br Introduction Given the growing acceptance of ergothionein

Introduction Given the growing acceptance of ergothioneine (ET) as a biologically important agent with potential therapeutic applications [[1], [2], [3], [4]] it is increasingly important to clearly establish the role of the organic cation transporter (novel) type 1 (OCTN1). OCTN1 was discovered in 1997 by Tamai and co-workers [5], which they later declared to be a multispecific pharmacokinetic transporter [6]. In 2005 OCTN1 was proposed to be a specific ET transporter by Grundemann et al. [7] who demonstrated its high selectivity for this compound over other previously reported and chemically similar substrates. Following this suggestion, there have been numerous reports that OCTN1 may serve alternate purposes. Most notably is the work published by the Indiveri group [8], using proteoliposomes containing OCTN1 from Escherichia coli overexpression. It was claimed to transport choline, Terazosin mg and acetylated cations at an efficiency higher than that of ET. Such findings have served as the basis for the assertion that OCTN1 is a physiological acetylcholine exporter (and choline importer) and is intimately involved in the non-neuronal cholinergic system [9]. It was suggested that anti-inflammatory effects associated with OCTN1 are mediated through acetycholine export. OCNT1 was claimed to be the missing link in the non-neuronal acetylcholine system; however Wessler and Kirkpatrick had previously identified organic cation transporter 1 and 2 (OCT1,2) as facilitating acetylcholine export in non-neuronal tissues [10]. Indiveri et al. formed their hypothesis using proteoliposomes, systems which they claim permit better resolution of kinetic and molecular functions over that of cell culture [11]. However results obtained with their system demonstrate very poor ET transport when compared with all other models of OCTN1 function. It seems odd that they demonstrated more effective uptake of tetraethylammonium (TEA) (6-fold greater) over ET [8] in direct contradiction to earlier work published by Grundemann et al. [7]. OCTN1 has also been claimed to transport cytarabine [12]. However the authors failed to demonstrate any competition between ET and cytarabine and suggested that OCTN1 has a distinct nucleoside binding site that does not overlap with the ET binding site. By contrast Tschirka et al. [13] suggested that this result was an artifact of the transfection system, as they were unable to find any transport of cytarabine, deoxycytidine, gemcytibine or deoxyadenosine in their OCTN1 model. Recently, Bai et al. [14] suggested that OCTN1 contributes to the placental transport of sulpiride based upon a small reduction in sulpiride uptake when co-incubated with a 4-fold greater concentration of ET; however they failed to demonstrate any inhibition of sulpiride uptake in BeWo cells on application of an excess of ET. Evidence exists that gabapentin and ET interact to activate OCTN1, i.e. pre-incubation of cells with ET stimulates uptake of gabapentin and vice versa. It was even suggested that gabapentin is exchanged for ET to enhance ET reuptake at the apical surface of the kidney [15]. Ingestion of ET-rich shiitake mushrooms was found to increase renal clearance of gabapentin by 19%, prolonging the time to reach maximal gabapentin plasma concentration; however no other pharmacokinetic parameters of gabapentin were modified [16]. Tschirka et al., [13] however, identified gabapentin as a weak substrate for OCTN1. OCTN1 has also been implicated as a metformin transporter by Nakamichi et al. [17]; however, evidence has since been accumulated to suggest otherwise. Arner et al. [18] demonstrated that OCTN1 knockdown did not impair biguanide driven lipolysis, and likewise Tschirka et al. [13] demonstrated the lack of metformin transport by OCTN1 in their overexpression model. With all the confusion surrounding the substrates of OCNT1 and its physiological role, more studies are needed to re-establish the role of this compound in the body and rule out any potentially important alternate functions. The work here examined the kinetics of ET uptake by OCTN1 natively expressed in HeLa cells, previously demonstrated to express OCTN1 [19] as well as investigating the competitive inhibition of ET uptake by various other compounds.