Augmentation of Reduced Folate Carrier-Mediated Folate/Antifolate Transport through an Antiport Mechanism with 5-Aminoimidazole-4-Carboxamide Riboside Monophosphate

Abstract

5-Aminoimidazole-4-carboxamide riboside (AICAR), an agent with diverse pharmacological properties, augments transport of folates and antifolates. This report further characterizes this phenomenon and defines the mechanism by which it occurs. Exposure of HeLa cells to AICAR resulted in augmentation of methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate initial rates and net uptake in cells that express the reduced folate carrier (RFC). This did not occur in cells that express only the proton-coupled folate transporter and accumulated folates by this mechanism. Transport stimulation correlated with the accumulation of 5-aminoimidazole-4-carboxamide ribotide monophosphate (ZMP), the monophosphate derivative of AICAR, within cells as established by liquid chromatography. When ZMP formation was blocked with 5-iodotubercidin, an inhibitor of adenosine kinase, folate transport stimulation by AICAR was absent. When cells first accumulated ZMP and were then exposed to 5-iodotubercidin or AICAR-free buffer, the ZMP level markedly decreased and folate transport stimulation was abolished. Extracellular ZMP inhibited RFCmediated folate influx, and the presence of intracellular ZMP correlated with inhibition of folate efflux. The data indicate that intracellular ZMP trans-stimulates folate influx and inhibits folate efflux, which, together, produce a marked augmentation in the net cellular folate level. This interaction among ZMP, folates, and RFC, a folate/organic phosphate antiporter, is consistent with a classic exchange reaction. The transmembrane gradient for one transport substrate (ZMP) drives the uphill transport of another (folate) via a carrier used by both substrates, a phenomenon intrinsic to the energetics of RFC-mediated folate transport.

The Major Facilitative Folate Transporters Solute Carrier 19A1 and Solute Carrier 46A1: Biology and Role in Antifolate Chemotherapy of Cancer

“…In support of this model are reports that MTX transport is competitively inhibited by structurally diverse organic anions such as adenine nucleotides and thiamine phosphates (Goldman, 1971). Further, thiamine pyrophosphate and ZMP (AICA ribonucleotide) are bona fide RFC substrates which, when present within cells, trans-stimulate folate influx by RFC while inhibiting (anti) folate export via this mechanism (Zhao et al, 2001b(Zhao et al, , 2002Visentin et al, 2012b). Although RFC generates only small transmembrane chemical gradients, when considered in light of the dianionic nature of folates and membrane potentials, RFC generates substantial electrochemical potentials across the plasma membrane (Goldman et al, 1968;Goldman, 1971).…”

“…The original finding that AICA ribonucleoside regulates hRFC transport (McGuire et al, 2006) now appears to be unrelated to the activating effect of AICA ribonucleotide (ZMP) on AMP-activated protein kinase (AMPK), but rather reflects trans-stimulation of hRFC by intracellular ZMP (Visentin et al, 2012b) (described previously).…”

Inhibition of the Proton-Coupled Folate Transporter (PCFT-SLC46A1) by Bicarbonate and Other Anions

“…R1-11-RFC-6 and R1-11-PCFT-4 are stable transfectants of R1-11 cells that express RFC and PCFT, respectively, at a level similar to that of HeLa cells (Zhao et al, 2008). R1-11-PCFT-h is a stable transfectant of R1-11 cells with levels of PCFT expression much higher than that of HeLa cells (Visentin et al, 2012). Cells were grown in RPMI medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin.…”

Methotrexate and 5-aminoimidazole-4-carboxamide riboside exert synergistic anticancer action against human breast cancer and hepatocellular carcinoma

“…Visentin et al have reported that pretreatment of HeLa cells with AICA riboside results in augmentation of MTX initial rates and net uptake in cells [36] . There may be an interaction between MTX and AICA riboside, and further studies on the influence of AICA riboside on the antitumor efficacy and pharmacokinetics of MTX are needed.…”