Signalni put cikličnog guanozin monofosfata u Leydig-ovim ćelijama

Abstract

<p>Biosinteza testosterona, glavnog mu&scaron;kog polnog&nbsp;hormona, u Leydig-ovim ćelijama testisa je&nbsp;primarno regulisana cAMP signalnim putem, ali se i&nbsp;drugi signalni putevi uključuju kao modulatorni.&nbsp;Obzirom da nema preciznih podataka o uče&scaron;ću&nbsp;cGMP signalnog puta u regulaciji androgeneze&nbsp;Leydig-ovih ćelija, ova studija je dizajnirana da&nbsp;defini&scaron;e: (1) funkcionalnost cGMP signalnog puta,&nbsp;kao i mehanizme preko kojih se ovaj signalni put&nbsp;upliće u regulaciju procesa sinteze testosterona; (2)&nbsp;funkcionisanje cGMP puta u uslovima poremećene&nbsp;homeostaze testosterona tj. eventualne&nbsp;mehanizme povratne sprege od testosterona na&nbsp;cGMP signalni puta u Leydig-ovim ćelijama testisa&nbsp;adultnih pacova. Homeostaza Leydig-ovih ćelija i&nbsp;funkcionalnost NO-cGMP signalnog puta je praćena&nbsp;kori&scaron;ćenjem pet in vivoekperimentalih modela: dva&nbsp;u kojima je mimikrirana poremećena homeostaza&nbsp;cGMP signalnog puta (akutni i hronični tretman&nbsp;Sildenafil-citratom, Vijagrom) i tri u kojima je&nbsp;mimikrirana poremećena homeostaza testosterona&nbsp;(androgenizacija; hipogonadalni-hipgonadizam;&nbsp;sistemska blokada androgenog receptora). Rezultati&nbsp;su pokazali da Leydig-ove ćelije ekspresuju&nbsp;funkcionalne komponente cGMP signalnog puta&nbsp;(GUCY1, GUCY2, PRKG1, PRKG2 i PDE5) i da cGMP&nbsp;stimuli&scaron;e androgenezu Leydig-ovih ćelija&nbsp;povećavajući transport holesterola u mitohondrije&nbsp;preko PRKG1 zavisne fosforilacije StAR proteina.&nbsp;Akutni&nbsp; in vivotretman sa Sildenafil-citratom je&nbsp;povećao zapreminu TIF-a, kao i &nbsp;koncentraciju&nbsp;androgena u TIF-u, testikularnom tkivu i serumu, a&nbsp;ovaj stimulatorni efekat je ostvaren angažovanjem&nbsp;cGMP-PRKG1 signalnog puta. Hronični&nbsp; in vivo&nbsp;tretman Sildenafil-citratom je povećao nivo&nbsp;cirkuli&scaron;ućih androgena i bazalnu androgenu&nbsp;produkciju Leydig-ovih ćelija ukjučivanjem i cAMP i&nbsp;cGMP signalnih puteva i &nbsp;njihovom zajedničkom&nbsp;aktivacijom StAR proteina. Sa druge strane, visoki&nbsp;nivo sistemskog testosterona je smanjio&nbsp;membranski potencijal mitohondrija (∆&psi;m&nbsp;),&nbsp;stimulisao ekspresiju pro-apoptotičke NOS2, a&nbsp;samim tim i produkciju NO, a inhibisao ekspresiju&nbsp;anti-apoptotičke PRKG1, &scaron;to je uz povećanu&nbsp;razgradnju cGMP-a stimulacijom ekspresije PDE5,&nbsp;omogućilo prevladavanje pro-apoptotičnih nad&nbsp;anti-apoptočnim signalima i apoptozu Leydig-ovih&nbsp;ćelija. Sistemskom blokadom androgenih receptora&nbsp;poni&scaron;teni su pro-apoptotički efekti testosterona.&nbsp;Sumirano, može se zaključiti da je cGMP signalni&nbsp;put u Leydig-ovim ćelijama adultnih pacova&nbsp;funkcionalan i da reguli&scaron;e proces biosinteze&nbsp;testosterona, a da testosteron modulacijom NO-cGMP signalnog puta predstavlja potencijalni&nbsp;autokrini regulator testikularne anrogeneze i da u&nbsp;uslovima visokog nivoa sistemskog testosterona&nbsp;doprinosi uspostavljanju &bdquo;androgene homeostaze&ldquo;&nbsp;preko regulacije brojnosti populacije Leydig-ovih&nbsp;ćelija.</p>

<p>Biosynthesis of testosterone, the main male sex&nbsp;hormone, in testicular Leydig cells is dominantly&nbsp;regulated by cAMP signaling but also several other&nbsp;pathways also contribute to this process including&nbsp;nitric oxide (NO)-cGMP signaling pathway. Although&nbsp;NO-cGMP signaling pathway is operative, its&nbsp;presence and the role in steroidogenesis have been&nbsp;incompletely characterized. For that reason, this&nbsp;study was designed to define: (1) status of cGMP&nbsp;signaling pathway in Leydig cells, and the&nbsp;mechanism by which cGMP could control&nbsp;steroidogenesis, as well as, (2) the possible&nbsp;autocrine role of testosterone on NO-cGMP&nbsp;pathway in Leydig cells. To addressed these issue&nbsp;several different experimental approaches were&nbsp;used: two&nbsp; in vivomodels that mimicked disturbed&nbsp;homeostasis of NO-cGMP signaling pathway (acute&nbsp;and chronic treatment with the inhibitor of&nbsp;phosphodiesterase 5 (PDE5), Sildenafil citrate,&nbsp;Viagra) and three&nbsp; in vivomodels that mimicked&nbsp;disturbed homeostasis &nbsp;of testosterone (treatment&nbsp;of eugonadal rats with testosterone enanthate; an&nbsp;experimental model of hypogonadotropic&nbsp;hypogonadism together with testosterone&nbsp;replacement; and treatment with androgen&nbsp;receptor antagonist). Additionally ex vivostudies on&nbsp;purified Leydig cells were applied. The results&nbsp;showed that in Leydig cells expressed functional&nbsp;components of cGMP signaling pathway (GUCY1,&nbsp;GUCY2, PRKG1, PRKG2 and PDE5) and that cGMP&nbsp;stimulates androgenesis in Leydig cells by increasing&nbsp;transport of cholesterol into the mitochondria&nbsp;through PRKG1-dependent StAR protein&nbsp;phosphorylation. Acute&nbsp; in vivoInhibition of PDE5&nbsp;increased TIF volume, as well as, concentration of&nbsp;androgens in TIF, testicular tissue and serum, and&nbsp;this stimulatory effect was achieved by engaging&nbsp;cGMP-PRKG1 signaling pathway. Chronic&nbsp; in vivo&nbsp;PDE5 inhibition increased the level of circulating&nbsp;androgens and basal androgen production of Leydig&nbsp;cells by engagement of cAMP- and cGMP-signaling&nbsp;pathways and their mutual activation of StAR&nbsp;protein. Data obtained in second part provide novel&nbsp;evidence for the role of testosterone in the down-regulation of Leydig cell functionality by regulating&nbsp;the NO-cGMP signaling pathway. The high systemic&nbsp;testosterone level reduced mitochondrial&nbsp;<br />membrane potential (∆&psi;m&nbsp;), stimulated expression&nbsp;of pro-apoptotic NOS2, and thus the &nbsp;production of&nbsp;NO, but inhibited expression of anti-apoptotic&nbsp;PRKG1 and increased degradation of cGMP by&nbsp;stimulation of PDE5 expression. This signaling&nbsp;scenario leads to decreased Leydig cell&nbsp;steroidogenesis and was associated with increased&nbsp;apoptosis of Leydig cells. Systemic androgen&nbsp;receptor blockade abrogated the pro-apoptotic&nbsp;effects of testosterone. Furthermore, in&nbsp;hypogonadal animals with low serum testosterone,&nbsp;the NOS2 protein level and NO production from&nbsp;Leydig cells were significantly reduced. The&nbsp; ex vivo&nbsp;results indicate that testosterone gradually up-regulated Nos2 gene expression and NO production&nbsp;in primary Leydig cell cultures. These findings&nbsp;suggest that up-regulation of&nbsp; Nos2gene expression&nbsp;is a direct effect of testosterone on Leydig cells.In&nbsp;summary, it can be concluded that cGMP signaling&nbsp;pathway is functional in Leydig cells of adult rats&nbsp;and is involved in regulation of testosterone&nbsp;biosynthesis. Testosterone, on the other side, acts&nbsp;as autocrine regulator of testicular androgenesis&nbsp;and important regulator of abundance of Leydig cell&nbsp;population.</p>