EXPRESSION OF INDUCIBLE NITRIC-OXIDE SYNTHASE IN SMOOTH-MUSCLE CELLS FROM RAT PENILE CORPORA CAVERNOSA

Nitric oxide (NO), the main mediator of penile erection, is assumed to be synthesized in the penis by the neuronal constitutive nitric oxide synthase (nNOS). However, nNOS has not been identified in the penile smooth muscle, the target of NO action. The other NOS isozymes, the in ducible NOS (INOS) and the endothelial NOS (eNOS) have not been report ed in any penile tissue. The smooth muscle vascular and trabecular tis sue from rat corpora cavernosa is represented in vitro by cell culture s designated RPSMC. To determine whether iNOS can be expressed in peni le smooth muscle, RPSMC were treated with different lymphokines and/or bacterial lipopolysaccharide (LPS). The selected inducer, LPS/interfe ron, elicited at 48 hours up to a 50-fold increase in nitrites in the medium; the increase continued by 72 hours. The induction was inhibite d by L-N omega-nitroarginine methyl ester (L-NAME), aminoguanidine, ac tinomycin D, cycloheximide, transforming growth factor-beta 1 (TGF-bet a 1), and dexamethasone, but was resistant to nifedipine and platelet- derived growth factor-AB (PDGF-AB). iNOS induction increased with cell passage. The [H-3] L-arginine/citrulline measurement of NO synthesis with intact cells confirmed these results. Incubations of soluble and particulate fractions showed that the cytosol contained most of the ac tivity (Km = 43 mu M), which was partially inhibited by ethyleneglycal -bis-tetraacetic acid (EGTA). The 4.4-kb iNOS mRNA peaked at a late pe riod (24-30 hours) and remained high for up to 72 hours. iNOS mRNA ind uction was strongly inhibited by actinomycin D and dexamethasone, part ially inhibited by TGF-beta 1, inhibited slightly by PDGF-AB, and unaf fected by nifedipine. These results show that iNOS can be expressed in RPSMC in a cell passage-dependent fashion that has so far not been re ported for other cell lines, and that the induction reaches much highe r levels than in rat or human vascular smooth muscle cells. The expres sion pattern is also distinctive for the penile cells in time course o f induction, Ca2+ dependence, response to certain agents, and mRNA sta bility.