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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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Related in: MedlinePlus

ET-1 stimulates recruitment of endogenous GRK2, but not GRK5 to the plasma membrane. MSMCs were challenged with ET-1 (50 nM) for 3 min, before fixation and processing for immunocytochemical detection of GRKs 2 and 5 (see Methods). Representative confocal images show the distribution of GRK2 and GRK5 in MSMCs. Left-hand panels are images of untreated cells, whereas the right-hand panels show: (A) GRK2 recruitment to the plasma membrane after ET-1 challenge (indicated by arrows); (B) Lack of movement of GRK5 after ET-1 challenge; (C) Redistribution of GRK5 from the nucleus to the cytoplasm after ionomycin (2 µM, 15 min) treatment. Data are representative of experiments from cells isolated from three separate animals.
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CVP310F6: ET-1 stimulates recruitment of endogenous GRK2, but not GRK5 to the plasma membrane. MSMCs were challenged with ET-1 (50 nM) for 3 min, before fixation and processing for immunocytochemical detection of GRKs 2 and 5 (see Methods). Representative confocal images show the distribution of GRK2 and GRK5 in MSMCs. Left-hand panels are images of untreated cells, whereas the right-hand panels show: (A) GRK2 recruitment to the plasma membrane after ET-1 challenge (indicated by arrows); (B) Lack of movement of GRK5 after ET-1 challenge; (C) Redistribution of GRK5 from the nucleus to the cytoplasm after ionomycin (2 µM, 15 min) treatment. Data are representative of experiments from cells isolated from three separate animals.

Mentions: Immnuoblotting data showed that GRKs 2, 5, 6 are expressed in rat MSMCs, however, in agreement with the previous findings16 we were unable to detect GRK3 expression (see Supplementary material online, Figure S1). Immunocytochemical evidence highlights an anticipated cytoplasmic distribution of GRK2 (Figure 6A), whereas GRK5 expression appears to be primarily nuclear (Figure 6B and C).


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)

ET-1 stimulates recruitment of endogenous GRK2, but not GRK5 to the plasma membrane. MSMCs were challenged with ET-1 (50 nM) for 3 min, before fixation and processing for immunocytochemical detection of GRKs 2 and 5 (see Methods). Representative confocal images show the distribution of GRK2 and GRK5 in MSMCs. Left-hand panels are images of untreated cells, whereas the right-hand panels show: (A) GRK2 recruitment to the plasma membrane after ET-1 challenge (indicated by arrows); (B) Lack of movement of GRK5 after ET-1 challenge; (C) Redistribution of GRK5 from the nucleus to the cytoplasm after ionomycin (2 µM, 15 min) treatment. Data are representative of experiments from cells isolated from three separate animals.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2802200&req=5

CVP310F6: ET-1 stimulates recruitment of endogenous GRK2, but not GRK5 to the plasma membrane. MSMCs were challenged with ET-1 (50 nM) for 3 min, before fixation and processing for immunocytochemical detection of GRKs 2 and 5 (see Methods). Representative confocal images show the distribution of GRK2 and GRK5 in MSMCs. Left-hand panels are images of untreated cells, whereas the right-hand panels show: (A) GRK2 recruitment to the plasma membrane after ET-1 challenge (indicated by arrows); (B) Lack of movement of GRK5 after ET-1 challenge; (C) Redistribution of GRK5 from the nucleus to the cytoplasm after ionomycin (2 µM, 15 min) treatment. Data are representative of experiments from cells isolated from three separate animals.
Mentions: Immnuoblotting data showed that GRKs 2, 5, 6 are expressed in rat MSMCs, however, in agreement with the previous findings16 we were unable to detect GRK3 expression (see Supplementary material online, Figure S1). Immunocytochemical evidence highlights an anticipated cytoplasmic distribution of GRK2 (Figure 6A), whereas GRK5 expression appears to be primarily nuclear (Figure 6B and C).

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