Effect of flumazenil on the memory-enhancing properties of (−)-nicotine in rodents

The effect of the benzodiazepine-receptor antagonist flumazenil on the facilitatory effect of (?)-nicotine on memory in septal-lesioned rats in a spatial task and in the inhibitory avoidance test in mice was investigated. In the two-platform spatial discrimination test, septallesioned rats exhibited a significant number of errors in comparison to sham animals, an effect that can be reversed by the administration of (?)-nicotine during the training phase. Flumazenil did not affect the performance of septal-lesioned rats but it blocked the facilitatory effect of (–)-nicotine on lesioned rats. In the inhibitory avoidance test in mice,(?)-nicotine as well as flumazenil facilitated retention of the test at 0.62 and 10 μmol/kg, respectively. However, a low-noneffective dose of flumazenil blocked the memory enhancing effect of (?)-nicotine. The blockade of the facilitatory effect of (?)-nicotine by flumazenil in normal and septal-lesioned animals suggests that the cognitive effect of (?)-nicotine requires the activation of benzodiazepine receptors. © 1994 Wiley-Liss, Inc.     

Enhancement of the discriminative stimulus effects of phencyclidine by the tetracycline antibiotics doxycycline and minocycline in rats

RATIONALE: Tetracycline antibiotics share some neuroprotective and CNS effects with N-methyl- D-aspartate (NMDA) receptor antagonists. OBJECTIVES: The acute effects of two tetracycline antibiotics were compared to those of the prototypic NMDA antagonist phencyclidine (PCP). METHODS: The effects of minocycline (10-56 mg/kg) and doxycycline (10-56 mg/kg) were evaluated in Sprague-Dawley rats trained to discriminate 2.0 mg/kg IP of PCP from saline under a fixed ratio schedule of food presentation. RESULTS: Even though minocycline and doxycycline did not substitute for PCP, pretreatment with 32 mg/kg of either drug produced leftward shifts in the PCP dose-response curve. The 32 mg/kg dose of minocycline also produced a leftward shift in the dose-response curve for dizocilpine (MK-801), another NMDA channel blocker, in the same subjects. CONCLUSIONS: Tetracycline antibiotics may interact either directly or indirectly with NMDA receptors. This suggests that they might be utilized in treatment of brain disorders in which NMDA receptor over-activation has been implicated.     

Modulation of the discriminative stimulus produced by pentylenetetrazol by centrally administered drugs

Pentylenetetrazol is anxiogenic in humans and produces an interoceptive discriminative stimulus in rats which is mimicked by anxiogenic drugs and other treatments and antagonized by anxiolytic drugs. It was proposed that the discriminative stimulus of pentylenetetrazol originates centrally. This hypothesis was tested by injecting small amounts of anxiogenic or anxiolytic drugs into the brain and comparing their ability to mimic or block, respectively, the response to pentylenetetrazol, observed after systemic injection. Food-restricted rats were trained in a two-lever operant task to discriminate the interoceptive discriminative stimulus produced by pentylenetetrazol. Intraperitoneal or intracerebroventricular injection of Ro 5-3663 was substituted in a dose-dependent manner for the stimulus produced by systemically administered pentylenetetrazol. Diazepam injected systemically, blocked the pentylenetetrazol-like stimulus associated with Ro 5-3663 administered systemically or centrally. Midazolam injected intracerebroventricularly and in a dose-dependent manner, antagonized the discriminative stimulus produced by systemic injection of pentylenetetrazol. When injected into the amygdala, midazolam also antagonized in a dose-dependent manner the pentylenetetrazol-induced stimulus. Thus, these data suggest that there are sites in the CNS for both the initiation of a pentylenetetrazol-like stimulus by Ro 5-3663 and the antagonism of the stimulus produced by pentylenetetrazol by midazolam.     

Interaction of the discriminative stimulus properties of diazepam and ethanol in pigeons

One group of pigeons (n = 5) was trained to discriminate between the effects induced by 5.6 mg/kg of diazepam (DZP) and the vehicle whereas other pigeons (n = 5) had to discriminate between 3.0 g/kg of ethanol (ETOH) and the vehicle, administered intragastrically (IG) 10 and 40 min prior to the training sessions respectively. Once trained, the pigeons were tested with either diazepam or ethanol alone and in combination. The birds trained to discriminate between DZP and the vehicle mostly performed non-drug associated responses when tested with ETOH (0.56 to 3.0 g/kg). Tests with other doses of DZP (0.3 to 3.0 mg/kg) in the diazepam-trained birds resulted in an ED50 value of 1.4 mg/kg. The birds trained to discriminate between ETOH and the vehicle generalized DZP to ETOH, the ED50 value for diazepam being 3.0 mg/kg. Tests with other doses of ETOH (0.56 to 2.0 g/kg) in this latter group resulted in an ED50 value of 1.3 g/kg. Tests with combinations of DZP and ETOH produced a shift of the dose-response curves to the left indicating drug additivity. The discrimination of 5.6 mg/kg of IG administered DZP but not that of ETOH (3.0 g/kg) was attenuated by injections of the analeptic bemegride (ED50 = 5.5 mg/kg), thus suggesting a difference in the cueing processes of the two drugs. When tested singly, bemegride induced non-drug responding or complete suppression of responding in the birds at the doses of 3.0 and 10.0 mg/kg respectively. In conclusion, the discriminable effects of DZP and ETOH are additive or even supra-additive, but the stimulus properties of the two drugs are not identical.     

Cerebral uptake of 11C—Ro 15—1788 and its acid metabolite 11C—Ro 15—3890; PET study in healthy volunteers

The benzodiazepine antagonist Ro 15–1788 and its acid metabolic product Ro 15–3890 were labelled with ¹¹C and used in a PET study to examine their ability to penetrate the blood-brain barrier in man. After intravenous administration to healthy volunteers, ¹¹C-Ro 15–1788 immediately accumulated to a large extent in the brain, where about 5 per cent of the dose injected was still present after 20 min. ¹¹C-Ro 15–3890, on the other hand, did not accumulate significantly in the central nervous system. At 20 min after the injection less than 0·1 per cent of the radioactivity injected was present in the brain. The results allow the conclusion that the acid metabolite of Ro 15–1788 formed by peripheral metabolism does not significantly contribute to brain radioactivity levels after intravenous injection of ¹¹C-Ro 15–1788 to man. The results also demonstrate the potential of the PET technique to analyse pharmacokinetic aspects of drug transport into the central nervous system in man.     

Modulation of the ethanol-like discriminative stimulus effects of diazepam and phencyclidine by L-type voltage-gated calcium-channel ligands in rats

Rationale: Administration of voltage-gated calcium-channel (VGCC) modulators with ethanol can result in enhancement or attenuation of some behavioral effects of ethanol, including its discriminative stimulus effects. Objectives: The present study used a drug- discrimination paradigm to characterize modulation of the ethanol-like discriminative stimulus effects of a γ-amino-butyric acid (GABA)_A and N-methyl-d-aspartate (NMDA) ligand by administration of VGCC ligands. Methods: Two groups of adult male Long-Evans rats were trained to discriminate either 1.0 g/kg ethanol (n=8) or 2.0 g/kg ethanol (n=9) from water under a fixed-ratio (FR) 20 schedule of food presentation. Following training, ethanol substitution tests were conducted with cumulative doses of the GABA_A-positive modulator diazepam (0.3–10 mg/kg, i.p.) (DZP) and the uncompetitive NMDA antagonist phencyclidine (0.3–5.6 mg/kg, i.p.) (PCP). Next, a single dose of the VGCC antagonist nimodipine, nifedipine, isradipine, or the VGCC agonist (–)-BAY k 8644 (0.3 mg/kg, i.p.) was administered prior to a cumulative DZP or PCP dose–response determination. Results: None of the VGCC modulators produced robust or consistent alterations in the ethanol-like discriminative stimulus effects of DZP in animals trained with either 1.0 g/kg or 2.0 g/kg ethanol. However, the ethanol-like discriminative stimulus effects of PCP were significantly enhanced in the presence of the VGCC antagonists and attenuated in the presence of the agonist in animals trained with 2.0 g/kg ethanol. Conclusions: Overall, these data show that VGCC modulation is not a robust component of ethanol-like discriminative stimulus effects of DZP in animals trained with 1.0 g/kg or 2.0 g/kg ethanol. However, the ethanol-like effects of PCP, particularly at higher training doses, appear to be modulated by dihydropyridine-sensitive VGCCs. Received: 26 August 1999 / Final version: 22 October 1999     

The discriminative stimulus effects of ethanol are mediated by NMDA and GABAA receptors in specific limbic brain regions

 This study was conducted to assess the involvement of N-methyl-d-aspartate (NMDA) and γ-aminobutyric acid (GABA) receptor systems, located in specific limbic brain regions, in the discriminative stimulus effects of ethanol. Male Long-Evans rats were trained to discriminate between intraperitoneal (IP) injections of ethanol (1 g/kg) and saline on a two-lever drug discrimination task. The rats were then implanted with bilateral injector guides aimed at the nucleus accumbens core (AcbC), prelimbic cortex (PrLC), hippocampus area CA1 (CA1), or extended amygdala (i.e., at the border of the central and basolateral nuclei). Infusions of the non-competitive NMDA antagonist MK 801 in the AcbC or CA1 resulted in dose-dependent full substitution for IP ethanol. MK 801 infusion in the PrLC or amygdala failed to substitute for ethanol. Injection of the competitive NMDA antagonist CPP in the AcbC also failed to substitute for ethanol. Co-infusion of MK 801 in the hippocampus potentiated the effects of MK 801 in the AcbC, whereas NMDA infusion in the hippocampus attenuated the ability of MK 801 in the AcbC to substitute for ethanol. The direct GABA_A agonist muscimol resulted in dose-dependent full substitution for IP ethanol when it was injected into the AcbC or amygdala, but failed to substitute when administered in the PrLC. Co-infusion of MK 801, but not CPP, potentiated the effects of muscimol in the AcbC. These results demonstrate that ethanol’s discriminative stimulus function is mediated centrally by NMDA and GABA_A receptors located in specific limbic brain regions. The data also suggest that the discriminative stimulus effects of ethanol are mediated by interactions between ionotropic GABA_A and NMDA receptors in the nucleus accumbens, and by interactions among brain regions. Received: 2 December 1997 / Final version: 24 January 1998     

Effects of Ro 15-4513, fluoxetine and desipramine on the intake of ethanol, water and food by the alcohol-preferring (P) and -nonpreferring (NP) lines of rats

The effects of the IP administration of RO 15-4513 (1,2 and 4 mg/kg), fluoxetine (5 and 10 mg/kg) and desipramine (5 and 10 mg/kg) on the intake of 10% ethanol, H₂O and food were determined in the selectively bred alcohol-preferring (P) and -nonpreferring (NP) lines of rats with daily access to fluids being limited to single 2-hour sessions. The imidazobenzodiazepine Ro 15-4513 (a partial inverse benzodiazepine agonist) significantly reduced the intake of 10% ethanol by the P rats to 50–60% of control levels in the first 30 minutes without altering food or H₂O intake. The attenuating actions of 2 mg/kg Ro 15-4513 on ethanol intake could be completely blocked by the central benzodiazepine receptor antagonist Ro 15-1788 (10 mg/kg). Ro 15-1788, by itself, produced no effects on alcohol and H₂O consumption. The 5 mg/kg dose of fluoxetine significantly reduced 10% ethanol intake by the P rats to 20% of control values without altering either H₂O or food consumption. The 10 mg/kg dose of fluoxetine further reduced ethanol intake by the P rats, but this dose also reduced daily food intake to approximately 70% of normal. Desipramine at both doses significantly (p<0.05) reduced both ethanol and food uptake by the P rats and had a tendency to reduce H₂O consumption as well. In general, the three drugs had effects in the NP rats similar to those observed for the P group, although the effects on 10% ethanol intake were difficult to compare because of the low, variable intake of alcohol by the NP group. The data are consistent with the involvement of serotonin and the GABA-benzodiazepine receptor complex in alcohol drinking behavior.     

ABT-089 [2-methyl-3-(2-(s)-pyrrolidinylmethoxy) pyridine dihydrochloride]: Discriminative stimulus properties and electrophysiological actions

ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy) pyridine dihydrochloride] is a novel cholinergic channel modulator (ChCM) with cognitive-enhancing and neuroprotective activities. Experiments were conducted to determine the discriminative stimulus properties of ABT-089 in comparison with (–)-nicotine. While rats were able to discriminate (–)-nicotine (1.9 μmol/kg s.c.) from saline in 43 ± 1.9 days, they were not able to discriminate ABT-089 (19 or 62 μmol/kg s.c.) from a saline solution after 64 days of training. In rats trained to discriminate 1.9 μmol/kg (–)-nicotine from saline, ABT-089 induced a reduced maximal generalization (up to 52% with 6.2–190 μmol/kg, s.c.) and was 100 times less potent than (–)-nicotine to induce the cue. A-94224.3, the R-enantiomer of ABT-089, induced a saline response at 62 μmol/kg in nicotine-trained rats. The effects of (–)-nicotine and ABT-089 were completely blocked by the cholinergic channel blocker, mecamylamine (15 μmol/kg). Consistent with its oral bioavailability, ABT-089 exhibited comparable effects in (–)-nicotine-trained rats after oral administration. Electrophysiological studies in dopamine-containing neurons in the ventral tegmental area indicate that ABT-089 is a weak partial agonist that can also block the excitatory actions of (–)-nicotine. The low intrinsic efficacy of ABT-089 to cross-generalize with (–)-nicotine is consistent with the minimal activity of the compound at the ganglia-like α3 subtype. Drug Dev. Res. 40:259–266, 1997. © 1997 Wiley-Liss, Inc.     

Sex differences in the discriminative stimulus effects of m-chlorophenylpiperazine and ethanol withdrawal

Rationale: The serotonergic system plays a role in regulation of anxiety and ethanol withdrawal (EW). Nevertheless, few studies have assessed sex differences in serotonergic effects on EW. Objectives: This study examined sex differences in the anxiogenic stimu-li induced by a serotonin (5-HT)_1b/2 agonist, meta- chlorophenylpiperazine (mCPP), prior to ethanol and during EW. Methods: Gonadectomized or sham-operated adult male and female rats and 17β-estradiol (2.5 mg, 21-day release, s.c.) -replaced ovariectomized (OVX) rats were trained to discriminate mCPP (1.2 mg/kg, i.p.) from saline in a two-lever choice task for food. Latency to the first lever press and mCPP lever selection were measured following mCPP (0–1.2 mg/kg). Rats then received chronic ethanol-containing liquid diet (6.5%) for 10 days and were tested for mCPP lever selection 12 h and 36 h after removal of ethanol. Results: Fewer sham female and β-estradiol-replaced OVX rats selected the mCPP lever than male or OVX rats, and showed an increased initiation latency after mCPP injection. During EW (12 h and 36 h), fewer sham female and β-estradiol-replaced OVX rats responded on the mCPP-lever after saline injection as well as after mCPP challenge than male or OVX rats. Castration did not alter any response of male rats to mCPP. Conclusions: (1) mCPP discrimination is a useful measure of EW in male and female rats; and (2) sham female and β-estradiol-replaced OVX rats are less sensitive to the discriminative stimulus prior to and during EW, but more sensitive to impaired behavioral initiation induced by mCPP than male or OVX rats. Received: 3 August 1999 / Final version: 23 November 1999     

NMR structural characterization of cecropin A(1–8) – magainin 2(1–12) and cecropin A(1–8) – melittin(1–12) hybrid peptides

Abstract: In order to elucidate the structure–antibiotic activity relationships of the peptides, the three-dimensional structures of two hybrid peptides, CA(1–8) – MA(1–12) and CA(1–8) – ME(1–12) in trifluoroethanol-containing aqueous solution were investigated by NMR spectroscopy. Both CA(1–8) – MA(1–12) and CA(1–8) – ME(1–12) have strong antibacterial activity but only CA(1–8) – ME(1–12) has hemolytic activity against human erythrocytes. CA(1–8) – MA(1–12) has a hydrophobic 3₁₀-helix of only two turns combined with one short helix in the N-terminus with a flexible hinge section in between. CA(1–8) – MA(1–12) has a severely bent structure in the middle of the peptide. These structural features as well as the low hydrophobicity of CA(1–8) – MA(1–12) seem to be crucial for the selective lysis against the membrane of prokaryotic cells. CA(1–8) – ME(1–12) has an α-helical structure of about three turns in the melittin domain and a flexible structure with one turn in the cecropin domain connected with a flexible hinge section in between, and these might be the structural features required for membrane distruption against prokaryotic and eukaryotic cells. The central hinge region (Gly9-Ile10-Gly11) in an amphipathic antibacterial peptide is considered to play an important role in providing the conformational flexibility required for ion channel formation of the C-terminal hydrophobic α-helix on cell membrane.     

Effects of the cholinergic channel activator ABT-418 on cortical EEG: Comparison with (−)-nicotine

ABT-418[(S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole] is a novel cholinergic channel activator (ChCA) and, like the prototypic ChCA (−)-nicotine, has cognition enhancing and anxiolytic effects. It appears, however, to have fewer central nervous system (CNS) or peripheral side effects compared to (−)-nicotine. The purpose of this study was to determine whether ABT-418 also is distinguishable from (−)-nicotine on neocortical EEG parameters affected by neuronal nicotinic acetylcholine receptor (nAChR) stimulation. Acute administration of ABT-418 (0.62–6.2 μmol/kg) did not have a significant effect on EEG amplitude using FFT frequency band analysis. In contrast, acute administration of (−)-nicotine (1.9 μmol/kg) produced a sustained EEG desynchronization which was reflected in significantly lowered EEG amplitude, an effect indicative of generalized cortical stimulation. Like (−)-nicotine, however, ABT-418 (0.62–6.2 μmol/kg) dose-dependently reduced the incidence of spontaneous 6–8 Hz neocortical spike wave discharges (high voltage spindles, HVS) in awake 12-month-old rats. The HVS effect of ABT-418 was blocked by the nicotinic antagonist mecamylamine (5.0 μmol/kg), consistent with the inhibition of HVS discharges being due to activation of nAChRs. (−)-Nicotine (6.0 μmol/kg/day) reduced total slow wave sleep (SWS) time during 15 days of subcutaneous infusion. In contrast, ABT-418 (14.0 μmol/kg/day) did not affect SWS time on day 1, but did reduce SWS by day 15 of infusion. On day 15, total SWS duration was reduced 26% and 20% by (−)-nicotine and ABT-418, respectively. The fewer effects of ABT-418 on neocortical EEG and sleep distinguishes this compound from (−)-nicotine. This study suggests that the previously reported behavioral effects ABT-418 may not be a simple consequence of neocortical activation, as ABT-418 did not induce EEG activation at doses which have been found to evoke behavioral responses. © 1996 Wiley-Liss, Inc.     

Saturation analysis of specific 11C Ro 15-1788 binding to the human neocortex using positron emission tomography

The ¹¹C-labelled benzodiazepine antagonist Ro 15–1788 (flumazenil) and positron emission tomography (PET) were used to determine quantitative characteristics of benzodiazepine receptor binding in the neocortex of healthy young men. Saturating doses of unlabelled flumazenil administered i.v., before or together with the ligand-reduced ¹¹C-flumazenil accumulation in the neocortex by about 90 per cent. Saturating doses of unlabelled flumazenil had little effect on the accumulation of radioactivity in the benzodiazepine receptor-poor regions such as pons or white matter. By giving graded doses of unlabelled flumazenil together with the tracer, saturation isotherms were obtained allowing the calculation of receptor density (B_max) and equilibrium dissociation constant (K_d) values on the basis of certain assumptions B_max values were in the order of 90 pmol/g and K_d values in the order of 10 nM in the neocortex. Scatchard and Hill plots of the radioactivity data indicated that ¹¹C-flumazenil binds to saturable sites of a homogeneous population. The data indicate that intravenous doses of 1 or 2 mg flumazenil result in a benzodiazepine receptor occupancy of about 50 per cent. The method described should be useful for studying regional differences in benzodiazepine receptor characteristics in the living human brain in healthy subjects and neuropsychiatric disorders, and also in relation to treatment with drugs interacting with benzodiazepine receptors.     

Interactions of Ro15-4513, Ro15-1788 (flumazenil) and ethanol on measures of exploration and locomotion in rats

The present study investigated the role of the GABA_A-benzodiazepine (BDZ) receptor complex in mediating ethanol (ETOH)-induced increases in exploration (head-dipping) and locomotion of rats in a holeboard test. Male Sprague-Dawley rats were selected based on low basal exploratory rates to increase the likelihood that ETOH would increase these behaviors. The effects of the BDZ partial inverse agonist, Ro15-4513 (2.5 mg/kg), and the BDZ receptor antagonist, Ro15-1788 (flumazenil) (8.0 mg/kg), alone, and in combination with ETOH (0.25, 0.50 and 0.75 g/kg, IP) were investigated. The 0.25 and 0.50 g/kg doses of ETOH markedly increased both exploration and locomotion in low exploratory rats. The ETOH-induced increases were prevented by Ro15-4513 on both measures at a dose that produced no observable intrinsic action; however, this apparent lack of intrinsic activity on exploration may have been related to the low basal rates of responding in the subjects. The BDZ antagonist, flumazenil, completely reversed the antagonistic action produced by Ro15-4513 of the ETOH-induced stimulant effects on locomotion, however, flumazenil exerted only a marginal statistically significant effect on Ro15-4513's actions on head-dipping. When flumazenil was given alone, it increased head-dipping, but was without effect on locomotion. Flumazenil did not affect ETOH-induced increases in locomotion; however, ETOH and flumazenil appeared to show agonistic effects on exlporation. The different effects exerted by flumazenil alone, and in combination with ETOH on head-dipping and locomotion suggest that the actions of flumazenil on these behaviors are mediated through separate mechanisms. The research further suggests that both the anxiolytic and locomotor activational effects of ETOH are mediated through the GABA_A-BDZ receptor complex.I would like to dedicate this paper to the late Dr. Richard G. Lister, a friend, teacher, colleague and exceptional scientist who contributed substantially to the area of alcohol abuse and alcoholism. Over the past years, Dr. Lister investigated the interaction of alcohol with benzodiazepine inverse agonists and benzodiazepine receptor antagonists. His seminal and more recent papers played an important role delineating the neuromechanisms of alcohol in relation to the GABA-benzodiazepine receptor complex. Richard, we will miss you.     

Complex discriminative stimulus properties of (+)lysergic acid diethylamide (LSD) in C57Bl/6J mice

Rationale The drug discrimination procedure is the most frequently used in vivo model of hallucinogen activity. Historically, most drug discrimination studies have been conducted in the rat. With the development of genetically modified mice, a powerful new tool has become available for investigating the mechanisms of drug-induced behavior. The current paper is part of an ongoing effort to determine the utility of the drug discrimination technique for evaluating hallucinogenic drugs in mice.Objective To establish the training procedures and characterize the stimulus properties of (+)lysergic acid diethylamide (LSD) in mice.Methods Using a two-lever drug discrimination procedure, C57Bl/6J mice were trained to discriminate 0.45 mg/kg LSD vs saline on a VI30 sec schedule of reinforcement, with vanilla-flavored Ensure serving as the reinforcer.Results As in rats, acquisition was orderly, but the training dose was nearly five-fold higher for mice than rats. LSD lever selection was dose-dependent. Time-course studies revealed a rapid loss of the LSD stimulus effects. The 5-HT_2A/2C receptor agonist, 2,5-dimethoxy-4-bromoamphetamine [(–)DOB] (1.0 mg/kg), substituted fully for LSD and the 5-HT_1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) (1.6 mg/kg), substituted partially for LSD. Pretreatment with the 5-HT_2A receptor-selective antagonist, MDL 100907, or the 5-HT_1A-selective antagonist WAY 100635, showed that each antagonist only partially blocked LSD discrimination. Substitution of 1.0 mg/kg (–)DOB for LSD was fully blocked by pretreatment with MDL 100907 but unaltered by WAY 100635 pretreatment.Conclusions These data suggest that in mice the stimulus effects of LSD have both a 5-HT_2A receptor and a 5-HT_1A receptor component.     

−(−)Gossypol promotes the apoptosis of bladder cancer cells in vitro

Dysregulation of Bcl2 family member proteins has been associated with poor chemotherapeutic response in bladder cancer, suggesting that agents targeting these crucial proteins may provide an interventional strategy to slow or halt bladder cancer progression and metastasis. In this study, we investigated whether the cottonseed polyphenol, -(-)gossypol, a BH3 mimetic, can reduce the expression of pro-survival, or increase the expression of pro-apoptotic, Bcl2 family proteins and thereby effectively sensitize otherwise resistant bladder cancer cells to the standard chemotherapeutic drugs gemcitabine, paclitaxel and carboplatin. These studies show that gossypol induced apoptosis in both chemosensitive UM-UC2 and chemoresistant resistant UM-UC9 bladder cancer cells in vitro in a dose and time dependent manner via a caspase mediated death signaling pathway. Moreover, in combined treatments, gossypol synergized with gemcitabine and carboplatin to induce apoptosis in chemoresistant bladder cancer cells. This effect was associated with the down-regulation the Bcl-xl and Mcl-1 pro-survival Bcl2 family proteins and up-regulation of the Bim and Puma BH3-only Bcl2 family proteins. Overall, these studies show that gossypol sensitizes bladder cancer cells to standard chemotherapeutic drugs and may provide a promising new strategy for bladder cancer treatment.     

Further investigation of the stimulus properties of chlordiazepoxide and zolpidem. Agonism and antagonism by two novel benzodiazepines

The effects of Ro 16-6028 and Ro 17-1812, two novel benzodiazepines with mixed agonist and antagonist properties, were studied in rats trained to discriminate either chlordiazepoxide or the benzodiazepine receptor ligand zolpidem. In rats discriminating 5 mg/kg chlordiazepoxide from saline both Ro 16-6028 and Ro 17-1812 produced responding on the drug lever. At a dose of 10 mg/kg both compounds gave rise to 100% responding on the lever associated with chlordiazepoxide. The dose-response curve produced by Ro 16-6028 was flatter than that for Ro 17-1812, however. The discriminative cue produced by 2 mg/kg zolpidem did not generalise to either Ro 16-6028 or Ro 17-1812. In contrast, both of those compounds antagonised the zolpidem cue and also antagonised the reduction in response rates produced by zolpidem. These results are consistent with previous findings that Ro 16-6028 and Ro 17-1812 may exert anxiolytic-like effects typical of benzodiazepines while antagonising the depressant actions of benzodiazepine receptor ligands. The results are also consistent with the suggestion that the discriminative cues produced by chlordiazepoxide and zolpidem may be mediated, at least partially, by activity at different sub-types of benzodiazepine receptors.     

Effects of combining ethanol (EtOH) with gamma-hydroxybutyrate (GHB) on the discriminative stimulus, locomotor, and motor-impairing functions of GHB in mice

Rationale Gamma-hydroxybutyrate (GHB) is a drug of abuse that is often coabused with ethanol (EtOH) and has been implicated as a date rape agent in conjunction with EtOH. Much information is lacking regarding the manner in which GHB interacts with EtOH. Objective This study was designed to further characterize the behavioral effects of GHB alone and in combination with EtOH in male Swiss–Webster mice. Methods The effects of GHB (0.1–1.0 g/kg) and EtOH (2.0–5.0 g/kg) alone, as well as the effects of GHB in combination with EtOH, were examined using an automated locomotor activity procedure, a functional observational battery (FOB) and a GHB drug discrimination procedure. Results GHB decreased, whereas EtOH had little effect on locomotor activity. In the FOB, EtOH dose-dependently decreased activity in combination with 0.3 g/kg GHB. Alone, each drug had little effect on the righting reflex, but combining ineffective doses of GHB and EtOH significantly impaired righting. GHB and EtOH decreased forelimb grip strength. Combinations of ineffective doses of GHB and EtOH decreased forelimb grip strength when given together. GHB and EtOH impaired inverted screen performance, and EtOH increased the impairing effects of low, but not high, doses of GHB. GHB and EtOH increased hind limb splay, and EtOH increased the effects of 0.1 and 0.3 g/kg GHB on splay. GHB and EtOH decreased body temperature, and EtOH augmented the temperature-decreasing effects of GHB. EtOH produced less than 50% GHB-like discriminative stimulus effects, and GHB failed to alter the GHB-like discriminative stimulus effects of EtOH. Conclusions Overall, EtOH increased the effects of GHB on several gross measures of behavior and only partially occasioned the discriminative stimulus properties of GHB.     

Neuropharmacological reassessment of the discriminative stimulus properties ofd-lysergic acid diethylamide (LSD)

The neuropharmacological mechanisms underlying the behavioral effects ofd-lysergic acid diethylamide (LSD) were assessed by comparing the discriminative stimulus properties of LSD with those of agonists and antagonists that act selectively at putative serotonin (5-hydroxytryptamine; 5-HT) receptor subtypes (5-HT₁ and 5-HT₂). Male Sprague-Dawley rats (N=23) were trained to discriminate LSD (0.08 mg/kg) from saline and given substitution tests with the following agents: 8-hydroxy-2(di-n-propylamino) tetralin (8-OHDPAT; 0.02–0.64 mg/kg), Ru 24969 (0.2–3.2 mg/kg),m-chlorophenylpiperazine (MCPP; 0.1–1.6 mg/kg), 1-(m-trifluoromethylphenyl)piperazine (TFMPP; 0.1–1.6 mg/kg), and quipazine (0.2–3.2 mg/kg). Only quipazine mimicked LSD. In combination tests, BC 105 (0.2–3.2 mg/kg), 2-bromolysergic acid diethylamide (BOL; 0.1–1.6 mg/kg), Ly 53857 (0.4–3.2 mg/kg), metergoline (0.05–0.8 mg/kg), ketanserin (0.2–3.2 mg/kg), and pipenperone (0.0025–0.08 mg/kg), all of which act as 5-HT₂ antagonists, blocked the LSD cue; only spiperone (0.02–0.32 mg/kg) was without effect. Although commonalities may exist among 5-HT agonists, the present results demonstrate that such agonists are not identical. Since putative 5-HT₁ agonists do not mimic LSD and the LSD cue is potently blocked by 5-HT₂ antagonists, it appears that 5-HT₂ neuronal systems are of greater importance than 5-HT₁ systems in mediating the discriminative stimulus and, perhaps, other effects of LSD.     

The discriminative stimulus properties of U50,488 and morphine are not shared by fedotozine

Fedotozine is a kappa opioid receptor agonist having antinociceptive properties but devoid of diuretic effects. The aim of the study was to evaluate the discriminative stimulus effects of fedotozine at doses previously reported to produce maximal effects in in vivo assays measuring kappa-mediated analgesia. By using a two-lever drug discrimination task, two groups of rats were trained to discriminate either a 3 mg/kg i.p. dose of the kappa opioid agonist, U50,488, or a 5 mg/kg i.p. dose of the mu opioid agonist, morphine, from saline. Once trained, rats were used to conduct tests of stimulus generalization with morphine, U50,488 and fedotozine along with another kappa agonist, CI-977, and another mu agonist, fentanyl. The stimulus effect of U50,488 was shared by CI-977 but not by morphine. Conversely, the stimulus effect of morphine was shared by fentanyl but not by U50,488. Fedotozine (1–10 mg/kg) failed to substitute to either U50,488 or morphine. These results indicate that, when administered at doses fully effective in producing antinociception, the interoceptive stimulus effects of fedotozine, if any, can be distinguished from those produced by U50,488 and morphine.