Morris Gavin E, Nelson Carl P, Standen Nicholas B, Challiss R A John, Willets Jonathon M
Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, UK.
Cardiovasc Res. 2010 Feb 1;85(3):424-33. doi: 10.1093/cvr/cvp310. Epub 2009 Sep 11.
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).
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.
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.
内皮素（ET）受体信号传导延长会导致血管收缩，并可能引发高血压、血管平滑肌肥大和增生。通常，G蛋白偶联受体信号传导受G蛋白偶联受体激酶（GRK）负调控，以防止信号传导延长或异常。本研究调查了GRK是否调节成年Wistar大鼠肠系膜动脉平滑肌细胞（MSMCs）中的ET受体收缩信号。
使用共聚焦显微镜，在转染了PLCδ1的pleckstrin同源结构域（eGFP-PH）并加载Fura-Red的大鼠MSMCs中评估ET-1刺激的磷脂酶C（PLC）活性和[Ca2+]i的变化。应用ET-1（30秒）刺激了瞬时浓度依赖性的eGFP-PH从质膜向细胞质的转位以及[Ca2+]i的分级增加。ET-1介导的PLC信号传导被A型内皮素受体（ET(A)R）拮抗剂BQ123阻断。为了表征ET(A)R脱敏，用最大有效浓度的ET-1（50 nM，30秒）刺激细胞，随后进行可变的洗脱期，然后再次相同应用ET-1。这种短暂暴露于ET-1显著降低了ET(A)R对再次刺激的反应性，即使将激动剂刺激之间的时间间隔增加到60分钟，逆转也不完全。为了评估GRK在ET(A)R脱敏中的作用，MSMCs与eGFP-PH和催化无活性的（D110A，K220R）GRK2、（D110A，K220R）GRK3、（K215R）GRK5或（K215R）GRK6构建体共转染。（D110A，K220R）GRK2的表达显著减弱了ET(A)R脱敏，而其他构建体无效。靶向GRK2的小干扰RNA敲低同样减弱了ET(A)R脱敏。最后，免疫细胞化学数据表明，ET(A)R激活将内源性GRK2从细胞质募集到膜上。
这些研究确定GRK2是阻力动脉中ET(A)R反应性的关键调节因子，突出了这种GRK同工酶在调节与血管疾病相关的血管收缩信号通路中的潜在重要性。
