Marginal cells of the stria vascularis of gerbils take up glucose via the facilitated transporter GLUT: application of autofluorescence.
Strial marginal cells are known to take up and metabolize glucose as their main source of metabolic energy. The membrane transport mechanisms for glucose uptake into strial marginal cells, however, are largely unknown. Two types of glucose transporters in mammalian cells have been described, the facilitated glucose transporter GLUT and the sodium/glucose cotransporter SGLT. The goal of the present study was to determine which of these represent the main glucose uptake mechanism in strial marginal cells. Glucose uptake into strial marginal cells was assessed by monitoring the cellular concentration of the reduced form of nicotinamide adenine dinucleotide (NADH) fluorometrically. The relation between the autofluorescence from marginal cells and cellular metabolism was verified as follows. The autofluorescence (excitation: 340 nm, emission: 450-490 nm) decreased when oxidative phosphorylation in the mitochondria was uncoupled with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and increased when cytochrome oxidase was inhibited with cyanide. These effects indicate that the autofluorescence is dependent on the mitochondrial metabolic state, and more specifically on the level of NADH in mitochondria. Glucose removal from the bath solution elicited a 39% decrease in the autofluorescence intensity within 5 min. Similarly, cytochalasin B (10 microM) reduced the fluorescence intensity by 34% in 5 min. In contrast, neither phlorizin (0.1 mM) nor Na+ removal from the bath solution caused any appreciable change in the autofluorescence intensity. These results indicate that glucose depletion affects the metabolic state of the marginal cell within a few minutes, and that marginal cells take up glucose via GLUT, but not via SGLT. Since the excitation and emission wavelengths of several fluorescent dyes used in physiological studies (e.g., Fura-2 and SBFI) are similar to those of NADH, possible effects of autofluorescence on recording signals should always be taken into account when these dyes are utilized.
Takeuchi
S
S
Department of Physiology, Kochi Medical School, Nankoku, Japan. takeuchi@kochi-ms.ac.jp
Ando
M
M
D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D003486:Cyanides / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors; D005453:Fluorescence; D005470:Fluorometry; D005849:Gerbillinae; D005947:Glucose / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000502:physiology*; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D013316:Stria Vascularis / Q000166:cytology / Q000378:metabolism*; D014466:Ultraviolet Rays; D014475:Uncoupling Agents / Q000494:pharmacology
D003486:Cyanides; D009004:Monosaccharide Transport Proteins; D014475:Uncoupling Agents; D009243:NAD; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D003576:Electron Transport Complex IV; D005947:Glucose