Homocysteine and copper interact to promote type 5 phosphodiesterase expression in rabbit cavernosal smooth muscle cells: mediation by superoxide and inhibition with sildenafil

13 Freeland Place, Bristol BS8 4NP, UK Tel: +44-01173-739-049 Fax: +44-01924-881-228 E-mail: [email protected]. Aim: The pro-erectile actions of nitric oxide are reduced by type 5 phosphodiesterase (PDE5) that hydrolyses cyclic guanosine monophosphate to inactive guanosine monophosphate. An upregulation of PDE5 might therefore promote erectile dysfunction (ED). As there is evidence that homocysteine and copper interact to promote ED, their effects on PDE5 expression in cavernosal vascular smooth muscle cells (CVSMCs) were studied. Superoxide (O(2)(.-)) derived from nicotinamide adenine dinucleotide phosphate oxidase was also studied, as homocysteine and copper generate O(2)(.-), and O(2)(.-) upregulates PDE5 expression. Methods: CVSMCs derived from rabbit penis were incubated with homocysteine or copper chloride with or without superoxide dismutase (SOD), catalase, sildenafil citrate, or apocynin (nicotinamide adenine dinucleotide phosphate inhibitor) for 16 h. The expression of PDE5 and of glyceraldehyde-3-phosphate dehydrogenase (internal standard) was assessed using Western blot analysis. In parallel, O(2)(.-) was measured spectrophotometrically. Results: CuCl(2) alone (up to 10 mumol/L) and homocysteine alone (up to 100 mumol/L) had no effect on O(2)(.-) formation in CVSMCs compared to controls. In combination, however, homocysteine and CuCl(2) markedly increased O(2)(.-) formation, an effect blocked by SOD, catalase, apocynin, and sildenafil (1 mumol/L) when co-incubated over the same time course. PDE5 expression was also significantly increased in CVSMCs incubated with homocysteine and CuCl(2), compared to controls. This effect was also negated by 16 h co-incubation with SOD, catalase, apocynin, and sildenafil. Conclusion: This represents a novel pathogenic mechanism underlying ED, and indicates that the therapeutic actions of prolonged sildenafil use are mediated in part through inhibition of this pathway.