RESUMO
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
GRP94 is a member of the heat shock protein family normally confined to the endoplasmic reticulum that sometimes escapes the KDEL-mediated retention system. It is overexpressed in some gastric and other gastrointestinal carcinomas, but little is known about the physiological role of GRP94 in gastric mucosa. We investigated the membrane presence of GRP94 in parietal cells, which secrete acid into the gastric lumen, using subcellular fractionation, selective solubilization of membrane proteins, Western blotting, and radio-ligand binding and provided evidence of functional GRP94 expression at the surface of gastric mucosa parietal cells anchored to the basolateral domain. Our results show that GRP94 is not an integral membrane protein since 50 mM Na2CO3 treatment dissociates part of it from the membrane. However, 100 mM Na2CO3 treatment did not extract all GRP94 from the membrane, which indicates that it is strongly associated with it. The presence of GRP94 in isolated plasma membrane was demonstrated by Western blotting and its functionality by radio-ligand binding experiments. Both the K(D) value obtained in saturation experiments with N-ethylcarboxamido-[3H]adenosine at 4°C, at the nanomolar range, and the inhibition constant of its binding by radicicol, the most specific GRP94 inhibitor, indicate that active receptor regions are exposed at the membrane surface. Western blotting of plasma membrane subfractions showed that GRP94 is mainly expressed in the basolateral membrane of gastric parietal cells, while its presence in the apical domain is negligible, thereby inferring a role for GRP94 in processes operating in this membrane domain.
Assuntos
Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células Parietais Gástricas/metabolismo , Adenosina-5'-(N-etilcarboxamida)/química , Adenosina-5'-(N-etilcarboxamida)/metabolismo , Animais , Carbonatos/química , Membrana Celular/metabolismo , Feminino , Mucosa Gástrica/citologia , Mucosa Gástrica/metabolismo , Proteínas de Choque Térmico HSP70/química , Cinética , Macrolídeos/química , Macrolídeos/metabolismo , Masculino , Proteínas de Membrana/química , Células Parietais Gástricas/citologia , Ligação Proteica , CoelhosRESUMO
Although the human homologue of SND p102, p100 coactivator, was initially described as a nuclear protein, the p100 coactivator protein family members have non-nuclear localization in mammalian cells with active lipid handling, storage, and secretion. However, their role in lipid homeostasis remains unresolved. Here, we investigate the distribution of the rat homologue SND p102 (also called SND1) and its association with newly formed lipid droplets in the liver parenchyma and cultured hepatocytes. Sucrose gradient fractionation showed that SND p102 cofractionated with endoplasmic reticulum and Golgi markers. Such cofractionation was not altered in regenerating steatotic rat liver. However, SND p102 was also detected in lipid droplets from regenerating liver, showing a specific directionalization to the least dense ones. Confocal microscopy of cultured hepatocytes confirmed the findings of gradient fractionation. In addition, p100 coactivator was consistently encountered in microsomes and lipid droplets in control and oleate-treated HepG2 cells. The total amount of SND p102 in hepatocytes was similar in both conditions, suggesting a specific translocation of the protein. Our findings indicate that SND p102 and the human p100 coactivator have a ubiquitous cytoplasmic distribution in hepatocytes and that steatogenic conditions promote the targeting of SND p102 from other cell compartments to specific low density lipid droplets.
Assuntos
Fígado Gorduroso/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Animais , Endonucleases , Retículo Endoplasmático/metabolismo , Feminino , Imunofluorescência , Complexo de Golgi/metabolismo , Humanos , Ratos , Ratos Sprague-DawleyRESUMO
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Although the human homologue of SND p102, p100 coactivator, was initially described as a nuclear protein, the p100 coactivator protein family members have non-nuclear localization in mammalian cells with active lipid handling, storage, and secretion. However, their role in lipid homeostasis remains unresolved. Here, we investigate the distribution of the rat homologue SND p102 (also called SND1) and its association with newly formed lipid droplets in the liver parenchyma and cultured hepatocytes. Sucrose gradient fractionation showed that SND p102 cofractionated with endoplasmic reticulum and Golgi markers. Such cofractionation was not altered in regenerating steatotic rat liver. However, SND p102 was also detected in lipid droplets from regenerating liver, showing a specific directionalization to the least dense ones. Confocal microscopy of cultured hepatocytes confirmed the findings of gradient fractionation. In addition, p100 coactivator was consistently encountered in microsomes and lipid droplets in control and oleate-treated HepG2 cells. The total amount of SND p102 in hepatocytes was similar in both conditions, suggesting a specific translocation of the protein. Our findings indicate that SND p102 and the human p100 coactivator have a ubiquitous cytoplasmic distribution in hepatocytes and that steatogenic conditions promote the targeting of SND p102 from other cell compartments to specific low density lipid droplets (AU)
