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


RESULTS
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.


CONCLUSIONS
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.
Morris
Gavin E
GE
Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, UK.
Nelson
Carl P
CP
Everitt
Diane
D
Brighton
Paul J
PJ
Standen
Nicholas B
NB
Challiss
R A John
RA
Willets
Jonathon M
JM
D000818:Animals; D019390:Arrestins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020013:Calcium Signaling; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D008638:Mesenteric Arteries / Q000187:drug effects / Q000502:physiology; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D058470:Receptors, Purinergic P2Y / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014544:Uridine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000071557:beta-Arrestins
D019390:Arrestins; D034741:RNA, Small Interfering; D058470:Receptors, Purinergic P2Y; D058473:Receptors, Purinergic P2Y2; D000071557:beta-Arrestins; C496569:Grk2 protein, rat; D054769:G-Protein-Coupled Receptor Kinase 2; D014544:Uridine Triphosphate