RESUMEN
The new neuropeptide Y (NPY) Y5 receptor antagonist CGP 71683A displayed high affinity for the cloned rat NPY Y5 subtype, but > 1, 000-fold lower affinity for the cloned rat NPY Y1, Y2, and Y4 subtypes. In LMTK cells transfected with the human NPY Y5 receptor, CGP 71683A was without intrinsic activity and antagonized NPY-induced Ca2+ transients. CGP 71683A was given intraperitoneally (dose range 1-100 mg/kg) to a series of animal models of high hypothalamic NPY levels. In lean satiated rats CGP 71683A significantly antagonized the increase in food intake induced by intracerebroventricular injection of NPY. In 24-h fasted and streptozotocin diabetic rats CGP 71683A dose-dependently inhibited food intake. During the dark phase, CGP 71683A dose-dependently inhibited food intake in free-feeding lean rats without affecting the normal pattern of food intake or inducing taste aversion. In free-feeding lean rats, intraperitoneal administration of CGP 71683A for 28 d inhibited food intake dose-dependently with a maximum reduction observed on days 3 and 4. Despite the return of food intake to control levels, body weight and the peripheral fat mass remained significantly reduced. The data demonstrate that the NPY Y5 receptor subtype plays a role in NPY-induced food intake, but also suggest that, with chronic blockade, counterregulatory mechanisms are induced to restore appetite.
Asunto(s)
Regulación del Apetito/fisiología , Naftalenos/farmacología , Neuropéptido Y/metabolismo , Pirimidinas/farmacología , Receptores de Neuropéptido Y/fisiología , Animales , Unión Competitiva , Glucemia/metabolismo , Composición Corporal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Calcio/metabolismo , Línea Celular , Condicionamiento Psicológico/efectos de los fármacos , Diabetes Mellitus Experimental/fisiopatología , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Humanos , Hipotálamo/metabolismo , Hipotálamo/fisiología , Insulina/sangre , Insulina/farmacología , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Receptores de Neuropéptido Y/metabolismo , Triglicéridos/sangreRESUMEN
In the insulin producing cell line RINm5F distribution of serine/threonine specific protein phosphatases type 1 (PP1) and 2A (PP2A) was studied. Using different agents which inhibit or stimulate PP1 and PP2A we found that in membrane and nuclear fractions phosphatase activity was inhibited by okadaic acid (OA), protamine, heparin, and inhibitor-2 in a concentration-dependent manner. C2-ceramide had no effect. In the cytosolic fraction the inhibitory effect of okadaic acid was tenfold higher. Protamine stimulated phosphatase activity at low concentrations and became inhibitory at higher concentrations. Inhibitor-2 and heparin caused a decrease in phosphatase activity whereas C2-ceramide led to a slight activation. The data suggest that in membrane and nuclear fractions of RINmSF cells predominantly PP1 is present, whereas in the cytosol PP1 as well as PP2A can be detected.
Asunto(s)
Islotes Pancreáticos/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Animales , Biomarcadores/química , Fraccionamiento Celular , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN , Inhibidores Enzimáticos/farmacología , Heparina/farmacología , Chaperonas de Histonas , Ácido Ocadaico/farmacología , Fosfoproteínas/metabolismo , Fosforilasa a/metabolismo , Protaminas/farmacología , Proteínas/farmacología , Ratas , Esfingosina/análogos & derivados , Esfingosina/farmacología , Factores de Transcripción , Células Tumorales CultivadasRESUMEN
The binding of glucagon to its hepatic receptor is known to result in a number of effects, including the intracellular accumulation of cAMP, the mobilization of intracellular Ca2+, and the endocytosis of glucagon and its receptor into intracellular vesicles. In this study, we begin to define the functional role of the COOH-terminal tail of the human glucagon receptor in glucagon-stimulated signal transduction and receptor internalization. We have created and expressed in Chinese hamster ovary (CHO) cells five truncation mutants in which the COOH-terminal 24, 56, 62, 67, and 73 amino acids have been removed. Cells expressing relevant truncated receptors were assayed for cell surface expression by immunofluorescence, for ligand-binding properties, for cAMP and Ca2+-mediated signal transduction properties, and for receptor endocytosis. In addition, a mutant receptor containing seven serine-to-alanine mutations in the COOH-terminal tail was studied. Our results reveal the following: 1) a region of the COOH-terminal tail that is required for proper cell surface expression, 2) the COOH-terminal 62 amino acids, which comprise the majority of the tail, are not required for ligand binding, cAMP accumulation, or Ca2+ mobilization, and 3) phosphorylation of the COOH-terminal tail is crucial for glucagon-stimulated receptor endocytosis.
Asunto(s)
Glucagón/fisiología , Receptores de Glucagón/fisiología , Secuencia de Aminoácidos , Animales , Células CHO , Calcio/fisiología , Cricetinae , Citoplasma/fisiología , Endocitosis , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes , Eliminación de Secuencia , Serina/química , Transducción de Señal , Relación Estructura-ActividadRESUMEN
It has been shown that okadaic acid (OA) diminishes insulin secretion of rat pancreatic islets in response to glucose, glyceraldehyde and KCl. Glucose, glyceraldehyde and KCl cause release of insulin by depolarization and subsequent opening of L-type calcium channels. Calcium entry into cells is thought to be related to protein phosphorylation. To evaluate whether or not OA mediated inhibition of insulin secretion in response to depolarization might be due to an interference with calcium uptake, we studied its effect on KCl (30 mM)-induced increases of cytosolic calcium and discharge of insulin in the insulin secreting clonal tumor cell line RINm5F. OA inhibited KCl-stimulated insulin release in concentrations > or = 1 microM. In intact RINm5F cells similar concentrations of OA decreased the activity of protein phosphates PP-1/PP-2A and inhibited the depolarization-induced rise of cytosolic calcium ([Ca2+]i). The latter action could also be achieved with the protein phosphatase inhibitor calyculin A, whereas the OA analogue 1-nor-okadaone, which is without effect on phosphatases, did not affect [Ca2+]i or insulin release. It is concluded that depression of depolarization-induced insulin secretion by OA is due to inhibition of calcium entry along voltage dependent calcium channels. The data also suggest that in RINm5F cells protein phosphatases PP-1/PP-2A are related to the function of voltage-dependent calcium channels.
Asunto(s)
Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Insulinoma/metabolismo , Ácido Ocadaico/farmacología , Neoplasias Pancreáticas/metabolismo , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Cloruro de Potasio/farmacología , Animales , Insulina/metabolismo , Secreción de Insulina , Potenciales de la Membrana/efectos de los fármacos , Fosforilasa Fosfatasa/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
The glucagon receptor is a member of the G protein-coupled receptor superfamily. Since several G protein-coupled receptors undergo phosphorylation in response to agonist, we investigated the phosphorylation of the glucagon receptor following the addition of glucagon to a Chinese hamster ovary cell line expressing the human glucagon receptor (CHO/hGR). Glucagon induced a rapid, time and concentration-dependent phosphorylation of its receptor on serine residues. Neither forskolin nor phorbol ester increased receptor phosphorylation, suggesting that cAMP-dependent protein kinase and protein kinase C do not catalyze this phosphorylation event. Furthermore, two mutant cell lines expressing glucagon receptors with successively truncated receptor cytoplasmic tails were tested. A strong correlation between the number of potential phosphorylation sites, receptor phosphorylation and receptor internalization was observed, suggesting that phosphorylation of the glucagon receptor in CHO/hGR cells is functionally linked to its internalization.