CLONING OF RAT AN HUMAN INDUCIBLE PENILE NITRIC-OXIDE SYNTHASE - APPLICATION FOR GENE-THERAPY OF ERECTILE DYSFUNCTION

Erectile dysfunction is mainly due to the inability of the cavernosal smooth muscle of the penis to undergo complete relaxation. In the agin g rat model, erectile dysfunction is accompanied by a reduction of pen ile smooth muscle compliance and, in very old animals, by a decrease i n penile nitric oxide synthase (NOS), which is responsible for the syn thesis of the mediator oi: penile erection, nitric oxide (NO). We have investigated whether the stimulation of penile NOS expression by loca l induction or gene therapy can mitigate erectile dysfunction in the a ged rat. A mix of iNOS (inducible NOS) inducers was continuously deliv ered to the penises of 5- (''adult''), 20- (''old''), and 30- (''very old'') mo-old rats for 3-6 days, and the erectile response to electric al field stimulation of the cavernosal nerve was measured. The erectil e dysfunction observed in old and very old rats as compared to adult a nimals was ameliorated by treatment with iNOS inducers. Penile iNOS wa s detectable in the penis of these rats by Western blot, NADPH diaphor ase, and NOS activity assays. Inducible NOS was inducible in vitro in both rat and human corpora cavernosal tissue and in rat penile smooth muscle cells (RPSMC), as shown by Western blots. However, NO synthesis in cavernosal tissue upon iNOS protein induction remained low, indica ting that the increased NOS levels were under physiological control. T he iNOS cDNA was cloned from induced RPSMC mRNA and generated by rever se transcriptase polymerase chain reaction (RT-PCR) from induced human penile smooth muscle cells and corporal tissue. The coding regions fr om both the rat (RPiNOS) and human (HPiNOS)penile iNOS showed several amino acid differences from their analogous isoform in nonpenile tissu es, RPiNOS cDNA injected into the penis mitigated the aging-associated erectile dysfunction. The iNOS construct was detected in cavernosal t issue by PCR,and its expression by RT-PCR and Western blots. These res ults open the way for the possible use of NOS isoforms in the manageme nt of erectile dysfunction.