Human gamma-aminobutyric acid A receptor alpha2 gene moderates the acute effects of alcohol and brain mRNA expression.

Gamma-aminobutyric acid A (GABA(A)) receptors moderate several of the behavioral effects of alcohol. In fact, recent studies have shown an association between the gene for the alpha2-subunit of the GABA(A) receptor (GABRA2) and alcoholism. In the present study, we examined the functional relevance of the GABRA2 gene in alcohol dependence by assessing brain GABRA2 mRNA and GABA(A)alpha2-subunit protein levels in post-mortem prefrontal cortical tissue collected from control and alcohol-dependent individuals. In addition, using an endophenotype approach, we tested whether the GABRA2 gene moderates sensitivity to the acute effects of alcohol in two independent samples from distinct human alcohol challenge studies. Results indicated that GABRA2 mRNA levels significantly differed by GABRA2 genotype. GABRA2 single nucleotide polymorphisms (rs573400, rs279871 and rs279858) were significantly associated with sensitivity to the acute effects of alcohol. Specifically, there was a significant main effect of GABRA2 x breath alcohol concentration on several measures of subjective responses to alcohol, including the hedonic value of alcohol. Importantly, reanalysis of a previous intravenous alcohol administration study confirmed the results of the oral alcohol challenge study. In summary, these results extend previous findings and provide new insights into the putative biobehavioral mechanisms that may moderate the association between the GABRA2 gene, sensitivity to the acute effects of alcohol and ultimately alcohol dependence.

Methamphetamine-induced rapid decrease in dopamine transporter function: role of dopamine and hyperthermia.

Single and multiple high-dose administrations of methamphetamine (METH) differentially decrease dopamine (DA) transporter (DAT) function, as assessed by measuring [(3)H]DA uptake into rat striatal synaptosomes prepared 1 h after treatment. Prevention of METH-induced hyperthermia attenuated the decrease in DAT activity induced by multiple injections of the stimulant. Likewise, this decrease was attenuated by previous depletion of striatal DA levels using alpha-methyl-p-tyrosine (alphaMT) or pretreatment with the D1 and D2 antagonists SCH-23390 and eticlopride, respectively. However, METH-induced hyperthermia was also blocked by alphaMT and eticlopride. Reinstatement of hyperthermia to alphaMT- or eticlopride-pretreated rats partially restored the METH-induced decrease in DAT activity. In contrast, neither prevention of METH-induced hyperthermia depletion of DA, nor DA antagonists altered the decrease in DAT function induced by a single administration of METH. Pretreatment with the antioxidant N-t-butyl-alpha-phenylnitrone prevented part of the decrease in DAT function associated with multiple, but not a single, METH injections. Although not tested directly, additional data presented here suggest that the reduction in DAT activity induced by a single METH administration constitutes a part of the total reduction observed immediately after multiple administrations. Taken together, the results indicate that DA, hyperthermia, and oxygen radicals contribute to a component of the rapid decrease in DAT function induced by multiple injections of METH but do not appear to be associated with the reduction induced by a single administration of the stimulant.

The effects of methamphetamine on serotonin transporter activity: role of dopamine and hyperthermia.

Multiple administrations of methamphetamine (METH) rapidly decreased serotonin (5HT) transporter (SERT) function in rat striatum and hippocampus. The purpose of this study was to identify the mechanisms/ factors contributing to this METH-induced decrease in SERT function. Multiple high-dose METH injections rapidly decreased 5HT uptake without altering binding of the 5HT transporter ligand paroxetine. Hyperthermia contributed to this deficit in transporter function in striatum and hippocampus, as prevention of METH-induced hyperthermia attenuated this decrease. A role for dopamine (DA) was suggested by findings that pretreatment with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine, the D1 antagonist SCH-23390, or the D2 antagonist eticlopride attenuated the METH-induced decrease in striatal, but not hippocampal, SERT activity. These effects were independent of the ability of these DA-antagonizing drugs to prevent METH-induced hyperthermia. These results suggest that DA contributes to the decrease in SERT function caused by multiple METH injections in the striatum, but not hippocampus, and that hyperthermia facilitates these deficits in SERT function in both brain regions. In contrast, the response of SERT to a single administration of METH was DA and hyperthermia independent. These findings suggest that the mechanisms/ factors involved in decreasing SERT activity after a single administration of METH are distinct from that caused by multiple administrations.

Differential effects of methamphetamine on Na(+)/Cl(-)-dependent transporters.

It has been demonstrated that methamphetamine (METH) administration affects Na(+)/Cl(-)-dependent transporters; for example, METH treatment rapidly and reversibly decreases dopamine (DA) and serotonin (5HT) transporter function in rat striatum in vivo, as assessed in synaptosomes prepared from METH-treated rats. Because acute effects of METH on other transporters within this family have been less studied, the responses of norepinephrine (NE) and gamma-aminobutyric acid (GABA) transporters to METH administration(s) were determined. Both single and multiple METH administrations inhibited hippocampal NE uptake 1 h after METH treatment(s). In contrast, striatal GABA uptake was not affected by either treatment paradigm. The effects observed after both single and multiple METH administrations on NE transporters were attributable to increases in K(m,) with no changes in V(max); effects that were eliminated by repeated washing of the synaptosomes. These 'washout' data suggest that residual METH introduced by the in vivo subcutaneous injection(s) directly reduced NE transporter activity in the in vitro assay and that, unlike DA and 5HT transporters, METH did not indirectly alter NE transporter function. Taken together, these data demonstrate differences in the responses of NE, GABA, DA, and 5HT transporters to METH treatment.

Differential effects of psychostimulants and related agents on dopaminergic and serotonergic transporter function.

High-dose administrations of amphetamine, methamphetamine, cathinone, methcathinone or methylenedioxymethamphetamine rapidly decrease dopamine and serotonin transporter function in vivo, as assessed in striatal synaptosomes obtained from drug-treated rats. In contrast, high-dose injections of fenfluramine, cocaine or methylphenidate had little or no effect on the activity of these transporters. Interestingly, the capacity of these agents to directly alter dopamine and serotonin uptake, as assessed in vitro by direct application to rat striatal synaptosomes, did not predict their potential to modulate transporter activity following in vivo administration. These findings demonstrate heretofore-unreported differences in the effects of these agents on monoamine transporter function, and a distinction between drug effects after direct application in vitro vs. administration in vivo.

Oxygen radicals differentially affect Na+/Cl(-)-dependent transporters.

Incubation with the oxygen radical-generating enzyme, xanthine oxidase, dramatically reduced striatal dopamine transporter activity, but was unexpectedly without effect on rat hippocampal norepinephrine uptake. To determine whether environmental differences between the striatum and hippocampus contributed to this lack of oxidative effect on norepinephrine transporters, synaptosomal gamma-aminobutyric acid (GABA) uptake was assessed in both regions. Xanthine oxidase similarly decreased [3H]GABA uptake in both the striatum and hippocampus, supporting the conclusion that environmental differences did not account for the lack of effect on norepinephrine transport. These data suggest that norepinephrine transporters are less vulnerable than other Na+/Cl(-)-dependent transporters to oxidative inactivation.

Makaluvamines vary in ability to induce dose-dependent DNA cleavage via topoisomerase II interaction.

The makaluvamines are marine natural products that were originally isolated because of their cytotoxicity in a cell-based mechanism screen. They have significant anti-cancer activity in animal models. There is, however, disagreement in the literature as to whether these compounds target topoisomerase II via a clinically relevant mechanism. This work shows that the makaluvamines can induce dose-dependent DNA cleavage via topoisomerase II. For most of the makaluvamines the levels of cleavage are significantly below those achieved by equimolar concentrations of etoposide. To some extent these results might explain the discrepancies present in the literature.

Differential regional effects of methamphetamine on the activities of tryptophan and tyrosine hydroxylase.

Administration of high doses of methamphetamine (METH) produces both short- and long-term enzymatic deficits in central monoaminergic systems. To determine whether a correlative relationship exists between these acute and long-term consequences of METH treatment, in the present study we examined the regional effects of METH on tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH) activities in various regions of the caudate nucleus, nucleus accumbens, and globus pallidus. A single METH administration decreased TPH activity 1 h after treatment in the globus pallidus, in the nucleus accumbens, and throughout the caudate; in the anterior caudate, the ventral-medial was more affected than the dorsal-lateral region. In contrast, TH activity was not decreased in either the caudate or the globus pallidus after a single METH administration; however, it was altered in the nucleus accumbens. Seven days after multiple METH administrations, TH and TPH activities were decreased in most caudate regions but not in the nucleus accumbens or globus pallidus. These data demonstrate that (1) the effects of METH on TPH and TH vary regionally; and (2) the short-term and long-term regional responses of TPH to METH in the caudate and globus pallidus correlated. In contrast, METH-induced acute TH responses did not predict the long-term changes in TH activity.

Disposition of codeine in female human hair after multiple-dose administration.

Studies of the disposition of codeine into the hair of female subjects (n = 7) were performed after a multiple-dose protocol. Caucasian female subjects with dark-brown-to-black hair were administered a total dose of 450 mg codeine over 5 days (30 mg was administered three times a day for 5 days). Analyses of codeine and metabolites in plasma, urine, and hair were performed by positive ion chemical ionization gas chromatography-mass spectrometry (GC-MS) on a Finnigan Magnum" mass spectrometer. Hair was plucked from the scalp for 5 weeks and subsequently cut from the scalp for up to 10 weeks. Prior to analysis, plucked hair specimens were cut into three segments: (a) a proximal, 1-cm segment, which contained the root, (b) the next distal, 3-cm segment, which was closest to the scalp, and (c) a segment containing all remaining hair to the natural hair tip. in female subjects, the mean (plus or minus standard error of the mean) hair concentration of codeine in the proximal, 1-cm segment from plucked hair was 2.7 ng/mg (+/- 0.55) at 12 h after the last codeine dose; 0.44 ng/mg (+/- 0.20) still remained in this segment at 5 weeks. Codeine was detected in the next 3-cm hair segment (above the scalp) at 1 week; the average codeine concentration for 10 weeks in the 3-cm, distal segment was 0.54 ng/mg (+/- O.05). No codeine was detected in the remaining hair segment (to the tip) at any time. In male subjects studied previously, the mean hair concentration of codeine in the proximal, 1-cm hair segment was 2.6 ng/mg (+/- 0.34) at 12 h after the last codeine dose, and no codeine was detected at 5 weeks. Codeine was not detected in the next 3-cm hair segment (above the scalp) until 3 weeks, and the average mean codeine concentration for 10 weeks was 0.09 ng/mg (+/- 0.01). Differences noted between male and female subjects in distal codeine hair concentrations were not explained by plasma pharmacokinetics.