Ernesto Solis

Bath salts components mephedrone and methylenedioxypyrovalerone (MDPV) act synergistically at the human dopamine transporter

Bath salts is the street name for drug combinations that contain synthetic cathinone analogues, among them possibly mephedrone (MEPH) and certainly methylenedioxypyrovalerone (MDPV). In animal studies, cathinone and certain cathinone analogues release dopamine (DA), similar to the action of amphetamine (AMPH) and methamphetamine (METH). AMPH and METH act on the human DA transporter (hDAT); thus, we investigated MEPH and MDPV acting at hDAT.

Baths Salts, Spice, and Related Designer Drugs: The Science Behind the Headlines

The abuse of synthetic psychoactive substances known as “designer drugs,” or “new psychoactive substances” (NPS), is increasing at an alarming rate. NPS are purchased as alternatives to traditional illicit drugs of abuse and are manufactured to circumvent laws regulating the sale and use of controlled substances. Synthetic cathinones (i.e., “bath salts”) and synthetic cannabinoids (i.e., “spice”) are two types of NPS that have received substantial media attention. Although low recreational doses of bath salts or spice compounds can produce desirable effects, high doses or chronic exposure often leads to dangerous medical consequences, including psychosis, violent behaviors, tachycardia, hyperthermia, and even death. Despite the popularity of NPS, there is a paucity of scientific data about these drugs. Here we provide a brief up-to-date review describing the mechanisms of action and neurobiological effects of synthetic cathinones and cannabinoids.

Deconstruction of the abused synthetic cathinone methylenedioxypyrovalerone (MDPV) and an examination of effects at the human dopamine transporter.

Synthetic cathinones, β-keto analogues of amphetamine (or, more correctly, of phenylalkylamines), represent a new and growing class of abused substances. Several such analogues have been demonstrated to act as dopamine (DA) releasing agents. Methylenedioxypyrovalerone (MDPV) was the first synthetic cathinone shown to act as a cocaine-like DA reuptake inhibitor. MDPV and seven deconstructed analogues were examined to determine which of MDPVs structural features account(s) for uptake inhibition. In voltage-clamped (-60 mV) Xenopus oocytes transfected with the human DA transporter (hDAT), all analogues elicited inhibitor-like behavior shown as hDAT-mediated outward currents. Using hDAT-expressing mammalian cells we determined the affinities of MDPV and its analogues to inhibit uptake of [3H]DA by hDAT that varied over a broad range (IC50 values ca. 135 to >25,000 nM). The methylenedioxy group of MDPV made a minimal contribution to affinity, the carbonyl group and a tertiary amine are more important, and the extended α-alkyl group seems most important. Either a tertiary amine, or the extended α-alkyl group (but not both), are required for the potent nature of MDPV as an hDAT inhibitor.

A Conserved Asparagine Residue in Transmembrane Segment 1 (TM1) of Serotonin Transporter Dictates Chloride-coupled Neurotransmitter Transport

Na+- and Cl−-dependent uptake of neurotransmitters via transporters of the SLC6 family, including the human serotonin transporter (SLC6A4), is critical for efficient synaptic transmission. Although residues in the human serotonin transporter involved in direct Cl− coordination of human serotonin transport have been identified, the role of Cl− in the transport mechanism remains unclear. Through a combination of mutagenesis, chemical modification, substrate and charge flux measurements, and molecular modeling studies, we reveal an unexpected role for the highly conserved transmembrane segment 1 residue Asn-101 in coupling Cl− binding to concentrative neurotransmitter uptake.

4-(4-(Dimethylamino)phenyl)-1-methylpyridinium (APP+) Is a Fluorescent Substrate for the Human Serotonin Transporter

Monoamine transporters terminate synaptic neurotransmission and are molecular targets for antidepressants and psychostimulants. Fluorescent reporters can monitor real-time transport and are amenable for high-throughput screening. However, until now, their use has mostly been successful to study the catecholamine transporters but not the serotonin (5HT) transporter. Here, we use fluorescence microscopy, electrophysiology, pharmacology, and molecular modeling to compare fluorescent analogs of 1-methyl-4-phenylpyridinium (MPP+) as reporters for the human serotonin transporter (hSERT) in single cells. The fluorescent substrate 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP+) exhibits superior fluorescence uptake in hSERT-expressing HEK293 cells than other MPP+ analogs tested. APP+ uptake is Na+- and Cl−-dependent, displaced by 5HT, and inhibited by fluoxetine, suggesting APP+ specifically monitors hSERT activity. ASP+, which was previously used to study catecholamine transporters, is 10 times less potent than APP+ at inhibiting 5HT uptake and has minimal hSERT-mediated uptake. Furthermore, in hSERT-expressing oocytes voltage-clamped to −60 mV, APP+ induced fluoxetine-sensitive hSERT-mediated inward currents, indicating APP+ is a substrate, whereas ASP+ induced hSERT-mediated outward currents and counteracted 5HT-induced hSERT currents, indicating ASP+ possesses activity as an inhibitor. Extra-precise ligand receptor docking of APP+ and ASP+ in an hSERT homology model showed both ASP+ and APP+ docked favorably within the active region; accordingly, comparable concentrations are required to elicit their opposite electrophysiological responses. We conclude APP+ is better suited than ASP+ to study hSERT transport fluorometrically.

S(+)amphetamine induces a persistent leak in the human dopamine transporter: molecular stent hypothesis.

BACKGROUND AND PURPOSE Wherever they are located, dopamine transporters (DATs) clear dopamine (DA) from the extracellular milieu to help regulate dopaminergic signalling. Exposure to amphetamine (AMPH) increases extracellular DA in the synaptic cleft, which has been ascribed to DAT reverse transport. Increased extracellular DA prolongs postsynaptic activity and reinforces abuse and hedonic behaviour.

Electrical coupling between the human serotonin transporter and voltage-gated Ca2+ channels

Monoamine transporters have been implicated in dopamine or serotonin release in response to abused drugs such as methamphetamine or ecstasy (MDMA). In addition, monoamine transporters show substrate-induced inward currents that may modulate excitability and Ca(2+) mobilization, which could also contribute to neurotransmitter release. How monoamine transporters modulate Ca(2+) permeability is currently unknown. We investigate the functional interaction between the human serotonin transporter (hSERT) and voltage-gated Ca(2+) channels (CaV). We introduce an excitable expression system consisting of cultured muscle cells genetically engineered to express hSERT. Both 5HT and S(+)MDMA depolarize these cells and activate the excitation-contraction (EC)-coupling mechanism. However, hSERT substrates fail to activate EC-coupling in CaV1.1-null muscle cells, thus implicating Ca(2+) channels. CaV1.3 and CaV2.2 channels are natively expressed in neurons. When these channels are co-expressed with hSERT in HEK293T cells, only cells expressing the lower-threshold L-type CaV1.3 channel show Ca(2+) transients evoked by 5HT or S(+)MDMA. In addition, the electrical coupling between hSERT and CaV1.3 takes place at physiological 5HT concentrations. The electrical coupling between monoamine neurotransmitter transporters and Ca(2+) channels such as CaV1.3 is a novel mechanism by which endogenous substrates (neurotransmitters) or exogenous substrates (like ecstasy) could modulate Ca(2+)-driven signals in excitable cells.

Amphetamine activates calcium channels through dopamine transporter-mediated depolarization.

Amphetamine (AMPH) and its more potent enantiomer S(+)AMPH are psychostimulants used therapeutically to treat attention deficit hyperactivity disorder and have significant abuse liability. AMPH is a dopamine transporter (DAT) substrate that inhibits dopamine (DA) uptake and is implicated in DA release. Furthermore, AMPH activates ionic currents through DAT that modify cell excitability presumably by modulating voltage-gated channel activity. Indeed, several studies suggest that monoamine transporter-induced depolarization opens voltage-gated Ca(2+) channels (CaV), which would constitute an additional AMPH mechanism of action. In this study we co-express human DAT (hDAT) with Ca(2+) channels that have decreasing sensitivity to membrane depolarization (CaV1.3, CaV1.2 or CaV2.2). Although S(+)AMPH is more potent than DA in transport-competition assays and inward-current generation, at saturating concentrations both substrates indirectly activate voltage-gated L-type Ca(2+) channels (CaV1.3 and CaV1.2) but not the N-type Ca(2+) channel (CaV2.2). Furthermore, the potency to achieve hDAT-CaV electrical coupling is dominated by the substrate affinity on hDAT, with negligible influence of L-type channel voltage sensitivity. In contrast, the maximal coupling-strength (defined as Ca(2+) signal change per unit hDAT current) is influenced by CaV voltage sensitivity, which is greater in CaV1.3- than in CaV1.2-expressing cells. Moreover, relative to DA, S(+)AMPH showed greater coupling-strength at concentrations that induced relatively small hDAT-mediated currents. Therefore S(+)AMPH is not only more potent than DA at inducing hDAT-mediated L-type Ca(2+) channel currents but is a better depolarizing agent since it produces tighter electrical coupling between hDAT-mediated depolarization and L-type Ca(2+) channel activation.

Fentanyl-Induced Brain Hypoxia Triggers Brain Hyperglycemia and Biphasic Changes in Brain Temperature

Fentanyl is a potent synthetic opioid used extensively in humans for general anesthesia and analgesia. Fentanyl has emerged as a recreational drug, often in combination with heroin, and can result in lethality during overdose. Fentanyl is well characterized as an anesthetic, but the basic physiological effects of fentanyl in the brain when taken as a drug of abuse are largely unknown. We used high-speed amperometry in freely moving rats to examine the effects of intravenous fentanyl at doses within the range of possible human intake (3–40 μg/kg) on oxygen and glucose levels in nucleus accumbens (NAc). Fentanyl induced a rapid, dose-dependent decrease in NAc oxygen followed by a more delayed and prolonged increase in NAc glucose. Fentanyl induced similar oxygen decreases in the basolateral amygdala, indicating that brain hypoxia could be a generalized phenomenon. We used oxygen recordings in the subcutaneous space to confirm that fentanyl-induced brain hypoxia results from decreases in blood oxygen levels caused by drug-induced respiratory depression. Temperature recordings in the NAc, muscle, and skin showed that fentanyl induces biphasic changes in brain temperature, with an initial decrease that results primarily from peripheral vasodilation, and a subsequent increase driven by metabolic brain activation. The initial vasodilation appears caused by respiratory depression-induced hypoxia and a subsequent rise in CO2 that drives fentanyl-induced increases in NAc glucose. Together, these data suggest that fentanyl-induced respiratory depression triggers brain hypoxia and subsequent hyperglycemia, both of which precede slower changes in brain temperature and metabolic brain activity.

N -Alkylated Analogs of 4-Methylamphetamine (4-MA) Differentially Affect Monoamine Transporters and Abuse Liability

Clandestine chemists synthesize novel stimulant drugs by exploiting structural templates known to target monoamine transporters for dopamine, norepinephrine, and serotonin (DAT, NET, and SERT, respectively). 4-Methylamphetamine (4-MA) is an emerging drug of abuse that interacts with transporters, but limited structure–activity data are available for its analogs. Here we employed uptake and release assays in rat brain synaptosomes, voltage-clamp current measurements in cells expressing transporters, and calcium flux assays in cells coexpressing transporters and calcium channels to study the effects of increasing N-alkyl chain length of 4-MA on interactions at DAT, NET, and SERT. In addition, we performed intracranial self-stimulation in rats to understand how the chemical modifications affect abuse liability. All 4-MA analogs inhibited uptake at DAT, NET, and SERT, but lengthening the amine substituent from methyl to ethyl, propyl, and butyl produced a stepwise decrease in potency. N-methyl 4-MA was an efficacious substrate-type releaser at DAT that evoked an inward depolarizing current and calcium influx, whereas other analogs did not exhibit these effects. N-methyl and N-ethyl 4-MA were substrates at NET, whereas N-propyl and N-butyl 4-MA were not. All analogs acted as SERT substrates, though N-butyl 4-MA had very weak effects. Intracranial self-stimulation in rats showed that elongating the N-alkyl chain decreased abuse-related effects in vivo that appeared to parallel reductions in DAT activity. Overall, converging lines of evidence show that lengthening the N-alkyl substituent of 4-MA reduces potency to inhibit transporters, eliminates substrate activity at DAT and NET, and decreases abuse liability of the compounds.