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Roles of GRK and PDE4 activities in the regulation of beta2 adrenergic signaling.

Xin W, Tran TM, Richter W, Clark RB, Rich TC - J. Gen. Physiol. (2008)

Bottom Line: We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels.This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 muM isoproterenol triggered transient increases in cAMP levels near the plasma membrane.Pretreatment of cells with 10 muM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 muM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 muM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine and Center for Lung Biology, University of South Alabama, Mobile, AL 36688, USA.

ABSTRACT
An important focus in cell biology is understanding how different feedback mechanisms regulate G protein-coupled receptor systems. Toward this end we investigated the regulation of endogenous beta(2) adrenergic receptors (beta2ARs) and phosphodiesterases (PDEs) by measuring cAMP signals in single HEK-293 cells. We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 muM isoproterenol triggered transient increases in cAMP levels near the plasma membrane. Pretreatment of cells with 10 muM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 muM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 muM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included. Taken together, these data indicate that either GRK-mediated desensitization of beta2ARs or PKA-mediated stimulation of PDE4 activity is sufficient to cause declines in cAMP signals. In addition, the data indicate that GRK-mediated desensitization is primarily responsible for a sustained suppression of beta2AR signaling. To better understand the interplay between receptor desensitization and PDE4 activity in controlling cAMP signals, we developed a mathematical model of this system. Simulations of cAMP signals using this model are consistent with the experimental data and demonstrate the importance of receptor levels, receptor desensitization, basal adenylyl cyclase activity, and regulation of PDE activity in controlling cAMP signals, and hence, on the overall sensitivity of the system.

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GRKs mediate the sustained suppression of isoproterenol-induced cAMP signals. HEK-293 cells were exposed to 1 μM isoproterenol, followed by a 5-min washout, and a rechallenge with isoproterenol at the times indicated. Shown are representative traces of cells exposed to control intracellular solution (A) or cells exposed to intracellular solution containing PKI (B), 59-74E (C), or PKI + 59-74E (D). (E) Ratio of the peak currents elicited by exposure to isoproterenol (second exposure/first exposure).
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fig4: GRKs mediate the sustained suppression of isoproterenol-induced cAMP signals. HEK-293 cells were exposed to 1 μM isoproterenol, followed by a 5-min washout, and a rechallenge with isoproterenol at the times indicated. Shown are representative traces of cells exposed to control intracellular solution (A) or cells exposed to intracellular solution containing PKI (B), 59-74E (C), or PKI + 59-74E (D). (E) Ratio of the peak currents elicited by exposure to isoproterenol (second exposure/first exposure).

Mentions: We then tested the effects of these pharmacological agents on signals elicited during a two-pulse stimulation with isoproterenol. During this protocol, cells were exposed to 1 μM isoproterenol for 5 min, washed for 5 min, and then reexposed to 1 μM isoproterenol (Fig. 4). There was little or no response to the second stimulation with isoproterenol when either vehicle or 20 nM PKI was included in the pipette solution (Fig. 4, A, B, and E). In contrast, there was a substantial response to the second pulse of isoproterenol when 59-74E was included in the pipette solution (Fig. 4, C and E). Interestingly, when both PKI and 59-74E were included in the pipette solution, there was substantial response to the second pulse of isoproterenol (Fig. 4 D), but the relative amplitude of the second response was not significantly greater than with 59-74E alone (Fig. 4 E). The incomplete recovery may be due to incomplete inhibition of GRK activity with 59-74E, or to the presence of a GRK(s) that is not significantly inhibited by 59-74E. Based upon these observations, we propose that GRK-mediated desensitization is primarily responsible for the sustained suppression of cAMP signals during the 5-min wash period.


Roles of GRK and PDE4 activities in the regulation of beta2 adrenergic signaling.

Xin W, Tran TM, Richter W, Clark RB, Rich TC - J. Gen. Physiol. (2008)

GRKs mediate the sustained suppression of isoproterenol-induced cAMP signals. HEK-293 cells were exposed to 1 μM isoproterenol, followed by a 5-min washout, and a rechallenge with isoproterenol at the times indicated. Shown are representative traces of cells exposed to control intracellular solution (A) or cells exposed to intracellular solution containing PKI (B), 59-74E (C), or PKI + 59-74E (D). (E) Ratio of the peak currents elicited by exposure to isoproterenol (second exposure/first exposure).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279169&req=5

fig4: GRKs mediate the sustained suppression of isoproterenol-induced cAMP signals. HEK-293 cells were exposed to 1 μM isoproterenol, followed by a 5-min washout, and a rechallenge with isoproterenol at the times indicated. Shown are representative traces of cells exposed to control intracellular solution (A) or cells exposed to intracellular solution containing PKI (B), 59-74E (C), or PKI + 59-74E (D). (E) Ratio of the peak currents elicited by exposure to isoproterenol (second exposure/first exposure).
Mentions: We then tested the effects of these pharmacological agents on signals elicited during a two-pulse stimulation with isoproterenol. During this protocol, cells were exposed to 1 μM isoproterenol for 5 min, washed for 5 min, and then reexposed to 1 μM isoproterenol (Fig. 4). There was little or no response to the second stimulation with isoproterenol when either vehicle or 20 nM PKI was included in the pipette solution (Fig. 4, A, B, and E). In contrast, there was a substantial response to the second pulse of isoproterenol when 59-74E was included in the pipette solution (Fig. 4, C and E). Interestingly, when both PKI and 59-74E were included in the pipette solution, there was substantial response to the second pulse of isoproterenol (Fig. 4 D), but the relative amplitude of the second response was not significantly greater than with 59-74E alone (Fig. 4 E). The incomplete recovery may be due to incomplete inhibition of GRK activity with 59-74E, or to the presence of a GRK(s) that is not significantly inhibited by 59-74E. Based upon these observations, we propose that GRK-mediated desensitization is primarily responsible for the sustained suppression of cAMP signals during the 5-min wash period.

Bottom Line: We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels.This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 muM isoproterenol triggered transient increases in cAMP levels near the plasma membrane.Pretreatment of cells with 10 muM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 muM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 muM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine and Center for Lung Biology, University of South Alabama, Mobile, AL 36688, USA.

ABSTRACT
An important focus in cell biology is understanding how different feedback mechanisms regulate G protein-coupled receptor systems. Toward this end we investigated the regulation of endogenous beta(2) adrenergic receptors (beta2ARs) and phosphodiesterases (PDEs) by measuring cAMP signals in single HEK-293 cells. We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 muM isoproterenol triggered transient increases in cAMP levels near the plasma membrane. Pretreatment of cells with 10 muM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 muM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 muM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included. Taken together, these data indicate that either GRK-mediated desensitization of beta2ARs or PKA-mediated stimulation of PDE4 activity is sufficient to cause declines in cAMP signals. In addition, the data indicate that GRK-mediated desensitization is primarily responsible for a sustained suppression of beta2AR signaling. To better understand the interplay between receptor desensitization and PDE4 activity in controlling cAMP signals, we developed a mathematical model of this system. Simulations of cAMP signals using this model are consistent with the experimental data and demonstrate the importance of receptor levels, receptor desensitization, basal adenylyl cyclase activity, and regulation of PDE activity in controlling cAMP signals, and hence, on the overall sensitivity of the system.

Show MeSH
Related in: MedlinePlus