The part played by catechol-O-methyltransferase in the plasma kinetics of 3,4-dihydroxyphenylglycol and 3,4-dihydroxyphenylalanine in the anaesthetized rabbit

Summary The present study, carried out in anaesthetized rabbits, aimed at determining the effects of catechol-O-methytransferase (COMT) inhibition on the plasma kinetics of infused 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxyphenylalanine (DOPA) as well as on endogenous plasma noradrenaline, DOPEG, DOPA and 3-methoxy-4-hydroxyphenylglycol (MOPEG). The plasma kinetics of infused MOPEG were also evaluated. To block the function of COMT, 3,4-dihydroxy-4-methyl-5-nitrobenzophenone (Ro 40-7592) was given intravenously. Dose-finding, experiments, in which the drug-induced fall in endogenous plasma MOPEG was used to quantify COMT inhibition, indicated that a Ro 40-7592 dose of 3 mg/kg followed by 1.5 mg/kg every 30 min was sufficient to obtain a virtually complete inhibition of COMT.More than 150 min of COMT inhibition were required for endogenous MOPEG to disappear from plasma, since the plasma half-life of MOPEG was 54 min. COMT inhibition produced marked increases in the plasma levels of endogenous DOPA (1.7-fold) and DOPEG (3.9-fold) and did not alter endogenous plasma noradrenaline. The results concerning the effect of COMT inhibition on the plasma kinetics of infused DOPA and DOPEG were as follows: the plasma clearance of DOPA was not altered, whereas that of DOPEG fell by 41%; the plasma half-life of DOPA increased from 4.9 to 13.0 min and that of DOPEG from 4.8 to 31.0 min; there was an increase in the volume of distribution of DOPA (2 to 3-fold) and DOPEG (4 to 5-fold).Hence, COMT inhibition was much more effective in increasing endogenous plasma DOPA and DOPEG than in increasing the plasma concentrations of infused DOPA and DOPEG, suggesting that endogenously formed DOPA and DOPEG are more extensively metabolized by COMT than infused DOPA and DOPEG. Moreover, as the increase in the plasma half-lives of DOPA and DOPEG induced by COMT inhibition was mainly due to an increase in the volume of distribution, it can be concluded that the action of COMT limits the distribution of infused DOPA and DOPEG within the body.This study was supported by the Deutsche Forschungsgemeinschaft (GR 490/5). A preliminary account of the results was presented to the German Society for Pharmacology and Toxicology (Friedgen 1992)Correspondence to K.-H. Graefe at the above address     

Pre- and postganglionic stimulation-induced noradrenaline overflow is markedly facilitated by a prejunctional β2-adrenoceptor-mediated control mechanism in the pithed rat

The aim of the study was to further explore the prejunctional -adrenoceptor-mediated control mechanism of noradrenaline release from sympathetic nerves in response to preganglionic nerve stimulation (PNS) and local nerve stimulation of the portal vein, respectively, in the pithed rat.Baseline values as well as the increments of mean arterial blood pressure (-BP), heart rate (-HR) and plasma noradrenaline levels (-NA) in response to four PNS episodes (0.8 Hz, 3 ms, 75 V for 45 s at 20 min intervals), respectively, were evaluated. Fenoterol administration (0.25 mg/kg, i.v.) reduced significantly the basal blood pressure but did not alter d-BP in response to PNS. Basal heart rate markedly increased after fenoterol without any further change in heart rate induced by PNS.The ₁-selective antagonist CGP 20712A attenuated -BP in response to PNS and prevented the fenoterol-induced increase in basal heart rate. The ₂-selective antagonist ICI 118,551 per se did not change the blood pressure and heart rate values, but antagonized the fenoterol-induced decrease in basal blood pressure.Fenoterol enhanced plasma -NA in response to PNS by 105% in comparison to the corresponding control value. This effect of fenoterol could be blocked by pretreatment with ICI 118,551 but not with CGP 20712A (a selective ₁-adrenoceptor antagonist) which per se did not significantly change plasma -NA.Repeated local stimulation of the portal vein (S1–S 3, 2 Hz, 3 ms, 10 mA, for 120 s at 30 min intervals) increased portal plasma noradrenaline without changing mean blood pressure and heart rate in pithed rats. Fenoterol enhanced the increase in portal-vein plasma noradrenaline evoked by nerve stimulation by 110%. Pretreatment with ICI 118,551 antagonized this effect of fenoterol, but had per se no effect on the portal vein nerve stimulation-evoked increase in portal plasma noradrenaline.It is concluded that the increase in plasma noradrenaline evoked both by pre- and postganglionic nerve stimulation can be markedly enhanced by activation of a facilitatory prejunctional ₂-adrenoceptor control mechanism. Correspondence to: V.I. Tarizzo     

Generalization of the discriminative stimulus properties of x0394; 9 in rats

Rats were trained in a water maze to discriminate between IP injections of 3 mg/kg ⁹- (⁹⁽¹¹⁾-THC) and its vehicle. Both ⁸- and ⁹⁽¹¹⁾ were generalized to the training drug. In contrast to our observations in rhesus monkeys, where ⁹-THC is at least 100 times less potent than ⁹-THC, ⁹⁽¹¹⁾ was found to be only seven times less potent in the rat. Relative potencies, expressed as the dosage at which 50% of the animals gave drug responses (ED₅₀) were 1.8 mg/kg and 12.2 mg/kg for ⁹-THC and ⁹⁽¹¹⁾ respectively. Twenty-four hours after receiving 7×ED₅₀=12 mg/kg ⁹ the tests showed intermediate results when conducted with the training dosage; 4×ED₅₀=50 mg/kg ⁹-THC 48 h prior to the training dosage of 3 mg/kg ⁹-THC completely blocked drug-appropriate responses. Coinjection of ED₅₀ dosages of ⁹- and ⁹⁽¹¹⁾-THC led to 90% drug responses, demonstrating the additivity of the cannabis-like effect of both cannabinoids. Differences in the individual sensitivity of the rats to the tested cannabinoids were observed. Findings are interpreted in terms of the receptor mechanism for cannabis-like activity.This paper is dedicated to the memory of Dr. M. Binder who died on February 15, 1984     

Evidence for saturation of catechol-0-methyltransferase by low concentrations of noradrenaline in perfused lungs of rats

Previous studies on the pulmonary removal and metabolism of catecholamines in rat lungs have shown that, when the lungs are perfused with a low concentration (1 nmol/1) of noradrenaline, the amine is metabolized by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), but is predominantly O-methylated, and the activities of COMT and MAO are 0.357 min^–1 and 0.186 min^–1, respectively. The aim of the present study was to examine the changes in the metabolic profile of noradrenaline in rat lungs over a range of concentrations, and to examine the kinetics of the pulmonary O-methylation of noradrenaline and adrenaline.In isolated lungs perfused with ³H-noradrenaline, there was a progressive decrease in the proportion of O-methylated metabolites and a corresponding increase in the proportion of deaminated metabolites, as the noradrenaline concentration in the perfusion solution was increased from 1 to 10 to 100 to 1000 nmol/l. Experiments designed to determine the rate of uptake of noradrenaline in lungs perfused with 1 nmol/l ³H-noradrenaline, under conditions of MAO inhibited, COMT inhibited and COMT and MAO inhibited, showed that the results were compatible with co-existence of COMT and MAO in the pulmonary endothelial cells. Hence, it appeared that the changing metabolic profile with amine concentration in the previous series of experiments was not due to saturation of noradrenaline uptake into cells that contained COMT but not MAO.Further experiments to examine the kinetics of O-methylation of noradrenaline and adrenaline (MAO inhibited) showed that the O-methylation of these amines in the lungs was predominantly saturable, with half-saturation occurring at concentrations (9.8 nmol/I and 19.4 nmol/l, respectively) that were two orders of magnitude lower than those required to half-saturate uptake₁ of the amines. Saturation of O-methylation by these low concentrations of noradrenaline (1) provides the explanation for the change in the metabolic profile of noradrenaline described above and (ii) appears to occur because V_{max uptake} V_{max COMT} for the metabolizing system consisting of non-neuronal uptake₁ + COMT in the lungs, as has been described previously for the system consisting of uptake₂ + COMT in extraneuronal sites in rat heart. The results show that the metabolic profile of catecholamines in the pulmonary circulation will reflect that occurring at physiological levels only if studies are carried out with very low amine concentrations.Abbreviations COMT Catechol-O-methyltransferase - DOMA 3,4-dihydroxymandelic acid - DOPEG 3 4-dihydroxyphenylglycol - ECS Extracellular space - HSOC Half-saturating outside concentration - K_{m uptake} Half-saturation constant for uptake - k_COMT Rate constant for O-methylation - k_MAO Rate constant for deamination - k_{out NA} Rate constant for efflux of noradrenaline - MAO Monoamine oxidase - MB-COMT Membrane-bound - COMT NMN Normetanephrine - OMDA O-methylated deaminated metabolites - S-COMT Soluble COMT - T/M_NA Tissue to medium ratio of noradrenaline - U-0521 3,4-dihydroxy-2-methylpropiophenone - V_max Maximal rate of uptake or O-methylation - V_st-st Steady-state rate of metabolite formation - V_uptake Rate of uptake Preliminary results of part of this study were presented to the Seventh Meeting on Adrenergic Mechanisms, Porto, Portugal (Bryan 1990)     

A kinetic investigation of the pulmonary metabolism of dopamine in rats shows marked differences compared with noradrenaline

The aim of this study was to investigate the deamination of dopamine in the intact pulmonary circulation of isolated lungs of the rat. The first part of the study showed that dopamine is not converted to noradrenaline by dopamine--hydroxylase (DBH) when dopamine is perfused through isolated lung preparations with monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) inhibited. Hence, it was not necessary to inhibit DBH in subsequent experiments.The metabolite profile for deamination of dopamine in the lungs was examined by determining whether MAO and semicarbazide-sensitive amine oxidases (SSAO) contribute to the deamination of dopamine (and noradrenaline), and by determining the activity of MAO (k_MAO) for the metabolism of dopamine. Lungs were perfused with I nmol/l ³H-dopamine or ³H-noradrenaline with COMT inhibited and, in experiments to determine the contribution of SSAO to deamination, with MAO inhibited. Inhibition of MAO reduced the deamination of dopamine and noradrenaline by 99.8% and 98.6%, respectively, indicating that MAO, and not SSAO, was responsible for deamination of the catecholamines in the lungs. The k_MAO value for deamination of dopamine was 3.89 min^–1. Further experiments were carried out to determine the contributions of MAO-A and MAO-B to the deamination of dopamine in lungs perfused with 1 nmol/l ³H-dopamine and 100 nmol/1 lazabemide or 300 nmol/I Ro41-1049, respectively. The values of k_MAO-A and k_MAO-B were 3.05 min^–1 and 0.626 min^–1, respectively.It was concluded that, in rat lungs, MAO-A contributed 78–84% and MAO-B 16–22% to the total deamination of dopamine and SSAO had no significant role in its pulmonary metabolism. These relative contributions of MAO-A and MAO-B to the deamination of dopamine are very similar to those that have been determined previously for noradrenaline, but the rate constant for deamination of dopamine is 26-fold greater than that for noradrenaline in rat lungs.Abbreviations COMT Catechol-O-methyltransferase - DBH Dopamine-\-hydroxylase - DOPEG 3,4-dihydroxyphenylglycol - DOMA 3,4-dihydroxyman delic acid - DOPAC 3,4-dihydroxyphenylacetic acid - DOPET 3,4-dihydroxphenylethanol - ECS Extracellular space - K_m Michaelis or half-saturation constant - k_COMT Rate constant for O-methylation by COMT - k_deam Rate constant for total deamination - k_MAO Rate constant for deamination by MAO - MAO Monoamine oxidase - MB-COMT Membrane-bound COMT - SSAO Semicarbazidesensitive amine oxidases - S-COMT Soluble COMT - T/M Tissue to medium concentration ratio of dopamine or noradrenaline - V_max Maximal rate - V_{st - st} Steady-state rate of metabolite formation     

Thermodynamics of Solutions I: Benzoic Acid and Acetylsalicylic Acid as Models for Drug Substances and the Prediction of Solubility

Purpose. To investigate the solution process of drug substances (exemplified by benzoic acid, BA, and acetylsalicylic acid, ASA), particularly the interrelation between enthalpic and entropic terms of Gibbs energy, in different solvents. To develop an approach for the estimation of standard solution enthalpies based on a self-consistent thermochemical scale. Method. Two independent methods, solubility experiments (concentrations of saturated solutions) and solution calorimetry (standard solution enthalpies) in aliphatic alcohols and individual organic solvents were used. Correlation between the thermodynamic functions in various solvents were analyzed by standard statistical methods. Multiple regression analysis between H ⁰ _sol values and the parameters of the solvents was run on the Koppel-Palm equation. Results. Based on experimental data, a compensation effect between thermodynamic functions was observed. Correlation was found between H ⁰ _sol (BA) and H ⁰ _sol (ASA) [where the H ⁰ _sol (BA)-values were used as a self-consistent thermochemical scale]. Furthermore, H ⁰ _sol correlated with the Koppel-Palm basicity of the solvents. Conclusions. The model based on solubility and solution experiments might be useful for the prediction of solubility or solvation of drug substances in different media. The regression equation based on the self-consistent thermochemical scale makes it possible to approximate the ability to solvate a drug substance in comparison with structure-relative substances.     

Effect of cannabinoids on the turnover rate of acetylcholine in rat hippocampus,striatum and cortex

Summary The effects of ⁹-tetrahydrocannabinol, (⁹THC) the major psychoactive compound of marijuana, and cannabidiol (CBD), a non-psychoactive component, on the acetylcholine (ACh) concentration and the turnover rate of ACh (TR_ACh) have been studied in various regions of the rat brain. Neither ⁹THC doses from 0.2 to 10 mg/kg nor CBD (10 or 20 mg/kg) alter the ACh concentration in the brain areas examined 30 min, after the intravenous injection. However, ⁹-THC (doses from 0.2 to 10 mg/kg) causes a marked dose-related decrease in the TR_ACh in hippocampus whereas CBD is without effect in this brain region even when 20 mg/kg is given. Furthermore, high doses of ⁹-THC (5 mg/kg) and CBD (20 mg/kg) that produce a significant decrease in the TR_ACh of striatum fail to change the TR_ACh in parietal cortex. The low doses of ⁹-THC required to reduce hippocampal TR_ACh suggest that an action on these cholinergic mechanisms may play a role in the psychotomimetic activity of ⁹-THC.     

Discriminative effects of combinations of Δ9-Tetrahydrocannabinol and pentobarbital in pigeons

The purpose of the present study is to examine potentially additive effects of pentobarbital and ⁹-Tetrahydrocannabinol ⁹-THC using a drug discrimination paradigm. Three groups of pigeons were trained to discriminate between the effects induced by i.m. administrations of either (a) 0.25 mg/kg ⁹-THC and vehicle, (b) 4 mg/kg pentobarbital and saline, and (c) ⁹-THC and pentobarbital. Test probes under extinction conditions produced orderly dose generalization gradients among the drug-vs nondrug-trained animals. ED₅₀ for pentobarbital was 1.60 mg/kg and ED₅₀ for ⁹-THC was 0.10 mg/kg. Tests in birds discriminating between pentobarbital and ⁹-THC suggested a sharpening of the drug cue effects; tests with the vehicles resulted in approximately a random key selection (33%–66%) while tests with combinations of the two training drugs suggested that ⁹-THC was the more salient cue in this group. Tests with combinations of various doses of pentobarbital and ⁹-THC in the drug-vs nondrug-trained birds did not increase responding on the respective drug-training associated key. Thus the cue effects of pentobarbital and ⁹-THC were not summational under these experimental conditions. In conclusion, rather low doses of pentobarbital and ⁹-THC are effective as discriminative cues in pigeons and the cues thus induced are different. Combinations of the two drugs are not necessarily summational, and the pentobarbital vs the ⁹-THC discriminations augmented the discriminable effects of the two drugs. In addition, the analeptic drug, bemegride, antagonizes the pentobarbital (4 mg/kg) stimulus in the group trained to discriminate between this barbiturate and saline, which agrees with earlier drug antagonism data obtained among mammals (gerbils and rats), required to discriminate between barbiturates and the nondrug condition.     

Evidence for separate neuronal mechanisms for the discriminative stimulus and catalepsy induced by Δ9-THC in the rat

The cataleptogenic effect of ⁹-THC was compared to its discriminative stimulus effects in rats. The ED₅₀s for the discriminative stimulus and catalepsy were 0.8 and 4.0 mg/kg, respectively, while their time courses were very similar. The ED₅₀ of ⁹-THC for catalepsy in experimentally naive rats was not different from that in rats trained with the drug discrimination procedure, indicating that the cataleptogenic effect was not appreciably attenuated by long-term exposure to low doses of ⁹-THC. Pharmacologically, the catalepsy produced by ⁹-THC more closely resembled that of haloperidol than of morphine, since anticholinergic pretreatment eliminated the ⁹-THC-induced catalepsy while pre-treatment with naloxone had no effect. Although the cataleptogenic effect of ⁹-THC could be pharmacologically manipulated by anticholinergic pre-treatment, its discriminative stimulus effects were not changed in the same animals. These results demonstrate that distinctive mechanisms of action exist for these cannabinoid-induced behaviors.     

Clinical effects and plasma levels of Δ9-Tetrahydrocannabinol (Δ9-THC) in heavy and light users of cannabis

⁹-Tetrahydrocannabinol (⁹-THC) was administered in a crossover design by smoking and IV injection to groups of heavy and light users of marihuana. Plasma concentrations of ⁹-THC were similar for the groups after IV injection of 5.0 mg ⁹-THC, but the AUC_{0–240 min} showed a trend towards lower values for the heavy user group. To achieve a maximum desired high, both groups smoked similar amounts (about 13 mg) of ⁹-THC. Heavy users tended to have higher plasma levels than light users. The systemic availability of smoked ⁹-THC was significantly higher for the heavy users (heavy users 23±16% vs 10±7% for light users). These results also indicate that heavy cannabis users smoke more efficiently than casual smokers.Both light and heavy users showed more clinical effect following IV administration than after smoking. The response of the heavy users, both with respect to effect on heart and high, was quite comparable to that of light users.The present study does not suggest that tolerance readily develops in heavy users.     

Δ9-THC as a discriminative stimulus in rats and pigeons: Generalization to THC metabolites and SP-111

In a drug discrimination paradigm pigeons and rats were trained with an operant procedure to discriminate between the presence and absence of the effects of ⁹-THC (1.0 and 3.0 mg/kg, injected IM 90 min and I.P. 30 min before the start of the session). Once trained, various THC metabolites as well as a water-soluble derivative of THC (SP-111), were substituted for ⁹-THC to test for generalization to the training drug. Generalization to ⁹-THC occurred with the 11-hydroxy metabolites and the potency order was 11-OH-⁹-THC >11-OH-⁸-THC ⁹-THC. Among the other metabolites tested (8-OH-⁹-THC, 8, 11-di-OH-⁹-THC, 8-OH-⁹-THC, 8, 11-di-OH-⁹-THC), it was only 11-di-OH-⁹-THC that completely substituted for ⁹-THC in pigeons, albeit at very high dose levels (rats were not tested with these metabolites). SP-111 generalized to ⁹-THC in both species. However, the onset of action of SP-111 was slower than that for ⁹-THC, especially in pigeons. These studies show the importance of obtaining complete dose-effect determinations over time when assessing structure-activity relationships with drug-discrimination procedures.A brief account of the results, which are summarized in Neuropharmacology 18:1023–1024, 1979), was presented at the British Association for Psychopharmacology Summer Conference, July 15–17, Birmingham, England, 1979     

Effect of catechol-O-methyl-transferase (COMT) inhibition on the vascular and metabolic responses to noradrenaline,isoprenaline and sympathetic nerve stimulation in canine subcutaneous adipose tissue

Summary The effect of COMT-inhibitors (U-0521, H 22/54) on lipolysis and on vascular responses induced by noradrenaline, isoprenaline and nerve stimulation in canine adipose tissue in situ has been studied.COMT inhibition, before or after -adrenoceptor blockade, did not influence the -adrenoceptor mediated vasoconstriction due to nerve stimulation (2–5 Hz). Vasoconstriction due to close i.a. noradrenaline was unaffected at a dose of 2×10^–10 moles but at a dose of 10^–9 moles the peak vasoconstriction was enhanced but the duration of the response was not changed. Prior -adrenoceptor blockade did not alter the responses to U-0521.After -adrenoceptor blockade with Hydergine (120–300 g i.a.) a vasodilatation was induced by noradrenaline 2×10^–10 mole. This response and that produced by isoprenaline (2×10^–11 or 10^–10 moles) were enhanced after U-0521-treatment, but the vasodilating response to nerve stimulation (2 Hz) after -adrenoceptor blockade was unaffected.Lipolysis induced by nerve stimulation or noradrenaline in vivo was increased 50% and that induced by isoprenaline was increased 100% by COMT inhibition. U-0521 did not alter basal lipolysis in vitro but enhanced lipolysis induced by 0.1 M noradrenaline significantly, both in the presence and in the absence of theophylline.The present results shows that COMT influences the effective concentration of isoprenaline more than that of noradrenaline. In the vasculature the effects of exogenous noradrenaline is influenced to a larger degree than those induced by nerve stimulation. However, COMT blockade increased lipolysis induced by equieffective doses of exogenous noradrenaline and nerve stimulation to an equal extent. These results are in agreement with the idea that COMT is of physiological importance mainly at receptor sites that are not in close contact with sympathetic nerve endings and supports the idea that vascular -adrenoceptors are innervated receptors. Vascular -adrenoceptors on the other hand seem to be humoral receptors unrelated to sympathetic nerve endings and preferentially stimulated by circulating noradrenaline. The -adrenoceptors on the fat cells may be of both types, i.e. innervated and humoral.     

The activity of the neuronal and extraneuronal catecholamine-metabolizing enzymes of the perfused rat heart

Summary In a comparative study the neuronal and extraneuronal metabolism of several ³H-catecholamines (all of which were tritiated in the C-7 position of the side chain only) was determined in isolated rat hearts perfused at a concentration of the 3H-amines of 50 nmol/1. While the neuronal MAO activity was determined after inhibition of extraneuronal uptake (100 mol/1 OMI) and COMT (10 mol/1 U-0521), the extraneuronal MAO activity was estimated after inhibition of neuronal uptake (30 mol/1 cocaine) and COMT. The extraneuronal COMT activity was determined under conditions of inhibition of both neuronal uptake and MAO (pretreatment with pargyline). Hearts were perfused with the 3H-catecholamines until the rate of appearance of the various ³H-metabolites in the venous effluent has reached a steady state. From these rates (v _st-st) and the steady-state content of the unchanged ³H-catecholamines in the tissue (S _i), the rate constants (V _max/K _m) for the unsaturated intracellular enzymes COMT (_COMT) and MAO (_MAO) were calculated. The _COMTvalues for all four catecholamines, (–)-noradrenaline, dopamine, (–)-adrenaline and (±)-isoprenaline exhibit a range from 0.24 to 0.78 min^–1; the metabolism of the catecholamines by the COMT differs: (-)-noradrenaline = dopamine < (–)-adrenaline < (±)-isoprenaline. The extraneuronal MAO activity was low for all three catecholamines, (–)-adrenaline, (–)-noradrenaline and dopamine (range of _MAOfrom 0.05 to 0.28 min^–1) and declined in the order: (–)-adrenaline < (–)-noradrenaline < dopamine. The neuronal MAO activity for (–)-adrenaline, (–)-noradrenaline and dopamine was slightly higher than that in the extraneuronal cells (range of kMAO from 0.08 to 0.35 min^–1), but the ranking order showed the same pattern: (–)-adrenaline < (–)-noradrenaline = dopamine.Abbreviations MAO monoamine oxidase - COMT catechol-Omethyltransferase - NMN normetanephrine - MN metanephrine - MT 3-methoxytyramine - OMI 3-O-methyl-isoprenaline - DOPEG dihydroxyphenylglycol - DOPET dihydroxyphenylethanol - DOMA dihydroxymandelic acid - DOPAC dihydroxyphenylacetic acid - U-0521 3,4-dihydroxy-2-methyl propiophenone     

CB<Subscript>1</Subscript> receptor mediation of cannabinoid behavioral effects in male and female rats

Rationale Cannabinoids have been shown to produce greater behavioral effects in female than male rats. Although central nervous system CB₁ receptors are known to mediate cannabinoid-induced behavioral effects in male rats, it is not known whether the same is true for females.Objective To determine if cannabinoid-induced antinociception and catalepsy are similarly mediated by central CB₁ receptors in male and female rats.Methods The ability of SR141716A, a CB₁ receptor selective antagonist, administered ICV (1–1000 g) or IT (1–600 g) to block 10 mg/kg IP ⁹-THC-induced antinociception (paw pressure) and catalepsy (bar test), was compared in male and female rats.Results ⁹-THC alone produced slightly greater antinociception, and significantly greater catalepsy in females than males. When administered ICV, SR141716A partially antagonized ⁹-THC-induced antinociception in both females and males. IT SR141716A also antagonized ⁹-THC-induced antinociception in both sexes; it was slightly more potent in males but equally effective in males and females. SR141716A antagonized ⁹-THC-induced catalepsy in a similar manner in males and females when given ICV or IT.Conclusions These results confirm that ⁹-THC-induced behavioral effects are mediated by central CB₁ receptors in male and female rats.     

Stimulus effects of Δ9-THC and its interaction with naltrexone and catecholamine blockers in rats

Rats trained in a T-shaped maze to discriminate the effects of i.p. injections of ⁹-tetrahydrocannabinol (⁹-THC, 4mg/kg) and the effects of the vehicle were tested for antagonism and generalization to the ⁹-THC stimulus by naltrexone (4 mg/kg), haloperidol (0.32 mg/kg), propranolol (20 mg/kg), and phenoxybenzamine (10 mg/kg). None of these drugs blocked the ⁹-THC stimulus, nor were they found to generalize to ⁹-THC.     

Influence of polymorphism on the surface energetics of salmeterol xinafoate crystallized from supercritical fluids

Purpose. To characterize the surface thermodynamic properties of two polymorphic forms (I and II) of salmeterol xinafoate (SX) prepared from supercritical fluids and a commercial micronized SX (form I) sample (MSX). Methods. Inverse gas chromatographic analysis was conducted on the SX samples at 30, 40, 50, and 60°C using the following probes at infinite dilution: nonpolar probes (NPs; alkane C5-C9 series); and polar probes (PPs; i.e., dichloromethane, chloroform, acetone, ethyl acetate, diethyl ether, and tetrahydrofuran). Surface thermodynamic parameters of adsorption and Hansen solubility parameters were calculated from the retention times of the probes. Results. The free energies of adsorption (-G_A) of the three samples obtained at various temperatures follow this order: SX-II > MSX SX-I for the NPs; and SX-II > MSX > SX-I for the PPs. For both NPs and PPs, SX-II exhibits a less negative enthalpy of adsorption (H_A) and a much less negative entropy of adsorption (S_A) than MSX and SX-I, suggesting that the high -G_A of SX-II is contributed by a considerably reduced entropy loss. The dispersive component of surface free energy (_s ^D) is the highest for MSX but the lowest for SX-II at all temperatures studied, whereas the specific component of surface free energy of adsorption (-G_A ^SP) is higher for SX-II than for SX-I. That SX-II displays the highest -G_A for the NP but the lowest _s ^D of all the SX samples may be explained by the additional -G_A change associated with an increased mobility of the probe molecules on the less stable and more disordered SX-II surface. The acid and base parameters, K_A and K_D, that were derived from H_A ^SP reveal significant differences in the relative acid and base properties among the samples. The calculated Hansen solubility parameters (_D, _P, and _H) indicate that the surface of SX-II is the most polar and most energetic of all the three samples in terms of specific interactions (mostly hydrogen bonding). Conclusions. The metastable SX-II polymorph possesses a higher surface free energy, higher surface entropy, and a more polar surface than the stable SX-I polymorph.     

Effects of chronic Δ9-tetrahyrocannabinol administration on schedule-controlled behavior of pigeons: Cross-tolerance to pentobarbital and barbital

Pigeons responding under a variable-interval (VI) 75-s schedule of food presentation were used to study cross-tolerance from ⁹-tetrahyrocannabinol (⁹-THC) to pentobarbital and barbital. After initial dose-effect functions for pentobarbital and barbital were determined, the birds received ⁹-THC injections for 6 weeks. This chronic administration regimen resulted in a greater than 100-fold tolerance to ⁹-THC. Redetermination of the pentobarbital and barbital dose-effect functions during the chronic ⁹-THC regimen revealed statistically significant shifts to the right for the pentobarbital (0.191 log unit) and barbital (0.078 log unit) dose-effect curves. All six birds showed tolerance to pentobarbital, while four of the six showed tolerance to barbital. Blood barbital levels before and after chronic ⁹-THC administration did not differ significantly. Tolerance to ⁹-THC was more prolonged and of much greater magnitude than the cross-tolerance to pentobarbital or barbital. The results demonstrate that cross-tolerance can develop from ⁹-THC to a barbiturate that normally undergoes little metabolism.     

Effect of K+ channel blockers on the α2-adrenoceptor-coupled regulation of electrically evoked noradrenaline release in hippocampus

Summary The question whether presynaptic ₂-adrenoceptors regulating noradrenaline release in hippocampus directly couple to tetraethylammonium chloride (TEA) or -dendrotoxin (-DTX)-sensitive K⁺ channels was investigated. Hippocampal slices, prelabelled with [³H] noradrenaline, were superfused in the presence of (+)-oxaprotiline and electrically stimulated with 4 pulses delivered at 100 Hz, in order to avoid autoinhibition due to released noradrenaline.TEA enhanced the evoked [³H]noradrenaline release in rabbit hippocampus in a concentration-dependent manner, yielding an approximately 4-fold increase at 30 mmol/l, whereas the spontaneous outflow of tritium was only slightly affected at this concentration. The ₂-adrenoceptor agonist clonidine, at 10–100 nmol/l inhibited the evoked [³H]noradrenaline release between 77% and 96%. The inhibitory effect of the ₂-agonist was distinctly diminished in the presence of 30 mmol/l TEA but was restored in low Ca²⁺/high Mg²⁺ buffer. Therefore, the diminution of the ₂-agonist effect by TEA observed in experiments with normal Ca²⁺ can be explained by an increase of the Ca²⁺ availability for the release process due to the prolongation of action potentials. In rabbit hippocampus -DTX (10–200 nmol/l) did neither affect the evoked release of [³H]noradrenaline nor its ₂-agonist-induced modulation. However, in rat hippocampus -DTX significantly increased the evoked transmitter release and diminished the effect of clonidine.Taken together, the present data for the rabbit hippocampus exclude the possibility that activation of presynaptic ₂-adrenoceptors inhibits depolarization-evoked [³H]noradrenaline release by inducing an outward K⁺ current through TEA- or -DTX-sensitive K⁺ channels. However, there are species differences between the rabbit and the rat so that in the rat the ₂-adrenoceptors could actually be coupled to K⁺ channels — provided that the release-enhancing properties of -DTX do not account for the ₂-antagonism observed.Correspondence to C. Allgaier at the above address     

Δ9-THC-induced cognitive deficits in mice are reversed by the GABAA antagonist bicuculline

Rationale The results of recent in vitro studies have underscored the important role that activation of CB₁ receptors has on GABAergic activity in brain areas associated with memory.Objectives The primary purpose of this study was to test the hypothesis that the memory disruptive effects of ⁹-tetrahydrocannabinol (⁹-THC) in vivo are mediated through GABAergic systems. Conversely, we also evaluated whether blocking CB₁ receptor signaling would alter memory deficits elicited by GABA agonists.Methods The GABA_A antagonist bicuculline and GABA_B antagonist CGP 36742 were evaluated for their ability to ameliorate ⁹-THC-induced deficits in a mouse working memory Morris water maze task. Mice were also assessed in a T-maze task, as well as non-cognitive behavioral assays. Additionally, the effects of GABA_A and GABA_B agonists were assessed in either CB₁ (–/–) mice or wild type mice treated with the CB₁ antagonist SR 141716.Results Memory deficits resulting from 10 mg/kg ⁹-THC in the Morris water maze were completely reversed by bicuculline, though unaffected by CGP 36742. Bicuculline also blocked the disruptive effects of ⁹-THC in the T-maze, but failed to alter non-mnemonic effects of ⁹-THC. Although CB₁ (–/–) mice exhibited supersensitivity to muscimol-induced water maze deficits compared with wild type control mice, muscimol elicited virtually identical effects in SR 141716-treated and vehicle-treated wild type mice.Conclusions This is the first demonstration of which we are aware showing that GABA_A receptors may play a necessary role in ⁹-THC-induced memory impairment in whole animals.