Cyclic AMP produces desensitization of prostacyclin and adenosine A2 receptors in hybrid cell lines but does not affect Gs function

Keen, Mary; Kelly, Eamonn; Krane, Anke; Austin, Andrew; Wiltshire, Robin; Taylor, Nick; Docherty, Kevin; MacDermot, John

doi:10.1016/0167-4889(92)90039-e

Cited by 15 publications

(3 citation statements)

References 12 publications

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“…Agonist-dependent phosphorylation of the A 2b receptor has not yet been demonstrated. We have shown previously that the short term agonist-dependent homologous desensitization of A 2a and A 2b receptor-stimulated adenylyl cyclase in NG108 -15 cells is not mediated by cAMP-dependent protein kinase (24). Thus, if phosphorylation of these two A 2 receptor subtypes underlies their desensitization in NG108 -15 cells, a GRK-mediated process seems likely.…”

Section: Discussionmentioning

confidence: 90%

A Dominant Negative Mutant of the G Protein-Coupled Receptor Kinase 2 Selectively Attenuates Adenosine A₂Receptor Desensitization

1997

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G protein-coupled receptor kinases (GRKs) are thought to be important in mediating the agonist-induced phosphorylation and consequent desensitization of G protein-coupled receptor responses. NG108-15 mouse neuroblastoma X rat glioma cells express a wide range of G protein-coupled receptors and significant levels of GRK2. Therefore, to determine the role of GRK2 in agonist-induced desensitization of various G(s)-coupled receptors in NG108-15 cells, we stably transfected cells with a dominant negative mutant GRK2 construct (Lys220Arg). In homogenates prepared from cells overexpressing the dominant negative mutant GRK2, the acute stimulation of adenylyl cyclase by various receptor and nonreceptor agonists was the same as in control cells stably transfected with plasmid only. NG108-15 cells express both A2a and A2b adenosine receptors, which mediate activation of adenylyl cyclase, with both of these responses being subject to agonist-induced desensitization with a t1/2 of 15-20 min. In dominant negative mutant GRK2 cells, the rates of desensitization of A2a and A2b receptor-stimulated adenylyl cyclase were markedly slower than in plasmid transfected controls, with the latter being similar to wild-type cells. After a 20-min treatment with an adenosine agonist, the desensitization of A2a and A2b receptor-stimulated adenylyl cyclase in dominant negative mutant GRK2 cells was less than half that seen in plasmid transfected control cells. On the other hand, the agonist-induced desensitization of secretin and IP-prostanoid receptor-stimulated adenylyl cyclase was the same in dominant negative mutant GRK2 cells as in plasmid transfected control cells. These results indicate that in intact cells, GRK2 may mediate the desensitization of adenosine A2 receptors. Furthermore, there seems to be selectivity of GRK2 action between G(s)-coupled receptors because the agonist-induced desensitization of secretin and IP-prostanoid receptor-stimulated adenylyl cyclase was not affected by dominant negative mutant GRK2 overexpression.

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Section: Discussionmentioning

confidence: 90%

A Dominant Negative Mutant of the G Protein-Coupled Receptor Kinase 2 Selectively Attenuates Adenosine A₂Receptor Desensitization

1997

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“…Also, the present study suggests that PKA is selectively regulating secretin receptor signalling at the level of the receptor itself, as adenylyl cyclase responses to forskolin, iloprost and NECA were una ected by PKA inhibition. It has been reported that forskolin-mediated PKA activation can heterologously regulate A 2 adenosine and prostanoid IP receptor desensitization, however, these e ects were only observed after 17 h of forskolin treatment and were consistent with a decrease in receptor expression (Keen et al, 1992;Krane et al, 1994). As secretin partly exerts its physiological e ects via stimulation of adenylyl cyclase with subsequent activation of PKA (Ulrich et al, 1998), feedback inhibition by PKA is likely to play an important role in physiological regulation of secretin receptor responsiveness.…”

Section: British Journal Of Pharmacology Vol 135 (8)mentioning

confidence: 94%

Second messenger‐dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness

Ghadessy

Kelly

2002

British J Pharmacology

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1 The present study investigated the role of second messenger-dependent protein kinase A (PKA) and C (PKC) in the regulation of endogenous secretin receptor responsiveness in NG108-15 mouse neuroblastoma6rat glioma hybrid cells. 2 In whole cell cyclic AMP accumulation studies, activation of PKC either by phorbol 12-myristate 13-acetate (PMA) or by purinoceptor stimulation using uridine 5'-triphosphate (UTP) decreased secretin receptor responsiveness. PKC activation also inhibited forskolin-stimulated cyclic AMP accumulation but did not a ect cyclic AMP responses mediated by the prostanoid-IP receptor agonist iloprost, or the A 2 adenosine receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA). 3 In additivity experiments, saturating concentrations of secretin and iloprost were found to be additive in terms of cyclic AMP accumulation, whereas saturating concentrations of NECA and iloprost together were not. This suggests compartmentalization of G s -coupling components in NG108-15 cells and possible heterologous regulation of secretin receptor responsiveness at the level of adenylyl cyclase activation. 4 Cells exposed to the PKA inhibitor H-89, exhibited a time-dependent increase in secretin receptor responsiveness compared to control cells. This e ect was selective since cyclic AMP responses to forskolin, iloprost and NECA were not a ected by H-89 treatment. Furthermore, treatment with the protein synthesis inhibitor cycloheximide produced a time-dependent increase in secretin receptor responsiveness. 5 Together these results indicate that endogenous secretin receptor responsiveness is regulated by PKC, PKA and protein neosynthesis in NG108-15 cells.

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“…A problem with the clinical utility of Ado is its potential to produce A 2 -adenosine-receptor desensitization after sustained infusion. Although desensitization of the A 2 -adenosine receptor has not been demonstrated in the pulmonary circulation, the downregulation of G s ␣ protein, inhibition of adenylyl cyclase, and activation of phosphodiesterase have been observed in other cell lines as mechanisms of A 2 -adenosine-receptor desensitization (2,11,12). We therefore sought to determine whether prolonged administration of Ado agonists results in sustained vasodilation or receptor desensitization in the pulmonary circulation.…”

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confidence: 99%

5-(N-ethylcarboxamido)adenosine desensitizes the A_2b-adenosine receptor in lung circulation

Haynes

Obiako

Babál

et al. 1999

American Journal of Physiology-Heart and Circulatory Physiology

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The adenosine agonist 5-( N-ethylcarboxamido)adenosine (NECA) induces vasodilation in the pulmonary circulation via A2-adenosine-receptor activation. We addressed whether prolonged treatment with NECA desensitizes in A2-adenosine- receptor function in isolated lung and pulmonary artery smooth muscle cells (PASMC). In lung microcirculation preconstricted with a hypoxic gas, initial administration of NECA caused a 57% vasodilatory response after 3–4 min. Readministration of NECA after 45 min resulted in minimal vasodilation. The highest accumulation of PASMC cAMP occurred 3–5 min after NECA, coincident with NECA-induced vasodilation. In PASMCs treated with NECA for 45 min, cAMP did not increase. Isoproterenol- and indolidan-induced vasodilation remained intact in NECA-desensitized lungs. In NECA-desensitized PASMCs, isoproterenol-induced cAMP accumulation was decreased, suggesting a common mechanism of desensitization. cAMP accumulation was decreased in cholera toxin-treated NECA-desensitized PASMCs compared with cholera toxin-treated control PASMCs, demonstrating that Gsα-adenylyl cyclase signaling contributes to desensitization. The A2a-adenosine-receptor agonist CGS-21680C neither increased cAMP accumulation in PASMCs nor attenuated NECA-induced vasodilation. These data support that the A2b-adenosine receptor is responsible for pulmonary vasodilation and desensitization through mechanisms(s) involving Gsα-adenylyl cyclase signaling.

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Cyclic AMP produces desensitization of prostacyclin and adenosine A2 receptors in hybrid cell lines but does not affect Gs function

Cited by 15 publications

References 12 publications

A Dominant Negative Mutant of the G Protein-Coupled Receptor Kinase 2 Selectively Attenuates Adenosine A₂Receptor Desensitization

A Dominant Negative Mutant of the G Protein-Coupled Receptor Kinase 2 Selectively Attenuates Adenosine A₂Receptor Desensitization

Second messenger‐dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness

5-(N-ethylcarboxamido)adenosine desensitizes the A_2b-adenosine receptor in lung circulation

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Cyclic AMP produces desensitization of prostacyclin and adenosine A2 receptors in hybrid cell lines but does not affect Gs function

Cited by 15 publications

References 12 publications

A Dominant Negative Mutant of the G Protein-Coupled Receptor Kinase 2 Selectively Attenuates Adenosine A2Receptor Desensitization

A Dominant Negative Mutant of the G Protein-Coupled Receptor Kinase 2 Selectively Attenuates Adenosine A2Receptor Desensitization

Second messenger‐dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness

5-(N-ethylcarboxamido)adenosine desensitizes the A2b-adenosine receptor in lung circulation

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A Dominant Negative Mutant of the G Protein-Coupled Receptor Kinase 2 Selectively Attenuates Adenosine A₂Receptor Desensitization

A Dominant Negative Mutant of the G Protein-Coupled Receptor Kinase 2 Selectively Attenuates Adenosine A₂Receptor Desensitization

5-(N-ethylcarboxamido)adenosine desensitizes the A_2b-adenosine receptor in lung circulation