Abstract
We fractionated leukocytes from three donors into >90% pure samples of granulocytes, lymphocytes, and monocytes and tested them for transcriptional and translational expression of three physiologically-proven lactate transporters, monocarboxylate transporter 1(MCT1), MCT2, and MCT4, using RT-PCR and affinity-purified rabbit antibody (Ab) to the C-terminal segment of each human MCT. Transcripts of all three MCTs were identified in each leukocyte fraction by RT-PCR and proven by sequencing of fragments extracted after isolation on agarose gels. Transporter protein of the appropriate size was demonstrated for each of the monocarboxylate transporters MCTs in lymphocytes and monocytes by Western blot, while lower-molecular-weight bands were found in granulocytes and are presumed to be degraded forms, because they were blocked by antibody-antigen (Ab-Ag) preincubation. IHC demonstrated all three MCTs in methanol-fixed droplets of all three leukocyte fractions; stain was abolished on omission of the primary Ab. Plasmalemmal staining occurred with all MCTs in all leukocyte fractions. Because the K(m) for lactate increases approximately fivefold at each step, with MCT2<1<4, leukocytes must use the full range of lactate binding to survive in acidic and hypoxic environments. Except for MCT4 in lymphocytes, all the MCTs also stained leukocyte cytoplasm, often with distinct granularity. Nuclear membrane staining was also seen with MCT1 and MCT2, while platelet plasmalemma stained only with MCT2.