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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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Pharmacological profiling of the molecular mechanisms underlying transient isoproterenol-induced cAMP signals. Responses were elicited by exposure of CNG channel–expressing cells to 1 μM isoproterenol. (A) The decline in the cAMP transients was abolished when 10 μM rolipram (a PDE4 inhibitor) was included in the patch pipette. The decline in the response remained largely intact when 20 nM PKI (a PKA inhibitor, B) or 10 μM St-Ht31 (an AKAP-PKA disruptor, C) were included in the patch pipette, indicating the PKA was not solely responsible for the decline in the signal. (D) An 18–24-h pretreatment with PTX (an inhibitor of Gi) had little effect on the overall kinetics of the signal. (E) 3 μM 59-74E (a GRK inhibitor) did not prevent the decline in the cAMP transients. However, the decline in the response was largely eliminated when both PKI and 59-74E (F) or St-Ht31 and 59-74E (G) were included in the patch pipette. (H) Data were quantified by measuring the percentage of current remaining 3 min after the peak current.
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fig3: Pharmacological profiling of the molecular mechanisms underlying transient isoproterenol-induced cAMP signals. Responses were elicited by exposure of CNG channel–expressing cells to 1 μM isoproterenol. (A) The decline in the cAMP transients was abolished when 10 μM rolipram (a PDE4 inhibitor) was included in the patch pipette. The decline in the response remained largely intact when 20 nM PKI (a PKA inhibitor, B) or 10 μM St-Ht31 (an AKAP-PKA disruptor, C) were included in the patch pipette, indicating the PKA was not solely responsible for the decline in the signal. (D) An 18–24-h pretreatment with PTX (an inhibitor of Gi) had little effect on the overall kinetics of the signal. (E) 3 μM 59-74E (a GRK inhibitor) did not prevent the decline in the cAMP transients. However, the decline in the response was largely eliminated when both PKI and 59-74E (F) or St-Ht31 and 59-74E (G) were included in the patch pipette. (H) Data were quantified by measuring the percentage of current remaining 3 min after the peak current.

Mentions: To further elucidate the molecular mechanisms underlying near-membrane cAMP signals, we monitored isoproterenol-induced C460W/E583M channel activity in the presence of rolipram, PKI (a peptide inhibitor of PKA), PTX (an inhibitor of Gi), St-Ht31 (a peptide that disrupts AKAP-PKA complexes), and 59-74E (a peptide inhibitor of GRK characterized by Winstel et al., 2005). Inhibitors were included in the pipette solution to ensure that we exposed the intracellular space to known inhibitor concentrations. Representative traces of cAMP signals in the presence of these compounds are shown in Fig. 3 (A–G). The percentage of current remaining 3 min after the peak current was used to quantify the extent to which the different compounds altered the decay in cAMP signals (Fig. 3 H). Including rolipram (10 μM) in the pipette solution abolished the decline in the isoproterenol-induced cAMP signal, demonstrating the significance of PDE4 activity in shaping near-membrane cAMP signals. It is important to note that rolipram will inhibit both basal and stimulated PDE4 activity (∼99% of all PDE4 activity at 10 μM rolipram and 1 μM cAMP); whereas PKI will only inhibit PKA-mediated processes such as the stimulation of PDE4 activity, leaving basal PDE4 activity intact. When either PKI (20 nM) or St-Ht31 (10 μM) were included in the pipette solution, no significant effects on isoproterenol-induced signals were observed (Fig. 3, B and C). These results were in stark contrast to our previous observations that both PKI and St-Ht31 prevented the decline in PGE1-induced cAMP signals in the same cells (Rich et al., 2007). Thus, it was apparent that mechanisms other than stimulation of PDE4 activity contribute to shaping the time course of isoproterenol-induced cAMP signals.


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)

Pharmacological profiling of the molecular mechanisms underlying transient isoproterenol-induced cAMP signals. Responses were elicited by exposure of CNG channel–expressing cells to 1 μM isoproterenol. (A) The decline in the cAMP transients was abolished when 10 μM rolipram (a PDE4 inhibitor) was included in the patch pipette. The decline in the response remained largely intact when 20 nM PKI (a PKA inhibitor, B) or 10 μM St-Ht31 (an AKAP-PKA disruptor, C) were included in the patch pipette, indicating the PKA was not solely responsible for the decline in the signal. (D) An 18–24-h pretreatment with PTX (an inhibitor of Gi) had little effect on the overall kinetics of the signal. (E) 3 μM 59-74E (a GRK inhibitor) did not prevent the decline in the cAMP transients. However, the decline in the response was largely eliminated when both PKI and 59-74E (F) or St-Ht31 and 59-74E (G) were included in the patch pipette. (H) Data were quantified by measuring the percentage of current remaining 3 min after the peak current.
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Related In: Results  -  Collection

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

fig3: Pharmacological profiling of the molecular mechanisms underlying transient isoproterenol-induced cAMP signals. Responses were elicited by exposure of CNG channel–expressing cells to 1 μM isoproterenol. (A) The decline in the cAMP transients was abolished when 10 μM rolipram (a PDE4 inhibitor) was included in the patch pipette. The decline in the response remained largely intact when 20 nM PKI (a PKA inhibitor, B) or 10 μM St-Ht31 (an AKAP-PKA disruptor, C) were included in the patch pipette, indicating the PKA was not solely responsible for the decline in the signal. (D) An 18–24-h pretreatment with PTX (an inhibitor of Gi) had little effect on the overall kinetics of the signal. (E) 3 μM 59-74E (a GRK inhibitor) did not prevent the decline in the cAMP transients. However, the decline in the response was largely eliminated when both PKI and 59-74E (F) or St-Ht31 and 59-74E (G) were included in the patch pipette. (H) Data were quantified by measuring the percentage of current remaining 3 min after the peak current.
Mentions: To further elucidate the molecular mechanisms underlying near-membrane cAMP signals, we monitored isoproterenol-induced C460W/E583M channel activity in the presence of rolipram, PKI (a peptide inhibitor of PKA), PTX (an inhibitor of Gi), St-Ht31 (a peptide that disrupts AKAP-PKA complexes), and 59-74E (a peptide inhibitor of GRK characterized by Winstel et al., 2005). Inhibitors were included in the pipette solution to ensure that we exposed the intracellular space to known inhibitor concentrations. Representative traces of cAMP signals in the presence of these compounds are shown in Fig. 3 (A–G). The percentage of current remaining 3 min after the peak current was used to quantify the extent to which the different compounds altered the decay in cAMP signals (Fig. 3 H). Including rolipram (10 μM) in the pipette solution abolished the decline in the isoproterenol-induced cAMP signal, demonstrating the significance of PDE4 activity in shaping near-membrane cAMP signals. It is important to note that rolipram will inhibit both basal and stimulated PDE4 activity (∼99% of all PDE4 activity at 10 μM rolipram and 1 μM cAMP); whereas PKI will only inhibit PKA-mediated processes such as the stimulation of PDE4 activity, leaving basal PDE4 activity intact. When either PKI (20 nM) or St-Ht31 (10 μM) were included in the pipette solution, no significant effects on isoproterenol-induced signals were observed (Fig. 3, B and C). These results were in stark contrast to our previous observations that both PKI and St-Ht31 prevented the decline in PGE1-induced cAMP signals in the same cells (Rich et al., 2007). Thus, it was apparent that mechanisms other than stimulation of PDE4 activity contribute to shaping the time course of isoproterenol-induced cAMP signals.

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