Differential inhibitory effects of drugs acting at the noradrenaline and 5-hydroxytryptamine transporters in rat and human neocortical synaptosomes.

BACKGROUND AND PURPOSE: Although the amino acid sequences of rat and human 5-hydroxytryptamine (5-HT) and noradrenaline (NA) transporters (i.e. SERT and NET) are highly homologous, species differences exist in the inhibitory effects of drugs acting at these transporters. Therefore, comparison of the potencies of drugs acting at SERT and NET in native human and rat neocortex may serve to more accurately predict their clinical profile. EXPERIMENTAL APPROACH: Synaptosomes prepared from fresh human and rat neocortical tissues were used for [(3)H]-5-HT and [(3)H]-NA saturation and competition uptake experiments. The drugs tested included NA reuptake inhibitors (desipramine, atomoxetine and (S,S)-reboxetine), 5-HT reuptake blockers (citalopram, fluoxetine and fluvoxamine) and dual 5-HT/NA reuptake inhibitors (duloxetine and milnacipran). KEY RESULTS: In saturation experiments on synaptosomal [(3)H]-5-HT and [(3)H]-NA uptake, the dissociation constants did not indicate species differences although a smaller density of both SERT and NET was observed in human tissues. In competition experiments with the various drugs, marked species differences in their potencies were observed, especially at SERT. The rank order of selectivity ratios (SERT/NET) in human neocortex was as follows: citalopram >or= duloxetine = fluvoxamine >or= fluoxetine > milnacipran > desipramine = atomoxetine > (S,S)-reboxetine. Significant species differences in these ratios were observed for duloxetine, atomoxetine and desipramine. CONCLUSIONS AND IMPLICATIONS: This study provides the first compilation of drug potency at native human neocortical SERT and NET. The significant species differences (viz., human vs. rat) in drug potency suggest that the general use of rodent data should be limited to predict clinical efficacy or profile.

Differential modulation of K(+)-evoked (3)H-neurotransmitter release from human neocortex by gabapentin and pregabalin.

Anticonvulsant, analgesic, and anxiolytic effects have been observed both in preclinical and clinical studies with gabapentin (GBP) and pregabalin (PGB). These drugs appear to act by binding to the alpha(2)delta subunit of voltage-sensitive Ca(2+) channels (VSCC), resulting in the inhibition of neurotransmitter release. In this study, we examined the effects of GBP and PGB (mostly 100 microM, corresponding to relatively high preclinical/clinical plasma levels) on the release of neurotransmitters in human neocortical slices. These slices were prelabeled with (3)H-dopamine ((3)H-DA), (3)H-choline (to release (3)H-acetylcholine ((3)H-ACh)), (3)H-noradrenaline ((3)H-NA), and (3)H-serotonin ((3)H-5-HT), and stimulated twice in superfusion experiments by elevation of extracellular K(+) in the presence and absence of GBP and PGB. The alpha(2)delta ligands produced significant inhibitions of K(+)-evoked (3)H-ACh, (3)H-NA, and (3)H-5-HT release between 22% and 56% without affecting (3)H-DA release. Neither drug reduced (3)H-NA release in the presence of L: -isoleucine, a putative alpha(2)delta antagonist. Interestingly, this antagonism did not occur using the enantiomer, D: -isoleucine. These results suggest that GBP and PGB are not general inhibitors of VSCC and neurotransmitter release. Such alpha(2)delta ligands appear to be selective modulators of the release of certain, but not all, neurotransmitters. This differential modulation of neurotransmission presumably contributes to their clinical profile.

Modulation of K+-evoked [3H]-noradrenaline release from rat and human brain slices by gabapentin: involvement of KATP channels.

To elucidate the mechanism of action of the anticonvulsant gabapentin (GBP), we compared its effects on K+-evoked [3H]-noradrenaline ([3H]-NA) release from rat hippocampal and human neocortical slices with those of the KATP channel opener pinacidil and the Na+ channel blockers phenytoin, carbamazepine and lamotrigine. Rat hippocampal and human neocortical slices were loaded with [3H]-NA and superfused. [3H]-NA release was evoked by increasing the extracellular [K+] from 3 to 15 mM. GBP decreased [3H]-NA release from rat hippocampal with a pIC50 of 5.59 and a maximum inhibition of 44%. Concentration-dependent inhibition was also seen in human neocortical slices (39% inhibition with 100 microM GBP). These inhibitory effects were antagonized by the KATP channel antagonist glibenclamide, yielding a pA2 of 7.50 in the rat. The KATP channel opener pinacidil (10 microM), like GBP, decreased [3H]-NA release from rat hippocampal slices by 27% and this effect was also antagonized by glibenclamide. In human neocortical slices the inhibition by pinacidil (10 microM) was 31%. Although phenytoin (10 microM), carbamazepine (100 microM) and lamotrigine (10 microM) also decreased [3H]-NA release (by 25%, 57% and 22%, respectively), glibenclamide did not antagonize the effects of these classical Na+ channel blockers. These findings suggest that GBP inhibits K+-evoked [3H]-NA release through activation of KATP channels. To establish whether the KATP channels under investigation were located on noradrenergic nerve terminals or on other neuronal elements, the effects of GBP were compared in the absence and in the presence of tetrodotoxin (TTX 0.32 microM) throughout superfusion. Since the functional elimination of the perikarya of interneurons by TTX reduced the inhibitory effect of GBP, the KATP channels mediating the effect of GBP may be located on nerve terminals, probably on both noradrenergic and glutamatergic nerve endings.

Stimulus-dependent modulation of [(3)H]norepinephrine release from rat neocortical slices by gabapentin and pregabalin.

Gabapentin (GBP; Neurontin) has proven efficacy in several neurological and psychiatric disorders yet its mechanism of action remains elusive. This drug, and the related compounds pregabalin [PGB; CI-1008, S-(+)-3-isobutylgaba] and its enantiomer R-(-)-3-isobutylgaba, were tested in an in vitro superfusion model of stimulation-evoked neurotransmitter release using rat neocortical slices prelabeled with [(3)H]norepinephrine ([(3)H]NE). The variables addressed were stimulus type (i.e., electrical, K(+), veratridine) and intensity, concentration dependence, onset and reversibility of action, and commonality of mechanism. Both GBP and PGB inhibited electrically and K(+)-evoked [(3)H]NE release, but not that induced by veratridine. Inhibition by these drugs was most pronounced with the K(+) stimulus, allowing determination of concentration-effect relationships (viz., 25 mM K(+) stimulus: GBP IC(50) = 8.9 microM, PGB IC(50) = 11.8 microM). R-(-)-3-Isobutylgaba was less effective than PGB to decrease stimulation-evoked [(3)H]NE release. Other experiments with GBP demonstrated the dependence of [(3)H]NE release inhibition on optimal stimulus intensity. The inhibitory effect of GBP increased with longer slice exposure time before stimulation, and reversed upon washout. Combination experiments with GBP and PGB indicated a similar mechanism of action to inhibit K(+)-evoked [(3)H]NE release. GBP and PGB are concluded to act in a comparable, if not identical, manner to preferentially attenuate [(3)H]NE release evoked by stimuli effecting mild and prolonged depolarizations. This type of modulation of neurotransmitter release may be integral to the clinical pharmacology of these drugs.

Modulation of K(+)-induced synaptosomal calcium influx by gabapentin.

Gabapentin, a drug useful in several neurological and psychiatric disorders, decreased K(+) (15 mM)-induced [Ca(2+)](i) increase in Fura-PE3-loaded rat neocortical synaptosomes (IC(50)=9.7 microM; submaximal inhibition of 28.6%). This effect may indicate a selective modulation of presynaptic Ca(2+) influx in response to depolarizing (pathological) conditions causing excessive neurotransmitter release.

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.

Inhibition of K(+)-evoked glutamate release from rat neocortical and hippocampal slices by gabapentin.

Gabapentin (Neurontin((R))) has preclinical and clinical efficacy as an anticonvulsant, antihyperalgesic, anxiolytic, and neuroprotective drug. Since L-glutamic acid (GLU) is involved in various CNS (central nervous system) disorders, gabapentin may attenuate the release of this neurotransmitter possibly by interacting with the auxiliary alpha(2)delta subunit of voltage-sensitive calcium channels (VSCC). The effects of gabapentin, pregabalin (S-(+)-3-isobutylgaba) and its enantiomer R-(-)-3-isobutylgaba, and N- and P/Q-type VSCC-targeting peptide ligands (omega-conotoxin MVIIA, omega-conotoxin MVIIC, omega-agatoxin TK) were assessed in vitro on K(+)-evoked (endogenous) GLU release from rat neocortical and hippocampal slices. Gabapentin and pregabalin decreased GLU release by 11-26% with R-(-)-3-isobutylgaba being less effective than pregabalin. The reference N- and P/Q-type VSCC-targeting ligands reduced GLU release by 19-55% to implicate these VSCC in this Ca(2+)-dependent process. The inhibitory effect of gabapentin and related compounds on GLU release may reflect a subtle modulation of VSCC function which normalizes pathological changes in neurotransmitter release.

Structure-activity relationship of N-methyl-N-aralkyl-peptidylamines as novel N-type calcium channel blockers.

Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown efficacy in several animal models of stroke and pain. In the process of searching for small molecule N-type calcium channel blockers, we have identified a series of N-methyl-N-aralkyl-peptidylamines with potent functional activity at N-type VSCCs. The most active compound discovered in this series is PD 173212 (11, IC50 = 36 nM in the IMR-32 assays). SAR and pharmacological evaluation of this series are described.

Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas.

We describe the first potent and selective blocker of the class E Ca2+channel. SNX-482, a novel 41 amino acid peptide present in the venom of the African tarantula, Hysterocrates gigas, was identified through its ability to inhibit human class E Ca2+ channels stably expressed in a mammalian cell line. An IC50 of 15-30 nM was obtained for block of the class E Ca2+ channel, using either patch clamp electrophysiology or K+-evoked Ca2+ flux. At low nanomolar concentrations, SNX-482 also blocked a native resistant or R-type Ca2+ current in rat neurohypophyseal nerve terminals, but concentrations of 200-500 nM had no effect on R-type Ca2+ currents in several types of rat central neurons. The peptide has the sequence GVDKAGCRYMFGGCSVNDDCCPRLGCHSLFSYCAWDLTFSD-OH and is homologous to the spider peptides grammatoxin S1A and hanatoxin, both peptides with very different ion channel blocking selectivities. No effect of SNX-482 was observed on the following ion channel activities: Na+ or K+ currents in several cultured cell types (up to 500 nM); K+ current through cloned potassium channels Kv1.1 and Kv1. 4 expressed in Xenopus oocytes (up to 140 nM); Ca2+ flux through L- and T-type Ca2+ channels in an anterior pituitary cell line (GH3, up to 500 nM); and Ba2+ current through class A Ca2+ channels expressed in Xenopus oocytes (up to 280 nM). A weak effect was noted on Ca2+ current through cloned and stably expressed class B Ca2+ channels (IC50 > 500 nM). The unique selectivity of SNX-482 suggests its usefulness in studying the diversity, function, and pharmacology of class E and/or R-type Ca2+ channels.

Reduction of 3,4-diaminopyridine-induced biogenic amine synthesis and release in rat brain by gabapentin.

The anticonvulsant drug gabapentin has been shown recently to exhibit anxiolytic and analgesic actions in animals. Such actions have been postulated in part to reflect effects on biogenic amine neuronal activity. Therefore the effects of gabapentin on biogenic amine neuronal activity were assessed by measuring the synthesis of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in rat brain and on the release of [3H] NE from rat hippocampal slices both in the presence and absence of the depolarizing agent 3,4-diaminopyridine (DAP). Gabapentin (30 and 100 mg/kg, i.p.) did not alter the basal synthesis rates of NE and DA as assessed by the unchanged accumulation of L-dihydroxyphenylalanine (DOPA) in the NE-enriched hippocampus and cortex and in the DA-enriched striatum and mesolimbic areas. Gabapentin also did not alter 5-HT synthesis as determined by the unaltered accumulation of 5-hydroxytryptophan (5-HTP) in the same brain areas. DAP (2 mg/kg, i.p.) induced a modest but significant increase in DOPA accumulation in the hippocampal, mesolimbic and striatal regions. This DAP-induced increase in DOPA accumulation was antagonized significantly in the hippocampus and mesolimbic regions by gabapentin at 30 and 100 mg/kg and in striatum by 100 mg/kg; a 10 mg/kg dose was inactive. DAP increased selectively 5-HT synthesis in hippocampus and this effect was blocked by gabapentin. These findings indicate that the increased synthesis of biogenic amines induced by DAP is antagonized by gabapentin. In support of the in vivo studies, gabapentin was also shown to inhibit the DAP-evoked release of [3H]NE from hippocampal slices. Although the underlying mechanism for these effects is unclear, the present findings nevertheless demonstrate that gabapentin has inhibitory effects on stimulated NE, DA and 5-HT neurons that may be involved in explaining in part the CNS effects of this drug.

Assessment of endothelin receptor subtype-mediated increases of [Ca2+]i in distinct rat cell types using fluorimetric imaging.

Activation of endothelin (ET) receptor subtypes by various agonists causes an increase in [Ca2+]i in different cell types. This effect can be readily monitored in a 96-well plate format by detecting 1-s fluorescence changes of cell-permeant, Ca(2+)-sensitive dyes (e.g., Calcium Green-1 AM) using a fluorimetric imaging plate reader. This device was used to assess the ET receptor subtypes in primary cultures of rat mixed neocortical neuronal/glial cells and aortic smooth-muscle cells. Pharmacologic experiments with several ET receptor agonists and antagonists indicated that the ETA receptor subtype was functionally responsive in the smooth-muscle cells and that the ETB receptor subtype had a similar role in the mixed neuronal/glial cells.

A summary of mechanistic hypotheses of gabapentin pharmacology.

Although the cellular mechanisms of pharmacological actions of gabapentin (Neurontin) remain incompletely described, several hypotheses have been proposed. It is possible that different mechanisms account for anticonvulsant, antinociceptive, anxiolytic and neuroprotective activity in animal models. Gabapentin is an amino acid, with a mechanism that differs from those of other anticonvulsant drugs such as phenytoin, carbamazepine or valproate. Radiotracer studies with [14C]gabapentin suggest that gabapentin is rapidly accessible to brain cell cytosol. Several hypotheses of cellular mechanisms have been proposed to explain the pharmacology of gabapentin: 1. Gabapentin crosses several membrane barriers in the body via a specific amino acid transporter (system L) and competes with leucine, isoleucine, valine and phenylalanine for transport. 2. Gabapentin increases the concentration and probably the rate of synthesis of GABA in brain, which may enhance non-vesicular GABA release during seizures. 3. Gabapentin binds with high affinity to a novel binding site in brain tissues that is associated with an auxiliary subunit of voltage-sensitive Ca2+ channels. Recent electrophysiology results suggest that gabapentin may modulate certain types of Ca2+ current. 4. Gabapentin reduces the release of several monoamine neurotransmitters. 5. Electrophysiology suggests that gabapentin inhibits voltage-activated Na+ channels, but other results contradict these findings. 6. Gabapentin increases serotonin concentrations in human whole blood, which may be relevant to neurobehavioral actions. 7. Gabapentin prevents neuronal death in several models including those designed to mimic amyotrophic lateral sclerosis (ALS). This may occur by inhibition of glutamate synthesis by branched-chain amino acid aminotransferase (BCAA-t).

P-type Ca2+ channels trigger stimulus-evoked [3H]acetylcholine release from mammalian motor endplates.

In the present experiments it was tested whether omega-agatoxin-IVA, a peptide blocking P-type voltage-dependent Ca2+ channels, inhibits the evoked release of newly synthesized [3H]acetylcholine from the rat phrenic nerve. Release of [3H]acetylcholine was evoked by electrical stimulation of the isolated phrenic nerve (100 or 750 pulses at 5 Hz). omega-Agatoxin-IVA inhibited evoked [3H]acetylcholine release in a concentration-related manner; inhibition started at a concentration of 30 nM with complete block occurring at 500 nM. In conclusion, the present experiments demonstrate that omega-agatoxin-IVA-sensitive P-type Ca2+ channels are critically involved in the regulation of stimulus-induced transmitter release at mammalian motor endplates.

Conodipine-M, a novel phospholipase A2 isolated from the venom of the marine snail Conus magus.

We describe the purification and first biochemical characterization of an enzymatic activity in venom from the marine snail Conus magus. This enzyme, named conodipine-M, is a novel phospholipase A2 with a molecular mass of 13.6 kDa and is comprised of two polypeptide chains linked by one or more disulfide bonds. The amino acid sequence of conodipine-M shows little if any homology to other previously sequenced phospholipase A2 enzymes (PLA2s). Conodipine-M thus represents a new group of PLA2s. This is remarkable, since conodipine-M displays a number of properties that are similar to those of previously characterized 14-kDa PLA2s. The enzyme shows little, if any, phospholipase A1, diacyglycerol lipase, triacylglycerol lipase, or lysophospholipase activities. Conodipine-M hydrolyzes the sn-2 ester of various preparations of phospholipid only in the presence of calcium and with specific activities that are comparable to those of well known 14-kDa snake venom and pancreatic PLA2s. The Conus enzyme binds tightly to vesicles of the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol and catalyzes the hydrolysis of this substrate in a processive fashion. Conodipine-M does not significantly discriminate against phospholipids with unsaturated versus saturated fatty acids at the sn-2 position or with different polar head groups. Linoleoyl amide and a phospholipid analog containing an alkylphosphono group at the sn-2 position are potent inhibitors of conodipine-M. We suggest that the functional resemblance of conodipine-M to other PLA2s might be explained by the utilization of similar catalytic residues.

Synthesis and pharmacological evaluation of phenylacetamides as sodium-channel blockers.

The synthesis and structure-activity relationships of a series of phenylacetamides related to N-[3-(2,6-dimethyl-1-piperidinyl)propyl]-alpha-phenylbenzeneacetamide (1) (PD85639) acting at the voltage-dependent Na+ channel are described. All structural variations for this study were made in the phenylacetic acid portion of these molecules, and the compounds were synthesized by coupling the appropriately substituted phenylacetic acid derivative with 3-[1-(2,6-dimethyl)piperidinyl]-propanamine using standard methods of amide formation. Compounds were tested as inhibitors of [3H]batrachtoxinin binding in rat neocortical membranes and also as inhibitors of veratridine-induced Na+ influx in Chinese hamster ovary cells expressing type IIA Na+ channels. Diphenylacetic acid derivatives with halogenated aromatic rings (12-15) were very potent in both assays, while alkoxy and alkyl substitution did not affect activity (16 and 17). Selected compounds were tested as potential neuroprotective agents in two cell culture assays involving inhibition of veratridine-induced and hypoxia-induced lactate dehydrogenase release. Compound 15 was equipotent with flunarizine, a reference compound in both neuroprotection assays.

Characteristics of [125I]omega-conotoxin MVIIA binding to rat neocortical membranes.

[125I]omega-Conotoxin MVIIA (omega-CTM) binding to N-type voltage-sensitive calcium channels (VSCCs) was characterized using rat neocortical membranes. [125I]omega-CTM bound rapidly and with high affinity; these parameters were similar to binding using omega-conotoxin GVIA ([125I]omega-CTG). Unlike [125I]omega-CTG, however, [125I]omega-CTM readily dissociated from its binding site. Monovalent and divalent cations, polyamines, and aminoglycosides inhibited [125I]omega-CTM binding. Since [125I]omega-CTM appears to bind to the same site as [125I]omega-CTG in mammalian neurons, the reversibility of [125I]omega-CTM binding makes this ligand preferable for equilibrium binding analyses.

K(+)-stimulated 45Ca2+ flux into rat neocortical mini-slices is blocked by omega-Aga-IVA and the dual Na+/Ca2+ channel blockers lidoflazine and flunarizine.

High-threshold neuronal voltage-sensitive Ca2+ channels (VSCCs) have been classified into at least three subtypes, including L, N, and P, based on biophysical and pharmacological criteria. We examined K(+)-induced 45Ca2+ flux into rat neocortical mini-slices to determine which of these subtype(s) might be involved in this phenomenon. Neither the L-type Ca2+ channel antagonist isradipine at 10 microM nor the N-type antagonist omega-conotoxin GVIA at 1 microM were effective antagonists of 45Ca2+ flux in this model. However, the P-type Ca2+ channel antagonist, omega-Aga-IVA, blocked 70% of flux at 200 nM, with an IC50 of 17 nM, strongly implicating P-type Ca2+ channel involvement in K(+)-stimulated Ca2+ entry into mammalian nerve terminals. About 30% of the flux response was resistant to the action of omega-Aga-IVA, suggesting that a still uncharacterized subtype of VSCC is involved in Ca2+ entry into mammalian nerve terminals. Both the omega-Aga-IVA sensitive and insensitive components of 45Ca2+ flux were blocked by the diphenylalkylpiperazines, lidoflazine and flunarizine (IC50 = 6.4 microM and 11 microM, respectively), which have dual Na+/Ca2+ channel blocking actions.

The non-dihydropyridine L-type voltage-sensitive calcium channel activator FPL 64176 enhances K(+)-evoked efflux of [3H]norepinephrine from rat neocortical slices.

The non-dihydropyridine FPL 64176 (methyl-2,5-dimethyl-4-(2-phenylmethyl)benzoyl-[1-H]pyrrole-3-carboxy la te) was tested for an interaction with neuronal L-type voltage-sensitive calcium channels (L-VSCCs) by using a [3H]isradipine ([3H]ISR) binding assay, and for its ability to enhance K(+)-evoked [3H]norepinephrine ([3H]NE) release from rat neocortical slices. The classical L-VSCC activator, the dihydropyridine (DHP) BAY K 8644, was also used for comparative purposes. FPL 64176 and BAY K 8644 both produced a similar concentration-dependent enhancement of 15 mM K(+)-evoked [3H]NE release which could be completely blocked by the L-VSCC blocker ISR (0.1 microM). FPL 64176, in contrast to BAY K 8644, was a very weak inhibitor of [3H]ISR binding to L-VSCCs. These findings indicate that FPL 64176 is a novel non-dihydropyridine L-VSCC activator, most probably by acting on a site different from the DHP binding site.

Differential profile of the CCKB receptor antagonist CI-988 and diazepam in the four-plate test.

The cholecystokininB receptor antagonist CI-988 ([R-(R*,R*)]-4-[[2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2- [[(tricyclo[3.3.1.1(3,7)]dec-2-yloxy)carbonyl]amino]- propyl]amino]-1-phenylethyl]amino]-4-oxobutanoic acid compound with 1-deoxy-1-(methylamino)-D-glucitol (1:1)) and the benzodiazepine receptor agonist diazepam were tested for potential anxiolytic effects on punished exploratory behavior in the four-plate test using mice. Diazepam (0.31-5 mg/kg PO) increased the number of shocks taken in a dose-dependent manner, an effect blocked by the benzodiazepine receptor antagonist flumazenil. CI-988 (0.00001-1 mg/kg PO) tended to increase the number of delivered shocks over the chosen dose range; this effect was, however, not dose-related or as large as that produced by diazepam. A limited testing of the 5-hydroxytryptamine3 receptor antagonist ondansetron (0.1 and 1 mg/kg PO) suggested an effect similar to CI-988. These results indicate that distinct and contrasting dose-response profiles exist for these classical and atypical drugs in an animal model of anxiety based on electric shock.