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Cardiovascular Research 2004 63(4):739-746; doi:10.1016/j.cardiores.2004.05.015
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Facilitation of noradrenaline release by activation of adenosine A2A receptors triggers both phospholipase C and adenylate cyclase pathways in rat tail artery

Paula Fresco, Carmen Diniz and Jorge Gonçalves*

Serviço de Farmacologia, CEQOFF/FCT, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, P 4050-047 Porto, Portugal

* Corresponding author. Tel.: +351-222078932; fax: +351-222078969. Email address: jorge.goncalves{at}ff.up.pt

Received 5 November 2003; revised 13 May 2004; accepted 19 May 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The present work is aimed at elucidating the signalling pathway(s) triggered by activation of A2A receptors involved in the facilitation of noradrenaline release in rat tail artery as an attempt to clarify their role in the cardiovascular system. Methods: Electrically evoked (5 Hz, 100 pulses, 1 ms) tritium overflow was evaluated in preparations of rat tail artery, pre-incubated with [3H]-noradrenaline (0.1 µM), in the absence or in the presence of adenosine receptor agonists and antagonists and/or activators and inhibitors of phospholipase C (PLC)-protein kinase C (PKC) and of adenylate cyclase (AC)-cyclic adenosine-3',5'-monophosphate (cAMP)-protein kinase A (PKA) pathways. Results: Activation of A2A receptors by 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 100 nM) enhanced tritium overflow, an effect prevented by the A2A receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3]-1,2,4-triazolo[1,5]pyrimidine (SCH 58261; 20 nM), by the protein kinase A (PKA) inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89; 1 µM), or the PKC inhibitor 2-(8-[(dimethylamino)methyl-6,7,8,9-tetrahydropyrido[1,2]indol-3-yl]-3-(1-methylindol-3-yl)maleimide (Ro 32-0432; 1 µM). The PKC activator phorbol 12-myristate 13-acetate (PMA; 1 µM) and the PKA activator 8-bromo-cAMP (0.5 mM) also enhanced tritium overflow. The effect caused by PMA was blunted both by Ro 32-0432 and by H-89 whereas that caused by 8-bromo-cAMP was only prevented by H-89. Conclusions: In rat tail artery, the A2A receptor-mediated facilitation of noradrenaline release requires activation of both PKC and PKA, and PKA activation seems to occur downstream of PKC activation.

KEYWORDS Adenosine; Receptors; Protein kinase C; Protein kinase A; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Adenosine exerts its biological effects at the cardiovascular system through activation of specific membrane receptors known as A1, A2A, A2B and A3 [1,2] present either peripherally [2] or at the central nervous system [3,4]. Inhibition of release of vasoconstrictor neurotransmitters, through activation of presynaptic A1 receptors [5–7], and vasodilatation, mediated by postsynaptic A2A receptors [8–10], are the most extensively studied and accepted functions mediated by adenosine receptors. However, cardiovascular effects of adenosine may not be limited to these functions. Indeed, in some vascular beds (pulmonary [11] and renal arteries [12]) adenosine exerts a vasoconstriction, mediated by postsynaptic A1 receptors and may facilitate noradrenaline release, through activation of presynaptic A2A receptors (guinea-pig pulmonary [13], rabbit ear [14] and rat tail [15,16] arteries).

The A2A receptor-mediated enhancement of noradrenaline release seems to be the result of a disinhibition rather than a simple facilitation since facilitation of noradrenaline release requires an ongoing activation of release-inhibitory receptors [16]. The signal transduction pathways triggered by activation of A2A receptors are still not completely understood. A2A receptors are generally accepted to couple to the Gs-adenylate cyclase (AC)-protein kinase A (PKA) pathway [17–19], but may also couple to pathways involving G-proteins other than Gs (Golf [20,21], G{alpha}15/16 [22], Gi/o [23]) or even to AC-cyclic adenosine-3',5'-monophosphate (cAMP)-PKA independent signal transduction pathways. In striatal nerve terminals, A2A receptor-mediated modulation of GABA [24] or of acetylcholine release [25] seems to involve protein kinase C (PKC), as well as, in hippocampal nerve terminals, modulation of GABA release [26,27] and, in epididymal portion of vas deferens, facilitation of noradrenaline release [28]. The present work aimed at elucidating the signalling pathway(s) triggered by activation of the A2A receptors involved in the facilitation of noradrenaline release in rat tail artery as an attempt to clarify the role of this presynaptic facilitatory adenosine receptor in the control of cardiovascular functions.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animals and tissue preparation
Adult male Wistar rats (230–390 g; CRIFFA, Barcelona, Spain) were used. Animals were kept under standard laboratory conditions: light/dark cycles of 12/12 h, temperature of 20–22 °C, and free access to water and pellet food. The handling and care of all animals were conducted according to the European Union guidelines for animal research (86/609/EEC; in agreement with the NIH guidelines) and the Portuguese law (Portarias n. 1005/92 and n. 1131/97).

Animals were killed by cervical dislocation and exsanguination. The ventral tail artery was dissected out and cleaned of connective tissue. Four tissue preparations were obtained from each artery.

2.2. Transmitter overflow experiments
Experiments were carried out as previously described [16]. Briefly, tissue preparations were incubated in 2 ml medium containing 0.1 µM [3H]-noradrenaline, for 40 min at 37 °C. Individual preparations were placed in superfusion chambers, between platinum electrodes, and superfused with [3H]-noradrenaline-free medium at a constant rate of 1 ml min–1. Successive 5-min samples of the superfusate were collected from t=55 min onwards (t=0 being the onset of superfusion). At the end of the experiments, tissue preparations were homogenised in 2.5 ml perchloric acid (0.2 M) and tritium content determined in superfusate samples and in tissues, by liquid scintillation spectrometry (LS 6500, Beckman Instruments, Fullerton, USA).

The medium contained (mM): NaCl 118.6, KCl 4.7, CaCl2 2.52, MgSO4 1.23, NaHCO3 25.0, glucose 10.0, ascorbic acid 0.3 and dissodium EDTA 0.031; it was saturated with 95% O2–5% CO2 and kept at 37 °C. Desipramine (400 nM; to inhibit neuronal uptake of noradrenaline) and, in some experiments, yohimbine (1 µM, to block {alpha}2-autoreceptors) were added at the beginning of superfusion and kept throughout.

Three periods of electrical stimulation were applied (Stimulator II, Hugo Sachs Elektronik, March-Hugstetten, Germany; constant current mode; rectangular pulses; 1 ms width; current strength 50 mA; voltage drop between electrodes 18 V cm–1; 100 pulses at 5 Hz). The first period, starting at t=30 min (S0), was not used for determination of tritium outflow. The subsequent periods (S1 and S2) started at t=60 and t=90 min. CGS 21680, PMA, 8-bromo-cAMP and forskolin were added 5 min before S2 and kept until the end of the stimulation period. Adenosine receptor antagonists were added 20 min before S2 and kept until the end of the experiment; 9-CP-Ade, U 73122, U 73343, H-89, KT 5720, chelerythrine and Ro 32-0432 were added 10 min before S2 and kept until the end of the stimulation period. From each animal, no more than two tissue preparations were submitted to identical treatments.

2.3. Data evaluation
Tritium outflow was expressed as the fraction of tritium tissue content at the onset of the respective collection period (fractional rate of outflow; per minute). The electrically evoked overflow of tritium was calculated as the difference between "total tritium outflow during and after stimulation" and the estimated "basal outflow" and expressed as percentage of the tissue tritium content at the time of stimulation. The effects of drugs on basal tritium outflow were estimated by the ratios b2/b1 and expressed as percentage of the mean ratio obtained in the appropriate control; b2 was the fractional rate of outflow in the 5-min period before S2, and b1 the fractional rate of outflow in the 5-min period before S1. The effects of drugs added after S1 on electrically evoked tritium overflow were evaluated as ratios of the overflow elicited by S2 and the overflow elicited by S1 (S2/S1). S2/S1 ratios obtained in individual experiments in which a test compound A was added after S1 were calculated as a percentage of the respective mean ratio in the appropriate control group (solvent instead of A). When the interaction of A, added after S1, and a drug B added after S1, was studied, the "appropriate control" was a group in which B alone was used [29].

2.4. Drugs
The following drugs were used: levo-[ring-2,5,6-3H]-noradrenaline, specific activity 46.8 Ci mmol–1 was from DuPont NEN (Garal, Lisboa, Portugal); 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680), chelerythrine hydrochloride, desipramine hydrochloride, yohimbine hydrochloride, 3,7-dimethyl-1-propargylxanthine (DMPX), 9-cyclopentyladenine (9-CP-Ade), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 7β-deacetyl-7β-({gamma}-N-methylpiperazino)butyrylforskolin (forskolin), 8-bromoadenosine-3',5'-cyclic monophosphate (8-bromo-cAMP), N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89), phorbol 12-myristate 13-acetate hydrochloride (PMA), 2-(8-[(dimethylamino)methyl-6,7,8,9-tetrahydropyrido [1,2]indol-3-yl]-3-(1-methylindol-3-yl)maleimide hydrochloride (Ro 32-0432), (1-[6-([17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl]-2,5-pyrrolidinedione (U 73343) and (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:3',2',1'-kl]pyrrolo[3,4][1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT 5720) were from Sigma Aldrich (Alcobendas, Spain); 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U 73122), 4-(2[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3][1,3,5triazin-5-ylamino]ethyl)phenol (ZM 241385) and 4,4',4'',4'''-[carbonylbis(imino-5,1,3-benzenetryl-bis(carbonylimino))]tetrakis-1,3-benzenedisulfonic acid octasodium salt (NF 449) were from Tocris (Bristol, UK); 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3]-1,2,4-triazolo[1,5]pyrimidine (SCH 58261) was generously offered by Dr. Scott Weiss (Vernalis, UK). All other reagents used were of analytical grade. Unless otherwise stated, stock solutions were made up in dimethylsulphoxide (DMSO) or distilled water and diluted in medium immediately before use (concentration of DMSO in the medium was always lower than 0.01%). All stock solutions were stored as frozen aliquots at –20 °C. Solvents were added to the superfusion medium in parallel control experiments.

2.5. Statistics
Results are expressed as mean±SEM and n denotes the number of tissue preparations. Differences between means were tested for significance using the unpaired Student's t test or one-way analysis of variance (ANOVA) followed by the multiple comparisons Dunnett's t test. A value of P<0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Electrically evoked tritium overflow experiments
In the absence of drugs (except 400 nM desipramine, present in the superfusion medium of all experiments), the fractional rate of tritium outflow from tissue preparations pre-incubated with [3H]-noradrenaline immediately before S1 (b1) was 0.098±0.007% min–1 (n=84); tritium overflow elicited by S1 was 0.407±0.115% (n=84) of the tritium content of the tissue. An ongoing {alpha}2-autoinhibition was reported to occur in these conditions [16]. Basal outflow and electrically evoked tritium overflow remained constant throughout the experiments with b2/b1 and S2/S1 values close to unity (0.91±0.02, n=84; and 0.97±0.03, n=84, respectively). Drugs added after S1 did not change either basal tritium outflow or tritium overflow elicited by S2, except the selective PKA inhibitor H-89 (1 µM) [30] that increased tritium overflow to 161±21% (n=15; P<0.05).

3.2. Pharmacological characterization of the facilitatory adenosine receptors in rat tail artery
CGS 21680 (100 nM), a selective A2A receptor agonist [31], enhanced tritium overflow from postganglionic sympathetic nerve terminals in rat tail artery (Fig. 1), as previously described [15,16]. The CGS 21680-induced enhancement of tritium overflow was prevented by the selective A2 receptor antagonist DMPX (100 nM) [32] but was not influenced by the selective A1 receptor antagonist DPCPX (30 nM; Fig. 1) [33].


Figure 1
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  		Fig. 1 Effects of CGS 21680 on the evoked tritium overflow in rat tail artery in the absence or in the presence of the selective A2A receptor antagonists SCH 58261 (20 nM) and ZM 241385 (20 nM), of the A2 receptor antagonist DMPX (100 nM) or of A1 receptor antagonist DPCPX (30 nM). S1 and S2 are the overflows evoked by trains of 100 pulses at 5 Hz (30-min intervals). CGS 21680 (100 nM) was added 5 min before S2 and kept until the end of the stimulation period. Antagonists were added 20 min before S2 and kept until the end of the experiment. Ordinate, S2/S1 values expressed as percentage of the appropriate average S2/S1 control value. Results are expressed as mean±SEM of 10–14 tissue preparations. Significant differences from the appropriate control (Student's t test), *P<0.05; from the effect of CGS 21680 alone (one-way ANOVA followed by Dunnet's t test), +P<0.05.

 
Confirmation of the A2 receptor subtype involved in the CGS 21680-induced enhancement of tritium overflow facilitation was made using the selective A2A receptor antagonists ZM 241385 [34] (63-fold selectivity for A2A versus A2B [35]) and SCH 58261 (selectivity for A2A versus A2B higher than 8000-fold [35]). As shown in Fig. 1, the facilitatory effect of CGS 21680 (100 nM) was prevented by either ZM 241385 (20 nM) or SCH 58261 (20 nM).

The facilitatory effect of CGS 21680 100 nM was also blocked by NF 449 (10 µM; 95±10%, n=6), accepted to be a selective Gs{alpha} antagonist [36].

3.3 Pharmacological characterization of the transducing systems coupled to facilitatory adenosine A2A receptors in rat tail artery
A2A receptors are recognized to be coupled to the AC-cAMP-PKA transduction pathway. To confirm that activation of this signal transduction pathway leads to an enhancement of tritium overflow in rat tail artery, the effects of the direct activator of AC, forskolin [37], and of the cell permeable cAMP analogue, 8-bromo-cAMP [38], were tested. Both forskolin (1 µM) and 8-bromo-cAMP (0.5 mM) enhanced tritium overflow elicited by electrical stimulation (Fig. 2A). The AC inhibitor, 9-CP-Ade (100 µM) [39, 40], or the PKA inhibitors, H-89 (1 µM) and KT 5720 (1 µM) [41], prevented the enhancement of tritium overflow caused by forskolin and that caused by 8-bromo-cAMP, respectively (Fig. 2A).


Figure 2
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  		Fig. 2 Effects of drugs that interfere with the AC-cAMP-PKA pathway (A) and of the A2A receptor agonist CGS 21680 (B) on the evoked tritium overflow in rat tail artery in the absence or in the presence of the AC inhibitor, 9-CP-Ade (100 µM) or the PKA inhibitors, H-89 (1 µM) and KT 5720 (1 µM). Forskolin (1 µM), 8-bromo-cAMP (0.5 mM) and CGS 21680 (100 nM) were added 5 min before S2. 9-CP-Ade, H-89 and KT 5720 were added 10 min before S2 and were kept until the end of the stimulation period. S1 and S2 are overflows evoked by trains of 100 pulses at 5 Hz (30-min intervals). Ordinate, S2/S1 values expressed as percentage of the corresponding average S2/S1 control value. Results are expressed as mean±SEM of 5–11 tissue preparations. Significant differences from the appropriate control (Student's t test), *P<0.05; from the effect of the activator alone (Student's t test), #P<0.05; from the effect of CGS 21680 alone (one-way ANOVA followed by Dunnet's t test), +P<0.05.

 
Involvement of the AC-cAMP-PKA transduction pathway on the A2A receptor-mediated enhancement of tritium overflow was investigated by testing the effects of AC and PKA inhibition. As observed with forskolin, enhancement of tritium overflow caused by 100 nM CGS 21680 was prevented by 9-CP-Ade (100 µM), H-89 (1 µM) or KT 5720 (1 µM), as depicted in Fig. 2B.

Several pieces of evidence indicate that facilitation of neurotransmitter release mediated by A2A receptors may be dependent on PKC activation [25–28]. To investigate whether PKC activation leads to an enhancement of tritium overflow in rat tail artery, the effect of the cell permeable PKC activator, PMA, was tested. PMA (1 µM) enhanced tritium overflow, which was prevented by the PKC inhibitor, Ro 32-0432 (1 µM; Fig. 3) [42]. Chelerythrine (5 µM), another PKC inhibitor [43], also totally prevented the enhancement of tritium overflow elicited by CGS 21680 (to 92±8%, n=13, P<0.05). A putative involvement of the PLC–PKC transduction pathway on the A2A receptor-mediated enhancement of tritium overflow was supported by the observation that the effects of CGS 21680 (100 nM) were blunted by the PKC inhibitor Ro 32-0432 (1 µM) and by the PLC inhibitor U 73122 (5 µM; Fig. 3) [44] whereas U 73343 (5 µM), the inactive analogue of the PLC inhibitor U 73122 [44], did not influence the enhancement of electrically evoked tritium overflow caused by CGS 21680 (159±19%, n=8, P>0.05). Taken together these data suggest that A2A receptor-mediated enhancement of tritium overflow involves activation of both AC-cAMP-PKA and PLC–PKC signal transduction pathways.


Figure 3
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  		Fig. 3 Influence of the A2A receptor agonist CGS 21680 and of the PKC activator PMA on the evoked tritium overflow in rat tail artery in the absence or in the presence of the PLC inhibitor, U 73122, or the PKC inhibitor, Ro 32-042. CGS 21680 (100 nM) and PMA (1 µM) were added 5 min before S2 and kept until the end of the stimulation period. Ro 32-042 (1 µM) and U 73122 (5 µM) were added 10 min before S2 and kept until the end of the stimulation period. S1 and S2 are overflows evoked by trains of 100 pulses at 5 Hz (30-min intervals). Ordinate, S2/S1 values expressed as percentage of the corresponding average S2/S1 control value. Results are expressed as mean±SEM of 5–11 tissue preparations. Significant differences from the appropriate control (Student's t test), *P<0.05; from the effect of the activator alone (Student's t test), #P<0.05; from the effect of CGS 21680 alone (one-way ANOVA followed by Dunnet's t test), +P<0.05.

 
The possibility that the effect of PKC may be related to inactivation of inhibitory autoreceptors was tested by experiments carried out in the presence of 1 µM yohimbine ({alpha}2-adrenoceptor antagonist), conditions where no autoinhibition is occurring. An activator of PKC (PMA 1 µM), in these conditions, did not facilitate noradrenaline release (97±4%, n=10) in opposition to the facilitatory effect of PMA obtained in the absence of yohimbine (158±15%, n=10).

In order to gather more information on the sequence of events of the signalling cascade triggered by activation of A2A receptors, the effects of PKC inhibition upon PKA activation and of PKA inhibition upon PKC activation were investigated. Fig. 4 shows that the enhancement of evoked tritium overflow caused by 8-bromo-cAMP (0.5 mM) was not changed by the PKC inhibitor Ro 32-0432 (1 µM), whereas enhancement of tritium overflow caused by PMA (1 µM) was blunted by the PKA inhibitors H-89 (1 µM) and KT 5720 (1 µM). In these conditions the PKA inhibitor H-89 (1 µM) also blunted the effects of forskolin (1 µM). These results strongly suggest that PKC activation occurs prior to PKA activation.


Figure 4
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  		Fig. 4 Influence of activators of PKC, PKA or AC on the evoked tritium overflow in rat tail artery in the absence and in the presence of the PKA inhibitors, H-89 and KT 5720, or of the PKC inhibitor, Ro 32-0432. PMA (1 µM), the membrane-permeable cAMP analogue 8-bromo-cAMP (0.5 mM) and the AC activator forskolin (1 µM) were added 5 min before S2 and kept until the end of the stimulation period. H-89 (1 µM), KT 5720 (1 µM) and Ro 32-0432 (1 µM) were added 10 min before S2 and kept until the end of the stimulation period. S1 and S2 are the overflows evoked by trains of 100 pulses at 5 Hz (30-min intervals). Ordinate, S2/S1 values expressed as percentage of the corresponding average S2/S1 control value. Results are expressed as mean±SEM of five to eight tissue preparations. Significant differences from the appropriate control (Student's t test), *P<0.05; from the effect of the activator alone (one-way ANOVA followed by Dunnet's t test), xP<0.05; from the effect of the activator alone (Student's t test), #P<0.05.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study the evoked overflow of tritium elicited by electrical stimulation of tissue preparations of rat tail artery pre-incubated with [3H]-noradrenaline was assumed to reflect action potential-evoked neuronal noradrenaline release, and drug-induced changes in the evoked tritium overflow were assumed to reflect changes in neuronal release of noradrenaline, as in previous studies [16,28].

Noradrenaline release was enhanced by the adenosine A2A receptor agonist CGS 21680, an effect not altered by the A1 receptor antagonist DPCPX but blocked by the A2A receptor antagonists ZM 241385 and SCH 58261. Therefore, it was assumed that the effects of CGS 21680 were due to a selective activation of A2A receptors. Evidence for a facilitatory effect of adenosine mediated by A2A receptors has been previously described in rat tail artery [15,16] and in other vascular tissues: guinea-pig pulmonary artery [13], rabbit ear [14] and saphenous [45] arteries.

cAMP-dependent pathways have been implicated in the facilitation of electrically evoked noradrenaline release from vessels: tail artery [46], mesenteric artery and vein [19], and A2A receptors are generally accepted as being coupled to the Gs protein and linked to the AC-cAMP-PKA pathway [2,18] which is compatible with the blockade of the A2A-receptor mediated facilitation by the suramin analogue NF 449. Our experiments confirm that the AC-cAMP-PKA pathway is indeed involved in the A2A receptor-mediated facilitation of noradrenaline release in rat tail artery. Like the A2A receptor agonist, CGS 21680, the direct AC activator, forskolin, or the cAMP analogue, 8-bromo cAMP (known to activate PKA), facilitated noradrenaline release, an effect inhibited by the selective PKA inhibitor, H-89.

A2A receptors may use other transduction pathways, namely, the PLC–PKC pathway, since the effects mediated by A2A receptors were totally [26] or partially [25,27] prevented by inhibition of PKC. Our experiments also provide evidence supporting the view that PLC–PKC is involved in the A2A receptor-mediated facilitation of noradrenaline release in rat tail artery: facilitation of noradrenaline release caused by either the PKC activator, PMA, or by CGS 21680 was inhibited by the selective PKC inhibitor Ro 32-0432. Furthermore, the effect of CGS 21680 was also inhibited by the PLC inhibitor U 73122 but not by U 73443, an analogue unable to inhibit PLC [44]. Therefore, our findings support the view that facilitation of noradrenaline release mediated by A2A receptors may involve activation of both AC-cAMP-PKA and PLC–PKC pathways in rat tail artery.

Coupling of A2A receptor-mediated activation to both PKA and PKC has been previously described in distinct tissues/cells such as striatal nerve terminals [25], hippocampal nerve terminals [27] or PC12 cells [47]. However, it remained unclear whether PKC activity was directly controlled by A2A receptors or was a consequence of the increased cAMP levels and, therefore, PLC–PKC pathway would be activated downstream AC-cAMP-PKA activation. The present study indicates the opposite, i.e. that AC-cAMP-PKA pathway is activated downstream PLC–PKC activation. The arguments supporting this view are (i) facilitation of noradrenaline release caused by the A2A receptor agonist CGS 21680 was inhibited either by 1 µM H-89, 1 µM Ro-32-4032 or 5 µM U-73122, concentrations regularly used to obtain a selective inhibition of PKA [30], PKC [42] or PLC [44], respectively; (ii) facilitation of noradrenaline release caused by the PKC activator, PMA, was blunted by inhibition of PKA with H-89; and (iii) facilitation of noradrenaline release caused by the PKA activator, 8-bromo-cAMP, was not altered by the PKC inhibitor, Ro 32-0432, in the same concentration that inhibited the facilitatory effect of the PKC activator PMA and of the A2A receptor agonist CGS 21680.

The present findings in rat tail artery extend previous observations in other sympathetic innervated tissues (rat vas deferens) [28] that demonstrated that activation of the adenosine A2A receptors involved in the facilitation of noradrenaline release is mediated through PKC activation. However, in contrast to the present study, involvement of PKA-dependent mechanisms was excluded [28].

We have previously showed that the A2A receptor-mediated facilitation of noradrenaline release in rat tail artery requires, or at least is much amplified by, an ongoing activation of release-inhibitory receptors [16], i.e. the enhancement of noradrenaline release elicited by A2A receptors seems to be the result of a disinhibition. Such an interaction was also shown to occur in rat vas deferens [28] and, in a similar way, for the AT1 and B2 receptor-mediated facilitation of noradrenaline release in mouse atria [48]. The mechanism proposed was that, once activated, the prejunctional facilitatory receptors would stimulate PKC and disrupt the ongoing autoinhibition by inactivating a protein involved in the Gi/o-mediated prejunctional inhibition [48]. The PKC target protein was proposed to be the N-type calcium channels, a phenomenon termed PKC/G-protein cross-talk [49]. Although our findings indicate involvement of PKC on the A2A receptor-mediated facilitation of noradrenaline release, the PKC/G protein cross-talk does not seem to be the mechanism involved in rat tail artery because the facilitatory A2A receptors and the release-inhibitory receptors share the AC-cAMP-PKA pathway and, therefore, may interact at an earlier step of the signalling cascade. In fact, the effect of an activator of PKC was abolished in the absence of Gi/o activation (blockade of {alpha}2-autoreceptors).

An alternative target could be AC itself: the ongoing activation of release-inhibitory receptors would lead to AC inhibition and to a decrease in PKA activity, whereas activation of A2A receptors would suppress AC inhibition, by a PKC-mediated mechanism. Such interaction was described in rat pancreatic islets where the ability of {alpha}2-adrenoceptor agonists to reduce cAMP production was decreased by PKC activation [50].

Adenosine has been viewed as a major modulator of cardiovascular functions by exerting combined pre- (A1 receptor-mediated inhibition of release of vasoconstrictor transmitters) and postsynaptic (A2 receptor-mediated vasodilatation) actions that favour an increase of local blood flow in case of ischaemia preventing, at least partially, its deleterious effects [51]. The findings that adenosine may also be involved in the facilitation (or in the disinhibition) of release of vasoconstrictor transmitters (such as noradrenaline) and the possibility that adenosine may potentiate vasoconstriction through activation of postsynaptic A1 receptors [11,12] question the simplistic view of the role of the adenosine modulation in cardiovascular system. Moreover, the need for further studies in order to understand how all the effects of adenosine are integrated in physiological and pathophysiological processes such as regulation of blood flow under normal conditions and ischaemia is reinforced.

In conclusion, the present study confirms the occurrence of an A2A receptor-mediated facilitation of noradrenaline release in a vascular tissue (rat tail artery). It further presents the original finding of a coupling of A2A receptors to the AC-cAMP-PKA pathway downstream PLC–PKC activation, a mechanism distinct from the transduction pathways described in other preparations (see Fig. 5).


Figure 5
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  		Fig. 5 Putative model for the signalling transduction pathway triggered by adenosine A2A receptor activation. Facilitation of noradrenaline (NA) release by A2A receptor activation seems to trigger the AC-cAMP-PKA pathway activation downstream PLC-PKC activation.

 


   Acknowledgements
 
The authors are deeply grateful for the generous gift of SCH 58261 by Dr. Scott Weiss (Vernalis, UK). The authors also thank Associação Nacional das Farmácias for the scintillation spectrometry equipment and M.C. Pereira for technical assistance.

This work was supported by Fundação para a Ciência e Tecnologia (FCT: I&D no. 226/94, POCTI-QCA III, POCTI/36545/FCB/2000) and FEDER.


   Notes
 
Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Olsson R.A., Pearson J.D. Cardiovascular purinoceptors. Physiol. Rev. (1990) 70:761–845.[Free Full Text]
  2. Ralevic V., Burnstock G. Receptors for purines and pyrimidines. Pharmacol. Rev. (1998) 115:648–652.
  3. Barraco R.A., Clough-Helfman C., Goodwin B.P., Anderson G.F. Evidence for presynaptic adenosine A2a receptors associated with norepinephrine release and their desensitization in the rat nucleus tractus solitarius. J. Neurochem. (1995) 65:1604–1611.[Web of Science][Medline]
  4. Monopoli A., Casati C., Lozza G., Forlani A., Ongini E. Cardiovascular pharmacology of the A2A adenosine receptor antagonist, SCH 58261, in the rat. J. Pharmacol. Exp. Ther. (1998) 285:9–15.[Abstract/Free Full Text]
  5. Vaz-da-Silva M.J., Guimarães S., Moura D. Adenosine and the endothelium-dependent modulation of 3H-noradrenaline release in the canine pulmonary artery. Naunyn-Schmiedeberg's Arch. Pharmacol. (1995) 352:640–645.[Web of Science][Medline]
  6. Rongen G.A., Lenders J.W.M., Lambrou J., et al. Presynaptic inhibition of norepinephrine release from sympathetic nerve endings by endogenous adenosine. Hypertension (1996) 27:933–938.[Abstract/Free Full Text]
  7. Ralevic V. Sympathoinhibition by adenosine A1 receptors, but not P2 receptors, in the hamster mesenteric arterial bed. Eur. J. Pharmacol. (2000) 387:287–293.[CrossRef][Web of Science][Medline]
  8. Hiley C.R., Bottrill F.E., Warnock J., Richardson P.J. Effects of pH on responses to adenosine, CGS 21680, carbachol and nitroprusside in the isolated perfused superior mesenteric arterial bed of the rat. Br. J. Pharmacol. (1995) 116:2641–2646.[Web of Science][Medline]
  9. Flood A., Headrick J.P. Functional characterization of coronary vascular adenosine receptors in the mouse. Br. J. Pharmacol. (2001) 133:1063–1072.[CrossRef][Web of Science][Medline]
  10. Rump L.C., Jabbari T.J., von Kügelgen I., Oberhauser V. Adenosine mediates nitric-oxide-independent renal vasodilatation by activation of A2A receptors. J. Hypertens. (1999) 17:1987–1993.[CrossRef][Web of Science][Medline]
  11. Szentmiklósi J., Ujfalusi A., Cseppentó Á., Nosztray K., Kovács P., Szabó J.Z.S. Adenosine receptors mediate both contractile and relaxant effects of adenosine in main pulmonary artery of guinea pigs. Naunyn-Schmiedeberg's Arch. Pharmacol. (1995) 351:417–425.[Web of Science][Medline]
  12. Hansen P.B., Schnermann J. Vasoconstrictor and vasodilator effects of adenosine in the kidney. Am. J. Physiol. Renal Physiol. (2003) 285:F590–F599.[Abstract/Free Full Text]
  13. Wiklund N.P., Cederqvist B., Gustafsson L.E. Adenosine enhancement of adrenergic neuroeffector transmission in guinea-pig pulmonary artery. Br. J. Pharmacol. (1989) 96:425–433.[Web of Science][Medline]
  14. Maynard K.I., Burnstock G. Evoked noradrenaline release in the rabbit ear artery: enhancement by purines, attenuation by neuropeptide Y and lack of effect of calcitonin gene-related peptide. Br. J. Pharmacol. (1994) 112:123–126.[Web of Science][Medline]
  15. Gonçalves J., Queiroz G. Purinoceptor modulation of noradrenaline release in rat tail artery: tonic modulation mediated by inhibitory P2Y- and facilitatory A2A-purinoceptors. Br. J. Pharmacol. (1996) 117:156–160.[Web of Science][Medline]
  16. Fresco P., Diniz C., Queiroz G., Gonçalves J. Release inhibitory receptor activation favours the A2A-adenosine receptor-mediated facilitation of noradrenaline release in isolated rat tail artery. Br. J. Pharmacol. (2002) 136:230–236.[CrossRef][Web of Science][Medline]
  17. Fredholm B.B., Abbracchio M.P., Burnstock G., et al. Nomenclature and classification of purinoceptors. Pharmacol. Rev. (1994) 46:143–156.[Web of Science][Medline]
  18. Fredholm B.B., Ijzerman A.P., Jacobson K.A., Klotz K.-N., Linden J. Nomenclature and classification of adenosine receptors. Pharmacol. Rev. (2001) 53:527–552.[Abstract/Free Full Text]
  19. Mutafova-Yambolieva V.N., Smyth L., Bobalova J. Involvement of cyclic AMP-mediated pathway in neural release of noradrenaline in canine isolated mesenteric artery and vein. Cardiovasc. Res. (2003) 57:217–224.[Abstract/Free Full Text]
  20. Kull B., Svenningsson P., Fredholm B.B. Adenosine A2A receptors are colocalized with an activate Golf in rat striatum. Mol. Pharmacol. (2000) 58:771–777.[Abstract/Free Full Text]
  21. Corvol J.C., Studler J.M., Schonn J.S., Girault J.A., Hervé D. G{alpha}olf is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum. J. Neurochem. (2001) 76:1585–1588.[CrossRef][Web of Science][Medline]
  22. Offermanns S., Simon M.I. G{alpha}15 and G{alpha}16 couple a wide variety of receptors to phospholipase C. J. Biol. Chem. (1995) 270:15175–15180.[Abstract/Free Full Text]
  23. Cunha R.A., Constantino M.D., Ribeiro J.A. G protein coupling of CGS 21680 binding sites in the rat hippocampus and cortex is different from that of A1 and striatal A2A receptors. Naunyn-Schmiedeberg's Arch. Pharmacol. (1999) 359:295–302.[CrossRef][Web of Science][Medline]
  24. Kirk I.P., Richardson P.J. Inhibition of striatal GABA release by the adenosine A2a receptor is not mediated by increases in cyclic AMP. J. Neurochem. (1995) 64:2801–2809.[Web of Science][Medline]
  25. Gubitz A.K., Widdowson L., Kurokawa M., Kirkpatrick K.A., Richardson P.J. Dual signalling by the adenosine A2a receptor involves activation of both N- and P-type calcium channels by different G proteins and protein kinases in the same striatal nerve terminals. J. Neurochem. (1996) 67:374–381.[Web of Science][Medline]
  26. Cunha R.A., Ribeiro J.A. Adenosine A2A receptor facilitation of synaptic transmission in the CA1 area of the rat hippocampus requires protein kinase C but not protein kinase A activation. Neurosci. Lett. (2000) 289:127–130.[CrossRef][Web of Science][Medline]
  27. Cunha R.A., Ribeiro J.A. Purinergic modulation of [3H]GABA release from rat hippocampal nerve terminals. Neuropharmacology (2000) 39:1156–1167.[CrossRef][Web of Science][Medline]
  28. Queiroz G., Talaia C., Gonçalves J. Adenosine A2A receptor-mediated facilitation of noradrenaline release involves protein kinase C activation and attenuation of presynaptic inhibitory receptor-mediated effects in the rat vas deferens. J. Neurochem. (2003) 85:740–748.[Web of Science][Medline]
  29. von Kügelgen I., Stoffel D., Starke K. P2-purinoceptor-mediated inhibition of noradrenaline release in rat atria. Br. J. Pharmacol. (1995) 115:247–254.[Web of Science][Medline]
  30. Chijiwa T., Mishima A., Hagiwara M., et al. Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolisulfonamide (H-89), of PC12D pheochromocytoma cells. J. Biol. Chem. (1990) 265:5267–5272.[Abstract/Free Full Text]
  31. Lupica C.R., Cass W.A., Zahniser N.R., Dunwiddie T.V. Effects of the selective adenosine A2 receptor agonist CGS 21680 on in vitro electrophysiology, cAMP formation and dopamine release in rat hippocampus and striatum. J. Pharmacol. Exp. Ther. (1990) 252:1134–1141.[Abstract/Free Full Text]
  32. Daly J.W., Padgett W.L., Shamim M.T. Analogues of caffeine and theophylline: effect of structural alterations on affinity at adenosine receptors. J. Med. Chem. (1986) 29:1305–1308.[CrossRef][Web of Science][Medline]
  33. Bruns R.F., Fergus J.H., Badger E.W., et al. Binding of the A1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes. Naunyn-Schmiedeberg's Arch. Pharmacol. (1987) 335:59–63.[CrossRef][Web of Science][Medline]
  34. Poucher S.M., Keddie J.R., Singh P., et al. The in vitro pharmacology of ZM 241385, a potent, non-xanthine, A2a selective adenosine receptor antagonist. Br. J. Pharmacol. (1995) 115:1096–1102.[Web of Science][Medline]
  35. Ongini E., Dionisotti S., Gessi S., Irenius E., Fredholm B.B. Comparison of CGS 15943, ZM 241385 and SCH 58261 as antagonists at human adenosine receptors. Naunyn-Schmiedeberg's Arch. Pharmacol. (1999) 359:7–10.[CrossRef][Web of Science][Medline]
  36. Hohenegger M., Waldhoer M., Beindl W., et al. Gs{alpha}-selective G protein antagonists. Proc. Natl. Acad. Sci. U. S. A. (1998) 95:346–351.[Abstract/Free Full Text]
  37. Seamon K.B., Daly J.W. Forskolin: its biological and chemical properties. Adv. Cycl. Nucleotide Protein Phosporyl. Res. (1986) 20:1–150.
  38. Hei Y.J., MacDonnel K.L., McNeill J.H., Diamond J. Lack of correlation between activation of cyclic AMP-dependent protein kinase and inhibition of contraction of rat vas deferens by cyclic AMP analogs. Mol. Pharmacol. (1991) 39:233–238.[Abstract]
  39. Johnson R.A., Désaubry L., Bianchi G., et al. Isozyme-dependent sensitivity of adenylyl cyclases to P-site-mediated inhibition by adenine nucleosides and nucleoside 3'-polyphosphates. J. Biol. Chem. (1997) 272:8962–8966.[Abstract/Free Full Text]
  40. Ibrahimi A., Abumrad N., Maghareie H., et al. Adenylyl cyclase P-site ligands accelerate differentiation in Ob1771 preadipocytes. Am. J. Physiol. (1999) 276:C487–C496.[Web of Science][Medline]
  41. Cabell L., Audesirk G. Effects of selective inhibition of protein kinase C, cyclic AMP-dependent protein kinase, and Ca2+-calmodulin-dependent protein kinase on neurite development in cultured rat hippocampal neurons. Int. J. Dev. Neurosci. (1993) 11:357–368.[CrossRef][Web of Science][Medline]
  42. Wilkinson S.E., Parker P.J., Nixon J.S. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem. J. (1993) 294:335–337.[Web of Science][Medline]
  43. Herbert J.M., Augerau J.M., Gleye J., Maffrand J.P. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. (1990) 172:993–999.[CrossRef][Web of Science][Medline]
  44. Smith R.J., Sam L.M., Justen J.M., Bundy G.L., Bala G.A., Bleasdale J.E. Receptor coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitory of phospholipase C-dependent processes on cell responsiveness. J. Pharmacol. Exp. Ther. (1990) 253:688–697.[Abstract/Free Full Text]
  45. Todorov L.D., Bjur R.A., Westfall D.P. Inhibitory and facilitatory effects of purines on transmitter release from sympathetic nerves. J. Pharmacol. Exp. Ther. (1994) 268:985–989.[Abstract/Free Full Text]
  46. Ouedraogo S., Stoclet J.C., Bucher B. Effects of cyclic AMP and analogues on neurogenic transmission in the rat tail artery. Br. J. Pharmacol. (1994) 111:625–631.[Web of Science][Medline]
  47. Huang N.-K., Lin Y.-W., Huang C.-L., Messing R.O., Chern Y. Activation of protein kinase A and atypical protein kinase C by A2A adenosine receptors antagonizes apoptosis due to serum deprivation in PC12 cells. J. Biol. Chem. (2001) 276:13838–13846.[Abstract/Free Full Text]
  48. Cox S.L., Shelb V., Trendelenburg A.U., Starke K. Enhancement of noradrenaline release by angiotensin II and bradykinin in mouse atria: evidence for cross-talk between Gq/11 protein- and Gi/o protein-coupled receptors. Br. J. Pharmacol. (2000) 129:1095–1102.[CrossRef][Web of Science][Medline]
  49. Cooper C.B., Arnot M.I., Feng Z.-P., Jarvis S.E., Hamid J., Zamponi G.W. Cross-talk between G-protein and protein kinase C modulation of N-type calcium channels is dependent on the G-protein β subunit isoform. J. Biol. Chem. (2000) 275:40777–40781.[Abstract/Free Full Text]
  50. El-Mansoury A.M., Morgan N.G. Activation of protein kinase C modulates {alpha}2-adrenergic signalling in rat pancreatic islets. Cell Signal. (1998) 10:637–643.[CrossRef][Web of Science][Medline]
  51. Rongen G.A., Floras J.S., Lenders J.W.M., Thien T., Smits P. Cardiovascular pharmacology of purines. Clin. Sci. (1997) 92:13–24.[Web of Science][Medline]

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