- Joined
- Mar 18, 2012
- Messages
- 2,759
Chronic treatment with sildenafil stimulates Leydig cell and testosterone secretion
Chronic treatment with sildenafil stimulates Leydig cell and testosterone secretion , Karina Lidianne Alcantara Saraiva, Int. J. Exp. Path. (2009), 90, 454–462
Discussion
The relevance of cGMP system on steroidogenesis can be demonstrated by the use of Sildenafil. Particularly, in the testis, this cyclic nucleotide is involved in spermatozoa motility, development of testicular germ cells, relaxation of peritubular lamina propria cells, testosterone production in Leydig cells and dilatation of testicular blood vessels (Middendorff et al. 2000).
The steroid-secreting Leydig cells regulate the development and activity of the male reproductive tract and external sex characteristics, and may enhance spermatogenesis in the seminiferous tubules. Steroid hormones are synthesized from cholesterol in a multi-step enzymatic process, in which the StAR appears to transfer cholesterol from cellular stores to the inner mitochondrial membrane to be converted to pregnenolone by the cytochrome P450scc enzyme. The pregnenolone is transported and enzymatically converted to testosterone precursors in the SER, before the testosterone synthesis (Clark & Cochrum 2007; Yang et al. 2007).
This is the first research that demonstrated the effects of the chronic treatment with Sildenafil on Leydig cells steroidogenesis. These cells consist of abundant smooth endoplasmic reticulum, numerous mitochondria with tubular cristae and a variable number of lipid droplets, which participate actively in hormonal biosynthesis. Sildenafil treated Leydig cells showed a vesicular SER and some large vacuoles scattered throughout the cytoplasm that are features of an active secreting cell. According to Ohata (1979), these vacuoles are derived from testosterone-containing vesicles of the SER and could migrate towards the cell surface to open into the extra-cellular space. The function of membranous whorles is still uncertain. It is hypothesized that this organelle stores cholesterol used in testosterone synthesis. Interestingly, in Sildenafil treated Leydig cells, several vesicles were observed in the whorles’ periphery, which could be related to the hormonal precursors transport.
Isolated Leydig cells presented similar ultrastructural characteristics; however, they showed an SER with a trabecular appearance. This morphological alteration could be related to the isolation procedure, as these cells were digested in an enzymatic solution, probably affecting the filaments network.
All the ultrastructural alterations observed in this work could be related to an enhancement of the testosterone synthesis, resultant from PDE5 inhibition and cGMP accumulation. To confirm this hypothesis, an immunocytochemical and hormonal study was performed. The results indicated an enhanced labelling to StAR, P450scc and testosterone in isolated hCG-stimulated Leydig cell from the treated group. Also, a significant increase in the plasmatic testosterone was achieved in treated group compared with that in control. The PDE5 inhibitors promote their pharmacological effects by inhibiting phosphodiesterase type 5, an enzyme responsible for the degradation of cGMP. The raised levels of this cyclic nucleotide affect many intracellular functions (Glenn et al. 2007). This drug mechanism could be involved in the activation of the steroidogenic pathway and testosterone secretion, as recently PDE5 was identified in Leydig cells (Scipioni et al. 2005). Steroid hormones are synthesized from cholesterol in the gonads in response to pituitary hormones, such as hCG via the classical first messenger . second messenger pathway. To delineate the mechanism of hCG-mediated induction of testosterone synthesis, a study was performed to investigate the changes in 3b-HSD-1, P450scc and StAR expression in mouse Leydig cells and an enhanced expression of these enzymes was detected (Yang et al. 2007). In the present work, after the chronic treatment with Sildenafil, isolated and hCG-stimulated Leydig cells presented an augmented immunolocalization of StAR, P450scc and testosterone. These data indicate that Sildenafil and hCG would possibly act synergistically to increase cGMP levels via different mechanisms. The hCG increases intracellular cGMP production by activating guanylyl cyclase system (Lin et al. 1979; Mukhopadhyay & Leideberger 1988), and Sildenafil induces an accumulation of cGMP by inhibiting the PDE5, or could act via NO or ANP-dependent mechanism (Preston et al. 2004).
The cyclic GMP is synthesized by two classes of enzymes called guanylyl cyclases, and both generate cGMP from intracellular GTP. The particulate guanylyl cyclases are membrane bound receptors that bind natriuretic and guanylin peptides. The sGC is a heme-containing, heterodimeric nitric oxide receptor. It consists of two subunits, a and b, which make up the active enzyme. The cGMP acts directly with effectors, such as cGMP-dependent protein kinases, cyclic nucleotide-gated channels, and cGMP-regulated phosphodiesterases (Wong & Garbers 1992; Krumenacker & Murad 2006).
Andric et al. (2007) described that the stimulation of both guanylyl cyclases and inhibition of phosphodiesterase 5 in vitro were accompanied by elevations in cGMP and androgen production. However, the inhibition of sGC and protein kinase G (PKG) significantly reduced the StAR phosphorilation, leading to a decreased steroidogenesis. Thus, the authors suggested that cGMP participates in Leydig cell steroidogenesis through the PKG-dependent modification of the StAR protein.
A study investigated the effects of 4b-PMA, a potent activator of protein kinase C (PKC), on atrial peptide-induced testosterone production and cGMP accumulation in Leydig cells. This compound inhibited testosterone production and markedly reduced the amount of cGMP through an enhanced phosphodiesterase activity, stimulated by PKC. The testosterone production and cGMP formation was completely reversed in the presence of a phosphodiesterase inhibitor. The cause of the decreased hormone production after 4b-PMA treatment was related to the diminished accumulation of the second messenger (Mukhopadhyay & Leideberger 1988).
Similarly, the present work shows that the chronic treatment with Sildenafil increased the testosterone biosynthesis in Leydig cells, probably through cGMP accumulation due to PDE5 inhibition.
The results obtained in the present study are in agreement with those of Carosa et al. (2004). Men suffering from erectile dysfunction have androgen levels significantly lower than normal ones. These authors demonstrated the effects of two different type V phosphodiesterase inhibitors treatments, Sidenafil (50 mg) and Tadalafil (20 mg) on the reversibility of testosterone reduction. This pharmacological therapy was able to enhance testosterone and reduce LH levels, probably due to the higher frequency of full sexual intercourse. However, the authors did not rule out the possibility of a direct effect of the PDE5 inhibitors on the on the human testis, as this tissue expresses quite a high level of PDE5. Khurana and Pandey (1993) demonstrated that the atrial natriuretic factor, brain natriuretic peptide and C-type natriuretic peptide (CNP), induced the production of intermediate precursors of testosterone biosynthesis, in purified mouse Leydig cells. All of these natriuretic peptides stimulated the steroidogenesis pathways by a transmembrane signal transduction mechanism involving the generation and accumulation of intracellular cGMP. This stimulatory effect begins at the first step involved in the conversion of cholesterol to pregnenolone.
The pulmonary vasodilator effects of Sildenafil on acute and chronic hypoxia-induced pulmonary hypertension in rats are potentiated by atrial natriuretic peptide (ANP), possibly by a synergistic effect on the increase in plasma cGMP synthesis (Preston et al. 2004). Similarly, ANP could be involved in cGMP synthesis in the Leydig cell, which would be accumulated in response to Sildenafil treatment. Conversely, the NO exerts an inhibitory effect on Leydig cell steroidogenesis, which is not mediated by the sGC as it fails to increase cGMP production (Del Punta et al. 1996). According to Middendorff et al. (1997), NO and CNP have been shown to influence testosterone release. While CNP increase testosterone via a promiscuous activation of cAMP-dependent protein kinase by cGMP in mice, a decrease on testosterone levels was detected in response to NO, as this agent could inhibit P450scc enzyme by directly binding to its heme iron.
Androgens have been shown to exert direct vasodilator effects in arteries from both humans and experimental animals. Oka et al. (2007) demonstrated that Dehydroepiandrosterone, a testosterone precursor, completely blocked hypoxic pulmonary vasoconstriction, which was accompanied by opening potassium channels, upregulation of sGC protein expression . activity and improved pulmonary artery vasodilator responsiveness to NO. In addition, Seyrek et al. (2007) have recently demonstrated that supraphysiological concentrations of testosterone act via ATP-sensitive K+ channel opening to induce relaxation in radial artery in vitro.
Therefore, the present study did not rule out the possible involvement of the enhanced steroid hormones biosynthesis, after the chronic treatment with Sildenafil, on pulmonary hypertension improvement.
In conclusion, the chronic treatment with Sildenafil exerted a stimulatory effect on testosterone production, probably via cGMP accumulation. This increased hormonal synthesis should be taken into account, as this therapeutic scheme has been extensively used worldwide in patients with pulmonary hypertension disorders. Additional experiments are in development in our laboratory to describe the possible effects on sperm parameters and fertilizing capacity.
From,
Sildenafil treatment in vivo stimulates Leydig cell steroidogenesis via the cAMP/cGMP signaling pathway, Silvana A. Andric, AJP - Endo October 2010 vol. 299 no. 4 E544-E550
Abstract
Sildenafil citrate (Viagra), a cGMP-selective phosphodiesterase (PDE) inhibitor, is widely used to treat erectile dysfunction and pulmonary arterial hypertension. In contrast to its well established action on erectile dysfunction, little is known on the action of sildenafil on cGMP/cAMP signaling and testicular steroidogenesis. This study was designed to assess the effects of prolonged sildenafil treatment on NO synthase-dependent signaling and steroidogenic function of rat Leydig cells.
Male adult rats were treated with Viagra (1.25 mg/kg body wt) daily for 30 days. In our studies, serum testosterone and ex vivo testosterone production significantly increased in sildenafil-treated animals. Human chorionic gonadotropin-stimulated testosterone production and cAMP accumulation were also significantly higher in Leydig cells obtained from sildenafil-treated rats. The expression of soluble guanylyl cyclase (GUCY1) subunits (Gucy1a1, Gucy1b1) significantly increased; cAMP-specific Pde4a, cGMP-specific Pde6c, and dual Pde1c and Nos2 were inhibited and expression of Nos3, protein kinase G1 (Pkg1), and Pde5 remained unchanged. Treatment of purified Leydig cells with NO donor caused a dose-dependent increase in both testosterone and cGMP production. Testosterone and cGMP production was significantly higher in Leydig cells obtained from sildenafil-treated animals. The stimulatory effect of NO donor was significantly enhanced by saturating concentrations of hCG in both Leydig cells obtained from control and sildenafil-treated animals. Occurrence of mature steroidogenic acute regulatory protein also increased in sildenafil treated animals in accord with increased cAMP and cGMP production. In summary, inhibition of PDE activity during prolonged sildenafil treatment increased serum testosterone level and Leydig cells' steroidogenic capacity by coordinated stimulatory action on cAMP and cGMP signaling pathway.......
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2741156/
Chronic treatment with sildenafil stimulates Leydig cell and testosterone secretion , Karina Lidianne Alcantara Saraiva, Int. J. Exp. Path. (2009), 90, 454–462
Discussion
The relevance of cGMP system on steroidogenesis can be demonstrated by the use of Sildenafil. Particularly, in the testis, this cyclic nucleotide is involved in spermatozoa motility, development of testicular germ cells, relaxation of peritubular lamina propria cells, testosterone production in Leydig cells and dilatation of testicular blood vessels (Middendorff et al. 2000).
The steroid-secreting Leydig cells regulate the development and activity of the male reproductive tract and external sex characteristics, and may enhance spermatogenesis in the seminiferous tubules. Steroid hormones are synthesized from cholesterol in a multi-step enzymatic process, in which the StAR appears to transfer cholesterol from cellular stores to the inner mitochondrial membrane to be converted to pregnenolone by the cytochrome P450scc enzyme. The pregnenolone is transported and enzymatically converted to testosterone precursors in the SER, before the testosterone synthesis (Clark & Cochrum 2007; Yang et al. 2007).
This is the first research that demonstrated the effects of the chronic treatment with Sildenafil on Leydig cells steroidogenesis. These cells consist of abundant smooth endoplasmic reticulum, numerous mitochondria with tubular cristae and a variable number of lipid droplets, which participate actively in hormonal biosynthesis. Sildenafil treated Leydig cells showed a vesicular SER and some large vacuoles scattered throughout the cytoplasm that are features of an active secreting cell. According to Ohata (1979), these vacuoles are derived from testosterone-containing vesicles of the SER and could migrate towards the cell surface to open into the extra-cellular space. The function of membranous whorles is still uncertain. It is hypothesized that this organelle stores cholesterol used in testosterone synthesis. Interestingly, in Sildenafil treated Leydig cells, several vesicles were observed in the whorles’ periphery, which could be related to the hormonal precursors transport.
Isolated Leydig cells presented similar ultrastructural characteristics; however, they showed an SER with a trabecular appearance. This morphological alteration could be related to the isolation procedure, as these cells were digested in an enzymatic solution, probably affecting the filaments network.
All the ultrastructural alterations observed in this work could be related to an enhancement of the testosterone synthesis, resultant from PDE5 inhibition and cGMP accumulation. To confirm this hypothesis, an immunocytochemical and hormonal study was performed. The results indicated an enhanced labelling to StAR, P450scc and testosterone in isolated hCG-stimulated Leydig cell from the treated group. Also, a significant increase in the plasmatic testosterone was achieved in treated group compared with that in control. The PDE5 inhibitors promote their pharmacological effects by inhibiting phosphodiesterase type 5, an enzyme responsible for the degradation of cGMP. The raised levels of this cyclic nucleotide affect many intracellular functions (Glenn et al. 2007). This drug mechanism could be involved in the activation of the steroidogenic pathway and testosterone secretion, as recently PDE5 was identified in Leydig cells (Scipioni et al. 2005). Steroid hormones are synthesized from cholesterol in the gonads in response to pituitary hormones, such as hCG via the classical first messenger . second messenger pathway. To delineate the mechanism of hCG-mediated induction of testosterone synthesis, a study was performed to investigate the changes in 3b-HSD-1, P450scc and StAR expression in mouse Leydig cells and an enhanced expression of these enzymes was detected (Yang et al. 2007). In the present work, after the chronic treatment with Sildenafil, isolated and hCG-stimulated Leydig cells presented an augmented immunolocalization of StAR, P450scc and testosterone. These data indicate that Sildenafil and hCG would possibly act synergistically to increase cGMP levels via different mechanisms. The hCG increases intracellular cGMP production by activating guanylyl cyclase system (Lin et al. 1979; Mukhopadhyay & Leideberger 1988), and Sildenafil induces an accumulation of cGMP by inhibiting the PDE5, or could act via NO or ANP-dependent mechanism (Preston et al. 2004).
The cyclic GMP is synthesized by two classes of enzymes called guanylyl cyclases, and both generate cGMP from intracellular GTP. The particulate guanylyl cyclases are membrane bound receptors that bind natriuretic and guanylin peptides. The sGC is a heme-containing, heterodimeric nitric oxide receptor. It consists of two subunits, a and b, which make up the active enzyme. The cGMP acts directly with effectors, such as cGMP-dependent protein kinases, cyclic nucleotide-gated channels, and cGMP-regulated phosphodiesterases (Wong & Garbers 1992; Krumenacker & Murad 2006).
Andric et al. (2007) described that the stimulation of both guanylyl cyclases and inhibition of phosphodiesterase 5 in vitro were accompanied by elevations in cGMP and androgen production. However, the inhibition of sGC and protein kinase G (PKG) significantly reduced the StAR phosphorilation, leading to a decreased steroidogenesis. Thus, the authors suggested that cGMP participates in Leydig cell steroidogenesis through the PKG-dependent modification of the StAR protein.
A study investigated the effects of 4b-PMA, a potent activator of protein kinase C (PKC), on atrial peptide-induced testosterone production and cGMP accumulation in Leydig cells. This compound inhibited testosterone production and markedly reduced the amount of cGMP through an enhanced phosphodiesterase activity, stimulated by PKC. The testosterone production and cGMP formation was completely reversed in the presence of a phosphodiesterase inhibitor. The cause of the decreased hormone production after 4b-PMA treatment was related to the diminished accumulation of the second messenger (Mukhopadhyay & Leideberger 1988).
Similarly, the present work shows that the chronic treatment with Sildenafil increased the testosterone biosynthesis in Leydig cells, probably through cGMP accumulation due to PDE5 inhibition.
The results obtained in the present study are in agreement with those of Carosa et al. (2004). Men suffering from erectile dysfunction have androgen levels significantly lower than normal ones. These authors demonstrated the effects of two different type V phosphodiesterase inhibitors treatments, Sidenafil (50 mg) and Tadalafil (20 mg) on the reversibility of testosterone reduction. This pharmacological therapy was able to enhance testosterone and reduce LH levels, probably due to the higher frequency of full sexual intercourse. However, the authors did not rule out the possibility of a direct effect of the PDE5 inhibitors on the on the human testis, as this tissue expresses quite a high level of PDE5. Khurana and Pandey (1993) demonstrated that the atrial natriuretic factor, brain natriuretic peptide and C-type natriuretic peptide (CNP), induced the production of intermediate precursors of testosterone biosynthesis, in purified mouse Leydig cells. All of these natriuretic peptides stimulated the steroidogenesis pathways by a transmembrane signal transduction mechanism involving the generation and accumulation of intracellular cGMP. This stimulatory effect begins at the first step involved in the conversion of cholesterol to pregnenolone.
The pulmonary vasodilator effects of Sildenafil on acute and chronic hypoxia-induced pulmonary hypertension in rats are potentiated by atrial natriuretic peptide (ANP), possibly by a synergistic effect on the increase in plasma cGMP synthesis (Preston et al. 2004). Similarly, ANP could be involved in cGMP synthesis in the Leydig cell, which would be accumulated in response to Sildenafil treatment. Conversely, the NO exerts an inhibitory effect on Leydig cell steroidogenesis, which is not mediated by the sGC as it fails to increase cGMP production (Del Punta et al. 1996). According to Middendorff et al. (1997), NO and CNP have been shown to influence testosterone release. While CNP increase testosterone via a promiscuous activation of cAMP-dependent protein kinase by cGMP in mice, a decrease on testosterone levels was detected in response to NO, as this agent could inhibit P450scc enzyme by directly binding to its heme iron.
Androgens have been shown to exert direct vasodilator effects in arteries from both humans and experimental animals. Oka et al. (2007) demonstrated that Dehydroepiandrosterone, a testosterone precursor, completely blocked hypoxic pulmonary vasoconstriction, which was accompanied by opening potassium channels, upregulation of sGC protein expression . activity and improved pulmonary artery vasodilator responsiveness to NO. In addition, Seyrek et al. (2007) have recently demonstrated that supraphysiological concentrations of testosterone act via ATP-sensitive K+ channel opening to induce relaxation in radial artery in vitro.
Therefore, the present study did not rule out the possible involvement of the enhanced steroid hormones biosynthesis, after the chronic treatment with Sildenafil, on pulmonary hypertension improvement.
In conclusion, the chronic treatment with Sildenafil exerted a stimulatory effect on testosterone production, probably via cGMP accumulation. This increased hormonal synthesis should be taken into account, as this therapeutic scheme has been extensively used worldwide in patients with pulmonary hypertension disorders. Additional experiments are in development in our laboratory to describe the possible effects on sperm parameters and fertilizing capacity.
From,
Sildenafil treatment in vivo stimulates Leydig cell steroidogenesis via the cAMP/cGMP signaling pathway, Silvana A. Andric, AJP - Endo October 2010 vol. 299 no. 4 E544-E550
Abstract
Sildenafil citrate (Viagra), a cGMP-selective phosphodiesterase (PDE) inhibitor, is widely used to treat erectile dysfunction and pulmonary arterial hypertension. In contrast to its well established action on erectile dysfunction, little is known on the action of sildenafil on cGMP/cAMP signaling and testicular steroidogenesis. This study was designed to assess the effects of prolonged sildenafil treatment on NO synthase-dependent signaling and steroidogenic function of rat Leydig cells.
Male adult rats were treated with Viagra (1.25 mg/kg body wt) daily for 30 days. In our studies, serum testosterone and ex vivo testosterone production significantly increased in sildenafil-treated animals. Human chorionic gonadotropin-stimulated testosterone production and cAMP accumulation were also significantly higher in Leydig cells obtained from sildenafil-treated rats. The expression of soluble guanylyl cyclase (GUCY1) subunits (Gucy1a1, Gucy1b1) significantly increased; cAMP-specific Pde4a, cGMP-specific Pde6c, and dual Pde1c and Nos2 were inhibited and expression of Nos3, protein kinase G1 (Pkg1), and Pde5 remained unchanged. Treatment of purified Leydig cells with NO donor caused a dose-dependent increase in both testosterone and cGMP production. Testosterone and cGMP production was significantly higher in Leydig cells obtained from sildenafil-treated animals. The stimulatory effect of NO donor was significantly enhanced by saturating concentrations of hCG in both Leydig cells obtained from control and sildenafil-treated animals. Occurrence of mature steroidogenic acute regulatory protein also increased in sildenafil treated animals in accord with increased cAMP and cGMP production. In summary, inhibition of PDE activity during prolonged sildenafil treatment increased serum testosterone level and Leydig cells' steroidogenic capacity by coordinated stimulatory action on cAMP and cGMP signaling pathway.......
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2741156/
Last edited:
I stickied it in the Study Corner.