RESUMEN
BACKGROUND: Matrix metalloproteinases (MMPs) have roles in inflammation and other processes relevant to the architectural disturbances seen in the gastric mucosa in response to Helicobacter pylori infection. Upregulation of MMPs has been reported in H pylori infection, but there are no detailed reports regarding altered production of their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). AIMS: To investigate changes in the abundance of TIMPs in human gastric corpus mucosa and murine stomach in Helicobacter infection, and to study cellular sources in man. METHODS: Gastric corpus biopsy samples were assessed for abundance of mRNA or protein for TIMP-1 to -4 by real-time quantitative PCR or western blotting, respectively. Antral and corpus biopsies were processed for histology, H pylori status and inflammatory scoring. Cellular sources of TIMP-1, -3 and -4 were examined by indirect immunohistochemistry. Circulating gastrin was measured by radioimmunoassay. Also, abundance of TIMP-1, -3 and -4 mRNA in the stomach of Helicobacter felis infected mice post-infection was compared with that of uninfected control animals. RESULTS: Compared with uninfected patients, mRNA and protein for TIMP-1, -3 and -4 were significantly more abundant in the gastric corpus of H pylori infected subjects. Gastric TIMP expression did not differ significantly between hyper- and normogastrinaemic subjects within the H pylori negative and positive groups. There was no difference in mRNA abundance for MMP-3 or -8. Immunohistochemistry showed TIMP proteins localised to gastric epithelial, stromal cells and inflammatory cells. Murine H felis infection was associated with upregulation of TIMP-1 and -3 mRNA. CONCLUSIONS: Helicobacter infection is associated with upregulation of specific TIMPs (TIMP-1 and -3) in glandular epithelium and stroma. It is suggested that increased expression of specific protease inhibitors in the corpus mucosa may exert important effects on extracellular matrix remodelling and influence the outcome of H pylori infection.
Asunto(s)
Mucosa Gástrica/metabolismo , Gastritis/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Animales , Gastrinas/sangre , Gastritis/sangre , Gastritis/microbiología , Gastritis/patología , Infecciones por Helicobacter/sangre , Infecciones por Helicobacter/patología , Humanos , Ratones , Ratones Transgénicos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Índice de Severidad de la Enfermedad , Inhibidores Tisulares de Metaloproteinasas/genética , Regulación hacia ArribaRESUMEN
Food intake is regulated by signals from the gastrointestinal tract. Both stimulation and inhibition of food intake may be mediated by upper gastrointestinal tract hormones, e.g. ghrelin and cholecystokinin that act at least partly via vagal afferent neurones. We now report that vagal afferent neurones in both rat and man express melanin-concentrating hormone and its receptor, melanin-concentrating hormone-1R. In nodose ganglia from rats fasted for 24 h, RT-PCR revealed the expression of both melanin-concentrating hormone and melanin-concentrating hormone-1R, whereas in ganglia from animals fed ad libitum expression was virtually undetectable. Immunohistochemical studies also revealed expression of melanin-concentrating hormone and melanin-concentrating hormone-1R in nodose ganglion neurones of fasted rats, but signals were weak in rats fed ad libitum. Melanin-concentrating hormone and melanin-concentrating hormone-1R were expressed in the same neurones, a high proportion of which also expressed the cholecystokinin-1 receptor. When fasted rats were refed, there was down-regulation of melanin-concentrating hormone and melanin-concentrating hormone-1R expression over a period of 5 h. Similar effects were produced by administration of cholecystokinin to fasted rats. The cholecystokinin-1 receptor antagonist lorglumide inhibited food-induced down-regulation of melanin-concentrating hormone and melanin-concentrating hormone-1R. We conclude that the satiety hormone cholecystokinin acts on vagal afferent neurones to inhibit expression of melanin-concentrating hormone and its receptor. Since the melanin-concentrating hormone system is associated with stimulation of food intake this effect of cholecystokinin may contribute to its action as a satiety hormone.
Asunto(s)
Vías Aferentes/fisiología , Hormonas Hipotalámicas/fisiología , Melaninas/fisiología , Neuronas/fisiología , Hormonas Hipofisarias/fisiología , Receptores de Somatostatina/fisiología , Nervio Vago/fisiología , Animales , Ayuno , Conducta Alimentaria , Masculino , Ganglio Nudoso/fisiología , Ratas , Ratas Wistar , Respuesta de SaciedadRESUMEN
Transgenic mice (hGAS) that overexpress human progastrin are more susceptible than wild-type mice (FVB/N) to the induction of colonic aberrant crypt foci (ACF) and adenomas by the chemical carcinogen azoxymethane. We have previously shown significantly increased levels of colonic mitosis in hGAS compared with FVB/N mice after gamma-radiation. To investigate whether the effects of progastrin observed in hGAS colon require the presence of other forms of circulating gastrin, we have crossed hGAS (hg(+/+)) with gastrin knockout (G(-/-)) mice to generate mice that express progastrin and no murine gastrin (G(-/-)hg(+/+)). After azoxymethane, G(-/-)hg(+/+) mice developed significantly more ACF than control G(-/-)hg(-/-) mice (which do not express any forms of gastrin). G(-/-)hg(+/+) mice also exhibited significantly increased colonic mitosis both before and after exposure to 8 Gray Gy gamma-radiation or 50 mg/kg azoxymethane compared with G(-/-)hg(-/-). Treatment of G(-/-)hg(-/-) mice with synthetic progastrin (residues 21-101 of human preprogastrin) or G17 extended at its COOH terminus corresponding to the COOH-terminal 26-amino-acid residues of human preprogastrin (residues 76-101, G17-CFP) resulted in continued colonic epithelial mitosis after gamma-radiation, whereas glycine-extended gastrin-17 and the COOH-terminal tryptic fragment of progastrin [human preprogastrin-(96-101)] had no effect. Immunoneutralization with an antibody against G17-CFP before gamma-radiation significantly decreased colonic mitosis in G(-/-)hg(+/+) mice to levels similar to G(-/-)hg(-/-). We conclude that progastrin does not require the presence of other forms of gastrin to exert proliferative effects on colonic epithelia and that the portion of the peptide responsible for these effects is contained within amino acid residues 76-101 of human preprogastrin.
Asunto(s)
Colon/citología , Células Epiteliales/efectos de los fármacos , Gastrinas/farmacología , Mitógenos , Mitosis/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Precursores de Proteínas/farmacología , Secuencia de Aminoácidos , Animales , Antimetabolitos , Apoptosis/efectos de los fármacos , Azoximetano/farmacología , Bromodesoxiuridina , Carcinógenos/farmacología , Colon/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Rayos gamma , Gastrinas/química , Gastrinas/genética , Genotipo , Humanos , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , Fragmentos de Péptidos/química , Precursores de Proteínas/químicaRESUMEN
There is evidence for interactions between leptin and cholecystokinin in controlling food intake. Since cholecystokinin acts on vagal afferent neurones, we asked whether the leptin receptor was also expressed by these neurones. Primers for different forms of the leptin receptor were used in reverse transcriptase-polymerase chain reaction (RT-PCR) of rat and human nodose ganglia. RT-PCR yielded products corresponding to the long (functional) form as well as short forms of the rat leptin receptor. Moreover, RT-PCR revealed the long form of the leptin receptor in a human nodose ganglion. The identities of RT-PCR products were confirmed by sequencing. Primers corresponding to leptin itself did not give RT-PCR products in nodose ganglia. Immunocytochemical studies revealed leptin-receptor immunoreactivity in neuronal cell bodies. Many neurones co-expressed the leptin and cholecystokinin type A receptors, or leptin receptor and cocaine- and amphetamine-related transcript. We conclude that vagal afferent neurones that express the cholecystokinin type A receptor and cocaine- and amphetamine-related transcript, may also express the long form of the leptin receptor providing a neurochemical basis for observations of interactions between cholecystokinin and leptin.
Asunto(s)
Regulación del Apetito/fisiología , Proteínas Portadoras/genética , Leptina/metabolismo , Neuronas Aferentes/metabolismo , Ganglio Nudoso/metabolismo , Receptores de Superficie Celular , Aferentes Viscerales/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/genética , Péptido Relacionado con Gen de Calcitonina/metabolismo , Proteínas Portadoras/metabolismo , Colecistoquinina/metabolismo , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente , Humanos , Hibridación in Situ , Microscopía Confocal , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas Aferentes/citología , Ganglio Nudoso/citología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptor de Colecistoquinina A , Receptores de Colecistoquinina/genética , Receptores de Colecistoquinina/metabolismo , Receptores de Leptina , Homología de Secuencia de Ácido Nucleico , Aferentes Viscerales/citologíaRESUMEN
1. The acidic interior of neuroendocrine secretory vesicles provides both an energy gradient for amine-proton exchangers (VMATs) to concentrate small transmitter molecules, for example catecholamines, and an optimal pH for the prohormone convertases which cleave hormone precursors. There is evidence that VMAT activity modulates prohormone cleavage, but in the absence of measurements of pH in secretory vesicles in intact cells, it has not been possible to establish whether these effects are attributable to raised intravesicular pH due to proton transport through VMATs. 2. Clones were generated of the hamster insulinoma cell line HIT-T15 expressing a pH-sensitive form of green fluorescent protein (GFP-F64L/S65T) targeted to secretory vesicles, with and without co-expression of VMAT2. In order to study prohormone cleavage, further clones were generated that expressed preprogastrin with and without co-expression of VMAT2. 3. Confocal microscopy of GFP fluorescence indicated that the pH in the secretory vesicles was 5.6 in control cells, compared with 6.6 in cells expressing VMAT2; the latter was reduced to 5.8 by the VMAT inhibitor reserpine. 4. Using a pulse-chase labelling protocol, cleavage of 34-residue gastrin (G34) was found to be inhibited by co-expression with VMAT2, and this was reversed by reserpine. Similar effects on vesicle pH and G34 cleavage were produced by ammonium chloride. 5. We conclude that VMAT expression confers the linked abilities to store biogenic amines and modulate secretory vesicle pH over a range influencing prohormone cleavage and therefore determining the identity of regulatory peptide secretory products.
Asunto(s)
Álcalis/metabolismo , Hidrógeno/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Neuropéptidos , Sistemas Neurosecretores/metabolismo , Vesículas Secretoras/metabolismo , Cloruro de Amonio/farmacología , Animales , Cricetinae , Gastrinas/antagonistas & inhibidores , Gastrinas/efectos de los fármacos , Gastrinas/metabolismo , Gastrinas/fisiología , Proteínas Fluorescentes Verdes , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Proteínas Luminiscentes , Glicoproteínas de Membrana/antagonistas & inhibidores , Microscopía Confocal , Sistemas Neurosecretores/citología , Precursores de Proteínas/antagonistas & inhibidores , Precursores de Proteínas/efectos de los fármacos , Precursores de Proteínas/metabolismo , Precursores de Proteínas/fisiología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Reserpina/farmacología , Células Tumorales Cultivadas , Proteínas de Transporte Vesicular de Aminas Biógenas , Proteínas de Transporte Vesicular de MonoaminasRESUMEN
Gastric epithelial organization and function are controlled and maintained by a variety of endocrine and paracrine mediators. Peptides encoded by the gastrin gene are an important part of this system because targeted deletion of the gene, or of the gastrin-CCKB receptor gene, leads to decreased numbers of parietal cells and decreased gastric acid secretion. Recent studies indicate that the gastrin precursor, preprogastrin, gives rise to a variety of products, each with a distinctive spectrum of biological activity. The conversion of progastrin to smaller peptides is regulated by multiple mechanisms including prohormone phosphorylation and secretory vesicle pH. Progastrin itself stimulates colonic epithelial proliferation; biosynthetic intermediates (Gly-gastrins) stimulate colonic epithelial proliferation and gastric epithelial differentiation; and C-terminally amidated gastrins stimulate colonic proliferation, gastric epithelial proliferation and differentiation, and acid secretion. The effects of progastrin-derived peptides on gastric epithelial function are mediated in part by release of paracrine factors that include histamine, epidermal growth factor (EGF)-receptor ligands, and Reg. The importance of the appropriate regulation of this system is shown by the observation that prolonged moderate hypergastrinemia in transgenic mice leads to remodelling of the gastric epithelium, and in the presence of Helicobacter, to gastric cancer.
Asunto(s)
Gastrinas/biosíntesis , Gastrinas/metabolismo , Células Parietales Gástricas/metabolismo , Animales , HumanosRESUMEN
Vesicular monoamine transporter 2 is important for the accumulation of monoamine neurotransmitters into synaptic vesicles and histamine transport into secretory vesicles of the enterochromaffin-like cell of the gastric corpus. In this study we have investigated the mechanisms regulating the transcriptional activation of the rat vesicular monoamine transporter 2 (VMAT2) promoter in gastric epithelial cells. Maintenance of basal levels of transcription was dependent on the presence of SP1, cAMP-response element (CRE), and overlapping AP2/SP1 consensus sequences within the region of promoter from -86 to +1 base pairs (bp). Gastrin stimulation increased transcriptional activity, and responsiveness was shown to be dependent on the CRE (-33 to -26 bp) and AP2/SP1 (-61 to -48 bp) consensus sites but independent of the SP1 site at -86 to -81 bp. Gastrin-induced transcription was dependent on the cooperative interaction of an uncharacterized nuclear factor of approximately 23.3 kDa that bound to the putative AP2/SP1 site, CRE-binding protein (CREB), and CREB-binding protein/p300. Gastrin stimulation resulted in the increased binding of phosphorylated CREB to the promoter, but it did not result in the increased binding of the AP2/SP1-binding protein. The gastrin responsiveness of the promoter was shown to be dependent on both the protein kinase C and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-signaling pathways, which may converge on the AP2/SP1-binding protein.
Asunto(s)
Células Epiteliales/metabolismo , Mucosa Gástrica/metabolismo , Gastrinas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Neuropéptidos , Regiones Promotoras Genéticas , Activación Transcripcional , Proteínas E1A de Adenovirus/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Luciferasas/metabolismo , Sistema de Señalización de MAP Quinasas , Datos de Secuencia Molecular , Plásmidos/metabolismo , Unión Proteica , Proteína Quinasa C/metabolismo , Ratas , Factor de Transcripción AP-2 , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Rayos Ultravioleta , Proteínas de Transporte Vesicular de Aminas Biógenas , Proteínas de Transporte Vesicular de MonoaminasRESUMEN
The mechanisms by which neuroendocrine stimulants regulate CCK gene transcription are unclear. We examined promoter activation by pituitary adenylate cyclase-activating polypeptide (PACAP), a known CCK secretagogue, in the enteroendocrine cell line STC-1. The promoter region from -70 to -87 bp, relative to the transcriptional start site, contains a composite calcium/cyclic AMP response element (CRE)/activator protein 1 (AP1) site that may bind CRE binding protein (CREB) and AP1. PACAP (with IBMX) stimulated expression of an 87-bp construct 3.35+/-0.36-fold but had no effect on a -70 construct. The effect was blocked by the protein kinase A inhibitor H-89 and by a dominant-negative CREB plasmid. Mutation of the CRE/AP1 site to a canonical CRE site did not affect the response to PACAP, but mutation to a canonical AP1 site prevented it. CREB phosphorylation was increased after PACAP treatment. Electrophoretic mobility shift assay and supershift analysis revealed that CREB and not AP1 bound to the CRE/AP1 site and that PACAP increased the proportion of phosphorylated CREB that was bound. We conclude that PACAP increases CCK gene expression via a cAMP-mediated pathway involving CREB phosphorylation by protein kinase A and activation of a composite CRE/AP1 site.
Asunto(s)
Colecistoquinina/genética , Neuropéptidos/genética , Sulfonamidas , Transcripción Genética/fisiología , Animales , Calcio/metabolismo , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Electroforesis , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Genes Reporteros , Isoquinolinas/farmacología , Luciferasas/genética , Ratones , Mutagénesis/fisiología , Tumores Neuroendocrinos , Sondas de Oligonucleótidos , Fosforilación , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Regiones Promotoras Genéticas/fisiología , Ratas , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Células Tumorales CultivadasRESUMEN
1. Gastrointestinal endocrine cells produce biogenic amines which are transported into secretory vesicles by one of two proton-amine exchangers, vesicular monoamine transporters type 1 and 2 (VMAT1 and 2). We report here the presence of VMAT1 in rat gastrin (G) cells and the relevance of VMAT1 function for the modulation of progastrin processing by biogenic and dietary amines. 2. In immunocytochemical studies VMAT1, but not VMAT2, was localized to subpopulations of G cells and enterochromaffin (EC) cells; neither was found in antral D cells. The expression of VMAT1 in antral mucosa was confirmed by Northern blot analysis, which revealed an mRNA band of approximately 3.2 kb, and by Western blot analysis, which revealed a major protein of 55 kDa. 3. In pulse-chase labelling experiments, the conversion of the amidated gastrin G34 to G17 was inhibited by biogenic amine precursors (L-DOPA and 5-hydroxytryptophan). This inhibition was stereospecific and sensitive to reserpine (50 nM), which blocks VMAT1 and VMAT2, but resistant to tetrabenazine, which is a selective inhibitor of VMAT2. 4. Dietary amines such as tyramine and tryptamine also inhibited G34 cleavage. This effect was associated with a loss of the electron-dense core of G cell secretory vesicles. It was not stereospecific or reserpine sensitive, but was correlated with hydrophobicity. 5. Thus rat antral G cells can express VMAT1; transport of biogenic amines into secretory vesicles by VMAT1 is associated with inhibition of G34 cleavage, perhaps by raising intravesicular pH. Dietary amines also modulate cleavage of progastrin-derived peptides, but do so by a VMAT1-independent mechanism; they may act as weak bases that passively permeate secretory vesicle membranes and raise intravesicular pH.
Asunto(s)
Sistema Digestivo/metabolismo , Glándulas Endocrinas/metabolismo , Gastrinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Neuropéptidos , Aminoácidos/administración & dosificación , Aminoácidos/fisiología , Animales , Sistema Cromafín/citología , Sistema Cromafín/metabolismo , Dieta , Sistema Digestivo/citología , Glándulas Endocrinas/citología , Inmunohistoquímica , Masculino , Glicoproteínas de Membrana/biosíntesis , Antro Pilórico/metabolismo , Ratas , Ratas Wistar , Proteínas de Transporte Vesicular de Aminas Biógenas , Proteínas de Transporte Vesicular de MonoaminasRESUMEN
BACKGROUND & AIMS: The RegIalpha gene (Reg) encodes a secretory protein proposed to regulate islet beta-cell and gastric mucous cell growth. Reg is expressed in rat gastric enterochromaffin-like (ECL) cells. The aim of this study was to examine Reg expression in human corpus and to determine the identity of Reg in ECL cell carcinoid tumors in hypergastrinemic patients. METHODS: Reg messenger RNA (mRNA) abundance was quantified by Northern blot in extracts of gastric corpus from patients with and without ECL cell tumors and in AR4-2J cells stimulated by gastrin; cellular origins were determined by immunocytochemistry. Mutations of Reg were determined by reverse-transcription polymerase chain reaction, cloning, and sequencing, and the mutated protein was expressed in HIT-T15 cells. RESULTS: Reg mRNA abundance was increased approximately threefold in the corpus of hypergastrinemic patients compared with controls, and was enriched in 3 of 7 ECL cell carcinoid tumors but not in non-endocrine cell gastric polyps. In AR4-2J cells, gastrin stimulated Reg mRNA abundance; this was eliminated by the gastrin/cholecystokinin B antagonist L-740,093 (10(-9) mol/L). Immunocytochemistry indicated that Reg was located in both chief cells and ECL cells in human corpus. Mutations of Reg were identified in 3 of 5 patients with ECL cell carcinoid tumors; in 2 cases, mutation of the initiator methionine residue led to exclusion of the protein from the secretory pathway. CONCLUSIONS: Gastrin regulates Reg mRNA abundance in human corpus. Mutations of Reg that prevent secretion are associated with ECL cell carcinoids, suggesting a function as an autocrine or paracrine tumor suppressor.
Asunto(s)
Proteínas de Unión al Calcio/genética , Tumor Carcinoide/genética , Tumor Carcinoide/patología , Células Enterocromafines/patología , Gastrinas/sangre , Mutación/fisiología , Proteínas del Tejido Nervioso , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Adulto , Anciano , Anciano de 80 o más Años , Proteínas de Unión al Calcio/metabolismo , Tumor Carcinoide/sangre , Tumor Carcinoide/metabolismo , Neoplasias de las Glándulas Endocrinas/sangre , Femenino , Mucosa Gástrica/metabolismo , Gastrinas/fisiología , Humanos , Litostatina , Masculino , Metionina/genética , Persona de Mediana Edad , Biosíntesis de Proteínas/fisiología , ARN Mensajero/sangre , Neoplasias Gástricas/sangre , Neoplasias Gástricas/metabolismo , Distribución Tisular/fisiologíaRESUMEN
Uptake and storage of monoamines in secretory granules is accomplished by vesicular monoamine transporters, and it is likely that vesicular monoamine transporter 2 (VMAT2) is important for histamine transport in vivo. In the present study we have used the pre-B-cell line Ea3.123 to investigate the mechanisms involved in the transcriptional activation of the VMAT2 gene. In Ea3.123 cells, VMAT2 mRNA abundance was increased following mobilization of intracellular calcium, and this increased mRNA expression was paralleled by changes in l-histidine decarboxylase mRNA, suggesting that VMAT2 may be responsible for sequestration of histamine into secretory vesicles in this cell line. We cloned the 5'-flanking region of the VMAT2 gene and determined its transcriptional start site by primer extension of rat VMAT2 mRNA. There was no TATA or TATA-like sequence upstream of this region; instead there were GC-rich elements, Ca2+/cAMP-response-element- and SP1-binding motifs. Approx. 900 bp upstream of the transcriptional start site was a purine-pyrimidine repeat sequence that may form a Z-DNA structure. A series of 5'-deletional VMAT2-promoter segments cloned upstream of a luciferase reporter were capable of driving transcription and indicated the presence of multiple regulatory elements, while stimulation with ionomycin or PMA resulted in an increased level of the transcriptional activity of the 5'-promoter segments studied.
Asunto(s)
Linfocitos B/metabolismo , Células Madre Hematopoyéticas/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Neuropéptidos , Activación Transcripcional , Animales , Linfocitos B/citología , Secuencia de Bases , Monoaminas Biogénicas/metabolismo , Línea Celular , Clonación Molecular , Gránulos Citoplasmáticos , Regulación de la Expresión Génica , Hematopoyesis , Células Madre Hematopoyéticas/citología , Histidina Descarboxilasa/genética , Ionomicina/farmacología , Glicoproteínas de Membrana/biosíntesis , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/análisis , Ratas , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética , Proteínas de Transporte Vesicular de Aminas Biógenas , Proteínas de Transporte Vesicular de MonoaminasRESUMEN
BACKGROUND & AIMS: Amidated gastrins are acid secretagogues and growth factors. Their precursor, progastrin, is a growth factor but not a secretagogue. Cleavage of progastrin at Arg94/95 determines the expression of these two alternative patterns of biological activity. We examined the hypothesis that cleavage at Arg94/95 is regulated by phosphorylation of the adjacent Ser96 residue. METHODS: Hamster insulinoma cells were stably transfected with wild-type rat preprogastrin and phosphorylation site mutants; biosynthesis was studied by a pulse-chase protocol. RESULTS: Rates of cleavage at Arg94/95 were increased in Ser96-->Ala compared with wild-type progastrin. Mutation of Glu98 to Ala inhibited incorporation of [32P]phosphate into progastrin and increased the rate of cleavage at Arg94/95. Conversely, mutation of Ser96 to Asp reduced rates of cleavage at Arg94/95. Depletion of calcium stores decreased phosphorylation of Ser96 and increased cleavage at Arg94/95. Modulation of Ser96 phosphorylation also directly influenced the ratio of progastrin-cleavage products (progastrin/CFP; G17Gly/G34Gly) secreted into the medium. CONCLUSIONS: Phosphorylation of progastrin is dependent on calcium stores, determines prohormone cleavage rates, and thereby controls the production of the alternative active products of preprogastrin translation.
Asunto(s)
Gastrinas/genética , Gastrinas/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/fisiología , Caseína Quinasas , Cricetinae , Insulinoma/metabolismo , Insulinoma/patología , Mutación/fisiología , Fragmentos de Péptidos/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Ratas , Transfección , Células Tumorales CultivadasRESUMEN
Gut endocrine cells possess the capacity to take up and decarboxylate biogenic amine precursors. Decarboxylation is mediated by aromatic L-amino acid decarboxylase (AADC) which is encoded by mRNAs differing in their 5' untranslated regions (UTR) depending on the usage of alternative first exons, 1a and 1b, each with its own acceptor site (-13 and -8 bases relative to the translation start site, respectively). We describe here a novel splice variant of exon 1a-AADC mRNA in rat antral mucosa. Both exon 1a and 1b mRNAs were expressed in rat antral mucosa, but the 1a form was spliced into the acceptor site usually associated with exon 1b (-8). An enteroendocrine cell line (STC-1) expressed exon 1a or exon 1b mRNAs spliced into the -8 acceptor site of exon 2. Transient transfection of a range of cell lines with reporter constructs revealed that all three 5' UTRs efficiently supported expression of the Luciferase reporter. There is therefore a novel, functional 5' UTR of AADC mRNA in rat antral mucosa; alternative AADC splice variants could provide the capacity for control at the level of mRNA translation.
Asunto(s)
Descarboxilasas de Aminoácido-L-Aromático/genética , Mucosa Gástrica/enzimología , Antro Pilórico/enzimología , ARN Mensajero/análisis , Animales , Descarboxilasas de Aminoácido-L-Aromático/biosíntesis , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Complementario/análisis , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Ratas , Ratas WistarRESUMEN
1. The acid secretagogue effect of gastrin is mainly mediated by the release of enterochromaffin-like (ECL) cell histamine, but the mechanism of muscarinic stimulation of acid secretion remains unclear. The results of studying aminopyrine uptake in isolated parietal cells, and histamine release in isolated ECL cells suggest that muscarinic agents may act both directly on the parietal cell and indirectly via histamine release from ECL cells. 2. We examined parietal and ECL cell responses to the muscarinic agent carbamylcholine (carbachol) in conscious rats and in rat isolated vascularly perfused stomachs. 3. Intravenous carbachol stimulated acid secretion in conscious gastric fistula rats and increased H+K+ ATPase mRNA abundance, indicating activation of parietal cells. In these experiments there was no increase in portal venous histamine, or in oxyntic mucosal histidine decarboxylase (HDC) enzyme activity and HDC mRNA abundance. 4. In rat isolated stomachs stimulated with carbachol in the dose range 10 nM(-1) mM only the 1 microM concentration increased venous histamine significantly. 5. We concluded that the muscarinic agent carbachol stimulates acid secretion and H+K+ ATPase mRNA in vivo by a direct effect on the parietal cell, that does not depend on the release of ECL cell histamine.
Asunto(s)
Carbacol/farmacología , Ácido Gástrico/metabolismo , Mucosa Gástrica/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Células Parietales Gástricas/efectos de los fármacos , Animales , Mucosa Gástrica/enzimología , Mucosa Gástrica/metabolismo , Gastrinas/administración & dosificación , Gastrinas/sangre , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Histamina/sangre , Liberación de Histamina/efectos de los fármacos , Histidina Descarboxilasa/genética , Histidina Descarboxilasa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Multiple gastric carcinoids are a well-recognized complication of hypergastrinemia associated with chronic atrophic gastritis. However, the management of large tumors (>2 cm in diameter) remains uncertain, with the decision between antrectomy or total gastrectomy being empirical. This report describes the investigation of a patient with chronic atrophic gastritis and multiple large gastric carcinoid tumors. Before surgery, octreotide was infused for 72 hours to suppress enterochromaffin-like (ECL) cell and gastrin cell function. The infusion decreased plasma gastrin and gastrin synthesis; moreover, there were marked reductions in markers of ECL cell function, e.g., histidine decarboxylase and chromogranin A messenger RNA abundance, in carcinoid tumor tissue and macroscopically normal corpus mucosa. An antrectomy was performed, after which the patient made an uneventful recovery. Six months after surgery, a single residual polyp, enriched with smooth muscle cells but not ECL cells, was removed. One year after antrectomy, the remaining stomach was normal. The response of ECL cell markers in carcinoid tissue to octreotide suggested that these cells were under neuroendocrine control and, therefore, predicted a beneficial outcome for antrectomy. It is suggested that an octreotide supression test coupled with assay of histidine decarboxylase or chromogranin A gene expression is useful in the assessment of gastric carcinoid tumors.
Asunto(s)
Tumor Carcinoide/cirugía , Células Similares a las Enterocromafines/efectos de los fármacos , Octreótido , Antro Pilórico/cirugía , Neoplasias Gástricas/cirugía , Adulto , Tumor Carcinoide/metabolismo , Cromogranina A , Cromograninas/genética , Células Similares a las Enterocromafines/fisiología , Femenino , Humanos , Octreótido/farmacología , ARN Mensajero/análisis , Neoplasias Gástricas/metabolismoAsunto(s)
Proteínas de Unión al ADN/metabolismo , Células Similares a las Enterocromafines/enzimología , Células Similares a las Enterocromafines/patología , Histidina Descarboxilasa/genética , Proteínas de Transporte de Membrana , Neuropéptidos , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Anemia/enzimología , Anemia/patología , Animales , Cromogranina A , Cromograninas/genética , Proteínas de Unión al ADN/genética , Factor de Transcripción GATA4 , Factor de Transcripción GATA6 , Regulación de la Expresión Génica , Histidina Descarboxilasa/metabolismo , Humanos , Hiperplasia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Ratas , Factores de Transcripción/genética , Proteínas de Transporte Vesicular de Aminas BiógenasRESUMEN
The aim of this study was to investigate whether gastrin regulates morphological changes and alpha-subunit gene expression in parietal cells through the gastrin/CCK-B receptor on enterochromaffin-like cells by histamine release. Treatment with 100 mg/kg of YM022, a potent and selective gastrin/CCK-B receptor antagonist, for one week in rats did not alter mRNA levels of histidine decarboxylase or H+, K+-ATPase. However, parietal cell morphology predominantly changed to the resting form, although the serum gastrin concentration was significantly increased. Additional treatment with YM022 and oral omeprazole, 100 mg/kg, for one week markedly suppressed the increases of mRNA levels of histidine decarboxylase and H+, K+-ATPase and completely blocked the morphological transformation of the parietal cells to a stimulated form induced by treatment with omeprazole alone. This indicates that the morphological transformation of parietal cells to an activated form with a subsequent increase in H+, K+-ATPase synthesis caused by hypergastrinemia is mediated by increased histidine decarboxylase gene expression in enterochromaffin-like cells via gastrin/CCK-B receptors.
Asunto(s)
Ácido Gástrico/metabolismo , Gastrinas/fisiología , Células Parietales Gástricas/efectos de los fármacos , Receptores de Colecistoquinina/fisiología , Actinas/biosíntesis , Animales , Benzodiazepinas/farmacología , Inhibidores Enzimáticos/farmacología , Gastrinas/sangre , Expresión Génica , ATPasa Intercambiadora de Hidrógeno-Potásio/biosíntesis , Liberación de Histamina , Histidina Descarboxilasa/antagonistas & inhibidores , Histidina Descarboxilasa/biosíntesis , Antagonistas de Hormonas/farmacología , Masculino , Omeprazol/farmacología , Inhibidores de la Bomba de Protones , ARN Mensajero/genética , Ratas , Ratas Wistar , Receptores de Colecistoquinina/antagonistas & inhibidoresRESUMEN
The cholecystokinin receptors expressed by vagal afferent neurons mediate the effect of cholecystokinin in inhibiting food intake and gastric emptying. We have determined the relative abundance of cholecystokininA, gastrin-cholecystokininB and gastrin-cholecystokininC receptor populations in the rat vagus by autoradiography using [125I]Bolton Hunter-cholecystokinin-8, [125I]Bolton Hunter-heptadecapeptide gastrin and [125I]Leu(15)2-17Glycine-extended heptadecapeptide gastrin, together with the selective antagonists devazepide and L-740093. The results indicate approximately three-fold higher abundance of cholecystokininA compared with gastrin-cholecystokininB receptors, and no significant representation of gastrin-cholecystokininC receptors. Topical capsaicin applied to the vagal nerve trunk abolished the accumulation of sites binding both [125I]Bolton Hunter-labelled cholecystokinin-8 and heptadecapeptide gastrin indicating that both cholecystokininA and gastrin-cholecystokininB receptor populations were present on afferent fibres. The molecular identity of the receptors expressed by rat and human nodose ganglia was examined using the reverse transcription polymerase chain reaction. Products of the predicted size for the cholecystokininA and gastrin-cholecystokininB receptors were identified. The human and rat cholecystokininA receptor products were cloned and the sequences were found to be 99% homologous to those published for receptors expressed by rat pancreas and human gall bladder. We conclude that cholecystokininA and gastrin-colecystokininB receptors are synthesized by nodose ganglion cells, and that the receptor proteins are transported to the periphery along afferent fibres. While there is a clear role for vagal cholecystokininA receptors, the function of vagal afferent gastrin-cholecystokininB receptors remains to be determined.
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Vías Aferentes/metabolismo , Neuronas/metabolismo , Receptores de Colecistoquinina/metabolismo , Nervio Vago/metabolismo , Animales , Secuencia de Bases , Femenino , Humanos , Masculino , Microscopía Electrónica , Datos de Secuencia Molecular , Ratas , Ratas Wistar , Receptores de Colecistoquinina/efectos de los fármacosRESUMEN
1. Inhibition of gastric acid secretion by proton pump inhibitors like omeprazole increases the synthesis and secretion of the pyloric antral hormone gastrin. We report here how omeprazole influences the conversion of the gastrin precursor to its final products, and the abundance of mRNAs encoding proteins associated with progastrin processing in rat antral mucosa. 2. Progastrin processing was studied using a pulse-chase protocol in antral mucosa, incubated in vitro, from rats treated with omeprazole for up to 5 days. Labelled peptides were detected by on-line scintillation counting after immunoprecipitation and HPLC. The mRNAs encoding prohormone-processing enzymes were identified by Northern blot, polymerase chain reaction or ribonuclease protection assay, and their cellular origins identified by immunocytochemistry. 3. Cleavage of [3H]- and [35S]-labelled progastrins at Arg-94-95 or Arg-57-58, and amidation at Phe-92 were not influenced by pretreatment with omeprazole. In contrast, cleavage of G34 (the thirty-four amino acid amidated gastrin) at Lys-74-75 to give G17 (the seventeen amino acid amidated gastrin), and of G34-Gly to G17-Gly (G34 and G17 with COOH-terminal glycine), was increased 3-fold after treatment with omeprazole for either 1 or 5 days. 4. Approximately 20% of newly synthesized amidated and Gly-extended gastrins were secreted within 240 min of the labelling period in omeprazole-treated samples, but secretion of labelled gastrins from control tissue was undetectable over a comparable period. 5. The amidating enzyme, peptidyglycine alpha-amidating mono-oxygenase (PAM), the prohormone convertases PC1/3, PC2, PC5 and the PC2 chaperone 7B2 were localized to rat antral gastrin cells by immunocytochemistry. The relative abundance of mRNA species encoding 7B2, PC5 and PAM were unchanged after treatment with omeprazole for 5 days, whereas gastrin, PC1/3 and PC2 mRNAs are known to increase at this time. 6. The main consequence of increased cleavage at Lys-74-75 is the production of G17 and G17-Gly at the expense of G34 and G34-Gly, respectively. The latter have longer plasma half-lives, and so their increased cleavage may serve to limit the rise in plasma gastrin concentration after inhibition of acid secretion. Changes in the abundance of mRNAs encoding prohormone-processing enzymes cannot account for the rapidity of the changes in cleavage of progastrin at Lys residues after omeprazole.
Asunto(s)
Ácido Gástrico/fisiología , Mucosa Gástrica/metabolismo , Gastrinas/biosíntesis , Gastrinas/metabolismo , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Subtilisinas/biosíntesis , Animales , Furina , Masculino , Omeprazol/farmacología , Biosíntesis de Proteínas , Precursores de Proteínas/biosíntesis , Antro Pilórico , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: GATA transcription factors may regulate gene expression in developing tissues, including gut epithelium. In the stomach, their expression has been linked to regulation of proton pump genes. However, GATA consensus sequences also occur in the promoter of the histidine decarboxylase gene, located in enterochrommafin-like cells. The aim of this study was to determine if GATA factors are located in gastric endocrine cells and to examine their expression during development and in response to changes in the gastric luminal environment. METHODS: Polymerase chain reaction cloning, Northern blot, and gel shift assays were used to examine GATA expression in gastric endocrine cells; changes in GATA messenger RNA during development and in response to fasting, feeding, and gastric achlorhydria were determined by Northern blot. RESULTS: GATA-6 was expressed strongly in rodent gastric endocrine cell fractions, in a human ECL cell tumor, and in an endocrine cell line (STC-1) derived from gut epithelium; proteins from STC-1 cells bound specifically to GATA consensus sequences in the human histidine decarboxylase promoter. GATA messenger RNA abundance was up-regulated during terminal differentiation of the rat stomach and on feeding after a fast. CONCLUSIONS: The GATA-6 transcription factor is expressed in gastric endocrine cells and is a potential regulator of gastric differentiation and of genes involved in the response to feeding.