adenosine-induced nitric oxide production in human endothelial cells: a novel calcium-insensitive...

Early activation of the p42/p44MAPK pathway mediatesadenosine-induced nitric oxide production in humanendothelial cells: a novel calcium-insensitivemechanism

AMANDA W. WYATT, JOERN R. STEINERT, CAROLINE P. D. WHEELER-JONES,*ANTHONY J. MORGAN, DAVID SUGDEN,† JEREMY D. PEARSON, LUIS SOBREVIA,‡

AND GIOVANNI E. MANN1

Centre for Cardiovascular Biology and Medicine, GKT School of Biomedical Sciences, King’s CollegeLondon, Guy’s Campus, London SE1 1UL, UK; *Department of Veterinary Basic Science, RoyalVeterinary College, London NW1 0UT, UK; †Endocrinology and Reproduction Research Group,GKT School of Biomedical Sciences, King’s College London, Guy’s Campus, London SE1 1UL, UK;and ‡Cellular and Molecular Physiology Laboratory, Faculty of Biological Sciences, University ofConcepcion, Concepcion, Chile

ABSTRACT Adenosine is released from the myocar-dium, endothelial cells, and skeletal muscle in ischemiaand is an important regulator of coronary blood flow.We have already shown that acute (2 min) activation ofA2a purinoceptors stimulates NO production in humanfetal umbilical vein endothelial cells (1) and now reporta key role for p42/p44 mitogen-activated protein ki-nases (p42/p44MAPK) in the regulation of the L-argi-nine-nitric oxide (NO) signaling pathway. Expressionof mRNA for the A2a-, A2b-, and A3-adenosine receptorsubtypes was abundant whereas A1-adenosine receptormRNA levels were negligible. Activation of A2a purino-ceptors by adenosine (10 �M) or the A2a receptoragonist CGS21680 (100 nM) resulted in an increase inL-arginine transport and NO release that was not medi-ated by changes in intracellular Ca2�, pH, or cAMP.Stimulation of endothelial cells with adenosine wasassociated with a membrane hyperpolarization andphosphorylation of p42/p44MAPK. L-NAME abolishedthe adenosine-induced hyperpolarization and stimula-tion of L-arginine transport whereas sodium nitroprus-side activated an outward potassium current. Genistein(10 �M) and PD98059 (10 �M), an inhibitor of MAPKkinase 1/2 (MEK1/2), inhibited adenosine-stimulatedL-arginine transport, NO production, and phosphoryla-tion of p42/p44MAPK. We found no evidence for acti-vation of eNOS via the serine/threonine kinase Akt/PKB (protein kinase B) in adenosine-stimulated cells.Our results provide the first evidence that adenosinestimulates the endothelial cell L-arginine-NO pathway ina Ca2�-insensitive manner involving p42/p44MAPK, withrelease of NO leading to a membrane hyperpolariza-tion and activation of L-arginine transport.—Wyatt,A. W., Steinert, J. R., Wheeler-Jones, C. P. D., Morgan,A. J., Sugden, D., Pearson, J. D., Sobrevia, L., Mann,G. E. Early activation of the p42/p44MAPK pathwaymediates adenosine-induced nitric oxide production in

human endothelial cells: a novel calcium-insensitivemechanism. FASEB J. 16, 1584–1594 (2002)

Key Words: mitogen-activated protein kinases � NO � L-argi-nine � A2a purinoceptors

Adenosine is an endogenously produced purine nu-cleoside generated from either the actions of 5� nucle-otidases on AMP or specific hydrolases on S-adenosyl-homocysteine (2). It is an important regulator of bloodflow and has been shown to decrease coronary resis-tance under basal and restricted blood flow conditions(3). In many cell types, adenosine elicits vasodilationvia direct stimulation of G-protein-coupled purinocep-tors located on vascular smooth muscle cells by modu-lating intracellular cAMP levels (2). Activation of A1and A3 receptor subtypes leads to inactivation of adeny-lyl cyclase and a decrease in cAMP levels; activation ofA2a and A2b receptor subtypes enhances cAMP accumu-lation, resulting in vasorelaxation (2). Although aden-osine is classically regarded as an endothelium-inde-pendent vasodilator, accumulating evidence implicatesendothelium-derived nitric oxide (NO) in adenosine-mediated vasodilation (1, 4–9).

Our previous studies in human umbilical vein endo-thelial cells (1) and studies by others in porcine carotidartery endothelial cells (10) have established that acti-vation of A2a purinoceptors by adenosine stimulatesNO release. Our experiments in umbilical vein endo-thelial cells demonstrated that adenosine acutely stim-ulates influx of l-arginine (substrate for eNOS) via asodium-independent y� transport system, whose activity

1 Correspondence: Centre for Cardiovascular Biology andMedicine, GKT School of Biomedical Sciences, King’s Col-lege London, Guy’s Campus, London SE1 1UL, UK. [email protected]

1584 0892-6638/02/0016-1584 © FASEB

was enhanced in response to membrane hyperpolariza-tion (1). Although adenosine has been reported toincrease endothelial cell proliferation most likely viathe activation of p42/p44MAPK (11–13), the signaltransduction pathway(s) mediating adenosine-inducedactivation of eNOS have not yet been elucidated.

In endothelial cells, constitutive nitric oxide synthase(eNOS) is classically activated by agonists that raiseintracellular Ca2� ([Ca2�]i) levels such as histamineand bradykinin. Recent evidence suggests that eNOScan also be activated in a Ca2�-insensitive manner inresponse to fluid shear stress (14, 15) and 17�-estradiol(16). In response to fluid shear stress, eNOS can bephosphorylated by the serine/threonine protein kinaseAkt (protein kinase B, PKB) in a phosphatidylinositol3-kinase (PI3-kinase) -dependent manner, allowing en-zyme activation at basal levels of Ca2� (17, 18). Activa-tion of eNOS in response to 17�-estradiol has beenreported to be mediated via p42/p44MAPK in a calcium-dependent manner (19). As recent evidence implicatesAkt/PKB and p42/p44MAPK in phosphorylation ofeNOS (20), we investigated the role of these kinases inmodulation of the endothelial l-arginine-NO pathwayby adenosine.

Acute stimulation of NO production by adenosineoccurred independent of changes in intracellular Ca2�,pH, or cAMP but was associated with phosphorylationof p42/p44MAPK. Inhibition of protein tyrosine kinasesand MAPK kinase (MEK1/2, upstream activator ofp42/44MAPK) abolished A2a purinoceptor-stimulatedincreases in l-arginine transport, NO production,and p42/44MAPK phosphorylation. A preliminary ac-count of part of this work has been published inabstract form (21).

MATERIALS AND METHODS

Endothelial cell culture

Endothelial cells were isolated from human umbilical veins(HUVEC) by collagenase (0.5 mg mL�1) digestion andcultured in medium 199 containing 10% (v/v) fetal calfserum (FCS), 10% (v/v) newborn calf serum, 5 mM l-glutamine, penicillin (100 units mL�1), streptomycin (100units mL�1), porcine heparin (90 �g mL�1), and endothelialcell growth factor (20 �g mL�1). As described (22), HUVECcultures were identified by their typical cobblestone morphol-ogy and positive staining for von Willebrand factor (data notshown) (23). All experiments were performed using passage2 cells.

RT-PCR analysis

Confluent endothelial cells in 24-well microtiter plates werewashed twice with phosphate-buffered saline (PBS, 37°C) and200 �L of ice-cold lysis buffer containing 100 mM Tris-HCl,pH 7.5, 500 mM LiCl, 10 mM EDTA, 1% LiDS, and 5 mMDTT was added to disrupt the cells. Poly A� mRNA in eachlysate was isolated using magnetic oligo (dT)25 beads andcDNA was synthesized immediately. mRNA was added to oligo(dT)18 (1 �g) and random 10-MERS (1 �g); the mixture washeated (70°C, 5 min) to remove secondary RNA structures

and cooled immediately on ice. DTT (20 mM), dATP, dCTP,dTTP, and dGTP (0.5 mM), 40 U recombinant ribonucleaseinhibitor (Rnasin), and avian Moloney murine leukemia virusreverse transcriptase (MMLV-RT) (10 U) were added. Tubeswere incubated for 1 h at 37°C, followed by 15 min at 42°C.MMLV-RT was inactivated by heating (98°C, 3 min). ThecDNA generated was diluted 1:10 with tRNA (10 �g/mL) andstored at �85°C. The adenosine receptor subtype primerswere A1-adenosine receptor subtype; sense primer: 5�-AATTGC TGT GGA CCG CTA CCT C-3�, antisense primer:5�-CGA CAC CTT CTT GTT GAG CTG-3�; A2a-adenosinereceptor subtype: sense primer 5�-TTG ACC GCT ACA TTGCCA TCC G-3� antisense primer 5�-GAA GAT CCG CAA ATAGAC ACC-3�; A2b-adenosine receptor subtype, sense primer:5�-ACC AAC TAC TTC CTG GTG TCC-3�; antisense primer:5�-GCA GCT TTC ATT CGT GGT TCC-3�; A3-adenosinereceptor subtype: sense primer: 5�-ATC GCT GTG GAC CGATAC TTG-3�; antisense primer: 5�-AAT GCA CCT GTC TCTTTG GAG 3�. Predicted sizes of PCR products were A1, 349bp; A2a, 305 bp; A2b, 374 bp; A3, 353 bp. PCR tubes contained1 �L of cDNA (1:10 dilution), 100 �M of each deoxynucleo-side 5�-triphosphate, 0.5 �M of appropriate forward andreverse primer, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCL(pH 8.3), 0.5% glycerol, 0.1% triton X-100, and 1 U Taq DNApolymerase. The thermal cycling conditions for all PCRreaction were 94°C, 1 min, 60°C, 1 min, and 72°C, 2 min for35–40 cycles. The PCR products were separated by agarosegel electrophoresis (1.8% w/v) and bands were visualized byethidium bromide (0.5 �g/mL) staining. Adenosine receptorsubtype PCR products were cut from agarose gels and puri-fied using a Qiaquick gel extraction kit. The identity of eachPCR product was confirmed by direct sequencing on an ABIautomated sequencer.

Effects of A2a purinoceptor agonists and inhibitors ofp42/p44MAPK and adenylyl cyclase on L-arginine transport

Confluent HUVEC in 96-well microtiter plates were preequili-brated for 30 min in Krebs buffer containing (mM): 131NaCl, 5.6 KCl, 25 NaHCO3; 1 NaH2PO4, 2.5 CaCl2, 1 MgCl2,5 D-glucose, 20 HEPES, pH 7.4 supplemented with l-arginine(100 �M), and the phosphodiesterase inhibitor 3-isobutyl-methylxanhine (IBMX, 1 �M), then stimulated for 2 min withadenosine (10 �M), the A2a purinoceptor agonist 2-p-(2-carboxyethyl) phenethylamino-5�-N-ethylcarboxamido-aden-osine (CGS21680, 100 nM) or forskolin (1 �M). Basal andagonist-stimulated rates of l-[3H]arginine (100 �M) trans-port were measured over the last 30 s of a 2 min incubationperiod; as in our previous studies (1), we excluded thepotential influence of adenosine transport by preincubatingcells with the adenosine transport inhibitor nitrobenzylthio-inosine (NBMPR, 10 �M, 30 min). In other experiments, cellswere pretreated with the protein tyrosine kinase inhibitorgenistein (10 �M, 30 min), its less active analog daidzein (10�M, 30 min), an inhibitor of MEK1/2, PD98059 (2�-amino-3�-methoxyflavone, 10 �M, 30 min), or adenylyl cyclaseSQ22536 (9-(tetrahydro-2-furanyl)-9H purin-6-amine, 100�M, 30 min). In some experiments, cells were preincubatedwith the eNOS inhibitor NG-nitro-l-arginine-methyl ester(l-NAME, 100 �M, 30 min), calcium-free Krebs supplementedwith ethylene glycol bis (2-aminoethylether)-N,N,N�,N�-tet-raacetic acid (EGTA, 1 mM), or exposed to 80 mM KCl toevoke a membrane depolarization.

Single-cell measurement of [Ca2�]i and pHi

Intracellular calcium ([Ca2�]i) and pH were measured usingratiometric fluorescent dyes. HUVEC were seeded onto glass

1585ACTIVATION OF THE L-ARGININE-NO PATHWAY BY ADENOSINE

no. 1 coverslips and cultured for 2–3 days before use. Tomeasure single-cell [Ca2�]i changes, cells were loaded with 1�M Fura-2/AM for 60 min at room temperature in 20% FCS,80% DMEM, and fluorescence ratios measured using eitherimaging (350/380 nm) or spectrophotometric (340/380 nm)analysis (24, 25). To determine pHi, cells were loaded undersimilar conditions but using 2 �M BCECF/AM. After loading,cells were maintained in a HEPES-buffered balanced saltsolution (HBSS) of the following composition (mM): 145NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, 5 glucose, 1%(w/v) bovine serum albumin (BSA), pH 7.4. For experimen-tal runs, the same solution was used but with a lower BSAconcentration (0.1%). Coverslips were mounted on a ther-mostatted stage (30 or 37°C) of an inverted epifluorescencemicroscope equipped with a 40� objective (N.A. 1.3) andsuperfused with HBSS by gravity feed. For imaging, excitationwas via an LEP dual filter wheel system (Ludl, Hawthorne,NY) equipped with various neutral density and interferencefilters: 350 and 380 nm (Fura-2); 440 and 490 nm (BCECF).Emission measured either �400 nm (Fura-2) or �505 nm(BCECF), which was captured on a 14 bit cooled CCD cameraHamamatsu C4880–80 controlled by Openlab software (Im-provision, Coventry UK). An image pair was captured every1–3 s. [Ca2�]i and pHi are expressed qualitatively as the ratioof fluorescence at 350/380 nm or 490/440 nm, respectively.For single-cell photometric measurements of [Ca2�]i, excita-tion was via a 75W xenon arc lamp and fluorescence wasmonitored at �450 nm. For Fura-2 loaded cells, autofluores-cence was estimated by addition of 2 mM Mn2� plus 10 �Mhistamine.

Measurement of intracellular cAMP levels

Confluent HUVEC in 24-well plates were incubated for 30min with warmed (37°C) Krebs solution containing 100 �Ml-arginine and rolipram (50 �M, cAMP-specific phosphodi-esterase inhibitor). cAMP levels were determined under basalconditions and in cells challenged for 2 min with adenosine(10 �M), CGS21680 (100 nM) or forskolin (1 �M). Cells wereextracted in 65% ethanol and dried before cAMP analysisusing an enzyme immunoassay kit.

Measurement of intracellular cGMP accumulation

As described previously (1, 22), basal and stimulated cGMPaccumulation in HUVEC monolayers was abolished by theNOS inhibitor l-NAME, and thus cGMP levels were used asan index of NO production. Cells were preincubated for 30min in Krebs buffer containing l-arginine (100 �M) andIBMX (1 �M). The preincubation medium was removed andcells were stimulated with adenosine (10 �M, 2 min) or theA2a receptor agonist CGS21680 (100 nM, 2 min). HCl (0.1M)cell extracts were stored at �20°C for radioimmunoassay ofcGMP levels (22). Basal and A2a purinoceptor-stimulatedcGMP levels were also measured in cells pretreated withinhibitors of eNOS (l-NAME, 100 �M, 30 min), adenylylcyclase (SQ22536, 100 �M, 30 min), tyrosine kinases(genistein, 10 �M, 30 min), a less active analog of genistein(daidzein, 10 �M, 30 min), MEK1/2 (PD98059, 10 �M, 30min), PI3-kinase (wortmannin, 20 nM, 30 min or LY294002,10 �M, 30 min). In some experiments, cGMP accumulationwas assayed in HUVEC exposed to nominally Ca2�-free Krebsbuffer or 80 mM KCl.

Whole-cell patch clamp analysis of K� currents

Ionic currents were measured using the whole-cell recordingmode of the patch clamp technique with a RK-400 (Biologi-

cal, France) amplifier (26). Recording pipettes (2–5 M�)were filled with an intracellular solution containing (mM)140 KCl, 2 MgCl2, 15 HEPES, and Nystatin (0.2 mg mL�1).The bath solution contained (mM) 137 NaCl, 5.4 KCl, 2CaCl2, 15 HEPES, 5 D-glucose, pH 7.4. Voltage pulses wereapplied in steps of 20 mV (�100–100 mV) for 400 ms and theholding potential was set to �60 mV. Voltage pulse genera-tion, data acquisition, and analysis were performed usingsoftware written by J. Dempster (University of Strathclyde,Glasgow, UK). All traces shown were leak subtracted. Currentrecordings were obtained from HUVEC challenged withadenosine (10 �M) after pretreatment in the absence orpresence of l-NAME (100 �M, 30 min) or challenged withthe NO donor sodium nitroprusside (10 �M).

Immunoblotting

Confluent passage 2 HUVEC in 60 mm dishes were deprivedof serum for 12–16 h. Monolayers were washed twice withKrebs buffer (37°C), then preincubated with Krebs buffercontaining 100 �M l-arginine in the absence or presence ofa protein tyrosine kinase inhibitor, genistein (10 �M, 30 min)or the MEK1/2 inhibitors PD98059 (10 �M, 30 min) andU0126 (1,4-diamino-2,3-dicyano-1,4-bis[aminophenylthio]butadiene, 1 �M, 30 min). The concentrations of the struc-turally distinct MEK1/2 inhibitors used are consistent withour previous reports (27, 28) and have no inhibitory effect onthe p38MAPK or JNK pathways (29, 30). Cells were preincu-bated with a soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, 10 �M, 30 min),l-NAME (100 �M, 30 min), ZM241385 (100 nM, 30 min, A2apurinoceptor antagonist), or the PI3-kinase inhibitorLY249002 (10 �M, 30 min). HUVEC were subsequentlychallenged with adenosine (10 �M, 2 min) and the reactionwas stopped by washing monolayers with ice-cold PBS con-taining 200 �M sodium orthovanadate. Cells were lysed andextracts were immunoblotted using a polyclonal antibodyraised against dually phosphorylated (threonine 183 andtyrosine 185) p42/p44MAPK, total p42/p44MAPK, or serine-phosphorylated Akt/PKB, with protein bands detected byenhanced chemiluminescence (27). The polyclonal p42/p44MAPK antibody raised against the dually phosphorylatedproteins provides a good indicator of p42/p44MAPK activity,since we previously correlated p42/p44MAPK activity inHUVEC using an in gel kinase assay with dual phosphoryla-tion of p42/p44MAPK (31). To ascertain equal protein load-ing, membranes were stained with Ponceau red (0.1% w/v in5% acetic acid). The density of Western blots was analyzedusing ScnImage software (Scion Corporation, Frederick, MD).

Materials

All cell culture reagents, collagenase type II from Clostridiumhistolyticum, adenosine, histamine, IBMX, rolipram, wortman-nin, ODQ, genistein, daidzein, SQ22536, CGS21680, N6-cyclopentyladenosine (CPA), N-ethylcarboxamidoadenosine(NECA), and (2-chloro-N6-(3-iodobenzyl)-5�-(N-methylcar-bamoyl)adenosine (Cl-IB-MECA) were from Sigma (Poole,Dorset, UK); A2a purinoceptor antagonist 4-(2-[7-amino-2-(2-furyl)]1,2,4-triazolo[2,3-a] [1,3,5]triazin-5-ylamino]ethyl)phe-nol (ZM241385) from AstraZeneca Pharmaceuticals (Maccles-field, Cheshire, UK); PD98059, LY294002, and U0126 fromCalbiochem (Beeston, Nottingham, UK); Fura-2 acetoxy-methyl ester and BCECF from Molecular Probes (CambridgeBioscience, UK). l-[2,3-3H]arginine (36.1 Ci mmol�1) 3�,5�cyclic GMP-TME[tyrosine-125I], ECL reagents and the cAMPEIA kit were purchased from Amersham plc (Buckingham-shire, UK). Antibodies against the dually phosphorylated and

1586 Vol. 16 October 2002 WYATT ET AL.The FASEB Journal

total p42/p44MAPK and all reagents for the RT-PCR were fromPromega (Southampton, UK); the anti-phosphoserine Akt/PKB antibody and positive control were from New EnglandBiolabs (Hertfordshire, UK). The magnetic oligo (dT)25beads (Dynabeads) were from Dynal (Wirral, UK) and theQiaquick gel extraction kit from Qiagen (Teddington, Mid-dlesex, UK).

Statistics

Data are expressed as means se, where n indicates thenumber of different umbilical vein endothelial cell cultureswith at least 2–6 replicate measurements per experimentalcondition in each cell culture. Statistical analyses were carriedout using ordinary ANOVA where applicable and a two-tailedStudent’s t test for unpaired data, with P � 0.05 consideredstatistically significant.

RESULTS

RT-PCR analysis of adenosine receptor subtypesin human fetal endothelial cells

We have previously reported that A2a- but not A1-adenosine receptor agonists stimulate the l-argi-nine-NO pathway in human umbilical vein endothelialcells (1). To extend our pharmacological findings, wehave used RT-PCR analyses to identify adenosine recep-tor subtypes in HUVEC. A2a-, A2b-, and A3-adenosinereceptor subtype mRNA was readily detected, whereasexpression of the A1-adenosine receptor subtype wasnegligible (Fig. 1). Sequence analysis of the A2a-, A2b-,and A3-adenosine receptor subtype PCR productsshowed a 99–100% homology with known humansequences (data not shown).

Effects of [Ca2�]o removal or elevated K� onL-arginine transport and cGMP production

Adenosine (10 �M, 2 min) -stimulated l-arginine trans-port [pmol (�g protein)�1 min�1: 1.450.4 vs.3.70.4, n4, P�0.05] was significantly inhibited bythe NOS inhibitor l-NAME (20.9, n4, P�0.05).Adenosine-stimulated increases in cGMP levels [pmol(106 cells)�1 5 min�1: 2.20.2 vs. 4.80.1, n4,P�0.01] were mimicked by an A1/2 purinoceptor ago-nist NECA [100 nM, 2 min: 3.30.2 pmol (106 cells)�1

5 min�1, P � 0.05], unaffected by an A3 agonistCl-IB-MECA [100 nM, 2 min: 2.50.2 pmol (106

cells)�1 5 min�1], and abolished by l-NAME(2.00.06, n4). As in our earlier study (1), pretreat-ment of HUVEC with a selective A2a purinoceptorantagonist (ZM241385) prevented both CGS21680(data not shown) and NECA [2.40.04 pmol (106

cells)�1 5 min�1]-induced cGMP accumulation. Prein-cubation of HUVEC in nominally Ca2�-free buffer hadno significant effect on basal, adenosine- or CGS21680-stimulated rates of l-arginine transport or cGMP pro-duction (Table 1). Depolarization of cells with 80 mMK� reduced stimulated rates of l-arginine transport buthad no significant effect on cGMP levels (Table 1).l-arginine transport and cGMP production were alsounaffected by NBMPR (data not shown), an inhibitor ofthe equilibrative es nucleoside transporter (32, 33).

Effects of adenosine receptor agonists and histamineon [Ca2�]i and pHi

Given that eNOS can be stimulated directly by Ca2�

(34), it was pertinent to examine the effects of adeno-sine receptor activation on potential changes in[Ca2�]i. Despite a significant stimulation of NO pro-duction (Table 1), adenosine (10 �M) and CGS21680(100 nM) failed to stimulate a detectable increase in[Ca2�]i, even though histamine evoked a characteristicbiphasic response in the same cells (Fig. 2A and inset).Similar data were obtained in cells challenged initiallywith histamine, followed by acute exposure to adeno-sine or CGS21680 (data not shown). Careful examina-tion of the 350/380 nm ratio images did not reveal anyhighly localized [Ca2�]i increases in response to aden-osine that might have escaped detection when measur-ing global cytosolic [Ca2�]i. Nevertheless, subcellularCa2� wave initiation loci were readily detected duringthe early phase of a histamine-stimulated Ca2� spike.Acute exposure of HUVEC to specific A1 (CPA, 100nM), A1/2 (NECA, 100 nM), or A3 (Cl-IB-MECA, 100nM) purinoceptor agonists failed to elevate cytosolic[Ca2�]i (Fig. 2B–D).

In addition to [Ca2�]i, changes in pHi have beensuggested to modulate eNOS activity, i.e., alkalinizationof the cytosol promotes NO release in some endothelialcells (35). Although histamine (10 �M) evoked atransient increase in the 490/440 nm ratio (data notshown) reflecting an increase in pHi, adenosine (10�M) caused negligible changes in pHi (490/440 nm �

Figure 1. RT-PCR analysis of adenosine receptor subtypes inhuman umbilical vein endothelial cells. HUVEC mRNA wasisolated as described in Materials and Methods. cDNA wasgenerated from the mRNA and amplified by PCR usingspecific adenosine receptor primers, with PCR products sep-arated on a 1.8% agarose gel and visualized by ethidiumbromide staining. Images are representative of 3 different cellcultures. C control (HUVEC mRNA not subjected toreverse transcription); P plasmid containing the corre-sponding adenosine receptor sequence; HUVEC cDNA.


ratio 0.060.004, n98 cells). As expected, an ammo-nium pulse (20 mM) caused a profound alkalinization(490/440 nm � ratio 0.570.01, n98 cells, P�0.001),followed by an acid rebound on ammonium with-drawal.

Effects of adenosine, CGS21680, and forskolinon intracellular cAMP levels

As activation of A2a purinoceptors has been shown toincrease intracellular cAMP levels in endothelial cells(36), we investigated whether adenosine (10 �M, 2min) or CGS21680 (100 nM, 2 min) increased cAMPlevels in HUVEC. Although activation of adenylyl cy-clase by forskolin (1 �M, 2 min) increased cAMPaccumulation (% of control: 23115, n4, P�0.05),treatment of cells with either adenosine or CGS21680had no effect on intracellular cAMP levels (% ofcontrol: 11311, n4 and 11719, n4, respectively).

Basal cAMP levels ranged between 7.5 and 14 pmol (106

cells)�1 5 min�1. Forskolin increased basal rates ofl-arginine transport without altering intracellularcGMP levels (Fig. 3A). Pretreatment of endothelialcells with the adenylyl cyclase inhibitor SQ22536 hadno effect on either adenosine-induced increases inl-arginine transport or cGMP accumulation (Fig. 3B).

Effects of PI3-kinase inhibitors on adenosine-inducedstimulation of the L-arginine-NO pathway

As eNOS has been reported to be regulated in aCa2�-independent manner via PI3-kinase/Akt-medi-ated phosphorylation (17, 18), we investigated whetheradenosine-stimulated NO production required activa-tion of PI3-kinase and Akt/PKB. Inhibition of PI3-kinase with wortmannin or LY294002 had no effect onadenosine-mediated increases in cGMP accumulation(Fig. 4A) or phosphorylation of Akt/PKB (Fig. 4B).

Figure 2. Activation of adeno-sine receptors does not elevate[Ca2�]i in human umbilicalvein endothelial cells. HUVECwere maintained in Ca2�-con-taining solution and basal andagonist-stimulated peak and pla-teau [Ca2�]i levels were moni-tored in single cells using imag-ing (350/380 nm fluorescenceratio, A) or spectrophotometricanalysis (340/380 nm ratio,B–D). A) [Ca2�]i in cells chal-lenged with adenosine (10 �M)and histamine (10 �M); inset:cells challenged with the A2apurinoceptor agonist CGS21680(100 nM) and histamine (10�M). Images are pseudo-col-ored to show ratio and are mod-ulated by the brightness of thesum of the 350 and 380 nmimages with a gamma correctionof 1.5 and filtered with a 3 � 3median filter. B–D) Cells challenged initially with either an A1 (100 nM CPA), A1/2 (100 nM NECA), or A3 (100 nM Cl-IB-MECA)purinoceptor agonist, then histamine (10 �M). Data are representative of similar experiments in 3 different cell cultures.

TABLE 1. Effect of extracellular calcium removal and membrane depolarization on adenosine- and CGS21680-induced L-argininetransport and cGMP productiona

l-arginine transport (pmol (�g protein)�1min�1) Intracellular cGMP (pmol (106 cells)�15 min�1)

Normal �[Ca2�]o 80 mM K� Normal �[Ca2�]o 80 mM K�

Control 1.8 0.1 2.1 0.1 1.7 0.2 3.4 0.4 2.5 0.5 2.5 0.4Adenosine 2.9 0.2* 3.1 0.3* 1.5 0.04 6.1 0.6** 5.2 0.6* 4.5 0.6*CGS21680 3.2 0.1* 3.4 0.1* 2.1 0.7 6.4 1.1** 5.5 0.6* 5.5 0.5*

a Confluent HUVEC were washed twice with Krebs (37°C) and incubated in Ca2� free Krebs buffer containing 100 �M l-arginine and EGTA(1 mM) or elevated KCl (80 mM) before stimulation with adenosine (10 �M, 2 min) or CGS21680 (100 nM, 2 min). Transport of 100 �Ml-[3H]arginine was determined for the last 30 s of a 2 min incubation period and intracellular cGMP levels were determined in HCl cell extractsby radioimmunoassay. Values denote the means se of triplicate measurements in each of 4 different cell cultures; * P � 0.05, ** P � 0.01vs. respective control.


Effect of genistein, PD98059, and U0126 onadenosine-induced stimulation of L-arginine transport,cGMP accumulation, and activation of p42/p44MAPK

Preincubation of HUVEC with genistein, a broad spec-trum tyrosine kinase inhibitor, or the MEK1/2 inhibi-tor PD98059 had no effect on basal rates of l-argininetransport or cGMP accumulation (Table 2). In thesesame experiments, adenosine (10 �M, 2 min) evoked atwofold increase in l-arginine transport and cGMPaccumulation, which was abolished in cells pretreatedwith either genistein (10 �M) or PD98059 (10 �M) butunaffected by daidzein (10 �M), a less active analog ofgenistein.

In parallel experiments, we found that adenosine (10�M) caused a time-dependent (15 s–5 min) increase inp42/p44MAPK phosphorylation (Fig. 5A), which wasattenuated by the selective A2a receptor antagonistZM241385 (Fig. 5B). The increase in p42/p44MAPK

phosphorylation in response to adenosine was not theresult of enhanced protein levels of p42/p44MAPK, sincetotal p42/p44MAPK protein levels were unchanged (Fig.5A). Our ability to detect basal phosphorylation ofp42/p44MAPK is consistent with our previous studieswith HUVEC in which we suggested that removal ofmedia and addition of agonist could result in shearstress and subsequent phosphorylation of p42/p44MAPK

(27, 37). As shown in Fig. 6A–C, adenosine-inducedincreases in p42/p44MAPK phosphorylation were abol-ished by pretreatment of cells with genistein (10 �M),PD98059 (10 �M), and U0126 (1 �M). The inhibitoryeffects of PD98059 and U0126 on p42/p44MAPK activa-tion and/or cGMP accumulation could not be attrib-uted to actions on cyclooxygenases (38), since indo-methacin (10 �M) had no effect on adenosine-stimulated cGMP accumulation or p42/p44MAPK

phosphorylation (data not shown). Treatment of cellswith LY294002 had no effect on adenosine-induced

Figure 3. Role of adenylyl cyclase in adenosine andCGS21680-stimulated l-arginine transport and intracellularcGMP accumulation. A) Basal and agonist-stimulated l-argi-nine transport and cGMP production were determined inHUVEC challenged with adenosine (10 �M, 2 min),CGS21680 (100 nM, 2 min), or forskolin (1 �M, 2 min).l-arginine transport was measured over the final 30 s of a 2min incubation period and cGMP production in HCl extractswas determined by radioimmunoassay. B) HUVEC monolay-ers were incubated with adenosine (10 �M, 2 min) in theabsence or presence of an adenylyl cyclase inhibitor SQ22536(100 �M, 30 min) and l-arginine transport and cGMPaccumulation were determined. Basal l-arginine transportand cGMP levels ranged between 1.3–2.3 pmol (�g pro-tein)�1 min�1 and 8.1–9.8 pmol (106)�1 5 min�1, respec-tively. Values denote the means se of triplicate measure-ments in 4 different cell cultures, *P � 0.05 vs. control.

Figure 4. Lack of involvement of PI3-kinase and Akt/PKB inadenosine-stimulated cGMP production. A) HUVEC werepreincubated with the PI3-kinase inhibitors LY294002 (10�M, 30 min) or wortmannin (20 nM, 30 min) before acuteexposure to adenosine (10 �M, 2 min). Intracellular cGMPlevels were determined by radioimmunoassay. Values denotethe means se of triplicate measurements in 4 different cellcultures. Basal cGMP levels ranged between 1 and 2.5 pmol(106)�1 5 min�1. B) quiescent HUVEC were stimulated withadenosine (10 �M, 2 min); lysates were separated by SDS-PAGE, transferred to membranes, and probed with an anti-phosphoserine Akt/PKB antibody. A commercially availablepositive control for phosphorylated Akt/PKB obtained fromNIH/3T3 cells treated with platelet-derived growth factor(PDGF, 100 �g mL�1, 10 min) was also analyzed. Blot isrepresentative of similar experiments in 5 different cellcultures.


p42/p44MAPK phosphorylation, confirming that adeno-sine does not signal via PI3-kinases in HUVEC (Fig. 7A).

Effect of eNOS inhibition on adenosine-inducedp42/p44MAPK phosphorylation

Although adenosine-induced stimulation of l-argininetransport and cGMP accumulation was prevented byl-NAME, inhibition of eNOS had no effect on aden-osine-stimulated p42/p44MAPK phosphorylation (Fig.7B). Moreover, pretreatment of cells with ODQ, aninhibitor of soluble guanylyl cyclase, had only a mar-ginal effect on p42/p44MAPK phosphorylation (Fig. 7C)but, as expected, abolished adenosine-stimulated cGMPaccumulation (data not shown).

Effects of adenosine on whole-cell potassium currents

As adenosine-stimulated l-arginine transport is sensi-tive to changes in membrane potential (1), we deter-mined whether adenosine acutely modulates mem-brane currents in HUVEC using the whole-cell patchclamp technique. Adenosine (10 �M) activated anoutward K� current (Fig. 8A), which was inhibited bytetraethylammonium (10 mM; data not shown) andl-NAME (Fig. 8B). Activation of this K� current byadenosine was mimicked by the NO donor sodiumnitroprusside (Fig. 8C), suggesting that a NO-mediatedmembrane hyperpolarization may account for the stim-ulation of l-arginine transport in HUVEC challengedwith adenosine.

DISCUSSION

The present study provides the first evidence that acuteA2a purinoceptor activation stimulates the l-argi-

nine-NO pathway in fetal endothelial cells via Ca2�-,pH-, and cAMP-independent mechanisms involvingphosphorylation of p42/p44MAPK. Release of NO (or adownstream mediator) leads to a membrane hyperpo-larization and stimulation of l-arginine influx, which inHUVEC is mediated predominantly via the Na�-inde-pendent cationic amino acid transport system y� desig-nated CAT-1 (1, 39). Stimulation of eNOS activity inresponse to A2a purinoceptor activation was not medi-ated via PI3 kinase and Akt/PKB, as previously docu-mented for shear stress and estradiol-mediated NOproduction (17, 18, 40).

We have previously shown that adenosine-inducedactivation of l-arginine transport and NO production

TABLE 2. Involvement of protein tyrosine kinases andp42/p44MAPK in adenosine-induced activationof the L-arginine–NO pathwaya

Condition

l-arginine(pmol

(�g protein)�1

min1)

Intracellular cGMP(pmol (106 cells)�1

5min�1)

Control 2.2 0.3 1.8 0.3Genistein (10 �M) 2.4 0.6 2.4 0.3Daidzein (10 �M) 2 0.4 2.3 0.2PD98059 (10 �M) 2.6 0.5 2.4 0.4Adenosine (10 �M) 4.1 0.2† 4.8 0.06†

� Genistein (10 �M) 2.7 0.4* 2 0.4*� Daidzein (10 �M) 4.2 0.2† 5.1 1.8†� PD98059 (10 �M) 2.2 0.3* 2.2 0.2*

a Confluent HUVEC were preincubated in the absence or pres-ence of genistein (10 �M, 30 min), daidzein (10 �M, 30 min), orPD98059 (10 �M, 30 min) and stimulated with adenosine (10 �M, 2min). l-arginine transport was measured over the final 30 s of theincubation period and cGMP levels were determined in HCl cellextracts by radioimmunoassay. Data denote the means se ofduplicate measurements in each of 8 different cell cultures; † P �0.01 compared to control, * P � 0.01 compared to adenosine alone.

Figure 5. Acute effects of adenosine on p42/p44MAPK phos-phorylation is mediated by A2a purinoceptor activation. A)Quiescent HUVEC were stimulated with adenosine (10 �M)for 15 s, 30 s, 1 min, 2 min, and 5 min and cell lysates wereseparated by SDS-PAGE, transferred to membranes, andprobed using the p42/p44MAPK antibody against the duallyphosphorylated isoforms and an antibody against total p42/p44MAPK protein. Blot is representative of experiments in 3different cell cultures. B) Quiescent HUVEC were stimulatedwith adenosine (10 �M, 2 min) after pretreatment of cellswith Krebs or ZM241385 (A2a antagonist, 100 nM, 30 min).Cell lysates were immunoblotted for dually phosphorylatedp42/p44MAPK and equal protein loading was assessed byPonceau red staining (not shown). Analysis of the foldchanges in band density over control is illustrated below theblot. Data denote the means se of density measurementsfrom 4 different cell cultures, *P � 0.05 compared to control.


in HUVEC is mediated via A2a purinoceptors (1) andhave now established that this cell type possesses mRNAfor the A2a-, A2b-, and A3 purinoceptor subtypes withlittle or no detectable mRNA for the A1 purinoceptor.

Our findings are supported by a recent study withHUVEC reporting that mRNA levels for the A2a puri-noceptor are 10-fold greater than the A2b purinoceptor(41). In agreement with our findings, the latter studyfailed to detect mRNA for the A1 purinoceptor. More-over, A2a- and A2b purinoceptors have been reported inporcine and human coronary artery endothelial cells byRT-PCR and Western blot analysis, establishing a correla-tion between mRNA levels and protein expression (42).

In agreement with the present study and an earlierreport by Sobrevia et al. (1), a primary role for A2apurinoceptors in adenosine-stimulated NO synthesishas been documented in human iliac and porcinecarotid artery endothelial cells (10), guinea pig coro-nary vasculature (4), hamster aorta (43), and porcinecoronary artery endothelial cells (44). Moreover, acti-vation of A1 purinoceptors markedly attenuates aden-osine-stimulated NO production in iliac and carotidartery endothelial cells (10). The only report implicat-ing A1 purinoceptors in adenosine-mediated NO pro-duction in HUVEC used much higher adenosine con-centrations (5 mM) (45) than the present study (10�M), which may have led to activation of differentsignaling pathways.

Classically, agonists that increase NO production inendothelial cells do so via an elevation in [Ca2�]i and,in the case of bradykinin, an intracellular alkalinization

Figure 6. Effect of genistein, PD98059, and U0126 on aden-osine-induced activation of p42/p44MAPK. Quiescent endo-thelial cells were stimulated with adenosine (Ado, 10 �M, 2min) in the absence or presence of genistein (10 �M, 30 min;A), PD98059 (10 �M, 30 min; B), and U0126 (1 �M, 30 min;C). Cell lysates were analyzed by SDS-PAGE and blots wereprobed using a p42/p44MAPK antibody raised against thedually phosphorylated isoforms. Equal protein loading wasassessed by Ponceau red staining (data not shown). Blots inpanels A, B, and C are representative of experiments in 4, 6,and 3 different cell cultures, respectively, and the foldchanges in band density over control for blots in panels A andB are summarized below the respective blots. Data denote themeans se of density measurements from 4 and 6 differentcell cultures, *P � 0.05 compared to control.

Figure 7. Characterization of adenosine-induced activation ofp42/p44MAPK phosphorylation. Quiescent cells were preincu-bated with either a PI3-kinase inhibitor LY294002 (10 �M, 30min; A), l-NAME (100 �M, 30 min; B), or ODQ (a solubleguanylyl cyclase inhibitor, 10 �M, 30 min; C), then chal-lenged with adenosine (10 �M, 2 min). Immunoblots wereprobed using the p42/p44MAPK antibody raised against thedually phosphorylated isoforms. Equal protein loading wasassessed by Ponceau red staining (not shown). Blots arerepresentative of experiments in 4 different cell cultures.


(35). However, adenosine- and CGS21680-mediatedNO synthesis in HUVEC occurred without measurablechanges in cytosolic Ca2� or pH, and chelation ofextracellular Ca2� had no effect on A2a purinoceptor-stimulated l-arginine transport or NO production.These findings agree with recent evidence that shearstress (46) and 17�-estradiol-induced NO accumulationin HUVEC occurs via a Ca2�-insensitive mechanism(16). Recent reports have further established thateNOS can be serine phosphorylated in response toshear stress via Akt/PKB, which itself is regulated byPI3-kinase (17, 18). The involvement of Akt/PKB andPI3-kinase in A2a purinoceptor-mediated responses inHUVEC seems unlikely, since we found that cGMPaccumulation was unaffected by the PI3-kinase inhibi-

tors LY294002 or wortmannin and Akt/PKB was notserine phosphorylated in response to adenosine.

Activation of A2 purinoceptors is known to increasecAMP levels in vascular smooth muscle and endothelialcells (2), and elevated cAMP levels have been reportedto stimulate endothelium-derived NO production (47).In the present study, however, adenosine- andCGS21680-stimulated NO release was not accompaniedby an increase in intracellular cAMP levels. Moreover,inhibition of adenylyl cyclase with SQ22536 had noeffect on A2a purinoceptor-stimulated l-arginine trans-port or cGMP accumulation (see Fig. 3B). Our findingsagree with previous reports showing that cAMP levels inHUVEC and coronary arterioles are not altered byadenosine (36, 48). Direct activation of adenylyl cyclasewith forskolin increased cAMP levels and l-argininetransport but had no effect on cGMP production (seeFig. 3A). As cAMP activates large conductance K�

channels in endothelial cells (49), it is possible thatmembrane hyperpolarization might explain the forsko-lin-mediated increase in l-arginine transport. Forskolinhas been reported to potentiate agonist (bradykininand histamine) –stimulated but not basal cGMP accu-mulation in endothelial cells (50), consistent with ourobservation that forskolin did not alter basal cGMPlevels.

A role for protein tyrosine kinases in shear stress and17�-estradiol-mediated activation of eNOS has beenreported (14, 51). In endothelial cells, acute stimula-tion of NO production in response to estradiol can beinhibited by herbimycin (16) and the MEK1/2 inhibi-tor PD98059 (19), suggesting that protein tyrosinekinases and p42/p44MAPK are involved in the signalingpathway regulating NO production in endothelial cells.Our study of HUVEC provides further evidence thatadenosine-stimulated activation of p42/p44MAPK is pre-vented by genistein and the structurally distinctMEK1/2 inhibitors PD98059 and U0126 (Fig. 6). Aden-osine-stimulated phosphorylation of p42/p44MAPK wasprevented by the selective A2a-adenosine receptor an-tagonist ZM241385 but unaffected by nitrobenzylthio-inosine, an inhibitor of the es nucleoside transporter(data not shown), confirming that phosphorylation ofp42/p44MAPK is a result of A2a purinoceptor activationrather than a consequence of adenosine transport. Ourfindings are consistent with an earlier report thatstimulation of A2a purinoceptors in HUVEC leads toactivation of the p42/p44MAPK pathway and cell prolif-eration (13). A2b-Adenosine receptors have been impli-cated in the activation p42/p44MAPK in HEK-293 cells,with activation mediated via Gq/11 receptor couplingdependent on activation of Ras (52). We can exclude arole for A2b receptors in the activation of p42/p44MAPK

in HUVEC since 1) phosphorylation is reduced to basallevels by the selective A2a purinoceptor antagonistZM241385 and activated by the A2a purinoceptor ago-nist CGS21680 (100 nM, 2 min; data not shown), and 2)receptor coupling to Gq/11 leads to activation of phos-pholipase C and subsequent release of store Ca2�,

Figure 8. Effects of adenosine and sodium nitroprusside onoutward K� currents. Whole-cell currents were recorded fromsingle cells in the absence or presence of A) adenosine (10�M), B) adenosine (10 �M) � l-NAME (100 �M, pretreat-ment for 30 min), or C) sodium nitroprusside (SNP, 10 �M).Voltage was applied in steps of 20 mV between �100 mV and�100 mV. Values denote the means se of current/voltagerelationships in 3–4 different cell cultures with 4 replicatemeasurements per condition, *P � 0.05 vs. correspondingcontrol or control � l-NAME.


which was not observed in our single-cell fluorescenceexperiments (Fig. 2).

V EGF has been shown to stimulate NO productionin endothelial cells, resulting in an increase in intracel-lular cGMP levels that in turn lead to the phosphoryla-tion of p42/p44MAPK (53). However, in our experi-ments adenosine-stimulated NO production requiredactivation of p42/p44MAPK. Since adenosine-stimulatedp42/p44MAPK phosphorylation was not inhibited byl-NAME and ODQ only partially attenuated the re-sponse, this suggests that p42/p44MAPK phosphoryla-tion precedes NO release and cGMP accumulation.These findings implicate p42/p44MAPK as upstreamregulators of NO production in response to A2a puri-noceptor activation in HUVEC. The observation thatODQ led to a slight decrease in p42/p44MAPK phos-phorylation may stem from nonspecific effects of thisinhibitor since l-NAME, which reduces NO productionand cGMP levels, had no effect on adenosine-inducedp42/p44MAPK phosphorylation. Incubation of cells withl-NAME before adenosine stimulation abolished theup-regulation of l-arginine transport, suggesting thatNO (or a downstream mediator) may cause the in-creased transport rate in HUVEC. Our observation isconsistent with the report that acute exposure of bovineaortic endothelial cells to NO donors stimulatesl-arginine transport via the cationic amino acidtransporter CAT-1 (54).

l-arginine transport is sensitive to changes in mem-brane potential (22, 55–57) and there is evidence forthe activation of ion channels by NO (58). In thepresent study, adenosine activated an outward K� cur-rent, which was abolished by l-NAME and mimicked bya NO donor (Fig. 8). These findings suggest that NOgenerated after stimulation of A2a purinoceptors acti-vates this current. Alternatively, as reported in HEK293cells, cGMP-activated protein kinase may lead to phos-phorylation of a large conductance BKCa channel (59),leading to membrane hyperpolarization and an in-creased driving force for l-arginine entry.

In conclusion, we have shown that acute activation ofA2a purinoceptors in fetal endothelial cells stimulatesl-arginine transport and cGMP accumulation indepen-dent of changes in cytosolic Ca2�, pH, or cAMP, butinvolving protein tyrosine kinases and p42/p44MAPK

phosphorylation. The resulting increase in NO produc-tion (or a downstream protein) then leads to increasesin l-arginine transport, most likely via a NO-inducedactivation of an outward K� current and membranehyperpolarization.

This work was supported by the Medical Research Council,Wellcome Trust (040727/Z/94; 052953/Z/97), British HeartFoundation, Fondo Nacional de Desarrollo Cientifico y Tec-nologico (1000354 and 7000354, Chile), and Direccion de Investi-gacion, Universidad de Concepcion (DIUC 201.084.003–1, Chile).We are grateful to Dr. Rebecca Houliston for her assistance inthe initial immunoblotting experiments and to Dr. AlbertFerro for his advice in measuring cAMP levels, and themidwives of the Guy’s Hospital Maternity Ward for collectingthe umbilical cords.

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Received for publication December 21, 2001.Revised for publication May 30, 2002.


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