1 The P1-purinoceptors mediating relaxation of the rat duodenum and inhibition of contraction of the rat urinary bladder were characterized by use of adenosine and its analogues 5'-N-ethylcarboxamidoadenosine (NECA), N6-cyclopentyladenosine (CPA) and 2-p-((carboxyethyl)phenethylamino) -5'-carboxamidoadenosine (CGS 21680), as well as the Al-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). The stable analogue of adenosine 5'-triphosphate (ATP), adenylyl 5'-(fi,y-methylene)diphosphonate (AMPPCP), was also used as previous work had indicated that it has a direct action on some P1 receptors in addition to its P2-purinoceptor activity. 2 In the rat duodenum, the order of potency of the adenosine agonists was NECA > CPA > AMPPCP = adenosine > CGS 21680, and DPCPX antagonized CPA and AMPPCP at a concentration of 1 nm whereas equivalent antagonism of NECA and adenosine required a concentration of 1 ym. This suggests the presence of a mixture of A1 and A2 receptors in this tissue, with CPA and AMPPCP acting on the A1 and NECA and adenosine acting on the A2 receptors. 3 In the rat bladder, the order of potency of the adenosine agonists for inhibition of carbachol-induced contractions was NECA > adenosine > CPA = CGS 21680, and a concentration of DPCPX of 1 $M was required to antagonize responses to NECA and adenosine. This suggests the presence of A2 receptors in this tissue. ATP and AMPPCP each caused contractions which were not enhanced by DPCPX (1 M) which suggests that in this tissue AMPPCP was acting only via P2 receptors and had no P1 agonist activity. That AMPPCP was active on the A1 receptors in the duodenum but inactive on the A2 receptors in the bladder implies that it has selectivity for the A1 subtype.4 That CGS 21680, which has been reported to bind selectively to the high affinity A2a subclass of A2 receptors, had a very low potency on the A2 receptors in the duodenum and in the bladder suggests that these receptors are of the low affinity A2b subclass.

1 P1-purinoceptors mediating relaxation of the rat duodenum longitudinal muscle and contraction of the rat duodenum muscularis mucosae were characterized by the use of adenosine and its analogues, 5'-N-ethylcarboxamidoadenosine (NECA), N6-cyclopentyl-adenosine (CPA), N6-(phenylisopropyl)adenosine (R-PIA), 2-chloroadenosine (2-CADO) and 2-p-((carboxyethyl)phenethylamino)-5'-carboxamidoadenosine (CGS21680), as well as the P1-purinoceptor antagonist 8-phenyltheophylline (8-PT) and the Alselective antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX Schild analysis for DPCPX versus NECA, however, gave a slope of 0.674 suggesting that NECA was acting on both Al and A2 receptors. CGS21680, a selective A2a agonist, was much less potent than adenosine suggesting that the A2 receptors are of the A2b subtype. 3 In the rat duodenum muscularis mucosae, the order of potency of the adenosine agonists was NECA >R-PIA=CPA>2-CADO>adenosine, and DPCPX antagonized responses to CPA and NECA at a concentration of 1 ,UM. CGS21680, at a concentration of 10 gM, had no effect on this tissue. This suggests the presence of A2 receptors in this tissue and that they are of the A2b subtype. 4 These results are in agreement with previous studies in the whole duodenum showing the presence of A1 and A2b receptors causing relaxation, and this shows that the longitudinal muscle dominates the response of the whole tissue. In addition, a contractile A2b receptor has been revealed on the muscularis mucosae, the first time this subtype has been reported to elicit an excitatory response in a smooth muscle preparation.

1The ontogeny of responses to purines and analogues of smooth muscle preparations was studied in rat duodenum and rat urinary bladder. 2 Responses to adenosine and to adenosine 5'-triphosphate (ATP) mediated by P1-and P2-purinoceptors respectively were present as early as postnatal day 2, the earliest day studied.3 In rat bladder, adenosine was inhibitory and ATP and adenosine 5'-(fy-methylene) triphosphonate (AMP-PCP) were excitatory, acting on the P2X subtype of P2-purinoceptors. Adenosine was more potent in the neonate than in the adult, while the potency of the nucleotides initially increased with age but then declined, being highest between postnatal days 10 and 25. 4 In rat duodenum also, adenosine was inhibitory, its potency being less than the adult before day 15. 5 ATP at low concentrations was inhibitory in rat duodenum at every age studied and its potency increased with age, but higher concentrations of ATP (3 um and above) were excitatory until day 15. Both relaxations and contractions were mediated by the P2Y subtype of P2-purinoceptors. These ATP-induced contractions were not inhibited by indomethacin (25 pM) or by tetrodotoxin (1 pM) and are therefore not due to prostaglandin synthesis or to ATP-induced release of transmitter substances from nerves. 6 These results show that responses to adenosine and to adenine nucleotides are present from birth and vary with age, and that the changes seen indicate a differential development for P1-, P2X-and P2y-purinoceptors.

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1 Previous results obtained with the rat colon muscularis mucosae, which contracts in response to adenosine and adenosine 5'-triphosphate (ATP), had suggested that adenylyl 5'4fy-methylene)diphosphonate (AMPPCP), a stable ATP analogue, acted on Pl-purinoceptors rather than, as expected, on P2-purinoceptors. This possibility has been examined in two tissues in which adenosine and ATP both cause relaxation, the guinea-pig taenia caeci and the rat duodenum. 2 ATP, 2-methylthio-ATP (2-MeSATP), AMPPCP, adenosine 5'-(a4,-methylene)triphosphonate (AMPCPP) and adenosine each relaxed the taenia caeci and the duodenum, and the order of potency of the nucleotides in each tissue was 2-MeSATP > ATP > AMPCPP > AMPPCP, indicating that these effects were mediated by P2y-purinoceptors. 3 The P, antagonist 8-(p-sulphophenyl)theophyuine (8-SPT) (100pM) did not affect the responses to ATP, 2-MeSATP or AMPCPP in either tissue, but inhibited the responses of adenosine and of AMPPCP in both tissues. In the duodenum a lower concentration of 8-SPT caused a parallel shift to the right of the concentration-response curve to adenosine and to AMPPCP but to different extents, with AMPPCP being inhibited more powerfully than adenosine. A dose-ratio of around 5 was observed for adenosine and AMPPCP at concentrations of 8-SPT of 20gM and 2pM respectively, but Schild analysis resulted in plots with slopes greater than unity. In the taenia caeci, however, 8-SPT inhibited adenosine more powerfully than AMPPCP, and a range of concentrations (10-20gM) only caused a two fold shift in the concentration-response curve for AMPPCP, although the concentration-response curve to adenosine was shifted in a concentration-dependent manner and Schild analysis gave a pA2 value of 5.13 with a slope of 0.90. 4 As has been shown in other tissues, including the guinea-pig taenia caeci, ATP (100pMm) was rapidly dephosphorylated by enzymes present in the rat duodenum, with less than 10% remaining after 20min incubation, whereas AMPPCP (100pMm) was resistant to degradation, with greater than 90% remaining at the same time point. 5 AMPPCP therefore has pronounced but variable agonist actions on P,-purinoceptors, and appears to act entirely via these receptors on the rat duodenum although in the guinea-pig taenia caeci this action is less important and it acts largely via P2y-purinoceptors. These Pl-purinoceptor effects of AMPPCP are direct and are not due to its degradation to adenosine.