Paul Leff

The two-state model of receptor activation

Over the past few years, the concept that the activation of G protein-coupled receptors and transmitter-gated ion channels depends on a conformational change has received increasingly widespread acceptance. As a result, these two structurally distinct families of receptors can now be considered to obey a similar two-state mechanism. However, traditional receptor theory has largely overlooked this concept. In this article, Paul Leff explains and illustrates the predictions of the two-state model of receptor activation and discusses its impact on the analysis and interpretation of agonist-receptor interactions.

A three-state receptor model of agonist action.

The concept that receptors can exist in multiple conformational states is becoming a physical reality. A fundamental question is how many active states need to be proposed in order to account for pharmacological observations, in particular, the finding that the same receptor type can exhibit a different agonist pharmacology when coupled to different effector pathways. In this article, Paul Leff, Clare Scaramellini, Clare Law and Ken McKechnie propose and develop a three-state receptor model in which two active conformations are assumed to exist. They show that this relatively simple theoretical model provides a basis for predicting variable agonist and inverse agonist behaviour in different systems containing the same receptor, and that it is able to account for emerging data obtained on promiscuously coupled receptors. It is argued that, while these new theoretical considerations challenge the fundamental assumptions and concepts of traditional receptor theory, the general principles of pharmacological receptor classification are largely preserved.

How should P2X purinoceptors be classified pharmacologically

When ATP is released as a neurotransmitter from central and peripheral nerves it acts at P2X purinoceptors to produce postsynaptic depolarization and excitation. The P2X purinoceptor was originally classified on the basis of the relative agonist potencies of ATP and a number of its structural analogues. However, it is now clear that the potency of some agonists is greatly decreased by breakdown by ectonucleotidase enzymes, leading to an incorrect determination of agonist potency order. In this article, Charles Kennedy and Paul Leff discuss recent results that indicate that the established classification of P2X purinoceptors is no longer valid and needs redefinition.

Further concerns over Cheng-Prusoff analysis

In his article in last months issue of TiPS (14, 89-91), Douglas Craig discussed the application of the Cheng-Prusoff relationship to the analysis of antagonists in functional experiments. Craig described how the translation of this method from biochemistry to pharmacology can be accompanied by errors due to theoretical misunderstanding. He also emphasized the lower level of rigour associated with this means of estimating antagonist affinity constants compared with Schild analysis. In this article, Paul Leff and Iain Dougall extend Craigs arguments by describing some additional ways in which the application of the Cheng-Prusoff method to pharmacological experiments are likely to result in misinterpretation and erroneous estimates of antagonist affinity.

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 α,β‐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 α,β‐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 > 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 α,β‐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‐β,γ‐methylene ATP was employed (pKB = 5.17). 7 Under the same conditions, suramin was found to have no effect on KCl‐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 P2x‐receptor antagonist.

FPL 66096: a novel, highly potent and selective antagonist at human platelet P2T-purinoceptors.

1 ADP‐dependent platelet aggregation is mediated by the P2T‐purinoceptor and is specifically inhibited by ATP, which is a competitive P2T‐purinoceptor antagonist. However, ATP functions as an agonist at other P2‐purinoceptor subtypes in other tissues and is, therefore, non‐selective. This paper describes the effects of the novel ATP analogue, FPL 66096 (2‐propylthio‐D‐β,γ‐difluoromethylene ATP), on ADP‐induced and ADP‐independent aggregation of human washed platelets and in standard preparations containing P2X‐ (rabbit ear artery) and P2Y‐purinoceptors (guinea‐pig aorta). 2 In suspensions of human washed platelets, FPL 66096 (1‐100nM) produced concentration‐dependent rightward displacement of concentration‐effect (E/[A]) curves obtained for ADP‐induced platelet aggregation. Logistic fitting of E/[A] data indicated that the effect of FPL 66096 was consistent with simple competition with a pKB value of 8.66. FPL 66096 (10–1000 nm) had no effect on aggregation produced by the thromboxane A2‐mimetic, U46619 (0.1–10 μm) when the response to this agent was rendered ADP‐independent by inclusion of the non‐selective P2‐purinoceptor antagonist, suramin (100 μ). 3 The antiaggregatory potency of FPL 66096 was not influenced by increasing the incubation time from 2 to 15min nor by inclusion of the P1‐purinoceptor antagonist 8‐sulphophenyltheophylline at a concentration (300 μm) that produced a 68 fold rightward displacement of the anti‐aggregatory E/[A] curve for the P1‐purinoceptor agonist, 5′‐N‐ethylcarboxamidoadenosine (0.1–1000 μm). 4 FLP 66096 behaved as a weak (pA50 3.68) but full P2x‐purinoceptor agonist in preparations of the rabbit isolated ear artery and as a weak, competitive antagonist (apparent pKB 4.71) at P2Y‐purinoceptors in the guinea‐pig isolated aorta, indicating a selectivity of at least 9000 fold for the P2T‐subtype. In the latter preparation, non‐specific relaxations were produced by concentrations of FPL 66096 ≥ 10 μm. 5 These results indicate that FPL 66096 is a P2T‐purinoceptor antagonist of unprecedented potency and selectivity and that its effects are consistent with simple competition at the P2T‐purinoceptor. Therefore, FPL 66096 represents a novel pharmacological tool in the classification of P2‐purinoceptors and in the elucidation of the mechanisms involved in activation of platelets by ADP.

P2Y1‐receptors in human platelets which are pharmacologically distinct from P2YADP‐receptors

In the present study we have classified the receptor(s) mediating increases in intracellular calcium concentration ([Ca2+]i) in human washed platelets and compared the pharmacological profile obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y1‐receptor. The P2Y1‐receptor antagonist, adenosine‐3′‐phosphate‐5′‐phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar affinities (pKB of 5.8 and 6.0, respectively). The selective P2YADP antagonist, AR‐C66096, exhibited partial agonism in the Jurkat cells with an affinity (pKA) of 4.9. This value is consistent with its known P2Y1‐receptor activity. In platelets, AR‐C66096 at a concentration (0.1 μM) approximately 100 fold greater than its known P2YADP receptor affinity, had no effect on ADP‐induced increases in [Ca2+]i. The ability of adenine nucleotide analogues to elevate [Ca2+]i in the Jurkat cells was also determined. The rank order of agonist potency (p[A]50) was: 2‐MeSADP (8.3)>2‐ClATP (7.8)>ADP (7.5)=2‐MeSATP (7.4)>ATPγS (6.5)>ATP (6.2), with ATP appearing to be a partial agonist. The same rank order of potency was observed when similar experiments were performed in platelets. However, the absolute potencies of all the agonists and the intrinsic activities of both ATPγS and ATP were lower in platelets. The operational model of agonism was used to test whether the agonist concentration‐effect profiles obtained in these two cell types could be explained on the basis of differences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y1‐receptor system. The hypothesis that the observed partial agonist behaviour of ATP was the result of receptor activation by contaminating ADP with concomitant receptor blockade by ATP, was tested in the platelet system. This hypothesis was supported by a theoretical analysis, which yielded an affinity value for ATP similar to that obtained previously at P2Y1‐receptors. In summary, the results of this study indicate that human washed platelets contain P2Y1‐receptors which mediate increases in [Ca2+]i and that this receptor population is pharmacologically distinct from P2YADP‐receptors.

ADP can induce aggregation of human platelets via both P2Y1 and P2T receptors

In the present study we have investigated the roles of P2Y1 and P2T receptor subtypes in adenosine 5′‐diphosphate (ADP)‐induced aggregation of human platelets in heparinized platelet rich plasma. The response to ADP can be characterized as the initial rate or the maximum or final extent of aggregation. The response profile is determined by the concentration of ADP used, being transient at lower and sustained at higher concentrations. The P2Y1 receptor antagonist, adenosine‐3′‐phosphate‐5′‐phosphate (A3P5P) competitively antagonized the initial rate of aggregation (pKB 5.47) and transformed the response profile to a slowly developing but sustained response. Both maximum and final extents were also inhibited by A3P5P although not in a competitive manner (Schild slope <1). The P2T receptor antagonist, AR‐C67085, competitively antagonized the final extent of aggregation (pKB 8.54), transforming the response profile to one of rapid, transient aggregation. Its effect on maximum extent (the most widely used index of aggregation) was complex, and further supported the involvement of both receptor subtypes in the aggregation response. ADP‐induced aggregation is a complex phenomenon, the nature of which is determined by the relative occupancy of the two receptor subtypes. While P2Y1 receptor activation causes a rapid and transient aggregation, the extent of sustained aggregation is determined by the level of P2T receptor occupancy. Hence, detailed analysis of the aggregation response is essential to correctly define the purinergic pharmacology of the platelet and interpretation of results is critically dependent on the response index chosen.

Agonist-antagonist interactions at angiotensin receptors: application of a two-state receptor model

Interactions between agonists and antagonists at angiotensin receptors are characterized by a number of features: variation of antagonist dynamics between apparent simple competition, insurmountable antagonism and, occasionally, augmentation; the tendency for insurmountable antagonism to be saturable; slow recovery of agonist responses following agonist-induced tachyphylaxis; and the ability of competitive antagonists to accelerate recovery from the latter intervention. Some of these phenomena have also been observed in studies of 5-HT2 receptors where they were attributed to the operation of a two-state model with an allosteric site. In this article, Mark Robertson and colleagues propose that the properties of angiotensin AT1 receptors may be explained by a similar model, but without the need to evoke an allosteric site.

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‐α,β‐methylene ATP (d‐α,β‐meATP), d‐β,γ‐methylene ATP (d‐β,γ‐meATP), l‐β,γ‐methylene ATP (l‐β,γ‐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‐α,β‐meATP, l‐β,γ‐meATP, d‐β,γ‐meATP and 2‐MeSATP. Responses to ATP were inhibited by desensitization but a significant resistant component was still apparent. 3 d‐α,β‐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‐β,γ‐meATP. The agonist potency order was: d‐α,β‐meATP > l‐β,γ‐meATP > d‐β,γ‐meATP ≥ 2‐MeSATP > 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.