatives; Transferases; Non-specific serine/threonine protein kinase; Rho-kinase; Smooth muscle; Esterases; Phosphoric diester hydrolases; In vitro; NAD(P)H oxidase; type 5 PDE; Hyperoxides; Nitric oxide; NADPH oxidase; Hydrolases; Oxidoreductases; vascular smooth muscle cell; superoxide; Prostaglandin derivatives
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/bjp.2008.300

Background and purpose:
To determine whether there is an association between vascular NADPH oxidase (NOX), superoxide, the small GTPase Rac1 and PDE type 5 (PDE5) in human vascular smooth muscle cell (hVSMCs).
Experimental approach:
hVSMCs were incubated with xanthine–xanthine oxidase (X‐XO; a superoxide generating system) or the thromboxane A2 analogue, U46619 (±superoxide dismutase (SOD) or apocynin) for 16 h. The expression of PDE5 and NOX‐1 was assessed using Western blotting and superoxide measured. The role of Rac1 in superoxide generation was assessed by overexpressing either the dominant‐negative or constitutively active Rac isoforms. The effects of iloprost, DETA‐NONOate and the Rho‐kinase inhibitor, Y27632, on PDE5 and NOX‐1 expression were also studied.
Key results:
Following 16 h incubation, U46619 and X‐XO promoted the expression of PDE5 and NOX‐1, an effect blocked by SOD or apocynin when co‐incubated over the same time course. X‐XO and U46619 both promoted the formation of superoxide. Overexpression of dominant‐negative Rac1 or addition of iloprost, DETA‐NONOate or Y27632 completely blocked both superoxide release and PDE5 protein expression and activity.
Conclusions and implications:
These data demonstrate that superoxide derived from NOX upregulates the expression of PDE5 in human VSMCs. As PDE5 hydrolyses cyclic GMP, this effect may blunt the vasculoprotective actions of NO.
British Journal of Pharmacology (2008) 155, 847–856; doi:10.1038/bjp.2008.300; published online 28 July 2008