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Biomedical subjects

P Leff

Publications and source records attributed to P Leff.

At least 73 records · Page 4Linked to original sources
PubMed1987

Resultant action of cimetidine in a cardiac adenylate cyclase assay: its elucidation by concentration-ratios analysis.

Cimetidine is widely used as a ligand for the classification of histamine H2-receptors in peripheral and central tissues. It demonstrates apparently simple competitive antagonism in isolated tissue assays and in brain adenylate cyclase assays. In this study its action in a cardiac adenylate cyclase assay was compared with its effects in the isolated guinea pig right atrial preparation. Using either histamine or impromidine, cimetidine expressed similar affinity (pKB = 6.1) in the intact tissue assay. In washed homogenates of guinea pig ventricle cimetidine appeared to antagonize competitively the stimulation by histamine of adenylate cyclase but the estimated pKB was 6.7. In contrast, the pKB for tiotidine was similar in both assays (7.7). Application of a recently developed concentration-ratios method indicated that cimetidine expressed a resultant action in the cyclase assay consisting of competitive H2-receptor antagonism and an additional inhibitory property involving a postreceptor action. The concentration-ratios analysis allowed the purely competitive component of cimetidine action to be quantified in the cyclase assay giving a pKB of 6.0.

Adenylyl Cyclases↗
PubMed1987

Resultant pharmacological actions of verapamil: quantification of competitive 5-hydroxytryptamine antagonism in combination with calcium antagonism.

According to previous reports, the effect of verapamil on 5-hydroxytryptamine (5-HT) responses in the rabbit aorta is likely to represent a resultant of 5-HT2-receptor antagonism and Ca++-channel blockade. In practice, verapamil produced rightward displacement and depression of 5-HT-concentration-effect curves in this tissue. Under these conditions, verapamil's competitive property may not be justifiably quantified by analyzing curve displacements. In order to detect and quantify this property, a recently developed concentration-ratios method was applied. This method depends on the effect of the test substance, verapamil here, on the expression of competition by a standard antagonist, spiperone in this study. Analysis of the concentration-ratios produced by spiperone in the presence of verapamil permitted receptor competition by verapamil to be detected. Quantification of this property indicated a pKB of 5.73. This estimate of affinity did not account for the total rightward displacement of 5-HT-concentration-effect curves that verapamil produced, indicating, presumably, that concomitant Ca++-antagonism contributed to these displacements. Also, this estimate of verapamil's 5-HT2-receptor affinity is lower than that derived elsewhere from an analysis of the overall antagonistic effect of verapamil on 5-HT responses in the rabbit aorta. These results illustrate the potentially general utility of the concentration-ratios methods in elucidating resultant pharmacological actions.

Animals↗
PubMed1986

Potential errors in agonist dissociation constant estimation caused by desensitization.

A simple model of receptor desensitization is developed and analysed to predict the consequences of acute receptor loss on the pharmacological quantification of agonist action. The model incorporates the desensitization scheme of Katz and co-workers (1957) into the operational model of agonism (Black & Leff, 1983) and, therefore, it assumes the occupancy theory of agonist action. Pharmacological effect-time profiles are simulated which illustrate (i) the extent to which overt fade may be detectable under different conditions and (ii) the extent to which measured pharmacological effects deviate from those which would be measured in the absence of desensitization. It is shown that the resulting agonist concentration-effect curves may be displaced rightwards from their equilibrium positions and that the agonist dissociation constants estimated from them may be overestimated. Such errors are predicted to occur regardless of whether or not fade is detectable in the effect-time profiles. Estimates of agonist efficacy appears to be unaffected by desensitization. The results of this analysis are discussed with respect to their implications for receptor classification using agonist dissociation constant estimates and for the development of agonist drugs.

Kinetics↗
PubMed1986

Pharmacological analysis of the interaction between Bay K 8644 and 5-HT in rabbit aorta.

Bay K 8644 potentiated and augmented 5-hydroxytryptamine (5-HT)-induced contractions in the rabbit, isolated aorta preparation, as manifested in leftward shift and increase in the asymptote of 5-HT E/[A] (effect vs concentration) curves. The operational model of agonism (Black & Leff, 1983) was used to analyse this interaction and the concomitant effects of irreversible receptor alkylation by phenoxybenzamine. The competitive effects of spiperone in the presence and absence of Bay K 8644 were also examined. From these analyses it is concluded that Bay K 8644 elicits its potentiating effects by increasing the efficacy of 5-HT at the 5-HT2 receptor with no alteration in affinity. This is consistent with the known effect of Bay K 8644 of causing an increase in the functional concentration of plasmalemmal calcium channels coupled to the 5-HT2 receptors in this preparation. The positively co-operative shape of the 5-HT E/[A] curves obtained in the aorta and the quantitative nature of their potentiation by Bay K 8644 indicated that the coupling of 5-HT2 receptor occupancy to intracellular calcium concentration is linear and that the co-operativity resides in the subsequent relation between intracellular calcium and pharmacological effect. Bay K 8644 may serve as a probe for differentiating between the types of calcium channels that transduce 5-HT receptor-mediated effects in different systems. Such information would be useful in the classification of agonist interactions with 5-HT receptors.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Peripheral 5-HT2-like receptors. Can they be classified with the available antagonists?

Interactions between 5-hydroxytryptamine (5-HT) and the so-called 5-HT2 receptor antagonists ketanserin, spiperone, trazodone and methysergide were studied in isolated preparations of the rabbit aorta, rat jugular vein, and rat caudal artery. Trazodone and spiperone were apparently simple competitive antagonists since they produced antagonism that was surmountable over the concentration range studied and, in each tissue, their apparent affinity appeared to be independent of the antagonist concentration. Furthermore, concentration-ratios obtained with the two antagonists in combination suggested that antagonism was additive, implying mutual competition with a single population of 5-HT receptors. Ketanserin was a non-surmountable antagonist of 5-HT in the rat caudal artery and methysergide demonstrated surmountable, competitive antagonism only in the rabbit aorta. Antagonist dissociation constants estimated for apparently competitive interactions showed that ketanserin, spiperone and trazodone expressed affinities which differed according to the tissue used. In the case of trazodone, affinity estimates differed by as much as 12 fold. These discrepancies were independent of the 5-HT receptor agonist used and could not be attributed to an inadequate equilibration of the antagonist. These results can be interpreted in two ways: either the receptors in the different tissues are heterogeneous or the antagonists used here must be considered as unreliable probes for the classification of 5-HT2-like receptors.

Animals↗
PubMed1986

The classification of peripheral 5-HT2-like receptors using tryptamine agonist and antagonist analogues.

In a previous study, we attempted to verify the classification of 5-hydroxytryptamine2 (5-HT2) receptors in three vascular tissues, by use of the conventional antagonists, ketanserin, spiperone, methysergide and trazodone. However, it was not possible to conclude homogeneity of the receptor type in the three tissues due to the inconsistent behaviour of these antagonists, in particular, their apparently variable affinities between the tissues. These results led to the reliability of the conventional antagonists being questioned as receptor probes. In the present study, a set of tryptamine analogues were investigated in two of the tissues, the rabbit aorta and the rat jugular vein. Unlike the conventional antagonists, these compounds bear a close chemical relation to the natural agonist, 5-HT. In both tissues, alpha, alpha-dimethyltryptamine demonstrated apparently simple competitive antagonism of 5-HT-induced constrictions. Its affinity was estimated to be the same in each case. The affinities and relative efficacies of 5-HT, 5-cyanotryptamine, N,N-dimethyltryptamine and N-benzyl-5-methoxytryptamine were also found to be indistinguishable between the two tissues. Unlike the conventional 5-HT2 receptor antagonists, these tryptamine analogues failed to distinguish between the 5-HT receptors in the rabbit aorta and rat jugular vein implying that they truly belong to the same class. In view of this result, it is suggested that simple tryptamine analogues are more reliable probes for 5-HT receptor classification than ligands which bear little or no chemical relation to the natural agonist.

Animals↗
PubMed1986

Analysis of competitive antagonism when this property occurs as part of a pharmacological resultant.

In this paper, pharmacological resultant is defined as the net effect of a single compound resulting from the simultaneous expression of two or more specific actions. The principles of concentration-ratio analysis are extended to develop a method for detecting and quantifying competitive antagonism when this property is a component of a pharmacological resultant. The method is general to the extent that it allows analysis of competitive antagonism in combination with all types of post-receptor intervention. Essentially it depends on the altered expression of competition by a reference antagonist. It incorporates tests for validating its application and it is independent of agonist concentration-effect curve shape: in these respects the method is analogous to Schild plot-analysis of simple competition. The methodology for the practical application of the analysis is exemplified by studying the net effect of a combination of a phosphodiesterase inhibitor (isobutylmethylxanthine) and histamine H2-receptor antagonist (metiamide) on histamine-stimulated tachycardia in guinea-pig, isolated, right atrium. Cimetidine was used as the reference antagonist. The equation used in this analysis is similar in form to one recently described by Hughes & Mackay (1985) to elucidate the situation when competitive antagonism occurs in combination with functional interactions. The relation between their method and the present analysis is discussed.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Quantification of H2-agonism by clonidine and dimaprit in an adenylate cyclase assay.

In homogenates of guinea-pig ventricle clonidine and dimaprit both stimulate adenylate cyclase and exhibit "bell-shaped' E/[A] curves. The two properties (stimulatory and inhibitory) could be resolved using a theoretical model assuming a "down line' auto-inhibitory mechanism. In the case of clonidine a further depressive property could be seen in the presence of high concentrations of the selective H2-receptor antagonist tiotidine which is not explicable in terms of this model. The results suggest that clonidine has a direct agonistic effect on H2-receptors in guinea-pig heart. However, like dimaprit, clonidine appears to be a partial agonist because its expression is confounded by a secondary inhibitory property(ies).

Adenylyl Cyclases↗
PubMed1985

Further analysis of anomalous pKB values for histamine H2-receptor antagonists on the mouse isolated stomach assay.

Agonist-antagonist interactions at histamine receptors have been re-examined using improved techniques, on the mouse isolated, lumen-perfused, stomach gastric acid assay. Using histamine as agonist, pKB values have been estimated for burimamide, metiamide, cimetidine, ranitidine, oxmetidine and famotidine on both the gastric and guinea-pig isolated right atrium assays. With the exception of oxmetidine on the atrial assay, these compounds behaved as competitive antagonists on both assays. Oxmetidine significantly depressed basal rate on the atrial assay and the Schild plot slope parameter (0.81) was significantly less than one. The pKB values estimated on the gastric assay were lower than those on the atrial assay. However, the difference between the values on the gastric and atrial assays was not constant. The difference between the two assays for famotidine was not significant. We conclude that the apparent varying selectivity of the antagonists for gastric and atrial histamine H2-receptors may be explained by the differential loss of antagonists into the gastric secretion from the receptor compartment and that there is no need to postulate heterogeneity of histamine H2-receptors.

Animals↗
PubMed1985

Pharmacological analysis of the pentagastrin-tiotidine interaction in the mouse isolated stomach.

The pentagastrin-tiotidine interaction has been analysed, using improved techniques, in the mouse isolated, lumen-perfused, stomach assay. For comparison and quantification of the H2-receptor blocking activity of tiotidine, histamine-tiotidine interactions have also been analysed in the mouse stomach and guinea-pig isolated right atrial preparation. Tiotidine behaved as a competitive antagonist of histamine both in the guinea-pig right atrium (pKB 7.57) and mouse stomach (pKB 6.96). The difference in pKB was attributed to the loss of tiotidine into the gastric secretion. On the stomach assay, pentagastrin concentration-effect curves were significantly flatter with lower maximal responses than those obtained to histamine. In addition the profile of inhibition observed with tiotidine was different in that the pentagastrin curve maxima were depressed with only a small concomitant dextrad shift. A mathematical model has been developed which accounts for the differences in agonist concentration-effect curves and describes in a quantitative manner the expectations for the competitive antagonism of endogenous histamine assumed to be released by pentagastrin. Fitting of the pentagastrin-tiotidine data to this model provided a reasonable goodness-of-fit. The results are discussed in terms of the role of endogenous histamine in gastrin-stimulated acid secretion. We conclude that the results are consistent with the hypothesis that pentagastrin stimulates acid secretion via the release of endogenous histamine under the present experimental conditions.

Animals↗
PubMed1985

Application of the operational model of agonism to establish conditions when functional antagonism may be used to estimate agonist dissociation constants.

The operational model of agonism (Black & Leff, 1983) has been used to analyse comparatively functional antagonism and irreversible antagonism as methods for estimating agonist dissociation constants (KAs). A general condition is established in terms of the model parameters which defines the type of experimental interventions at the receptor and the post-receptor level that allow valid KA estimation. It is shown that functional antagonism and other post-receptor interventions may produce changes in agonist-concentration effect curves which are qualitatively indistinguishable but quantitatively distinct, from those produced by irreversible antagonism. Experimental data obtained with the guinea-pig tracheal strip preparation are in keeping with the theoretical predictions and show how studies using functional antagonism may overestimate agonist affinity. In general, functional antagonism, unlike irreversible antagonism, is in principle an unreliable method for quantifying agonism.

Animals↗
PubMed1985

An operational model of pharmacological agonism: the effect of E/[A] curve shape on agonist dissociation constant estimation.

An operational model of pharmacological agonism has been analysed to predict the behaviour of rectangular hyperbolic and non-hyperbolic agonist-concentration effect, E/[A], curves with variation in receptor concentration, [Ro]. Irreversible antagonism is predicted to cause E/[A] curve gradient changes in non-hyperbolic cases but not in hyperbolic cases; in both cases estimation of agonist dissociation constants (KAS) is theoretically valid. 5-Hydroxytryptamine (5-HT) produced "steep' E/[A] curves in contracting the rabbit isolated aorta preparation. Irreversible antagonism by phenoxybenzamine (Pbz) produced a flattened E/[A] curve, consistent with theoretical predictions. Fitting 5-HT E/[A] curves in the presence and absence of Pbz to the model provided an estimate of KA for 5-HT which was not significantly different from the estimate obtained using Furchgott's null method. The operational model of agonism appears to account qualitatively and quantitatively for the effects of [Ro] changes on hyperbolic and non-hyperbolic E/[A] curves. Under conditions where irreversible antagonism may be used to estimate KAS, fitting the operational model directly to E/[A] data represents a valid, economical and analytically simple alternative to the conventional null method.

Animals↗
PubMed1985

Pharmacological analysis of beta-adrenoceptor-mediated agonism in the guinea-pig, isolated, right atrium.

The recently developed, operational model of pharmacological agonism defines the efficacy of agonists by tau = [Ro]/KE, where [Ro] is the total functional concentration of receptors and KE is the concentration of agonist-occupied receptors for half-maximal effect. Theoretically, variations in [Ro] and KE affect tau and in turn, E/[A] curve profiles similarly. Using the beta-adrenoceptor mediated chronotropic responses of the guinea-pig isolated right atrial preparation we have investigated the consequences of experimental [Ro] and KE variation. Bromoacetylalprenolol menthane (M-75) produced displacements of isoprenaline and dichloroisoprenaline E/[A] curves consistent with [Ro] reduction. Cholera toxin produced displacements consistent with decreases in KE. The operational model provides a simple conceptual framework for the prediction and interpretation of changes in E/[A] curve profile resulting from experimental interventions at the post-receptor (KE) level as well as at the receptor ([Ro]) level.

Adrenergic beta-Antagonists↗
PubMed1983

Operational models of pharmacological agonism.

The traditional receptor-stimulus model of agonism began with a description of drug action based on the law of mass action and has developed by a series of modifications, each accounting for new experimental evidence. By contrast, in this paper an approach to modelling agonism is taken that begins with the observation that experimental agonist-concentration effect, E/[A], curves are commonly hyperbolic and develops using the deduction that the relation between occupancy and effect must be hyperbolic if the law of mass action applies at the agonist-receptor level. The result is a general model that explicitly describes agonism by three parameters: an agonist-receptor dissociation constant, KA; the total receptor concentration, [R0]; and a parameter, KE, defining the transduction of agonist-receptor complex, AR, into pharmacological effect. The ratio, [R0]/KE, described here as the 'transducer ratio', tau, is a logical definition for the efficacy of an agonist in a system. The model may be extended to account for non-hyperbolic E/[A] curves with no loss of meaning. Analysis shows that an explicit formulation of the traditional receptor-stimulus model is one particular form of the general model but that it is not the simplest. An alternative model is proposed, representing the cognitive and transducer functions of a receptor, that describes agonist action with one fewer parameter than the traditional model. In addition, this model provides a chemical definition of intrinsic efficacy making this parameter experimentally accessible in principle. The alternative models are compared and contrasted with regard to their practical and conceptual utilities in experimental pharmacology.

Kinetics↗
PubMed1981

Steady-state kinetic studies on benzylamine oxidase from pig plasma.

Steady-state kinetic studies on the enzyme benzylamine oxidase from pig plasma are described. Eadie-Hofstee plots with benzylamine as the varying substrate are non-linear; examination of this data indicates that the observed effects are probably due to the amine substrate participating in at least two reactions with enzyme. Ammonia and imidazole modify the activity of the enzyme; under specified conditions of pH or modifier concentration, the effect on the activity can be either activation or inhibition. Eadie-Hofstee plots of the data establish that the modifier also participates in at least three reactions with the enzyme. Eadie-Hofstee plots at pH 9 with oxygen as the varying substrate are linear, which allows kinetic parameters to be determined. From studies on the effect of ammonia and imidazole on these parameters, information has been derived on how these modifiers affect component steps of the catalytic cycle.

Ammonium Chloride↗
PubMed1980

Deviations from Michaelis-Menten kinetics. The possibility of complicated curves for simple kinetic schemes and the computer fitting of experimental data for acetylcholinesterase, acid phosphatase, adenosine deaminase, arylsulphatase, benzylamine oxidase, chymotrypsin, fumarase, galactose dehydrogenase, beta-galactosidase, lactate dehydrogenase, peroxidase and xanthine oxidase.

The possible graph shapes for one-site/two-state and substrate-modifier models are discussed. The two-state model is a version of the Monod-Wyman-Changeux model and gives a rate equation with 240 denominator terms. Discussion in terms of K and V effects is not possible. A simplified version of the mechanism can be shown to give v-versus-[S] curves that are either sigmoid or non-sigmoid. They may show substrate inhibition or no final maximum, and the double-reciprocal plots can be concave up or down. The corresponding binding model is determined by only two constants and gives a linear double-reciprocal plot. The substrate-modifier mechanism is a simple example of a mechanism where inclusion of catalytic steps leads to a genuine increase in degree of the rate equation. The v-versus-[S] curve can show such complexities as two maxima and a minimum, and the double-reciprocal plot can cross its asymptote twice, proving the rate equation to be 4:4. A simplified version is 3:3, and analysis shows that at least 18 of the 27 double-reciprocal plots that can arise with 3:3 functions are possible with this particular mechanism. Representative double-reciprocal and Scatchard plots are presented for several sets of rate-constant values. It is concluded that relatively simple mechanisms give pseudo-steady-state rate equations of high degree and considerable complexity. With extended ranges of substrate concentrations there is every reason to believe that experimental data would show the sort of deviations from Michaelis-Menten kinetics seen with calculated curves for such simple mechanisms. Narrow ranges of substrate concentration, on the other hand, would lead to inflexions and curvature being overlooked. It is not possible to discuss such deviations from Michaelis-Menten kinetics in terms of kinetic constants such as Km and V, and, in general, it is also difficult to see any simple way to explain intuitively such features as sigmoidicity, substrate inhibition, double-reciprocal convexity and decrease in degree by cancellation of common factors between numerator and denominator of rate equations. These conclusions apply with even more force when catalytic steps are included, for then the rate equations, are for multi-site mechanisms, of higher degree, allowing increasingly complex curve shapes. A number of enzymes were studied and initial-rate data were fitted by computer.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholinesterase↗