AZD3965 inhibition of lactate membrane transport

To determine the selectivity of AZD3965, the effect on lactate transport mediated by MCT3 or MCT4 was determined. Human MCT1, MCT3 or MCT4 were expressed in the MCT1, MCT2, MCT3 and MCT4 null rat pancreatic INS1 cell line. Lactate transport was measured using the pH- sensitive fluorescent dye BCECF ((2’,7’-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester)) to detect the rapid decrease in intracellular pH that accompanies proton-linked lactate transport as described by Murray et al. [30]. Briefly, cells were loaded with 1 mM BCECF and incubated with compound or vehicle for 1 hr in Tyrodes buffer pH7.4. The ratio of fluorescence at 535 nm after excitation at 490 nm/440 nm was measured after addition of exogenous L-lactate using the Flexstation scanning fluorometer. The change in pHi was determined from a calibration curve using buffer containing nigericin to equilibrate internal and external pH [30].