Journal of Pharmacological and Toxicological Methods 70 (2014) 152162

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Journal of Pharmacological and Toxicological Methods

j ourna l homepage: www.e lsev ie r .com/ locate / jpharmtoxOriginal articleQuantitative determination of 2B-adrenoceptor-evoked myosin lightchain phosphorylation in vascular smooth muscle cellsSusann Bjrk a,1, Anna Huhtinen a,1,, Anne Vuorenp a,b, Mika Scheinin a,c

a Department of Pharmacology, Drug Development and Therapeutics, Institute of Biomedicine, University of Turku, Turku, Finlandb Department of Biosciences, bo Akademi University, Turku, Finlandc Unit of Clinical Pharmacology, Turku University Hospital, Turku, FinlandAbbreviations: pMLC20, myosin light chain phosphory Corresponding author at: Department of Pharmac

Therapeutics, Institute of Biomedicine, University of T20520 Turku, Finland. Tel.: +358 2 333 7359; fax: +358

E-mail address: anna.huhtinen@utu.fi (A. Huhtinen).1 Authors contributed equally.

http://dx.doi.org/10.1016/j.vascn.2014.07.0041056-8719/ 2014 Elsevier Inc. All rights reserved.a b s t r a c ta r t i c l e i n f oArticle history:

Received 15 January 2014Accepted 15 July 2014Available online 7 August 2014

Keywords:alpha2-AdrenoceptorContractionFunctional assayMethodsMyosin light chain phosphorylationQuantitative immunostainingVascular smooth muscle cell96-well plate

Introduction: Phosphorylation of myosin light chains is a biochemical readout of smooth muscle cellcontraction. 2-Adrenoceptor agonists and antagonists may have important applications in cardiovasculardrug development. To assess 2-adrenoceptor-mediated drug effects on vascular smooth muscle contraction,we developed a cell-based assay for the quantitative determination of myosin light chain phosphorylation(pMLC20) in cultured A7r5 smooth muscle cells from rat aorta, transfected to express the human 2B-adrenoceptor (A7r5-2B cell line). Methods: In a 96-well format, confluent and serum-starved cells (+/inhibitor preincubation) were treated with receptor ligands for 5120 s and the evoked pMLC20 response wasmonitoredwith a quantitative in-cell immunoassay, employing time-resolved fluorescence technology.Westernblotting, immunofluorescent labelling and intracellular calcium concentration measurements were used forassay validation. Results: The 2-adrenoceptor agonist dexmedetomidine induced rapid, transient anddose-dependent (EC50 3065 nM) myosin light chain phosphorylation, peaking at 2045 s with an Emax valueof approximately 60% over vehicle control. The endogenous agonist arginine vasopressin produced responsesthat were comparable to those evoked by dexmedetomidine. Blockers of 2-adrenoceptors, myosin light

chain kinase, Gi-proteins, G subunits, L-type calcium channels and phospholipase C antagonized thedexmedetomidine-evokedmyosin light chain phosphorylation,whereas blockers of protein kinase C and proteinkinase A potentiated the response to dexmedetomidine. Discussion: The novel method is suitable as a ligandprofiling tool to assess the capacity of ligands to evoke or inhibit vascular smooth muscle cell contraction andfor investigating the intracellular pathways involved in this process. The assay now allows the quantitativedetermination of pMLC20 signal induction or inhibition in vascular smoothmuscle cells and is superior to conven-tional Western blotting due to the reduced number of cells required and the potential for measurement ofdetailed time curves, multiple treatments and replicates on each plate. 2014 Elsevier Inc. All rights reserved.1. Introduction

The 2-adrenoceptors, a subclass of the family of G-protein coupledreceptors, are potential targets for cardiovascular drug developmentbecause they mediate important actions of noradrenaline and adrena-line in the regulation of vascular tone and blood pressure. The vasculareffects of 2-adrenoceptor agonists are complex, as they reflect theresults of the interplay between a central sympatholytic effect andpre- and postsynaptic 2-adrenoceptor activation (Gilsbach & Hein,2012). The intravenous administration of an 2-agonist is associatedlation.ology, Drug Development andurku, Kiinamyllynkatu 10, FI-2 333 7216.with a biphasic blood pressure response: initial vasoconstrictionand transient hypertension mediated by peripheral 2B-adrenoceptorsin vascular smooth muscle (Link et al., 1996; Paris et al., 2003; Talke,Lobo, & Brown, 2003), followed by sympathetic nervous systeminhibition, bradycardia and prolonged hypotensionmediated by central2A-adrenoceptors (Ebert, Hall, Barney, Uhrich, & Colinco, 2000;Macmillan, Hein, Smith, Piascik, & Limbird, 1996). Therefore, central2A-adrenoceptors are currently employed as targets of antihyperten-sive agents (Aantaa & Jalonen, 2006), but vascular 2-adrenoceptorshave so far not been exploited as cardiovascular drug targets. Aperipherally acting subtype-nonselective 2-adrenoceptor antagonistwas recently shown to effectively counteract the vasoconstrictionevoked by the potent 2-adrenoceptor agonist dexmedetomidine(Honkavaara et al., 2012). However, chronic subtype-nonselective2-adrenoceptor blockade is likely to lead to adverse cardiovasculareffects as a consequence of loss of presynaptic 2A-autoreceptor func-tion. This situation emphasizes the need to discover novel antagonists

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153S. Bjrk et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 152162of the 2B-adrenoceptor subtype, and calls for relevant functionalassays to test the capacity of potential drug candidates to evoke orinhibit vascular smooth muscle cell contraction and vasoconstriction.Cell-based functional assays are commonly employed in modern drugdiscovery and development to identify lead molecules and to searchfor desired properties in potential drug candidates (Kenakin, 2009).

Increased intracellular concentrations of calcium ions in smoothmuscle cells activate the formation of calcium-calmodulin complexes,leading to activation of myosin light chain kinase. This phosphorylatesmyosin light chains, allowing these to form cross-bridges with actinfilaments, which is the molecular basis of smooth muscle cell contrac-tion (Small & Sobieszek, 1977; Somlyo & Somlyo, 1968). Most studieson 2-adrenoceptor-mediated vascular smooth muscle contractionhave employed isometric tension measurements on blood vessels, andthe results have varied greatly depending on the luminal diameter ofthe vessel and its anatomical origin, with 2-adrenoceptors beingmore prominent in small arteries and veins but not in large arteriessuch as the aorta (Nielsen et al., 1989; Parkinson & Hughes, 1995;Roberts, 2001, 2003). In particular, the vascular responses relating todexmedetomidine appear to be very complex; in endothelium-denuded arteries, the drug has induced contraction, whereas in intactvessels the relaxing and contracting effects of dexmedetomidine appearto oppose each other (Kim et al., 2009; Wong, Man, Vanhoutte, & Ng,2010). In addition, dexmedetomidine has induced endothelium-dependent relaxation at low concentrations in contracted small arteriesand contraction at higher concentrations (Wong et al., 2010). Studies oncultured vascular smooth muscle cells have been focused on themonitoring of intracellular calcium levels (Chotani et al., 2004;Hughes, Parkinson, & Wijetunge, 1996; Mironneau & Macrez-Lepretre,1995). Even if monitoring of myosin light chain phosphorylation byWestern blotting has been applied to investigate other signallingpathways in isolated vascular smooth muscle cells, we found no suchreports related to2-adrenoceptors. Therefore, we have nowdevelopeda cell-based functional assay with a biochemical readout of the contrac-tion process, i.e. phosphorylation of Ser19 of myosin light chains(pMLC20), to be used as a tool for the pharmacological characterizationof the capacity of 2-adrenoceptor agonists and antagonists to regulatevascular smooth muscle cell contraction. For this purpose, we chose toemploy the most commonly used vascular smooth muscle cell line, rataortic A7r5 cells (Kimes & Brandt, 1976), transfected to express thehuman 2B-adrenoceptor subtype (Huhtinen & Scheinin, 2008), andapproaches already validated in earlier studies (Aguilar, Zielnik,Tracey, & Mitchell, 2010; Schroter, Griffin, Weiser, Feng, & LoGrasso,2008).

2. Materials and methods

2.1. Materials

A7r5 cells were obtained from the American Type Culture Collection(ATCC;Manassas, VA, USA). Fetal bovine serumwas fromPAA Laborato-ries GmbH (Pasching, Austria), Nonidet P40 was from Calbiochem(Novabiochem, La Jolla, CA, USA). Dulbecco's modified Eagle's medium(DMEM), penicillin, streptomycin, trypsin, pertussis toxin, Geneticin(G418 disulphate salt solution), bovine serum albumin, poly-L-lysine,formaldehyde, arginine vasopressin, brimonidine, oxymetazoline,nifedipine, nimodipine, phosphatase inhibitor cocktail 3, anti--smoothmuscle actin antibody, HRP-conjugated anti-mouse secondaryantibody, pluronic acid and goat serum were from Sigma Aldrich(St. Louis, MO, USA). GF109203X, ML 7 hydrochloride, U73122, galleinand rauwolscine were from Tocris Bioscience (Bristol, UK). Fluor546-linked anti-mouse-IgG secondary antibody (goat) and Fura-2, AM wasfromMolecular Probes (Eugene, OR, USA). Protease inhibitor (CompleteMini EDTA free) was from Roche Diagnostics GmbH (Mannheim,Germany). DELFIA assay buffer, Enhancement Solution, Isoplate-96white TC and Eu-N1 anti-mouse antibody were from PerkinElmer Lifeand Analytical Sciences (Boston, MA, USA). Monoclonal mouse anti-pSer19-MLC antibody was from Cell Signalling Technology (Beverly,MA, USA). Vectashield with DAPI was from Vector Laboratories (Burlin-game, CA, USA). Dexmedetomidine and atipamezole were kind giftsfrom Orion Pharma (Turku, Finland). Other chemicals and reagentswere obtained from commercial suppliers.

2.2. Cell culture

A7r5-2B cells transfected to stably express the human 2B-adrenoceptor were cultured and maintained as described previously(Huhtinen & Scheinin, 2008). Briefly, the cells were cultured in DMEMsupplemented with 10% heat-inactivated fetal bovine serum, 100 U/mlpenicillin and 100 g/ml streptomycin. Cells were grown to approxi-mately 90% confluence in 75 cm2 culture flasks. Cultures weremaintained at 37 C in a humidified atmosphere containing 5% CO2.The medium was changed every 3 days and the cells were passagedapproximately once a week by dissociation with a solution of 0.025%trypsin and 0.1% EDTA.

2.3. Immunofluorescent labelling of phosphorylated myosin light chains

Immunofluorescent labelling of A7r5-2B cells was done (withminor modifications) as described previously (Huhtinen & Scheinin,2008). Briefly, A7r5-2B cells, cultured on poly-L-lysine-treatedcoverslips, were treated with 1 M dexmedetomidine (final) orDMEM (control) for 1045 s and fixed with 3.7% formaldehyde for20 min. The cells were subsequently thoroughly (56 times) washedwith phosphate buffered saline (supplemented with 0.9 mM CaCl2and 0.5 mM MgCl2) and immunolabelled either directly or stored at4 C. Non-specific binding was blocked by incubating the cells withblocking buffer (0.2% Nonidet P40, 2% goat serum, 1% BSA inphosphate-buffered saline). Phosphorylated myosin light chains werelabelled with a monoclonal phospho-myosin light chain (pSer19)antibody (1:50) in blocking buffer for 1 h at RT. The secondary antibody,Alexa Fluor 546 (1:1000), was applied for 45 min at RT. Nuclei werestained with DAPI (included in the Vectashield mounting medium).Cells were observed with a fluorescence microscope (Zeiss AxioVert200 M, AxioVision 4.8.1 software, Plan-NEOFLUAR 40/0.75 objective).

2.4. Protein extraction and Western immunoblotting

A7r5-2B cells were plated in 60 mm dishes and grown to conflu-ence (elongated shape), then serum-starved o/n inDMEMsupplementedwith 0.5% serum. Dishes were taken out of the incubator and the cellstreated instantly at RT with 1 M (final) dexmedetomidine (vehicle,other drugs or a combination of drugs) added carefully on top of themedium for 1045 s. For each time point, 2 samples were treated withdrugs and 2 samples served as controls and were treated with vehicle.The medium was aspirated, the plates were placed on ice and the cellswere lysed by scraping them into ice-cold RIPA buffer containingphosphatase and protease inhibitors. Samples were maintained atconstant agitation for 20 min at 4 C and then centrifuged (20 min,12,000 rpm, 4 C). The supernatant was frozen at 20 C and proteinconcentrations were determined with a conventional protein assay kitaccording to the manufacturer's instructions (BioRad DC ProteinAssay). Approximately 30 g of protein per sample was taken for SDS-PAGE, sample buffer was added (final 2% SDS, 5% -mercaptoethanol,10% glycerol, 0.01% bromophenol blue and 0.0625 M Tris-HCl; pH 6.8),and the samples were boiled for 5 min. They were then loaded onto 15or 18% conventional acrylamide SDS-PAGE gels with a 4% stacking gel.SDS-PAGE was carried out in running buffer (25 mM Tris, 192 mMglycine, 0.1% SDS; pH 8.3) using 60 V until the samples reached theseparating gel and then at 150V in a BioRadMini Protean ElectrophoresisCell. The separated proteins in the gel were then transferred to a PVDFmembrane (pre-soaked in MeOH for 2 min) in ice-cold transfer buffer

154 S. Bjrk et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 152162(25 mM Tris, 192 mM glycine, 20% MeOH; pH 8.3) at 15 V for 50 minusing a BioRad Trans Blot SD Semi-dry Transfer Cell. The membranewas washed for 5 min in PBS and then incubated in 0.5% formaldehydefor 1 h to improve the retention of phosphoproteins to the membrane(Takeya, Loutzenhiser, Shiraishi, Loutzenhiser, & Walsh, 2008), andthen washed twice in TBS for 5 min. Membranes were then blocked inOdyssey blocking buffer (OBB, Li-Cor Biosciences) for 1 h at RT. Incuba-tionwithmouse anti-pSer19-MLC antibody (1:2000) and anti--smoothmuscle actin antibody (1:20,000) in OBBwas then performed o/n at 4 C.Next morning, the membranes were washed seven times (total, 25 min)in TBST (TBS with 0.02% Tween-20) at RT on a bench-top shaker.Membranes were then blocked again with 5% milk in TBST for 30 minat RT and finally incubated with the HRP-conjugated anti-mousesecondary antibody diluted 1:1000 in 5% milk/TBST for 1 h at RT.Membranes were then again washed seven times with TBST (total,40 min). Chemiluminescence was developed with the Amersham ECLWestern blotting reagent.

2.5. Measurement of intracellular calcium levels

A7r5-2B cells were cultured on poly-L-lysine coated coverslips for48 h before experiments. Cells were washed twice with imaging buffer(138 mM NaCl, 5 mM KCl, 0.4 mM KH2PO4, 4 mM NaHCO3, 1.2 mMMgCl2, 2 mM TES, 10 mM glucose, 1 mM CaCl2 and 1 mM probenecid;pH 7.4). Cells were incubated with loading buffer (2 M Fura-2, AM,0.01% pluronic acid in imaging buffer) for 30 min at 37 C and thenwashed with imaging buffer. The coverslips were placed in a perfusionchamber that was mounted on a fluorescence microscope (ZeissAxiovert 35, SensiCam CCD camera). The 340 and 380 nm excitationfilters were used and emission was measured at 510 nm. The lightsource was an XBO 75 W/2 xenon lamp. The shutter was controlled bya Lambda 10-2 control device (Sutter Instruments, Novato, CA, USA),and images were collected with a SensiCam CCD camera (PCO/CDImaging, Kelheim, Germany). When a stable baseline was obtained(after 1 min), the cells were stimulated with agonists at RT.Pre-treatment with nimodipine (1 M) lasted 5 min. The F340/F380ratio was used to evaluate the increases in intracellular calcium levels.

2.6. Myosin light chain phosphorylation: 96-well assay

A7r5-2B cells were plated in sterile 96-well plates and grown toconfluence (elongated shape), then serum-starved o/n in 50 l DMEM/0.5% FBS. For studieswith agonists, plateswere taken out of the incubatorand instantly treated at RT by adding 50 l of dexmedetomidine,brimonidine or oxymetazoline solution (DMEM for controls) carefullyon top of the medium for 5120 s with a 12-well multichannel pipette.Receptor antagonists (atipamezole and rauwolscine; 100 M final)were added simultaneously with dexmedetomidine. For studies withinhibitors, cells were pre-treated with 50 l of each tested inhibitor inDMEM (vehicle for controls) at 37 C. The employed concentrations ofthe inhibitors were based on published reports and were as follows:30 M ML-7 (10 min), 200 ng/ml PTX (o/n), 100 M gallein (20 min),20 M nifedipine (20 min), 1 M U73122 (15 min), 10 M GF 109203X(1020 min), 10 MG 6976 (20 min) and 10 MH-89 (15 min). Plateswere taken out of the incubator and instantly treated withdexmedetomidine as above. The layout of the plates for the inhibitorexperiments was as follows: columns 13 were control samples withoutinhibitor, columns 46were dexmedetomidine-treated samples withoutinhibitor, columns 79were control samples with inhibitor and columns1012 were dexmedetomidine-treated samples with inhibitor.Treatment times were: 5 (row A), 10, 20, 30, 45, 60, 90 and 120 s(row H). This allowed for normalization of every sample to its owncontrols for each separate time point. Cells were then immediatelyfixed by adding 3 (3.7% final) formaldehyde (20 min at RT) on topof the samples using a calibrated digital multichannel pipette with arepeated function to ensure accuracy in treatment timing. The successiveprotocol was modified from a previous report (Aguilar et al., 2010).Afterfixation, the cellswerewashed three timeswith PBS-H (PBS supple-mentedwith 20mMHepes), permeabilizedwith 0.1% TritonX-100+0.1M glycine/PBS-H for 20 min at RT and then washed three times withPBS-H. Cells were blocked with 2% BSA/PBS-H for 30 min at RT undergentle agitation and then labelledwithmouse anti-pSer19-MLC antibody(1:2000) in DELFIA assay buffer (supplemented with BSA to a finalconcentration of 1%) for 1 h at RT under gentle agitation. Cells werewashed five times (total 20 min) with TBST (TBS with 0.1% Tween20).Subsequently, cells were incubated with secondary Eu-N1- anti-mouseantibody at a final concentration of 300 ng/ml, diluted in DELFIA assaybuffer (supplemented with 1% BSA and 2 mM CaCl2), for 45 min at RTunder agitation. Cells were washed eight times with TBST (total25 min). Enhancement solution was added and plates were shakenvigorously for 15 min on a DELFIA plate shaker (Wallac, Turku,Finland). Time-resolved fluorescence was read with a Victor V2 multi-label plate reader (615/8.5 nm; Wallac, Turku, Finland).

2.7. Data analysis

For Western blot densitometry, bands were analysed with Image-J1.45 s (National Institutes of Health, Bethesda, MD, USA). Immunofluo-rescent labelling was quantitated as follows. Images were taken fromfour randomly chosen spots from both vehicle and dexmedetomidine-treated cells using the same exposure and light intensity. Fluorescenceintensities were quantitated with Image-J and the averaged signalswere compared. The images for measurement of intracellular calciumwere processed using Axon Imaging Workbench 5.1 software (AxonInstruments, Foster City, CA, USA). In the myosin light chain phosphor-ylation assay, each sample had three replicates and Cook's distanceestimation (Cook, 1977) was used to identify outlying replicates. Cook'sdistance was calculated according to the formula (meanmean_i)2/standard error (mean)2, where the mean was the average of all repli-cates and mean_i was the average of all replicates except replicate i.Replicates with a Cook's distance greater than 0.8 were omitted fromfurther analysis and pMLC20 responses were calculated as follows. Thearea under the pMLC20 response curve (AUC) was determined up to45 s for the different compounds used in the experiments according tothe trapezoidal rule:

AUC Xt45 s

t0 s 1=2 y1 y2 t2t1

where y1, y2 = averaged Europium counts from successive samples, t1,t2 = successive time points in an experiment. The AUC values of differ-ent treatmentswere compared to the AUC of 1 Mdexmedetomidine inthe same experiment and pMLC20 responses were expressed as apercentage of 1 M dexmedetomidine. Graphs of individual experi-ments were plotted using GraphPad Prism programs (GraphPad Soft-ware, San Diego, CA, USA). Statistical analyses were carried out withtwo-tailed, one-sample t tests. P values smaller than 0.05 were consid-ered to be statistically significant.

3. Results

3.1. Assay validation with Western blotting, immunofluorescent labellingand intracellular calcium measurements

Confluent and serum-starved A7r5-2B cells were tested for theoccurrence of agonist-induced phosphorylation of myosin light chains(pMLC20) by Western blotting. The upper panel in Fig. 1A shows cellstreated with 1 M dexmedetomidine or vehicle for 10 or 20 s, and thelower panel shows cells treated for 30 or 45 s. An antibody againstphosphorylated serine 19 of myosin light chains detected pMLC20bands of the expected molecular mass (18 kDa, the lower band inboth panels), while smooth muscle-actin was used as loading control(the upper band in both panels). Actin-normalized results are shown in

Fig. 1. Detection of myosin light chain phosphorylation (pMLC20) and intracellular calcium levels in A7r5-2B cells. (A) Western blot membranes showing pMLC20 immunoreactivity inconfluent and serum-starved A7r5-2B cells treatedwith vehicle or 1 Mdexmedetomidine (Dex) for 10 s, 20 s, 30 s or 45 s. (B)Western blot densitometry analysis of pMLC20 expressedas a percentage of vehicle controls. Individual samples were normalized to their actin levels. Results are means SEM of three individual experiments performed in duplicate.(C, D) Immunofluorescence imaging of pMLC20 in confluent and serum-starvedA7r5-2B cells treated for 30 swith (C) vehicle or (D)1 Mdexmedetomidine. (E) The cellswere stimulatedwith 100 nM dexmedetomidine and the change in fluorescence ratio was measured. Dexmedetomidine-induced increases in intracellular calcium were measured in A7r5-2B (meantraces of 46 measurements) and A7r5 wild-type (wt) cells, as well as in A7r5-2B cells pre-treated with the L-type calcium channel inhibitor nimodipine. (F) Dexmedetomidine(10 and 100 nM) evoked increases in intracellular calcium were compared to those evoked by 100 nM oxymetazoline (Oxy), brimonidine (Bri) and arginine vasopressin (AVP). Resultsare expressed as means SEM of the 340/380 increase in fluorescence intensity.

155S. Bjrk et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 152162Fig. 1B. Dexmedetomidine treatment increased the pMLC20 signal(expressed as a percentage of vehicle-treated controls) to 121 4%(10 s), 111 11% (20 s), 141 7% (30 s) and 128 28% (45 s). Thetime point at which the maximal dexmedetomidine response wasdetected varied from one experiment to another, making it impossibleto select one treatment time point for further experiments. Therefore,this method was not directly amenable for pharmacological determina-tions because of the vast number of samples it would have required.

Fluorescence microscopy was used to visualize and determinepMLC20 levels in confluent and serum-starved A7r5-2B cells treatedas above. Similar to the Western blotting method, the time point ofthe maximal dexmedetomidine response varied from one experimentto another. Visual inspection of all samples used for the quantitativeanalysis presented here indicated that the maximal pMLC20 responseto dexmedetomidine stimulation in this experiment occurred at 30 s(data not shown), and this time point was chosen for quantitation.Dexmedetomidine treatment increased the pMLC20 immunoreactivityto 158 9% of vehicle-treated controls. Even if the basal pMLC20 signalin vehicle-treated cells was relatively high (Fig. 1C), dexmedetomidinetreatment clearly increased the fluorescence intensity (Fig. 1D).

To validate the assay parameters with a readout other than pMLC20,we employed measurements of intracellular calcium levels in A7r5-2Bcells. Stimulation with 100 nM of dexmedetomidine induced anapproximately 140% increase in the 340/380 nm fluorescence ratio,reflecting free intracellular calcium, peaking approximately 10 s afterdexmedetomidine addition. The endogenous agonist, arginine vaso-pressin (100 nM) induced a comparable increase (136%) in the 340/380 nm fluorescence ratio. Non-transfected wild-type A7r5 cellsshowed no calcium responses to dexmedetomidine, indicating thatthe response was mediated by 2B-adrenoceptors (Fig. 1E). Anothertwo 2-adrenoceptor agonists, oxymetazoline and brimonidine,as well as a lower concentration of dexmedetomidine (10 nM), pro-duced similar but smaller increases in intracellular calcium (Fig. 1F;p b 0.0001). Additionally, pre-treatment with the L-type calciumchannel inhibitor nimodipine (1 M) abolished the dexmedetomidine-induced increase in intracellular calcium (Fig. 1E).

image of Fig.1

156 S. Bjrk et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 1521623.2. Quantitation of myosin light chain phosphorylation with 96-well assay

3.2.1. Effects of agonists and antagonistsPhosphorylation of myosin light chains can be considered a

biochemical readout of smooth muscle cell contraction (Small &Sobieszek, 1977; Somlyo & Somlyo, 1968). Therefore, a cell-basedquantitative 96-well assay measuring myosin light chain phosphoryla-tion was developed to monitor 2B-adrenoceptor-evoked contractionof A7r5-2B vascular smooth muscle cells. The results of individualexperiments described in this section are illustrated in Fig. 2 and theresults of all experiments are summarized in Fig. 4A. Dexmedetomidinewas chosen as a reference agonist, since it is the most selective (2/1-adrenoceptor selectivity ratio 1620) 2-adrenoceptor agonist currentlyavailable (Virtanen, Savola, Saano, & Nyman, 1988). It is a potent, full2-adrenoceptor agonist in terms of receptor-mediated G-proteinFig. 2. Agonist-inducedmyosin light chain phosphorylation (pMLC20) in A7r5-2B cells. Conflue5120 s or with (A) arginine vasopressin (AVP, 100 nM), (B) with different concentrations of(E) oxymetazoline (Oxy, 1 M). A7r5-2B cells were treated with dexmedetomidine (1 M) ina 10-min preincubation with the myosin light chain kinase inhibitor ML-7 (30 M). Normalizeof individual experiments repeated at least three times in triplicate (duplicate for 2B). The horactivation in the employed A7r5-2B cell line (Huhtinen & Scheinin,2008). Dexmedetomidine-induced myosin light chain phosphorylationwas assessed over 5120 s. Typically, the dexmedetomidine responsepeaked at 2045 s with an Emax value of 59 5% (n= 29) over vehiclecontrols (Fig. 2A). This is in linewith the results obtained fromWesternblotting and immunofluorescent labelling. As a control for cell respon-siveness, a set of samples treated with dexmedetomidine was includedin all experiments and all other compounds were related to the pMLC20response evoked by 1 M of dexmedetomidine. The dexmedetomidineresponse was dose-dependent (Fig. 2B, where the number of replicatesis 2); treatmentwith 100 nMdexmedetomidinewas associatedwith anAUC value of 75 11% (compared with the response to 1 Mdexmedetomidine, defined as 100%), and the AUC after 30 nM was20 7%, after 10 nM 11 5% and after 1 nM it was 3 1% (Fig. 4A).Concentration-response curveswere generatedusing either the averagent and serum-starved A7r5-2B cells were treated with 1 Mdexmedetomidine (Dex) fordexmedetomidine (1 nM1 M) or with the 2-agonists (D) brimonidine (Bri, 1 M) orthe presence or absence of (C) the 2-antagonist atipamezole (Ati, 100 M) or (F) afterd data are expressed as a percentage over vehicle controls. The graphs are representativeizontal bar in 2A indicates the timescale used for calculating AUC values.

image of Fig.2

157S. Bjrk et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 152162peak (Emax) values or the area under the curve (AUC) as the measure ofresponse (n=6; Fig. 4B).The calculated EC50 (Emax) average was 30 nM(95% CI, 6157 nM) and the EC50 (AUC) averagewas 65 nM (95% CI, 30140 nM).

The 2-adrenoceptor antagonist atipamezole (Fig. 2C) blocked thedexmedetomidine-evoked pMLC20 response by 87 11% (Fig. 4A),indicating that the dexmedetomidine response was mediated by 2-adrenoceptors. Rauwolscine, another 2-adrenoceptor antagonist,blocked the dexmedetomidine-evoked pMLC20 response by 73 6%(data not shown). Treatment of wild-type (non-transfected) A7r5cells with dexmedetomidine had no effect, indicating that the pMLC20response seen in A7r5-2B cells was mediated by 2B-adrenoceptors(data not shown). Stimulation of endogenously expressed vasopressinV1 receptors with 100 nM of arginine vasopressin (Fig. 2A) evoked apMLC20 response of 15429% (comparedwith 1 Mdexmedetomidine;Fig. 4A) in A7r5-2B cells. Two additional 2-adrenoceptor agonists,brimonidine (Fig. 2D) and oxymetazoline (Fig. 2E), at 1 M, evokedapproximately similar pMLC20 responses as dexmedetomidine (120 14% and 108 31% compared to dexmedetomidine, respectively;Fig. 4A). However, a comparison of 100 nM dexmedetomidine,brimonidine and oxymetazoline revealed that similar to the calciumexperiments, brimonidine and oxymetazoline at this concentrationevoked smaller pMLC20 responses, with Emax values of 31 2% and29 2% over vehicle control, respectively, compared to 100 nM ofdexmedetomidine (46 3%; n = 3). In an AUC comparison, thepMLC20 responses to 100 nM brimonidine and 100 nM oxymetazolinewere 39 15% and 37 9%, when compared to the AUC value of100 nMdexmedetomidine (100%). To ensure that the observed responseactually was phosphorylation of myosin light chains, an inhibitor ofmyosin light chain kinase (ML-7) was applied (Fig. 2F). As expected,pre-treatment of the cells with ML-7 completely abolished thedexmedetomidine-evoked pMLC20 response (Fig. 4A).

3.2.2. Effects of inhibitorsTo explore the intracellular pathways involved in the 2B-

adrenoceptor mediated phosphorylation of myosin light chains, A7r5-2B cells were exposed to different inhibitor compounds. The resultsof individual experiments described in this section are illustrated inFig. 3 and the results of all experiments are summarized in Fig. 4A.Exposure to pertussis toxin eliminates the capacity of Gi-typeG-proteins to be activated by their cognate receptors (Bokoch, Katada,Northup, Hewlett, & Gilman, 1983). Pre-treatment with pertussistoxin (Fig. 3A) almost totally abolished the dexmedetomidine-evokedpMLC20 response (by 83 8%; Fig. 4A), suggesting that thedexmedetomidine response is Gi-protein mediated. G-protein activa-tion is followed by the dissociation of the subunit from the subunit(Gilman, 1987), which enables the dissociated subunits to interactwith distinct effector proteins. Gallein is an inhibitor of subunit-dependent signalling (Lehmann, Seneviratne, & Smrcka, 2008). In cellspre-treated with gallein (Fig. 3B), the dexmedetomidine-evokedpMLC20 response was almost completely abolished (by 93 6%;Fig. 4A), indicating an important role for subunits in the pMLC20response. Pre-treatment with nifedipine (Fig. 3C), an L-type calciumchannel blocker, attenuated the dexmedetomidine-evoked pMLC20response by 66 12% (Fig. 4A), suggesting the involvement of L-typecalcium channels. The phospholipase C inhibitor U73122 (Fig. 3D)completely abolished the dexmedetomidine-evoked pMLC20 response(Fig. 4A), indicating that phospholipase C is also involved in the intra-cellular pathways leading to the phosphorylation ofmyosin light chains.Unexpectedly, the protein kinase C inhibitors GF109203X (Fig. 3E) andG6976 (Fig. 3F) very markedly potentiated the dexmedetomidine-induced pMLC20 response to 323 74% and 300 51% of the responseto dexmedetomidine alone (Fig. 4A). Nifedipine also blocked thecombined effects of dexmedetomidine and protein kinase C inhibition(Fig. 3G and Fig. 4A), indicating that increased influx of calcium ionsvia L-type calcium channels is necessary for the potentiation of thedexmedetomidine-induced pMLC20 response following pre-treatmentwith the protein kinase C inhibitors. Finally, blockade of protein kinaseA with H-89 (Fig. 3H) augmented the pMLC20 response to 211 22%(Fig. 4A). Control experimentswere performedwith the different inhib-itors alone and all combination treatment results were normalized totheir own controls (inhibitor alone). The effects of the inhibitors aloneon the extent of basal MLC phosphorylation (without agonist) wereevaluated with t-tests for the AUC over 45 s after drug addition. Thechange in AUC was statistically non-significant for most of the testedinhibitors (ML-7, +7%; PTX, +2%; U73122, 6%; G6976, 14% andnifedipine, +7%), but significant decreases in pMLC were observedafter GF109203X (PKC inhibitor), H-89 (PKA inhibitor) and gallein(G inhibitor: 17%, 33% and 16%; p b 0.05, p b 0.01 andp b 0.05, respectively).

4. Discussion

Smooth muscle contraction is initiated by myosin light chainphosphorylation (pMLC20) at Ser19 by myosin light chain kinase(Small & Sobieszek, 1977; Somlyo & Somlyo, 1968). A linear correlationbetween the extent of MLC20 phosphorylation and muscle contractilityhas been established (Takashima, 2009), and therefore, phosphoryla-tion ofMLC20 at Ser19 has obvious face validity as a biochemical readoutof smooth muscle cell contraction. Traditionally, vascular smoothmuscle contraction has been investigated by tension measurementson isolated blood vessels or by analysing myosin light chain phosphor-ylationbyWesternblotting. These techniques, however, are not suitablefor screening a large number of ligands or for studies with inhibitors.We describe here the use of a cell-based functional assay for thequantitative determination of pMLC20 that can be used for ligand profil-ing purposes of agonist-induced vascular smooth muscle contraction.Assay validationwas performedwith single-cell analysis of intracellularcalcium responses, with Western blotting and with immunostaining.

The reproducibility and robustness of the pMLC20 readout are notexcellent. Even in this relatively simple cell-based model system, thereare many factors at play, some of them unidentified and difficult tocontrol. One of the confounders is the physiology related to 2-adrenoceptors,which is discussed inmore detail below. Another impor-tant factor is cell stress. Numerous parameters were optimized for theassay, such as the number of cells per well (104) required for the cellmonolayer to reach confluence in three days and the effect of black orwhite plates with regard to fluorescence scattering between wells. Aswe wanted to avoid cell stress, in particular since cell stress results inincreased pMLC20, we evaluated the importance of maintaining thesamples at 37 C during treatments by using a heating block, as wellas by monitoring the temperature of the dexmedetomidine/controlliquids added into the wells. Another stress-inducing factor was theremoval of media prior to, e.g. agonist addition, and therefore, wealways added media on top of the existing liquid. Furthermore, wenoticed that dexmedetomidine eagerly adsorbs onto differentmaterialsand hence the use of disposable plastic containers was necessary toavoid carryover effects. Despite the optimization attempts, theresponses to dexmedetomidine showed marked variability regardingboth the amplitude of the response as well as the time course. Thatthe response was observed to peak at different time points after agonistaddition is very limiting with regard to attaining classic pharmacologicalassay parameters. This phenomenon was, however, noticed in all threemethods used, which supports the notion that the pMLC20 responseneeds to be followed over time to be able to detect themaximal response.This may be an inherent property of the experimental model: atransfected receptor in a sensitive vascular smooth muscle cell line andinsufficient technical control of all relevant parameters.

The pMLC20 response is almost instantaneous, it takes only secondsfrom agonist stimulation to maximal phosphorylation of MLC20(Takashima, 2009). Initially, we followed the time course of thepMLC20 response upon dexmedetomidine stimulation in A7r5-2B

Fig. 3. Effects of different inhibitors on agonist-induced myosin light chain phosphorylation (pMLC20) in A7r5-2B cells. Confluent and serum-starved A7r5-2B cells were treated with1 M dexmedetomidine (DEX) for 5120 s in the presence or absence of 1020 min preincubation (o/n for PTX) with the following inhibitors: (A) pertussis toxin (PTX, 200 ng/ml),(B) gallein (100 M), (C) nifedipine (20 M), (D) U73122 (1 M), (E) GF109203X (GF, 10 M), (F) G6976 (G, 10 M), (G) nifedipine (20 M) + GF109203X (10 M) and (H) H-89(10 M). Normalized data are expressed as a percentage over vehicle controls. Note that panels EH are from preincubationswith inhibitors dissolved in DMSO. Graphs are representativeof individual experiments repeated at least three times in triplicate.

158 S. Bjrk et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 152162

image of Fig.3

Fig. 4. Effects of agonists, antagonists and inhibitors on the phosphorylation of myosinlight chains (pMLC20) in A7r5-2B cells, in relation to the pMLC20 response (AUC) inducedby the 2-agonist 1 M dexmedetomidine (100%). (A) Agonists included argininevasopressin (AVP, 100 nM), brimonidine (BRI, 1 M), oxymetazoline (OXY, 1 M) anddexmedetomidine (DEX, 1 nM1 M). The antagonist atipamezole (ATI, 100 M) wasadded simultaneously with dexmedetomidine. Pre-treatment times for inhibitors were1020 min (o/n for PTX) and inhibitors used were: ML-7 (myosin light chain kinaseinhibitor, 30 M), pertussis toxin (PTX; Gi protein inhibitor, 200 ng/ml), gallein (Gsubunit inhibitor, 100 M), nifedipine (calcium channel blocker, 20 M), U73122(phospholipase C inhibitor, 1 M), GF109203X (GF) and G6976 (G; protein kinase Cinhibitors, 10 M) and H-89 (protein kinase A inhibitor, 10 M). Results are AUCmeans SEM (up to 45 s) of at least three individual experiments performed in triplicate.*, p b 0.05; **, p b 0.01; ***, p b 0.001 compared to 1 M dexmedetomidine. Combinedtreatments with dexmedetomidine and GF and G failed to reach statistical significance(p = 0.050.10) because of the small number of observations. (B) Dose-response curvesgenerated using either the average peak (Emax) values or area under the curve (AUC)values as the measure of response. Results are means from six individual experimentsperformed in duplicate.

159S. Bjrk et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 152162cells over 5 min. Typically, the response was fully developed 2045 safter agonist addition, and pMLC20 levels returned to baseline by twominutes, highlighting the importance of quick and accurate samplehandling. Simultaneous termination of the reactions was performedby adding fixative with a digital multichannel pipette, making itpossible to fix an entire assay plate within a few seconds. Differentexternal stimuli (pipetting, shaking the plate) may increase the basallevel of pMLC20; therefore, only simultaneously treated samples arecomparable and it is important to design the layout of an assay platein such a way that each sample has all necessary controls in the samerow. Had we normalized all data to one control time point, e.g. the2 min time point, it would have resulted in a significantly higher Emaxsince at this point the stress response is largely overnow, variation isalso introduced to the control cells by disturbing themwith the additionof vehicle.We propose that the reproducibility, signal-to-noise ratio andmagnitude of the responses would greatly benefit from an automatedsystem, where the plates would be firmly attached, kept in a built-inincubator and simultaneous and controlled liquid additions would beaccomplished with an automated 96-channel liquid handling device.Such a robotic system would allow identical drug delivery conditionsin each well and eliminate problems related to reproducibility such aschanges in temperature, homogeneity of drug solutions, liquid and aircurrents and tremor. In our evaluation, data normalization of thepMLC20 responses was not necessary since protein levels were constantas confirmed by total myosin light chain content determination (datanot shown). As a specificity control for the pMLC20 antibody, the specificinhibitor of myosin light chain kinase, ML-7, completely abolished theagonist-evoked signal.

Different stimuli, such as changes in temperature, pressure and flow,can evoke the contraction of vascular smooth muscle cells in theirnatural surroundings in the blood vessel wall (Bevan & Joyce, 1990;Shepherd, Rusch, & Vanhoutte, 1983). To reduce the effects of suchstimuli on the basal levels of pMLC20, handling of the assay plates waskept at a minimum. Even so, A7r5-2B cells exhibited relatively highbasal levels of pMLC20, as exemplified by strong pMLC20 bands in theWestern blot control samples. In line with this, cultured rat aorticsmooth muscle cells were reported to have a basal pMLC20 of 34% andphosphorylation was increased to 7080% upon stimulation with thestrong endogenous vasoconstrictors endothelin-1 and angiotensin II(Woodsome, Polzin, Kitazawa, Eto, & Kitazawa, 2006). For 2-adrenoceptor-mediated constriction of strips of rabbit saphenousveins in vitro, the reported maximal pMLC20 responses are 2530% oftotal MLC (Aburto, Lajoie, & Morgan, 1993). Even at its most powerful,2-adrenoceptor-mediated constriction evoked by high-efficacyagonists is smaller compared to, e.g. 1-adrenoceptor-evoked constric-tion (Crassous, Flavahan, & Flavahan, 2009; Flavahan, 2005; Flavahan,Rimele, Cooke, & Vanhoutte, 1984). As increased intracellular calciumis needed for smooth muscle contraction evoked by 2-adrenoceptors,the interplay between postjunctional 1- and 2-adrenoceptors maybe important on the physiological level. Noradrenaline and adrenalineactivate both 1- and 2-adrenoceptors, and due to the 1-adrenoceptor-mediated increase in intracellular calcium, concurrent2-adrenoceptor-mediated constriction is potentiated (Jantschak &Pertz, 2012).

Vascular 2-adrenoceptors play an essential physiological role inmodulating blood vessel diameter and blood flow, but different vascularbeds are variably regulated by the different 2-adrenoceptor subtypes.2A-Adrenoceptors appear to contribute to the control of vasculartone in the carotid (Villalon et al., 2004), mesenteric (Paiva, Morato,Moura, & Guimaraes, 1999) and nasal mucosal (Corboz et al., 2003)vascular beds. In superficial veins, constriction by 2C-adrenoceptorsappears to predominate (Gavin, Colgan, Moore, Shanik, & Docherty,1997; Gornemann et al., 2007; Rizzo et al., 2001). However, in vivostudies in conscious mice suggest that 2B-adrenoceptors in smallarteries play a major role in regulating total peripheral vascularresistance (Link et al., 1996; Paris et al., 2003; Talke et al., 2003). Thecomplex pharmacology of vascular 2-adrenoceptors led us to developan assay on a more simple level in order to assess the capacity of 2-adrenoceptor agonists and antagonists to regulate vascular smoothmuscle cell contraction. It is to be kept in mind that this is a cell-basedmodel system and the results are thus to be interpreted accordingly.

Dexmedetomidine is a full agonist of the 2B-adrenoceptor(Peltonen, Pihlavisto, & Scheinin, 1998). Upon intravenous administra-tion, it produces a transient increase in blood pressure in humans andother mammals (Ebert et al., 2000; Honkavaara et al., 2012; Snapiret al., 2006). Therefore, dexmedetomidine was chosen as the referenceagonist to which the effects of other compounds were compared.

image of Fig.4

160 S. Bjrk et al. / Journal of Pharmacological and Toxicological Methods 70 (2014) 152162Arginine vasopressin was chosen to represent natural agonists, stimu-lating endogenously expressed vasopressin V1 receptors in A7r5-2Bcells (Thibonnier, Bayer, Simonson, & Kester, 1991). The time courseand maximal effect of vasopressin-evoked pMLC20 responses werecomparable to those evoked by dexmedetomidine.

From the responses to dexmedetomidine (1 nM1 M), curves weregenerated using either the peak response (Emax) or the AUC values asthe measure of response. EC50 values calculated from these curveswere comparable to the reported [35S]GTPS EC50 value of 10 nM fordexmedetomidine in the same cells (Huhtinen & Scheinin, 2008). Thesomewhat higher EC50 values derived from the present assay mayreflect the effects of non-equilibrium conditions, which tend to under-estimate drug potency (Charlton & Vauquelin, 2010). The otheremployed 2-adrenoceptor agonists, oxymetazoline and brimonidine,have previously been shown to be partial agonists of the 2B-adrenoceptor (Huhtinen & Scheinin, 2008; Olli-Lhdesmki, Tiger,Vainio, Scheinin, & Kallio, 2004). They were less potent thandexmedetomidine in the present calcium measurements. In thepMLC20 assay, both oxymetazoline and brimonidine evoked similarpMLC20 responses as dexmedetomidine, although with lesser potency,suggesting that full agonism may not be a requirement for a full-scalepMLC20 response. A large receptor reserve in these transfected cellsmay be the explanation.

The assay also allows for preincubation with inhibitor compounds,and to evaluate this feature, we investigated a panel of G-protein, kinaseand lipase inhibitors expected to be involved in pathways linked tovascular2B-adrenoceptor-evokedmyosin light chain phosphorylation.These events are not fully known,which attests to the complexity of2-adrenoceptor physiology and pharmacology, as these receptors appearto regulate a multitude of possible effector pathways in different celltypes and tissues (Cotecchia et al., 1990; Dorn, Oswald, McCluskey,Kuhel, & Liggett, 1997; Eason, Kurose, Holt, Raymond, & Liggett, 1992;Hughes et al., 1996; Lepretre & Mironneau, 1994; Mironneau &Macrez-Lepretre, 1995). Classically, 2-adrenoceptors have beenassociated with pertussis toxin-sensitive Gi-dependent inhibition ofadenylyl cyclase activity, but this alone cannot account for all themechanisms involved in contraction. Studies on vascular smoothmuscle cells have shown that 2-adrenoceptor activation increasesintracellular calcium levels (Chotani et al., 2004), and pertussis toxin-sensitive, Gi-mediated activation of L-type calcium channels has beensuggested as a mechanism (Hughes et al., 1996; Lepretre &Mironneau, 1994; Mironneau & Macrez-Lepretre, 1995). The existenceof such a pathway was confirmed in studies using isometric tensionmeasurements on blood vessels (Parkinson & Hughes, 1995; Roberts,2001), and elevated intracellular calcium levels are indeed needed forpMLC20 formation (Aburto et al., 1993). Our current results are inagreement with these proposedmechanisms, as blocking the activationof Gi-proteins with pertussis toxin resulted in almost complete abolish-ment of the pMLC20 response evoked by dexmedetomidine, andaddition of the L-type calcium channel blocker nifedipine resulted in a66% decrease of pMLC20. Stimulation of phospholipase C activity bypertussis toxin-sensitive G-proteins in fibroblasts was reported for2A- (Chabre, Conklin, Brandon, Bourne, & Limbird, 1994) and 2C-adrenoceptors (Cotecchia et al., 1990). In erythroleukemia (HEL) cellsendogenously expressing the 2A-subtype, as well as in transfectedfibroblasts, coupling to phospholipase C was suggested to be mediatedby G subunits and was followed by an inositol-1,4,5-trisphosphate-induced increase in intracellular calcium (Dorn et al., 1997). To furtherevaluate the involvement of G-mediated signalling in our testsystem, we employed the -inhibitor gallein, which decreased thedexmedetomidine-evoked pMLC20 response by 93%, strongly indicatingmediation by G-signalling. Furthermore, we propose involvement ofG-mediated activation of phospholipase C, since its inhibition withU73122 resulted in complete abolishment of the pMLC20 response.Both intracellular and extracellular sources of calcium ions thus appearto be essential for the process.Activation of phospholipase C results in the formation of inositol-1,4,5-trisphosphate and diacylglycerol, the latter activating proteinkinase C (Berridge & Irvine, 1984). The tested protein kinase C inhibitorsvery markedly potentiated the dexmedetomidine-induced pMLC20response, which might be due to increased responsiveness of theinositol-lipid signalling pathways, as reported for other smooth musclecells (Pfeilschifter, Ochsner, Whitebread, & De Gasparo, 1989; Zhong,Murtazina, Phillips, Ku, & Sanborn, 2008). Blockade of protein kinase Aalso augmented the pMLC20 response, although the interpretation ofthis effect is complicated by a reduction of basal pMLC20 levels(by 33%) by the PKA inhibitor H-89. At high agonist concentrations,2B-adrenoceptors also mediate Gs-dependent activation of adenylylcyclases (Pohjanoksa et al., 1997). Elevated cAMP levels in smoothmuscle cells inhibit myosin light chain kinase through activation ofprotein kinase A, resulting in smooth muscle relaxation (Adelstein,Conti, Hathaway, & Klee, 1978). It is conceivable that inhibition ofsuch a Gs-dependent protein kinase A-mediated pathway could explainthe increased pMLC20 response to dexmedetomidine. However, H-89has also been shown to block several other kinases, which also hampersthe interpretation of the results obtained with this inhibitor (Davies,Reddy, Caivano, & Cohen, 2000).

Commercially available inhibitors are commonly employed todeduce the involvement of specific proteins in the intracellular signal-ling cascades of receptors. Nevertheless, as noted above, inhibitorsmay give inconsistent results because of the complexity of the involvedpathways, and their toxicity, solubility and lack of specificity imposeadditional significant limitations (Davies et al., 2000). For example,when gallein and U73122 were not properly dissolved in our initialexperiments, these inhibitors erroneously appeared to produce noeffects. In addition, fresh inhibitor solutions should be used in all exper-iments, and the cells should be inspected to confirm their viability afterinhibitor treatments. Many inhibitors required the use of DMSO assolvent, e.g. the PKC and PKA inhibitors used in Fig. 3EH. We noticedrepeatedly that preincubation with a DMSO-containing solutionattenuated the dexmedetomidine-induced pMLC20 response, andtherefore, whenever possible, water or ethanol was used to dissolveinhibitors. Addition of DMSO at 0.21% has been reported to increaseintracellular calcium levels in many types of cultured cells (Morley &Whitfield, 1993), and might provide an explanation to the attenuateddexmedetomidine response.

We have demonstrated here that the novel assay is capable ofmonitoring myosin light chain phosphorylation evoked by activationof 2B-adrenoceptors in cultured vascular smooth muscle cells. Thisquantitative cell-based method allows the assessment of the capacityof ligands to evoke or inhibit vascular smooth muscle cell contractionand to investigate the intracellular pathways involved. This method isapplicable not only to transfected receptors but also to endogenouslyexpressed receptors, as shown for the vasopressin V1-receptor.Monitoring of a biochemical marker of agonist-evoked smooth musclecontraction in a complex, homeostatic system provides a more detailedimpression of the process compared to, e.g. intracellular calciummeasurements, which represent a more simplified picture.

Acknowledgments

We thankDr. Hector Aguilar and Dr. Alan Smrcka for valuable adviceand Professor Kid Trnquist for providing samples of inhibitors. UllaUoti is thanked for skilful technical assistance. We thank Drs. KidTrnquist, Christoffer Lf and Jyrki Kukkonen for assistance and adviceon measurements of intracellular calcium. We thank Dr. JoukoSandholm and the Cell Imaging Core at the Turku Centre for Biotechnol-ogy for assistance with fluorescence microscopy. This study has beenfinanced by the Turku Graduate School of Biomedical Sciences,TYKSLAB, the Academy of Finland, the Sigrid Juslius Foundation, theVarsinais-Suomi Regional Fund of the Finnish Cultural Foundation, theOtto A. Malm Foundation, the Ida Montin Foundation, the Waldemar

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Quantitative determination of 2B-adrenoceptor-evoked myosin light chain phosphorylation in vascular smooth muscle cells1. Introduction2. Materials and methods2.1. Materials2.2. Cell culture2.3. Immunofluorescent labelling of phosphorylated myosin light chains2.4. Protein extraction and Western immunoblotting2.5. Measurement of intracellular calcium levels2.6. Myosin light chain phosphorylation: 96-well assay2.7. Data analysis

3. Results3.1. Assay validation with Western blotting, immunofluorescent labelling and intracellular calcium measurements3.2. Quantitation of myosin light chain phosphorylation with 96-well assay3.2.1. Effects of agonists and antagonists3.2.2. Effects of inhibitors

4. DiscussionAcknowledgmentsReferences