RESUMO
Type 1 diabetes (T1D) affects gastrointestinal (GI) motility, favoring gastroparesis, constipation, and fecal incontinence, which are more prevalent in women. The mechanisms are unknown. Given the G-protein-coupled estrogen receptor's (GPER) role in GI motility, we investigated sex-related diabetes-induced epigenetic changes in GPER. We assessed GPER mRNA and protein expression levels using qPCR and Western blot analyses, and quantified the changes in nuclear DNA methyltransferases and histone modifications (H3K4me3, H3Ac, and H3K27Ac) by ELISA kits. Targeted bisulfite and chromatin immunoprecipitation assays were used to evaluate DNA methylation and histone modifications around the GPER promoter by chromatin immunoprecipitation assays in gastric and colonic smooth muscle tissues of male and female control (CTR) and non-obese diabetic (NOD) mice. GPER expression was downregulated in NOD, with sex-dependent variations. In the gastric smooth muscle, not in colonic smooth muscle, downregulation coincided with differences in methylation ratios between regions 1 and 2 of the GPER promoter of NOD. DNA methylation was higher in NOD male colonic smooth muscle than in NOD females. H3K4me3 and H3ac enrichment decreased in NOD gastric smooth muscle. H3K4me3 levels diminished in the colonic smooth muscle of NOD. H3K27ac levels were unaffected, but enrichment decreased in NOD male gastric smooth muscle; however, it increased in the NOD male colonic smooth muscle and decreased in the female NOD colonic smooth muscle. Male NOD colonic smooth muscle exhibited decreased H3K27ac levels, not female, whereas female NOD colonic smooth muscle demonstrated diminished enrichment of H3ac at the GPER promoter, contrary to male NOD. Sex-specific epigenetic mechanisms contribute to T1D-mediated suppression of GPER expression in the GI tract. These insights advance our understanding of T1D complications and suggest promising avenues for targeted therapeutic interventions.
Assuntos
Colo , Metilação de DNA , Epigênese Genética , Histonas , Músculo Liso , Regiões Promotoras Genéticas , Receptores Acoplados a Proteínas G , Animais , Feminino , Masculino , Camundongos , Colo/metabolismo , Colo/patologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/genética , Histonas/metabolismo , Camundongos Endogâmicos NOD , Músculo Liso/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Estômago/patologiaRESUMO
Type 2 diabetes mellitus (T2D) causes gastroparesis, delayed intestinal transit, and constipation, for unknown reasons. Complications are predominant in women than men (particularly pregnant and postmenopausal women), suggesting a female hormone-mediated mechanism. Low G-protein coupled estrogen receptor (GPER) expression from epigenetic modifications may explain it. We explored sexually differentiated GPER expression and gastrointestinal symptoms related to GPER alterations in wild-type (WT) and T2D mice (db/db). We also created smooth muscle-specific GPER knockout (GPER KO) mice to phenotypically explore the effect of GPER deficiency on gastrointestinal motility. GPER mRNA and protein expression, DNA methylation and histone modifications were measured from stomach and colon samples of db/db and WT mice. Changes in gut motility were also evaluated as daily fecal pellet production patterns. We found that WT female tissues have the highest GPER mRNA and protein expressions. The expression is lowest in all db/db. GPER downregulation is associated with promoter hypermethylation and reduced enrichment of H3K4me3 and H3K27ac marks around the GPER promoter. We also observed sex-specific disparities in fecal pellet production patterns of the GPER KO mice compared to WT. We thus, conclude that T2D impairs gut GPER expression, and epigenetic sex-specific mechanisms matter in the downregulation.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Masculino , Camundongos , Feminino , Humanos , Animais , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Experimental/genética , Estrogênios , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Músculo Liso/metabolismo , Epigênese Genética , RNA MensageiroRESUMO
For many patients, diabetes Mellitus and Malignancy are frequently encountered comorbidities. Diabetes affects approximately 10.5% of the global population, while malignancy accounts for 29.4 million cases each year. These troubling statistics indicate that current treatment approaches for these diseases are insufficient. Alternative therapeutic strategies that consider unique signaling pathways in diabetic and malignancy patients could provide improved therapeutic outcomes. The G-protein-coupled estrogen receptor (GPER) is receiving attention for its role in disease pathogenesis and treatment outcomes. This review aims to critically examine GPER' s comparative role in diabetes mellitus and malignancy, identify research gaps that need to be filled, and highlight GPER's potential as a therapeutic target for diabetes and malignancy management. There is a scarcity of data on GPER expression patterns in diabetic models; however, for diabetes mellitus, altered expression of transport and signaling proteins has been linked to GPER signaling. In contrast, GPER expression in various malignancy types appears to be complex and debatable at the moment. Current data show inconclusive patterns of GPER expression in various malignancies, with some indicating upregulation and others demonstrating downregulation. Further research should be conducted to investigate GPER expression patterns and their relationship with signaling pathways in diabetes mellitus and various malignancies. We conclude that GPER has therapeutic potential for chronic diseases such as diabetes mellitus and malignancy.
Assuntos
Diabetes Mellitus , Neoplasias , Humanos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Estrogênios , Diabetes Mellitus/metabolismo , Proteínas de Ligação ao GTP/metabolismoRESUMO
In addition to the α, ß, and γ subunits of ENaC, human salt-sensing taste receptor cells (TRCs) also express the δ-subunit. At present, it is not clear if the expression and function of the ENaC δ-subunit in human salt-sensing TRCs is also modulated by the ENaC regulatory hormones and intracellular signaling effectors known to modulate salt responses in rodent TRCs. Here, we used molecular techniques to demonstrate that the G-protein-coupled estrogen receptor (GPER1), the transient receptor potential cation channel subfamily V member 1 (TRPV1), and components of the renin-angiotensin-aldosterone system (RAAS) are expressed in δ-ENaC-positive cultured adult human fungiform (HBO) taste cells. Our results suggest that RAAS components function in a complex with ENaC and TRPV1 to modulate salt sensing and thus salt intake in humans. Early, but often prolonged, symptoms of COVID-19 infection are the loss of taste, smell, and chemesthesis. The SARS-CoV-2 spike protein contains two subunits, S1 and S2. S1 contains a receptor-binding domain, which is responsible for recognizing and binding to the ACE2 receptor, a component of RAAS. Our results show that the binding of a mutated S1 protein to ACE2 decreases ACE2 expression in HBO cells. We hypothesize that changes in ACE2 receptor expression can alter the balance between the two major RAAS pathways, ACE1/Ang II/AT1R and ACE2/Ang-(1-7)/MASR1, leading to changes in ENaC expression and responses to NaCl in salt-sensing human fungiform taste cells.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Canais Epiteliais de Sódio/metabolismo , Adulto , Animais , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/genética , Sistema Renina-Angiotensina , Cloreto de Sódio/farmacologia , Canais de Cátion TRPV/genética , Papilas Gustativas/metabolismoRESUMO
BACKGROUND: l-amino acids, such as monosodium glutamate (MSG), activate the umami receptor T1R1/T1R3. We previously showed increased peristalsis in response to activation of T1R1/T1R3 by MSG in mouse colon. However, the expression and function of these receptors in the different regions of the stomach are not clear. METHODS: Mouse gastric smooth muscle cells (SMCs) were isolated and cultured in Dulbecco's Modified Eagle Medium. Expression of T1R1 and T1R3 was measured by RT-PCR and Western blot. The effect of MSG with and without inosine monophosphate (IMP, an allosteric activator of T1R1/T1R3) on acetylcholine (ACh)-induced contraction was measured in muscle strips and isolated SMCs by scanning micrometry. The effect of MSG with or without IMP on activation of G proteins and ACh-induced Ca2+ release was measured in SMCs. KEY RESULTS: Monosodium glutamate inhibited ACh-induced contractions in muscle strips from both antrum and fundus and the effect of MSG was augmented by IMP; the effects were concentration-dependent and not affected by the nitric oxide synthase inhibitor, L-NNA, or tetrodotoxin suggesting a direct effect on SMCs. In isolated gastric SMCs, T1R1 and T1R3 transcripts and protein were identified. Addition of MSG with or without IMP inhibited ACh-induced Ca2+ release and muscle contraction; the effect on contraction was blocked by pertussis toxin suggesting activation of Gαi proteins. MSG in the presence of IMP selectively activated Gαi2 . CONCLUSIONS AND INFERENCES: Umami receptors (T1R1/T1R3) are present on SMCs of the stomach, and activation of these receptors induces muscle relaxation by decreasing [Ca2+ ]i via Gαi2 .
Assuntos
Músculo Liso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Estômago , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Actin polymerization plays an important role in smooth muscle contraction. Integrin-linked kinase (ILK) was shown to mediate actin polymerization in airway smooth muscle. The role of ILK in actin polymerization in response to m2 receptor activation was not in gastric smooth muscle. METHODS: Phosphorylation of paxillin, neuronal Wiskott-Aldrich syndrome protein (N-WASp), and association of paxillin with GEF proteins (Cool2/αPix [Cool2/PAK-interacting exchange factor alpha], Cool1/ßPix [Cool1/PAK-interacting exchange factor beta], and DOCK 180 [Dedicator of cytokinesis]) and N-WASp with Arp2/3 complex were measured by western blot. Activation of Cdc42 was determined using an antibody for activated Cdc42. Actin polymerization was measured as an increase in F-actin/G-actin ratio. RESULTS: Phosphorylation of paxillin, an association of paxillin with GEF proteins, Cdc42 activity, and actin polymerization were increased in response to m2 receptor activation in gastric smooth muscle cells. The increases in paxillin phosphorylation, Cdc42 activity, and actin polymerization were inhibited by a PI3Kγ inhibitor (AS-605240), ILK siRNA, and ILK dominant negative mutant (ILK [R211]). Increase in actin polymerization was also inhibited by Cdc42 dominant negative mutant (Cdc42 [T17N]). Increases in the association of paxillin with GEF proteins, phosphorylation of N-WASp and its association with Arp2/3 complex were inhibited by ILK (R211). CONCLUSION: In gastric smooth muscle cells, activation of PI3Kγ by muscarinic m2 receptors causes ILK-dependent phosphorylation of paxillin, an association of paxillin with Cdc42 GEF proteins and activation of Cdc42, which, in turn, causes phosphorylation of N-WASp and its association with Arp2/3 complex leading to actin polymerization.
Assuntos
Actinas/metabolismo , Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor Muscarínico M2/metabolismo , Estômago/fisiologia , Animais , Contração Muscular/fisiologia , Polimerização , CoelhosRESUMO
BACKGROUND: Isovaleric acid (IVA) is a 5-carbon branched-chain fatty acid present in fermented foods and produced in the colon by bacterial fermentation of leucine. We previously reported that the shorter, straight-chain fatty acids acetate, propionate and butyrate differentially affect colonic motility; however, the effect of branched-chain fatty acids on gut smooth muscle and motility is unknown. AIMS: To determine the effect of IVA on contractility of colonic smooth muscle. METHODS: Murine colonic segments were placed in a longitudinal orientation in organ baths in Krebs buffer and fastened to force transducers. Segments were contracted with acetylcholine (ACh), and the effects of IVA on ACh-induced contraction were measured in the absence and presence of tetrodotoxin (TTx) or inhibitors of nitric oxide synthase [L-N-nitroarginine (L-NNA)] or adenylate cyclase (SQ22536). The effect of IVA on ACh-induced contraction was also measured in isolated muscle cells in the presence or absence of SQ22536 or protein kinase A (PKA) inhibitor (H-89). Direct activation of PKA was measured in isolated muscle cells. RESULTS: In colonic segments, ACh-induced contraction was inhibited by IVA in a concentration-dependent fashion; the IVA response was not affected by TTx or L-NNA but inhibited by SQ22536. Similarly, in isolated colonic muscle cells, ACh-induced contraction was inhibited by IVA in a concentration-dependent fashion and the effect blocked by SQ22536 and H-89. IVA also increased PKA activity in isolated smooth muscle cells. CONCLUSIONS: The branched-chain fatty acid IVA acts directly on colonic smooth muscle and causes muscle relaxation via the PKA pathway.
Assuntos
Colo/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Ácidos Graxos Voláteis/farmacologia , Relaxamento Muscular/fisiologia , Músculo Liso/metabolismo , Ácidos Pentanoicos/farmacologia , Animais , Colo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Hemiterpenos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The insulinotropic effects of the incretin hormone, glucagon-like peptide-1 (GLP-1) are mediated via GLP-1 receptors (GLP-1R) present on pancreatic ß cells. GLP-1 causes a decrease in the motility of stomach and intestine which involves both central and peripheral nervous systems. The expression and function of GLP-1R in gastrointestinal smooth muscle, however, are not clear. Muscle strips and isolated muscle cells were prepared from mouse colon and the effect of GLP-1(7-36) amide on acetylcholine (ACh)-induced contraction was measured. Muscle cells in culture were used to identify the expression of GLP-1R and the signaling pathways activated by GLP-1(7-36) amide. GLP-1R was expressed in the mucosal and non-mucosal tissue preparations derived from colon, and in smooth muscle cell cultures devoid of other cells such as enteric neurons. In colonic muscle strips, the addition of GLP-1(7-36) amide caused dose-dependent inhibition of acetylcholine-induced contractions. The effect of GLP-1(7-36) amide was partly inhibited by the neuronal blocker tetrodotoxin and nitric oxide (NO) synthase inhibitor l-NNA suggesting both NO-dependent neural and NO-independent direct effects on smooth muscle. In isolated colonic smooth muscle cells, GLP-1(7-36) amide caused an increase in Gαs activity, cAMP levels, and PKA activity, and inhibited ACh-induced contraction. The effect of GLP-1(7-36) amide on Gαs activity and cAMP levels was blocked by NF449, an inhibitor of Gαs, and the effect of GLP-1(7-36) amide on contraction was blocked by NF449 and myristoylated PKI, an inhibitor of PKA. We conclude that colonic smooth muscle cells express GLP-1R, and GLP-1(7-36) amide inhibits acetylcholine-induced contraction via GLP-1R coupled to the Gαs/cAMP/PKA pathway.
Assuntos
Acetilcolina/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Contração Muscular , Músculo Liso/metabolismo , Transdução de Sinais , Animais , Colo/metabolismo , Colo/fisiologia , AMP Cíclico/metabolismo , Expressão Gênica , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/fisiologia , Camundongos , Músculo Liso/fisiologiaRESUMO
In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-ß/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-ß1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, ß (α and ß PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation.
Assuntos
Actinas/metabolismo , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Multimerização Proteica/fisiologia , Estômago/fisiologia , Animais , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Cadeias Leves de Miosina/metabolismo , Fosforilação/fisiologia , Coelhos , Receptor Muscarínico M3 , Transdução de Sinais/fisiologiaRESUMO
The pathogenesis of diabetes-associated motility disorders are multifactorial and attributed to abnormalities in extrinsic and intrinsic innervation, and a decrease in the number of interstitial cells of Cajal, and nNOS expression and activity. Here we studied the effect of hyperglycemia on smooth muscle function. Using smooth muscles from the fundus of ob/ob mice and of wild type (WT) mice treated with 30 mM glucose (HG), we identified the molecular mechanism by which hyperglycemia upregulates RhoA/Rho kinase pathway and muscle contraction. RhoA expression, Rho kinase activity and muscle contraction were increased, while miR-133a expression was decreased in smooth muscle of ob/ob mice and in smooth muscle treated with HG. Intraperitoneal injections of pre-miR-133a decreased RhoA expression in WT mice and reversed the increase in RhoA expression in ob/ob mice. Intraperitoneal injections of antagomiR-133a increased RhoA expression in WT mice and augmented the increase in RhoA expression in ob/ob mice. The effect of pre-miR-133a or antagomiR-133a in vitro in smooth muscle treated with HG was similar to that obtained in vivo, suggesting that the expression of RhoA is negatively regulated by miR-133a and a decrease in miR-133a expression in diabetes causes an increase in RhoA expression. Oxidative stress (levels of reactive oxygen species and hydrogen peroxide, and expression of superoxide dismutase 1 and NADPH oxidase 4) was increased in smooth muscle of ob/ob mice and in HG-treated smooth muscle. Treatment of ob/ob mice with N-acetylcysteine (NAC) in vivo or addition of NAC in vitro to HG-treated smooth muscle reversed the effect of glucose on the expression of miR-133a and RhoA, Rho kinase activity and muscle contraction. NAC treatment also reversed the decrease in gastric emptying in ob/ob mice. We conclude that oxidative stress in diabetes causes a decrease in miR-133a expression leading to an increase in RhoA/Rho kinase pathway and muscle contraction.
Assuntos
Contração Muscular/fisiologia , Músculo Liso/fisiologia , Estresse Oxidativo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Acetilcisteína/farmacologia , Animais , Western Blotting , Células Cultivadas , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatologia , Sequestradores de Radicais Livres/farmacologia , Mucosa Gástrica/metabolismo , Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Hiperglicemia/genética , Hiperglicemia/metabolismo , Hiperglicemia/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Obesos , MicroRNAs/genética , Músculo Liso/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Estômago/fisiologia , Regulação para Cima , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
During postnatal development rats demonstrate an age-dependent increase in NaCl chorda tympani (CT) responses and the number of functional apical amiloride-sensitive epithelial Na+ channels (ENaCs) in salt sensing fungiform (FF) taste receptor cells (TRCs). Currently, the intracellular signals that regulate the postnatal development of salt taste have not been identified. We investigated the effect of cAMP, a downstream signal for arginine vasopressin (AVP) action, on the postnatal development of NaCl responses in 19-23 day old rats. ENaC-dependent NaCl CT responses were monitored after lingual application of 8-chlorophenylthio-cAMP (8-CPT-cAMP) under open-circuit conditions and under ±60 mV lingual voltage clamp. Behavioral responses were tested using 2 bottle/24h NaCl preference tests. The effect of [deamino-Cys1, D-Arg8]-vasopressin (dDAVP, a specific V2R agonist) was investigated on ENaC subunit trafficking in rat FF TRCs and on cAMP generation in cultured adult human FF taste cells (HBO cells). Our results show that in 19-23 day old rats, the ENaC-dependent maximum NaCl CT response was a saturating sigmoidal function of 8-CPT-cAMP concentration. 8-CPT-cAMP increased the voltage-sensitivity of the NaCl CT response and the apical Na+ response conductance. Intravenous injections of dDAVP increased ENaC expression and γ-ENaC trafficking from cytosolic compartment to the apical compartment in rat FF TRCs. In HBO cells dDAVP increased intracellular cAMP and cAMP increased trafficking of γ- and δ-ENaC from cytosolic compartment to the apical compartment 10 min post-cAMP treatment. Control 19-23 day old rats were indifferent to NaCl, but showed clear preference for appetitive NaCl concentrations after 8-CPT-cAMP treatment. Relative to adult rats, 14 day old rats demonstrated significantly less V2R antibody binding in circumvallate TRCs. We conclude that an age-dependent increase in V2R expression produces an AVP-induced incremental increase in cAMP that modulates the postnatal increase in TRC ENaC and the neural and behavioral responses to NaCl.
Assuntos
Nervo da Corda do Tímpano/efeitos dos fármacos , AMP Cíclico/farmacologia , Cloreto de Sódio/farmacologia , Paladar/efeitos dos fármacos , Adulto , Fatores Etários , Animais , Western Blotting , Células Cultivadas , Nervo da Corda do Tímpano/fisiologia , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Desamino Arginina Vasopressina/farmacologia , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Preferências Alimentares/efeitos dos fármacos , Preferências Alimentares/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Microscopia Confocal , Ratos Sprague-Dawley , Receptores de Vasopressinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Paladar/fisiologia , Papilas Gustativas/efeitos dos fármacos , Papilas Gustativas/metabolismo , Papilas Gustativas/fisiologia , Tionucleotídeos/metabolismo , Tionucleotídeos/farmacologiaRESUMO
In addition to the T2R bitter taste receptors, neuronal nicotinic acetylcholine receptors (nAChRs) have recently been shown to be involved in the bitter taste transduction of nicotine, acetylcholine and ethanol. However, at present it is not clear if nAChRs are expressed in enteroendocrine cells other than beta cells of the pancreas and enterochromaffin cells, and if they play a role in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of nAChRs in enteroendocrine STC-1 cells. Our studies using RT-PCR, qRT-PCR, immunohistochemical and Western blotting techniques demonstrate that STC-1 cells express several α and ß nAChR subunits. Exposing STC-1 cells to nicotine acutely (24h) or chronically (4 days) induced a differential increase in the expression of nAChR subunit mRNA and protein in a dose- and time-dependent fashion. Mecamylamine, a non-selective antagonist of nAChRs, inhibited the nicotine-induced increase in mRNA expression of nAChRs. Exposing STC-1 cells to nicotine increased intracellular Ca2+ in a dose-dependent manner that was inhibited in the presence of mecamylamine or dihydro-ß-erythroidine, a α4ß2 nAChR antagonist. Brain-derived neurotrophic factor (BDNF) mRNA and protein were detected in STC-1 cells using RT-PCR, specific BDNF antibody, and enzyme-linked immunosorbent assay. Acute nicotine exposure (30 min) decreased the cellular content of BDNF in STC-1 cells. The nicotine-induced decrease in BDNF was inhibited in the presence of mecamylamine. We also detected α3 and ß4 mRNA in intestinal mucosal cells and α3 protein expression in intestinal enteroendocrine cells. We conclude that STC-1 cells and intestinal enteroendocrine cells express nAChRs. In STC-1 cells nAChR expression is modulated by exposure to nicotine in a dose- and time-dependent manner. Nicotine interacts with nAChRs and inhibits BDNF expression in STC-1 cells.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Nicotina/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Receptores Nicotínicos/biossíntese , Animais , Fator Neurotrófico Derivado do Encéfalo/antagonistas & inibidores , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Cálcio/metabolismo , Linhagem Celular , Di-Hidro-beta-Eritroidina/administração & dosagem , Células Enterocromafins/metabolismo , Células Enteroendócrinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mecamilamina/administração & dosagem , Mecamilamina/metabolismo , Camundongos , Nicotina/administração & dosagem , Nicotina/antagonistas & inibidores , RNA Mensageiro/biossíntese , Receptores Acoplados a Proteínas G/genética , Receptores Nicotínicos/genéticaRESUMO
BACKGROUND: The contractility of colonic smooth muscle is dysregulated due to immune/inflammatory responses in inflammatory bowel diseases. Inflammation in vitro induces up-regulation of regulator of G-protein signaling 4 (RGS4) expression in colonic smooth muscle cells. AIMS: To characterize the immune/inflammatory responses and RGS4 expression pattern in colonic smooth muscle after induction of colitis. METHODS: Colitis was induced in rabbits by intrarectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Innate/adaptive immune response RT-qPCR array was performed using colonic circular muscle strips. At 1-9 weeks after colonic intramuscular microinjection of lentivirus, the distal and proximal colons were collected, and muscle strips and dispersed muscle cells were prepared from circular muscle layer. Expression levels of RGS4 and NFκB signaling components were determined by Western blot analysis. The biological consequences of RGS4 knockdown were assessed by measurement of muscle contraction and phospholipase C (PLC)-ß activity in response to acetylcholine (ACh). RESULTS: Contraction in response to ACh was significantly inhibited in the inflamed colonic circular smooth muscle cells. RGS4, IL-1, IL-6, IL-8, CCL3, CD1D, and ITGB2 were significantly up-regulated, while IL-18, CXCR4, CD86, and C3 were significantly down-regulated in the inflamed muscle strips. RGS4 protein expression in the inflamed smooth muscles was dramatically increased. RGS4 stable knockdown in vivo augmented ACh-stimulated PLC-ß activity and contraction in colonic smooth muscle cells. CONCLUSION: Inflamed smooth muscle exhibits up-regulation of IL-1-related signaling components, Th1 cytokines and RGS4, and inhibition of contraction. Stable knockdown of endogenous RGS4 in colonic smooth muscle increases PLC-ß activity and contractile responses.
Assuntos
Colo/efeitos dos fármacos , Inflamação/induzido quimicamente , Contração Muscular/imunologia , Músculo Liso/patologia , Ácido Trinitrobenzenossulfônico/toxicidade , Animais , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/imunologia , Inflamação/patologia , Coelhos , Regulação para CimaRESUMO
Inhibitory neurotransmitters, chiefly nitric oxide and vasoactive intestinal peptide, increase cyclic nucleotide levels and inhibit muscle contraction via inhibition of myosin light chain (MLC) kinase and activation of MLC phosphatase (MLCP). H2S produced as an endogenous signaling molecule synthesized mainly from l-cysteine via cystathionine-γ-lyase (CSE) and cystathionine-ß-synthase (CBS) regulates muscle contraction. The aim of this study was to analyze the expression of CSE and H2S function in the regulation of MLCP activity, 20-kDa regulatory light chain of myosin II (MLC20) phosphorylation, and contraction in isolated gastric smooth muscle cells. Both mRNA expression and protein expression of CSE, but not CBS, were detected in smooth muscle cells of rabbit, human, and mouse stomach. l-cysteine, an activator of CSE, and NaHS, a donor of H2S, inhibited carbachol-induced Rho kinase and PKC activity, Rho kinase-sensitive phosphorylation of MYPT1, PKC-sensitive phosphorylation of CPI-17, and MLC20 phosphorylation and sustained muscle contraction. The inhibitory effects of l-cysteine, but not NaHS, were blocked upon suppression of CSE expression by siRNA or inhibition of its activity by dl-propargylglycine (PPG) suggesting that the effect of l-cysteine is mediated via activation of CSE. Glibenclamide, an inhibitor of KATP channels, had no effect on the inhibition of contraction by H2S. Both l-cysteine and NaHS had no effect on basal cAMP and cGMP levels but augmented forskolin-induced cAMP and SNP-induced cGMP formation. We conclude that both endogenous and exogenous H2S inhibit muscle contraction, and the mechanism involves inhibition of Rho kinase and PKC activities and stimulation of MLCP activity leading to MLC20 dephosphorylation and inhibition of muscle contraction.
Assuntos
Sulfeto de Hidrogênio/metabolismo , Contração Muscular , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais , Estômago/enzimologia , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Relação Dose-Resposta a Droga , Humanos , Sulfeto de Hidrogênio/farmacologia , Camundongos Endogâmicos C57BL , Contração Muscular/efeitos dos fármacos , Proteínas Musculares/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Doadores de Óxido Nítrico/farmacologia , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Coelhos , Transdução de Sinais/efeitos dos fármacos , Estômago/citologia , Estômago/efeitos dos fármacos , Transfecção , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
The effect of proinflammatory cytokines on the expression and activity of soluble guanylyl cyclase (sGC) and cGMP-phosphodiesterases (PDEs) was determined in intestinal longitudinal smooth muscle. In control muscle cells, cGMP levels are regulated via activation of sGC and PDE5; the activity of the latter is regulated via feedback phosphorylation by cGMP-dependent protein kinase. In muscle cells isolated from muscle strips cultured with interleukin-1ß (IL-1ß) or tumor necrosis factor α (TNF-α) or obtained from the colon of TNBS (2,4,6-trinitrobenzene sulfonic acid)-treated mice, expression of inducible nitric oxide synthase (iNOS) was induced and sGC was S-nitrosylated, resulting in attenuation of nitric oxide (NO)-induced sGC activity and cGMP formation. The effect of cytokines on sGC S-nitrosylation and activity was blocked by the iNOS inhibitor 1400W [N-([3-(aminomethyl)phenyl]methyl)ethanimidamide dihydrochloride]. The effect of cytokines on cGMP levels measured in the absence of IBMX (3-isobutyl-1-methylxanthine), however, was partly reversed by 1400W or PDE1 inhibitor vinpocetine and completely reversed by a combination of 1400W and vinpocetine. Expression of PDE1A was induced and was accompanied by an increase in PDE1A activity in muscle cells isolated from muscle strips cultured with IL-1ß or TNF-α or obtained from the colon of TNBS-treated mice; the effect of cytokines on PDE1 expression and activity was blocked by MG132 (benzyl N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate), an inhibitor of nuclear factor κB activity. NO-induced muscle relaxation was inhibited in longitudinal muscle cells isolated from muscle strips cultured with IL-1ß or TNF-α or obtained from the colon of TNBS-treated mice, and this inhibition was completely reversed by the combination of both 1400W and vinpocetine. Inhibition of smooth muscle relaxation during inflammation reflects the combined effects of decreased sGC activity via S-nitrosylation and increased cGMP hydrolysis via PDE1 expression.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/biossíntese , Regulação Enzimológica da Expressão Gênica , Guanilato Ciclase/biossíntese , Relaxamento Muscular/fisiologia , Músculo Liso/enzimologia , Óxido Nítrico Sintase Tipo II/biossíntese , Receptores Citoplasmáticos e Nucleares/biossíntese , Animais , Citocinas/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Guanilil Ciclase SolúvelRESUMO
PURPOSE: We examined the role of NMDAR in the regulation of bladder hypertrophy and function in a rat model of cyclophosphamide induced cystitis. MATERIALS AND METHODS: Cystitis was induced by intraperitoneal injection of cyclophosphamide (150 mg/kg body weight). NMDAR phosphorylation (activity) and signal transduction pathways were examined by direct measurement and by specific inhibitors in vivo. Bladder hypertrophy was measured by bladder weight/body weight and type I collagen expression. Bladder function was examined by metabolic recording, conscious cystometry and detrusor muscle strip contractility in response to carbachol. RESULTS: NMDAR activity measured by the phosphorylation level of the NMDAR1 (NR1) subunit was expressed in the spinal cord but not in the bladder at 48 hours of cystitis. NMDAR inhibition with dizocilpine (MK-801) reduced the cystitis induced increment of bladder weight and type I collagen up-regulation in the bladder. NMDAR regulated type I collagen up-regulation was mediated by the PI3K/Akt pathway. NMDAR inhibition also attenuated cystitis induced urinary frequency measured by metabolic cage and cystometry. Cystitis decreased the responsiveness of detrusor muscle strips to carbachol, which was reversed by MK-801 in vivo. Unlike MK-801 the NMDAR antagonist D-AP5, which could not block central NMDAR activity, had no effect on bladder hypertrophy, type I collagen up-regulation or Akt activation caused by cystitis in the bladder. CONCLUSIONS: Findings suggest that NMDAR activity has a role in cystitis induced bladder hypertrophy and overactivity. NMDAR mediated Akt activation may underlie the mechanism of bladder dysfunction.
Assuntos
Cistite/tratamento farmacológico , Cistite/fisiopatologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Bexiga Urinária/patologia , Bexiga Urinária/fisiopatologia , Animais , Ciclofosfamida/administração & dosagem , Cistite/induzido quimicamente , Hipertrofia/tratamento farmacológico , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiologia , Bexiga Urinária/efeitos dos fármacosRESUMO
Activation of plasma membrane TGR5 receptors in enteroendocrine cells by bile acids is known to regulate gastrointestinal secretion and motility and glucose homeostasis. The endocrine functions of the gut are modulated by microenvironment of the distal gut predominantly by sulfur-reducing bacteria of the microbiota that produce H2S. However, the mechanisms involved in the release of peptide hormones, GLP-1 and PYY in response to TGR5 activation by bile acids and the effect of H2S on bile acid-induced release of GLP-1 and PYY are unclear. In the present study, we have identified the signaling pathways activated by the bile acid receptor TGR5 to mediate GLP-1 and PYY release and the mechanism of inhibition of their release by H2S in enteroendocrine cells. The TGR5 ligand oleanolic acid (OA) stimulated Gαs and cAMP formation, and caused GLP-1 and PYY release. OA-induced cAMP formation and peptide release were blocked by TGR5 siRNA. OA also caused an increase in PI hydrolysis and intracellular Ca(2+). Increase in PI hydrolysis was abolished in cells transfected with PLC-ε siRNA. 8-pCPT-2'-O-Me-cAMP, a selective activator of Epac, stimulated PI hydrolysis, and GLP-1 and PYY release. L-Cysteine, which activates endogenous H2S producing enzymes cystathionine-γ-lyase and cystathionine-ß-synthase, and NaHS and GYY4137, which generate H2S, inhibited PI hydrolysis and GLP-1 and PYY release in response to OA or 8-pCPT-2'-O-Me-cAMP. Propargylglycine, an inhibitor of CSE, reversed the effect of L-cysteine on PI hydrolysis and GLP-1 and PYY release. We conclude: (i) activation of Gαs-coupled TGR5 receptors causes stimulation of PI hydrolysis, and release of GLP-1 and PYY via a PKA-independent, cAMP-dependent mechanism involving Epac/PLC-ε/Ca(2+) pathway, and (ii) H2S has potent inhibitory effects on GLP-1 and PYY release in response to TGR5 activation, and the mechanism involves inhibition of PLC-ε/Ca(2+) pathway.
RESUMO
Intraluminal nutrients in the gut affect the peristaltic reflex, although the mechanism is not well defined. Recent evidence supports the presence of taste receptors and their signaling components in enteroendocrine cells, although their function is unclear. This study aimed to determine if nutrients modify colonic motility through activation of taste receptors. Colonic sections were immunostained for the umami taste receptor T1R1/T1R3, which mediates the response to umami ligands, such as monosodium glutamate (MSG), in taste cells. Ascending contraction, descending relaxation, and calcitonin gene-related peptide release were measured in three-chamber flat-sheet preparations of rat colon in response to MSG alone or with inosine 5'-monophosphate (IMP). Velocity of artificial fecal pellet propulsion was measured by video recording in guinea pig distal colon. T1R1/T1R3 receptors were present in enteroendocrine cells of colonic sections from human, rat, mouse, and guinea pig. MSG initiated ascending contraction and descending relaxation components of the peristaltic reflex and calcitonin gene-related peptide release in flat-sheet preparations. IMP augmented the MSG-induced effects, suggesting activation of T1R1/T1R3 receptors. In T1R1(-/-) mice, mucosal stroking, but not MSG, elicited a peristaltic reflex. Intraluminal perfusion of MSG enhanced the velocity of artificial fecal pellet propulsion, which was also augmented by IMP. Propulsion was also increased by l-cysteine, but not l-tryptophan, supporting a role of T1R1/T1R3 receptors. We conclude that T1R1/T1R3 activation by luminal MSG or l-cysteine elicits a peristaltic reflex and CGRP release and increases the velocity of pellet propulsion in distal colon. This mechanism may explain how nutrients regulate colonic propulsion.
Assuntos
Colo/efeitos dos fármacos , Motilidade Gastrointestinal/efeitos dos fármacos , Peristaltismo/efeitos dos fármacos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Paladar/efeitos dos fármacos , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cisteína/farmacologia , Feminino , Aditivos Alimentares , Cobaias , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos Sprague-Dawley , Glutamato de Sódio/farmacologia , Triptofano/farmacologiaRESUMO
This study identified a distinctive pattern of expression and activity of adenylyl cyclase (AC) and phosphodiesterase (PDE) isoforms in mouse colonic longitudinal smooth muscle cells and determined the changes in their expression and/or activity in response to proinflammatory cytokines (IL-1ß and TNF-α) in vitro and 2,4,6 trinitrobenzene sulphonic acid (TNBS)-induced colonic inflammation in vivo. AC5/6 and PDE4D5, expressed in circular muscle cells, were also expressed in longitudinal smooth muscle. cAMP formation was tightly regulated via feedback phosphorylation of AC5/6 and PDE4D5 by PKA. Inhibition of PKA activity by myristoylated PKI blocked phosphorylation of AC5/6 and PDE4D5 and enhanced cAMP formation. TNBS treatment in vivo and IL-1ß and TNF-α in vitro induced inducible nitric oxide synthase (iNOS) expression, stimulated ERK1/2 activity, caused iNOS-mediated S-nitrosylation and inhibition of AC5/6, and induced phosphorylation of PDE4D5 and stimulated its activity. The resultant decrease in AC5/6 activity and increase in PDE4D5 activity decreased cAMP formation and smooth muscle relaxation. S-nitrosylation and inhibition of AC5/6 activity were reversed by the iNOS inhibitor 1400W, whereas phosphorylation and activation of PDE4D5 were reversed by the phosphatidylinositol 3-kinase inhibitor LY294002 and the ERK1/2 inhibitor PD98059. The effects of IL-1ß or TNF-α on forskolin-stimulated cAMP formation and smooth muscle relaxation reflected inhibition of AC5/6 activity and activation of PDE4D5 and were partly reversed by 1400W or PD98059 and completely reversed by a combination of the two inhibitors. The changes in the cAMP/PKA signaling and smooth muscle relaxation contribute to colonic dysmotility during inflammation.
Assuntos
Adenilil Ciclases/metabolismo , Colite/enzimologia , Colo/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Citocinas/metabolismo , Motilidade Gastrointestinal , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Inibidores de Adenilil Ciclases , Animais , Colite/induzido quimicamente , Colite/imunologia , Colite/fisiopatologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/fisiopatologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Relaxamento Muscular , Músculo Liso/efeitos dos fármacos , Músculo Liso/imunologia , Músculo Liso/fisiopatologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Fosforilação , Transdução de Sinais , Ácido Trinitrobenzenossulfônico , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
The signaling pathways mediating sustained contraction of mouse colonic longitudinal smooth muscle and the mechanisms involved in hypercontractility of this muscle layer in response to cytokines and TNBS-induced colitis have not been fully explored. In control longitudinal smooth muscle cells, ACh acting via m3 receptors activated sequentially Gα12, RhoGEF (LARG), and the RhoA/Rho kinase pathway. There was abundant expression of MYPT1, minimal expression of CPI-17, and a notable absence of a PKC/CPI-17 pathway. LARG expression was increased in longitudinal muscle cells isolated from muscle strips cultured for 24 h with IL-1ß or TNF-α or obtained from the colon of TNBS-treated mice. The increase in LARG expression was accompanied by a significant increase in ACh-stimulated Rho kinase and ZIP kinase activities, and sustained muscle contraction. The increase in LARG expression, Rho kinase and ZIP kinase activities, and sustained muscle contraction was abolished in cells pretreated with the Jun kinase inhibitor, SP600125. Expression of the MLCP activator, telokin, and MLCP activity were also decreased in longitudinal muscle cells from TNBS-treated mice or from strips treated with IL-1ß or TNF-α. In contrast, previous studies had shown that sustained contraction in circular smooth muscle is mediated by sequential activation of Gα13, p115RhoGEF, and dual RhoA-dependent pathways involving phosphorylation of MYPT1 and CPI-17. In colonic circular smooth muscle cells isolated from TNBS-treated mice or from strips treated with IL-1ß or TNF-α, CPI-17 expression and sustained muscle contraction were decreased. The disparate changes in the two muscle layers contribute to intestinal dysmotility during inflammation.