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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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Simulated effects of increased basal AC activity on β2AR-mediated cAMP signaling. (A and B)Increasing basal adenylyl cyclase activity from control levels of 0.005 μM/s to 0.05 μM/s increased both the basal PKA and PDE activities, in essence priming the negative feedback loop. The effects of this priming phenomena were (B) smaller cAMP signals leading to (A) decreased peak currents through CNG channels. In addition, the overall kinetics of the signal were slowed with increased basal AC activity, and, at the highest basal AC activity shown, were no longer transient.
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fig8: Simulated effects of increased basal AC activity on β2AR-mediated cAMP signaling. (A and B)Increasing basal adenylyl cyclase activity from control levels of 0.005 μM/s to 0.05 μM/s increased both the basal PKA and PDE activities, in essence priming the negative feedback loop. The effects of this priming phenomena were (B) smaller cAMP signals leading to (A) decreased peak currents through CNG channels. In addition, the overall kinetics of the signal were slowed with increased basal AC activity, and, at the highest basal AC activity shown, were no longer transient.

Mentions: We next used this model to examine the effects of increasing basal adenylyl cyclase activity on the responsiveness of the system (Fig. 8). We let the system reach equilibrium at each level of basal adenylyl cyclase activity (in the absence of receptor activation) to determine the initial conditions (e.g., initial cAMP levels, PKA and PDE activities, etc.). Interestingly, in the presence of small (twofold) increases in basal adenylyl cyclase activity, receptor-mediated cAMP signals were markedly slower with smaller amplitudes. These effects were due to the increased basal cAMP levels activating PKA, and, in turn, PKA stimulating PDE4 activity. In essence, increased basal adenylyl cyclase activity primed PDE-mediated feedback on subsequent cAMP signals. 10-fold increases in basal adenylyl cyclase activity triggered even further reductions in the peak cAMP levels. The higher basal adenylyl cyclase activity also largely eliminated the transient nature of the response, again due to increased PKA and PDE activity.


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)

Simulated effects of increased basal AC activity on β2AR-mediated cAMP signaling. (A and B)Increasing basal adenylyl cyclase activity from control levels of 0.005 μM/s to 0.05 μM/s increased both the basal PKA and PDE activities, in essence priming the negative feedback loop. The effects of this priming phenomena were (B) smaller cAMP signals leading to (A) decreased peak currents through CNG channels. In addition, the overall kinetics of the signal were slowed with increased basal AC activity, and, at the highest basal AC activity shown, were no longer transient.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Simulated effects of increased basal AC activity on β2AR-mediated cAMP signaling. (A and B)Increasing basal adenylyl cyclase activity from control levels of 0.005 μM/s to 0.05 μM/s increased both the basal PKA and PDE activities, in essence priming the negative feedback loop. The effects of this priming phenomena were (B) smaller cAMP signals leading to (A) decreased peak currents through CNG channels. In addition, the overall kinetics of the signal were slowed with increased basal AC activity, and, at the highest basal AC activity shown, were no longer transient.
Mentions: We next used this model to examine the effects of increasing basal adenylyl cyclase activity on the responsiveness of the system (Fig. 8). We let the system reach equilibrium at each level of basal adenylyl cyclase activity (in the absence of receptor activation) to determine the initial conditions (e.g., initial cAMP levels, PKA and PDE activities, etc.). Interestingly, in the presence of small (twofold) increases in basal adenylyl cyclase activity, receptor-mediated cAMP signals were markedly slower with smaller amplitudes. These effects were due to the increased basal cAMP levels activating PKA, and, in turn, PKA stimulating PDE4 activity. In essence, increased basal adenylyl cyclase activity primed PDE-mediated feedback on subsequent cAMP signals. 10-fold increases in basal adenylyl cyclase activity triggered even further reductions in the peak cAMP levels. The higher basal adenylyl cyclase activity also largely eliminated the transient nature of the response, again due to increased PKA and PDE activity.

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