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Abstract:The aim of the present study was to assess the acute motility effects and desensitizing activity of the stable ATP analogue and P 2X purinoceptor agonist a,b-methylene ATP (a,b-meATP) and the effect of a,b-meATP desensitization on nerve-mediated cholinergic responses in the guinea-pig ileum in vitro. It was confirmed that a,b-meATP (1-30 mM) causes neurally-mediated, cholinergic (tetrodotoxin-and atropine-sensitive) longitudinal contractions. These responses were not influenced by the ganglionic blocking drug hexamethonium (50 mM), or a combination of the adrenergic neurone blocking drug guanethidine (3 mM), the opioid receptor antagonist naloxone (0.5 mM) and the nitric oxide synthase inhibitor N Gnitro-L-arginine (L-NOARG; 100 mM), but were strongly reduced or abolished by the P 2 purinoceptor antagonist PPADS (30 mM) or by tachyphylaxis evoked by 10 mM a,b-meATP. The contractile effect of a,b-meATP (3 mM) was moderately inhibited by 10 mM and strongly suppressed by 30 mM of NF 279, an antagonist predominantly affecting P2X 1 purinoceptors, but left uninfluenced by the P2X 5,7 receptor antagonist Brilliant blue G. No relaxant effect of a,b-meATP was detected in the concentration range of 1-30 mM. Tachyphylaxis to a,b-meATP (1-10 mM) caused a moderate inhibition of the cholinergic (atropine-sensitive) contractile response of the ileum to electrical field stimulation (5 Hz for 5 sec.). This reduction was unaltered in the presence of guanethidine, naloxone and L-NOARG. Responses to nicotine (1 or 2 mM) were not reduced by a,b-meATP tachyphylaxis. It is suggested that a,b-meATP-sensitive P 2X purinoceptors are involved in the prejunctional modulation of cholinergic neurotransmission between the myenteric plexus and longitudinal smooth muscle in the guinea-pig small intestine.a,b-Methylene ATP (a,b-meATP) is an analogue of adenosine-5'-triphoshate (ATP) with a greater metabolic stability than the parent compound. Though its spectrum of agonist activity at various purinoceptors is somewhat different from that of ATP itself (Lambrecht 2000 for review) a,b-meATP is one of the traditional drugs used for studying purinergic mechanisms (Ralevic & Burnstock 1998;Lambrecht 2000). In addition to stimulating some subtypes of P 2X purinoceptors, a,b-meATP can induce powerful tachyphylaxis on these receptors, which has been exploited in the identification of endogenous ATP as neurotransmitter, e.g. in the urinary bladder (Ralevic & Burnstock 1998).Our recent work indicated that the P 2 purinoceptor antagonist PPADS and suramin, two P 2 purinoceptor antagonists of limited subtype selectivity reduced cholinergic longitudinal contractions of the guinea-pig small intestine in vitro (Barthó et al. 1997). Several lines of evidence suggest that this effect reflect an involvement of PPADS-and suramin-sensitive P 2 purinoceptors in the mediation/positive modulation of cholinergic contractions, rather than a nonspecific action (Barthó et al. 1997). In the present work we attempt to approach this problem with a,b-meATP tachyphyl...

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Adenosine 5ø-triphosphate (ATP) may be an important neurotransmitter in the gastrointestinal tract. The present study examined the motor effects of exogenous ATP on longitudinally-oriented preparations of the guinea-pig isolated ileum and the influence of drugs on the ATP-induced responses. High micromolar concentrations of ATP caused two types of contraction, a phasic, cholinergic response and a tonic, tetrodotoxin-resistant contraction. The phasic contraction was reduced by hexamethonium (5¿10 ª5 M), but left uninfluenced by capsaicin tachyphylaxis or tachyphylaxis to a,bmethylene ATP. The tonic response was resistant to atropine, hexamethonium, capsaicin, w-conotoxin GVIA, or pretreatment with a,b-methylene ATP. Both types of ATP-induced contraction were diminished or abolished by the P 2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2ø,4ø-disulphonic acid (PPADS, 3¿10ª6 and 3¿10 ª5 M, respectively). In the precontracted, atropine-treated ileum ATP (10 ª6 -10 ª4 M) caused guanethidine-resistant relaxation. This response was not influenced by tetrodotoxin, w-conotoxin GVIA, or N G -nitro-L-arginine, but was abolished by apamin (10 ª7 M), and inhibited by PPADS (3¿10 ª5 M) or reactive blue 2 (10 ª5 M), in a surmountable manner. A high degree of tachyphylaxis was observed with the relaxant effect of ATP (10 ª5 -10 ª4 M). A high concentration (3¿10 ª4 M) of PPADS failed to influence ileum contractions to exogenous acetylcholine or histamine. It is concluded that, in addition to its direct contractile action in the guinea-pig ileum, ATP can activate (partly preganglionic) cholinergic neurones, an effect whose mechanism is largely different from that of a,b-methylene ATP. ATP also causes relaxation by a direct, probably P 2Y -receptormediated effect on the smooth muscle. All motor effects of ATP are inhibited by the antagonist PPADS.

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