Functional evidence for a monocarboxylate transporter (MCT) in strial marginal cells and molecular evidence for MCT1 and MCT2 in stria vascularis.
The transport of lactate, pyruvate and other monocarboxylates across plasma membranes of metabolically active cells such as strial marginal cells (SMC) may be important under aerobic conditions as well as under ischemic and hypoxic conditions. This study addresses the question whether SMC from the gerbil contain a membrane transport mechanism for monocarboxylates. The type of transporter was identified in functional studies by monitoring uptake of monocarboxylates into SMC through measurement of the cytosolic pH (pHi) with the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Further, subtypes of the functionally identified transporter which are present in stria vascularis were identified as transcripts by cloning and sequencing the reverse-transcription polymerase chain reaction (RT-PCR) products. All functional experiments were performed under nominally HCO3--free conditions. The monocarboxylates acetate and pyruvate (each 20 mM) induced an acidification of pHi. In contrast, the dicarboxylate malonate (20 mM) had no significant effect on pHi. Alpha-cyano-4-hydroxycinnamate (CHC; 5 mM), a blocker of H+/monocarboxylate cotransporter (MCT), reduced reversibly the acidification induced by 5 mM pyruvate. In contrast, 1 microM DIDS, a blocker of band-3 protein, had no significant effect on the acidification induced by 20 mM acetate. The presence of the transcripts for each of the MCT subtypes, MCT1 and MCT2, was determined by RT-PCR of stria vascularis from gerbil. RT-PCR performed with primers for the MCT1 and MCT2 subtypes on total RNA from stria vascularis revealed PCR products of the predicted sizes. Sequence analysis confirmed that amplified MCT1 and MCT2 cDNA fragments encoded a nucleotide sequence of MCT1 and MCT2, respectively. These observations suggest that SMC contain a MCT and that stria vascularis contains RNA for the subtypes MCT1 and MCT2 subtypes.
Shimozono
M
M
Cell Physiology Laboratory, Boys Town National Research Hospital, Omaha, NE 68131, USA.
Scofield
M A
MA
Wangemann
P
P
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D003373:Coumaric Acids; D006224:Cricetinae; D003600:Cytosol; D018076:DNA, Complementary / Q000096:biosynthesis; D004587:Electrophoresis, Agar Gel; D005849:Gerbillinae; D006863:Hydrogen-Ion Concentration; D019344:Lactic Acid / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D027501:Monocarboxylic Acid Transporters; D016133:Polymerase Chain Reaction; D019289:Pyruvic Acid / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013316:Stria Vascularis / Q000378:metabolism*
D002352:Carrier Proteins; D003373:Coumaric Acids; D018076:DNA, Complementary; D008565:Membrane Proteins; D027501:Monocarboxylic Acid Transporters; C007175:alpha-cyano-4-hydroxycinnamate; D019344:Lactic Acid; D012313:RNA; D019289:Pyruvic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid