Histamine H3A receptor-mediated inhibition of noradrenaline release in the mouse brain cortex

Summary Mouse brain cortex slices preincubated with ³H-noradrenaline were superfused with physiological salt solution containing desipramine plus a drug with ₂-adrenoceptor antagonist properties, and the effects of histamine receptor ligands on the electrically (0.3 Hz) evoked tritium overflow were studied. The evoked overflow (from slices superfused with phentolamine) was inhibited by histamine (pIC₃₅ 6.53), the H₃ receptor agonist R-(–)--methylhistamine (7.47) and its S-(+)-enantiomer (5.82) but not influenced by the H₁ receptor agonist 2-(2-thiazolyl)-ethylamine 3.2 mol/l and the H₂ receptor agonist dimaprit 10 mol/l. The inhibitory effect of histamine was not affected by the H₁ receptor antagonist dimetindene 1 mol/l and the H₂ receptor antagonist ranitidine 10 ol/l. The concentration-response curve of histamine (determined in the presence of rauwolscine) was shifted to the right by the H₃ receptor antagonists thioperamide (apparent pA₂ 8.67), impromidine (7.30) and burimamide (6.82) as well as by dimaprit (6.16). The pA₂ values of the four drugs were compared with their affinities for H_3A and H_3B binding sites in rat brain membranes (West et al. 1990 Mol Pharmacol 38:610); a significant correlation was obtained for the H_3A, but not for the H_3B sites. The results suggest that noradrenaline release in the mouse brain cortex is inhibited by histamine via H_3A receptors and that dimaprit is an H₃ receptor antagonist of moderate potency. Send offprint requests to E. Schlicker at the above address     

Cannabinoid CB1 receptor-mediated inhibition of noradrenaline release in the human and guinea-pig hippocampus

We examined the question of whether cannabinoid receptors modulating noradrenaline release are detectable in the brain of humans and experimental animals. For this purpose, hippocampal slices from humans, guinea-pigs, rats and mice and cerebellar, cerebrocortical and hypothalamic slices from guinea-pigs were incubated with [³H]noradrenaline and then superfused. Tritium overflow was evoked either electrically (0.3 or 1Hz) or by introduction of Ca²⁺ ions (1.3μM) into Ca²⁺-free, K⁺-rich medium (25μM) containing tetrodotoxin 1μM. Furthermore, the cAMP accumulation stimulated by forskolin 10μM was determined in guinea-pig hippocampal membranes. We used the following drugs: the cannabinoid receptor agonists (–)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP-55,940) and R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone (WIN 55,212-2), the inactive S(–)-enantiomer of the latter (WIN 55,212-3) and the CB₁ receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR 141716). The electrically evoked tritium overflow from guinea-pig hippocampal slices was reduced by WIN 55,212-2 (pIC_30% 6.5) but not affected by WIN 55,212-3 up to 10μM. The concentration-response curve of WIN 55,212-2 was shifted to the right by SR 141716 (0.032μM) (apparent pA₂ 8.2), which by itself did not affect the evoked overflow. WIN 55,212-2 1μM also inhibited the Ca²⁺-evoked tritium overflow in guinea-pig hippocampal slices and the electrically evoked overflow in guinea-pig cerebellar, cerebrocortical and hypothalamic slices as well as in human hippocampal slices but not in rat and mouse hippocampal slices. SR 141716 (0.32μM) markedly attenuated the WIN 55,212-2-induced inhibition in guinea-pig and human brain slices. SR 141716 0.32μM by itself increased the electrically evoked tritium overflow in guinea-pig hippocampal slices but failed to do so in slices from the other brain regions of the guinea-pig and in human hippocampal slices. The cAMP accumulation stimulated by forskolin was reduced by CP-55,940 and WIN 55,212-2. The concentration-response curve of CP 55,940 was shifted to the right by SR 141716 (0.1μM; apparent pA₂ 8.3), which by itself did not affect cAMP accumulation. In conclusion, cannabinoid receptors of the CB₁ subtype occur in the human hippocampus, where they may contribute to the psychotropic effects of cannabis, and in the guinea-pig hippocampus, cerebellum, cerebral cortex and hypothalamus. The CB₁ receptor in the guinea-pig hippocampus is located presynaptically, is activated by endogenous cannabinoids and may be negatively coupled to adenylyl cyclase. Received: 5 June 1997 / Accepted: 6 August 1997     

Histamine H3 receptor-mediated inhibition of serotonin release in the rat brain cortex

Summary Rat brain cortex slices preincubated with ³H-serotonin were superfused with physiological salt solution (containing citalopram, an inhibitor of serotonin uptake) and the effect of histamine on the electrically (3 Hz) evoked ³H overflow was studied. Histamine decreased the evoked overflow in a concentration-dependent manner. The inhibitory effect of histamine was antagonized by impromidine and burimamide, but was not affected by pheniramine, ranitidine, metitepine and phentolamine. Given alone, impromidine facilitated the evoked overflow, whereas burimamide, pheniramine and ranitidine had no effect. The results suggest that histamine inhibits serotonin release in the rat brain cortex via histamine H3 receptors, which may be located presynaptically. Send offprint requests to E. Schlicker at the above address     

Cannabinoid CB1 receptor-mediated inhibition of NMDA- and kainate-stimulated noradrenaline and dopamine release in the brain

Guinea-pig hippocampal slices preincubated with [³H]noradrenaline were superfused with medium containing desipramine and rauwolscine and rat striatal slices preincubated with [³H]dopamine were superfused with medium containing nomifensine; the effect of cannabinoid receptor ligands on tritium overflow stimulated by NMDA or kainate was examined. Furthermore, the affinity of the drugs for cannabinoid CB₁ receptors was determined in rat brain cortex membranes using [³H]SR 141716. In guinea-pig hippocampal slices preincubated with [³H]noradrenaline, tritium overflow stimulated by NMDA 100 μM and 1000 μM and by kainate 1000 μM was inhibited by the cannabinoid receptor agonists CP-55,940 and/or WIN 55,212-2. The CB₁ receptor antagonist SR 141716 increased the NMDA (1000 μM)-stimulated tritium overflow but did not affect tritium overflow stimulated by NMDA 100 μM or kainate 1000 μM. The inhibitory effect of WIN 55,212-2 on the NMDA (100 μM)- and kainate (1000 μM)-evoked tritium overflow was antagonized by SR 141716. In rat striatal slices preincubated with [³H]dopamine, WIN 55,212-2 inhibited the NMDA (1000 μM)-stimulated tritium overflow. SR 141716, which, by itself, did not affect tritium overflow, counteracted the inhibitory effect of WIN 55,212-2. [ ³ H]SR 141716 binding to rat cortical membranes was inhibited by SR 141716, CP-55,940 and WIN 55,212-2 (pK _i 8.53, 7.34 and 5.93, respectively) but not affected by desipramine, rauwolscine and nomifensine (pK _i < 5). In conclusion, activation of CB₁ receptors inhibits the NMDA- and kainate-stimulated noradrenaline release in guinea-pig hippocampus and the NMDA-stimulated dopamine release in rat striatum. The explanation for the facilitatory effect of SR 141716 might be that it acts as an inverse agonist at CB₁ receptors or that these receptors are activated by endogenous cannabinoids. Received: 25 February 1999 / Accepted: 12 April 1999     

Inhibition of noradrenaline release in the rat brain cortex via presynaptic H3 receptors

Summary The effects of histamine and related drugs on the evoked tritium overflow from superfused rat brain cortex slices preincubated with³H-noradrenaline were determined. Tritium overflow was stimulated electrically (3 Hz; slices superfused with normal physiological salt solution) or by introduction of CaCl₂ 1.3 mmol/l (slices superfused with Ca²⁺-free medium containing K⁺ 20 mmol/l).Histamine slightly decreased the electrically evoked^H overflow in slices superfused in the presence of desipramine. The degree of inhibition obtained with histamine was doubled when both desipramine and phentolamine were present in the superfusion medium (pIC₁₅ 6.46). Under the latter condition, the evoked overflow was inhibited by the H₃ receptor agonist R-(–)--methylhistamine and its S-(+) enantiomer (pIC₁₅ 7.36 and 5.09, respectively), but was not affected by the H₂ receptor agonist dimaprit and the H₁ receptoragonist 2-thiazolylethylamine (both at up to 32 µmol/l). The concentration-response curve of histamine was shifted to the right by the H3 receptor antagonists thioperamide, impromidine and burimamide (apparent pA₂ 8.37, 6.86 and 7.05, respectively), by the H₂ receptor antagonist ranitidine (apparent pA₂ 4.27) and was not affected by the H₁ receptor antagonist dimetindene (32 µmol/l). The inhibitory effect of R-(–)--methylhistamine on the evoked overflow was also counteracted by thioperamide. Given alone, none of the five histamine receptor antagonists affected the evoked overflow. In the absence of desipramine plus phentolamine, impromidine and burimamide facilitated the electrically evoked³H overflow whereas thioperamide had no effect. The facilitatory effects of impromidine and burimamide were abolished by phentolamine, but not affected by desipramine. The concentration-response curve of noradrenaline for its inhibitory effect on the evoked overflow was shifted to the right by impromidine and burimamide, but not influenced by thioperamide (apparent pA₂ 5.24, 5.04 and <6.5, respectively; experiments carried out in the presence of desipramine). In slices superfused with Ca²⁺-free K⁺-rich medium containing tetrodotoxin, desipramine plus phentolamine, the tritium overflow evoked by introduction of Ca²⁺ was inhibited by histamine; the concentration-response curve of histamine was shifted to the right by thioperamide.The present study shows that the inhibitory effect of histamine on noradrenaline release in the rat brain cortex involves presynaptic H₃ receptors and that the degree of inhibition is increased in the presence of phentolamine. The H₃ receptor antagonists impromidine and burimamide are weak ₂-adrenoceptor antagonists. Send offprint requests to E. Schlicker at the above address     

Differential control of Ca2+-dependent [3H]noradrenaline release from rat brain slices through presynaptic opiate receptors and alpha-adrenoceptors

The inhibitory actions of the Ca2+ antagonist Cd2+, morphine and noradrenaline (exogenously added + endogenously released) on electrically evoked release of [3H]noradrenaline from superfused rat neocortical slices were strongly reduced when release was enhanced by 4-aminopyridine. In the presence of 4-aminopyridine the release inhibiting effects of these drugs were restored by lowering the extracellular Ca2+ concentration. When release was enhanced by prolonging the pulse duration, only the release inhibiting effect of noradrenaline was reduced but the effects of Cd2+ and morphine were unchanged. Irrespective of the pulse duration, blockade of presynaptic alpha-adrenoceptors with phentolamine did not affect the release inhibiting effects of Cd2+ and morphine. The inhibitory effects of morphine and noradrenaline remained unchanged in Cl--free medium. Furthermore, these drugs strongly reduced the [3H]noradrenaline release induced by 20 mM K+ in the presence of tetrodotoxin. The results suggest that activation of presynaptic opiate-receptors inhibits Ca2+ entry through voltage-sensitive Ca2+ channels, whereas presynaptic alpha-adrenoceptors affect a step in the secretory process subsequent to Ca2+ influx. Moreover, the involvement of (direct) changes in Na+, K+ or Cl- permeability appears unlikely for both receptor systems.     

Electrically induced release of endogenous noradrenaline and dopamine from brain slices: pseudo-one-pulse stimulation utilized to study presynaptic autoinhibition

Summary Slices of rat hypothalamus (noradrenaline experiments) or rabbit caudate nucleus (dopamine experiments) were prepared, superfused, and field-stimulated using series of monophasic rectangular pulses. Noradrenaline, dopamine and the main dopamine metabolite, dihydroxyphenylacetic acetic acid (DOPAC), were determined using HPLC with electrochemical detection. Electrical stimulation was performed using the following protocols: 1) 4 pulses delivered at 100 Hz; this type of stimulation is referred to as pseudo-one-pulse stimulation (POP); its short duration of only 32 ms does not allow the development of autoinhibition; 2) 2 bursts of 4 pulses at 100 Hz, delivered 1 s apart (2-POP-stimulation); 3) 8 pulses at 1 Hz (dopamine experiments only); 4) 36 pulses at 3 Hz. Noradrenaline experiments. The ₂-adrenoceptor antagonist yohimbine (1 mol/l) did not enhance noradrenaline overflow following POP stimulation, but enhanced the overflow following 2-POP-stimulation by about 50% and that following 36-pulse-stimulation by almost 100%. Dopamine experiments. The D₂-dopamine receptor antagonist sulpiride (3 mol/l) facilitated the overflow of dopamine elicited with 2-POP-stimulation (66%), 8 pulses/1 Hz (92%), and 36 pulses/3 Hz (140%). It did not significantly facilitate the overflow of dopamine following POP-stimulation (19%). The overflow of DOPAC was not, or only slightly, increased by electrical stimulation, and its spontaneous outflow was more than three times higher than that of dopamine. Furthermore, the electrically induced overflow of dopamine did not exceed the outflow of DOPAC at any of the stimulation conditions employed.The results of the present study bear out important claims of the autoreceptor theory and confirm the data obtained in previous experiments using labelled transmitters. Correspondence to E. A. Singer at the above address     

A cumulative dose-response technique for the characterization of presynaptic receptors modulating [3H]noradrenaline release from rat brain slices

A cumulative dose-response technique was developed for the characterization of presynaptic receptors involved in the modulation of [³H]noradrenaline (NA) release from rat hippocampus slices, using continuous K⁺ (20 mM) depolarization. The results obtained with this technique were compared with those obtained using a repetitive K⁺ stimulation procedure. The release of [³H]NA induced by continuous K⁺ stimulation as well as that caused by repetitive K⁺ stimulation was strongly Ca²⁺-dependent and consisted for more than 90% of unmetabolized [³H]NA. Using continuous K⁺ stimulation it was demonstrated that the presynaptic inhibition of ³H-NA release by exogenous NA reached a maximum 10 min after addition of NA. The inhibitory effect of NA appeared to be independent of the time of addition, suggesting that the sensitivity of the presynaptic α-adrenoceptors remained unchanged during the experiment. Cumulative dose-response curves were recorded by the successive addition, at 10 min intervals, of increasing concentrations of NA. It was shown that continuous stimulation and repetitive K⁺ stimulation were basically similar with regard to the characteristics of the resulting [³H]NA release as well as its presynaptic α-adrenoceptor-mediated modulation by exogeneous NA. However, the cumulative dose-response technique, which can be carried out only using continuous K⁺ stimulation, makes it possible to determine more rapidly and also more accurately the apparent affinities and intrinsic activities of drugs towards receptors involved in the modulation of neurotransmitter release from brain slices.     

Evidence for M1 muscarinic cholinoceptors mediating facilitation of noradrenaline release in guinea-pig carotid artery

Summary The muscarinic agonists acetylcholine (150 mol/l), carbachol (1–10 mol/l) and McN-A-343 (1–50 mol/l, selective for M₁ receptors) increased, in a concentration-dependent manner, the electrically-evoked tritium overflow from guinea-pig carotid arteries preincubated with [³H]-noradrenaline. The increase caused by acetylcholine was not modified by hexamethonium (300 mol/l) but was reduced by the muscarinic receptor antagonists methylatropinium (0.5 and 1 nmol/l, nonselective), pirenzepine (1 and 5 mol/l, M₁-selective), methoctramine (1 and 5 mol/l, M₂-selective) and pfluoro-hexahydro-sila-difenidol (0.1–1 mol/l, M₃-selective). The order of potencies (expressed as negative logarithms of concentrations that reduced by 50% the facilitatory effect of acetylcholine) was: methylatropinium (9.93) > pirenzepine (8.83) > p-fluoro-hexahydro-siladifenidol (6.81) methoctramine (6.20). These results demonstrate the existence of facilitatory M₁ receptors modulating noradrenaline release in blood vessels. Correspondence to M. Salaices at the above address     

Presynaptic muscarinic receptor subtypes involved in the inhibition of acetylcholine and noradrenaline release in bovine cerebral arteries

Summary Experiments were performed in bovine cerebral arteries preincubated with [³H]-choline or [³H]-noradrenaline to analyze the presynaptic muscarinic receptors involved in inhibition of acetylcholine and noradrenaline release induced by electrical stimulation (4 Hz, 200 mA, 0.3 ms, 1 min). For this purpose, the actions of several muscarinic receptor antagonists on the ³H overflow and on the carbacol-induced inhibition of this overflow were assessed. The evoked [³H]-acetylcholine release and [³H]-noradrenaline release were markedly reduced by the presence of tetrodotoxin, Ca²⁺-free medium, and the inhibitor of both choline transport and choline acetyltransferase, AF64A. Chemical sympathetic denervation with 6-hydroxydopamine (6-OHDA) decreased the uptake of[³H]-noradrenaline, and AF64A reduced mainly the uptake of [³H]-choline, but also of [³H]-noradrenaline. Carbachol reduced the evoked [³H]-noradrenaline and [³H]-acetylcholine release; the IC₅₀ values were 0.37 and 0.43 mol/l, respectively.Atropine and 4-DAMP, but not AF DX 116, methoctramine or pirenzepine, increased the evoked [³H]-acetylcholine release. However, these muscarinic antagonists failed to modify the evoked [³H]-noradrenaline release. Carbachol inhibited the release of both acetylcholine and noradrenaline. The inhibition was blocked by the antagonists. The rank orders of potency (based on plC₅₀ values) were, in the case of [³H]-acetylcholine release, atropine > 4-DAMP >AF-DX 116 >- pirenzepine >- methoctramine, and, in the case of [³H]-noradrenaline release, atropine > 4-DAMP > AF-DX 116 >- methoctramine >-pirenzepine. These results suggest (1) that the prosynaptic receptors that modulate endogenous acetylcholine release are likely of the M₃ subtype, whilst those involved on the effect of the exogenous agonist Carbachol are of M₂ subtype, and (2) that those which inhibit noradrenaline release are probably a mixture of M₂ and M₃ subtypes as well. The autoinhibition of the acetylcholine release was funtionally active under our experimental conditions, while noradrenaline release does not appear to be modulated by muscarinic receptors in physiological conditions.Send offprint requests to G. Balfagón at the above address     

N-methyl-d-aspartate (NMDA)-stimulated noradrenaline (NA) release in rat brain cortex is modulated by presynaptic H3-receptors

In superfused rat brain cortex slices and synaptosomes preincubated with [³H]noradrenaline the effect of agonists or antagonists at presynaptic H₃ receptors on NMDA-evoked [³H]noradrenaline release was investigated. In experiments on slices, histamine and the preferential H₃ receptor agonist R-(–)--methylhistamine inhibited NMDA-evoked tritium overflow (IC₂₀ values 0.27 mol/l or 0.032 mol/l, respectively); S-(+)--methylhistamine (up to 10 mol/l) as well as the selective H₁ receptor agonist (2-(2-thiazolyl)ethylamine) and the selective H₂ receptor agonist dimaprit (each up to 10 mol/l) were ineffective. The H₃ receptor antagonist thioperamide abolished the inhibitory effect of histamine whereas the preferential H₁ receptor antagonist dimetindene and the preferential H₂ receptor antagonist ranitidine were ineffective. In experiments on synaptosomes, histamine and R-(–)--methylhistamine inhibited NMDA-evoked tritium overflow, whereas 2-(2-thiazolyl)ethylamine or dimaprit had no effect. The inhibitory effect of histamine was abolished by thioperamide. When tritium overflow was stimulated by NMDA in the presence of -conotoxin GVIA (which by itself decreased the response to NMDA by about 55%), R-(–)--methylhistamine did not inhibit NMDA-evoked overflow. It is concluded that NMDA-evoked noradrenaline release in the cerebral cortex can be modulated by inhibitory H₃ receptors. NMDA receptors and H₃ receptors are both located presynaptically and may interact at the same noradrenergic varicosity. An unimpaired function of the N-type voltage-sensitive calcium channel probably is a prerequisite for the inhibition of NMDA-evoked noradrenaline release by H₃ receptor stimulation. Correspondence to: M. Göthert at the above address     

P2-Receptor-mediated inhibition of noradrenaline release in the rat pancreas

The aim of the study was to find out whether, and if so through which receptors, nucleotides modulate the release of noradrenaline in the rat pancreas. Segments of the pancreas were preincubated with [³H]-noradrenaline, superfused with medium containing desipramine (1μM) and yohimbine (1μM), and stimulated electrically, in most experiments by 60 pulses/1Hz. The adenosine A₁-receptor agonist N⁶-cyclopentyl-adenosine (CPA; EC₅₀ 32nM), the non-subtype-selective adenosine receptor agonists adenosine (EC₅₀ 15μM) and 5’-N-ethylcarboxamidoadenosine (NECA; EC₅₀ 135nM), and the nucleotides ATP (EC₅₀ 13μM), adenosine-5’-O-(3-thiotriphosphate) (ATPγS; EC₅₀ 19μM) and adenosine-5’-O-(2-thiodiphosphate) (ADPβS; EC₅₀ 16μM) decreased the evoked overflow of tritium. The adenosine A_2A-agonist 2-p-(2-carboxyethyl)-phenethylamino-5’-N-ethylcarboxamido-adenosine (CGS 21680) caused no change. The concentration-response curve of CPA was shifted to the right by the A₁-antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 10nM; pK_d 9.1) but, like the concentration-response curve of adenosine, hardly affected by the P2-receptor antagonist cibacron blue 3GA (30μM). Combined administration of a high concentration of DPCPX (1μM) and 8-phenyltheophylline (10μM) abolished the effects of CPA and NECA. The concentration-response curves of ATP and ADPβS were shifted to the right by both DPCPX (10nM; pK_d 8.7 and 8.9, respectively) and cibacron blue 3GA (30μM; pK_d 5.0 and 5.2, respectively). The antagonist effects of DPCPX (10nM) and cibacron blue 3GA (30μM) against ATP were additive in a manner compatible with the blockade of two separate receptors for ATP. In the presence of the high concentration of DPCPX (1μM) and 8-phenyltheophylline (10μM), ATP and ADPβS still decreased evoked tritium overflow, and this decrease was attenuated by additional administration of cibacron blue 3GA (30μM). The P2-antagonists cibacron blue 3GA, reactive blue 2, reactive red 2, and to a limited extent also suramin and 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulphonate (XAMR0721), increased the evoked overflow of tritium by up to 114%. Pyridoxalphosphate-6-azophenyl-2’,4’-disulphonate (PPADS) caused no change. The results indicate that the postganglionic sympathetic axons of the rat pancreas possess A₁-adenosine and P2-receptors. Both receptors mediate an inhibition of noradrenaline release. The presynaptic P2-receptors are activated by an endogenous ligand, presumably ATP, during appropriate trains of action potentials. This is the first demonstration of presynaptic P2-receptors at postganglionic sympathetic neurons that are located in prevertebral ganglia. Received: 6 November 1997 / Accepted: 6 January 1998     

P2-Receptor-mediated inhibition of noradrenaline release in the rat hippocampus

Experiments on hippocampal slices were carried out in order to find out whether the release of noradrenaline in the hippocampus can be modulated through P2-receptors. The slices were preincubated with [³H]-nor-adrenaline, superfused with medium containing desipramine (1 μM), and stimulated electrically, in most experiments by 4 pulses/100 Hz. The adenosine A₁-receptor agonist N⁶-cyclopentyl-adenosine (CPA) and the nucleotides ATP, adenosine-5’-O-(3-thiotriphosphate) (ATPγS) and adenosine-5’-O-(2-thiodiphosphate) (ADPβS) decreased the evoked overflow of tritium by up to 55 %. The adenosine A_2a-agonist 2-p-(2-carboxyethyl)-phenethylamino-5’-N-ethylcarboxamido-adenosine (CGS 21680; 0.003-0.3 μM) caused no change. The concentration-response curve of CPA was shifted to the right by the A₁-antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 3 nM) but not by the P2-receptor antagonists cibacron blue 3GA (30 μM) and reactive blue 2 (30 μM); the apparent pK_B value of DPCPX against CPA was 9.0. In contrast, the concentration-response curve of ATP was shifted to the right by DPCPX (3 nM), apparent pK_B 8.7, as well as by ciba-cron blue 3GA (30 μM), apparent pK_B 5.2, and reactive blue 2 (30 μM), apparent pK_B 5.6; the antagonist effects of DPCPX and cibacron blue 3GA were additive in a manner compatible with the blockade of two separate receptors for ATP. The same pattern was obtained with ATPγS: its concentration-response curve was shifted to the right by DPCPX as well as by cibacron blue 3GA and reactive blue 2. Suramin (300 μM) antagonized neither the effect of ATP nor that of ATPγS. The 5’-nucleotidase inhibitor α,β-methylene-ADP (100 μM) did not change the effect of ATP. Only cibacron blue 3GA (30 μM) but not reactive blue 2 (30 μM), given alone, consistently caused a small increase of the evoked overflow of tritium. Hippocampal slices degraded exogenous ATP, and this degradation was reduced by cibacron blue 3GA (30 μM), reactive blue 2 (30 μM) and suramin (300 μM). The results indicate that the noradrenergic terminal axons of the rat hippocampus possess P2-receptors in addition to the known A₁-adenosine receptors. The presynaptic P2-receptors mediate an inhibition of noradrenaline release, are activated by nucleotides but not nucleosides, and are blocked by cibacron blue 3GA and reactive blue 2. ATP and ATPγS act at both the A₁- and the P2-receptors. An autoreceptor function of cerebral presynaptic P2-receptors remains doubtful. Received: 20 November 1996 / Accepted: 10 February 1997     

ACTH increases noradrenaline release in the rabbit heart

Summary The effects of ACTH on the release of noradrenaline and the increase of heart rate produced by sympathetic nerve stimulation (1 Hz) were studied in isolated perfused rabbit hearts. ACTH-(1–24) 0.1–100 nmol/l increased the stimulation-evoked overflow of noradrenaline concentration-dependently, reversibly and up to two-fold. The basal outflow of noradrenaline, the basal heart rate and the stimulation-evoked increase in heart rate were not changed. Human ACTH-(1–39) also increased the evoked overflow of noradrenaline. The effect of ACTH-(1–24) 0.3 nmol/l persisted after blockade of -adrenoceptors with propranolol and blockade of neuronal catecholamine uptake by cocaine. ACTH-(1–24) 3 nmol/l did not change the removal of noradrenaline from the perfusion fluid, when hearts were perfused with medium containing 59 nmol/l noradrenaline. The results show that ACTH increases the action potential-evoked release of noradrenaline from cardiac postganglionic sympathetic neurones, probably by activating specific presynaptic ACTH receptors. The high potency of ACTH suggests that these presynaptic receptors may be activated in vivo by circulating ACTH under certain pathophysiological conditions.Send offprint requests to B. Szabo at the above address     

Kainate receptors are involved in the glutamate-induced indirect,purinergic inhibition of [3H]-noradrenaline release in rabbit brain cortex

Activation of ionotropic but not of metabotropic glutamate receptors causes an indirect inhibition of the release of noradrenaline in slices of rabbit brain cortex. The inhibition is mediated by adenosine which activates presynaptic adenosine A₁-receptors. The present study characterizes the ionotropic receptor types through which glutamate itself produces this indirect inhibition. Rabbit brain cortex slices were preincubated with [³H]-noradrenaline, superfused with medium containing desipramine (1 M) and stimulated electrically by trains of 6 pulses at 100 Hz.Glutamate (100–3000 M) reduced the electrically evoked overflow of tritium by up to 58 %. The effect did not differ 20 min and 60 min after addition of glutamate. Adenosine deaminase (1 U ml^-1) as well as 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 30 M) and d--glutamylamino-methanesulfonate (GAMS; 30 M), both of which block kainate receptors, attenuated the glutamate-induced inhibition. The NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP5; 100 M) and the AMPA receptor antagonist 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX; 30 M) did not change the effect of glutamate. Given alone, CNQX and GAMS, but not AP5 and NBQX, slightly increased the evoked overflow of tritium; the increases were abolished in the presence of adenosine deaminase.The results indicate that activation of kainate but not NMDA and AMPA receptors is involved in the indirect, adenosine-mediated inhibition by exogenous glutamate of the release of noradrenaline in rabbit brain cortex slices. Moreover, as shown by the increase caused by CNQX and GAMS, endogenous excitatory amino acids inhibit the release of noradrenaline through the kainate receptor-adenosine mechanism and thus contribute to the purinergic inhibitory control of noradrenaline release in the brain.     

Nociceptin inhibits noradrenaline release in the mouse brain cortex via presynaptic ORL1 receptors

A fourth type of opioid receptor, termed ORL₁, has been cloned and nociceptin (also known as orphanin FQ) has been identified as an endogenous ligand at this receptor. We examined whether nociceptin affects the release of noradrenaline in the brain. For this purpose, cerebral cortex slices from the mouse, rat or guinea-pig were preincubated with [³H]noradrenaline and then superfused with medium containing desipramine and rauwolscine. Tritium overflow was evoked electrically (0.3 Hz) or by introduction of Ca²⁺ 1.3 mM into Ca²⁺-free K⁺-rich (15 mM) medium. Nociceptin 1 μM reduced the electrically evoked tritium overflow from mouse, rat and guinea-pig brain cortex slices by 80, 71 and 36%, respectively. Naloxone 10 μM did not change the effect of nociceptin. All subsequent experiments were performed on mouse brain cortex slices and in the presence of naloxone 10 μM. The concentration-response curve of nociceptin (maximum inhibition by 80%, pEC₅₀ 7.5) was shifted to the right by the non-selective ORL₁ receptor antagonist naloxone benzoylhydrazone and the selective ORL₁ receptor antagonist [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ (pA₂ 6.6 and 7.2, respectively). Naloxone benzoylhydrazone did not affect the evoked overflow by itself whereas [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ caused an inhibition by maximally 35% (pEC₅₀ 7.0; intrinsic activity α 0.45). The inhibitory effect of [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ was counteracted by naloxone benzoylhydrazone. Nociceptin also reduced the Ca ²⁺ -evoked tritium overflow in mouse brain cortex slices superfused in the presence of tetrodotoxin. This effect was also antagonized by naloxone benzoylhydrazone, which, by itself, did not affect the evoked tritium overflow. In conclusion, nociceptin inhibits noradrenaline release more markedly in the mouse than in the rat or guinea-pig brain cortex. The effect of nociceptin in the mouse brain cortex involves ORL₁ receptors, which are located presynaptically on noradrenergic neurones. Received: 19 June 1998 / Accepted: 17 July 1998     

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

Summary Slices 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 ³ H-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 ³ H-dopamine were superfused with medium containing nomifensine 1 mol/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 ³ H-choline were superfused either with physostigmine-free medium or with medium containing physostigmine 1 mol/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 mol/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. Send offprint requests to N. Limberger at the above address     

Neuropeptide Y differentiates between exocytotic and nonexocytotic noradrenaline release in guinea-pig heart

Summary The overflow of neuropeptide Y (NPY; radioimmunoassay), noradrenaline and dihydroxyphenylethylenglycol (DOPEG; high pressure liquid chromatography) from guinea-pig perfused hearts was investigated in relationship to exocytotic and nonexocytotic release mechanisms. Exocytotic release: Electrical stimulation of the left stellate ganglion (12 Hz; 1 min) evoked a calcium-dependent overflow of noradrenaline and NPY, that was accompanied by a minor and prolonged increase in DOPEG overflow. This increase in DOPEG overflow was attenuated by blockade of neuronal amine re-uptake. In the presence of calcium, a closely related co-release of noradrenaline and NPY was also observed during administration of veratridine (10 M); it was completely prevented by tetrodotoxin (1 M). Nonexocytotic release: In the absence of extracellular calcium, veratridine (30 M) induced noradrenaline overflow only when combined with the reserpine-like agent Ro 4-1284 (10 M). This overflow was accompanied by efflux of DOPEG, but not of NPY. Similarily, tyramine (1–100 M) induced a calcium-independent concomitant overflow of both noradrenaline and DOPEG, but not of NPY. During anoxic and glucose-free perfusion a predominantly calcium-independent overflow of noradrenaline was observed; only in the presence of extracellular calcium was this overflow accompanied by a minor overflow of NPY. Noradrenaline overflow, induced by veratridine plus Ro 4-1284 (in the absence of calcium), by tyramine, or by anoxia, was suppressed by blockade of neuronal amine re-uptake, and was, therefore, mediated by reversed transmembrane amine transport by the neuronal uptake₁ carrier.The results indicate that NPY is co-released with noradrenaline only during calcium-dependent exocytosis. On the other hand, whenever, noradrenaline is released by non-exocytotic (calcium-independent and carrier-mediated) release mechanisms, no substantial NPY overflow is observed. The simultaneous determination of noradrenaline and NPY overflow, therefore, allows a differentiation between exocytotic and nonexocytotic noradrenaline release, and NPY may be utilized as a marker of exocytotic noradrenaline release.This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 320 — Herzfunktion und ihre Regulation)Presented in part at the 62nd Scintific Sessions of the American Heart Association, New Orleans/USA, November 1989     

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     

Cannabinoid CB<Subscript>1</Subscript> receptor-mediated inhibition of noradrenaline release in guinea-pig vessels,but not in rat and mouse aorta

Cannabinoids exert complex effects on blood pressure related to their interference with cardiovascular centres in the central nervous system and to their direct influence on vascular muscle, vascular endothelium and heart. In view of the relative lack of information on the occurrence of CB₁ receptors on the vascular postganglionic sympathetic nerve fibres, the aim of the present study was to examine whether cannabinoid receptor ligands affect the electrically evoked tritium overflow in superfused vessels (tissue pieces) from the guinea-pig, the rat and the mouse preincubated with ³H-noradrenaline. The cannabinoid receptor agonist WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3-de]1,4-benzoxazinyl](1-naphthalenyl) methanone) inhibited the evoked tritium overflow in the guinea-pig aorta, but not in that of the rat or mouse. The concentration–response curve of WIN 55,212-2 was shifted to the right by the CB₁ receptor antagonist rimonabant, yielding an apparent pA₂ value of 7.9. The most pronounced (near-maximum) inhibition obtained at the highest WIN 55,212-2 concentration applied (3.2 μM) amounted to 40%. WIN 55,212-2 also inhibited the evoked overflow in guinea-pig pulmonary artery, basilar artery and portal vein, again in a manner sensitive to antagonism by rimonabant. The latter did not affect the evoked overflow by itself in the four vessels, but did increase the electrically evoked tritium overflow from superfused guinea-pig hippocampal slices preincubated with ³H-choline and from superfused guinea-pig retina discs preincubated with ³H-noradrenaline (labelling dopaminergic cells in this tissue). The inhibitory effect of 3.2 μM WIN 55,212-2 on the evoked overflow from the guineapig aorta was comparable in size to that obtained with agonists at the histamine H₃, κ opioid (KOP) and ORL₁ (NOP) receptor (1 or 10 μM, producing the respective near-maximum effects) whereas prostaglandin E₂ 1 μM caused a higher near-maximum inhibition of 70%. Prostaglandin E₂ also induced an inhibition by 65 and 80% in the rat and mouse aorta respectively, indicating that the present conditions are basically suitable for detecting presynaptic receptor-mediated inhibition of noradrenaline release. The results show that the postganglionic sympathetic nerve fibres in the guineapig aorta, but not in the rat or mouse aorta, are endowed with presynaptic inhibitory cannabinoid CB₁ receptors; such receptors also occur in guineapig pulmonary artery, basilar artery and portal vein. These CB₁ receptors are not subject to an endogenous tone and the extent of inhibition obtainable via these receptors is within the same range as that of several other presynaptic heteroreceptors, but markedly lower than that obtainable via receptors for prostaglandin E₂.