Intracellular cAMP Concentration Research Articles

The Effect of Simulated Physiological Oocyte Maturation (SPOM) and L-Carnitine on Bovine Oocyte Developmental Competence.

Simulated Physiological Oocyte Maturation (SPOM) mimics in vitro the physiological events of oocyte maturation in the presence of cAMP modulators. These modulators increase the intracellular concentrations of cAMP, which inhibits the immediate resumption of meiosis and gives the oocyte more time to gain optimal developmental competence. In addition, L-carnitine helps to increase the energy supply of cells through the β-oxidation of fatty acids. This study aimed to investigate the effect of SPOM and L-carnitine supplementation during In Vitro Maturation (IVM) and In Vitro Culture (IVC) on the developmental competence of bovine oocytes. Ovarian Cumulus Complexes (COCs) were cultured in the presence or absence of forskolin+IBMX during the first 2 hr of IVM (pre-IVM) with or without L-carnitine (LC) during IVM or IVC in six experimental groups as follows: I) pre-IVM (pre-IVM group), II) pre-IVM with L-carnitine supplementation during IVM (pre-IVM/LC group), III) L-carnitine supplementation during IVM (IVM/LC group), IV) L-carnitine supplementation during in vitro culture (IVC/LC group), V) pre-IVM+ IVC/LC group, and VI) no treatment during IVM and IVC (Control group). The cleavage and blastocyst rates, the blastocysts' total cells number, and the expression of Nanog, Bax, Oct4, Cdx2, and Ifnt genes in resulting blastocysts were assessed. To assess differences among experimental groups, a one-way analysis of variance was initially employed, followed by post hoc Fisher LSD. The difference between groups was considered statistically significant when p<0.05. The cleavage and blastocyst rates in the Pre-IVM and Pre-IVM/LC groups was higher than control group and other groups (p≤0.05) except for IVC/LC and IVM/LC groups, respectively. The number of blastocyst's Inner Cell Mass (ICM) in pre-IVM and Pre-IVM/LC groups as well as the ratio of ICM/TE were higher than control group (p<0.05). The expression of OCT4, CDX2, and IFNT increased in both the pre-IVM and pre-IVM/LC groups compared to the control group (p<0.05). In conclusion, the application of SPOM-adapted IVM and L-carnitine during IVM of bovine oocyte improves the quantity and quality of the resulting embryos.

Pharmacological effect of Berotek and the leading mechanisms of Lung Pathology in miners Central Kazakhstan

In modern production conditions, industrial aerosols are still among the most common harmful occupational factors that determine the increased risk of developing occupational respiratory diseases among workers at industrial enterprises. The dust respiratory diseases are significantly common among workers in the mining and coal industries of Kazakhstan. In recent years, there has been a clear increase in the incidence of chronic dust bronchitis. The particular attention should be paid to such types of metal dust as dust of beryllium, vanadium, molybdenum, tungsten, cobalt and their compounds, under the influence of which not only peculiar damage to the lungs are observed, but also pronounced changes in other organs and systems. Dust of these metals, according to numerous authors, has a general toxic and toxic-allergic effect. An urgent problem of modern occupational medicine is the study of the role of exogenous and endogenous factors in the development of occupational pathology of the bronchopulmonary system. To identify groups at increased risk of developing dust pathology of the bronchopulmonary system, it is necessary to comprehensively study clinical and genetic factors, as well as determine the most significant diagnostic markers for the development of this pathology. An important advantage of inhaled anticholinergic drugs is the minimal frequency and severity of adverse events.The broncholytic effect is expressed in the form of inhibition of phosphodiesterase isoforms with a further increase in intracellular cAMP content and relaxation of smooth muscles of the respiratory tract. Immunomodulatory and anti-inflammatory effects of bronchodilators have also been established.

Glucocorticoids Rapidly Modulate CaV1.2-Mediated Calcium Signals through Kv2.1 Channel Clusters in Hippocampal Neurons.

The precise regulation of Ca2+ signals plays a crucial role in the physiological functions of neurons. Here, we investigated the rapid effect of glucocorticoids on Ca2+ signals in cultured hippocampal neurons from both female and male rats. In cultured hippocampal neurons, glucocorticoids inhibited the spontaneous somatic Ca2+ spikes generated by Kv2.1-organized Ca2+ microdomains. Furthermore, glucocorticoids rapidly reduced the cell surface expressions of Kv2.1 and CaV1.2 channels in hippocampal neurons. In HEK293 cells transfected with Kv2.1 alone, glucocorticoids significantly reduced the surface expression of Kv2.1 with little effect on K+ currents. In HEK293 cells transfected with CaV1.2 alone, glucocorticoids inhibited CaV1.2 currents but had no effect on the cell surface expression of CaV1.2. Notably, in the presence of wild-type Kv2.1, glucocorticoids caused a decrease in the surface expression of CaV1.2 channels in HEK293 cells. However, this effect was not observed in the presence of nonclustering Kv2.1S586A mutant channels. Live-cell imaging showed that glucocorticoids rapidly decreased Kv2.1 clusters on the plasma membrane. Correspondingly, Western blot results indicated a significant increase in the cytoplasmic level of Kv2.1, suggesting the endocytosis of Kv2.1 clusters. Glucocorticoids rapidly decreased the intracellular cAMP concentration and the phosphorylation level of PKA in hippocampal neurons. The PKA inhibitor H89 mimicked the effect of glucocorticoids on Kv2.1, while the PKA agonist forskolin abrogated the effect. In conclusion, glucocorticoids rapidly suppress CaV1.2-mediated Ca2+ signals in hippocampal neurons by promoting the endocytosis of Kv2.1 channel clusters through reducing PKA activity.

PDE4B Missense Variant Increases Susceptibility to Post-traumatic Stress Disorder-Relevant Phenotypes in Mice.

Large-scale genome-wide association studies (GWASs) have associated intronic variants in PDE4B, encoding cAMP-specific phosphodiesterase-4B (PDE4B), with increased risk for post-traumatic stress disorder (PTSD), as well as schizophrenia and substance use disorders that are often comorbid with it. However, the pathophysiological mechanisms of genetic risk involving PDE4B are poorly understood. To examine the effects of PDE4B variation on phenotypes with translational relevance to psychiatric disorders, we focused on PDE4B missense variant M220T, which is present in the human genome as rare coding variant rs775201287. When expressed in HEK-293 cells, PDE4B1-M220T exhibited an attenuated response to a forskolin-elicited increase in the intracellular cAMP concentration. In behavioral tests, homozygous Pde4b M220T male mice with a C57BL/6JJcl background exhibited increased reactivity to novel environments, startle hyperreactivity, prepulse inhibition deficits, altered cued fear conditioning, and enhanced spatial memory, accompanied by an increase in cAMP signaling pathway-regulated expression of BDNF in the hippocampus. In response to a traumatic event (10 tone-shock pairings), neuronal activity was decreased in the cortex but enhanced in the amygdala and hippocampus of Pde4b M220T mice. At 24 h post-trauma, Pde4b M220T mice exhibited increased startle hyperreactivity and decreased plasma corticosterone levels, similar to phenotypes exhibited by PTSD patients. Trauma-exposed Pde4b M220T mice also exhibited a slower decay in freezing at 15 and 30 d post-trauma, demonstrating enhanced persistence of traumatic memories, similar to that exhibited by PTSD patients. These findings provide substantive mouse model evidence linking PDE4B variation to PTSD-relevant phenotypes and thus highlight how genetic variation of PDE4B may contribute to PTSD risk.

The multidrug resistance protein 4 is expressed and functionally active in isolated bladder from pig.

Multidrug resistance proteins type 4 (MRP4) and 5 (MRP5) play pivotal roles in the transport of cyclic nucleotides in various tissues. However, their specific functions within the lower urinary tract remain relatively unexplored. This study aimed to investigate the effect of pharmacological inhibition of MRPs on cyclic nucleotide signaling in isolated pig bladder. The relaxation responses of the bladder were assessed in the presence of the MRP inhibitor, MK571. The temporal changes in intra- and extracellular levels of cAMP and cGMP in stimulated tissues were determined by mass spectrometry. The gene (ABCC4) and protein (MRP4) expression were also determined. MK571 administration resulted in a modest relaxation effect of approximately 26% in carbachol-precontracted bladders. The relaxation induced by phosphodiesterase inhibitors such as cilostazol, tadalafil, and sildenafil was significantly potentiated in the presence of MK571. In contrast, no significant potentiation was observed in the relaxation induced by substances elevating cAMP levels or stimulators of soluble guanylate cyclase. Following forskolin stimulation, both intracellular and extracellular cAMP concentrations increased by approximately 15.8-fold and 12-fold, respectively. Similarly, stimulation with tadalafil + BAY 41-2272 resulted in roughly 8.2-fold and 3.4-fold increases in intracellular and extracellular cGMP concentrations, respectively. The presence of MK571 reduced only the extracellular levels of cGMP. This study reveals the presence and function of MRP4 transporters within the porcine bladder and paves the way for future research exploring the role of this transporter in both underactive and overactive bladder disorders.NEW & NOTEWORTHY This study investigates the impact of pharmacological inhibition of MRP4 and MRP5 transporters on cyclic nucleotide signaling in isolated pig bladders. MK571 administration led to modest relaxation, with enhanced effects observed in the presence of phosphodiesterase inhibitors. However, substances elevating cAMP levels remained unaffected. MK571 selectively reduced extracellular cGMP levels. These findings shed light on the role of MRP4 transporters in the porcine bladder, opening avenues for further research into bladder disorders.

Apigenin potentiates glucose-stimulated insulin secretion through the PKA-MEK kinase signaling pathway independent of K-ATP channels

AimApigenin, a natural bioflavonoid, is reported as an anti-diabetic agent since it possesses the ability to inhibit α-glucosidase activity, cause stimulation of insulin action and secretion, manage ROS, and prevent diabetes complications. Apigenin was identified as a new insulin secretagogue that enhances glucose-stimulated insulin secretion and seems like a better antidiabetic drug candidate. Here we explored the insulinotropic mechanism(s) of apigenin in vitro in mice islets and in vivo in diabetic rats. MethodsSize-matched pancreatic islets were divided into groups and incubated in the presence or absence of apigenin and agonists or antagonists of major insulin signaling pathways. The secreted insulin was measured by ELISA. The intracellular cAMP was estimated by cAMP acetylation assay. The acute and chronic effects of apigenin were evaluated in diabetic rats. Resultsapigenin dose-dependently enhanced insulin secretion in isolated mice islets, and its insulinotropic effect was exerted at high glucose concentrations distinctly different from glibenclamide. Furthermore, apigenin amplified glucose-induced insulin secretion in depolarized and glibenclamide-treated islets. Apigenin showed no effect on intracellular cAMP concentration; however, an additive effect was observed by apigenin in both forskolin and IBMX-induced insulin secretion. Interestingly, H89, a PKA inhibitor, and U0126, a MEK kinase inhibitor, significantly inhibited apigenin-induced insulin secretion; however, no significant effect was observed by using ESI-05, an epac2 inhibitor. Apigenin improved glucose tolerance and increased glucose-stimulated plasma insulin levels in diabetic rats. Apigenin also lowered blood glucose in diabetic rats upon chronic treatment. ConclusionApigenin exerts glucose-stimulated insulin secretion by modulating the PKA-MEK kinase signaling cascade independent of K-ATP channels.

Norepinephrine regulates TNF expression via the A1AR-p38 MAPK-Relish pathway in granulocytes of oyster Crassostrea gigas

Norepinephrine (NE) is involved in regulating cytokine expression and phagocytosis of immune cells in the innate immunity of vertebrates. In the present study, the modulation mechanism of NE on the biosynthesis of TNFs in oyster granulocytes was explored. The transcripts of CgTNF-1, CgTNF-2 and CgTNF-3 were highly expressed in granulocytes, and they were significantly up-regulated after LPS stimulation, while down-regulated after NE treatment. The phagocytic rate and apoptosis index of oyster granulocytes were also triggered by LPS stimulation and suppressed by NE treatment. The mRNA expressions of CgMAPK14 and CgRelish were significantly induced after NE treatment, and the translocation of CgRelish from cytoplasm to nucleus was observed. The concentration of intracellular Ca2+ in granulocytes was significantly up-regulated upon NE incubation, and this trend reverted after the treatment with DOX (specific antagonist for NE receptor, CgA1AR-1). No obvious significance was observed in intracellular cAMP concentrations in the PBS, NE and NE + DOX groups. Once CgA1AR-1 was blocked by DOX, the mRNA expressions of CgMAPK14 and CgRelish were significantly inhibited, and the translocation of CgRelish from cytoplasm to nucleus was also dramatically suppressed, while the mRNA expression of CgTNF-1 and the apoptosis index increased significantly to the same level with those in LPS group, respectively. These results collectively suggested that NE modulated TNF expression in oyster granulocyte through A1AR-p38 MAPK-Relish signaling pathway.

Prostaglandin E2 inhibits the differentiation of T regulatory cells by Peroxisome Proliferator-Activated Receptor-Gamma during allergic rhinitis.

Allergic rhinitis (AR) represents a significant global health concern that can give rise to numerous diseases and result in labor productivity. T regulatory (Treg) cells are pivotal players in the pathogenesis of AR, and their deficiencies are closely related to Prostaglandin E2 (PGE2). However, the downstream mechanisms of this relationship remain poorly understood. This study aims to investigate the inhibitory mechanisms through which PGE2 impacts the differentiation of Treg cells. We compared the differentiation of Treg cells from naïve CD4+ T cells of AR patients and healthy controls, with or without the presence of PGE2 by flow cytometry. Intracellular cAMP concentration, mRNA and protein levels of cyclic-AMP dependent protein kinase A (PKA), as well as their downstream target, Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) were examined in Treg cells from AR and healthy donors. AR mouse model was established by pollen administration. PGE2 suppressed the differentiation of Treg cells from human naïve CD4+ T cells through the EP4 receptor. Furthermore, in AR patients and AR mouse, the expression of EP4 receptor were observed enhanced. The PGE2-EP4 signal was carried out by activating cAMP-PKA signaling pathway. Subsequently, phospholated PKA would suppress PPAR-γ expression. Treatment of Pioglitazone, a PPAR-γ agonist, was demonstrated to rescue the differentiation of Treg and help alleviate inflammation in the AR mouse model. In AR disease, the PGE2-EP4 signaling exerts an inhibitory effect on Treg differentiation by influencing the cAMP-PKA pathway and its downstream target PPAR-γ.

Anti-hair loss effect of a shampoo containing caffeine and adenosine.

Hair loss is a widespread health problem that affects numerous individuals and is associated with age, lack of sleep, stress, endocrine problems, and other problems. Caffeine exerts various pharmacological effects, particularly after ingestion. The caffeine-induced inhibition of phosphodiesterases can increase intracellular cAMP concentrations, ultimately resulting in stimulatory effects on cell metabolism and proliferation. Hence, caffeine has been confirmed to inhibit hair loss caused by premature termination of the hair growth phase. Adenosine also improves hair loss by stimulating hair growth and thickening hair shafts. However, further empirical evidence is required to comprehensively assess the efficacy of hair loss treatment and prevention using a formulation of caffeine and adenosine in specific proportions in shampoos. This study aimed to evaluate a shampoo with caffeine and adenosine as a daily scalp care product for hair loss in 77 subjects aged 18-60 years. The overall and local hair densities were assessed using professional cameras and dermoscopes at different magnifications and distances. Five hairs that came off the participant's head were randomly selected to measure hair diameter. The self-assessment questionnaires were filled on third month of product use. The combination of caffeine and adenosine in the shampoo significantly enhanced hair density compared to that of the baseline. The results revealed a significant reduction in hair loss. The hair diameters of the subjects did not change significantly. Most of the participants (71.05%) were satisfied with their hair after using the product. Shampoos containing caffeine and adenosine have been demonstrated to exert therapeutic benefits for reducing hair loss.

Molecular characterization of GPR84 in domestic cats

G protein-coupled receptor 84 (GPR84) was cloned as an orphan receptor, and medium-chain fatty acids were then revealed as endogenous ligands. GPR84 is expressed in immune cells and is believed to protect liver function from lipotoxicity caused by overeating and high-fat diet intake. This study aimed to present the molecular characterization of GPR84 in domestic cats. The deduced amino acid sequence of the feline GPR84 shows high sequence homology (83–89 %) with the orthologues from other mammalians by cDNA cloning of feline GPR84. Remarkably high mRNA expression was observed in the bone marrow by Q-PCR analysis. The inhibition of intracellular cAMP concentration was observed in cells transfected with feline GPR84 and treated with medium-chain fatty acids. Immunostaining of GPR84 and free fatty acid receptor 2 (FFAR2)/GPR43 in the bone marrow, where high mRNA expression was observed, showed reactions in macrophages and myeloid cells. To clarify whether the receptor formed homo/hetero-merization, GPR84 and FFARs were analyzed using Nano-Luc binary technology and NanoLuc bioluminescence resonance energy transfer technologies, which revealed that GPR84 formed more heteromers with FFAR2 than homomers with each other. In addition, when GPR84 and FFAR2/GPR43 were cotransfected in the cell, their localization on the cell membrane was reduced compared with that when single receptors were transfected. These results indicated that GPR84 is a functional receptor protein that is expressed in cat tissues and may have a protein–protein interaction with FFAR2/GPR43 on the cell membrane.

Transcriptional and Metabolic Response of a Strain of Escherichia coli PTS- to a Perturbation of the Energetic Level by Modification of [ATP]/[ADP] Ratio.

The intracellular [ATP]/[ADP] ratio is crucial for Escherichia coli's cellular functions, impacting transport, phosphorylation, signaling, and stress responses. Overexpression of F1-ATPase genes in E. coli increases glucose consumption, lowers energy levels, and triggers transcriptional responses in central carbon metabolism genes, particularly glycolytic ones, enhancing carbon flux. In this contribution, we report the impact of the perturbation of the energetic level in a PTS- mutant of E. coli by modifying the [ATP]/[ADP] ratio by uncoupling the cytoplasmic activity of the F1 subunit of the ATP synthase. The disruption of [ATP]/[ADP] ratio in the evolved strain of E. coli PB12 (PTS-) was achieved by the expression of the atpAGD operon encoding the soluble portion of ATP synthase F1-ATPase (strain PB12AGD+). The analysis of the physiological and metabolic response of the PTS- strain to the ATP disruption was determined using RT-qPCR of 96 genes involved in glucose and acetate transport, glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), TCA cycle and glyoxylate shunt, several anaplerotic, respiratory chain, and fermentative pathways genes, sigma factors, and global regulators. The apt mutant exhibited reduced growth despite increased glucose transport due to decreased energy levels. It heightened stress response capabilities under glucose-induced energetic starvation, suggesting that the carbon flux from glycolysis is distributed toward the pentose phosphate and the Entner-Duodoroff pathway with the concomitant. Increase acetate transport, production, and utilization in response to the reduction in the [ATP]/[ADP] ratio. Upregulation of several genes encoding the TCA cycle and the glyoxylate shunt as several respiratory genes indicates increased respiratory capabilities, coupled possibly with increased availability of electron donor compounds from the TCA cycle, as this mutant increased respiratory capability by 240% more than in the PB12. The reduction in the intracellular concentration of cAMP in the atp mutant resulted in a reduced number of upregulated genes compared to PB12, suggesting that the mutant remains a robust genetic background despite the severe disruption in its energetic level.

Effect of polymorphisms in the FAM151B gene on testis-related trait parameters in Hu sheep

The family with sequence similarity 151 member B (FAM151B) gene family is widely distributed in animals. This gene family encodes proteins belonging to the phosphodiesterase superfamily which plays a role in hydrolyzing intracellular second messengers, and affects the proliferation of Sertoli cells in the testis by degrading intracellular cAMP or cGMP content, thereby affecting the sperm production capacity of the testis. In this study, mutations in the FAM151B gene were screened in 491 Hu sheep to analyze the association among FAM151B gene polymorphisms testicular traits, and sperm-related parameters. A synonymous mutation (5: g.77976636 C>T), was detected in FAM151B, resulting in three genotypes, namely, CC, TT, and TC, with genotypic frequencies of 0.33, 0.05, and 0.62, respectively. This synonymous mutation was found to be significantly correlated with testicular weight, long testicular circumference, short testicular circumference, epididymal weight, and sperm count (p <0.05). In addition, individuals with CC genotypes had significant reproductive performance advantages compared with those with TC and TT genotypes. The current study shows that the FAM151B gene may play important roles in testicular development, and its SNPs are associated with testicular parameters, and sperm count, which provides important indicators for ram selection at early stage.

The regulatory effects of (p)ppGpp and indole on cAMP synthesis in Escherichia coli cells.

Bacterial stress adaptive response is formed due to changes in the cell gene expression profile in response to alterations in environmental conditions through the functioning of regulatory networks. The mutual influence of network signaling molecules represented by cells' natural metabolites, including indole and second messengers (p) ppGpp and cAMP, is hitherto not well understood, being the aim of this study. E. coli parent strain BW25141 ((p) ppGpp+) and deletion knockout BW25141ΔrelAΔspoT which is unable to synthesize (p)ppGpp ((p)ppGpp0) were cultivated in M9 medium supplemented with different glucose concentrations (5.6 and 22.2 mM) in the presence of tryptophan as a substrate for indole synthesis and in its absence. The glucose content was determined with the glucose oxidase method; the indole content, by means of HPLC; and the cAMP concentration, by ELISA. The onset of an increase in initially low intracellular cAMP content coincided with the depletion of glucose in the medium. Maximum cAMP accumulation in the cells was proportional to the concentration of initially added glucose. At the same time, the (p) ppGpp0 mutant showed a decrease in maximum cAMP levels compared to the (p)ppGpp+ parent, which was the most pronounced in the medium with 22.2 mM glucose. So, (p)ppGpp was able to positively regulate cAMP formation. The promoter of the tryptophanase operon responsible for indole biosynthesis is known to be under the positive control of catabolic repression. Therefore, in the cells of the (p)ppGpp+ strain grown in the tryptophan-free medium that were characterized by a low rate of spontaneous indole formation, its synthesis significantly increased in response to the rising cAMP level just after glucose depletion. However, this was not observed in the (p)ppGpp0 mutant cells with reduced cAMP accumulation. When tryptophan was added to the medium, both of these strains demonstrated high indole production, which was accompanied by a decrease in cAMP accumulation compared to the tryptophan-free control. Thus, under glucose depletion, (p)ppGpp can positively regulate the accumulation of both cAMP and indole, while the latter, in its turn, has a negative effect on cAMP formation.

The cyclic AMP (cAMP) phosphodiesterase CpdA required for growth, biofilm formation, motility and pathogenicity of Edwardsiella piscicida

Edwardsiella piscicida is a severe fish pathogen with wide host range, causing the huge economic losses in the aquaculture industry. Cyclic adenosine monophosphate (cAMP) as an important second messenger regulates the physiological and behavioral responses to environmental cues in eukaryotic and prokaryotic. The intracellular level of cAMP for effective activity is tightly controlled by the synthesis of adenylate cyclase, excretion and degradation of phosphodiesterase. In this study, we identified and characterized a class III cAMP phosphodiesterase, named as CpdA, in the E. piscicida. To investigate the role of CpdA in the physiology and pathogenicity, we constructed the in-frame deletion mutant of cpdA of E. piscicida, TX01ΔcpdA. The results showed that TX01ΔcpdA accumulated the higher intracellular cAMP concentration than TX01, indicating that CpdA exerted the hydrolysis of cAMP. In addition, compared to the TX01, the TX01ΔcpdA slowed growth rate, diminished biofilm formation and lost motility. More importantly, pathogenicity analysis confirmed that TX01ΔcpdA significantly impaired the ability of invading the epithelial cells, reproduction in macrophages, tissues dissemination and lethality for healthy tilapias. The most of lost properties of TX01ΔcpdA were restored partially or fully by the introduction of cpdA gene. These results suggest that cpdA is required for regulation of the physiology and virulence of E. piscicida.

Bile acids regulate SF-1 to alter cholesterol balance in adrenocortical cells via S1PR2

Glucocorticoid synthesis typically occurs in adrenal cortex and is influenced by cholesterol balance, since cholesterol is the sole precursor of steroids. Bile acids as the signaling molecules, have been shown to promote steroidogenesis in steroidogenic cells. However, whether bile acids directly regulate cholesterol balance remains elusive. In this study, we prepared cholestatic mouse models and cultured human adrenocortical cells (H295R) treated with taurochenodeoxycholic acid (TCDCA) to determine transcription levels of cholesterol metabolism associated genes and cholesterol concentrations in adrenocortical cells. Results showed that common bile duct ligation (CBDL) and chenodeoxycholic acid (CDCA) feeding elevated the mRNA levels of Abca1, Cyp51, Hmgcr, Srb1, and Mc2r in adrenals of mice. Meanwhile, the concentrations of total cholesterol and cholesteryl ester in adrenals of CBDL and CDCA-fed mice were dramatically lowered. The total and phosphorylation levels of HSL in adrenal glands of CBDL mice were also enhanced. Similarly, TCDCA treatment in H295R cells decreased intracellular concentrations of total cholesterol and cholesteryl ester and increased transcription levels of SRB1, MC2R, and HSL as well. Inhibition of bile acids’ receptor sphingosine 1‐phosphate receptor 2 (S1PR2), extracellular signal‐regulated kinase (ERK) phosphorylation, and steroidogenic factor 1 (SF-1) respectively successfully abolished effect of TCDCA on H295R cells. SF-1s was found to be phosphorylated at Thr75 in TCDCA-treated H295R cells. While a mild increase of intracellular cAMP concentration was detected upon TCDCA treatment, inhibition of PKA activity with Rp-Isomer in H295R cells failed to decrease the expression of SF-1 and its target genes. Our findings suggest that conjugated bile acids affect cholesterol balance through regulation of SF-1 in adrenocortical cells so as to provide an adequate cholesterol supply for glucocorticoid synthesis, which improves and enriches our understanding of the mechanism whereby bile acids regulate cholesterol balance to affect adrenal function.

Effect and mechanism of Zexie Decoction in promoting white adipose tissue browning/brown adipose tissue activation based on GLP-1R/cAMP/PKA/CREB pathway

This study investigated the mechanism of Zexie Decoction(ZXD) in promoting white adipose tissue browning/brown adipose tissue activation based on the GLP-1R/cAMP/PKA/CREB pathway. A hyperlipidemia model was induced by a western diet(WD) in mice, and the mice were divided into a control group, a model group(WD), and low-, medium-, and high-dose ZXD groups. An adipogenesis model was induced in 3T3-L1 cells in vitro, and with forskolin(FSK) used as a positive control, low-, medium-, and high-dose ZXD groups were set up. Immunohistochemistry and immunofluorescence results showed that compared with the WD group, ZXD promoted the expression of UCP1 in white and brown adipose tissues, and also upregulated UCP1, CPT1β, PPARα, and other genes in the cells. Western blot analysis showed a dose-dependent increase in the protein expression of PGC-1α, UCP1, and PPARα with ZXD treatment, indicating that ZXD could promote the white adipose tissue browning/brown adipose tissue activation. Hematoxylin-eosin(HE) staining results showed that after ZXD treatment, white and brown adipocytes were significantly reduced in size, and the mRNA expression of ATGL, HSL, MGL, and PLIN1 was significantly upregulated as compared with the results in the WD group. Oil red O staining and biochemical assays indicated that ZXD improved lipid accumulation and promoted lipolysis. Immunohistochemistry and immunofluorescence staining for p-CREB revealed that ZXD reversed the decreased expression of p-CREB caused by WD. In vitro intervention with ZXD increased the protein expression of CREB, p-CREB, and p-PKA substrate, and increased the mRNA level of CREB. ELISA detected an increase in intracellular cAMP concentration with ZXD treatment. Molecular docking analysis showed that multiple active components in Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma could form stable hydrogen bond interactions with GLP-1R. In conclusion, ZXD promotes white adipose tissue browning/brown adipose tissue activation both in vivo and in vitro, and its mechanism of action may be related to the GLP-1R/cAMP/PKA/CREB pathway.

Adaptive laboratory evolution of Rhodococcus rhodochrous DSM6263 for chlorophenol degradation under hypersaline condition

BackgroundNormally, a salt amount greater than 3.5% (w/v) is defined as hypersaline. Large amounts of hypersaline wastewater containing organic pollutants need to be treated before it can be discharged into the environment. The most critical aspect of the biological treatment of saline wastewater is the inhibitory/toxic effect exerted on bacterial metabolism by high salt concentrations. Although efforts have been dedicated to improving the performance through the use of salt-tolerant or halophilic bacteria, the diversities of the strains and the range of substrate spectrum remain limited, especially in chlorophenol wastewater treatment.ResultsIn this study, a salt-tolerant chlorophenol-degrading strain was generated from Rhodococcus rhodochrous DSM6263, an original aniline degrader, by adaptive laboratory evolution. The evolved strain R. rhodochrous CP-8 could tolerant 8% NaCl with 4-chlorophenol degradation capacity. The synonymous mutation in phosphodiesterase of strain CP-8 may retard the hydrolysis of cyclic adenosine monophosphate (cAMP), which is a key factor reported in the osmoregulation. The experimentally verified up-regulation of intracellular cAMP level in the evolved strain CP-8 contributes to the improvement of growth phenotype under high osmotic condition. Additionally, a point mutant of the catechol 1,2-dioxygenase, CatAN211S, was revealed to show the 1.9-fold increment on activity, which the mechanism was well explained by molecular docking analysis.ConclusionsThis study developed one chlorophenol-degrading strain with extraordinary capacity of salt tolerance, which showed great application potential in hypersaline chlorophenol wastewater treatment. The synonymous mutation in phosphodiesterase resulted in the change of intracellular cAMP concentration and then increase the osmotic tolerance in the evolved strain. The catechol 1,2-dioxygenase mutant with improved activity also facilitated chlorophenol removal since it is the key enzyme in the degradation pathway.

Polyamines modulate mouse sperm motility

Polyamines are polycationic molecules which contains two or more amino groups (-NH3 +) highly charged at physiological pH, and among them we found spermine, spermidine, putrescine, and cadaverine. They interact with proteins, nucleic acids, modulate Ca2+, K+, and Na+ channels, and protect sperm from oxidative stress. In this work, we evaluate the effect of spermine, spermidine, and putrescine on the total, progressive and kinematic parameters of motility, capacitation, acrosome reaction, also in presence and absence of the dbcAMP, an analogue of the cAMP, and the IBMX, a phosphodiesterase inhibitor. In addition, we evaluated the intracellular concentrations of cAMP [cAMP]i, and performed an in silico analysis between polyamines and the sAC from mouse to predict the possible interaction among them. Our results showed that all polyamines decrease drastically the total, progressive and the kinetic parameters of sperm motility, decrease the capacitation, and only spermidine and putrescine impeded the acquisition of acrosome reaction. Moreover, the effect of polyamines was attenuated but not countered by the addition of db-cAMP and IBMX, suggesting a possible inhibition of the sAC. Also, the presence of polyamines induced a decrease of the [cAMP]i, and the in silico analysis predicted a strong interaction among polyamines and the sAC. Overall, the evidence suggests that probably the polyamines interact and inhibit the activity of the sAC.

SAT001 Membrane Progesterone Receptors Induce Glycogenolysis In Uterine Epithelial Cells

Abstract Disclosure: M. Berg: None. M. Dean: None. In humans and cattle, 40-60% of pregnancies fail, with most losses occurring prior to or during implantation. Throughout this time, the developing embryo needs glucose to survive. The demand for glucose increases dramatically at the morula stage as the embryo enters the uterus. Yet, how glucose is regulated to match the changing needs of the embryo is unclear. Glycogen is composed of thousands of glucose molecules and is present in the uterine epithelium of many species. Our lab has shown that the glycogen content of the bovine uterine epithelium was lower during the luteal phase than at estrus. Therefore, our objective was to elucidate the role of progesterone in glycogenolysis of uterine epithelial cells. Bovine uterine epithelial (BUTE) cells were treated with insulin-like growth factor 1 (IGF1) to stimulate glycogenesis, followed by treatment for 48 hours. Progesterone decreased glycogen levels in BUTE cells by 99% (P=0.0002). RU486 did not block progesterone's effect, indicating that the effect of progesterone was not mediated by nuclear progesterone receptors (nPR). Thus, we hypothesized that the effect of progesterone is mediated by membrane progesterone receptors (mPRs). RT-PCR confirmed that BUTE cells expressed all five mPRs (α, β, γ, δ, and ε). Like progesterone, a specific mPR agonist (Org OD 02-0) decreased glycogen levels in BUTE cells by 99% (P&amp;lt;0.0001). Indicating a potential for mPRs to regulate glycogen in vivo, immunohistochemistry showed that the bovine uterine epithelium expressed high levels of mPRα. These results validate that progesterone is acting through mPRs to stimulate glycogen breakdown in BUTE cells. Once progesterone binds to the mPR, two signaling pathways can be activated, cAMP and AMPK. Neither progesterone nor Org OD 02-0 changed intracellular cAMP concentrations. In agreement, the adenylyl cyclase activator forskolin increased cAMP concentration but did not decrease glycogen. BUTE cells treated with progesterone had increased pAMPK levels at 24 hours compared to the control (P=0.0001). BUTE cells treated with an AMPK activator (D942) had a decrease in glycogen (P=0.0051). Supporting these results in vivo, pAMPK levels in the uterine epithelium were high in the bovine uterine epithelium during the luteal phase when glycogen levels were low. In a human model, progesterone and Org OD 02-0 decreased glycogen levels to a similar extent in Ishikawa cells that express mPRs but not nPRs (P=0.0027). In conclusion, progesterone acting through the mPR and AMPK to stimulate glycogenolysis may play a crucial role in providing glucose to endometrial tissue or the growing embryo. Presentation: Saturday, June 17, 2023

OR2H2 Activates CAMKKβ-AMPK-Autophagy Signaling Axis and Suppresses Senescence in VK2/E6E7 Cells.

Olfactory receptors are expressed in multiple extra-nasal tissues and these ectopic olfactory receptors mediate tissue-specific functions and regulate cellular physiology. Ectopic olfactory receptors may play key roles in tissues constantly exposed to odorants, thus the functionality of these receptors in genital tissues is of particular interest. The functionality of ectopic olfactory receptors expressed in VK2/E6E7 human vaginal epithelial cells was investigated. OR2H2 was the most highly expressed olfactory receptor expressed in VK2/E6E7 cells, and activation of OR2H2 by aldehyde 13-13, a ligand of OR2H2, increased the intracellular calcium and cAMP concentrations. Immunoblotting demonstrated that activation of OR2H2 by aldehyde 13-13 stimulated the CAMKKβ-AMPK-mTORC1-autophagy signaling axis, and that these effects were negated by OR2H2 knockdown. AMPK is known to regulate senescence; consequently, we investigated further the effect of aldehyde 13-13 on senescence. In H2O2-induced senescent cells, activation of OR2H2 by aldehyde 13-13 restored proliferation, and reduced the expression of senescence markers, P16 and P19. Additionally, aldehyde 13-13 induced apoptosis of H2O2-induced senescent cells, compared with non-senescent normal cells. In vivo, aldehyde 13-13 increased the lifespan of Caenorhabditis elegans and budding yeast. These findings demonstrate that OR2H2 is a functional receptor in VK2/E6E7 cells, and that activation of OR2H2 activates the AMPK-autophagy axis, and suppresses cellular aging and senescence, which may increase cellular health.