194 Nucleotide signalling is mediated through different classes of recep- tor, including the ionotropic P2X and metabotropic P2Y receptor families.5 Vascular smooth muscle cell contractile responses are mediated through the P2X1 subtype and several P2Y subtypes (i.e. P2Y2,4,6). P2X1-receptors cause a rapid inﬂux of Ca2+ to initiate smooth muscle contraction. The G protein-coupled receptors (GPCRs) P2Y2, P2Y4, and P2Y6 couple with Gaq/11 proteins to acti- vate phospholipase C (PLC), generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. This initiates the mobilization of intracellular Ca2+ stores, activation of protein kinase C, and induces contrac- tion.1,5 Both P2X- and P2Y-mediated pathways are known to contrib- ute signiﬁcantly to the control of vascular tone.3,6,7 Considering the potentially important roles that P2Y-receptors may play in the aetiology of vascular disease, very little is currently known regarding the regulation of their signalling in vascular smooth muscle. P2Y-receptors, like most GPCRs, are known to undergo desensitization,8,9 a process which can prevent prolonged/ inappropriate receptor-mediated signalling.10 Desensitization of agonist-occupied GPCRs is usually initiated by the recruitment of G protein-coupled receptor kinases (GRKs), which phosphorylate key serine/threonine residues within the third intracellular (i3) loop and/ or C-terminal tail of the GPCR.10 GRK-mediated phosphorylation promotes the binding of non-visual arrestins (arrestin2 and/or arrestin3), which sterically inhibit GPCR-G protein interactions, and promote receptor internalization.10,11 Of the seven GRK isoen- zymes (GRK1 – 7) and four arrestin isoforms (arrestin1 – 4) expressed in mammals, GRK2, GRK3, GRK5, and GRK6, and arrestin2 (b-arrestin-1) and arrestin3 (b-arrestin-2) are commonly reported to be ubiquitously expressed.10 Here, we have characterized the desensitization and resensitization of P2Y-receptor responses in mesenteric arterial smooth muscle cells (MSMCs), a widely documen- ted model of systemic resistance arteries.12 Furthermore, we have combined confocal imaging with biochemical and molecular biological manipulation of individual GRK and arrestin subtypes to identify their roles in the regulation of endogenous P2Y-receptor-stimulated PLC signalling and contraction of MSMCs. 2. Methods 2.1 Isolation and culture of MSMCs Adult male Wistar rats were killed by cervical dislocation, a method approved under the United Kingdom Animals (Scientiﬁc Procedures) Act 1986. The investigation also conforms to the Guide for Care and Use of Laboratory Animals US (NIH Publication No. 85-23, revised 1996). Smooth muscle cells were isolated from small branches of mesen- teric artery by enzymatic dissociation as previously described.12 Smooth muscle cells were separated by gentle trituration in 231 medium (Cascade Biologics, Nottingham, UK), supplemented with smooth muscle growth supplement, 100 IU/mL penicillin, 100 mg/mL streptomy- cin, and 2.5 mg/mL amphotericin B. Cells plated onto coverslips were maintained at 378C and 5% CO2 in humidiﬁed conditions. 2.2 Myography Contractile force recordings were made from 2 to 4 mm ring segments of third-order mesenteric arteries mounted in a Mulvany-Halpern wire myo- graph (JP Trading, Denmark). The bath solution contained (mM): NaCl 134, KCl 6, MgCl2 1, CaCl2 1.8, glucose 4, mannitol 6, HEPES 10, pH 7.4. NaCl was reduced to 81 mM and replaced with 60 mM K+ for the high K+ solution. All bathing solutions contained L-NAME (20 mM) to G.E. Morris et al. prevent endogenous nitric oxide production. Solutions and drugs were added directly to the organ bath, maintained at 378C. 2.3 Relative contributions of P2Y receptors to MSMC Ca21 signalling MSMCs were plated into 96-well multiplates. Monolayers ( 90% con- ﬂuency) were loaded with the Ca2+-sensitive dye Fluo4-AM (3 mM) at room temperature for 60 min. Selective P2Y2/4/6-receptor agonists (Tocris, Bristol, UK) were added and changes in intracellular Ca2+ levels ([Ca2+]i) were determined as the relative change in ﬂuorescence using a NovoStar imaging system (BMG Labtech, Aylesbury, UK). 2.4 Single cell confocal imaging UTP-stimulated PLC activity was determined in MSMCs transfected (24 – 48 h after isolation) with the pleckstrin-homology domain of phospho- lipase Cd1 tagged with enhanced green ﬂuorescent protein (eGFP-PH, 0.5 mg an extensively characterized IP3 biosensor13) using confocal imaging exactly as previously described14 (see Supplementary material online). Changes in cytosolic eGFP-PH ﬂuorescence are displayed as the ﬂuorescence emission (F )/initial basal ﬂuorescence (F0) (F/F0). Additional Ca2+ experiments in the absence of eGFP-PH were conducted using Fluo-4-AM (3 mM, 60 min) and imaged as for eGFP. 2.5 Manipulating cellular GRK and arrestin levels To assess the role that GRKs play in the regulation of P2Y-receptor signalling, MSMCs were co-transfected with eGFP-PH (0.5 mg), and the following dominant-negative, catalytically inactive GRK mutants: D110A,K220RGRK2, D110A,K220RGRK3, K215RGRK5, K215RGRK6 (0.5 mg),14 or negative control plasmid (pcDNA3). Additionally, GRK2 expression was depleted using anti-GRK2 small interfering (si)RNA (10 nM) [with non-targeting siRNA as negative-control (NC)].14 Our previous data demonstrated that co-transfection rates are .90%,14 therefore we have assumed that eGFP-expressing cells are successfully co-transfected. To examine the role that arrestins play in the regulation of P2Y-receptor sig- nalling, MSMCs were transfected with anti-arrestin2 (5′-GCCACUGACU CGGCUACAAtt-3′; 100 nM), anti-arrestin3 (5′-GCCUUCUGUGCCAA AUCUAtt-3′; 100 nM), or NC siRNA (100 nM) (Applied Biosystems, UK). Arrestin depletion was assessed 48 h after nucleofection (Lonza AG, Cologne, Germany) by immunoblotting using an anti-arrestin2 anti- body (A1CT). Arrestin expression was quantiﬁed using the GeneGnome image analysis system (Syngene, Cambridge, UK). For signalling studies, MSMCs were transfected using Lipofectamine2000 (Invitrogen, Paisley, UK) according to manufacturer’s instructions and PLC activity assessed as described above. 2.6 Manipulating cellular P2Y receptor levels An siRNA approach was also applied to delineate the respective roles of P2Y2- and P2Y4-receptors in UTP-mediated signalling. MSMCs were trans- fected with differing concentrations of anti-P2Y2 (5′-GAACUGACACUG UGAGGAAtt-3′), or anti-P2Y4 (5′-CGUCUACUUCAGUUCGGCAtt-3′) siRNAs. To validate the siRNAs and to quantify P2Y2/P2Y4 knockdown, rat aortic smooth muscle cells were nucleofected and after 48 h lysed, mRNA isolated and transcribed into cDNA, prior to PCR ampliﬁcation using a LightCycler instrument (Roche Applied Science, Burgess Hill, UK). Data are presented as DCT values (calculated as CT (scrambled siRNA-transfected cells)2CT (anti-P2Y siRNA-transfected cells)). A more detailed method is provided in the accompanying Supplementary Material. 2.7 Data and statistical analysis Data presented are from cells obtained from at least three separate prep- arations, expressed as means + SEM. Data were analysed using one-way