Deviations from receptor theory?
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Animals↗
Biomedical subjects
P Leff
Publications and source records attributed to P Leff.
Classification of platelet and vascular prostaglandin D2 (DP) receptors: estimation of affinities and relative efficacies for a series of novel bicyclic ligands. With an appendix on goodness-of-fit analyses.
1. The DP receptors located on platelets and vasculature were examined in a human washed platelet preparation and in isolated rings of rabbit external jugular vein. 2. A series of eight novel bicyclic compounds were studied for their effects in the two assays. Seven produced agonism, inhibition of aggregation or vascular relaxation, and one compound was 'silent' in both assays. 3. The operational model of agonism (Black & Leff, 1983) was fitted simultaneously to concentration-effect curve data for the seven agonist compounds. The affinity and efficacy estimates so obtained were tested for similarity between the two tissues by analysis of variance, showing that the model could be fitted to both sets of data by assuming the same relative affinity and efficacy values. However, absolute affinity estimates were consistently lower in the vascular preparation. 4. Analysis of two of the seven agonists as antagonists was also possible. This provided pKB estimates which supported the agonist affinity estimates. The eighth compound was also analysed as an antagonist. It, like the other seven, demonstrated a difference in affinity between the two tissues. 5. The results of this study support the view that platelet and vascular DP receptors are similar, assuming that the systematic difference in affinity estimates for the series of compounds between the two tissues is the consequence of receptor micro-environment and/or accessory binding site differences.
Algorithms↗
Adenosine 5'-(2-fluorodiphosphate) is not a selective P2Y purinoceptor agonist in the rabbit jugular vein.
The relaxant properties of the putative selective P2Y agonist adenosine 5'-(2-fluorodiphosphate) (ADP-beta-F) and its structural analog adenosine 5'-(2-thiodiphosphate) have been investigated in the rabbit precontracted jugular vein preparation. In tissues with intact endothelium, ADP-beta-F produced a multiphasic agonist concentration/effect curve made up of two vasorelaxant components which were kinetically and pharmacologically distinct. The higher potency phase (p[A50] 5.58 +/- 0.13), characterized by slow, tonic responses, was retained after endothelial denudation and blocked by the selective P1 purinoceptor antagonist 8-sulphophenyltheophylline. The lower potency phase (p[A50] 3.98 +/- 0.07), characterized by fast, phasic responses, was abolished by endothelial denudation and is presumed to be mediated at P2Y purinoceptors. By contrast, the agonist concentration/effect curve to adenosine 5'-(2-dithiophosphate) in endothelium-intact tissues appear monophasic and was unaffected by 8-sulphophenyltheophylline (p[A50] 6.86 +/- 0.12), although endothelial denudation revealed a secondary P1-induced relaxant component (p[A50] 5.73 +/- 0.20). This study demonstrates that in the rabbit jugular vein, relaxant responses to ADP-beta-F are mediated primarily by activation of P1 purinoceptors, and it is, therefore, invalid to regard ADP-beta-F as a selective probe for P2Y purinoceptors, whereas adenosine 5'-(2-thiodiphosphate) does show some selectivity for this receptor.
Adenosine Diphosphate↗
Further subclassification of ATP receptors based on agonist studies.
Recent studies using agonist analogues of ATP and other nucleotides have generated some surprising observations which may have ramifications for the classification of P2 receptors, particularly for those responses currently attributed to P2Y receptor activation. 2-MethylthioATP (2-MeSATP), the conventional P2Y receptor agonist, does not interact with ATP in the expected fashion in various models of endothelial function, suggesting that it acts by a different mechanism. Furthermore, in certain cell types where responses to ATP are mediated by phospholipase C activation, 2-MeSATP has little or no activity. Interestingly, the pyrimidine uridine triphosphate (UTP) invariably shows similar potency to ATP in systems where 2-MeSATP is inactive. In this article Steve O'Connor and colleagues discuss these data and their significance, and propose that separate receptors may be responsible: one sensitive to 2-MeSATP and the other, a 'nucleotide' receptor, sensitive to UTP.
Animals↗
Estimation of the efficacy and affinity of the beta 2-adrenoceptor agonist salmeterol in guinea-pig trachea.
1. We have attempted to estimate the affinity and efficacy of the new beta 2-adrenoceptor agonist salmeterol in the guinea-pig isolated trachea and to compare these estimates with those obtained for salbutamol. 2. The kinetics of the relaxation to salmeterol were considerably slower than those to salbutamol and the responses were poorly defined. Nevertheless, in experiments employing a cumulative curve design both salmeterol and salbutamol exhibited partial agonist behaviour. Operational model-fitting of these data indicated that the intrinsic efficacies of the two agonists were indistinguishable but that salmeterol had an affinity (pKA = 7.4) some 30 fold greater than salbutamol (pKA = 5.9). 3. Single dose experiments, using maximally effective concentrations of salmeterol or salbutamol were carried out in an attempt to quantify more reliably the relative efficacies of the two agonists. In these experiments salbutamol was found to have an efficacy (2.5) approximately 3 fold greater than that of salmeterol (0.8). 4. Full/partial agonist interaction studies were carried out to provide another estimate of the affinity of salmeterol. Isoprenaline and adrenaline were employed as full agonists in these experiments and the affinity (pKB) estimates obtained for salmeterol against these agonists were 7.3 and 7.4 respectively. These affinity estimates were similar to the pKA value (7.4) obtained by operational model-fitting. 5. Salmeterol has slightly lower efficacy (3 fold) but markedly greater affinity (30 fold) than salbutamol at beta 2-receptors. The slightly lower efficacy of salmeterol is unlikely to have clinical implications for this drug as a bronchodilator beta 2-adrenoceptor agonist in asthma.
Adrenergic beta-Agonists↗
Estimation of agonist affinity and efficacy by direct, operational model-fitting.
A few years ago Black and Leff (1983) presented a theoretical model for agonist action that can be fitted directly to experimental agonist concentration-effect data in order to estimate affinities and relative efficacies of agonists. The model has been successfully applied to data obtained using experimental designs commonly employed in pharmacologic experiments. This article describes the methodology used to fit the model equations to data for full and partial agonists, shows how affinity and efficacy estimates are calculated for these values, and shows how experimental design influences these calculations.
Models, Biological↗
Errors in agonist affinity estimation: do they and should they occur in isolated tissue experiments?
Two plausible theories of agonist action - the isomerization and ternary complex mechanisms - predict that agonist affinity measured using the inactivation method may be subject to overestimation. Moreover, the greater the intrinsic efficacy, the greater the predicted error. But are these predictions correct, and are they borne out by experimental data? Paul Leff and colleagues argue that accurate agonist affinity constants can be measured despite these predictions, but that effort must be made to detect any potential errors that might be anticipated from theory.
Animals↗
Inhibition of collagen- and ADP-induced platelet aggregation by substance P in vivo: involvement of endothelium-derived relaxing factor.
We examined the effect of substance P, a potent stimulator of endothelium-derived relaxing factor (EDRF) release, on responses to collagen and adenosine 3',5'-diphosphate (ADP) in an in vivo model of platelet aggregation. Substance P inhibited platelet aggregation induced in vivo by both collagen and ADP. This anti-platelet effect was particularly pronounced against collagen-induced aggregation and was prevented by prior administration of haemoglobin (Hb), a known inhibitor of EDRF-mediated responses. Collagen-induced platelet aggregation in vitro was unaffected by a concentration of substance P equivalent to that achieved in plasma following in vivo administration. This study provides a clear demonstration of the anti-platelet activity of EDRF in vivo and an indication that its effectiveness may depend on the aggregating agent used.
Adenosine Diphosphate↗
Pharmacological estimation of agonist affinity: detection of errors that may be caused by the operation of receptor isomerisation or ternary complex mechanisms.
1. Recent theoretical studies have questioned the pharmacological estimation of agonist affinity. They showed that when receptor isomerisation or ternary complex mechanisms operate, the receptor inactivation method can substantially overestimate affinity, whereas methods for partial agonist analysis are more accurate. We previously suggested that the operation of such mechanisms and therefore the presence of errors could be detected by analysing the same partial agonist by the receptor inactivation and comparative methods. This paper describes the practical application of this test. 2. The ternary complex mechanism was simulated for a partial agonist under various conditions relating receptor (R) and transducer (T) concentrations, one of which also corresponds to the receptor isomerisation mechanism. The theoretical data so generated were then analysed by the inactivation and comparative methods to quantify the magnitude of error of affinity estimation that could occur. 3. This analysis showed that for a partial agonist with approximately 85% of the activity of a full agonist, the inactivation method could produce an affinity (pKA) estimate up to 0.7 log10 units higher than that produced by the comparative method. This difference would occur when the total receptor concentration ([R0]) is less than or equal to the total transducer concentration ([T0]). It also showed that the overestimation of affinity by the inactivation method was accompanied by drastic overestimation of Em, the maximal effect parameter. 4. The test was then exemplified using the muscarinic receptor system in the guinea-pig isolated left atrial preparation, where there is evidence that a ternary complex mechanism operates. The test agonist was pilocarpine, which produced on average 83% of the activity of the full agonist, carbachol. Pilocarpine was analysed in comparison with carbachol and by receptor inactivation in the same tissue resulting in small and statistically insignificant differences in Em (96.7% and 97.3% respectively) and pKA (5.03 and 4.95 respectively). 5. In conclusion, in this experimental system, there was no evidence for the errors in agonist affinity estimation predicted by theory. Although this conclusion only applies to this system and application of the test to others is necessary to establish the generality of the present results, further examination of the theoretical basis for the predicted errors is required.
Animals↗
Characterization of P2x-receptors in rabbit isolated ear artery.
1. The isolated central ear artery of the rabbit contracts in response to adenosine 5'-triphosphate (ATP) and analogues, effects proposed to be mediated by stimulation of P2x-receptors. We have extended the characterization of the purinoceptor in this tissue by examining the effects of a series of receptor agonists. The study was designed in such a way as to avoid factors which normally limit attempts to classify receptors on the basis of agonist potency orders. 2. D-alpha, beta-methylene ATP (D-alpha, beta-meATP), D-beta, gamma-methylene ATP (D-beta, gamma-meATP), L-beta, gamma-methylene ATP (L-beta, gamma-meATP), 2-methylthio-D-ATP (2-MeSATP) and ATP produced concentration-related contractions of the ear artery with similar maximum responses, suggesting that they were full agonists. Selective desensitization of P2x-receptors abolished or greatly reduced responses to D-alpha, beta-meATP, L-beta, gamma-meATP, D-beta, gamma-meATP. Responses to ATP were inhibited by by desensitization but a significant resistant component was still apparent. 3. D-alpha, beta-meATP was the most potent agonist tested (pA50 6.47 +/- 0.04) being 2138 times more potent than ATP and approximately 9 times more potent than L-beta, gamma-meATP. The agonist potency order was: D-alpha, beta-meATP greater than L-beta, gamma-meATP greater than D-beta, gamma-meATP greater than or equal to 2-MeSATP greater than ATP. This is generally consistent with the order proposed for P2x-receptors. The relative potencies of P2x-agonists in the rabbit ear artery show both similarities to and differences from data obtained in other smooth muscle preparations.
Adenosine Triphosphate↗
Suramin is a slowly-equilibrating but competitive antagonist at P2x-receptors in the rabbit isolated ear artery.
1. The antagonist dynamics of suramin were investigated at P2x-receptors in isolated rings of endothelium-denuded ear artery from New Zealand White (NZW) rabbits. 2. alpha, beta-Methylene adenosine 5'-triphosphate (ATP) concentration-effect curves were constructed cumulatively in a paired curve design in the absence and presence of increasing concentrations of suramin, incubated for 45 min. The slope of the resulting Schild plot was significantly greater than unity (1.50 +/- 0.08). 3. Assuming that slow equilibration by suramin explains the steep Schild plot, further experiments were conducted using short (15 min) and long (3 h) incubation times. The resulting Schild plot slopes were 1.66 +/- 0.36 and 1.06 +/- 0.13 respectively confirming the assumption. However, after 3 h incubation, suramin also caused depression of alpha, beta-methylene ATP curves. 4. In an attempt to minimize the depressant effect of suramin, a kinetic study was designed to calculate the minimum incubation times for each concentration of suramin used in the Schild analysis to achieve effectively complete equilibrium. Theoretically fractional occupancy for the antagonist is given by (r - 1)/r, where r is the dose-ratio. A plot of (r - 1)/r against time allowed the apparent 'on' and 'off' rate constants to be calculated. 5. With the resulting rate constant estimates, an optimised antagonism study was carried out in which incubation times were chosen such that greater than 95% occupancy by suramin could be achieved without agonist curve depression at each concentration of suramin used. 6. Under these conditions, suramin fulfilled all criteria for simple competition: parallel rightward displacement of alpha,beta-methylene ATP curves and a Schild plot slope of unity (1.00 + 0.09). The resulting pKB estimate was 4.79 + 0.05. This estimate of affinity was shown to be independent of the agonist used in another experiment in which L-beta-methylene ATP was employed (pKB = 5.17). 7. Under the same conditions, suramin was found to have no effect on KCI-induced contractions and only slight effects on phenylephrine- and histamine-induced responses.8. This analysis provides the first evidence that suramin is a genuine competitive P21-receptor antagonist.
Adenosine Triphosphate↗
Do pharmacological methods for the quantification of agonists work when the ternary complex mechanism operates?
It is well established that many receptors couple to G-proteins in order to subserve their pharmacological or physiological effects. In those systems it is possible that a ternary complex mechanism operates in which initiation of an effect depends on the concentration of agonist-receptor-G-protein complex formed. Such systems may be considered to obey a receptor-transducer model (Black & Leff, 1983, Proc. R. Soc. B220, 141). A theoretical analysis of this model is presented which seeks to determine how the operation of the ternary complex mechanism affects the quantification of agonists by conventional pharmacological methods. Previous analyses have concluded that pharmacological models may or may not accommodate the ternary complex mechanism depending upon the relationship between the relative concentrations of receptor and transducer units, [R0], and [T0] respectively. The present study extends these in two ways. It considers the impact of the ternary complex mechanism on agonist quantification under a more complete range of conditions relating [R0] and [T0], and it does so with regard to the analysis of partial agonists (by the comparative method) as well as of full agonists (by the method of receptor inactivation). The following predictions are made: (i) reliable estimates of affinity and efficacy can be made using the comparative method under the conditions [R0] much greater than [T0] and [R0] much less than [T0] whereas the inactivation method only works under the former condition; (ii) errors occur in the estimation of affinity and efficacy by both methods when [R0] = [T0] although better estimates are produced by the comparative method; (iii) when errors occur in the absolute estimation of affinity and efficacy, the orders of affinity and efficacy determined by the comparative method will generally be correct but this is not the case for the inactivation method; (iv) in general, the comparative method for agonist quantification appears to produce more reliable information for the purposes of receptor classification and medicinal chemistry than does the receptor inactivation method.
Chemical Phenomena↗
The classification of prostaglandin DP-receptors in platelets and vasculature using BW A868C, a novel, selective and potent competitive antagonist.
1. BW A868C, a novel compound, behaved as a simple competitive antagonist in a human washed platelet aggregation assay. Anti-aggregatory concentration-effect curves to BW 245C were displaced in a parallel manner. The shifts accorded with a Schild plot slope of unity and a pKB of 9.26. 2. Inhibition of platelet aggregation by prostaglandin D2 (PGD2) was antagonized with a similar potency, as were the relaxation effects of BW 245C and PGD2 in the rabbit jugular vein. BW A868C can, therefore, be classified as a DP-receptor antagonist. 3. Actions of BW A868C at other prostaglandin receptors (IP, EP1, EP2, TP and FP) were excluded at concentrations up to 1,000 times higher than the DP-receptor affinity. 4. Analyses of BW 245C- and PGD2-mediated effects were complicated by additional agonist receptor interactions which were revealed by BW A868C. In rabbit jugular vein a resistant phase of agonism was detectable, indicating that both agonists exerted effects through another receptor (possibly EP2). Also, PGD2, in addition to its anti-aggregatory effect on platelets, demonstrated a pro-aggregatory action in the presence of BW A868C. 5. The contractile effects of PGD2 in guinea-pig tracheal strips were resistant to 10 microM BW A868C indicating that they were not mediated through DP-receptors. 6. To our knowledge this is the first account of a well-classified competitive antagonist at the DP-receptor. Its potency and selectivity make it an important new tool in prostanoid receptor classification and identification.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Estimation of affinities and efficacies for kappa-receptor agonists in guinea-pig ileum.
1. Ethylketocyclazocine (EKC) and U50,488H have been employed widely as kappa-receptor agonists in the study of opioid receptor systems. However, the quantification of their agonism in terms of affinities and relative efficacies has not been investigated. 2. In this study, operational model-fitting was used to analyse the effects of irreversible receptor alkylation by beta-chlornaltrexamine (beta-CNA) on the kappa-receptor mediated effects of EKC and U50,488H in the isolated, coaxially stimulated ileum of the guinea-pig. 3. EKC produced monophasic inhibitory concentration-effect curves which were readily amenable to analysis. In contrast U50,488H produced biphasic curves characterized by a higher potency phase of agonism that was susceptible to antagonism by 16-methylcyprenorphine (RX8008M) and a lower potency phase that was apparently non-opioid in nature. 4. Analysis of the kappa-receptor-mediated effects of both agonists indicated that EKC has fifteen fold higher affinity than U50,488H and that the two agonists possess similar intrinsic efficacies.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
ADP-beta-F is not a selective P2y-receptor agonist in rabbit jugular vein.
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Adenosine Diphosphate↗
The biology and pharmacology of PGD2.
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Animals↗
Analysis of agonist action using the operational model.
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Animals↗
Differences in agonist dissociation constant estimates for 5-HT at 5-HT2-receptors: a problem of acute desensitization?
1. The agonist dissociation constant for 5-hydroxytryptamine (5-HT) was estimated in the guinea-pig isolated trachea by the method of receptor inactivation. The value obtained (pKA = 6.45) was significantly lower than estimates previously obtained in the rabbit aorta and rat jugular vein, although all three tissues are supposed to contain the same 5-HT2 class of receptor. 2. The antagonist dissociation constant for alpha,alpha-dimethyltryptamine was also estimated in the guinea-pig trachea. The pKB value (5.43) was not significantly different from previous estimates in the rabbit aorta and rat jugular vein, consistent with receptor homogeneity between the three tissues. 3. The effect-time profiles corresponding to individual 5-HT applications were more transient in the guinea-pig trachea than in the rabbit aorta. This difference could be accounted for using a simple model of acute receptor desensitization (Leff, 1986), assuming that the conversion of active agonist-receptor complexes into inactive ones was faster in the guinea-pig trachea than in the rabbit aorta. 4. Computer simulation of the desensitization model showed that the discrepancy of pKA estimates for 5-HT between the rabbit aorta and guinea-pig trachea could also be explained using the same rate constant difference that accounted for the difference in effect-time profiles. This analysis indicated that the estimate made in the trachea was erroneously low, whereas that made in the aorta was concluded to be correct. 5. The apparent association between transience of response and pKA estimates is discussed with particular attention to the reliability of agonist affinity estimates in receptor classification.
Animals↗