Protein Aggregates Seem to Play a Key Role Among the Parameters Influencing the Antigenicity of Interferon Alpha (IFN-α) in Normal and Transgenic Mice

Purpose. During long-term treatment of various malignant or viral diseases with IFN- up to 20% of patients develop anti-IFN- antibodies for as yet unknown reasons. Methods. To address this issue, a mouse model using Balb/C mice was established and the relevance of several potentially anti-IFN- antibodies inducing factors was studied. Results. The model revealed that both a higher frequency of injections and a higher dosage of IFN- were more immunogenic and that the route of administration affected the antibody response to IFN-. The intrinsic immunostimulatory activity of IFN- itself also enhanced the immune response. IFN- protein aggregates (IFN--IFN- and human serum albumin (HSA)-IFN- aggregates), which were recently identified in all marketed IFN- products, were significantly more immunogenic than IFN- monomers. These aggregates broke the tolerance against human IFN- monomers in human IFN- transgenic mice. Conclusions. Based on these animal studies it is proposed that the immune response to IFN- in humans is most probably elicited by a combination of several factors among which IFN- protein aggregates seem to play a key role.     

Viscoelastic Properties of Polyacrylic Acid Gels in Mixed Solvents

The objective of this study is to investigate the viscoelastic properties of Carbopol 934P polymeric systems in a variety of mixtures of pharmaceutical solvents. Carbopol 934P neutralized with a 1:1 equivalent ratio of triethanolamine was dissolved in various binary or ternary solvent mixtures consisting of propylene glycol, glycerol formal, and water. Dynamic moduli G and G, complex viscosities, and , and loss tangent, tan, were examined over a frequency range of 10-3 to 10 Hz using an oscillatory viscoelastic rheometer at 30°C. The results indicated that for 0.5-1.5 wt% neutralized Carbopol in ternary mixtures, G and G increased by 3-4 orders of magnitude and the phase angle decreased from 80 to 25° when the water content in the solvent mixture increased from 10 to 80 wt%. These studies also indicated that the addition of water to nonaqueous Carbopol 934P polymer systems transforms them from low-viscosity solutions to gels with significant elastic behavior involving physical interaction and entanglement of polymer segments with solvents.     

Heroin increases the density and sensitivity of platelet α2-adrenoceptors in human addicts

The density of platelet ₂-adrenoceptors, quantified by means of the binding of [³H]clonidine, and the aggregation response induced by adrenaline, was investigated in ten heroin addicts. The number of binding sites for [³H]clonidine was significantly increased during heroin abuse. Concomitantly, there was a potentiation of the adrenaline-induced platelet aggregation, which suggested that continuous heroin use (opiate dependence) is associated with supersensitive platelet ₂-adrenoceptors in human addicts. Spontaneous heroin withdrawal further increased in the same addicts the density of platelet ₂-adrenoceptors. These results suggest that platelet ₂-adrenoceptors may be used as a model to study receptor mechanisms of opiate dependence and withdrawal in humans     

Possible subdivision of postsynaptic α-adrenoceptors mediating pressor responses in the pithed rat

Summary Additional evidence has been obtained indicating a possible subclassification of postsynaptic -adrenoceptors into ₁ and ₂-subtypes. The pressor responses to the -adrenoceptor agonists L-phenylephrine and guanfacine were quantified after i.v. administration to pithed rats. The -sympatholytic drug yohimbine (1 mg/kg) displaced both dose-response curves to the right, but the effect was greatest for guanfacine. After prazosin (0.1 mg/kg) a 53-fold shift to the right was noticed for the dose-response characteristic of L-phenylephrine. Prazosin antagonized the effect of only the higher doses of guanfacine. The findings indicate that L-phenylephrine and prazosin preferentially interact with ₁-adrenoceptors as agonist and antagonist, respectively. Yohimbine proved less selective than prazosin, but preferentially blocks postjunctional ₂-adrenoceptors in the vascular wall. The results obtained with guanfacine may be interpreted to indicate that this drug acts on ₂-adrenoceptors at lower doses and additionally stimulates ₁-adrenoceptors at higher ones. Preliminary findings with corynanthine and rauwolscine support this interpretation.     

Synthesis of some aralkyldimethylhydrazine derivatives as blocking agents of adrenergic neurons

N-benzyl-N,N-dimethyl-N-acetylhydrazinium chloride, N-(o-bromobenzyl)-N,N-dimethyl-N-acetylhydrazinium bromide, and N-(o-chlorobenzyl)-N,N-dimethyl-N-acetylhydrazinium bromide, which are blocking agents for adrenergic neurones, have been synthesized. N-(o-bromobenzyl)-N,N-dimethyl-N-acetylhydrazinium bromide has the most pronounced inhibiting activity.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 6, pp. 13–15, June, 1967.     

Relationship of metabolism of 2′-, 3′- and 5′-adenine nucleotides to presynaptic inhibition of transmitter release in rat vas deferens

Summary In the isolated rat vas deferens stimulated at 0.2 Hz, a series of 2, 3-, and 5-substituted adenine nucleotides all inhibited the twitch responses, their actions being potentiated by the nucleoside transport inhibitors, HNBTGR, NBMPR and dipyridamole.The metabolism of these nucleotides was examined utilising HPLC analysis of the bathing medium after exposure to 30 M nucleoside or nucleotide for 5 min. 5-AMP, 5-ADP, 5-ATP, and NAD⁺ were all partially hydrolysed to adenosine, the relative extent of this being 5-AMP>5-ADP=5-ATPNAD⁺. However, the other nucleotides examined were not detectably converted to adenosine or to adenosine deamination products.These results indicate that the 2-, 3- and 5-substituted nucleotides studied act at a P₁-purinoceptor in rat vas deferens to inhibit neurotransmission and, with the exception of 5-AMP, 5-ADP, 5-ATP and NAD⁺, all appear to act directly at this receptor. However, the 5-adenine nucleotides (AMP, ADP and ATP) and NAD⁺ all appear to act at least partially indirectly subsequent to their hydrolysis to adenosine.Abbreviations. The following abbreviations are used ADA adenosine deaminase (EC 3.5.4.4) - 5-ADP adenosine 5-diphosphate - 2,5-ADP adenosine 2,5-diphosphate - 3 5-ADP, adenosine 3,5-diphosphate - 2-, 3 or 5-AMP adenosine 2-, 3-, or 5-monophosphate - 5-ATP adenosine 5-triphosphate - cNADP⁺ -nicotinamide dinucleotide 2,3-cyclic monophosphate - CoA coenzyme A - HNBTGR 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine - NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ -nicotinamide adenine dinucleotide phosphate - NBMPR 6-(4-nitrobenzylthio)-purine riboside     

Metabolism and presynaptic inhibitory activity of 2′,3′ and 5′-adenine nucleotides in rat vas deferens

Summary In the isolated rat vas deferens stimulated at 0.2 Hz, [¹⁴C]labelled 5-AMP, 5-ADP and 5-ATP (10 M) inhibited twitch responses, were broken down to [¹⁴C]adenosine in the medium and incorporated into [¹⁴C]adenine ribonucleotides in the tissue. Pretreatment of tissues with 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine (NBTGR), a potent inhibitor of adenosine transport, potentiated the presynaptic inhibitory action of these 5 nucleotides and reduced their incorporation in [¹⁴C]adenine nucleotides, but did not alter the appearance of [¹⁴C]adenosine in the medium.A series of 2, 3 and 5-substituted adenine nucleotides (10 M) inhibited the twitch responses of the vas deferens stimulated at 0.2 Hz. This effect was potentiated by NBTGR. Addition of exogenous adenosine deaminase very significantly reduced the inhibitory actions of adenosine, 5-AMP, 5-ADP and 5-ATP and also reduced those of 2, 5-ADP, NAD⁺ and dePCoA. The inhibitory actions of the other 2, 3 and 5 adenine nucleotides studied were not altered by exogenous adenosine deaminase.These results indicated that the presynaptic inhibitory actions of 5-AMP, 5-ADP and 5-ATP in rat vas deferens predominantly result from their prior hydrolysis to adenosine whereas the 2, 3 and 5-substituted adenine nucleotides appear to act mainly directly to inhibit transmitter release.Abbreviations. The following abbreviations are used 5-ADP 5-adenosine diphosphate - 2,5-ADP 2,5-adenosine diphosphate - 3,5-ADP 3,5-adenosine diphosphate - 2,3 or 5-AMP 2,3 or 5-adenosine monophosphate - 5-ATP 5-adenosine triphosphate - CoA coenzyme A - 2,3-cAMP 2,3-cyclic adenosine monophosphate - cNADP⁺ -nicotinamide dinucleotide 2,3-cyclic monophosphate - dePCoA dephosphocoenzyme A - NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ -nicotinamide adenine dinucleotide phosphate - NBTGR 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine - oxid CoA oxidized-coenzyme A     

Solasodine glycosides of Solanum unguiculatum (A.) Rich

Besides solasonine, three new glycosides, namely, 3-O--L-rhamnopyranosyl-(13)-solasodine, 3-O--L-rhamnopyranosyl-(12)--L-rhamnopyranosyl-(14)--D-galactopyranosyl solasodine, and 3-O--L-rhamnopyranosyl-(12)--D-galactopyranosyl solasodine, were isolated fromSolanum unguiculatum (A.) Rich. Their structures were determined on the basis of chemical and spectral methods.     

Synthesis and antimicrobial action of α-(5-nitrofuryl-2)quinoxaline and its derivatives

Conclusions -(5-Nitrofuryl-2) quinoxaline, and its -cyano- and -oxyderivative were synthesized by nitration of the respective furylquinoxalines with a nitrating mixture. When 2-oxy-3-(furyl-2) quinoxaline is subjected to nitration with excess of nitric acid a dinitro-derivative is formed which is presumably 6-nitro-3-(5-nitrofuryl-2)-2-oxyquinoxaline. The antibacterial, tuberculostatic, and fungistatic activities of the -(furyl-2)- and -(5-nitrofuryl-2) quinoxalines and their derivatives were studied in vitro.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 10, pp. 14–17, October, 1968.     

Effect of ethanol on the metabolism of lithocholic acid in rat liver

Summary Lithocholic acid 24-C¹⁴ is converted by 20000x g-supernatant of rat liver homogenate into several products of higher polarity. 3-6-dihydroxy-5-cholanic acid is the main metabolite, whereas 7-hydroxylation occurs only to a small extent. Besides of the hydroxylations conjugation with taurine and the formation of a 3-sulfate ester of lithocholic acid can be demonstrated. Addition of ethanol to the enzymatic system results in an inhibition of the formation of 3,6-dihydroxy-5-cholanic acid, whereas no effect can be observed with respect to the formation of the other products. This inhibition is present also in 20000x g-supernatant obtained from rats pretreated with ethanol 100 min before being preparation from ethanol-pretreated rats amounts to 68% of the controls. The chrome P-450 mediated oxidative mechanism, which has been shown to be involved in microsomal 6-hydroxylation of bile acids.This work was supported by the Deutsche Forschungsgemeinschaft.     

Presynaptic dopamine DA2-receptors in rabbit jejunal arteries

Summary Excitatory junction potentials (e.j.ps) evoked by nerve stimulation with 15 pulses at 1 Hz were recorded from muscle cells of rabbit isolated jejunal arteries. LY 171555 1 mol/l, SKF 38393 10 mol/l, dopamine 10 ol/l and clonidine 0.1 mol/l depressed all e j.ps in the train. The percentage inhibition was inversely related to the number of pulses. S- and R-sulpiride, 10 mol/l, domperidone 1 mol/l, SCH 23390 1 mol/l and rauwolscine 1 mol/l did not change, or even depressed the first e j.ps. Of these compounds only S- and R-sulpiride, 10 mol/l and rauwolscine 1 mol/l facilitated the late e.j.ps. The percentage facilitation increased with the number of pulses until a maximum was reached; rauwolscine 1 ol/l had the largest effect. S- and R-sulpiride, 10 mol/l, as well as domperidone 1 ol/l antagonized the action of LY 171555 1 mol/l. S-Sulpiride was more potent than its R-isomer. SCH 23390 1 mol/l and rauwolscine 1 mol/l blunted the effect of SKF 38393 10 mol/l. Rauwolscine 1 mol/l slightly reduced the inhibition by dopamine 10 mol/l; S-sulpiride 10 mol/l was antagonistic only in the presence of rauwolscine 1 mol/l. When rauwolscine 1 mol/l, prazosin 0.1 mol/l, propranolol 1 mol/l and cocaine 10 mol/l was added to the medium, dopamine 10 mol/l continued to produce the same depression of e j.ps, as in the absence of these compounds. Under such conditions S-sulpiride 10 mol/l also counteracted dopamine 10 gmol/l. Rauwolscine 1 mol/l prevented the effect of clonidine 0.1 mol/l. The antagonists were not absolutely selective against only one type of agonist. We suggest that both presynaptic DA₂- and postsynaptic DA₁-receptors are present in rabbit jejunal arteries. The activation of either receptor-type may depress the e j.ps. Dopamine interferes with neuroeffector transmission due to ₂-adrenoceptor agonist properties; its DA₂-effect is unmasked only after ₂-adrenoceptor blockade. There was no evidence for a co-transmitter function of dopamine. Send offprint requests to P. Illes at the above address     

Evaluation of P2-purinoceptor antagonists at two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli

The guinea-pig taenia coli possesses two relaxation-mediating receptors for nucleotides: a prototypic P_2Y-purinoceptor, which is activated by adenosine 5-O-(2-thiodiphosphate) (ADPßS), and a separate receptor for ,-methylene ATP (,-MeATP). Effects of several as yet incompletely characterized P₂-purinoceptor antagonists at these receptors were examined.The concentration-relaxation curve of ADPßS was shifted to the right by reactive blue 2, suramin, 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulphonic acid (XAMR0721; at 1000 M only), pyridoxalphosphate-6-azophenyl-2,5-disulphonic acid (iso-PPADS), pyridoxal 5-phosphate, trypan blue and Evans blue (at 320 M only). Schild plots for the antagonism of reactive blue 2, suramin, iso-PPADS and pyridoxal 5-phosphate against ADPßS had slopes <1. The concentration-relaxation curve of ,-MeATP was shifted to the right by reactive blue 2, suramin, XAMR0721, iso-PPADS, pyridoxal 5-phosphate and trypan blue but not by Evans blue (320 M). Schild plots for the antagonism of suramin, XAMR0721 and iso-PPADS against ,-MeATP had slopes >1. Only XAMR0721 differed clearly in potency against the two nucleotides: it was considerably more potent against ,-MeATP than against ADPßS. 2-Methylthio ATP (MeSATP; 1 M) and ATP (100 M) were degraded by pieces of taenia coli. All antagonists except trypan blue attenuated the degradation of either or one of the two nucleotides.The selective effect of XAMR0721 against ,-MeATP confirms the existence of two relaxation-mediating P₂-purinoceptors in guinea-pig taenia coli. Comparison of the apparent affinities of the antagonists for the two taenia coli receptors with affinities for the P_2X-purinoceptor of the rat vas deferens shows that reactive blue 2, suramin, iso-PPADS, pyridoxal 5-phosphate and trypan blue have little selectivity for any of the three receptors. XAMR0721, which has been shown to possess relatively high affinity for the P_2Y-purinoceptor in turkey erythrocytes, was very weak at the P_2Y-receptor of the taenia, thus supporting the existence of pharmacologic P_2Y-receptor subtypes. Evans blue, with little effect in the taenia coli but a marked effect in the rat vas deferens, is the most selective P_2X-(versus P_2Y-) purinoceptor antagonist presently known, although its effect on the degradation of nucleotides must be kept in mind.     

Plasma levels and β-blocking effect of α-hydroxymetoprolol—Metabolite of metoprolol—in the dog

The plasma levels and the - blocking effect of metoprolol and its active metabolite - hydroxymetoprolol have been studied after i.v. bolus injections of the substances to dogs. For both substances the - blockade increased with the dose, and there was a linear relationship between percent reduction in exercise heart rate and the logarithm of plasma concentration. The dose of the metabolite, however, had to be 5 times higher than that of metoprolol to induce the same degree of - blockade. Because of differences in the volume of distribution, 2.0 liters/kg for - OH-metoprolol and 3.5 liters/kg for metoprolol, the 5 times higher dose of - OH-metoprolol resulted in 10 times higher plasma levels of the metabolite than of metoprolol. - OH-Metoprolol was more slowly eliminated (t_1/27.0 hr, total body clearance 3.5 ml-kg^–1-min^–1) than metoprolol (t_1/22.0 hr, total body clearance 20.0 ml-kg^–1-min^–1). Approximately 5% of an i.v. dose of metoprolol was metabolized to - OH-metoprolol. The half-life of the endogenously formed metabolite was the same as after an i.v. dose of the compound.     

Postjunctional α-adrenoceptors

Summary The postsynaptic -adrenoceptors in rat aorta and in pithed rat were investigated according to their sensitivity to nine -adrenergic agonists and to the selective antagonists yohimbine (₂) and prazosin (₁) and the non-selective one, phentolamine. In addition, in radioligand binding studies, the affinity and selectivity of the drugs were determined on rat cerebral cortex using [³H] yohimbine and [³H] prazosin.On rat aorta, prazosin is 1,000 times more potent than yohimbine against each -adrenoceptor agonist, whether ₁- or ₂-selective. Rat aorta probably contains only ₁-adrenoceptors.Pressor effects in pithed rats are mediated by post-junctional ₁- and ₂-adrenoceptors. The dose-response curve for -methylnorepinephrine in the presence of prazosin, using Hofstee's plots, revealed ₁- and ₂-adrenoceptors, respective proportions being 80.5 and 19.5%     

Cyanine-related compounds: a novel class of potent inhibitors of extraneuronal noradrenaline transport

Summary The neurotransmitter noradrenaline is removed from the extracellular space by neuronal and extraneuronal transport mechanisms. In the past, further functional and biochemical characterisation of the corticosterone-sensitive extraneuronal transporter was hampered by the lack of highly potent inhibitors. Here we describe a new class of selective and highly potent inhibitors of the extraneuronal noradrenaline transporter.Clonal Caki-1 cells possess the human type of extraneuronal noradrenaline carrier. The effect of various steroids and steroid-like compounds on initial rates of specific ³H-noradrenaline transport in Caki-1 cells was investigated. None of these steroids had an inhibitory potency higher than that of corticosterone which hitherto was generally accepted as the most potent inhibitor of the extraneuronal noradrenaline transport. On the other hand, a variety of quinoline and isoquinoline derivatives interacted with the extraneuronal noradrenaline transporter. Several cationic quinolines that belong to the chemical class of the cyanine dyes turned out to be very potent inhibitors of ³H-noradrenaline transport in Caki-1 cells. The isocyanines, 1,1-diisopropyl-2,4-cyanine (disprocynium24) and 1-methyl-1-isopropyl-2,4-cyanine as well as the pseudoisocyanines 1,1-diethyl-2,2-cyanine (decynium22) and 1-isopropyl-l-ethyl-2,2-cyanine (iprecynium22) were most potent with IC₅₀'s of 14, 62, 16, and 18 nmol/l, respectively. The inhibitory potency on extraneuronal noradrenaline transport of 1-methyl-l-isopropyl-2,4-cyanine was determined also in isolated organs, namely the isolated incubated rabbit aorta and the isolated perfused rat heart. The IC₅₀'s were 740 and 100 nmol/l, respectively. By contrast, the desipramine-sensitive neuronal type of noradrenaline transporter in PC 12 cells was hardly affected by the cyanine-related compounds. Decynium22 (3 mol/l) inhibited the neuronal noradrenaline transporter of clonal PC12 cells by 14% only.Cyanine-related compounds potently and selectively inhibit the extraneuronal transport mechanism for noradrenaline. They are expected to facilitate the functional and biochemical characterisation of the extraneuronal noradrenaline transporter.Supported by the Deutsche Forschungsgemeinschaft (SFB 176) and the Universitätsbund Würzburg Correspondence to: H. Russ at the above address     

Rewarding properties of β-endorphin as measured by conditioned place preference

The role of -endorphin as a possible mediator in the reinforcing properties of opiates was investigated using a conditioned place preference paradigm. Heroin, a synthetic opiate known to have reinforcing properties, produced a strong preference for an environment previously paired with heroin injection at all doses tested (0.25, 0.5, 1.0, 2.0 mg/kg SC). No such place preference was observed following saline injections. Rats also showed dose-dependent place preference for the environment paired with -endorphin when injected intracerebroventricularly (significant dose was 2.5 g). At higher doses (5.0 and 10.0 g) rats showed no preference for the paired environment, but were catatonic. Pretreatment with naloxone (0.04, 0.2, 1.0 mg/kg SC) attenuated the rewarding effect of -endorphin (2.5 g) at all doses tested. The lowest dose of naloxone which had no aversive effect when tested alone could also significantly block the positive effect of -endorphin. The reinforcing dose of -endorphin (2.5 g) also produced an increase in locomotor activity, when tested in photocell cages. This suggests that the hyperactivity induced by -endorphin may contribute to the preference for an environment previously paired with the same drug. The reinforcing effect of -endorphin is most probably mediated by the mu and/or delta opioid subtype receptor, since -endorphin has a high affinity for these receptors. These results demonstrate positive reinforcing properties of -endorphin in the central nervous system.     

Enzyme kinetics of zearalenone biotransformation: pH and cofactor effects

The aim of the present study was to investigate the hepatic biotransformation of the mycotoxin zearalenone (ZEA) in vitro using subcellular fractions of pig livers. The dependencies of the enzymatic reactions involved on the enzyme velocity, on the cofactor and on pH were analysed in both the microsomal fraction and the post-mitochondrial cell fraction. Finally, the inhibitory effects of various endogenous substrates on the enzymes involved (3- and 3-hydroxysteroid dehydrogenase) were examined. Significant differences were observed between the individual subcellular fractions in terms of prevailing metabolites and absolute amounts of the metabolites produced. Moreover, this study also demonstrated that the reactions for both subcellular fractions of porcine liver are dependent on the cofactor, as -zearalenol (-ZOL) formation increased in the presence of NADPH, whereas -zearalenol (-ZOL) production only increased in the presence of NADH (P<0.001). The optimal pH for -ZOL production was pH 5.6 and that for -ZOL formation pH 7.4. Subsequent inhibition studies showed significant inhibitory effects for 5-androstanedione>androstanedione>pregnenolone on -ZOL formation, whereas -ZOL production was only inhibited by pregnenolone. Finally, the contributions of 3- and 3-hydroxysteroid dehydrogenase during the bioconversion of ZEA are discussed in the context of these experiments.     

A pharmacokinetic model-independent approach for estimating dose required to give desired steady-state trough concentrations of drug in plasma

A maintenance dose designed to give a desired minimum concentration of drug in plasma at steady-state can be determined in a model-independent manner assuming that concentration-time data needed for the calculation are obtained after absorption and distribution are complete. Using a few concentration-time points obtained after the first dose, numerical values of and Z, a parameter consisting of different pharmacokinetic parameters for different models, can be obtained. An administration interval () can be chosen based on . Using the values of , Z, and , a maintenance dose is calculated. This approach will allow calculation of a maintenance dose when drug is present in plasma at the time the first monitored dose is given.     

Synthesis and pharmacological study of some alkylamini esters and amides of α,α-diarylaliphatic acids

Conclusions Sixteen alkylamino esters and amides of ,-diphenyl--ethoxyacetic acid and ,-diphenylpropionic acid have been synthesized and investigated pharmacologically. All the compounds prepared possess spasmolytic and cholinolytic properties. Derivatives of ,-diphenyl--ethoxyacetic acid are also characterized by the presence of analgesic and anti-cough action. Loading the alcoholic or acid part of the molecule, or replacement of the ester bond by an amide bond, leads to weakening of activity.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 1, pp. 9–14, January, 1970.     

Presynaptic α2-autoreceptors in brain cortex: α2D in the rat and α2A in the rabbit

Summary Presynaptic ₂-autoreceptors in rat and rabbit brain cortex were compared by means of antagonists and agonists. Brain cortex slices were preincubated with [³H]-noradrenaline and then superfused and stimulated by 3 (rat) or 4 (rabbit) pulses at a frequency of 100 Hz.The ₂-adrenoceptor agonist bromoxidine (UK 14 304) reduced the electrically evoked overflow of tritium with EC₅₀ values of 4.5 nmol/l in the rat and 0.7 nmol/l in the rabbit. The antagonists phentolamine, 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole (BRL 44408), rauwolscine, 1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo(c,f)imidazo(1,5-a)azepine (BRL 41992), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4, 5-tetrahydro-3-benzazepine (SKF 104078), imiloxan, prazosin and corynanthine did not per se increase the evoked overflow of tritium but shifted the concentration-inhibition curve of bromoxidine to the right in a manner compatible with competitive antagonism. Up to 4 concentrations of each antagonist were used to determine its dissociation constant K_D. The K_D values correlated only weakly between the rat and the rabbit. Dissociation constants K_A of bromoxidine were calculated from equieffective concentrations in unpretreated brain slices and slices in which part of the ₂-adrenoceptors had been irreversibly blocked by phenoxybenzamine. The K_A value was 123 nmol/l in the rat and 7.2 nmol/l in the rabbit.The results confirm the species difference between rat and rabbit brain presynaptic ₂-autoreceptors. Comparison with data from the literature indicates that the rat brain autoreceptors can be equated with the _2D subtype as defined by radioligand binding, whereas the rabbit brain autoreceptors conform to the _2A subtype. For example, the antagonist affinities for the rat autoreceptors correlate with their binding affinities for the gene product of ₂-RG20, the putative rat _2D-adrenoceptor gene (r = 0.97; P<0.01), but not with their binding affinities for the gene product of ₂-C10, the putative human _2A-adrenoceptor gene. Conversely, the rabbit autoreceptors correlate with the ₂-C10 (r = 0.98; P<0.001) but not with the ₂-RG20 gene product. Since presynaptic ₂-autoreceptors are also _2D in rat submaxillary gland and perhaps vas deferens and _2A in rabbit pulmonary artery, the possibility arises that the majority of ₂-autoreceptors generally are _2D in the rat and _2A in the rabbit. Moreover, receptors of the _2A/D group generally may be the main mammalian ₂-autoreceptors.Correspondence to: N. Limberger at the above address