RESUMEN
The enzyme responsible for gastric acidification is a heterodimeric (alpha and beta subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes electroneutral exchange of exoplasmic K(+) for cytoplasmic H(3)O(+). The molecular mechanisms of the catalytic exchange reaction are imperfectly understood, and await clarification of the precise topology of the enzyme with respect to the secretory membrane. Antibodies directed against H,K-ATPase subunits have been useful in confirming hydropathy plot predictions of HKalpha and HKbeta secondary structure. The monoclonal antibody HK 12.18, which labels gastric mucosal parietal cells by immunocytochemistry, and which binds to a single M(r) approximately 94,000 polypeptide by SDS-PAGE immunoblot of gastric microsomes, has been widely used as a specific marker of parietal cells in clinical and cell biological studies of acid secretion, and as a specific HKalpha probe in biochemical studies. However, the uncertain location of the HK 12.18 epitope has limited the antibody's usefulness as a topology probe. In this study, HK 12. 18 immune reactivity with native H,K-ATPase tryptic peptides, HKalpha cDNA fragments expressed in bacteria, and overlapping synthetic HKalpha tridecapeptides, was used to identify the HK 12.18 epitope as seven consecutive amino acids (Asp(682)-Met-Asp-Pro-Ser-Glu-Leu(688)) in the cytoplasmic middle third of HKalpha.
Asunto(s)
Anticuerpos Monoclonales/química , Citoplasma/inmunología , Epítopos/inmunología , ATPasa Intercambiadora de Hidrógeno-Potásio/inmunología , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Epítopos/química , Ratones , Ratones Endogámicos BALB C , Estómago/enzimología , PorcinosRESUMEN
Clinical studies and in vitro data from isolated parietal cells suggest that acute Helicobacter pylori infection inhibits acid secretion. Gastric acidification is mediated by H(+)-K(+)-ATPase, an integral protein of parietal cell apical membranes. To test the hypothesis that H. pylori downregulates H(+)-K(+)-ATPase alpha-subunit (HKalpha) gene expression and to identify potential intracellular signaling pathways mediating such regulation, we transfected human gastric adenocarcinoma (AGS) cells with human and rat HKalpha 5'-flanking DNA fused to a luciferase reporter plasmid. Histamine caused dose-dependent, cimetidine-sensitive (10(-4) M) increases in cAMP, free intracellular Ca(2+), and HKalpha promoter activation in AGS cells. H. pylori infection of transfected AGS cells dose dependently inhibited basal and histamine-stimulated HKalpha promoter activity by 80% and 66%, respectively. H. pylori dose dependently inhibited phorbol myristate acetate-induced (10(-7) M) and staurosporine- (10(-7) M) and calphostin C-sensitive (5 x 10(-8) M) activation of HKalpha promoter. Also, H. pylori inhibited epidermal growth factor (EGF) (10(-8) M), genistein-sensitive (5 x 10(-5) M) activation of HKalpha promoter, reducing activity to 60% of basal level. These data suggest that H. pylori inhibits HKalpha gene expression via intracellular pathways involving protein kinases A and C and protein tyrosine kinase, AGS cells have functional histamine H(2) and EGF receptors, and transiently transfected AGS cells are a useful model for studying regulation of HKalpha gene expression.
Asunto(s)
Expresión Génica/fisiología , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , Helicobacter pylori/fisiología , Isoenzimas/genética , Estómago/enzimología , Animales , Calcio/metabolismo , Cimetidina/farmacología , AMP Cíclico/metabolismo , Regulación hacia Abajo , Infecciones por Helicobacter/genética , Histamina/farmacología , Humanos , Membranas Intracelulares/metabolismo , Regiones Promotoras Genéticas/fisiología , Ratas , Transfección , Células Tumorales CultivadasRESUMEN
The H,K-ATPase responsible for gastric acidification is a heterodimeric (alpha and beta subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes the electroneutral exchange of K+ for protons, is stimulated by K+ and is inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1, 2-alpha]pyridine-3-acetonitrile (SCH 28080). Hydropathy analysis of the catalytic alpha subunit has been interpreted in terms of four N-terminal transmembrane domains, a cytoplasmically oriented segment containing ATP binding and phosphorylation sites, and a C-terminal region with four or six putative transmembrane domains. Several lines of evidence implicate the C-terminal region of P-type ATPases in cation-binding and occlusion, conformational changes, and interactions with the beta subunit (HKbeta), making the definition of topology a prerequisite for understanding the structural basis of these functions. Influenza haemagglutinin epitopes (YPYDVPDYA; flu tag) were inserted in predicted hydrophilic segments of the alpha subunit (HKalpha) to establish the membrane orientation of two amino acids with different predicted topologies in the C-terminal four- and six-transmembrane models. Wild-type and mutated HKalpha and HKbeta cDNA species were expressed in insect cells (Sf9) via recombinant baculovirus infection, and expression of H,K-ATPase was verified by immunoblotting with HKalpha- and HKbeta-specific and flu-tag-specific antibodies. Functional assays showed K+-stimulated, SCH 28080-sensitive ATPase activity, confirming neo-native topology in H,K-ATPase heterodimers expressed in Sf9 cells. The topology of flu tags was determined by microsomal protease protection assays in Sf9 cells and immunolabelling of HKalpha and HKbeta in intact and permeabilized Sf9 cells. In addition, MS of native H,K-ATPase tryptic peptides identified cytoplasmically oriented HKalpha residues. The results indicated cytoplasmic exposure of Leu844 and Phe996, and luminal exposure of Pro898, leading to a revised secondary structure model of the C-terminal third of HKalpha.
Asunto(s)
Epítopos/inmunología , ATPasa Intercambiadora de Hidrógeno-Potásio/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Dimerización , Endopeptidasas/metabolismo , Epítopos/química , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , ATPasa Intercambiadora de Hidrógeno-Potásio/inmunología , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Imidazoles/farmacología , Inmunohistoquímica , Microsomas/efectos de los fármacos , Microsomas/enzimología , Microsomas/metabolismo , Modelos Moleculares , Peso Molecular , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Potasio/farmacología , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Spodoptera/citología , Spodoptera/genética , Porcinos , Transducción GenéticaRESUMEN
Integrin alpha4beta1 mediates leukocyte recruitment, activation, mediator release, and apoptosis inhibition, and it plays a central role in inflammatory pathophysiology. High-affinity, selective inhibitors of alpha4beta1, based on the Leu-Asp-Val (LDV) sequence from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin, are described that employ a novel N-terminal peptide "cap" strategy. One inhibitor, BIO-1211, was approximately 10(6)-fold more potent than the starting peptide and exhibited tight-binding properties (koff = 1.4 x 10(-4) s-1, KD = 70 pM), a remarkable finding for a noncovalent, small-molecule inhibitor of a protein receptor. BIO-1211 was also 200-fold selective for the activated form of alpha4beta1, and it stimulated expression of ligand-induced epitopes on the integrin beta1 subunit, a property consistent with occupancy of the receptor's ligand-binding site. Pretreatment of allergic sheep with a 3-mg nebulized dose of BIO-1211 inhibited early and late airway responses following antigen challenge and prevented development of nonspecific airway hyperresponsiveness to carbachol. These results show that highly selective and potent small-molecule antagonists can be identified to integrins with primary specificity for peptide domains other than Arg-Gly-Asp (RGD); they confirm the generality of integrins as small molecule targets; and they validate alpha4beta1 as a therapeutic target for asthma.
Asunto(s)
Antialérgicos/síntesis química , Hiperreactividad Bronquial/prevención & control , Integrinas/antagonistas & inhibidores , Oligopéptidos/síntesis química , Receptores Mensajeros de Linfocitos/antagonistas & inhibidores , Animales , Antialérgicos/química , Antialérgicos/metabolismo , Antialérgicos/farmacología , Sitios de Unión , Hiperreactividad Bronquial/inducido químicamente , Hiperreactividad Bronquial/inmunología , Carbacol/toxicidad , Adhesión Celular/efectos de los fármacos , Línea Celular , Diseño de Fármacos , Epítopos , Fibronectinas/química , Fibronectinas/fisiología , Humanos , Integrina alfa4beta1 , Integrinas/metabolismo , Células Jurkat , Cinética , Ligandos , Oligopéptidos/química , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Receptores Mensajeros de Linfocitos/metabolismo , Ovinos , Relación Estructura-Actividad , Molécula 1 de Adhesión Celular Vascular/fisiologíaRESUMEN
Helisoma neurons require a factor(s) present in conditioned medium (CM), for successful neurite outgrowth in vitro. A approximately 300 kDa Helisoma extracellular matrix (ECM) protein has been identified in CM and is necessary for neurite initiation. Here we show that purified approximately 300 kDa ECM protein supports outgrowth. Furthermore, anti- approximately 300 kDa Fab fragments cause a rapid, dose-dependent decrease in outgrowth when added to neurons already growing in CM, culminating in growth cone collapse and neurite retraction at 200 micrograms/ml. Collapsing growth cones rapidly lost lamellipodia and filopodia transformed into long filamentous strands. Contortion of microtubules in retracting neurites into serpentine shapes, apparently by compressive forces, suggests that large-scale microtubule depolymerization is not a prerequisite for growth cone retraction. These results imply that substrate-bound approximately 300 kDa CM protein is necessary and sufficient for CM-stimulated growth cone initiation and neurite elongation from Helisoma neurons.