Age related changes of cAMP and MAPK signaling in Leydig cells of Wistar rats.

Here, we chronologically analyzed age-associated changes of cAMP- and MAPK-signaling in Leydig cells (LCs) in relation with decreased testosterone (T) production. In Wistar rats, decreased serum T observed in 12 to 24-month-old rats was not related to decreased serum LH concentration but to reduced luteinizing hormone receptor (Lhr/LHR) and time-coordinated reduction of steroidogenic gene expression (decreased Cyp11a1, Cyp17a1 in 12-month-old rats followed by decreased Star/StAR, Hsd3b/HSD3B, Hsd17b4, and increased Cyp19a1 later in life). The predecessors of age-related changes noted in LCs from 6 to 12-month-old rats were increased level of soluble adenylate cyclase (Adcy/AC) 10, increased JNK phosphorylation but suppressed P38 MAPK. At approximately the same time changed mRNA abundance for transcription factors important for steroidogenesis was detected (increased Nur77 and decreased Sf1, Dax1). Aging caused biphasic expression pattern of ERK1/2 and Nur77: increased in 12-month but decreased in LCs from 24-month-old rats. Further, decreased basal cAMP level observed from 12 to 24th month coincidence with increased expression of cAMP-specific phosphodiesterase (Pde)4a, Pde4b and regulatory subunit of protein kinase A (Prkar/PKAR). Exposing of senescent LCs to permeable cAMP-analog improved transcription of Sf1, Nur77, Star, Cyp11a1,Cyp17a1, but without effect on aging pattern of Dax1, Pde4a/b, Prkar2a, Lhr and MAPK genes. Collectively, results indicated that age-related LC dysfunction is accompanied with changes in MAPK and cAMP signaling and coordinated reduction in the expression of many of the genes that participate in T synthesis. The predecessors of aged-related changes are increased ratio of pJNK/JNK, AC10 and decreased P38 level in LCs from 6-month-old rats.

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.

Doxazosin (Doxa) is an α1-selective adrenergic receptor (ADR) antagonist widely used, alone or in combination, to treat high blood pressure, benign prostatic hyperplasia symptoms, and recently has been suggested as a potential drug for prostate cancer prevention/treatment. This study was designed to evaluate the effect of in vivo Doxa po-application, in clinically relevant dose, on: (i) steroidogenic machinery homeostasis; (ii) cAMP/cGMP signalling; (iii) transcription profile of ADR in Leydig cells of adult rats. The results showed that po-application of Doxa for once (1×Doxa), or for two (2×Doxa) or 10 (10×Doxa) consecutive days significantly disturbed steroidogenic machinery homeostasis in Leydig cells. Doxa po-application significantly decreased circulating luteinizing hormone and androgens levels. The level of androgens in testicular interstitial fluid and that extracted from testes obtained from 1×Doxa/2×Doxa rats decreased, although it remained unchanged in 10×Doxa rats. Similarly, the ex vivo basal androgen production followed in testes isolated from 1×Doxa/2×Doxa rats decreased, while remained unchanged in 10×Doxa rats. Differently, ex vivo testosterone production and steroidogenic capacity of Leydig cells isolated from 1×Doxa/2×Doxa rats was stimulated, while 10×Doxa had opposite effect. In the same cells, cAMP content/release showed similar stimulatory effect, but back to control level in Leydig cells of 10×Doxa. 1×Doxa/2×Doxa decreased transcripts for cAMP specific phosphodiesterases Pde7b/Pde8b, whereas 10×Doxa increased Pde4d. All types of treatment reduced the expression of genes encoding protein kinase A (PRKA) regulatory subunit (Prkar2b), whereas only 10×Doxa stimulated catalytic subunit (Prkaca). Doxa application more affected cGMP signalling: stimulated transcription of constitutive nitric oxide synthases (Nos1, Nos3) in time-dependent manner, whereas reduced inducible Nos2. 10×Doxa increased guanylyl cyclase 1 transcript and PRKG1 protein in Leydig cells. Orally applied Doxa significantly disturbed the transcriptional 'signature' of steroidogenic machinery, cAMP/cGMP signalling and ADRs and ß-ADRs kinases in Leydig cells, thus giving new molecular insights into the role of cAMP/cGMP/adrenalin signalling in Leydig cells homeostasis.

Structural complexity of the testis and PKG I / StAR interaction regulate the Leydig cell adaptive response to repeated immobilization stress.

The role of the structural complexity of the testis and the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway was analysed in adult male rats exposed to acute and repeated immobilization stress (IMO). In whole testis preparations, exposure to acute and repeated IMO caused an increase in NO production. In contrast, NO production was inhibited in interstitial cell preparations after exposure to all types of stress. In purified Leydig cell preparations, NO production was inhibited only after exposure to prolonged IMO. These findings indicate that biologically active compounds released from various testicular compartments exert both stimulatory and inhibitory effects on NO production. TaqMan Low Density Array of rat phosphodiesterases revealed a decrease in the expression of cGMP-specific phosphodiesterase 5 (PDE5) in Leydig cells of animals exposed to repeated IMO. In contrast, the expression of cGMP-dependent protein kinase type I (PKG I), total and phosphorylated steroidogenic acute regulatory protein (StAR), and PKG I/StAR immunoprecipitated complex was increased during repeated exposure to IMO. The increase in both total and phosphorylated StAR formation was effectively blocked by inhibition of PKG I in vitro. Thus, increased expressions of PKG I and StAR complex, accompanied by decreased PDE5 activity, suggest that the NO-cGMP signalling pathway and consequent activation of the StAR protein regulate the adaptive response of Leydig cells to repeated IMO stress.

The adaptive response of adult rat Leydig cells to repeated immobilization stress: the role of protein kinase A and steroidogenic acute regulatory protein.

The ability of immobilization stress (IMO) to decrease Leydig cell steroidogenesis and serum androgen concentration has been previously observed, but the possible mechanism(s) involved in the adaptation to prolonged or repeated stress have not been identified. In this study, we investigated whether the Leydig cells obtained from adult rats subjected to acute (15 min, 30 min or 2 h) and repeated (2 or 10 days, 2 h daily) IMO show adaptive mechanism(s) in response to stress-impaired steroidogenesis. The results showed that basal and human chorionic gonadotropin-stimulated cAMP production by Leydig cells isolated from rats exposed to both acute and repeated IMO was significantly reduced. Despite the reduced cAMP production, immunoblot analysis revealed increased immunoreactivity for both protein kinase A (PKA) and steroidogenic acute regulatory (StAR) protein in Leydig cells obtained from rats repeatedly exposed to IMO. Also, the phosphorylation and production of mature StAR protein was evident during exposure of rats to repeated IMO treatment. Treatment with cholesterol, the steroid substrate transported into mitochondria by StAR, significantly increased androgen and progesterone production by Leydig cells isolated from rats exposed to repeated IMO. In contrast, when other steroid substrates (22(R)-OH-cholesterol, pregnenolone, progesterone, Delta4-androstenedione) were present in the culture media, Leydig cell steroidogenesis was still reduced by IMO. Thus, PKA-mediated phosphorylation of StAR protein is an important mechanism in the adaptive response of Leydig cells to repeated IMO.

Spontaneous and receptor-controlled soluble guanylyl cyclase activity in anterior pituitary cells.

Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH(3) immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH(3) cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH(3) cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-gamma further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC(50)s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.

Dependence of soluble guanylyl cyclase activity on calcium signaling in pituitary cells.

The role of nitric oxide (NO) in the stimulation of soluble guanylyl cyclase (sGC) is well established, but the mechanism by which the enzyme is inactivated during the prolonged NO stimulation has not been characterized. In this paper we studied the interactions between NO and intracellular Ca(2+) in the control of sGC in rat anterior pituitary cells. Experiments were done in cultured cells, which expressed neuronal and endothelial NO synthases, and in cells with elevated NO levels induced by the expression of inducible NO synthase and by the addition of several NO donors. Basal sGC-dependent cGMP production was stimulated by the increase in NO levels in a time-dependent manner. In contrast, depolarization of cells by high K(+) and Bay K 8644, an L-type Ca(2+) channel agonist, inhibited sGC activity. Depolarization-induced down-regulation of sGC activity was also observed in cells with inhibited cGMP-dependent phosphodiesterases but not in cells bathed in Ca(2+)-deficient medium. This inhibition was independent from the pattern of Ca(2+) signaling (oscillatory versus nonoscillatory) and NO levels, and was determined by averaged concentration of intracellular Ca(2+). These results indicate that inactivation of sGC by intracellular Ca(2+) serves as a negative feedback to break the stimulatory action of NO on enzyme activity in intact pituitary cells.

Acute effects of polychlorinated biphenyl-containing and -free transformer fluids on rat testicular steroidogenesis.

Polychlorinated biphenyl (PCB)-based transformer fluids belong to a class of environmentally persistent mixtures with known toxic effects. Here, we studied the acute effects of Askarel (which contains Aroclor 1260) and two substitute transformer fluids (the silicone oil-based DC561 and the mineral oil-based ENOL C) on rat testicular steroidogenesis. Single intraperitoneal (ip; 10 mg/kg body weight) or bilateral intratesticular (itt; 25 microg/testis) injections of Askarel markedly decreased serum androgen levels 24 hr after administration. In acute testicular cultures from these animals, chorionic gonadotropin-stimulated progesterone and androgen productions were severely attenuated. When itt was injected or added in vitro, Askarel inhibited 3ss-hydroxysteroid dehydrogenase (3ssHSD), stimulated 17[alpha]-hydroxylase/lyase (P450c17), and did not affect 17ss-hydroxysteroid dehydrogenase in testicular postmitochondrial fractions. The ip-injected Askarel did not affect 3ssHSD, but inhibited P450c17, suggesting that a more intensive metabolism of peripherally injected Askarel reduces the circulating levels of active ingredients below the threshold needed for inhibition of 3ssHSD and generates a derivative that inhibits P450c17. In contrast to Askarel, itt-injection (25 microg/testis) of DC561 and ENOL C did not affect in vivo and in vitro steroidogenesis. These findings show the acute effects of Askarel, but not silicone and mineral oils, on testicular steroidogenesis.

Inhibition of rat testicular androgenesis by a polychlorinated biphenyl mixture aroclor 1248.

Polychlorinated biphenyls (PCBs) are complex mixtures of congeners that exhibit carcinogenic and toxicant activities in a variety of mammalian tissues. Here, we studied the acute in vivo and in vitro effects of a commercially used PCB product, Aroclor 1248 (A1248), a mixture of tri-, tetra-, and pentachloro congeners. Single intraperitoneal (i.p.) or bilateral intratesticular (i.t.) injections of A1248 decreased serum androgen levels in both groups 24 h after injection. Chorionic gonadotropin-stimulated androgen production by acute testicular cultures from both groups was also reduced, and progesterone production was attenuated in cultures from i.t.-treated animals. The capacity of the postmitochondrial fractions from testes of i.t.-treated animals to convert pregnenolone to progesterone and progesterone to testosterone was reduced as well. In vitro studies revealed that a 10- to 15-min exposure of postmitochondrial testicular fractions and intact interstitial cells from normal animals to A1248 in a subnanomolar concentration range was sufficient to attenuate the conversion of pregnenolone to progesterone and progesterone to testosterone. At micromolar concentrations, A1248 added in vitro also inhibited the conversion of Delta(4)-androstendione to testosterone without affecting the viability of interstitial cells. These results indicate that A1248 down-regulates the testicular androgenesis by an acute inhibition of 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase/lyase, and 17beta-hydroxysteroid dehydrogenase activities.

Inhibitory effects of stress-activated nitric oxide on antioxidant enzymes and testicular steroidogenesis.

The messenger role of nitric oxide (NO) in immobilization stress-induced inhibition of testicular steroidogenesis has been previously suggested. In accord with this, here, we show that the intratesticular injection of isosorbide dinitrate (ISDN; 2x2.5 mg/testis), an NO donor, mimicked the action of stress on serum testosterone concentrations and hCG-stimulated testosterone production in rat testicular tissue. When added in vitro, ISDN inhibited testicular 3beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase/lyase. Immobilization stress and injections of ISDN also decreased the activity of catalase, glutathione peroxidase, glutathione transferase, and glutathione reductase in the interstitial compartment of testis. When stressed rats were treated concomitantly with bilateral intratesticular injections of N(omega)-nitro-L-arginine methyl ester, a non-selective NOS inhibitor (2x600 microg/testis), the activities of antioxidative enzymes, as well as serum testosterone concentration, were partially normalized. These results indicate that stress-induced stimulation of the testicular NO signalling pathway leads to inhibition of both steroidogenic and antioxidant enzymes.

Characterization of a plasma membrane calcium oscillator in rat pituitary somatotrophs.

In excitable cells, oscillations in intracellular free calcium concentrations ([Ca(2+)](i)) can arise from action-potential-driven Ca(2+) influx, and such signals can have either a localized or global form, depending on the coupling of voltage-gated Ca(2+) influx to intracellular Ca(2+) release pathway. Here we show that rat pituitary somatotrophs generate spontaneous [Ca(2+)](i) oscillations, which rise from fluctuations in the influx of external Ca(2+) and propagate within the cytoplasm and nucleus. The addition of caffeine and ryanodine, modulators of ryanodine-receptor channels, and the depletion of intracellular Ca(2+) stores by thapsigargin and ionomycin did not affect the global nature of spontaneous [Ca(2+)](i) signals. Bay K 8644, an L-type Ca(2+) channel agonist, initiated [Ca(2+)](i) signaling in quiescent cells, increased the amplitude of [Ca(2+)](i) spikes in spontaneously active cells, and stimulated growth hormone secretion in perifused pituitary cells. Nifedipine, a blocker of L-type Ca(2+) channels, decreased the amplitude of spikes and basal growth hormone secretion, whereas Ni(2+), a blocker of T-type Ca(2+) channels, abolished spontaneous [Ca(2+)](i) oscillations. Spiking was also abolished by the removal of extracellular Na(+) and by the addition of 10 mM Ca(2+), Mg(2+), or Sr(2+), the blockers of cyclic nucleotide-gated channels. Reverse transcriptase-polymerase chain reaction and Southern blot analyses indicated the expression of mRNAs for these channels in mixed pituitary cells and purified somatotrophs. Growth hormone-releasing hormone, an agonist that stimulated cAMP and cGMP productions in a dose-dependent manner, initiated spiking in quiescent cells and increased the frequency of spiking in spontaneously active cells. These results indicate that in somatotrophs a cyclic nucleotide-controlled plasma membrane Ca(2+) oscillator is capable of generating global Ca(2+) signals spontaneously and in response to agonist stimulation. The Ca(2+)-signaling activity of this oscillator is dependent on voltage-gated Ca(2+) influx but not on Ca(2+) release from intracellular stores.

Involvement of inducible nitric oxide synthase in stress-impaired testicular steroidogenesis.

The immobilization stress induces an acute inhibition of testicular steroidogenesis that is mediated by the nitric oxide (NO) signaling pathway. Here we compared the effects of 2-h immobilization stress on in vivo and in vitro rat steroidogenesis at two time points, 0 h and 6 h after the end of the stress session. As expected, serum androgens and human chorionic gonadotropin (hCG)-stimulated progesterone and testosterone production by testicular tissue were inhibited at 0 h, and also at the 6-h time point. Both the acute and sustained inhibitions of in vitro steroidogenesis were accompanied by a significant increase in nitrite, a stable oxidation product of NO. To clarify which subtype of NO synthase (NOS) (constitutive (cNOS) or inducible (iNOS)) participates in down-regulation of testicular steroidogenesis, aminoguanidine hydrochloride (AG), a selective iNOS inhibitor, was employed. Intratesticular injection of AG prevented the sustained, but not the acute, stress-induced decrease in serum testosterone. When added in vitro, it also prevented the sustained decrease in steroid production and increase in nitrite production by testicular tissue, both in a dose-dependent manner and with EC microM. Furthermore, AG added in vivo and in vitro effectively blocked the sustained decrease in 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17alpha-hydroxylase/C17-20 lyase (P450c17) activities. In all concentrations employed, AG did not affect serum androgens and in vitro steroid and nitrite production in unstressed animals. These results indicate that the NO signaling pathway participates in acute and sustained stress-induced down-regulation of testicular steroidogenesis, presumably through its direct action on 3betaHSD and P450c17. The acute NO production is controlled by cNOS and the sustained production of this messenger is controlled by iNOS.