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Endothelin signalling in arterial smooth muscle is tightly regulated by G protein-coupled receptor kinase 2.

Morris GE, Nelson CP, Standen NB, Challiss RA, Willets JM - Cardiovasc. Res. (2009)

Bottom Line: This brief exposure to ET-1 markedly decreased ET(A)R responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min.Finally, immunocyotchemical data showed that ET(A)R activation recruited endogenous GRK2 from cytoplasm to membrane.These studies identify GRK2 as a key regulator of ET(A)R responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

View Article: PubMed Central - PubMed

Affiliation: Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, UK.

ABSTRACT

Aims: Prolonged endothelin (ET) receptor signalling causes vasoconstriction and can lead to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. Usually, G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs), preventing prolonged or inappropriate signalling. This study investigated whether GRKs regulate ET receptor contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs).

Methods and results: ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca2+]i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLCdelta1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca2+]i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ET(A)R) antagonist, BQ123. To characterize ET(A)R desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ET(A)R responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ET(A)R desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive (D110A,K220R)GRK2, (D110A,K220R)GRK3, (K215R)GRK5, or (K215R)GRK6 constructs. (D110A,K220R)GRK2 expression significantly attenuated ET(A)R desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ET(A)R desensitization. Finally, immunocyotchemical data showed that ET(A)R activation recruited endogenous GRK2 from cytoplasm to membrane.

Conclusion: These studies identify GRK2 as a key regulator of ET(A)R responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

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Depletion of endogenous GRK2 inhibits ETAR desensitization. MSMCs were co-transfected with 0.5 µg eGFP-PH and either negative-control or anti-GRK2 (10 nM) siRNAs. Cells were loaded with Fura-Red and subjected to the standard R1/R2 desensitization protocol (see Methods). (A) Representative images of changes in IP3 and Ca2+ in MSMCs expressing either control siRNA or anti-GRK2 siRNA at both the R1 and R2 stimulation points are shown. Representative traces from single cells transfected with control siRNA (B) or anti-GRK2 siRNA (C). ETAR desensitization was determined as the relative change in R2 response compared with R1 for both eGFP-PH (black traces) and Fura-Red (broken traces). Cumulative data (D) show means ± SEM from 7–9 cells from four or more animals. Statistical significance is indicated as **P < 0.01; ***P < 0.001 (one-way ANOVA, unpaired t-test).
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CVP310F4: Depletion of endogenous GRK2 inhibits ETAR desensitization. MSMCs were co-transfected with 0.5 µg eGFP-PH and either negative-control or anti-GRK2 (10 nM) siRNAs. Cells were loaded with Fura-Red and subjected to the standard R1/R2 desensitization protocol (see Methods). (A) Representative images of changes in IP3 and Ca2+ in MSMCs expressing either control siRNA or anti-GRK2 siRNA at both the R1 and R2 stimulation points are shown. Representative traces from single cells transfected with control siRNA (B) or anti-GRK2 siRNA (C). ETAR desensitization was determined as the relative change in R2 response compared with R1 for both eGFP-PH (black traces) and Fura-Red (broken traces). Cumulative data (D) show means ± SEM from 7–9 cells from four or more animals. Statistical significance is indicated as **P < 0.01; ***P < 0.001 (one-way ANOVA, unpaired t-test).

Mentions: To examine the effect of siRNA-mediated GRK2 knockdown on ETAR desensitization, MSMCs were co-transfected with eGFP-PH (0.5 µg) and negative-control (10 nM) or anti-GRK2 (10 nM) siRNAs and subjected to the standard R1/R2 desensitization protocol. In the presence of negative-control siRNA, R2 responses were decreased by ≥80% for eGFP-PH and by ≥60% for [Ca2+]i signals compared with R1, consistent with the degree of receptor desensitization observed in untransfected cells (Figure 4A, B, D). In contrast, in cells transfected with anti-GRK2 siRNA, R2 and R1 responses were similar to those previously seen in D110A,K220RGRK2-transfected MSMCs, with a 50% decrease in eGFP-PH and ∼30% decrease in Ca2+ signals (Figure 4A, C, D). To assess whether quantitatively similar effects of manipulating cellular GRK2 are seen with respect to the DAG/PKC arm of the ETAR signalling pathway, we have assessed ET-1-stimulated translocation of the Ca2+ and DAG-sensitive PKCα and DAG-sensitive PKCε isoenzymes. Applying the standard desensitization protocol to eGFP-PKCα- (Figure 5A, C) or eGFP-PKCε (Figure 5B, C)-transfected MSMCs showed that a significantly greater recovery with respect to PKC recruitment responses was also observed when cellular GRK2 levels were selectively diminished.


Endothelin signalling in arterial smooth muscle is tightly regulated by G protein-coupled receptor kinase 2.

Morris GE, Nelson CP, Standen NB, Challiss RA, Willets JM - Cardiovasc. Res. (2009)

Depletion of endogenous GRK2 inhibits ETAR desensitization. MSMCs were co-transfected with 0.5 µg eGFP-PH and either negative-control or anti-GRK2 (10 nM) siRNAs. Cells were loaded with Fura-Red and subjected to the standard R1/R2 desensitization protocol (see Methods). (A) Representative images of changes in IP3 and Ca2+ in MSMCs expressing either control siRNA or anti-GRK2 siRNA at both the R1 and R2 stimulation points are shown. Representative traces from single cells transfected with control siRNA (B) or anti-GRK2 siRNA (C). ETAR desensitization was determined as the relative change in R2 response compared with R1 for both eGFP-PH (black traces) and Fura-Red (broken traces). Cumulative data (D) show means ± SEM from 7–9 cells from four or more animals. Statistical significance is indicated as **P < 0.01; ***P < 0.001 (one-way ANOVA, unpaired t-test).
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CVP310F4: Depletion of endogenous GRK2 inhibits ETAR desensitization. MSMCs were co-transfected with 0.5 µg eGFP-PH and either negative-control or anti-GRK2 (10 nM) siRNAs. Cells were loaded with Fura-Red and subjected to the standard R1/R2 desensitization protocol (see Methods). (A) Representative images of changes in IP3 and Ca2+ in MSMCs expressing either control siRNA or anti-GRK2 siRNA at both the R1 and R2 stimulation points are shown. Representative traces from single cells transfected with control siRNA (B) or anti-GRK2 siRNA (C). ETAR desensitization was determined as the relative change in R2 response compared with R1 for both eGFP-PH (black traces) and Fura-Red (broken traces). Cumulative data (D) show means ± SEM from 7–9 cells from four or more animals. Statistical significance is indicated as **P < 0.01; ***P < 0.001 (one-way ANOVA, unpaired t-test).
Mentions: To examine the effect of siRNA-mediated GRK2 knockdown on ETAR desensitization, MSMCs were co-transfected with eGFP-PH (0.5 µg) and negative-control (10 nM) or anti-GRK2 (10 nM) siRNAs and subjected to the standard R1/R2 desensitization protocol. In the presence of negative-control siRNA, R2 responses were decreased by ≥80% for eGFP-PH and by ≥60% for [Ca2+]i signals compared with R1, consistent with the degree of receptor desensitization observed in untransfected cells (Figure 4A, B, D). In contrast, in cells transfected with anti-GRK2 siRNA, R2 and R1 responses were similar to those previously seen in D110A,K220RGRK2-transfected MSMCs, with a 50% decrease in eGFP-PH and ∼30% decrease in Ca2+ signals (Figure 4A, C, D). To assess whether quantitatively similar effects of manipulating cellular GRK2 are seen with respect to the DAG/PKC arm of the ETAR signalling pathway, we have assessed ET-1-stimulated translocation of the Ca2+ and DAG-sensitive PKCα and DAG-sensitive PKCε isoenzymes. Applying the standard desensitization protocol to eGFP-PKCα- (Figure 5A, C) or eGFP-PKCε (Figure 5B, C)-transfected MSMCs showed that a significantly greater recovery with respect to PKC recruitment responses was also observed when cellular GRK2 levels were selectively diminished.

Bottom Line: This brief exposure to ET-1 markedly decreased ET(A)R responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min.Finally, immunocyotchemical data showed that ET(A)R activation recruited endogenous GRK2 from cytoplasm to membrane.These studies identify GRK2 as a key regulator of ET(A)R responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

View Article: PubMed Central - PubMed

Affiliation: Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, UK.

ABSTRACT

Aims: Prolonged endothelin (ET) receptor signalling causes vasoconstriction and can lead to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. Usually, G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs), preventing prolonged or inappropriate signalling. This study investigated whether GRKs regulate ET receptor contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs).

Methods and results: ET-1-stimulated phospholipase C (PLC) activity and changes in [Ca2+]i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLCdelta1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca2+]i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ET(A)R) antagonist, BQ123. To characterize ET(A)R desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ET(A)R responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ET(A)R desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive (D110A,K220R)GRK2, (D110A,K220R)GRK3, (K215R)GRK5, or (K215R)GRK6 constructs. (D110A,K220R)GRK2 expression significantly attenuated ET(A)R desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ET(A)R desensitization. Finally, immunocyotchemical data showed that ET(A)R activation recruited endogenous GRK2 from cytoplasm to membrane.

Conclusion: These studies identify GRK2 as a key regulator of ET(A)R responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

Show MeSH
Related in: MedlinePlus