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Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a novel approach for the treatment of psychotic symptoms associated with schizophrenia and other neuropsychiatric disorders. We recently reported that the selective M4 PAM VU0152100 produced an antipsychotic drug-like profile in rodents after amphetamine challenge. Previous studies suggest that enhanced cholinergic activity may also improve cognitive function and reverse deficits observed with reduced signaling through the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) in the central nervous system. Prior to this study, the M1 mAChR subtype was viewed as the primary candidate for these actions relative to the other mAChR subtypes. Here we describe the discovery of a novel M4 PAM, VU0467154, with enhanced in vitro potency and improved pharmacokinetic properties relative to other M4 PAMs, enabling a more extensive characterization of M4 actions in rodent models. We used VU0467154 to test the hypothesis that selective potentiation of M4 receptor signaling could ameliorate the behavioral, cognitive, and neurochemical impairments induced by the noncompetitive NMDAR antagonist MK-801. VU0467154 produced a robust dose-dependent reversal of MK-801-induced hyperlocomotion and deficits in preclinical models of associative learning and memory functions, including the touchscreen pairwise visual discrimination task in wild-type mice, but failed to reverse these stimulant-induced deficits in M4 KO mice. VU0467154 also enhanced the acquisition of both contextual and cue-mediated fear conditioning when administered alone in wild-type mice. These novel findings suggest that M4 PAMs may provide a strategy for addressing the more complex affective and cognitive disruptions associated with schizophrenia and other neuropsychiatric disorders.
BACKGROUND AND PURPOSE - Designer β-keto amphetamines (e.g. cathinones, 'bath salts' and 'research chemicals') have become popular recreational drugs, but their pharmacology is poorly characterized.
EXPERIMENTAL APPROACH - We determined the potencies of cathinones to inhibit DA, NA and 5-HT transport into transporter-transfected HEK 293 cells, DA and 5-HT efflux from monoamine-preloaded cells, and monoamine receptor binding affinity.
KEY RESULTS - Mephedrone, methylone, ethylone, butylone and naphyrone acted as non-selective monoamine uptake inhibitors, similar to cocaine. Mephedrone, methylone, ethylone and butylone also induced the release of 5-HT, similar to 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and other entactogens. Cathinone, methcathinone and flephedrone, similar to amphetamine and methamphetamine, acted as preferential DA and NA uptake inhibitors and induced the release of DA. Pyrovalerone and 3,4-methylenedioxypyrovalerone (MDPV) were highly potent and selective DA and NA transporter inhibitors but unlike amphetamines did not evoke the release of monoamines. The non-β-keto amphetamines are trace amine-associated receptor 1 ligands, whereas the cathinones are not. All the cathinones showed high blood-brain barrier permeability in an in vitro model; mephedrone and MDPV exhibited particularly high permeability.
CONCLUSIONS AND IMPLICATIONS - Cathinones have considerable pharmacological differences that form the basis of their suggested classification into three groups. The predominant action of all cathinones on the DA transporter is probably associated with a considerable risk of addiction.
© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.
Amphetamine (AMPH) poses a serious hazard to public health. Defining the molecular targets of AMPH is essential to developing treatments for psychostimulant abuse. AMPH elicits its behavioral effects primarily by increasing extracellular dopamine (DA) levels through the reversal of the DA transporter (DAT) cycle and, as a consequence, altering DA signaling. In Caenorhabditis elegans, an excess of synaptic DA results in a loss of motility in water, termed swimming-induced paralysis (SWIP). Here we demonstrate that AMPH produces SWIP in a time- and dose-dependent manner in wild-type (wt) animals but has a reduced ability to generate SWIP in DAT knock out worms (dat-1). To determine whether D1-like and/or D2-like receptors are involved in AMPH-induced SWIP, we performed experiments in DOP-1 and DOP-4, and DOP-2, and DOP-3 receptor knockout animals, respectively. AMPH administration resulted in a reduced ability to induce SWIP in animals lacking DOP-3, DOP-4, and DOP-2 receptors. In contrast, in worms lacking DOP-1 receptors, AMPH-induced SWIP occurred at wt levels. Using microamperometry on C. elegans DA neurons, we determined that in contrast to wt cells, AMPH failed to promote DA efflux in dat-1 DA neurons. These data suggest that DA efflux is critical to sustaining SWIP behavior by signaling through DOP-3, DOP-4, and DOP-2. In a double mutant lacking both DAT-1 and DOP-1 expression, we found no ability of AMPH to induce SWIP or DA efflux. This result supports the paradigm that DA efflux through C. elegans DAT is required for AMPH-induced behaviors and does not require DOP-1 signaling.
The behavioral effects of psychomotor stimulants such as amphetamine (AMPH) arise from their ability to elicit increases in extracellular dopamine (DA). These AMPH-induced increases are achieved by DA transporter (DAT)-mediated transmitter efflux. Recently, we have shown that AMPH self-administration is reduced in rats that have been depleted of insulin with the diabetogenic agent streptozotocin (STZ). In vitro studies suggest that hypoinsulinemia may regulate the actions of AMPH by inhibiting the insulin downstream effectors phosphotidylinositol 3-kinase (PI3K) and protein kinase B (PKB, or Akt), which we have previously shown are able to fine-tune DAT cell-surface expression. Here, we demonstrate that striatal Akt function, as well as DAT cell-surface expression, are significantly reduced by STZ. In addition, our data show that the release of DA, determined by high-speed chronoamperometry (HSCA) in the striatum, in response to AMPH, is severely impaired in these insulin-deficient rats. Importantly, selective inhibition of PI3K with LY294002 within the striatum results in a profound reduction in the subsequent potential for AMPH to evoke DA efflux. Consistent with our biochemical and in vivo electrochemical data, findings from functional magnetic resonance imaging experiments reveal that the ability of AMPH to elicit positive blood oxygen level-dependent signal changes in the striatum is significantly blunted in STZ-treated rats. Finally, local infusion of insulin into the striatum of STZ-treated animals significantly recovers the ability of AMPH to stimulate DA release as measured by high-speed chronoamperometry. The present studies establish that PI3K signaling regulates the neurochemical actions of AMPH-like psychomotor stimulants. These data suggest that insulin signaling pathways may represent a novel mechanism for regulating DA transmission, one which may be targeted for the treatment of AMPH abuse and potentially other dopaminergic disorders.
Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the distal C terminus of DAT and colocalized with DAT in dopaminergic neurons. CaMKIIalpha stimulated dopamine efflux via DAT in response to amphetamine in heterologous cells and in dopaminergic neurons. CaMKIIalpha phosphorylated serines in the distal N terminus of DAT in vitro, and mutation of these serines eliminated the stimulatory effects of CaMKIIalpha. A mutation of the DAT C terminus impairing CaMKIIalpha binding also impaired amphetamine-induced dopamine efflux. An in vivo role for CaMKII was supported by chronoamperometry measurements showing reduced amphetamine-induced dopamine efflux in response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIalpha binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux.
Ras proto-oncogene mutations have been implicated in the pathogenesis of many malignancies, including leukemia. While both human and animal studies have linked several chemical carcinogens to specific ras mutations, little data exist regarding the association of ras mutations with parental exposures and risk of childhood leukemia. Using data from a large case-control study of childhood acute lymphoblastic leukemia (ALL; age <15 years) conducted by the Children's Cancer Group, we used a case-case comparison approach to examine whether reported parental exposure to hydrocarbons at work or use of specific medications are related to ras gene mutations in the leukemia cells of children with ALL. DNA was extracted from archived bone marrow slides or cryopreserved marrow samples for 837 ALL cases. We examined mutations in K-ras and N-ras genes at codons 12, 13, and 61 by PCR and allele-specific oligonucleotide hybridization and confirmed them by DNA sequencing. We interviewed mothers and, if available, fathers by telephone to collect exposure information. Odds ratios (ORs) and 95% confidence intervals (CIs) were derived from logistic regression to examine the association of parental exposures with ras mutations. A total of 127 (15.2%) cases had ras mutations (K-ras 4.7% and N-ras 10.68%). Both maternal (OR 3.2, 95% CI 1.7-6.1) and paternal (OR 2.0, 95% CI 1.1-3.7) reported use of mind-altering drugs were associated with N-ras mutations. Paternal use of amphetamines or diet pills was associated with N-ras mutations (OR 4.1, 95% CI 1.1-15.0); no association was observed with maternal use. Maternal exposure to solvents (OR 3.1, 95% CI 1.0-9.7) and plastic materials (OR 6.9, 95% CI 1.2-39.7) during pregnancy and plastic materials after pregnancy (OR 8.3, 95% CI 1.4-48.8) were related to K-ras mutation. Maternal ever exposure to oil and coal products before case diagnosis (OR 2.3, 95% CI 1.1-4.8) and during the postnatal period (OR 2.2, 95% CI 1.0-5.5) and paternal exposure to plastic materials before index pregnancy (OR 2.4, 95% CI 1.1-5.1) and other hydrocarbons during the postnatal period (OR 1.8, 95% CI 1.0-1.3) were associated with N-ras mutations. This study suggests that parental exposure to specific chemicals may be associated with distinct ras mutations in children who develop ALL.
Although it is clear that amphetamine-induced dopamine (DA) release mediated by the dopamine transporter (DAT) is integral to the behavioral actions of this psychostimulant, the mechanism of this release is not clear. In this study, we explored the requirement for intracellular Ca(2+) in amphetamine-induced DA efflux and currents mediated by the human DAT. The patch-clamp technique in the whole-cell configuration was used on Na(+) and DA-preloaded human embryonic kidney 293 cells stably transfected with the human DAT (hDAT cells). Chelation of intracellular Ca(2+) by inclusion of 50 microM BAPTA in the whole-cell pipette reduced the voltage-dependent amphetamine-induced hDAT current, with the greatest effect seen at positive voltages. Likewise, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) reduced amphetamine-induced DA efflux as measured by amperometry. Furthermore, preincubation of the cells with 50 microM BAPTA acetoxy methyl ester (AM) or thapsigargin also blocked amphetamine-induced release of preloaded N-methyl-4-[(3)H]phenylpyridinium from superfused hDAT cells. BAPTA-AM also reduced DA release from striatal synaptosomes. Amphetamine also led to an increase in intracellular Ca(2+) that was blocked by prior treatment with 5 microM thapsigargin or 10 microM cocaine. These studies demonstrate that amphetamine-induced DAT-mediated currents and substrate efflux require internal Ca(2+) and that amphetamine can stimulate dopamine efflux by regulating cytoplasmic Ca(2+) levels through its interaction with DAT.
Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22-normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a "reluctant" state to a "willing" state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake.
Activation of the cerebral cortex is seen during hallucinations. The 5-HT(2A/C) agonist 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) is a potent hallucinogen that has been proposed to act by targeting 5-HT(2A) heteroceptors on thalamocortical neurons and eliciting release of glutamate from these cells, which in turn drives cortical neurons. We used in vivo microdialysis to determine if DOI increases extracellular glutamate levels. Systemic administration of DOI significantly increased extracellular glutamate levels in the somatosensory cortex of the freely-moving rat. Similarly, intracortical administration of DOI by reverse dialysis increased cortical extracellular glutamate levels. No consistent changes in either extracellular GABA or glycine levels were observed in response to DOI. The increase in glutamate levels elicited by intracortical DOI was blocked by treatment with the selective 5-HT(2A) antagonist MDL 100,907. These data are consistent with the hypothesis that 5-HT(2A) receptor-mediated regulation of glutamate release is the mechanism through which hallucinogens activate the cerebral cortex.
BACKGROUND - Few studies have examined the risk of childhood acute lymphoblastic leukemia (ALL) associated with parental medication use. As part of a large case-control study conducted by the Children's Cancer Group, we evaluated the association between maternal and paternal medication use and the risk of ALL in offspring.
METHODS - Information on selected medication use in the year before and during the index pregnancy was obtained by telephone interview. Participants included 1842 children of 14 years or younger with newly diagnosed and immunophenotypically defined ALL and 1986 individually matched controls. Data were analyzed using logistic regression models and stratified by immunophenotypes of ALL and age at diagnosis of cases.
RESULTS - After adjusting for potential confounders and other medication use, we found that maternal use of vitamins (odds ratio [OR] = 0.7, 99% confidence interval [CI]: 0.5-1.0) and iron supplements (OR = 0.8, 99% CI: 0.7-1.0) only during the index pregnancy was associated with a decreased risk of ALL. Parental use of amphetamines or diet pills and mind-altering drugs before and during the index pregnancy was related to an increased risk of childhood ALL, particularly among children where both parents reported using these drugs (OR = 2.8, 99% CI: 0.5-15.6 for amphetamines or diet pills, OR = 1.8, 99% CI: 1.1-3.0 for mind-altering drugs). Stratified analyses showed that maternal use of antihistamines or allergic remedies and parental use of mind-altering drugs were strongly associated with infant ALL, whereas patterns of association between childhood ALL and parental medication use did not influence markedly the immunophenotypic subgroup of ALL.
CONCLUSIONS - The findings of this study suggest that certain parental medication use immediately before and during the index pregnancy may influence risk of ALL in offspring.
Copyright 2002 American Cancer Society.