IJUP08 - Universidade do Porto

Adenosine regulates its own release from myenteric neurons via
A2A receptors coupled to adenylate cyclase / cAMP pathway
J. Duarte, R. Dias, M. Duarte-Araújo, T. Magalhães-Cardoso & P. Correia-de-Sá
Laboratório de Farmacologia e Neurobiologia, Unidade Multidisciplinar de Investigação
Biomédica (UMIB), Instituto de Ciências Biomédicas Abel Salazar - Universidade do Porto
(ICBAS-UP), Portugal.
Adenosine is a ubiquitous component of cells, which acts as a homeostatic regulator in the nervous
system [1]. In the gastrointestinal tract, besides the inhibitory effect of adenosine mediated by
neuronal A1 receptors [2], endogenous adenosine plays a predominant facilitatory action on ACh
release from myenteric neurons of the rat ileum, via the activation of prejunctional facilitatory A2A
receptors [3]. Myenteric neurons are the main source of adenosine released per se in response to
electrical stimulation [4]; parallel formation of adenosine from the hydrolysis of released ATP (via
ectonucleotidases pathway) may also occur during prolonged periods of stimulation [3]. It remains,
however, to be elucidated whether adenosine plays a role regulating its own extracellular levels.
The experiments were performed at 37ºC on longitudinal muscle-myenteric plexus (LM-MP) of rat
ileum, superfused with gassed (95% O2 + 5% CO2) Tyrode´s solution. LM-MP preparations were
stimulated (10 Hz, 3000 pulses, 1 ms, 100 V) in the presence of test drugs. Samples were collected
before and after stimulus application and retained for nucleoside analysis by HPLC.
Electrical stimulation of the LM-MP increased (74±6%, n=6) the outflow of adenosine (ADO)
above control levels. Blockade of action potentials generation with tetrodotoxin (1 µM) or
omission of Ca 2+ (plus EGTA, 1 mM) in the buffer essentially abolished the release of ADO.
Blockade of smooth muscle contractions by inhibiting Ca 2+ influx through L-type channels, with
nifedipine (1 µM), was devoid of effect on ADO outflow. Application of the adenosine kinase
inhibitor, 5’-iodotubericidin (ITU, 10 µM), led to an increase in stimulation-induced ADO
accumulation by 51±6%, (n=8). The nucleoside transport inhibitor, dipyridamole (DIPY, 0.5 µM),
was more effective in inhibiting (-59±9%, n=4) ADO accumulation in the stimulated LM-MP as
compared with the ecto-5’-nucleotidase inhibitor, concanavalin A (Con A, 0.1 mg/ml, -27±1%,
n=5). Activation of A2A receptors with CGS 21680C (3 nM) increased stimulation-induced ADO
outflow by 129±10% (n=5). The facilitatory effect of CGS 21680C (3 nM) was prevented by the
selective A2A antagonist, ZM 241385 (50 nM) and by DIPY (0.5 µM), while Con A (0.1 mg/ml)
was unable to change the effect of CGS 21680C (3 nM). The adenylate cyclase activator, forskolin
(FSK, 3 µM), mimicked (158±14%, n=4) the facilitatory effect of CGS 21680C (3 nM) on the
release of the nucleoside; the effect of FSK (3 µM) was also attenuated by DIPY (0.5 µM).
These results indicate that although ADO may be formed by the extracellular catabolism of
released adenine nucleotides via the ectonucleotidase pathway, this might not represent the major
source of endogenous ADO in the rat myenteric plexus. Normally, Ca 2+ -dependence release of
neuromediators indicates a vesicular storage and exocytotic release. Since there is no evidence for
vesicular storage and release of ADO, the intracellular Ca 2+ requirement for ADO release may
reflect actions on intracellular processes that modify metabolic production of the nucleoside or
promote its efflux through equilibrative transporters. Data showed that ADO mediates a positive
feedback mechanism regulating its own transport from myenteric motoneurons into the
extracellular space via the activation of A2A receptors coupled to adenylate cyclase / cyclic AMP
pathway.
[1] Cunha (2001) Neurochem. Int., 38, 107-125; [2] De Man et al. (2003) Br. J. Pharmacol. 139, 172-184; [3]
Duarte-Araújo et al. (2004) Br. J.Pharmacol. 141, 925-934; [4] Correia-de-Sá et al. (2006) Auton Neurosci.
126-127, 211-224.
This work was supported by FCT (FEDER funding, POCTI/45549/FCB/2002) and PTDC/74462/CVT/2006
projects).
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