Ian Kennedy

NEW INSIGHTS ON P2X PURINOCEPTORS

Significant advances in understanding of P2X purinoceptor pharmacology have been made in the last few years. The limitations of nucleotide agonists as drug tools have now been amply demonstrated. Fortunately, inhibitors of the degrading ecto-ATPase enzymes are becoming available and it has become apparent that the complete removal of all divalent cations can be used experimentally in some systems to prevent nucleotide breakdown. Despite these issues, convincing evidence for P2X receptor heterogeneity, from data with agonists, has recently been reported.A number of new antagonists at P2X purinoceptors have also recently been described which to some degree appear to be more specific and useful than earlier antagonists like suramin. It is now apparent that suramin is a poor antagonist of ATP in many tissues because it potently inhibits ATPase activity at similar concentrations to those at which it blocks the P2X purinoceptor.Advances in the use of radiolabelled nucleotides as radioligands for binding studies has allowed the demonstration of P2X purinoceptors in a variety of tissues throughout the body including the brain. These studies have also provided evidence for receptor heterogeneity. Excitingly, two P2X purinoceptor genes have been cloned but operational studies suggest that more than two types exist. The cloning studies have also demonstrated a unique structure for the P2X purinoceptor which differentiates it from all other ligand-gated ion channel receptors. Further studies on P2X purinoceptor operation and structure are needed to help resolve controversies alluded to regarding the characterization and classification of nucleotide receptors. Hopefully such studies will also lead to a better understanding of the physiological and pathological importance of ATP and its activation of P2X purinoceptors. This will require the identification of better drug tools, in particular antagonists which may also provide the basis for novel therapeutic agents.

The use of antagonists to characterize the receptors mediating depolarization of the rat isolated vagus nerve by α,β‐methylene adenosine 5′‐triphosphate

1 We have previously found that the P2x‐purinoceptor agonist, α,β‐methylene adenosine 5′‐triphosphate (α,β‐methylene ATP), depolarizes the rat cervical vagus nerve, measured with a ‘grease‐gap’ extracellular recording technique. This effect was attenuated by the P2 purinoceptor antagonist, suramin. In the present study we have investigated in more detail the antagonism produced by suramin and have also investigated the actions of two other putative P2 purinoceptor antagonists, cibacron blue and pyridoxal‐phosphate‐6‐azophenyl‐2′, 5′‐disulphonic acid (iso‐PPADS). Furthermore, we have studied the interactions between suramin and cibacron blue or iso‐PPADS in an attempt to determine whether these antagonists act at a common receptor site. 2 Suramin (1 × 10−5–1 × 10−4 m) produced reversible, concentration‐related rightward displacements of the concentration‐effect curve to α,β‐methylene ATP. Schild analysis of this antagonism yielded a pA2 value of 5.90 with a slope value of 0.47. 3 Cibacron blue (3 × 10−5–1 × 10−4 m) also antagonized depolarizations induced by α,β‐methylene ATP. The antagonistic effects of cibacron blue were slow to reach equilibrium but could be readily reversed on washout. At low concentations for antagonism, cibacron blue (1 × 10−5 m and 3 × 10−5 m) produced enhancement of the maximal response to α,β‐methylene ATP. At the highest concentration tested (1 × 10−4 m) the concentration‐effect curve to α,β‐methylene ATP was shifted to the right in a parallel manner, yielding a pKB estimate of 4.96. 4 Iso‐PPADS (1 × 10−6–1 × 10−5 m) produced a concentration‐related depression in the maxima of the concentration‐effect curves to α,β‐methylene ATP. Analysis of these data by a double reciprocal plot yielded a pKB estimate of 6.02. This profile of insurmountable antagonism could not be attributed to irreversible binding of iso‐PPADS to the receptor since the effect of iso‐PPADS could be reversed on washing, albeit slowly. 5 In the presence of suramin (1 × 10−4 m), cibacron blue (1 × 10−4 m) produced no further rightward displacement of the α,β‐methylene ATP concentration‐effect curve. The mean agonist concentration‐ratios in the presence of suramin or cibacron blue alone (11.7 and 10.3, respectively) were not significantly different from the mean concentration‐ratio in the presence of both antagonists (11.8). This finding suggests that high concentrations of α,β‐methylene ATP activate a receptor population which is resistant to blockade by either antagonist. 6 The antagonistic effect of iso‐PPADS (1 × 10−5 m) was partially attenuated by suramin (1 × 10−4 m). It is possible that this interaction reflects a slow dissociation of iso‐PPADS from the receptor with which suramin and α,β‐methylene ATP interact. 7 Suramin, cibacron blue or iso‐PPADS had no marked effect on depolarization produced by 5‐hydroxytryptamine (5‐HT, 1 × 10−7–3 × 10−5 m), indicating their specificity in antagonizing responses to α,β‐methylene ATP. 8 Responses to α,β‐methylene ATP were not antagonized by 8‐para‐sulphophenyltheophylline (3 × 10−5 m), ondansetron (1 × 10−7 m), bicuculline (1 × 10−5 m), phentolamine (1 × 10−6 m) or hexamethonium (1 × 10−4 m), which are antagonists at P1‐purinoceptors, 5‐HT3 receptors, GABAA receptors, α‐adrenoceptors and nicotinic cholinoceptors, respectively, thereby excluding the involvement of these receptors. Indomethacin (3 × 10−6 m) had no effect on responses to α,β‐methylene ATP. 9 The results obtained with three purinoceptor antagonists confirm and extend our original supposition that α,β‐methylene ATP‐induced depolarization of the rat vagus nerve is mediated predominantly via P2 purinoceptors, thought to be of the P2x subtype. The finding that responses induced by high concentrations of agonist were resistant to blockade by suramin and cibacron blue, but could be attenuated by iso‐PPADS, adds further weight to our speculation that the purinoceptor population in the rat vagus nerve is heterogeneous.

Characterization of purinoceptors mediating depolarization of rat isolated vagus nerve

1 As part of a broader study to characterize neuronal purinoceptors, the effects of adenosine 5′‐triphosphate (ATP) and a range of ATP analogues were investigated on the extracellularly recorded membrane potential of the rat isolated vagus nerve, using a ‘grease‐gap’ technique. 2 ATP evoked depolarization of the rat vagus nerve. The concentration‐effect curve to ATP was not monophasic: at the lower concentrations (1 × 10−5–1 × 10−3 m) the curve was shallow (<50% of the near maximal response to 5‐hydroxytryptamine (5‐HT)) whilst at higher concentrations the relationship between concentration and amplitude of depolarization was steeper (> 135% of the response to 5‐HT at the highest concentration tested, 1 × 10−2 m). On washout of the high drug concentrations large after‐hyperpolarizations were often observed. 3 α,β‐methylene ATP (1 × 10−6–3 × 10−4 m), β,γ‐methylene ATP (1 × 10−6–1 × 10−3 m), and 5′‐adenylylimidodiphosphate (β,γ‐imido ATP; 1 × 10−6–1 × 10−3 m) were all more potent than ATP and produced large depolarizations of the rat vagus nerve at the highest concentrations tested (> 150% of the response to 5‐HT). The overall rank order of potency was α,β‐methylene ATP > β,γ‐methylene ATP = β,γ‐imido ATP > ATP. 4 In contrast, 2‐methylthio ATP (1 × 10−6–1 × 10−3 m) produced relatively small depolarizations (< 100% of the response to 5‐HT). As was the case with low concentrations of ATP, the concentration‐effect curve to 2‐methylthio ATP was very shallow. 5 Adenosine 5′‐diphosphate (ADP), adenosine 5′‐monophosphate (AMP), adenosine and adenosine 5′‐O‐(2‐thiodiphosphate) (ADP‐β‐s; all 1 × 10−6–1 × 10−3 m) evoked only small depolarizations of the vagus nerve, amounting to 47 ± 2.5%, 40.8 ± 7.8%, 33.7 ± 3.3% and 62.4 ± 12.7% of the response to 5‐HT, respectively. Uridine 5′‐triphosphate (UTP; 1 × 10−6–1 × 10−3 m) was inactive. 6 The P2 purinoceptor antagonist, suramin (1 × 10−5 m–1 × 10−4 m), antagonized responses to α,β‐methylene ATP. The nature of this antagonism was not, however, consistent with simple competitive kinetics between agonist and antagonist. Depolarizations produced by β,γ‐methylene ATP and β,γ‐imido ATP were also attenuated by suramin (1 × 10−4 m), but in contrast, suramin had no effect on responses to ADP, 2‐methylthio ATP, ADP‐β‐S or 5‐HT. 7 In addition to its antagonist effects, suramin (10−4 m) markedly increased the maximum amplitude of the depolarization produced by ATP. 8 It is concluded that a heterogeneous receptor population mediates depolarization of the rat vagus nerve by purine nucleotides. Importantly, the large amplitude depolarizations to α,β‐methylene ATP, β,γ‐methylene ATP and β,γ‐imido ATP are mediated via receptors that share many characteristics of the classical P2x receptor. In contrast, the relatively small depolarizing effects of ADP, ADP‐β‐S and 2‐methylthio ATP were suramin‐resistant. Although it appears that other purinoceptors are present, these data suggest that the rat vagus nerve may serve as a useful preparation for studying the pharmacology of neuronal P2x receptors.

Evidence for the presence of two types of P2 purinoceptor in the guinea-pig ileal longitudinal smooth muscle preparation

The effects of some agonists acting at P2 purinoceptors on guinea-pig isolated ileum longitudinal smooth muscle have been examined. The preparation contracted in response to ATP, alpha,beta-methylene ATP and 2-methylthio ATP, but not UTP. In this respect, alpha,beta-methylene ATP and 2-methylthio ATP were approximately equipotent and both were 10-50 times more active than ATP. Responses to alpha,beta-methylene ATP, but not 2-methylthio ATP or ATP, were antagonised by atropine and tetrodotoxin, suggesting that alpha,beta-methylene ATP activates cholinergic nerves in the ileum, whilst the other two compounds act on the smooth muscle. Two other purine nucleotide analogues, beta,gamma-methylene ATP and beta,gamma-imido ATP, did not cause contraction. However, both compounds antagonised responses to alpha,beta-methylene ATP, but not those to 2-methylthio ATP. Suramin antagonised responses to both alpha,beta-methylene ATP and 2-methylthio ATP, whilst Cibacron blue was without effect on responses to either agonist. We conclude that the purinoceptor on cholinergic nerves has some of the characteristics of the P2x purinoceptor, whilst the purinoceptor on ileal smooth muscle has some of the characteristics of the P2Y purinoceptor. However, further work will be necessary before definitive classification is possible.

Effects of divalent cations on the potency of ATP and related agonists in the rat isolated vagus nerve: implications for P2 purinoceptor classification.

1 By use of a ‘grease‐gap’ technique, the depolarizing effects of adenosine 5′‐triphosphate (ATP) and ATP analogues on the rat isolated vagus nerve were determined in normal and in Ca2+/Mg2+‐free (+ 1 × 10−3 m ethylenediamine tetraacetic acid) physiological salt solution (PSS). 2 In normal PSS, ATP produced concentration‐dependent depolarization responses but the concentration‐effect curve to ATP was incomplete and a maximum effect was not achieved. The threshold concentration for depolarization was 1 × 10−5 m and at the highest concentration tested (1 × 10−3 m) the peak amplitude of the response to ATP only amounted to 71% of the depolarization produced by a near maximal response to 5‐hydroxytryptamine (5‐HT, 1 × 10−5 m). 3 In Ca2+/Mg2+‐free PSS, ATP produced depolarization responses at much lower concentrations and of markedly larger amplitude. Under these conditions, the threshold concentration for depolarization was 1–3 × 10−7 m and the maximal response to ATP amounted to 526% of the response to 5‐HT (1 × 10−5 m) in normal PSS. The concentration‐effect curve to ATP was sigmoid, with a defined maximum effect and a mean EC50 value of 1.2 × 10−6 m. 4 In contrast to the effects on responses to ATP, the absence of divalent cations in the PSS did not modify the effective concentrations of either α,β‐methylene ATP or 5‐HT. However, the maximum responses to both α,β‐methylene ATP and 5‐HT were significantly increased in Ca2+/Mg2+‐free PSS. 5 The depolarizing effects of several analogues of ATP were determined in Ca2+/Mg2+‐free PSS. ATP‐γ‐S and 2‐methylthioATP were of similar potency to ATP (respective equi‐effective molar ratios (EMRs) of 1.9 and 1.3, where ATP= 1) and similar maximum responses were obtained. α,β‐Methylene ATP, β,γ‐methylene ATP and β,γ‐imido ATP were considerably less potent than ATP, analysis yielding mean EMRs of 48.9, 85.0 and 60.0, respectively. Maximum responses to these latter three agonists were not obtained at the highest concentrations tested (1 × 10−4‐3 × 10−4 m). Benzoyl ATP, adenosine 5′‐O‐(2‐thiodiphosphate) and adenosine diphosphate produced only small depolarizing responses at high concentrations (> 1 × 10−4 m). Adenosine monophosphate, adenosine and uridine 5′‐triphosphate each had little or no depolarizing effect in Ca2+/Mg2+‐free PSS. 6 These data demonstrate that in the absence of divalent cations the excitatory actions of some, but not all, purine nucleotides in the rat vagus nerve are markedly potentiated. In Ca2+/Mg2+‐free PSS, the rank order of agonist potencies was ATP = 2‐methylthioATP = ATP‐γ‐S>> α,β‐methylene ATP = β,γ−imido ATP = β,γ‐methylene ATP. These findings are in stark contrast to our previous observations in normal PSS where the rank order of agonist potencies for these nucleotides was α,β‐methylene ATP > ATP‐γ‐S > β,γ‐imido ATP = β,γ‐methylene ATP > 2‐methylthioATP > ATP. 7 We suggest that the two different rank orders of potency can be explained by differential metabolism involving Ca2+/Mg2+‐dependent ectonucleotidases. If so, these data indicate that ATP and 2‐methylthioATP are inherently more potent than α,β‐methylene ATP as agonists at neuronal P2X purinoceptors in the rat vagus nerve. The possible implications of these findings to the present system for subclassifying P2 purinoceptors are profound.

College of Medicine replies to its critics

Cassidy’s article and the rapid responses contain errors and misunderstandings.1 2 3 4 The college aims to promote a more politically and professionally transparent, patient centred, and sustainable approach to healthcare, using whatever social or therapeutic approaches are safe, effective, and empowering for patients. The college is calling for a more compassionate NHS, where practice based on good evidence has …

Points: Appropriate technology obstetrics.

Dr R D S WATSON and Dr R F FLETCHER (Dudley Road Hospital, Birmingham B18 7QH) write: Dr G R Scott and colleagues (18 August, p 399) reported that thyrotoxic patients with atrial fibrillation who became hypothyroid after radio iodine (600 mBq iodine-131) were more likely to revert spontaneously to sinus rhythm than those whose thyroid function became normal; thev suggested that use of larger doses of radio iodine might be considered. No information was given concerning the use of antithyroid drugs either before administration of iodine-131 or between administration and induction of a euthyroid state. Did their patients remain thyrotoxic for up to three months while awaiting the effects of iodine-131 ? Scott et al suggest that hypothyroidism is positively beneficial in the presence of atrial fibrillation. We suggest that this objective could be achieved more reliably and quickly by giving carbimazole as well. Other important treatments which should be considered are cardioversion and anticoagulation.

Colour in Rhodesia

SIR,-I am sure many doctors in Britain will support Dr. Shees plea for Birmingham University to revoke its decision on Salisbury Medical School (28 March, p. 817). Quite apart from the phariseeism of the Birmingham students, one wonders whether Government pressure had anything to do with the decision. Your leading article on colour in Rhodesia (21 March, p. 705) oversimplifies the issue. It is difficult to see what effective protest can be made which will not backfire on the African doctors and patients, as has happened so often in South Africa. Salisbury is an exciting medical school whose importance is vital for the future of medicine in Central Africa-not Rhodesia. It will be virtually impossible for African medical students there ever to leave the country, let alone find places in medical schools which can never fit them quite so well for work in their own environment. The teaching staff at Salisbury are wide awake to the medical problems of developing Africa, and acutely aware of the personal, social, and political difficulties facing their African students. To embarrass them in this way is a great folly of which Birmingham should be deeply ashamed. Will a Salisbury graduate cease to be a doctor as soon as he steps over the Rhodesian border? Africa has few enough doctors. Nothing Birmingham can do will compensate for the loss of these graduates from Salisbury.-I am, etc., IAN KENNEDY. Exeter, Devon.