Fast and slow interactions of n-alkanols with human 5-HT3A receptors: Implications for anesthetic mechanisms.

This is part of a continuing patch-clamp study exploring molecular actions of anesthetics and systematically varied related substances on 5-HT3A receptors as prototypes of ligand-gated ion channels. Specifically, n-alkanols, related to but simpler in structure than propofol, were studied to explore the complex actions of this leading intravenous anesthetic. Outside-out patches excised from HEK 293 cells heterologously expressing human 5-HT3A receptors were superfused with even-numbered n-alkanols (ethanol through n-tetradecanol) of different concentrations. Fast solution exchange for varying durations allowed separation of drug actions by their kinetics. Compared with propofol the electrophysiological responses to n-alkanols were not much simpler. n-Alkanols produced fast and slow inhibition or potentiation of current amplitudes, and acceleration of current rise and decay time constants, depending on exposure time, concentration, and chain-length of the drug. Inhibition dominated, characterized by fast and slow processes with time constants separated by two orders of magnitude which were similar for different n-alkanols and for propofol. Absolute interaction energies for ethanol to n-dodecanol (relative to xenon) ranged from -10.8 to -37.3kJmol(-1). No two n-alkanols act completely alike. Potency increases with chain length (until cutoff) mainly because of methylene groups interacting with protein sites rather than because of their tendency to escape from the aqueous phase. Similar wash-in time constants for n-alkanols and propofol suggest similar mechanisms, dominated by the kinetics of conformational state changes rather than by binding reactions.

Effects of opioids on human serotonin transporters.

The serotonin (5-hydroxtryptamine, 5-HT) system plays a role in analgesia and emesis. The aim of this study was to test whether opioids or ketamine inhibit the human 5-HT transporter and whether this increases free plasma 5-HT concentrations. HEK293 cells, stably transfected with the human 5-HT transporter cDNA, were incubated with morphine, hydromorphone, fentanyl, alfentanil, pethidine (meperidine), tramadol, ketamine, and the reference substance citalopram (specific 5-HT transporter inhibitor). The uptake of [(3)H]5-HT was measured by liquid scintillation counting. In a second series of experiments, study drugs were incubated with plasma of ten healthy blood donors and change of 5-HT plasma-concentrations were measured (ELISA). The end point was the inhibition of the 5-HT transporter by different analgesics either in HEK293 cells or in human platelets ex vivo. Tramadol, pethidine, and ketamine suppressed [(3)H]5-HT uptake dose-dependently with an IC50 of 1, 20.9, and 230 µM, respectively. These drugs also prevented 5-HT uptake in platelets with an increase in free plasma 5-HT. Free 5-HT concentrations in human plasma were increased by citalopram 1 µM, tramadol 20 µM, pethidine 30 µM, and ketamine 100 µM to 280 [248/312]%, 269 [188/349]%, and 149 [122/174]%, respectively, compared to controls without any co-incubation (means [95 % CI]; all p < 0.005). No change in both experimental settings was observed for the other opioids. Tramadol and pethidine inhibited the 5-HT transporter in HEK293 cells and platelets. This inhibition may contribute to serotonergic effects when these opioids are given in combination, e.g., with monoamine oxidase inhibitors or selective serotonin reuptake inhibitors.

Which agonist properties are important for the activation of 5-HT3A receptors?

PURPOSE: Why do anesthetics not activate excitatory ligand-gated ion channels such as 5-HT3 receptors in contrast to inhibitory ligand-gated ion channels? This study examines the actions of structural closely-related 5-HT derivatives and 5-HT constituent parts on 5-HT3A receptors with the aim of finding simpler if not minimal agonists and thus determining requirements for successful agonist action. EXPERIMENTAL APPROACH: Responses to 5-HT derivatives of human 5-HT3A receptors stably expressed in HEK 293 cells have been examined with the patch-clamp technique in the outside-out configuration combined with a fast solution exchange system. RESULTS: Phenol, pyrrole and alkyl amines, constituents of 5-HT, even at high concentrations, cannot activate 5-HT3A receptors but they can inhibit them. To date, tyramines are the smallest known agonists. However, an aromatic ring is not required for activation as acetylcholine is also an agonist of similar strength. CONCLUSION: Simultaneous interactions of adequate strength at two separate subsites within the 5-HT binding domain appear to be essential for successful agonist function. Anesthetics either fail to achieve this or the activation they produce is so weak that it is masked by a comparatively very strong inhibition.

The effects of morphine on human 5-HT3A receptors.

5-HT3 receptors are ligand-gated ion channels that are involved in the modulation of emesis and pain. In this study, we investigated whether the opioid analgesic, morphine, exerts specific effects on human 5-HT3 receptors. Whole-cell patches from HEK-293 cells stably transfected with the human 5-HT3A receptor cDNA were used to determine the effects of morphine on the 5-HT-induced currents using the patch clamp technique. At negative membrane potentials, 5-HT induced inward currents in a concentration-dependent manner. The 5-HT3 receptor antagonist, ondansetron, (0.3 nM) reversibly inhibited the 5-HT-induced signals. Morphine reversibly suppressed 5-HT-induced peak currents as a function of concentration (IC50 = 1.1 microM, Hill coefficient = 1.2). The block by morphine decreased with increasing 5-HT concentrations, suggesting a competitive effect. In addition, the activation, as well as the inactivation, kinetics of the currents were significantly slowed in the presence of morphine. The morphine antagonist, naloxone, also inhibited 5-HT-induced currents (e.g., at 3 microM by 17%). The effects of morphine and naloxone were not additive. The potency of morphine and the competitivity of the blocking effect points to a specific mechanism at a receptor site rather than an unspecific membrane effect.

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.

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.

The Effects of isoflurane on acetylcholine receptor channels: 3. Effects of conservative polar-to-nonpolar mutations within the channel pore.

We performed macroscopic and single-channel current measurements on wild-type (WT) and two mutant muscle-type nicotinic acetylcholine (ACh) receptor channels transiently expressed in HEK-293 cells. The mutants contained polar-to-nonpolar substitutions at the 10' (alpha(2)S10'A beta T10'A gamma delta) and 6' positions (alpha(2)S6'A beta gamma delta S6'A) in the M2 pore region of the channel. We studied the behavior of these channels in the absence and presence of the volatile general anesthetic isoflurane. Both mutations changed the gating behavior of the channel. A comparison of the alpha(2)S10'A beta T10'A gamma delta mutant to WT receptors revealed faster desensitization kinetics, increased sensitivity to ACh, a higher efficacy for activation by the partial nicotinic agonist decamethonium, and a greater number of openings per burst. A comparison of the alpha(2)S6'A beta gamma delta S6'A mutant to WT receptors also revealed increased sensitivity to ACh and an increased burst duration at the single-channel level with ACh as agonist. The alpha(2)S10'A beta T10'A gamma delta mutation increased the sensitivity of the ACh receptor to isoflurane, whereas the alpha(2)S6'A beta gamma delta S6'A mutation did not. These changes were probably not caused by the differential effects of the mutation on channel gating and desensitization. The increased sensitivity of the alpha(2)S10'A beta T10'A gamma delta receptor to isoflurane is state-dependent; the mutation changes the affinity of the closed state but not that of the open state of the channel.

Modified 5-HT3A receptor function by co-expression of alternatively spliced human 5-HT3A receptor isoforms.

Serotonin (5-HT) exerts fast excitatory responses by activation of 5-HT3 receptors, irrespective of whether they are homomerically composed of 5-HT3A subunits or heteromerically assembled of 5-HT3A and 5-HT3B subunits. Here we describe a short, truncated (h5-HT3AT) and a long (h5-HT3AL) splice variant of the human 5-HT3A (hS-HT3A) receptor subunit. The deduced protein of the short isoform consists of 238 amino acids (aa) with a single transmembrane domain (M1). Compared to the known 5-HT3A receptor, the long isoform contains 32 additional aa in the extracellular loop between M2 and M3. Both splice variants are co-expressed together with the 5-HT3A subunit in the amygdala and hippocampus, whereas in the placenta only the short variant is co-expressed. Both splice variants, when expressed in transfected human embryonic kidney (HEK) 293 cells, are not able to form functional homomeric receptors, but modify 5-HT response at heteromeric h5-HT3A receptors. Co-expression of the short variant considerably decelerates the desensitization of the 5-HT3 receptor; thus, heteromeric assemblies of h5-HT3A and the h5-HT3AT subunit exhibit 5-HT-induced cation fluxes which are much larger than those of homomeric hS-HT3A receptors. In contrast, heteromeric complexes containing the h5-HT3AL subunit display reduced cation fluxes. In conclusion, the splice variants increase the functional diversity of 5-HT3 receptors.

Recombinant human 5-HT3A receptors in outside-out patches of HEK 293 cells: basic properties and barbiturate effects.

The patch-clamp technique was used on excised (outside-out) patches to characterize h5-HT3A receptors stably transfected in HEK 293 cells and to compare the effects of the barbiturate anaesthetics methohexital and pentobarbital on this ligand-gated cation channel. At negative membrane potentials 5-HT induced inward currents in a concentration-dependent manner (EC50=8.6 microM, Hill coefficient =1.5). The mean peak current induced by 30 microM 5-HT was -110 pA at -100 mV. The 5-HT3A receptor antagonist ondansetron (0.3 nM) reversibly inhibited the 5-HT (30 microM) signal by 70% and at 3 nM it abolished the response. Methohexital and pentobarbital inhibited 5-HT-induced (30 microM) currents in a concentration-dependent manner. The maximal inhibition with a given methohexital or pentobarbital concentration was reached when the respective drug was applied 45 s prior to and during the 2-s 5-HT pulse (IC50 values=95 microM and 127 microM, Hill coefficient = -1.0 and -1.6, respectively). Although the barbiturates were, thus, equipotent, their effects differed substantially with respect to the dependence on the time schedule of application to the patches: the potency of methohexital was virtually maximal when the drug was applied exclusively 45 s before the agonist pulse, but its inhibitory potency decreased considerably when it was exclusively applied during the 2-s 5-HT pulse (IC50=380 microM). Conversely, pentobarbital was almost maximally potent in inhibiting the 5-HT signal when it was exclusively coapplied with this agonist, but its inhibitory potency was considerably lower (IC50 approximately 500 microM) when applied exclusively 45 s before 5-HT. Another difference between both barbiturates involves the rate of inactivation of 5-HT3 receptor-mediated currents: whereas high concentrations of methohexital (> or = 300 microM) were necessary to induce moderate (< or = twofold) acceleration of this parameter, pentobarbital produced such an effect at all concentrations and the extent of acceleration increased with increasing concentration (1.5- to fivefold). In conclusion, two barbiturates, chemically closely related but of different lipophilicity, clearly differ with respect to the kinetics of their effect on 5-HT3 receptor channels; one possible explanation involves drug access to an amphipathic site of action via both an aqueous and a hydrophobic pathway. Pentobarbital, in contrast to methohexital, inhibits hS-HT3A receptor-mediated currents at anaesthetic concentrations (approximately 90 microM).

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.

Inhibition by steroids of [14C]-guanidinium flux through the voltage-gated sodium channel and the cation channel of the 5-HT3 receptor of N1E-115 neuroblastoma cells.

Effects of some naturally occurring steroids and synthetic analogues on the cation flux through the cation channel of the 5-HT3 receptor and the voltage-gated and tetrodotoxin-sensitive sodium channel were studied in N1E-115 mouse neuroblastoma cells by measuring the 2-min influx of the organic cation [14C]-guanidinium. The cation fluxes in intact cells were either induced by 2 min exposure of the cells to 5-hydroxytryptamine (5-HT, 100microM) or to veratridine (1 mM). Influx of [14C]-guanidinium through both channels was concentration-dependently inhibited by all compounds studied. The rank order of potency for inhibition of the 5-HT3 receptor-induced cation flux was clomiphene approximately/= cyproterone acetate > estradiol > progesterone approximately/= allotetrahydrodeoxycorticosterone > alfaxalone approximately/= testosterone > aldosterone > dexamethasone. With the exception of dexamethasone and testosterone, which were more potent at the voltage-dependent sodium channel, and progesterone and testosterone, which were about nearly equipotent in inhibiting both cation channels, the steroids were twofold (alfaxalone, allotetrahydrodeoxycorticosterone) to 107-fold (cyproterone acetate) more potent at the 5-HT3 receptor channel than at the voltage-gated sodium channel. The potencies of the steroids (and the synthetic analogues) for inhibition of the 5-HT3 receptor-induced [14C]-guanidinium influx were correlated with their lipophilicity (log P values). A similar correlation between log P values and pIC50 values for the steroid-induced inhibition of the veratridine-evoked cation influx through the voltage-gated sodium channel was only found when cyproterone acetate (a compound with extremely low inhibitory potency at this channel) was not included in the regression analysis. The results indicate that both the 5-HT3 receptor channel and the voltage-gated sodium channel are targets for steroids. The relationship between most of the compounds in inhibiting both cation channels and lipophilicity is compatible with a common mechanistic principle in steroid-induced inhibition of the two channels, i.e. a nonspecific hydrophobic interaction with certain membrane lipids in the neighbourhood of the two channels.

Inhibition of 5-HT3 receptor cation channels by ifenprodil in excised patches of N1E-115 cells.

The patch-clamp technique was applied in out-side-out patches of N1E-115 mouse neuroblastoma cells to investigate the effects of ifenprodil [(+/-) erythreo-ifenprodil tartratel, a drug with neuroprotective properties in cerebral ischemia, on the inward currents through 5-HT3 receptor channels. A high time resolution was achieved by using a rapid solution exchange system (exchange rate <1 ms). Ifenprodil inhibited the peak currents evoked by 30 microM 5-HT in a concentration-dependent but voltage-independent manner. The effect was most potent when ifenprodil was continuously applied to the patches 45 s before and during the 2-s administration of 5-HT (IC50=16 microM) and it was only slightly less potent when it was applied during the 45 s prior to 5-HT only (IC50=29 microM). When applied in this manner, ifenprodil also produced a concentration-dependent increase of the onset time constant (tauON) of the 5-HT (30 microM)-induced currents. When the drug was exclusively co-applied with 5-HT, ifenprodil was least potent in inhibiting the peak currents (IC50=98 microM), and it had no effect on the current onset kinetics. All protocols of ifenprodil application accelerated current inactivation as reflected by a decrease of the current inactivation time constant (tauOFF). All effects of ifenprodil were reversible after washout periods of 2-5 min. In conclusion, the potency of ifenprodil in inhibiting the inward current through 5-HT3 receptor channels is strongly dependent on the application protocol: presence of the drug before the agonist-induced activation of the 5-HT3 receptor channels is necessary for a relatively potent inhibition of the 5-HT-induced peak current and is a prerequisite for the prolongation of tauON; in addition, a weak but fast inhibitory effect on the current amplitude and decay constant of the 5-HT-induced current was revealed by the experiments in which ifenprodil was exclusively present during exposure to 5-HT. Three alternatives compatible with the components of the ifenprodil effect have been discussed: (1) different effects of the two enantiomers, (2) action via two different mechanisms, and (3) operation via a single mechanism only.

Interactions of general anesthetics within the pore of an ion channel.

(1) We review the electrophysiological evidence that the ion channel pore is the site at which general anesthetics bind to inhibit muscle-type acetylcholine receptor channels. (2) The amphipathic character of a pore certainly offers a suitable environment for the binding of amphipathic anesthetics. (3) The absence of direct information about the binding sites of these rather non-specific drugs, forces us to rely on indirect information provided by kinetic experiments. (4) We also discuss the implications of these findings for the interaction of general anesthetics with other ion channels.

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.

Mechanisms of barbiturate inhibition of acetylcholine receptor channels.

We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts in 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 microM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 microM, n = 1.09; for barbital, Ki = 1900 microM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. < 0.1 ms at the Ki). Pentobarbital binds > or = 10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at > 100 microM pentobarbital.

Increasing effect of ethanol on 5-HT3 receptor-mediated 14C-guanidinium influx in N1E-115 neuroblastoma cells.

N1E-115 mouse neuroblastoma cells were used to study the influence of ethanol on the 5-HT- and veratridine-induced influx of 14C-guanidinium via the 5-HT3 receptor channel and the fast sodium channel, respectively. Ethanol (10-100 mM) concentration-dependently increased the 5-HT-induced 14C-guanidinium influx, leaving the basal and veratridine (100 microM)-induced influx unaffected. The increasing effect of ethanol (100 mM) was observed at all 5-HT concentrations investigated; accordingly, ethanol increased the maximum response to 5-HT. Whereas in the absence of ethanol the concentration-response curve for 5-HT was bell-shaped, this was no longer the case when ethanol (100 mM) was present in the incubation buffer; the descending branch of the concentration-response curve for 5-HT at concentrations above 300 microM was virtually no longer observed. When, in the presence of substance P (10 microM) the 5-HT-induced 14C-guanidinium influx was already enhanced, the ability of ethanol (100 mM) to increase the 5-HT-induced influx was considerably diminished (by 72%). Preincubation of N1E-115 cells with 5-HT caused a decay of the subsequent 5-HT response ("desensitization") which was dependent on the duration of preincubation; ethanol (100 mM) did not affect the rate of this decay of the 5-HT response. The 5-HT (30 microM)-induced 14C-guanidinium influx was also increased by methanol (100 mM) and n-propanol (100 mM). The rank order of the increasing effect of the n-alkanols (at 100 mM) was: methanol < ethanol < n-propanol; i.e. the degree of enhancement increased with the lipophilicity of the alcohols.(ABSTRACT TRUNCATED AT 250 WORDS)

5-HT3 receptor antagonism by anpirtoline, a mixed 5-HT1 receptor agonist/5-HT3 receptor antagonist.

1. The aim of this study was to provide evidence that anpirtoline, which is an agonist at 5-HT1B and 5-HT1D receptors and also displays submicromolar affinity for 5-HT1A recognition sites, in addition, acts as an antagonist at 5-HT3 receptors. 2. In radioligand binding studies on rat brain cortical membranes, anpirtoline inhibited specific binding of [3H]-(S)-zacopride to 5-HT3 receptor recognition sites (pKi: 7.53). 3. In N1E-115 neuroblastoma cells in which [14C]-guanidinium was used as a tool to measure cation influx through the 5-HT3 receptor channel, the 5-HT-induced influx was concentration-dependently inhibited by anpirtoline. In this respect, anpirtoline mimicked other 5-HT3 receptor antagonists; the rank order of potency was ondansetron > anpirtoline > metoclopramide. 4. The concentration-response curve for 5-HT as a stimulator of [14C]-guanidinium influx was shifted to the right by anpirtoline (apparent pA2: 7.78). 5. In urethane-anaesthetized rats, anpirtoline inhibited (at lower potency than zacopride and tropisetron) the 5-HT- or phenylbiguanide-induced bradycardia (Bezold-Jarisch reflex), but did not induce this reflex by itself. 6. Intravenous infusion of cisplatin in the domestic pig caused a consistent emetic response which was antagonized by anpirtoline. 7. It is concluded that anpirtoline, which was previously characterized as a 5-HT1 receptor agonist also proved to be a 5-HT3 receptor antagonist in several experimental models and, hence, exhibits a unique pattern of properties at different 5-HT receptors.