AimsProlonged 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 resultsMesenteric 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 (Rmax: 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; Rmax = 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.ConclusionThese 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.

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.

The aim of this study was to investigate the selectivity of the ATP‐sensitive potassium (KATP) channel inhibitor U‐37883A (4‐morpholinecarboximidine‐N‐1‐adamantyl‐N′‐1‐cyclohexyl). Membrane currents through KATP channels were recorded in single muscle cells enzymatically isolated from rat mesenteric artery, cardiac ventricle and skeletal muscle (flexor digitorum brevis). KATP currents were induced either by cell dialysis with 0.1 mM ATP and 0.1 mM ADP, or by application of synthetic potassium channel openers (levcromakalim or pinacidil). U‐37883A inhibited KATP currents in smooth muscle cells from rat mesenteric artery. Half inhibition of 10 μM levcromakalim‐induced currents occurred at a concentration of 3.5 μM. Relaxations of rat mesenteric vessels caused by levcromakalim were reversed by U‐37883A. 1 μM levcromakalim‐induced relaxations were inhibited at a similar concentration of U‐37883A (half inhibition, 1.1 μM) to levcromakalim‐induced KATP currents. KATP currents activated by 100 μM pinacidil were also studied in single myocytes from rat mesenteric artery, skeletal muscle and cardiac ventricle. 10 μM U‐37883A substantially inhibited KATP currents in vascular cells, but had little effect in skeletal or cardiac myocytes. Higher concentrations of U‐37883A (100 μM) caused a modest decrease in KATP currents in skeletal and cardiac muscle. The sulphonylurea KATP channel antagonist glibenclamide (10 μM) abolished currents in all muscle types. The effect of U‐37883A on vascular inward rectifier (KIR) and voltage‐dependent potassium (KV) currents was also examined. While 10 μM U‐37883A had little effect on these currents, some inhibition was apparent at higher concentrations (100 μM) of the compound. We conclude that U‐37883A inhibits KATP channels in arterial smooth muscle more effectively than in cardiac and skeletal muscle. Furthermore, this compound is selective for KATP channels over KV and KIR channels in smooth muscle cells. British Journal of Pharmacology (1999) 128, 909–916; doi:

The techniques of small vessel isometric myography and patch clamp were used to investigate the action of neomycin on K+-induced isometric force and voltage-gated Ca2+ channel currents in rat arterial smooth muscle. Neomycin and the dihydropyridine (DHP) Ca2+ channel antagonist (-)202-791 concentration-dependently and reversibly inhibited 40 mM K+-induced isometric force in rings of rat mesenteric and basilar arteries (IC50 values of 70 microM and 1. 2 nM, respectively, n = 10 and 4). Elevation of [Ca2+]o by a factor of 2 significantly reduced the IC50 values for inhibition of K+-induced force for both neomycin and (-)202-791 (192 microM and 3. 7 nM, respectively, n = 6 and 4), but did not affect the Hill coefficient of the concentration/effect relationships. In patch-clamp experiments using freshly isolated basilar arterial myocytes, the voltage-gated inward current carried by Ba2+ was reversibly and concentration-dependently inhibited by neomycin (IC50 32 microM, n = 3). The concentration/effect curve for inhibition of the inward Ba2+ current by neomycin was significantly shifted to the right when [Ba2+]o was raised from 1.8 mM to 10 mM (IC50 144 microM, n = 8). Our findings suggest that neomycin relaxes high-K+-induced force in rat isolated mesenteric and basilar arteries largely by inhibition of voltage-dependent and DHP-sensitive Ca2+ channels.

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