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In isolated mouse detrusor ATP has been shown to elicit contraction (via P2X receptors) and relaxation in carbachol or pre-contracted preparations (Boland et al., 1993) The order o f potency for relaxation was 2meSATP> ATP> p,y-me-L-ATP and was not antagonised by S-(sulphophenyl) theophylline (8-SPT), indicative of a P2Y type

receptor.

ATP mediated relaxation of rat detmsor following desensitisation o f P2X receptors using a,p-me-ATP has been described (Bolego et a l, 1995a) Furthermore the G- protein activator, guanosine 5 ' -0 -3 -thiotriphosphate (GTPyS) significantly potentiated and the G-protein blocker, guanosine 5 ' -O-2-thiodiphosphate (GDPyS) abolished the relaxant response to ATP and 2-MeS-ATP. Evidence for P2Y receptors that mediate

excitatory responses in addition to P2X responses have also been observed in the rat bladder. UTP contracts the rat bladder, presumably either via UTP sensitive P2 receptors, P2Y2 or P2Y4 (Bolego et a l, 1995b). Evidence for a population of ADPpS-

sensitive receptors that mediate contraction via release o f calcium from intracellular stores and therefore likely mediated via a P2Y receptor, has also been presented (Naramatsu e? fl/., 1997).

In the marmoset urinary bladder evidence for multiple postjunctional P2 receptor subtypes has been reported (McMurray et al, 1998). A biphasic response to ATP in which the contractile component was abolished by desensitisation with 10 pM a,P-me- ATP revealed a clear relaxation phase. The potency order for the relaxation phase was ATP = 2-MeSATP > ADP » a,P-me-ATP. Furthermore the relaxant response was unaffected by the PI receptor antagonist 8-SPT or the nitric oxide synthase inhibitor, L-

NOARG, but was blocked by Cibacron blue which is regarded as a P2Y antagonist, at least on native receptors subtypes. The relaxant response was also blocked by the G- protein inactivator GDPpS and was antagonised by A^tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of cyclic AMP-dependent protein kinase A (PKA) consistent with the presence o f a P2Y G-protein coupled receptor that acts via stimulation of adenylate cyclase.

L 6 .0 Th e Ph a r m a c o l o g ya n d Mo l e c u l a r Bi o l o g y o f P 1 -r e c e p t o r s

Following release, ATP is rapidly degraded in a stepwise manner by extracellular ATPases to ADP, AMP and finally adenosine. These three degradation products are all pharmacophores at purinoceptors. ADP and AMP are weakly active at P2 and PI receptors respectively and since they are both rapidly degraded to adenosine it seems unlikely that they have a major physiological effect. Adenosine, however is the endogenous ligand for the PI-purinoceptors, and inhibits or relaxes agonist and nerve- mediated contractions in detrusor smooth muscle preparations. The following section presents an overview o f the current understanding of PI (adenosine) receptors, evidence for the existence o f PI receptors in the bladder and the reported actions of adenosine and its structural analogues in detrusor smooth muscle.

Adenosine (PI) receptors were initially subdivided on the basis of whether they inhibit or stimulate adenylate cyclase, and therefore, decrease or increase intracellular cyclic AMP (cAMP) levels. Those receptors mediating the inhibition of adenylate cyclase were classed Ai receptors, while those mediating stimulation o f adenylate cyclase were classed A% receptors (Van Calker et a l, 1979).

It has since been discovered that adenosine can bind to numerous other effector systems, such as channels, Ca^^ channels, phospholipase C (PLC), phospholipase A (PLA) and guanylate cyclase (see Fredholm et a l, 1994). The nomenclature system has therefore been amended so that the A1/A2 classification no longer implies modulation of

The A2 receptor has now been further subdivided into Aia and A2B receptors (Daly et

al., 1983; Bruns et al, 1986; Bruns et al, 1987). On the basis of cloning and functional characterisation studies in the rat striatum a third adenosine receptor, the Agreceptor has been identified Zhou et a l (1992). Ai, A2A and A2B receptors have now also been

cloned, confirming the existence of the two distinct PI receptor subtypes (see Collis & Hourani, 1993; Fredholm et al, 1994; Olah & Stiles, 1995).

Adenosine has been chemically modified in a number of ways which has produced a range of analogues which have been used to classify PI receptors in functional studies (Jacobson, 1990). C2-substitution o f the adenine moiety results in analogues such as 2- chloroadenosine (2-CADO), 2-phenylaminoadenosine (CVl 808) and 2-p- ((carboxyethyl)phenylethylamino)-5 ’ -N-ethylcarboxyamidoadenosine (CGS 21680); N^-substitutions of the adenine moiety gives analogues such as N^- (phenylisopropyl)adenosine (PI A), N^-cyclohexyladenosine (CHA) and N^- cyclopentyladenosine (CPA) which are potent agonists at Ai receptors. Structural alterations at the ribose moiety are rarely tolerated by PI receptors, however 5’-N- ethylcarboxyamidoadenosine (NECA) has been used as a non-selective PI receptor agonist (Bruns et a l, 1986). The potency o f adenosine can also be influenced by uptake and deamination, and these ‘termination mechanisms’ are discussed in detail later. There is no evidence, however, for the uptake or deamination o f analogues such as CPA or NECA. For example, in the guinea-pig trachea (Brown & Collis, 1982) and the guinea-pig taenia caeci (Prentice et a l, 1995) uptake blockers do not enhance responses to NECA, and NECA is known not to be deaminated (Raberger et al, 1977). CPA is

also not deaminated and the effects of N^-substituted analogues are also not enhanced by uptake blockers (Brown & Collis, I 982).