Uncoupling of the platelet α2-adrenoceptor adenylate cyclase system by N-ethylmaleimide and the resulting effect on receptor density and sensitivity

Ja, García-Sevilla; Areso, Pilar; Mt, Giralt; Ulibarri, Isabel

doi:10.1016/0014-2999(88)90354-8

Cited by 14 publications

(4 citation statements)

References 9 publications

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“…There is evidence to suggest that agonist radioligands (e.g., [3H]clonidine and [3H]adrenaline) label preferentially the high-affinity state of the a2-adrenoceptor, whereas antagonists (e.g., [3H]yohimbine and [3H]rauwolscine) label both the high-and low-affinity states of this receptor (U'Prichard et al, 1983;Asakura et al, 1985). In human platdets, only the high-affinity population of the at2-adrenoceptor appears to be coupled with functional responses (Garcia-Sevilla et al,, 1988); therefore the binding of 3Hlabelled agonists is important in the study of pathological processes in which defects in a2-adrenoceptor function are postulated (e.g., endogenous depression, .…”

mentioning

confidence: 99%

Characterization and Regional Distribution of α₂‐Adrenoceptors in Postmortem Human Brain Using the Full Agonist [³H]UK 14304

Meana

Barturen

Garcı́a-Sevilla

1989

Journal of Neurochemistry

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The full agonist [3H]UK 14304 [5-bromo-6-(2-imidazolin-2-yl-amino)-quinoxaline] was used to characterize alpha 2-adrenoceptors in postmortem human brain. The binding at 25 degrees C was rapid (t1/2, 4.6 min) and reversible (t1/2, 14.1 min), and the KD determined from the kinetic studies was 0.48 nM. In frontal cortex, the rank order of potency of adrenergic drugs competing with [3H]UK 14304 or [3H]clonidine showed the specificity for an alpha 2A-adrenoceptor: UK 14304 approximately equal to yohimbine approximately equal to oxymetazoline approximately equal to clonidine greater than phentolamine approximately equal to (-)-adrenaline greater than idazoxan approximately equal to (-)-noradrenaline greater than phenylephrine greater than (+/-)-adrenaline much greater than corynanthine greater than prazosin much greater than (+/-)-propranolol. GTP induced a threefold decrease in the affinity of [3H]UK 14304, with no alteration in the maximum number of binding sites, suggesting that the radioligand labelled the high-affinity state of the alpha 2-adrenoceptor. In the frontal cortex, analyses of saturation curves indicated the existence of a single population of noninteracting sites for [3H]UK 14304 (KD = 0.35 +/- 0.13 nM; Bmax = 74 +/- 9 fmol/mg of protein). In other brain regions (hypothalamus, hippocampus, cerebellum, brainstem, caudate nucleus, and amygdala) the Bmax ranged from 68 +/- 7 to 28 +/- 4 fmol/mg of protein. No significant changes in the KD values were found in the different regions examined. The binding of [3H]UK 14304 was not affected by age, sex or postmortem delay.(ABSTRACT TRUNCATED AT 250 WORDS)

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confidence: 99%

Characterization and Regional Distribution of α₂‐Adrenoceptors in Postmortem Human Brain Using the Full Agonist [³H]UK 14304

Meana

Barturen

Garcı́a-Sevilla

1989

Journal of Neurochemistry

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“…However, the thiol groups of adenylate cyclase system are of different sensitivity to thioloxidation (Drummond 1981). Thus, low concentrations of NEM selectively inhibited the function of Ni-protein in human platelets ( Garcia-Sevilla et al 1988) and rat myocardium, whereas little or no effect on the catalytic unit or the N s -protein was observed (Jakobs et al 1982). From our data indicating that the action of glucagon on cAMP level is not affected by diamide and NEM, it is unlikely that under the conditions of the present study, the potentiation of forskolin-mediated increase of cAMP by diamide and NEM is due to their interference with the forskolin action on the catalytic unit and/or N s -protein.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, the existence of an N s - (Svoboda, Garcia-Morales, Dufrane, Sener, Valverde, Christophe and Malaisse 1985) and an Nj-protein in the adenylate cyclase system which possesses thiol groups has been reported in rat pancreatic islets (Katada and Ui 1981). In human platelets the oxidation/alkylation of thiol groups of the Ni-protein was found to alter its inhibitory action on the catalytic unit (Jakobs, Lasch, Aktories, Minuth and Schultz 1982;Garcia-Sevilla, Areso, Giralt and Ulibarri 1988). In pancreatic islets, the intracellular redox state is thought to be related to calcium net uptake as well as insulin release (Ammon and Mark 1985) since both could be inhibited by thiolreagents.…”

Section: Introductionmentioning

confidence: 99%

Potentiation of Forskolin-Induced Increase of cAMP by Diamide and N-Ethylmaleimide in Rat Pancreatic Islets

Anazodo

Müller²,

Safayhi³

et al. 1990

Horm Metab Res

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In isolated rat pancreatic islets, the effect of diamide and N-ethylmaleimide (NEM) on forskolin- as well as on glucagon-induced elevation of cAMP was studied. Forskolin and glucagon increased cAMP levels in batch-incubated islets. Diamide and NEM further augmented forskolin-induced increase of cAMP levels, whereas glucagon-stimulated elevation of cAMP was not affected. From our data it is likely that under the conditions of the present study, the thiols related to the Ns-protein and the catalytic unit are insensitive to oxidation and alkylation. The potentiation of forskolin-induced cAMP production in intact islet cells by diamide and NEM may be due to inactivation of the thiols related to the Ni-protein.

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“…Modification of this cysteine uncouples the G-protein from the activated receptor, abolishes signal generation and lowers the affinity of the receptor for its agonists. The affinity of the a,,-AR for [3H]UK 14,304 was about 30% lower after 30 min incubation with 100 PM NEM [30].…”

Section: Discussionmentioning

confidence: 99%

α_2A‐Adrenergic receptors activate protein kinase C in human platelets via a pertussis toxin‐sensitive G‐protein

Nieuwiand¹,

Wijburg²,

Willigen³

et al. 1994

FEBS Letters

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Abstract4,4'-Diisothiocyanato-stilbene-2,2'-dlsulfonic acid (DIDS) stimulates human platelets via cc ,,-adrenerglc receptor-mediated activation of protein kmase C (PKC) independent of the phospholipase C pathway. Here we show, that in permeabilized platelets activation of PKC by DIDS (20 PM), measured as "P incorporation in pleckstrin, is completely inhibited by guanosine 5'-(2-0-thio)diphosphate (200 fiM), an inhibitor of heterotrimeric G-proteins. Also pertussin toxin (4 ,&ml), which ADP-ribosylates the a-subunits of G,'s and G,, prevents pleckstrin phosphorylation by DIDS. N-Ethylmaleimide (SOpM), which uncouples G, from a,,-adrenoceptors, inhibits pleckstrin phosphorylation by DIDS in intact platelets. Activation of PKC by 55 nM phorbol 12-myristate 13-acetate and 500 nM platelet-activating factor are not disturbed by NEM. DIDS inhibits by 40 f 5% (n = 4) the pertussis toxin-catalyzed ["P]ADP-ribosylation of a 41 kDa protein fraction previously shown to contain the a-subunits of G,a-1, G,a-2 and G,a-3. Thus, the a,,-adrenergic receptor activates PKC via a G-protein of the G,-family.

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Uncoupling of the platelet α2-adrenoceptor adenylate cyclase system by N-ethylmaleimide and the resulting effect on receptor density and sensitivity

Cited by 14 publications

References 9 publications

Characterization and Regional Distribution of α₂‐Adrenoceptors in Postmortem Human Brain Using the Full Agonist [³H]UK 14304

Characterization and Regional Distribution of α₂‐Adrenoceptors in Postmortem Human Brain Using the Full Agonist [³H]UK 14304

Potentiation of Forskolin-Induced Increase of cAMP by Diamide and N-Ethylmaleimide in Rat Pancreatic Islets

α_2A‐Adrenergic receptors activate protein kinase C in human platelets via a pertussis toxin‐sensitive G‐protein

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Uncoupling of the platelet α2-adrenoceptor adenylate cyclase system by N-ethylmaleimide and the resulting effect on receptor density and sensitivity

Cited by 14 publications

References 9 publications

Characterization and Regional Distribution of α2‐Adrenoceptors in Postmortem Human Brain Using the Full Agonist [3H]UK 14304

Characterization and Regional Distribution of α2‐Adrenoceptors in Postmortem Human Brain Using the Full Agonist [3H]UK 14304

Potentiation of Forskolin-Induced Increase of cAMP by Diamide and N-Ethylmaleimide in Rat Pancreatic Islets

α2A‐Adrenergic receptors activate protein kinase C in human platelets via a pertussis toxin‐sensitive G‐protein

Contact Info

Product

Resources

About

Characterization and Regional Distribution of α₂‐Adrenoceptors in Postmortem Human Brain Using the Full Agonist [³H]UK 14304

Characterization and Regional Distribution of α₂‐Adrenoceptors in Postmortem Human Brain Using the Full Agonist [³H]UK 14304

α_2A‐Adrenergic receptors activate protein kinase C in human platelets via a pertussis toxin‐sensitive G‐protein