Characterization of α2-adrenoceptors in a plasma membrane enriched fraction from the insulin-secreting cell line RINm5F

Ullrich, Susanne; Wollheim, Claes B.

doi:10.1530/acta.0.1210525

Cited by 14 publications

(12 citation statements)

References 18 publications

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“…Previously, pharmacological characterizations of a 2 -AR-mediated inhibition of insulin secretion and catecholamine binding to insulin-secreting cells corroborated the expression of a 2A -ARs (25). The analysis of mRNA from purified b cells revealed that a 2A -AR and a 2C -AR are expressed in rat insulin-secreting cells (26).…”

Section: Discussionmentioning

confidence: 83%

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Inhibition of insulin secretion via distinct signaling pathways in alpha2-adrenoceptor knockout mice

Peterhoff

Sieg²,

Brede³

et al. 2003

European Journal of Endocrinology

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Objective: Adrenaline inhibits insulin secretion through activation of a 2 -adrenoceptors (ARs). These receptors are linked to pertussis toxin-sensitive G proteins. Agonist binding leads to inhibition of adenylyl cyclase, inhibition of Ca 2þ channels and activation of K þ channels. Recently, three distinct subtypes of a 2 -AR were described, a 2A -AR, a 2B -AR and a 2C -AR. At present, it is unknown which of these a 2 -AR subtype(s) may regulate insulin secretion. We used mice deficient in a 2 -ARs to analyze the coupling and role of individual a 2 -AR subtypes in insulin-secreting b cells. Methods: The inhibitory effect of adrenaline on insulin secretion was measured in freshly isolated and cultured wild type (wt) and a 2 -AR knockout (KO) mouse islets in order to examine the receptor subtypes which mediate adrenaline-induced inhibition of insulin secretion. Adenylyl cyclase activity was measured in isolated cultured islets. Membrane potential was measured using the amphotericin B permeabilized patch clamp method in isolated and cultured single islet cells. Results: In wt, a 2A -and a 2C -AR KO mouse islets, adrenaline, 1 mmol/l, inhibited secretion by 83, 80 and 100% respectively. In contrast, in a 2A/2C -AR double KO mouse islets, adrenaline had no effect on stimulated secretion indicating that both a 2A -AR and a 2C -AR, but not a 2B -AR, are functionally expressed in mouse islets. Surprisingly, glucose (16.7 mmol/l)-induced secretion in the presence of 1 mmol/l forskolin was greatly impaired in a 2A -AR KO islets. However, when cAMP levels were increased further by the combination of forskolin (5 mmol/l) and 3-isobutyl-1-methylxanthine (100 mmol/l), secretion was stimulated 2.7-fold (8.5-fold in wt islets). Adrenaline lowered the concentration of cAMP in wt and a 2C -AR KO mouse islets by 74%. Adrenaline also hyperpolarized wt and a 2C -AR KO b cells. In contrast, adrenaline did not inhibit adenylyl cyclase in islets of a 2A -AR KO mice, nor did it hyperpolarize a 2A -AR KO b cells. Conclusion: Adrenaline inhibits insulin release through a 2A -and a 2C -ARs via distinct intracellular signaling pathways.

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Section: Discussionmentioning

confidence: 83%

“…a high affinity to clonidine and a-yohimbine and a low affinity to prazosin (25). However, it is not known whether adrenaline exerts its different effects through the same or different isoforms of a 2 -ARs in insulinsecreting cells.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of insulin secretion via distinct signaling pathways in alpha2-adrenoceptor knockout mice

Peterhoff

Sieg²,

Brede³

et al. 2003

European Journal of Endocrinology

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“…Although pharmacological studies are consistent with the interpretation that the 2A AR subtype attenuates glucose-stimulated insulin release from pancreatic cells (Ullrich & Wollheim 1985, 1989a, Niddam et al 1990, and both 2A AR and 2C AR are expressed in a FACS-purified rat -cell population (Chan et al 1997), the lack of 2 AR subtype-specific ligands has prevented definitive identification of the subtype involved in suppression of insulin release. The development of mice null for each of these subtypes (Link et al 1995, 1996, Altman et al 1999) has helped to elucidate the subtype-specific role of each 2 AR subtype in vivo.…”

Section: Introductionmentioning

confidence: 85%

Proteomic exploration of pancreatic islets in mice null for the α2A adrenergic receptor

Friedman²,

Hill³

et al. 2005

Journal of Molecular Endocrinology

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The present studies extend recent findings that mice null for the 2A adrenergic receptor ( 2A AR KO mice) lack suppression of exogenous secretagogue-stimulated insulin secretion in response to 2 AR agonists by evaluating the endogenous secretagogue, glucose, ex vivo, and providing in vivo data that baseline insulin levels are elevated and baseline glucose levels are decreased in 2A AR KO mice. These latter findings reveal that the 2A AR subtype regulates glucose-stimulated insulin release in response to endogenous catecholamines in vivo. The changes in 2A AR responsiveness and resultant changes in insulin/glucose homeostasis encouraged us to utilize proteomics strategies to identify possible 2A AR downstream signaling molecules or other resultant changes due to perturbation of 2A AR expression. Although agonist stimulation of islets from wild type (WT) mice did not significantly alter islet protein profiles, several proteins were enriched in islets from 2A AR KO mice when compared with those from WT mice, including an enzyme participating in insulin protein processing. The present studies document the important role of the 2A AR subtype in tonic suppression of insulin release in response to endogenous catecholamines as well as exogenous 2 agonists and provide insights into pleiotropic changes that result from loss of 2A AR expression and tonic suppression of insulin release.

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“…Purified plasma membranes were prepared according to the method of Ullrich and Wollheim (18). Briefly, the monolayers were washed in homogenization buffer (250 mmol/1 sucrose, 5 mmol/1 HEPES, pH 7.4, 0.5 mmol/1 EDTA, and 0.5 mmol/1 phenylmethylsulfonyl fluoride [PMSF]), and harvested in the same buffer.…”

Section: Methodsmentioning

confidence: 99%

“…Consequently, as a step toward answering these questions, we have compared the effects of two activated receptors, those for norepinephrine and galanin, on the rates of receptor-G-protein interaction (by GTP7S binding) and the rates of activation and turnover of G-proteins (by measurement of GTPase activity) in the same 3-cell membranes. RINm5F cell membranes were used because of the already extensive characterization of the effects of galanin and other inhibitors (7)(8)(9)(10)(11)(12)(17)(18)(19)(20)(21) in this cell type. We also compared the ability of norepinephrine and galanin to facilitate ADP ribosylation of the a-subunits of G v and G o by cholera toxin (CTX).…”

mentioning

confidence: 99%

The α2-Adrenergic Receptor Is More Effective Than the Galanin Receptor in Activating G-Proteins in RINm5F β-cell Membranes

Gillison

Straub²,

Sharp³

1997

Diabetes

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Activated receptors for galanin and norepinephrine, and for several other agonists, inhibit insulin release from pancreatic beta-cells via pertussis toxin-sensitive Gi- and Go-proteins and by acting on at least four cellular mechanisms. These mechanisms include repolarization via activation of the ATP-sensitive potassium (K ATP) channel, inhibition of adenylyl cyclase, and inhibition by unknown mechanism at a "distal" site. For norepinephrine and galanin there is also inhibition of the L-type Ca2+ channel. Consequently, during simultaneous activation by multiple agonists, the effectiveness with which a receptor interacts with the G-proteins will, to some extent, determine the responses. This could have important consequences for the beta-cell. Therefore, the G-protein interactions of two activated receptors, those for norepinephrine and galanin, were compared in the same beta-cell membranes. Measurements were made of the rates of receptor-G-protein interaction (by GTPgammaS binding) and of the rates of turnover of G-proteins (by GTPase activity). A comparison was also made of the ability of norepinephrine and galanin to facilitate ADP ribosylation of the alpha-subunits of Gi and Go by cholera toxin (CTX). Such CTX-induced ADP ribosylation of Gi and Go occurs during G-protein interaction with an activated receptor. By measurement of the number of receptors in the membrane preparation used, the relative effectiveness of the two receptors was assessed. The alpha2-adrenergic receptor was found to be markedly more effective than the galanin receptor in activating G-proteins.

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Characterization of α2-adrenoceptors in a plasma membrane enriched fraction from the insulin-secreting cell line RINm5F

Cited by 14 publications

References 18 publications

Inhibition of insulin secretion via distinct signaling pathways in alpha2-adrenoceptor knockout mice

Inhibition of insulin secretion via distinct signaling pathways in alpha2-adrenoceptor knockout mice

Proteomic exploration of pancreatic islets in mice null for the α2A adrenergic receptor

The α2-Adrenergic Receptor Is More Effective Than the Galanin Receptor in Activating G-Proteins in RINm5F β-cell Membranes

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