Sodium nitroprusside attenuates hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats through the inhibition of overexpression of AT1 receptor, cell cycle proteins, and c-Src/growth factor receptor signaling pathways.

We recently showed that sodium nitroprusside (SNP), a NO donor, attenuated hypertension in spontaneously hypertensive rats (SHR). Since hypertension is associated with enhanced proliferation and hypertrophy of vascular smooth muscle cells (VSMC), the present study examines whether in vivo treatment of SHR with SNP could also inhibit the augmented proliferation of VSMC and explore the signaling mechanisms. Treatment of 8 week old SHR and Wistar Kyoto rats with SNP twice a week for 2 weeks inhibited the enhanced proliferation of VSMC from SHR, the enhanced expression of angiotensin II type 1 (AT1) receptor, and enhanced activation of c-Src and growth factor receptors and ERK1/2 signaling pathways. In addition, SNP also inhibited the overexpression of cell cycle proteins including cyclins D1, Cdk4, and phosphorylated pRB and restored the downregulated Cdk inhibitors p21Cip1 and p27Kip1 expression towards control levels. Furthermore, SNP-induced inhibition of enhanced levels of the AT1 receptor and enhanced proliferation was reversed by L-NAME, an inhibitor of nitric oxide synthase. These results suggest that the SNP-induced antiproliferative effect may be mediated through the inhibition of enhanced expression of the AT1 receptor, cell cycle proteins and activation of c-Src, growth factor receptors, and MAP kinase signaling.

Resveratrol prevents the development of high blood pressure in spontaneously hypertensive rats through the inhibition of enhanced expression of Giα proteins 1.

Resveratrol (RV), a polyphenolic component of red wine, has been shown to attenuate high blood pressure (BP) in spontaneously hypertensive rats (SHRs). We previously found that the enhanced expression of Giα proteins plays a role in the pathogenesis of hypertension in SHRs. In the present study, we investigated whether this RV-induced decrease in BP in SHRs can be attributed to the ability of RV to inhibit the enhanced expression of Giα proteins and the upstream signaling molecules implicated in the overexpression of Giα proteins. Administration of RV (50 mg/kg per day) to prehypertensive 2-week-old SHRs for 6 weeks prevented the development of high BP and inhibited the enhanced expression of Giα proteins, the enhanced levels of superoxide anion (O2-) and NADPH oxidase activity, the enhanced activation (phosphorylation) of c-Src and growth factor receptors, as well as the enhanced levels of extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) exhibited by vascular smooth muscle cells isolated from SHRs. In conclusion, these results indicate that RV attenuates the development of high BP in SHRs through the inhibition of enhanced levels of Giα proteins, oxidative stress, and the upstream signaling molecules that contribute to the overexpression of Giα proteins. These findings suggest that RV could potentially be used as a therapeutic agent in the treatment of cardiovascular complications including hypertension.

Adjudin-mediated germ cell depletion alters the anti-oxidant status of adult rat testis.

Successful spermatogenesis is dependent on the proper attachment of developing germ cells to Sertoli cells. Manipulation of these interactions by drugs like Adjudin can hamper the development of germ cells and lead to conditions of temporary infertility. Although studies have shown the contraceptive potential of Adjudin, much is not known about its action in the testis. In this study, we sought to investigate the effect of Adjudin on the oxidative status of mammalian testis. Adult male rats were administered with a single dose of Adjudin (50 mg/kg body weight) by oral gavage and were killed at 1, 2, 4, 7, 15, or 30 days of treatment. Adjudin caused a significant increase in the production of hydrogen peroxide and lipid peroxidation from 4 to 7 days after treatment. There was a significant decrease in the activities of anti-oxidant enzymes superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase from 4 to 7 days after treatment with Adjudin. However, the state of oxidative stress was less pronounced from 15 to 30 days after Adjudin treatment. The level of androgen binding protein (ABP) remained unchanged following Adjudin treatment. These results show that there is an induction of oxidative stress accompanying adherens junction restructuring which suggests a role for reactive oxygen species in the regulation of these testicular junctions. However, transient elevation in reactive oxygen species levels did not affect androgen transport.

Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes.

Maintaining the integrity of sperm DNA is vital to reproduction and male fertility. Sperm contain a number of molecules and pathways for the repair of base excision, base mismatches and DNA strand breaks. The presence of Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues has recently been shown in male germ cells, specifically during stage VII of spermatogenesis. High PARP expression has been reported in mature spermatozoa and in proven fertile men. Whenever there are strand breaks in sperm DNA due to oxidative stress, chromatin remodeling or cell death, PARP is activated. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Therefore, cleaved PARP (cPARP) may be considered a marker of apoptosis. The presence of higher levels of cPARP in sperm of infertile men adds a new proof for the correlation between apoptosis and male infertility. This review describes the possible biological significance of PARP in mammalian cells with the focus on male reproduction. The review elaborates on the role played by PARP during spermatogenesis, sperm maturation in ejaculated spermatozoa and the potential role of PARP as new marker of sperm damage. PARP could provide new strategies to preserve fertility in cancer patients subjected to genotoxic stresses and may be a key to better male reproductive health.

Inhibition of overexpression of Giα proteins and nitroxidative stress contribute to sodium nitroprusside-induced attenuation of high blood pressure in SHR.

We earlier showed that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit enhanced expression of Giα proteins which was attributed to the decreased levels of nitric oxide (NO), because elevation of the intracellular levels of NO by NO donors; sodium nitroprusside (SNP) and S-Nitroso-N-acetyl-DL-penicillamine (SNAP), attenuated the enhanced expression of Giα proteins. Since the enhanced expression of Giα proteins is implicated in the pathogenesis of hypertension, the present study was undertaken to investigate if treatment of SHR with SNP could also attenuate the development of high blood pressure (BP) and explore the underlying molecular mechanisms. Intraperitoneal injection of SNP at a concentration of 0.5 mg/kg body weight twice a week for 2 weeks into SHR attenuated the high blood pressure by about 80 mmHg without affecting the BP in WKY rats. SNP treatment also attenuated the enhanced levels of superoxide anion (O2- ), hydrogen peroxide (H2 O2 ), peroxynitrite (ONOO- ), and NADPH oxidase activity in VSMC from SHR to control levels. In addition, the overexpression of different subunits of NADPH oxidase; Nox-1, Nox-2, Nox-4, P22phox , and P47phox , and Giα proteins in VSMC from SHR were also attenuated by SNP treatment. On the other hand, SNP treatment augmented the decreased levels of intracellular NO, eNOS, and cGMP in VSMC from SHR. These results suggest that SNP treatment attenuates the development of high BP in SHR through the elevation of intracellular levels of cGMP and inhibition of the enhanced levels of Giα proteins and nitroxidative stress.

Rab4A GTPase catenin interactions are involved in cell junction dynamics in the testis.

A plethora of evidence has recently accumulated to suggest that Rab guanosine triphosphates (GTPases) may have functions other than those originally proposed in vesicle formation, movement, docking, and fusion. Studies have shown, for example, that Rab proteins interact with actin filaments and microtubules, illustrating cross-talk between intracellular transport and cytoskeletal dynamics. In this report, we show that Rab4A associates with adherens junction signaling proteins in the testis. By immunoprecipitation, Rab4A was found to interact with alpha- and beta-catenin as well as with actin, vimentin, alpha- and beta-tubulin, and protein kinase C (PKC)-alpha and -epsilon. Additionally, administration of Adjudin to adult rats up-regulated the Rab4A level, which coincided with the loss of spermatocytes, round and elongating/elongated spermatids from the seminiferous epithelium. More importantly, the ability of Rab4A to associate with alpha- and beta-catenin increased during Adjudin-induced junction restructuring in the testis, illustrating that Rab4A-catenin interactions are likely to be involved in the disassembly of Sertoli-germ cell contacts. Taken collectively, these results suggest that Rab4A participates in adherens junction dynamics.

Nitric oxide attenuates overexpression of Giα proteins in vascular smooth muscle cells from SHR: Role of ROS and ROS-mediated signaling.

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit decreased levels of nitric oxide (NO) that may be responsible for the overexpression of Giα proteins that has been shown as a contributing factor for the pathogenesis of hypertension in SHR. The present study was undertaken to investigate if increasing the intracellular levels of NO by NO donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP) could attenuate the enhanced expression of Giα proteins in VSMC from SHR and explore the underlying mechanisms responsible for this response. The expression of Giα proteins and phosphorylation of ERK1/2, growth factor receptors and c-Src was determined by Western blotting using specific antibodies. Treatment of VSMC from SHR with SNAP for 24 hrs decreased the enhanced expression of Giα-2 and Giα-3 proteins and hyperproliferation that was not reversed by 1H (1, 2, 4) oxadiazole (4, 3-a) quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, however, PD98059, a MEK inhibitor restored the SNAP-induced decreased expression of Giα proteins towards control levels. In addition, the increased production of superoxide anion, NAD(P)H oxidase activity, overexpression of AT1 receptor, Nox4, p22phox and p47phox proteins, enhanced levels of TBARS and protein carbonyl, increased phosphorylation of PDGF-R, EGF-R, c-Src and ERK1/2 in VSMC from SHR were all decreased to control levels by SNAP treatment. These results suggest that NO decreased the enhanced expression of Giα-2/3 proteins and hyperproliferation of VSMC from SHR by cGMP-independent mechanism and involves ROS and ROS-mediated transactivation of EGF-R/PDGF-R and MAP kinase signaling pathways.

Natriuretic peptide receptor-C attenuates hypertension in spontaneously hypertensive rats: role of nitroxidative stress and Gi proteins.

C-Atrial natriuretic peptide (ANP)4-23, a ring deleted analog of ANP that specifically interacts with natriuretic peptide receptor-C (NPR-C), has been shown to decrease the enhanced expression of Giα proteins implicated in the pathogenesis of hypertension. In the present study, we investigated whether in vivo treatment of spontaneously hypertensive rats (SHRs) with C-ANP4-23 could attenuate the development of high blood pressure (BP) and explored the underlying mechanisms responsible for this response. Intraperitoneal injection of C-ANP4-23 at the concentration of 2 or 10 nmol/kg body weight to prehypertensive SHRs attenuated the development of high BP, and at 8 weeks it was decreased by ≈20 and 50 mm Hg, respectively; however, this treatment did not affect BP in Wistar-Kyoto rats. C-ANP4-23 treatment of adult SHRs for 2 weeks also attenuated high BP, heart rate, and restored the impaired vasorelaxation toward control levels. In addition, the enhanced levels of superoxide anion (O2(-)), peroxynitrite, NADPH oxidase activity, and the enhanced expression of Giα proteins, NOX4, p47(phox), nitrotyrosine, and decreased levels of endothelial nitric oxide synthase (eNOS or NOS3) and NO in SHRs were attenuated by C-ANP4-23 treatment; however, the altered levels of NPR-A/NPR-C were not affected by this treatment. In conclusion, these results indicate that NPR-C activation by C-ANP4-23 attenuates the development of high BP in SHRs through the inhibition of enhanced levels of Giα proteins and nitroxidative stress and not through eNOS/cGMP pathway and suggest that NPR-C ligand may have the potential to be used as therapeutic agent in the treatment of cardiovascular complications including hypertension.

Impact of inflammation on male fertility.

The male uro-genital tract is susceptible to gram-negative bacterial infections that produce a state of inflammation, particularly in the testis and epididymis. Development of germline stem cells into motile spermatozoa takes place in these organs and thus any impairment therein has a direct effect on male fertility. A number of factors are known to impair male fertility including environmental and chemical factors, lifestyle, and infections. The last is a little-known and poorly understood cause of male sub-/infertility. The presence of the pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF- alpha), interleukin-1alpha (IL-1alpha) and interleukin-1beta (IL-1beta) in the male uro-genital tract following bacterial infections suggests that such infections could have cytokine-mediated anti-fertility effects. Furthermore, inflammation has been associated with elevated levels of reactive oxygen species and oxidative stress both of which affect male fertility. The present article summarizes the effects of inflammation on the testis, epididymis and spermatozoa. We review the correlations between inflammation and oxidative stress vis-à-vis spermatogenesis and discuss the implications of infections on male fertility/infertility and assisted reproductive technologies for the male.

Interleukin 1 alpha (IL1A) is a novel regulator of the blood-testis barrier in the rat.

Throughout spermatogenesis, leptotene spermatocytes must traverse the blood-testis barrier (BTB) at stages VIII-XI to gain entry into the adluminal compartment for continued development. However, the mechanism underlying BTB restructuring remains somewhat elusive. In this study, interleukin 1 alpha (IL1A) was administered intratesticularly to adult rats in order to assess its effects on spermatogenesis. IL1A was shown to perturb Sertoli-germ cell adhesion, resulting in germ cell loss from approximately 50% of seminiferous tubules by 15 days posttreatment. Equally important, the functional integrity of the BTB was compromised when inulin-fluorescein isothiocyanate was detected in the adluminal compartment of the seminiferous epithelium following its administration via the jugular vein. Interestingly, IL1A did not affect the steady-state levels of proteins that confer BTB function, namely OCLN, CLDN1, F11R, TJP1, and CDH2. Instead, the localizations of OCLN, F11R, and TJP1 in the seminiferous epithelium were altered; these proteins appeared to move away from sites of cell-cell contact. Moreover, IL1A was shown to perturb the orderly arrangement of filamentous actin at the BTB and apical ectoplasmic specialization with distinct areas illustrating loss of actin filaments. Taken collectively, these results suggest that IL1A-induced BTB disruption is not mediated via the reduction of target protein levels. Instead, IL1A's primary cellular target appears to be the Sertoli cell actin cytoskeleton. It is possible that localized production of IL1A by Sertoli and/or germ cells in vivo results in BTB restructuring, and this may facilitate the movement of leptotene spermatocytes across the BTB.

Adjudin-mediated junction restructuring in the seminiferous epithelium leads to displacement of soluble guanylate cyclase from adherens junctions.

A plethora of evidence supports the role of cyclic nucleotides in junction restructuring. For instance, studies have shown cGMP to be a key regulator of junction assembly and disassembly in different in vitro and in vivo systems. In this study, we examine the role of soluble guanylate cyclase (sGC) in junction restructuring in the seminiferous epithelium of the rat testis. First, the interaction of soluble guanylate cyclase beta1 (sGCbeta1; sGC is a heterodimer comprised of an alpha and a beta subunit) with proteins that constitute adherens and tight junctions in the testis was demonstrated. By immunoprecipitation, sGCbeta1 was found to associate with occludin, JAM-A, and ZO-1, as well as with cadherin, catenin, nectin, afadin, ponsin, and espin, suggestive of its role in cell junction dynamics. These results were corroborated in part by immunohistochemistry experiments, which revealed that the localization of sGCbeta1 was largely restricted to the site of the apical and basal ectoplasmic specialization. Next, the role of sGC in junction dynamics was addressed by using an in vivo model of junction restructuring. Administration of Adjudin--a chemical entity known to specifically perturb adhesion between Sertoli and germ cells (i.e., round and elongate(ing) spermatids and most spermatocytes)--resulted in a approximately 1.5-fold increase in sGCbeta1, coinciding with the loss of germ cells from the epithelium. More importantly, the ability of sGCbeta1 to associate with cadherin increased approximately three-fold during Adjudin-mediated restructuring of Sertoli-germ cell junctions, whereas its interaction with tight junction proteins (i.e., occludin and ZO-1) decreased. Taken collectively, these results suggest that sGC participates in the remodeling of cell junctions during spermatogenesis.