Abstract
1) The pharmacological actions of adenine and uracil nucleotides are reviewed. The current knowledge of purinoceptor subtypes involved in mediating the actions of these nucleotides and related analogues are summarised and their relevance in smooth muscle preparations discussed. 2) The receptor(s) mediating contraction at which adenosine 5'-triphosphate (ATP) and related analogues, and uridine 5'-triphosphate (UTP) act was studied in the rat colon muscularis mucosae. 2a) ATP and adenosine have been shown to contract the rat colon muscularis mucosae, and the receptors at which they act have been classified as P2Y- and A1-purinoceptors respectively. UTP also contracts this tissue, and desensitisation and antagonism by suramin was used to investigate the receptors by which it acts, in the light of suggestions that specific pyrimidinoceptors may exist for UTP, or that P2U-purinoceptors exist which are responsive to both ATP and UTP but not to some ATP analogues such as 2-methylthioadenosine 5-triphosphate (2-MeSATP). 2b) ATP, UTP and adenosine each contracted the rat colon muscularis mucosae in a concentration-dependent manner over the concentration range 0.3-300μM, although maximal responses to ATP and UTP were not obtained. ATP was approximately four times as potent as UTP and approximately equipotent with adenosine although the maximal response to adenosine appeared to be less than to ATP or UTP. 2c) Desensitisation of the tissue with ATP (200μM) given immediately before each concentration of the agonists reduced subsequent contractions induced by ATP itself and also to UTP, but did not significantly reduce contractions induced by adenosine. Desensitisation of the tissue with UTP (200μM) also reduced contractions induced by ATP and UTP but not by adenosine, whereas desensitisation with adenosine (200uM) reduced contractions induced by adenosine itself but not by ATP or UTP. 2d) Desensitisation of the tissue with 2-MeSATP (200μM), which is a more potent agonist than ATP at P2y-purinoceptors, greatly reduced the responses to ATP and to UTP, but had no effect on responses induced by adenosine. Attempts to desensitize the tissue with adenosine 5'-(a,B-methylene)triphosphonate (AMPCPP), which is a more potent agonist than ATP at P2x-purinoceptors but is less potent at P2Y-purinoceptors, were unsuccessful. 2e) The non-selective P2 antagonist suramin (100μM), inhibited contractions induced by both ATP and UTP to a similar extent, shifting their concentration-response curves to the right. 2f) ATP and UTP induced contractions were observed in neonatal (2 and 5 day old) and adult rat colon muscularis mucosae, and at each of these ages ATP and UTP showed a similar potency relative to each other. 2g) Equal antagonism of ATP and UTP by suramin suggests that ATP and UTP are acting at the same receptor in the rat colon muscularis mucosae. Furthermore, these results show that cross-desensitisation to ATP and UTP occurred and was specific for these agonists rather than being due to a general decrease in the ability of the muscle to contract. This further implies that ATP and UTP act at the same receptor, which does not support the existence of specific pyrimidinoceptors but which could be taken as evidence for the existence of a P2u-purinoceptor on this tissue. However, the ability of 2-MeSATP, which is inactive at the P2u-purinoceptor, also selectively to desensitize this receptor indicates instead that ATP and UTP are both acting at a purinoceptor of the P2y subtype in this tissue. An alternative explanation of this data could be heterologous desensitisation of both P2y- and P2u-purinoceptors. 3) The receptors at which ATP and related analogues, and uridine 5'-triphosphate (UTP) act was studied in the rat whole duodenum. 3a) Previous studies have shown that the rat duodenum relaxes to adenosine and ATP via P1 and P2Y-purinoceptors respectively, but in preliminary studies UTP was found to contract this tissue. Suramin and a number of nucleotides were therefore used to investigate this response further. 3b) 2-MeSATP, ATP, UTP, adenosine 5'-O-(3-thiotriphosphate) (ATP-γ-S), adenosine 5-diphosphate (ADP), uridine 5-diphosphate (UDP) and guanosine 5'-triphosphate (GTP) each relaxed the duodenum, with an agonist potency order of 2-MeSATP>ATP=ADP>AMPCPP=ATP-γ-S»GTP»UTP=UDP, consistent with the presence of a P2y-purinoceptor mediating relaxation. 3c) ATP-γ-S, UTP and UDP each contracted the duodenum with an agonist potency order of ATP-y-S>UTP>UDP, although maximal responses to these agonists were not obtained at a concentration of 267μM (ATP-γ-S) and 300μM (UTP and UDP). No contractions were observed with any of the other agonists at concentrations of up to 300uM. 3d) Indomethacin (25μM) did not inhibit the contractions induced by UTP, indicating that they were not mediated via production of prostaglandins. 3e) Suramin (100μM and 1mM) inhibited relaxations induced by ATP, shifting the concentration-response curve to the right, with the maximal response to ATP being decreased by the higher concentration of suramin (1mM). Suramin (1mM) inhibited relaxations induced by ATP-γ-S, shifting the concentration-response curve to the right, and completely abolished contractions induced by ATP-γ-S. In contrast, suramin (100μM and 1mM) had no effect on contractions induced by UTP. Contractions induced by UTP were, however, less sustained in the presence of suramin, which also affected the basal tone of some tissues when precontracted with carbachol (0.1μM). In the presence of suramin (1mM), no contractions to ATP were observed. 3f) These results confirm that in the rat duodenum there is a P2Y-purinoceptor that mediates relaxation in response to a number of purine nucleotides, and at which the pyrimidine nucleotides UTP and UDP are almost inactive. There are also receptors at which UTP and ATP-γ-S act to cause contraction. Suramin discriminates between the contractile effects of these two agonists, which may indicate the presence of a suramin-insensitive pyrimidinoceptor as well as a suramin-sensitive receptor for ATP-γ-S. An alternative explanation is that the differential effects of suramin are via its action as an antagonist in addition to its action as an ectonucleotidase inhibitor.