Hydroxyurea, a drug widely used for treating myeloproliferative diseases, has also been approved for the treatment of sickle cell disease by raising fetal hemoglobin (HbF). We have shown that nitric oxide (NO) and the soluble guanylyl cyclase (sGC) pathways are involved in hydroxyurea induction of HbF levels in erythroid progenitor cells (EPCs). We demonstrate now that during erythroid differentiation, endothelial NO synthase mRNA and protein levels decline steadily, as does the production of NO derivatives and cyclic adenosine monophosphate (cAMP) levels, but guanosine 3′,5′-cyclic monophosphate (cGMP) levels are stable. Hydroxyurea increased intracellular cGMP levels and cAMP levels in EPCs. The NO donor, DEANONOate, induced much higher cGMP levels, but reduced cAMP levels. Hydroxyurea (1 mM) induced production of approximately 45 pM cGMP/minute/ng of purified sGC, similar to induction by 1 μM DEANONOate. We found that hydroxyurea and ProliNONOate produced iron-nitrosyl derivatives of sGC. Thus, we confirm that hydroxyurea can directly interact with the deoxy-heme of sGC, presumably by a free-radical nitroxide pathway, and activate cGMP production. These data add to an expanding appreciation of the role of hydroxyurea as an inducer of the NO/cGMP pathway in EPCs. These mechanisms may also be involved in the cytostatic effects of hydroxyurea, as well as the induction of HbF.

Polychlorinated biphenyls (PCBs) are complex mixtures of congeners that exhibit carcinogenic and toxicant activities in a variety of mammalian tissue

Sildenafil citrate (Viagra), a cGMP-selective phosphodiesterase (PDE) inhibitor, is widely used to treat erectile dysfunction and pulmonary arterial hypertension. In contrast to its well established action on erectile dysfunction, little is known on the action of sildenafil on cGMP/cAMP signaling and testicular steroidogenesis. This study was designed to assess the effects of prolonged sildenafil treatment on NO synthase-dependent signaling and steroidogenic function of rat Leydig cells. Male adult rats were treated with Viagra (1.25 mg/kg body wt) daily for 30 days. In our studies, serum testosterone and ex vivo testosterone production significantly increased in sildenafil-treated animals. Human chorionic gonadotropin-stimulated testosterone production and cAMP accumulation were also significantly higher in Leydig cells obtained from sildenafil-treated rats. The expression of soluble guanylyl cyclase (GUCY1) subunits ( Gucy1a1, Gucy1b1) significantly increased; cAMP-specific Pde4a, cGMP-specific Pde6c, and dual Pde1c and Nos2 were inhibited and expression of Nos3, protein kinase G1 ( Pkg1), and Pde5 remained unchanged. Treatment of purified Leydig cells with NO donor caused a dose-dependent increase in both testosterone and cGMP production. Testosterone and cGMP production was significantly higher in Leydig cells obtained from sildenafil-treated animals. The stimulatory effect of NO donor was significantly enhanced by saturating concentrations of hCG in both Leydig cells obtained from control and sildenafil-treated animals. Occurrence of mature steroidogenic acute regulatory protein also increased in sildenafil treated animals in accord with increased cAMP and cGMP production. In summary, inhibition of PDE activity during prolonged sildenafil treatment increased serum testosterone level and Leydig cells' steroidogenic capacity by coordinated stimulatory action on cAMP and cGMP signaling pathway.

Testosterone, acting as a systemic and local factor, is one of the major regulatory molecules that initiate and maintain testicular function. In the present study, different experimental approaches were used to evaluate the role of testosterone in regulation of the nitric oxide (NO)-cGMP pathway in Leydig cells derived from normal and hypogonadotropic male rats treated with testosterone for 24 h and 2 wk. Real-time quantitative PCR and Western blot analysis revealed increased inducible NO synthase (NOS2) expression followed by increased NO secretion from Leydig cells ex vivo after continuous treatment with testosterone for 2 wk in vivo. The cGMP-specific phosphodiesterases Pde5, Pde6, and Pde9 were up-regulated, whereas PRKG1 protein was decreased after a 2-wk testosterone treatment. Induction of Nos2 and Pde5 in Leydig cells was blocked by androgen receptor antagonist. In experimental hypogonadotropic hypogonadism, expression of NOS2 was significantly reduced, and treatment with testosterone increased NOS2 expression above control levels. PDE5 protein level was unchanged in hypogonadal rats, whereas treatment of hypogonadal rats with testosterone significantly increased it. In contrast, hypogonadism and testosterone replacement reduced PRKG1 protein in Leydig cells. In vitro treatment with testosterone caused gradually increased Nos2 gene expression followed by increased nitrite and cGMP production by purified Leydig cells. In summary, testosterone up-regulated NO signaling via increased NOS2 expression and contributed to down-regulation of cGMP signaling in Leydig cells. Thus, testosterone-induced modulation of NO-cGMP signaling may serve as a potent autocrine regulator of testicular steroidogenesis.