Sildenafil inhibits the up-regulation of phosphodiesterase type 5 elicited with nicotine and tumour necrosis factor-α in cavernosal vascular smooth muscle cells:: mediation by superoxide

OBJECTIVES To determine whether there is an association between vascular phosphodiesterase type 5 (PDE-5) and NADPH oxidase (NOX) in cavernosal vascular smooth muscle cells (CVSMCs), and to study the actions of the PDE-5 inhibitor sildenafil; the pro-erectile actions of nitric oxide (NO) are reduced by PDE-5 which hydrolyses cGMP to inactive GMP, thus an up-regulation of PDE-5 and over-production of O-2(-) derived from NOX might promote erectile dysfunction (ED). MATERIALS AND METHODS To study the effects of nicotine and tumour necrosis factor-alpha (TNF-alpha) on superoxide (O-2(-)) production and PDE-5 expression, CVSMCs from rabbit penis were incubated with nicotine or TNF-alpha, and superoxide dismutase (SOD), catalase, sildenafil citrate, or apocynin (NADPH inhibitor) for 16 h. The expression of PDE-5 and of glyceraldehyde-3-phosphate dehydrogenase (internal standard) was assessed using Western blotting. O-2(-) was measured spectrophotometrically. RESULTS After a 16-h incubation, both nicotine (maximal at 10 mu M) and TNF-alpha (10 ng/mL) significantly increased O-2(-) formation in CVSMCs; this effect was blocked by co-incubating with SOD, catalase, and sildenafil (1 mu M). Apocynin also inhibited O-2(-) formation when added after 16-h incubation with nicotine (10 mu M) or TNF-alpha. PDE-5 expression was also significantly increased in CVSMCs incubated with nicotine and TNF-alpha. This effect was negated by 16-h co-incubation with SOD, catalase, apocynin, and sildenafil. CONCLUSION Nicotine and TNF-alpha up-regulate PDE-5 expression in CVSMCs through an a priori up-regulation of NOX and formation of O-2(-). As PDE-5 hydrolyses cGMP, this effect might 'blunt' the pro-erectile actions of NO. Sildenafil inhibits O-2(-) formation, and 'normalizes' PDE-5 expression. This represents a novel pathogenic mechanism underlying ED, and a novel mechanism of action of sildenafil.