RESUMO
This review examines the etiology and pathogenesis of idiopathic achalasia. This disease is clinically characterized by dysphagia of solids and liquids due to the presence of simultaneous or absent esophageal contractions and impaired or absent relaxation of the lower esophageal sphincter. It includes a review of (a) etiology and pathogenesis of this inflammatory process that damage the ganglion cells of the Auerbach plexus that is limited to the esophagus; (b) genetic abnormalities and polymorphisms associated with this disease that may help explain its heterogeneity expressed by the different motility abnormalities of its phenotypes as well as differences in its clinical progression. These different genetic abnormalities may be responsible for the slow progression of types I or II phenotypes; (c) indirect evidence of viruses present in these patients that may initiate its development; (d) the abnormalities of the muscle layer that may be responsible for the dilation of the body of the esophagus that ultimately causes the sigmoid-like esophagus in the very last phase of this disease. This progression to the end-stage phase tends to occur in about 5% of patients. And, (e) the chronic inflammatory abnormalities in the squamous mucosa that may be the cause of the dysplastic and neoplastic changes that may lead to squamous cell carcinoma whose incidence in this disease is increased. These mucosal abnormalities are usually present in patients with markedly dilated body of the esophagus and severe food stasis.
Assuntos
Acalasia Esofágica/etiologia , Carcinoma de Células Escamosas/complicações , Acalasia Esofágica/fisiopatologia , Neoplasias Esofágicas/complicações , HumanosRESUMO
The pathogenesis of gastroesophageal reflux disease (GERD) remains elusive, but recent evidence suggests that early secretion of inflammatory cytokines and chemokines by the mucosa leads to influx of immune cells followed by tissue damage. We previously showed that exposure of esophageal mucosa to HCl causes ATP release, resulting in activation of acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT), the enzyme responsible for the production of platelet-activating factor (PAF). In addition, HCl causes release of IL-8 from the esophageal mucosa. We demonstrate that esophageal epithelial cells secrete proinflammatory mediators in response to HCl and that this response is mediated by ATP. Monolayers of the human esophageal epithelial cell line HET-1A were exposed to acidified cell culture medium (pH 5) for 12 min, a total of seven times over 48 h, to simulate the recurrent acid exposure clinically occurring in GERD. HCl upregulated mRNA and protein expression for the acid-sensing transient receptor potential cation channel, subfamily vanilloid member 1 (TRPV1), lyso-PAF AT, IL-8, eotaxin-1, -2, and -3, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1. The chemokine profile secreted by HET-1A cells in response to repeated HCl exposure parallels similar findings in erosive esophagitis patients. In HET-1A cells, the TRPV1 agonist capsaicin reproduced these findings for mRNA of the inflammatory mediators lyso-PAF AT, IL-8, and eotaxin-1. These effects were blocked by the TRPV1 antagonists iodoresiniferatoxin and JNJ-17203212. These effects were imitated by direct application of ATP and blocked by the nonselective ATP antagonist suramin. We conclude that HCl/TRPV-induced ATP release upregulated secretion of various chemoattractants by esophageal epithelial cells. These chemoattractants are selective for leukocyte subsets involved in acute inflammatory responses and allergic inflammation. The data support the validity of HET-1A cells as a model of the response of the human esophageal mucosa in GERD.
Assuntos
Trifosfato de Adenosina/metabolismo , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Esôfago/efeitos dos fármacos , Refluxo Gastroesofágico/metabolismo , Ácido Clorídrico/farmacologia , Canais de Cátion TRPV/metabolismo , Idoso , Western Blotting , Técnicas de Cultura de Células , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Esôfago/metabolismo , Expressão Gênica , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Canais de Cátion TRPV/genética , Regulação para CimaRESUMO
We have shown that a novel NADPH oxidase isoform, NOX5-S, is the major isoform of NADPH oxidases in an esophageal adenocarcinoma (EA) cell line, FLO, and is overexpressed in Barrett's mucosa with high-grade dysplasia. NOX5-S is responsible for acid-induced reactive oxygen species production. In this study, we found that mRNA levels of NOX5-S were significantly higher in FLO EA cells than in the normal human esophageal squamous cell line HET-1A or in a Barrett cell line, BAR-T. The mRNA levels of NOX5-S were also significantly increased in EA tissues. The data suggest that NOX5-S may be important in the development of EA. Mechanisms of functional regulation of NOX5-S are not fully understood. We show that small G protein Rac1 was present in HET-1A cells, BAR-T cells, and EA cell lines FLO and OE33. Rac1 protein levels were significantly higher in FLO and OE33 cells than in HET-1A or BAR-T cells. Knockdown of Rac1 with Rac1 small interfering RNA significantly decreased acid-induced increase in H(2)O(2) production in FLO EA cells. Overexpression of constitutively active Rac1 significantly increased H(2)O(2) production, an increase that was blocked by knockdown of NOX5-S. By immunofluorescence staining and immunoprecipitation, we found that NOX5-S was present in the cytosol of FLO EA cells and colocalized with Rac1 and SERCA1/2 Ca(2+)-ATPase which is located in the endoplasmic reticulum membrane. We conclude that Rac1 may be important in activation of NOX5-S in FLO EA cells.
Assuntos
Adenocarcinoma/enzimologia , Esôfago de Barrett/enzimologia , Neoplasias Esofágicas/enzimologia , Proteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Análise de Variância , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Linhagem Celular Tumoral , Citosol/enzimologia , Retículo Endoplasmático/enzimologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Imunofluorescência , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Ácido Clorídrico/metabolismo , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Imunoprecipitação , Proteínas de Membrana/genética , Microscopia de Fluorescência , NADPH Oxidase 5 , NADPH Oxidases/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Regulação para Cima , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
In esophageal mucosa, HCl causes TRPV1-mediated release of calcitonin gene-related peptide (CGRP) and substance P (SP) from submucosal neurons and of platelet-activating factor (PAF) from epithelial cells. CGRP and SP release was unaffected by PAF antagonists but reduced by the purinergic antagonist suramin. ATP caused CGRP and SP release from esophageal mucosa, confirming a role of ATP in the release. The human esophageal epithelial cell line HET-1A was used to identify epithelial cells as the site of ATP release. HCl caused ATP release from HET-1A, which was reduced by the TRPV1 antagonist 5-iodoresiniferatoxin. Real-time PCR demonstrated the presence of mRNA for several P2X and P2Y purinergic receptors in epithelial cells. HCl also increased activity of lyso-PAF acetyl-CoA transferase (lyso-PAF AT), the enzyme responsible for production of PAF. The increase was blocked by suramin. ATP caused a similar increase, confirming ATP as a mediator for the TRPV1-induced increase in enzyme activity. Repeated exposure of HET-1A cells to HCl over 2 days caused upregulation of mRNA and protein expression for lyso-PAF AT. Suramin blocked this response. Repeated exposure to ATP caused a similar mRNA increase, confirming ATP as a mediator for upregulation of the enzyme. Thus, HCl-induced activation of TRPV1 causes ATP release from esophageal epithelial cells that causes release of CGRP and SP from esophageal submucosal neurons and activation of lyso-PAF AT, the enzyme responsible for the production of PAF in epithelial cells. Repeated application of HCl or of ATP causes upregulation of lyso-PAF AT in epithelial cells.
Assuntos
Trifosfato de Adenosina/metabolismo , Esôfago/citologia , Esôfago/metabolismo , Ácido Clorídrico/farmacologia , Canais de Cátion TRPV/metabolismo , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Antineoplásicos/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Linhagem Celular , Humanos , Mucosa/citologia , Mucosa/metabolismo , Fosforilação/fisiologia , Fator de Ativação de Plaquetas/antagonistas & inibidores , Fator de Ativação de Plaquetas/genética , Fator de Ativação de Plaquetas/metabolismo , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Purinérgicos/genética , Receptores Purinérgicos/metabolismo , Substância P/metabolismo , Suramina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have previously shown that NADPH oxidase NOX5-S generates reactive oxygen species (ROS) when Barrett's metaplastic cells are exposed to acid. Besides metaplastic cells, other H(2)O(2)-producing cells (e.g., inflammatory cells) present in BE mucosa may produce additional ROS, which may also affect metaplastic cells contributing to esophageal tumorigenesis. In this study, we investigate whether exogenous H(2)O(2) stimulates cell proliferation by increasing NOX5-S expression. Low dose (10(-13) M) of H(2)O(2) significantly increased thymidine incorporation, NOX5-S mRNA, and protein expression in a Barrett's EA cell line FLO. H(2)O(2)-induced increase in NOX5-S expression was significantly inhibited by knockdown of nuclear factor (NF)-κB1 p50 with p50 small interfering RNA (siRNA) in EA cell lines FLO and OE33. H(2)O(2) significantly increased p65 phosphorylation and the luciferase activity in FLO cells transfected with a NF-κB activation reporter plasmid pNF-κB-Luc. H(2)O(2)-induced increase in luciferase activity in FLO cells was significantly decreased by knockdown of extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase (MAPK). Overexpression of p50 and p65 remarkably increased the luciferase activity in FLO cells transfected with a NOX5-S reporter plasmid NOX5-LP. In addition, H(2)O(2)-induced thymidine incorporation in FLO cells was significantly decreased by the MAPK kinase 1/2 inhibitor 2'-amino-3'methoxyflavone (PD98059) and ERK2 siRNA but not by ERK1 siRNA. Likewise, H(2)O(2)-induced increase in NOX5-S expression was significantly decreased by ERK2 siRNA in FLO and OE33 cells. We conclude that a low dose of H(2)O(2) increases cell proliferation. H(2)O(2)-induced increase in cell proliferation may depend on sequential activation of ERK2 MAPK, NF-κB1 p50, and NOX5-S.
Assuntos
Adenocarcinoma/patologia , Esôfago de Barrett/patologia , Proliferação de Células , Neoplasias Esofágicas/patologia , Peróxido de Hidrogênio/metabolismo , Western Blotting , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Luciferases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NADPH Oxidase 5 , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Plasmídeos/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidina/metabolismo , TransfecçãoRESUMO
BACKGROUND AND AIMS: Mechanisms of the progression from Barrett's oesophagus to oesophageal adenocarcinoma (OA) are not fully understood. Bile acids may have an important role in this progression. This study aimed at examining the role of NADPH oxidase NOX5-S and a novel bile acid receptor TGR5 in taurodeoxycholic acid (TDCA)-induced increase in cell proliferation. METHODS: Human Barrett's cell line BAR-T and OA cell line FLO were transfected by the Lipofectamine 2000 or Amaxa-Nucleofector-System. mRNAs were measured by real-time PCR. H(2)O(2) was measured by a fluorescent assay. Cell proliferation was determined by measurement of thymidine incorporation. RESULTS: NOX5-S was present in FLO cells. TDCA significantly increased NOX5-S expression, H(2)O(2) production and thymidine incorporation in FLO and BAR-T cells. This increase in thymidine incorporation was significantly reduced by knockdown of NOX5-S. TGR5 mRNA and protein levels were significantly higher in OA tissues than in normal oesophageal mucosa or Barrett's mucosa. Knockdown of TGR5 markedly inhibited TDCA-induced increase in NOX5-S expression, H(2)O(2) production and thymidine incorporation in FLO and BAR-T cells. Overexpression of TGR5 significantly enhanced the effects of TDCA in FLO cells. TGR5 receptors were coupled with Galphaq and Galphai3 proteins, but only Galphaq mediated TDCA-induced increase in NOX5-S expression, H(2)O(2) production and thymidine incorporation in FLO cells. CONCLUSIONS: TDCA-induced increase in cell proliferation depends on upregulation of NOX5-S expression in BAR-T and FLO cells. TDCA-induced NOX5-S expression may be mediated by activation of the TGR5 receptor and Galphaq protein. These data may provide potential targets to prevent and/or treat Barrett's OA.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas de Neoplasias/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Adenocarcinoma/etiologia , Colagogos e Coleréticos , Neoplasias Esofágicas/etiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/metabolismo , NADPH Oxidase 5 , NADPH Oxidases/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ácido Taurodesoxicólico , Células Tumorais CultivadasRESUMO
The slow transit time of the colon in females with constipation is due to impairment of agonist-induced contraction. The impairment is associated with downregulation of G proteins that mediate contraction and upregulation of Gs proteins that mediate relaxation. These changes are caused by overexpression of progesterone (P4) receptors in the colon, rendering its muscle cells sensitive to physiological P4 concentrations. Downregulation of Gq/11 is mediated by P4 receptor B (PR-B). We examined whether upregulation of Gs proteins increased the inhibition of contraction and whether the increase is mediated by the P4 receptor A (PR-A). These studies were conducted in colon-isolated colon muscle cells from human control and slow-transit constipation (STC) females and from guinea pigs. Muscle cell contraction was induced by CCK-8. Inhibition of contraction was induced by vasoactive intestinal polypeptide (VIP), and 8'bromo-c'AMP (8B-c'AMP) G protein levels were determined by Western blot. VIP-induced inhibition of contraction was greater in muscle cells from STC and P4-treated muscle cells. There were no differences in the inhibition induced by 8B-c'AMP between muscle cells from STC and P4-treated controls. The increased VIP-induced inhibition of muscle cells treated with P4 was blocked by pretreatment with PR-A antibodies and unaffected by PR-B antibodies. These antibodies had no effect on 8B-c'AMP induced-inhibition. The P4 upregulation of Gs proteins was blocked by PR-A antibodies and unaffected by PR-B antibodies. Similar results were obtained in muscle cells from guinea pig colons. We concluded that P4 upregulation of Gs proteins increases VIP-induced inhibition of contraction mediated by PR-A.
Assuntos
Colo/citologia , Contração Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Receptores de Progesterona/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Adulto , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Células Cultivadas , Colo/metabolismo , Constipação Intestinal/metabolismo , AMP Cíclico/metabolismo , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Cobaias , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Progesterona/farmacologia , Receptores de Progesterona/imunologia , Sincalida/farmacologiaRESUMO
Transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) receptors were identified in human esophageal squamous epithelial cell line HET-1A by RT-PCR and by Western blot. In fura-2 AM-loaded cells, the TRPV1 agonist capsaicin caused a fourfold cytosolic calcium increase, supporting a role of TRPV1 as a capsaicin-activated cation channel. Capsaicin increased production of platelet activating factor (PAF), an important inflammatory mediator that acts as a chemoattractant and activator of immune cells. The increase was reduced by the p38 MAP kinase (p38) inhibitor SB203580, by the cytosolic phospholipase A2 (cPLA(2)) inhibitor AACOCF3, and by the lyso-PAF acetyltransferase inhibitor sanguinarin, indicating that capsaicin-induced PAF production may be mediated by activation of cPLA(2), p38, and lyso-PAF acetyltransferase. To establish a sequential signaling pathway, we examined the phosphorylation of p38 and cPLA(2) by Western blot. Capsaicin induced phosphorylation of p38 and cPLA(2). Capsaicin-induced p38 phosphorylation was not affected by AACOCF3. Conversely, capsaicin-induced cPLA(2) phosphorylation was blocked by SB203580, indicating that capsaicin-induced PAF production depends on sequential activation of p38 and cPLA(2). To investigate how p38 phosphorylation may result from TRPV1-mediated calcium influx, we examined a possible role of calmodulin kinase (CaM-K). p38 phosphorylation was stimulated by the calcium ionophore A23187 and by capsaicin, and the response to both agonists was reduced by a CaM inhibitor and by CaM-KII inhibitors, indicating that calcium induced activation of CaM and CaM-KII results in P38 phosphorylation. Acetyl-CoA transferase activity increased in response to capsaicin and was inhibited by SB203580, indicating that p38 phosphorylation in turn causes activation of acetyl-CoA transferase to produce PAF. Thus epithelial cells produce PAF in response to TRPV1-mediated calcium elevation.
Assuntos
Células Epiteliais/metabolismo , Esôfago/citologia , Fator de Ativação de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Canais de Cátion TRPV/metabolismo , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Capsaicina/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Piridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fármacos do Sistema Sensorial/farmacologia , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPV/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Exposure of esophageal mucosa to hydrochloric acid (HCl) is a crucial factor in the pathogenesis of reflux disease. We examined supernatant of HCl-exposed rabbit mucosa for inflammatory mediators enhancing migration of leukocytes and production of H(2)O(2) as an indicator of leukocyte activation. A tubular segment of rabbit esophageal mucosa was tied at both ends to form a sac, which was filled with HCl-acidified Krebs buffer at pH 5 (or plain Krebs buffer as control) and kept oxygenated at 37 degrees C. The medium around the sac (supernatant) was collected after 3 h. Rabbit peripheral blood leukocytes (PBL) were isolated, and sac supernatant was used to investigate PBL migration and H(2)O(2) production. HCl-exposed esophageal mucosa released substance P (SP), CGRP, platelet-activating factor (PAF), and IL-8 into the supernatant. PBL migration increased in response to IL-8 or to supernatant of the HCl-filled mucosal sac. Supernatant-induced PBL migration was inhibited by IL-8 antibodies and by antagonists for PAF (CV3988) or neurokinin 1 (i.e., SP), but not by a CGRP antagonist. Supernatant of the HCl-filled mucosal sac increased H(2)O(2) release by PBL that was significantly reduced by CV3988 and by a SP antagonist but was not affected by IL-8 antibodies or by a CGRP antagonist. We conclude that IL-8, PAF, and SP are important inflammatory mediators released by esophageal mucosa in response to acid that promote PBL migration. In addition, PAF and SP induce production of H(2)O(2) by PBL. These findings provide a direct link between acid exposure and recruitment and activation of immune cells in esophageal mucosa.
Assuntos
Esôfago/efeitos dos fármacos , Ácido Clorídrico/toxicidade , Peróxido de Hidrogênio/metabolismo , Inflamação/induzido quimicamente , Leucócitos/metabolismo , Mucosa/metabolismo , Animais , Esôfago/fisiologia , Regulação da Expressão Gênica/fisiologia , Inflamação/metabolismo , Interleucina-1/genética , Interleucina-1/metabolismo , CoelhosRESUMO
Inactivation of tumor suppressor gene p16 may play an important role in the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). Hypermethylation of p16 gene promoter is an important mechanism inactivating p16. However, the mechanisms of p16 hypermethylation in EA are not known. Therefore, we examined whether acid increases methylation of p16 gene promoter and whether NADPH oxidase NOX5-S mediates acid-induced p16 hypermethylation in a Barrett's cell line BAR-T and an EA cell line OE33. We found that NOX5-S was present in BAR-T and OE33 cells. Acid-induced increase in H(2)O(2) production and cell proliferation was significantly reduced by knockdown of NOX5-S. Exogenous H(2)O(2) remarkably increased p16 promoter methylation and cell proliferation. In addition, acid treatment significantly increased p16 promoter methylation and decreased p16 mRNA level. Knockdown of NOX5-S significantly increased p16 mRNA, inhibited acid-induced downregulation of p16 mRNA, and blocked acid-induced increase in p16 methylation and cell proliferation. Conversely, overexpression of NOX5-S significantly decreased p16 mRNA and increased p16 methylation and cell proliferation. In conclusion, NOX5-S is present in BAR-T cells and OE33 cells and mediates acid-induced H(2)O(2) production and cell proliferation. NOX5-S is also involved in acid-induced hypermethylation of p16 gene promoter and downregulation of p16 mRNA. It is possible that acid reflux present in BE patients may activate NOX5-S and increase production of reactive oxygen species, which in turn increase p16 promoter methylation, downregulate p16 expression, and increase cell proliferation, thereby contributing to the progression from BE to EA.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Esofágicas/metabolismo , Regulação da Expressão Gênica/fisiologia , Genes p16/fisiologia , Proteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana/genética , Metilação , NADPH Oxidase 5 , NADPH Oxidases/genética , Regiões Promotoras Genéticas , Espécies Reativas de OxigênioRESUMO
Progesterone (P4) inhibits the gastrointestinal muscle contraction by downregulating Galpha(q/11) proteins that mediate contraction, by upregulating Galpha(s) proteins that mediate relaxation, and by altering the pattern of cyclooxygenase (COX) enzymes and prostaglandins. We aimed to examine whether P4 treatment of guinea pigs in vivo affects basal colon motility [basal motility index (MI)] by altering the levels and actions of PGF(2alpha) and PGE(2). Guinea pigs were treated with intramuscular (IM) P4 for 4 days. The BASAL MI, the PGF(2alpha)-induced contraction, and PGE(2)-induced inhibition of contraction were examined in muscle strips and cells. The levels of PGF(2alpha) and PGE(2) were measured by radioimmunoassay. Treatment with P4 reduced the basal MI, the levels of PGF(2alpha), and PGF(2alpha)-induced contraction. P4 increased PGE(2) levels, and PGE(2) induced relaxation. Pretreatment with IM RU-486 (10 mg/kg per day), a P4 receptor antagonist, 1 h before P4 blocked the actions of P4. The PGF(2alpha) antagonist Al-1180 abolished basal MI and PGF(2alpha)-induced contraction. N-ethylmaleimide, which blocks unoccupied membrane receptors, blocked Ach and VIP actions but had no effect on PGF(2alpha) and PGE(2) effects. A COX-1 inhibitor decreased and a COX-2 inhibitor increased PGF(2alpha) levels; GTPgammaS increased and GDPbetaS decreased the levels of PGF(2alpha). Galpha(q/11) protein antibodies (Abs) reduced PGF(2alpha) levels, and Galpha(i3) Abs blocked its motor actions. Galphas Abs increased PGF(2alpha) but decreased PGE(2) levels. We concluded that P4 decreases basal MI by reducing PGF(2alpha) levels caused by downregulation of Galpha(q/11) and that PGF(2alpha)-induced contraction was blocked by downregulating Galpha(i3). P4 also decreased the basal MI by increasing PGE(2) levels, and PGE(2) induced relaxation by upregulating Galpha(s) proteins.
Assuntos
Colo/efeitos dos fármacos , Colo/fisiologia , Motilidade Gastrointestinal/efeitos dos fármacos , Progesterona/farmacologia , Prostaglandinas/metabolismo , Acetilcolina/administração & dosagem , Acetilcolina/farmacologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprosta/administração & dosagem , Dinoprosta/antagonistas & inibidores , Dinoprosta/metabolismo , Dinoprosta/farmacologia , Dinoprostona/metabolismo , Dinoprostona/farmacologia , Etilmaleimida/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/imunologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/imunologia , Motilidade Gastrointestinal/fisiologia , Cobaias , Técnicas In Vitro , Masculino , Mifepristona/administração & dosagem , Mifepristona/farmacologia , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Toxina Pertussis/farmacologia , Progesterona/administração & dosagem , Prostaglandinas/administração & dosagem , Prostaglandinas/farmacologia , Receptores de Prostaglandina/antagonistas & inibidores , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
To test whether transient receptor potential channel vanilloid subfamily member-1 (TRPV1) mediates acid-induced inflammation in the esophagus, a tubular segment of esophageal mucosa was tied at both ends, forming a sac. The sac was filled with 0.01 N HCl (or Krebs buffer for control) and kept in oxygenated Krebs buffer at 37 degrees C. The medium around the sac (supernatant) was collected after 3 h. Supernatant of the HCl-filled sac abolished contraction of esophageal circular muscle strips in response to electric field stimulation. Contraction was similarly abolished by supernatant of mucosal sac filled with the TRPV1 agonist capsaicin (10(-6) M). These effects were reversed by the selective TRPV1 antagonist 5'-iodoresiniferatoxin (IRTX) and by the platelet-activating factor (PAF) receptor antagonist CV9388. Substance P and CGRP levels in mucosa and in supernatant increased in response to HCl, and these increases were abolished by IRTX and by tetrodotoxin (TTX) but not affected by CV9388, indicating that substance P and CGRP are neurally released and PAF independent. In contrast, the increase in PAF was blocked by IRTX but not by TTX. Presence of TRPV1 receptor was confirmed by RT-PCR and by Western blot analysis in whole mucosa and in esophageal epithelial cells enzymatically isolated and sorted by flow cytometry or immunoprecipitated with cytokeratin antibodies. In epithelial cells PAF increased in response to HCl, and the increase was abolished by IRTX. We conclude that HCl-induced activation of TRPV1 receptors in esophageal mucosa causes release of substance P and CGRP from neurons and release of PAF from epithelial cells.
Assuntos
Células Epiteliais/metabolismo , Esôfago/metabolismo , Ácido Clorídrico/metabolismo , Mucosa/metabolismo , Contração Muscular , Plexo Submucoso/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Capsaicina/farmacologia , Gatos , Diterpenos/farmacologia , Estimulação Elétrica , Células Epiteliais/efeitos dos fármacos , Esôfago/efeitos dos fármacos , Esôfago/inervação , Técnicas In Vitro , Masculino , Mucosa/efeitos dos fármacos , Mucosa/inervação , Contração Muscular/efeitos dos fármacos , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Estimulação Química , Plexo Submucoso/efeitos dos fármacos , Substância P/metabolismo , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/genética , Tetrodotoxina/farmacologiaRESUMO
Eosinophilic esophagitis (EoE) is a clinico-pathological entity with esophageal symptoms and dense esophageal eosinophilic infiltration throughout the esophagus that may persist despite treatment with proton pump inhibitors. This eosinophilic infiltration is usually absent in the stomach, small intestine and colon, although there are a number of reports of patients with a multi-organ involvement. EoE is associated with abnormalities involving TH2-dependent immunity, with multiple environmental factors strongly contributing to disease expression. The layer of the esophagus affected by the eosinophilic infiltration causes the specific symptoms. Esophageal involvement results mostly in dysphagia for solids that can be severe enough to cause recurrent esophageal obstruction with typical endoscopic features suggesting esophageal remodeling and pathological changes of eosinophilic infiltration of the mucosa, sub-epithelial fibrosis and muscle hypertrophy. This disease is frequently associated with other allergic conditions such as allergic asthma, allergic dermatitis and eosinophilia. The treatment of patients with EoE depends on the severity of the symptoms and of the inflammatory process as well as to their response to a gradual step-up treatment. The first line of treatment consists of steroid containing local inhalers. If unresponsive they are then treated with oral steroids. Intravenous interleukin blockers seem to have a consistent positive therapeutic effect.
RESUMO
The following on esophageal disease provides updated information the mucosal defense against acid and acid-pepsin injury; the roles of platelet activating factor, mast cells, proinflammatory cytokines, and chemokines in inflammation; differences and similarities in erosive and nonerosive esophagitis; acid and vanilloid receptors in esophageal mucosa; and bile acid receptors in esophageal epithelium.
Assuntos
Doenças do Esôfago/fisiopatologia , Inflamação/fisiopatologia , Mastócitos/imunologia , Humanos , Fator de Ativação de Plaquetas/fisiologiaRESUMO
We have focused on understanding the onset of gastroesophageal reflux disease by examining the mucosal response to the presence of acid in the esophageal lumen. Upon exposure to HCl, inflammation of the esophagus begins with activation of the transient receptor potential channel vanilloid subfamily member-1 (TRPV1) in the mucosa, and production of IL-8, substance P (SP), calcitonin gene related peptide (CGRP) and platelet activating factor (PAF). Production of SP and CGRP, but not PAF, is abolished by the neural blocker tetrodotoxin suggesting that SP and CGRP are neurally released and that PAF arises from non neural pathways. Epithelial cells contain TRPV1 receptor mRNA and protein and respond to HCl and to the TRPV1 agonist capsaicin with production of PAF. PAF, SP and IL-8 act as chemokines, inducing migration of peripheral blood leukocytes. PAF and SP activate peripheral blood leukocytes inducing the production of H(2)O(2). In circular muscle, PAF causes production of IL-6, and IL-6 causes production of additional H(2)O(2), through activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Among these, NADPH oxidase 5 cDNA is significantly up-regulated by exposure to PAF; H(2)O(2) content of esophageal and lower esophageal sphincter circular muscle is elevated in human esophagitis, causing dysfunction of esophageal circular muscle contraction and reduction in esophageal sphincter tone. Thus esophageal keratinocytes, that constitute the first barrier to the refluxate, may also serve as the initiating cell type in esophageal inflammation, secreting inflammatory mediators and pro-inflammatory cytokines and affecting leukocyte recruitment and activity.
RESUMO
Gastroesophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE to EA are not fully understood. Besides acid reflux, bile acid reflux may also play an important role in the progression from BE to EA. In this study, we examined the role of phosphatidylinositol-specific phospholipase C (PI-PLC) and a novel NADPH oxidase NOX5-S in bile acid-induced increase in cell proliferation. We found that taurodeoxycholic acid (TDCA) significantly increased NOX5-S expression, hydrogen peroxide (H(2)O(2)) production, and cell proliferation in EA cells. The TDCA-induced increase in cell proliferation was significantly reduced by U73122, an inhibitor of PI-PLC. PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, and PI-PLCgamma2, but not PI-PLCbeta2 and PI-PLCdelta1, were detectable in FLO cells by Western blot analysis. Knockdown of PI-PLCgamma2 or extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein (MAP) kinase with small interfering RNAs (siRNA) significantly decreased TDCA-induced NOX5-S expression, H(2)O(2) production, and cell proliferation. In contrast, knockdown of PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, or ERK1 MAP kinase had no significant effect. TDCA significantly increased ERK2 phosphorylation, an increase that was reduced by U73122 or PI-PLCgamma2 siRNA. We conclude that TDCA-induced increase in NOX5-S expression and cell proliferation may depend on sequential activation of PI-PLCgamma2 and ERK2 MAP kinase in EA cells. It is possible that bile acid reflux present in patients with BE may increase reactive oxygen species production and cell proliferation via activation of PI-PLCgamma2, ERK2 MAP kinase, and NADPH oxidase NOX5-S, thereby contributing to the development of EA.
Assuntos
Ácidos e Sais Biliares/metabolismo , Proteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Fosfolipase C gama/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Refluxo Biliar/metabolismo , Refluxo Biliar/patologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Colagogos e Coleréticos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Estrenos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , NADPH Oxidase 5 , NADPH Oxidases/genética , Inibidores de Fosfodiesterase/farmacologia , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/genética , Pirrolidinonas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Taurodesoxicólico/farmacologiaRESUMO
Gallbladder disease is prevalent during pregnancy. It has been suggested that this complication of pregnancy is attributable to increased bile cholesterol (Ch) induced by estrogens and to gallbladder hypomotility caused by increasing levels of progesterone (P4). Studies on nonpregnant gallbladders have shown that increased levels of bile Ch contribute to both gallstone formation and bile stasis. These studies investigated the effects of high levels of plasma membrane Ch on P4 on gallbladder muscle cells from human and guinea pigs. Contraction was studied in intact and permeabilized muscle cells. G proteins were determined by Western blot, and 3H-P4 incorporation by muscle cells was measured in the beta-scintillation counter. High levels of caveolar Ch blocked the effects induced by P4 treatment for 6 h. They suppressed the expected P4 inhibition of GTP-gammaS (a G protein activator)-induced contraction and changes in G proteins by downregulating Gi3 and upregulating Gs protein levels. Ch inhibited these P4 actions at the caveolar 3 (CAV-3) level, since the P4 effects were antagonized by treatment with CAV-3 antibody, by reducing CAV-3 expression through CAV-3 siRNA. CAV-3 antibody and siRNA reduced caveolar Ch levels. High caveolar levels of Ch and CAV-3 antibody blocked the incorporation of 3H-P4 into caveolae. Treatment with GDP-betaS (a G protein antagonist) had no effect on P4 actions. High caveolar Ch levels blocked the P4 effects on muscle contraction and G protein changes probably because both Ch and P4 require CAV-3 proteins for their transport across the plasma membrane.
Assuntos
Colesterol/química , Vesícula Biliar/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Progesterona/farmacologia , Animais , Caveolina 3/genética , Caveolina 3/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/fisiologia , Inativação Gênica , Genômica , Cobaias , Humanos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/citologia , Miócitos de Músculo Liso/química , RNA Interferente PequenoRESUMO
Colon muscle strips and cells from female patients with slow-transit constipation (STC) exhibit impaired motility, signal transduction abnormalities characterized by downregulation of Gq/11 and upregulation of Gs proteins, decreased cyclooxygenase (COX)-1 and thromboxane (Tx)B2 levels, increased COX-2 and PGE2 levels, and overexpression of progesterone receptors (PGR). Progesterone (P4) treatment of normal cells reproduced these motility and signal transduction abnormalities. The purpose of the study was to examine whether overexpression of PGR-B reproduces these abnormalities by rendering the cells more sensitive to physiological concentrations of P4. Cultured human colon muscle was transfected with a plasmid DNA expressing PGR-B. The mRNAs of PGR, COX-1, COX-2, and Gq/11 were determined by quantitative real-time PCR. Their protein expression was determined by Western blot, and prostaglandins were measured by radioimmunoassay. Cultured muscle cells maintained their phenotypic features determined with myosin light chain (MLC) and h-caldesmon antibodies. Control and transfected muscle cells responded to 10(-6) M P4. In contrast, muscle cells transfected with PGR-B responded to lower P4 concentration (10(-7) M). This P4 concentration reduced MLC phosphorylation induced by CCK-8 (10(-8) M), downregulated Gq/11, and decreased COX-1 and TxB2 levels. It upregulated Gs proteins. It also increased COX-2 and PGE2 levels. We conclude that overexpression of PGR-B renders the cells more sensitive to physiological concentrations of P4. These results are consistent with the hypothesis that overexpression of PGR-B contributes to the motility and signal transduction abnormalities observed in female patients with STC and normal serum levels of P4.
Assuntos
Colo/metabolismo , Miócitos de Músculo Liso/metabolismo , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Transdução de Sinais , Proteínas de Ligação a Calmodulina/metabolismo , Células Cultivadas , Colo/enzimologia , Constipação Intestinal/metabolismo , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Pessoa de Meia-Idade , Miócitos de Músculo Liso/enzimologia , Cadeias Leves de Miosina/metabolismo , Fenótipo , Fosforilação , Prostaglandinas/metabolismo , RNA Mensageiro/metabolismo , Receptores de Progesterona/genética , Sincalida/metabolismo , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
We have shown that NADPH oxidase NOX5-S is overexpressed in Barrett's esophageal adenocarcinoma (EA) cells and may contribute to the progression from Barrett's esophagus (BE) to EA presumably by increasing cell proliferation and decreasing apoptosis (Fu X, Beer DG, Behar J, Wands J, Lambeth D, Cao W. J Biol Chem 281: 20368-20382, 2006). The mechanism(s) of NOX5-S overexpression in EA, however, is not fully understood. In SEG1 EA cells we found that acid treatment significantly increased platelet-activating factor (PAF) production, which in turn markedly increased NOX5-S expression and hydrogen peroxide (H(2)O(2)) production. Knockdown of NOX5-S by NOX5-S small interfering RNA (siRNA) blocked PAF-dependent H(2)O(2) production. PAF-dependent induction of NOX5-S expression and H(2)O(2) production were significantly decreased by the MAPK kinase 1 inhibitor PD-98059, by the cytosolic phospholipase A(2) (cPLA(2)) inhibitor AACOCF3, and by STAT5 downregulation with STAT5 siRNA. PAF significantly increased the phosphorylation of ERK1/2 MAPK, cPLA(2), and STAT5. Using inhibitors, we demonstrated that PAF-induced STAT5 phosphorylation depends on activation of ERK1/2 MAPK and cPLA(2), whereas PAF-induced cPLA(2) phosphorylation was associated with activation of ERK1/2 MAPK. Given that STAT5 bound to the c-sis-inducible element (TTCTGGTAA) of the NOX5-S promoter, overexpression of STAT5 significantly increased NOX5-S promoter activity. We conclude that acid-induced NOX5-S expression and H(2)O(2) production is mediated in part by production of PAF in SEG1 EA cells, and that PAF-induced increase in NOX5-S expression depends on sequential activation of ERK MAP kinases, cPLA(2), and STAT5 in these cells.
Assuntos
Adenocarcinoma/metabolismo , Esôfago de Barrett/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Ácidos Araquidônicos/farmacologia , Esôfago de Barrett/enzimologia , Esôfago de Barrett/genética , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/genética , Flavonoides/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Proteínas de Membrana/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NADPH Oxidase 5 , NADPH Oxidases/genética , Fosfolipases A2 Citosólicas/antagonistas & inibidores , Fosfolipases A2 Citosólicas/metabolismo , Éteres Fosfolipídicos/farmacologia , Fosforilação , Inibidores da Agregação Plaquetária/farmacologia , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT5/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Regulação para CimaRESUMO
The gallbladder (GB) maintains tonic contraction modulated by neurohormonal inputs but generated by myogenic mechanisms. The aim of these studies was to examine the role of prostaglandins in the genesis of GB myogenic tension. Muscle strips and cells were treated with prostaglandin agonists, antagonists, cyclooxygenase (COX) inhibitors, and small interference RNA (siRNA). The results show that PGE2, thromboxane A2 (TxA2), and PGF(2alpha) cause a dose-dependent contraction of muscle strips and cells. However, only TxA2 and PGE2 (E prostanoid 1 receptor type) antagonists induced a dose-dependent decrease in tonic tension. A COX-1 inhibitor decreased partially the tonic contraction and TxB2 (TxA2 stable metabolite) levels; a COX-2 inhibitor lowered the tonic contraction partially and reduced PGE2 levels. Both inhibitors and the nonselective COX inhibitor indomethacin abolished the tonic contraction. Transfection of human GB muscle strips with COX-1 siRNA partially lowered the tonic contraction and reduced COX-1 protein expression and TxB2 levels; COX-2 siRNA also partially reduced the tonic contraction, the protein expression of COX-2, and PGE2. Stretching muscle strips by 1, 2, 3, and 4 g increased the active tension, TxB2, and PGE2 levels; a COX-1 inhibitor prevented the increase in tension and TxB2; and a COX-2 inhibitor inhibited the expected rise in tonic contraction and PGE2. Indomethacin blocked the rise in tension and TxB2 and PGE2 levels. We conclude that PGE2 generated by COX-2 and TxA2 generated by COX-1 contributes to the maintenance of GB tonic contraction and that variations in tonic contraction are associated with concomitant changes in PGE2 and TxA2 levels.