Christine M. Brown

EFFECTS OF ADENOSINE 5′‐TRIPHOSPHATE (ATP) AND β‐γ‐METHYLENE ATP ON THE RAT URINARY BLADDER

1 High concentrations of adenosine 5′‐triphosphate (ATP, 100 to 1000 μm) were required to cause contraction of the rat urinary bladder, while adenosine and adenosine 5′‐monophosphate (AMP, 1 to 50/im) produced relaxation. 2 One hundred fold lower concentrations of β‐γ‐methylene ATP, which is resistant to degradation to AMP and adenosine, caused dose‐dependent, phasic contractions which mimicked atropine‐resistant responses to nerve stimulation. 3 Adenosine and AMP caused dose‐dependent inhibition of carbachol‐induced contractions; theophylline competitively antagonized this inhibition but not the contractile responses to β‐γ‐methylene ATP, ATP or atropine‐resistant nerve stimulation. 4 These results suggest that the insensitivity of the rat bladder to ATP is due to its rapid degradation to AMP and adenosine and support the hypothesis that the bladder receives a purinergic excitatory innervation.

An Introduction to Purinergic Receptors

Adenosine 5′-triphosphate (ATP) was primarily considered as an intracellular energy source for many years. However, during the past fifty years purine nucleotides and nucleosides have been shown to have potent extracellular actions on excitable membranes which may be involved in physiological regulatory processes (Berne, 1963; Burnstock, 1972, 1975, 1979; Baer and Drummond, 1979). While knowledge of purinergic receptors is still in its infancy compared to the voluminous work on the classical adrenergic and cholinergic receptors, much progress is being made on their pharmacological characterisation, the ionic basis of their actions and their biochemical identity. The historical aspects of the purinergic receptor field will be briefly summarised below, while details of their distribution, physiological roles and chemistry will be dealt with in the chapters which follow.

α2-Adrenoceptors: more subtypes but fewer functional differences

The proliferation of receptor subtypes based on differences in amino acid sequence does not necessarily coincide with functional differences. The number of alpha 2-adrenoceptor subtypes, as defined by ligand-binding and molecular studies, has been increasing in the past few years, which suggests the possibility of distinct physiological and pathological pathways that could be targeted by new selective drugs. However, the evidence from functional studies has been less convincing. This could be due to the lack of sufficiently selective ligands or to the similarity between the activated state of receptor subtypes. Species differences and the local receptor environment are also important determinants of the pharmacological profile of a particular subtype. The pharmacology of the putative subtypes of alpha 2-adrenoceptors and their function are discussed in this review by Alison MacKinnon, Mike Spedding and Christine Brown.

The structural conformation of the polyphosphate chain of the ATP molecule is critical for its promotion of prostaglandin biosynthesis

The adenine nucleotide ATP relaxed the tracheal strip of the guinea pig, while the methylene isosteres of ATP, alpha, beta-methylene ATP (APCPP) and beta, gamma-methylene ATP (APPCP) in concentrations up to 100 micrograms were either inactive or produced a small contraction. The relaxation of the tracheal strip elicited by ATP was completely abolished by treatment of the tissue with indomethacin, a prostaglandin synthesis inhibitor. The relaxations induced by ATP in the guinea-pig taenia coli were followed by a rebound contraction which could be blocked by indomethacin. The two methylene analogues of ATP, produced relaxations in the taenia coli which were not followed by a rebound contraction. It is concluded that in the guinea-pig tracheal chain and taenia coli preparations ATP stimulated prostaglandin synthesis while the analogues modified in the polyphosphate chain were unable to do so.

EVIDENCE IN SUPPORT OF THE P1/P2 PURINOCEPTOR HYPOTHESIS IN THE GUINEA‐PIG TAENIA COLI

1 The relaxations induced by adenosine 5′‐triphosphate (ATP), adenosine 5′‐diphosphate (ADP), adenosine 5′‐monophosphate (AMP) and adenosine on the carbachol‐contracted taenia coli of the guinea‐pig have been studied. ATP and ADP produce similar responses which differ in nature and time course from those of AMP and adenosine. 2 Theophylline, at concentrations (25–200μm) lower than those which produce significant phosphodiesterase inhibition, blocks the effects of AMP and adenosine but fails to antagonize the responses elicited by ATP and ADP. The antagonism of adenosine by theophylline appears to be competitive. 3 Apamin (1–100 nM) blocks the inhibitory effects of ATP and ADP but fails to antagonize the responses to AMP and adenosine. The antagonism by apamin is non‐competitive. 4 The results indicate that ATP and adenosine relax the taenia coli by activating different receptors and are consistent with the P1 P2 purinoceptor hypothesis.

EVIDENCE FOR STEREOSPECIFICITY OF THE P1-PURINOCEPTOR

1 The effects of adenosine, 5′‐N‐ethylcarboxamidoadenosine (NECA), 2‐chloroadenosine, 2‐azidoadenosine, and their l‐enantiomers were examined on driven left atria, trachea and transmurally stimulated ileum of the guinea‐pig. 2 In each tissue the order of potency of the d‐enantiomers for producing inhibitory effects was NECA > 2‐chloroadenosine > 2‐azidoadenosine > adenosine. 3 The log concentration‐response curve of each agonist was shifted to the right in the presence of the P1‐purinoceptor antagonist, theophylline. 4 Dipyridamole, which blocks adenosine uptake, potentiated the effects of adenosine but not those of the d‐enantiomers of adenosine analogues. 5 The greater potency of the adenosine analogues therefore, is at least partly due to their resistance to tissue uptake and subsequent enzymatic destruction. 6 The l‐enantiomers of adenosine and its analogues did not produce inhibitory responses in the driven left atria or transmurally stimulated ileum. At high concentrations relaxations of the tracheal muscle were obtained, with the potency series l‐NECA > 2‐chloro‐l‐adenosine > 2‐azido‐l‐adenosine > l‐adenosine. 7 It is concluded that the postsynaptic P1‐purinoceptors in the guinea‐pig atria and trachea and the presynaptic P1‐purinoceptors on cholinergic nerve terminals in guinea‐pig ileum are stereospecific for the d‐enantiomers of adenosine and its analogues.

Adenosine contracts the isolated rat tail artery by releasing endogenous-5-hydroxytryptamine

Adenosine (10(-4)-10(-3) M) contracted the isolated rat tail artery. This effect exhibited rapidly developing tachyphylaxis. Methysergide (10(-7) M) prevented the contractile response to adenosine and 5-hydroxytryptamine (5-HT) without affecting that to noradrenaline. Pretreatment of rats with parachlorophenylalanine (PCPA) abolished the contraction to adenosine (5 X 10(-4) M). Responses to 5-HT and noradrenaline were not significantly affected. These results indicate that the contractile response of the rat tail artery induced by adenosine is probably mediated by endogenous 5-HT.

The guinea-pig ileum preparation as a model for 5-HT1A receptors: anomalous effects with RS-30199-193.

1 Agents that have high and selective affinity for the 5‐HT1A site such as 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT) and N,N‐dipropyl‐5‐carboxamidotryptamine (DP5CT) inhibited the responses to field stimulation in guinea‐pig ileum preparations; the inhibitory effects were antagonized by methiothepin and spiperone, consistent with effects at the 5‐HT1A site. 2 The inhibitory effects of DP5CT were pronounced in Tyrode solution containing low Ca2+(0.9 mm), but were much less apparent in Tyrode solution containing 1.8 or 5.4 mm Ca2+. 3 Responses to DP5CT were abolished by pretreatment with phorbol dibutyrate (3 μm), whereas the responses to UK14304 were only slightly inhibited. 4 Buspirone and ipsapirone (1 μm) inhibited the responses to field stimulation, and the effects were resistant to idazoxan, but inhibited by 8‐OH‐DPAT or spiperone. 5 RS‐30199–193 (5‐chloro‐2‐methyl‐1,2,3,4,8,9,10,10a‐octahydronaphth‐[1,8‐cd]‐azepine hydrochloride) an azepine with high affinity for the 5‐HT1A site in rat cerebral cortex in binding experiments, augmented contractions, but did not antagonize the responses to DP5CT or to 8‐OH‐DPAT. 6 The hybrid compound of RS‐30199–193 with buspirone, RS‐64459–193 (5‐chloro‐2‐[4‐(8‐azaspiro[4,5] decane‐7,9‐dione)‐but‐1‐yl]‐1,2,3,4,8,9,10,10a‐octahydronaphth[1,8‐cd]‐3‐azepine hydrochloride) maintained high affinity for the 5‐HT1A binding site in rat brain and both inhibited the response to field stimulation and antagonized the responses to 8‐OH‐DPAT and DP5CT. Thus the buspirone side chain when added to RS‐30199–193 appears either to induce affinity for a distinct subset of receptors in the guinea‐pig ileum or is required for functional effectiveness at the 5‐HT1A receptor.

The α2-adrenoceptor antagonist SK & F 104078 has high affinity for 5-HT1A and 5-HT2 receptors

Abstract The affinity of SK & F 104078, a putative selective postjunctional α 2 -adrenoceptor antagonists, was determined at 5-HT 1A and 5-HT 2 receptors in rat brain. SK & F 104078 had moderate affinity towards α 2 -adrenoceptors (pK i 6.7) but displayed higher affinity at 5-HT 1A (pK i 8.1) and 5-HT 2 (pK i 7.6) receptors. If SK & F 104078 is used in functional studies to define heterogeneity within of α 2 -adrenoceptors, care must be taken to define the role of 5-HT 1A and 5-HT 2 receptors in the response being measured.