Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP-dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated crossactivation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI-deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.

Mast-cell products can stimulate fibroblast proliferation, implying that these cells are key players in fibrosis. One mast-cell product, the serine protease tryptase, is known to activate protease-activated receptor 2 (PAR2) and cause proliferation of fibroblasts. We found that recombinant tryptase, human mastcell (HMC-1) supernatant, which contains tryptase, and the PAR2-activating peptide SLIGKV exert fibroproliferative actions in human fibroblasts. Here we report insights into this action, which after activation of PAR2 leads to increased expression of cyclooxygenase 2 (COX2), a key enzyme in the biosynthesis of prostaglandins, and consequently to enhanced prostaglandin synthesis. Subsequent cell proliferation is mediated by the prostaglandin 15-deoxy-⌬ 12,14 -prostaglandin J2, which acts via the nuclear peroxisome proliferator-activated receptor ␥ (PPAR␥). Fibroblast proliferation induced by tryptase and PAR2 agonist peptide can be blocked by antagonists of COX2 and PPAR␥, implying that the proliferative effect of tryptase is PAR2-initiated but depends on COX2, 15-deoxy-⌬ 12,14 -prostaglandin J2, and PPAR␥. This previously uncharacterized pathway could be of relevance for human fibrotic diseases. For instance, increased numbers of activated mast cells are correlated with fibrosis in testes of infertile men. In these cases all components of the signaling pathway of tryptase were detected as well as expression of COX2. Therefore, our study describes as-yet-unknown interactions between mast cells and fibroblasts, which could be relevant for human fibrotic diseases.

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Besides the hypothalamus and pituitary, melatonin action at the testicular level has been recently suggested. Therefore, we investigated in the Syrian hamster, a well-characterized seasonal breeder, melatonin action on Leydig cells, testicular expression of melatonergic receptors, and possible interactions between melatonin receptors and the previously identified testicular serotoninergic and CRH systems. In isolated Leydig cells from active testes of adult hamsters kept in a long-day (14 h light, 10 h dark) photoperiod and from regressed testes of adult animals exposed to a short-day photoperiod during 16 wk (6 h light, 18 h dark), melatonin significantly reduced human chorionic gonadotropin-stimulated production of cAMP and the main androgens: testosterone and androstane-3alpha,17beta-diol, respectively, and decreased the expression of steroidogenic acute regulatory protein, P450 side chain cleavage, 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase. In Leydig cells exposed to a short-day photoperiod during 16 wk, melatonin stimulated the conversion of testosterone into 5alpha-reduced androgens by inducing 5alpha-reductase isoform 1, and controlled androstane-3alpha,17beta-diol production by inhibiting 3alpha-hydroxysteroid dehydrogenase expression. Melatonin subtype (mel1a) receptors were detected in Leydig cells. Although the local serotonin system did not mediate melatonin action on androgen production, melatonergic effect on steroidogenesis involved the interaction between mel1a receptors and the inhibitory CRH system. Moreover, melatonin significantly increased CRH mRNA levels and production in hamster Leydig cells expressing CRH subtype 1 receptors. Our studies indicate that melatonin may act as a local inhibitor of human chorionic gonadotropin-stimulated cAMP and androgen production through mel1a receptors, down-regulation of steroidogenic acute regulatory protein, and key steroidogenic enzymes expression and its interaction with the local CRH system.