Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, UK.
Cardiovasc Res. 2011 Jan 1;89(1):193-203. doi: 10.1093/cvr/cvq249. Epub 2010 Aug 12.
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 EC(50) UTP concentration (30 µM, 5 min) was added 5 min before (R(1)) and after (R(2)) the addition of a maximal UTP concentration (R(max): 100 µM, 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 Ca(2+)-sensitive dyes. A similar protocol (R(1)/R(2) = 10 µM; R(max) = 100 µM, 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 IP(3)/Ca(2+) 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 P2Y(2)-receptor responsiveness in resistance arteries, emphasizing their potential importance in regulating vasoconstrictor signalling pathways implicated in vascular disease.
P2Y 受体信号的延长可导致血管收缩、高血压、血管平滑肌肥大和增生。G 蛋白偶联受体信号受 G 蛋白偶联受体激酶 (GRK) 和阻滞蛋白的负调控，以防止信号的延长或不当。本研究旨在探讨 GRK 和阻滞蛋白是否调节尿嘧啶 5'-三磷酸 (UTP)刺激的成年 Wistar 大鼠肠系膜动脉平滑肌细胞 (MSMC)的收缩信号。
肠系膜动脉对 UTP 刺激有收缩反应：当加入 30µM（5min）的 UTP 时，EC50 浓度（5min 前为 R1，5min 后为 R2），当加入 100µM（5min）的最大 UTP 浓度（Rmax）时，R2 反应相对于 R1 降低，表明脱敏。在转染了肌醇 1,4,5-三磷酸生物传感器和/或加载了 Ca2+敏感染料的分离的 MSMC 中，研究了 UTP 诱导的 PLC 信号的 P2Y 受体脱敏。用类似的方案（R1/R2=10µM；Rmax=100µM，应用 30s）发现与 R1 反应相比，R2 明显减少。用显性负性 GRK 或针对特定 GRK/阻滞蛋白的 siRNA 转染 MSMC，以探讨它们在 P2Y 受体脱敏中的各自作用。GRK2 抑制，但不是 GRK3、GRK5 或 GRK6，减弱了 P2Y 受体脱敏。阻滞蛋白 2 的 siRNA 介导的敲低减弱了 UTP 刺激的 P2Y 受体脱敏，而阻滞蛋白 3 的耗竭则没有。P2Y(2)-受体的特异性 siRNA 敲低几乎完全消除了 UTP 刺激的 IP3/Ca2+信号，强烈表明我们的研究专门描述了这种嘌呤能受体亚型。
这些新的数据突出了 GRK2 和阻滞蛋白 2 作为 UTP 刺激的 P2Y(2)-受体反应性在阻力血管中的重要调节因子的作用，强调了它们在调节血管收缩信号通路中的潜在重要性，这些信号通路与血管疾病有关。
