scholarly journals Occupancy of G alpha s-linked receptors uncouples chemoattractant receptors from their stimulus-transduction mechanisms in the neutrophil

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 1052-1057
Author(s):  
BN Cronstein ◽  
KA Haines ◽  
S Kolasinski ◽  
J Reibman
Keyword(s):  
Protein Kinase ◽  
Adrenergic Receptors ◽  
Plasma Membranes ◽  
Kinase Inhibitor ◽  
Neutrophil Function ◽  
Gtpase Activity ◽  
Dibutyryl Camp ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Transduction Mechanisms

Adenosine and adrenergic agonists modulate neutrophil function by ligating their specific receptors (adenosine A2 and beta-adrenergic) on the neutrophil. When occupied, adenosine A2 and beta-adrenergic receptors stimulate, presumably via G alpha s, an increase in intracellular 3′, 5′ cyclic adenosine monophosphate (cAMP). cAMP affects cellular functions, in part, via protein kinase-mediated phosphorylation. Therefore, we determined whether inhibition of protein kinase A activity by KT5720 (10 mumol/L) reversed the inhibition of FMLP-stimulated O2- generation by 5′N-ethylcarboxamidoadenosine (NECA), the most potent adenosine A2 agonist, and by isoproterenol a potent beta-adrenergic agonist. KT5720 did not affect O2- generation stimulated by FMLP (125% +/- 13% of control, n = 5). However, KT5720 completely reversed inhibition of O2- generation by dibutyryl cAMP (DbcAMP, 1 mmol/L, from 26% +/- 5% to 84% +/- 25% of control, n = 5, P less than .004), but not by NECA (1 mumol/L, 26% +/- 5% v 33% +/- 7% of control, n = 5) or isoproterenol (10 mumol/L, 20% +/- 8% to 38% +/- 6% of control, n = 5). Nearly identical results were obtained using the less specific protein kinase inhibitor H-7. To determine whether occupancy of adenosine A2 or beta-adrenergic receptors inhibits neutrophil (PMN) activation by uncoupling chemoattractant receptors from G proteins, we determined the effect of NECA and isoproterenol on guanosine triphosphatase (GTPase) activity, a parameter that reflects G protein “activation,” of plasma membranes derived from human PMNs. Control GTPase activity was 138.9 pmol/mg protein/min; NECA (1 nmol/L to 1 mumol/L) and isoproterenol (10 nmol/L to 10 mumol/L) alone did not significantly affect GTPase activity. FMLP (0.1 mumol/L) increased GTPase activity by 31.9 +/- .9 pmol/mg/min, an increment that was markedly inhibited to approximately 50% of control by NECA (IC50 = 3 nmol/L, P less than .001, n = 5) and isoproterenol (IC50 = 30 nmol/L, P less than .001, n = 5). Neither cAMP nor dibutyryl cAMP (10 mumol/L and 1 mmol/L) affected resting or stimulated GTPase activity. In addition, neither adenosine nor DbcAMP affected protein phosphorylation in resting or stimulated neutrophils. Our studies are consistent with the hypothesis that ligation of G alpha s-linked receptors uncouples chemoattractant receptors from their signal-transduction mechanisms rather than inhibiting neutrophil function via cAMP-mediated effects.

scholarly journals Occupancy of G alpha s-linked receptors uncouples chemoattractant receptors from their stimulus-transduction mechanisms in the neutrophil

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 1052-1057 ◽  
Author(s):  
BN Cronstein ◽  
KA Haines ◽  
S Kolasinski ◽  
J Reibman
Keyword(s):  
Protein Kinase ◽  
Adrenergic Receptors ◽  
Plasma Membranes ◽  
Kinase Inhibitor ◽  
Neutrophil Function ◽  
Gtpase Activity ◽  
Dibutyryl Camp ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Transduction Mechanisms

Abstract Adenosine and adrenergic agonists modulate neutrophil function by ligating their specific receptors (adenosine A2 and beta-adrenergic) on the neutrophil. When occupied, adenosine A2 and beta-adrenergic receptors stimulate, presumably via G alpha s, an increase in intracellular 3′, 5′ cyclic adenosine monophosphate (cAMP). cAMP affects cellular functions, in part, via protein kinase-mediated phosphorylation. Therefore, we determined whether inhibition of protein kinase A activity by KT5720 (10 mumol/L) reversed the inhibition of FMLP-stimulated O2- generation by 5′N-ethylcarboxamidoadenosine (NECA), the most potent adenosine A2 agonist, and by isoproterenol a potent beta-adrenergic agonist. KT5720 did not affect O2- generation stimulated by FMLP (125% +/- 13% of control, n = 5). However, KT5720 completely reversed inhibition of O2- generation by dibutyryl cAMP (DbcAMP, 1 mmol/L, from 26% +/- 5% to 84% +/- 25% of control, n = 5, P less than .004), but not by NECA (1 mumol/L, 26% +/- 5% v 33% +/- 7% of control, n = 5) or isoproterenol (10 mumol/L, 20% +/- 8% to 38% +/- 6% of control, n = 5). Nearly identical results were obtained using the less specific protein kinase inhibitor H-7. To determine whether occupancy of adenosine A2 or beta-adrenergic receptors inhibits neutrophil (PMN) activation by uncoupling chemoattractant receptors from G proteins, we determined the effect of NECA and isoproterenol on guanosine triphosphatase (GTPase) activity, a parameter that reflects G protein “activation,” of plasma membranes derived from human PMNs. Control GTPase activity was 138.9 pmol/mg protein/min; NECA (1 nmol/L to 1 mumol/L) and isoproterenol (10 nmol/L to 10 mumol/L) alone did not significantly affect GTPase activity. FMLP (0.1 mumol/L) increased GTPase activity by 31.9 +/- .9 pmol/mg/min, an increment that was markedly inhibited to approximately 50% of control by NECA (IC50 = 3 nmol/L, P less than .001, n = 5) and isoproterenol (IC50 = 30 nmol/L, P less than .001, n = 5). Neither cAMP nor dibutyryl cAMP (10 mumol/L and 1 mmol/L) affected resting or stimulated GTPase activity. In addition, neither adenosine nor DbcAMP affected protein phosphorylation in resting or stimulated neutrophils. Our studies are consistent with the hypothesis that ligation of G alpha s-linked receptors uncouples chemoattractant receptors from their signal-transduction mechanisms rather than inhibiting neutrophil function via cAMP-mediated effects.

Beta-adrenergic modulation of insulin binding in skeletal muscle

1986 ◽  
Vol 250 (2) ◽  
pp. E198-E204
Author(s):  
B. Webster ◽  
S. R. Vigna ◽  
T. Paquette ◽  
D. J. Koerker
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Protein Phosphatase ◽  
Adrenergic Receptors ◽  
Plasma Membranes ◽  
Acute Exercise ◽  
Insulin Binding ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Adrenergic Antagonist

Both a high physiological concentration (13.1 nM) of epinephrine (E) and acute exercise (AEx) have previously been shown to increase 125I-insulin binding in skeletal muscle. To investigate the site and mechanism of the effect of epinephrine on binding and the possible link between epinephrine- and AEx-enhanced insulin binding, we measured insulin binding in three different preparations: 1) crude membranes derived from whole soleus muscle incubated in vitro with 13.1 nM E, 2) crude membranes with E present in the binding assay, and 3) purified plasma membranes with E present. Epinephrine enhanced binding in all three preparations by 169, 144, and 164%, respectively, at low concentrations of insulin but had little effect at high concentrations. Epinephrine, therefore appears to have its effect at the plasma membrane. Propranolol (10 microM), a beta-adrenergic antagonist, blocked E-enhanced insulin binding and when added to crude membranes made from soleus and extensor digitorum longus muscle of AEx rats reversed the increase in binding seen with exercise. This indicates that E-enhanced insulin binding is mediated by beta-adrenergic receptors and that AEx enhances insulin binding via beta-adrenergic receptors. Sodium orthovanadate (3 mM), a phosphotyrosyl-protein phosphatase inhibitor, also inhibited the increase in insulin binding due to E, implying that E may increase insulin binding by activating a phosphotyrosyl-protein phosphatase which decreases the phosphorylation of a plasma membrane protein, presumably the insulin receptor.

Desensitization of beta-adrenergic receptors in adipocytes causes increased insulin sensitivity of glucose transport

1996 ◽  
Vol 271 (2) ◽  
pp. E271-E276 ◽  
Author(s):  
A. Green ◽  
R. M. Carroll ◽  
S. B. Dobias
Keyword(s):  
Insulin Sensitivity ◽  
Glucose Transport ◽  
Adrenergic Receptors ◽  
Plasma Membranes ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
High Dose ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Adipocyte Plasma Membranes

To determine the effect of desensitization of adipocyte beta-adrenergic receptors on insulin sensitivity, rats were continuously infused with isoproterenol (50 or 100 micrograms.kg-1.h-1) for 3 days by osmotic minipumps. Epididymal adipocytes were isolated. The cells from treated animals were desensitized to isoproterenol, as determined by response of lipolysis (glycerol release). Binding of [125I]iodocyanopindolol was decreased by approximately 80% in adipocyte plasma membranes isolated from treated rats, indicating that beta-adrenergic receptors were downregulated. Cellular concentrations of Gn alpha and Gi alpha were not altered. Insulin sensitivity was determined by measuring the effect of insulin on glucose transport (2-deoxy-[3H]glucose uptake). Cells from the isoproterenol-infused rats were markedly more sensitive to insulin than those from control rats. This was evidenced by an approximately 50% increase in maximal glucose transport rate in cells from the high-dose isoproterenol-treated rats and by an approximately 40% decrease in the half-maximal effective concentration of insulin in both groups. 125I-labeled insulin binding to adipocytes was not altered by the isoproterenol infusions, indicating that desensitization of beta-adrenergic receptors results in tighter coupling between insulin receptors and stimulation of glucose transport.

Beta-adrenoceptor metabolism in wild-type, Gs, and protein kinase A-variant S49 cells

1990 ◽  
Vol 259 (1) ◽  
pp. C41-C46 ◽  
Author(s):  
J. R. Jasper ◽  
H. J. Motulsky ◽  
L. C. Mahan ◽  
P. A. Insel
Keyword(s):  
Protein Kinase ◽  
Adrenergic Receptors ◽  
Protein Kinase Activity ◽  
Wild Type ◽  
Beta Adrenergic Receptors ◽  
Dependent Protein Kinase ◽  
Beta Adrenergic ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Gs Alpha

To determine the role of the stimulatory guanine nucleotide-binding protein, Gs, and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase in the basal metabolism of beta-adrenergic receptors in S49 lymphoma cells, we measured the return of receptor number and function after irreversible blockade of receptors. After inactivation of receptors with the irreversible ligand N8-(bromoacetyl)-N'-[3-(4-indolyoxy)-2-hydroxypropyl]-(2)-1,8-diam ino-p- methane (BIM), beta-adrenergic receptors (defined as [125I]iodocyanopindolol binding sites) reappeared in a biphasic manner, the faster phase having a half-time (t 1/2) of 3-8 h (approximately 50% of the sites) and the slower phase greater than 40 h. Although the slow phase is not readily explained, recovery of binding sites during the first 10 h matched recovery of receptor function after BIM treatment (as measured by stimulation of cAMP accumulation) and recovery of receptor sites after downregulation induced by the agonist isoproterenol. Thus quantifying receptor recovery during the first 10 h after BIM treatment appears to be a reasonable method for examining basal receptor metabolism in S49 cells. Measured in this way, metabolism of beta-adrenergic receptors is very similar in wild-type S49 and the following variant clones: cyc- (absent Gs alpha), UNC and H21a (defective Gs alpha), and kin- (lacking cAMP-dependent protein kinase activity). Although previous data have demonstrated that agonist-promoted downregulation of beta-adrenergic receptors requires functional receptor-Gs coupling, the current data suggest that neither Gs nor cAMP-dependent protein kinase activity plays an important role in the regulation of basal metabolism of beta-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in beta-adrenergic receptors in dog livers during endotoxic shock

1983 ◽  
Vol 244 (5) ◽  
pp. R718-R723 ◽  
Author(s):  
M. S. Liu ◽  
S. Ghosh
Keyword(s):  
Adrenergic Receptors ◽  
Plasma Membranes ◽  
Endotoxic Shock ◽  
Scatchard Analysis ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Endotoxin Administration ◽  
Liver Plasma Membranes ◽  
Dog Liver

The effects of endotoxin administration on beta-adrenergic receptors in dog liver plasma membranes were studied using [3H]dihydroalprenolol as a radioactive ligand. The Scatchard analysis revealed a one-component binding characteristic both in control and endotoxin-injected dogs. The Kd (dissociation constant) value was increased by 60% (4.2 +/- 0.5 nM for control vs. 6.7 +/- 0.5 nM for endotoxic; P less than 0.01) and the Bmax (maximum binding capacity) was decreased by 38% (600 +/- 60 and 370 +/- 70 fmol/mg protein for control and endotoxic, respectively; P less than 0.01) 2 h following endotoxin administration. The competitive inhibition studies show that the apparent Kd values for (-)-isoproterenol, (-)-epinephrine, and (-)-norepinephrine were increased by 14, 51, and 5 times, respectively, 2 h postendotoxin. In addition, endotoxin in vitro had a dose-dependent inhibitory effect on the specific binding of [3H]dihydroalprenolol, and it also reduced the number of beta-receptors. These data demonstrate that endotoxin, both in vivo and in vitro, decreased the binding affinity and the number of beta-adrenergic receptors in dog liver plasma membranes. A modification of the beta-adrenergic receptors in dog livers induced by endotoxin administration may play an important role in the development of hepatic glucose dyshomeostasis during shock.

Abstract 407: Beta-blockers Reverse Beta Adrenergic Receptors Desensitization Under Hypoxia

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Yu Sun ◽  
Manveen K Gupta ◽  
Kate Stenson ◽  
Sathyamangla Prasad
Keyword(s):  
G Protein ◽  
Adrenergic Receptors ◽  
Plasma Membranes ◽  
Significant Loss ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Β Blocker ◽  
Β Blockers ◽  
Underlying Mechanisms ◽  
Grk2 Expression

Hypoxia to heart or brain is a primary cause of heart failure or stroke. Studies have shown that hypoxia increases the beta-adrenergic receptors (βARs) phosphorylation and dysfunction (Cheong et. al., 2016). These observations provide evidence that βARs can directly be regulated by hypoxia but less is known about the underlying mechanisms. We postulated that hypoxia shifts the homeostasis between kinase and phosphatase driven mechanisms may underlie βAR dysfunction. β2AR HEK 293 cells were exposed to hypoxia (2% O 2 ) and assessed the mechanisms underlying desensitization (G-protein coupled receptor kinases, GRKs) and resensitization (Protein phosphatase 2A, PP2A). Six hours of hypoxia treatment resulted in increase of βAR phosphorylation and GRK2 expression, and interestingly, the internalization of phosphorylated β2AR is β-arrestin independent. Assessment of βAR phosphorylation in the plasma membrane and endosomal fractions surprisingly, showed marked increase in β2AR phosphorylation in the endosomal fraction. Furthermore, we also observed that receptor associated PP2A activity was inhibited in the endosomes following hypoxia with minimal changes of activity at the plasma membranes. At the same time, PI3Kγ activity markedly upregulated in the endosomes along with an increase of I2PP2A phosphorylation. Similarly subjecting normal mice to 20 hours of hypoxia resulted in significant cardiac dysfunction (% FS: Normoxia 38.83% vs. Hypoxia 32.38%, P=0.0055; % EF: Normoxia 69.71% vs. Hypoxia 60.76%,P=0.0105) and was associated with significant increase in β2AR phosphorylation associated with significant loss in βAR function as measured by G-protein coupling adenylyl cyclase activity. Given that β-blockers confer beneficial effects, we tested whether β-blocker (propranolol) would prevent βARs phosphorylation under hypoxia or normoxia. Consistently, β-blocker treatment in normoxia results in increased β2AR phosphorylation however, remarkably β-blocker treatment in hypoxia results in loss of β2AR phosphorylation, reduction in GRK2 expression and increase in βAR-associated PP2A. These studies show that agonist-independent hypoxia-driven β2AR dysfunction can be ameliorated by β-blockers and the underlying mechanisms for this unexpected findings will be discussed in the presentation. These findings have significant clinical implications as understanding these mechanisms could provide novel insights into the benefits provided by β-blockers.

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