Morphine and δ9-tetrahydrocannabinol: Tolerance to the stimulus effects

Morphine and saline served as discriminative Stimuli for one group of rats in a 2-lever discrimination task. ⁹-THC and vehicle were discriminative stimuli for a second group. Depression of one lever resulted in reinforcement following the administration of morphine or ⁹-THC and the opposite lever was reinforced after vehicle. A high degree of discriminated responding occurred with both drugs. During daily supplemental injections of morphine or ⁹-THC up to several times the training dose for a period of 2 months, subjects still discriminated morphine or ⁹-THC from vehicle. However, the degree of discrimination was reduced indicating a limited tolerance to either drug. Naloxone precipitated narcoticlike withdrawal symptoms in rats chronically treated with either morphine or ⁹-THC, indicating that there is an interaction between chronic administration of ⁹-THC and the narcotic antagonist, naloxone.     

GABAergic modulation of D-1 dopamine receptor-mediated 3H-acetylcholine release from rabbit retina

Summary Dopamine evokes calcium-dependent release of ³H-acetylcholine from superfused rabbit retina labeled in vitro with ³H-choline, through activation of a D-1 dopamine receptor. This study investigates the activation of this receptor by endogenous dopamine and the modulation of the spontaneous and dopamine-evoked release of ³H-acetylcholine from rabbit retina labeled with ³H-choline by GABAergic agonists and antagonists. Endogenous dopamine, released from dopaminergic amacrine neurons by the indirect amines tyramine or D-amphetamine evoked the calcium-dependent release of ³H-acetylcholine from rabbit retina. The release of ³H-acetylcholine elicited by tyramine (10 M) or D-amphetamine (10 M) was attenuated by the selective D-1 antagonist SCH 23390 (0.1 M) and by the dopamine uptake inhibitor nomifensine (3 M). At concentrations of 1 mM and 1 M respectively, GABA and muscimol inhibited the spontaneous release of tritium from rabbit retina labeled in vitro with ³H-choline. Picrotoxin and bicuculline (10 M) increased the spontaneous release of tritium. GABA and the GABA agonist muscimol (0.01–100 M) inhibited in a concentration-dependent manner the release of ³H-acetylcholine elicited by 100 M dopamine with IC₅₀ values of 4.5 M and 0.02 M respectively. The inhibition of dopamine-evoked ³H-acetylcholine release by GABA (10 M) and muscimol (0.1 M) was antagonized by the GABA antagonists bicuculline and picrotoxin. Picrotoxin and bicuculline (10 M) increased the spontaneous release of tritium, and potentiated the release of ³H-acetylcholine evoked by 100 M dopamine consistant with a tonic, inhibitory GABAergic input to the cholinergic amacrine neurons in rabbit retina. Dopamine-evoked acetylcholine release in rabbit retina may be of physiological importance as D-1 dopamine receptor-mediated increases in ³H-acetylcholine release from rabbit retina can be elicited by endogenous dopamine. In addition, activation of GABA receptor sites modulates the spontaneous and dopamine-evoked acetylcholine release from rabbit retina. Send offprint requests to M. L. Dubocovich at the above address     

Enhancement of δ- but not μ-opiate agonist binding by calcium

Summary Present evidence for distinction of 2 types of opiate receptor sites in rat brain homogenates originates from different relative affinities of morphine-like alkaloids and enkephalins to -or enkephalin and - or morphine-receptor sites. We now report that Ca²⁺ in a physiological dose range (0.5–3 mM) enhances the binding of ³H-enkephalin in hypotonically treated rat brain membranes, whereas specific binding of ³H-morphine-like alkaloids is not affected. Furthermore, the potency of [d-Ala², d-Leu⁵]-enkephalin to inhibit [³H]-diprenorphine and [³H]-ethylketazocine binding increased in the presence of Ca²⁺, whereas an increase in potency of [d-Ala², d-Leu⁵]-enkephalin to inhibit binding of -receptor ligands was not observed. Kinetic analysis revealed that Ca²⁺ decreased the rate of dissociation of [d-Ala², d-Leu⁵]-enkephalin without affecting the rate of association, thereby increasing the affinity. However, in saturation binding studies, performed in diencephalic membranes, in which [d-Ala², d-Leu⁵]-enkephalin binds predominantly to -receptors, Ca²⁺ also increased the binding affinity of [³H]-[d-Ala², d-Leu⁵]-enkephalin. Double reciprocal analysis suggested a mixed competitive-noncompetitive type of inhibition of [d-Ala², d-Leu⁵]-enkephalin binding by dihydromorphine. Thus, the interactions of - and -opiate ligands with -receptors may involve topographically different, but closely related binding sites, located on a single receptor molecule.Abbreviations DADL [d-Ala², d-Leu⁵]-enkephalin - DHM dihydromorphine - met-enkephalin methionine-enkephalin - leu-enkephalin leucine-enkephaline - FK 33-824 [d-Ala², MePhe⁴, Met(O)-ol]-enkephalin - EGTA ethyleneglycol-bis-(-aminoethylether) N, N'-tetraacetic acid - TRIS Tris (hydroxymethyl)-aminomethan     

Release of ATP in rat vas deferens: origin and role of calcium

Release of endogenous ATP elicited by electrical (neural) stimulation and exogenous agonists was studied in the rat isolated vas deferens. The aims were to dissect neural and postjunctional contributions to the nerve activity-evoked overflow of ATP and to clarify the role of transmitter receptors and calcium in postjunctional ATP release.In tissues preincubated with [³H]-noradrenaline, electrical stimulation (100 pulses/10 Hz) elicited contraction and an overflow of tritium and ATP. Contractions as well as ATP overflow were reduced by prazosin 0.3 M and even more so by prazosin 0.3 M combined with suramin 300 M. They were also reduced by nifedipine 10 M and even more so by nifedipine 10 M combined with ryanodine 20 M (the additional effect of ryanodine on ATP overflow was not significant). In tissues not pretreated with [³H]-noradrenaline, exogenous noradrenaline 10 M and ,-methylene ATP 10 M elicited contraction and an overflow of ATP. Responses to noradrenaline were blocked by prazosin 0.3 M but not suramin 300 M and were greatly reduced by nifedipine 10 M and in Ca²⁺-free medium. Responses to ,-methylene ATP were blocked by suramin 300 M but not prazosin 0.3 M were reduced by nifedipine 10 M (effect on ATP overflow not significant) and were reduced even more in Ca²⁺-free medium. Neuropeptide Y 0.3 M caused only very small contraction and ATP overflow. The electrically as well as the agonist-evoked ATP overflow correlated well with the contraction responses except in experiments with suramin which retarded the removal, by vas deferens tissue, of ATP from the medium.Itsis concluded that the overflow of ATP from rat vas deferens elicited by electrical (neural) stimulation is at least 90% postjunctional, presumably smooth muscle, in origin. ATP is released from postjunctional cells as a consequence of both ₁-adrenoceptor and P₂-purinoceptor activation. Ca²⁺ is a second messenger in the postjunctional ATP release response; its major part enters through L-type channels. A purely neural overflow of ATP was not isolated under the conditions of the experiments. Correspondence to: R. Bültmann at the above address     

The binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties

Summary Four groups of narcotic analgesic drugs have been assessed for their opiate activities by using three binding assays and three pharmacological bioassays. In the binding assays, their inhibition constants (K _I, nM) were determined against the binding of the -ligand, [³H]-[d-Ala ² ,MePhe ⁴ , Gly-ol⁵]enkephalin, of the -ligand, [³H]-[d-Ala ² ,d-Leu ⁵]enkephalin and of the -ligand, [³H]-(±)-ethylketazocine after suppression of - and -binding by 100 nM of the unlabelled -ligand and 100 nM of the unlabelled -ligand. The pharmacological agonist or antagonist activities were assayed on the guinea-pig ileum, mouse vas deferens and rat vas deferens.The first group of compounds were pure agonists in all three pharmacological bioassays. The majority of the compounds showed preference to -binding but phenazocine and particularly etorphine had also high affinities to the - and -binding sites.The second group consisted of N-allyl and N-cyclopropylmethyl homologues of the morphine, 3-hydroxymorphinan and normetazocine series which had agonist and antagonist activities in the guinea-pig ileum and mouse vas deferens but were pure antagonists in the rat vas deferens. In the binding assays, -binding and -binding were prominent.The third group was made up by the ketazocine-like compounds which in the guinea-pig ileum and mouse vas deferens were pure agonists and in the rat vas deferens pure antagonists. The binding spectrum showed particularly high binding to the -binding site.The fourth group was the antagonists which were devoid of agonist activity with the exception of diprenorphine and Mr 2266 which had retained some agonism. The binding spectrum showed considerable variation, naloxone in low concentration being a selective -antagonist, Mr 2266 having high affinities to the - and -binding sites and diprenorphine having considerable affinities to the -, - and -binding sites.Since each of the four groups of compounds, whether pure agonists, agonist-antagonists, ketazocine-like drugs or pure antagonists, shows independent varittions in the affinities to the - and -binding sites, their different pharmacological behaviour cannot be solely due to difference in the binding spectra.     

Effects of (−)δ9-trans-tetrahydrocannabinol (δ9-THC) on memory,attention and subjective state

In a double blind study, cross-over design, on 37 volunteers several effects of an orally administered dosage of 15 mgs (–)- ⁹-trans-tetrahydrocannabinol ( ⁹-THC) were assessed. The results did bear out the hypotheses of this experiment that the impairment of attention and information storage in the long-term memory as well as depersonalization and temporal disintegration phenomena induced by ⁹-THC are interrelated. Furthermore, the sequence of information retrieval from memory was found to be changed by ⁹-THC.     

Cocaine and heroin (‘speedball’) self-administration: the involvement of nucleus accumbens dopamine and <Emphasis Type="Italic">μ</Emphasis>-opiate,but not <Emphasis Type="Italic">δ</Emphasis>-opiate receptors

Rationale The combined administration of heroin and cocaine (speedball) is common among intravenous drug users. Dopamine receptors in the nucleus accumbens play a key role in cocaine self-administration; however, their role in speedball self-administration is unknown, as is the role of opiate receptors in this region.Objectives The effect of blocking dopamine D1, D2, -opiate or -opiate receptors in the nucleus accumbens on the intravenous self-administration of combined heroin and cocaine was examined in rats.Methods Rats with bilateral cannulae implanted into the nucleus accumbens were trained to self-administer intravenous speedball (ratio of cocaine/heroin, 17:1) under a progressive ratio (PR) schedule. Prior to their self-administration session, rats were then microinjected with the dopamine D1 receptor antagonist SCH 23390 (1 and 6 nmol side^–1), the D2 receptor antagonist raclopride (3 and 10 nmol side^–1), the -opiate receptor antagonist CTOP (0.1, 0.3 and 1.0 nmol side^–1), the -opiate receptor antagonist naltrindole (1.0, 3.0 and 10 nmol side^–1) or a cocktail of SCH 23390 (1 nmol side^–1) and CTOP (0.1 nmol side^–1) into the nucleus accumbens.Results Microinjection of SCH 23390, raclopride or CTOP into the nucleus accumbens produced dose-dependent decreases in breakpoints under the PR schedule, while naltrindole was without effect. The highest dose of SCH 23390 also significantly reduced locomotor activity measured during speedball self-administration. The combination of SCH 23390 and CTOP significantly reduced breakpoints, while not affecting locomotor activity.Conclusions These results indicate that dopamine and -opiate receptors, but not -opiate receptors, in the nucleus accumbens are involved in the reinforcing effects of speedball. Combined administration of D1 and -opiate receptor antagonists may be more selective at reducing the reinforcing effects of speedball self-administration than either drug alone.     

Photoelectric measurement of neurogenic vasoconstriction in jejunal branches of the rabbit mesenteric artery reveals the presence of presynaptic opioid δ-eceptors

Summary The perivascular nerves of rabbit mesenteric arteries were stimulated with 15 pulses at 2 Hz, and decreases in external diameter were measured by means of a photoelectric device. Both extra- and intraluminally added [Met⁵]-enkephalin 1 mol/l depressed vasoconstriction, although with the second mode of application a larger inhibition occurred. Therefore, in the subsequent experiments all opioids were added into the lumen. [Met⁵]enkephalin 0.1 mol/l had no effect. [d-Pen², l-Pen⁵]enkephalin 3 mol/l was less potent than [Met⁵]enkephalin 1 mol/l. ICI 174864 1 mol/l was also without effect when given alone, but antagonized the action of [Met⁵]enkephalin 1 mol/l.Ethylketocyclazocine, dynorphin A(1–13), normorphine and DAGO, all 1 mol/l, were ineffective. [Met⁵]enkephalin 1 mol/l did not change the vasoconstriction evoked by the application of noradrenaline (0.1 –3 mol/l). It is concluded that in the mesenteric artery action potential-induced transmitter release, and in consequence vasoconstriction can be inhibited by the activation of presynaptic opioid -receptors. Send offprint requests to P. Illes at the above address     

The effect of opiates on arterial baroreceptor reflex function in the rabbit

Summary The contribution of exogenous and endogenous opioid peptides to the central modulation of the baroreceptor reflex was investigated in rabbits.Baroreceptor sensitivity was assessed in pentobarbitone anaesthetised animals by measuring heart period in response to rises in arterial pressure after bolus intravenous injections of phenylephrine and falls induced by intravenous sodium nitroprusside and controlled haemorrhage. The slope of the linear relationship between arterial pressure and heart period was used as an index of baroreflex sensitivity.Thirty minutes after the intracisternal administration of 50 g/kg RX783016 (a -opiate receptor agonist) baroreceptor sensitivity was reduced to all three methods of blood pressure manipulation. Ketazocine (50 g/kg) a -opiate agonist and [d-Ala ² , d-Leu ⁵ ] enkephalin (1 g/kg) a -opiate agonist, 15 min after intracisternal injection caused an increase in baroreflex sensitivity in response to a rise in pressure and a reduction in response to a fall.Intravenous injection of naloxone (80 g/kg) caused an increase in varoreflex gain. However, a higher dose (200 g/kg) was required to attenuate the effects of RX783016 and [d-Ala ² , d-Leu ⁵ ] enkephalin but not ketazocine. No change in baseline arterial pressure or heart rate occurred after the opiates or naloxone. It appears that exogenous and endogenous opiates modify baroreceptor reflex function, through a mechanism which involves central opiate receptors of the - and -types.     

One-Dimensional and Two-Dimensional 1H- and 13C-Nuclear Magnetic Resonance (NMR) Analysis of Vitamin E Raw Materials or Analytical Reference Standards

Two-dimensional spectral analysis (COSY, HETCOR) was utilized to make the complete ¹³C- and ¹H-NMR assignments for -, -, --, and -tocopherol as well as for the acetate and succinate esters of -tocopherol. ¹³C-NMR was found to be especially useful in distinguishing between the various tocopherols and distinguishing between the d-isomer and the d,l-racemic mixture. HETCOR spectra were also found to be useful for the qualitative identification of mixtures of the tocopherols and sesame oil. Using a procedure designed to minimize errors arising from spin relaxation and nuclear Overhauser effects, ¹³C-NMR peak integrals were used to quantitate -tocopherol and -tocopherol in the presence of sesame oil using benzoic acid as the standard for calibration of the quantitation. The NMR results were compared to a capillary column gas chromatographic analysis of the individual -tocopherol and -tocopherol reference materials.Chris W. Myers: Recipient of the Analysis and Pharmaceutical Quality Section Undergraduate Award in Pharmaceutical Analysis from the APQ Section of the American Association of Pharmaceutical Scientists.     

Distinct distribution of opioid receptor types in rat lumbar spinal cord

Summary The distribution of opiate binding sites was studied in sections of rat lumbar spinal cord under conditions selective for , and receptors. While the levels of binding sites were highest in the substantia gelatinosa, elevated levels were also observed in laminae III, IV, V and VIII. In contrast, binding was notable only in lamina I. The levels of typical sites were low, and were concentrated in the substantia gelatinosa. An additional, atypical site was detected using ³H-diprenorphine in the presence of , and receptor blocking agents, and this site was also concentrated in the substantia gelatinosa. Send offprint requests to B. J. Morris at the above address     

The modulation of [3H]noradrenaline and [3H]serotonin release from rat brain synaptosomes is not mediated by the α2B-adrenoceptor subtype

Summary The present study aimed at relating the presynaptic ₂-adrenoceptors, known to modulate noradrenaline and serotonin release, with the recently described _2A- and _2B-adrenoceptor subtypes. The effects of the agonist oxymetazoline (selective for _2A subtype) and of three adrenoceptor antagonists (idazoxan, 1-(2-pyrimidinyl)piperazine (PmP) and prazosin, the last one known to be _2B selective) were evaluated on [³H]noradrenaline and [^H]serotonin release in superfused synaptosomes from rat brain cortex. These drugs were also tested in [³H]yohimbine binding to human platelet membranes (containing only _2A receptors) and to neonatal rat lung membranes (containing only _2B receptors).The affinity pattern of these compounds at _2A-adrenoceptors in binding studies was oxymetazoline > = idazoxan > PmP > prazosin; at _2B-adrenoceptors it was idazoxan > = prazosin > PmP = oxymetazoline. Oxymetazoline inhibited with high and similar potencies the K⁺-evoked [³H]noradrenaline and [³H]serotonin release, IC₅₀ 18 and 7 nM, respectively; in the same conditions, the IC₅₀ values of noradrenaline were 42 and 168 nM, respectively. The antagonist affinity pattern (antagonism against noradrenaline) was idazoxan > PmP > prazosin, either on [³H]serotonin release.These results indicate that presynaptic ₂ auto- or heteroreceptors do not belong to the _2B subtype and suggest that the modulation of noradrenaline and serotonin release may be mediated by the _2A-adrenoceptor subtype. Send offprint requests to M. Gobbi at the above address     

Releasing activities of d-fenfluramine and fluoxetine and fluoxetine on rat hippocampal synaptosomes preloaded with [3H]serotonin

Summary Rat hippocampal synaptosomes preloaded with [³H]serotonin and maintained in a superfusion apparatus were exposed for 3 min to d-fenfluramine or fluoxetine. Both drugs evoked a tritium overflow which was reserpine-sensitive requiring the presence of intact synaptic vesicles. However the two drugs displayed different characteristics: 1) the overflow was immediate with dfenfluramine whereas the releasing activity of fluoxetine showed a delay of about 2 min; 2) d-fenfluramine-induced overflow was already apparent at 0.15 mol/l whereas the minimal effective concentration of fluoxetine was 2.5 mol/l. Their concentration-effect curves were differently shaped, the effect of d-fenfluramine being saturable at 5–20 mol/l (EC₅₀ about 1 gmol/l) while no saturation was observed with fluoxetine up to 10 mol/l; 3) only 1907o of the tritium overflow evoked by fluoxetine (2.5–10 mol/l) consisted of true [³H]serotonin, compared with 7001o when 0.5 mol/l d-fenfluramine was used; 4) the releasing action of 0.5 mol/l d-fenfluramine was completely Ca⁺⁺-dependent, while at higher dfenfluramine concentrations the Ca⁺⁺-independent overflow became more important. The fluoxetine induced overflow was mainly. (70010) Ca⁺⁺-independent; 5) the releasing acitvity of d-fenfluramine was mainly (80%) blocked by the serotonin uptake blockers indalpine, midalcipram and also fluoxetine whereas fluoxetine-induced overflow was insensitive to inhibition of the serotonin carrier.In conclusion, the releasing activity of d-fenfluramine is already present at a very low concentration (0.5 mol/l) and at this concentration its mechanism of action was Ca⁺⁺-dependent, together with the requirement of a functional serotonin carrier. These data therefore do not support the hypothesis of a simple. displacement of 5-HT from its storage vesicles but suggest an exocytotic release possibly triggered by interaction of d-fenfluramine with intracellular receptors. A direct releasing activity is also shown for fluoxetine, very marked at 5–10 mol/l; such effect is different from that of d-fenfluramine and is probably due to the overflow of 5-hydroxyindoleacetic acid, formed in the synaptosomes after the fluoxetine-induced displacement of serotonin from its storage vesicles. The active concentrations of fluoxetine on serotonin release are compatible with those found in rat brain at doses inducing an anorectic activity. Send offprint requests to M. Gobbi at the above address     

Biliary Excretion of 17β-Estradiol 17β-d-Glucuronide Is Predominantly Mediated by cMOAT/MRP2

Purpose. The mechanism for the biliary excretion of 17-estradiol170-d-glucuronide (E₂17G), a cholestatic metabolite of estradiol, isstill controversial. The purpose of the present study is to examine thetransport of E₂17G across the bile canalicular membrane. Methods. We examined the uptake of [³H]E₂17G by isolatedcanalicular membrane vesicles (CMVs) prepared from Sprague-Dawley (SD)rats and Eisai Hyperbilirubinemic rats (EHBR) whose canalicularmultispecific organic anion transporter/multidrug resistance associatedprotein 2 (cMOAT/MRP2) function is hereditarily defective. Also,in vivo biliary excretion of intravenously administered [³H]E₂17Gwas examined. Results. In CMVs prepared from SD rats, but not from EHBR, amarked ATP-dependent uptake of [³H]E₂17G was observed.Moreover, E₂17G competitively inhibited the ATP-dependent uptake of[³H]2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, nosignificant inhibitory effect of verapamil (100 M) and PSC-833 (5 M) onthe uptake of [³H]E₂17G was observed. In vivo, the biliary excretionof intravenously administered [³H]E₂17G was severely impaired inEHBR while the biliary excretion of [³H]E₂17G in SD rats wasreduced by administering a cholestatic dose (10 mol/kg) unlabeledE₂17G, but not by PSC-833 (3 mg/kg). Conclusions. The transport of E₂17G across the bile canalicularmembrane is predominantly mediated by cMOAT/MRP2.     

Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx

The aim of this study was to determine whether ⁴⁵Ca²⁺ influx could be used as a quantitative measure of channel activation for functional characterisation of P2X purinoceptors in cell lines. In undifferentiated PC12 cells, grown in suspension, ATP (EC₅₀ = 45 M), ATPTS (EC₅₀ = 50 M) and 2-meSATP (EC₅₀ = 81 M) but not meATP (I mM) stimulated ⁴⁵Ca²⁺ influx 2–5 fold. This effect did not appear to be due to activation of P2U or P2Y purinoceptors since 1 mM UTP, ADP or ADPS did not produce any significant effect. Similarly, the effects of ATP were not apparently mediated through activation of P2Z purinoceptors since dibenzylATP behaved as a weak (EC₅₀ = 191 M) partial agonist (Maximal effect 29.5% of ATP maximum) and there was no detectable ATP-stimulated ethidium bromide uptake in the PC12 cells.ATP-stimulated ⁴⁵Ca²⁺ influx was not affected by nifedipine suggesting that it was not secondary to activation of L-type calcium channels and rather reflected influx through a P2X purinoceptor present in these cells. The ATP-stimulated ⁴⁵Ca²⁺ influx could be reduced by monovalent cations, presumably as a result of direct competition for influx through the cation channel, with the following rank order of potency :- guanidinium (EC₅₀ = 16 mM) > sodium > Tris > choline > N-methyl-D-glucamine = sucrose.A number of P2 purinoceptor antagonists inhibited ATP-stimulated ⁴⁵Ca²⁺ influx. Pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (3–300 M), pyridoxal 5phosphate (3–300 M) and d-tubocurarine (30–300 M) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC₅₀. Suramin (100–300 M) and cibacron blue (30–300 M) produced a surmountable antagonism while DIDS (4,4-diisothiocyana-tostilbene-2,2disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 M. The general properties of the P2X purinoceptor population identified in these cells were consistent with them being P2X₂ purinoceptors. These findings suggest that ATP-stimulated ⁴⁵Ca²⁺ influx may be used as a reliable and quantitative functional assay for characterisation of P2X purinoceptor subtypes in cell lines.     

Subtype specificity of γ-aminobutyric acid type A receptor antagonism by clozapine

Clozapine, an atypical neuroleptic, functionally antagonizes the -aminobutyric acid-induced chloride uptake via the main central inhibitory receptor, -aminobutyric acid type A (GABA_A) receptor, in brain vesicles. GABA_A antagonism by micromolar concentrations of clozapine is more efficient in rat cerebrocortical and hippocampal membranes than in cerebellar membranes, as evidenced by clozapine reversal GABA-inhibition of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding. A typical neuroleptic, haloperidol, failed to antagonize GABA in any of these brain regions, while the specific GABA_A antagonist 2-(3-carboxy-2,3-propyl)-3-amino-6-p-methoxyphenylpyrazinium bromide (SR 95531) was efficient in all three brain regions. Clozapine action on [³⁵S]TBPS binding was unaffected by the benzodiazepine receptor antagonist flumazenil. Clozapine inhibited the binding of [³H]muscimol and [³H]SR 95531 to the GABA recognition site, but this effect only partially correlated with the regional differences in and the potency of clozapine antagonism of GABA-inhibition of [³⁵S]TBPS binding, suggesting that also other than GABA sites may mediate clozapine actions. Autoradiography of [35S]TBPS binding revealed GABA antagonism by clozapine in most brain regions. Main exceptions were cerebellar granule cell and molecular layers, olfactory bulb external plexiform and glomerular layers and primary olfactory cortex, where clozapine antagonized GABA inhibition less than average, and lateral hypothalamic and preoptic areas where its antagonism was greater than average. Recombinant 622 receptors, the predominant 6 subunit-containing receptor subtype in cerebellar granule cells, failed to show GABA antagonism by clozapine up to 100 M. In contrast, recombinant 122 receptors, forming the predominant receptor subtype in the brain, were clozapine sensitive. Recombinant 622 and 632 receptors resulted in clozapine-insensitive receptors, whereas 612 receptors were clozapine sensitive. The efficacy of clozapine to antagonize GABA in 1x2 receptors decreased in the order of 112>122>132. The results indicate that clozapine antagonizes the function of most GABA_A receptor subtypes, and that the interaction is determined by the interaction of the and subunit variants. GABA antagonism is a unique property of clozapine, not shared by haloperidol, which might be involved in the pharmacological mechanism for the increased seizure susceptibility associated with clozapine treatment.     

Dopamine inhibits prostaglandin F2α-induced depolarization of rabbit jejunal arteries via activation of DA1-receptors

Summary In rabbit jejunal arteries, the membrane potential of single smooth muscle cells decreased on the application of noradrenaline 3 mol/1. LY 171555 1 mol/1 did not change, whereas SKF 38393 10 mol/1 reversed the effect of noradrenaline. When prostaglandin F₂ (PGF₂) was used to evoke depolarization in the presence of prazosin 0.1 mol/1, rauwolscine 1 mol/1 and propranolol 1 mol/1, both SKF 38393 10 mol/1 and dopamine 10 mol/1 repolarized the membrane. SCH 23390 1 mol/1 antagonized the effects of SKF 38393 10 mol/1 and dopamine 10 mol/1. Thus, the change in membrane potential is mediated by a DA₁-recep-tor.     

Binding of 3H-clonidine to an α-adrenoceptor in membranes of guinea-pig ileum

Summary The binding of ³H-clonidine to membrane particles from guinea-pig ileum was investigated. The specific binding, i.e. the binding that could be inhibited by high concentrations of unlabeled clonidine or noradrenaline, was of high affinity, K _D3 nM. The number of sites was approximately 25 fmol/mg protein. Rate constants of association and dissociation were 5.3×10⁷ M^–1 min^–1 and 0.18 min^–1, respectively. Affinites of various drugs to the binding site were determined by measuring their effect on the binding of ³H-clonidine. The affinity of adrenergic agonists decreased in the order clonidine = tramazoline > (–)-erythro--methylnoradrenaline > (–)-noradrenaline (–)-phenylephrine. (–)-Noradrenaline had about 20 times more affinity than the (+)-isomer. The affinity of -adrenoceptor antagonists decreased in the order phentolamine > rauwolscine = yohimbine > WB 4101 > pseudoyohimbine > prazosin = corynanthine. Yohimbine and rauwolscine had about 100 times more affinity than their stereoisomer corynanthine. Serotonin 10 M and metiamide 10 M did not affect the binding, and propranolol inhibited it only at high concentrations. — The results indicate that ³H-clonidine labels an ₂-adrenoceptor in guinea-pig ileum. The orders of affinity of -adrenoceptor agonists and antagonists agree well with their orders of potency in functional tests, namely as modulators of cholinergic transmission in the guinea-pig ileum and as modulators of noradrenaline release in the rabbit pulmonary artery. An -adrenoceptor should be classified as ₂ when the affinities of clonidine, tramazoline and -methylnoradrenaline greatly exceed the affinity of phenylephrine, and when the affinities of rauwolscine and yohimbine exceed those of prazosin and corynanthine.     

The steady-state concentration gradient for 3H-noradrenaline generated by uptake1 in the extracellular space of the rat vas deferens incubated with this amine

Summary The rat vas deferens was incubated with 0.2 mol/l ³H-noradrenaline for 60 min, washed out with amine-free solution for 100 min and then prepared for autoradiography (same tissues as presented by Azevedo et al. (1990) Naunyn-Schmiedeberg's Arch Pharmacol 342: 245 – 248). The autoradiography images were then digitized, and grain density was determined as a function of the distance from the surface of the tissue. When neither monoamine oxidase nor vesicular uptake was impaired, i. e. under control conditions, grain density declined monophasically exponentially towards the centre of the tissue. This decline amounted to 0.017 m^–1 or 0.124 varicosity^–1, since the average distance between varicosities was calculated to be 7.4 m. After inhibition of monoamine oxidase and vesicular uptake the rate constant was significantly reduced, and the grain density in close proximity of the surface of the tissue was also reduced.It is proposed that the distribution of grain density observed in controls reflects the steady-state concentration gradient that is generated by uptake₁ during the incubation with ³H-noradrenaline.During spontaneous efflux of ³H-noradrenaline one has to distinguish between re-uptake of the ³H-amine into the leaking varicosity and uptake en passant (during diffusion through the extracellular space). On the basis of the present results, the extent of uptake en passant was calculated (with a computer-assisted model) for the spontaneous efflux of heterogeneously distributed ³H-noradrenaline (after wash-out). Uptake en passant into varicosities located between the source of efflux and the medium amounted to about 55% of the net leakage of ³H-noradrenaline from all varicosities. Earlier experiments had indicated that the sum of the two uptake processes was responsible for the neuronal uptake of 90% of the gross leakage of ³H-noradrenaline from varicosities. Hence, the following appears to take place: about 78% of the gross leakage of noradrenaline from a varicosity are subject to re-uptake into the same varicosity. During diffusion to the medium, about 55% of the ³H-noradrenaline escaping re-uptake is then subject to neuronal uptake en passant. Send offprint requests to E. Schömig at the above address     

Effect of nicotine and tacrine on acetylcholine release from rat cerebral cortical slices

Summary The effect of nicotine (1–10 M) and tacrine (9-amino-1,2,3,4-tetrahydroacridine; THA) on stimulation evoked release of [³H]acetylcholine from the rat brain slice preparation preincubated with [³H]choline was investigated.In these preparations, nicotine enhanced while tacrine inhibited evoked [³H]acetylcholine release. These effects were blocked by (+)tubocurarine (1 M) and atropine (0.1 M) respectively. In the presence of idazoxan (0.3 M) plus atropine (0.1 M), nicotine (3 M) continued to enhance evoked [³H]acetylcholine release while the inhibitory effect of tacrine (1 M) on evoked [³H]acetylcholine release was reversed to an enhancement. Under these circumstances the effects of both nicotine and tacrine were blocked by (+)tubocurarine (1 M).These findings demonstrate that tacrine can both inhibit or enhance [³H]acetylcholine release, most likely through its activity as a cholinesterase inhibitor. Under normal circumstances following tacrine the predominant effect of the elevated levels of acetylcholine will be activation of inhibitory presynaptic muscarine receptors on cholinergic nerves and an inhibition of evoked [³H]acetylcholine release. Under conditions where both presynaptic inhibitory muscarine and ₂-adrenoceptors are blocked, the elevated levels of acetylcholine produced by tacrine will lead to the activation of facilitatory presynaptic nicotine cholinoceptors on cholinergic nerves and an enhancement of evoked [³H]acetylcholine release. Send offprint requests to R. Loiacono at the above address