Endocytosis of beta-adrenergic ligands by rat liver. Comparison of beta-adrenergic receptor and adenylate cyclase distribution in endosome and plasma-membrane fractions.
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ABSTRACT:
SUBMITTER: Hadjiivanova N
PROVIDER: S-EPMC1144238 | biostudies-other | 1984 Sep
REPOSITORIES: biostudies-other
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Project description:Nuclei from purified human peripheral lymphocytes were prepared by incubations with Triton X-100 to disrupt the cells, followed by sucrose-density gradient centrifugation. The nuclei were pure as judged by phase-contrast microscopy and had low contents of non-nuclear marker enzymes. In addition, nuclei prepared from lymphocytes surface-labelled with 125I had only 2-7% of the radioactivity bound to intact lymphocytes. At 3.3 mM-Ca2+ and 100 micronM-ATP a fluoride-sensitive adenylate cyclase was demonstrated in nuclei prepared in 0.2% Triton X-100 or 0.33% Triton X-100. There was linear accumulation of cyclic AMP for 10 min in both preparations. The apparent Km for ATP was 90 micronM. Adenylate cyclase activity was augmented by 1.0 mM-Mn2+ and inhibited at higher concentrations. Ca2+ showed two peaks of stimulation, at 1.0-2.5 mM- and above 10 mM-Ca2+. Mg2+ was inhibitory at all concentrations. EDTA OR EGTA only slightly decreased adenylate cyclase activity, suggesting that another metal ion may be necessary for activity. Adenylate cyclase activity was stimulated by 10mM-isoproterenol and 10 micronM-adrenaline in the presence of a phosphodiesterase inhibitor. Phytohaemagglutinin and prostaglandin E1 alone or in combination with isoproterenol had no effect on nuclear adenylate cyclase activity in either nuclei preparation. These results indicate that human lymphocyte nuclei contain one or several adenylate cyclases which differ from adenylate cyclases found in other subcellular fractions of these cells with regard to their bivalentcation requirements and responsiveness to pharmacological agents.
| S-EPMC1164631 | biostudies-other
Project description:Human peripheral lymphocytes were broken in a Dounce homogenizer and subcellular fractions enriched in plasma membranes or microsomal particles and mitochondria were isolated by centrifugation through a discontinuous sucrose gradient. Various agents that promote cyclic AMP accumulation in intact lymphocytes were compared in their ability to stimulate adenylate cyclase activity in the individual fractions. Plasma-membrane-rich fractions that were essentially free of other subcellular particles as judged by electron microscopy and marker enzyme measurements responded to fluoride, but weakly or not at all to prostaglandin E1 and other prostaglandins. Microsomal and mitochondrial-rich fractions responded markedly to both prostaglandin E1 and fluoride. In some, but not all, experiments phytohaemagglutinin produced a modest increase in enzyme activity in plasma-membrane-rich fractions. Catecholamines, histamine, parathyrin, glucagon and corticotropin produced little or no response. In the absence of theophylline, adenosine (1-10 micronM) stimulated basal enzyme activity, although at higher concentrations the responses to prostaglandin E1 and fluoride were inhibited. GTP (1-100 micronM) and GMP(5-1000 micronM) respectively inhibited or stimulated the response to fluoride, whereas the converse was true with prostaglandin E1.
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Project description:After fractionation of rabbit bone marrow into dividing (early) and non-dividing (late) erythroid cells, the adenylate cyclase activity of membrane ghosts was assayed in the presence of guanine nucleotides ((GTP and its analogue p[NH]ppG (guanosine 5'-[beta, gamma-imido]triphosphate))), the beta-adrenergic agonist L-isoprenaline (L-isoproterenol) and the antagonist L-propranolol. Both GTP and p[NH]ppG increased the adenylate cyclase activity of early and late erythroblasts, whereas the stimulating effect of the beta-adrenergic drug L-isoprenaline was limited to the immature dividing bone-marrow cells. The effect of L-isoprenaline was completely inhibited by the antagonist L-propranolol, confirming that the response was due to stimulation of beta-adrenergic receptors on the plasma membrane. The lack of response of non-dividing erythroblasts to beta-adrenergic stimuli is not due to loss of beta-receptors, since both dividing and non-dividing cells bind the selective ligand [125I]iodohydroxybenzylpindolol with almost equal affinities, the apparent dissociation constants, Kd, being 0.91 X 10(-8)M and 1.0 X 10(-8) M respectively. The number of beta-adrenergic receptors per cell was 2-fold higher in the dividing cells. No significant change in binding affinity for GTP and p[NH]ppG during erythroblast development was observed: the dissociation constants of both guanine nucleotides were almost identical with early and late erythroblast membrane preparations [2-3 (X 10(-7) M]. With dividing cells, however, in the presence of L-isoprenaline the dissociation constants of GTP and p[NH]ppG were lower (6 X 10(-8) M). The dose-response curves for isoprenaline competition in binding of [125I]iodohydroxybenzylpindolol by dividing cells showed that the EC50 (effective concentration for half maximum activity) value for isoprenaline was higher in the presence of p[NH]ppG. With non-dividing cells the EC50 value for isoprenaline was equal in the presence and in the absence of p[NH]ppG and similar to that observed with dividing-cell membranes in the presence of the nucleotide. Thus differentiation of rabbit bone-marrow erythroid cells seems to be accompanied by uncoupling of the beta-adrenergic receptors from the adenylate cyclase catalytic protein as well as by a decrease in the number of receptors per cell, but not by changes in the catecholamine and guanine-nucleotide-binding affinities.
| S-EPMC1154257 | biostudies-other
Project description:We have studied the properties of beta-adrenergic receptors and of their interaction with adenylate cyclase in the chick myocardium during embryogenesis. Between 4.5 and 7.5 days in ovo the number of receptors determined by (-)-[3H]dihydroalprenolol ([3H]DHA) binding is constant at approx. 0.36 pmol of receptor/mg of protein. By day 9 the density decreases significantly to 0.22 pmol of receptor/mg of protein. At day 12.5--13.5 the number was 0.14--0.18 pmol of receptor/mg of protein. This number did not change further up to day 16. The same results were obtained with guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) added to the assay mixtures. There was no significant change in receptor affinity for the antagonist [3H]DHA between days 5.5 and 13. Despite the decrease in numbers of beta-adrenergic receptors, there was no change in basal, p[NH]ppG-, isoprenaline- or isoprenaline-plus-p[NH]ppG-stimulated adenylate cyclase activity between days 3 and 12 of development. We conclude that beta-adrenergic receptors and adenylate cyclase are not co-ordinately regulated during early embryonic development of the chick heart. Some of the beta-adrenergic receptors present very early in the ontogeny of cardiac tissue appear not to be coupled to adenylate cyclase since their loss is not reflected in decreased activation of the enzyme.
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Project description:The subcellular distribution of the alpha 2-adrenergic receptor, pertussis-toxin substrates (Gi, the inhibitory G-protein) and adenylate cyclase was determined in human platelets. The alpha 2-adrenergic receptor and pertussis-toxin substrate activity codistribute with surface membranes identified by a novel fluorescent-lectin method. The platelet granule fractions did not contain detectable Gi. Only 2-4% of the total pertussis-toxin substrate activity appears in soluble fractions, and this amount was not increased upon addition of purified beta gamma units or after pretreatment of platelets with adrenaline. There is no evidence for compartmentation of the alpha 2-adrenergic receptor or Gi to account for the low-affinity component of agonist binding to the alpha 2-adrenergic receptor in human platelet membranes. Translocation of Gi from plasma membrane to platelet cytosol or granules does not appear to play any significant role in the mechanism of alpha 2-receptor-mediated platelet activation.
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Project description:Desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes results in a 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of beta-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37 degrees C, pH 8.0. In both preparations alkaline phosphatase treatment significantly decreased desensitization of agonist-stimulated adenylate cyclase activity by 40-75% (P less than 0.05). Similar results were obtained after alkaline phosphatase treatment of membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized duck erythrocytes. Moreover, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with 12-O-tetradecanoylphorbol 13-acetate returned agonist-stimulated adenylate cyclase activity to near control values. In all experiments, inclusion of 20 mM-sodium phosphate to inhibit alkaline phosphatase during treatment of membranes attenuated the enzyme's effect on agonist-stimulated adenylate cyclase activity. In addition, alkaline phosphatase treatment of membranes from control and isoprenaline-desensitized turkey erythrocytes increased the mobility of beta-adrenergic-receptor proteins, specifically photoaffinity-labelled with [125I]iodocyanopindolol-diazirine, on SDS/polyacrylamide-gel electrophoresis. The increased mobility of the beta-adrenergic-receptor proteins after alkaline phosphatase treatment of membranes was again inhibited by 20 mM-phosphate. These results provide additional evidence for a direct role for phosphorylation in desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes.
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Project description:Possible modifications of the beta-adrenergic effector system during the development of bovine perirenal brown adipose tissue (BAT) in utero and its transformation into white-like adipose tissue after birth were studied. The parameters assessed were the level of expression of beta 1-, beta 2- and beta 3-adrenergic receptor (AR) mRNAs and the response of the plasma-membrane adenylate cyclase to (-)-isoprenaline and to the beta 3-agonist BRL 37344. The beta 3-AR mRNA was found to be expressed very early in utero, i.e. before the third month of foetal life. Then it increased dramatically (9-fold) between month 6 of foetal life and birth. A high beta 3-AR mRNA level was maintained after birth up to an age of 3 months. After conversion of BAT into white-like adipose tissue, i.e. in the adult bovine, the beta 3-AR mRNA expression became small or not detectable, and the beta 1-AR mRNA, which was expressed much less than the beta 3-AR mRNA in foetal life, became predominant. A response of the adenylate cyclase to (-)-isoprenaline was observed in foetal life (3.1-fold stimulation). It decreased after birth (1.8-fold stimulation) and then remained constant until adulthood. A response to BRL 37344 was also observed in foetal life (1.8-fold stimulation). It was maintained after birth, but disappeared in the adult. A possible relationship between the beta-AR expression and the adenylate cyclase response to (-)-isoprenaline on the one hand and the uncoupling-protein expression on the other is discussed. The bovine might represent a good model to understand the transition from brown to white fat in the human.
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Project description:We used a simple experimental approach to clarify some contradictory predictions of the collision coupling and equilibrium models (e.g. ternary complex, two-state ternary complex or quinternary complex), which describe G-protein-mediated beta-adrenergic receptor signalling in essentially different manners. Analysis of the steady-state coupling of beta-adrenoceptors to adenylate cyclase in turkey erythrocyte membranes showed that: (1) in the absence of an agonist, Gpp(NH)p (a hydrolysis-resistant analogue of GTP) can activate adenylate cyclase very slowly; (2) this activity reaches a steady state in approx. 5 h, the extent of activity depending on the concentration of the nucleotide; (3) isoprenaline-activated steady-state adenylate cyclase can be inactivated by propranolol (a competitive antagonist that relaxes the receptor activation), in the presence of Gpp(NH)p (which provides a virtual absence of GTPase) and millimolar concentrations of Mg2+ (the rate of this inactivation is relatively fast); (4) increasing the concentration of Gpp(NH)p can saturate the steady-state activity of adenylate cyclase. The saturated enzyme activity was lower than that induced by isoprenaline under the same conditions. This additional agonist-induced activation was reversible. In the light of these results, we conclude that agonist can also activate the guanine nucleotide-saturated system in the absence of GTPase by a mechanism other than guanine nucleotide exchange. We explain these phenomena in the framework of a quinternary complex model as an agonist-induced and receptor-mediated dissociation of guanine nucleotide-saturated residual heterotrimer, the equilibrium concentration of which is not necessarily zero. These results, which suggest a continuous interaction between receptor and G-protein, can hardly be accommodated by the collision coupling model that was originally suggested for the present experimental system and then applied to many other G-protein systems. Therefore we attempt to unify the equilibrium and collision coupling approaches to provide a consistent theoretical basis for the G-protein-mediated beta-adrenergic receptor signalling in turkey erythrocyte membranes.
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Project description:Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide; it takes part in the regulation of various physiological processes, such as feeding, reproduction, catecholamine synthesis, thermoregulation, motor activity, brain development and neuronal survival. Since PACAP plays important regulatory roles, we hypothesized that the level of PACAP in blood is associated with expression of other proteins, which are involved in different metabolic pathways. The objective of the present study was to compare plasma protein profiles of cows with high and low plasma PACAP levels. Differential proteome analyses were performed by two-dimensional gel electrophoresis (2D-PAGE) followed by tryptic digestion and protein identification by liquid chromatography−mass spectrometry (LC-MS). A total of 210 protein spots were detected, and 16 protein spots showed statistically significant differences (p < 0.05) in the expression levels between groups. Ten spots showed a higher intensity in the high-PACAP-concentration group, while six spots were more abundant in the low-PACAP-concentration group. The functions of the differentially expressed proteins indicate that the PACAP level of plasma is related to the lipid metabolism and immune status of cattle.
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Project description:To understand the trafficking of endocytosed hemoglobin (Hb) in Leishmania, we investigated the characteristics of in vitro fusion between endosomes containing biotinylated Hb (BHb) and avidin-horseradish peroxidase (AHRP). We showed that early endosome fusion in Leishmania is temperature and cytosol dependent and is inhibited by ATP depletion, ATPgammaS, GTPgammaS and N-ethylmaleimide treatment. The Rab5 homolog from Leishmania donovani, LdRab5, was cloned and expressed. Our results showed that homotypic fusion between the early endosomes in Leishmania is Rab5 dependent. Early endosomes containing BHb fused efficiently with late endosomes in a process regulated by Rab7, whereas no fusion between early and late endosomes was detected using fluid phase markers. Pre-treatment of early endosomes containing BHb with monoclonal antibody specific for the C-terminus of the Hb receptor (HbR) or the addition of the C-terminal cytoplasmic fragment of the HbR specifically inhibited the fusion with late endosomes, suggesting that signal(s) mediated through the HbR cytoplasmic tail promotes the fusion of early endosomes containing Hb with late endosomes.
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