Cardiovascular and Locomotor Effects of Binary Mixtures of Common "Bath Salts" Constituents: Studies with Methylone, MDPV, and Caffeine in Rats.

Background and Purpose: The use of "Bath Salts" drug preparations has been associated with high rates of toxicity and death. Preparations often contain mixtures of drugs including multiple synthetic cathinones or synthetic cathinones and caffeine; however, little is known about whether interactions among "Bath Salts" constituents contribute to the adverse effects often reported in users. Experimental Approach: This study used adult male Sprague-Dawley rats to characterize the cardiovascular effects, locomotor effects, and pharmacokinetics of methylone, MDPV, and caffeine, administered alone and as binary mixtures. Dose-addition analyses were used to determine the effect levels predicted for a strictly additive interaction for each dose pair. Key Results: Methylone, MDPV, and caffeine increased heart rate and locomotion, with methylone producing the largest increase in heart rate, MDPV producing the largest increase in locomotor activity, and caffeine being the least effective in stimulating heart rate and locomotor activity. MDPV and caffeine increased mean arterial pressure, with caffeine being more effective than MDPV. The nature of the interactions between methylone and MDPV tended toward sub-additivity for all endpoints, whereas interactions between MDPV or methylone and caffeine tended to be additive or sub-additive for cardiovascular endpoints, and additive or supra-additive for increases in locomotion. No pharmacokinetic interactions were observed between individual constituents, but methylone displayed non-linear pharmacokinetics at the largest dose evaluated. Conclusion and Implications: These findings demonstrate that the composition of "Bath Salts" preparations can impact both cardiovascular and locomotor effects and suggest that such interactions among constituent drugs could contribute to the "Bath Salts" toxidrome reported by human users.

Ethanol potentiates dopamine uptake and increases cell surface distribution of dopamine transporters expressed in SK-N-SH and HEK-293 cells.

Ethanol increases dopaminergic release in the reward and reinforcement areas of the brain. The primary protein responsible for terminating dopamine (DA) neurotransmission is the plasma membrane-bound dopamine transporter (DAT). In vitro electrophysiological and biochemical studies in Xenopus laevis oocytes have previously shown ethanol potentiates DAT function and increases transporter-binding sites. The potentiating effect of ethanol on the transporter is eliminated in Xenopus oocytes by the DAT mutation glycine 130 to threonine. However, ethanol's action on DAT functional regulation has yet to be examined in mammalian cell expression systems. To further understand the molecular mechanisms of ethanol's action on DAT, we determined the direct mechanistic action of short-term (< or =2 h) ethanol exposure on transporter function and cell surface distribution in non-neuronal human embryonic kidney cells-293 (HEK-293) and neuronal SK-N-SH neuroblastoma cells expressing the transporter. Wild-type or G130T mutant DAT were overexpressed in HEK-293 and SK-N-SH cells. Ethanol potentiated DAT mediated [(3)H]DA uptake in a dose (25, 50, 100 mM), but not time dependent manner in cells expressing wild-type DAT. Ethanol-induced potentiation of uptake was significantly reduced in cells expressing the G130T mutant. Analysis of DA uptake kinetic parameters indicates 100-mM ethanol exposure increased [(3)H]DA uptake velocity (V(max)), while affinity for DA (K(m)) remained unchanged. The effect of ethanol on wild-type DAT surface expression was measured by biotinylation cell surface labeling. DAT surface expression increased 40%-50% after 1-h, 100-mM ethanol exposure. These studies show ethanol potentiates DAT functional regulation in both neuronal and non-neuronal cells, suggesting a direct mechanistic action of ethanol on transporter trafficking in mammalian systems. Our findings demonstrate ethanol's action on DAT function and regulation is consistent across multiple model systems.

Antagonistic effects of dopaminergic signaling and ethanol on protein kinase A-mediated phosphorylation of DARPP-32 and the NR1 subunit of the NMDA receptor.

BACKGROUND: This article extends our initial investigation of the interactions between dopamine and glutamate receptor systems after acute exposure to ethanol. DARPP-32 (dopamine and cyclic adenosine monophosphate-regulated phosphoprotein of approximate molecular weight 32 kDa) is an important regulator of protein phosphatase-1 that in turn regulates a large number of effectors, including the NMDA receptor. METHODS: We measured the protein kinase A (PKA)-mediated phosphorylation of DARPP-32 and the NR1 subunit of the NMDA receptor. Initially, corpus striatum was assayed after intraperitoneal treatment of mice with the D1 agonist SKF82958, the D2 agonist and anticraving drug bromocriptine, or ethanol. In other experiments we blocked D1 receptors with the selective D1 antagonist SCH23390 or blocked D2 receptors with the selective D2 antagonist eticlopride. Finally, we examined combinations of some dopaminergic drugs with and without ethanol. RESULTS: SKF82958 alone significantly increased PKA-mediated phosphorylation of both DARPP-32 and NR1. Bromocriptine alone had no effect on pDARPP-32 or on pNR1. However, when D1 receptors were blocked, bromocriptine reduced the PKA-mediated phosphorylation of both DARPP-32 and NR1. Coincident treatment with bromocriptine and ethanol reversed both of these effects with D1 receptors blocked. The combination of eticlopride (D2 blocker) and SF82958 (D1 agonist) did not significantly alter either pDARPP-32 or pNR1. CONCLUSIONS: These data demonstrate antagonistic effects of acute ethanol exposure on D1 signaling in vivo and the potential of acute in vivo challenge protocols to help fill gaps in the understanding of ethanol's effects on protein phosphorylation.