Norbert Limberger

Species differences in presynaptic serotonin autoreceptors : mainly 5-HT1B but possibly in addition 5-HT1D in the rat, 5-HT1D in the rabbit and guinea-pig brain cortex

SummaryThe pharmacological properties of presynaptic serotonin autoreceptors were compared in slices of rat, rabbit, and guinea-pig brain cortex. The slices were preincubated with 3H-serotonin and then superfused with medium containing fluvoxamine 3 μmol/l and stimulated four times by trains of four pulses delivered at 100 Hz. Cumulative concentration-response curves were determined and used for the calculation of agonist EC50 values and maximal effects and antagonist KB values.Unlabelled serotonin itself and the serotonin receptor agonists 5-carboxamidotryptamine (5-CT), 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) and (±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) reduced the stimulation-evoked overflow of tritium with a rank order of potency 5-CT = RU 24969 > serotonin > 8-OH-DPAT in the rat and 5-CT > serotonin > RU 24969 > 8-OH-DPAT in the rabbit and guinea-pig. Ipsapirone caused no change. Metitepine and metergoline antagonized the effect of 5-CT; the KB values were lower in the rabbit and guinea-pig than in the rat. Yohimbine at up to 1 μmol/1 did not reduce the evoked overflow of tritium and did not antagonize the inhibitory effect of 5-CT in the rat but reduced the evoked overflow in the rabbit and counteracted the effect of 5-CT in the guinea-pig. (−)-Propranolol, conversely, reduced the evoked overflow of tritium in the rat but neither reduced the evoked overflow nor antagonized the effect of 5-CT in the rabbit and guinea-pig. Isamoltane did not significantly change the effect of 5-CT in any species. In the rat, it also failed to antagonize the inhibitory effect of 8-OH-DPAT but did antagonize the effect of RU 24969. The inhibition caused by 8-OH-DPAT persisted in the presence of idazoxan but was attenuated by metitepine in all species.The experimental conditions used permit the determination of the constants of agonist and antagonist action undistorted by autoinhibition. The results confirm the view that the serotonin axons of rat brain possess 5-HT1B autoreceptors. They show by direct comparison under identical conditions that the autoreceptors in rabbit and guinea-pig are very similar to each other but differ markedly from those in the rat. The results give additional credence to previous suggestions that, in the rabbit and guinea-pig, the autoreceptors are 5-HT1D. The serotonin axons of rat brain cortex may possess 5-1D in addition to 5-HT1B autoreceptors. In many previous studies agonist potencies at, and antagonist affinities for, presynaptic serotonin autoreceptors have been underestimated due to the use of too intense stimuli to elicit serotonin release.

A Search for Receptors Modulating the Release of γ-[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices

Abstract: Various putative striatal transmitters and related compounds were studied for their effects on the release of γ‐aminobutyric acid (GABA) from slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused and stimulated electrically at 5 or 20 Hz. Aminooxyacetic acid was present throughout. The main changes observed were the following. The basal and, less consistently, the electrically evoked overflow of [3H]GABA were enhanced by 3,4‐dihydroxyphenylethylamine (dopamine), an effect not blocked by cis‐flupentixol or domperidone and not mimicked by apomorphine and D1‐selective agonists. The electrically evoked overflow was diminished by 5‐hydroxytryptamine (serotonin); the inhibition was prevented by methiothepin. The basal but not the electrically evoked overflow was enhanced by carbachol; acetylcholine and nicotine also accelerated the basal out‐flow whereas oxotremorine caused no consistent change; the effects of carbachol and acetylcholine were blocked by hexamethonium but not by atropine or by tetrodotoxin. These findings indicate that the GABA neurons in the caudate nucleus may be stimulated by dopamine, although the receptor type involved remains unclear; inhibited by serotonin; and stimulated by acetylcholine acting via a nicotine receptor. However, all drug effects observed were relatively small. No evidence was obtained for autoreceptors, α2‐adrenoceptors or receptors for opioids, adenosine or substance P at the GABA neurons.

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

SummaryPresynaptic α2-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 α2-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 KD. The KD values correlated only weakly between the rat and the rabbit. Dissociation constants KA of bromoxidine were calculated from equieffective concentrations in unpretreated brain slices and slices in which part of the α2-adrenoceptors had been irreversibly blocked by phenoxybenzamine. The KA 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 α2-autoreceptors. Comparison with data from the literature indicates that the rat brain autoreceptors can be equated with the α2D subtype as defined by radioligand binding, whereas the rabbit brain autoreceptors conform to the α2A subtype. For example, the antagonist affinities for the rat autoreceptors correlate with their binding affinities for the gene product of α2-RG20, the putative rat α2D-adrenoceptor gene (r = 0.97; P<0.01), but not with their binding affinities for the gene product of α2-C10, the putative human α2A-adrenoceptor gene. Conversely, the rabbit autoreceptors correlate with the α2-C10 (r = 0.98; P<0.001) but not with the α2-RG20 gene product. Since presynaptic α2-autoreceptors are also α2D in rat submaxillary gland and perhaps vas deferens and α2A in rabbit pulmonary artery, the possibility arises that the majority of α2-autoreceptors generally are α2D in the rat and α2A in the rabbit. Moreover, receptors of the α2A/D group generally may be the main mammalian α2-autoreceptors.

“Real time” measurement of endogenous dopamine release during short trains of pulses in slices of rat neostriatum and nucleus accumbens : role of autoinhibition

SummaryRelease of endogenous dopamine elicited in slices of rat neostriatum or nucleus accumbens by a single electric pulse or by trains of 4 or 10 pulses was examined using fast cyclic voltammetry.Single electric pulses gave rise to a marked and transient increase in the extracellular concentration of dopamine in the neostriatum (by 0.43 μmol/l) and nucleus accumbens (by 0.39 μmol/l). The overflow elicited by subsequent pulses delivered at a frequency of 0.2 Hz caused separate but much smaller peaks of dopamine concentration, whereas the overflow elicited by subsequent pulses delivered at 1 Hz caused only a shoulder in the descending limb of the peak due to pulse 1. Four pulses at 5 Hz produced a monophasic response that was higher than the single pulse-evoked peak. Nomifensine 1 μmol/l greatly increased and prolonged the evoked overflow of dopamine. In the absence of nomifensine, metoclopramide 0.3 μmol/l did not change the response to a single pulse or 4 pulses delivered at 0.2 Hz but increased the response to 4 or 10 pulses at 1 Hz and to 4 pulses at 5 Hz. In the presence of nomifensine, metoclopramide increased the response to a single pulse as well as, to a greater extent, the response to 4 pulses at 0.2 Hz and 4 pulses at 1 Hz. Sulpiride 1 μmol/l produced effects similar to those of metoclopramide in the neostriatum in the presence of nomifensine. During trains of pulses at 0.2 or 1 Hz, metoclopramide and sulpiride did not increase (or increased only slightly in the presence of nomifensine) the initial peak that reflected dopamine overflow elicited by pulse 1, but increased greatly the subsequent peaks (0.2 Hz) or the sholder (1 Hz) that reflected the overflow due to the subsequent pulses.The study demonstrates the release of dopamine in the neostriatum and nucleus accumbens with high temporal resolution so that, at least at low frequency, the release elicited by each pulse in a train can be recognized. As previously concluded from experiments with 3H-dopamine, single pulses elicit a large release whereas subsequent pulses delivered at 0.2 to 5 Hz elicit much smaller release. Presynaptic autoinhibition develops immediately after pulse 1 in trains of appropriately spaced pulses. However, it is only partly responsible for the marked fall in release after pulse 1; other, unknown factors contribute to the decline.

Presynaptic α2-adrenoceptor, opioid κ-receptor and adenosine A1-receptor interactions on noradrenaline release in rabbit brain cortex

SummaryThe interaction of presynaptic, release-inhibiting α2-adrenoceptors, opioid κ-receptors and adenosine A1-receptors was studied in slices of the occipito-parietal cortex of the rabbit. The slices were preincubated with 3H-noradrenaline and then superfused and stimulated electrically twice for 2 min each (S1, S2). The stimulation-evoked overflow of tritium was taken to reflect action potential-evoked release of noradrenaline. One of two release-modulating compounds to be examined for interaction was kept in the medium throughout superfusion, the other one was added before S2. In many experiments, the stimulation parameters were adjusted (frequency 0.5–7 Hz; voltage drop 2–5 V/cm) in order to obtain similar reference release (S1) values despite the presence of the first release-modulating compound.The selective κ-receptor agonist ethylketocyclazocine (EK) attenuated markedly the release-inhibiting effects of the α2-adrenoceptor-selective agonists clonidine and α-methylnoradrenaline as well as the release-facilitating effect of the α2-adrenoceptor-selective antagonist yohimbine. The attenuation occurred both when the parameters of electrical stimulation were kept constant and when they were adjusted to obtain similar S1 release values. The selective A1-receptor agonist R-N6-phenylisopropyladenosine (PIA) also attenuated the effects of clonidine and yohimbine. Conversely, clonidine attenuated and yohimbine enhanced the release-inhibiting effect of PIA. Yohimbine also enhanced the release-facilitating effect of the adenosine receptor antagonist 8-phenyltheophylline. Again, these changes occurred both at constant stimulation parameters and when stimulation parameters were adjusted. EK attenuated the release-inhibiting effect of PIA, and conversely PIA attenuated the effect of EK, both at constant and at adjusted parameters of electrical stimulation. The release-inhibiting effects of tetrodotoxin and Cd2+ remained unchanged in the presence of clonidine or EK.These results demonstrate mutual interactions between presynaptic a2-, opioid K- and adenosine A1-receptors. As soon as any one of the three systems is activated, the inhibition due to activation of either of the two remaining systems is blunted. The interactions are not a consequence of the change in release per se that the first receptor ligand inevitably produces. α-Adrenoceptors interact with opioid κ-receptors in a similar manner, independently of the chemical nature (imidazoline or phenylethylamine derivative) of the α-agonist used. The interaction is specific for release-modulating receptors and does not extend to Na+ ar Ca2+ channel blockers. It may occur at the level of the receptors themselves or at the post-receptor transduction mechanisms.

PRESYNAPTIC ALPHA 2A-ADRENOCEPTORS INHIBIT THE RELEASE OF ENDOGENOUS DOPAMINE IN RABBIT CAUDATE NUCLEUS SLICES

Abstractα2-Adrenoceptors modulating the release of dopamine were identified and characterized in slices of the head of the rabbit caudate nucleus. Release of endogenous dopamine was measured by fast cyclic voltammetry as the increase in the extracellular concentration of dopamine elicited by electrical stimulation. The electrochemical signal was identified as dopamine by means of the oxidation potential, the voltammogram and the fact that the signal was not changed by desipramine, which inhibits the high affinity uptake of noradrenaline, but was greatly increased by nomifensine, which in addition inhibits the high affinity uptake of dopamine.Stimulation by 6 pulses/100 Hz increased the extracellular concentration of dopamine by about 85 nM. The selective α2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced this release with an EC50 of 173 nM and by maximally 75%. The α2-adrenoceptor agonists clonidine and oxymetazoline only tended to cause a decrease. Six drugs, including oxymetazoline, were tested as antagonists against UK 14,304. Their order of antagonist potency (pKD values in brackets) was rauwolscine (8.0) > oxymetazoline (7.5) > 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101; 7.3) > phentolamine (7.1) > corynanthine (5.1) ≈ prazosin (< 6). Given alone, the antagonists did not change the release of dopamine elicited by 6 pulses/100 Hz, and the same was true for the dopamine receptor antagonist sulpiride. When caudate slices were stimulated by 10 pulses/1 Hz, sulpiride increased the release of dopamine. Desipramine and rauwolscine, in contrast, again caused no change.It is concluded that dopaminergic axons in the rabbit caudate nucleus possess release-inhibiting α2-adrenoceptors. The antagonist affinities indicate that they belong to the α2A subtype. In this, they agree with all presynaptic α2-autoreceptors studied so far in rabbits as well as with the α2-heteroreceptors modulating the release of serotonin in rabbit brain cortex, suggesting that at least the majority of presynaptic α2-adrenoceptors in the rabbit are α2A. The agonist sensitivity of the caudate presynaptic α2-adrenoceptors is low in comparison with cerebrocortical presynaptic α2-autoreceptors, possibly due to absence of a receptor reserve.

Transmitter release patterns of noradrenergic, dopaminergic and cholinergic axons in rabbit brain slices during short pulse trains, and the operation of presynaptic autoreceptors

SummarySlices of rabbit brain were field-stimulated either by single electrical pulses or by trains of 4 or 8 pulses at 1 or 100 Hz in order to study transmitter release patterns and the autoinhibition of transmitter release. The slices were preincubated with 3H-noradrenaline (cortex), 3H-dopamine (caudate nucleus) or 3H-choline (caudate nucleus).Slices preincubated with 3H-noradrenaline were superfused with medium containing desipramine 1 gmol/l. The overflow of tritium elicited by single pulses amounted to 0 .19% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was similar, whereas that elicited by 4 pulses/100 Hz was 5.1-fold higher. Yohimbine 101000 nmol/l increased up to 2.5-fold the overflow evoked by 4 pulses/1 Hz but did not change the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with 3H-dopamine were superfused with medium containing nomifensine 1 μmol/l. The overflow of tritium elicited by single pulses was 0.39% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was 1.3-fold and the overflow elicited by 4 pulses/100 Hz 1.4-fold higher. Domperidone 1–100 nmol/l and sulpiride 10–1000 nmol/1 increased up to 2.4-fold the overflow evoked by 4 pulses/ 1 Hz but increased only slightly the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with 3H-choline were superfused either with physostigmine-free medium or with medium containing physostigmine 1 μmol/l. In physostigmine-free medium, atropine did not increase the evoked overflow of tritium at any stimulation condition. In physostigmine-containing medium, the overflow elicited by single pulses was 0.18% of the tritium content of the tissue. The overflow elicited by 8 pulses/1 Hz was 2.0-fold and the overflow elicited by 8 pulses/100 Hz 2.2-fold higher. Atropine 2–200 nmol/1 increased up to 2.4-fold the overflow evoked by 8 pulses/1 Hz but increased only slightly the overflow evoked bysingle pulses or 8 pulses/100 Hz. In physostigmine-free medium, sulpiride 10–1000 nmol/1 did not change the single-pulse-evoked overflow of tritium in the absence but increased it in the presence of nomifensine 1 μmol/l.Single pulses elicit a large release of 3H-noradrenaline, 3H-dopamine and 3H-acetylcholine under the conditions of these experiments. Release elicited by single pulses is not subject to autoinhibition except for a small inhibition by spontaneously released transmitter in the case of dopaminergic and cholinergic axons. When 3 or 7 further pulses follow the first one at intervals of 1 s, they elicit much smaller release. At least a great part of the fall is due to autoreceptor mediated inhibition (for 3H-acetylcholine release in the presence of physostigmine only). When 3 or 7 further pulses follow at intervals of 10 ms, they elicit release that is either similar to that evoked by the first pulse (3H-noradrenaline) or much smaller (3H-dopamine, 3H-acetylcholine). However, the fall is not due to stimulation-dependent, auto-receptor-mediated inhibition; autoinhibition does not develop in these short high-frequency trains. Overall, the results are in accord with the autoreceptor theory. They demonstrate the role of autoinhibition in determining the transmitter release patterns of central noradrenergic, dopaminergic and cholinergic neurones.

Pharmacological characterization of presynaptic α2-autoreceptors in rat submaxillary gland and heart atrium

1 The pharmacological properties of presynaptic α2‐autoreceptors were studied in rat isolated submaxillary glands and atria. Tissue pieces were preincubated with [3H]‐noradrenaline, then superfused with medium containing desipramine, and stimulated electrically. In one series of experiments, pEC30 values of 12 α‐adrenoceptor antagonists were determined, i.e., negative logarithms of concentrations that increased the electrically evoked overflow of tritium by 30%. In another series, pKD values of 9 α‐adrenoceptor antagonists against the release‐inhibiting effect of 5‐bromo‐6‐(2‐imidazolin‐2‐ylamino)‐quinoxaline (UK 14304), and of 3 antagonists against the release‐inhibiting effect of methoxamine, were determined. 2 In submaxillary glands, the pEC30 values of the antagonists correlated well with their pKD values against UK 14304 (r = 0.93). The same was true for atria (r = 0.92). 3 In submaxillary glands, the pKD values of 3 antagonists against UK 14304 were very similar to their pKD values against methoxamine, with a maximal difference of 0.4. The same was true for atria where the maximal difference was 0.3. 4 The pEC30 values obtained in submaxillary glands correlated significantly with those obtained in atria (r = 0.81). The same was true for the pKD values (r = 0.79). However, the pEC30 and pKD values also indicated consistent differences between the two tissues. 5 It is concluded that the sites of action of the imidazoline UK 14304 (α2‐selective), the phenylethylamine noradrenaline, and the phenylethylamine methoxamine (α1‐selective) are exclusively α2‐adrenoceptors. There is no indication for presynaptic α1‐adrenoceptors or for an effect of UK 14304 mediated by presynaptic imidazoline receptors. The α2‐autoreceptor population in the submaxillary gland differs from that in the atrium. 6 Comparison with studies from the literature indicates that the submaxillary autoreceptors are closely similar to the α2D radioligand binding site found in the bovine pineal gland and probably the rat submaxillary gland. The atrial autoreceptors also conform best to this site, but the agreement is more limited; the atrial autoreceptors may represent a type related to, but distinct from, the α2D site, or a mixture of different types.

Release-inhibiting α2-adrenoceptors at serotonergic axons in rat and rabbit brain cortex: evidence for pharmacological identity with α2-autoreceptors

The pharmacological properties of the presynaptic α2-adrenoceptors modulating the release of serotonin in rat and rabbit brain cortex (α2-heteroreceptors) were compared with the properties of presynaptic α2-autoreceptors in the same brain area. Brain cortex slices were preincubated with [3H]-serotonin or [3H]-noradrenaline and then superfused and stimulated by brief high-frequency pulse trains.The α2-adrenoceptor agonist bromoxidine reduced the electrically evoked overflow of tritium in experiments with both [3H]-noradrenaline and [3H]-serotonin and in brain slices from either species. The antagonists phentolamine, idazoxan, (+)-mianserin, rauwolscine, 5-chloro-4(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-amine (ORG 20350), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), (−)-mianserin and corynanthine caused parallel shifts of the concentration-inhibition curves of bromoxidine to the right. Negative logarithms of antagonist dissociation constants pKd were calculated from the shifts. In the rat, the α2-autoreceptor pKd value of each single antagonist was similar to its α2-heteroreceptor pKd value, maximal difference 0.4, giving a close correlation, r = 0.97 (P<0.001). In the rabbit equally, the α2-autoreceptor pKd value of each single antagonist was similar to its α2-heteroreceptor pKd value, maximal difference 0.4, again yielding a close correlation, r = 0.96 (P < 0.001). However, antagonist pKd values at rat α2-autoreceptors differed from those at rabbit α2-autoreceptors, r = 0.70 (P > 0.05), and antagonist pKd values at rat α2-heteroreceptors differed from those at rabbit α2-heteroreceptors, r = 0.64 (P > 0.05). Comparison with radioligand binding experiments from the literature indicated that, in the rat, both auto- and heteroreceptors conformed best to the α2D subtype (r ⪖ 0.97, P < 0.01 for pKd correlation with binding sites in rat submaxillary gland) whereas, in the rabbit, they conformed best to the α2A subtype (r ⪖ 0.93, P < 0.01 for pKd correlation with binding sites in HT29 cells).It is concluded that, in both the rat and the rabbit, the α2-adrenoceptors modulating the release of serotonin are pharmacologically identical with the presynaptic α2-autoreceptors. However, rat α2-autoreceptors and -heteroreceptors differ pharmacologically from rabbit α2-autoreceptors and -heteroreceptors. Presynaptic α2-auto-as well as -heteroreceptors are α2D in the rat and α2A in the rabbit.

Estimation ofpA2 values at presynaptic α2-autoreceptors in rabbit and rat brain cortex in the absence of autoinhibition

SummaryAn attempt was made to determinepA2 values of antagonists at the presynaptic, release-inhibiting α2-autoreceptorsof rabbit and rat brain cortex under conditions when there was very little released noradrenaline in the autoreceptor biophase and, hence,pA2 values were not distorted by endogenous autoinhibition. Cortex slices were preincubated with3H-noradrenaline and then superfused and stimulated by trains of 4 pulses delivered at 100 Hz or, in a few cases, by trains of 36 pulses at 3 Hz. The α-adrenoceptor agonists clonidine, noradrenaline, and α-methylnoradren-aline concentration-dependently decreased the stimulation-evoked overflow of tritium. The a-adrenoceptor antagonists yohimbine, rauwolscine and idazoxan did not increase the overflow of tritium elicited by 4 pulses/100 Hz in rabbit brain slices and increased it only slightly in rat brain slices. In contrast, the antagonists increased markedly the overflow at 36 pulses/3 Hz. All antagonists caused parallel shifts to the right of the concentration-response curves of clonidine, noradrenaline, and α-methylnoradrenaline.pA2 values were calculated either from linear regression of log [agonist concentration ratio − 1] on log [antagonist concentration] or from sigmoid curve fitting. The slopes of the linear regression lines were close to unity, and thepA2 values calculated by the two methods agreed well. There was no consistent preferential antagonism of any antagonist to any agonist.pA2 values determined with stimulation by 4 pulses/100 Hz were by 0.53–0.80 log units higher than those determined with stimulation by 36 pulses/3 Hz. ThepA2 values (4 pulses/100 Hz) of yohimbine and rauwolscine in rabbit brain slices (approximately 7.9 and 8.2, respectively), were slightly higher than in rat brain slices (approximately 7.6 and 7.7, respectively), whereas thepA2 value of idazoxan in the rabbit. (about 7.1) was lower than itspA2 value in the rat (about 8.0). The experiments confirm thatpA2 values determined under conditions of autoinhibition are too low. Stimulation with short (30 ms) bursts of pulses permits the estimation ofpA2 values at presynaptic a2-autoreceptors without (rabbit) or almost without (rat) the complication of autoinhibition. The values suggest that α2-adrenoceptors in rabbit brain cortex differ slightly from those in rat brain cortex.