L-648,051, sodium 4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)- propylsulfonyl]-gamma-oxo-benzenebutanoate: a leukotriene D4 receptor antagonist

L-648,051, sodium 4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy) propylsulfonyl]-gamma-oxo-benzenebutanoate is a selective and competitive inhibitor of [3H]leukotriene D4 (KB value of 4.0 microM) and to a lesser extent [3H]leukotriene C4 (Ki value of 36.7 microM) binding in guinea pig lung homogenates. Functionally, it selectively antagonized contractions of guinea pig trachea induced by leukotrienes C4, D4, E4, and F4 in concentrations that did not antagonize contractions induced by acetylcholine, histamine, serotonin, prostaglandin F2 alpha, or U-44069 (endoperoxide analogue). Schild plot analysis indicated that L-648,051 competitively antagonized contractions of guinea pig ileum induced by leukotriene D4 (pA2 7.7) and contractions of trachea induced by leukotrienes D4, E4, and F4 (pA2 7.3, 7.4, and 7.5, respectively). Contractions of guinea pig trachea induced by leukotriene C4 were inhibited in a noncompetitive fashion (Schild plot slope, 0.45). Developed contractions of trachea induced by the leukotrienes were rapidly reversed by L-648,051 greater than FPL-55712 greater than L-649,923. Intravenous L-648,051 selectively blocked bronchoconstriction induced in anaesthetized guinea pigs by intravenous leukotrienes C4, D4, and E4 but not that induced by arachidonic acid, serotonin, U-44069, or acetylcholine. The compound displayed poor activity following intraduodenal administration. The profile of activity for L-648,051 indicates that it may be a useful topical agent for studying the role of leukotrienes in diseases such as bronchial asthma.     

Characterisation of CGRP receptors in the human isolated middle meningeal artery

Although the understanding of migraine pathophysiology is still incomplete, there seems to be little doubt that dilatation of cranial blood vessels, including meningeal arteries, is involved in the headache phase of migraine. Since calcitonin gene-related peptide (CGRP) has been implicated in this vasodilatation, the present study set out to compare the relaxant effects of the endogenous ligand h-alphaCGRP, and [ethylamide-Cys(2,7)]h-alphaCGRP ([Cys(Et)(2,7)]h-alphaCGRP), a CGRP(2) receptor agonist, on human isolated middle meningeal artery segments, precontracted with KCl. Classical Schild plot analysis was used to characterise the receptor population in this artery using BIBN4096BS and h-alphaCGRP(8-37) as antagonists. h-alphaCGRP relaxed arterial segments more potently than [Cys(Et)(2,7)]h-alphaCGRP (pEC(50): 8.51+/-0.16 and 7.48+/-0.24, respectively), while the maximal responses to these agonists were not significantly different. BIBN4096BS equipotently blocked the relaxations induced by both agonists with a pA(2) of approximately 10 and with a Schild plot slope not significantly different from unity. h-alphaCGRP(8-37) also antagonised the response to h-alphaCGRP with a pA(2) of 6.46+/-0.16 and a Schild plot slope not different from unity. Furthermore, the results obtained from RT-PCR studies confirmed the presence of all the essential components required for a functional CGRP(1) receptor in these arteries. Considering the high antagonist potency of BIBN4096BS, coupled to the lower agonist potency of [Cys (Et)(2,7)]h-alphaCGRP, it is reasonable to suggest a predominant role of CGRP(1) receptors in the human middle meningeal artery. This view is reinforced by Schild plot analysis, which revealed a slope of unity in all experiments, giving further evidence for a homogeneous CGRP receptor population in this vascular preparation.     

An analysis of the in vivo interactions between chemical convulsants and anticonvulsants

The interactions between pentylenetetrazol (PTZ), picrotoxin (PIC), or bicuculline (BIC) and diazepam, phenobarbital, or valproate were subjected to Schild plot analysis. Log dose-probit response curves for minimal clonic seizures were determined for three chemical convulsants in the absence and in the presence of various concentrations of three anticonvulsants. The calculated median convulsant doses were subjected to Schild plot analysis and the pA2 values determined. A comparison of the pA2 values for the various convulsant/anticonvulsant combinations suggested the following conclusions: (i) the sequence of events leading to minimal clonic seizures evoked by PTZ or PIC involves a common receptor, (ii) BIC acts through a different receptor, and (iii) Schild plot analysis of the antagonism between convulsant and anticonvulsant is in agreement with their antagonism in vitro studies. Thus, Schild plot analysis can be useful in the evaluation of anticonvulsant activity in vivo and may offer some insight into the potential clinical usefulness of anticonvulsant substances.     

Pharmacological study of the effects of leukotrienes C4, D4, E4 & F4 on guinea pig trachealis: interaction with FPL-55712

Leukotrienes D4 greater than C4 greater than E4 greater than F4 produced qualitatively similar contractions of guinea-pig trachealis, which were antagonized by the SRS-antagonist FPL-55712. Schild analyses indicated that FPL-55712 when tested in a low concentration range (0.57 - 5.7 X 10(-6) M) was a competitive antagonist of LTC4, LTE4 and LTF4 (slope not significantly different from one). The interaction of FPL-55712 with LTD4 may be noncompetitive (slope less than 1). Comparison of the calculated dissociation constants (-log KB) indicated that FPL-55712 was more effective at blocking LTE4 and LTF4 compared to LTC4 and LTD4. In the presence of higher concentrations of FPL-55712 (1.9 X 10(-5) M) the antagonism of LTC4 became noncompetitive. These findings indicate that important differences exist in the interaction of FPL-55712 with the various peptido leukotrienes in guinea pig trachealis. Discovery of more selective antagonists will be needed to determine if multiple receptor subtypes are present in this tissue.     

L-649,923, sodium (beta S*, gamma R*)-4-(3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propylthio)- gamma-hydroxy-beta-methylbenzenebutanoate, a selective, orally active leukotriene receptor antagonist

L-649,923, Sodium (beta S*, gamma R*)-4-(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propylthio)- gamma- hydroxy-beta-methylbenzenebutanoate is a selective and competitive inhibitor of [3H]leukotriene D4 (Ki value of 400 nM) and to a lesser extent [3H]leukotriene C4 (Ki value of 8.6 microM) binding in guinea-pig lung homogenates. Functionally, it selectively antagonized contractions of guinea pig trachea induced by leukotriene C4, D4, E4, and F4 but not those induced by acetylcholine, histamine, serotonin, prostaglandin F2 alpha, or U-44069 (stable endoperoxide analogue). Schild plot analysis indicated a competitive inhibition of contractions of guinea-pig ileum induced by leukotriene D4 (pA2 8.1) and contractions of guinea-pig trachea induced by leukotrienes E4 and F4 (pA2 7.1 and 6.9, respectively). In contrast, contractions of guinea-pig trachea induced by leukotrienes C4 (pA2 7.2; slope 0.6) and D4 (pA2 7.2; slope 0.7) were inhibited in a noncompetitive fashion. In vivo, intravenously administered L-649,923 selectively blocked bronchoconstriction induced in anesthetized guinea pigs by leukotriene C4 and D4 (ED50 values i.v. 0.38 and 0.26 mg/kg, respectively) but not that induced by histamine, arachidonic acid, serotonin, U-44069, or acetylcholine. Following intraduodenal administration, L-649,923, blocked leukotriene D4 induced bronchoconstriction (5 and 10 mg/kg). The present findings indicate that selective antagonists, such as L-649,923, may be useful for defining the role of leukotrienes in diseases such as bronchial asthma.     

The Dissociation Rate of Unlabelled Drugs from Receptor Sites: A Poorly Investigated,Yet Important Aspect in Receptor Research. Studies on the Serotonin-S2 Receptor

Abstract

The labelling by ³H-spiperone of serotonin-S₂ receptors in rat frontal cortex tissue adsorbed to glass fibre filters was investigated. For 12 unlabelled serotonin antagonists the dissociation time from serotonin-S₂ receptors was measured using rat frontal cortex tissue preparations adsorbed to glass fibre filters. The dissociation half-time varied from 4.8 min for pipamperone to 160 min for ritanserin. The drug-receptor dissociation time was not related to a particular class of chemical structure, or to the lipophilicity or the acid dissociation constant of the drugs. The essential requirement of experimental determination of the drug-receptor dissociation time for each drug individually is illustrated. The possible applications of the knowledge of the drug-receptor dissociation time in in vitro and in vivo receptor studies, in pharmacological and pharmaco-kinetic studies and in drug design and receptor modelling is discussed. For various serotonin-S₂ antagonists, the type of inhibition produced by the drug on ³H-ketanserin binding to serotonin-S₂ receptors was determined using suspensions of rat frontal cortical tissue. The observed patterns of inhibition were clearly related to the drug-receptor dissociation times: rapidly dissociating drugs produced competitive inhibition, drugs with dissociation half-times between 15-30 min produced mixed type inhibition, and the very slowly dissociating ritanserin produced non-competitive inhibition.     

Estimation of the affinity of naloxone at supraspinal and spinal opioid receptors in vivo: studies with receptor selective agonists

The apparent affinity of naloxone at cerebral and spinal sites was estimated using selective mu [D-Ala2, Gly-o15]-enkephalin (DAGO) and delta [D-Pen2, D-Pen5]enkephalin] (DPDPE) opioid agonists in the mouse warm water tail-withdrawal test in vivo; the mu agonist morphine was employed as a reference compound. The approach was to determine the naloxone pA2 using a time-dependent method with both agonist and antagonist given intracerebroventricularly (i.c.v.) or intrathecally (i.th.); naloxone was always given 5 min before the agonist. Complete time-response curves were determined for each agonist at each site in the absence, and in the presence, of a single, fixed i.c.v. or i.th. dose of naloxone. From these i.c.v. or i.th. pairs of time-response curves, pairs of dose-response lines were constructed at various times; these lines showed decreasing displacement with time, indicative of the disappearance of naloxone. The graph of log (dose ratio-1) vs. time was linear with negative slope, in agreement with the time-dependent form of the equation for competitive antagonism. From this plot, the apparent pA2 and naloxone half-life was calculated at each site and against each agonist. The affinity of naloxone was not significantly different when compared between agonists after i.c.v. administration. A small difference was seen between the affinity of i.th. naloxone against DPDPE and DAGO; the i.th. naloxone pA2 against morphine, however, was not different than that for DPDPE and DAGO. The naloxone half-life varied between 6.6 and 16.9 min, values close to those previously reported for this compound. These results suggest that the agonists studied may produce their i.c.v. analgesic effects at the same receptor type or that alternatively, the naloxone pA2 may be fortuitously similar for mu and delta receptors in vivo. Additionally, while the affinity of naloxone appears different for the receptors activated by i.th. DAGO and DPDPE, further work may be necessary before firm conclusions regarding the nature of the spinal analgesic receptor(s) can be drawn.     

Comparative characteristics of the discriminative and analgesic effects of morphine

Experiments on mice were made to study and compare the discriminative and analgesic effects of morphine. The time-course of tolerance to the drug effects was found to be different. The Schild method permitted one to determine significantly different characteristics of naloxone antagonism (pA2) as regards the analgesic and discriminative effects of morphine. The data obtained attest to the different mechanisms of the analgesic and discriminative effects of morphine.     

Pharmacologic methods for identification of receptors

Determinations of apparent equilibrium dissociation constants of drug-receptor interactions are made from both functional and radioligand binding studies. In each type of study, reversible reactions are assumed and the mass action law is applied. Functional studies are frequently used to determine the dissociation constant of a competitive antagonist but are less frequently used to obtain this constant for agonist compounds since the latter determination requires an experimental procedure that irreversibly inactivates a fraction of the receptors. In the present report, values of dissociation constant for prototype agonists and antagonists, determined from binding and from functional studies, are examined in two classical isolated preparations, rabbit aorta and guinea-pig ileum. In each preparation the dissociation constants from binding and functional experiments agree well for the antagonists but differ markedly for the agonists. Further, the dissociation constant values from binding are seen to be greater for the agonists than for the antagonists. When a chronic treatment regimen in the rabbit resulted in a pronounced change in the functional dissociation constant of subsequently administered norepinephrine, there was no significant change in either the binding constant of this agonist or in the pA2 value of the alpha antagonist, phentolamine. These, and the previously described results, are shown to be compatible with a simple two-state receptor model in which agonists bind with high and low affinity to each state while antagonists do not distinguish between the states. In this model, the ratio of low to high affinity states accounts for the failure of the binding procedure to detect changes in the agonists dissociation constant that are highly significant in the functional study. Whereas the model is based on data for these two classical preparations only, and may not be more generally applicable, the findings demonstrate the necessity for employing both functional and radioligand binding experiments when characterizing drug receptors.     

Electrostatic dependence of the thrombin-thrombomodulin interaction

The rate constants for the binding interaction between thrombin and a fully active fragment of its anticoagulant cofactor, thrombomodulin, have been determined by surface plasmon resonance. At physiological ionic strength, the k(a) was 6.7x10(6) M(-1) s(-1 )and the dissociation rate constant was 0.033 s(-1). These extremely fast association and dissociation rates resulted in an overall binding equilibrium constant of 4.9 nM, which is similar to previously reported values. Changing the ionic strength from 100 mM to 250 mM NaCl caused a tenfold decrease in the association rate while the dissociation rate did not change significantly. A similar effect was observed with tetramethylammonium chloride. A Debye-Hückel plot of the data had a slope of -6 and an intercept at 0 ionic strength of 10(9) M(-1) s(-1). The same slope and intercept were obtained for data that was collected in the presence of glycerol to slow the association rates. These results show that the thrombin-TM456 interaction is extremely rapid and nearly completely electrostatically steered. An association model is presented in which TM456 approaches thrombin along the direction of the thrombin molecular dipole.     

Efffect of noscapine,the antitussive opioid alkaloid,on bradykinin-induced smooth muscle contraction in the isolated ileum of the guinea-pig

Bradykinin (BK) and related kinins are autocoid peptides that play integral roles in many pathophysiological processes such as cough. In this study, the inhibitory effect of noscapine, the antitussive opioid alkaloid, on BK receptors, was tested in the guinea-pig ileum. Contractions of the isolated ileum of the guinea-pig in response to BK were inhibited by noscapine (10-1,000 nM) in a concentration-dependent manner. Concentration-response curves (CRCs) to BK were slightly shifted to the right with a concomitant decrease in the maximum effect. A pA2 value of 6.68 was calculated for noscapine. The slope of the Schild plot of the antagonism was found to be 0.56. Noscapine had no effect on contractions induced by KCl, acetylcholine, histamine, 5-hydroxy tryptamine or angiotensin II. In conclusion, noscapine has a specific antagonistic effect on BK receptors and the mode of inhibition was found to be non-competitive.     

Antagonism by nalmefene of systemic and intrathecal morphine-induced analgesia in mice

Nalmefene is an orally active opiate antagonist structurally related to naloxone and naltrexone. In this study using two different strains of mice (Swiss Cox and ICR), the antagonist activity of nalmefene given subcutaneously (sc) was quantified by determination of the apparent pA2 values against the antinociceptive activity (tail flick and hot plate tests) of morphine given sc or intrathecally (lumbar spinal cord). The apparent pA2 values (constrained to a slope of -1) were 8.06 (7.79-8.33) in Swiss Cox mice and 7.81 (7.62-8.00) in ICR mice in the tail flick test with sc morphine. These values were larger than the corresponding value for naloxone in ICR mice, 7.35 (7.10-7.60). The hot plate test provided similar results: the apparent pA2 values for nalmefene with sc morphine were 8.14 (7.89-8.39) in Swiss Cox mice and 7.81 (7.65-7.97) in ICR mice, values which were different from naloxone 7.33 (7.23-7.42) in ICR mice. Apparent pA2 values for nalmefene with intrathecal morphine were not different from those for naloxone in the tail flick test. Thus, these sets of results suggest that it may be worthwhile to further determine whether systemic nalmefene might possibly possess an advantage over naloxone in antagonizing systemic side effects of morphine arising from local spinal morphine administration.     

Conservative whole‐organ scaling contrasts with highly labile suborgan scaling differences among compound eyes of closely related Formica ants

Static allometries determine how organ size scales in relation to body mass. The extent to which these allometric relationships are free to evolve, and how they differ among closely related species, has been debated extensively and remains unclear; changes in intercept appear common, but changes in slope are far rarer. Here, we compare the scaling relationships that govern the structure of compound eyes of four closely related ant species from the genus Formica. Comparison among these species revealed changes in intercept but not slope in the allometric scaling relationships governing eye area, facet number, and mean facet diameter. Moreover, the scaling between facet diameter and number was conserved across all four species. In contrast, facet diameters from distinct regions of the compound eye differed in both intercept and slope within a single species and when comparing homologous regions among species. Thus, even when species are conservative in the scaling of whole organs, they can differ substantially in regional scaling within organs. This, at least partly, explains how species can produce organs that adhere to genus wide scaling relationships while still being able to invest differentially in particular regions of organs to produce specific features that match their ecology.     

pA2 and receptor differentiation: a statistical analysis of competitive antagonism.

The pA₂ of a competitive antagonist was defined by Schild as the negative logarithm of the molar concentration of an antagonist which reduces the effect of a dose of agonist to that of half the dose. The relationship of the pA₂ to the dissociation constant, K, of the antagonist-receptor complex is discussed within the framework of competitive theory. Various methods for the determination of pA₂, and the accuracy and precision of these methods, are presented. Special problems associated with the determination of pA₂in vivo are also discussed, with particular attention being given to narcotic analgesics and their antagonists.     

Kinetics of the reaction of prostaglandin H synthase compound II with ascorbic acid

The reduction of prostaglandin H synthase compound II by ascorbic acid in the presence of diethyldithiocarbamate was studied in 0.1 M phosphate buffer (pH 8.0) at 4.0 +/- 0.5 degrees C, by rapid scan spectrometry and transient state kinetics. A saturation effect and nonzero intercept were observed in the plot of pseudo-first-order rate constant versus ascorbic acid concentration. The saturation behavior suggests formation of a complex between prostaglandin H synthase compound II and ascorbic acid, whereas the nonzero intercept is attributable to the reaction of compound II of prostaglandin H synthase with diethyldithiocarbamate present in the system as a stabilizing agent. A rate equation has been derived which includes all pathways for the conversion of prostaglandin H synthase compound II back to native enzyme. Kinetic parameters for the reduction of compound II by ascorbic acid were obtained. They are the second-order rate constant of (1.4 +/- 0.5) X 10(5) M-1, S-1, for the formation of the compound II-ascorbic acid complex, the first-order rate constant of (14 +/- 4) S-1 for the oxidation-reduction reaction of the complex and its dissociation, and a parameter, Km of 92 +/- 10 microM analogous to the Michaelis-Menten constant. Thus we demonstrate that a quantitative kinetic study on the prostaglandin H synthase reactions can be performed in the presence of diethyldithiocarbamate.     

Displacement Reactions Employing Heterologous Tracer Ligands in Peptide Receptor Studies: A Review

Abstract

Heterologous tracer ligands used in displacement studies with peptide-receptor systems may become unsuitable to these aims for several reasons. (1) The forms of binding isotherms for the tracer and for the ligand under investigation are different. The Schild plot and similar schemes are then not applicable. Possible modifications of the computational methods are indicated. (2) The rate of dissociation from the receptor is slowed, until almost irreversible. In such cases, there is no chance for displacement studies. (3) Large discrepancy in rate constants, or dramatically different distribution coefficients between binding system and water, for the two ligands mimic irreversible binding to receptors in a pharmacological experiment. Adjustment of ligand concentrations (control of association rate) may help in some instances.     

Steady-state kinetic mechanism of rat tyrosine hydroxylase.

The steady-state kinetic mechanism for rat tyrosine hydroxylase has been determined by using recombinant enzyme expressed in insect tissue culture cells. Variation of any two of the three substrates, tyrosine, 6-methyltetrahydropterin, and oxygen, together at nonsaturating concentrations of the third gives a pattern of intersecting lines in a double-reciprocal plot. Varying tyrosine and oxygen together results in a rapid equilibrium pattern, while the other substrate pairs both fit a sequential mechanism. When tyrosine and 6-methyltetrahydropterin are varied at a fixed ratio at different oxygen concentrations, the intercept replot is linear and the slope replot is nonlinear with a zero intercept, consistent with rapid equilibrium binding of oxygen. All the replots when oxygen is varied in a fixed ratio with either tyrosine or 6-methyltetrahydropterin are nonlinear with finite intercepts. 6-Methyl-7,8-dihydropterin and norepinephrine are competitive inhibitors versus 6-methyltetrahydropterin and noncompetitive inhibitors versus tyrosine. 3-Iodotyrosine, a competitive inhibitor versus tyrosine, shows uncompetitive inhibition versus 6-methyltetrahydropterin. At high concentrations, tyrosine is a competitive inhibitor versus 6-methyltetrahydropterin. These results are consistent with an ordered kinetic mechanism with the order of binding being 6-methyltetrahydropterin, oxygen, and tyrosine and with formation of a dead-end enzyme-tyrosine complex. There is no significant primary kinetic isotope effect on the V/K values or on the Vmax value with [3,5-2H2]tyrosine as substrate. No burst of dihydroxyphenylalanine production is seen during the first turnover. These results rule out product release and carbon-hydrogen bond cleavage as rate-limiting steps.     

Data-driven analysis in drug discovery

In the process of drug discovery for new chemical entities, application of appropriate pharmacological models often is not possible because the molecular mechanism of the compound is not yet elucidated. Therefore, a data-driven approach using generic tools designed to quantify characteristic patterns of concentration-response curves is required. This article outlines the options available for quantifying agonist and antagonist activity. Specifically, for agonists, the use of the Operational model for the determination of functional effects (equimolar potency ratios for full agonists, calculation of relative efficacy) is described. For antagonists, the measurement of pKB (-log of the equilibrium dissociation constant of the antagonist-receptor complex) for orthosteric antagonists that do not alter basal response (simple competitive antagonists), increase basal response (partial agonists), and decrease basal response (in constitutively active systems; inverse agonists) is discussed. In addition, this article considers methods to discern orthosteric receptor antagonism from allosteric antagonism whereby the agonist and antagonist bind to separate sites and interact through a conformational change in the receptor. Methods for the measurement of the pKB for allosteric modulators as well as co-operativity constants for these modulators is described.     

Aprotinin can inhibit the proteolytic activity of thrombin. A fluorescence and an enzymatic study.

Aprotinin has been shown to reduce blood loss and blood requirement when administered prior to surgery and this therapeutic benefit appears to be related to its specificity as a protease inhibitor. The inhibition of plasmin by aprotinin is well characterized, but little is known of its effect on thrombin. In preliminary experiments, we showed that aprotinin can prevent platelet aggregation induced by thrombin. Follow-up studies have now been performed in order to clarify the effect of aprotinin on thrombin. A fluorescence study of the direct binding of aprotinin to human alpha-thrombin was analysed according to the Michaelis-Menten model and a dissociation constant of 30 x 10(-6) mol.l-1 was determined. Aprotinin can displace p-aminobenzamidine, a fluorescent-probe molecule which binds to the active site of serine proteases, showing that the active site of thrombin was involved. Aprotinin also inhibited the ability of thrombin to induce a fibrin clot from purified fibrinogen and to induce the hydrolysis of the chromogenic substrate H-D-phenylalanylpipecolylarginine-p-nitroanalidehydrochloride++ + (S-2238). With S-2238, double-reciprocal plots show that the inhibition is competitive with a Ki of 61 microM and a Km of 1.72 microM. Aprotinin was a potent inhibitor of thrombin-induced aggregation. A Schild plot of the aggregation data yielded a slope of 0.97 +/- 0.12 and an apparent dissociation constant of 57.0 +/- 13.1 microM (mean +/- SEM). Thus, the inhibition of thrombin-induced platelet aggregation by aprotinin fits a model of competitive inhibition. Conclusions are that, in addition to a possible direct effect of aprotinin on platelets, the inhibition of thrombin-induced platelet activation by aprotinin can be also explained, in part, by a direct effect of the inhibitor on the thrombin molecule itself. This supports the concept that a proteolytic step is involved in the platelet response to thrombin. Finally, evidence is in favour of the participation of Trp245 in the fluorescence response of thrombin on binding to aprotinin.     

Antinociceptive effects of intrathecally administered human beta-endorphin in the rat and cat

Rats chronically implanted with intrathecal catheters displayed a dose-dependent increase in the hot-plate and tail-flick response latencies following the injection of human beta-endorphin into the lumbar spinal subarachnoid space through the indwelling catheter. beta-Endorphin was approximately 25 times more potent than morphine on a molar basis. Matching morphine and beta-endorphin doses such that approximately equal submaximal submaximal effects occurred, it was observed that the antinociception produced by beta-endorphin lasted approximately three times longer than that produced by morphine. Experiments with intrathecal injection of beta-endorphin into the spinal subarachnoid space of cats fitted with intrathecal catheters also revealed a potent antinociceptive effect which was completely antagonized by naloxone. In the rats, naloxone administered systemically in doses of 10--100 microgram/kg produced a parallel shift in the dose-response curves of both nociceptive measures suggesting a competitive antagonism. Using a dose ratio analysis, an in vivo pA2 of 7.1 for naloxone was obtained. These data and those derived from previous work based on the pA2 suggest that the interaction of morphine, certain pentapeptides, and beta-endorphin is the same with regard to the spinal opiate receptor population mediating behaviorally defined analgesia.