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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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Simulations demonstrate the effects of receptor levels on [cAMP] in the absence and presence of PDE4 activity. Solid circles represent normalized peak cAMP levels (from model simulations) in response to a 5-min stimulation with isoproterenol. Open circles represent cAMP levels measured at the end of the second stimulation with isoproterenol (following a 5-min stimulation with 1 μM isoproterenol followed by a 5-min wash). (A–F) The effects of receptor desensitization in the following scenarios: (A) high receptor levels (100 μM) and PDE4 activity inhibited (10 μM rolipram); (B) low receptor levels (1 μM) and PDE activity inhibited; (C) high receptor levels with basal PDE activity (20 nM PKI to inhibit PKA activity); (D) low receptor levels with basal PDE activity; (E) high receptor levels and PKA-mediated regulation of PDE activity intact; (F) low receptor levels and PKA-mediated regulation of PDE activity intact.
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fig9: Simulations demonstrate the effects of receptor levels on [cAMP] in the absence and presence of PDE4 activity. Solid circles represent normalized peak cAMP levels (from model simulations) in response to a 5-min stimulation with isoproterenol. Open circles represent cAMP levels measured at the end of the second stimulation with isoproterenol (following a 5-min stimulation with 1 μM isoproterenol followed by a 5-min wash). (A–F) The effects of receptor desensitization in the following scenarios: (A) high receptor levels (100 μM) and PDE4 activity inhibited (10 μM rolipram); (B) low receptor levels (1 μM) and PDE activity inhibited; (C) high receptor levels with basal PDE activity (20 nM PKI to inhibit PKA activity); (D) low receptor levels with basal PDE activity; (E) high receptor levels and PKA-mediated regulation of PDE activity intact; (F) low receptor levels and PKA-mediated regulation of PDE activity intact.

Mentions: Lastly, we used this model to investigate the interactions between receptor levels, GRK-mediated receptor desensitization, and PKA-mediated stimulation of PDE4 activity in regulating isoproterenol-induced cAMP signals by comparing peak cAMP levels elicited during the first and second stimulation of the two pulse protocol (Fig. 9). The black circles depict peak cAMP levels in response to different isoproterenol concentrations measured during the first isoproterenol pulse. The open circles depict the cAMP levels in response to different isoproterenol concentrations measured at the end of the second isoproterenol pulse (following a 5-min pulse to 1 μM isoproterenol and 5-min wash). It should be noted that, as stated above, the effects of PKA-mediated desensitization of β2ARs were not modeled; thus, cAMP levels may be overestimated at low ligand concentrations.


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)

Simulations demonstrate the effects of receptor levels on [cAMP] in the absence and presence of PDE4 activity. Solid circles represent normalized peak cAMP levels (from model simulations) in response to a 5-min stimulation with isoproterenol. Open circles represent cAMP levels measured at the end of the second stimulation with isoproterenol (following a 5-min stimulation with 1 μM isoproterenol followed by a 5-min wash). (A–F) The effects of receptor desensitization in the following scenarios: (A) high receptor levels (100 μM) and PDE4 activity inhibited (10 μM rolipram); (B) low receptor levels (1 μM) and PDE activity inhibited; (C) high receptor levels with basal PDE activity (20 nM PKI to inhibit PKA activity); (D) low receptor levels with basal PDE activity; (E) high receptor levels and PKA-mediated regulation of PDE activity intact; (F) low receptor levels and PKA-mediated regulation of PDE activity intact.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Simulations demonstrate the effects of receptor levels on [cAMP] in the absence and presence of PDE4 activity. Solid circles represent normalized peak cAMP levels (from model simulations) in response to a 5-min stimulation with isoproterenol. Open circles represent cAMP levels measured at the end of the second stimulation with isoproterenol (following a 5-min stimulation with 1 μM isoproterenol followed by a 5-min wash). (A–F) The effects of receptor desensitization in the following scenarios: (A) high receptor levels (100 μM) and PDE4 activity inhibited (10 μM rolipram); (B) low receptor levels (1 μM) and PDE activity inhibited; (C) high receptor levels with basal PDE activity (20 nM PKI to inhibit PKA activity); (D) low receptor levels with basal PDE activity; (E) high receptor levels and PKA-mediated regulation of PDE activity intact; (F) low receptor levels and PKA-mediated regulation of PDE activity intact.
Mentions: Lastly, we used this model to investigate the interactions between receptor levels, GRK-mediated receptor desensitization, and PKA-mediated stimulation of PDE4 activity in regulating isoproterenol-induced cAMP signals by comparing peak cAMP levels elicited during the first and second stimulation of the two pulse protocol (Fig. 9). The black circles depict peak cAMP levels in response to different isoproterenol concentrations measured during the first isoproterenol pulse. The open circles depict the cAMP levels in response to different isoproterenol concentrations measured at the end of the second isoproterenol pulse (following a 5-min pulse to 1 μM isoproterenol and 5-min wash). It should be noted that, as stated above, the effects of PKA-mediated desensitization of β2ARs were not modeled; thus, cAMP levels may be overestimated at low ligand concentrations.

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