EX VIVO EXPRESSION OF NITRIC OXIDE SYNTHASE ISOFORMS (eNOS/iNOS) AND CALMODULIN IN HUMAN PENILE CAVERNOSAL CELLS

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Abstract
Nitric oxide (NO) synthesized by nitric oxide synthase (NOS) is recognized as the central mediator of penile erection. This process appears to be mediated mainly by neuronal NOS (nNOS), which is localized to the nonadrenergic, noncholinergic innervation of the penis. However, the role of non-neuronal penile constituents (specifically the cavernosal smooth muscle), as well as other NOS isoforms in NO production in the human penis is not well understood. The present study evaluates the expression of non-neuronal (inducible and endothelial) isoforms of NOS in human penile cavernosal smooth muscle cells in culture. Primary culture was initiated with explants of human corpora cavernosa. For gene expression studies, total RNA was extracted from cavernosal cells and subjected to reverse transcriptase polymerase chain reaction (RT-PCR). For NADPH-diaphorase histochemistry, the cells were incubated with 1 mM beta-NADPH and 0.5 mM nitrobluetetrazolium at 37C for 3 hours. For indirect immunofluorescence and electron microscopy, cells were incubated overnight at 4C with specific primary (eNOS; calmodulin) and secondary antibodies. A conventional avidin biotin complex technique was used for electron microscopy. The mRNA expression studies revealed that these cells express both endothelial (eNOS) and inducible (iNOS) forms. Localization studies showed positive signals for NADPH-diaphorase, eNOS, and calmodulin. The electron microscopic evaluation confirmed the localization of eNOS to the cytoplasm and small vesicles in the cells. These findings support the hypothesis that human cavernosal smooth muscle cells express both endothelial and inducible forms of NOS, which may significantly contribute to NO production in the penile architecture during the erectile process.