SUBSTRATE AND INHIBITOR SPECIFICITIES OF THE MONOCARBOXYLATE TRANSPORTERS OF SINGLE-RAT HEART-CELLS

We have used the intracellular pH-sensitive fluorescent dye 2',7'-bis( carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to characterize the subs trate and inhibitor specificity of monocarboxylate transport into isol ated rat heart cells. Further evidence was obtained for the presence o f two lactate carriers present in heart cells (Wang et al., Biochem. J . 290: 249-258, 1993) both distinct from the recently cloned monocarbo xylate transporter isoform 1 (MCT-1) found in many other cell types. O nly one isoform was potently inhibited by alpha-cyano-4-hydroxycinnama te [CHC; inhibitor constant (K-i) 190 mu M] and the stilbene disulfona tes 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (K-i 79 mu M) and 4,4'-dinitrostilbene-2,2'-disulfonate (K-i of cis- and trans- isomers 38 and 171 mu M, respectively; neither isomer inhibits MCT-1). The se cond carrier had a K-i of similar to 3 mM for CHC and 0.5-2 mM for the stilbene disulfonates. Thus, unlike in many other tissues, in rat hea rt cells these inhibitors are not effective at blocking lactate transp ort totally unless used at very high concentrations. Both carriers wer e inhibited by 3-isobutyl-1-methylxanthine (K-i 340 mu M) and neither by 5-nitro-2-(3-phenylpropylamino)benzoate (a potent inhibitor of MCT- 1). The overall Michaelis constant (K-m) and maximum reaction rate (V- max) for transport of a variety of substituted monocarboxylates (C2-C5 ) were determined, although it was not possible to elucidate the kinet ic parameters of the two isoforms. Of physiological interest, the keto ne bodies D-beta-hydroxybutyrate and acetoacetate had K-m values of 10 and 5.4 mM, respectively. V-max values were similar to those of L-lac tate and pyruvate and indicate that transport could limit rates of uti lization of ketone bodies. No stereoselectivity for Lover D-isomers of 2-chloro or 2-hydroxy acids was observed.