N-Type calcium channels control sympathetic neurotransmission in human heart atrium.

BACKGROUND: Because knowledge about the type of calcium channels involved in action potential-induced norepinephrine release from the human peripheral sympathetic nervous system is sparse, we investigated which types of calcium channels are functionally important in the sympathetic nerves of human cardiac tissue. METHODS AND RESULTS: In superfused segments of human right atrial appendages, the type of calcium channels that control [(3)H]norepinephrine release evoked by transmural electrical stimulation was determined. [(3)H]norepinephrine release was almost abolished by 0.2 micromol/L omega-conotoxin GVIA (a selective blocker of N-type channels) but was not modified by 0.1 micromol/L omega-agatoxin IVA (a selective blocker of P- and Q-type channels). Mibefradil (a T-type and N-type calcium channel blocker) at concentrations of 0.3 to 3 micromol/L reduced the evoked tritium overflow in a frequency- and calcium-dependent manner, whereas 0.1 to 10 micromol/L amlodipine, diltiazem, and verapamil (selective blockers of L-type channels) were ineffective. CONCLUSIONS: Norepinephrine release from cardiac sympathetic nerves is triggered by Ca(2+) influx via N-type but not L- and P/Q-type calcium channels. The inhibitory effect of mibefradil on norepinephrine release at clinically relevant concentrations is probably due to its blocking action on N-type Ca(2+) channels. This property of mibefradil is unique among the calcium channel blockers that have been or still are therapeutically applied and may considerably contribute to its slight negative chronotropic effect in vivo.

Cannabinoid CB1 receptor-mediated inhibition of noradrenaline release in guinea-pig vessels, but not in rat and mouse aorta.

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

Pharmacological properties of the naturally occurring Phe-124-Cys variant of the human 5-HT1B receptor: changes in ligand binding, G-protein coupling and second messenger formation.

The aim of this study was to analyse whether substitution of phenylalanine in position 124 of the human (h) 5-HT1B receptor by cysteine, a naturally occurring variant of this receptor, modifies not only ligand binding, but also G-protein coupling and second messenger formation. Stably transfected rat C6 glioma cells, which express either the h5-HT1B variant receptor (VR) or the wild-type receptor (WTR) were used. In saturation experiments with [3H]5-carboxamidotryptamine ([3H]5-CT), the maximum binding (Bmax) of the VR amounted to only 60% of that to WTR. In competition experiments with 1 nM [3H]5-CT, the following 5-HT receptor ligands exhibited a higher affinity for the mutant receptor than for the WTR: L-694,247, 5-CT, 5-HT, sumatriptan (agonists listed at decreasing order of potency) and SB-224289 (a selective h5-HT1B receptor inverse agonist with competitive antagonistic properties). In contrast, the mixed 5-HT1B/1D receptor antagonist GR-127935 exhibited equal affinity for both isoforms. The efficacy of L-694,247, 5-CT, 5-HT and sumatriptan in stimulating [35S]GTPgammaS binding (a measure of G protein coupling) to membranes of cells expressing the VR was approximately 50-65% lower compared to membranes of cells expressing the WTR, but their potency was 2.8-3.6-fold higher. SB-224289, which decreased [35S]GTPgammaS binding when given alone, but not GR-127935, was more potent in antagonizing the stimulatory effect of 5-CT on [35S]GTPgammaS binding to membranes expressing the VR compared to membranes expressing the WTR. In whole cells expressing the VR, 5-CT and sumatriptan inhibited the forskolin-stimulated cAMP accumulation 3.2-fold more potently than in cells expressing the WTR. In conclusion, our data suggest that the Phe-124-Cys mutation modifies the pharmacological properties of the h5-HT1B receptor and may account for pharmacogenetic differences in the action of h5-HT1B receptor ligands. Thus, the sumatriptan-induced vasospasm which occurs at low incidence as a side-effect in migraine therapy may be related to the expression of the (124-Cys)h5-HT1B receptor in patients with additional pathogenetic factors such as coronary heart disease.

Modified ligand binding to the naturally occurring Cys-124 variant of the human serotonin 5-HT1B receptor.

A substitution of phenylalanine by cysteine in position 124 is the only known naturally occurring variant of the human 5-HT1B (h5-HT1B) receptor. The present study was designed to evaluate the potential influence of the Cys-124 variant on pharmacological properties of the receptor and to test for an involvement of the mutation in the genetic predisposition to schizophrenia or bipolar affective disorder. Binding of [3H]5-carboxamidotryptamine ([3H]5-CT) and its competition with serotonin receptor agonists and antagonists were determined in COS-7 cells transfected with the wild-type or the variant h5-HT1B receptor cDNA. In saturation experiments with [3H]5-CT, the maximum binding (Bmax) of the variant receptor was approximately 30% of the wild-type receptor. In competition experiments with 1 nM [3H]5-CT, the following serotonin receptor ligands exhibited a two to three times higher affinity for the mutant than for the wild-type receptor: dihydroergotamine, L-694,247, SB-216641, 5-CT, 5-HT, sumatriptan, RU24969 and methysergide (compounds listed at decreasing order of potency at the wild-type receptor). In contrast, the serotonin receptor antagonist ketanserin exhibited higher binding affinity for the wild-type than for the mutant h5-HT1B receptor and GR127939, (-)propranolol and BRL-15572 showed equal affinity for both types of receptor. Mutation screening of schizophrenic and bipolar patients revealed no relationship between the variant receptor and development of disease. In conclusion, our data suggest that the Cys-124 variant significantly affects the pharmacological properties of the h5-HT1B receptor. Carriers of the variant may exhibit differences in response to drugs acting on the h5-HT1B receptor or may develop side-effects to such agents.

Investigation into the age-dependence of release of serotonin and noradrenaline in the rat brain cortex and of autoreceptor-mediated modulation of release.

Slices of cerebral cortex from young adult and aged rats, preincubated with [3H]serotonin or [3H]noradrenaline were superfused and the electrically (3 Hz) evoked overflow of tritium and its modulation by the respective agonists and antagonists were studied. The electrically evoked overflow in slices preincubated with [3H]serotonin from 2.5-year-old rats was smaller (by about 35%) than in slices from 3-month-old animals. No difference was found for the inhibition by serotonin and the facilitation by metitepine of the evoked overflow and for the metitepine-induced shift of the concentration-response curve for serotonin to the right. There was no difference between slices preincubated with [3H]noradrenaline from 3 months, 1, 2 and 2.5-year-old rats, with respect to the electrically evoked overflow, its inhibition and facilitation by noradrenaline and phentolamine, respectively, and the phentolamine-induced shift of the concentration-response curve for noradrenaline to the right. The present results suggest that the release of serotonin is reduced in the cerebral cortex of aged rats whereas the release of noradrenaline and the autoreceptor-mediated modulation of the release of either monoamine is not altered.

Exon-intron organization of the human 5-HT3A receptor gene.

The gene structure of the human 5-HT3A receptor gene was analyzed by exon to exon polymerase chain reaction and subsequent sequencing. The results were confirmed by restriction analysis and genomic Southern blotting. The coding region of the human gene was found to be split by eight introns at identical positions as in the murine 5-HT3A receptor gene. All exon-intron boundaries exhibited fully conserved splice donor and acceptor consensus sequences. The alternative splice acceptor in intron eight of the murine gene was not found in the human counterpart. The length of particular introns differs markedly from the murine gene. With the exception of intron 5, all human introns are longer than their murine counterparts. From the start to the stop codon the human gene stretches over about 14.5 kb. The human exon sequences confirm one of three published human 5-HT3A receptor cDNA sequences. Knowledge of the gene structure, including 1.9 kb of the 5' noncoding region, all introns and the exon-intron boundaries of the human 5-HT3A receptor gene should facilitate investigation of its potential role in psychiatric disorders.

5-HT3 receptors in outside-out patches of N1E-115 neuroblastoma cells: basic properties and effects of pentobarbital.

A fast solution exchange system (Dilger and Brett, 1990; Biophysics Journal 57: 723-731) with an exchange rate < 1 msec was used to study 5-HT3 (5-HT; 5-hydroxytryptamine) receptor-mediated currents in superfused outside-out patches of N1E-115 mouse neuroblastoma cells. At negative membrane potentials, 5-HT induced inward currents in a concentration-dependent manner (IC50 = 3.8 microM, Hill coefficient = 1.8). The mean peak current at a near-maximally effective 5-HT concentration of 30 microM was 20.6 pA. The 5-HT3 receptor antagonist ondansetron (0.3 nM) reversibly inhibited the 5-HT (30 microM) signal by approximately 50%. The currents induced during application of 30 microM 5-HT for 2 sec were characterized by inward rectification, a monophasic onset (tau ON = 37.5 msec) and, after reaching a peak, a monophasic decay (desensitization; tau OFF = 391 msec). Onset and decay were slower at lower 5-HT concentrations. The recovery of fully desensitized patches required a washout period of 45 sec. Pentobarbital inhibited 5-HT-induced (30 microM) currents in a concentration-dependent manner. The maximally obtainable inhibition with a given pentobarbital concentration was reached already when it was exclusively coapplied with 5-HT (IC50 = 135 microM. Hill coefficient = -0.7), since additional preexposure for at least 45 sec did not alter the concentration-response curve of pentobarbital. In conclusion, outside-out patches of N1E-115 cells are suitable to study the kinetic properties of 5-HT3 receptor channels. The results obtained in this model with pentobarbital are compatible with the suggestion that the inhibitory action of pentobarbital on 5-HT3 receptors is dependent on the agonist-activated (open) channel.

Inhibition of 5-HT3 receptors by propofol: equilibrium and kinetic measurements.

Patch-clamp/rapid solution exchange experiments as well as tracer ([14C]-guanidinium) influx measurements were applied to investigate effects of propofol on 5-HT3 receptor channels and compare the results with those obtained with pentobarbital. Currents induced by 30 microM 5-HT were recorded in outside-out patches from N1E-115 cells. Application of propofol 45 s before and during 5-HT application inhibited peak-currents and integrated current responses in a concentration-dependent manner (IC50 values=14.5 and 10.5 microM; Hill coefficients -1.5 and -1.3, respectively). The inhibitory effect of propofol in the current measurements was similar to the propofol-induced inhibition in tracer influx experiments in whole N1E-115 cells (Barann et al., 1993. Naunyn-Schmiedeberg's Archives of Pharmacology 347, 125-132). Pentobarbital-induced inhibition of 5-HT3 receptors in both patch-clamp (Barann et al., 1997. Neuropharmacology 36, 655-664) and tracer influx measurements indicated a lower potency and lower slope (IC50 values=130 and 55 microM; Hill coefficients -0.8 and -0.7, respectively) compared to propofol. Propofol, in contrast to pentobarbital, showed nearly the full potency when applied to the patches exclusively 45 s before 5-HT. Propofol was least effective when administered exclusively during 5-HT. The onset of inhibition of 5-HT-induced peak currents by propofol had a time constant of 220 ms, similar to the kinetics of 5-HT-induced desensitization.

Facilitatory effect of adrenocorticotropic hormone and related peptides on Ca2+-dependent noradrenaline release from sympathetic nerves.

Strips of rabbit pulmonary artery and aorta were incubated with [3H]noradrenaline and subsequently superfused. Tritium overflow from strips superfused with physiological salt solution was stimulated either electrically (usually at a frequency of 2 Hz) or by tyramine 1 mumol/l and overflow from strips superfused with Ca2+-free solution containing K+ 54.7 mmol/l was stimulated by introduction of Ca2+ 1.6 mmol/l. In most of the experiments (stimulation by electrical impulses or CaCl2) neuronal and extraneuronal uptake and beta-adrenoceptors were blocked by cocaine, corticosterone and propranolol, respectively. The electrically evoked overflow of 3H-labelled substances from pulmonary artery and aorta was increased by adrenocorticotropic hormone. In the pulmonary artery, the adrenocorticotropic hormone--induced increase in impulse--evoked overflow (and contraction) was the more pronounced, the lower the frequency of stimulation (6, 2 and 0.66 Hz: 360 impulses). The electrically evoked overflow of 3H-labelled substances was also increased by alpha-melanocyte-stimulating hormone and porcine adrenocorticotropic hormone, but was not affected by adrenocorticotropic hormone. Adrenocorticotropic hormone also facilitated the Ca2+-evoked overflow of 3H-labelled substances promoted by high K+, but it did not affect the Ca2+-independent tyramine-evoked overflow. Adrenocorticotropic hormone did not alter the percentages of [3H]noradrenaline and 3H-labelled metabolites contained in electrically or tyramine-evoked overflow of 3H-labelled substances. In conclusion, adrenocorticotropic hormone and fragments of adrenocorticotropic hormone cause an increase in stimulation-evoked, Ca2+-dependent noradrenaline release from postganglionic sympathetic nerve fibres, probably by activating presynaptic receptors for adrenocorticotropic hormone.

Identification of presynaptic beta 2-adrenoceptors on the sympathetic nerve fibres of the human pulmonary artery.

Strips of human pulmonary arteries from patients undergoing surgery for lung tumour were incubated with [3H]-noradrenaline. Subsequently, they were superfused with physiological salt solution containing cocaine and corticosterone. Tritium overflow from the strips was stimulated by transmural electrical impulses (2 Hz). The electrically evoked overflow of tritium consisted of 91% unmetabolized [3H]-noradrenaline, and this percentage was not altered by isoprenaline. Adrenaline (in the presence of rauwolscine), isoprenaline and the preferential beta 2-adrenoceptor agonist, procaterol, concentration-dependently increased the electrically evoked tritium overflow. Prenalterol, a beta-adrenoceptor agonist with moderate preference for beta 1-adrenoceptors, was considerably less active than the previously mentioned agonists; noradrenaline (in the presence of rauwolscine) was ineffective. The concentration-response curve of procaterol was shifted to the right by the preferential beta 2-adrenoceptor antagonist ICI 118-551 but was not affected by the beta 1-selective antagonist, atenolol. Propranolol, but not atenolol, produced a shift to the right of the concentration-response curve of isoprenaline. It is concluded that the sympathetic nerve fibres of the human pulmonary artery are endowed with facilitatory presynaptic beta 2-adrenoceptors.

Stimulation of noradrenaline release in human cerebral cortex mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors.

1. Human brain cortical slices from patients undergoing neurosurgery for treatment of epilepsy resistant to antiepileptic drugs were used to identify and characterize N-methyl-D-aspartate (NMDA) and non-NMDA receptors mediating stimulation of noradrenaline release. The slices preincubated with [3H]-noradrenaline were superfused with Krebs-Henseleit solution with or without Mg2+ (1.2 mmol l-1) and were stimulated by 2-min exposure to NMDA, kainic acid or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). 2. In slices superfused without Mg2+, NMDA induced a concentration-dependent tritium overflow. 3. The NMDA-evoked tritium overflow was almost abolished by tetrodotoxin (TTX), Mg2+ or by omission of Ca2+ from the superfusion fluid. 2-Amino-5-phosphonopentanoic acid (AP5; a competitive NMDA receptor antagonist) or dizocilpine (formerly MK-801; an antagonist at the phencyclidine receptor within the NMDA-gated ion channel) inhibited the NMDA-evoked tritium overflow. The stimulatory effect of NMDA was not significantly enhanced by glycine added to the superfusion fluid but was reduced by 7-chlorokynurenic acid (an antagonist at the glycine site coupled to the NMDA receptor). 4. In slices superfused with solution containing Mg2+, kainic acid or AMPA induced a concentration-dependent tritium overflow which was susceptible to blockade by TTX. 5. The kainic acid-evoked tritium overflow was not affected by DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP37849; a competitive NMDA receptor antagonist), but was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; an antagonist at glutamate receptors of the non-NMDA type). 6. The AMPA-evoked tritium overflow was also inhibited by CNQX.2n

Characterization of 5-HT3 receptors of N1E-115 neuroblastoma cells by use of the influx of the organic cation [14C]-guanidinium.

1. The 5-HT3 receptor-mediated cation influx into N1E-115 mouse neuroblastoma cells has been studied by the use of the organic cation [14C]-guanidinium. 2. 5-Hydroxytryptamine (5-HT, 30 microM) caused a time-dependent influx of [14C]-guanidinium which, in contrast to the influx elicited by veratridine (100 microM), was not inhibited by tetrodotoxin (TTX, 10 microM). The 5-HT-induced influx was potentiated by substance P and inhibited by ondansetron. 3. 5-HT and the selective 5-HT3 receptor agonists, m-chloro-phenylbiguanide, phenylbiguanide and 2-methyl-5-HT caused bell-shaped concentration-response curves; the rank order of potency was m-chloro-phenylbiguanide > 5-HT > phenylbiguanide = 2-methyl-5-HT. Among these agonists, 5-HT elicited the highest influx of [14C]-guanidinium. 5-Methoxytryptamine, an agonist at 5-HT4 receptors, showed no effect. 4. The [14C]-guanidinium influx induced by 100 microM 5-HT was not affected by methysergide (10 microM) and ketanserin (10 microM) but was inhibited by 5-HT3 receptor antagonists with the following rank order of potency: ICS 205-930 > ondansetron > MDL 72222 >> metoclopramide. 5. The 5-HT-induced [14C]-guanidinium influx was increased in the absence of Ca2+ and/or Na+ and by a reduction of the temperature from 36 degrees to 20 degrees C. 6. Preincubation with 5-HT (100 microM) caused a time-dependent and rapidly reversible decrease of the 5-HT-induced [14C]-guanidinium influx. 7. It is concluded that [14C]-guanidinium influx measurement in N1E-115 cells is a convenient method to study properties of the cation channel of the 5-HT3 receptor. This influx is independent of the fast sodium channel.

Modulation of noradrenaline release from the sympathetic nerves of the human saphenous vein and pulmonary artery by presynaptic EP3- and DP-receptors.

1. Spirally cut strips of the human saphenous vein and pulmonary artery were used to determine the pharmacological properties of the presynaptic prostanoid receptors involved in the modulation of sympathetic [3H]-noradrenaline release. Strips preincubated with [3H]-noradenaline were superfused with physiological salt solution containing inhibitors of uptake1 and uptake2 and rauwolscine to eliminate involvement of presynaptic alpha 2-adrenoceptors. Tritium overflow was evoked by transmural electrical stimulation (standard frequency: 2 Hz). 2. In the saphenous vein, prostaglandin E2 (PGE2) inhibited the electrically-evoked tritium overflow; at the highest concentration investigated, tritium overflow was inhibited by more than 75% and the pEC50 value was 7.00. These effects were mimicked by prostaglandin E1, the EP1/EP3 receptor agonist, sulprostone and the EP2/EP3 receptor agonist, misoprostol with the rank order (pEC50): sulprostone (8.60) > PGE1 (7.25) > misoprostol (6.96). This rank order of potency suggests that the inhibitory effect of the drugs is mediated by presynaptic EP3-receptors. In contrast, PGF2 alpha did not inhibit evoked tritium overflow; the IP/EP1 receptor agonist iloprost and the stable thromboxane A2 analogue U 46619 (9, 11-dideoxy-11 alpha,9 alpha-epoxy-methanoprostaglandin F2 alpha) produced inhibition only at concentrations above 1 microM. 3. The EP1-receptor antagonist, AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) had no effect on the evoked tritium overflow nor did it modify the inhibitory effect of PGE2, further excluding involvement of inhibitory presynaptic EP1-receptors. 4. PGD2 caused a facilitation of evoked tritium overflow in the saphenous vein; this facilitation is probably mediated by presynaptic DP-receptors, since it was abolished by the selective DP-receptor antagonist, BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethylamino)hydantoin).5. In the pulmonary artery, sulprostone (pECm value 8.35), misoprostol (7.70) and PGE2 (6.80)inhibited electrically-evoked tritium overflow. This rank order of potency is consistent with the involvement of inhibitory presynaptic EP3-receptors.6. These results suggest that the sympathetic nerve fibres of both human saphenous vein and pulmonary artery are endowed with presynaptic inhibitory EP3 receptors. The EP3-receptors do not interact with the alpha 2-autoreceptors. In addition, the human saphenous vein seems to be endowed with presynaptic facilitatory DP-receptors.

Influence of sodium substitutes on 5-HT-mediated effects at mouse 5-HT3 receptors.

1 The influence of sodium ion substitutes on the 5-hydroxytryptamine (5-HT)-induced flux of the organic cation [14C]guanidinium through the ion channel of the mouse 5-HT3 receptor and on the competition of 5-HT with the selective 5-HT3 receptor antagonist [3H]GR 65630 was studied, unless stated otherwise, in mouse neuroblastoma N1E-115 cells. 2 Under physiological conditions (135 mm sodium), 5-HT induced a concentration-dependent [14C]guanidinium influx with an EC50 (1.3 microm) similar to that in electrophysiological studies. 3 The stepwise replacement of sodium by increasing concentrations of the organic cation hydroxyethyl trimethylammonium (choline) concentration dependently caused both a rightward shift of the 5-HT concentration-response curve and an increase in the maximum effect of 5-HT. Complete replacement of sodium resulted in a 34-fold lower potency of 5-HT and an almost two times higher maximal response. A low potency of 5-HT in choline buffer was also observed in other 5-HT3 receptor-expressing rodent cell lines (NG 108-15 or NCB 20). 4 Replacement of Na+ by Li+ left the potency and maximal effects of 5-HT almost unchanged. Replacement by tris (hydroxymethyl) methylamine (Tris), tetramethylammonium (TMA) or N-methyl-d-glucamine (NMDG) caused an increase in maximal response to 5-HT similar to that caused by choline. The potency of 5-HT was only slightly reduced by Tris, to a high degree decreased by TMA (comparable to the decrease by choline), but not influenced by NMDG. 5 The potency of 5-HT in inhibiting [3H]GR65630 binding to intact cells was 35-fold lower when sodium was completely replaced by choline, but remained unchanged after replacement by NMDG. 6 The results are compatible with the suggestion that choline competes with 5-HT for the 5-HT3 receptor; the increase in maximal response may be partly due to a choline-mediated delay of the 5-HT-induced desensitization. For studies of 5-HT-evoked [14C]guanidinium flux through 5-HT3 receptor channels, NMDG appears to be an 'ideal' sodium substituent since it increases the signal-to-noise ratio without interfering with 5-HT binding.

Inhibition of neuronal Ca(2+) influx by gabapentin and subsequent reduction of neurotransmitter release from rat neocortical slices.

Cytosolic calcium ion concentrations ([Ca(2+)](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 [(3)H]-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 [Ca(2+)](i) increase in synaptosomes (IC(50)=14 microM; maximal inhibition by 36%). The inhibitory effect of gabapentin was abolished in the presence of the P/Q-type Ca(2+) channel blocker omega-agatoxin IVA, but not by the N-type Ca(2+) channel antagonist omega-conotoxin GVIA. Gabapentin (100 microM) decreased the K(+)-evoked release of endogenous aspartate and glutamate in neocortical slices by 16 and 18%, respectively. Gabapentin reduced the K(+)-evoked [(3)H]-noradrenaline release in neocortical slices (IC(50)=48 microM; 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 [(3)H]-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 [Ca(2+)](i) increase in excitatory amino acid nerve terminals by inhibiting P/Q-type Ca(2+) channels; this decreased Ca(2+) 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.

Dual interaction of agmatine with the rat alpha(2D)-adrenoceptor: competitive antagonism and allosteric activation.

In segments of rat vena cava preincubated with [(3)H]-noradrenaline and superfused with physiological salt solution, the influence of agmatine on the electrically evoked [(3)H]-noradrenaline release, the EP(3) prostaglandin receptor-mediated and the alpha(2D)-adrenoceptor-mediated inhibition of evoked [(3)H]-noradrenaline release was investigated. Agmatine (0.1-10 microM) by itself was without effect on evoked [(3)H]-noradrenaline release. In the presence of 10 microM agmatine, the prostaglandin E(2)(PGE(2))-induced EP(3)-receptor-mediated inhibition of [(3)H]-noradrenaline release was not modified, whereas the alpha(2D)-adrenoceptor-mediated inhibition of [(3)H]-noradrenaline release induced by noradrenaline, moxonidine or clonidine was more pronounced than in the absence of agmatine. However, 1 mM agmatine antagonized the moxonidine-induced inhibition of [(3)H]-noradrenaline release. Agmatine concentration-dependently inhibited the binding of [(3)H]-clonidine and [(3)H]-rauwolscine to rat brain cortex membranes (K(i) values 6 microM and 12 microM, respectively). In addition, 30 and 100 microM agmatine increased the rate of association and decreased the rate of dissociation of [(3)H]-clonidine resulting in an increased affinity of the radioligand for the alpha(2D)-adrenoceptors. [(14)C]-agmatine labelled specific binding sites on rat brain cortex membranes. In competition experiments. [(14)C]-agmatine was inhibited from binding to its specific recognition sites by unlabelled agmatine, but not by rauwolscine and moxonidine. In conclusion, the present data indicate that agmatine both acts as an antagonist at the ligand recognition site of the alpha(2D)-adrenoceptor and enhances the effects of alpha(2)-adrenoceptor agonists probably by binding to an allosteric binding site of the alpha(2D)-adrenoceptor which seems to be labelled by [(14)C]-agmatine.

Presynaptic imidazoline receptors and non-adrenoceptor [3H]-idazoxan binding sites in human cardiovascular tissues.

1 In segments of human right atrial appendages and pulmonary arteries preincubated with [3H]-noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone, the involvement of imidazoline receptors in the modulation of [3H]-noradrenaline release was investigated. 2 In human atrial appendages, the guanidines aganodine and DTG (1,3-di(2-tolyl)guanidine) which activate presynaptic imidazoline receptors, inhibited electrically-evoked [3H]-noradrenaline release. The inhibition was not affected by blockade of alpha 2-adrenoceptors with 1 microM rauwolscine, but antagonized by extremely high concentrations of this drug (10 and/or 30 microM; apparent pA2 against aganodine and DTG: 5.55 and 5.21, respectively). 3 In the presence of 1 microM rauwolscine, [3H]-noradrenaline release in human atrial appendages was also inhibited by the imidazolines idazoxan and cirazoline, but not by agmatine and noradrenaline. The inhibitory effects of 100 microM idazoxan and 30 microM cirazoline were abolished by 30 microM rauwolscine. 4 In the atrial appendages, the rank order of potency of all guidelines and imidazolines for their inhibitory effect on electrically-evoked [3H]-noradrenaline release in the presence of 1 microM rauwolscine was: aganodine > or = BDF 6143 [4-chloro-2-(2-imidazolin-2-yl-amino)-isoindoline] > DTG > or = clonidine > cirazoline > idazoxan (BDF 6143 and clonidine were previously studied under identical conditions). This potency order corresponded to that previously determined at the presynaptic imidazoline receptors in the rabbit aorta. 5 When, in the experiments in the human pulmonary artery, rauwolscine was absent from the superfusion fluid, the concentration-response curve for BDF 6143 (a mixed alpha 2-adrenoceptor antagonist/imidazoline receptor agonist) for its facilitatory effect on electrically-evoked [3H]-noradrenaline release was bell-shaped. In the presence of 1 microM rauwolscine, BDF 6143 and cirazoline concentration-dependently inhibited the evoked [3H]-noradrenaline release. 6 In human atrial appendages, non-adrenoceptor [3H]-idazoxan binding sites were identified and characterized. The binding of [3H]-idazoxan was specific, reversible, saturable and of high affinity (KD: 25.4 nM). The specific binding of [3H]-idazoxan (defined by cirazoline 0.1 mM) to membranes of human atrial appendages was concentration-dependently inhibited by several imidazolines and guanidines, but not by rauwolscine and agmatine. In most cases, the competition curves were best fitted to a two-site model. 7 The rank order of affinity for the high affinity site (in a few cases for the only detectable site; cirazoline = idazoxan > BDF 6143>DTG> or = clonidine) is compatible with the pharmacological properties of I2-imidazoline binding sites, but is clearly different from the rank order of potency for inhibiting evoked noradrenaline release from sympathetic nerves in the same tissue. 8 It is concluded that noradrenaline release in the human atrium and, less well established, in the pulmonary artery is inhibited via presynaptic imidazoline receptors. These presynaptic imidazoline receptors appear to be related to those previously characterized in rabbit aorta and pulmonary artery, but differ clearly from I1 and I2 imidazoline binding sites.

Anpirtoline, a novel, highly potent 5-HT1B receptor agonist with antinociceptive/antidepressant-like actions in rodents.

1. The purpose of the present study was to relate the effects of the novel drug, anpirtoline, on 5-hydroxytryptamine (5-HT) receptor subtypes to its antinociceptive and antidepressant-like actions in rodents. 2. Binding assays with rat brain membranes have shown that anpirtoline bound with a much higher affinity to 5-HT1B receptor (Ki = 28 nM) than to 5-HT1A (Ki = 150 nM) and 5-HT2 (Ki = 1.49 microM) receptors. 3. Like 5-HT, anpirtoline concentration-dependently inhibited forskolin-stimulated adenylate cyclase activity in homogenates from the rat substantia nigra. Both effects were not additive, and could be prevented by 5-HT1B receptor antagonists such as propranolol and penbutolol. 4. In superfused rat and pig brain cortex slices preincubated with [3H]-5-HT, the electrically evoked tritium overflow was inhibited by anpirtoline and 5-HT. Whereas 5-HT was equipotent in both tissues (EC50 = 69 nM), anpirtoline was markedly less potent in pig brain cortex slices (EC50 = 1190 nM) than in rat brain cortex slices (EC50 = 55 nM). The concentration-response curve for anpirtoline was shifted to the right by metitepine in both preparations. 5. In the social behaviour deficit test, anpirtoline and trifluoromethylphenyl-piperazine were effective in reversing the isolation-induced impairments in mice, an effect shown only by compounds with agonist properties at the 5-HT1B receptor. 6. In the electrostimulated pain test using mice, anpirtoline dose-dependently increased the pain threshold with an ED50 of 0.52 mg kg-1, i.p. The antinociceptive activity of anpirtoline was abolished by pretreatment with cyproheptadine or propranolol.7. In the forced swimming test in rats, anpirtoline induced a dose-related increase in swimming activity. With an ED50 value of 4.6mgkg-1, i.p., anpirtoline was 4 times more potent than the two standard compounds imipramine and desipramine. The decrease of immobility time or the increase of active periods in this model of behavioural despair is suggested to be characteristic of antidepressant drugs.8. Anpirtoline exhibits both antinociceptive and antidepressant-like activities in animals. It is probable that anpirtoline elicits these pharmacological effects via its agonist effect on 5-HT1B and 5-HT1A receptors.

Novel histamine H(3)-receptor antagonists and partial agonists with a non-aminergic structure.

We determined the affinities of eight novel histamine H(3)-receptor ligands (ethers and carbamates) for H(3)-receptor binding sites and their agonistic/antagonistic effects in two functional H(3)-receptor models. The compounds differ from histamine in that the ethylamine chain is replaced by a propyloxy chain; in the three ethers mentioned below (FUB 335, 373 and 407), R is n-pentyl, 3-methylbutyl and 3,3-dimethylbutyl, respectively. The compounds monophasically inhibited [(3)H]-N(alpha)-methylhistamine binding to mouse cerebral cortex membranes (pK(i) 7.51 - 9.53). The concentration-response curve of histamine for its inhibitory effect on the electrically evoked [(3)H]-noradrenaline overflow from mouse cortex slices was shifted to the right by these compounds (apparent pA(2) 6.61 - 8.00). Only FUB 373 and 407 inhibited the evoked overflow by themselves (intrinsic activities 0.3 and 0.4); these effects were counteracted by the H(3)-receptor antagonist clobenpropit. [(35)S]-GTPgammaS binding to mouse cortex membranes was stimulated by the H(3)-receptor agonist (R)-alpha-methylhistamine in a manner sensitive to clobenpropit. Among the novel compounds only FUB 373 and 407 stimulated [(35)S]-GTPgammaS binding (intrinsic activities 0.6 and 0.4). In conclusion, the novel compounds are partial H(3)-receptor agonists (FUB 373 and 407) or H(3)-receptor antagonists; comparison with FUB 335 shows that the transition from antagonist to agonist is caused by a slight structural change. A protonated N atom in the side chain is not necessary for agonism at H(3) receptors, proposing a receptor-ligand interaction different from that of classical agonists.

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): 9-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.