Comparison of the ORL1 receptor-mediated inhibition of noradrenaline release in human and rat neocortical slices

The effects of nociceptin/orphanin (N/OFQ) and the selective ORL1 antagonist J-113397 (1-[(3R,4R)-1-cyclo-octylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one) were studied on electrically-evoked release of [(3)H]-noradrenaline ([(3)H]-NA) from human and rat neocortical slices. Specimens of human tissue were obtained during neurosurgery. Slices were preincubated with 0.1 microM [(3)H]-NA, superfused in the presence of desipramine, idazoxan, and naloxone (1 microM each), and stimulated electrically up to three times under conditions (4 pulses, 100 Hz, 2 ms, 60 mA) that prevent inhibition of evoked [(3)H]-NA release by endogenous modulators accumulating during ongoing stimulation. N/OFQ decreased electrically-evoked [(3)H]-NA release in both human and rat neocortical slices in a concentration-dependent manner. The respective pEC(50) values were 7.74 [CI(95): 7.47, 8.04] and 7.64 [CI(95): 7.48, 7.77], and the maximal inhibitions were 36.9% [CI(95): 32.4%, 41.8%] and 66.4% [CI(95): 61.7%, 72.7%]. N/OFQ (1 microM) inhibited K(+) (15 mM)-evoked [(3)H]-NA release from neocortical slices of both species by a similar magnitude, either in the presence or absence of tetrodotoxin. The nonpeptide ORL1 antagonist J-113397 competitively attenuated, with similar potency, the inhibition of electrically-evoked [(3)H]-NA release by N/OFQ in both species (pA(2) values: human, 8.16 [CI(95): 7.64, 8.64]; rat, 8.47 [CI(95): 8.27, 8.67]). J-113397 (0.1 microM) by itself did not alter either the evoked or spontaneous [(3)H]-NA release, suggesting that presynaptic ORL1 receptors are not activated by endogenous N/OFQ under the stimulation conditions employed. This study provides the first evidence that N/OFQ modulates [(3)H]-NA release in human neocortex via specific ORL1 receptors most likely located on noradrenergic axon terminals.     

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     

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     

ORL1 receptor-mediated inhibition by nociceptin of noradrenaline release from perivascular sympathetic nerve endings of the rat tail artery

In the present study the effect of the opioid heptadecapeptide nociceptin, also termed orphanin FQ, an endogenous ligand for the orphan receptor named ORL₁ (opioid receptor-like 1) receptor, was investigated on [³H]noradrenaline release induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery in the absence and presence of an α₂-adrenoceptor antagonist, rauwolscine 3 μM. Nociceptin inhibited the electrically-evoked tritiated noradrenaline release in a concentration-dependent manner from rat tail arteries. This inhibitory effect of nociceptin was enhanced in the presence of the α₂-adrenoceptor antagonist rauwolscine (maximum inhibition by 25% and 50% in the absence and presence of rauwolscine, respectively). At a supramaximal concentration (10 μM), the inhibitory action of DAGO, a selective μ-opioid receptor agonist, was less pronounced than that of nociceptin. The inhibitory effect of nociceptin was counteracted by naloxone benzoylhydrazone (3 μM) which by itself did not change the stimulation-evoked noradrenaline overflow. Naloxone (10 μM), a non-selective opioid receptor antagonist, did not affect the inhibitory effect of nociceptin whereas it abolished that of DAGO. In conclusion, these results suggest that nociceptin modulates noradrenergic neurotransmission by acting on prejunctional ORL₁ receptors located on nerve terminals innervating the rat tail artery. They also demonstrate that prejunctional ORL₁ receptors interact with prejunctional α₂-adrenoceptors. The physiological significance of this phenomenon remains to be determined. Received: 27 August 1998 / Accepted: 29 September 1998     

H2 receptor-mediated facilitation and H3 receptor-mediated inhibition of noradrenaline release in the guinea-pig brain

The effect of histamine and related drugs on the tritium overflow evoked electrically (0.3 Hz) or by introduction of Ca²⁺ ions into Ca²⁺-free K⁺-rich (25 mmol/l) medium containing tetrodotoxin was studied in superfused guinea-pig brain cortex, cerebellum, hippocampus or hypothalamus slices and in mouse brain cortex slices preincubated with ³H-noradrenaline. The electrically evoked tritium overflow in guinea-pig cortex slices was inhibited by histamine; the H₃ receptor antagonist clobenpropit reversed the effect of histamine to a slight facilitation. The facilitatory effect of histamine (obtained in the presence of clobenpropit) was not affected by the H₁ receptor antagonist mepyramine but abolished by the H₂ receptor antagonist ranitidine. In the absence of clobenpropit, ranitidine augmented the inhibitory effect of histamine. In slices superfused in the presence of ranitidine, the evoked overflow was inhibited by histamine and, more potently, by the H₃ receptor agonist R-α-methylhistamine in a concentration-dependent manner (maximum inhibitory effect obtained for both agonists 30–35%). The concentration-response curve of histamine was shifted to the right by the H₃ receptor antagonist thioperamide. R-α-Methylhistamine inhibited the electrically evoked tritium overflow also in guinea-pig cerebellar, hippocampal and hypothalamic slices. In cortex slices superfused in the presence of clobenpropit, the H₂ receptor agonists impromidine and, less potently, R-sopromidine facilitated the evoked overflow in a concentration-dependent manner. S-Sopromidine only tended to increase the evoked overflow. The effect of impromidine was counteracted by the H₂ receptor antagonists ranitidine and cimetidine. The extent of the maximum facilitatory effect of impromidine (by 15–20%) was about the same when (i) the Ca²⁺ concentration in the medium was reduced from 1.3 to 0.98 mmol/l, (ii) the time of exposure to impromidine was reduced from 28 to 8 min or (iii) cerebellar, hippocampal or hypothalamic slices were used instead of cortical slices. The Ca²⁺-induced tritium overflow in guinea-pig cortex slices was inhibited by histamine (in the presence of ranitidine); this effect was abolished by clobenpropit. In slices superfused in the presence of clobenpropit, impromidine failed to facilitate the Ca²⁺-evoked tritium overflow. The electrically evoked tritium overflow in mouse brain cortex slices was inhibited by histamine by about 60% (both in the absence or presence of ranitidine). The inhibitory effect of histamine was abolished (but not reversed) by clobenpropit. In conclusion, noradrenaline release in the guinea-pig brain cortex is inhibited via presynaptic H₃ receptors and facilitated via H₂ receptors not located presynaptically. In the mouse brain cortex, only inhibitory H₃ receptors occur. The extent of the H₃ receptor-mediated effect is more marked in the mouse than in the guinea-pig brain cortex. Received: 25 September 1997 / Accepted: 17 November 1997     

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     

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 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     

Evidence for presynaptic cannabinoid CB(1) receptor-mediated inhibition of noradrenaline release in the guinea pig lung.

Using neurochemical method, evidence was obtained that cannabinoid CB(1) receptors are localized on noradrenergic terminals and their stimulation by WIN-55,212-2 reduces the release of [3H]noradrenaline evoked by axonal activity in a frequency-dependent manner. At stimulation rates of 1 and 3 Hz, there was significant inhibition of noradrenaline release, with IC(50) of WIN-55,212-2 41.5+/-2.6 and 320.5+/-28.2 nM, for 1 and 3 Hz, respectively. Cannabinoid CB(1) receptor antagonist SR 141716A completely prevented WIN-55,212-2 from reducing the release. The release of noradrenaline is negatively modulated by presynaptic alpha(2)-adrenoceptors. Because BRL-44408, an alpha(2B)-adrenoceptor, and prazosin, an alpha(1)- and alpha(2B)-adrenoceptor antagonist, both increased the release of [(3)H]noradrenaline, it seems likely that the alpha(2B) subtype is responsible for the negative feedback modulation of noradrenaline release. In the presence of alpha(2)-adrenoceptor antagonism, cannabinoid CB(1) receptor activation by WIN-55,212-2 was much more effective in inhibiting the release of [(3)H]noradrenaline. Using a specific antibody against the C-terminus of the rat cannabinoid CB(1) receptor and also against neuropeptide Y, ultrastructural evidence was obtained that cannabinoid CB(1) receptors are exclusively localized on neuropeptide Y-positive noradrenergic varicosities.Since the sympathetic innervation of the human airway smooth muscle is sparse, and mainly the circulating adrenaline relaxes the airways via activation of beta(2)-adrenoceptor localized on the smooth muscle, it is suggested that inhibition of noradrenaline release by cannabinoids, and the subsequent bronchospasm, may be limited to those cases when noradrenaline released from sympathetic varicosities is involved in airway relaxation.     

Evidence for P2-purinoceptor-mediated inhibition of noradrenaline release in rat brain cortex.

1. Some postganglionic sympathetic axons possess P2Y-like P2-purinoceptors which, when activated, decrease the release of noradrenaline. We examined the question of whether such receptors also occur at the noradrenergic axons in the rat brain cortex. Slices of the brain cortex were preincubated with [3H]-noradrenaline, then superfused with medium containing desipramine (1 microM) and stimulated electrically, in most experiments by trains of 4 pulses/100 Hz. 2. The selective adenosine A1-receptor agonist, N6-cyclopentyl-adenosine (CPA; 0.03-3 microM) as well as the non-subtype-selective agonist 5'-N-ethylcarboxamido-adenosine (NECA; 0.3-3 microM) reduced the evoked overflow of tritium, whereas the adenosine A2a-receptor agonist, 2-p-(2-carbonylethyl)-phenethylamino-5'-N-ethylcarboxamido-a denosine (CGS-21680; 0.003-30 microM) and the adenosine A3-receptor agonist N6-2-(4-aminophenyl)ethyl-adenosine (APNEA; 0.03-3 microM) caused no change. Of the nucleotides tested, ATP (30-300 microM), adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S; 30-300 microM), adenosine-5'-O-(2-thiodiphosphate) (ADP beta S; 30-300 microM), P1,P4-di(adenosine-5')-tetraphosphate (Ap4A; 30-300 microM) and the preferential P2Y-purinoceptor agonist, 2-methylthio-ATP (300 microM) decreased the evoked overflow of tritium. The P2X-purinoceptor agonist, alpha,beta-methylene-ATP (3-300 microM) caused no change. 3. The A1-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM) attenuated the effects of the nucleosides CPA (apparent pKB value 9.8) and NECA as well as of the nucleotides ATP (apparent pKB 9.3), ATP gamma S (apparent pKB 9.2) and ADP beta S (apparent pKB 8.7).(ABSTRACT TRUNCATED AT 250 WORDS)     

Brain prostanoid TP receptor-mediated adrenal noradrenaline secretion and EP3 receptor-mediated sympathetic noradrenaline release in rats

Sympathetic nerves release noradrenaline, whereas adrenal medullary chromaffin cells secrete noradrenaline and adrenaline. Therefore, plasma noradrenaline reflects the secretion from adrenal medulla in addition to the release from sympathetic nerves, however the exact mechanisms of adrenal noradrenaline secretion remain to be elucidated. The present study was designated to characterize the source of plasma noradrenaline induced by intracerebroventricularly (i.c.v.) administered bombesin and prostaglandin E2 in urethane-anesthetized rats. Bombesin (1.0 nmol/animal, i.c.v.) elevated plasma noradrenaline and adrenaline, while prostaglandin E2 (0.3 nmol/animal, i.c.v.) elevated only plasma noradrenaline. The bombesin-induced elevations of both catecholamines were attenuated by pretreatments with furegrelate (an inhibitor of thromboxane A2 synthase) [250 and 500 microg (0.9 and 1.8 micromol)/animal, i.c.v.)] and [(+)-S-145] [(+)-(1R,2R,3S,4S)-(5Z)-7-(3-[4-3H]-phenylsulphonyl-aminobicyclo[2.2.1]hept-2-yl)hept-5-enoic acid sodium salt] (an antagonist of prostanoid TP receptors) [100 and 250 microg (250 and 625 nmol)/animal)], and abolished by acute bilateral adrenalectomy. On the other hand, the prostaglandin E2-induced elevation of plasma noradrenaline was not influenced by acute bilateral adrenalectomy. These results suggest that adrenal noradrenaline secretion and sympathetic noradrenaline release are mediated by differential central mechanisms; brain prostanoid TP receptors activated by bombesin are involved in the adrenal noradrenaline secretion, while brain prostanoid EP (probably EP3) receptors activated by prostaglandin E2 are involved in the sympathetic noradrenaline release in rats. Brain prostanoid TP receptors activated by bombesin are also involved in the adrenal adrenaline secretion.     

Endogenous noradrenaline impairs the prostaglandin-induced inhibition of noradrenaline release

Summary The effects of prostaglandin E₂ (PGE₂) on electrically evoked noradrenaline release in rat brain cortex were studied under conditions under which autoinhibition of release was avoided. When stimulation was carried out with 36 pulses at 3 Hz, 1 mol/1 PGE2, produced about 50% inhibition of release. In the presence of the ₂-adrenoceptor antagonist yohimbine (1 gmol/1) the effect of PGE₂ was markedly increased. When release was elicited by 3 pulses/100 Hz the period of stimulation was too short to permit development of autoinhibition by released noradrenaline. Then the concentration-response-curve for PGE₂ was very similar to that obtained under the above conditions (36 pulses/3 Hz, in the presence of yohimbine). These data suggest that both the ₂-adrenoceptor and the PGE₂-receptor are linked to a common pathway. Since indometacin (10 mol/1) did not enhance evoked transmitter release, an influence of endogenous PG's on in vitro release of noradrenaline from rat brain cortex slices can be excluded.Abbreviation PG prostaglandin Send offprint requests to C. Allgaier at the above address     

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 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.     

Role of K+ channels in M2 muscarinic receptor-mediated inhibition of noradrenaline release from the rat stomach

Previously we reported the cholinergic M2 muscarinic receptor-mediated inhibition of noradrenaline release from the rat stomach (K. Yokotani, Y. Osumi. J Pharmacol Exp Ther. 1993;264:54-60). In the present study, we investigated the role of K+ channels in oxotremorine (a muscarinic receptor agonist)-induced inhibition of noradrenaline release using isolated, vascularly perfused rat stomach. The gastric postganglionic sympathetic nerves were electrically stimulated twice at 2.5 Hz for 1 min and test reagents were added during the second stimulation. The electrically evoked release of noradrenaline was augmented by tetraethylammonium and 4-aminopyridine (non-selective K+ channel blockers) and also by charybdotoxin (a blocker of big conductance Ca2+-activated K+ channel). On the other hand, apamin (a selective blocker of small conductance Ca2+-activated K+ channels) and glibenclamide (an ATP-activated K+ channel blocker) had no effect on the evoked noradrenaline release. Oxotremorine-induced inhibition of noradrenaline release was attenuated by tetraethylammonium and 4-aminopyridine, while the inhibition was not influenced by charybdotoxin, apamin, and glibenclamide. These results suggest that tetraethylammonium- and 4-aminopyridine-sensitive K+ channels (probably voltage-activated K+ channels) are involved in the muscarinic receptor-mediated inhibition of noradrenaline release from the rat stomach.     

Protein kinase C involvement in maintenance and modulation of noradrenaline release in the mouse brain cortex.

1. The role of protein kinase C in the modulation of noradrenaline release was investigated in mouse cortical slices which were pre-incubated with [3H]-noradrenaline. The aim was to investigate the hypothesis that protein kinase C is activated during high levels of transmitter release to maintain transmitter output. 2. The protein kinase C activators, phorbol myristate acetate (0.01-0.3 microM) and to a greater extent 4 beta-phorbol 12,13-dibutyrate (0.01-0.3 microM) significantly enhanced stimulation-induced noradrenaline release whereas 4 alpha-phorbol 12,13-dibutyrate (0.1 microM) which does not activate protein kinase C was without effect. The effect of the protein kinase C activator, phorbol myristate acetate, on noradrenaline release was attenuated by the protein kinase C inhibitor, polymyxin B (21 microM) which by itself inhibited stimulation-induced noradrenaline release. 3. Protein kinase C was down-regulated by 10 h exposure of the cortical slices to 4 beta-phorbol 12,13-dibutyrate (1 microM). In this case the facilitatory effect of 4 beta-phorbol 12,13-dibutyrate (0.1 microM) on noradrenaline release was abolished as was the inhibitory effect produced by polymyxin B. This indicates that polymyxin B was acting selectively at protein kinase C. 4. The inhibitory effect of polymyxin B on noradrenaline release, when expressed as a percentage of the appropriate frequency control, was constant at 1, 5 and 10 Hz. Furthermore, the ratio of release at 5 Hz to that at 10 Hz was not altered by protein kinase C down-regulation, indicating that there is no additional effect of protein kinase C at higher stimulation frequencies. 5. When transmitter release was elevated by blocking alpha 2-adrenoceptor auto-inhibition with idazoxan (0.1 microM) or K+ channels with tetraethylammonium (300 microM), the elevation in transmitter release was significantly attenuated by protein kinase C down-regulation, suggesting an involvement of protein kinase C. 6. We conclude that protein kinase C is involved in the modulation of noradrenaline release over a wide range of stimulation frequencies, in addition to a role when noradrenaline release is elevated by presynaptic mechanisms.     

Cultured chick sympathetic neurons: prostanoid EP1 receptor-mediated facilitation of noradrenaline release

Prostanoid EP receptor-mediated modulation of noradrenaline release from cultured chick sympathetic neurons was investigated. Transmitter release from dissociated cell cultures of embryonic paravertebral ganglia, loaded with [³H]-noradrenaline, was elicited either by electrical field stimulation (36 pulses/3 Hz) or by elevating the extracellular concentration of K⁺ (to 30 mM; for 2 min).Prostaglandin E₂ (PGE₂; 0.01–3 M) enhanced electrically evolved [³H]-noradrenaline release in a concentration-dependent manner with a maximal increase by about 50% at 1 M. Also iloprost (0.1–3 M) increased transmitter release concentration-dependently, whereas misoprostol (0.1–3 M) had no effect. Indometacin (10 M) influenced neither evoked release per se nor the enhancement caused by PGE₂. AH6809 (3 M), a selective EP₁ receptor antagonist, blocked the enhancement caused by both PGE₂ and iloprost. K⁺-evoked noradrenaline release, which was virtually insensitive to tetrodotoxin (0.3 M), was increased by PGE₂ to an extent comparable to that observed after electrical stimulation.In summary, the present data indicate that PGE₂ facilitates noradrenaline release from cultured chick sympathetic neurons by a receptor which shows the pharmacological profile of the EP₁ subtype and is probably located at the processes of the neuron.     

Cannabinoid receptor-mediated inhibition of dopamine release in the retina

The possible occurrence of cannabinoid (CB) receptors was studied on superfused guinea-pig retinal discs preincubated with [3H]dopamine or [³H]noradrenaline. Tritium overflow was evoked either electrically (3 Hz) or by re-introduction of Ca²⁺, 1.3 mM after superfusion with Ca²⁺-free medium containing K⁺ 30 rnM. The accumulation of [³H]dopamine ([³H]DA) and [³H]noradrenaline ([³H]NA) was inhibited by the selective inhibitor of the neuronal dopamine transporter GBR-12909 (pIC_50% 7.29 and 7.41, respectively) but not by the selective inhibitor of the neuronal noradrenaline transporter desipramine (1 M). The electrically or Ca²⁺-evoked tritium overflow in retinal discs preincubated with [³H]DA or [³H]NA was reduced by the CB receptor agonists CP-55,940 and WIN 55,212-2 (pIC_50% in discs preincubated with [³H]NA, electrical stimulation: 7.03 and 6.70, respectively) but not affected by the inactive S(–)enantiomer of the latter, WIN 55,212-3 (up to 10 M). The concentration-response curve of WIN 55,212-2 was shifted to the right by the CB₁ receptor antagonist SR 141716 (apparent pA₂: 8.29) which, by itself, increased the evoked overflow. The facilitatory effect of SR 141716 was not affected by GBR-12909 and the dopamine receptor antagonist haloperidol. In conclusion, the dopaminergic neurones of the guinea-pig retina can be labelled by both [³H]DA and [³H]NA. Transmitter release from the dopaminergic neurones is inhibited by activation of cannabinoid receptors of the CB₁ type, which appear to be tonically activated by an endogenous CB receptor ligand.