Takeuchi S, Ando M
Department of Physiology, Kochi Medical School, Nankoku, Japan.
Hear Res. 1997 Dec;114(1-2):69-74. doi: 10.1016/s0378-5955(97)00157-3.
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.
已知血管纹边缘细胞摄取并代谢葡萄糖作为其主要的代谢能量来源。然而，葡萄糖进入血管纹边缘细胞的膜转运机制在很大程度上尚不清楚。哺乳动物细胞中有两种类型的葡萄糖转运体，即易化葡萄糖转运体GLUT和钠/葡萄糖协同转运体SGLT。本研究的目的是确定其中哪一种代表血管纹边缘细胞中主要的葡萄糖摄取机制。通过荧光法监测烟酰胺腺嘌呤二核苷酸（NADH）还原形式的细胞浓度来评估葡萄糖进入血管纹边缘细胞的摄取情况。边缘细胞的自发荧光与细胞代谢之间的关系如下得到验证。当线粒体中的氧化磷酸化用羰基氰3-氯苯腙（CCCP）解偶联时，自发荧光（激发：340nm，发射：450-490nm）降低，而当细胞色素氧化酶用氰化物抑制时，自发荧光增加。这些效应表明自发荧光依赖于线粒体的代谢状态，更具体地说，依赖于线粒体中NADH的水平。从浴液中去除葡萄糖在5分钟内使自发荧光强度降低了39%。同样，细胞松弛素B（10μM）在5分钟内使荧光强度降低了34%。相比之下，根皮苷（0.1mM）或从浴液中去除Na+均未引起自发荧光强度的任何明显变化。这些结果表明，葡萄糖耗竭在几分钟内影响边缘细胞的代谢状态，并且边缘细胞通过GLUT摄取葡萄糖，而不是通过SGLT。由于生理学研究中使用的几种荧光染料（如Fura-2和SBFI）的激发和发射波长与NADH的相似，因此在使用这些染料时应始终考虑自发荧光对记录信号的可能影响。
