G protein-coupled receptor kinase 2 and arrestin2 regulate arterial smooth muscle P2Y-purinoceptor signalling

Abstract

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). Mesenteric arteries contracted in response to UTP challenge: When an EC₅₀ UTP concentration (30 µM, 5 min) was added 5 min before (R₁) and after (R₂) the addition of a maximal UTP concentration (R_max: 100 µM, 5 min), R₂ responses were decreased relative to R₁, 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 (R₁/R₂ = 10 µM; R_max = 100 µM, applied for 30 s) revealed markedly reduced R₂ when compared with R₁ 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 P2Y₂-receptor almost completely abolished UTP-stimulated IP₃/Ca²⁺ signalling, strongly suggesting that our study is specifically characterizing this purinoceptor subtype. These new data highlight roles of GRK2 and arrestin2 as important regulators of UTP-stimulated P2Y₂-receptor responsiveness in resistance arteries, emphasizing their potential importance in regulating vasoconstrictor signalling pathways implicated in vascular disease.