Activation of beta-adrenergic receptor kinase during myocardial ischemia

During myocardial ischemia, a local release of noradrenaline coincides with an increased density of beta-adrenergic receptors. The functional activity of these receptors, however, is mainly determined by their state of phosphorylation. The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates and thereby inactivates beta-adrenergic receptors after stimulation by receptor agonists, facilitating the binding of the inhibitor protein beta-arrestin to the receptors. beta ARK activation involves a translocation of the enzyme to the membrane. In the present study, we investigated the density and the functional activity of beta-adrenergic receptors, the enzymatic activity of beta ARK in membranes and cytosol, the mRNA levels of beta ARK-1, and the expression of beta-arrestin during stop-flow and low-flow ischemia in the isolated perfused rat heart. After 60 minutes of stop-flow ischemia, beta-adrenergic receptor density was upregulated, but beta-agonist-mediated adenylate cyclase activity was blunted. Simultaneously, beta ARK activity in the particulate fraction was significantly induced. The increase in beta ARK activity was reversible after inhibition of ischemia-evoked noradrenaline release by desipramine. Also, exposure to externally given noradrenaline increased beta ARK activity in the particulate fraction. Cytosolic beta ARK activity remained largely unchanged during stop-flow or low-flow ischemia. The steady state concentration of beta ARK-1 mRNA increased after 20 minutes of stop-flow ischemia and then returned to baseline values after another 20 minutes. Cardiac ischemia did not alter beta-arrestin levels. During myocardial ischemia, an increase in the number of beta-adrenergic receptors is paralleled by increased membrane activity of the receptor kinase beta ARK. This increased membrane activity may contribute to enhanced receptor phosphorylation and inactivation.     

Beta-adrenergic signal transduction in the failing and hypertrophied myocardium

A strong sympathetic activation has been observed in heart failure and is the cause of beta-adrenergic desensitization in this condition. On the receptor level there is downregulation of beta1-adrenergic receptors and uncoupling of beta2-adrenoceptors. The latter mechanism has been related to an increased activity and gene expression of beta-adrenoceptor kinase in failing myocardium, leading to phosphorylation and uncoupling of receptors. beta3-Adrenoceptors mediate negative inotropic effects, but alterations in these receptors are not known. In addition, an increase in inhibitory G protein alpha subunits (Gi alpha) has been suggested to be causally linked to adenylyl cyclase desensitization in heart failure. In contrast, the catalytic subunit of adenylyl cyclase, stimulatory G protein alpha and betagamma subunits, have been observed to be unchanged. Recent evidence shows that increases in Gi alpha also depress adenylyl cyclase in compensated cardiac hypertrophy both in monogenic and polygenic and in secondary hypertension. These increases of Gi alpha can suppress adenylyl cyclase in the absence of beta-adrenergic receptor downregulation. Since cardiac hypertrophy in pressure overload is a strong predictor of cardiac failure, these observations indicate that adenylyl cyclase desensitization by Gi alpha may be a pathophysiologically relevant mechanism contributing to the progression from compensated cardiac hypertrophy to heart failure.     

Insulin stimulates sequestration of beta-adrenergic receptors and enhanced association of beta-adrenergic receptors with Grb2 via tyrosine 350

G-protein-linked receptors, such as the beta2-adrenergic receptor, are substrates for growth factor receptors with intrinsic tyrosine kinase activity (Karoor, V., Baltensperger, K., Paul, H., Czech, M. P., and Malbon C. C. (1995) J. Biol. Chem. 270, 25305-25308). In the present work, the counter-regulatory action of insulin on catecholamine action is shown to stimulate enhanced sequestration of beta2-adrenergic receptors in either DDT1MF-2 smooth muscle cells or Chinese hamster ovary cells stably expressing beta2-adrenergic receptors. Both insulin and insulin-like growth factor-1 stimulate internalization of beta-adrenergic receptors, contributing to the counter-regulatory effects of these growth factors on catecholamine action. In combination with beta-adrenergic agonists, insulin stimulates internalization of 50-60% of the complement of beta-adrenergic receptors. Insulin administration in vitro and in vivo stimulates phosphorylation of Tyr-350 of the beta-adrenergic receptor, creating an Src homology 2 domain available for binding of the adaptor molecule Grb2. The association of Grb2 with beta-adrenergic receptors was established using antibodies to Grb2 as well as a Grb2-glutathione S-transferase fusion protein. Insulin treatment of cells provokes binding of Grb2 to beta2-adrenergic receptors. Insulin also stimulates association of phosphatidylinositol 3-kinase and dynamin, via the Src homology 3 domain of Grb2. Both these interactions as well as internalization of the beta-adrenergic receptor are shown to be enhanced by insulin, beta-agonist, or both. The Tyr-350 --> Phe mutant form of the beta2-adrenergic receptor, lacking the site for tyrosine phosphorylation, fails to bind Grb2 in response to insulin, fails to display internalization of beta2-adrenergic receptor in response to insulin, and is no longer subject to the counter-regulatory effects of insulin on cyclic AMP accumulation. These data are the first to demonstrate the ability of a growth factor insulin to counter-regulate G-protein-linked receptor, the beta-adrenergic receptor, via a new mechanism, i.e. internalization.     

Mechanism underlying histamine-induced desensitization of amylase secretion in rat parotid glands

1. Histamine acted on H2 receptors in rat parotid tissues and induced the amylase secretion. Immunoblot analysis by using anti-H2 receptor protein antiserum demonstrated that histamine induced the increase and decrease in the amounts of H2 receptor proteins in basolateral and intracellular membranes, respectively. 2. Short-term treatment with histamine resulted in decreases in amylase secretion, the density of H2 receptors and their affinity for the agonists during further incubation with histamine, but showed an unaltered secretory response to isoproterenol, indicating that the histamine-induced desensitization was confined to H2 receptors. 3. This treatment triggered a 20% decrease in the histamine-stimulated adenylate cyclase activity and a 40% decrease in the phosphorylation level of Gi2alpha protein in the tissues, resulting in an increase in pertussis toxin (IAP)-catalyzed ADP-ribosylation of the protein. An enhancement of cholera toxin-catalyzed ADP-ribosylation of Gs protein was observed only during the first incubation with histamine. 4. This treatment triggered a 30% decrease and a 60% increase in the histamine-stimulated activities of protein kinase A and protein phosphatase 2A in the tissues, respectively. 5. Pretreatment with okadaic acid completely blocked the histamine-induced decrease in amylase secretion and increase in IAP-catalyzed ADP-ribosylation of Gi protein. The levels of Gi2alpha and Gs alpha proteins in the tissues were not modified by histamine treatment and the level of Gi2alpha protein was not affected by pretreatment with okadaic acid, as assessed by immunoblot analyses with anti-Gi2alpha and anti-Gs alpha protein antiserum. 6. The regulation of Gi2alpha protein phosphorylation in parotid tissues plays an important role in the histamine-induced desensitization of amylase secretion.     

Gi-mediated activation of the p21ras-mitogen-activated protein kinase pathway by alpha 2-adrenergic receptors expressed in fibroblasts

The alpha 2-adrenergic receptors are linked to inhibition of adenylylcyclase and, under certain circumstances, to stimulation of phospholipid hydrolysis via pertussis toxin-sensitive G proteins. Here we show that alpha 2-adrenergic receptors can couple to an alternative signaling pathway. When expressed in Rat-1 cells, stimulation of the alpha 2A receptor, which couples to Gi2 and Gi3, causes rapid, transient activation of the protooncogene product p21ras as measured by an increase in the amount of bound GTP. Furthermore, alpha 2A receptor stimulation causes rapid phosphorylation of the p42 mitogen-activated protein (MAP) kinase. Pertussis toxin completely inhibits both p21ras activation and MAP kinase phosphorylation, but both responses appear to be independent of adenylylcyclase inhibition or phospholipase stimulation. Thus, alpha 2-adrenergic receptors can couple to the p21ras-MAP kinase pathway via Gi, which may explain the mitogenic potential of alpha 2 agonists in certain cell types; together with previous results, these findings further suggest that activation of this pivotal signaling pathway may be a common event in the action of Gi-coupled receptors.     

Agonist-mediated downregulation of G alpha i via the alpha 2-adrenergic receptor is targeted by receptor-Gi interaction and is independent of receptor signaling and regulation

One mechanism of long-term agonist-promoted desensitization of alpha2AR function is downregulation of the cellular levels of the alpha subunit of the inhibitory G protein, Gi. In transfected CHO cells expressing the human alpha2AAR, a 40.1 +/- 3.3% downregulation of Galphai2 protein occurred after 24 h of exposure of the cells to epinephrine, which was not accompanied by a decrease in Galphai2 mRNA. The essential step that targets Gi for degradation by agonist occupancy of the receptor was explored using mutated alpha2AAR lacking specific structural or functional elements. These consisted of 5HT1A receptor and beta2AR sequences substituted at residues 113-149 of the second intracellular loop and 218-235 and 355-371 of the N- and C-terminal regions of the third intracellular loop (altered Gi and Gs coupling), deletion of Ser296-299 (absent GRK phosphorylation), and substitution of Cys442 (absent palmitoylation and receptor downregulation). Of these mutants, only those with diminished Gi coupling displayed a loss of agonist-promoted Gi downregulation, thus excluding Gs coupling and receptor downregulation, palmitoylation, and phosphorylation as necessary events. Furthermore, coupling-impaired receptors consisting of mutations in the second or third loops ablated Gi downregulation, suggesting that a discreet structural motif of the receptor is unlikely to represent a key element in the process. While pertussis toxin ablated Gi downregulation, blocking downstream intracellular consequences of alpha2AAR activation or mimicking these pathways by heterologous means failed to implicate cAMP/adenylyl cyclase, phospholipase C, phospholipase D, or MAP kinase pathways in alpha2AAR-mediated Gi downregulation. Taken together, agonist-promoted Gi downregulation requires physical alpha2AAR-Gi interaction which targets Gi for degradation in a manner that is independent of alpha2AAR trafficking, regulation, or second messengers.     

Nuclear matrix proteins and their potential applications to diagnostic pathology

The sympathetic nervous system controls lipolysis in fat by activation of four adrenergic receptors: beta1, beta2, beta3, and alpha2. During pregnancy, maternal metabolism presents anabolic and catabolic phases, characterized by modifications of fat responsiveness to catecholamines. The contributions of the four adrenergic receptors to adipocyte responsiveness during pregnancy have never been studied. Our aim was to evaluate the influence of pregnancy on adrenergic receptor-mediated lipolysis in rabbit white adipocytes. Functional studies were performed using subtype-selective and non-selective adrenergic receptor agonists. Overall adrenergic responsiveness was measured with the physiological agonist epinephrine. Non-adrenergic agents were used to evaluate different steps of the lipolytic cascade. The alpha2- and beta1/beta2-adrenergic receptor numbers were determined with selective radioligands. Non-adrenergic agents revealed that pregnancy induced an intracytoplasmic modification of the lipolytic cascade in inguinal but not in retroperitoneal adipocytes. Pregnancy induced an increase in beta1- and specially beta3-mediated lipolysis. The amounts of adipocyte beta1/beta2- and alpha2-adrenergic receptors were increased in pregnant rabbits. Epinephrine effects revealed an increased contribution of alpha2-adrenergic receptor-mediated antilipolysis in adipocytes from pregnant rabbits. These results indicate that pregnancy regulates adipocyte responsiveness to catecholamines mainly via the alpha2- and beta3-adrenergic pathways. Pregnancy induces an intracytoplasmic modification of the lipolytic cascade, probably via hormone-sensitive lipase, with differences according to fat location.-Bousquet-Mélou, A., C. Mu?oz, J. Galitzky, M. Berlan, and M. Lafontan. Pregnancy modifies the alpha2-beta-adrenergic receptor functional balance in rabbit fat cells.     

Chronic morphine administration increases beta-adrenergic receptor kinase (beta ARK) levels in the rat locus coeruleus

Based on the established role of beta-adrenergic receptor kinase (beta ARK) and beta-arrestin in the desensitization of several G protein-coupled receptors, we investigated the effect of chronic morphine administration on beta ARK and beta-arrestin levels in selected brain areas. Levels of beta ARK were measured by blot immunolabeling analysis using antibodies specific for two known forms of beta ARK, i.e., beta ARK1 and beta ARK2. It was found that chronic morphine treatment produced an approximately 35% increase in levels of beta ARK1 immunoreactivity in the locus coeruleus, but not in several other brain regions studied. In contrast, chronic morphine treatment failed to alter levels of beta ARK2 immunoreactivity in any of the brain regions studied. Levels of beta-arrestin immunoreactivity, measured using an antiserum that recognizes two major forms of this protein in brain, were also found to increase (by approximately 20%) in the locus coeruleus. It is proposed that chronic morphine regulation of beta ARK1 and beta-arrestin levels may contribute to opioid-receptor tolerance that is known to occur in this brain region.     

Signal transduction through trimeric G proteins in megakaryoblastic cell lines

The biogenesis of trimeric G proteins was investigated by measurement of the expression of alpha-subunits in the megakaryoblastic cell lines MEG-01, DAMI, and CHRF-288-11, representing stages of increasing maturation, and compared with platelets. Megakaryoblasts and platelets contained approximately equal amounts of Gi alpha-1/2, Gi alpha-3, Gq alpha, and G12 alpha protein. Maturation was accompanied by (1) downregulation of mRNA for Gs alpha and disappearance of iloprost-induced Ca2+ mobilization, (2) upregulation of the long form of Gs alpha protein (Gs alpha-L) and an increase in iloprost-induced cAMP formation, and (3) upregulation of G16 alpha mRNA and G16 alpha protein and appearance of thromboxane A2-induced signaling (Ca2+ mobilization and stimulation of prostaglandin I2-induced cAMP formation). Gz alpha protein was absent in the megakaryoblasts despite weak expression of Gz alpha mRNA in DAMI and relatively high levels of Gz alpha mRNA and Gz alpha protein in platelets. These findings reveal major changes in G protein-mediated signal transduction during megakaryocytopoiesis and indicate that G16 alpha couples the thromboxane receptor to phospholipase C beta.     

Differential coupling of alpha1-, alpha2-, and beta-adrenergic receptors to mitogen-activated protein kinase pathways and differentiation in transfected PC12 cells

Three adrenergic receptor families that selectively activate three different G proteins (alpha1/Gq/11, alpha2/Gi, and beta/Gs) were used to study mitogen-activated protein kinase (MAPK) activation and differentiation in PC12 cells. PC12 cells were stably transfected with alpha1A-, alpha2A-, or beta1-adrenergic receptors (ARs) in an inducible expression vector, and subclones were characterized. Norepinephrine stimulated inositol phosphate formation in alpha1A-transfected cells, inhibited cyclic adenosine 3'5'-monophosphate (cAMP) formation in alpha2A-transfected cells, and stimulated cAMP formation in beta1-transfected cells. Nerve growth factor activated extracellular signal-regulated kinases (ERKs) in all cell lines; however, norepinephrine activated ERKs only in alpha1A- and beta1-transfected cells but not in alpha2A-transfected cells. Norepinephrine also activated c-Jun NH2-terminal kinase and p38 MAPK in alpha1A-transfected cells but not in beta1- or alpha2A-transfected cells. Norepinephrine caused differentiation of PC12 cells expressing alpha1A-ARs but not those expressing beta1- or alpha2A-ARs. However, norepinephrine acted synergistically with nerve growth factor in promoting differentiation of cells expressing beta1-ARs. Whereas ERKs are activated by Gi- but not Gs-linked receptors in many fibroblastic cell lines, we observed the opposite in PC12 cells. The results show that activation of the different G protein signaling pathways has different effects on MAPKs and differentiation in PC12 cells, with Gq signaling pathways activating all three major MAPK pathways.     

The effect of combretastatin A-4 disodium phosphate in a C3H mouse mammary carcinoma and a variety of murine spontaneous tumors

OBJECTIVE: Human myometrium contains both beta1-adrenergic and beta2-adrenergic receptors. This study was designed to assess the importance of each beta-adrenergic receptor subtype in relaxation of human myometrial muscle strips. STUDY DESIGN: Radioligand binding studies were used to establish the presence of each beta-adrenergic receptor subtype, whereas highly selective beta1-antagonists and beta2-antagonists were used to assess the contribution of beta-adrenergic receptor subtypes to myometrial relaxation after exposure to (-)-isoproterenol. RESULTS: Membranes prepared from myometrium contained 82% +/- 4% beta2-adrenergic receptors. After contraction produced by exposure to potassium chloride (35 mmol/L), isoproterenol produced relaxation with half maximal effect at 0.02 micromol/L and a maximal relaxation of 52% +/- 3%. Beta1-antagonist CGP-20712A had no significant effect, whereas beta2-antagonist ICI-118551 produced a characteristic rightward shift of the isoproterenol concentration-relaxation relationship. CONCLUSIONS: Although both beta1-adrenergic receptors and beta2-adrenergic receptors are present in human myometrial tissue at term, relaxation by nonselective beta-agonist isoproterenol is mediated exclusively by beta2-adrenergic receptors.     

Treatment of articular cartilage defects of the knee with autologous chondrocyte implantation

alpha 1-Adrenergic receptor-mediated responses are overwhelming in adult rat hepatocytes. Inversely, beta-responses are predominant over alpha 1-responses in the hepatocytes that have been cultured at a low cell density (10(4) cells/cm2) for 24 h. The insulin-EGF-induced DNA synthesis in the beta-response-dominant hepatocytes was doubled by beta-agonists or cAMP-generating agents added far behind (16-20 h) the addition of insulin/EGF; i.e., immediately before the entry into the S-phase of the cell cycle. Agonists of alpha 1-adrenergic or other Ca2+, mobilising receptors added to the alpha 1-response-dominant hepatocytes increased DNA synthesis only if they were added within 1-2 h after the addition of insulin/EGF, at the early stage of G1-phase. Agonists of "non-dominant" receptors were rather antagonistic to agonists of "dominant" receptors. Thus, agonists of alpha 1-adrenergic (and other Ca2+ mobilising) receptors and agonists of beta-adrenergic (and other cAMP-generating) receptors acted as comitogens in their own particular manners in the presence of growth factors in hepatocytes in which the respective receptor functions were dominant.     

Global increases in seizure susceptibility in mice lacking 5-HT2C receptors: a behavioral analysis

OBJECTIVE: Vasodilation by beta-adrenergic receptors of smooth muscle cells appears to be impaired early after the onset of hypercholesteremia. The aim of this study was to analyze the modulation of beta-adrenergic receptor density and adenylyl cyclase activity in the presence of moderately elevated concentrations of LDL. The effects of beta 1- and beta 2-adrenergic receptor antagonists on LDL-induced receptor changes were studied. METHODS AND RESULTS: Media explants of porcine coronary arteries were incubated with moderately elevated LDL concentrations (0.7-3.9 mmol/l). The density of beta-adrenergic receptors was determined in plasma membranes using the radioligand [125I]iodocyanopinodolol. LDL (3.9 mmol/l) resulted in a decrease of beta-adrenergic receptor density (control 137 +/- 5 vs. 89 +/- 7 fmol/mg protein, P < 0.01). After removal of LDL and cultivation for an additional 3 days beta-adrenergic receptors increased to 129 +/- 5 fmol/mg. In the presence of the beta 1- or beta 2-adrenergic receptor antagonists the LDL-mediated decrease was inhibited. Addition of metoprolol after 3 days of LDL incubation caused a restoration of receptor density. The basal, isoproterenol- and forskolin-stimulated adenylyl cyclase activities were increased after LDL incubation by 180, 110 or 80%, respectively. CONCLUSION: Moderately elevated LDL levels decreased beta-adrenergic receptor density while adenylyl cyclase activity was simultaneously increased. beta 1- or beta 2-adrenergic receptor antagonists prevented this receptor decrease and might preserve the beta-adrenergic receptor density in the presence of moderately elevated LDL levels.     

Galphai is not required for chemotaxis mediated by Gi-coupled receptors

Pertussis toxin inhibits chemotaxis of neutrophils by preventing chemoattractant receptors from activating trimeric G proteins in the Gi subfamily. In HEK293 cells expressing recombinant receptors, directional migration toward appropriate agonist ligands requires release of free G protein betagamma subunits and can be triggered by agonists for receptors coupled to Gi but not by agonists for receptors coupled to two other G proteins, Gs and Gq. Because activation of any G protein presumably releases free Gbetagamma, we tested the hypothesis that chemotaxis also requires activated alpha subunits (Galphai) of Gi proteins. HEK293 cells were stably cotransfected with the Gi-coupled receptor for interleukin-8, CXCR1, and with a chimeric Galpha, Galphaqz5, which resembles Galphai in susceptibility to activation by Gi-coupled receptors but cannot regulate the Galphai effector, adenylyl cyclase. These cells, unlike cells expressing CXCR1 alone, migrated toward interleukin-8 even after treatment with pertussis toxin, which prevents activation of endogenous Galphai but not that of Galphaqz5. We infer that chemotaxis does not require activation of Galphai. Because chemotaxis is mediated by Gbetagamma subunits released when Gi-coupled receptors activate Galphaqz5, but not when Gq- or Gs-coupled receptors activate their respective G proteins, we propose that Gi-coupled receptors transmit a necessary chemotactic signal that is independent of Galphai.     

Endogenous norepinephrine regulates tumor necrosis factor-alpha production from macrophages in vitro

Evidence for the extraneuronal accumulation of norepinephrine has been demonstrated to occur in macrophage (M phi), yet the physiologic role of this system remains undefined. We have assessed the response of murine peritoneal M phi to adrenergic antagonists. We have also defined a physiologic role of a M phi-associated pool of the nonspecific adrenergic agonist norepinephrine. We investigated the constitutive involvement of alpha-adrenergic and beta-adrenergic receptors in LPS-induced TNF-alpha production. CFA-elicited M phis were incubated with LPS (1 microgram/ml) in the presence or absence of adrenergic agonists and/or antagonists. Although stimulation of alpha-adrenergic receptors increased TNF production and gene expression, beta-adrenergic receptors decreased it. Interestingly, when adrenergic antagonists along with LPS alone were added to M phi, they generated the response opposite to that produced by their suitable agonist, suggesting a role for endogenous norepinephrine in M phi. Thus, although alpha 2-adrenergic antagonists attenuated TNF production, beta-adrenergic antagonists augmented TNF expression in a concentration-dependent manner. Norepinephrine and epinephrine were found in M phi as determined by HPLC and LPS stimulation induced a significant decrease in their content. M phis were also incubated with LPS or medium only, washed, and then challenged 12 h later with LPS. When given a second LPS stimulation, M phis were found to have an increased response to alpha 2-adrenergic agonists and decreased response to alpha 2-adrenergic antagonists. Therefore, M phi-associated norepinephrine appears to regulate LPS-induced TNF production in an autocrine fashion.     

Binding of G protein beta gamma-subunits to pleckstrin homology domains

Ligand-induced activation of many receptors leads to dissociation of the alpha- and beta gamma-subunit complexes of heterotrimeric G proteins, both of which regulate a variety of effector molecules involved in cellular signaling processes. In one case, a cytosolic enzyme, the beta-adrenergic receptor kinase (beta ARK) binds to the dissociated, prenylated, membrane-anchored beta gamma-subunits of heterotrimeric G proteins (G beta gamma) and is thereby targeted to its membrane-bound receptor substrate. Quite recently, numerous proteins involved in cellular signal transduction have been shown to contain sequences homologous with a "domain" originally identified in the protein "pleckstrin" (pleckstrin homology domain; PH domain) and subsequently found in the G beta gamma interaction region of the beta ARK sequence. Here we demonstrate that glutathione S-transferase-fusion proteins, containing sequences encompassing the PH domain of nine proteins from this group, bind G beta gamma to varying extents. Binding of G beta gamma to these fusion proteins was documented either by a direct binding assay or by ability to block G beta gamma-mediated membrane translocation of beta ARK1. G beta gamma binding to these fusion proteins was inhibited by the alpha subunit of Go (Go alpha), indicating that the binding of G beta gamma to G alpha and the PH domain-containing fusion proteins is mutually exclusive. Studies with a series of truncated PH domains derived from the Ras-guanine-nucleotide-releasing factor indicate that the G beta gamma binding domain includes only the C-terminal portion of the PH domain and sequences just distal to this. Protein-protein interactions between G beta gamma and PH domain-containing proteins may play a significant role in cellular signaling analogous to that previously demonstrated for Src homology 2 and 3 domains.