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

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 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. 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.

Endothelin-I and angiotensin II inhibit arterial voltage-gated K+ channels through different protein kinase C isoenzymes.

AIMS: Voltage-gated K+ (Kv) channels of arterial smooth muscle (ASM) modulate arterial tone and are inhibited by vasoconstrictors through protein kinase C (PKC). We aimed to determine whether endothelin-1 (ET-1) and angiotensin II (AngII), which cause similar inhibition of Kv, use the same signalling pathway and PKC isoenzyme to exert their effects on Kv and to compare the involvement of PKC isoenzymes in contractile responses to these agents. METHODS AND RESULTS: Kv currents recorded using the patch clamp technique with freshly isolated rat mesenteric ASM cells were inhibited by ET-1 or AngII. Inclusion of a PKCepsilon inhibitor peptide in the intracellular solution substantially reduced inhibition by AngII, but did not affect that by ET-1. Kv inhibition by ET-1 was reduced by the conventional PKC inhibitor Gö 6976 but not by the PKCbeta inhibitor LY333531. Selective peptide inhibitors of PKCalpha and PKCepsilon were linked to a Tat carrier peptide to make them membrane permeable and used to show that inhibition of PKCalpha prevented ET-1 inhibition of Kv current, but did not affect that by AngII. In contrast, inhibition of PKCepsilon prevented Kv inhibition by AngII but not by ET-1. The Tat-linked inhibitor peptides were also used to investigate the involvement of PKCalpha and PKCepsilon in the contractile responses of mesenteric arterial rings, showing that ET-1 contractions were substantially reduced by inhibition of PKCalpha, but unaffected by inhibition of PKCepsilon. AngII contractions were unaffected by inhibition of PKCalpha but substantially reduced by inhibition of PKCepsilon. CONCLUSION: ET-1 inhibits Kv channels of mesenteric ASM through activation of PKCalpha, while AngII does so through PKCepsilon. This implies that ET-1 and AngII target Kv channels of ASM through different pathways of PKC-interacting proteins, so each vasoconstrictor enables its distinct PKC isoenzyme to interact functionally with the Kv channel.