1 a 1B -adrenoceptors are localized at a steady state in the plasma membrane in untreated cells, and internalize to intracellular vesicles when exposed to agonist. Flow cytometry analysis with an anti-Nterminus-antibody (1B-N1-C, ) facilitated the quanti®cation of cell surface a 1B -adrenoceptor. Also, the cellular distribution of a 1B -adrenoceptors was visually monitored by immunocytochemical confocal microscopy. 2 Utilizing this combined approach, we have examined the molecular mechanism for cellular tracking of a 1B -adrenoceptors, including the process of sorting of the synthesized receptor protein to the cell surface, and the agonist-induced internalization. The two processes were separately examined by using a 1B -adrenoceptor inducible DDT 1 MF-2 cells for the sorting process and CHO cells stably expressing a 1B -adrenoceptors for the agonist-promoted internalization. 3 We examined the eects of cytochalasin D and mycalolide B (actin depolymerization agents), demecolcine (a microtubule disrupting agent), brefeldin A (an inhibitor of vesicular transport and Golgi function), ba®lomycin A1 (a speci®c inhibitor of the vacuolar proton pump) or hyperosmotic sucrose treatment (that may inhibit clathrin-mediated endocytosis) on these processes. 4 We found that the agonist-promoted internalization was blocked by cytochalasin D and mycalolide B, while the cell surface sorting process was speci®cally blocked by brefeldin A, indicating that the two processes involve dierent components of the cellular endocytic machinery. 5 The experimental approach as exempli®ed in this study would provide a valuable system to study further the molecular mechanism(s) of cellular tracking of G protein-coupled receptors.
Abstract-The effects of development upon adrenergic regulation of glycogenolysis were characterized using isolated hepatocytes from 3 different age groups of male rats (6 week-old, 8 week-old and 30 week-old).The phosphorylase a response in isolated hepatocytes to alpha-adrenergic stimulation decreased moderately with advancing age; whereas, that to beta-adrenergic stimulation declined more rapidly and almost disappeared at the age of 30 weeks. This developmental alteration in relative contribution of alpha and beta-adrenergic regulation of phosphorylase was further confirmed by the experiments with specific antagonists. Also, the dramatic decrease of beta-adrenergic response on glycogen phosphorylase activity was found to be closely associated with a similar change of cAMP generation. In addition, the glucagon effect on cAMP production was found to be declined with advancing animal age. These results demonstrate that the glycogenolytic response of isolated rat hepatocytes to catecholamines can be mediated by different pathways according to the age of the animal; thus, juvenile male rats exhibit both the alpha and beta-adrenergic mechanism for activation of phosphorylase and the maturation is associated with a modest decline of alpha receptor-mediated effect and a dramatic attenuation of a beta-adrenergic/cAMP response.The regulation of liver glycogen me tabolism is one of the factors which plays an important role in the control of carbohydrate metabolism (1). The rate-limiting enzyme for the regulation of glycogenolysis is glycogen phosphorylase.This enzyme exists in an interconvertible active and inactive form and are controlled by a variety of hormones and neural stimulation (2, 3). In particular, catecholamines and glucagon have profound effects on the process (4).Hepatic glycogenolysis induced by catecholamines occur through both alpha and beta-adrenergic receptor mediated pathways (4, 5). The beta-adrenergic activation of glycogen phosphorylase is blocked by propranolol and is presumed to result from an increase of intracellular cAMP. The alpha-adrenergic pathway, on the other hand, activates phosphorylase without detectable increases in intracellular cAMP levels or the activation of cAM P-dependent protein kinase, and is effectively blocked by phentolamine, phenoxybenzamine and prazosin, but is unaffected by propranolol. According to the current lines of evidence, the alpha-receptors governing hepatic glycogenolysis in isolated rat hepatocytes belong to the alpha, -subclass (6, 7), and this alpha-adrenergic receptor-mediated pathway involves a rise in cytosolic Ca2+ which will stimulate phosphorylase kinase and lead to the activation of glycogen phosphorylase (8 10). (18)(19)(20).Developmental maturity and the aging process have been known to be associated with reduced responsiveness of the hepatic beta-adrenergic receptor/cAM P system as reflected in assays of adenylate cyclase activity and cAM P accumulation (21-24).In addition, the alpha-adrenergic receptor-mediated pathway has been known to predomina...
Rat hepatocytes contain several types of Ca2`-linked receptors, all of which stimulate glycogen breakdown by increasing cytosolic free Ca2" concentration ([Ca2+jc). In vivo desensitization of this Ca2" messenger system was studied in hepatocytes isolated from either pheochromocytoma (PHEO)-harboring and chronically norepinephrine (NE)-infused rats. Homologous desensitization for alpha,-adrenergic receptor-mediated phosphorylase activation developed in the early stage of PHEO rats (3-4 wk after implantation), whereas, in the later stage of tumor development or in the NE-infused rats, phosphorylase responses to all Ca2+-mobilizing stimulations were subsensitive (heterologous desensitization). In the homologous desensitization, the [Ca2"Ic response to alpha,-adrenergic stimulation was selectively reduced. We found, using the phenoxybenzamine inactivation method, that there was a linear relationship between alpha, receptor density and the [Ca2"ic response; consequently, the blunted [Ca2"jc response to alpha,-adrenergic stimulation could not be explained by the 34% downregulation of alpha, receptors seen in these rats.These results indicated that uncoupling at a step proximal to alpha, receptor-stimulated [Ca2"Jc increase is also of primary importance in homologous desensitization of phosphorylase activation. On the other hand, heterologous desensitization also involved alteration(s) at steps distal to the rise in [Ca2"Ic.Our data demonstrate that prolonged exposure to catecholamines results in desensitization of the [Ca2+Jc mobilization pathway and may involve multiple mechanisms.
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