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1. We studied whether cannabinoid CB1 receptor gene disruption (to yield CB1-/- mice) affects the electrically evoked tritium overflow from vas deferens and atrial pieces preincubated with [3H]-noradrenaline (NA) ('noradrenaline release') and from cerebral cortex slices preincubated with [3H]-choline ('acetylcholine release'). 2. NA release was higher by 37% in vas deferens from CB1-/- mice than in vas deferens from CB1+/+ mice. The cannabinoid receptor agonist WIN 55,212-2 inhibited, and the CB1 receptor inverse agonist/antagonist SR 141716, increased NA release in vas deferens from CB1+/+ mice without affecting it in vas deferens from CB1-/- mice. 3. Atrial NA release did not differ between CB1+/+ and CB1-/- mice nor did WIN 55,212-2 affect NA release in either strain. 4. Cortical acetylcholine (Ach) release did not differ between CB1+/+ and CB1-/- mice. WIN 55,212-2 inhibited, but SR 141716 did not affect, Ach release in the cortex from CB1+/+ mice. Both drugs did not alter Ach release in the cortex from CB1-/- mice. 5. Tritium content did not differ between CB1+/+ and CB1-/- mice in any preparation. 6. In conclusion, the increase in NA release associated with CB1 receptor deficiency in the vas deferens, which cannot be ascribed to an alteration of tritium content of the preparations, suggests an endogenous tone at the CB1 receptors of CB1+/+ mice in this tissue. Furthermore, the effect of WIN 55,212-2 on NA release in the vas deferens and on cortical Ach release involves CB1 receptors, whereas the involvement of non-CB1-non-CB2 receptors can be excluded.

The modulation of dopamine release from the guinea-pig retina was studied using maximally effective concentrations of 10 agonists acting on G(i)-, G(s)- or G(q)-protein-coupled receptors (PCRs). Retinal discs were preincubated with [(3)H]noradrenaline and superfused; tritium overflow was evoked electrically. The following compounds acting on G(i)-PCRs reduced the tritium overflow, which represents quasi-physiological dopamine release under the experimental conditions of our study: the dopamine and alpha(2)-adrenoceptor agonist B-HT 920 by 95%, the muscarinic agonist oxotremorine by 96%, melatonin by 94%, the cannabinoid agonist WIN 55,212-2 by 71% and histamine by 66%. Tritium overflow was not affected by serotonin or by agonists acting on G(s)-PCRs (ACTH1-24 and the beta-adrenoceptor agonist procaterol) and G(q)-PCRs (angiotensin II and bradykinin). The effects of B-HT 920, oxotremorine and melatonin were studied in more detail using appropriate antagonists. The inhibitory effect of a submaximally active concentration of B-HT 920 was counteracted by the dopamine D(2/3) antagonist haloperidol but not affected by the alpha(2)-adrenoceptor antagonist phentolamine. The muscarinic antagonist atropine shifted to the right the concentration-response curve of oxotremorine (pA(2) 8.7) and the melatonin MT(2) antagonist 4-P-PDOT produced a rightward shift of the concentration-response curve of melatonin (pA(2) 10.6). Melatonin was also studied in superfused brain slices (from the guinea-pig) preincubated with [(3)H]noradrenaline. The electrically evoked tritium overflow in cerebrocortical, hippocampal and hypothalamic slices (representing quasi-physiological noradrenaline release) and in striatal slices (representing quasi-physiological dopamine release) was not affected by melatonin at a concentration that causes the maximum effect in retinal discs. In conclusion, dopamine release in the guinea-pig retina is inhibited via G(i)-PCRs including dopamine (D(2/3)), muscarinic and melatonin (MT(2)) receptors but not affected via any of the G(s)- or G(q)-PCRs under study. Unlike in the retina, melatonin fails to inhibit monoamine release in four brain regions of the guinea-pig.

The effect of the newly synthetized H1 agonists 2-(3-trifluoromethylphenyl)histamine (2-TFMPH) and 2-(3-bromophenyl)histamine (2-BPH) and of the reference compound 2-(2-thiazolyl)ethylamine (2-TEA) on diastolic blood pressure and heart rate was studied in pithed and vagotomized rats. 2-TFMPH and 2-BPH were at least equipotent with 2-TEA in producinga dimethindene-sensitive, short-lasting vasodepressor response. At the highest dose, 2-TFMPH and 2-BPH produced an additional small vasopressor response, which was followed by a long-lasting vasodepressor effect not counteracted by dimethindene and ranitidine 2-TEA, at the highest dose, induced an additional vasopressor response abolished by prazosin plus rauwolscine. Basal heart rate was increased by 2-TEA (in a desipramine-sensitive manner) but not affected by 2-TFMPH or 2-BPH. In conclusion, 2-TFMPH and 2-BPH are potent H1 agonists, devoid of an indirect sympathomimetic effect; at high doses, they produce a vasodepressor response not mediated via histamine receptors.

In pithed and vagotomized rats, the electrically induced vasopressor response was not affected by 2-(2-thiazolyl)ethylamine and dimaprit but was inhibited by Nα-methylhistamine (NαMH) in a thioperamidesensitive manner. NαMH did not affect the vasopressor response to exogenously added noradrenaline. NαMH produced a biphasic effect on basal diastolic blood pressure, i.e. a vasodepressor response (abolished by dimethindene plus ranitidine) and a subsequent vasopressor effect (counteracted by dimethindene or adrenalectomy). Basal heart rate was increased by NαMH (effect abolished by propranolol, dimethindene or adrenalectomy). In conclusion, presynaptic H3, but not H1 or H2, receptors are detectable in the resistance vessels of the rat. NαMH allows the characterization of another four histamine receptor-mediated effects, i.e vasodepressor effects mediated via H1 and H2 receptors and an increase in blood pressure and heart rate mediated via catecholamine-releasing H1, receptors in the adrenal medulla.

The endocannabinoid anandamide exerts complex effects on the cardiovascular system [Malinowska et al., Br J Pharmacol, 2011]. Thus, rapid intravenous injection of anandamide and its stable analogue methanandamide in anaesthetized rats and mice induces typical triphasic changes. Phase I, the so-called BezoldJarisch reflex, is characterized by a rapid bradycardia and hypotension associated with a decrease in cardiac contractility and an increase in total peripheral resistance. Phase II is a brief pressor response associated with an increase in cardiac contractility and blood flow in the mesenteric and renal arteries. Phase III comprises a prolonged (lasting up to 10 min) and marked decrease in blood pressure, mesenteric and renal blood flow, a marked decrease in cardiac contractility and a slight decrease in heart rate and total peripheral resistance. Phase I is mediated via vanilloid TRPV1 receptors since it was induced also by the TRPV1 receptor agonist capsaicin (but not by the CB1 receptor agonist CP55940), blocked by the TRPV1 receptor antagonists capsazepine and ruthenium red (but not by the CB1 receptor antagonist rimonabant) and absent in TRPV1 / mice. Phase II consists of a peripheral component that is most probably located in blood vessels, is sensitive to nifedipine, ruthenium red and pentobarbitone and, hence, probably represents a Ca-dependent mode of action and a central component. The paraventricular nucleus of the hypothalamus (PVN) may contribute to the latter since a pressor effect was obtained after injection of anandamide, methanandamide or CP55940 (intraventricularly or directly into the PVN). This effect only occurred when the peripheral CB1 receptors were blocked by administration of CB1 receptor antagonists, including AM6545 that does not reach the brain. It was sensitive to adrenalectomy and counteracted by antagonists of CB1, NMDA, thromboxane A2 and b2adrenergic receptors [AM251 (directly into the PVN), MK-801, SQ29548 and ICI118551, respectively] suggesting that the latter four receptors are involved in the events ultimately leading to the anandamide-induced adrenal secretion of catecholamines. Importantly, the hypotensive effect of intravenously administered CP55940 was reversed into a hypertensive one in the presence of AM6545 and both responses were abolished by chemical lesion of the PVN by kainic acid. Phase III is mediated mainly by presynaptic inhibitory CB1 receptors located on the postganglionic (although a preganglionic localization cannot be excluded) sympathetic nerve endings innervating resistance vessels and heart. The CB1 receptor antagonist-sensitive inhibitory effect of these receptors was detected after electrical stimulation of the preganglionic sympathetic nerve fibres in pithed rabbits or rats or following injection of nicotine but not phenylephrine/noradrenaline or isoprenaline (increasing blood pressure and heart rate, respectively). The prolonged hypotension might also be related to the activation of CB1 and/or vanilloid TRPV1 receptors in the spinal cord or to a direct vasodilatation via O-1918 sensitive receptors. In conscious rats anandamide and methanandamide induced a brief pressor (phase II) response connected with renal and mesenteric vasoconstriction and hindquarters vasodilatation. In addition, higher doses of anandamide elicited an initial bradycardia, hypotension and hindquarters vasoconstriction (phase I). However, unlike in anaesthetized rats, none of the cannabinoids led to a prolonged hypotension (phase III). Cardiovascular effects of anandamide are changed under pathophysiological conditions. Thus, in anaesthetized rats myocardial ischemia enhanced phase I and reduced phase III whereas hypertension enhanced phase III. Acknowledgement: B.M. and E.S. would like to express their gratitude to Professor Manfred Gothert for his continuous encouragement extending over a time period of decades.

Cannabis (marijuana) is not only a frequently abused drug but also has the potential for the development of useful agents for the treatment of emesis, anorexia and multiple sclerosis. In this article, the effects of modulation of transmitter release by cannabinoids in both the CNS and the PNS of various species, including humans, will be discussed. Cannabinoids inhibit neurotransmitter release via specific presynaptic cannabinoid CB1 receptors. Studies using either the CB1 receptor antagonist and inverse agonist SR141716 or CB1-receptor-deficient mice suggest that numerous presynaptic cannabinoid receptors are tonically activated by endogenous cannabinoids and/or are constitutively active. CB1-receptor-mediated inhibition of transmitter release might explain, for example, reinforcing properties and memory impairment caused by cannabinoids.

We have shown previously that histaprodifen and its Nalpha-substituted analogues methylhistaprodifen and dimethylhistaprodifen are highly potent H1-receptor agonists in vivo. The aim of the present study was to examine the influence of four newly synthesized histaprodifen analogues, 3-fluoro-methylhistaprodifen (1), Nalpha-imidazolylethylhistaprodifen (2), bis-histaprodifen (3) and Nalpha-methyl-bis-histaprodifen (4), on the cardiovascular system in the pithed and in the anaesthetized rat. In pithed and vagotomized rats, diastolic blood pressure (which was increased to 80-85 mmHg by vasopressin infusion) was decreased dose dependently by methylhistaprodifen (the reference compound) and by compounds 1-4. The maximum decrease was about 47-50 mmHg for methylhistaprodifen and compounds 1, 2 and 3. Their potencies, expressed as pED50 (the negative logarithm of the dose in mole per kilogram body weight that decreased diastolic blood pressure by 25 mmHg), were 8.31, 8.23, 8.26 and 7.84, respectively. With compound 4 the maximal effect was not achieved at doses up to 1 micromol/kg (the latter dose decreased blood pressure by about 30 mmHg; pED50 approximately 6.5). The vasodepressor effect of the five compounds was attenuated by the H1-receptor antagonist dimetindene (1 micromol/kg) but was not changed by combined administration of the H2- and H3-receptor antagonists ranitidine and thioperamide (1 micromol/kg each), by combined administration of the alpha1- and alpha2-adrenoceptor antagonists prazosin and rauwolscine (1 micromol/kg each) or by the beta-adrenoceptor antagonist propranolol (3 micromol/kg). In anaesthetized rats methylhistaprodifen and compounds 1-4 induced almost the same fall in blood pressure as in pithed and vagotomized animals; the effects were sensitive to blockade by dimetindene (1 micromol/kg). Higher doses of compounds 1 and 2 (1 micromol/kg) increased heart rate in pithed and vagotomized rats in a manner sensitive to propranolol (3 micromol/kg) but insensitive to dimetindene (3 micromol/kg). The same dose of methylhistaprodifen and of compounds 3 and 4 failed to affect heart rate. We conclude that the agonistic potency of compounds 1 and 2 at H1-receptors in the cardiovascular system of the rat equals that of methylhistaprodifen, the most potent histamine H1-receptor agonist available until recently. Compounds 1 and 2 exhibit sympathomimetic activity at high doses.

In this article, the effect of nociceptin (orphanin FQ) on transmitter release in the central nervous system in vitro and in vivo is reviewed. Nociceptin inhibits the electrically or K(+)-evoked noradrenaline, dopamine, serotonin, and glutamate release in brain slices from guinea-pig, rat, and mouse. This effect is usually naloxone-resistant but antagonized by OP(4) receptor antagonists like [Phe(1)psi(CH(2)-NH)Gly(2)]-nociceptin(1-13)NH(2). In the rat in vivo, nociceptin diminishes acetylcholine release in the striatum, reduces dopamine release, and prevents the stimulatory effect of morphine on this transmitter in the nucleus accumbens and also elevates extracellular glutamate and gamma-aminobutyric acid levels in mesencephalic dopaminergic areas. The effect of nociceptin on the mesencephalic dopaminergic system might explain its actions on motor behavior.

The amount of serotonin (5-hydroxytryptamine; 5-HT) released from the varicosities of the serotoninergic axon terminals in response to invading action potentials at a given frequency is by no means constant. As generally accepted now, exocytotic release of 5-HT can be significantly modified by presynaptic receptors, i.e. receptors located on the serotoninergic axon terminals (for reviews, see Moret 1985; Middlemiss 1988; Starke et al. 1989; Gothert 1990). When such receptors are stimulated by 5-HT released from the serotoninergic axon terminals, they are termed presynaptic 5-HT autoreceptors. When receptors on the serotoninergic axon terminals are activated by other transmitters released from non-serotoninergic neurons, they are denoted as presynaptic heteroreceptors. In the latter case, the axon terminals of these neurons may form axo-axonic synapses with the serotoninergic terminals, or the interacting neighbouring neurons may release their transmitter non-synaptically into the extracellular space and, thus, may influence larger neuronal territories including the serotoninergic axon terminals (Beaudet and Descarries 1978; Tork 1990).

A novel compound, SB-236057 (1'-ethyl-5-(2'-methyl-4'-(5-methyl-1,3,4-oxadiazol-2-yl)biphenyl-4-carbonyl)-2,3,6,7-tetrahydrospiro[furo]2,3-f]indole-3,4'-piperidine]) has been shown to have high affinity for human 5-hydroxytryptamine 1B (5-HT 1B ) receptors (pK i = 8.2) and displays over 75 or more-fold selectivity for the human 5-HT 1B receptor over other 5-HT receptors, including the human 5-HT 1D receptor, and a range of other receptors, ion channels and enzymes. In functional studies using [ 35 S]GTPγS binding, SB-236057 displayed negative intrinsic activity (pEC 50 = 8.0) at human 5-HT 1B receptors stably expressed in Chinese Hamster Ovary (CHO) cells and caused a rightward shift of agonist concentration response curves consistent with competitive antagonism (pA 2 = 8.9). SB-236057 potentiated [ 3 H]5-HT release from electrically stimulated guinea pig or human cortical slices. SB-236057 also abolished the inhibitory effect of exogenously superfused 5-HT on electrically-stimulated release from slices of the guinea pig cortex. These studies using SB-236057 confirm that, in both the guinea pig and human cerebral cortex, the terminal 5-HT autoreceptor is of the 5-HT 1B subtype.

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 microM and 1000 microM and by kainate 1000 microM 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 microM)-stimulated tritium overflow but did not affect tritium overflow stimulated by NMDA 100 microM or kainate 1000 microM. The inhibitory effect of WIN 55,212-2 on the NMDA (100 microM)- and kainate (1000 microM)-evoked tritium overflow was antagonized by SR 141716. In rat striatal slices preincubated with [3H]dopamine, WIN 55,212-2 inhibited the NMDA (1000 microM)-stimulated tritium overflow. SR 141716, which, by itself, did not affect tritium overflow, counteracted the inhibitory effect of WIN 55,212-2. [3H]SR 141716 binding to rat cortical membranes was inhibited by SR 141716, CP-55,940 and WIN 55,212-2 (pKi 8.53, 7.34 and 5.93, respectively) but not affected by desipramine, rauwolscine and nomifensine (pKi5). 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.

Mr 2266 [(-)-(1R,5R,9R)-5,9-diethyl-2-(3-furylmethyl)-2'-hydroxy-6,7-benzomorpha n] is an antagonist at kappa-opioid receptors and at ORL1 receptors as well. The aim of our study was to examine whether the known stereoselective antagonism of Mr 2266 at kappa-opioid receptors also extends to ORL1 receptors. In mouse brain cortex membranes, the binding of the ORL1 receptor agonist [3H]nociceptin was equipotently inhibited by Mr 2266 and its enantiomer Mr 2267 (pK(i) 4.82 and 5.14, respectively), whereas the binding of the kappa-opioid receptor agonist [3H]U-69,593 was inhibited by Mr 2266 more potently (pK(i) 9.11) than by its enantiomer Mr 2267 (pK(i)7.15). In mouse brain cortex slices preincubated with [3H]noradrenaline, the concentration-response curve of nociceptin for inhibition of the electrically evoked overflow of tritium was equipotently shifted to the right by Mr 2266 and Mr 2267 (pA2 5.77 and 5.64, respectively). On the other hand, the inhibitory effect of U-69,593 on the electrically evoked overflow of tritium in guinea-pig brain cortex slices preincubated with [3H]noradrenaline was more potently antagonized by Mr 2266 (pA2 8.81) than by Mr 2267 (pA2 7.15). These data show that the stereoselective antagonism of Mr 2266 at kappa-opioid receptors does not extend to ORL1 receptors.

We determined the affinities of five newly synthesized histamine H3-receptor antagonists in an H3-receptor binding assay and their potencies in a functional H3-receptor model. Furthermore, we determined their potencies in a histamine H2- and H1-receptor model. The compounds differ from histamine in that the ethylamine side chain is replaced by an aryl-substituted propyloxy chain and they differ from one another by varying substituents of the aryl rest. Iodoproxyfan, a highly potent and selective antagonist at H3 receptors, is structurally related to these five compounds. The specific binding of [3H]-Nalpha-methylhistamine to rat brain cortex membranes was monophasically displaced by each of the five compounds at pKi values ranging from 8.24 to 9.27. Inhibition by histamine of the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]noradrenaline was antagonized by all compounds and the concentration-response curve was shifted to the right with apparent pA2 values ranging from 7.78 to 9.39. The five compounds under study possess negligible potencies at histamine H2 and H1 receptors studied in the guinea-pig right atrium and ileum, respectively (pD'2 or pKp values < or = 5.2). The present paper shows that the five compounds under study possess high affinities and potencies at histamine H3 receptors, four out of the five compounds in this respect being equipotent with iodoproxyfan. Like iodoproxyfan, the five compounds show an at least 1000-fold selectivity for H3 as compared to H2 and H1 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 microM reduced the electrically evoked tritium overflow from mouse, rat and guinea-pig brain cortex slices by 80, 71 and 36%, respectively. Naloxone 10 microM did not change the effect of nociceptin. All subsequent experiments were performed on mouse brain cortex slices and in the presence of naloxone 10 microM. 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 [Phe1psi(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 [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 caused an inhibition by maximally 35% (pEC50 7.0; intrinsic activity alpha 0.45). The inhibitory effect of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)NH2 was counteracted by naloxone benzoylhydrazone. Nociceptin also reduced the Ca2+-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.

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 1 Hz) or by introduction of Ca2+ ions (1.3 mM) [corrected] into Ca(2+)-free, K(+)-rich medium (25 mM) [corrected] containing tetrodotoxin 1 microM. Furthermore, the cAMP accumulation stimulated by forskolin 10 microM 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)cyclo-hexanol (CP-55,940) and R(+)-[2,3-dihydro-5-methyl-3- [(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]- (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 (pIC30% 6.5) but not affected by WIN 55,212-3 up to 10 microM. The concentration-response curve of WIN 55,212-2 was shifted to the right by SR 141716 (0.032-microM) (apparent pA2 8.2), which by itself did not affect the evoked overflow. WIN 55,212-2 1 microM also inhibited the Ca(2+)-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 (0.32 microM) markedly attenuated the WIN 55,212-2-induced inhibition in guinea-pig and human brain slices. SR 141716 0.32 microM 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 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 (0.1 microM; 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.

We examined the possible existence of prejunctional histamine H3 receptors on sympathetic nerve fibers innervating rat tail artery. The stimulation-evoked tritium outflow from isolated vessels preincubated with [3H]-noradrenaline and perfused/superfused in the presence of the alpha2-adrenoceptor antagonist rauwolscine, 3 microM, was inhibited by histamine 10 microM (by 8%) and the H3 agonists R-(-)-alpha-methylhistamine, 10 microM (by 18%), and imetit, 0.1-10 microM (by < or =20%). The inhibitory effect of imetit, which did not occur in the absence of rauwolscine, was counteracted by thioperamide, 1 microM. In the presence of rauwolscine, 3 microM, the inhibitory effect of imetit also occurred when the current strength or the Ca2+ concentration in the medium was reduced to compensate for the increase in tritium overflow elicited by rauwolscine, indicating that the inhibitory action of imetit is not associated with the increase in noradrenaline release produced by rauwolscine. In spontaneously hypertensive rats (SHRs), imetit also inhibited the overflow of tritium. This inhibitory effect was comparable to that observed in Wistar-Kyoto (WKY) rats and indicates that the sympathetic nerves of the rat tail artery in SHRs, like those in normotensive rats, are endowed with prejunctional histamine H3 receptors.

The present study was carried out to clarify whether the imidazolines clonidine, moxonidine and cirazoline as well as the guanidine aganodine inhibit noradrenaline release in the rat and rabbit brain via imidazoline receptors, alpha 2-adrenoceptors and/or histamine H3 receptors. Slices or synaptosomes from the rat or the rabbit brain were incubated with 3H-noradrenaline and exposed to phenoxybenzamine, which irreversibly blocks presynaptic alpha 2-adrenoceptors and, at considerably lower potency, imidazoline receptors. Tritium overflow in the superfused preparations was evoked electrically (3 Hz; slices) or by K+ 15 mmol/l (synaptosomes). Noradrenaline and rauwolscine, which possess low affinity, if any, for imidazoline receptors, were used as reference drugs. The evoked overflow in rat brain cortex slices and synaptosomes and in rat medulla oblongata slices, not exposed to phenoxybenzamine, was inhibited by clonidine, moxonidine and noradrenaline. Phenoxybenzamine markedly attenuated the effect of each drug to about the same extent. In rabbit brain cortex slices, not exposed to phenoxybenzamine, the evoked overflow was inhibited by clonidine, moxonidine, aganodine and noradrenaline, facilitated by BDF 6143 (4-chloro-2-(2-imidazoline-2-yl-amino)-isoindoline), idazoxan and rauwolscine and not affected by cirazoline. In slices exposed to phenoxybenzamine, the inhibitory effects of the imidazolines, of aganodine and of noradrenaline were again attenuated by about the same high degree, the facilitatory effects of BDF 6143, idazoxan and rauwolscine were abolished and cirazoline produced a slight inhibition of the evoked overflow. The latter effect was not affected by high concentrations of rauwolscine and idazoxan (at which these drugs act antagonistic at imidazoline receptors in other models). The specific binding of 3H-N alpha-methylhistamine to H3 receptors in rat brain cortex membranes was displaced only by high concentrations of moxonidine (pKi = 6.16) and at even lower affinity by aganodine, BDF 6143, cirazoline, clonidine and idazoxan (pKi5). Histamine, which was used as a reference drug, proved to be very potent (pKi = 8.20). In conclusion, imidazolines affect noradrenaline release in the rat and rabbit brain cortex and medulla oblongata via alpha 2-adrenoceptors but not via imidazoline receptors resembling the presynaptic imidazoline receptors previously identified in peripheral tissues of the rabbit. In addition, the involvement of I1- or I2-imidazoline binding sites or of H3 receptors is very improbable in view of the low affinity of aganodine, moxonidine and/or clonidine for these recognition sites and/or incompatibility of the rank order of their affinities with the potencies of the drugs in inhibiting noradrenaline release.

Hippocampal slices were prepared from brain tissue of patients undergoing neurosurgery for epilepsy. The slices were incubated with 3H-serotonin (3H-5-HT) and then superfused with physiological salt solution containing 6-nitroquipazine. Tritium overflow was evoked either electrically (3 Hz) or by K+ 25 mM. The electrically evoked overflow of tritium was almost abolished by tetrodotoxin or by omission of Ca2+ ions. 5-HT 0.1 and 1 microM reduced the evoked overflow by 38 and 55%, respectively. The effect of 5-HT 1 microM was abolished by the 5-HT1/2 receptor antagonist methiothepin 1 microM, which, by itself, increased the evoked overflow by 59%. Tritium overflow evoked by high K+ in slices superfused with medium containing tetrodotoxin was reduced by 5-HT 1 microM by 49%. This effect was markedly attenuated by methiothepin 0.32 microM, which, by itself, tended to increase the evoked overflow. The results show that the serotoninergic neurones of the human hippocampus are endowed with presynaptic inhibitory autoreceptors.

We determined the affinities of 16 newly synthesized H3 receptor antagonists in an H3 receptor binding assay and the potencies of 12 of these compounds at functional H3 receptors in the mouse brain cortex and guinea-pig ileum. The compounds differ from histamine in that the C-C-N side chain is replaced by a chain of the structure C-C-C-O. The two major aims of the study were (1) to investigate whether the two functional H3 receptors are pharmacologically different and (2) to derive structure-activity relationships. The specific binding of 3H-Na-methylhistamine to rat brain cortex membranes was monophasically displaced by each of the 16 compounds at pKi values ranging from 7.30 to 9.48. In superfused mouse brain cortex slices preincubated with 3H-noradrenaline, the electrically evoked tritium overflow was slightly decreased by iodoproxyfan and its deiodo analogue; this effect was counteracted by the H3 receptor antagonist clobenpropit. The other compounds did not affect the evoked tritium overflow by themselves. The concentration-response curve of histamine for its inhibitory effect on the electrically evoked tritium overflow was shifted to the right by the 12 compounds with apparent pA2 values ranging from 7.02 to 9.00. The 12 compounds also shifted to the right the concentration-response curve of R-a-methylhistamine for its inhibitory effect on the electrically induced contraction in guinea-pig ileum strips; the apparent pA2 values ranged from 5.97 to 9.00. Iodoproxyfan decreased the electrically induced contraction by itself and this effect was counteracted by the H3 receptor antagonist thioperamide. The apparent pA2 values in the two functional H3 receptor models showed a highly significant correlation (r = 0.882; P < 0.001). Highly significant correlations were also obtained when the pKi values of the compounds in the binding assay were compared to their apparent pA2 values in the mouse brain (r = 0.799; P < 0.004) and in the guinea-pig ileum (r = 0.851; P < 0.001). In each of the three experimental models, iodoproxyfan was the most potent compound; its deiodo analogue was less potent by more than 1.1 log units. The present results show that the compounds under study possess moderate to high affinity and/or (partial) H3 receptor antagonist potency. The two functional H3 receptors in the mouse brain cortex and the guinea-pig ileum may be slightly different; further studies are necessary to clarify whether this difference is due to H3 receptor heterogeneity, species variants or differences in the efficiency of receptor coupling. The marked difference in the affinity/potency between iodoproxyfan and its deiodo analogue may suggest that a highly lipophilic residue in that part of the molecule favours a high affinity/antagonistic potency at H3 receptors.

1. The influence of beta 1-, beta 2-, and beta 3-adrenoceptor agonists and of CGP 12177 and cyanopindolol on heart rate and diastolic blood pressure was studied in the pithed rat. 2. The beta 1-adrenoceptor agonist, prenalterol, increased heart rate and the beta 2-adrenoceptor agonist, fenoterol, caused a fall in blood pressure. The effect of prenalterol was antagonized by the beta 1-adrenoceptor antagonist, CGP 20712 0.1 mumol kg-1 and the action of fenoterol was attenuated by the beta 2-adrenoceptor antagonist, ICI 118551 0.1 mumol kg-1. Both effects were markedly diminished by the non-selective beta-adrenoceptor antagonist, bupranolol 0.1 mumol kg-1. 3. The non-selective beta-adrenoceptor agonist, isoprenaline, three beta 3-agonists as well as CGP 12177 and cyanopindolol elicited a positive chronotropic effect, exhibiting the following pED delta 60 values (negative log values of the doses increasing heart rate by 60 beats min-1): isoprenaline 10.4, CGP 12177 8.3, cyanopindolol 7.2, BRL 37344 6.9, ZD 2079 5.2 and CL 316243 < 5. 4. CGP 20712 0.1 mumol kg-1, given together with ICI 118551 0.1 mumol kg-1, markedly attenuated the positive chronotropic effect of isoprenaline, BRL 37344, ZD 2079 and CL 316243 without affecting the increase in heart rate produced by CGP 12177 and cyanopindolol. 5. The positive chronotropic effect of CGP 12177 and cyanopindolol was attenuated by CGP 20712, 1 and 10 mumol kg-1 and bupranolol, 10 mumol kg-1 but was not affected by ICI 118551, 10 mumol kg-1. The effect of CGP 12177 was also not changed by BRL 37344 1 mumol kg-1, ZD 2079 10 mumol kg-1, CL 316243 10 mumol kg-1, the alpha 1-adrenoceptor antagonist, prazosin 1 mumol kg-1 and the 5-hydroxytryptamine 5-HT2A receptor antagonist, ketanserin 3 mumol kg-1. 6. CGP 12177 0.002 mumol kg-1 and cyanopindolol 0.003 mumol kg-1 shifted to the right the dose-response curve of prenalterol for its positive chronotropic effect. The -log values of the doses causing a twofold shift to the right were 9.6 and 9.5, respectively. 7. Isoprenaline 0.00001-0.001 mumol kg-1, BRL 37344 0.01-1 mumol kg-1 and CGP 12177 0.1 mumol kg-1 caused a fall in diastolic blood pressure which was markedly attenuated by combined administration of CGP 20712 and ICI 118551, 0.1 mumol kg-1 each. 8. CGP 12177 0.01 and 0.1 mumol kg-1 and cyanopindolol 1 mumol kg-1 elicited an increase in diastolic blood pressure. CGP 20712, ICI 118551, bupranolol and, in the case of CGP 12177, also BRL 37344, ZD 2079, CL 316243, prazosin and ketanserin did not influence this effect. 9. In conclusion, the positive chronotropic effect of CGP 12177 and cyanopindolol is not mediated via beta 1-, beta 2-, beta 3-, alpha 1-adrenoceptors or 5-HT2A receptors. This effect may involve atypical beta-adrenoceptors, similar or identical to those described by Kaumann (1989) in isolated heart preparations.

We determined the affinity and/or potency of two metabolites of clozapine (clozapine-N-oxide and N-desmethylclozapine) and of five atypical neuroleptics, chemically related (olanzapine) or unrelated to clozapine (remoxipride, risperidone, thioridazine, zotepine), at H3 receptors. The specific binding of 3H-N alpha-methylhistamine to rat brain cortex homogenates was inhibited by the seven compounds; the pKi values were: N-desmethylclozapine (5.33); clozapine-N-oxide (4.18); olanzapine (5.45); thioridazine (4.91); zotepine (4.75); remoxipride (4.51) and risperidone (4.43). Three compounds were examined in a functional H3 receptor model as well. The electrically evoked tritium overflow from superfused mouse brain cortex slices, which represents quasi-physiological noradrenaline release, was not affected by N-desmethylclozapine (3.2 and 10 microM), clozapine-N-oxide (3.2-100 microM) and olanzapine (3.2-32 microM). On the other hand, the three compounds shifted to the right the concentration-response curve of histamine for its inhibitory effect on the evoked overflow; the apparent pA2 values were 5.84, 4.21 and 5.80, respectively. The present study shows that five atypical neuroleptics of different chemical classes and the two major metabolites of clozapine possess a lower affinity and/or antagonistic potency at H3 receptors than clozapine itself (pKi 6.15, pA2 6.33; Kathmann M, Schlicker E, Göthert M (1994). Psychopharmacology 116: 464-468).

We determined the affinity and/or potency of the novel H3 receptor antagonist iodoproxyfan at alpha 2 and 5-HT3 receptors. Iodoproxyfan and rauwolscine (a reference alpha 2 ligand) (i) monophasically displaced 3H-rauwolscine binding to rat brain cortex membranes (pKi 6.79 and 8.59); (ii) facilitated the electrically evoked tritium overflow from superfused mouse brain cortex slices preincubated with 3H-noradrenaline (pEC50 6.46 and 7.91) and (iii) produced rightward shifts of the concentration-response curve (CRC) of (unlabelled) noradrenaline for its inhibitory effect on the evoked overflow (pA2 6.65 and 7.88). In the guinea-pig ileum, iodoproxyfan 6.3 mumol/l failed to evoke a contraction by itself but depressed the maximum of the CRC of 5-hydroxytryptamine (pD'2 5.24). Tropisetron (a reference 5-HT3 antagonist) produced rightward shifts of the CRC of 5-hydroxytryptamine (pA2 7.84). In conclusion, the affinity/potency of iodoproxyfan at H3 receptors (range 8.3-9.7 [1]) exceeds that at alpha 2 receptors by at least 1.5 log units and that at 5-HT3 receptors by at least 3 log units.

Mouse or rat brain cortex slices were preincubated with 3H-noradrenaline and superfused with physiological salt solution containing desipramine. We studied the effects of prostaglandin E2 (PGE2), prostaglandin D2 (PGD2) and related drugs on the electrically evoked (50 mA, 2 ms, 0.3 Hz) tritium overflow. PGE2 inhibited the electrically evoked tritium overflow from mouse brain cortex slices; the maximum effect of PGE2 (79010) was attenuated by the α2-adrenoceptor agonist talipexole (to 52010) and enhanced by the α2-adrenoceptor antagonist rauwolscine (to 92%). Rauwolscine was added to the superfusion medium in all subsequent experiments. The effect of PGE2 was readily reversible upon withdrawal from the medium and remained constant upon prolonged exposure of the tissue to the prostanoid. Studies with EP receptor agonists, mimicking the inhibitory effect of PGE2, showed the following potencies (pIC50): sulprostone (8.22); misoprostol (8.00); PGE2 (7.74); PGEZ (7.61); iloprost (5.86). The concentration-response curve of PGE2 was marginally shifted to the right by the EP1 receptor antagonist AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid; apparent pA2 3.97) and by the TP receptor antagonist vapiprost (4.50). AH 6809, by itself, did not affect the evoked overflow whereas vapiprost increased it. PGD2 inhibited the evoked overflow at high concentrations (pIC50 4.90); this effect was not altered by the DP receptor antagonist BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2hydroxyethylamino)hydantoin), which, by itself, did not affect the evoked overflow. Indometacin slightly increased the evoked overflow and tended to increase the inhibitory effect of PGE2. PGE2 inhibited the electrically evoked tritium overflow also in rat brain cortex slices. The maximum effect (obtained in the presence of rauwolscine) was 61%; the pIC30 value was 7.67. The present study suggests that PGE2 inhibits noradrenaline release from mouse brain cortex via EP3 receptors and that its maximum effect is more marked in the mouse than in the rat. The inhibitory effect of PGD2 (in the mouse brain) does not involve DP receptors and may also be related to EP3 receptors. The EP3 receptors interact with a2-adrenoceptors and may be activated by endogenous prostanoids.

The present study was aimed at the identification of mechanisms following the activation of histamine H3 receptors. Mouse brain cortex slices preincubated with 3H-noradrenaline were superfused and the (H3 receptor-mediated) effect of histamine on the electrically evoked tritium overflow was studied under a variety of conditions. The extent of inhibition produced by histamine was inversely related to the frequency of stimulation used to evoke tritium overflow and to the Ca2+ concentration in the superfusion medium. An activator (levcromakalim) and blocker (glibenclamide) of ATP-dependent K+ channels did not affect the electrically evoked tritium overflow and its inhibition by histamine. A blocker of voltage-sensitive K+ channels, tetraethylammonium (TEA), increased the evoked overflow and attenuated the inhibitory effect of histamine. TEA also reduced the inhibitory effect of noradrenaline and prostaglandin E2 on the evoked overflow. When the facilitatory effect of TEA on the evoked overflow was compensated for by reducing the Ca 2+ concentration in the superfusion medium, TEA did no longer attenuate the effect of histamine. Exposure of the slices to the SH group-alkylating agent N-ethylmaleimide increased the evoked overflow and attenuated the inhibitory effect of histamine; both effects were counteracted by the SH group-protecting agent dithiothreitol, which, by itself, did not affect the evoked overflow and its inhibition by histamine. Mouse brain cortex membranes were used to study the effect of the H3 receptor agonist R-(−)α-methylhistamine on the basal cAMP accumulation and on the accumulation stimulated by forskolin or noradrenaline. R-(−)-α-Methylhistamine did not affect basal cAMP accumulation but, at high concentrations, inhibited the forskolin- and noradrenaline-stimulated cAMP accumulation. S-(+)-α-Methylhistamine (which is 100 times less potent than R-(−)-α-methylhistamine at H3 receptors) was equipotent with the R-(−)-enantiomer in inhibiting the forskolin-stimulated CAMP accumulation. The inhibition by R-(−)-α-methylhistamine was not affected by the H3 receptor antagonist clobenpropit but was counteracted by the α2-adrenoceptor antagonist rauwolscine. The present results suggest that the histamine-induced inhibition of noradrenaline release depends on the availability of extracellular Ca2+ ions for stimulus-release coupling; in particular, a decrease in Ca 2+ influx into the varicosities may contribute to this inhibition. The H3 receptors (which may be coupled to a G protein) do not appear to be coupled to adenylate cyclase, to ATP-dependent K+ channels or to (TEA-sensitive) voltage-regulated K+ channels. α-Methylhistamine, in addition to its main action as a stereoselective H3 receptor agonist, proved to be weakly potent as an α2-adrenoceptor agonist.

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-(-)-alpha-methylhistamine inhibited NMDA-evoked tritium overflow (IC20 values 0.27 mumol/l or 0.032 mumol/l, respectively); S-(+)-alpha-methylhistamine (up to 10 mumol/l) as well as the selective H1 receptor agonist (2-(2-thiazolyl)ethylamine and the selective H2 receptor agonist dimaprit (each up to 10 mumol/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-(-)-alpha-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 omega-conotoxin GVIA (which by itself decreased the response to NMDA by about 55%), R-(-)-alpha-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.

We have investigated binding and functional effects of a new peptide YY analogue, [Pro34]peptide YY, at Y1 and Y2-like subtypes of receptors for peptide YY and neuropeptide Y. In binding studies [Pro34]peptide YY had a similarly high affinity as peptide YY to human Y1-like receptors in SK-N-MC cells, a human neuroblastoma cell line of presumed neurogenic origin, and HEL cells, a human cell line derived from a patient with Hodgkin's disease. In functional studies [Pro34]peptide YY stimulated Ca2+ elevations in both Y1-like receptor cell lines with similar potency and efficacy as peptide YY. In contrast to peptide YY [Pro34]peptide YY was 1000-fold less potent in binding to Y2-like receptors in porcine splenic membranes and lacked agonistic effects in another Y2-like receptor-mediated model system, i.e. inhibition of [3H]serotonin release from rat cerebral cortical slices. Thus, [Pro34]peptide YY is a highly Y1-selective full agonist of peptide YY/neuropeptide Y receptors. [Pro34]peptide YY could be useful for studying the importance of Y receptor subtypes in mediating peptide YY physiological actions.

Presynaptic H3 receptors occur on histaminergic neurones of the CNS (autoreceptors) and on non-histaminergic neurones of the central and autonomic nervous system (heteroreceptors). H3 heteroreceptors, most probably located on the postganglionic sympathetic nerve fibres innervating the resistance vessels and the heart, have been identified in the model of the pithed rat. Furthermore, we could show in superfusion experiments that H3 heteroreceptors also occur on the sympathetic neurones supplying the human saphenous vein and the vasculature of the pig retina and on the serotoninergic, dopaminergic and noradrenergic neurones in the brain of various mammalian species, including man. The effects of three recently described H3 receptor ligands were studied in superfused mouse brain cortex slices. The potency of the novel H3 receptor agonist imetit exceeded that of R-(-)-alpha-methylhistamine (the reference H3 receptor agonist) by one log unit and that of histamine by almost two log units. Clobenpropit was shown to be a competitive H3 receptor antagonist, exhibiting a pA2 as high as 9.6 (exceeding the pA2 of the reference H3 receptor antagonist thioperamide by one log unit). The irreversible antagonism of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was also studied. Interactions of the H3 heteroreceptor with the dopamine autoreceptor in mouse striatal slices and the alpha 2-autoreceptor in mouse brain cortex slices could be demonstrated. Activation of alpha 2-autoreceptors decreases the H3 receptor-mediated effect. Blockade of alpha 2-autoreceptors increases the H3 receptor-mediated effect only if the alpha 2-autoreceptors are simultaneously activated by endogenous noradrenaline. The H3 receptor-mediated inhibition of noradrenaline release in mouse brain cortex slices was attenuated by the K+ channel blocker tetraethylammonium but this attenuation was abolished by reduction of the Ca2+ concentration in the medium (to compensate for the facilitatory effect of tetraethylammonium on noradrenaline release). Accordingly, we assume that the H3 receptors are not coupled to voltage-sensitive K+ channels. Pertussis toxin and N-ethylmaleimide attenuated the H3 receptor-mediated effect in the mouse brain cortex, suggesting that the H3 receptors are coupled to a G protein (eg Gi or Go). However, negative coupling to an adenylate cyclase does not appear to exist since an H3 receptor-mediated inhibition of cAMP accumulation was not obtained in mouse brain cortex membranes. H3 receptor ligands are currently undergoing clinical testing and might become new remedies for the treatment of disease of the gastrointestinal and bronchial system and the CNS.

We analyzed the facilitatory effect of the 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide (mCPBG) on the electrically evoked noradrenaline release in superfused mouse brain tissue. In addition, we determined the affinities of mCPBG and two other 5-HT receptor ligands, namely 2-methyl-5-hydroxytryptamine (2-methyl-5-HT; also a 5-HT3 receptor agonist) and 5-carboxamidotryptamine (5-CT; a 5-HT1 receptor agonist) for alpha 2 binding sites. The latter two 5-HT receptor agonists were included because of the claimed involvement of alpha 2-adrenoceptors in their effects on noradrenaline release. In superfusion experiments on mouse brain cortex slices preincubated with 3H-noradrenaline, tritium overflow evoked by 2-min periods of electrical field stimulation (3 Hz) was facilitated by mCPBG and, in addition, by rauwolscine (alpha 2-adrenoceptor antagonist) and tetraethylammonium (K+ channel blocker) (which were examined for comparison). The effect of mCPBG was not affected by the 5-HT3 receptor antagonist tropisetron or by desipramine but was abolished by rauwolscine. In slices superfused with medium containing desipramine, the concentration-response curve of unlabelled noradrenaline for its inhibitory effect on the electrically (0.3 Hz) evoked overflow was shifted to the right by mCPBG and rauwolscine (apparent pA2 5.35 and 7.88, respectively). In another series of superfusion experiments, 4 electrical pulses, administered at 100 Hz, were used to evoke tritium overflow. Tritium overflow evoked by this stimulation procedure (under which an endogenous tone of noradrenaline does not develop) was not affected by mCPBG and rauwolscine but still increased by tetraethylammonium.(ABSTRACT TRUNCATED AT 250 WORDS)

We determined the affinities of nordimaprit, homodimaprit, clobenpropit and imetit for H3 binding sites (labelled by 3H-N alpha-methylhistamine) in rat brain cortex homogenates and their potencies at presynaptic H3A receptors on noradrenergic nerve endings in mouse brain cortex slices. 3H-N alpha-Methylhistamine bound saturably to rat brain cortex homogenates with a Kd of 0.70 nmol/l and a Bmax of 98 fmol/mg protein. Binding of 3H-N alpha-methylhistamine was displaced monophasically by dimaprit (pKi 6.55), nordimaprit (5.94), homodimaprit (6.44), clobenpropit (9.16), imetit (9.83), R-(-)-alpha-methylhistamine (8.87) and histamine (8.20), and biphasically by burimamide (pKi high 7.73, pKi low 5.97). In superfused mouse brain cortex slices preincubated with 3H-noradrenaline, the electrically (0.3 Hz) evoked tritium overflow was inhibited by imetit (pIC35 8.93), R-(-)-alpha-methylhistamine (7.87) and histamine (7.03). The effect of histamine was attenuated by nordimaprit, homodimaprit, clobenpropit and N-ethoxycarbonyl-2- ethoxy-1,2-dihydroquinoline (EEDQ); EEDQ (but not nordimaprit, homodimaprit and clobenpropit) attenuated the effect of histamine also in slices pre-exposed to the drug 60-30 min prior to superfusion. The concentration-response curve of histamine was shifted to the right by homodimaprit and clobenpropit; Schild plots yielded straight lines with a slope of unity for both drugs (pA2 5.94 and 9.55, respectively). Nordimaprit depressed the maximum effect of histamine (pD'2 5.55) and also slightly increased the concentration of histamine producing the half-maximum effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Slices from human neocortex preincubated with [3H]serotonin ([3H]5-HT) were superfused and stimulated electrically to investigate whether the alpha 2-adrenoceptors on serotonergic terminals can be stimulated by endogenous noradrenaline (NA) released from neighboring noradrenergic fibers. The stimulation-evoked 3H overflow, representing action potential-induced, exocytotic release of 5-HT, was depressed by the NA uptake blocker (+)-oxaprotiline. Rauwolscine (a mixed alpha 2-adrenoceptor antagonist/5-HT autoreceptor agonist) or phentolamine [a combined alpha-adrenoceptor/5-HT autoreceptor antagonist; the latter drug in the presence of (+)-oxaprotiline] enhanced the release when the 5-HT autoreceptors had previously been blocked by metitepine. Under hypothermia the release of 5-HT was found to be decreased and that of NA to be increased; under these conditions idazoxan (an alpha 2-adrenoceptor antagonist) enhanced the release of 5-HT. In neocortex slices from rats (+)-oxaprotiline similarly depressed the release of 5-HT (measured with the same methods) as in human tissue. When rats were pretreated with 6-hydroxydopamine, the inhibitory effect of exogenous NA on 5-HT release was increased, and in slices from rats pretreated with desipramine, it was decreased. In conclusion, alpha 2-heteroreceptors can be activated by endogenous NA released from neighboring noradrenergic fibers. Because regulatory processes analogous to those in rats probably occur in humans as well, an up- or down-regulation of alpha 2-heteroreceptors in depressed patients with a (pathological) decrease or a (therapeutic) enhancement of the noradrenergic neurotransmission may also be assumed to occur.

In pithed and vagotomized rats the effects of the H3 receptor agonist R-(-)-alpha-methylhistamine, the H1 receptor agonist 2-(2-thiazolyl)ethylamine and the H2 receptor agonist dimaprit on basal diastolic blood pressure, basal heart rate and the electrically induced rise in heart rate were examined. Basal diastolic blood pressure was not altered by low, but increased by high doses of R-(-)-alpha-methylhistamine; the latter effect was not affected by selective H1, H2 or H3 receptor antagonists and by prazosin, but was attenuated by rauwolscine. Rauwolscine also unmasked a vasodepressor response to R-(-)-alpha-methylhistamine not affected by the H3 receptor antagonist thioperamide, but counteracted by the H1 receptor antagonist dimetindene or the H2 receptor antagonist ranitidine. The vasodepressor responses to 2-(2-thiazolyl)ethylamine and dimaprit were antagonized by dimetindene and ranitidine, respectively. The vasodepressor response to 2-(2-thiazolyl)ethylamine was not altered by indomethacin, but reduced by an inhibitor of endothelial nitric oxide synthase, N omega-nitro-L-arginine methyl ester (which, by itself, markedly increased blood pressure). Both drug tools did not alter the effect of dimaprit. Basal heart rate was not affected by 2-(2-thiazolyl)ethylamine (examined after administration of propranolol), dimaprit and R-(-)-alpha-methylhistamine. The electrically induced increase in heart rate (studied in animals which had received rauwolscine) was decreased by R-(-)-alpha-methylhistamine but not affected by 2-(2-thiazolyl)ethylamine and dimaprit. The effect of R-(-)-alpha-methylhistamine was abolished by thioperamide. R-(-)-alpha-methylhistamine did not influence the increase in heart rate produced by isoprenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

In superfused mouse striatal slices preincubated with [3H]dopamine 25 nmol/l, the electrically (3 Hz) evoked tritium overflow was inhibited by histamine 10 mumol/l by 18%. The degree of inhibition was increased to 38% by haloperidol but not affected by (1) atropine, (2) reducing the stimulation frequency to 0.3 Hz or (3) increasing the concentration of [3H]dopamine (used for preincubation) to 100 nmol/l. The effect of histamine was mimicked by the H3 agonist R-(-)-alpha-methylhistamine; it was not affected by the H1 antagonist dimetindene and the H2 antagonist ranitidine but abolished by the H3 antagonist thioperamide. Tritium overflow evoked by Ca2+ ions (introduced into Ca(2+)-free, K(+)-rich medium containing tetrodotoxin) was not affected by histamine 10 mumol/l in the absence, but inhibited (by 30%) in the presence of haloperidol; the effect of histamine was abolished by thioperamide. In conclusion, the dopaminergic nerve terminals in the mouse striatum are endowed with presynaptic H3 receptors. Simultaneous blockade of dopamine autoreceptors increases the extent of the H3 receptor-mediated inhibition of dopamine release.

1. In segments of the rat vena cava preincubated with [3H]-noradrenaline and superfused with physiological salt solution (containing desipramine and corticosterone), we studied the effects of prostaglandins of the D, E and F series, of a prostacyclin analogue and a thromboxane-mimetic and of subtype-selective prostaglandin E-receptor (EP-receptor) ligands on the electrically (0.66 Hz)-evoked tritium overflow. 2. The electrically-evoked tritium overflow was inhibited by prostaglandin E2 (maximum inhibition by about 80%; pIC40 7.49). The effect of prostaglandin E2 was not affected by rauwolscine, which, by itself, increased the evoked overflow; the alpha 2-adrenoceptor antagonist was added to the superfusion medium in all subsequent experiments. Indomethacin failed to affect either the evoked tritium overflow or its inhibition by prostaglandin E2. 3. The inhibitory effect of prostaglandin E2 on the electrically-evoked tritium overflow was not altered by the EP1-receptor antagonist. AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) at a concentration at least 30 fold higher than its pA2 value at EP1-receptors. The following compounds mimicked the effect of prostaglandin E2 showing the following rank order of potencies: misoprostol (EP2-/EP3-receptor agonist) congruent to sulprostone (EP1-/EP3-receptor agonist) congruent to prostaglandin E1 = prostaglandin E2iloprost (EP1-/IP-receptor agonist) = prostaglandin F2 alpha. The evoked overflow was not affected by high concentrations of prostaglandin D2 or the thromboxane-mimetic U46619 (9,11-dideoxy-11 alpha, 9 alpha-epoxy-methano-prostaglandin F2 alpha). 4. The present results suggest that the postganglionic sympathetic nerve fibres innervating the rat vena cava are endowed with presynaptic EP3-receptors.They are not tonically activated by endogenously formed products of cyclo-oxygenase and do not interact with the presynaptic M2-adrenoceptors.

Mouse brain cortex slices preincubated with 3H-noradrenaline were superfused with physiological salt solution containing desipramine plus a drug with alpha 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-(-)-alpha-methylhistamine (7.47) and its S-(+)-enantiomer (5.82) but not influenced by the H1 receptor agonist 2-(2-thiazolyl)-ethylamine 3.2 mumol/l and the H2 receptor agonist dimaprit 10 mumol/l. The inhibitory effect of histamine was not affected by the H1 receptor antagonist dimetindene 1 mumol/l and the H2 receptor antagonist ranitidine 10 mumol/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 H3A and H3B binding sites in rat brain membranes (West et al. 1990 Mol Pharmacol 38:610); a significant correlation was obtained for the H3A, but not for the H3B sites. The results suggest that noradrenaline release in the mouse brain cortex is inhibited by histamine via H3A receptors and that dimaprit is an H3 receptor antagonist of moderate potency.

In pithed rats, we studied the effects of prostaglandin E2 and of subtype-selective prostaglandin E receptor (EP receptor) ligands on the rise in blood pressure induced by electrical stimulation of the preganglionic sympathetic nerves. Prostaglandin E2, the EP1/EP3 receptor agonist sulprostone and the EP2/EP3 receptor agonist misoprostol inhibited the electrically induced increase in diastolic blood pressure (rank order of potencies sulprostone ≥ misoprostol ≥ prostaglandin E2); the rise in blood pressure induced by exogenously added noradrenaline was not affected by these compounds. The inhibitory effect of sulprostone on the electrically induced vasopressor response was not significantly changed by indomethacin. Iloprost (an agonist at EP1 and prostacyclin receptors (IP receptors)) failed to affect the electrically evoked increase in blood pressure. The present study suggests that prostaglandin E2 inhibits the release of catecholamines in pithed rats via prostanoid receptors of the EP3 subtype, probably located presynaptically on the postganglionic sympathetic nerve fibres.

In rat brain cortex slices 3 H-noradrenaline release was induced by activation of NMDA receptors, which at least in part are located presynaptically. The NMDA-evoked 3 H-noradrenaline release was modulated by both presynaptic α 2 -autoreceptors and histamine H 3 heteroreceptors on the noradrenergic nerve terminals.

Presynaptic serotonin (5-HT) receptors on the postganglionic sympathetic nerves, which mediate inhibition of noradrenaline release in blood vessels of various species and which interact with the presynaptic α2-autoreceptors, are heterogeneous. In the rat vena cava, they are of the 5-HT1B subtype, in the pig coronary artery they belong to a novel, so far unknown class of 5-HT receptors, and in the human saphenous vein they could be classified as 5-HT1D. These results point to marked species differences and the need to carry out experiments in human vascular preparations. Presynaptic 5-HT receptors may be involved in the mechanism of action of the new antimigraine drug sumatriptan.

In rat brain cortex slices and synaptosomes, the depolarization induced 3 H-5-hydroxytryptamine ( 3 H-5-HT) release was inhibited by neuropeptide Y (NPY) and related peptides; presynaptic NPY (presumably Y 2 ) receptors are probably involved in this effect. Application of subtype-selective histamine receptor agonists and antagonists in rat brain cortex slices or synaptosomes and in pig retina discs revealed that the depolarization-induced 3 H-5-HT and 3 H-noradrenaline ( 3 H-NA) release are modulated by inhibitory histamine H 3 receptors. In rat brain cortex synaptosomes superfused in the presence of glycine, N-methyl-D-aspartate (NMDA) or L-glutamate induced Ca 2+ -dependent, Mg 2+ -sensitive 3 H-NA release, indicating that the NA nerve terminals are endowed with excitatory NMDA heteroreceptors; the NMDA-evoked 3 H-NA release in rat brain cortex slices can be modulated by presynaptic ∝ 2 -autoreceptors and histamine H 3 heteroreceptors.

Rat brain cortex slices or synaptosomes preincubated with 3H-serotonin were superfused with physiological salt solution (which, in the case of slices, contained citalopram, an inhibitor of serotonin uptake), and the effects of histamine and related drugs on the evoked tritium overflow were studied. The electrically (3 Hz) evoked tritium overflow from slices was inhibited by histamine and the H3 receptor agonists R-(-)-alpha-methylhistamine and N alpha-methylhistamine (pIC12.5 values: 6.41, 7.28 and 6.12, respectively), but not affected by the H1 receptor agonist 2-(2-thiazolyl)ethylamine and the H2 receptor agonist dimaprit (each at 10 mumol/l). The concentration-response curve for histamine was shifted to the right by the H3 receptor antagonists impromidine, burimamide and thioperamide (apparent pA2 values: 7.45, 5.97 and 7.88, respectively); the concentration-response curve of serotonin for its inhibitory effect on the electrically evoked overflow was not affected by the three drugs (apparent pA2 values: less than 5.5, less than 5.5 and less than 6.5). Given alone, impromidine, thioperamide and a low concentration of burimamide facilitated the electrically evoked overflow. In slices superfused with K(+)-rich, Ca2(+)-free solution containing tetrodotoxin throughout and in synaptosomes superfused with Ca2(+)-free solution, histamine inhibited the overflow evoked by introduction of Ca2+ (in synaptosomes, simultaneously with an increased amount of K+). In either tissue, the effect of histamine was counteracted by thioperamide. The results provide evidence that exogenous and probably also endogenous histamine inhibits serotonin release in the rat brain cortex via presynaptic histamine H3 receptors.

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 3H-dopamine 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-(-)-alpha-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 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Slices and synaptosomes from human cerebral cortex (which had to be removed to reach deeply located tumours) and, for comparison, synaptosomes from guinea-pig and rat cerebral cortex were preincubated with [3H]5-hydroxytryptamine and superfused with physiological salt solution containing an inhibitor of 5-hydroxytryptamine uptake. The effects of alpha-adrenoceptor agonists and antagonists on the electrically (slices) or potassium-evoked (synaptosomes) tritium overflow were studied. In human cerebral cortical slices, the electrically-evoked [3H] overflow was inhibited by noradrenaline (pIC25 value: 6.35); the non-selective alpha-adrenoceptor antagonist phentolamine, at a concentration of 0.32 mumol/l, strongly antagonized the inhibitory effect of noradrenaline (apparent pA2 value: 8.19) but did not affect the evoked overflow by itself. In synaptosomes from humans, guinea-pigs and rats, noradrenaline also inhibited the K(+)-evoked [3H] overflow in a concentration dependent manner; the alpha 2-adrenoceptor clonidine (1 mumol/l), but not the alpha 1-adrenoceptor agonist methoxamine (1 mumol/l), mimicked the effects of noradrenaline; the effect of noradrenaline (0.3 mumol/l) was abolished by the alpha 2-adrenoceptor antagonist idazoxan (0.5 mumol/l), but not by the alpha 1-adrenoceptor antagonist prazosin (1 mumol/l). It is concluded that release-inhibiting adrenoceptors of the alpha 2-subtype exist on 5-hydroxytryptamine terminals innervating the cerebral cortex in human and guinea-pig brain.

The alpha-adrenoceptor-mediated effects of the newly developed antihypertensive drug moxonidine and of clonidine were compared in four in vitro models and in anesthetized rabbits. Inhibition of the electrically induced twitch response in the isolated rat vas deferens by moxonidine and clonidine (pEC50 values 7.8 and 8.5) was counteracted by yohimbine. The maximum degree of inhibition obtained with both drugs was identical. The contractile response of the rat anococcygeal muscle to moxonidine and clonidine (pEC50 6.2 and 6.9) was antagonized by prazosin. The maximum degree of contraction obtained with moxonidine was similar in degree to that of phenylephrine, whereas that of clonidine was lower. Moxonidine and clonidine inhibited the electrically evoked [3H]norepinephrine (NE) overflow in strips of rabbit pulmonary artery (pEC50 7.1 and 6.6) and in rat brain cortex slices (pEC50 6.9 and 7.7); the effect of moxonidine was counteracted by rauwolscine. After intracisternal injection in anesthetized rabbits, moxonidine lowered blood pressure (BP) more potently than did clonidine. Intracisternal pretreatment of rabbits with 6-hydroxydopamine strongly attenuated the hypotensive effect of moxonidine without affecting that of clonidine. The hypotensive effect of moxonidine was abolished by pretreatment with reserpine and alpha-methyl-p-tyrosine (alpha-MPT). In conclusion, in isolated tissues of the rat, moxonidine is as selective as clonidine as an alpha-adrenoceptor agonist. The hypotensive effect of moxonidine, unlike that of clonidine, appears to involve central presynaptic alpha 2-autoreceptors in large part.

Rat brain cortex slices preincubated with 3H-noradrenaline were superfused with Krebs-Henseleit solution with or without Mg2+. In the absence of Mg2+ ions, NMDA evoked 3H-noradrenaline overflow above basal efflux; this effect was concentration-dependently inhibited by Mg2+ (IC50: 19 mumol/l). Despite the presence of 1.2 mmol/l Mg2+, which is known to block cation influx through the ion channel coupled to the NMDA receptor, NMDA evoked 3H-noradrenaline release if the membrane was permanently kept depolarized by 20 or 25 mmol/l K+, 1 mumol/l veratridine or 200 mumol/1 3,4-diaminopyridine; the stimulant effect of NMDA was counteracted by 2-amino-5-phosphonovaleric acid (2-APV), a competitive antagonist at the NMDA receptor and by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK 801), an antagonist acting at the cation channel associated with the NMDA receptor. In contrast, no stimulatory effect of NMDA in the presence of 1.2 mmol/l Mg2+ was observed when the membrane of the nerve terminals was intermittently depolarized by electrical impulses of 2 ms duration at a frequency of 1-3 Hz. It is concluded that continuous depolarization of the nerve membrane counteracts the blocking effect of Mg2+ on cation influx through the NMDA receptor-associated ion channel. Under this condition, noradrenaline release can be stimulated by NMDA receptor activation even in the presence of physiological Mg2+ concentrations.

The effects of histamine, thioperamide and EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) at the noradrenaline release-modulating H3 receptor in the mouse brain were examined. In superfused mouse brain cortex slices preincubated with 3H-noradrenaline, the inhibitory effect of histamine on the electrically (0.3 Hz) evoked tritium overflow was virtually identical when the time of exposure was 30, 80 or 130 min; after withdrawal of histamine, the evoked overflow recovered within 80 min. The attenuation of the effect of histamine by thioperamide was reversible within 50 min after withdrawal of the antagonist, whereas the attenuation produced by EEDQ remained constant for at least 80 min. In conclusion, the effects of histamine and thioperamide at the H3 receptor are readily reversible, whereas EEDQ appears to be an irreversible antagonist; desensitization of the H3 receptor does not occur.

Rat brain cortex slices preincubated with 3H-serotonin were superfused with physiological salt solution containing the serotonin uptake blocker DU 24565 (6-nitroquipazine). The effects of (+/-)-cyanopindolol and its enantiomers, of ICI 118,551 (erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobut an-2-ol) and of isoprenaline on the electrically (3 Hz) evoked 3H overflow were studied. (+/-)-Cyanopindolol increased the evoked 3H overflow; this effect was prevented by preexposure to the previously characterized serotonin receptor antagonist metitepin. The concentration-response curve of unlabelled serotonin for its inhibitory effect on the electrically evoked 3H overflow was shifted to the right by (+/-)-cyanopindolol (apparent pA2 value: 8.29), whereas that of noradrenaline (determined in the absence of DU 24565) was not affected (apparent pA2 value: less than 6.0). The concentration-response curve of serotonin was also shifted to the right by (-)-cyanopindolol (apparent pA2 value: 8.30) and (+)-cyanopindolol (6.83) but not by ICI 118,551 (less than 5.5). Isoprenaline (up to 10 mumol/l; examined in the absence of DU 24565) did not influence the electrically evoked 3H overflow. The present results show that the presynaptic serotonin autoreceptor is blocked by cyanopindolol in a stereoselective way. This drug is 20 times more potent than metitepin as an antagonist at the presynaptic serotonin autoreceptor, and, in contrast to the latter, it does not act as an antagonist at the presynaptic alpha 2-adrenoceptor on the serotoninergic neurone.