Carolina Mondillo

Interaction between arachidonic acid and cAMP signaling pathways enhances steroidogenesis and StAR gene expression in MA-10 Leydig tumor cells

Previous studies have demonstrated that trophic hormone stimulation induced cyclic AMP (cAMP) formation and arachidonic acid (AA) release from phospholipids and that both these compounds were required for steroid biosynthesis and steroidogenic acute regulatory (StAR) gene expression in MA-10 mouse Leydig tumor cells. The present study further investigates the synergistic effects of the AA and cAMP interaction on steroidogenesis. To demonstrate cAMP-induced AA release, MA-10 cells were pre-loaded with 3H-AA and subsequently treated with dibutyryl cyclic AMP (dbcAMP). Stimulation with dbcAMP significantly induced AA release in MA-10 cells to a level 145.7% higher than that of controls. Lowering intracellular cAMP concentration by expressing a cAMP-phosphodiesterase significantly reduced human chorionic gonadotrophin (hCG)-induced AA release. The dbcAMP-induced AA release was inhibited significantly by the phospholipase A(2) (PLA(2)) inhibitor dexamethasone (Dex) and also by the protein kinase A (PKA) inhibitor H89, suggesting the involvement of PKA phosphorylation and/or PLA(2) activation in cAMP-induced AA release. The effect of the interaction between AA and cAMP on StAR gene expression and steroid production was also investigated. While 0.2 mM dbcAMP induced only very low levels of StAR protein, StAR mRNA, StAR promoter activity and steroid production, all of these parameters increased dramatically as AA concentration in the culture medium was increased from 0 to 200 microM. Importantly, AA was not able to induce a significant increase in steroidogenesis at any concentration when used in the absence of dbcAMP. However, when used in concert with submaximal concentrations of dbcAMP (0.05 mm to 0.5 mm), AA was capable of stimulating StAR gene expression and increasing steroid production significantly. The results from this study demonstrate that AA and cAMP act in a highly synergistic manner to increase the sensitivity of steroid production to trophic hormone stimulation and probably do so by increasing StAR gene expression.

Involvement of nitric oxide synthase in the mechanism of histamine-induced inhibition of Leydig cell steroidogenesis via histamine receptor subtypes in Sprague-Dawley rats.

Abstract This study was conducted to shed light on the so far unexplored intracellular mechanisms underlying negative modulation of Leydig cell steroidogenesis by histamine (HA). Using the MA-10 cell line and highly purified rat Leydig cells as experimental models, we examined the effect of the amine on biochemical steps known to be modulated by HA or involved in LH/hCG action. In agreement with previous findings, HA at 10 μM showed a potent inhibitory effect on hCG-stimulated steroid synthesis, regardless of the gonadotropin concentration used. Moreover, HA decreased not only LH/hCG-induced cAMP production but also steroid synthesis stimulated by the permeable cAMP analog dibutyryl cAMP (db-cAMP). Considering the post-cAMP sites of HA action, it is shown herein that HA markedly inhibited db-cAMP-stimulated steroidogenic acute regulatory (STAR) protein expression, as well as steps catalyzed by P450-dependent enzymes, mainly the conversion of cholesterol to pregnenolone by cholesterol side-chain cleavage enzyme (CYP11A). The antisteroidogenic action of HA was blocked by addition of the phospholipase C (PLC) inhibitor U73122, and HA significantly augmented inositol triphosphate (IP3) production, suggesting a major role for the PLC/IP3 pathway in HA-induced inhibition of Leydig cell function. Finally, HA increased nitric oxide synthase (NOS) activity, and the NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) markedly attenuated the effect of the amine on steroid synthesis. On the basis of our findings, HA antagonizes the gonadotropin action in Leydig cells at steps before and after cAMP formation. NOS activation is the main intracellular mechanism by which HA exerts its antisteroidogenic effects.

Dual Role of Histamine in Modulation of Leydig Cell Steroidogenesis via HRH1 and HRH2 Receptor Subtypes

Abstract Although several reports indicate effects of histamine (HA) on female reproductive functions, scant literature exists to suggest a physiological role of HA in the male gonad. In the present study, we report a dual concentration-dependent effect of HA on steroidogenesis in MA-10 murine Leydig cells and purified rat Leydig cells. Although 1 nM HA can stimulate steroid production and significantly increase the response to LH/hCG in these cells, 10 μM HA exerts an inhibitory effect. We also provide confirming evidence for the existence of functional HRH1 and HRH2 receptors in both experimental models. The use of HRH1 and HRH2 selective agonists and antagonists led us to suggest that HRH2 activation would be largely responsible for stimulation of steroidogenesis, while HRH1 activation is required for inhibition of steroid synthesis. Our results regarding signal transduction pathways associated with these receptors indicate the coupling of HRH2 to the adenylate cyclase system through direct interaction with a Gs protein. Moreover, we show HRH1 activation mediates increases in inositol phosphate production, possibly due to coupling of this receptor to Gq protein and phospholipase C activation. The data compiled in this report clearly indicate that HA can modulate Leydig cell steroidogenesis in the testis and suggest a possible new physiological site of action for HA. Given that many drugs binding to HRH1, HRH2, or both, are widely prescribed for the treatment of diverse HA-related pathologies, it seems necessary to increase the knowledge regarding histaminergic regulation of testicular functions, to avoid possible unexpected side effects of such substances in the testis.

Effects of nitric oxide on aldosterone synthesis and nitric oxide synthase activity in glomerulosa cells from bovine adrenal gland

This study investigated the effects of two NO-releasing agents, diethylenetriamine-NO (deta-NO) and sodium nitroprusside (SNP), on basal, ACTH-, and angiotensin II (AngII)-stimulated aldosterone production in glomerulosa cells from bovine adrenal gland. NO donors inhibited basal and ACTH- or AngII-stimulated aldosterone synthesis in a concentration-dependent manner. Deta-NO and SNP also provoked a concentration-dependent stimulation of cGMP production. However, cGMP was not responsible for the inhibition of aldosterone secretion, because a cGMP analog did not reproduce the inhibitory effect. Moreover, soluble guanylyl cyclase or protein kinase G inhibitors did not revert the inhibitory effect of NO on aldosterone production. NO donors did not modify ACTH-stimulated cAMP production or AngII-stimulated PLC activity stimulation, but inhibited 22[R] hydroxycholesterol- or pregnenolone-stimulated aldosteronogenesis. NO can be synthesized in bovine glomerulosa cells because nitrite production was determined and characterization of NOS activity was also performed. Nitrite accumulation was not modified in the presence of ACTH, AngII, or other factors used to induce iNOS. NOS activity that showed a Michaelis-Menten kinetic was NADPH- and calcium-dependent and was inhibited by two competitive inhibitors, L-NAME and L-NMMA. These results show that NO inhibits aldosterone production in glomerulosa cells acting on P450scc and other P450-dependent steroidogenic enzymes, and these cells display NOS activity suggesting that NO can be produced by constitutive NOS isozymes.

H4 histamine receptors inhibit steroidogenesis and proliferation in Leydig cells

The histamine H4 receptor (HRH4), discovered only 13 years ago, is considered a promising drug target for allergy, inflammation, autoimmune disorders and cancer, as reflected by a steadily growing number of scientific publications and patent applications. Although the presence of HRH4 has been evidenced in the testis, its specific localization or its role has not been established. Herein, we sought to identify the possible involvement of HRH4 in the regulation of Leydig cell function. We first evaluated its expression in MA-10 Leydig tumor cells and then assessed the effects of two HRH4 agonists on steroidogenesis and proliferation. We found that HRH4 is functionally expressed in MA-10 cells, and that its activation leads to the inhibition of LH/human chorionic gonadotropin-induced cAMP production and StAR protein expression. Furthermore, we observed decreased cell proliferation after a 24-h HRH4 agonist treatment. We then detected for the sites of HRH4 expression in the normal rat testis, and detected HRH4 immunostaining in the Leydig cells of rats aged 7-240 days, while 21-day-old rats also presented HRH4 expression in male gametes. Finally, we evaluated the effect of HRH4 activation on the proliferation of normal progenitor and immature rat Leydig cell culture, and both proved to be susceptible to the anti-proliferative effect of HRH4 agonists. Given the importance of histamine (2-(1H-imidazol-4-yl)ethanamine) in human (patho)physiology, continued efforts are directed at elucidating the emerging properties of HRH4 and its ligands. This study reveals new sites of HRH4 expression, and should be considered in the design of selective HRH4 agonists for therapeutic purposes.

Novel Role for Histamine Through Classical H1 and H2 Receptors: Regulation of Leydig Cell Steroidogenesis and its Implications for Male Reproductive Function

Most of the physiological functions of HA described to date have been linked to the well-characterized H1 and H2 receptors. Nevertheless, extensive research is continuously going on to elucidate new roles for these receptors. In this respect, recent reports have indicated expression of H1 and H2 receptors in germinal and peritubular cells of the testis, as well as in macrophages and Leydig cells. Interestingly, HA plays a role as autocrine/paracrine modulator of Leydig cell steroidogenesis in several experimental models, both in vivo and in vitro. It was demonstrated very recently that this modulatory effect is concentration-dependent and biphasic: while H1 receptor activation would be responsible for HA-mediated negative modulation of steroidogenesis, H2 receptor activation would lead to stimulation of steroid synthesis. Because antihistamine drugs target HA receptors, the novel role of HA as modulator of testicular steroidogenesis will surely attract more attention to possible unexpected side-effects of such drugs, which might alter the local balance and in turn enhance or decrease androgen production. Considering that HA has been implicated in spermatogenesis, penile erection and sexual behavior as well as steroidogenesis, it appears that the amine plays an integral role in the regulation of male reproductive functions which certainly deserves further investigation.