sodium-lactate and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

Monocarboxylate transporter (MCT) 4 is the major monocarboxylate transporter isoform present in white skeletal muscle and is responsible for the efflux of lactic acid produced by glycolysis. Here we report the characterisation of MCT4 expressed in Xenopus oocytes. The protein was correctly targeted to the plasma membrane and rates of substrate transport were determined from the rate of intracellular acidification monitored with the pH-sensitive dye 2', 7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In order to validate the technique, the kinetics of monocarboxylate transport were measured in oocytes expressing MCT1. Km values determined for L-lactate, D-lactate and pyruvate of 4.4, > 60 and 2.1 mM, respectively, were similar to those determined previously in tumour cells. Comparison of the time course of [14C]lactate accumulation with the rate of intracellular acidification monitored with BCECF suggests that the latter reflects pH changes close to the plasma membrane associated with transport, whilst the former may include diffusion-limited movement of lactate into the bulk cytosol. Km values of MCT4 for these substrates were found to be 28, 519 and 153 mM, respectively, and for a range of other monocarboxylates values were at least an order of magnitude higher than for MCT1. Vmax values appeared to be similar for all substrates. K0.5 values of MCT4 (determined at 30 mM L-lactate) for inhibition by alpha-cyano-4-hydroxycinnamate (991 microM), phloretin (41 microM), 5-nitro-2-(3-phenylpropylamino)benzoate (240 microM), p-chloromercuribenzene sulphonate (21 microM) and 3-isobutyl-1-methylxanthine (970 microM, partial inhibition) were also substantially higher than for MCT1. No inhibition of MCT4 by 2 mM 4,4'-diisothiocyanostilbene-2,2'-disulphonate was observed. The properties of MCT4 are consistent with published data on giant sarcolemmal vesicles in which MCT4 is the dominant MCT isoform, and are appropriate for the proposed role of MCT4 in mediating the efflux from the cell of glycolytically derived lactic acid but not pyruvate.

Topics: Animals; Biological Transport, Active; Carrier Proteins; Cells, Cultured; Fluoresceins; Fluorescent Dyes; Humans; Kinetics; Lactic Acid; Monocarboxylic Acid Transporters; Muscle Proteins; Muscle, Skeletal; Oocytes; Protein Isoforms; Substrate Specificity; Xenopus

A number of methods have been developed to measure intracellular pH (pHi) because of its importance in intracellular events. A major advance in accurate pHi measurement was the development of the ratiometric fluorescent indicator dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). We have used a fluorescence multi-well plate reader and a ratiometric method for determining pHi in primary cultures of rabbit corneal epithelial (CE) cells with BCECF. Fluorescence was measured at excitation wavelengths of 485 +/- 11 nm and 395 +/- 12.5 nm, with emission detected at 530 +/- 15 nm. Cells grown in multi-well plates were loaded with 4 microM BCECF for 30 min at 37 degrees C. Resting pHi was 7.34 +/- 0.03 (2 cultures, N = 5 wells). Changes in pHi determined with the fluorescence multi-well plate reader after the addition and removal of NH4Cl or sodium lactate were comparable to changes in cells analyzed with a digitized fluorescence imaging system. A concentration-response relationship involving changes in pHi was easily demonstrated in CE cells after treatment with ionomycin, a calcium ionopore. Low doses of ionomycin (2.5-5 microM), produced a prolonged acidification; 7.5 microM ionomycin produced a transient acidification; and 10 microM ionomycin resulted in a slight alkalinization. We conclude that accurate pHi measurements can be obtained with a ratiometric method with BCECF in a multi-well plate reader. This technology may simplify screening studies evaluating effects of hormones, growth factors, or toxicants on pHi homeostasis.

Topics: Ammonium Chloride; Animals; Buffers; Cells, Cultured; Cornea; Epithelium; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Ionomycin; Ionophores; Monensin; Nigericin; Rabbits; Sodium Lactate; Spectrometry, Fluorescence