� Corresponding author. Tel.: +351-222

78969.

E-mail address: gloria@ff.up.pt (G. Que

0028-3908/$ - see front matter # 2004 Else

doi:10.1016/j.neuropharm.2004.03.015

0-78932; fax: +351-2220-

iroz).

vier Ltd. All rights reserved.

Neuropharmacology 47 (2004) 216–224

www.elsevier.com/locate/neuropharm

Coupling to protein kinases A and C of adenosine A2B receptorsinvolved in the facilitation of noradrenaline release in the prostatic

portion of rat vas deferens

Gloria Queiroz �, Clara Quintas, Carlos Talaia, Jorge Goncalves

Laboratorio de Farmacologia, CEQOFFUP, Faculdade de Farmacia, Universidade do Porto, Rua Anıbal Cunha, 164, 4050-047 Porto, Portugal

Received 4 November 2003; received in revised form 13 February 2004; accepted 18 March 2004

Abstract

In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1–30 lM), but not the A2A

agonist CGS 21680 (0.001–10 lM), caused a facilitation of electrically evoked noradrenaline release (up to 43� 4%), when inhibi-tory adenosine A1 receptors were blocked.NECA-elicited facilitation of noradrenaline release was prevented by the A2B receptor-antagonist MRS 1754, enhanced by pre-

venting cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rpand attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C.The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adeno-

sine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade ofa1-adrenoceptors and P2X receptors with prazosin and NF023, respectively.It was concluded that adenosine A2B receptors are involved in a facilitation of noradrenaline release in the prostatic portion of

rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to becoupled to the adenylyl cyclase–protein kinase A pathway but activation of the protein kinase C by protein kinase A, may alsocontribute to the adenosine A2B receptor-mediated facilitation of noradrenaline release.# 2004 Elsevier Ltd. All rights reserved.

Keywords: Adenosine A2B receptors; Noradrenaline release; Signalling pathway; Protein kinase C; Protein kinase A; Vas deferens

1. Introduction

Adenosine is a local regulator of many physiological

functions. It acts via receptors coupled to regulatory

G-proteins that include four subtypes, named A1, A2A,

A2B and A3, cloned from several mammalian species

(Fredholm et al., 2001). The availability of selective

ligands has allowed the pharmacological characteriza-

tion of A1, A2A and A3 receptors (Klots et al., 2000)

and the study of their physiological roles. However, the

lack of selective ligands for adenosine A2B receptors

has hindered their study in spite of their ubiquitous

distribution (Stehle et al., 1992; Dixon et al., 1996).

Recently, important functions for adenosine A2B

receptors have been discovered, namely their impli-cation in mast cell activation and asthma, vasodilata-tion, regulation of cell growth, intestinal functionand modulation of neurosecretion (Feoktistov andBiaggioni, 1997; Haas and Selbach, 2000). Unlike otheradenosine receptors, no selective agonists for adenosineA2B receptors have been found and 50-(N-ethylcarbox-amido)-adenosine (NECA) remains the most potentA2B agonist so far (Brackett and Daly, 1994; Brunset al., 1986; Feoktistov and Biaggioni, 1998). In pre-parations where adenosine receptor subtypes co-exist,the lack of selectivity of NECA makes the characteriza-tion of effects mediated by adenosine A2B receptors dif-ficult what may be minimised by the combined use ofNECA with the selective adenosine A2B receptor-antagonists recently developed (Kim et al., 2000).

G. Queiroz et al. / Neuropharmacology 47 (2004) 216–224 217

In the rat vas deferens, postganglionic sympathetictransmission is modulated by adenosine that can either

inhibit or facilitate noradrenaline release through acti-vation of prejunctional adenosine A1 or A2 receptors,respectively (Queiroz et al., 2002). In the prostatic por-tion, adenosine A2 receptors seem to have a pharmaco-logical profile similar to the adenosine A2B receptors(Queiroz et al., 2002) which presence has been demon-

strated by molecular biology and immunohistochemicalstudies (Dixon et al., 1996; Diniz et al., 2003).The present study was undertaken in the prostatic

portion of rat vas deferens, in order: (i) to clarifyweather adenosine A2 receptors involved in the modu-lation of noradrenaline release belong to the A2B sub-type by using the most selective antagonists available;(ii) to investigate if a putative adenosine A2B receptor-mediated modulation of noradrenaline release would

be activated by endogenous adenosine and/or wasdependent on the release of other modulators, namelyprostaglandins or nitric oxide; (iii) to identify the intra-cellular signalling pathway activated by the adenosineA2B receptors.

2. Methods

Adult male Wistar rats (300–350 g; IBMC, Porto,

Portugal) were used. Handling and care of animalswere conducted according to the EU guiding principlesin animal research (86/609/EU). Animals were killedby cervical dislocation and prostatic halves of vas defe-rens were dissected out, cleaned of connective tissueand divided in portions of 20� 5 mg weight. Tissuepreparations were incubated in 2-ml medium contain-

ing [3H]-noradrenaline (0.1 lM; specific activity of

46.8 Ci mmol�1) for 40 min at 37vC. Individual pre-

parations were then transferred to 0.3-ml superfusionchambers, where they were held by a polypropylenemesh between platinum plate electrodes 4 mm apart,

and superfused with [3H]-noradrenaline free medium at1 ml min�1. A stimulator I (Hugo Sacks Elektronic,March-Hungstetten, Germany), operating in theconstant current mode, was used for electrical fieldstimulation (rectangular pulses: 1 ms; width: 50 mAcurrent strength). The stimulation periods consisted of100 pulses at 8 Hz. The first period, applied at

t ¼ 30 min (t ¼ 0 min was the onset of superfusion)was not used for determination of tritium overflow.Unless stated otherwise, subsequent stimulation peri-ods were applied at t ¼ 60 (S1), t ¼ 90 (S2), t ¼ 120 (S3)and t ¼ 150 (S4) min. In most of the experiments onlyS1 and S2 were applied. Superfusate samples were col-

lected at 5-min intervals from t ¼ 55 min onwards. Atthe end of the experiments, tritium was determined insuperfusate samples and in tissues by scintillation spec-

trometry (Beckman LS 6500, Beckman Instruments,Fullerton, USA).The incubation and superfusion medium contained

(mM): NaCl 118.6, KCl 4.70, CaCl2 2.52, MgSO4 1.23,NaHCO3 25.0, glucose 10.0, ascorbic acid 0.3 anddisodium EDTA 0.031; it was saturated with 5% CO2

in O2 and maintained at 37vC. Unless otherwise stated,

desipramine (400 nM; to inhibit neuronal uptakeof noradrenaline) and 8-cyclopentyl-1,3-dipropylxanthine(DPCPX, 100 nM; to block adenosine A1 receptors) werepresent from the beginning of superfusion and keptthroughout. In some experiments, indomethacin,prazosin, 8,80-[carbonylbis(imino-3,1-phenylenecarbony-limino)]bis-1,3,5-naphthalene-trisulphonic acid (NF023),rolipram, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquin-olinesulfonamide (H-89) or 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U–73122) were also present throughout. Adenosine receptoragonists were added to the superfusion medium, atincreasing concentrations, 6 min before S2, S3 andS4 and kept until the end of the respective stimulationperiod. Forskolin and 8-bromoadenosine-30,50-cyclicmonophosphate (8-Br-cAMP) were added to the super-fusion medium 10 min, and phorbol 12-myristate13-acetate (PMA) 15 min before S2 and were keptuntil the end of the respective stimulation period.Antagonists and other enzyme inhibitors were added20 min, S-(4-nitrobenzyl)-6-thioinosine (NBTI) wasadded 30 min before S2 and all were kept until the end ofthe experiment.The tritium outflow was calculated as fraction of the

tissue tritium content at the onset of the respective col-lection period (fractional rate of outflow, min�1). Drugeffects on basal tritium outflow were estimated by thebn/b1 ratios and were expressed as percentage ofchange from the respective mean ratio obtained in theappropriate control; bn was the fractional rate of out-flow in the 5-min period before S2, S3 and S4 (b2, b3and b4, respectively) and b1 was the fractional rate ofoutflow in the 5-min period before S1. The overflow oftritium evoked by electrical stimulation was estimatedby subtracting basal outflow from total outflowobserved during and in the 10-min period subsequentto each stimulation period, and was expressed as a per-centage of the total level of tritium present in the tissueat the onset of the stimulation. Effects of drugs addedafter S1 on tritium overflow were evaluated as ratios ofthe overflow elicited by S2, S3 and S4 (Sn) and the over-flow elicited by S1 (Sn/S1). Sn/S1 values obtained inindividual experiments in which a test compound Awas added after S1 were calculated as a percentage ofchange (increase or decrease) from the respective meanratio obtained in the appropriate control group (sol-vent instead of A). When interaction of A, added afterS1, and a drug B either added after S1 or at the begin-

218 G. Queiroz et al. / Neuropharmacology 47 (2004) 216–224

ning of superfusion, was studied, the ‘‘appropriate con-trol’’ was a group in which B alone was used.The following drugs were used: l-[ring-2,5,6-3H]-nor-

adrenaline, specific activity 46.8 Ci mmol�1 was fromDuPont NEN (Garal, Lisboa, Portugal); adenosinedeaminase type VI (ADA; EC 3.5.4.4), angiotensin II(human), bisindolylmaleimide XI hydrochloride (Ro32-0432), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquino-linesulfonamide dihydrochloride (H-89), calphostin C,2-p-(2-carboxyethyl)-phenethylamino-50-N-ethylcarbox-amidoadenosine hydrochloride (CGS 21680), 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)-xanthine (MRS 1754), N6-cyclopentyladenosine (CPA),8-cyclopentyl-1,3-di-(n-propylxanthine) (DPCPX), desi-pramine hydrochloride, 50-N-ethylcarboxamidoadeno-sine (NECA), enprofylline, indomethacin, a,b-methyle-neadenosine 50-diphosphate (AOPCP), NG-nitro-l-argi-nine methyl ester hydrochloride (l-NAME), S-(4-nitrobenzyl)-6-thioinosine (NBTI), phorbol 12-myr-istate 13-acetate (PMA), prazosin hydrochloride, werefrom Sigma (Sintra, Portugal); (R)-adenosine, cyclic30,50-(hydrogenphosphorothioate) triethylammonium(cAMPS-Rp), 8-bromoadenosine-30,50-cyclic mono-phosphate sodium salt (8-Br-cAMP), 8,80-[carbonylbis-(imino-3,1-phenylenecarbonylimino)]bis-1,3,5-naphtha-lene-trisulphonic acid, hexasodium salt (NF023), 1,9-dideoxyforskolin, 6-N,N-diethyl-d-b,c-dibromomethy-lene 50-triphosphate (ARL 67156), forskolin, (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg:30,20,10-kl]pyrrolo[3,4-i]-[1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT5720), (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3fg:30,20,10-kl]pyrrolo[3,4-i][1,6]-benzodiazocine-10-carboxylicacid, methyl ester (KT 5823), 1-[6-[[(17b)-3-methoxy-estra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), 1H-[1,2,4]-oxadiazolo[4,3-a]quinox-

alin-1-one (ODQ) and rolipram were from Tocris(Bristol, UK), 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyr-azolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261)was provided by Scott Weiss (Vernalis, UK). Solutions ofdrugs were prepared with dimethylsulphoxide or waterand diluted with medium immediately before use.Results are presented as means� S:E:M:; n is the

number of tissue preparations. Effect of drugs on basaltritium outflow and evoked tritium overflow was testedfor significance by analysis of variance (ANOVA)followed by Dunnett’s multiple comparison test; Pvalues lower than 0.05 were taken to indicate signifi-cant differences.

3. Results

3.1. General observations

Electrical stimulation with trains of 100 pulses at 8Hz increased tritium outflow from the prostatic portionof rat vas deferens. In experiments only with desipra-mine present throughout, tritium overflow elicited byS1 was 0:178� 0:011% of the tritium content of thetissue (n ¼ 4) and b1 amounted to 0:098� 0:013% ofthe tritium content of the tissue per min (n ¼ 4). Whenthe selective adenosine A1 receptor-antagonist DPCPX(100 nM) was also present throughout superfusion,which occurred in most of the experiments, basal tri-tium outflow and evoked tritium overflow were not dif-ferent from those observed in experiments withdesipramine alone (see Table 1). When, in addition todesipramine and DPCPX, the type IV phosphodiester-ase inhibitor rolipram (10 lM), the protein kinase Ainhibitor H-89 (1 lM) or the phospholipase C inhibitorU-73122 (5 lM) were also present throughout super-fusion, b1 but not S1, was increased (see Table 1). Basaloutflow and tritium overflow remained constant

Table 1

Basal tritium outflow (b1) and electrically evoked tritium overflow (S1) in the prostatic portion of rat vas deferens

Drugs present before S1 (in addition

to DMI and DPCPX)

B

(

asal tritium outflow

b1; percentage of tissue tritium min�1)

Evoked tritium overflow

(S1; percentage of tissue tritium)

Solvent 0

:131� 0:003 (60) 0:180� 0:006 (60)

Indomethacin (100 lM) 0

:159� 0:008 (5) 0:178� 0:006 (5)

Prazosin (0.1 lM) 0

:155� 0:008 (4) 0:223� 0:018 (4)

NF023 (20 lM) 0

:131� 0:012 (4) 0:225� 0:027 (4)

NF023 ð20 lMÞ þ Prazosin ð0:1 lMÞ 0

:161� 0:006 (8) 0:224� 0:016 (8)

Rolipram (10 lM) 0

:166� 0:018 (10)�� 0:176� 0:023 (10)

H-89 (1 lM) 0

:231� 0:013 (10)�� 0:192� 0:015 (10)

U-73122 (5 lM) 0

:289� 0:016 (6)�� 0:189� 0:011 (6)

Preparations were incubated with [3H]-noradrenaline and superfused with medium containing desipramine (DMI, 400 nM) and DPCPX (100 nM)

and the drugs as indicated in the left column. S1 was applied after 60 min of superfusion and consisted of 100 pulses/8 Hz; b1 refers to tritium

outflow during the 5-min period immediately before S1. Evoked tritium overflow was obtained by subtracting the estimated basal outflow from

total outflow observed during and in the 10 min subsequent to the first stimulation period (S1), and was expressed as a percentage of the total

level of tritium present in the tissue at the onset of the stimulation. Values are means� S:E:M: for (n) tissue preparations. Significant differences

from basal outflow or tritium overflow in the presence of solvent: �� P < 0:01.

G. Queiroz et al. / Neuropharmacology 47 (2004) 216–224 219

throughout the experiment with bn/b1 and Sn/S1 valuesclose to unity (not shown). None of the drugs addedafter S1 changed basal tritium outflow, except the ade-nosine uptake inhibitor NBTI (5 lM) that increasedbasal outflow by 21� 2% (n ¼ 30; P < 0:01).

3.2. Effect of adenosine receptor agonists on tritiumoverflow

Initial experiments were carried out in order to char-acterize the effects of adenosine A2 receptors on modu-lation of tritium overflow. In the presence of theadenosine A1 receptor-antagonist DPCPX (100 nM),the non-selective adenosine receptor agonist NECA(0.1–30 lM) but not the adenosine A1 receptor agonistCPA (0.1–10 lM) or the adenosine A2A receptor ago-nist CGS 21680 (0.001–10 lM), enhanced tritium over-flow in a concentration dependent manner (Fig. 1). Thehighest enhancement caused by NECA was about43� 4%, and the EC50, the concentration that causedhalf of that enhancement, was about 2 lM (calculatedby interpolation from the nearest points of the concen-tration–response curve; Fig. 1). The adenosine A2B

receptor-antagonists enprofylline (30 lM) and MRS1754 (10 nM), but not the adenosine A2A receptor-antagonist SCH 58261 (20 nM), attenuated theenhancement on tritium overflow caused by NECA(Fig. 2A).The enhancement of tritium overflow caused by

NECA could be a consequence of a postjunctionalrelease of prostaglandins (Driessen and Starke, 1994)

or nitric oxide (Pastorino et al., 1998; Vetri et al.,2000), therefore this possibility was also investigated.Inhibition of prostaglandin synthesis with indometha-cin (100 lM) or simultaneous blockade of a2-adreno-ceptors and P2X-receptors with prazosin (100 nM) plusNF023 (20 lM), respectively, did not change theenhancement of tritium overflow caused by 10 lMNECA (Fig. 2A). Neither the nitric oxide synthaseinhibitor l-NAME (100–300 lM) nor the guanylylcyclase inhibitor ODQ (10 lM) or the protein kinase Ginhibitor KT 5823 (10 lM) changed the effect ofNECA (see Fig. 2B).

3.3. Intracellular signalling of adenosine A2B receptor

The adenosine A2B receptors are usually coupled tothe Gs–adenylyl cyclase–protein kinase A pathway (seeFredholm et al., 2001). However, several studies indi-cate that adenosine A2B receptors can also be coupledto the Gq–phospholipase C–protein kinase C pathway(Linden et al., 1999). Involvement of these two signal-ling pathways in the enhancement of tritium overflowcaused by NECA was studied by using a pharmaco-logical approach.

of the adenosine receptor agonists, CG

Fig. 1. Effects S 21680 (open

squares), CPA (open triangles) and NECA (closed circles), on electri-

cally evoked tritium overflow from prostatic portion of rat vas defe-

rens. Tissues were electrically stimulated with five trains of 100 pulses

at 8 Hz (S0–S4). Adenosine receptor agonists were added 6 min

before Sn (S2–S4) at increasing concentrations and were kept until the

end of respective stimulation period. DPCPX (100 nM) was present

from the beginning to the end of superfusion. Ordinates, tritium

overflow expressed as percentage of increase from the respective con-

trol (see Methods). Abscissa, concentration of agonists. Values are

means� S:E:M: from 4 to 8 tissue preparations. Significant differ-

ences from respective control: �P < 0:05 and ��P < 0:01.

Fig. 2. Effect of NECA on tritium overflow: (A) in the absence and

in the presence of the adenosine A2B receptor-antagonists enprofylline

and MRS 1754, adenosine A2A antagonist SCH 58261, prostaglandin

synthesis inhibitor indomethacin and the P2X-receptor-antagonist

NF023 plus the a1-adrenoceptor antagonist prazosin; (B) in the

absence and in the presence of the nitric oxide synthase inhibitor

l-NAME, guanylyl cyclase inhibitor ODQ and protein kinase G

inhibitor KT 5832. Tissues were electrically stimulated with three

trains of 100 pulses at 8 Hz (S0–S2). NECA was added 6 min before S2and kept up to the end of the stimulation period. Enprofylline, MRS

1754, L-NAME, ODQ and KT 5823 were added 20 min before S2 and

kept throughout superfusion. Indomethacin, NF023 and prazosin

were added at the beginning of superfusion and kept throughout.

DPCPX (100 nM) was present from the beginning to the end of

superfusion. Ordinates, tritium overflow expressed as percentage of

increase from the respective control (see Methods). Values are

means� S:E:M: from 4 to 12 tissue preparations. Significant differ-

ences from respective control: ��P < 0:01; from the effect of NECA

alone: þþP < 0:01.

220 G. Queiroz et al. / Neuropharmacology 47 (2004) 216–224

The facilitatory effect of NECA (10 lM) on tritiumoverflow was enhanced by rolipram (10 lM), an inhibi-tor of type IV phosphodiesterase (enzyme that metabo-lises cyclic-AMP) and abolished by the cell permeablecyclic-AMP antagonist cAMPS-Rp (30 lM) and by theprotein kinase A inhibitors H-89 (1 lM) and KT 5720(1 lM). Furthermore, enhancement of tritium overflowcaused by NECA was attenuated by the protein kinaseC inhibitor Ro 32-0432 (1 lM), but it was not changedby the phospholipase C inhibitor U-73122 (5 lM; Fig. 3)in a concentration that attenuated the enhancement oftritium overflow caused by 30 nM angiontensin II from63� 5% (n ¼ 4) to 26� 5% (n ¼ 6; P < 0:01).The adenylyl cyclase activator, forskolin (1 lM), but

not its inactive analogue 1,9-dideoxyforskolin (10 lM),mimicked the effect of NECA on tritium overflow, aneffect that was also potentiated by rolipram and atte-nuated by inhibition of either protein kinase A or C(see Table 2). Direct activation of protein kinase Awith the cell permeable and enzymatically stable cyclic-AMP analogue 8-Br-cAMP (0.1 mM) also enhancedtritium overflow and the effect was abolished by H-89(1 lM) and attenuated by Ro 32-0432 (1 lM; Table 2).The enhancement of tritium overflow caused by the

protein kinase C activator PMA (1 lM; Table 2) wasprevented by the protein kinase C inhibitor Ro 32-0432(1 lM), but not by the protein kinase A inhibitor H-89(1 lM; Table 2).The effect of another highly potent and selective pro-

tein kinase C inhibitor, calphostin C, that acts on theregulatory domain of protein kinase C (Kobayashiet al., 1989) was tested to confirm the effects of Ro 32-0432. Calphostin C (0.1 lM), in a concentration thatalmost abolished the enhancement of tritium overflowcaused by PMA (Table 2), also attenuated the effect of8-Br-cAMP (Table 2) and NECA (see Fig. 3).

3.4. Effect of endogenous adenosine on tritium overflow

It was previously observed that inhibition of adeno-sine uptake with NBTI enhanced electrically evokedtritium overflow from the prostatic portion of rat vasdeferens (Queiroz et al., 2002). The enhancement of tri-tium overflow caused by NBTI (5 lM) was even moresignificant when adenosine A1 receptors were blockedwith 100 nM DPCPX and was highly attenuated by theadenosine A2B receptor-antagonists enprofylline (30lM) and MRS 1754 (10 nM), but not by the adenosineA2A receptor-antagonist SCH 58261 (20 nM; seeFig. 4A). Enhancement of tritium overflow caused byNBTI was abolished by adenosine deaminase (ADA, 2

ffect of NECA on tritium overflow in the absen

Fig. 3. E ce and in

the presence of the type IV phosphodiesterase inhibitor rolipram, the

cAMP inactive analogue cAMPS-Rp, protein kinase A inhibitors

H-89 and KT 5720, phospholipase C inhibitor U-73122 and protein

kinase C inhibitors Ro 32-0432 and calphostin C. Tissues were elec-

trically stimulated with three trains of 100 pulses at 8 Hz (S0–S2).

NECA was added 6 min before S2 and kept up to the end of the

stimulation period. KT 5720, cAMPS-Rp, Ro 32-0432 and calphostin

C were added 20 min before S2 and kept throughout superfusion.

Rolipram, H-89 and U-73122 were added at the beginning of super-

fusion and kept throughout. DPCPX (100 nM) was present from the

beginning to the end of superfusion. Ordinates, tritium overflow

expressed as percentage of increase from the respective control (see

Methods). Values are means� S:E:M: from 4 to 20 tissue prepara-

tions. Significant differences from respective control: ��P < 0:01; from

the effect of NECA in alone, þP < 0:05 and þþP < 0:01.

Table 2

Effects of forskolin, 8-Br-cAMP and PMA on tritium overflow in the

absence and in the presence of the protein kinase A and C inhibitors

Drugs added before S2 P

ercentage increase

of tritium overflow

n

Forskolin (1 lM) 4

3� 2�� 18

+Rolipram (10 lM) 7

2� 8��,++ 5

+cAMPS-Rp (30 lM) 1

5� 5++ 6

+H-89 (1 lM) 1

3� 5++ 5

+KT 5720 (1 lM) 1

� 6++ 6

+RO 32-0432 (1 lM) 2

5� 5��,+ 6

1,9-Dideoxiforskolin (10 lM) 8

� 6 5

8-Br-cAMP (0.1 mM) 4

2� 2�� 8

+H-89 (1 lM) 1

0� 9++ 5

+RO 32-0432 (1 lM) 2

6� 3��,+ 6

+Calphostin C (0.1 lM) 2

8� 4��,+ 5

PMA (1 lM) 4

9� 4�� 10

+H-89 (1 lM) 4

8� 8�� 6

+RO 32-0432 (1 lM) 8

� 4++ 5

+Calphostin C (0.1 lM) 1

6� 2�,++ 5

Tissues were electrically stimulated with three trains of 100 pulses at

8 Hz (S0–S2). Forskolin, 1,9-dideoxforskolin and 8-Br-cAMP were

added 10 min, and PMA was added 15 min before S2 and kept up to

the end of the stimulation period. KT 5720, Ro 32-0432 and calphos-

tin C were added 20 min before S2 and kept throughout. Rolipram

and H-89 when present were added throughout superfusion. DPCPX

(100 nM) was always present from the beginning to the end of super-

fusion. Values are the means� S:E:M: from (n) tissue preparations.

Significant differences from respective control: � P < 0:05 and�� P < 0:01; from the effect of forskolin, 8-Br-cAMP or PMA alone:+ P < 0:05 and ++ P < 0:01.

G. Queiroz et al. / Neuropharmacology 47 (2004) 216–224 221

U/ml), and attenuated by the 50-nucelotidase inhibitorAOPCP (50 lM) and by the ectonucleotidase inhibitorARL 67156 (50 lM; Fig. 4B). Blockade of a1-adreno-ceptors with prazosin (100 nM) or P2X-receptors withNF023 (20 lM) attenuated the enhancement of tritiumoverflow caused by NBTI (Fig. 4B).Like the effect of NECA, the enhancement of tritium

overflow caused by NBTI in the presence of DPCPX(100 nM) was increased by rolipram (10 lM), abol-ished by the protein kinase A inhibitor H-89 (1 lM)and attenuated by the protein kinase C inhibitors Ro32-0432 (1 lM) and calphostin C (0.1 lM; Fig. 5).

4. Discussion

The aim of the present study was to proceed with thestudy of prejunctional adenosine A2 receptors involvedin a facilitation of noradrenaline release in the prostaticportion of rat vas deferens (see Queiroz et al., 2002), toconfirm whether they belong to the A2B subtype andto elucidate the intracellular signalling mechanism(s) towhich they are coupled.The pharmacological characterization of adenosine

A2B receptors relies mainly on negative results, i.e. the

lack of effectiveness of agonists that are potent and

selective for other adenosine receptor types. CGS 21680

but not NECA, is ineffective on adenosine A2B receptors

but is as potent as NECA in activating A2A receptors,

with EC50 values in the low nanomolar range (Jarvis

et al., 1989; Alexander et al., 1996). Thus, in tissues that

express adenosine A2A and A2B receptors, CGS 21680

can be used to distinguish A2A from A2B receptor-medi-

ated effects of NECA (Feoktistov and Biaggioni, 1995).

The non-selective agonist NECA, in combination with

the selective antagonist of A1 receptors DPCPX (Lohse

et al., 1987), which is 20-fold selective for A1 versus A2B

receptors (Robeva et al., 1996), have also been success-

fully used to study effects mediated by adenosine A2B

receptors in tissues co-expressing both A1 and A2B

receptors (Murphy et al., 1995; Nicholls et al., 1996). In

the present study, both strategies were used to demon-

strate an involvement of adenosine A2B receptors on the

modulation of noradrenaline release in the prostatic

portion of rat vas deferens. Blockade of prejunctional

inhibitory adenosine A1 receptors, with DPCPX,

revealed the occurrence of a release-enhancing effect

mediated by adenosine receptors with an agonist profile

compatible to that of adenosine A2B receptors:

NECA > CPA > CGS 21680. The most potent A2B

receptor agonist NECA, enhanced noradrenaline release

with an EC50 similar to that of adenosine A2B receptor-

mediated stimulation of adenylyl cyclase in other tissues

Fig. 4. Effect of the adenosine uptake inhibitor NBTI on tritium

overflow: (A) in the absence and in the presence of DPCPX; in the

presence of DPCPX plus the adenosine A2B receptor-antagonists

enprofylline and MRS 1754 and the adenosine A2A receptor-antagon-

ist SCH 58261; (B) in the presence of DPCPX alone and DPCPX

plus: adenosine deaminase, 50-nucleotidase inhibitor AOPCP, ectonu-cleotidase inhibitor ARL 67156, a1-adrenoceptor antagonist prazosin,P2X-receptor-antagonist NF023 and prazosin plus NF023. Tissues

were electrically stimulated with three trains of 100 pulses at 8 Hz

(S0–S2). NBTI was added 30 min before S2 (applied at t ¼ 100 min)

and enprofylline, MRS 1754, SCH 58261, adenosine deaminase,

AOPCP and ARL 67154 were added 20 min before S2 and kept

throughout superfusion. DPCPX, NF023 and prazosin were added at

the beginning of superfusion and kept throughout. Ordinates, tritium

overflow expressed as percentage of increase from the respective con-

trol (see Methods). Values are means� S:E:M: from 4 to 17 tissue

preparations. Significant differences from respective control: �P <

0:05 and ��P < 0:01; from the effect of NBTI in the absence of

DPCPX, +P < 0:01; from the effect of NBTI in the presence of

DPCPX alone, þþP < 0:01.

t of the NBTI on the evoked tritium ov

Fig. 5. Effec erflow in the

absence and in the presence of the type IV phosphodiesterase inhibi-

tor rolipram, protein kinase A inhibitor H-89 and protein kinase C

inhibitors Ro 32-0432 and calphostin C. Tissues were electrically sti-

mulated with three trains of 100 pulses at 8 Hz (S0–S2). NBTI was

added 30 min before S2 (applied at t ¼ 100 min). Ro 32-0432 and cal-

phostin C were added 20 min before S2 and kept throughout.

DPCPX, rolipram and H-89 were added at the beginning of super-

fusion and kept throughout. Ordinates, tritium overflow expressed as

percentage of increase from the respective control (see Methods).

Values are means� S:E:M: from 6 to 12 tissue preparations. Signifi-

cant differences from respective control: ��P < 0:01; from the effect of

NBTI alone, þP < 0:05 and þþP < 0:01.

222 G. Queiroz et al. / Neuropharmacology 47 (2004) 216–224

and cells (about 2 lM; Brackett and Daly, 1994; Schulteand Fredholm, 2000).The effect of NECA on noradrenaline release was

attenuated by the A2B-antagonist enprofylline in a con-centration that is about four-times the dissociationconstant of the antagonist-receptor complex (KB ¼7 lM; Feoktistov and Biaggioni, 1995; Robeva et al.,1996; Auchampach et al., 1997) and also by MRS 1754a highly potent (Ki ¼ 2 nM) and selective adenosineA2B receptor-antagonist (8.5- and 310-fold selectiveversus A1 and A2A receptors, respectively; Kim et al.,2000). The adenosine A2A receptor-antagonist SCH58261 (Lindstrom et al., 1996) had no significant effecton the enhancement of noradrenaline release causedNECA. Taken together, the present results confirmthat, in the prostatic portion of rat vas deferens, theadenosine receptors involved on the release-enhancingeffect of NECA are mainly of A2B subtype, as pre-viously suggested (Queiroz et al., 2002).Substances released by activation of postjunctional

receptors can also modulate noradrenaline release. Forexample, in the vas deferens electrical field stimulationand activation of postjunctional a1-adrenoceptorsinduce prostaglandin release, which modulates sym-pathetic transmission through activation of presynapticinhibitory EP3-receptors (Driessen and Starke, 1994) orfacilitatory IP-receptors (Tam et al., 1997). Nitric oxideproduced and released by activation of a1-adrenocep-tors and P2X receptors (Vetri et al., 2000) or adenosineA2B receptors (Olanrewaju and Mustafa, 2000), mayalso facilitate release of sympathetic transmitters(Pastorino et al., 1998). These observations raised thepossibility that adenosine A2B receptor-mediated facili-tation of noradrenaline release could be due to post-junctional adenosine A2B receptor-mediated release ofprostaglandins and/or nitric oxide. This possibility wasexcluded because neither blockade of prostaglandinsynthesis with indomethacin, nor simultaneous block-ade of postjunctional a1-adrenoceptors and P2X recep-tors, changed the release-enhancing effect of NECA.The effect mediated by adenosine A2B receptors doesnot seem to involve nitric oxide since neither l-NAME,up to 300 lM, nor inhibition of the guanylyl cyclase–protein kinase G pathway, changed the effect of NECA.The adenosine uptake inhibitor NBTI facilitates nor-

adrenaline release in the prostatic portion of rat vasdeferens (see Queiroz et al., 2002; and in the presentstudy). The effect of NBTI was attenuated by the ade-nosine A2B receptor-antagonists enprofylline andMRS1754, indicating that it is mainly mediated by ade-nosine A2B receptors. This facilitation was abolished byadenosine deaminase indicating that it may result fromaccumulation of endogenous adenosine at the biophaseof A2B receptors. Endogenous adenosine seems to beformed from the extracellular catabolism of ATP (a co-transmitter in this tissue) because inhibition of ectonu-

cleotidase with ARL 67156 (Crack et al., 1995) or ofAMP catabolism, with the 50-nucleotidase inhibitorAOPCP (Cunha et al., 1992), attenuated the effect ofNBTI. ATP is also released from postjunctional sites,upon activation of a1-adrenoceptores and P2X recep-tors (see Kurz et al., 1994). Under the present experi-mental conditions, ATP released from postjunctionalsites, is the primary source of the adenosine involvedon the A2B receptor-mediated facilitation of noradrena-line release, since blockade of a1-adrenoceptores andP2X receptors, with prazosin and NF023, greatly atte-nuated the release-enhancing effects of NBTI.It is generally accepted that adenosine A2B receptors

are coupled to Gs proteins, because they activate ade-nylyl cyclase in almost every cell in which they areexpressed (Feoktistov and Biaggioni, 1997). In thepresent study, the intracellular signalling pathway acti-vated by these receptors was also investigated, pharma-cologically, because intraneuronal variations of secondmessengers are difficult to measure in this kind of prep-aration. However, this kind of approach has knownlimitations since inhibitors of the intracellular signal-ling pathways, namely kinase inhibitors that act on theATP site, may act as adenosine receptor antagonists(see Schulte and Fredholm, 2002). To avoid this limi-tation, inhibitors of protein kinase A and C with differ-ent mechanisms of action were tested, in order toexclude the interference of eventual non-kinase effectsof the compounds.Facilitation of noradrenaline caused by NECA was

enhanced by inhibition of the type IV phosphodiester-ase and abolished by the protein kinase A inhibitorsH-89 and KT 5720 that act on the catalytic site of theenzyme and by cAMPS-Rp that acts on the regulatorysite (Van Haastert et al., 1984). Like the effect ofNECA, the release-enhancing effect of endogenous ade-nosine revealed by NBTI (under conditions of adeno-sine A1 receptor blockade) was also enhanced byphosphodiesterase inhibition and abolished by proteinkinase A inhibition. Hence, it is likely that adenosineA2B receptors are coupled to a signalling pathway thatleads to protein kinase A activation (probably throughthe Gs–adenylyl cyclase–protein kinase A pathway).Several studies have reported that adenosine A2B

receptors can couple to Gq–phospholipase C pathway(Feoktistov and Biaggioni, 1995; Auchampach et al.,1997; Linden et al., 1999) that leads to activation ofprotein kinase C. In the prostatic portion of rat vasdeferens, protein kinase C may also be involved onthe adenosine A2B receptor-signalling as suggested bythe effects of the protein kinase C inhibitor Ro32-0432, that in a concentration that prevented therelease-enhancing effects of PMA, also attenuated theeffects of NECA and NBTI. The possibility that adeno-sine A2B receptors are coupled to protein kinase C viaactivation of phospholipase C is unlikely because the

G. Queiroz et al. / Neuropharmacology 47 (2004) 216–224 223

phospholipase C inhibitor U-73122 did not change theeffect of NECA under conditions that prevented therelease-enhancing effects of angiotensin II (which acti-vates receptors coupled to the Gq/11–phospholipase C–protein kinase C pathway; Boehm and Kubista, 2002).Protein kinase C activity may also be regulated by

other kinases such as protein kinase A (Borner et al.,1989; Wooten et al., 1996; Huang et al., 2001). There-fore, it is possible that involvement of protein kinase Con the adenosine A2B receptor-mediated effects is sec-ondary to protein kinase A activation. This hypothesisis supported by the following observations: (i) the pro-tein kinase C inhibitor Ro 32-0432 attenuated theenhancement of noradrenaline release caused by acti-vation of either adenylyl cyclase with forskolin or pro-tein kinase A with 8-BrcAMP; (ii) calphostin C, aselective protein kinase C inhibitor (Kobayashi et al.,1989) that acts by a different mechanism from Ro 32-0432 (Wilkinson et al., 1993), attenuated the enhance-ment of noradrenaline release caused by protein kinaseA activation with 8-Br-cAMP and (iii) to our knowl-edge, in the concentrations tested, neither Ro 32-0432nor calphostin C inhibit protein kinase A (Kobayashiet al., 1989; Davis et al., 1992).In conclusion, the present study shows the occur-

rence of an adenosine A2B receptor-mediated facili-tation of noradrenaline release in the prostatic portionof rat vas deferens. The adenosine A2B receptors can beactivated by endogenous adenosine formed by extra-cellular catabolism of nucleotides, mainly released frompostjunctional sites. Facilitation of noradrenalinerelease by adenosine A2B receptors seems to involvecoupling to the adenylyl cyclase–protein kinase A path-way but protein kinase C activated by a protein kinaseA-dependent mechanism may also contribute.

Acknowledgements

Supported by FCT (CEQOFFUP, I&D no. 226/94,POCTI-QCAIII and FEDER), by POCTI/36545/FCB/2000 and Associacao Nacional de Farmacias.We thank M.C. Pereira for the technical assistance

and to Scott Weiss (Vernalis, UK) for the compoundSCH 58261 used in this work.

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