Cyclic Adenosine Monophosphate Content Research Articles

Membrane Androgen Receptor ZIP9 Induces Croaker Ovarian Cell Apoptosis via Stimulatory G Protein Alpha Subunit and MAP Kinase Signaling.

Recent studies show that androgen-induced apoptosis in Atlantic croaker primary granulosa and theca (G/T) cells and in human breast and prostate cancer cell lines is mediated by the membrane androgen receptor ZIP9, which belongs to the SLC39A zinc transporter family. However, the apoptotic signaling pathways remain unclear because ZIP9 activates an inhibitory G protein in human cancer cells, whereas recombinant croaker ZIP9 activates a stimulatory G protein (Gs) in transfected cancer cells. We investigated androgen-dependent apoptotic pathways to identify the signaling pathways regulated through wild-type croaker ZIP9 in ovarian follicle cells. We show that the ZIP9-mediated apoptotic signaling pathway in croaker G/T cells shares several proapoptotic members with those in human cancer cells, but is activated through a Gsα subunit-dependent pathway. Testosterone treatment of croaker G/T cells increased intracellular zinc levels, mitogen-activated protein (MAP) kinase activity, caspase 3 activity, messenger RNA levels of proapoptotic members Bax, p53, and c-Jun N-terminal kinase, and the incidence of apoptosis, similar to findings in mammalian cancer cells, but also increased cyclic adenosine monophosphate concentrations. Transfection with small interfering RNA targeting croaker ZIP9 blocked testosterone-induced increase in bax, p53, and jnk expression. Testosterone-induced apoptosis and caspase 3 activation depended on the presence of extracellular zinc and were effectively blocked with cotreatment of inhibitors of the Gsα subunit, adenylyl cyclase, protein kinase A, and MAP kinase (Erk1/2) activation. These results indicate that ZIP9-mediated testosterone signaling in croaker G/T cells involves multiple pathways, some of which differ from those activated through ZIP9 in human cancer cells even though a similar apoptotic response is observed.

Butyric acid regulates progesterone and estradiol secretion via cAMP signaling pathway in porcine granulosa cells

Butyric acid (BA), one of the short chain fatty acids (SCFAs), has positive actions on the metabolism, inflammation, etc. However, whether it influences the reproductive physiology and if so the detail mechanism involved has not yet been determined. In this study, the porcine granulosa cells (PGCs) were treated with gradient concentrations of BA. After 24h culture, 0.05mM BA significantly stimulated the progesterone (P4) secretion (P<0.05), 5mM and 10mM BA significantly inhibited the P4 secretion (P<0.05). Simultaneously, BA up-regulated the estradiol (E2) secretion in a dose dependent manner, 5mM and 10mM BA significantly promoted the E2 level (P<0.05). In addition, 10mM BA significantly promoted the G-protein-coupled receptor 41/43 mRNA (P<0.05). Interestingly, 5mM BA treatment significantly down-regulated cyclic adenosine monophosphate (cAMP) content (P<0.05), steroidogenic acute regulatory (StAR), steroidogenic factor 1 (SF1), P450scc in the mRNA and/or protein level (P<0.05), and these actions were reversed by cAMP activator forskolin (FK). Moreover, the co-treatment of 5mM BA and bupivacaine (BPC, the cAMP inhibitor) significantly accumulated the inhibition action of BPC on cAMP, the secretion of P4, and the abundance of StAR mRNA (P<0.05), inhibited the up-regulation of 5mM BA on the E2 secretion (P<0.05). Further, the Global Proteome and KEGG pathway analysis found that 5mM BA significantly up-regulated the I3LM80 proteins (P<0.05), which is involved in the steroid biosynthesis signaling pathway. 5mM BA significantly decreased the F2Z5G3 protein level (P<0.05), and the cAMP signaling pathway. In conclusion, present findings for the first time demonstrated that BA could regulate the P4 and E2 hormone synthesis in PGCs via the cAMP signaling pathway.

Expanding the role of vasopressin antagonism in polycystic kidney diseases: From adults to children?

Polycystic kidney disease (PKD) encompasses a group of genetic disorders that are common causes of renal failure. The two classic forms of PKD are autosomal recessive polycystic kidney disease (ARPKD) and autosomal dominant polycystic kidney disease (ADPKD). Despite their clinical differences, ARPKD and ADPKD share many similarities. Altered intracellular Ca2+ and increased cyclic adenosine monophosphate (cAMP) concentrations have repetitively been described as central anomalies that may alter signaling pathways leading to cyst formation. The vasopressin V2 receptor (V2R) antagonist tolvaptan lowers cAMP in cystic tissues and slows renal cystic progression and kidney function decline when given over 3years in adult ADPKD patients. Tolvaptan is currently approved for the treatment of rapidly progressive disease in adult ADPKD patients. On the occasion of the recent initiation of a clinical trial with tolvaptan in pediatric ADPKD patients, we aim to describe the most important aspects in the literature regarding the AVP-cAMP axis and the clinical use of tolvaptan in PKD.

Decreased vasorelaxation induced by iloprost during acute inflammation in human internal mammary artery

Cyclooxygenase-2 (COX-2) induction in human internal mammary arteries (IMA) under inflammatory conditions has been associated with attenuated norepinephrine (NE)-induced vasoconstriction. This effect was associated with increased prostaglandin (PG) E2 and prostacyclin (PGI2) releases. The present study was designed to assess the role of these PG and their receptors (EP and IP, respectively) on the vascular reactivity during acute inflammation. Isolated IMA were cultured in the absence (Control conditions) or presence (Inflammatory conditions) of both interleukin-1 beta (IL-1β) and lipopolysaccharide (LPS). The vasorelaxation and the increased content of cyclic adenosine monophosphate (cAMP) induced by iloprost, a PGI2 analogue, were significantly reduced under inflammatory conditions and restored in preparations cultured with the IP antagonist (CAY10441). Decreased cAMP levels under inflammatory conditions are due to at least increased phosphodiesterase (PDE) 4B expression. On the other hand, PGE2, thromboxane analogues and EP agonists-induced vasoconstrictions were not affected under inflammatory conditions. No vasorelaxation was observed with PGD2, PGE2 or the EP2/4 agonists in pre-contracted IMA. Finally, using RT-qPCR and immunohistochemistry, the COX-2, IP receptor and PGI2 synthase (PGIS) were detected. A significant increase of COX-2 and moderate increase of IP mRNA expression was observed under inflammatory conditions, whereas PGIS mRNA level was not affected. This study demonstrates that PGI2/IP receptor signalling and PGI2-induced relaxation are impaired in human IMA during acute inflammation, whereas the responses induced by other prostanoids are not affected. These results could explain some of the mechanisms of vascular dysfunction reported in inflammatory conditions.

Cyclooxygenase-2 Signalling Pathway in the Cortex is Involved in the Pathophysiological Mechanisms in the Rat Model of Depression

This study was designed to investigate the effect of the cortical cyclooxygenase-2 (COX2) pathway on depressive behaviour in rats. Meloxicam, COX2 overexpressed lentivirus and COX2 RNAi lentivirus were administered to Sprague-Dawley rats subjected to chronic unpredictable mild stress (CUMS). Behaviour tests, biochemistry and immunohistochemistry methods, enzyme-linked immunosorbent assays, western blotting and reverse transcription polymerase chain reactions were used to evaluate the changes in rat behaviour and the cortical COX2 pathway. CUMS rats showed depressive–like behaviours. The superoxide dismutase activity and cyclic adenosine monophosphate (cAMP) contents were significantly decreased, the contents of malondialdehyde, prostaglandin E2 (PGE2) and inflammatory cytokines were significantly increased. The expressions of protein kinase A (PKA) and cAMP response element-binding protein (CREB) were decreased, and the levels of brain-derived neurotrophic factor (BDNF) and COX2 were significantly increased. Meloxicam and COX2 RNAi lentivirus significantly alleviated the abnormalities induced by CUMS, while COX2 overexpressed lentivirus aggravated these abnormalities. Our results indicated that the cortical COX2 pathway was activated in CUMS rats. Inhibition of COX2 activity/expression can obviously improve depressive behaviours in CUMS rats. Upregulating COX2 expression can increase the susceptibility of rats to CUMS. An imbalance in the cortical COX2-PGE2-cAMP/PKA-CREB-BDNF signalling pathway participates in the pathogenic mechanism of depression.

CAMP/PKA-CREB-BDNF signaling pathway in hippocampus mediates cyclooxygenase 2-induced learning/memory deficits of rats subjected to chronic unpredictable mild stress

To investigate the mechanism of cyclooxygenase 2 (COX2) in learning and memory impairments in rats subjected to chronic unpredictable mild stress (CUMS), meloxicam was used intragastrically to inhibit the activity of cyclooxygenase 2. Moreover, cyclooxygenase 2 over-expressing or RNA interfere lentivirus was injected intraventricularly to increase or decrease the enzyme's expression, respectively. The body weights and sucrose consumption were used to analyze depressive behaviors, while the Morris water maze and step-down-type passive avoidance tests were carried out to evaluate the learning-memory functions. The levels of inflammatory cytokines were measured to estimate inflammation and the contents of cyclic adenosine monophosphate (cAMP) were used to measure the levels of the second messenger. Changes in cyclooxygenase 2 mRNA levels were analyzed using reverse transcription polymerase chain reaction. Moreover, the expression of cyclooxygenase 2, brain-derived neurotrophic factor (BDNF), prostaglandins receptor 3 (EP3), protein kinase A (PKA), cAMP response element binding protein (CREB), and phosphorylated CREB were estimated using immunohistochemical staining or western blotting. The results showed that CUMS led to significant depressive-like behaviors and learning and memory dysfunctions. Also, the cAMP levels decreased significantly, while levels of inflammatory cytokines and prostaglandins E2 increased significantly. The expressions of PKA, BDNF, phosphorylated CREB/CREB declined and cyclooxygenase 2 was increased. Meloxicam and cyclooxygenase 2 RNA interfere lentivirus reversed the changes caused by CUMS while cyclooxygenase 2-overexpressing lentivirus worsened these abnormalities. The findings also showed that CUMS increased cyclooxygenase 2 expression, which can cause learning and memory impairments, mainly through activating the hippocampal neuronal cAMP/PKA-CREB-BDNF signaling pathways.

6-Hydroxy-5,7-dimethoxy-flavone suppresses the neutrophil respiratory burst via selective PDE4 inhibition to ameliorate acute lung injury

Over-activated neutrophils produce enormous oxidative stress and play a key role in the development of acute and chronic inflammatory diseases. 6-Hydroxy-5,7-dimethoxy-flavone (UFM24), a flavone isolated from the Annonaceae Uvaria flexuosa, showed inhibitory effects on human neutrophil activation and salutary effects on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. UFM24 potently inhibited superoxide anion (O2•−) generation, reactive oxidants, and CD11b expression, but not elastase release, in N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF)-activated human neutrophils. However, UFM24 failed to scavenge O2•− and inhibit the activity of subcellular NADPH oxidase. fMLF-induced phosphorylation of protein kinase B (Akt) was inhibited by UFM24. Noticeably, UFM24 increased cyclic adenosine monophosphate (cAMP) concentration and protein kinase (PK) A activity in activated human neutrophils. PKA inhibitors significantly reversed the inhibitory effects of UFM24, suggesting that the effects of UFM24 were through cAMP/PKA-dependent inhibition of Akt activation. Additionally, activity of cAMP-related phosphodiesterase (PDE)4, but not PDE3 or PDE7, was significantly reduced by UFM24. Furthermore, UFM24 attenuated neutrophil infiltration, myeloperoxidase activity, and pulmonary edema in LPS-induced ALI in mice. In conclusion, our data demonstrated that UFM24 inhibits oxidative burst in human neutrophils through inhibition of PDE4 activity. UFM24 also exhibited significant protection against endotoxin-induced ALI in mice. UFM24 has potential as an anti-inflammatory agent for treating neutrophilic lung damage.

The protection of meloxicam against chronic aluminium overload-induced liver injury in rats.

The present study was designed to observe the protective effect and mechanisms of meloxicam on liver injury caused by chronic aluminium exposure in rats. The histopathology was detected by hematoxylin-eosin staining. The levels of prostaglandin E2, cyclic adenosine monophosphate and inflammatory cytokines were detected by enzyme linked immunosorbent assay. The expressions of cyclooxygenases-2, prostaglandin E2 receptors and protein kinase A were measured by western blotting and immunohistochemistry. Our experimental results showed that aluminium overload significantly damaged the liver. Aluminium also significantly increased the expressions of cyclooxygenases-2, prostaglandin E2, cyclic adenosine monophosphate, protein kinase A and the prostaglandin E2 receptors (EP1,2,4) and the levels of inflammation and oxidative stress, while significantly decreased the EP3 expression in liver. The administration of meloxicam significantly improved the impairment of liver. The contents of prostaglandin E2 and cyclic adenosine monophosphate were significantly decreased by administration of meloxicam. The administration of meloxicam also significantly decreased the expressions of cyclooxygenases-2 and protein kinase A and the levels of inflammation and oxidative stress, while significantly increased the EP1,2,3,4 expressions in rat liver. Our results suggested that the imbalance of cyclooxygenases-2 and downstream prostaglandin E2 signaling pathway is involved in the injury of chronic aluminium-overload rat liver. The protective mechanism of meloxicam on aluminium-overload liver injury is attributed to reconstruct the balance of cyclooxygenases-2 and downstream prostaglandin E2 signaling pathway.

Time- and Dose-Dependent Effects of Desflurane in Sensitized Airways

Although the bronchodilatory actions of volatile anesthetics, such as halothane, isoflurane, and sevoflurane, have been well documented in previous studies, the properties of desflurane remain controversial. The aim of this study was to investigate the effects of desflurane at different concentrations and durations in an ovalbumin-sensitized guinea pig model of airway hyper-responsiveness. Ovalbumin-sensitized animals (n = 176) were randomly assigned to 5 groups according to the minimum alveolar concentration (MAC) of desflurane they received: 0.0, 0.5, 1.0, 1.5, and 2.0 MAC. Total lung resistance in vivo, airway smooth muscle tension in vitro, and intracellular cyclic adenosine monophosphate (AMP) levels were measured to evaluate the effects of desflurane. In 5 sensitized groups, total lung resistance increased from baseline to peak at approximately 8 minutes and then decreased slowly until about 17 minutes with extended administration of desflurane. Desflurane dose-dependently increased total lung resistance with or without incremental doses of acetylcholine and reduced muscle tension with increasing concentrations of carbacholine. Cyclic AMP levels were increased by desflurane: at the 60-minute time point, cyclic AMP concentrations (means ± SD) with 0.5 MAC (1.96 ± 0.40) and 1.0 MAC (2.11 ± 0.50) desflurane were higher than those at the 8-minute time point (1.11 ± 0.23 and 1.32 ± 0.32). Desflurane exerted time- and dose-dependent effects and could be used at 0.5 and 1.0 MAC concentrations without significant bronchoconstriction in ovalbumin-sensitized guinea pigs. Cyclic AMP-mediated airway smooth muscle relaxation might be one mechanism by which desflurane induces bronchodilation.

Zebrafish phosvitin-derived peptide Pt5 inhibits melanogenesis via cAMP pathway.

Zebrafish phosvitin-derived peptide Pt5, consisting of the C-terminal 55 residues of phosvitin, has been shown to have an antimicrobial-immunomodulatory activity comparable to phosvitin. Here, we showed clearly that Pt5 had the capacity to inhibit tyrosinase (TYR) activity and melanin biosynthesis, and this inhibition was independent of cell proliferation and cytotoxic effects. Incubation of fluorescein isothiocyanate (FITC)-labeled Pt5 with B16F10 melanoma cells revealed that Pt5 was localized in the cytoplasm of the cells. In addition, Pt5 inhibited the expression of TYR, tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), and microphthalmia-associated transcription factor (MITF) in B16F10 melanoma cells and reduced the intracellular cyclic adenosine monophosphate (cAMP) concentration in the cells, but it did not affect the cellular contents of pERK1/2 and β-catenin, suggesting that Pt5 regulates melanin biosynthesis via cAMP signaling pathway rather than Wnt and MAPK pathways. Collectively, these data indicate that Pt5 has the potential to be used as a melanogenesis inhibitor in medical and cosmetic industry, a novel role ever reported.

Transcriptome analysis of the potential roles of FOXL2 in chicken pre-hierarchical and pre-ovulatory granulosa cells

Forkheadbox L2 (FOXL2) is a transcription factor involved in mammalian ovarian development, especially in granulosa cell differentiation. However, this factor's function in mature chicken ovary is unclear. To explore the function of FOXL2 in chicken granulosa cells, we performed RNA-seq to compare the transcriptomes of pre-hierarchical (phGCs) and pre-ovulatory granulosa cells (poGCs) by FOXL2 overexpression. We observed that focal adhesion might be one of the key pathways activated during the differentiation of granulosa cells, and FOXL2 might be involved in follicle selection by regulating the expression of cytokines and the concentration of cyclic adenosine monophosphate (cAMP). Interestingly, we observed that FOXL2 played different roles in phGCs and poGCs, which might contribute to homeostasis in the chicken follicle by inducing differentiation of granulosa cells in pre-hierarchal follicles and preventing premature ovulation in pre-ovulatory follicles. Taken together, the results of our study establish a framework for understanding the potential functions of FOXL2 in the chicken granulosa cell.

Endogenous Parathyroid Hormone Promotes Fracture Healing by Increasing Expression of BMPR2 through cAMP/PKA/CREB Pathway in Mice

Background/Aims: Endogenous parathyroid hormone (PTH) plays an important role in fracture healing. This study investigated whether endogenous PTH regulates fracture healing by bone morphogenetic protein (BMP) and/or the transforming growth factor-β (TGF-β) signaling pathway. Methods: Eight-week-old wild-type (WT) and PTH-knockout (PTH KO) male mice were selected, and models of open right-femoral fracture were constructed. Fracture healing and callus characteristics of mice in the two groups were compared by X-ray, micro-computed tomography, histological, and immunohistochemical examinations. Bone marrow mesenchymal stem cells (BMMSCs) of 8-week-old WT and PTHKO male mice were obtained and induced into osteoblasts and chondrocytes. Results: We found that expression levels of Runt-related transcription factor (RUNX2), bone morphogenetic protein-receptor-type Ⅱ (BMPR2), phosphorylated Smad 1/5/8, and phosphorylated cyclic adenosine monophosphate-responsive element binding protein (CREB) in the callus of PTHKO mice were significantly decreased, whereas no significant difference in expression of SOX9, TGF-βR2,or pSMAD2/3 was observed between PTHKO and WT mice. Additionally, the activity of osteoblast alkaline phosphatase was low at 7 days post-induction, and was upregulated by addition of PTH or dibutyryl cyclic adenosine monophosphate (dbcAMP) to the cell culture. Furthermore, H89 (protein kinase A inhibitor)eliminated the simulating effects of PTH and dbcAMP, and a low concentration of cyclic adenosine monophosphate (cAMP) was observed in PTHKO mouse BMMSCs. Conclusion: These results suggested that endogenous PTH enhanced BMPR2 expression by a cAMP/PKA/CREB pathway in osteoblasts, and increased RUNX2 expression through transduction of the BMP/pSMAD1/5/8 signaling pathway.

Silver nanoparticles induce oocyte maturation in zebrafish (Danio rerio)

Public concern regarding silver nanoparticles (AgNPs) in the environment has been increasing since they can cause adverse effects in some aquatic species. However, few data are actually available on the effects of AgNPs on the germ cells. In the present study, we used the zebrafish ovarian follicle as a model to assess the potentially adverse effects of AgNPs on oocyte maturation (germinal vesicle breakdown, GVBD) in vitro. Similar to the maturation inducing hormone (17α, 20β-dihydroxy-4-pregnen-3-one), AgNPs induced GVBD, and reduced the total cyclic adenosine monophosphate (cAMP) concentration in zebrafish ovarian follicles. The results from transmission electron microscope observation and Hoechst 33342 staining clearly indicated that AgNPs induced apoptosis in ovarian follicle cells surrounding the oocyte. Similar to AgNPs, AgNO3 also induced GVBD, decreased cAMP concentration and induced apoptosis of ovarian follicle cells. However, the results from gene expression analysis showed that transcript levels of oxidative stress related genes were more sensitive to AgNPs than AgNO3. Further more, H2O2 has an ability to induce zebrafish oocytes maturation by induction of apoptosis in ovarian follicle cells. Taken together, the results from our study indicated that oxidative stress appeared to be one of important mechanisms in AgNP induced apoptosis in ovarian follicle cells, which further triggered the GVBD.

Lithium Reversibly Inhibits Schwann Cell Proliferation and Differentiation Without Inducing Myelin Loss.

This study was undertaken to examine the bioactivity, specificity, and reversibility of lithium's action on the growth, survival, proliferation, and differentiation of cultured Schwann cells (SCs). In isolated SCs, lithium promoted a state of cell cycle arrest that featured extensive cell enlargement and c-Jun downregulation in the absence of increased expression of myelin-associated markers. In addition, lithium effectively prevented mitogen-induced S-phase entry without impairing cell viability. When lithium was administered together with differentiating concentrations of cyclic adenosine monophosphate (cAMP) analogs, a dramatic inhibition of the expression of the master regulator of myelination Krox-20 was observed. Likewise, lithium antagonized the cAMP-dependent expression of various myelin markers such as protein zero, periaxin, and galactocerebroside and allowed SCs to maintain high levels of expression of immature SC markers even in the presence of high levels of cAMP and low levels of c-Jun. Most importantly, the inhibitory action of lithium on SC proliferation and differentiation was shown to be dose dependent, specific, and reversible upon removal of lithium compounds. In SC-neuron cultures, lithium suppressed myelin sheath formation while preserving axonal integrity, SC-axon contact, and basal lamina formation. Lithium was unique in its ability to prevent the onset of myelination without promoting myelin degradation or SC dedifferentiation. To conclude, our results underscored an unexpected antagonistic action of lithium on SC mitogenesis and myelin gene expression. We suggest that lithium represents an attractive pharmacological agent to safely and reversibly suppress the onset of SC proliferation, differentiation, and myelination while maintaining the integrity of pre-existing myelinated fibers.

Abstract 11162: Hypothermia Attenuates Postresuscitation Myocardial Dysfunction by Inhibiting Beta Adrenergic Signaling

Introduction: Therapeutic hypothermia reduce the severity of post-resuscitation myocardial dysfunction caused by epinephrine administered during cardiopulmonary resuscitation (CPR) , and the mechanism remains unclear. Hypothesis: hypothermia may reduce the concentration of cyclic adenosine monophosphate (cAMP), a second messenger of beta-adrenergic receptor activity. Methods: Thirty two male Sprague-Dawley rats were randomized to four groups: 1) normothermic placebo control; 2) normothermic epinephrine; 3) hypothermic placebo control; and 4) hypothermic epinephrine. Ventricular fibrillation was induced electrically and untreated for 8 mins. Hypothermia was initiated coincident with the start of CPR and maintained at 33 ± 0.2°C for 4 hrs after resuscitation and animals were then euthanized. Either placebo or epinephrine was administered 5 mins after the start of CPR and 3 mins before defibrillation. Post-resuscitation ejection fraction was measured hourly for 4 hrs. The hearts were then harvested for cAMP and Inositol 1,4,5-trisphosphate (IP3) assay analysis. Results: All animals were successfully resuscitated except one in normothermic placebo group. Epinephrine significantly increased coronary perfusion pressure during CPR (26.5±6.23mmHg vs 21.14±1.95 mmHg P&lt;0.05). Post-resuscitation myocardial function was significantly impaired in normothermic epinephrine group compared with other three groups (figure 1). The concentration of myocardial cAMP was double in normothermic epinephrine group (655.06±447.63 umol/L) when compared to that in hypothermic epinephrine group (302.51±97.98 umol/L P&lt;0.05). However, there was no difference in the concentration of IP3 in all four groups. Conclusions: The potential mechanism of myocardial protective effect of hypothermia may involve the reduced activities of β-receptors.

Crystal Structure of the Escherichia coli Fic Toxin-Like Protein in Complex with Its Cognate Antitoxin.

FIC domain proteins mediate post-translational modifications of target proteins, which typically results in their inactivation. Depending on the conservation of crucial active site residues, the FIC fold serves as structural scaffold for various enzymatic activities, mostly target adenylylation. The founding member of the vast Fic protein family, EcFicT, was identified in Escherichia coli some time ago. The G55R point mutant of EcFicT displays the “filamentation induced by cAMP” (Fic) phenotype at high 3',5'-cyclic adenosine monophosphate (cAMP) concentrations and elevated temperature, but the underlying molecular mechanism and any putative biochemical activity of EcFicT have remained unknown. EcFicT belongs to class I Fic toxin proteins that are encoded together with a small inhibitory protein (antitoxin), named EcFicA in E. coli. Here, we report the crystal structures of two mutant EcFicT/EcFicA complexes (EcFicTG55RA and EcFicTAE28G) both showing close resemblance with the structure of the AMP-transferase VbhT from Bartonella schoenbuchensis in complex with its cognate antitoxin VbhA. However, crucial differences in the active site of EcFicT compared to VbhT and other AMP-transferases rationalize the lack of evidence for adenylylation activity. Comprehensive bioinformatic analysis suggests that EcFicT has evolved from canonical AMP-transferases and has acquired a conserved binding site for a yet to be discovered novel substrate. The G55R mutation has no effect on structure or thermal stability of EcFicT, such that the molecular basis for its associated Fic phenotype remains elusive. We anticipate that this structure will inspire further bioinformatic and experimental analyses in order to characterize the enzymatic activity of EcFicT and help revealing its physiological role.

Testosterone improves erectile function through inhibition of reactive oxygen species generation in castrated rats.

Testosterone is overwhelmingly important in regulating erectile physiology. However, the associated molecular mechanisms are poorly understood. The purpose of this study was to explore the effects and mechanisms of testosterone in erectile dysfunction (ED) in castrated rats. Forty male Sprague-Dawley rats were randomized to four groups (control, sham-operated, castration and castration-with-testosterone-replacement). Reactive oxygen species (ROS) production was measured by dihydroethidium (DHE) staining. Erectile function was assessed by the recording of intracavernous pressure (ICP) and mean arterial blood pressure (MAP). Protein expression levels were examined by western blotting. We found that castration reduced erectile function and that testosterone restored it. Nitric oxide synthase (NOS) activity was decrease in the castrated rats, and testosterone administration attenuated this decrease (each p < 0.05). The testosterone, dihydrotestosterone, cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) concentrations were lower in the castrated rats, and testosterone restored these levels (each p < 0.05). Furthermore, the cyclooxygenase-2 (COX-2) and prostacyclin synthase (PTGIS) expression levels and phospho-endothelial nitric oxide synthase (p-eNOS, Ser1177)/endothelial nitric oxide synthase (eNOS) ratio were reduced in the castrated rats compared with the controls (each p < 0.05). In addition, the p40phox and p67phox expression levels were increased in the castrated rats, and testosterone reversed these changes (each p < 0.05). Overall, our results demonstrate that testosterone ameliorates ED after castration by reducing ROS production and increasing the activity of the eNOS/cGMP and COX-2/PTGIS/cAMP signaling pathways.

Exposure to an Extremely-Low-Frequency Magnetic Field Stimulates Adrenal Steroidogenesis via Inhibition of Phosphodiesterase Activity in a Mouse Adrenal Cell Line.

Extremely low-frequency magnetic fields (ELF-MFs) are generated by power lines and household electrical devices. In the last several decades, some evidence has shown an association between ELF-MF exposure and depression and/or anxiety in epidemiological and animal studies. The mechanism underlying ELF-MF-induced depression is considered to involve adrenal steroidogenesis, which is triggered by ELF-MF exposure. However, how ELF-MFs stimulate adrenal steroidogenesis is controversial. In the current study, we investigated the effect of ELF-MF exposure on the mouse adrenal cortex-derived Y-1 cell line and the human adrenal cortex-derived H295R cell line to clarify whether the ELF-MF stimulates adrenal steroidogenesis directly. ELF-MF exposure was found to significantly stimulate adrenal steroidogenesis (p < 0.01–0.05) and the expression of adrenal steroid synthetic enzymes (p < 0.05) in Y-1 cells, but the effect was weak in H295R cells. Y-1 cells exposed to an ELF-MF showed significant decreases in phosphodiesterase activity (p < 0.05) and intracellular Ca2+ concentration (p < 0.01) and significant increases in intracellular cyclic adenosine monophosphate (cAMP) concentration (p < 0.001–0.05) and cAMP response element-binding protein phosphorylation (p < 0.05). The increase in cAMP was not inhibited by treatment with NF449, an inhibitor of the Gs alpha subunit of G protein. Our results suggest that ELF-MF exposure stimulates adrenal steroidogenesis via an increase in intracellular cAMP caused by the inhibition of phosphodiesterase activity in Y-1 cells. The same mechanism may trigger the increase in adrenal steroid secretion in mice observed in our previous study.

Are aryl hydrocarbon receptor and G-protein–coupled receptor 30 involved in the regulation of seasonal testis activity in photosensitive rodent—the bank vole (Myodes glareolus)?

Within the reproductive system both aryl hydrocarbon receptor (AHR) and G-protein–coupled receptor 30 (GPR30) contribute to estrogen signaling and controlling of reproductive physiology. The specific question is whether and how AHR and GPR30 are involved in regulation of testis function in seasonally breeding rodents. Bank vole testes were obtained from animals reared under 18 hours light:6 hours dark (LD) and 6 hours light:18 hours dark (SD) conditions. Aryl hydrocarbon receptor and GPR30 expression were analyzed by quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry and/or immunofluorescent staining. In addition, the activity of enzymes involved in the intracellular signal transduction; extracellular signal-regulated kinase (ERK), protein kinase (PKA), matrix metalloproteinase 9 (MMP 9) and the concentrations of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and calcium (Ca2+) were examined by immunohistochemical, immunoenzymatic, and colorimetric assays, respectively. Aryl hydrocarbon receptor and GPR30 were expressed in testes of actively reproducing voles and regressed ones although their expression at the messenger RNA and AHR also at protein level appeared to be photoperiod-dependent. A specific cellular localization and expression of AHR and GPR30 correlated with the expression of ERK, PKA, and MMP 9. Moreover, we found robust differences in the levels of cAMP, cGMP, and Ca2+ in testicular homogenates between LD and SD voles. In the testes of LD voles, the levels of second messengers were always higher compared to SD. In vole testis, AHR and GPR30 can induce signaling pathways that involve ERK, PKA, MMP 9 and cAMP, cGMP, Ca2+. In addition, in AHR, signaling the engagement of both photoperiod and estrogens, whereas in GPR30, signaling only estrogens is reported. It is likely that in vole, because of a differential activity of signaling molecules, signal transduction via AHR rather than through GPR30 plays a role in regulation of seasonal changes of testis physiology.

Lead Inhibits Human Sperm Functions by Reducing the Levels of Intracellular Calcium, cAMP, and Tyrosine Phosphorylation.

It is well known that there has been a worldwide decrease in human male fertility in recent years. One of the main factors affecting this is environmental pollution. Lead is one of the major heavy metal contaminants that threaten the health of animals and human beings in China. It preferentially accumulates in male reproductive organs and can be up to 10 µM in human seminal plasma. Lead impairs mammalian spermatogenesis and sperm quality in vivo. It also inhibits sperm functions in vitro but the underlying mechanisms remain unclear. Therefore, we aimed to investigate the in vitro toxicity of lead on human sperm functions and to elucidate the underlying mechanisms. Semen samples were collected from 20 healthy volunteers with different careers and backgrounds living in Nanchang, Jiangxi. Human sperm suspensions were treated with different concentrations of lead acetate (0, 0.5, 2.5, 10, 50, and 100 µM) and the viability, motility, capacitation and progesterone-induced acrosome reaction were examined. Treatment with 10-100 µM lead acetate dose-dependently inhibited total and progressive motility measures, capacitation and progesterone-induced acrosome reaction. It also dose-dependently decreased the intracellular concentrations of cyclic adenosine monophosphate (cAMP) and calcium ([Ca(2+)]i), and reduced the tyrosine phosphorylation of sperm proteins, all of which are thought to be key factors in the regulation of sperm function. Our findings suggest that lead inhibits human sperm functions by reducing the levels of sperm intracellular cAMP, [Ca(2+)]i and tyrosine phosphorylation of sperm proteins in vitro.