Journal
CARDIOVASCULAR RESEARCH
Volume 89, Issue 1, Pages 193-203Publisher
OXFORD UNIV PRESS
DOI: 10.1093/cvr/cvq249
Keywords
UTP; G protein-coupled receptor kinase; Arrestin; P2Y-purinoceptor; Resistance artery
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Funding
- British Heart Foundation [PG06/161/22136, RG06/008/22062]
- British Heart Foundation [RG/06/008/22062] Funding Source: researchfish
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Aims Prolonged P2Y-receptor signalling can cause vasoconstriction leading to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs) and arrestin proteins, preventing prolonged or inappropriate signalling. This study investigates whether GRKs and arrestins regulate uridine 5'-triphosphate (UTP)-stimulated contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs). Methods and results Mesenteric arteries contracted in response to UTP challenge: When an EC50 UTP concentration (30 mu M, 5 min) was added 5 min before (R-1) and after (R-2) the addition of a maximal UTP concentration (R-max: 100 mM, 5 min), R-2 responses were decreased relative to R-1, indicating desensitization. UTP-induced P2Y-receptor desensitization of phospholipase C signalling was studied in isolated MSMCs transfected with an inositol 1,4,5-trisphosphate biosensor and/or loaded with Ca2+-sensitive dyes. A similar protocol (R-1/R-2 = 10 mu M; R-max = 100 mu M, applied for 30 s) revealed markedly reduced R-2 when compared with R-1 responses. MSMCs were transfected with dominant-negative GRKs or siRNAs targeting specific GRK/arrestins to probe their respective roles in P2Y-receptor desensitization. GRK2 inhibition, but not GRK3, GRK5, or GRK6, attenuated P2Y-receptor desensitization. siRNA-mediated knockdown of arrestin2 attenuated UTP-stimulated P2Y-receptor desensitization, whereas arrestin3 depletion did not. Specific siRNA knockdown of the P2Y(2)-receptor almost completely abolished UTP-stimulated IP3/Ca2+ signalling, strongly suggesting that our study is specifically characterizing this purinoceptor subtype. Conclusion These new data highlight roles of GRK2 and arrestin2 as important regulators of UTP-stimulated P2Y(2)-receptor responsiveness in resistance arteries, emphasizing their potential importance in regulating vasoconstrictor signalling pathways implicated in vascular disease.
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