M. Göthert

Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HT1B binding sites

Summary1.In rat brain cortex slices preincubated with [3H]5-HT, the potencies of 17 5-HT receptor agonists to inhibit the electrically evoked3H overflow and the affinities of 13 antagonists (including several β-adrenoceptor blocking agents) to antagonize competitively the inhibitory effect of unlabelled 5-HT on evoked3H overflow were determined.2.The affinities of the compounds for 5-HT1B and 5-HT2 binding sites in rat brain cortex membranes (labelled by [125I]cyanopindolol = [125I]-CYP in the presence of 30 μmol/l isoprenaline and [3H]ketanserin, respectively), for 5-HT1A binding sites in pig and rat brain cortex membranes (labelled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin = [3H]8-OH-DPAT) and for 5-HT1C binding sites in pig choroid plexus membranes (labelled by [3H]mesulergine) were also determined. The affinities of the drugs for the various 5-HT recognition sites ranged over 4–5 log units (the functional experiments revealed the same range of differences between the drugs).3.There were no significant correlations between the affinities of the drugs at 5-HT1C and 5-HT2 binding sites and their potencies or affinities, determined for the 5-HT autoreceptors. In contrast, significant correlations were found between the potencies or affinities of the drugs for the autoreceptors and their affinities at 5-HT1A or 5-HT1B binding sites; the best correlations were obtained with the 5-HT1B binding site.4.Some of the drugs investigated were not included in the correlation since their agonistic or antagonistic effects on the autoreceptors were weak and pEC30 or apparent pA2 values could not be determined (<5.5). Among these drugs, 8-OH-DPAT, TVX Q 7821 (2-(4-(4-(2-pyrimidin-yl)-1-piperazinyl)-butyl)-1,2-benzisothiazol-3(2H)one-1,1-dioxide) and spiperone showed a very low affinity for 5-HT1B binding sites (pKD<5.3), but a high affinity for 5-HT1A binding sites (pKD>7.2).5.In conclusion, the evidence indicates that the presynaptic 5-HT autoreceptor belongs to the 5-HT1B receptor subtype.

Inhibition of neuronal Ca2+ influx by gabapentin and pregabalin in the human neocortex

Gabapentin and pregabalin (S-(+)-3-isobutylgaba) produced concentration-dependent inhibitions of the K(+)-induced [Ca(2+)](i) increase in fura-2-loaded human neocortical synaptosomes (IC(50)=17 microM for both compounds; respective maximal inhibitions of 37 and 35%). The weaker enantiomer of pregabalin, R-(-)-3-isobutylgaba, was inactive. These findings were consistent with the potency of these drugs to inhibit [(3)H]-gabapentin binding to human neocortical membranes. The inhibitory effect of gabapentin on the K(+)-induced [Ca(2+)](i) increase was prevented by the P/Q-type voltage-gated Ca(2+) channel blocker omega-agatoxin IVA. The alpha 2 delta-1, alpha 2 delta-2, and alpha 2 delta-3 subunits of voltage-gated Ca(2+) channels, presumed sites of gabapentin and pregabalin action, were detected with immunoblots of human neocortical synaptosomes. The K(+)-evoked release of [(3)H]-noradrenaline from human neocortical slices was inhibited by gabapentin (maximal inhibition of 31%); this effect was prevented by the AMPA receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide). Gabapentin and pregabalin may bind to the Ca(2+) channel alpha 2 delta subunit to selectively attenuate depolarization-induced Ca(2+) influx of presynaptic P/Q-type Ca(2+) channels; this results in decreased glutamate/aspartate release from excitatory amino acid nerve terminals leading to a reduced activation of AMPA heteroreceptors on noradrenergic nerve terminals.

Histamine inhibits dopamine release in the mouse striatum via presynaptic H3 receptors

In superfused mouse striatal slices preincubated with [3H] dopamine 25 nmol/l, the electrically (3 Hz) evoked tritium overflow was inhibited by histamine 10 μmol/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-(−)-α-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 Ca2+free, K+-rich medium containing tetrodotoxin) was not affected by histamine 10 μmol/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.

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

SummaryRat 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.

Modulation of neurotransmitter release via histamine H3 heteroreceptors

Summary— 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‐(‐)‐α‐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 α2‐autoreceptor in mouse brain cortex slices could be demonstrated. Activation of α2‐autoreceptors decreases the H3 receptor‐mediated effect. Blockade of α2‐autoreceptors increases the H3 receptor‐mediated effect only if the α2‐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 diseases of the gastrointestinal and bronchial system and the CNS.

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

SummaryThe effects of histamine and related drugs on the evoked tritium overflow from superfused rat brain cortex slices preincubated with3H-noradrenaline were determined. Tritium overflow was stimulated electrically (3 Hz; slices superfused with normal physiological salt solution) or by introduction of CaCl2 1.3 mmol/l (slices superfused with Ca2+-free medium containing K+ 20 mmol/l).Histamine slightly decreased the electrically evokedH overflow in slices superfused in the presence of desipramine. The degree of inhibition obtained with histamine was doubled when both desipramine and phentolamine were present in the superfusion medium (pIC15 6.46). Under the latter condition, the evoked overflow was inhibited by the H3 receptor agonist R-(−)-α-methylhistamine and its S-(+) enantiomer (pIC15 7.36 and 5.09, respectively), but was not affected by the H2 receptor agonist dimaprit and the H1 receptoragonist 2-thiazolylethylamine (both at up to 32 µmol/l). The concentration-response curve of histamine was shifted to the right by the H3 receptor antagonists thioperamide, impromidine and burimamide (apparent pA2 8.37, 6.86 and 7.05, respectively), by the H2 receptor antagonist ranitidine (apparent pA2 4.27) and was not affected by the H1 receptor antagonist dimetindene (32 µmol/l). The inhibitory effect of R-(−)-α-methylhistamine on the evoked overflow was also counteracted by thioperamide. Given alone, none of the five histamine receptor antagonists affected the evoked overflow. In the absence of desipramine plus phentolamine, impromidine and burimamide facilitated the electrically evoked3H overflow whereas thioperamide had no effect. The facilitatory effects of impromidine and burimamide were abolished by phentolamine, but not affected by desipramine. The concentration-response curve of noradrenaline for its inhibitory effect on the evoked overflow was shifted to the right by impromidine and burimamide, but not influenced by thioperamide (apparent pA2 5.24, 5.04 and <6.5, respectively; experiments carried out in the presence of desipramine). In slices superfused with Ca2+-free K+-rich medium containing tetrodotoxin, desipramine plus phentolamine, the tritium overflow evoked by introduction of Ca2+ was inhibited by histamine; the concentration-response curve of histamine was shifted to the right by thioperamide.The present study shows that the inhibitory effect of histamine on noradrenaline release in the rat brain cortex involves presynaptic H3 receptors and that the degree of inhibition is increased in the presence of phentolamine. The H3 receptor antagonists impromidine and burimamide are weak α2-adrenoceptor antagonists.

Direct inhibition by cannabinoids of human 5‐HT3A receptors: probable involvement of an allosteric modulatory site

Excised outside‐out patches from HEK293 cells stably transfected with the human (h) 5‐HT3A receptor cDNA were used to determine the effects of cannabinoid receptor ligands on the 5‐HT‐induced current using the patch clamp technique. In addition, binding studies with radioligands for 5‐HT3 as well as for cannabinoid CB1 and CB2 receptors were carried out. The 5‐HT‐induced current was inhibited by the following cannabinoid receptor agonists (at decreasing order of potency): Δ9‐THC, WIN55,212‐2, anandamide, JWH‐015 and CP55940. The WIN55,212‐2‐induced inhibition was not altered by SR141716A, a CB1 receptor antagonist. WIN55,212‐3, an enantiomer of WIN55,212‐2, did not affect the 5‐HT‐induced current. WIN55,212‐2 did not change the EC50 value of 5‐HT in stimulating current, but reduced the maximum effect. The CB1 receptor ligand [3H]‐SR141716A and the CB1/CB2 receptor ligand [3H]‐CP55940 did not specifically bind to parental HEK293 cells. In competition experiments on membranes of HEK293 cells transfected with the h5‐HT3A receptor cDNA, WIN55,212‐2, CP55940, anandamide and SR141716A did not affect [3H]‐GR65630 binding, but 5‐HT caused a concentration dependent‐inhibition. In conclusion, cannabinoids stereoselectively inhibit currents through recombinant h5‐HT3A receptors independently of cannabinoid receptors. Probably the cannabinoids act allosterically at a modulatory site of the h5‐HT3A receptor. Thus the functional state of the receptor can be controlled by the endogenous ligand anandamide. This site is a potential target for new analgesic and antiemetic drugs.

Serotonin-receptor-mediated modulation of Ca2+-dependent 5-hydroxytryptamine release from neurones of the rat brain cortex

SummaryRat brain cortex slices were incubated with 3H-5-hydroxytryptamine and subsequently superfused. Tritium overflow from slices superfused with physiological salt solution was stimulated by rectangular electrical pulses (normal frequency 3 Hz), and overflow from slices superfused with Ca2+-free solution containing 25 mM K+ was stimulated by introduction of (usually) 1.3 mM CaCl2. In most of the experiments the neuronal uptake of 5-hydroxytryptamine (5-HT) was blocked by paroxetine.1.Electrically evoked 3H overflow was abolished by tetrodotoxin or omission of Ca2+ from the superfusion fluid, whereas Ca2+-evoked overflow was not affected by tetrodotoxin, but inhibited by Mg2+ ions.2.Both electrically and Ca2+-evoked 3H overflow were reduced by unlabelled 5-HT and increased by methiothepin; the potencies of the drugs in modifying evoked overflow were similar with both methods of stimulation. Methiothepin caused a substantial shift to the right of the concentration-response curve of unlabelled 5-HT for its inhibitory effect. By contrast, metergoline produced only a very slight shift of this curve.3.The inhibitory effect of unlabelled 5-HT on electrically evoked 3H overflow increased with decreasing Ca2+ concentration (1.3–0.65 mM; 3 Hz) or with decreasing frequency of stimulation (10–0.3 Hz; 1.3 mM Ca2+).4.When 3H overflow was stimulated by introduction of various Ca2+ concentrations (0.65–4.55 mM) into the Ca2+-free, K+-rich solution, the data obtained yielded a straight line in the double reciprocal plot (Km=2.5 mM Ca2+). Straight lines were also obtained, when the effects of unlabelled 5-HT and methiothepin on 3H overflow evoked by different Ca2+ concentrations were analyzed; these lines and that calculated for the controls had a common intercept on the ordinate, whereas the Km values were considerably different from each other (in the presence of unlabelled 5-HT and methiothepin, 5.4 and 1.2 mM Ca2+, respectively). It is concluded that the modification of 3H-5-HT release (caused by interaction of methiothepin and 5-HT with presynaptic 5-HT autoreceptors on serotoninergic neurones) is mediated by decreasing the availability of Ca2+ ions for stimulus-release coupling, probably by decreasing the affinity of the voltagesensitive permeability channel of the cell membrane for Ca2+ ions.

SB-216641 AND BRL-15572 - COMPOUNDS TO PHARMACOLOGICALLY DISCRIMINATE H5-HT1B AND H5-HT1D RECEPTORS

Abstract Despite only modest homology between h5-HT1B and h5-HT1D receptor amino acid sequences, these receptors display a remarkably similar pharmacology. To date there are few compounds which discriminate between these receptor subtypes and those with some degree of selectivity, such as ketanserin, have greater affinity for other 5-HT receptor subtypes. We now report on two compounds, SB-216641 (N-[3-(2-dimethylamino) ethoxy-4-methoxyphenyl]-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-yl)-(1,1’-biphenyl)-4-carboxamide) and BRL-15572 3-[4-(3-chlorophenyl) piperazin-1-yl]-1,1-diphenyl-2-propanol), which display high affinity and selectivity for h5-HT1B and h5-HT1D receptors, respectively. In receptor binding studies on human receptors expressed in CHO cells, SB-216641 has high affinity (pKi=9.0) for h5-HT1B receptors and has 25-fold lower affinity at h5-HT1D receptors. In contrast, BRL-15572 has 60-fold higher affinity for h5-HT1D (pKi=7.9) than 5-HT1B receptors. Similar affinities for these compounds were determined on native tissue 5-HT1B receptors in guinea-pig striatum. Functional activities of SB-216641 and BRL-15572 were measured in a [35S]GTPγS binding assay and in a cAMP accumulation assay on recombinant h5-HT1B and h5-HT1D receptors. Both compounds were partial agonists in these high receptor expression systems, with potencies and selectivities which correlated with their receptor binding affinities. In the cAMP accumulation assay, results from pKB measurements on the compounds again correlated with receptor binding affinities (SB-216641, pKB=9.3 and 7.3; BRL-15572, pKB=<6 and 7.1, for h5-HT1B and h5-HT1D receptors respectively). These compounds will be useful pharmacological agents to characterise 5-HT1B and 5-HT1D receptor mediated responses.

Inhibition of neuronal Ca2+ influx by gabapentin and subsequent reduction of neurotransmitter release from rat neocortical slices

Cytosolic calcium ion concentrations ([Ca2+]i) were measured in rat neocortical synaptosomes using fura‐2, and depolarization of synaptosomal membranes was induced by K+ (30 mM). The release of the endogenous excitatory amino acids glutamate and aspartate was evoked by K+ (50 mM) and determined by HPLC. The release of [3H]‐noradrenaline from rat neocortical synaptosomes or slices was evoked by K+ (15 and 25 mM) and measured by liquid scintillation counting. Gabapentin produced a concentration‐dependent inhibition of the K+‐induced [Ca2+]i increase in synaptosomes (IC50=14 μM; maximal inhibition by 36%). The inhibitory effect of gabapentin was abolished in the presence of the P/Q‐type Ca2+ channel blocker ω‐agatoxin IVA, but not by the N‐type Ca2+ channel antagonist ω‐conotoxin GVIA. Gabapentin (100 μM) decreased the K+‐evoked release of endogenous aspartate and glutamate in neocortical slices by 16 and 18%, respectively. Gabapentin reduced the K+‐evoked [3H]‐noradrenaline release in neocortical slices (IC50=48 μM; maximal inhibition of 46%) but not from synaptosomes. In the presence of the AMPA receptor antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 2,3‐dioxo‐6‐nitro‐1,2,3,4‐tetrahydro[f]quinoxaline‐7‐sulphonamide (NBQX), gabapentin did not reduce [3H]‐noradrenaline release. Gabapentin did, however, cause inhibition in the presence of the NMDA receptor antagonist DL‐(E)‐2‐amino‐4‐methyl‐5‐phosphono‐3‐pentanoic acid (CGP 37849). Gabapentin is concluded to reduce the depolarization‐induced [Ca2+]i increase in excitatory amino acid nerve terminals by inhibiting P/Q‐type Ca2+ channels; this decreased Ca2+ influx subsequently attenuates K+‐evoked excitatory amino acid release. The latter effect leads to a reduced activation of AMPA receptors which contribute to K+‐evoked noradrenaline release from noradrenergic varicosities, resulting in an indirect inhibition of noradrenaline release.