Calcium-Dependent Inhibition of Synaptosomal Serotonin Transport by the 2-Adrenoceptor Agonist 5-Bromo-N-[4,5- dihydro-1H-imidazol-2-yl]-6-quinoxalinamine (UK14304)

Termination of serotonergic transmission is the function of the plasma membrane 5-hydroxytryptamine (serotonin, 5-HT) transporter (SERT), which is also a high-affinity target in vivo for antidepressants, amphetamines, and cocaine. Studies show that SERT is regulated by protein kinaseand phosphataselinked pathways. In contrast, receptor-linked modulation of SERT is only minimally defined. Because noradrenergic stimulation is reported to influence 5-HT release, we explored possible presynaptic adrenoceptor-mediated regulation of SERT. In mouse forebrain synaptosomes, 2-adrenoceptor agonists, particularly 5-bromo-N-[4,5-dihydro-1H-imidazol-2-yl]-6-quinoxalinamine (UK14304), triggered a concentrationand timedependent decrease in 5-HT transport. In contrast, 5-HT uptake was unaffected by pharmacological 1-adrenoceptor activation. Kinetically, UK14304 significantly decreased the apparent substrate affinity, Km without altering transport capacity, Vmax. At concentrations of UK14304 supporting maximal inhibition of SERT in synaptosomes, no effect on SERT in transfected cells was observed, suggesting that UK14304 acts indirectly to reduce SERT activity. The effect of UK14304 on 5-HT uptake was not shared by other Na and Cl -dependent transporters. UK14304-mediated inhibition of SERT function was yohimbine-sensitive, as was inhibition triggered by norepinephrine, and was abolished in the absence of added Ca . Moreover, UK14304 effects were attenuated by voltage-sensitive Ca channel antagonists, consistent with a role for Ca in UK14304 effects. In agreement with altered 5-HT transport activity in vitro, in vivo chronoamperometry studies revealed that UK14304 significantly prolonged 5-HT clearance. Our findings suggest that UK14304 modulates SERT function in vitro and in vivo via signaling pathways, possibly supported by an influx of Ca through voltage-sensitive Ca channels. It is well established that perturbation in monoaminergic transmission is a contributing factor in depression (Malison et al., 1998; Frazer, 2000). Consistent with these findings, the therapeutic actions of the first generation of antidepressants, the tricyclics, most likely exert their therapeutic actions by blocking the neuronal uptake of 5-hydroxytryptamine (serotonin, 5-HT) and/or norepinephrine (NE) (Barker and Blakely, 1995). In the central nervous system (CNS), 5-HT, and NE systems are themselves intimately connected. Electron microscopic autoradiography studies have provided evidence that serotonergic neurons in the rat dorsal raphe receive direct input from noradrenergic neurons (Baraban and Aghajanian, 1981). In addition, in vivo microdialysis studies (Numazawa et al., 1995; Gobert et al., 1998) and release experiments in brain slices (Scheibner et al., 2001) and synaptosomes (Maura et al., 1992; Gobbi et al., 1993a) have demonstrated that 5-HT release is modulated by the activation of presynaptic 2-adrenoceptors. Such an interaction suggests a functional cross talk between the noradrenergic and the serotonergic systems in multiple