Sodium dependent 3H-noradrenaline release from rat neocortical slices in the absence of extracellular calcium presynaptic modulation by μ-opioid receptor and adenylate cyclase activation

Summary In Ca²⁺-free EGTA (1 mmol/l)-containing medium veratrine (3 mol/l) and ouabain (100 mol/l) strongly enhanced the efflux of ³H-noradrenaline from superfused rat brain neocortical slices prelabelled with the radioactive amine. In both cases ³H-noradrenaline release was prevented by tetrodotoxin (1 mol/l). These effects of veratrine and ouabain were virtually additive and independent of whether the noradrenaline uptake carrier was blocked with 1 mol/l desipramine or not. The adenylate cyclase activator forskolin (10 nmol/l–10 mol/l) strongly enhanced veratrine- and ouabain-induced ³H-noradrenaline release, without affecting spontaneous tritium efflux. The release induced by both stimuli was profoundly inhibited by the selective -opioid receptor agonist [d-Ala, MePhe⁴, Gly-ol⁵]enkaphalin (DAGO, 3 nmol/l–1 mol/l) in a concentration-dependent manner. The inhibitory effects of 1 mol/l DAGO were abolished by 1 mol/l naloxone. On the other hand, preincubation of the slices for 1 h with the -opioid receptor-selective irreversible ligand fentanyl isothiocyanate (1 pmol/l) did not change the inhibitory effects of DAGO.These data show that veratrine- and ouabain-induced ³H-noradrenaline release from central noradrenergic nerve terminals is facilitated by increasing intracellular cyclic AMP levels and reduced by activation of presynaptic -opioid receptors, indicating the involvement of exocytotic neurotransmitter release. The results provide further evidence for the hypothesis that under these conditions neurotransmitter release from central noradrenergic neurons is triggerred by a Na⁺-induced efflux of Ca²⁺ ions from intracellular stores.Abbreviations DAGO [d-Ala², McPhe⁴, Gly-ol⁵]enkephalin Send offprint requests to A. N. M. Schoffelmeer at the above address     

Histamine H3 receptor-mediated inhibition of noradrenaline release in pig retina discs

Summary Discs of pig retina were preincubated with ³H-noradrenaline, ³H-dopamine or ³H-serotonin and then superfused. Electrical field stimulation increased the outflow of tritium from discs preincubated with ³H-noradrenaline or ³H-dopamine, but no from discs preincubated with ³H-serotonin. The tritium content at the end of superfusion was similar in discs preincubated with ³H-noradrenaline or ³H-dopamine but about tenfold lower in discs preincubated with ³H-serotonin. The tritium content in discs preincubated with ³H-noradrenaline was markedly reduced when desipramine was present during preincubation but was not affected by selective inhibitors of dopamine and serotonin uptake. The tritium content in discs preincubated with 3Hdopamine and ³H-serotonin, in contrast, was reduced or tended to be reduced by a selective dopamine and serotonin uptake inhibitor, respectively.The electrically evoked overflow of tritium from discs preincubated with ³H-noradrenaline was abolished by tetrodotoxin or omission of Ca²⁺. In discs superfused with desipramine, the electrically evoked overflow was enhanced by phentolamine but not affected by histamine. When both desipramine and phentolamine were present in the superfusion medium, histamine inhibited the evoked overflow (pIC₁₅ 6.85). This effect was mimicked by the histamine H₃ receptor agonist R-(–)--methylhistamine as well as by its S-(+)-enantiomer (pIC₁₅ 7.85 and 5.30, respectively) but not by the H₁ receptor agonist 2-(2-thiazolyl)ethylamine and the H₂ receptor agonist dimaprit (each 10 mol/l). The inhibitory effect of histamine was abolished by the H₃ receptor antagonist thioperamide 0.32 mol/l and attenuated by impromidine 3.2 mol/l but not affected by the H₁ receptor antagonist dimetindene 3.2 mol/l and the H₂ receptor antagonist ranitidine 10 mol/l.The results suggest that, in the pig retina, noradrenaline is taken up into, and released from, noradrenergic neurones (most likely vascular postganglionic sympathetic nerve fibres, less probably tissue-specific noradrenergic neurones of the retina) and that noradrenaline release is subject to modulation via H₃ receptors and probably also a-adrenoceptors.Send offprint requests to E. Schlicker at the above address     

Differential effects of the stereoisomers of 3PPP in dopaminergic and cholinergic neurotransmission in superfused slices of the corpus striatum

Summary The two enantiomers of 3PPP were tested on the spontaneous and electrically-evoked release of ³H-dopamine from slices of the rabbit caudate nucleus and of ³H-acetylcholine (³H-ACh) from slices of the rat caudate nucleus.In caudate slices labelled with ³H-dopamine, exposure to (+)3PPP (0.1–1 M) facilitated the spontaneous outflow of radioactivity with a concomitant inhibition of the electrically-evoked release of ³H-dopamine. In the presence of cocaine 10 M, exposure to (+)3PPP (1 M) inhibited the electrically evoked release of ³H-dopamine without modifying the spontaneous outflow of radioactivity. This inhibitory effect was not significantly antagonized by S-sulpiride 0.01 M.Exposure to (+)3PPP 1 M inhibited the electrically-evoked release of ³H-ACh, and this effect was not modified by pretreatment with reserpine alone, or in combination with -methyl-p-tyrosine (-MT).In contrast to the (+) enantiomer, exposure to (-)3PPP (0.1–1 M) facilitated the electrically-evoked release of ³H-dopamine without affecting the spontaneous outflow of radioactivity. (-)3PPP antagonized the inhibitory effect of apomorphine on the electrically-evoked release of ³H-dopamine.Exposure to (-)3PPP 1 M did not modify the spontaneous or the electrically-evoked release of ³H-ACh. Yet, this concentration of (-)3PPP antagonized significantly the inhibitory effect of 0.03 M apomorphine, 1 M d-amphetamine, and 1 M (+)3PPP on the electrically-evoked release of ³H-ACh (-)3PPP (0.1–1 M) was about 100 times less potent than S-sulpiride at antagonizing the inhibitory effect of apomorphine on the electrically-evoked release of ³H-ACh.It is concluded that under in vitro conditions at the level of the dopamine receptor modulating the release of ³H-ACh from the cholinergic interneuron in the striatum, (+)3PPP behaves as a dopamine receptor agonist while (-)3PPP possesses dopamine receptor-antagonist properties. At the level of the dopaminergic nerve terminal, (-)3PPP facilitates the release of ³H-dopamine probably through the blockade of the dopamine autoreceptors. The dopamine autoreceptor agonists properties of (+)3PPP are difficult to establish in our model because of the dopamine releasing action of this enantiomer.Some of the results described in this publication have been presented at the British Pharmacological Society Meeting (Arbilla and Langer 1983).     

Interneurones are probably not involved in the presynaptic dopaminergic control of dopamine release in rabbit caudate nucleus

Summary Slices of rabbit caudate nucleus were preincubated with ³H-dopamine and then superfused. The influence of apomorphine and haloperidol on the overflow of tritium evoked by 20 mmol/l potassium was investigated in the presence and in the absence of tetrodotoxin. The potassium-evoked overflow was largely calcium-dependent and consisted mainly of ³H-dopamine. The dopamine receptor agonist apomorphine 0.01–1.0 mol/l reduced, whereas the antagonist haloperidol 0.1 mol/l enhanced the potassium-evoked overflow of tritium. The effects of apomorphine and haloperidol were as pronounced in the presence as in the absence of tetrodotoxin 0.3 mol/l. It is concluded that the presynaptic dopaminergic modulation of dopamine release is not mediated by a tetrodotoxin-sensitive interneuronal pathway.     

Noradrenaline release from rat sympathetic neurons evoked by P2-purinoceptor activation

The effects of ATP and analogues on the release of previously incorporated ³H-noradrenaline were studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Electrical field stimulation (40 mA at 3 Hz) of the neurons for 10 s markedly enhanced the outflow of tritium. ATP applied for 5 s to 2 min at concentrations of 0.01 to 1 mmol/l caused a time- and concentration-dependent overflow with half maximal effects at about 10 s and 100 mol/l, respectively. 2-Methylthio-ATP was equipotent to ATP in inducing ³H-overflow. ADP (100 mol/l), when applied for 2 min, also caused a small ³H-overflow, but , -methylene-ATP (100 mol/l), AMP (100 mol/l), R(–)N⁶-(2-phenylsiopropyl)-adenosine (R(–)-PIA; 10 mol/l) and 5-N-ethylcarboxamidoadenosine (NECA; 1 mol/l) did not. The ³H-overflow induced by 10 s applications of 100 mol/l ATP was abolished by suramin (100 mol/l) and reduced by about 70% by reactive blue 2 (3 mol/l). Electrically evoked overflow, in contrast, was slightly enhanced by suramin, but not modified by reactive blue 2. Xanthine amine congener (10 mol/l) and hexamethonium (10 mol/l) did not alter ATP-evoked release. Removal of extracellular Ca²⁺ from the medium reduced ATP- and electrically induced overflow by about 95%. Tetrodotoxin (1 mol/l) abolished electrically evoked ³H-overflow but inhibited ATP-induced overflow by only 70%. The ₂-adrenoceptor agonist UK 14,304 at a concentration of 1 mol/l diminished both electrically and ATP-evoked tritium overflow by approximately 70%. These results indicate that activation of P₂-purinoceptors stimulates noradrenaline release from rat sympathetic neurons. The release resembles electrically induced transmitter release, but additional mechanisms may contribute. Correspondence to: S. Boehm at the above address     

Effects of GABA,dopamine, and substance P on the release of newly synthesized 3H-5-hydroxytryptamine from rat substantia nigra in vitro

Summary The effects of GABA, substance P and dopamine on the release of newly synthesized ³H-5-HT were investigated, using slices of rat substantia nigra superfused with l-³H-tryptophan in vitro. GABA (50 M) had no inhibitory effect on the potassium-evoked-release of ³H-5-HT. Substance P (50 M) and eledoisin (50 M) stimulated the spontaneous release of ³H-5-HT. This effect seems to be indirect and is possibly mediated by dopaminergic neurones, since the dopamine antagonist drug -flupenthixol (1 M) abolished the substance P-evoked release of 5-HT. Furthermore, it was found that substance P (10 M) stimulated ³H-dopamine release from nigral slices in vitro and the dopaminergic agonist apomorphine (50 M) also stimulated ³H-5-HT release. Substance P may, therefore, activate nigral dopaminergic neurones which then release dopamine from their dendrites. The release of dopamine may in turn stimulate 5-HT release from terminals of the raphe-nigral pathway.     

Effects of the putative P-type calcium channel blocker,R,R-(−)-daurisoline on neurotransmitter release

The alkaloid and medicinal herb constituent, R,R-(–)-daurisoline, was originally reported to be a N-type Ca²⁺ channel blocker, but newer evidence indicates that it is a blocker of P-type Ca²⁺ channels. To clarify its specificity with respect to N- and P-channels, we compared its effects on the electrically induced release of endogenous glutamate, ³H-GABA and ³H-noradrenaline, from brain slices with those of -agatoxin IVA and -conotoxin GVIA. Like -agatoxin IVA (but with about 1000-fold lower potency), and unlike -conotoxin GVIA, R,R-(–)-daurisoline inhibited the release of ³H-GABA and glutamate, with IC₅₀ values of 8 and 18 M. However, inhibition particularly of ³H-GABA release was more complete than by -agatoxin IVA, indicating interaction with one or more additional voltage-sensitive Ca²⁺ channels, possibly the Q-type. Its potency to inhibit glutamate release elicited either electrically, by veratrine or by high concentrations of K⁺ was similar, in contrast to sodium channel blockkes. The effects of R,R-(–)-daurisoline on the release of ³H-noradrenaline, ³H-dopamine and ³H-acetylcholine were in agreement with previous knowledge from experiments with -agatoxin IVA suggesting an involvement of P-channels. A weak inhibition of ³H-noradrenaline release at 10 M, similar to that by -agatoxin IVA at 0.03 M, was occluded by ₂-antagonistic properties and could be unmasked in presence of rauwolscine. At 10 M, it also inhibited electrically evoked ³H-dopamine and ³H-5-hydroxytryptamine release and caused a marked spontaneous release of all three monoamines in a reserpine-like manner. Spontaneous and evoked release of ³H-acetylcholine was inhibited by about 25% at 10 M.In radioligand binding studies, R,R-(–)-daurisoline interacted with ₁ and ₂-adrenoceptors, 5-HT₂ and muscarinic cholinergic receptors with IC₅₀ values close to 1 M, and with opiate receptors even with 0.18 M. Atropine reduced the weak inhibitory effect of R,R-(–)-daurisoline on ³H-acetylcholine release somewhat, suggesting that it was brought about by both P channel blockade and cholinergic agonist activity. The effect on ³H-GABA release was unaffected by naloxone, indicating that the interaction of R,R-(–)-daurisoline with opiate receptors is antagonistic.The pattern of effects on neurotransmitter release observed with R,R-(–)-daurisoline resembles that of -agatoxin IVA and supports previous electrophysiological data suggesting that the compound blocks P-type voltage-sensitive Ca²⁺ channels. However, the more complete blockade of amino acid release by R,R-(–)-daurisoline suggests interaction with additional Ca⁺ channel subtypes. Although it does also possess other pharmacological properties, we think that the compound is suitable to test whether blockade of glutamate release via voltage-sensitive Ca²⁺ channels is a viable concept to obtain novel neuroprotective and/or anticonvulsant compounds.     

Further study of prerequisites for the enhancement by α-adrenoceptor antagonists of the release of noradrenaline

Summary Segments of the rabbit ear artery were preincubated with (–)-³H-noradrenaline and then perfused/superfused and stimulated by transmural electrical pulses. The outflow of ³H-noradrenaline and total tritium was determined.In the first series of experiments, stimulation periods of approximately constant length (50 s) were used (cocaine 5 M present). Thirteen pulses (0.25 Hz) elicited an overflow of ³H-noradrenaline of 0.024% of tissue tritium; 26 pulses (0.5 Hz) elicited an overflow of 0.059%, and 52 pulses (1 Hz) of 0.166%. Rauwolscine 1 M did not change the overflow evoked by 13 pulses, increased that evoked by 26 pulses and increased most markedly that evoked by 52 pulses. Phentolamine 1 M decreased the overflow at 13, did not change the overflow at 26, and increased the overflow at 52 pulses. Corynanthine 1 M decreased the overflow at 13, and did not change the overflow at 26 and 52 pulses. The effect of tetraethylammonium (TEA) 100 M was opposite to that of rauwolscine; it increased most markedly the overflow evoked by 13 pulses, increased less that evoked by 26 pulses, and least the overflow at 52 pulses.In the second series of experiments, the frequency of stimulation was kept constant (2 Hz). In the absence of cocaine, 10 pulses elicited an overflow of ³H-noradrenaline of 0.023% of tissue tritium; 20 pulses elicited an overflow of 0.043%, and 40 pulses of 0.089%. Phentolamine 1 M did not change the overflow evoked by 10 pulses, increased that evoked by 20 pulses, and increased most markedly that evoked by 40 pulses. TEA 100 M increased the evoked overflow at all pulse numbers. Similar results were obtained in the presence of cocaine 5 M.The results demonstrate that the enhancement by -adrenoceptor antagonists of the release of noradrenaline depends on the biophase concentration of noradrenaline. Under the present conditions, graded increases in biophase noradrenaline concentration led to graded increases in the effect of the antagonists. A second prerequisite for the release-enhancing effect appears to be a sufficient length of the pulse train. Under the present conditions, graded increases in train length up to about 20s led to graded increases in the effect of the antagonists, even though the average biophase concentration of noradrenaline did not change with the pulse train length. This pattern of effects of the -antagonists is not shared by at least one other release-enhancing drug, namely TEA.     

Activation of presynaptic α2-adrenoceptors attenuates the inhibitory effect of μ-opioid receptor agonists on noradrenaline release from brain slices

Summary ³H-noradrenaline release from rat neocortical slices induced by 15 mM K⁺ was concentration-dependently inhibited by morphine, [D-Ala²-D-Leu⁵] enkephalin (DADLE) and the calcium entry blocker Cd²⁺. Blockade of presynaptic ₂-adrenoceptors with phentolamine, almost doubling K⁺-induced ³H-noradrenaline release, slightly enhanced the relative inhibitory effects of morphine and DADLE, whereas that of Cd²⁺ remained unaffected. In contrast, activation of presynaptic ₂-adrenoceptors with clonidine (1 M) or TL-99 (1 M), inhibiting release by about 50%, completely abolished the inhibitory effects of morphine and DADLE without affecting that of Cd²⁺. When in the presence of 1 M clonidine adenylate cyclase was activated with forskolin (10 M), which restored release to the drug-free control level, the opioids still did not display their inhibitory effects. Therefore, -opioid receptor efficacy appears to be dependent on the degree of activation of ₂-adrenoceptors in central noradrenergic nerve terminals, probably through a local receptor interaction within the nerve terminal membrane.     

Adenosine but not an adenine nucleotide mediates tonic purinergic inhibition,as well as inhibition by glutamate,of noradrenaline release in rabbit brain cortex slices

Summary A possible contribution of adenine nucleotides to the endogenous purinergic, A₁-receptor-mediated inhibition of noradrenaline release was studied in rabbit occipito-parietal cortex slices. The slices were preincubated with [³H]-noradrenaline and then superfused and stimulated electrically, in most experiments by trains of 6 pulses/100 Hz. A few experiments were carried out in rat occipito-parietal cortex slices. The A₁-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1–100 nmol/l) as well as the enzyme adenosine deaminase (0.1–10 U/ml) increased the electrically evoked overflow of tritiated compounds. The maximal increase was by about 85% for both DPCPX and adenosine deaminase. The increases obtained with maximally effective concentrations of DPCPX and adenosine deaminase were not additive. The ₁-adrenoceptor-selective agonist methoxamine (10 but not 1 mol/l) reduced the evoked overflow. Its effect was antagonized by yohimbine 1 mol/l but then not attenuated further by DPCPX100 nmol/l.L-Glutamate (300 mol/l–2.3 mmol/l) also reduced the evoked overflow of tritium. Its effect was not changed by yohimbine 1 mol/l but greatly, and to the same extent, attenuated by DPCPX 100 mol/l and adenosine deaminase 3 U/ml. Neither the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine nor omission of Mg⁺⁺ changed the inhibition by glutamate. Glutamate did not alter the basal efflux of tritium from rabbit cortex slices under any experimental condition. In contrast, glutamate (100 mol/l and 1 mol/l) caused an immediate, marked and transient acceleration of tritium outflow from rat occipitoparietal cortex slices (medium without Mg⁺⁺). It is concluded that adenosine but not an adenine nucleotide mediates the tonic purinergic presynaptic inhibition of noradrenaline release in rabbit brain cortex. The marked degree of disinhibition by DPCPX and adenosine deaminase underscores the potential physiological role of this inhibition. The purinergic inhibitory tone is reinforced by glutamate, indicating that glutamate releases adenyl compounds in rabbit brain cortex. Again adenosine but not an adenine nucleotide mediates the indirect inhibition by glutamate of the release of noradrenaline. The noradrenaline-releasing effect that glutamate exerts in rat occipito-parietal cortex does not occur in rabbit occipito-parietal cortex. Methoxamine depresses the release of noradrenaline in rabbit brain cortex directly at presynaptic ₂-adrenoceptors rather than by release of purines.Correspondence to I. von Kügelgen 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     

Inhibition by dopamine of 3H-noradrenaline release elicited by nerve stimulation in the isolated cat's nictitating membrane

Summary After loading the isolated nerve-muscle preparation of the cat nictitating membrane with ³H-(±)-noradrenaline the effects of exogenous dopamine and (-)-noradrenaline were determined on ³H-transmitter overflow elicited by nerve stimulation in the presence of cocaine, 29 M. Dopamine, 0.20 M, and (-)-noradrenaline, 0.18 M, inhibited ³H-noradrenaline release elicited by nerve stimulation at 4 or 10 Hz. Similar results were obtained with apomorphine 0.03 or 0.1 M. Chlorpromazine, 1 M, or pimozide, 1 M, antagonized selectively the reduction in ³H-noradrenaline release obtained with dopamine or apomorphine, without affecting the inhibition obtained with (-)-noradrenaline. Phentolamine, 1 M, antagonized more effectively the inhibitory effects of (-)-noradrenaline than those of dopamine. Phenoxybenzamine, 0.29 M, prevented the inhibition of ³H-transmitter overflow obtained with (-)-noradrenaline, dopamine or apomorphine. In the absence of cocaine neither chlorpromazine nor pimozide were able to increase ³H-transmitter overflow during nerve stimulation. In contrast to these results, block of -adrenoceptors by phentolamine or phenoxybenzamine resulted in an increase ³H-transmitter overflow during nerve stimulation. Inhibition by dopamine of ³H-transmitter overflow appears to be mediated through dopamine receptors probably located in the outer surface of adrenergic nerve endings. These dopamine receptors differ from the prejunctional -adrenoceptors that mediate the negative feed-back regulatory mechanism for noradrenaline release by nerve stimulation. The prejunctional inhibitory dopamine receptors are not involved in an endogenously mediated regulatory mechanism for noradrenaline release by nerve stimulation under normal conditions. The possibility that these dopamine receptors are involved in the hypotension commonly observed in patients with chronic l-Dopa treatment is discussed.     

Veratridine-induced outward transport of 3H-noradrenaline from adrenergic nerves of the rat vas deferens

Summary 1. The neuronal release by 100 mol/l veratridine of preloaded ³H-noradrenaline was studied in the rat vas deferens, the MAO, COMT and vesicular uptake of which were inhibited. To prevent any exocytotic release of the ³-Hamine, all solutions were calcium-free. Veratridine induced an early and a late peak of tritium efflux. The early peak was abolished by the presence of 1 mol/l desipramine, the late peak was abolished by 1 mol/l tetrodotoxin (administered subsequently to the first peak). The administration of veratridine plus 1 mmol/l ouabain resulted in only the early peak of efflux. 2. The peak response to veratridine plus ouabain was increased by a very early administration of veratridine plus ouabain (after 40 min of wash-out instead of the usual 130 min) (i. e., when the relative size of the axoplasmic distribution compartment was increased). However, very high axoplasmic ³H-noradrenaline levels (after loading with 37 instead of the usual 0.2 mol/l) reduced the height of the peak (when expressed as a FRL). 3. Substantially similar responses to vcratridine plus ouabain were obtained after loading with ³H-noradrenaline, ³H-adrenaline or ³H-dopamine. 4. As the second peak of veratridine-induced release is ouabain-sensitive, it appears to be caused by exhaustion of neuronal ATP stores; this, in turn, raises the intravesicular pH and induces efflux of ³H-noradrenaline from the vesicles into the axoplasm. The first peak, on the other hand, represents outward transport of ³H-noradrenaline from the axoplasmic compartment. Evidently, a pronounced vesicular distribution of ³H-noradrenaline takes place even after inhibition by reserpine of the vesicular uptake. 5. In preparations with intact vesicular uptake (MAO and COMT inhibited) a plateauresponse was obtained; in the presence of 10 mol/l Ro 4-2184 (a reserpine-like compound) a peak response was restored after loading with 0.2 mol/l³H-noradrenaline, less so after loading with 37 mol/l. 6. It is confirmed that veratridine (plus ouabain) exerts a reserpine-like effect when applied to tissues with intact vesicular uptake and intact MAO.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxy mandelic acid - DOPEG dihydroxyphenylglycol - DOPAC dihydroxyphenylacetic acid - FRL fractional rate of loss - MAO monoamine oxidase - 5-HT 5-hydroxytryptamine with technical assistance of Marianne BablSupported by the Deutsche Forschungsgemeinschaft (Bo 521 and SFB 176) Send offprint requests to: H. Bönisch     

Effects of verapamil,diltiazem and ryosidine on the release of dopamine and acetylcholine in rabbit caudate nucleus slices

Summary Slices of the rabbit caudate nucleus were preincubated with ³H-dopamine or ³H-choline and then superfused with label-free medium. Release of ³H-dopamine and ³H-acetylcholine was elicited by either electrical stimulation at 8 (in one series 2) Hz, or an increase in the K⁺ concentration by 50 mmol/l, or addition of L-glutamate 1 mmol/l. Verapamil 1 mol/l, diltiazem 1 and 10 mol/l, and ryosidine 1 mol/l failed to the reduce the electrically-, K⁺- and glutamate-evoked overflow of tritium. Verapamil 1 mol/l and diltiazem 10 mol/l also failed to reduce the electricallyevoked overflow (2 Hz) when dopamine receptors, neuronal dopamine uptake, and neuronal choline uptake were blocked by domperidone, nomifensine and hemicholinium, respectively. Inhibition of the evoked overflow of tritium was only obtained when concentrations were increased to verapamil 10 mol/l, diltiazem 100 mol/l and ryosidine 10 mol/l. The inhibition was generally small. It was more evident for slices preincubated with ³H-choline than for those preincubated with ³H-dopamine, because in the latter verapamil, diltiazem and (much less) ryosidine accelerated the basal efflux of tritium. The inhibition of the K⁺-evoked overflow of tritium was probably due to blockade of Ca²⁺ channels because this overflow was Ca²⁺-dependent but tetrodotoxin-resistant. In contrast, the inhibition of the electrically- and glutamateevoked overflow possibly involved blockade of Na⁺ channels as well. The results indicate that three calcium antagonists from different chemical classes are very weak inhibitors of Ca²⁺ entry into, and hence transmitter release from, the terminal axons of central dopaminergic and cholinergic neurones. The function of the high affinity calcium antagonist binding sites that have been identified in brain remains unknown.     

Influence of N-allyl-normetazocine on acetylcholine release from brain slices: involvement of muscarinic receptors

Summary The effects of (±)N-allyl-normetazocine on the release of acetylcholine from different areas of guinea-pig and rat brain were investigated. 1. The drug did not modify the electrically (2 Hz) evoked tritium efflux from guinea-pig cerebral cortex, thalamus and caudate nucleus slices, preloaded with ³H-choline 0.1 mol/l and superfused with Krebs solution containing hemicholinium-3 10 mol/l. 2. (±)N-allyl-normetazocine 10 mol/l. enhanced the evoked ³H efflux from guinea-pig brain slices superfused with Krebs solution containing physostigmine 30 mol/l or oxotremorine 0.3 -1 gmol/l; the effect was naloxone-insensitive and was abolished by atropine 0.15 mol/l, but not by pirenzepine 1 mol/l. 3. (±)N-allyl-normetazocine 5 mol/l enhanced the electrically evoked release of endogenous acetylcholine as well, in a naloxone-insensitive way. 4. Both (±) and (+)N-allyl-normetazocine were without effect on ³H efflux from rat caudate nucleus slices electrically stimulated at 0.2 Hz frequency, after preloading with ³H-choline and during superfusion with hemicholinium-3. 5. The results are discussed in view of the antimuscarinic properties of the drug. Send offprint requests to A. Siniscalchi     

Dopaminergic modulation of hippocampal noradrenaline release

Summary ³H-Noradrenaline release in the rabbit hippocampus and its possible modulation via presynaptic dopamine receptors was studied. Hippocampal slices were preincubated with ³H-noradrenaline, continuously superfused in the presence of cocaine (30 mol/l) and subjected to electrical field stimulation. The electrically evoked tritium over-flow from the slices was reduced by 0.1 and 1 mol/l dopamine and apomorphine, but significantly enhanced by 10 mol/l apomorphine or by 0.1 and 1 mol/l bromocriptine. If the ₂-adrenoceptor antagonist yohimbine (0.1 mol/l) was present throughout superfusion, the inhibitory effects of dopamine and apomorphine were more pronounced and even 10 mol/l apomorphine and 1 mol/l bromocriptine inhibited noradrenaline release. Qualitatively similar observations were made in the presence of another ₂-antagonist, idazoxane (0.1 mol/l). In the presence of the D2-receptor antagonist domperidone (0.1 mol/l) the inhibitory effects of dopamine were almost abolished, whereas both apomorphine (>1 mol/l) and bromocriptine (>0.01 mol/l) greatly facilitated noradrenaline release. The D2-receptor agonist LY 171555 (0.1 and 1 mol/l) significantly reduced the evoked noradrenaline release whereas the D1-selective agonist SK & F 38393 was ineffective at similar concentrations. The effects of LY 171555 were abolished in the presence of domperidone (0.1 mol/l) but remained unchanged in the presence of yohimbine or idazoxane (0.1 mol/l, each).At 1 mol/l the D2-receptor antagonists domperidone and (-)sulpiride significantly increased the evoked noradrenaline release by about 10%. However, at this concentration, domperidone (but not (-)sulpiride) affected also basal tritium outflow. Bulbocapnine and the preferential D1-receptor antagonists SCH 23390 enhanced the evoked noradrenaline release already at 0.1 mol/l. Their marked facilitatory effects (50 to 60% increase at 1 mol/l) were reduced in the presence of idazoxane (0.1 mol/l) and almost abolished in the presence of 0.1 mol/l yohimbine, whereas the increase due to 1 mol/l (-)sulpiride persisted under these conditions.The evoked tritium efflux from rabbit hippocampal slices preincubated with ³H-serotonin was not affected by dopamine receptor agonists.From our results we conclude that hippocampal noradrenaline, but not serotonin release, is modulated via D2-dopamine receptors. In addition, our results provide evidence for more or less pronounced ₂-adrenoceptor agonistic properties of dopamine and ₂-adrenoceptor antagonistic properties of apomorphine, bromocriptine, SCH 23390 and bulbocapnine in this noradrenaline release model from CNS tissue.     

Alpha-receptor-mediated modulation of 3H-noradrenaline release from rat brain cortex synaptosomes

Summary The effects of oxymetazoline and noradrenaline (in the presence of desipramine) on the release of ³H-noradrenaline from rat brain cortex synaptosomes were studied using a superfusion technique. Both drugs (at 1M concentrations) were found to reduce the depolarization-induced (15 mM K⁺) release of ³H-noradrenaline. The release-modulating effect of noradrenaline was antagonized by phentolamine and yohimbine.The data provide direct evidence for the hypothesis that -receptors modulating the release of noradrenaline are localized on varicosities of central noradrenergic neurones.     

Mechanisms of the release of 3H-noradrenaline by dimethylphenylpiperazinium (DMPP) in the rat vas deferens

Summary In the rat vas deferens, DMPP is a substrate of uptake, (K_rn = 11.5 mol/I). After block of vesicular uptake, monoamine oxidase and catechol-O-methyl transferase, after loading of the tissue with ³H-noradrenaline, and in calcium-free solution (i. e., when axoplasmic ³H-noradrenaline levels were high and when depolarization-induced exocytotic release was impossible), DMPP induced a pronounced outward transport of ³H-noradrenaline. On the other hand, when, in similar experiments, vesicular uptake and monoamine oxidase were intact (i.e., when axoplasmic ³H-noradrenaline levels were low), DMPP induced very little outward transport of ³H-noradrenaline. This discrepancy indicates that DMPP has little ability to mobilize vesicularly stored ³H-amine.When the medium contained calcium (catechol-O-methyl transferase inhibited, all other mechanisms intact), 100 (but not 10) mol/l DMPP induced a hexamethonium-sensitive release of ³H-noradrenaline of short duration. Hence, in the presence of extracellular calcium, 100 mol/l DMPP elicits exocytotic release via activation of hexamethonium-sensitive nicotinic acetylcholine receptors.DMPP inhibits the monoamine oxidase of rat heart homogenate with an IC₅₀ of about 100 mol/l.Abbreviations COMT catechol-O-methyl transferase - DMPP dimethylphenylpiperazinium - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - MAO monoamine oxidase - NMN normetanephrine - OM-fraction column chromatographic fraction containing all O-methylated ³H-metabolites - OMDA fraction containing O-methylated and deaminated metabolites Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to U. Trendelenburg at the above address     

Blockade of γ-aminobutyric acid-receptors of the B-subtype inhibits the dopamine-induced enhancement of the release of cholecystokinin-like immunoreactivity from slices of rat dorsal caudatoputamen

Summary When slices of rat dorsal caudatoputamen (= neostriatum) are incubated in vitro, Choecystokinin-like immunoreactivity (CCK-LI) is released upon addition of veratridine (3.75 mol/l). This release is affected by dopamine and by -aminobutyric acid (GABA)-receptor agonists. Dopamine enhances the release by stimulating dopamine D₂-receptors and decreases it via D₁-receptors. GABA_A-receptor agonists enhance the veratridine-induced release of CCK-LI, while GABA_B-receptor agonists decrease it. In the present investigation, it was examined whether GABA-receptors are involved in the effect which dopamine exerts via D₂-receptors. The GABA_A-receptor antagonist bicuculline (10 mol/l)and the blocker of the GABA_A-receptor ionophore picrotoxin (1 mol/l) did not affect the dopamine (0.1 mol/1)-induced increase in the release of CCK-LI. However, the GABA_A-receptor agonist muscimol (1 mol/l) not only enhanced the release of CCK-LI, but also prevented a further enhancement by dopamine (0.1 mol/l). This effect of muscimol was blocked by bicuculline (10 mol/l). In the presence of -amino-n-valeric acid (0.1 mmol/l), which has been described to block GABA_B-receptors, dopamine no longer enhanced the veratridine-induced release of CCK-LI. -Amino-n-valeric acid also inhibited the pronounced enhancement of the release of CCK-LI caused by dopamine (0.1 mol/l) and 1 mol/l in the presence of the preferential D₁-receptor antagonist SCH 23390. The effect of -amino-n-valeric acid persisted in the presence of bicuculline (10 mol/l and 100 mol/l). (+)-Baclofen, a partial agonist at GABA_B-receptors, and the stereoisomer (–)-baclofen, a full agonist, also prevented the effect of dopamine on the veratridine-induced release of CCK-LI. The effects of both drugs may be due to desensitization of GABA_B-receptors, which has been described to develop quite rapidly. It is concluded that -amino-n-valeric acid blocks GABA_B-receptors and in this way prevents the enhancement of the veratridine-induced release of CCK-LI caused by dopamine via D₂-receptors. These data are interpreted as evidence that dopamine and GABA-neurons can directly or indirectly interact in the rat neostriatum. Send offprint requests to D. K. Meyer at the above address     

Influence of papaverine derivatives on phosphodiesterase activity,cyclic 3′,5′-AMP levels and relaxing effect on rabbit ileum

Summary The correlations between the relaxing effect of papaverine derivatives, inhibition of low K_m-phosphodiesterase (cAMP-PDE=EC 3.1.4.17) activity and cyclic 3,5-AMP (cAMP) levels in isolated rabbit ileum were investigated. There was a strong correlation between the relaxing effect, inhibition of PDE activity and cAMP content for eupaverine, ethylpapaverine and papaverine. Eupaverine was the most effective relaxing agent (I₅₀=7.5 M) and the most potent inhibitor of PDE activity (K_i=0.6 M), followed by ethylpapaverine (I₅₀=10 M); K_i=0.8 M) and papaverine (I₅₀=20 M; K_i=2 M). In contrast, there was a strong relaxing effect (I₅₀=6 M) but only slight inhibition of PDE activity (K_i=350 M) by tetrahydropapaveroline (THP). The adenylate cyclase stimulating effect of THP which was shown by others is most likely the reason for comparatively higher cAMP levels, which were found to be elevated about seven times over basal levels of 0.35 nmoles/g wet weight, and effective relaxation. Relaxation could be induced by exogenously added cAMP (I₅₀=45 M) and dibutyryl-cAMP (I₅₀=450 M). Our results support the assumption that smooth muscle relaxation in rabbit ileum is mediated by cAMP. Some of these observations have been published in abstract form (Schulz and Berndt, 1972).