MicroRNA expression is differentially altered by xenobiotic drugs in different human cell lines.

Several noncoding microRNAs (miR or miRNA) have been shown to regulate the expression of drug-metabolizing enzymes and transporters. Xenobiotic drug-induced changes in enzyme and transporter expression may be associated with the alteration of miRNA expression. Therefore, this study investigated the impact of 19 xenobiotic drugs (e.g. dexamethasone, vinblastine, bilobalide and cocaine) on the expression of ten miRNAs (miR-18a, -27a, -27b, -124a, -148a, -324-3p, -328, -451, -519c and -1291) in MCF-7, Caco-2, SH-SY5Y and BE(2)-M17 cell systems. The data revealed that miRNAs were differentially expressed in human cell lines and the change in miRNA expression was dependent on the drug, as well as the type of cells investigated. Notably, treatment with bilobalide led to a 10-fold increase of miR-27a and a 2-fold decrease of miR-148a in Caco-2 cells, but no change of miR-27a and a 2-fold increase of miR-148a in MCF-7 cells. Neuronal miR-124a was generally down-regulated by psychoactive drugs (e.g. cocaine, methadone and fluoxetine) in BE(2)-M17 and SH-SY5Y cells. Dexamethasone and vinblastine, inducers of drug-metabolizing enzymes and transporters, suppressed the expression of miR-27b, -148a and -451 that down-regulate the enzymes and transporters. These findings should provide increased understanding of the altered gene expression underlying drug disposition, multidrug resistance, drug-drug interactions and neuroplasticity.

d-LSD-induced c-Fos expression occurs in a population of oligodendrocytes in rat prefrontal cortex.

Induction of mRNA or protein for immediate-early genes, such as c-fos, is used to identify brain areas, specific cell types, and neuronal circuits that become activated in response to various stimuli including psychoactive drugs. The objective of the present study was to identify the cell types in the prefrontal cortex in which lysergic acid diethylamide (d-LSD) induces c-Fos expression. Systemic administration of d-LSD resulted in a dose-dependent increase in c-Fos immunoreactivity. Although c-Fos-positive cells were found in all cortical layers, they were most numerous in layers III, IV, and V. d-LSD-induced c-Fos immunoreactivity was found in cells co-labeled with anti-neuron-specific enolase or anti-oligodendrocyte Oligo1. The Oligo1-labeled cells had small, round bodies and nuclear diameters characteristic of oligodendrocytes. Studies using confocal microscopy confirmed colocalization of c-Fos-labeled nuclei in NeuN-labeled neurons. Astrocytes and microglia labeled with glial fibrillary acidic protein antibody and OX-42 antibody, respectively, did not display LSD-induced c-Fos expression. Pyramidal neurons labeled with anti-neurofilament antibody also did not show induction of c-Fos immunoreactivity after systemic d-LSD administration. The present study demonstrates that d-LSD induced expression of c-Fos in the prefrontal cortex occurs in subpopulations of neurons and in oligodendrocytes, but not in pyramidal neurons, astrocytes, and microglia.

Hallucinogen-like effects of N,N-dipropyltryptamine (DPT): possible mediation by serotonin 5-HT1A and 5-HT2A receptors in rodents.

N,N-dipropyltryptamine (DPT) is a synthetic tryptamine hallucinogen which has been used psychotherapeutically in humans, but has been studied preclinically only rarely. In the present studies, DPT was tested in a drug-elicited head-twitch assay in mice, and in rats trained to discriminate lysergic acid diethylamide (LSD), N,N-dimethyl-4-phosphoryloxytryptamine (psilocybin), or 3,4-methylenedioxymethamphetamine (MDMA). A separate group of rats was also trained to recognize DPT itself as a discriminative stimulus, and in all cases, the behavioral effects of DPT were challenged with the selective serotonin (5-HT)2A antagonist M100907, the 5-HT1A selective antagonist WAY-100635, or their combination. In the head-twitch assay, DPT elicited dose-dependent effects, producing a biphasic dose-effect curve. WAY-100635 produced a parallel rightward shift in the dose-effect curve for head twitches, indicative of surmountable antagonism, but the antagonist effects of M100907 were functionally insurmountable. DPT produced partial to full substitution when tested in rats trained to discriminate LSD, psilocybin or MDMA, and served as a discriminative stimulus. In all cases, the antagonist effects of M100907 were more profound than were those of WAY-100635. DPT is thus active in two rodent models relevant to 5-HT2 agonist activity. The effectiveness with which M100907 antagonizes the behavioral actions of this compound strongly suggest that the 5-HT2A receptor is an important site of action for DPT, but the modulatory actions of WAY-100635 also imply a 5-HT1A-mediated component to the actions of this compound.

Hallucinogen-like actions of 2,5-dimethoxy-4-(n)-propylthiophenethylamine (2C-T-7) in mice and rats.

RATIONALE: Few studies have examined the effects of 2,5-dimethoxy-4-(n)-propylthiophenethylamine (2C-T-7) in vivo. OBJECTIVES: 2C-T-7 was tested in a drug-elicited head twitch assay in mice and in several drug discrimination assays in rats; 2C-T-7 was compared to the phenylisopropylamine hallucinogen R(-)-1-(2,5-dimethoxy-4-methylphenyl)-2aminopropane (DOM) in both assays, with or without pretreatment with the selective 5-HT2A antagonist (+)-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol (M100907). Finally, the affinity of 2C-T-7 for three distinct 5-HT receptors was determined in rat brain. METHODS: Drug-elicited head twitches were quantified for 10 min following administration of various doses of either 2C-T-7 or R(-)-DOM, with and without pretreatments of 0.01 mg/kg M100907. In rats trained to discriminate lysergic acid diethylamide (LSD), 2C-T-7 and R(-)-DOM were tested for generalization. In further studies, rats were trained to discriminate 2C-T-7 from saline, then challenged with 0.05 mg/kg M100907. In competition binding studies, the affinity of 2C-T-7 was assessed at 5-HT2A receptors, 5-HT1A receptors, and 5-HT2C receptors. RESULTS: 2C-T-7 and R(-)-DOM induced similar head twitch responses in the mouse that were antagonized by M100907. In the rat, 2C-T-7 produced an intermediate degree of generalization (75%) to the LSD cue and served as a discriminative stimulus; these interoceptive effects were attenuated by M100907. Finally, 2C-T-7 had nanomolar affinity for 5-HT2A and 5-HT2C receptors and lower affinity for 5-HT1A receptors. CONCLUSIONS: 2C-T-7 is effective in two rodent models of 5-HT2 agonist activity and has affinity at receptors relevant to hallucinogen effects. The effectiveness with which M100907 antagonizes the behavioral actions of 2C-T-7 strongly suggests that the 5-HT2A receptor is an important site of action for this compound.

Characterization of a novel effect of serotonin 5-HT1A and 5-HT2A receptors: increasing cGMP levels in rat frontal cortex.

Elucidating the mechanisms of action of hallucinogens has become an increasingly important area of research as their abuse has grown in recent years. Although serotonin receptors appear to play a role in the behavioral effects of the phenethylamine and indoleamine hallucinogens, the signaling pathways activated by these agents are unclear. Here it is shown that administration of serotonin (5-hydroxytryptamine, 5-HT) increased cyclic guanosine monophosphate (cGMP) production in frontal cortical slices of rat brain. The effect of 5-HT was greater than that of N-methyl-D-aspartate (NMDA), a stimulant of cGMP formation in the central nervous system. The 5-HT(2A/2C) receptor phenethylamine agonist, 2,5-dimethoxy-4-methylamphetamine (DOM), increased cGMP content in the slices. Additionally 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), a 5-(HT1A/7) receptor agonist also increased cGMP production. Stimulation of cGMP formation by DOM was prevented by a 5-HT(2A/2C) receptor antagonist, pirenperone, as well as by a 5-HT2A receptor selective antagonist, MDL100907. A 5-HT2C receptor antagonist, SB242084, did not block the effect of DOM. Stimulation of cGMP production by DPAT was blocked by the 5-HT1A receptor antagonist, WAY100635. Stimulation of cGMP formation by serotonin could be prevented by pirenperone or WAY100635. In summary, activation of serotonin 5-HT1A and 5-HT2A receptors increase brain cGMP levels.

Role of serotonergic 5-HT2A receptors in the psychotomimetic actions of phencyclidine.

The psychotomimetic phencyclidine (PCP) alters various behavioural responses involving the serotonergic system including potentiating the discriminative stimulus effects of the phenethylamine hallucinogen, 2,5-dimethoxy-4-methylamphetamine (DOM). The present study was undertaken to test the hypothesis that PCP directly interacts with the 5-HT2A receptor. PC12 cells, a neuronal cell line, were stably transfected with the cDNA encoding the rat 5-HT2A receptor (PC12-5-HT2A). In these cells PCP and the related compounds, ketamine and dizocilpine, did not increase [3H]inositol phosphate generation nor did they alter 5-HT-stimulated phosphoinositide hydrolysis. These compounds also did not display appreciable affinity for the 5-HT2A receptor labelled with [3H]ketanserin. The present study indicates that the behavioural responses to PCP, ketamine and dizocilpine do not involve a direct interaction of these compounds with the 5-HT2A receptor.

Partial generalization of (-)DOM to fluvoxamine in the rat: implications for SSRI-induced mania and psychosis.

Recent reports have implicated selective serotonin-reuptake inhibitors in the induction of psychosis and mania when SSRIs are given in combination with neuroleptics. We hypothesize that the partial substitution of fluvoxamine for the hallucinogen, (-)DOM, in the rat provides evidence for a 5-HT(2)-mediated effect of fluvoxamine which may in turn account for the adverse effects observed in humans. Male Fischer-344 rats were trained with (-)DOM (0.56 mg/kg) as a discriminative stimulus using standard operant procedures. Tests of generalization were then conducted with fluvoxamine either alone or in combination with the 5-HT(1A) antagonist, WAY-100635, the 5-HT(2) antagonist, pirenperone, and the neuroleptics, fluphenazine, chlorpromazine, thioridazine, loxapine, risperidone, and clozapine. In rats trained with (-)DOM, fluvoxamine at a dose of 20 mg/kg yielded a maximum 58% (-)DOM-appropriate response. This partial generalization was potentiated by treatment with WAY-100635 and antagonized by pirenperone, loxapine, risperidone, and clozapine. The present data are compatible with a 5-HT(2)-mediated effect of fluvoxamine which may play a role in SSRI-induced mania and psychosis. It is predicted by the results of this study that the probability of these adverse effects will be increased by the concurrent use of antagonists at 5-HT(1A) receptors and decreased by neuroleptics with antagonistic activity at 5-HT(2) receptors.

Lysergic acid diethylamide and [-]-2,5-dimethoxy-4-methylamphetamine increase extracellular glutamate in rat prefrontal cortex.

The ability of hallucinogens to increase extracellular glutamate in the prefrontal cortex (PFC) was assessed by in vivo microdialysis. The hallucinogen lysergic acid diethylamide (LSD; 0.1 mg/kg, i.p.) caused a time-dependent increase in PFC glutamate that was blocked by the 5-HT(2A) antagonist M100907 (0.05 mg/kg, i.p.). Similarly, the 5-HT(2A/C) agonist [-]-2,5-dimethoxy-4-methylamphetamine (DOM; 0.6 mg/kg, i.p.), which is a phenethylamine hallucinogen, increased glutamate to 206% above saline-treated controls. When LSD (10 microM) was directly applied to the PFC by reverse dialysis, a rapid increase in PFC glutamate levels was observed. Glutamate levels in the PFC remained elevated after the drug infusion was discontinued. These data provide direct evidence in vivo for the hypothesis that an enhanced release of glutamate is a common mechanism in the action of hallucinogens.

Cellular mechanisms of serotonin 5-HT2A receptor-mediated cGMP formation: the essential role of glutamate.

The current study explores the mechanisms by which activation of serotonin(2A) (5-HT(2A)) receptors increase production of cyclic guanosine monophosphate (cGMP) in slices of rat frontal cortex. Contrary to results in cortical slices, stimulation of 5-HT(2A) receptors in cells stably expressing this serotonin receptor did not alter cGMP levels. In cortical slices, stimulation of cGMP formation by 2,5-dimethoxy-4-methylamphetamine (DOM), a 5-HT(2A/2C) receptor agonist, was blocked by tetanus toxin, a substance that prevents vesicular neurotransmitter release. However, this stimulation was not altered by tetrodotoxin, an agent that inhibits depolarization-induced neurotransmitter release. Addition of an N-methyl-d-aspartate (NMDA) receptor antagonist, d-AP-7, but not of an AMPA/kainate receptor antagonist CNQX, completely inhibited DOM-mediated cGMP production in the slices. Combined application of maximally effective concentrations of NMDA and DOM elicited a greater increase in cGMP content than either drug alone. The present study shows that 5-HT(2A) receptors do not directly stimulate cGMP formation, but rather that 5-HT(2A) receptor-mediated cGMP production is dependent on extracellular glutamate activating NMDA receptors. The results indicate that 5-HT(2A) receptor-mediated cGMP production could be at least partially attributed to potentiation of NMDA receptor-mediated cGMP formation.

Characterization of the discriminative stimulus properties of centrally administered (-)-DOM and LSD.

Despite the plausible assumption that the effects of hallucinogens predominantly arise in the central nervous system, most studies of these drugs in intact subjects have been conducted following systemic administration. The objective of the present investigation was to characterize the stimulus effects of (-)2,5-dimethoxy-4-methylamphetamine ((-)-DOM) following intracerebroventricular administration. Chronic indwelling cannulae were implanted into the lateral ventricle of male Fischer 344 rats trained to discriminate systemically administered (-)-DOM or lysergic acid diethylamide (LSD) from saline. Time-course and dose-response relationships for (-)-DOM and LSD administered intracerebroventricularly were established. For both LSD and (-)-DOM, central administration did not change the pretreatment times required for the maximal stimulus effects to occur. However, the onset of the stimulus effect was more rapid following intracerebroventricular administration. Following pretreatment periods that maximize drug-appropriate responding, central administration of (-)-DOM and LSD was approximately 2.4- and 1.5-times more potent, respectively, than systemic administration. The results of this study are consistent with the assumption that the stimulus effects of (-)-DOM and LSD are centrally mediated.

Agmatine attenuates methamphetamine-induced conditioned place preference in rats.

The polyamine agmatine modulates a variety of behavioral effects including the abuse-related effects of opioids and has been proposed as a potential medication candidate for the treatment of opioid abuse. However, little is known of the effects of agmatine on the abuse-related effects of other drugs of abuse. This study examined the effects of agmatine on the rewarding effects of methamphetamine in rats using a conditioned place preference paradigm. Methamphetamine (0.1-1.0mg/kg) dose-dependently increased the time spent in methamphetamine-paired side (place preference). Agmatine, at doses that did not produce place preference or aversion (10-32mg/kg), significantly decreased the development of methamphetamine-induced place preference when agmatine was administered in combination with methamphetamine during place conditioning. Agmatine also significantly decreased the expression of methamphetamine-induced place preference when an acute injection of agmatine was given immediately before test session. These doses of agmatine do not alter the motor activity in rats, suggesting that the observed attenuation of methamphetamine-induced place preference was not due to general behavioral disruption. Together, these data suggests that agmatine attenuates the rewarding effects of methamphetamine and may be able to modulate the abuse liability of methamphetamine.

Effects of imidazoline I(2) receptor agonists and morphine on schedule-controlled responding in rats.

Accumulating evidence indicates that imidazoline I(2) receptor agonists enhance the antinociceptive effects of opioids and therefore may be suitable for combination therapy with opioids for pain treatment. However, little is known of the effects of I(2) receptor agonists on other behavioral effects of opioids. This study used schedule-controlled responding and dose-addition analyses to examine interactions between the µ opioid receptor agonist morphine and two imidazoline I(2) receptor agonists, 2-BFI and BU224. In 8 rats responding under a fixed ratio 10 schedule of food presentation, morphine (3.2-17.8 mg/kg), 2-BFI (3.2-17.8 mg/kg), and BU224 (5.6-17.8 mg/kg) each dose-dependently decreased responding. The addition of fixed proportions of 2-BFI or BU224 shifted the morphine dose-effect curves leftward. The interactions between morphine and 2-BFI or BU224 were infra-additive when the same proportions of morphine and I(2) receptor agonists were mixed; however, the interaction between morphine and I(2) receptor agonists was additive when the drugs were mixed at other proportions. These results provide quantitative evidence that I(2) receptor agonists do not enhance the response rate-decreasing effect of morphine and suggest that the enhancement of morphine antinociception is selective. Together, these results further support the therapeutic potential of combining I(2) receptor agonists and opioids for pain control.

Morphine-induced antinociception in the rat: supra-additive interactions with imidazoline I2 receptor ligands.

Pain remains a significant clinical challenge and currently available analgesics are not adequate to meet clinical needs. Emerging evidence suggests the role of imidazoline I(2) receptors in pain modulation primarily from studies of the non-selective imidazoline receptor ligand, agmatine. However, little is known of the generality of the effect to selective I(2) receptor ligands. This study examined the antinociceptive effects of two selective I(2) receptor ligands 2-BFI and BU224 (>2000-fold selectivity for I(2) receptors over α(2) adrenoceptors) in a hypertonic (5%) saline-induced writhing test and analyzed their interaction with morphine using a dose-addition analysis. Morphine, 2-BFI and BU224 but not agmatine produced a dose-dependent antinociceptive effect. Both composite additive curve analyses and isobolographical plots revealed a supra-additive interaction between morphine and 2-BFI or BU224, whereas the interaction between 2-BFI and BU224 was additive. The antinociceptive effect of 2-BFI and BU224 was attenuated by the I(2) receptor antagonist/α(2) adrenoceptor antagonist idazoxan but not by the selective α(2) adrenoceptor antagonist yohimbine, suggesting an I(2) receptor-mediated mechanism. Agmatine enhanced the antinociceptive effect of morphine, 2-BFI and BU224 and the enhancement was prevented by yohimbine, suggesting that the effect was mediated by α(2) adrenoceptors. Taken together, these data represent the first report that selective I(2) receptor ligands have substantial antinociceptive activity and produce antinociceptive synergy with opioids in a rat model of acute pain. These data suggest that drugs acting on imidazoline I(2) receptors may be useful either alone or in combination with opioids for the treatment of pain.

Effects of imidazoline I2 receptor ligands on morphine- and tramadol-induced antinociception in rats.

Currently available analgesics cannot meet the increasing clinical needs and new analgesics with better therapeutic profiles are in great demand. The imidazoline I2 receptor is an emerging drug target for analgesics. However, few studies have examined the effects of selective I2 receptor ligands on the antinociceptive activity of opioids. This study examined the antinociceptive effects of the opioids morphine (0.1-10 mg/kg) and tramadol (3.2-56 mg/kg), the nonselective I2 receptor ligand agmatine (10-100 mg/kg), and the selective I2 receptor ligands 2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-BFI; 1-10 mg/kg) and 2-(4, 5-dihydroimidazol-2-yl) quinoline hydrochloride (BU224; 1-10mg/kg), alone and in combination, in a warm water tail withdrawal procedure in rats. Morphine and tramadol but not agmatine, 2-BFI or BU224 increased tail withdrawal latency in a dose-related manner at 48°C water. Agmatine and 2-BFI but not BU224 dose-dependently enhanced the antinociceptive effects of morphine and tramadol, shifting the dose-effect curves of morphine and tramadol leftward. The enhancement of agmatine and 2-BFI on morphine and tramadol antinociception was prevented by BU224. These results, combined with the fact that BU224 and 2-BFI share similar behavioral effects under other conditions, suggest that BU224 has lower efficacy than 2-BFI at I2 receptors, and that the enhancement of opioid antinociception by I2 receptor ligands depends on their efficacies.

Psychedelic 5-methoxy-N,N-dimethyltryptamine: metabolism, pharmacokinetics, drug interactions, and pharmacological actions.

5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) belongs to a group of naturally-occurring psychoactive indolealkylamine drugs. It acts as a nonselective serotonin (5-HT) agonist and causes many physiological and behavioral changes. 5-MeO-DMT is O-demethylated by polymorphic cytochrome P450 2D6 (CYP2D6) to an active metabolite, bufotenine, while it is mainly inactivated through the deamination pathway mediated by monoamine oxidase A (MAO-A). 5-MeO-DMT is often used with MAO-A inhibitors such as harmaline. Concurrent use of harmaline reduces 5-MeO-DMT deamination metabolism and leads to a prolonged and increased exposure to the parent drug 5-MeO-DMT, as well as the active metabolite bufotenine. Harmaline, 5-MeO-DMT and bufotenine act agonistically on serotonergic systems and may result in hyperserotonergic effects or serotonin toxicity. Interestingly, CYP2D6 also has important contribution to harmaline metabolism, and CYP2D6 genetic polymorphism may cause considerable variability in the metabolism, pharmacokinetics and dynamics of harmaline and its interaction with 5-MeO-DMT. Therefore, this review summarizes recent findings on biotransformation, pharmacokinetics, and pharmacological actions of 5-MeO-DMT. In addition, the pharmacokinetic and pharmacodynamic drug-drug interactions between harmaline and 5-MeO-DMT, potential involvement of CYP2D6 pharmacogenetics, and risks of 5-MeO-DMT intoxication are discussed.

The effect of chronic treatment with clozapine and haloperidol on stimulus control by DOM.

The present study investigated the effects of chronic treatment with the atypical antipsychotic, clozapine, or the typical antipsychotic, haloperidol, on the stimulus properties of 2,5-dimethoxy-4-methylamphetamine ([-]-DOM) in rats trained to discriminate [-]-DOM (0.3 mg/kg; 75 min pre-treatment time) from vehicle. As compared with control values, treatment with clozapine (25 mg/kg.d) for 7 d caused a statistically significant 57% reduction in [-]-DOM-appropriate responding. Unlike clozapine, treatment with haloperidol (1 mg/kg.d) for 7 d did not affect the stimulus properties of [-]-DOM. These findings demonstrate that a functionally significant decrease in 5-HT2A receptor-mediated activity is a unique component of the in-vivo response to chronic treatment with clozapine but not haloperidol and, therefore, might account for some of the clinical differences associated with atypical antipsychotics.