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Novel psychoactive substances, including synthetic substituted tryptamines, represent a potential public health threat. Additionally, some substituted tryptamines are being studied under medical guidance as potential treatments of psychiatric disorders. Characterizing the basic pharmacology of substituted tryptamines will aid in understanding differences in potential for harm or therapeutic use. Using human embryonic kidney cells stably expressing 5-hydroxytryptamine (5-HT)(1A), 5-HT(2A), and 5-HT(2C) receptors (5-HT(1A)R, 5-HT(2A)R, and 5HT(2C)R, respectively) or the serotonin transporter (SERT), we measured affinities, potencies and efficacies of 21 substituted tryptamines. With the exception of two 4-acetoxy compounds, substituted tryptamines exhibited affinities and potencies less than one micromolar at the 5-HT(2A)R, the primary target for psychedelic effects. In comparison, half or more exhibited low affinities/potencies at 5-HT(2C)R, 5-HT(1A)R, and SERT. Sorting by the ratio of 5-HT(2A) to 5-HT(2C), 5-HT(1A), or SERT affinity revealed chemical determinants of selectivity. We found that although 4-substituted compounds exhibited affinities that ranged across a factor of 100, they largely exhibited high selectivity for 5-HT(2A)Rs versus 5-HT(1A)Rs and 5-HT(2C)Rs. 5-substituted compounds exhibited high affinities for 5-HT(1A)Rs, low affinities for 5-HT(2C)Rs, and a range of affinities for 5-HT(2A)Rs, resulting in selectivity for 5-HT(2A)Rs versus 5-HT(2C)Rs but not versus 5-HT(1A)Rs. Additionally, a number of psychedelics bound to SERT, with non–ring-substituted tryptamines most consistently exhibiting binding. Interestingly, substituted tryptamines and known psychedelic standards exhibited a broad range of efficacies, which were lower as a class at 5-HT(2A)Rs compared with 5-HT(2C)Rs and 5-HT(1A)Rs. Conversely, coupling efficiency/amplification ratio was highest at 5-HT(2A)Rs in comparison with 5-HT(2C)Rs and 5-HT(1A)Rs. SIGNIFICANCE STATEMENT: Synthetic substituted tryptamines represent both potential public health threats and potential treatments of psychiatric disorders. The substituted tryptamines tested differed in affinities, potencies, and efficacies at 5-hydroxytryptamine (5-HT)(2A), 5-HT(2C), and 5HT(1A) receptors and the serotonin transporter (SERT). Several compounds were highly selective for and coupled very efficiently downstream of 5-HT(2A) versus 5-HT(1A) and 5-HT(2C) receptors, and some bound SERT. This basic pharmacology of substituted tryptamines helps us understand the pharmacologic basis of their potential for harm and as therapeutic agents.

Synthetic opioids, including fentanyl and its analogs, have therapeutic efficacy in analgesia and anesthesia. However, their illicit use in the United States has increased and contributed to the number one cause of death for adults 18–50 years old. Fentanyl and the heroin metabolite morphine induce respiratory depression that can be treated with the μ opioid receptor (MOR) antagonist naloxone. With higher or more rapid dosing, fentanyl, more than morphine, causes chest wall rigidity and can also induce rapid onset laryngospasm. Because non-MORs could mediate differing clinical manifestations, we examined the interactions of fentanyl and morphine at recombinant human neurotransmitter transporters, G protein–coupled receptors, and the N-methyl-D-aspartate glutamate receptor. Both drugs were agonists at MOR, κ, and δ opioid receptors. Morphine had little or no affinity at other human receptors and transporters (K(i) or IC(50) value >100 µM). However, fentanyl had K(i) values of 1407 and 1100 nM at α(1A) and α(1B) adrenoceptor subtypes, respectively, and K(i) values of 1049 and 1670 nM at dopamine D4.4 and D1 receptor subtypes, respectively; it also blocked [(3)H]neurotransmitter uptake by the vesicular monoamine transporter 2 (IC(50) = 911 nM). Pharmacokinetic models indicate that these Ki and IC(50) values are pharmacologically relevant. Fentanyl had little affinity for other receptors or transporters. Thus, noradrenergic disposition at specific receptor subtypes in relevant organs may play a role in respiratory and cardiothoracic effects of fentanyl. Data suggest that less selective fentanyl receptor pharmacology could play a role in the different clinical effects of morphine compared with fentanyl, including fentanyl-induced deaths after illicit use. SIGNIFICANCE STATEMENT: The synthetic opioid fentanyl induces different clinical effects, including rapid onset muscular rigidity, vocal cord closure, and rapid death, than the heroin metabolite morphine. Our data indicate for the first time that the two drugs have very different effects at recombinant human neurotransmitter receptors and transporters that might explain those clinical differences.

The non-medical use of opioids has become a national crisis in the USA. Developing non-opioid pharmacotherapies for controlling this opioid epidemic is urgent. Dopamine D

The use of new psychoactive substituted 2,5-dimethoxy-N-benzylphenethylamines is associated with abuse and toxicity in the United States and elsewhere and their pharmacology is not well known. This study compares the mechanisms of action of 2(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine (25D-NBOMe), 2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25E-NBOMe), 2-(2,5dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25H-NBOMe), 2-(((4-iodo-2,5dimethoxyphenethyl)amino)methyl)phenol (25I-NBOH); and 2-(2,5-dimethoxy-4-nitrophenyl)-N(2-methoxybenzyl)ethanamine) (25N-NBOMe) with hallucinogens and stimulants. Mammalian cells heterologously expressing 5-HT(1A), 5-HT(2A), 5-HT(2B) or 5-HT(2C) receptors, or dopamine, serotonin or norepinephrine transporters (DAT, SERT and NET, respectively) were used to assess drug affinities at radioligand binding sites. Potencies and efficacies were determined using [(35)S]GTPγS binding assays (5-HT(1A)), inositol-phosphate accumulation assays (5-HT(2A,) 5-HT(2B) and 5-HT(2C)), and uptake and release assays (transporters). The substituted phenethylamines were very low potency and low efficacy agonists at the 5-HT(1A) receptor. 25D-NBOMe, 25E-NBOMe, 25HNBOMe, 25I-NBOH and 25N-NBOMe had very high affinity for, and full efficacy at, 5HT(2A) and 5-HT(2C) receptors. In the 5-HT(2A) receptor functional assay, 25D-NBOMe, 25ENBOMe, 25I-NBOH and 25N-NBOMe had subnanomolar to low nanomolar potencies similar to (+)lysergic acid diethylamide (LSD) while 25H-NBOMe had lower potency, similar to serotonin. At the 5-HT(2C) receptor, four had very high potencies, similar to LSD and serotonin, while 25H-NBOMe had lower potency. At the 5-HT(2B) receptor, the compounds had lower affinity, potency and efficacy compared to 5-HT(2A) or 5-HT(2C.) The phenethylamines had low to mid micromolar affinities and potencies at the transporters. These results demonstrate that these –NBOMe and –NBOH substituted phenethylamines have a biochemical pharmacology consistent with hallucinogenic activity, with little psychostimulant activity.

While screening off-target effects of rigid (N)-methanocarba-adenosine 5′-methylamides as A3 adenosine receptor (AR) agonists, we discovered μM binding hits at the δ-opioid receptor (DOR) and translocator protein (TSPO). In an effort to increase OR and decrease AR affinity by structure activity analysis of this series, antagonist activity at κ-(K)OR appeared in 5′-esters (ethyl 24 and propyl 30), which retained TSPO interaction (μM). 7-Deaza modification of C2-(arylethynyl)-5′-esters but not 4′-truncation enhanced KOR affinity (MRS7299 28 and 29, Ki ≈ 40 nM), revealed μ-OR and DOR binding, and reduced AR affinity. Molecular docking and dynamics simulations located a putative KOR binding mode consistent with the observed affinities, placing C7 in a hydrophobic region. 3-Deaza modification permitted TSPO but not OR binding, and 1-deaza was permissive to both; ribose-restored analogues were inactive at both. Thus, we have repurposed a known AR nucleoside scaffold for OR antagonism, with a detailed hypothesis for KOR recognition.

Background Police academies need fit recruits to successfully engage in training activities. In a previous retrospective study, we documented that recruits with poor fitness at entry to the academy had significantly lower graduation rates, and we also suggested evidence-based entry-level fitness recommendations. Aims To validate our findings in a prospective cohort of police recruits. Methods Recruits entering Massachusetts municipal police academies during 2015–16 were followed prospectively until they dropped out, failed or successfully graduated their academy classes. Entry-level fitness was quantified at the start of each training class using: body composition, push-ups, sit-ups, sit-and-reach and 1.5-mile run time. The primary outcome of interest was the odds of failure (not successfully graduating from an academy). We used logistic regression to assess the probability of not graduating, based on entry-level fitness. Results On average, successful graduates were leaner and possessed better overall entry-level fitness. After adjusting for age, gender and body mass index, several fitness measures were strongly associated with academy failure: fewer sit-ups completed (OR 9.6 (95% CI 3.5–26.3) (≤15 versus 41–60)); fewer push-ups completed (OR 6.7 (95% CI 2.5–17.5) (≤20 versus 41–60)); and slower run times (OR 18.4 (95% CI 6.8–50.2) (1.5 miles in > 15 min 20 s versus 10 min 37 s to 12 min 33 s)). The prospective study results supported previously suggested minimum entry-level fitness (95% graduation rate) and target (98% graduation rate) recommendations. Conclusions Push-ups completed and 1.5-mile run time at police academy entry were successfully validated as predictors of successful academy graduation, while sit-ups were also a strong independent predictor in the prospective study.

Methamphetamine (MA) is highly addictive and neurotoxic, causing cell death in humans and in rodent models. MA, along with many of its analogs, is an agonist at the G protein-coupled trace amine-associated receptor 1 (TAAR1). TAAR1 activation protects against MA-induced degeneration of dopaminergic neurons, suggesting that TAAR1 plays a role in regulating MA-induced neurotoxicity. However, the mechanisms involved in TAAR1's role in neurotoxicity and cell death have not been described in detail. In this study, we investigated the apoptosis pathway in Taar1 wild-type (WT) and knockout (KO) mice and in cells expressing the recombinant receptor. Bcl-2, an antiapoptotic protein, was upregulated ∼3-fold in the midbrain area (substantial nigra and ventral tegmental area) in Taar1 KO compared with WT mice, and MA significantly increased Bcl-2 expression in WT mice but decreased Bcl-2 expression in KO mice. The proapoptotic protein Bax did not differ across genotype or in response to MA. Bcl-2 expression was significantly upregulated by the TAAR1 agonist RO5166017 ((S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine) in cells expressing the recombinant mouse TAAR1. Additionally, activation of TAAR1 by RO5166017 increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and protein kinase B (AKT), but only inhibition of ERK1/2 phosphorylation prevented TAAR1-induced increases in Bcl-2 levels, indicating that TAAR1 activation increases Bcl-2 through an ERK1/2-dependent pathway. All changes to ERK1/2 pathway intermediates were blocked by the TAAR1 antagonist, N-(3-ethoxyphenyl)-4-(1-pyrrolidinyl)-3-(trifluoromethyl) benzamide. These findings suggest that TAAR1 activation protects against MA-induced cell apoptosis and TAAR1 may play a role in cell death in neurodegenerative diseases. SIGNIFICANCE STATEMENT: Methamphetamine stimulates TAAR1, a G protein-coupled receptor. The role and mechanisms for TAAR1 in methamphetamine-induced neurotoxicity are not known. Here, we report that, in genetic mouse models and cells expressing the recombinant receptor, TAAR1 activates the ERK1/2 pathway but not the AKT pathway to upregulate the antiapoptotic protein Bcl-2, which protects cells from drug-induced toxicity.

Synthetic cathinones are components of “bath salts” and have physical and psychologic side effects, including hypertension, paranoia, and hallucinations. Here, we report interactions of 20 “bath salt” components with human dopamine, serotonin, and norepinephrine transporters [human dopamine transporter (hDAT), human serotonin transporter (hSERT), and human norepinephrine transporter (hNET), respectively] heterologously expressed in human embryonic kidney 293 cells. Transporter inhibitors had nanomolar to micromolar affinities (Ki values) at radioligand binding sites, with relative affinities of hDAT>hNET>hSERT for α-pyrrolidinopropiophenone (α-PPP), α-pyrrolidinobutiophenone, α-pyrrolidinohexiophenone, 1-phenyl-2-(1-pyrrolidinyl)-1-heptanone, 3,4-methylenedioxy-α-pyrrolidinopropiophenone, 3,4-methylenedioxy-α-pyrrolidinobutiophenone, 4-methyl-α-pyrrolidinopropiophenone, α-pyrrolidinovalerophenone, 4-methoxy-α-pyrrolidinovalerophenone, α-pyrrolidinopentiothiophenone (alpha-PVT), and α-methylaminovalerophenone, and hDAT>hSERT>hNET for methylenedioxypentedrone. Increasing the α-carbon chain length increased the affinity and potency of the α-pyrrolidinophenones. Uptake inhibitors had relative potencies of hDAT>hNET>hSERT except α-PPP and α-PVT, which had highest potencies at hNET. They did not induce [3H]neurotransmitter release. Substrates can enter presynaptic neurons via transporters, and the substrates methamphetamine and 3,4-methylenedioxymethylamphetamine are neurotoxic. We determined that 3-fluoro-, 4-bromo-, 4-chloro-methcathinone, and 4-fluoroamphetamine were substrates at all three transporters; 5,6-methylenedioxy-2-aminoindane (MDAI) and 4-methylethcathinone (4-MEC) were substrates primarily at hSERT and hNET; and 3,4-methylenedioxy-N-ethylcathinone (ethylone) and 5-methoxy-methylone were substrates only at hSERT and induced [3H]neurotransmitter release. Significant correlations between potencies for inhibition of uptake and for inducing release were observed for these and additional substrates. The excellent correlation of efficacy at stimulating release versus Ki/IC50 ratios suggested thresholds of binding/uptake ratios above which compounds were likely to be substrates. Based on their potencies at hDAT, most of these compounds have potential for abuse and addiction. 4-Bromomethcathinone, 4-MEC, 5-methoxy-methylone, ethylone, and MDAI, which have higher potencies at hSERT than hDAT, may have empathogen psychoactivity.

We have previously reported that dual 5-HT(1A) and 5-HT(7) receptor ligands might find utility as treatment options for various CNS related conditions including cognitive and anxiolytic impairments. We have also more recently reported that SYA16263 has antipsychotic-like properties with an absence of catalepsy in animal models ascribed to its ability to recruit β-arrestin to the D(2) receptor. However, SYA16263 also binds with very high affinity to 5-HT(1A)R (Ki = 1.1 nM) and a moderate affinity at 5-HT(7)R (Ki = 90 nM). Thus, it was of interest to exploit its pharmacophore elements in designing new dual receptor ligands. Using SYA16263 as the lead molecule, we have conducted a limited structure-affinity relationship (SAFIR) study by modifying various structural elements in the arylalkyl moiety, resulting in the identification of a new dual 5-HT(1A)R and 5-HT(7)R ligand, 6-chloro-2-methyl-2-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)-2,3-dihydro-1H-inden-1-one (21), which unlike SYA16263, has a sub-nanomolar (5-HT(1A)R, Ki = 0.74 nM) and a low nanomolar (5-HT(7)R, Ki = 8.4 nM ) affinity for these receptors. Interestingly, 21 is a full agonist at 5-HT(1A)R and antagonist at the 5-HT(7)R, functional characteristics which point to its potential as an antidepressant agent.

Substituted fentanyls are abused and cause rapid fatal overdose. As their pharmacology is not well characterized, we examined in vitro pharmacology and structure-activity relationships of 22 substituted fentanyls with modifications of the fentanyl propyl group, and conducted in silico receptor/ligand modeling. Affinities for mu, kappa, and delta opioid receptors (MOR, KOR, and DOR, respectively) heterologously expressed in mammalian cells were assessed in agonist radioligand binding assays. At MOR, furanyl fentanyl had higher affinity than fentanyl, while acryl, isobutyryl and cyclopropyl fentanyls had similar affinities. Comparing affinities, thiophene and methoxyacetyl fentanyls had highest selectivity for MOR (2520- and 2730-fold compared to KOR and DOR, respectively). Functional activities were assessed using [35S]GTPγS binding assays. At MOR, furanyl fentanyl had higher potency and 11 substituted fentanyls had similar high potencies compared to fentanyl. Eight compounds were full agonists of MOR and twelve compounds were partial agonists, with efficacies from 8.8% (phenyl fentanyl) to 60.2% (butyryl fentanyl). All efficacious compounds had selective functional potency for MOR. The predicted binding poses of flexible fentanyl and rigid morphine against MOR show partially overlapping binding pockets, with fentanyl maintaining additional interaction with the transmembrane (TM) 2 helix. Subsequent molecular dynamics simulations revealed a predominant fentanyl binding pose involving various TM interactions. The piperidine nitrogen of substituted fentanyls establishes a salt-bridge with the conserved D-1473.32 residue and the propanamide carbonyl group establishes a hydrogen bond with the indole side-chain (-NH) of W-3187.35. The simulation suggests theN-linked phenethyl group may regulate the rotameric switch of W-2936.48. The predicted binding pose, in conjunction with in vitro binding affinity, clarified the molecular basis of the binding/selectivity profile of furanyl fentanyl and other derivatives at the sequence level. In summary, substituted fentanyls with high MOR potencies, selectivities, and efficacies are likely to have abuse and overdose potential. The work presented here is a prototype to investigate fentanyl derivatives and their abuse potential.

Methamphetamine, a human vesicular monoamine transporter 2 (VMAT2) substrate, releases dopamine, serotonin, and norepinephrine from vesicles into the cytosol of presynaptic neurons and induces reverse transport by the monoamine transporters to increase extracellular neurotransmitters. Currently available radioligands for VMAT2 have considerable liabilities: The binding of [3H]dihydrotetrabenazine ([3H]DHTB) to a site on VMAT2 is not dependent on ATP, and [3H]reserpine binds almost irreversibly to VMAT2. Herein we demonstrate that several arylpiperidinylquinazolines (APQs) are potent inhibitors of [3H]reserpine binding at recombinant human VMAT2 expressed in HEK-293 cells. These compounds are biodiastereoselective and bioenantioselective. The lead radiolabeled APQ is unique because it binds reversibly to VMAT2 but does not bind the [3H]DHTB binding site. Furthermore, experimentation shows that several novel APQ ligands have high potency for inhibition of uptake by both HEK-VMAT2 cells and mouse striatal ve...

New psychoactive substances (NPSs), including substituted cathinones and other stimulants, are synthesized, sold on the Internet, and ingested without knowledge of their pharmacological activity and/or toxicity. In vitro pharmacology plays a role in therapeutic drug development, drug-protein in silico interaction modeling, and drug scheduling. The goal of this research was to determine mechanisms of action that may indicate NPS abuse liability. Affinities to displace the radioligand [125I]RTI-55 and potencies to inhibit [3H]neurotransmitter uptake for 22 cathinones, 6 benzofurans and another stimulant were characterized using human embryonic kidney cells stably expressing recombinant human transporters for dopamine, norepinephrine, or serotonin (hDAT, hNET, or hSERT, respectively). Selected compounds were tested for potencies and efficacies at inducing [3H]neurotransmitter release via the transporters. Computational modeling was conducted to explain plausible molecular interactions established by NPS and transporters. Most α-pyrrolidinophenones had high hDAT potencies and selectivities in uptake assays, with hDAT/hSERT uptake selectivity ratios of 83–360. Other substituted cathinones varied in their potencies and selectivities, with N-ethyl-hexedrone and N-ethyl-pentylone having highest hDAT potencies and N-propyl-pentedrone having highest hDAT selectivity. 4-Cl-ethcathinone and 3,4-methylenedioxy-N-propylcathinone had higher hSERT selectivity. Benzofurans generally had low hDAT selectivity, especially 1-(2,3-dihydrobenzofuran-5-yl)-N-methylpropan-2-amine, with 25-fold higher hSERT potency. Consistent with this selectivity, the benzofurans were releasers at hSERT. Modeling indicated key amino acids in the transporters’ binding pockets that influence drug affinities. The α-pyrrolidinophenones, with high hDAT selectivity, have high abuse potential. Lower hDAT selectivity among benzofurans suggests similarity to methylenedioxymethamphetamine, entactogens with lower stimulant activity.

Background Suboptimal recruit fitness may be a risk factor for poor performance, injury, illness, and lost time during police academy training. Aims To assess the probability of successful completion and graduation from a police academy as a function of recruits’ baseline fitness levels at the time of academy entry. Methods Retrospective study where all available records from recruit training courses held (2006–2012) at all Massachusetts municipal police academies were reviewed and analysed. Entry fitness levels were quantified from the following measures, as recorded at the start of each training class: body composition, push-ups, sit-ups, sit-and-reach, and 1.5-mile run-time. The primary outcome of interest was the odds of not successfully graduating from an academy. We used generalized linear mixed models in order to fit logistic regression models with random intercepts for assessing the probability of not graduating, based on entry-level fitness. The primary analyses were restricted to recruits with complete entry-level fitness data. Results The fitness measures most strongly associated with academy failure were lesser number of push-ups completed (odds ratio [OR] = 5.2, 95% confidence interval [CI] 2.3–11.7, for 20 versus 41–60 push-ups) and slower run times (OR = 3.8, 95% CI 1.8–7.8, [1.5 mile run time of ≥15′20″] versus [12′33″ to 10′37″]). Conclusions Baseline pushups and 1.5-mile run-time showed the best ability to predict successful academy graduation, especially when considered together. Future research should include prospective validation of entry-level fitness as a predictor of subsequent police academy success.

A quantitative trait locus (QTL) on proximal chromosome (Chr) 10 accounts for > 50% of the genetic variance in methamphetamine (MA) intake in mice selectively bred for high (MAHDR) and low (MALDR) voluntary MA drinking. The µ-opioid receptor (MOP-r) gene, Oprm1, resides at the proximal end of Chr 10, and buprenorphine reduces MA intake in MAHDR mice. However, this drug has only partial agonist effects at MOP-r. We investigated the impact of a full MOP-r agonist, morphine, on MA intake and saccharin intake, measured MOP-r density and affinity in several brain regions of the MA drinking lines and their C57BL/6J (B6) and DBA/2J (D2) progenitor strains, and measured MA intake in two congenic strains of mice to verify the QTL and reduce the QTL interval. Morphine reduced MA intake in the MAHDR line, but also reduced saccharin and total fluid intake. MOP-r density was lower in the medial prefrontal cortex of MAHDR, compared to MALDR, mice, but not in the nucleus accumbens or ventral midbrain; there were no MOP-r affinity differences. No significant differences in MOP-r density or affinity were found between the progenitor strains. Finally, Chr 10 congenic results were consistent with previous data suggesting that Oprm1 is not a quantitative trait gene, but is impacted by the gene network underlying MA intake. Stimulation of opioid pathways by a full agonist can reduce MA intake, but may also non-specifically affect consummatory behavior; thus, a partial agonist may be a better pharmacotherapeutic.

Mefloquine is used for the prevention and treatment of chloroquine-resistant malaria, but its use is associated with nightmares, hallucinations, and exacerbation of symptoms of post-traumatic stress disorder. We hypothesized that potential mechanisms of action for the adverse psychotropic effects of mefloquine resemble those of other known psychotomimetics.Using in vitro radioligand binding and functional assays, we examined the interaction of (+)- and (-)-mefloquine enantiomers, the non-psychotomimetic anti-malarial agent, chloroquine, and several hallucinogens and psychostimulants with recombinant human neurotransmitter receptors and transporters.Hallucinogens and mefloquine bound stereoselectively and with relatively high affinity (K i = 0.71-341 nM) to serotonin (5-HT) 2A but not 5-HT1A or 5-HT2C receptors. Mefloquine but not chloroquine was a partial 5-HT2A agonist and a full 5-HT2C agonist, stimulating inositol phosphate accumulation, with similar potency and efficacy as the hallucinogen dimethyltryptamine (DMT). 5-HT receptor antagonists blocked mefloquine's effects. Mefloquine had low or no affinity for dopamine D1, D2, D3, and D4.4 receptors, or dopamine and norepinephrine transporters. However, mefloquine was a very low potency antagonist at the D3 receptor and mefloquine but not chloroquine or hallucinogens blocked [(3)H]5-HT uptake by the 5-HT transporter.Mefloquine, but not chloroquine, shares an in vitro receptor interaction profile with some hallucinogens and this neurochemistry may be relevant to the adverse neuropsychiatric effects associated with mefloquine use by a small percentage of patients. Additionally, evaluating interactions with this panel of receptors and transporters may be useful for characterizing effects of other psychotropic drugs and for avoiding psychotomimetic effects for new pharmacotherapies, including antimalarial quinolines.

We have repurposed (N)-methanocarba adenosine derivatives (A3 adenosine receptor (AR) agonists) to enhance radioligand binding allosterically at the human dopamine (DA) transporter (DAT) and inhibit DA uptake. We extended the structure-activity relationship of this series with small N6-alkyl substitution, 5′-esters, deaza modifications of adenine, and ribose restored in place of methanocarba. C2-(5-halothien-2-yl)-ethynyl 5′-methyl 9 (MRS7292) and 5′-ethyl 10 (MRS7232) esters enhanced binding at DAT (EC50 ∼35 nM) and at norepinephrine transporter (NET). 9 and 10 were selective for DAT compared to A3AR in the mouse, but not human. At DAT, binding of two structurally dissimilar radioligands was enhanced; NET binding of only one radioligand was enhanced; SERT radioligand binding was minimally affected. 10 was more potent than cocaine at inhibiting DA uptake (IC50 = 107 nM). Ribose analogues were weaker in DAT interaction than corresponding bicyclics. Thus, we enhanced the neurotransmitter transporters activity of rigid nucleosides while reducing A3AR affinity.

Background Occupational exposures to ultrafine particles in the plume generated during laser hair removal procedures, the most commonly performed light based cosmetic procedure, have not been thoroughly characterized. Acute and chronic exposures to ambient ultrafine particles have been associated with a number of negative respiratory and cardiovascular health effects. Thus, the aim of this study was to measure airborne concentrations of particles in a diameter size range of 10 nm to 1 μm in procedure rooms during laser hair removal procedures. Methods TSI Model 3007 Condensation Particle Counters were used to quantify the particle count concentrations in the waiting and procedure rooms of a dermatology office. Particle concentrations were sampled before, during, and after laser hair removal procedures, and characteristics of each procedure were noted by the performing dermatologist. Results Twelve procedures were sampled over 4 days. Mean ultrafine particle concentrations in the waiting and procedure rooms were 14,957.4 particles/cm3 and 22,916.8 particles/cm3 (p

Neural processes influenced by γ-aminobutyric acid B (GABA(B)) receptors appear to contribute to acute ethanol sensitivity, including the difference between lines of mice bred for extreme sensitivity (FAST) or insensitivity (SLOW) to the locomotor stimulant effect of ethanol. One goal of the current study was to determine whether selection of the FAST and SLOW lines resulted in changes in GABA(B) receptor function, since the lines differ in sensitivity to the GABA(B) receptor agonist baclofen and baclofen attenuates the stimulant response to ethanol in FAST mice. A second goal was to determine whether the baclofen-induced reduction in ethanol stimulation in FAST mice is associated with an attenuation of the mesolimbic dopamine response to ethanol. In Experiment 1, the FAST and SLOW lines were found to not differ in GABA(B) receptor function (measured by baclofen-stimulated [(35)S]GTPγS binding) in whole brain or in several regional preparations, except in the striatum in one of the two replicate sets of selected lines. In Experiment 2, baclofen-induced attenuation of the locomotor stimulant response to ethanol in FAST mice was not accompanied by a reduction in dopamine levels in the nucleus accumbens, as measured by microdialysis. These data suggest that, overall, GABA(B) receptor function does not play an integral role in the genetic difference in ethanol sensitivity between the FAST and SLOW lines. Further, although GABA(B) receptors do modulate the locomotor stimulant response to ethanol in FAST mice, this effect does not appear to be due to a reduction in tonic dopamine signaling in the nucleus accumbens.

Thirty-two congeneric rigid adenine nucleoside derivatives containing a North (N)-methanocarba ribose substitution and a 2-arylethynyl group either enhanced (up to 760% of control) or inhibited [(125)I] methyl (1R,2S,3S)-3-(4-iodophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (RTI-55) binding at the human dopamine (DA) transporter (DAT) and inhibited DA uptake. Several nucleosides also enhanced [(3)H]mazindol [(±)-5-(4-chlorophenyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol] binding to the DAT. The combination of binding enhancement and functional inhibition suggests possible allosteric interaction with the tropanes. The structure-activity relationship of this novel class of DAT ligands was explored: small N(6)-substition (methyl or ethyl) was favored, while the N1 of the adenine ring was essential. Effective terminal aryl groups include thien-2-yl (compounds 9 and 16), with EC50 values of 35.1 and 9.1 nM, respectively, in [(125)I]RTI-55 binding enhancement, and 3,4-difluorophenyl as in the most potent DA uptake inhibitor (compound 6) with an IC50 value of 92 nM (3-fold more potent than cocaine), but not nitrogen heterocycles. Several compounds inhibited or enhanced binding at the norepinephrine transporter (NET) and serotonin transporter (SERT) and inhibited function in the micromolar range; truncation at the 4'-position in compound 23 allowed for weak inhibition of the SERT. We have not yet eliminated adenosine receptor affinity from this class of DAT modulators, but we identified modifications that remove DAT inhibition as an off-target effect of potent adenosine receptor agonists. Thus, we have identified a new class of allosteric DAT ligands, rigidified adenosine derivatives, and explored their initial structural requirements. They display a very atypical pharmacological profile, i.e., either enhancement by increasing affinity or inhibition of radioligand binding at the DAT, and in some cases the NET and SERT, and inhibition of neurotransmitter uptake.

The abuse liability profile of three synthetic hallucinogens, N,N-diisopropyltryptamine (DIPT), 5-N,N-diethyl-5-methoxytryptamine (5-MeO-DET), and 5-methoxy-α-methyltryptamine (5-MeO-AMT), was tested in rats trained to discriminate hallucinogenic and psychostimulant compounds, including cocaine, methamphetamine, 3,4-methylenedioxymethylamphetamine (MDMA), lysergic acid diethylamide (LSD), (-)-2,5-dimethoxy-4-methylamphetamine (DOM), and dimethyltryptamine (DMT). Because abused hallucinogens act at 5-hydroxytryptamine 1A (5-HT(1A)) and 5-HT(2A) receptors, and abused psychostimulants act at monoamine transporters, binding and functional activities of DIPT, 5-MeO-DET, and 5-MeO-AMT at these sites were also tested. DIPT fully substituted in rats trained to discriminate DMT (ED(50) = 1.71 mg/kg) and DOM (ED(50) = 1.94 mg/kg), but produced only 68% LSD-appropriate responding. 5-MeO-DET fully substituted for DMT (ED(50) = 0.41 mg/kg) and produced 59% MDMA-appropriate responding. 5-MeO-AMT did not fully substitute for any of the training drugs, but produced 67% LSD-appropriate responding. None of the compounds produced substitution in rats trained to discriminate cocaine or methamphetamine. All three compounds showed activity at 5-HT(1A) and 5-HT(2A) receptors as well as blockade of reuptake by the serotonin transporter. In addition, 5-MeO-AMT produced low levels of serotonin release and low potency blockade of dopamine uptake. DIPT, 5-MeO-DET, and 5-MeO-AMT produced behavioral and receptor effects similar to those of abused hallucinogens, but were not similar to those of psychostimulants. DIPT and 5-MeO-DET may have abuse liability similar to known hallucinogens and may be hazardous because high doses produced activity and lethality.

Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. We reported previously that osteoblastic cells express a functional serotonin (5-HT) signal transduction system, with mechanisms for responding to and regulating uptake of 5-HT. The clonal murine osteocytic cell line, MLO-Y4, demonstrates expression of the serotonin transporter (5-HTT), and the 5-HT1A, and 5-HT2A receptors by real-time RT-PCR and immunoblot analysis. Immunohistochemistry using antibodies for the 5-HTT, and the 5-HT1A and 5-HT2A receptors reveals expression of all three proteins in both osteoblasts and osteocytes in rat tibia. 5-HTT binding sites were demonstrated in the MLO-Y4 cells with nanomolar affinity for the stable cocaine analog [125I]RTI-55. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, show the highest potency to antagonize [125I]RTI-55 binding in the MLO-Y4 cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in MLO-Y4 cells was 2.85 pmol/15 min/well, with a Km value of 290 nM. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range. 5-HT rapidly stimulated PGE2 release from MLO-Y4 cells; the EC50 for 5-HT was 0.1 μM, with a 3-fold increase seen at 60 min. The rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase, is expressed in MLO-Y4 cells as well as osteoblastic MC3T3-E1 cells. Thus, osteocytes, as well as osteoblasts, are capable of 5-HT synthesis, and express functional receptor and transporter components of the 5-HT signal transduction system.

There is increasing evidence for a contribution of the neural system to the regulation of bone metabolism. The skeleton is richly innervated by both sympathetic and sensory neurons. While these nerves serve sensory and vascular functions, they are also being found to influence bone cell activities. The most convincing evidence for this has been provided by studies into the skeletal effects of the hormone leptin, which has been shown to centrally regulate bone mass, and through studies into the skeletal effects of hypothalamic neuropeptide Y2 and Y4 receptors. This paper discusses recent evidence for the neural regulation of bone metabolism and, in particular, the potential role of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT). Recent studies have demonstrated the presence of functional pathways in bone for both responding to and regulating the uptake of 5-HT. This is of high clinical relevance given the role of the serotonergic system in affective disorders, and the wide use of pharmacological agents that target the 5-HT system to manage these disorders. Initial data suggest that exposure to these agents at different stages during the lifespan may have significant effects on the skeleton.

The serotonin reuptake transporter (SERT) plays an important role in serotonin neurotransmission and in several psychopathological disorders such as depression and anxiety disorders. In this study, we investigated whether the ovarian steroids, estrogen (E) and progesterone (P) regulate SERT binding, intracellular distribution, and function using [(3)H]citalopram ligand binding with quantitative autoradiography, immunofluorescence histochemistry with confocal microscopy and [(3)H]serotonin uptake, respectively. Ovariectomized macaques received either placebo, E alone, P alone or E plus P for 28 days. In the raphe, E, P, and E+P treatments did not change SERT binding density. In several hypothalamic nuclei, [(3)H]citalopram binding was increased by E, P, and E+P. Immunofluorescent SERT in serotonin soma was intracellular and similar among treatments. In the hypothalamus, immunofluorescent SERT was located along the serotonergic axons and there was a significant proliferation of immunofluorescent fibers in hormone-treated animals. In addition, E and E+P treatment increased serotonin uptake in the basal ganglia. These findings suggest that ovarian hormones regulate SERT protein expression and distribution, perhaps via extracellular serotonin or mRNA stability, but not solely at the level of gene transcription. Further investigation on the possible action of ovarian steroids on the directionality of SERT transport is indicated.

Neurotransmitter regulation of bone metabolism has been the subject of increasing interest and investigation. Because serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we investigated the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines, including ROS 17/2.8, UMR 106-H5, and Py1a, showed mRNA expression for 5-HTT as well as the 5-HT(1A), 5-HT(1D), 5-HT(2A), and 5-HT(2B) receptors by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Protein expression of the 5-HT(1A), 5-HT(2A), and 5-HT(2B) receptors was confirmed by immunoblot. 5-HTT binding sites were assessed in ROS 17/2.8 and UMR 106-H5 cells by binding of the stable cocaine analog [125I]RTI-55, which showed a relatively high density of nanomolar affinity binding sites. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, showed the highest potency to antagonize [125I]RTI-55 binding in ROS and UMR cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in ROS cells was 110 pmol/10 min per well, with a K(m) value of 1.13 micromol/L. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC(50) values in the nanomolar range. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased almost eightfold by day 31. In mature rOB cultures, the estimated density of [125I]RTI-55 binding sites was 600 fmol/mg protein. Functional downregulation of transporter activity was assessed after PMA treatment, which caused a significant 40% reduction in the maximal uptake rate of [3H]5-HT, an effect that was prevented by pretreatment with staurosporine. The affinity of 5-HT for the transporter was significantly increased following PMA treatment. We assessed the functional significance of expression of the 5-HT receptors by investigating the interaction between 5-HT and parathyroid hormone (PTH) signaling. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR cells. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT.

Background: Ethanol withdrawal seizure-prone (WSP) and -resistant (WSR) mice have been genetically selected for differences in handling-induced convulsion severity during withdrawal from chronic ethanol administration. Importantly, drug-naive mice from these selected lines also differ in handling-induced convulsion severity. Different N-methyl-D-aspartate (NMDA) receptor subunit and splice variant associations confer varying sensitivities to ethanol, and may play a role in the different behavioral responses of the WSP and WSR mice. Methods: In situ hybridization of riboprobes was used to characterize NMDA receptor subunit and splice variant mRNA expression in cortex and hippocampus from WSP and WSR mice. In addition, immunoblotting and immunohistochemistry were used to examine the expression of specific NMDA receptor subunits and splice variants in hippocampus and cortex from the selected mouse lines. Results: In situ hybridization of riboprobes indicated that, in brain sections from both WSP and WSR mice, there was a differential regional distribution of mRNA for the mouse NR1, NR2A, NR2B, and NR2C NMDA receptor subunits. However, there were no differences between the selected lines in the hybridization of riboprobes to hippocampal subfields or cortical layers. In addition, hybridization of the probe for a 63-base N1-terminal cassette of ethanol-sensitive NR1 splice variants labeled both cortex and hippocampus. The level of hybridization did not differ across subfields of the hippocampus. Results from Western blot and immunohistochemical experiments also indicated that there were no differences between selected lines in NMDA receptor subunit protein expression. However, there was a correlation between mRNA and protein expression in hippocampus and cortex for each NMDA receptor subunit that was examined. Conclusions: The data suggest that at the level of both mRNA and protein, NMDA receptor subunit and splice variant expression can be uncoupled from convulsion severity in mice that have been selectively bred for symptoms of ethanol withdrawal.

Drug-induced efflux of substrates was characterized in C6 rat glioma cells stably expressing a recombinant human dopamine (DA) or serotonin (5-HT) transporter (C6-hDAT and C6-hSERT, respectively). In the absence of Ca2+, these cells spontaneously and rapidly released preloaded [3H]DA or [3H]5-HT, respectively, but maintained constant levels of [3H]N-methy-4-phenylpyridinium (MPP+) for up to 90 minutes. In C6-hSERT cells, transporter substrates such as methamphetamine, amphetamine, and dopamine induced relatively rapid release of [3H]MPP+, with t1/2 values of approximately 15 minutes, while the t1/2 value for serotonin was about 30 minutes. Similar results were obtained with C6-hDAT cells. Uptake blockers that are not substrates at the transporters had considerably greater t1/2 values, as compared to substrates, suggesting different mechanisms for altering transporter function. Dose-response curves for each drug, conducted at each drug's t1/2, indicated considerable differences in potency (EC50) at stimulating [3H]MPP+ release from C6-hSERT cells [3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester (RTI-55) > imipramine > 1-[2-diphenylmethoxy]ethyl-4-(3-phenylpropyl)-piperazine (GBR-12935) threo-(+/-)-methylphenidate > cocaine > mazindol > 2-beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) > (+)methamphetamine > amphetamine > DA > fenfluramine > norepinephrine (NE) > 5-HT]. A different rank order of potency was observed for the effects of drugs on [3H]MPP+ release from C6-hDAT cells [imipramine > RTI-55 > cocaine > mazindol > CFT > GBR-12935 > threo-(+/-)-methylphenidate > amphetamine > (+)methamphetamine > fenfluramine > DA > NE > 5-HT]. Based on efficacies for stimulating [3H]MPP+ release from C6-hDAT cells, drugs could be grouped into three categories, with substrates causing release of approximately 75% of loaded [3H]MPP+, cocaine analogues causing approximately 50% release, and other drugs causing an average release of approximately 25% of loaded [3H]MPP+. The results, taken together with results from previous reports, suggest that the transfected cell type contributes to the characteristics of transporter-mediated release, that drugs interact with different sites on the transporters in the uptake and release process, and that the mechanism of transporter-mediated release may not be a simple reversal of substrate uptake.

To determine if catechol-O-methyltransferase (COMT) metabolizes catecholamines within cell lines used for heterologous expression of plasmalemmal transporters and alters the measured characteristics of 3H-substrate transport, the uptake of monoamine transporter substrates was assessed in three cell lines (C6 glioma, L-M fibroblast, and HEK293 cells) that had been transfected with the recombinant human transporters. Uptake and cellular retention of 3H-catecholamines was increased by up to fourfold by two COMT inhibitors, tropolone and Ro 41-0960, with potencies similar to those for inhibition of COMT activity, whereas the uptake of two transporter substrates that are not substrates for COMT, [3H]serotonin and [3H]MPP+, was unaffected. Direct measurement of monoamine substrates by HPLC confirmed that tropolone (1 mM) increased the retention of the catecholamines dopamine and norepinephrine, but not the retention of serotonin in HEK293 cells. Saturation analysis of the uptake of [3H]dopamine by C6 cells expressing the dopamine transporter demonstrated that tropolone (1 mM) decreased the apparent Km of transport from 0.61 microM to 0.34 microM without significantly altering the maximal velocity of transport. These data suggest that endogenous COMT activity in mammalian cells may alter neurotransmitter deposition and thus the apparent kinetic characteristics of transport.

Psychoactive-substituted phenethylamines 2,5-dimethoxy-4-chlorophenethylamine (2C-C); 2,5-dimethoxy-4-methylphenethylamine (2C-D); 2,5-dimethoxy-4-ethylphenethylamine (2C-E); 2,5-dimethoxy-4-iodophenethylamine (2C-I); 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2); and 2,5-dimethoxy-4-chloroamphetamine (DOC) are used recreationally and may have deleterious side effects. This study compares the behavioral effects and the mechanisms of action of these substituted phenethylamines with those of hallucinogens and a stimulant. The effects of these compounds on mouse locomotor activity and in rats trained to discriminate dimethyltryptamine, (−)-DOM, (+)-LSD, (±)-MDMA, and S(+)-methamphetamine were assessed. Binding and functional activity of the phenethylamines at 5-HT1A, 5-HT2A, 5-HT2C receptors and monoamine transporters were assessed using cells heterologously expressing these proteins. The phenethylamines depressed mouse locomotor activity, although 2C-D and 2C-E stimulated activity at low doses. The phenethylamines except 2C-T-2 fully substituted for at least one hallucinogenic training compound, but none fully substituted for (+)-methamphetamine. At 5-HT1A receptors, only 2C-T-2 and 2C-I were partial-to-full very low potency agonists. In 5-HT2A arachidonic acid release assays, the phenethylamines were partial to full agonists except 2C-I which was an antagonist. All compounds were full agonists at 5-HT2A and 5-HT2C receptor inositol phosphate assays. Only 2C-I had moderate affinity for, and very low potency at, the serotonin transporter. The discriminative stimulus effects of 2C-C, 2C-D, 2C-E, 2C-I, and DOC were similar to those of several hallucinogens, but not methamphetamine. Additionally, the substituted phenethylamines were full agonists at 5-HT2A and 5-HT2C receptors, but for 2C-T-2, this was not sufficient to produce hallucinogen-like discriminative stimulus effects. Additionally, the 5-HT2A inositol phosphate pathway may be important in 2C-I's psychoactive properties.

The use of synthetic methcathinones, components of “bath salts,” is a world-wide health concern. These compounds, structurally similar to methamphetamine (METH) and 3,4-methylendioxymethamphetamine (MDMA), cause tachycardia, hallucinations and psychosis. We hypothesized that these potentially neurotoxic and abused compounds display differences in their transporter and receptor interactions as compared to amphetamine counterparts. 3,4-Methylenedioxypyrovalerone and naphyrone had high affinity for radioligand binding sites on recombinant human dopamine (hDAT), serotonin (hSERT) and norepinephrine (hNET) transporters, potently inhibited [ 3 H]neurotransmitter uptake, and, like cocaine, did not induce transporter-mediated release. Butylone was a lower affinity uptake inhibitor. In contrast, 4-fluoromethcathinone, mephedrone and methylone had higher inhibitory potency at uptake compared to binding and generally induced release of preloaded [ 3 H]neurotransmitter from hDAT, hSERT and hNET (highest potency at hNET), and thus are transporter substrates, similar to METH and MDMA. At hNET, 4-fluoromethcathinone was a more efficacious releaser than METH. These substituted methcathinones had low uptake inhibitory potency and low efficacy at inducing release via human vesicular monoamine transporters (hVMAT2). These compounds were low potency (1) h5-HT 1A receptor partial agonists, (2) h5-HT 2A receptor antagonists, (3) weak h5-HT 2C receptor antagonists. This is the first report on aspects of substituted methcathinone efficacies at serotonin (5-HT) receptors and in superfusion release assays. Additionally, the drugs had no affinity for dopamine receptors, and high-nanomolar to mid-micromolar affinity for hSigma1 receptors. Thus, direct interactions with hVMAT2 and serotonin, dopamine, and hSigma1 receptors may not explain psychoactive effects. The primary mechanisms of action may be as inhibitors or substrates of DAT, SERT and NET.

Objective The iboga alkaloids are a class of small molecules defined structurally on the basis of a common ibogamine skeleton, some of which modify opioid withdrawal and drug self-administration in humans and preclinical models. These compounds may represent an innovative approach to neurobiological investigation and development of addiction pharmacotherapy. In particular, the use of the prototypic iboga alkaloid ibogaine for opioid detoxification in humans raises the question of whether its effect is mediated by an opioid agonist action, or if it represents alternative and possibly novel mechanism of action. The aim of this study was to independently replicate and extend evidence regarding the activation of μ-opioid receptor (MOR)-related G proteins by iboga alkaloids. Methods Ibogaine, its major metabolite noribogaine, and 18-methoxycoronaridine (18-MC), a synthetic congener, were evaluated by agonist-stimulated guanosine-5´-O-(γ-thio)-triphosphate ([35S]GTPγS) binding in cells overexpressing the recombinant MOR, in rat thalamic membranes, and autoradiography in rat brain slices. Results And Significance In rat thalamic membranes ibogaine, noribogaine and 18-MC were MOR antagonists with functional Ke values ranging from 3 uM (ibogaine) to 13 uM (noribogaine and 18MC). Noribogaine and 18-MC did not stimulate [35S]GTPγS binding in Chinese hamster ovary cells expressing human or rat MORs, and had only limited partial agonist effects in human embryonic kidney cells expressing mouse MORs. Ibogaine did not did not stimulate [35S]GTPγS binding in any MOR expressing cells. Noribogaine did not stimulate [35S]GTPγS binding in brain slices using autoradiography. An MOR agonist action does not appear to account for the effect of these iboga alkaloids on opioid withdrawal. Taken together with existing evidence that their mechanism of action also differs from that of other non-opioids with clinical effects on opioid tolerance and withdrawal, these findings suggest a novel mechanism of action, and further justify the search for alternative targets of iboga alkaloids.

The dopamine transporter removes the neurotransmitter from the synapse, regulating dopamine availability. The transporter can be internalized and its function is blocked by cocaine and other ligands. Melittin inhibits dopamine transporter function and causes internalization of the recombinant transporter in stably transfected HEK-293 cells, but the specific pathways for internalization and disposition of the transporter are unknown. Here we report that melittin treatment increased both transporter internalization and colocalization with clathrin, effects that were blocked by pretreatment with cocaine. Density gradient centrifugation revealed that melittin treatment caused the dopamine transporter to associate with a density fraction containing the early endosome marker Rab 5A. Confocal microscopy revealed that melittin treatment also increased transporter colocalization with Rab 5A and decreased colocalization with the late endosome marker Rab 7 and the recycling endosome marker Rab 11. Following 60 min of melittin treatment, the transporter was trafficked back to the membrane. By comparison, phorbol ester treatment increased transporter colocalization with early endosome antigen 1 and Rab 7 in a time-dependent manner. Cocaine treatment alone does not affect transporter trafficking in these cells. Results indicate multiple dopamine transporter internalization and recycling pathways that depend on transporter-ligand interactions and post-translational modifications.

[1] The nearshore circulation induced by a focused pattern of surface gravity waves is studied at a beach adjacent to a major inlet with a large ebb tidal shoal. Using a coupled wave and wave-averaged nearshore circulation model, it is found that the nearshore circulation is significantly affected by the heterogeneous wave patterns caused by wave refraction over the ebb tidal shoal. The model is used to predict waves and currents during field experiments conducted near the mouth of San Francisco Bay and nearby Ocean Beach. The field measurements indicate strong spatial variations in current magnitude and direction and in wave height and direction along Ocean Beach and across the ebb tidal shoal. Numerical simulations suggest that wave refraction over the ebb tidal shoal causes wave focusing toward a narrow region at Ocean Beach. Due to the resulting spatial variation in nearshore wave height, wave-induced setup exhibits a strong alongshore nonuniformity, resulting in a dramatic change in the pressure field compared to a simulation with only tidal forcing. The analysis of momentum balances inside the surf zone shows that, under wave conditions with intensive wave focusing, the alongshore pressure gradient associated with alongshore nonuniform wave setup can be a dominant force driving circulation, inducing heterogeneous alongshore currents. Pressure-gradient-forced alongshore currents can exhibit flow reversals and flow convergence or divergence, in contrast to the uniform alongshore currents typically caused by tides or homogeneous waves.

Phospholipase A(2) releases the fatty acid arachidonic acid from membrane phospholipids. We used the purported phospholipase A(2) stimulator, melittin, to examine the effects of endogenous arachidonic acid signaling on dopamine transporter function and trafficking. In HEK-293 cells stably transfected with the dopamine transporter, melittin reduced uptake of [((3))H]dopamine. Additionally, measurements of fatty acid content demonstrated a melittin-induced release of membrane-incorporated arachidonic acid, but inhibitors of phospholipase C, phospholipase D, and phospholipase A(2) did not prevent the release. Subsequent experiments measuring [(125)I]RTI-55 binding to the dopamine transporter demonstrated a direct interaction of melittin, or a melittin-activated endogenous compound, with the transporter to inhibit antagonist binding. This effect was not specific to the dopamine transporter, as [(3)H]spiperone binding to the recombinant dopamine D(2) receptor was also inhibited by melittin treatment. Finally, melittin stimulated an increase in internalization of the dopamine transporter, and this effect was blocked by pretreatment with cocaine. Thus, melittin acts through multiple mechanisms to regulate cellular activity, including release of membrane-incorporated fatty acids and interaction with the dopamine transporter.

Rainbow trout (Oncorhynchus mykiss) are extremely sensitive to the neurotoxic activity of pyrethroid insecticides. One possible target for pyrethroids is the GABA(A) receptor of brain of the trout, the function of which can be tested by measurement of influx of 36Cl- into synaptoneurosomes, in response to the application of agonists. gamma-Aminobutyric acid produced a time- and concentration-dependent increase in influx of 36Cl- in synaptoneurosomes from the brain of the trout, which exhibited the pharmacology characteristic of a response mediated by activation of a GABAA receptor. Deltamethrin, (1R alpha S)-cis-cypermethrin and permethrin produced a dose-dependent increase in the basal uptake and a corresponding decrease in GABA-dependent influx, with a maximum inhibition of 70-82%. This effect of pyrethroid was stereospecific, of high potency and inhibited by tetrodotoxin (TTX) and t-butylbicyclophosphorothionate (TBPS). The sensitivity of the effect of the pyrethroid to TTX suggested an activation by pyrethroid of the voltage-dependent sodium channel. Veratridine, a sodium channel activator, elicited similar changes in the basal uptake of chloride, which were TTX-sensitive. Neither deltamethrin nor veratridine had a measurable effect on the efflux of 36Cl- from synaptoneurosomes. Thus, pyrethroid insecticides may interfere with the function of GABAA receptors indirectly through an interaction with the voltage-dependent sodium channel in the brain of the trout and consequently perturb chloride influx, possibly through a voltage-dependent chloride channel.

Five (1R-2-exo-3-exo)-3-(N-phenylcarbamoyl)ecgonine methyl ester analogues were synthesized and characterized by 1H and 13C NMR, IR, and thermospray MS. The compounds were synthesized in two or three steps as (-)-stereoisomers from (-)-ecgonine in good yield (56% overall). These cocaine derivatives were assessed for their ability to inhibit [3H]cocaine binding to rat striatal tissue and to inhibit [3H]dopamine uptake into synaptosomes prepared from the same tissue. The most potent of the analogues was (1R-2-exo-3-exo)-2-(carbomethoxy)-8-methyl-8-azabicyclo[3.2.1]octyl 3-N-(3'-nitrophenyl)carbamate. IC50 values for inhibition of cocaine binding and dopamine uptake were 37 and 178 nM, respectively. Amino derivatives were less active than the nitro and (1R-2-exo-3-exo)-2-(carbomethoxy)-8-methyl-8-azabicyclo [3.2.1]octyl 3-N-(4'-aminophenyl)carbamate had the lowest affinity for the receptor with IC50 values of 63 and greater than 100 microM in the aforementioned assays, respectively.

Themechanismsofinteractionbetweenlobelineandthedopaminetransporter(DAT)orthevesicular monoamine transporter (VMAT-2) are not clear. The goal of this study was toelucidatetheeffectsoflobelineonthesetransportersinacellsystemco-expressingtheDATand VMAT-2. Lobeline caused release of [ 3 H]dopamine to a similar extent as reserpine(VMAT-2 inhibitor), but was less efficacious than methamphetamine or dopamine. Addi-tionally, lobeline decreased the [ 3 H]dopamine-releasing effects of methamphetamine,unlike reserpine which increased release by methamphetamine. These results suggest thatlobeline has unique properties at the DAT and VMAT-2 which may make it useful as apharmacotherapeutic to treat methamphetamine abuse.Published by Elsevier Inc.* Corresponding author at: Research Service (RD-22), Veterans Affairs Medical Center, 3710 S.W. U.S. Veterans Hospital Road, Portland, OR97239, USA. Tel.: +1 503 721 7912; fax: +1 503 721 7839.E-mail address: janowsky@ohsu.edu (A. Janowsky). available at www.sciencedirect.comjournal homepage: www.elsevier.com/locate/biochempharm

Methamphetamine (METH) causes release of stored intracellular dopamine (DA). We explored the interactions of METH with the recombinant human vesicular monoamine (hVMAT2) and/or human DA transporters (hDAT) in transfected mammalian (HEK293) cells and compared the findings with those for DA. In 'static' release assays at 37 degrees C, less than 20% of pre-loaded [(3)H]DA was lost after 60 min, while nearly 80% of pre-loaded [(3)H]METH was lost at 37 degrees C under non-stimulated conditions. Results obtained by measuring substrate release using a superfusion apparatus revealed an even greater difference in substrate efflux. At pH 7.4, nearly all of the pre-loaded [(3)H]METH was lost after just 6 min, compared with the loss of 70-80% of pre-loaded [(3)H]DA (depending on cell type) after superfusion for 32 min. Increasing the extracellular pH from 7.4 to 8.6 had opposite effects on [(3)H]DA and [(3)H]METH retention. At pH 8.6, [(3)H]METH was retained more effectively by both hDAT and hDAT-hVMAT2 cells, compared with results obtained at extracellular pH 7.4. [(3)H]DA, however, was more effectively retained at pH 7.4 than at pH 8.6. These data suggest that DA and METH interact differently with the DAT and VMAT2, and require different H(+) concentrations to exert their effects.

Cocaine is a suspected cofactor in human immunodeficiency virus (HIV)-associated dementia but cocaine’s effects are not clear. Herein the authors describe investigations of the mechanisms by which cocaine increases HIV-1 invasion through brain microvascular endothelial cells (BMVECs). Cocaine binds to a site on BMVECs, which is not a biogenic amine transporter, a binding site for estrogen, or a muscarinic receptor and for which benztropine and tamoxifen have the highest affinity. Cocaine treatment of BMVECs disrupts intercellular junctions and induces cell ruffling, which could account for their increased permeability and decreased electrical resistance. HIV-1 enters BMVECs by macropinocytosis and is transported to lysosomes and inactivated. In cocaine-treated BMVECs, the virus enters and persists in large cytoplasmic “lakes.” Cocaine exposure of BMVECs up-regulates transcription of genes important in cytoskeleton organization, signal transduction, cell swelling, vesicular trafficking, and cell adhesion. The toxicity of cocaine for the blood-brain barrier may lead to increased virus neuroinvasion and neurovascular complications of cocaine abuse.

Many individuals are frightened by hair loss and are hesitant to speak about it. Many are unaware that stressors can causes hair loss and that hair care practices and habits can aggravate a hair loss situation. Intervention by the nurse in encouraging a person to have an adequate assessment and work-up can facilitate an accurate diagnosis. Supportive and appropriate therapy can then be arranged. The hair tells a story and can be associated with good health.