multiple signaling pathways

8/12/2019 Multiple Signaling Pathways

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0013-7227/91/1296-2845$03.00/0EndocrinologyCopyright 1991 by The Endocrine Society Vol. 129, No. 6Printed inU.S.A.

Multiple Signaling Pathways of Vi-Vascular VasopressinReceptors of A 7r5Cells*MARC THIBONNIER, ANDREA L. BAYER, MICHAEL S. SIMONSON, ANDMARK KESTERDepartment of Medicine, UniversityHospitalsofClevelanda ndCaseWestern Reserve U niversitySchool ofMedicine,Cleveland, Ohio 44106-4982

ABSTRACT. We explored the nature and time course of themultiple signal transduction pathways for V^vascular vasopres-sin (AVP) receptors of A7r6aortic smooth muscle cells in cultureby using radioligand binding techniques, intracellular calciummonitoring, and polyphosphoinositide and phospholipid anal-yses.Vi -vascular AVP receptors ofA7r6cells were characterized bythe agon ist radioligand [3H]AVPand the antagonist radioligand[3H]d(CH 2)8Tyr(Me)AVP. Affinity and capacity of agonist butnot antagonist binding were modulated by MgCk and aluminumfluoride, suggesting that the receptors are coupled to a guaninenucleotide regulatory protein.In fura-2-loaded A7r6 cells, AVP induced within seconds adose-dependent increase of free intracellular Ca ++([Ca++]i) con-sisting ofarapid transien t spike and a sustained increase lastingfor 3-5 min. The baseline [Ca ++]i was 136 18nM, the m aximum[Ca++]i response to AVP was 1,582 297 nM, and AVP ED 50was 1.87 0.15 nM. Diverse experiments performed with EG TA, ,2-bis(O-aminophenoxy)ethane-iV)iV,iV',iV'-tetraacetic acid

acetoxymethylester, Mn++ , ionomycin, terbutylbenzo hydroqui-none, and nicardipine suggested that the initial spike resultedfrom both intracellular Ca++ release from the endoplasmic retic-ulum and extracellular Ca++ influx, whereas the sustained phasedepended on dihydropyridine-insensitive extracellular Ca ++ in-flux. Experiments done with indomethacin and arachidonic acidindicated that AVP-induced extracellular Ca ++ influx was inpart dependent on phospholipase A2activation.In [3H]myoinositol and [3H]arachidonate-labeled A7r6cells,AVP stimulated inositol 1,4,5 trisphosphate and 1,2 diacylglyc-erol production via activation of phospholipase C. Also, AVPstimulated a transphosphatidylation reaction through activationof phospholipase D in A 7r5 cells labeled with [3H]l-O-alkyllysoglycerophosphocholine.Thus, the stimulation of Vi-vascular AVP receptors of A 7r6cells triggers several signaling pathways. The immediate andtransient [Ca++]i rise due to mobilization of intracellular andextracellular Ca++is associated with the activation of phospho-lipases A2and C, and the sustained activation of phospholipaseD. {Endocrinology 129:2845-2856, 1991)

\ 7A S0 PR ES S IN (AVP) binds to specific Vi-vascularV and V 2-renal membrane receptors which activatedistinct second messengers (1). We (2-8) and others ( 9-11) have described the characteristics of specific Vx-vascular AV P receptors presen t at th e surface of hum anplatelets whose activation leads to calcium mobilizationan d platelet aggregation. Membrane Vi-vascular AVPreceptors also have been characterized in hepatocytes(12), as well as in rat aortic (13) and mesenteric (14)smooth muscle cells.There is now evidence that many hormones and neu-rotransmitters act not only through the hydrolysis ofphosphatidylinositol 4,5-bisphosphate (PIP2) to inositol1,4,5 trisp hos pha te (Ins [l,4,5]P3) an d 1,2-diacylglycerol(DAG) but also through the breakdown ofphosphatidyl-

Received July 5,1991.Address all correspondence and requests for reprints to: Dr. MarcThibonnier, Division of Endocrinology and Hypertension, Departmentof Medicine, Case Western Reserve University School of Medicine,2109 Aldelbert R oad, Cleveland, Ohio 44106-4982.* This work was supported by NIH G rants R01-HL-39757, P01-HL-41618, and R29-AR-40225.

choline (PtdCho) (15). The hydrolysis of PtdCho byphospholipase A2 produces arachidonic acid (AA),whereas hydrolysis of PtdCh o by phospholipases C andDleads to the formation of the putative second messen-gers DAG and ph osph atidic acid (PA), respectively. AV Pstimulates a PIP2-specific phospho lipase C activity, gen-erating inositol phosphates and DAG, as shown in he-patocytes (15, 16). AVP rapidly but transiently elevatesIns[l,4,5]P 3,whereas DAG production is sustained andremains elevated even at time points where inositolphosphateshave returned to basallevels.Sustained DAGproduction has been linked to phospholipase C-inducedhydrolysis of phosphatidylcholine or phosphatidyletha-nolamine as well as sequential activation of phospholi-pase D and PA phosphohydrolase. The role of AVP inthe temporal production of DAG through such mecha-nismsneeds to be investigated further.

In order to obtain an integrated picture of the inter-actions of AVP Vi -vascular receptors with a guaninenucleotide regulatory protein, inositol phospholipid me-tabolism, calcium mobilization, and different phospholi-2845


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2846 AVP RECEPTORS OF A7r5 CELLS Endo1991Vol l29-No6pases activation, we explored the cascade of events frominitial binding to several intracellular events triggeredby AVP activation of Vi-vascular receptors of A7r5cells.More specifically, we documented: 1) the characteristicsof agonist and an tagonist binding; 2) the origins of intra -cellular Ca+ + ([Ca++]i) mobilized by AVP stimulation;and 3) to what extent AVP-stimulated A 7r5cells activatephospholipases A2, C, and D and generate the putativesecond messengers arachidonate, diglyceride, and PA,respectively. We also asked whether phosphatidylcholinecould serve as a substrate for phospholipases C and Dactivity.

Materials and M ethodsMaterials

AVP, Tris-HCl, and other reagents, unless stated otherwise,were from Sigma Chemical Co. (St. Louis, MO).[3H]AVP(SA,62 Ci/mM) and [3H]d(CH2)5Tyr(Me)AVP (SA, 56.2 Ci/mM)were from Dupont de Nemours (Wilmington, DE). Ionomycinand l,2-bis(O-aminophenoxy)ethane N,N,N',N'-tetraaceticacid acetoxymethylester (BAPTA) were from Calbiochem Corp.(San Diego, CA). The BCA protein assay reagents were fromPierce Chemical Co. (Rockford, IL). Fura-2 AM was fromMolecular Probes Inc. (Eugene, OR). 2,5-Di(tert-butyl)-l,4-benzohydroquinone, (tBuBHQ) was a generous gift from Dr.George Kass, Karolinska Institutet, (Huddinge, Sweden). Cellculture media were from Hazleton Dutchland (Denver, PA).Fetal bovine serum was from Hyclone (Logan, UT). [3H]1-O-alkyl lyso glycero-3-phosphocholine (SA, 146 Ci/mM) and [3H]arachidonic acid (SA, 240 Ci/mM) were from Amersham (Ar-lington Heights, IL). [3H]Myoinositol (SA,15Ci/mM) was fromARC (St. Louis, MO). Lipid standa rds were from Avanti P olarLipids (Pelham, AL). Silica gel 60 TLC plates (250 /tM thick-ness, heat activated at 110 C) were from EM Science (Gibbs-town, NJ).Culture ofA7r5vascularsmooth musclecells

A7r5 cells are an established smooth muscle cell line derivedfrom rat embryo aorta (17) in which AVP stimulates inositolphosph ate production, calcium m obilization, and electrical ac-tivity of ionic channels (18, 19). The A 7r5 cells were obtainedfrom the American Tissue Culture Collection (Rockville, MD).The cells were grown at 37 C in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal calf serum andantibiotics (100 U/ml penicillin and 100ng/n\\ streptomycin)under a 5% CO2atmosphere. A7r5cells were subcultured every7 days in 100-mm Petri dishes using 0.05% trypsin-0.02%EDTA, whereas media were renewed every 2-3 days.Preparation of membranes from culturedA7r5cells

Three days after tryp sinization, subconfluent monolayers ofA7r5cells were washed twice with physiological saline solution(PSS containing in mM: 140 NaCl, 4.6 KC1, 10 glucose, 10HEPES, pH 7.4) before dispersion with a cell scraper in 10 mlPSS.After rinsing with 10 ml PSS, the cells were concentratedby centrifugation at 2,000 xgfor 5 min at 4 C and resuspended

in 5 ml hypotonic buffer (5 mM Tris-H Cl, 5 mM EDT A, pH7.4) for homogenization by 10 strokes with a glass-Teflonpestle. The memb rane prepa rations were washed twice by cen-trifugation at 50,000 x g for 15 min at 4 C and resuspended inbuffer 50 mM Tris-HCl, 20% glycerol for storage at -70 C.Radioligandbinding experiments

Saturation experiments of AVP receptors of membrane prep-arations of A7r5cells were performed at 30 C in buffer 50 mMTris-HCl, 20% glycerol, pH 7.4, supplemented with increasingconcentrations of [3H]AVP (2.5 MM unlabeled AVP) and 10mM MgCl2 or [3H]d(CH2)5Tyr(Me)AVP [1 MM unlabeledd(CH2)5Tyr(Me)AVP] in duplicate tubes. After 30 min incu-bation, filtration was achieved through GF/B glass fiber filterssoaked in 1% polyethyleneimine (2-6) and set on a Brandelfiltration unit (Brandel, Gaithersburg, MD). The radioactivityretained on the filters was measured by liquid scintillationspectrometry (Beckman counter LS 5801, Beckman Instru-me nts, Palo Alto, CA; yield = 64 %). Affinity (Kd) and capacity(Bmax) of the AVP receptors were calculated by a nonlinearleast square analysis program (2). Protein concentration wasmeasured with Pierce's BCA reagent using ovalbumin as aninternal standard. Competition experiments were also per-formed a t 30 C for 30 min with mem brane preparation s of A7r6cells, one concentration of [3H]AVP (3-5 nM), and increasingconcentrations of AVP V rvascular and V2-renal agonists. IC 50values for competitors' inhibition of [3H]AVP binding weredetermined from dose-response curves and converted into in-hibition constant (Ki) values according to the equation of Chengand Prusoff (20).Measurements ofintracellular calcium

Serum-starved monolayer cultures of A7r5 cells were grownon ACLAR plastic coverslips (14 X 14 mm, Allied EngineeredPlastics, Pottsville, PA) as previously described (21) and wereassayed 2 days later. Cell monolayers (1-2 X 105 cells/cm2)were loaded in serum-free DMEM with fura-2 AM (1 JLIM/coverslip) for 40 min at 37 C. The permeable acetoxymethylester of fura-2 is hydrolyzed by cellular esterases upon enterin gthe cells, and the fura-2 formed is relatively impermeable andbecomes trapped inside the cells. The monolayers of A7r5cellswere washed, then transferred to serum-free, fura-2-free me-dium and incubated an additional 20 min at 37 C. The loadedmon olayers were stored on ice in buffer Krebs -Hens eleit-HE PE S (KHH , containing in mM: 130 NaCl, 5 KC1,1.25 CaCl2,0.8 MgSO4, 5.5 glucose, 20 HEPES, and 0.1% fatty acid freeBSA, pH 7.4) until used. The coverslips were placed into aquartz cuvette containing 2 ml KHH buffer and maintained at37 C with continuous stirring. When thermal equilibrium wasreached, the fluorescence signal was recorded with a CaseWestern Reserve University spectrofluorometer [340 nm exci-tation and500nm emission wavelengths as previously reported,(22)]. After recording the baseline signal for 5 min, increasingconcentrations of AVP were added to the cuvette to stimulatethe mobilization of intracellular calcium and the influx ofextracellular calcium. Fluorescence measurements were con-verted to intracellular calcium concentrations [Ca++"]i by deter-mining maximal fluorescence (Fmax) with the nonfluorescentCa++ ionophore, ionomycin (25 /XM , followed by minimal flu-


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AVP RECEPTORS OF A7r5 CELLS 2847orescence (Fmin) obtainedbyadditionof 1.25mM M nCl2.Thefollowing formula wasthen used: [Ca++]i = Kdx (F - Fm in )/(Fmnx - F),with Kdfor fura-2 = 224nM. A utofluorescencebythe cellsoragonistsaswellas fura-2 leak outsidethecells wasnegligible. In experiments where BAPTA was used, 10 fiMBAPTA acetoxymethylester wasadded andcoincubated withfura-2 inDMEMasdescribed ab ove.Measurement ofpolyphosphoinositideformation

The measurement of water-soluble inositol phosp hates (IPn)was performed as previously described using anion-exchangechromatography (23). Authentic [3Hlinositol[l]-phosphate(IPO, [3H]inositol[l,4]-bisphosphate (IP2), and [3H]inosi-tol[l,4,5]-trisphosphate (IP3) were used to standardize theDowex resin columnsandestablish recoveries.Radiolabelingand extraction of membrane phospholipids

As described previously for HL60 cellsand mesangial cells(23,24), A7r5cells were grownineither 60-mm Pe tri dishes andlabeled for 2 h with 1 /tCi/ml [3H]l-O-alkyl lyso glycero-3-phosphocholine ([3H]-alkyl lysoGPC) in calcium-free KHHbuffer for measurem ent of PA and phosphatidylethano l (PtdE t)orin 12-well cluster dishesandlabeledfor 3 hwith 1 ixCi/m\[3H]arachidonate incomplete KHHbuffer forDAG measure-ments.The radioisotopes were removedbyextensive washingon ice, and thecells were warmedat 37 C incompleteKHHbuffer. One micromolar AVP in 0.2% BSA was added forvariable times.ForPA and P tdE t experiments, AVP was addedin the presenceorabsenceof0.5%ethanol. The incubation wasterminated 15 min later with ice-cold acidified methanol,andthe cells were scraped and transferred into an equal vol ofchloroform. The cultures were scraped again in methanol toyield a chloroform/methanol/water ratio of1/2/0.8. After45minat4 C, the methanol and chloroform extracts were adjustedto yield two phases (chloroform/methanol/water, 1/1/0.9vol/vol/vol). The chloroform lipid extracts were separated, andthemethanol/water extracts were rewashed with chloroform.Thecombined chloroform extracts were dried under nitrogenandresuspended in 100fA90%chloroform/10% methan ol.Thesamples were spiked with a uthentic synthetic PA andPtdEtor1,2-diolein, then spotted onsilica gel 60 TLCplates. For thePA/PtdEt experiments, phospholipids were eluted from theorigin withamobile phase consisting of chloroform/methanol/acetic acid, 65/15/6, vo l/vol/vol, andforthe DAG experim ents,neutral lipids were separated by a benzene/diethyl ether/am-monia, 100/80/0.2, vol/vol/vol solvent system. W iththeaboveelution systems, PtdEt was well resolved from several phospho-lipid contam inants including bis-phosphatidic acid, cardiolipin,and phosphatidyl glycerol. Also,the DAG solvent systemsep-arated 1,2 DAGfrom 1,3 DAG, triacylglycerol, monoacylgly-cerol,and free fatty acids.Thelipids were visualizedbytolui-dino-2-naphthalene sulfonic acid spray and UV light. Theradiolabeled lipids which comigrate with the internal standard swere scraped and radioactivity determined in a liquid scintil-lation counter.Presentation ofdata

Each set of experiments wasperformed at least on threedifferent occasions, and each individual experiment wasrun in

triplicate. Resultsin thetext and tablesareexpressedasmean SEM. Statistical analysis used nonparametric tests includingWilcoxon signed-rank test and Friedman two-way analysisofvariance (Statview statistical packagefor theMacintosh com-puter).P values less than 0.05 were consideredas statisticallysignificant.Results

Identification and characterization of specific A VPreceptors ofA7r5cellsKinetics of ^HJAVP binding to plasma membranes ofA7r5 cells.The amount of [3H]AVP specific binding toA7r5plasma membranes is protein concentration depend-ent (Fig. 1A). Within a protein concentration range from0.10-0.60 mg/ml, a linear increase in [3H]AVP specificbinding was observed (r = 0.99, eight different proteinconcentrations, n = 3). Specific binding accounted for 58 3 % of total binding at the[3H]AVPconcentration used(4.85 0.30 nM). [

3H]AVP specific binding to plasmamembranes of A7r5 cells is time dependent and reachedan equilibrium value of 78 2 of total binding within

30 min of incubation at 30 C with 3.81 0.20 nM [3H]AVP (protein concentration = 0.61 0.20 mg/ml, n = 3,Fig. IB). The association kinetics could be fitted best bythe following equation, B = Beq(l - exp~K1*t) where B isspecific binding at a given time and Beqis specific bindingreached at equilibrium (Beq = 1906 cpm, Ki = 0.127min 1, r = 0.996). [3H]AVP specific binding to plasmamembranes of A7r5cells was rapidly reversible, as shownon Fig. 1C. To demonstrate reversibility, plasma mem-branes of A7r5cells (protein concentration = 0.32 0.10mg/ml) were incubated with 3.86 0.23 nM [3H]AVPfor 30 min; thereafter, a high concentration of unlabeledAVP (1 /XM)was added to the incubation mixture, andremaining [3H]AVP specific binding was measured atserial time intervals after addition of unlabeled AVP.Unlabeled AVP rapidly displaced [3H]AVP from thebinding sites. Forty-five minutes after addition of unla-beled AVP, no specific binding of[3H]AVPwas left. Thedissociation kinetic could be fitted best by the followingequation, B = BeqCexp 1 *1) where B is specific bindingat a given time after addition of unlabeled AVP and Beqis equilibrium specific binding reached before addition ofunlabeled AVP (Beq= 1006 cpm, K2 = 0.167 miiT1, r =0.996). Thus, the equilibrium dissociation constant, Kd,for [3H]AVPbinding determined from association anddissociation experiments was 1.31 nM.Concentration ofA7r5cellsf^HJAVP binding sites.Satu-ration equilibrium binding experiments were performedwith membrane preparations of A7r5 cells (protein con-centration = 0.25 0.06 mg/ml), and eight differentconcentrations (0.40-16 nM) of the agonist radioligand[3H]AVPin the presence or absence of 2.5 /xM unlabeled


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2848 AVP RECEPTORS OF A7r5 CELLS Endo1991Voll29No62500

2000

1500

1000

50 0

0.1 0.2 0.3 0.4 0.5Protein Concentration mg/ml)

0.6

2000

6. 1500

10008Q.~ 500

B

10 20 30Time min)

40 50

1200

S 900

S 600

30 0

10 40 500 30Time min)F I G . 1. A, [3H]AV P specific binding to A 7r6cell plasma m embranes asa function of protein concentration. B, [3H]AVP specific binding toA7r5cell plasma m embrane s as a function of time.C, Reversibility of[3H]AVP specific binding to A 7r6cell plasma m embrane s.AVP. As shown on Fig. 2A, analysis of[3H]AVPspecificbinding to V i-vascular receptors ofA7r5cells showed tha tdata could be fitted best by the following equation B =(Bmax*Lf)/(Lf + Kd) where B = concentration of boundtracer, Bm ax = total concentration of receptors, Lf =

85

I 65

2 45

2012 1 5

Free JH]d CH 2)5 Tyr Me)AV P nM)FiG.2. A, Saturation equilibrium specific binding of [3H]AVP to A7r6cellplasma membranes.Inset,S catchard linear transformation of thedata. B, Saturation equilibrium specific binding of [3H]d(CH 2)6Tyr(Me)AVPto A7r6cell plasm amembranes.Inset,Scatchardlineartransform ation of the data.concentration of unbound tracer, Kd = dissociation con-stant of the receptor. One single class of receptors wasidentified with a Kdof2.11 0.11 nM and aBm axof 306 10 fmol/mg protein (n =11).Tota l binding represented4 0.2% to 1.3 0.01% of total ligand concentrationwhen the tracer concentration was increased from 0.40 0.02 to 16 1.2 nM. Nonspecific binding increasedlinearly from 25 3% to 70 4% when ligand concen-tration was raised from 0.40 0.02 to 16 1.2 nM (r =0.998).Demonstration of the Vi-vascular nature of A VPreceptorsofA7r5cells

Saturation equilibrium binding experiments were alsoperformed with membrane preparations of A 7r5 cells(protein concentration = 0.35 0.05 mg/ml) and the Vi-vascular antagonist [3H]d(CH2)5Tyr(Me)AVP (eight dif-ferent concentrations from 0.4-12 nM) in the presence


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AVP RECEPTORS OF A7r5 CELLS 2849or absence of1nMunlabeled d(C H2)5Tyr(Me)AVP. Onesingle class of binding sites was identified with a Kd of0.31 0.05 nM and aBma x of 420 13 fmol/mg protein(n = 10, Fig. 2B). Th us, the excellent affinity of the V i-vascular antagonist for the AVP receptors of A 7r5 cellssupports their belonging to the Vi-vascular subtype.

Competitive inhibition of[3H]AVPagonist binding toVi-vascular AVP receptors of A7r5cells by various AVPVi and V2 analogs were carried out to confirm the Vi-vascular subtype of the A 7r5 AVP receptors. In competi-tion experiments, A7r5cell mem branes were incubated at30 C with one fixed con centration of [3H]AVP(3-5 nM)and increasing concentrations of AVP analogs. Analysisof the agonists' competition for [3H]AVP binding wasfitted best by one single site model. Displacement of [3H]AVP by unlabeled AVP confirmed the presence of onesingle class of binding sites (K ;= 1.53 0.32 nM, n = 3).The dissociation constants (K ;) of the different AVPanalogs are shown in Table 1. As indicated in Fig. 3,there was a significant correlation between the bindingdissociation constant values of these agonists and theircorresponding vasopressor activities (r = 0.91, P


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2850 AVP RECEPTORS OF A7r5 CELLS Endo1991Vol 129 No 6fluoride. Saturation equilibrium binding experimentswere also performed with m embran e prepa rations of A7r5cells and the Vi-vascular antagonist radioligand [3H]d(CH2)5Tyr(Me)AVP.Inthe absence of MgCl2, the affin-ity of the receptors for [3H]d(CH2)5Tyr(Me)AVP wasalready highandwasnotalteredbyadditionofalumin-ium fluoride (Table 2).Addition of MgCl2 aloneor inassociation with aluminium fluoridedid notmodifysig-nificantly the high affinity of thereceptorsfor the Vi-vascular antagonist. The total number of binding sitesavailable to [3H]d(CH2)5Tyr(Me)AVP wasindependentof MgCl2andaluminum fluoride.Intracellular calciumincreaseby activationof Vi-vascularAVPreceptors ofA7r5cells

An increase of [C a++]i , the hallmark of V:-vascularAVP receptor activation, was measuredin fura-2 loadedA7r5 cells.Thebasal [C a++ ]; in fura-2-loaded A7r5 cellswas 136 18nM(n = 34).S timulation ofA7r5 cellsbyAVP resultedin abiphasic increasein[C a++ ]; composedofa rapid (within2-5sec)andtran sien t spike, followedbyasmaller increase with returntobasal levels3-5 minafter addition of AVP (Fig. 4A).Preaddition of anequimolar dose of the Vi -vascular antago nistd(CH2)5Tyr(Me)AVP blocked AVP-induced increaseof[Ca++]i,but did not interfere with ionomycin mobiliza-tion ofCa++ , confirming theVi -vascular natur e of theAVP receptors involved (Fig. 4B).Aninitial stimulationB

1.25mM[Ca+t l0 1.25mMlCa+*]01600

5

1 |iM d CH2)5Tyr Mo)AVPII iM AVP

Ionomycin

I pM AVP

I nM AVP | iM AVP 1 10 100AVP Concentration nM)

FiG.4. [Ca++]i waveforms induced by AVP in A7r5 cells. A7r5 cellmonolayerson ACLAR coverslips were loaded with fura-2 andincu-batedinKHH buffer,pH7.4. Basal [Ca++]i was measured fluorometr-ically after reaching thermal equilibrium at 37 C. A, OnemicromolarAV P wasadded in thepresence of extracellular Ca++ ; B, 1f iM Vi-vascular antagonist d(CH2)5Tyr(Me)AVP followedby1nMAVP wereaddedinthe presenceofextracellular C a++ ; C, Two dosesof1nMAV Pwere addedat3-min intervalsin thepresenceofextracellular C a++ ;D,relationship between AVPdose and [Ca++]i waveform amplitude inA7r6cells.

with 1fiMAVP was followed byadramatic reductionof[Ca++]i responseto asubsequent identical challenge withAVP, suggesting down-regulation and /or desensitizationof the receptors (Fig. 4C).The[Ca++]i responseto AVPwas concentration dependentasshownonFig. 4D, withan ED 50= 1.87 0.15nM AVP(n = 3) calculated fromthe equation E = (Ema x*C)/(ED50 + C)whereE = re-sponse to AVP, Em ax = maximum response inducedbyAVP, ED 50= concentration of AVP inducing half-max-imum response, and C= concentrationofAVP (25).Themaximal [C a++ ]; response induced by 1 /*M AVP was1582297 nM(n = 15).Sourceof[Ca Jt increase induced by activationof Vi-vascularAVPreceptors ofA 5 cells

The increase in [Ca++]i inducedby AVP could resultfrom the release of intracellular stores, the influx ofCa++ ,thereduction ofC a++ efflux, or a combinationofthese mechanisms. When AVP was addedinthe absenceof extracellular Ca++ , the transient increase in [C a++ ] ;was blunted and the sustained phase was abolished (Fig.5A).Asimilar profile was noted when AVP wasaddedafter applicationof4mM EGTA (Fig. 5B). The maximalB

0 mM [ C a + + l 0 0 mM [Ca + + ) 0

EGTAII pM AVP

1 min D

}iMAV P

0 mM [ C a + + ] 0BAPTAj

Ionomycin

nMA VP C a + + Ionomycin 1 \i AVPFIG. 5. Relative contributionofthe mobilizationof intracellular Ca++storesand ofthe influxofextracellular C a++to the[C a++]i waveformsinducedbyAV PinA7r5cells.A, Onemicromolar AVPwasaddedinthe absence of extracellular Ca++ . B, A7r5cells incubated in the absenceof extracellular Ca++ were treated with4mM EGTAfor 30 secbeforeaddition of 1pMAVP. C,A7r5 cells were coloaded with fura-2 andBAPTAandresuspendedin theabsenceof extracellular Ca++ beforeadditionof1/iM AVP then extracellular Ca++andionomycin.D,A7r6cells were loaded with fura-2andresuspendedin theabsenceofextra-cellular Ca++ before additionofEGTA, then ionomycin and AVP.


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AVP RECEPTORS OF A 7r5 CELLS 2851[Ca++]i response induced by 1nMAVP in the absence ofextracellular Ca++ was 788 186 nM (n = 10). Theseexperiments suggest that the initial [C a++ ] ; peak ismostly due to mobilization of intracellular calcium,whereas the persistant elevation of [Ca++]i abolished byextracellular Ca++ depletion and EGTA is related to theinflux of extracellular Ca ++ . To confirm the intracellularorigin of the initial [C a++ ] ; spike induced by AVP, A7r5cells were loaded with 10 juM BAP TA , a high affinityCa++ chelator which does not interfere with the fluores-cence determination of [Ca++]i with fura-2. AVP (1/*M)failed to elevate [C a++ ] ; when added to A7r5cells loadedwith both fura-2 and BAPTA and incubated in Ca++ -freemedium (Fig. 5C). Subsequent addition of extracellularCa+ + and ionomycin confirmed that BAPTA loading didnot interfere with the fura-2 detection system (Fig. 5C).Further evidence that AVP releases Ca ++ from an intra-cellular pool was obtained by depletion of intracellularstores with ionomycin and EGTA before addition ofAVP. As shown on Fig. 5D, depletion of the intracellularCa+ +stores prevented any increase in [Ca++]i in responseto AVP. Taken together, the experiments presented inFigs. 4 and 5 suggest that the initial spike in [C a++ ];results from both intracellular release of Ca ++ and extra-cellular influx of Ca++ , whereas the sustained phase of[Ca++]i depends on extracellular calcium influx.Mobilization ofCa++ from the endoplasmic reticulumpool by activation of V\-vascularAVP receptors ofA^cells

To further analyze the intracellular pool of Ca + + re -leased by AVP, we used tBuBHQ, a relatively selectiveinhibitor of endoplasmic reticulum Ca++ -ATPase, whichprevents uptake of Ca ++ into the pool released by inosi-tol[l,4,5]P3 (26). In the absence of extracellular Ca++ ,tBuBHQ (25 /*M) caused a rapid increase in Ca++ , pre-sumably by depletion of the intracellular stores followedby a progressive decline linked to Ca ++ efflux (Fig. 6A).The subsequent addition of ionomycin provoked thereentry of Ca++ , indicating that tBuBHQ did not inter-fere with the Ca++ /fura-2 detection system. In the pres-ence of extracellular C a++ , the same patter n was observedbut with a greater influx of Ca++ after administration ofionomycin (Fig. 6B). The response to the addition ofAVP after tBuBHQ under conditions in which Ca ++stores can no longer be replenished by extracellular Ca ++influx was massively reduced, suggesting that AVP andtBuBHQ act on the same intracellular pool of Ca + + (Fig.6C).These experiments with tBuBH Q suggest that AVPreleases Ca++ from the endoplasmic reticulum pool, pre-sumably the same pool sensitive to Ins[l,4,5]P3 (27).Gatingo faplasma membrane Ca++channel byactivation ofVi-vascularAVP receptors ofA cells

Further evidence that AVP increases transmembranedivalent cation permeability was obtained by using

0 mM [Ca + + ) 0

tBuBHQB

Ionomycin

1 . 2 5 m M C a + + ] 0

1 min 1IonomycinttBuBHQ

t jiM AVPtBuBHQFIG. 6. AVP mobilization of Ca++ from the endoplasmic reticulum poolof A7r5 cells. tBuBHQ was added to fura-2-loaded A7r6 cells in theabsence (A) or presence (B) of extracellular calcium. tBuBHQ thenAVP were added in the absence of extracellular calcium (C).M n + + , a divalent cation which permeates Ca ++ channelsin other cells (26). Mn + +binds with high affinity to fura-2, but unlike Ca++ , M n + + quenches fura-2 fluorescence.When 10/xM M n+ + was added to fura-2-loaded A7r5cellsin Ca++-free medium, the fluorescence intensity declinedgradually, in relation with continuous influx of Mn + +(Fig. 7A). Addition of ionomycin, which facilitates Mn + +translocation, provoked a dramatic potentiation of fura-2 quenching. Chelation of extracellular M n + +with excessEGTA prevented the further decline in fluorescence butfailed to reverse this decline, suggesting tha t M n + + effluxfrom the cells was negligible (Fig. 7B). Therefore, in A 7r5cells measuring the rate of fura-2 quenching by exoge-nous addition of Mn + + can measure unidirectional diva-lent cation uptake. When Mn+ + was added immediatelybefore the addition of 1/JLMAVP, the transient increase


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2852 AVP RECEPTORS OF A7r5 CELLS Endo 99Vol 129 No 60 mM [ C a + + ] 0

10 nM M n + +

lonomycin 5r

0 mM [ C a + + ] 0 1.25mMlCa ++ )0

I tNicardipine 1\i AVPEGTA

5DC min

I IMAVP Ca ++

1 min0 m M[ C a++ ]0BAPTA;

0 m M[ Ca++ ] 0B A P T A j

O m M [ C a + + ] o 0 mM [Ca

1 pM AVP

FIG. 7. AVP gating of a plasma membrane Ca++ channel of A7r5cells.Fura-2-loaded A7r5 cells were suspended in calcium-free medium thenchallenged with Mn++ followed by ionomycin (A) or EDTA (B). Mn++was added before (C) or after AVP (D).in fura-2 fluorescence was blunted and marked quench-ing of fura-2 fluorescence occurred within seconds afterAVP addition (Fig. 7C). When Mn + + was added 1 minafter AVP,fura-2 fluorescence was also rapidly quenched(Fig. 7D). These results suggest that AVP increaseswithin seconds the influx of divalent cations.Vi-vascularAVP receptors ofA-jr5cells stimulate influxofextracellularCa++via a dihydropyridine-insensitiveCa++ channel

The sustained, extracellular Ca++ -dependent phase of[Ca++]i increase can be explained by the activation ofreceptor-gated Ca++ channels or of voltage-gated Ca++channels. Pretreatment with 10xM nicardipine failed tochange either the transient or the sustained phase of[Ca++]i induced by AVP, suggesting that AVP-induced[Ca++]i influx occurs via a dihydropyridine-insensitiveCa+ + channel, most likely through activation of a recep-tor-gated Ca++ channel (Fig. 8A). In the absence ofextracellular Ca++ , AVP causes only a transient andsmaller increase in [Ca++]i, but the rapid increase in[Ca++]i after repletion of extracellular Ca ++ suggests thatthe channels activated by AVP remain open (Fig. 8B).Clamping of intracellular Ca++ with BAPTA preventsthe transient increase in [Ca++]i by AVP, but extracel-lular Ca++ repletion still increases [Ca++]i (Fig. 8C).

I I1HMAVP Ca + + c a + + I J I M A V P

FIG. 8. AVP-stimulated influx of Ca++ is dihydropyridine insensitive.Fura-2-loaded A7r6 cells were stimulated by AVP in the presence ofcalcium and nicardipine (A). In calcium-free medium, AVP then Ca ++were subsequently added (B). AVP and calcium were added in altern ateorder in cells depleted of extra and intracellular calcium (C and D).Gating of AVP-sensitive Ca+ + channels was independentof intracellular Ca ++ as shown in BAPTA-clamped A7r5cells when extracellular C a++ was added before AVP (Fig.8D).Vi-vascularAV P receptorsofAnr*,cells stimulate influxofextracellularCa++viaa phospholipase A2sensitivemechanism

In the presence of extracellular Ca++ , pretreatment ofA7r5 cells with 20/xM indomethacin reduced the heightof the initial [C a++ ]; transient and shortened the sus-tained [Ca++]i increase induced by AVP (Fig. 9, A andB). In calcium-free medium, indomethacin preadditiondid not significantly alter the shape of the [Ca++]i tran-sient (data not shown). These results suggest that AVP-induced extracellular Ca ++ influx involves an arachidon-ate-derived second messenger and presumably phospho-lipase A2activation. This hy pothesis is further supportedby the fact that in the presence of extracellular Ca ++ ,addition of 10j*MAA induced an increase in [Ca++]i (Fig.9C). The subsequent response to AVP was of shortduration, suggesting that the AA-sensitive influx path-way desensitizes, thus preventing subsequent activationby AVP.AVP stimulates the formation ofinositolphosphates incells

AVP stimulated the accumulation of the various poly-phosphoinositides in a time-dependent manner (Fig.10A). In s[ l] P produ ction was stimulated only after 1minand was increased by181%at 5 min (12,563 2,532 cpm


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AVP RECEPTORS OF A7r6 CELLS 2853B

1.25mM Ca++ )0 1.25mM[Ca ++ )0

20nM Indomethacin

nM AVP1\i AVP

1 min

1 pM Arachidonic Ad d 1^M AVPFIG. 9. AVP-stimulated influx of Ca++ activates phospholipase A2. Inthe presence of extracellular calcium, fura-2-loaded A 7r6 cells werestimulated by AVP in the absence (A) or the presence (B) of indo-methacin or in the presence of AA (C).us. 6,960 1,253 cpm ). In s[l ,4] P2 increased rapidly andremained at a steady level (+142% at 5 min, 720 84cpm us. 508 64 cpm). Ins[l,4,5]P3 increment was rapidand dramatic (+234% at 5 min, 143 22 cpm us .61 12 cpm). Together, these results confirm that activationof Vi-vascular AVP receptors stimulates a PIP 2-specificphospholipase C, leading to a rapid and sustained pro-duction of Ins[l,4,5]P3.A VP stimulates the formation of DAG inA7r5cells

AVP stimulates the formation of DAG in A 7r5 cells ina biphasic manner. A rapid and transient peak was notedwithin the first min after application of 1nMAVP andwas followed by a secondary and sustained rise reachinga new plateau after 5 min (5504 603 cpm vs . 3158 362 cpm, Fig. 10B). This secondary increase in DAG maybe due to hydrolysis of alternate phospholipid substra tesbesides the polyphosphatidylinositols. Since DAG wasassessed in A7r5cells labeled to dynamic equilibrium with[3H]arachidonate and then washed free of exogenouslabel, [3H]DAG most likely reflects phospholipase C or

250 r

r 200

> 150

100

ln i.4,5)P3

lns i)P

lns i,4)P2

80 160ime sec)

240 320

150

S- 140

130

> 120

=6 110

100

90

1,2 OlacylGlycerol

0 5 10 15Time min)

FIG. 10. A, AVP stimulation of inositol phosphates production. A7r6cells preincubated with [3H]myoinositol were stimulated by AVP for15 sec to 5 min. The different radiolabeled phosphoinositides wereseparated by Dowex chromatography before measurement of 3H radio-activity (n = 6, *,P


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85 AVP RECEPTORS OF A7r5 CELLS Endo1991Vo 129No6TABLE3. AVP activation of phospholipase D in A7r5cells

ControlAVPControl +ethanolAVP + et-hanol

Phosphatidic acid8,45812,6637,3208,516

908 cpm/well 1,393 cpm/well" 879 cpm/well 854 cpm/well

Phosphatidylethanol8,153 1,168 cpm/well8,837 1,328 cpm/well7,126 922 cpm/well

16,667 1,833 cpm/well"A7r6 cells were labeled with [3H]l-O-alkyl lyso glycero-3-phospho-choline for 2 h then stimulated for 15 min by AVP in the presence orabsence of ethanol (n = 3 for each series). The chloroform extractswere run on TLC plates after spiking the samples with authenticsynthetic PA and PtdEt. The lipids were visualized by UV light aftertoluidino-2-naphthalene spraying, scraped, and their radioactivity de-termined in a liquid scintillation counter. P


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AVP RECEPTORS OF A7r5 CELLS 2855induced [Ca++]i increase are similar, therefore confirmingthe close tie between both events. AVP-induced [Ca++]imobilization is blocked by preaddition of the specific V\-vascular antagonist d(CH 2)5Tyr(Me)AVP, in agreementwith theVi-vascular subtype of AV P receptors involved.The rapid desensitization we observed after the secondAVP application w as also recently reported by Caram eloetal.(35) in vascular smooth muscle cells. These autho rsfound that the homologous AVP desensitization wasrapid, dose-dependent, and related to hormone receptoroccupancy and protein kinase C activation, but not toreceptor internalization.Our data suggest that the initial spike increase in[Ca++]i results from both intracellular calcium releasefrom the endoplasmic reticulum and influx of extracel-lular calcium. The secondary sustained increase in[Ca++]i is due to influx of extracellular calcium as it isabolished by EDTA and extracellular calcium depletion.The rapid rise of inositol trisphosphate suggests thatmobilization of intracellular calcium stores by AVP oc-curs via receptor-dependent activation of PIP 2-specificphospholipaseC.Experim ents with tBuB HQ , a relativelyselective inhibitor of the endoplasmic reticulumCa++ ATPase, are consistent with release of calcium byAVP from the endoplasmic reticulum pool sensitive toinositol trisphosphate. The initial and rapid productionof DAG induced by AVP provides corroborative evidencefor a phospholipase C signal transduc tion pathway whichis linked to the activation of protein kinase C. In A 7r5cells treated with tBuBHQ and stimulated by AVP, asteady return of [Ca++]i to basal levels was observed,suggesting that AVP-stimulated [Ca++]i transients arecontrolled in part by a hormone-dependent calcium ef-flux pathway which does not involve reuptake into theendoplasmic reticulum. Indeed, Doyle and Riiegg (36)have shown that AVP increased the efflux of 45Ca++ frompreloaded A7r6 cells, and Vigneet al. (37) have reportedthe presence ofaN a+ /C a + +antiporter in A7r5cells, whichwas sensitive to amiloride derivatives but insensitive toblockers of voltage-operated C a+ + channels.The magnitude and duration of the sustained phase of[Ca++]i increase produced by AVP in A 7r5cells is similarto what is observed with other peptides like angiotensinII and norepinephrine. The experiments performed withM n + + , a cation that permeates Ca ++ selective channels,demonstrate that AVP rapidly triggers Ca ++ uptakeacross A7r5 cell plasma membranes. Our data indicatethat calcium influx induced by AVP is independent ofvoltage-operated channels as it is unaltered by preaddi-tion of the dihydropyridine antagonist nicardipine. Thereduction of AVP-induced [Ca++]i increase in the pres-ence of extracellular C a+ + by indomethacin suggests thatphospholipase A2 and AA metabolites are also instru-mental in AVP stimulation of Ca + + influx. Measuring

AV P-stimulated release of radiolabeled arachidonic acidmetabolites by A-10 smooth muscle cells, Welsh et al.(38) observed that the activity of phospholipase A 2 ap -peared to be extracellular Ca++ -sensitive. Our data sug-gest that activation of the arachidonate pathways isinvolved in the AVP-induced influx of extracellular Ca++in A7r5cells.The activation of Vi-vascular AVP of A 7r5 cells stim-ulates a rapid hydrolysis of PIP 2in Ins[l,4,5]P3and DAGcontemporary to the initial [Ca++]j increase, suggestingcoupling to phospholipase C. Second messengers derivedfrom the hydrolysis of PtdCho may augment or sustainthis initial event. We have shown that AVP induces abiphasic elevation of DAG in A7r5 cells. The sustainedaccumulation of DAG may reflect phospholipase C hy-drolysis of phospholipid su bstrates besides the polyphos-phoinositides. In addition, the sustained DAG productionmay reflect sequential phospholipase D/PA phosphohy-drolase activities. We have dem onstrated conclusively anAVP-stimulated phospholipase D activity which usesPtdCho as a substrate. AVP stimulates the accumulationof PtdEt, a transphosphatidylation product of phospho-lipase D, which has been used in various cell models asa marker of phospholipase D activity. Phospholipase Dhydrolyzes phospholipid substrates to yield PA whichstimulates [Ca++]j increase and cell proliferation in othercell systems (23). Furthermore, AVP-stimulated phos-pholipase D may sustain protein kinase C activation, amodulator of contraction and proliferation, through PAphosphohydrolase-generated DAG formation (16, 39,40).Thus, we suggest that additional signal transductionpathways which hydrolyze PtdCho via either phospholi-pase C or D function as important regulators of smoothmuscle cell hom eostasis.

In conclusion, high affinity Vi-vascular AVP receptorsare present at the surface of A7r5 cells. Their activationleads to a dramatic rise in intracellular free calciumlasting a few minutes. Contemporary to the Ca+ + alter-ations is noted an activation of phospholipases A2, C,and D, critical events in Ca++ homeostasis and cellmitogenesis.Ac k n o wl e d g me n t s

We would like to thank Dr. George Dubyak for sharing with us hisexpertise on calcium signaling in A 7r5cells.References

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