Protein kinase G-mediated stimulation of basal Leydig cell steroidogenesis

Abstract

The androgen-secreting Leydig cells produce cGMP, but the pathways responsible for generation and actions of this intracellular messenger have been incompletely characterized in these cells. Here, we show the presence of mRNA transcripts for the membrane-bound and soluble guanylyl cyclases (sGC), the cGMP-specific phosphodiesterase 5, and the cGMP-dependent protein kinase I (PKG I) and PKG II in purified rat Leydig cells from adult animals. Stimulation of both guanylyl cyclases and inhibition of phosphodiesterase 5 in vitro were accompanied by elevations in cGMP and androgen production, whereas inhibition of sGC and PKG led to a decrease in steroidogenesis. The stimulatory action of cGMP on steroidogenesis was preserved in cells with inhibited cAMP-dependent protein kinases. Experiments with exogenously added substrates revealed the dependence of cGMP-induced progesterone and androgen synthesis on cholesterol but not on 22-OH cholesterol, pregnenolone, progesterone, and Δ4-androstenedione. Treatment with nitric oxide donor increased phosphorylation of the steroidogenic acute regulatory protein (StAR). In contrast, inhibition of sGC and PKG, but not protein kinase A, significantly reduced StAR phosphorylation. These results suggest that cGMP contributes to the control of basal steroidogenesis in Leydig cells through the PKG-dependent modification of the StAR protein.

Orally applied doxazosin disturbed testosterone homeostasis and changed the transcriptional profile of steroidogenic machinery, cAMP/cGMP signalling and adrenergic receptors in Leydig cells of adult rats

“…The proportion of Leydig cells present in culture was determined by staining for HSD3B activity (Payne et al, 1980), and was found to be 95.3 ± 2.7%, while the viability was more than 90%. The steroidogenic capacity of Leydig cells (estimated by dose-dependent stimulation with hCG) and the activity of steroidogenic enzymes (estimated by incubating cells with increasing concentrations of steroid substrates), were in line with those previously published by others (Akinbami et al, 1994) as well as our group (Andric et al, 2007(Andric et al, , 2010aKostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011. These data were not showed and served to us just as internal control.…”

“…To follow ex vivo steroids production, and the expression of steroidogenic machinery elements (steroidogenic enzymes, proteins related to the steroidogenesis, transcription factors), cAMP signalling elements (ADCY, PRKA subunits, cAMP specific PDEs), cGMP signalling elements (GUCY, PRKG isoforms, cGMP-specific and dual PDEs) and ADRs, we used primary cultures of purified Leydig cells obtained from control and Doxa-treated rats prepared as described previously by our group (Andric et al, 2007(Andric et al, , 2010aKostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011. Primary cultures of purified Leydig cells were prepared from suspensions of interstitial cells.…”

“…Primary cultures of purified Leydig cells were prepared from suspensions of interstitial cells. Suspensions of interstitial cells were prepared according to Anakwe et al (Anakwe et al, 1985) with some modifications described previously by our group (Andric et al, 2007(Andric et al, , 2010aKostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011. Testes were quickly removed, decapsulated and placed in a 50-mL plastic tube (two testes per tube) containing 3 mL of collagenase solution (1.25 mg/mL collagenase Type I; 1.5% BSA; 20 mM HEPES in DMEM/F12) and incubated for 15 min at 34°C in a shaking water bath oscillating at 120 cycles/min.…”

“…The 0.2% trypan blue dye exclusion test (Sigma Inc) was used to determine total cell counts and to ensure that greater than 95% of the cells were viable. This suspensions of interstitial cells (Klinefelter et al, 1987) was used to prepare primary cultures of purified Leydig cells (Andric et al, 2007(Andric et al, , 2010aKostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011 by centrifugation on a Percoll gradient consisting of four 2 mL layers of Percoll with densities of 1.090, 1.080, 1.065 and 1.045 g/mL (formed by mixing isotonic Percoll consisting of 109 concentrated DMEM/F12 enriched with 3% of BSA and the corresponding amount of Percoll and distilled water). A crude suspension of interstitial cells (approximately 35-40 9 10 6 cells), containing besides Leydig cells macrophages and endothelial cells (Klinefelter et al, 1987), was applied to each Percoll gradient and centrifuged at 500 g for 28 min at room temperature.…”

“…Hormones, cAMP/cGMP and NO measurement Progesterone (PROG), androgens (T+DHT), estradiol (E2) and LH levels were measured by radioimmunoassay (Andric et al, 2007(Andric et al, , 2010aKostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011. Progesterone measurements were assayed in duplicate, by RIA (the sensitivity: 6 pg per tube; the intra-assay coefficient of variation: 6.8%; the inter-assay coefficient of variation: 8.7%) using the anti-progesterone serum No.337 (Andric et al, 2007;Kostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011.…”