Antipsychotic drugs antagonize human serotonin type 3 receptor currents in a noncompetitive manner.

The serotonin type 3 (5-HT(3)) receptor is the only ligand-gated ion channel receptor for serotonin (5-HT). 5-HT(3) receptors play an important role in modulating the inhibitory action of dopamine in mesocorticolimbic brain regions. Neuroleptic drugs are commonly thought to exert their psychopharmacological action mainly through dopamine and serotonin type 2 (5-HT(2)) receptors. Except for clozapine, a direct pharmacological interaction of neuroleptics with 5-HT(3) receptors has not yet been described. Using the concentration-clamp technique, we investigated the effects of flupentixol, various phenothiazines, haloperidol, clozapine and risperidone on Na(+)-inward currents through 5-HT(3) receptors stably expressed in human embryonic kidney 293 cells, and through endogenous 5-HT(3) receptors of murine N1E-115 neuroblastoma cells. In addition, we studied their effects on Ca(2+) influx, measured as a change in intracellular Ca(2+) concentrations ([Ca(2+)](i)). All neuroleptic drugs, but not risperidone, antagonized Na(+)- and Ca(2+)-inward currents evoked by 5-HT (10 microM for 2 s and 1 microM, respectively) in a voltage-independent manner. Only clozapine was a competitive antagonist, while all other compounds turned out to be noncompetitive. Fluphenazine and haloperidol affected membrane anisotropy at concentrations below their IC(50) values, indicating that a change in membrane anisotropy might contribute to their antagonistic effect at the 5-HT(3) receptor. Only structure analogues of flupentixol and fluphenazine with a lipophilic side chain were potent antagonists against 5-HT-evoked Na(+) and Ca(2+) currents. Since 5-HT(3) receptors modulate mesolimbic and mesocortical dopaminergic activity, the functional antagonism of neuroleptics at 5-HT(3) receptors may contribute to their antipsychotic efficacy and may constitute a not yet recognized pharmacological principle of these drugs.

Alpha-thujone reduces 5-HT3 receptor activity by an effect on the agonist-reduced desensitization.

The convulsant effects of alpha-thujone, the psychotropic component of absinthe, were attributed to inhibitory actions at the GABAA receptor. Here, we investigated for the first time the 5-HT3 receptor as a potential site of the psychotropic actions of alpha-thujone. This cation permeable ligand-gated ion channel shows considerable homology to the GABAA receptor. We previously demonstrated that in homomeric assemblies of cloned human 5-HT,A receptor subunits. the endogenous agonist 5-HT induced desensitization via channel blockade. When the 5-HT3 B receptor subunit was co-expressed, the resulting heteromeric assemblies desensitized independent from channel blockade. In the present study, patch-clamp experiments revealed an inhibitory action of alpha-thujone on both homomeric and heteromeric 5-HT3 receptors. This inhibitory action was mediated via channel blockade. However, it was not alpha-thujone itself which blocked the channel. The present experiments suggested that, in homomeric receptors, alpha-thujone enhanced the inherent channel-blocking potency of the natural ligand. 5-HT. In heteromeric receptors, alpha-thujonerecruited an additional channel-blocking component of the agonist. By means of kinetic modeling, we simulated possible mechanisms by which alpha-thuljone decreased the 5-HT-induced responses. It is suggested that alpha-thujone reduced 5-HT3 receptor activity by an effect on mechanisms involved in receptor desensitization, which depend on receptor subunit composition. It remains to be shown if this inhibitory action on serotonergic responses contributes to behavioral effects of alpha-thujone.

Antidepressants are functional antagonists at the serotonin type 3 (5-HT3) receptor.

Antidepressants are commonly supposed to enhance serotonergic and/or noradrenergic neurotransmission by inhibition of neurotransmitter reuptake through binding to the respective neurotransmitter transporters or through inhibition of the monoamine oxidase. Using the concentration-clamp technique and measurements of intracellular Ca2+, we demonstrate that different classes of antidepressants act as functional antagonists at the human 5-HT3A receptor stably expressed in HEK 293 cells and at endogenous 5-HT3 receptors of rat hippocampal neurons and N1E-115 neuroblastoma cells. The tricyclic antidepressants desipramine, imipramine, and trimipramine, the serotonin reuptake inhibitor fluoxetine, the norepinephrine reuptake inhibitor reboxetine, and the noradrenergic and specific serotonergic antidepressant mirtazapine effectively reduced the serotonin-induced Na(+)- and Ca(2)(+)-currents in a dose-dependent fashion. This effect was voltage-independent and, with the exception of mirtazapine, noncompetitive. Desipramine, imipramine, trimipramine, and fluoxetine also accelerated receptor desensitization. Moclobemide and carbamazepine had no effect on the serotonin-induced cation current. By analyzing analogues of desipramine and carbamazepine, we found that a basic propylamine side chain increases the antagonistic potency of tricyclic compounds, whereas it is abolished by an uncharged carboxamide group. The antagonistic effects of antidepressants at the 5-HT3 receptor did not correlate with their effects on membrane fluidity. In conclusion, structurally different types of antidepressants modulate the function of this ligand-gated ion channel. This may represent a yet unrecognized pharmacological principle of antidepressants.

Alpha2-adrenoreceptor activation inhibits LTP and LTD in the basolateral amygdala: involvement of Gi/o-protein-mediated modulation of Ca2+-channels and inwardly rectifying K+-channels in LTD.

Activation of adrenoreceptors modulates synaptic transmission in the basolateral amygdala. Here, we investigated the effects of alpha2-adrenoreceptor activation on long-term depression and long-term potentiation in an in vitro slice preparation of the mouse basolateral amygdala. Field potentials and excitatory postsynaptic currents were evoked in the basolateral amygdala by stimulating the lateral amygdala. Norepinephrine (20 micro m) reduced synaptic transmission and completely blocked the induction of long-term potentiation and long-term depression. The alpha2-adrenoreceptor antagonist yohimbine (2 micro m) reversed this effect. The alpha2-adrenoreceptor agonist clonidine (10 micro m) mimicked the effects of norepinephrine. The Gi/o-protein inhibitor pertussis toxin (5 micro g/mL) reversed the effect of clonidine. Long-term depression was blocked in the presence of omega-conotoxin GVIA, but not omega-agatoxin IVA. Clonidine inhibited voltage-activated Ca2+ currents mediated via N- or P/Q-type Ca2+-channels. The inhibitory action of clonidine on long-term depression was reversed when inwardly rectifying K+-channels were blocked by Ba2+ (300 micro m). The present data suggest that alpha2-adrenoreceptor activation impairs the induction of long-term depression in the basolateral amygdala by a Gi/o-protein-mediated inhibition of presynaptic N-type Ca2+-channels and activation of inwardly-rectifying K+-channels.

Co-expression of the 5-HT3B serotonin receptor subunit alters the biophysics of the 5-HT3 receptor.

Homomeric complexes of 5-HT(3A) receptor subunits form a ligand-gated ion channel. This assembly does not fully reproduce the biophysical and pharmacological properties of native 5-HT(3) receptors which might contain the recently cloned 5-HT(3B) receptor subunit. In the present study, heteromeric assemblies containing human 5-HT(3A) and 5-HT(3B) subunits were expressed in HEK 293 cells to detail the functional diversity of 5-HT(3) receptors. We designed patch-clamp experiments with homomeric (5-HT(3A)) and heteromeric (5-HT(3AB)) receptors to emphasize the kinetics of channel activation and desensitization. Co-expression of the 5-HT(3B) receptor subunit reduced the sensitivity for 5-HT (5-HT(3A) receptor: EC(50) 3 micro M, Hill coefficient 1.8; 5-HT(3AB) receptor: EC(50) 25 micro M, Hill coefficient 0.9) and markedly altered receptor desensitization. Kinetic modeling suggested that homomeric receptors, but not heteromeric receptors, desensitize via an agonist-induced open-channel block. Furthermore, heteromeric 5-HT(3AB) receptor assemblies recovered much faster from desensitization than homomeric 5-HT(3A) receptor assemblies. Unexpectedly, the specific 5-HT(3) receptor agonist mCPBG induced an open-channel block at both homomeric and heteromeric receptors. Because receptor desensitization and resensitization massively affect amplitude, duration, and frequency of synaptic signaling, these findings are evidence in favor of a pivotal role of subunit composition of 5-HT(3) receptors in serotonergic transmission.

Isoflurane blocks synaptic plasticity in the mouse hippocampus.

BACKGROUND: The volatile anesthetic isoflurane depresses glutamatergic transmission. In this study, the authors investigated the effects of isoflurane on the induction of long-term potentiation (LTP) and long-term depression (LTD) in slices from the juvenile and adult mouse hippocampus. Both forms of synaptic plasticity involve the activation of glutamate receptors. METHODS: Field excitatory postsynaptic potentials and excitatory postsynaptic currents from neurons in the CA1 area were evoked by stimulation of the Schaffer collateral-commissural pathway. Two independent synaptic inputs were stimulated. Clinically relevant concentrations (0.2-0.3 mM) of isoflurane were added to the perfusion solution. RESULTS: Field excitatory postsynaptic potentials from slices of juvenile and adult mice were depressed to 37.3 +/- 6.1% and 58.3 +/- 7.4%, respectively, and excitatory postsynaptic currents were reduced to 36.7 +/- 5.4% by isoflurane. A brief tetanic stimulation (100 Hz, 1 s) induced stable LTP of field excitatory postsynaptic potentials. In the presence of isoflurane, tetanization failed to induce LTP. The effect of isoflurane on LTP induction was reversible and could be prevented by antagonizing gamma-aminobutyric acid type A receptors (GABAA). Low-frequency stimulation (1 Hz/900 pulses) induced LTD. In the presence of isoflurane, low-frequency stimulation failed to induce LTD. CONCLUSIONS: The prevention of the isoflurane-induced depression of LTP by the GABAA antagonist picrotoxin suggests an involvement of GABAA receptors. An enhancement of the efficacy of GABA-mediated inhibitory synaptic transmission prevents the depolarization of the postsynaptic membrane during tetanus, necessary for the induction of use-dependent alteration of synaptic strength. An impairment of these processes may be a cause for the transient loss of recall and cognitive impairment after anesthesia in juvenile and adult brains.

Isoflurane slows inactivation kinetics of rat recombinant alpha1beta2gamma2L GABA(A) receptors: enhancement of GABAergic transmission despite an open-channel block.

Recombinant alpha1beta2gamma2L gamma-aminobutyric acid (A) receptor (GABA(A)R) channels expressed in human embryonic kidney (HEK293) cells were used for patch-clamp experiments. The currents activated by brief pulses of GABA (10(-4) M) applied with a device for fast solution exchange to cells clamped in the whole-cell configuration mimicked GABA(A)R-mediated inhibitory postsynaptic currents. Isoflurane (ISO) at clinically relevant concentrations (0.6 mM) decreased the amplitude and prolonged the decay of the GABA-evoked response. To further detail the mechanism underlying the prolonged decay time, we made simulations based on these measurements. These simulations suggest that ISO slows the rate of GABA unbinding from the receptor. Under these conditions, ISO increases the GABA-induced charge transfer and, thus, could enhance GABAergic inhibition despite the concomitant open-channel block causing the decrease in the current amplitude.

Sevoflurane potentiates and blocks GABA-induced currents through recombinant alpha1beta2gamma2 GABAA receptors: implications for an enhanced GABAergic transmission.

BACKGROUND AND OBJECTIVE: The gamma-aminobutyric acidA receptor (GABAAR) is a target for anaesthetic agents. We investigated the interactions of sevoflurane with a recombinant GABAAR. Emphasis was on the mechanism of block, as relevant open-channel block by a volatile anaesthetic would possibly explain prolonged GABAergic postsynaptic currents. METHODS: The effect of sevoflurane on GABA-induced currents through recombinant alpha1beta2gamma2 GABAAR channels was studied (patch clamp; HEK293 cells). GABA 0.01 mM or 1 mM was applied alone or together with sevoflurane (0.05 mM to 5 mM). RESULTS: Currents elicited by GABA 0.01 mM were increased by low sevoflurane concentrations to 183% and decreased by high sevoflurane concentrations (> 1 mM) to 34% (P < 0.05). Ten- to 90%-rise times of the currents were reduced by sevoflurane concentration dependently. At GABA (1 mM), peak currents and 10-90%-rise times decreased with increasing sevoflurane concentrations. A transient current increase was induced by discontinuation of GABA and sevoflurane. Such rebound currents indicate a reversal of an open-channel block by sevoflurane. CONCLUSIONS: Sevoflurane (a) increases the apparent affinity of GABA to the GABAAR, as suggested by the decreased current rise times. This explains the enhancement of the currents induced by low GABA concentrations (0.01 mM). Additionally, sevoflurane (b) induces a picrotoxin-like open-channel block at the GABAAR. The reversal of the open-channel block elicits a delayed GABA response. These findings indicate at least two different sites of action of sevoflurane at this receptor that are both important for an enhanced GABAergic synaptic transmission.

Coadministered nitrous oxide enhances the effect of isoflurane on GABAergic transmission by an increase in open-channel block.

Clinically relevant concentrations of isoflurane (ISO) and nitrous oxide (N2O) enhance chloride currents induced by activating gamma-aminobutyric acid(A) receptors (GABA(A)R). Channel blocking by ISO overcomes the enhancing effect at higher concentrations. In this study, the effect of coadministered ISO and N2O on responses evoked by GABA in transfected human embryonic kidney 293 cells carrying alpha1beta2gamma2L GABA(A)R was investigated. Patch-clamp recordings from these cells were performed in the whole cell mode. A piezo-driven "liquid filament" drug application system was used to apply solutions of GABA, ISO, and N2O. Increasing the concentration of ISO in steps from 0.15 to 1.2 mM resulted in a bell-shaped concentration-response curve for GABA-induced currents. The maximum increase in current (1.51 +/- 0.14-fold) was seen at 0.45 mM ISO (about 1 minimum alveolar concentration, EC50). N2O (29.2 mM) increased GABA-evoked currents 1.54 +/- 0.10-fold. The enhancing effects of ISO and N2O on the GABAergic response were not additive. However, a transient current, associated with the rapid withdrawal of ISO from the receptor, was markedly increased by N2O. Such rebound currents probably reflect the transition from a "channel-blocked" to a "reopened" state. An open-channel block at ligand-gated receptors can prolong postsynaptic currents. Thus, we conclude that coadministered N2O could increase the enhancing effect of ISO on the GABAergic transmission by an increase in open-channel block at the GABA(A)R.

Nitrous oxide and xenon increase the efficacy of GABA at recombinant mammalian GABA(A) receptors.

We investigated the interactions between recombinant gamma-aminobutyric acid receptor complex (GABA(A)R) and nitrous oxide (N(2)O) or xenon (Xe). Human embryonic kidney cells (HEK 293) were transfected with rat cDNA for alpha(1)beta(2)gamma(2L) or for alpha(1)beta(2) recombinant GABA(A)R subunits. Patch clamp techniques were used in the whole-cell mode to evaluate the effect of N(2)O and Xe on GABA-induced currents. A piezo-driven "liquid filament switch" was used for fast application. Both N(2)O (100%, 29.2 mM) and Xe (100%, 3.9 mM) reversibly increased GABA-induced currents through the alpha(1)ss(2)gamma(2L) and the alpha(1)beta(2) GABA(A)R channels. The potentiating effect of N(2)O or Xe on peak currents was prominent at small GABA concentrations (10(-7) to 10(-5) M). The addition of N(2)O or Xe increased the efficacy of GABA (10(-7) to 10(-3) M). Both N(2)O and Xe significantly decreased the risetime((10%-90%)) of the currents elicited by small GABA concentrations. At the concentrations used, neither N(2)O nor Xe had an intrinsic effect. We conclude that, similar to other anesthetics, both N(2)O and Xe increase the efficacy of GABA at the GABA(A)R and enhance inhibitory GABAergic synaptic transmission.

Dual action of isoflurane on the gamma-aminobutyric acid (GABA)-mediated currents through recombinant alpha(1)beta(2)gamma(2L)-GABA(A)-receptor channels.

Isoflurane (ISO) increased the agonist-induced chloride flux through the gamma-aminobutyric acid A receptor (GABA(A)R). This may reflect an anesthetic-induced increase in the apparent agonist affinity. A dual effect of anesthetics was postulated for both the nicotinic acetylcholine receptor (nAChR) and the GABA(A)R. We tested the hypothesis that, in addition to a blocking effect, ISO increases gamma-aminobutyric acid (GABA)-gated currents through recombinant GABA(A)R channels. HEK293 cells were transfected with rat cDNA for alpha(1),beta(2),gamma(2L) subunits. Currents elicited by 1 mM or 0. 01 mM GABA, respectively, alone, or with increasing concentrations of ISO, were recorded by using standard patch clamp techniques. ISO reduced the peak current elicited by 1 mM GABA. Currents induced by 0.01 mM GABA were potentiated by small ISO (twofold at 0.5 mM ISO) and inhibited by larger concentrations. Withdrawal of ISO and GABA induced rebound currents, suggesting an open-channel block by ISO. These currents increased with increasing concentrations of ISO. At large concentrations of ISO, the inhibitory effect predominated and was caused by, at least partly, an open-channel block. At small concentrations of ISO, potentiation of the GABA-gated currents was more prominent. This dual action of ISO indicates different binding sites at the GABA(A)R. The balance between potentiation and block depends on the concentrations of both ISO and GABA.

Increasing isoflurane concentration may cause paradoxical increases in the EEG bispectral index in surgical patients.

We have studied the effects of increases in isoflurane concentration on the EEG bispectral index (BIS) in 70 patients anaesthetized with isoflurane-nitrous oxide-sufentanil for major abdominal surgery. During surgery, baseline BIS was recorded at 0.8% end-tidal isoflurane with nitrous oxide in oxygen (FIO2 0.35). After this, end-tidal isoflurane was increased to 1.6% for 15 min and decreased subsequently to 0.8% for 20 min to assess recovery. In 20 patients, BIS decreased from a mean value of 40 (SD 9) during baseline to 25 (10) at 1.6% isoflurane. In contrast, BIS did not change in 23 patients and increased in 27 patients from 35 (6) to 46 (8) as isoflurane was increased to 1.6%. In all patients, BIS recovered to baseline values at 0.8% isoflurane. The changes in BIS with increasing isoflurane concentration were not related to drugs or differences in physiological variables, which did not differ between groups. Patients with a decrease in BIS were significantly younger (38 (range 18-68) yr) than those with unchanged (55 (26-70) yr) or increased (60 (40-70) yr) BIS values (P < 0.001). It is possible that the paradoxical increase in BIS is related to continuous pre-burst EEG patterns consisting of high-frequency activity. This suggests that the use of BIS as a guide for isoflurane administration may be misleading in some patients undergoing surgical procedures.

Lovastatin decreases the receptor-mediated degradation of acetylated and oxidized LDLs in human blood monocytes during the early stage of differentiation into macrophages.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors are used therapeutically to upregulate the LDL receptor-mediated removal of plasma cholesterol by the liver. Several lines of evidence indicate that these drugs also exert direct effects on the metabolism of native and modified LDL in extrahepatic cells. We studied the effects of lovastatin (LOV) on the degradation of native, acetylated, and oxidized LDL, and on levels of mRNA encoding for the LDL, types I and II class A macrophage scavenger, and CD36 receptors in human blood monocytes at different stages of their maturation into adherent macrophages. LOV (10 micromol/L) reduced the degradation of acetylated LDL when added to freshly isolated cells cultured for 2 (81+/-4% of control, P<0.05) and 5 (76+/-6%, of control, P<0.05) days. The degradation of oxidized LDL was also reduced in cells treated with LOV for 2 days after seeding (51+/-3% of control, P<0. 001) but not in 5-day-old cells. LOV had no significant effect on the degradation of either acetylated or oxidized LDL when added to fully matured macrophages allowed to differentiate under control conditions for 7 days before incubations with 10 micromol/L LOV for an additional 2 days. In contrast, LOV increased the degradation of native LDL in these cells at all 3 stages of cell differentiation. LOV also reduced class A types I and II macrophage scavenger receptor and CD36 mRNA levels in 2- and 5-day-old cells but not in the more mature macrophages. These data suggest that 3-hydroxy-3-methylglutaryl-coenzyme A inhibitors may reduce the expression and function of the class A types I and II macrophage scavenger receptor and CD36 in monocytes, during the early stages of their differentiation into adherent macrophages. These effects, if operative in vivo, may slow down the development of the atherosclerotic plaque and thus contribute to the beneficial effects of these drugs.

Acetylated LDL endocytosis by the human monocytic Mono Mac 6sr cells is not mediated by the macrophage type I and II scavenger receptors.

We recently reported that the human monocytic Mono Mac 6sr cell line constitutively takes up and degrades acetylated (acLDL) and oxidized LDL through receptor-specific pathways. The present studies were undertaken to further characterize the acLDL binding site on a functional and molecular basis. The degradation of acLDL increased during differentiation of Mono Mac 6sr cells with lipopolysaccharide (10 ng/mL, 72 hours) and low concentrations of phorbol 12-myristate 13-acetate (PMA; 0.1 to 1.0 ng/mL, 72 hours). Higher doses of PMA (5 or 10 ng/mL), however, decreased acLDL degradation. Scatchard plots of acLDL binding in untreated and LPS-differentiated Mono Mac 6sr cells were nonlinear and suggested the presence of more than one binding site. Although the ligand specificity of the acLDL receptor in Mono Mac 6sr cells resembles that of the macrophage type I and type II scavenger receptors, we did not detect mRNA of either receptor type in untreated or differentiated Mono Mac 6sr cells by means of Northern blotting and reverse transcription polymerase chain reaction. Furthermore, ligand blotting with 125I-acLDL failed to detect the 220-kD types I and II scavenger receptor protein. Thus, Mono Mac 6sr cells express an acLDL receptor that is distinct from the type I and type II scavenger receptor found in human monocyte-derived macrophages but that, like the latter, is induced during monocytic differentiation.