RESUMO
Tideglusib is considered to be a promising alternative to nonyl alcohol-9 contraceptives. Previous studies have demonstrated that the rapid spermicidal effect of tideglusib at a high concentration (≥ 10 µM) may occur through detergent-like activity; however, the effect of low concentrations of tideglusib (< 5 µM) on sperm is unknown. We explored the intracellular mechanism of tideglusib (< 5 µM) on the immobilization of human sperm by exploring related signaling pathways in human sperm. After treatment with tideglusib (1.25 µM) for 2 h, sperm motility rate decreased to 0, while sperm membrane integrity rate was 70%. Protein tyrosine phosphorylation level and intracellular cyclic adenosine 3,5-monophosphate (cAMP) concentration decreased significantly compared to those in the control group. Isobutylmethylxanthine and 8-Bromo-cAMP relieved the inhibition of spermatozoa tyrosine phosphorylation, while tyrosine phosphorylation of sperm protein in the H89 and CALP1 treatment groups was significantly inhibited, and there was no difference in the tideglusib treatment group. H-89 and CALP1 reduced the level of serine phosphorylation of GSK-3α/ß (Ser21/9), while its level was enhanced by IBMX and 8-Bromo-cAMP. Our results show the existence of the GSK3-cAMP/PKA regulatory loop in human sperm, which may mediate the immobilization effect of tideglusib at low of concentrations (e.g., 1.25 µM) on sperm motility.
Assuntos
AMP Cíclico , Quinase 3 da Glicogênio Sintase , Humanos , Masculino , AMP Cíclico/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Motilidade dos Espermatozoides/fisiologia , Sêmen/metabolismo , Espermatozoides/metabolismo , Fosforilação , Tirosina/metabolismoRESUMO
Emerging data have indicated that long noncoding RNAs (lncRNA) are associated with the pathogenesis of endometriosis. However, few are associated with endometriosisassociated infertility. In addition, to the best of our knowledge, the role of lncRNAs in decidual formation during the window of implantation with endometriosis has not been reported to date. Based on our previous results, the aim of the present study was to explore the role of lncRNA long intergenic nonprotein coding RNA (LINC)01960201 in in vitro decidualization of endometrial stromal cells in endometriosis during the window of implantation, as well as to explore the biological function of LINC01960201, and the regulation of a disintegrin and metalloproteinase with thrombospondin motifs 7 (ADAMTS7), hsamicroRNA (miR)760 and hsamiR608 in the decidualization of endometrial stromal cells with endometriosis. Using miRanda, PITA and RNAhybrid, the present study predicted which miRs share the common target gene ADAMTS7 with LINC01960201 and the existence of regulatory targets. Dual luciferase vectors were constructed to extract the plasmids and measure the relative fluorescence values in order to estimate target regulatory association between LINC01960201, ADAMTS7 and miRs. Midsecretory endometrial tissues were collected from women with endometriosisassociated infertility. From these tissues, endometrial stromal cells were extracted and cultured as primary cultures. Medroxyprogesterone acetate (MPA) and 8Bromoadenosine 3',5'cyclic monophosphate (8BrcAMP) were added to induce in vitro decidualization, and to knockdown LINC01960201 and transfect a hsamiR608 mimic at the same time. Reverse transcriptionquantitative PCR and western blotting were conducted to compare the difference in gene expression between the experimental and negative control groups. No regulatory sites between LINC01960201 and hsamiR608 were identified; however, potential regulatory sites were detected between hsamiR608 and the 3'untranslated region (UTR) of ADAMTS7, whereas neither the 3'UTR of LINC01960201 or the 3'UTR of ADAMTS7 had any regulatory targets with hsamiR760. During the process of decidualization of endometrial stromal cells by in vitro induction, the expression of hsamiR608 in the knockdown group was significantly higher compared with that of the negative control group after LINC01960201knockdown, and the expression of ADAMTS7 in the transfection group was significantly lower compared with that of the negative control group after hsamiR608 mimic transfection. In conclusion, it was hypothesized that LINC01960201 played a notable regulatory role in the decidualization of endometrial stromal cells in women with endometriosis during the window of implantation, and its abnormal expression may lead to the decline of endometrial receptivity and recurrent abortions.
Assuntos
Substitutos Sanguíneos , Endometriose , Infertilidade , MicroRNAs , RNA Longo não Codificante , Gravidez , Feminino , Humanos , Endometriose/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteína ADAMTS7/genética , Acetato de Medroxiprogesterona/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Desintegrinas/genética , Desintegrinas/metabolismo , Substitutos Sanguíneos/metabolismo , Endométrio/metabolismo , Células Estromais/metabolismo , MicroRNAs/genética , Infertilidade/genética , Trombospondinas/genética , Regiões não Traduzidas , DecíduaRESUMO
BACKGROUND: Successful implantation is a complex process that is influenced by embryo quality, endometrial receptivity, immune factors, and the specific type of in vitro fertilization protocol used. DNA topoisomerase IIα (TOP2A) is a well-known protein involved in cell proliferation; however, its expression and effect on the endometrium in recurrent implantation failure (RIF) have not been fully elucidated. METHODS: The human endometrial tissues of healthy controls and patients with RIF were collected. A proteomic analysis was performed to evaluate the differentially expressed proteins between the RIF group and the fertile control group. The expression patterns of TOP2A in the human preimplantation endometrium of the patients with RIF were determined by immunohistochemical staining, Western blotting and qRT-PCR. TOP2A knockdown (sh-TOP2A) T-HESCs were generated using lentiviruses. The expression of TOP2A in T-HESCs was manipulated to investigate its role in decidualization. The TOP2A-related changes in decidualization were screened by mRNA sequencing in decidualized TOP2A knockdown and control T-HESCs and then confirmed by Western blotting and immunofluorescence staining. TOP2A-deficient mice were generated by injection of TOP2A-interfering adenovirus on GD2.5 and GD3.5. RESULTS: We performed a proteomic analysis of endometrial tissues to investigate the potential pathogenesis of RIF by comparing the patients with RIF and the matched controls and found that TOP2A might be a key protein in RIF. TOP2A is ubiquitously expressed in both stromal and glandular epithelial cells of the endometrium. The data indicate that TOP2A expression is significantly lower in the mid-secretory endometrium of women with RIF. TOP2A expression was downregulated under stimulation by 8-bromo-cAMP and MPA. Ablation of TOP2A resulted in upregulated expression of decidual biomarkers and morphological changes in the cells. Mechanistic analysis revealed that TOP2A regulates the NF-κB signaling pathway in decidualized T-HESCs. The TOP2A-deficient mice exhibited lower fetal weights. CONCLUSIONS: Our findings revealed that abnormal expression of TOP2A affects decidualization and changes the "window of implantation", leading to RIF. TOP2A participates in the processes of decidualization and embryo implantation, functioning at least in part through the NF-κB pathway. Regulating the expression of TOP2A in the endometrium may become a new strategy for the prevention and treatment of RIF.
Assuntos
DNA Topoisomerases Tipo II , Decídua , NF-kappa B , Proteínas de Ligação a Poli-ADP-Ribose , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Biomarcadores/metabolismo , DNA Topoisomerases Tipo II/genética , Decídua/metabolismo , Implantação do Embrião/genética , Endométrio/metabolismo , Feminino , Humanos , Camundongos , NF-kappa B/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteômica , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Células Estromais/metabolismoRESUMO
Acetaminophen is both widely used to treat children with fever and is also responsible for thousands being hospitalised annually. Historically the antipyretic actions of acetaminophen were attributed to the inhibition of cyclooxygenase (COX-1/2) enzymes and more recently a novel COX-1 variant (COX-3) located in the brain. However, the evidence for acetaminophen-mediated COX inhibition remains contentious. This study assesses the impact of acetaminophen and other putative COX-3 inhibitors on the release of fatty acids during lipolysis as an alternative mechanism by which antipyretics can reduce body temperature during fever. 3T3-L1 adipocytes, primary brown adipocytes and isolated mitochondria were exposed to COX-3 inhibitors and lipolysis and mitochondrial electron transport chain function assessed. Acetaminophen, aminopyrine and antipyrine at 1-10 mM caused a significant decrease (up to 70%; P < 0.01, from control) in lipolysis within 1, 3 and 24 h without affecting cell viability. The inhibition was observed regardless of where along its signalling pathway lipolysis was stimulated. All three compounds were found to significantly attenuate mitochondrial function by up to 30% for complex I and 40% for complex II (P < 0.01, from control). These novel observations combined with the known limited inhibition of the COX enzymes by acetaminophen suggest both the antipyretic and hypothermia induced by acetaminophen and related compounds could be attributed to the direct inhibition of lipolysis and mitochondrial function, rather than cyclooxygenase inhibition centrally. Further these observations could provide new drug targets for reducing fever with the added bonus of fewer individuals being hospitalized by accidental acetaminophen overdose.
Assuntos
Acetaminofen/farmacologia , Adipócitos/efeitos dos fármacos , Antipiréticos/farmacologia , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/fisiologia , Lipólise/efeitos dos fármacos , Células 3T3-L1 , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Adipócitos/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Aminopirina/farmacologia , Animais , Antipirina/farmacologia , Diferenciação Celular , Colforsina/metabolismo , Isoproterenol/farmacologia , Camundongos , Ratos , Ratos WistarRESUMO
The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). Pseudomonas putida has dozens of DGC/PDE-encoding genes in its genome, but the phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterize function and transcriptional regulation of a P. putida c-di-GMP-metabolizing enzyme, GcsA. GcsA consists of two per-ARNT-sim (PAS) domains, followed by a canonical conserved central sequence pattern (GGDEF) domain and a truncated EAL domain. In vitro analysis confirmed the DGC activity of GcsA. The phenotypic observation revealed that GcsA inhibited swimming motility in an FlgZ-dependent manner. In terms of transcriptional regulation, gcsA was found to be cooperatively regulated by c-di-GMP and cAMP via their effectors, FleQ and Crp respectively. The transcription of gcsA was promoted by c-di-GMP and inhibited by cAMP. In vitro binding analysis revealed that FleQ indirectly regulated the transcription of gcsA, while Crp directly regulated the transcription of gcsA by binding to its promoter. Besides, an inverse relationship between the cellular c-di-GMP and cAMP levels in P. putida was confirmed. These findings provide basic knowledge regarding the function and transcriptional regulation of GcsA and demonstrate a crosstalk between c-di-GMP and cAMP in the regulation of the expression of GcsA in P. putida.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fósforo-Oxigênio Liases/genética , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência Conservada , GMP Cíclico/metabolismo , Diester Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , Sistemas do Segundo MensageiroRESUMO
Recent reports show that protein kinase A (PKA), but not exchange protein activated by cAMP (EPAC), acts in a cell autonomous manner to constitutively reduce the angiogenic sprouting capacity of murine and human endothelial cells. Specificity in the cellular actions of individual cAMP-effectors can be achieved when a cyclic nucleotide phosphodiesterase (PDE) enzyme acts locally to control the "pool" of cAMP that activates the cAMP-effector. Here, we examined whether PDEs coordinate the actions of PKA during endothelial cell sprouting. Inhibiting each of the cAMP-hydrolyzing PDEs expressed in human endothelial cells revealed that phosphodiesterase 3 (PDE3) inhibition with cilostamide reduced angiogenic sprouting in vitro, while inhibitors of PDE2 and PDE4 family enzymes had no such effect. Identifying a critical role for PDE3B in the anti-angiogenic effects of cilostamide, silencing this PDE3 variant, but not PDE3A, markedly impaired sprouting. Importantly, using both in vitro and ex vivo models of angiogenesis, we show the hypo-sprouting phenotype induced by PDE3 inhibition or PDE3B silencing was reversed by PKA inhibition. Examination of the individual cellular events required for sprouting revealed that PDE3B and PKA each regulated angiogenic sprouting by controlling the invasive capacity of endothelial cells, more specifically, by regulating podosome rosette biogenesis and matrix degradation. In support of the idea that PDE3B acts to inhibit angiogenic sprouting by limiting PKA-mediated reductions in active cdc42, the effects of PDE3B and/or PKA on angiogenic sprouting were negated in cells with reduced cdc42 expression or activity. Since PDE3B and PKA were co-localized in a perinuclear region in human ECs, could be co-immunoprecipitated from lysates of these cells, and silencing PDE3B activated the perinuclear pool of PKA in these cells, we conclude that PDE3B-mediated hydrolysis of cAMP acts to limit the anti-angiogenic potential of PKA in ECs.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Células Endoteliais/metabolismo , Neovascularização Patológica/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , AMP Cíclico/genética , Humanos , Camundongos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Inibidores da Fosfodiesterase 3/farmacologiaRESUMO
Under physiologic conditions, conjunctival goblet cells (CGCs) secrete mucins into the tear film to preserve ocular surface homeostasis. Specialized proresolving mediators (SPMs), like resolvins (Rvs), regulate secretion from CGCs and actively terminate inflammation. The purpose of this study was to determine if RvD2 stimulated mucin secretion and to investigate the cellular signaling components. Goblet cells were cultured from rat conjunctiva. Secretion was measured by an enzyme-linked lectin assay, change in intracellular [Ca2+] ([Ca2+]i) using Fura-2, and cellular cAMP levels by ELISA. RvD2 (10-11-10-8 M) stimulated secretion, increased cellular cAMP levels and the [Ca2+]i. RvD2-stimulated increase in [Ca2+]i and secretion was blocked by Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis and the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride but not by the cAMP exchange protein inhibitor α-[2-(3-chlorophenyl)hydrazinylidene]-5-(1,1-dimethylethyl)-b-oxo-3-isoxazolepropanenitrile. Forskolin, 3-isobutyl-1-methylxanthine, and 8-bromo-cAMP (8-Br-cAMP) increased [Ca2+]i. Increasing cAMP with 8-Br-cAMP inhibited the increase in [Ca2+]i stimulated by the cAMP-independent agonist cholinergic agonist carbachol. In conclusion, RvD2 uses both cellular cAMP and [Ca2+]i to stimulate glycoconjugate secretion from CGCs, but the interaction of cAMP and [Ca2+]i is context dependent. Thus RvD2 likely assists in the maintenance of the mucous layer of the tear film to sustain ocular surface homeostasis and has potential as a novel treatment for dry eye disease.-Botten, N., Hodges, R. R., Li, D., Bair, J. A., Shatos, M. A., Utheim, T. P., Serhan, C. N., Dartt, D. A. Resolvin D2 elevates cAMP to increase intracellular [Ca2+] and stimulate secretion from conjunctival goblet cells.
Assuntos
Cálcio/metabolismo , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/metabolismo , AMP Cíclico/metabolismo , Ácidos Docosa-Hexaenoicos/fisiologia , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Células Cultivadas , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/metabolismo , Fura-2/metabolismo , Masculino , Mucinas/metabolismo , Ratos , Ratos Sprague-Dawley , Lágrimas/efeitos dos fármacos , Lágrimas/metabolismoRESUMO
Pharmacological agents that raise cAMP and activate protein kinase A (PKA) stimulate 26S proteasome activity, phosphorylation of subunit Rpn6, and intracellular degradation of misfolded proteins. We investigated whether a similar proteasome activation occurs in response to hormones and under various physiological conditions that raise cAMP. Treatment of mouse hepatocytes with glucagon, epinephrine, or forskolin stimulated Rpn6 phosphorylation and the 26S proteasomes' capacity to degrade ubiquitinated proteins and peptides. These agents promoted the selective degradation of short-lived proteins, which are misfolded and regulatory proteins, but not the bulk of cell proteins or lysosomal proteolysis. Proteasome activities and Rpn6 phosphorylation increased similarly in working hearts upon epinephrine treatment, in skeletal muscles of exercising humans, and in electrically stimulated rat muscles. In WT mouse kidney cells, but not in cells lacking PKA, treatment with antidiuretic hormone (vasopressin) stimulated within 5-minutes proteasomal activity, Rpn6 phosphorylation, and the selective degradation of short-lived cell proteins. In livers and muscles of mice fasted for 12-48 hours cAMP levels, Rpn6 phosphorylation, and proteasomal activities increased without any change in proteasomal content. Thus, in vivo cAMP-PKA-mediated proteasome activation is a common cellular response to diverse endocrine stimuli and rapidly enhances the capacity of target tissues to degrade regulatory and misfolded proteins (e.g., proteins damaged upon exercise). The increased destruction of preexistent regulatory proteins may help cells adapt their protein composition to new physiological conditions.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Epinefrina/farmacologia , Glucagon/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Rim , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Deficiências na Proteostase/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Proteínas Ubiquitinadas/metabolismoRESUMO
Human endometrium decidualization, a differentiation process involving biochemical and morphological changes, is a prerequisite for embryo implantation and successful pregnancy. Here, we show that the mammalian target of rapamycin (mTOR) is a crucial regulator of 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP)-induced decidualization in human endometrial stromal cells. The level of mSin1 in mTOR complex 2 (mTORC2) and DEPTOR in mTOR complex 1 (mTORC1) decreases during 8-Br-cAMP-induced decidualization, resulting in decreased mTORC2 activity and increased mTORC1 activity. Notably, DEPTOR displacement increases the association between raptor and insulin receptor substrate-1 (IRS-1), facilitating IRS-1 phosphorylation at serine 636/639. Finally, both S473 and T308 phosphorylation of Akt are reduced during decidualization, followed by a decrease in forkhead box O1 (FOXO1) phosphorylation and an increase in the mRNA levels of the decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1). Taken together, our findings reveal a critical role for mTOR in decidualization, involving the differential regulation of mTORC1 and mTORC2.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Decídua/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Adulto , Feminino , Proteína Forkhead Box O1/metabolismo , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pessoa de Meia-Idade , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Hypoxia or intermittent hypoxia (IH) have known to alter both synthesis and secretion of hormones. However, the effect of IH on the production of adrenal cortical steroid hormones is still unclear. The aim of present study was to explore the mechanism involved in the effect of IH on the production of corticosterone by rat ZFR cells. Male rats were exposed at 12% O2 and 88% N2 (8 hours per day) for 1, 2, or 4 days. The ZFR cells were incubated at 37 °C for 1 hour with or without ACTH, 8-Br-cAMP, calcium ion channel blockers, or steroidogenic precursors. The concentration of plasma corticosterone was increased time-dependently by administration of IH hypoxia. The basal levels of corticosterone production in cells were higher in the IH groups than in normoxic group. IH resulted in a time-dependent increase of corticosterone production in response to ACTH, 8-Br-cAMP, progesterone and deoxycorticosterone. The production of pregnenolone in response to 25-OH-C and that of progesterone in response to pregnenolone in ZFR cells were enhanced by 4-day IH. These results suggest that IH in rats increases the secretion of corticosterone via a mechanism at least in part associated with the activation of cAMP pathway and steroidogenic enzymes.
Assuntos
Corticosterona/biossíntese , Hipóxia/metabolismo , Zona Fasciculada/citologia , Zona Fasciculada/metabolismo , 11-beta-Hidroxiesteroide Desidrogenases/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Animais , Biomarcadores , Canais de Cálcio/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Corticosterona/sangue , Masculino , Pregnenolona/metabolismo , Ratos , Zona Fasciculada/efeitos dos fármacosRESUMO
The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H+-ATPase (V-ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V-ATPase subunits was demonstrated by RT-PCR analysis of CPE cells (CPECs) isolated by fluorescence-activated cell sorting. Immunofluorescence and electron microscopy localized the V-ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na+-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7.4, the cells failed to display significant concanamycin A-sensitive pHi recovery (i.e., V-ATPase activity). The recovery rate in the absence of Na+ amounted to <10% of the pHi recovery rate observed in the presence of Na+ Recovery of pHi was faster at pHo 7.8 and was abolished at pHo 7.0. The concanamycin A-sensitive pHi recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane-permeant cAMP analog 8-CPT-cAMP did not result in trafficking of the V-ATPase. In conclusion, we find evidence for the expression of a minor fraction of V-ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking-independent manner.
Assuntos
Membrana Celular/química , Plexo Corióideo/metabolismo , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Líquido Intracelular/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/administração & dosagem , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Encéfalo/fisiologia , Membrana Celular/metabolismo , Líquido Cefalorraquidiano/química , Líquido Cefalorraquidiano/enzimologia , Líquido Cefalorraquidiano/fisiologia , Plexo Corióideo/química , Plexo Corióideo/citologia , Plexo Corióideo/ultraestrutura , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Citometria de Fluxo , Líquido Intracelular/enzimologia , Líquido Intracelular/metabolismo , Macrolídeos/administração & dosagem , Macrolídeos/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Sódio/metabolismo , Tionucleotídeos/metabolismoRESUMO
In most conditions, glucose is the best carbon source for E. coli: it provides faster growth than other sugars, and is consumed first in sugar mixtures. Here we identify conditions in which E. coli strains grow slower on glucose than on other sugars, namely when a single amino acid (arginine, glutamate, or proline) is the sole nitrogen source. In sugar mixtures with these nitrogen sources, E. coli still consumes glucose first, but grows faster rather than slower after exhausting glucose, generating a reversed diauxic shift. We trace this counterintuitive behavior to a metabolic imbalance: levels of TCA-cycle metabolites including α-ketoglutarate are high, and levels of the key regulatory molecule cAMP are low. Growth rates were increased by experimentally increasing cAMP levels, either by adding external cAMP, by genetically perturbing the cAMP circuit or by inhibition of glucose uptake. Thus, the cAMP control circuitry seems to have a 'bug' that leads to slow growth under what may be an environmentally rare condition.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Metabolismo dos Carboidratos , Carboidratos/química , Ciclo do Ácido Cítrico , Escherichia coli/metabolismo , Glucose/metabolismo , Ácidos Cetoglutáricos/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/química , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Carbono/química , AMP Cíclico/metabolismo , Metabolismo Energético , Escherichia coli/crescimento & desenvolvimento , Glucose/química , Ácidos Cetoglutáricos/químicaRESUMO
BACKGROUND: Fibrolamellar hepatocellular carcinoma (FL-HCC) affects children without underlying liver disease. A consistent mutation in FL-HCCs leads to fusion of the genes encoding a heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). We sought to characterize the resultant chimeric protein and its effects in FL-HCC. METHODS: The expression pattern and subcellular localization of protein kinase A (PKA) subunits in FL-HCCs were compared to paired normal livers by quantitative polymerase chain reaction (qPCR), immunoblotting, and immunofluorescence. PKA activity was measured by radioactive kinase assay, and we determined whether the FL-HCC mutation is present in other primary liver tumors. RESULTS: The fusion transcript and chimeric protein were detected exclusively in FL-HCCs. DNAJB1-PRKACA was expressed 10-fold higher than the wild-type PRKACA transcript, resulting in overexpression of the mutant protein in tumors. Consequently, FL-HCCs possess elevated cAMP-stimulated PKA activity compared to normal livers, despite similar Kms between the mutant and wild-type kinases. CONCLUSION: FL-HCCs in children and young adults uniquely overexpress DNAJB1-PRKACA, which results in elevated cAMP-dependent PKA activity. These data suggest that aberrant PKA signaling contributes to liver tumorigenesis.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Carcinoma Hepatocelular/enzimologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Neoplasias Hepáticas/enzimologia , Mutação , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Carcinoma Hepatocelular/genética , Domínio Catalítico , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Cirrose Hepática/complicações , Neoplasias Hepáticas/genética , Metástase Linfática , Recidiva Local de NeoplasiaRESUMO
Tumor cell adhesion to the microvessel wall is a critical step during tumor metastasis. Vascular endothelial growth factor (VEGF), a secretion of tumor cells, can increase microvessel permeability and tumor cell adhesion in the microvessel. To test the hypothesis that inhibiting permeability increase can reduce tumor cell adhesion, we used in vivo fluorescence microscopy to measure both microvessel permeability and adhesion rates of human mammary carcinoma MDA-MB-231 cells in post-capillary venules of rat mesentery under the treatment of VEGF and a cAMP analog, 8-bromo-cAMP, which can decrease microvessel permeability. By immunostaining adherens junction proteins between endothelial cells forming the microvessel wall, we further investigated the structural mechanism by which cAMP abolishes VEGF-induced increase in microvessel permeability and tumor cell adhesion. Our results demonstrate that 1) Pretreatment of microvessels with cAMP can abolish VEGF-enhanced microvessel permeability and tumor cell adhesion; 2) Tumor cells prefer to adhere to the endothelial cell junctions instead of cell bodies; 3) VEGF increases microvessel permeability and tumor cell adhesion by compromising endothelial junctions while cAMP abolishes these effects of VEGF by reinforcing the junctions. These results suggest that strengthening the microvessel wall integrity can be a potential approach to inhibiting hematogenous tumor metastasis.
Assuntos
Capilares/fisiologia , Permeabilidade Capilar/fisiologia , Adesão Celular/fisiologia , Células Endoteliais/fisiologia , Endotélio Vascular/fisiologia , Microvasos/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Capilares/metabolismo , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Feminino , Humanos , Mesentério/irrigação sanguínea , Mesentério/metabolismo , Mesentério/fisiologia , Microvasos/metabolismo , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vênulas/metabolismo , Vênulas/fisiologiaRESUMO
Diabetes mellitus causes cardiac dysfunction and heart failure that is associated with metabolic abnormalities and autonomic impairment. Autonomic control of ventricular function occurs through regulation of cAMP-dependent protein kinase (PKA). The diabetic heart has suppressed ß-adrenergic responsiveness, partly attributable to receptor changes, yet little is known about how PKA signaling is directly affected. Control and streptozotocin-induced diabetic mice were therefore administered 8-bromo-cAMP (8Br-cAMP) acutely to activate PKA in a receptor-independent manner, and cardiac hemodynamic function and PKA signaling were evaluated. In response to 8Br-cAMP treatment, diabetic mice had impaired inotropic and lusitropic responses, thus demonstrating postreceptor defects. This impaired signaling was mediated by reduced PKA activity and PKA catalytic subunit content in the cytoplasm and myofilaments. Compartment-specific loss of PKA was reflected by reduced phosphorylation of discrete substrates. In response to 8Br-cAMP treatment, the glycolytic activator PFK-2 was robustly phosphorylated in control animals but not diabetics. Control adult cardiomyocytes cultured in lipid-supplemented media developed similar changes in PKA signaling, suggesting that lipotoxicity is a contributor to diabetes-induced ß-adrenergic signaling dysfunction. This work demonstrates that PKA signaling is impaired in diabetes and suggests that treating hyperlipidemia is vital for proper cardiac signaling and function.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Diabetes Mellitus Experimental/metabolismo , Miocárdio/enzimologia , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Domínio Catalítico , AMP Cíclico/metabolismo , Proteína Quinase Tipo II Dependente de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Modelos Animais de Doenças , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/patologia , Hemodinâmica , Lactatos/metabolismo , Lipídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Fosfofrutoquinase-2/metabolismo , Fosforilação , Transdução de SinaisRESUMO
The motility of cilia and flagella of eukaryotic cells is controlled by second messengers such as Ca(2+), cAMP, and cGMP. In this study, the cAMP-dependent control of flagellar bending of Chlamydomonas is investigated by applying cAMP through photolysis of 4,5-dimethoxy-2-nitrobenzyl adenosine 3',5'-cyclicmonophosphate (caged cAMP). When cAMP is applied to demembranated and reactivated cells, cells begin to swim with a larger helical path. This change is due to a larger turn about the axis normal to the anterior-posterior axis, indicating an increased imbalance in the propulsive forces generated by the cis-flagellum (flagellum nearer to the eyespot) and trans-flagellum (flagellum farther from the eyespot). Consistently, when cAMP is applied to isolated axonemes, some axonemes show attenuated motility whereas others do not. Axonemes from uni1 mutants, which have only trans-flagella, do not respond to cAMP. These observations indicate that cAMP controls the balance of the forces generated by cis- and trans-flagella in Chlamydomonas.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Axonema/fisiologia , Chlamydomonas/metabolismo , Cílios/fisiologia , Flagelos/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Chlamydomonas/ultraestrutura , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Sistemas do Segundo MensageiroRESUMO
8-Chloro-cyclic AMP (8-Cl-cAMP) is a cyclic AMP analog that induces growth inhibition and apoptosis in a broad spectrum of cancer cells. Previously, we found that 8-Cl-cAMP-induced growth inhibition is mediated by AMP-activated protein kinase (AMPK) as well as p38 mitogen-activated protein kinase (p38 MAPK). To identify downstream mediators of the 8-Cl-cAMP signaling, we performed co-immunoprecipitation combined with mass spectrometry using the anti-AMPK or p38 MAPK antibodies. Through this approach, SHC1 was identified as one of the binding partners of p38 MAPK. SHC1 phosphorylation was suppressed by 8-Cl-cAMP in HeLa and MCF7 cancer cells, which was mediated by its metabolites, 8-Cl-adenosine and 8-Cl-ATP; however, 8-Cl-cAMP showed no effect on SHC1 phosphorylation in normal human fibroblasts. SHC1 siRNA induced AMPK and p38 MAPK phosphorylation and growth inhibition in cancer cells, and SHC1 overexpression re-sensitized human foreskin fibroblasts to the 8-Cl-cAMP treatment. SHC1 phosphorylation was unaffected by Compound C (an AMPK inhibitor) and SB203580 (a p38 MAPK inhibitor), which suggests that SHC1 is upstream of AMPK and p38 MAPK in the 8-Cl-cAMP-stimulated signaling cascade. On the basis of these findings, we conclude that SHC1 functions as a sensor during the 8-Cl-cAMP-induced growth inhibition in SHC1-overexpressing cancer cells.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Proteínas Adaptadoras da Sinalização Shc/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Adenilato Quinase/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Espectrometria de Massas , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias/fisiopatologia , Fosforilação , RNA Interferente Pequeno/genética , Proteínas Adaptadoras da Sinalização Shc/genética , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: The neurobiological changes underlying depression resistant to treatments remain poorly understood, and failure to respond to selective serotonin reuptake inhibitors may result from abnormalities of neurotransmitter systems that excite serotonergic neurons, such as histamine. METHODS: Using behavioral (tail suspension test) and neurochemical (in vivo microdialysis, Western-blot analysis) approaches, here we report that antidepressant responses to selective serotonin reuptake inhibitors (citalopram or paroxetine) are abolished in mice unable to synthesize histamine due to either targeted disruption of histidine decarboxylase gene (HDC(-/-)) or injection of alpha-fluoromethylhistidine, a suicide inhibitor of this enzyme. RESULTS: In the tail suspension test, all classes of antidepressants tested reduced the immobility time of controls. Systemic reboxetine or imipramine reduced the immobility time of histamine-deprived mice as well, whereas selective serotonin reuptake inhibitors did not even though their serotonergic system is functional. In in vivo microdialysis experiments, citalopram significantly increased histamine extraneuronal levels in the cortex of freely moving mice, and methysergide, a serotonin 5-HT1/5-HT2 receptor antagonist, abolished this effect, thus suggesting the involvement of endogenous serotonin. CREB phosphorylation, which is implicated in the molecular mechanisms of antidepressant treatment, was abolished in histamine-deficient mice treated with citalopram. The CREB pathway is not impaired in HDC(-/-) mice, as administration of 8-bromoadenosine 3', 5'-cyclic monophosphate increased CREB phosphorylation, and in the tail suspension test it significantly reduced the time spent immobile by mice of both genotypes. CONCLUSIONS: Our results demonstrate that selective serotonin reuptake inhibitors selectively require the integrity of the brain histamine system to exert their preclinical responses.
Assuntos
Encéfalo/efeitos dos fármacos , Citalopram/farmacologia , Transtorno Depressivo/tratamento farmacológico , Histamina/metabolismo , Paroxetina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Antidepressivos/farmacologia , Encéfalo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Transtorno Depressivo/metabolismo , Transtorno Depressivo Resistente a Tratamento/metabolismo , Modelos Animais de Doenças , Feminino , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Masculino , Metilistidinas/metabolismo , Metisergida/farmacologia , Camundongos Knockout , Antagonistas da Serotonina/farmacologiaRESUMO
The effects of the Pseudomonas aeruginosa virulence factor pyocyanin (PCN) on the contractile function of porcine coronary arteries was investigated in vitro. Artery rings (5 mm) were suspended in organ baths containing Krebs' solution for the measurement of isometric tension. The effect of PCN on resting and precontracted coronary arteries was initially investigated with various agents. Arteries were precontracted with prostaglandin (PG) F2α or potassium chloride and endothelium-dependent relaxations were induced by various agents in the presence of PCN. Pyocyanin (0.1-10 µmol/L) evoked small-amplitude, dose-dependent contractions in resting porcine coronary arteries. In addition, PCN amplified the contractile response to PGF2α , but did not alter responses to carbachol. Pyocyanin (0.1-10 µmol/L) significantly inhibited endothelium-dependent relaxations evoked by neurokinin A. Pyocyanin also inhibited relaxations evoked by diethylamine nitric oxide (a nitric oxide donor), forskolin (an adenylate cyclase activator), dibuytyryl-cAMP (a cAMP analogue), 8-bromo-cGMP (a cGMP analogue) and P1075 (a KATP channel activator), but not isoprenaline (ß-adrenoceceptor agonist). These results indicate that physiological concentrations of PCN interfere with multiple intracellular processes involved in vascular smooth muscle relaxation, in particular pathways downstream of nitric oxide release. Thus, PCN may alter normal vascular function in patients infected with P. aeruginosa.
Assuntos
Vasos Coronários/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Óxido Nítrico/metabolismo , Piocianina/farmacologia , Vasodilatação/efeitos dos fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Colforsina/metabolismo , Vasos Coronários/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Dietilaminas/farmacologia , Dinoprosta/metabolismo , Feminino , Isoproterenol/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , SuínosRESUMO
Several studies suggest that acetyl-l-carnitine (ALC) might influence learning processes. Along this line of investigation, we have previously shown that ALC impaired sensitization and dishabituation induced by nociceptive stimulation of the dorsal skin of the leech Hirudo medicinalis, in the behavioural paradigm of the swim induction (SI). In previous works we showed that 5HT was involved in both sensitization and dishabituation of SI acting through the second messenger cAMP. In this work, we have reported that for given doses and temporal ranges ALC was able to block sensitization and to impair dishabituation mimicked by the injection of 5-HT or 8Br-cAMP, a membrane permeable analogue of cAMP. Our results show that a single treatment with 2mM ALC was the most effective concentration to block the onset of sensitization induced by 5-HT injection and its major effects occurred 11 days after ALC treatment. 2mM ALC also blocked sensitization induced by 8Br-cAMP injection, whereas, ALC did not completely abolish dishabituation induced by 5-HT or 8Br-cAMP injection at the tested concentrations and at every time point.