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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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Effects of PKA on isoproterenol (1 μM)-induced cAMP synthesis and hydrolysis in HEK-293 cells. (A) Time course of isoproterenol-induced stimulation of PDE4 activity in HEK-293 cell extracts. Cells were incubated with 1 μM isoproterenol for the indicated times. At the end of the incubation, cells were harvested and homogenates subjected to PDE activity assays using 1 μM cAMP as substrate. PDE activity was measured in the absence or presence of 10 μM rolipram. The rolipram-inhibited PDE4 activity is reported. It is clear that isoproterenol induced a two- to threefold increase in PDE4 activity. The increase in PDE4 activity was due to PKA-dependent phosphorylation of PDE4 and was prevented by pretreatment with PKA inhibitors (not depicted). (B) cAMP accumulation measured in HEK-293 cells using an enzyme immunoassay. Cells were stimulated with 10 μM isoproterenol for 5 min following pretreatment with vehicle (DMSO, 10 min), 10 μM H89 (10 min), 10 μM rolipram (5 min), or 10 μM H89 (10 min) and 10 μM rolipram (5 min). H89 had little or no effect on isoproterenol-induced cAMP accumulation in the presence of rolipram, indicating that PKA does not substantially stimulate receptor desensitization or inhibit the rate of cAMP synthesis following stimulation with saturating concentrations of isoproterenol. Data are the mean ± SEM of three (A) or four (B) separate experiments, each performed in triplicate.
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fig2: Effects of PKA on isoproterenol (1 μM)-induced cAMP synthesis and hydrolysis in HEK-293 cells. (A) Time course of isoproterenol-induced stimulation of PDE4 activity in HEK-293 cell extracts. Cells were incubated with 1 μM isoproterenol for the indicated times. At the end of the incubation, cells were harvested and homogenates subjected to PDE activity assays using 1 μM cAMP as substrate. PDE activity was measured in the absence or presence of 10 μM rolipram. The rolipram-inhibited PDE4 activity is reported. It is clear that isoproterenol induced a two- to threefold increase in PDE4 activity. The increase in PDE4 activity was due to PKA-dependent phosphorylation of PDE4 and was prevented by pretreatment with PKA inhibitors (not depicted). (B) cAMP accumulation measured in HEK-293 cells using an enzyme immunoassay. Cells were stimulated with 10 μM isoproterenol for 5 min following pretreatment with vehicle (DMSO, 10 min), 10 μM H89 (10 min), 10 μM rolipram (5 min), or 10 μM H89 (10 min) and 10 μM rolipram (5 min). H89 had little or no effect on isoproterenol-induced cAMP accumulation in the presence of rolipram, indicating that PKA does not substantially stimulate receptor desensitization or inhibit the rate of cAMP synthesis following stimulation with saturating concentrations of isoproterenol. Data are the mean ± SEM of three (A) or four (B) separate experiments, each performed in triplicate.

Mentions: We previously reported that PKA-mediated stimulation of PDE4 activity is primarily responsible for the decline in PGE1-induced cAMP signals in HEK-293 cells; that 5-min stimulation with PGE1, forskolin, or isoproterenol triggers a PKA-dependent, two- to threefold increase in PDE4 activity; and that this stimulation of PDE4 is responsible for the decline in PGE1-induced cAMP signals (Rich et al., 2001b, 2007). By analogy, it seemed likely that PKA-mediated stimulation of PDE4 activity also contributes to the decay of cAMP signals triggered by β-adrenergic agonists. To ensure that the time course of PDE4 activation was consistent with this hypothesis, we measured PDE4 activity in response to isoproterenol as described in the Materials and methods. Stimulation of HEK-293 cells with 1 μM isoproterenol triggered a threefold increase in PDE4 activity within 1 min, followed by a slight reduction in activity to a sustained level of twofold over basal (Fig. 2 A). PDE4 activation was inhibited by H89, a PKA inhibitor, in a dose-dependent manner (Rich et al., 2007). H89 did not affect the non-PDE4 activity, which contributes ∼50% of total PDE activity in these cells (unpublished data). Taken together, these data suggest that β-adrenergic agonists trigger PKA-mediated stimulation of PDE4 activity, which may contribute to the decay of transient 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)

Effects of PKA on isoproterenol (1 μM)-induced cAMP synthesis and hydrolysis in HEK-293 cells. (A) Time course of isoproterenol-induced stimulation of PDE4 activity in HEK-293 cell extracts. Cells were incubated with 1 μM isoproterenol for the indicated times. At the end of the incubation, cells were harvested and homogenates subjected to PDE activity assays using 1 μM cAMP as substrate. PDE activity was measured in the absence or presence of 10 μM rolipram. The rolipram-inhibited PDE4 activity is reported. It is clear that isoproterenol induced a two- to threefold increase in PDE4 activity. The increase in PDE4 activity was due to PKA-dependent phosphorylation of PDE4 and was prevented by pretreatment with PKA inhibitors (not depicted). (B) cAMP accumulation measured in HEK-293 cells using an enzyme immunoassay. Cells were stimulated with 10 μM isoproterenol for 5 min following pretreatment with vehicle (DMSO, 10 min), 10 μM H89 (10 min), 10 μM rolipram (5 min), or 10 μM H89 (10 min) and 10 μM rolipram (5 min). H89 had little or no effect on isoproterenol-induced cAMP accumulation in the presence of rolipram, indicating that PKA does not substantially stimulate receptor desensitization or inhibit the rate of cAMP synthesis following stimulation with saturating concentrations of isoproterenol. Data are the mean ± SEM of three (A) or four (B) separate experiments, each performed in triplicate.
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Related In: Results  -  Collection

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fig2: Effects of PKA on isoproterenol (1 μM)-induced cAMP synthesis and hydrolysis in HEK-293 cells. (A) Time course of isoproterenol-induced stimulation of PDE4 activity in HEK-293 cell extracts. Cells were incubated with 1 μM isoproterenol for the indicated times. At the end of the incubation, cells were harvested and homogenates subjected to PDE activity assays using 1 μM cAMP as substrate. PDE activity was measured in the absence or presence of 10 μM rolipram. The rolipram-inhibited PDE4 activity is reported. It is clear that isoproterenol induced a two- to threefold increase in PDE4 activity. The increase in PDE4 activity was due to PKA-dependent phosphorylation of PDE4 and was prevented by pretreatment with PKA inhibitors (not depicted). (B) cAMP accumulation measured in HEK-293 cells using an enzyme immunoassay. Cells were stimulated with 10 μM isoproterenol for 5 min following pretreatment with vehicle (DMSO, 10 min), 10 μM H89 (10 min), 10 μM rolipram (5 min), or 10 μM H89 (10 min) and 10 μM rolipram (5 min). H89 had little or no effect on isoproterenol-induced cAMP accumulation in the presence of rolipram, indicating that PKA does not substantially stimulate receptor desensitization or inhibit the rate of cAMP synthesis following stimulation with saturating concentrations of isoproterenol. Data are the mean ± SEM of three (A) or four (B) separate experiments, each performed in triplicate.
Mentions: We previously reported that PKA-mediated stimulation of PDE4 activity is primarily responsible for the decline in PGE1-induced cAMP signals in HEK-293 cells; that 5-min stimulation with PGE1, forskolin, or isoproterenol triggers a PKA-dependent, two- to threefold increase in PDE4 activity; and that this stimulation of PDE4 is responsible for the decline in PGE1-induced cAMP signals (Rich et al., 2001b, 2007). By analogy, it seemed likely that PKA-mediated stimulation of PDE4 activity also contributes to the decay of cAMP signals triggered by β-adrenergic agonists. To ensure that the time course of PDE4 activation was consistent with this hypothesis, we measured PDE4 activity in response to isoproterenol as described in the Materials and methods. Stimulation of HEK-293 cells with 1 μM isoproterenol triggered a threefold increase in PDE4 activity within 1 min, followed by a slight reduction in activity to a sustained level of twofold over basal (Fig. 2 A). PDE4 activation was inhibited by H89, a PKA inhibitor, in a dose-dependent manner (Rich et al., 2007). H89 did not affect the non-PDE4 activity, which contributes ∼50% of total PDE activity in these cells (unpublished data). Taken together, these data suggest that β-adrenergic agonists trigger PKA-mediated stimulation of PDE4 activity, which may contribute to the decay of transient 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