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Biomedical subjects

P Leff

Publications and source records attributed to P Leff.

At least 19 recordsLinked to original sources
PubMed2001

Orphanin-FQ/nociceptin inhibits kindling epileptogenesis and enhances hippocampal feed-forward inhibition.

The role of Orphanin-FQ/nociceptin in synaptic plasticity was assessed by its potency in modulating kindling epileptogenesis in vivo, and feed-forward inhibition in hippocampal recordings in vitro. In addition, a specific rabbit antiserum against this peptide was obtained and the immunohistochemical distribution of nociceptin was determined in rat brain slices. After the establishment of kindling epilepsy, by daily electrical stimulation of the piriform cortex, the i.c.v. injection of nociceptin, 20 min before the kindling stimulation, was not able to block the generation of the generalized seizures, nor to alter their duration. However, the i.c.v. injection of nociceptin, 20 min before each stimulation along the kindling process, depressed its development in a dose-dependent manner. This effect was specific since the nociceptin antagonist [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2, but not the broad-spectrum opiate antagonist, naloxone, was able to completely block nociceptin actions. The inhibitory role of nociceptin was assessed by in vitro recordings from entorhinal cortex-hippocampal slices. By single pulses applied over the Schaffer collaterals, we found that synaptic transmission was facilitated onto CA1, but using a paired-pulse protocol, we found that nociceptin potentiated feed-forward inhibition. The immunohistochemical data show that nociceptin is expressed in limbic cortical regions, including the piriform cortex and the hippocampus. Our results demonstrate that nociceptin exerts a modulatory role in limbic excitability and suggest that it provides an inhibitory control in the development of epilepsy by possibly inhibiting the spread of excitation through the system, by favoring feed-forward inhibition.

Animals↗
PubMed2000

Adventures in the pharmacological analysis of P2 receptors.

The pharmacological classification of P2 receptors owes its origin to the pioneering efforts of Geoff Burnstock and those who followed him, research that was conducted primarily in physiological experimental systems. Over recent years, the techniques of molecular biology have been increasingly applied in the study of P2 receptors while, at the same time, advances in their pharmacological analysis have been limited by a lack of potent and selective agonist or antagonist ligands. This has resulted in a classification scheme which is largely structural in nature, with relatively little contribution from pharmacology. Our endeavours in this area have been directed towards the discovery of ligands with which the pharmacological analysis and definition of P2 receptors could be advanced, the ultimate goal being the design of therapeutic agents. This article will describe some of our experiences in this challenging but rewarding area.

Animals↗
PubMed2000

ADP can induce aggregation of human platelets via both P2Y(1) and P(2T) receptors.

1. In the present study we have investigated the roles of P2Y(1) and P(2T) receptor subtypes in adenosine 5'-diphosphate (ADP)-induced aggregation of human platelets in heparinized platelet rich plasma. 2. 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. 3. The P2Y(1) receptor antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P) competitively antagonized the initial rate of aggregation (pK(B) 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). 4. The P(2T) receptor antagonist, AR-C67085, competitively antagonized the final extent of aggregation (pK(B) 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. 5. ADP-induced aggregation is a complex phenomenon, the nature of which is determined by the relative occupancy of the two receptor subtypes. While P2Y(1) receptor activation causes a rapid and transient aggregation, the extent of sustained aggregation is determined by the level of P(2T) 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.

Adenosine Diphosphate↗
PubMed1999

Antagonists of the platelet P2T receptor: a novel approach to antithrombotic therapy.

The platelet P2T receptor plays a major role in platelet aggregation, and its antagonists are predicted to have significant therapeutic potential as antithrombotic agents. We have explored analogues of adenosine triphosphate (ATP), which is a weak, nonselective but competitive P2T receptor antagonist. Modification of the polyphosphate side chain to prevent breakdown to the agonist adenosine diphosphate (ADP) and substitution of the adenine moiety to enhance affinity and selectivity for the P2T receptor led to the identification of 10e (AR-C67085MX), having an IC50 of 2.5 nM against ADP-induced aggregation of human platelets. Compound 10e was the first very potent antagonist of the P2T receptor, with a selectivity for that subtype of the P2 receptor family of >1000-fold. Further modification of the structure produced compound 10l (AR-C69931MX) having an IC50 of 0.4 nM. In vivo, at maximally effective antithrombotic doses, there is little prolongation of bleeding time (1.4-fold), which is in marked contrast to the 5-6-fold found with GPIIb/IIIa antagonists.

Adenosine Monophosphate↗
PubMed1998

A three-state receptor model: predictions of multiple agonist pharmacology for the same receptor type.

Recent studies have demonstrated that activation of the same G-protein coupled receptor can generate different agonist pharmacology depending on the signaling pathway(s) to which it couples. Two types of behavior have been exemplified; differences in affinity order, and differences in efficacy order with the same affinity order. The two-state model of receptor activation cannot explain these data, since a single active receptor state cannot couple differently to the two response pathways for different ligands. We have therefore extended the two-state model to a three-state model in which receptors exist in three states: an inactive state, R, and two different active states, R* and R**. The model has two modes, the 'intact mode', in which all the equilibria are linked; and the 'isolated mode' in which the two response pathways are isolated from each other, giving effectively two separate two-state systems. In the 'intact mode' the same agonist affinity order is predicted for both response pathways, but a different efficacy order. In the 'isolated mode', since the equilibria are no longer linked, the model predicts that a different affinity order may be obtained for the two pathways. Owing to the linkage of all the equilibria in the intact three-state model the level of constitutive activity through one pathway can affect the direction of agonism through the other pathway, resulting in the conversion of an inverse agonist into a positive agonist. This change in the direction of agonism is also predicted to occur when the two response pathways are isolated. The three-state model therefore predicts that agonists, acting at the same receptor, may show different affinity orders and different efficacy orders depending upon which response is measured and the assay system used, and also predicts that inverse agonism may be system dependent.

Animals↗
PubMed1998

P2Y1-receptors in human platelets which are pharmacologically distinct from P2Y(ADP)-receptors.

1. 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. 2. The P2Y1-receptor antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar affinities (pK(B) of 5.8 and 6.0, respectively). 3. The selective P2Y(ADP) antagonist, AR-C66096, exhibited partial agonism in the Jurkat cells with an affinity (pK(A)) of 4.9. This value is consistent with its known P2Y1-receptor activity. In platelets, AR-C66096 at a concentration (0.1 microM) approximately 100 fold greater than its known P2Y(ADP) receptor affinity, had no effect on ADP-induced increases in [Ca2+]i. 4. 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)>ATPgammaS (6.5)>ATP (6.2), with ATP appearing to be a partial agonist. 5. 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 ATPgammaS and ATP were lower in platelets. 6. 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. 7. 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. 8. 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 P2Y(ADP)-receptors.

Adenosine Diphosphate↗
PubMed1998

Effector pathway-dependent relative efficacy at serotonin type 2A and 2C receptors: evidence for agonist-directed trafficking of receptor stimulus.

There are many examples of a single receptor coupling directly to more than one cellular signal transduction pathway. Although traditional receptor theory allows for activation of multiple cellular effectors by agonists, it predicts that the relative degree of activation of each effector pathway by an agonist (relative efficacy) must be the same. In the current experiments, we demonstrate that agonists at the human serotonin2A (5-HT2A) and 5-HT2C receptors activate differentially two signal transduction pathways independently coupled to the receptors [phospholipase C (PLC)-mediated inositol phosphate (IP) accumulation and phospholipase A2 (PLA2)-mediated arachidonic acid (AA) release]. The relative efficacies of agonists differed depending on which signal transduction pathway was measured. Moreover, relative to 5-HT, some 5-HT2C agonists (e.g., 3-trifluoromethylphenyl-piperazine) preferentially activated the PLC-IP pathway, whereas others (e.g., lysergic acid diethylamide) favored the PLA2-AA pathway. In contrast, when two dependent responses were measured (IP accumulation and calcium mobilization), agonist relative efficacies were not different. These data strongly support the hypothesis termed "agonist-directed trafficking of receptor stimulus" recently proposed by Kenakin [Trends Pharmacol Sci 16:232-238 (1995)]. Concentration-response curves to 5-HT2C agonists were fit well by a three-state model of receptor activation, suggesting that two active receptor states may be sufficient to explain pathway-dependent agonist efficacy. Rational drug design that optimizes preferential effector activity within a group of receptor-selective drugs holds the promise of increased selectivity in clinically useful agents.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

Analysis of agonist-agonist interactions: the crucial influence of curve shape.

The two-receptor:one-transducer model (Leff, 1987) is here extended to analyze interactions between agonists displaying E[A] curves of different shapes, by incorporating slope factors into the separate and common parts of the transduction pathway. Interactions were modelled as the effect of one agonist, at fixed concentration, on the curve to the other. A variety of patterns of position and slope changes are predicted. These do not depend on the shape of the control curve, rather, they depend on the slope factors in the separate and common pathways. The following specific predictions are made: (1) when the common pathway is steep, curves undergo potentiation and flattening; (2) when the common pathway is flat, curves undergo right-shift and steepening; (3) when the common pathway is hyperbolic, curves undergo right-shift, with no slope change; (4) when the slope depends on the separate pathways, curves only undergo right-shift with no change in slope. The model provides a sound basis for classifying agonist interactions and for detecting additional, synergistic or antagonistic properties. This analysis indicates that methods based on dose-additivity or independence are less reliable for these purposes. The model provides a practical test, based on slope changes, to detect and quantify additional properties.

Dose-Response Relationship, Drug↗
PubMed1996

Partial agonist effects of BW A868C, a selective DP receptor antagonist, on Cl- secretion in dog tracheal epithelium.

We examined the interactions of prostaglandin D2, BW245C ((+/-)-5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)-hydantoin) a selective DP receptor agonist and BW A868C ((+/-)-3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2- hydroxyethylamino)-hydantoin) a selective DP receptor antagonist on Cl- secretion using dog isolated tracheal epithelial preparations in Ussing chambers. Both prostaglandin D2 and BW245C stimulated Cl- secretion as reflected by increased short-circuit current (Isc) in the epithelial cells where the latter was more potent than the former. BW A868C produced, consistently, weak but significant partial agonism on Cl- secretion in these preparations in addition to its expected antagonism at the DP receptors. A pKB estimate of 8.16 +/- 0.06 (n = 11) for BW A868C from its antagonism to BW245C was found to be comparable with its estimates of both p[A]50 (8.19 +/- 0.14, n = 5) and pKA (8.00 +/- 0.20, n = 5). In addition, no significant effect by BW A868C up to 1 microM on Cl- secretory responses to other prostanoids, such as prostaglandin E2, prostaglandin F2 alpha and 9 alpha, 11 beta-prostaglandin F2 alpha, was detected in the system. These results are consistent with previous findings that BW A868C is a selective antagonist at the DP receptors mediating Cl- secretion by epithelial cells. To our knowledge, this is a (the first) confirmation of partial agonist properties of BW A868C in an isolated tissue system.

Animals↗
PubMed1996

Interaction of BW A868C, a prostanoid DP-receptor antagonist, with two receptor subtypes in the rabbit isolated saphenous vein.

In isolated rings of rabbit saphenous vein (RbSV) pre-contracted with 40 mM KCl, Prostaglandin E2 (PGE2), BW245C (a DP-receptor agonist) and PGD2 caused concentration-dependent relaxations with mean potencies (EC50) of 0.5, 38 and 114 nM respectively. The DP-receptor antagonist, BW A868C, antagonized BW245C concentration-effect (E/[A]) curves, although the corresponding Schild plot had a slope less than unity and displayed a clear infection. Analysis of the data yielded two pKB estimates of 8.5 and 4.9, the higher estimate being consistent with antagonism at DP-receptors. The pA2 estimate of 5.1 obtained for BW A868C against PGE2 was not statistically different to the lower pKB of 4.9 obtained against BW245C, and is probably indicative of antagonism at the EP4-receptor. PGD2 mediated responses were also antagonized by BW A868C, however the resultant E/[A] curves were 'bell shaped' in nature. The weak EP4-receptor antagonist AH23848B, also antagonized BW245C and PGD2 responses, yielding pA2 estimates of 5.6 and 5.5 respectively. These results suggest that in the RbSV, BW245C and PGD2 are nonselective agonists mediating relaxations through DP- and EP4-receptors. BW A868C also displayed an affinity for DP-receptors in this preparation, in addition to a second receptor subtype, presumably the EP4-receptor.

Animals↗
PubMed1996

Synthesis of Hexahydrocyclopentimidazol-2-(1H)-one derivatives displaying selective DP-receptor agonist properties.

The rationale for investigating conformationally restricted analogues of BW245C as DP-receptor ligands and the syntheses of three such racemic bicyclic imidazolidinone analogues are described. Compounds 7 (BW587C), 8 (BW480C85), and 9 (BW572C85) were found to be potent inhibitors of human platelet aggregation and selective DP-receptor agonists in washed platelet and jugular vein isolated tissue assays.

Animals↗
PubMed1995

Physiological effects of inverse agonists in transgenic mice with myocardial overexpression of the beta 2-adrenoceptor.

G-protein-coupled receptors are thought to have an inactive conformation (R), requiring an agonist-induced conformational change for receptor/G-protein coupling. But new evidence suggests a two-state model in which receptors are in equilibrium between the inactive conformation (R), and a spontaneously active conformation (R*) that can couple to G protein in the absence of ligand (Fig. 1). Classic agonists have a high affinity for R* and increase the concentration of R*, whereas inverse agonists have a high affinity for R and decrease the concentration of R*. Neutral competitive antagonists have equal affinity for R and R* and do not displace the equilibrium, but can competitively antagonize the effects both of agonists and of inverse agonists. The lack of suitable in vivo model systems has restricted the evidence for the existence of inverse agonists to computer simulations and in vitro systems. We have used a transgenic mouse model in which there is such marked myocardial overexpression of beta 2-adrenoceptors that a significant population of spontaneously activated receptor (R*) is present, inducing a maximal response without agonist. We show that the beta 2-adrenoceptor ligand ICI-118,551 functions as an inverse agonist, providing evidence supporting the existence of inverse agonists and validating the two-state model of G-protein-coupled receptor activation.

Adrenergic beta-Agonists↗
PubMed1995

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.

Binding, Competitive↗
PubMed1995

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.

Adenosine Triphosphate↗