Evidence for a role of transporter-mediated currents in the depletion of brain serotonin induced by serotonin transporter substrates.

Serotonin (5-HT) transporter (SERT) substrates like fenfluramine and 3,4-methylenedioxymethamphetamine cause long-term depletion of brain 5-HT, while certain other substrates do not. The 5-HT deficits produced by SERT substrates are dependent upon transporter proteins, but the exact mechanisms responsible are unclear. Here, we compared the pharmacology of several SERT substrates: fenfluramine, d-fenfluramine, 1-(m-chlorophenyl)piperazine (mCPP) and 1-(m-trifluoromethylphenyl)piperainze (TFMPP), to establish relationships between acute drug mechanisms and the propensity for long-term 5-HT depletions. In vivo microdialysis was carried out in rat nucleus accumbens to examine acute 5-HT release and long-term depletion in the same subjects. In vitro assays were performed to measure efflux of [(3)H]5-HT in rat brain synaptosomes and transporter-mediated ionic currents in SERT-expressing Xenopus oocytes. When administered repeatedly to rats (6 mg/kg, i.p., four doses), all drugs produce large sustained elevations in extracellular 5-HT (>5-fold) with minimal effects on dopamine. Importantly, 2 weeks after dosing, only rats exposed to fenfluramine and d-fenfluramine display depletion of brain 5-HT. All test drugs evoke fluoxetine-sensitive efflux of [(3)H]5-HT from synaptosomes, but d-fenfluramine and its bioactive metabolite d-norfenfluramine induce significantly greater SERT-mediated currents than phenylpiperazines. Our data confirm that drug-induced 5-HT release probably does not mediate 5-HT depletion. However, the magnitude of transporter-mediated inward current may be a critical factor in the cascade of events leading to 5-HT deficits. This hypothesis warrants further study, especially given the growing popularity of designer drugs that target SERT.

Differential effects of opioid agonists on G protein expression in CHO cells expressing cloned human opioid receptors.

Recent evidence indicates that agonist ligands of G protein coupled receptors (GPCR) can activate different signaling systems. Such "agonist-directed" signaling also occurs with opioid receptors. Previous work from our laboratory showed that chronic morphine, but not DAMGO, up-regulates the expression of Galpha12 and that both morphine and DAMGO decreased Galphai3 expression in CHO cells expressing the cloned human mu opioid receptor. In this study, we tested the hypothesis that chronic opioid regulation of G protein expression is agonist-directed. Following a 20h treatment of CHO cells expressing the cloned human mu (hMOR-CHO), delta (hDOR-CHO) or kappa (hKOR-CHO) opioid receptors with various opioid agonists, we determined the expression level of Galpha12 and Galphai3 by Western blots. Among five mu agonists (morphine, etorphine, DADLE, DAMGO, herkinorin) tested with hMOR-CHO cells, only chronic morphine and etorphine up-regulated Galpha12 expression. All five mu agonists decreased Galphai3 expression. Among six delta agonists (SNC80, DPDPE, deltorphin-1, morphine, DADLE, etorphine) tested with hDOR-CHO cells, all six agonists down-regulated Galphai3 expression or moderately up-regulated Galpha12 expression. Among five kappa agonists, ((-)-ethylketocyclazocine, salvinorin A, U69,593, etorphine, (-)-U50,488) tested with hKOR-CHO cells, only chronic (-)-U50,488 and (-)-EKC up-regulated Galpha12 expression. All kappa agonists decreased Galphai3 expression. These data demonstrate that chronic opioid agonist regulation of G protein expression depends not only on the agonist tested, but also on the type of opioid receptor expressed in a common cellular host, providing additional evidence for agonist-directed signaling.