In the search for more specific

In the search for more specific inhibitors of uptake2, Iversen and Salt (1970) speculated that steroids may potentiate the actions of catecholamines on vascular smooth muscle by inhibiting uptake2-mediated catecholamine clearance of the transmitters. They went on to demonstrate that a variety of steroids, including corticosterone, deoxycorticosterone, and 17β-estradiol inhibit uptake2-mediated clearance of gdc-0980 and norepinephrine in heart tissue (Iversen and Salt, 1970). Subsequent studies have demonstrated that the ability of corticosteroids to enhance both the vasomotor actions of norepinephrine in vascular smooth muscle and the ionotropic and chronotropic actions in heart muscle were mediated by inhibition of uptake2-mediated norepinephrine clearance, thus explicitly linking rapid corticosteroid effects with a nongenomic mechanism (Goldie, 1976; Kalsner, 1969; Kalsner, 1974).
Based on overlapping substrate specificities and inhibitor sensitivities between uptake2 and the renal organic cation transporter, OCT2, Grundemann et al. hypothesized that the transporter responsible for uptake2 belonged to the same family of transporters, the ASF family. Based on this hypothesis, the group cloned a novel ASF transporter, the “extraneuronal monoamine transporter” (EMT), which displayed uptake2-like functional properties (Grundemann et al., 1998b). Cells stably expressing cloned EMT displayed corticosterone-sensitive uptake of the uptake2 substrates epinephrine, norepinephrine, 5-HT, tyramine, and MPP+. Like transport mediated by uptake2, EMT-mediated norepinephrine uptake was characterized by a high Km (510 μM) and Vmax (3.9 nmol/min/mg protein), indicating a much lower affinity, but higher capacity for substrate than that of “neuronal uptake”. Based upon these properties, EMT and neuronal uptake are expected to display similar rates of transport per cell (Grundemann et al., 1998b). In addition to identifying a key transporter mediating uptake2, Grundemann's group also demonstrated: a) that the concentrations of corticosterone required to block EMT-mediated uptake (Ki = 120 nM) lay in the physiological range of corticosteroid concentrations; and b) that, in addition to the heart and liver, EMT mRNA was also expressed in the human brain, indicating that, as in peripheral tissues, corticosterone may potentiate monoaminergic neurotransmission in the central nervous system by blocking uptake2-mediated clearance. During the same period of the cloning of EMT, another group cloned a third member of the organic cation transporter family, OCT3, from rat placenta, and demonstrated that it was identical to EMT (Kekuda et al., 1998; Wu et al., 1998). While both names are still in use, OCT3 is the more common designation. The OCTs (OCT1, OCT2 and OCT3) are structurally similar proteins, though OCT1 and OCT2 are more similar to each other (70% identical, 84% similar) than either is to OCT3 (50% identical, 70% similar to each) (Grundemann et al., 1999). Each transporter possesses 12 putative transmembrane domains (Kekuda et al., 1998). Functionally, OCT1, OCT2, and OCT3 all mediate bidirectional, Na+-independent transport of monoamines and other organic cations, though each transporter has a unique substrate specificity (for review, see Schomig et al., 2006). Importantly, transport mediated by all of the OCTs is inhibited by corticosterone, though sensitivity varies markedly among the three, and can vary from species to species (reviewed in Koepsell et al., 2007). Among the human transporters, OCT3 displays the greatest corticosterone sensitivity, with an IC50 of 0.2 μM, compared with 10 μM for OCT1 and 30 μM for OCT2 (Arndt et al., 2001; Grundemann et al., 1998a, Grundemann et al., 1998b; Hayer-Zillgen et al., 2002; Wu et al., 1998). In the rat, OCT1 is the least corticosterone-sensitive, with an IC50 of 150 μM, while OCT2 and OCT3 display similar corticosterone sensitivities (IC50 = 4 and 5 μM, respectively) (Schomig et al., 2006). Importantly, the measured sensitivity of the transporters also appears to depend on the preparation. We have demonstrated, in ex vivo studies of rat hypothalamic tissue, corticosterone inhibition of OCT3-mediated uptake at concentrations similar to those reported for human OCT3 (IC50 = 0.03–0.2 μM) (Gasser et al., 2006; Hill et al., 2011).