RESUMO
Aromatase, an estrogen synthase, exists in the gastric parietal cells of Wistar rats. The stomach synthesizes large amounts of estrogens and secretes them into the portal vein. We have been particularly studying gastric estrogen synthesis using Wistar rats. However, estrogen synthesis in the stomach of Sprague-Dawley (SD) rats, which are used as frequently as those of the Wistar strain, has not been clarified. We examined steroid synthesis in the stomach of SD rats using immunohistochemistry, in situ hybridization, Western blotting, real-time PCR, and LC-MS/MS. Aromatase also exists in the stomach of SD rats. Its distribution was not found to be different from that of Wistar rats. Results show that H+/K+-ATPase ß-subunit and aromatase colocalized in double immunofluorescence staining. Each steroid synthase downstream from progesterone was present in the gastric mucosa. These results suggest that steroid hormones are synthesized in the parietal cells in the same pathway as Wistar rats. Although mRNA expression of steroid synthases were higher in SD, no significant difference was found in the amount of protein and each steroid hormone level in the portal vein. Although differences between strains might exist in steroid hormone synthesis, results show that SD rats are as useful as Wistar rats for gastric estrogen synthesis experimentation.
Assuntos
Estrogênios/biossíntese , Células Parietais Gástricas/metabolismo , Ratos Sprague-Dawley , Animais , Aromatase/genética , Aromatase/metabolismo , Aromatase/fisiologia , Estrogênios/metabolismo , Mucosa Gástrica/citologia , Mucosa Gástrica/enzimologia , Mucosa Gástrica/metabolismo , Expressão Gênica , Masculino , Células Parietais Gástricas/enzimologia , Veia Porta/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos WistarRESUMO
Parietal cells of the gastric mucosa contain a complex and extensive secretory membrane system that harbors gastric H+, K+-adenosine triphosphatase (ATPase), the enzyme primarily responsible for gastric lumen acidification. Here, we describe the characterization of mice deficient in the H+, K+-ATPase α subunit (Atp4a-/-) to determine the role of this protein in the biosynthesis of this membrane system and the biology of the gastric mucosa. Atp4a-/- mice were produced by gene targeting. Wild-type (WT) and Atp4a-/- mice, paired for age, were examined at 10, 12, 14 and 16 weeks for histopathology, and the expression of mucin 2 (MUC2), α-methylacyl-CoA racemase (AMACR), Ki-67 and p53 proteins was analyzed by immunohistochemistry. For further information, phosphoinositide 3-kinase (PI3K), phosphorylated-protein kinase B (p-AKT), mechanistic target of rapamycin (mTOR), hypoxia-inducible factor 1α (HIF-1α), lactate dehydrogenase A (LDHA) and sirtuin 6 (SIRT6) were detected by Western blotting. Compared with the WT mice, hypochlorhydric Atp4a-/- mice developed parietal cell atrophy and significant antral inflammation (lymphocyte infiltration) and intestinal metaplasia (IM) with elevated MUC2 expression. Areas of dysplasia in the Atp4a-/- mouse stomach showed increased AMACR and Ki-67 expression. Consistent with elevated antral proliferation, tissue isolated from Atp4a-/- mice showed elevated p53 expression. Next, we examined the mechanism by which the deficiency of the H+, K+-ATPase α subunit has an effect on the gastric mucosa. We found that the expression of phosphorylated-PI3K, p-AKT, phosphorylated-mTOR, HIF-1α, LDHA and SIRT6 was significantly higher in tissue from the Atp4a-/- mice compared with the WT mice (P<0.05). The H+, K+-ATPase α subunit is required for acid-secretory activity of parietal cells in vivo, the normal development and cellular homeostasis of the gastric mucosa, and attainment of the normal structure of the secretory membranes. Chronic achlorhydria and hypergastrinemia in aged Atp4a-/- mice produced progressive hyperplasia and mucolytic and IM, and activated the Warburg effect via PI3K/AKT/mTOR signaling.
Assuntos
Acloridria/enzimologia , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/deficiência , Células Parietais Gástricas/enzimologia , Lesões Pré-Cancerosas/enzimologia , Neoplasias Gástricas/enzimologia , Acloridria/genética , Acloridria/patologia , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Doença Crônica , Metabolismo Energético , ATPase Trocadora de Hidrogênio-Potássio/genética , Metaplasia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucina-2/metabolismo , Células Parietais Gástricas/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Serina-Treonina Quinases TOR/metabolismo , Fatores de TempoRESUMO
Parietal cells in the gastric mucosa are known not only as cells playing major roles in food digestion but also as cells bearing endocrine function. In addition to their production of gastrin and ghrelin, it has been recently revealed that these cells are also involved in the synthesis and secretion of estrogens with their expression of aromatase in experimental animals. Although aromatase activity has been detected in human gastric cancer cells and related cell lines, much less study has been done to ascertain the expression of the enzymatic activity in normal gastric mucosa. It has not been established which cell type is responsible for estrogen production in human gastric glands consisting of epithelial cells of several types. The aim of this study is to define the expression of aromatase by parietal cells in human gastric glands using immunohistochemical techniques. We retrieved formalin-fixed paraffin embedded materials of gastric biopsies from 16 patients (nine men, seven women). Colocalization of aromatase and H+/K+-ATPase ß-subunit indicated that positive cells are parietal cells, but not chief cells and mucous cells. Furthermore, immunoreactivity of aromatase was detected within gastric glands irrespective of age or sex. These results suggest that human parietal cells synthesize estrogens within gastric mucosa and subsequently secrete them to the portal vein via gastric vein, as they do in rats. These estrogens might influence liver functions in humans. The estrogenic effects related to liver dysfunction might also be attributed to them.
Assuntos
Aromatase/análise , Aromatase/biossíntese , Mucosa Gástrica/enzimologia , Células Parietais Gástricas/enzimologia , Aromatase/metabolismo , Biópsia , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Células Parietais Gástricas/metabolismo , Células Parietais Gástricas/patologiaRESUMO
The A2B adenosine receptor (A2BR) mediates biological responses to extracellular adenosine in a wide variety of cell types. Adenosine deaminase (ADA) can degrade adenosine and bind extracellularly to adenosine receptors. Adenosine modulates chloride secretion in gastric glands and gastric mucosa parietal cells. A close functional link between surface A2BR and ADA has been found on cells of the immune system, but whether this occurs in the gastrointestinal tract is unknown. The goal of this study was to determine whether A2BR and ADA are coexpressed at the plasma membrane of the acid-secreting gastric mucosa parietal cells. We used isolated gastric parietal cells after purification by centrifugal elutriation. The membrane fraction was obtained by sucrose gradient centrifugation. A2BR mRNA expression was analyzed by RT-PCR. The surface expression of A2BR and ADA proteins was evaluated by Western blotting, flow cytometry and confocal microscopy. Our findings demonstrate that A2BR and ADA are expressed in cell membranes isolated from gastric parietal cells. They show a high degree of colocalization that is particularly evident in the surface of contact between parietal cells. The confocal microscopy data together with flow cytometry analysis suggest a tight association between A2BR and ADA that might be specifically linked to glandular secretory function.
Assuntos
Adenosina Desaminase/análise , Células Parietais Gástricas/química , Receptor A2B de Adenosina/análise , Animais , Western Blotting , Citometria de Fluxo , Microscopia Confocal , Células Parietais Gástricas/enzimologia , CoelhosRESUMO
A series of novel piperazine analogues bearing quinolin-8-yloxy-butan--ones/pyridin-2-yloxy-ethanones were synthesized by a simple and convenient approach based on various substituted piperazine incorporating quinoline and pyridine moieties. The analogues were evaluated for in vitro antioxidant activity against 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and ferrous ion radical scavenging activities and anti-inflammatory activity by inhibition of Vipera russelli venom (PLA2) and gastric K+/H(+)-ATPase activities. Most of the title compounds exhibited promising activity. Best antioxidant and PLA2-inhibiting activities were found for piperazine analogues with phenyl and nitro phenyl groups, whereas methoxy group on phenyl piperazine indicated selectivity for the H+/K(+)-ATPase.
Assuntos
Anti-Inflamatórios/síntese química , Antioxidantes/síntese química , Inibidores Enzimáticos/síntese química , Piperazinas/síntese química , Piridinas/química , Quinolinas/química , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Compostos de Bifenilo/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Radicais Livres/química , Fosfolipases A2 do Grupo II/antagonistas & inibidores , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Estrutura Molecular , Células Parietais Gástricas/efeitos dos fármacos , Células Parietais Gástricas/enzimologia , Picratos/química , Piperazinas/química , Piperazinas/farmacologia , OvinosRESUMO
Acute Helicobacter pylori infection of gastric epithelial cells induces CagA oncoprotein- and peptidoglycan (SLT)-dependent mobilization of NF-κB p50 homodimers that bind to H-K-ATPase α-subunit (HKα) promoter and repress HKα gene transcription. This process may facilitate gastric H. pylori colonization by induction of transient hypochlorhydria. We hypothesized that H. pylori also regulates HKα expression posttranscriptionally by miRNA interaction with HKα mRNA. In silico analysis of the HKα 3' untranslated region (UTR) identified miR-1289 as a highly conserved putative HKα-regulatory miRNA. H. pylori infection of AGS cells transfected with HKα 3' UTR-Luc reporter construct repressed luciferase activity by 70%, whereas ΔcagA or Δslt H. pylori infections partially abrogated repression. Transfection of AGS cells expressing HKα 3' UTR-Luc construct with an oligoribonucleotide mimetic of miR-1289 induced maximal repression (54%) of UTR activity within 30 min; UTR activity was unchanged by nontargeting siRNA transfection. Gastric biopsies from patients infected with cagA(+) H. pylori showed a significant increase in miR-1289 expression compared with uninfected patients or those infected with cagA(-) H. pylori. Finally, miR-1289 expression was necessary and sufficient to attenuate biopsy HKα protein expression in the absence of infection. Taken together, these data indicate that miR-1289 is upregulated by H. pylori in a CagA- and SLT-dependent manner and targets HKα 3' UTR, affecting HKα mRNA translation. The sensitivity of HKα mRNA 3' UTR to binding of miR-1289 identifies a novel regulatory mechanism of gastric acid secretion and offers new insights into mechanisms underlying transient H. pylori-induced hypochlorhydria.
Assuntos
Mucosa Gástrica/enzimologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Infecções por Helicobacter/enzimologia , Helicobacter pylori/metabolismo , MicroRNAs/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Células Parietais Gástricas/enzimologia , Processamento Pós-Transcricional do RNA , Regiões 3' não Traduzidas , Acloridria/enzimologia , Acloridria/microbiologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Linhagem Celular , Mucosa Gástrica/microbiologia , Regulação Enzimológica da Expressão Gênica , Genes Reporter , ATPase Trocadora de Hidrogênio-Potássio/genética , Infecções por Helicobacter/complicações , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Subunidade p50 de NF-kappa B/genética , Células Parietais Gástricas/microbiologia , Peptidoglicano/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Fatores de Tempo , Transfecção , VirulênciaRESUMO
Trametenolic acid B (TAB), the bioactive component in the Trametes lactinea (Berk.) Pat, was reported to possess cytotoxic activities and thrombin inhibiting effects. This study was performed to investigate the effects of TAB on H(+)/K(+)-ATPase and gastric cancer. The H(+)/K(+)-ATPase inhibitory activity was determined by gastric parietal cells. Compared to the normal control group, TAB (10, 20, 40 and 80 µg/mL) inhibited the H(+)/K(+)-ATPase activity by 15.97, 16.96, 24.86 and 16.25%, respectively. In the study, 36 Kunming mice were randomly divided into six groups: control, model, TAB-L (TAB, 5 mg/kg/day, i.g.), TAB-M (TAB, 20 mg/kg/day, i.g.), TAB-H (TAB, 40 mg/kg/day, i.g.) and omeprazole (OL, 10 mg/kg/day, i.g.). All mice except the control group were administrated with anhydrous alcohol (5.0 mL/kg, i.g.) for induced gastric-ulcer 1h after the 5th day. At the same time, the control mice were given the same volume of physiological saline. After 4h, TAB was evaluated for H(+)/K(+)-ATPase inhibitory activities of ulcerative gaster, gastric ulcer index and ulcer inhibition. In vitro, the anti-proliferation effect of TAB to gastric cancer cell (HGC-27) in acid environment was detected by MTT, and the apoptosis morphological changes were also observed by Hoechst 33258 dye assay. The results indicated that TAB inhibited moderately H(+)/K(+)-ATPase activity in vitro. Compared to the model group, TAB showed anti-ulcer effects in gastric tissue with the dosages of 20 and 5 mg/kg in vivo. Apart from that, TAB could selectively inhibit gastric cancer cell viability and reduce cell apoptosis against HGC-27 cells at low doses in acid environment.
Assuntos
ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Fitoterapia , Neoplasias Gástricas/tratamento farmacológico , Úlcera Gástrica/tratamento farmacológico , Estômago/efeitos dos fármacos , Trametes/química , Triterpenos/uso terapêutico , Animais , Antiulcerosos/química , Antiulcerosos/isolamento & purificação , Antiulcerosos/farmacologia , Antiulcerosos/uso terapêutico , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ácido Gástrico/metabolismo , Camundongos , Camundongos Endogâmicos , Omeprazol/farmacologia , Células Parietais Gástricas/efeitos dos fármacos , Células Parietais Gástricas/enzimologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Inibidores da Bomba de Prótons/química , Inibidores da Bomba de Prótons/isolamento & purificação , Inibidores da Bomba de Prótons/farmacologia , Inibidores da Bomba de Prótons/uso terapêutico , Distribuição Aleatória , Estômago/enzimologia , Neoplasias Gástricas/enzimologia , Úlcera Gástrica/enzimologia , Triterpenos/química , Triterpenos/isolamento & purificação , Triterpenos/farmacologiaRESUMO
OBJECTIVE: To investigate whether the ultrastructure and hydrogen potassium adenosine triphosphate (H+ /K+ ATPase) expression of human parietal cells were associated with aging. METHODS: In all, 50 participants who underwent gastroscopy due to dyspepsia were divided into two age groups, with 19 in the younger group (YG, aged 20-59 years) and 31 in the elder group (EG, aged ≥60 years). The ultrastructure of their parietal cell was determined by electron microscopy (EM), and the expressions of H+ /K+ ATPase α-subunit mRNA and ß-unit protein were detected. Furthermore, 24-h esophageal pH monitoring was performed in the two groups. RESULTS: EM images showed no distinct difference in the morphology and distribution of parietal cells or the acid secretion-related organelle between the two groups. There were no differences between YG and EG in the proportion of mitochondria and the tubulovesicular system area. The expressions of H+ /K+ ATPase α-subunit mRNA and ß-subunit protein showed no age-related alteration between YG and EG. The expression of H+ /K+ ATPase α-subunit mRNA in EG was higher than that in YG, whereas the expression of ß-subunit protein was significantly higher in those aged ≥80 years than in the YG. No significant difference was found in the 24-h esophageal pH monitoring between YG and EG. CONCLUSION: Acid secretion-related organelles in parietal cells do not degenerate with aging, the expression of H+ /K+ ATPase even shows a trend to increase, indicating the existence of intact molecular biological basis for acid secretion in healthy elderly individuals.
Assuntos
Envelhecimento/metabolismo , Ácido Gástrico/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/biossíntese , Células Parietais Gástricas/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Envelhecimento/patologia , Dispepsia/enzimologia , Dispepsia/patologia , Monitoramento do pH Esofágico , Mucosa Gástrica/enzimologia , Gastroscopia , Regulação Enzimológica da Expressão Gênica/fisiologia , ATPase Trocadora de Hidrogênio-Potássio/genética , Humanos , Microscopia Eletrônica , Pessoa de Meia-Idade , Células Parietais Gástricas/ultraestrutura , RNA Mensageiro/genética , Adulto JovemRESUMO
Gastric acid secretion by the H(+)-K(+)-ATPase at the apical surface of activated parietal cells requires luminal K(+) provided by the KCNQ1/KCNE2 K(+) channel. However, little is known about the trafficking and relative spatial distribution of KCNQ1 and H(+)-K(+)-ATPase in resting and activated parietal cells and the capacity of KCNQ1 to control acid secretion. Here we show that inhibition of KCNQ1 activity quickly curtails gastric acid secretion in vivo, even when the H(+)-K(+)-ATPase is permanently anchored in the apical membrane, demonstrating a key role of the K(+) channel in controlling acid secretion. Three-dimensional imaging analysis of isolated mouse gastric units revealed that the majority of KCNQ1 resides in an intracytoplasmic, Rab11-positive compartment in resting parietal cells, distinct from H(+)-K(+)-ATPase-enriched tubulovesicles. Upon activation, there was a significant redistribution of H(+)-K(+)-ATPase and KCNQ1 from intracytoplasmic compartments to the apical secretory canaliculi. Significantly, high Förster resonance energy transfer was detected between H(+)-K(+)-ATPase and KCNQ1 in activated, but not resting, parietal cells. These findings demonstrate that H(+)-K(+)-ATPase and KCNQ1 reside in independent intracytoplasmic membrane compartments, or membrane domains, and upon activation of parietal cells, both membrane proteins are transported, possibly via Rab11-positive recycling endosomes, to apical membranes, where the two molecules are closely physically opposed. In addition, these studies indicate that acid secretion is regulated by independent trafficking of KCNQ1 and H(+)-K(+)-ATPase.
Assuntos
Ácido Gástrico/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Canal de Potássio KCNQ1/metabolismo , Células Parietais Gástricas/enzimologia , Animais , Membrana Celular/enzimologia , Cromanos/farmacologia , Citoplasma/enzimologia , Endossomos/enzimologia , Transferência Ressonante de Energia de Fluorescência , Histamina/metabolismo , Canal de Potássio KCNQ1/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Células Parietais Gástricas/efeitos dos fármacos , Células Parietais Gástricas/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Transporte Proteico , Sulfonamidas/farmacologia , Fatores de Tempo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
UNLABELLED: Aromatase is a rate-limiting enzyme for estrogen biosynthesis and has been implicated in pathophysiological states of various diseases via estrogen production. This enzyme is known to be widely distributed in extragonadal and gonadal tissues including the stomach. In contrast to circulating estrogen, the functional role of gastric aromatase/estrogen has not been elucidated in detail, because there is no efficient methodology to investigate spatiotemporal changes of gastric aromatase/estrogen in vivo. Recently, (S)-(11)C-6-[(4-chlorophenyl)(1H-1,2,4-triazole-1-yl)methyl]-1-methyl-1H-benzotriazole ((11)C-labeled vorozole), based on a potent nonsteroidal aromatase inhibitor, has been developed as a tracer to investigate aromatase distribution in living animals and humans using a noninvasive PET technique. In the present study, we investigated gastric aromatase expression by means of PET with (11)C-vorozole. METHODS: After bolus injection of (11)C-vorozole into the tail vein, emission scans were obtained for 90 min on male and female rats under isoflurane anesthesia. Displacement studies with unlabeled vorozole and autoradiographic analysis were conducted for demonstration of specific binding. Immunohistochemistry was performed to confirm aromatase expression. RESULTS: PET scans revealed that (11)C-vorozole highly accumulated in the stomach and adrenal glands. Displacement studies and autoradiography demonstrated that aromatase was expressed in the stomach but that the accumulation of (11)C-vorozole in the adrenal glands might be through nonspecific binding. Immunohistochemical analysis revealed that aromatase is expressed in gastric parietal cells but not in adrenal glands. Moreover, the accumulation of (11)C-vorozole in the stomach was significantly increased in fatigued rats. CONCLUSION: These results suggest that the (11)C-vorozole PET technique is a useful tool for evaluation of gastric aromatase dynamics in vivo, which may provide important information for understanding the molecular mechanisms of gastric aromatase/estrogen-related pathophysiological processes and for the development of new drugs.
Assuntos
Aromatase/metabolismo , Células Parietais Gástricas/diagnóstico por imagem , Células Parietais Gástricas/enzimologia , Tomografia por Emissão de Pósitrons/métodos , Triazóis , Animais , Autorradiografia , Radioisótopos de Carbono , Fadiga/diagnóstico por imagem , Fadiga/metabolismo , Feminino , Imuno-Histoquímica , Masculino , Células Parietais Gástricas/metabolismo , Ratos , Ratos Sprague-Dawley , Imagem Corporal TotalRESUMO
The gastric parietal cell was the first system where a regulated membrane recycling hypothesis was proposed as the principal means for moving molecular transporters between cellular compartments. Upon stimulation, massive membrane flow from an endosomal compartment of tubulovesicle membranes to the apical secretory surface places the ATP-driven pumps in position to secrete a solution of strong acid in collaboration with several other membrane transporters. This review focuses on the membrane recycling pathway and proteins that support the recruitment and redistribution of H,K-ATPase-rich membranes, including those involved in signal transduction, membrane targeting, docking, and fusing, in addition to the integral role of the actin cytoskeleton and its associated proteins in the process of membrane recycling. Although much of the evidence discussed here comes from parietal cell studies, other physiological transport systems, as well as less complex cellular and in vitro models, are examined and cited for generality of principle.
Assuntos
ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Células Parietais Gástricas/enzimologia , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Endocitose/fisiologia , Ácido Gástrico/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Proteínas SNARE/metabolismo , Proteínas SNARE/fisiologia , Transdução de Sinais/fisiologia , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
The enzyme that acylates ghrelin was recently identified in mice as the fourth member of the membrane-bound O-acyltransferases superfamily (MBOAT4) and named ghrelin-O-acyltransferase (GOAT). Only one report showed GOAT mRNA expression in ghrelin-expressing cells of the mouse stomach. We investigated the distribution of GOAT protein in peripheral tissues and co-expression with endocrine markers in the gastric mucosa using a custom-made anti-GOAT antibody. Tissues were collected from male Sprague-Dawley rats and C57BL/6 mice. Western blot revealed two immunoreactive bands in rat and mouse gastric corpus mucosal proteins, a 50 kDa band corresponding to the GOAT protein and a 100 kDa band likely corresponding to a dimer. Western blot also detected GOAT in the plasma and levels were strongly increased after 24-h fasting in mice and slightly in rats. GOAT-immunoreactive cells were located in the gastric corpus mucosa and the anterior pituitary gland, whereas other peripheral tissues of rats and mice examined were negative. In mice, GOAT-immunoreactive cells were mainly distributed throughout the middle portion of the oxyntic glands, whereas in rats they were localized mainly in the lower portion of the glands. Double labeling showed that 95+/-1% of GOAT-immunoreactive cells in mice co-labeled with ghrelin, whereas in rats only 56+/-4% of GOAT-positive cells showed co-expression of ghrelin. The remainder of the GOAT-immunopositive cells in rats co-expressed histidine decarboxylase (44+/-3%). No co-localization was observed with somatostatin in rats or mice. These data suggest species differences between rats and mice in gastric GOAT expression perhaps resulting in a different role of the MBOAT4 enzyme in the rat stomach. Detection of GOAT in the plasma raises the possibility that ghrelin octanoylation may occur in the circulation and the fasting-induced increase in GOAT may contribute to the increase of acylated ghrelin after fasting.
Assuntos
Aciltransferases/metabolismo , Jejum/metabolismo , Células Parietais Gástricas/enzimologia , Aciltransferases/sangue , Animais , Anticorpos/imunologia , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-DawleyRESUMO
The lipid peroxidation state and the system functioning of antioxidant protection in parietal cells under rat chronic atrophic gastritis development was investigated. It was detected that the compensatory increase of superoxide dismutase and catalase activity did not affect the lipoperoxidation process and this resulted in accumulation of toxic TBA reactive substances and diene conjugates during the whole stages of the experimental pathology development. It was shown that the reserved power of the glutathione antioxidant system is sufficient to provide adoptable response in the acute period of the disease owing to increasing intracellular found of the reduced glutathione, but it is insufficient to prevent its decreasing in parietal cells in case of the chronic atrophic gastritis development. Our findings suggest that glutathione system is involved in processes of gastric atrophy. The obtained results testify about considerable system dysfunctions of lipid peroxidation and the antioxidant protection in processes of the rat experimental atrophic gastritis development.
Assuntos
Catalase/metabolismo , Gastrite Atrófica/enzimologia , Glucosefosfato Desidrogenase/metabolismo , Glutationa/metabolismo , Superóxido Dismutase/metabolismo , Administração Oral , Animais , Antioxidantes/análise , Doença Crônica , Ácido Desoxicólico/administração & dosagem , Ácido Desoxicólico/efeitos adversos , Modelos Animais de Doenças , Gastrite Atrófica/induzido quimicamente , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Glutationa Transferase/metabolismo , Peroxidação de Lipídeos , Masculino , Células Parietais Gástricas/enzimologia , Ratos , Salicilato de Sódio/administração & dosagem , Salicilato de Sódio/efeitos adversos , Tiobarbitúricos/análiseRESUMO
The parietal cell is responsible for secreting concentrated hydrochloric acid into the gastric lumen. To fulfill this task, it is equipped with a broad variety of functionally coupled apical and basolateral ion transport proteins. The concerted scientific effort over the last years by a variety of researchers has provided us with the molecular identity of many of these transport mechanisms, thereby contributing to the clarification of persistent controversies in the field. This article will briefly review the current model of parietal cell physiology and ion transport in particular and will update the existing models of apical and basolateral transport in the parietal cell.
Assuntos
Células Parietais Gástricas/fisiologia , Estômago/fisiologia , Adenilato Quinase/metabolismo , Antiporters/genética , Transporte Biológico , Proteínas de Transporte de Cátions/metabolismo , Digestão , Ácido Gástrico/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Íons/metabolismo , Modelos Biológicos , Células Parietais Gástricas/enzimologia , Transportadores de SulfatoRESUMO
Adenosine monophosphate (AMP)-activated protein kinase (AMPK) has been shown to be a metabolic energy regulator in various cells. Activation is a direct result of rising AMP concentration coupled with falling adenosine triphosphate (ATP). AMPK activation during metabolic stress consequently reduces cellular ATP consumption. The gastric parietal cell has a large abundance of mitochondria per cell volume due to the numerous energy-dependent transporters and channels responsible for acid secretion. We identified AMPK in the parietal cell as a metabolic energy regulator that can switch acid secretion off as cellular ATP levels fall. AMPK presence in murine gastric glands was evaluated by immunofluorescent localization. We used a digital imaging system to monitor acid secretion as observed by proton efflux from parietal cells in hand-dissected gastric glands loaded with the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein. Individual murine gastric glands were exposed to histamine, pentagastrin, or carbachol. AMPK was pharmacologically activated with 5-aminoimidazole-4-carboxamide-1-beta-D: -riboside (AICAR) monophosphate or inhibited with 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo[1,5-a] pyrimidine (compound C) or ATP. Acid secretion was evaluated under these conditions as the rate of intracellular pH recovery. In addition, whole-stomach pH measurements were performed. Immunofluorescent localization confirmed the presence of AMPK in gastric mucosa. Exposure to AICAR monophosphate significantly reduced secretagogue-induced acid secretion; addition of compound C or ATP restored acid secretion. Our results indicate that secretagogue-induced acid secretion could be significantly reduced with AMPK activation and restored with its deactivation. We therefore propose the AMPK as a cellular metabolic off switch for gastric acid secretion.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ácido Gástrico/metabolismo , Células Parietais Gástricas/enzimologia , Células Parietais Gástricas/metabolismo , Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Animais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Células Parietais Gástricas/efeitos dos fármacosRESUMO
1. In the present study, we evaluated the role of cyclo-oxygenase (COX)-1 and COX-2 on gastric acid secretion in rabbit isolated parietal cells and gastric glands by examining [(14)C]-aminopyrine uptake, prostaglandin (PG) E(2) synthesis and COX-1, COX-2 and proton pump expression at baseline and after treatment with various concentrations of specific COX-1 (SC-560), COX-2 (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methyl-sulphonyl)phenyl-2 (5H)-furanone; DFU) and non-specific COX (indomethacin) inhibitors. 2. In parietal cells, SC-560 and indomethacin, over the concentration range 10(-8) to 10(-4) mol/L, dose-dependently increased basal and 10(-4) mol/L histamine-stimulated aminopyrine uptake and inhibited PGE(2) synthesis, whereas DFU (10(-8) to 10(-5) mol/L) had no effect. However, at 10(-4) mol/L, DFU augmented histamine-stimulated aminopyrine uptake by 135% and inhibited PGE(2) synthesis by 39%, indicating an inhibition of COX-1 at this higher concentration. 3. The SC-560-, DFU- and indomethacin-induced augmentation of histamine-stimulated aminopyrine uptake was reduced to basal levels after 10(-5) mol/L lansoprazole treatment in parietal cells and gastric glands, whereas 10(-4) mol/L ranitidine only partially inhibited such augmentation. 4. Only COX-1 was detected in parietal cells. However, both COX-1 and COX-2 were expressed in gastric glands, with relative protein density of COX-1 being sixfold higher than that of COX-2. Protein levels of COX-1 in parietal cells and those of COX-1 and COX-2 in gastric glands remained unchanged, regardless of inhibitor treatment, either alone or with histamine. 5. Parietal cell proton pump expression was significantly enhanced by 10(-5) mol/L SC-560 and 10(-4) mol/L indomethacin (by 29 and 31%, respectively) and pump activity was enhanced by 61 and 65%, respectively. In contrast, 10(-5) mol/L DFU had no effect. 6. In conclusion, the data indicate that inhibition of COX-1- but not COX-2-derived PGE(2) synthesis is involved in augmentation of non-steroidal anti-inflammatory drug-induced gastric acid secretion in parietal cells by enhancing expression and activation of the proton pump.
Assuntos
Ciclo-Oxigenase 1/fisiologia , Ciclo-Oxigenase 2/fisiologia , Inibidores de Ciclo-Oxigenase/farmacologia , Ácido Gástrico/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/biossíntese , Células Parietais Gástricas/efeitos dos fármacos , Aminopirina/metabolismo , Animais , Células Cultivadas , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/biossíntese , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Concentração Inibidora 50 , Masculino , Células Parietais Gástricas/enzimologia , Células Parietais Gástricas/metabolismo , CoelhosRESUMO
A multidisciplinary study involving lectin histochemistry, IHC, immuno-lectin blotting, and immunogold was carried out to determine the distribution of sugar residues in the glycoproteins of Rana esculenta oxynticopeptic cells. We considered animals in two experimental conditions, fasting and fed. It is known that, in mammals, the tubulovesicular membranes are rich in proteins with several functions. The proton pump H(+),K(+)-ATPase, a heterodimeric complex with a catalytic alpha-subunit and a heavily glycosylated beta-subunit, responsible for acid secretion, is the most abundant. No data have been published regarding the localization and the structures of H(+),K(+)-ATPase in amphibians. In the water frog, the luminal membrane and tubulovesicular system of oxynticopeptic cells, which differ in morphology according to their functional stage, reacted with the primary gold-conjugated antibody against the H(+),K(+)-ATPase alpha-subunit. By lectin histochemistry and immunoblotting, in the oxynticopeptic cells of R. esculenta we detected the presence of N-linked glycans having fucosylated (poly)lactosamine chains, which could correspond to the oligosaccharide chains of the beta subunit. The latter are somewhat different from those described in mammals, and this is probably because of an adaptation to the different microenvironmental conditions in which the oxynticopeptic cells find themselves, in terms of their different habits and phylogeny.
Assuntos
Mucosa Gástrica/metabolismo , Glicoproteínas/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/biossíntese , Rana esculenta/metabolismo , Animais , Anticorpos Monoclonais , Ingestão de Alimentos , Células Endócrinas/enzimologia , Jejum , Mucosa Gástrica/ultraestrutura , Glicoproteínas/química , ATPase Trocadora de Hidrogênio-Potássio/imunologia , Histocitoquímica , Microscopia Imunoeletrônica , Oligossacarídeos/análise , Células Parietais Gástricas/enzimologia , Lectinas de Plantas , Subunidades Proteicas/biossíntese , Subunidades Proteicas/imunologiaRESUMO
Slc26a9 is a recently identified anion transporter that is abundantly expressed in gastric epithelial cells. To study its role in stomach physiology, gene targeting was used to prepare mice lacking Slc26a9. Homozygous mutant (Slc26a9(-/-)) mice appeared healthy and displayed normal growth. Slc26a9 deletion resulted in the loss of gastric acid secretion and a moderate reduction in the number of parietal cells in mutant mice at 5 weeks of age. Immunofluorescence labeling detected the H-K-ATPase exclusively on the apical pole of gastric parietal cells in Slc26a9(-/-) mice, in contrast to the predominant cytoplasmic localization in Slc26a9(+/+) mice. Light microscopy indicated that gastric glands were dilated, and electron micrographs displayed a distinct and striking absence of tubulovesicles in parietal cells and reductions in the numbers of parietal and zymogen cells in Slc26a9(-/-) stomach. Expression studies indicated that Slc26a9 can function as a chloride conductive pathway in oocytes as well as a Cl(-)/HCO(3)(-) exchanger in cultured cells, and localization studies in parietal cells detected its presence in tubulovesicles. We propose that Slc26a9 plays an essential role in gastric acid secretion via effects on the viability of tubulovesicles/secretory canaliculi and by regulating chloride secretion in parietal cells.
Assuntos
Antiporters/deficiência , Membrana Celular/patologia , Ácido Gástrico/metabolismo , Deleção de Genes , Células Parietais Gástricas/metabolismo , Células Parietais Gástricas/patologia , Animais , Animais Recém-Nascidos , Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Biomarcadores/metabolismo , Células COS , Chlorocebus aethiops , Imunofluorescência , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Concentração de Íons de Hidrogênio , Immunoblotting , Camundongos , Camundongos Knockout , Células Parietais Gástricas/enzimologia , Células Parietais Gástricas/ultraestrutura , Proteínas SLC4A , Transportadores de Sulfato , Titulometria , XenopusRESUMO
Epithelial cells of the gastrointestinal tract are an important barrier between the "milieu interne" and the luminal content of the gut. They perform transport of nutrients, salts, and water, which is essential for the maintenance of body homeostasis. In these epithelia, a variety of K(+) channels are expressed, allowing adaptation to different needs. This review provides an overview of the current literature that has led to a better understanding of the multifaceted function of gastrointestinal K(+) channels, thereby shedding light on pathophysiological implications of impaired channel function. For instance, in gastric mucosa, K(+) channel function is a prerequisite for acid secretion of parietal cells. In epithelial cells of small intestine, K(+) channels provide the driving force for electrogenic transport processes across the plasma membrane, and they are involved in cell volume regulation. Fine tuning of salt and water transport and of K(+) homeostasis occurs in colonic epithelia cells, where K(+) channels are involved in secretory and reabsorptive processes. Furthermore, there is growing evidence for changes in epithelial K(+) channel expression during cell proliferation, differentiation, apoptosis, and, under pathological conditions, carcinogenesis. In the future, integrative approaches using functional and postgenomic/proteomic techniques will help us to gain comprehensive insights into the role of K(+) channels of the gastrointestinal tract.
Assuntos
Células Epiteliais/metabolismo , Trato Gastrointestinal/metabolismo , Canais de Potássio/metabolismo , Animais , Células Epiteliais/enzimologia , Trato Gastrointestinal/enzimologia , Trato Gastrointestinal/fisiopatologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Humanos , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Pâncreas Exócrino/metabolismo , Células Parietais Gástricas/enzimologia , Canais de Potássio/classificação , Glândulas Salivares/metabolismoRESUMO
The gastric acid-secreting parietal cell exhibits profound morphological changes on stimulation. Studies in gastrin null (Gas-KO) mice indicate that maturation of parietal cell function depends on the hormone gastrin acting at the G-protein-coupled cholecystokinin 2 receptor. The relevant cellular mechanisms are unknown. The application of differential mRNA display to samples of the gastric corpus of wild-type (C57BL/6) and Gas-KO mice identified the cytoskeletal linker protein, ezrin, as a previously unsuspected target of gastrin. Gastrin administered in vivo or added to gastric glands in vitro increased ezrin abundance in Gas-KO parietal cells. In parietal cells of cultured gastric glands from wild-type mice treated with gastrin, histamine or carbachol, ezrin was localized to vesicular structures resembling secretory canaliculi. In contrast, in cultured parietal cells from Gas-KO mice, ezrin was typically distributed in the cytosol, and this did not change after incubation with gastrin, histamine or carbachol. However, priming with gastrin for approximately 24 h, either in vivo prior to cell culture or by addition to cultured gastric glands, induced the capacity for secretagogue-stimulated localization of ezrin to large vesicular structures in Gas-KO mice. Similarly, in a functional assay based on measurement of intracellular pH, cultured parietal cells from Gas-KO mice were refractory to gastrin unless primed. The priming effect of gastrin was not attributable to the paracrine mediator histamine, but was prevented by inhibitors of protein kinase C and transactivation of the epidermal growth factor receptor. We conclude that in gastrin null mice there is reduced ezrin expression and a defect in ezrin subcellular distribution in gastric parietal cells, and that both can be reversed by priming with gastrin.