O'Donnell Michael J, Leader John P
Department of Physiology, University of Otago, Dunedin, New Zealand.
Arch Insect Biochem Physiol. 2006 Nov;63(3):123-34. doi: 10.1002/arch.20148.
The effects of stimulants of fluid secretion on net transepithelial transport of the MRP2 substrate Texas Red and the p-glycoprotein substrate daunorubicin were examined in Malpighian tubules of Drosophila melanogaster. Fluid secretion rates were determined using the Ramsay assay and secreted fluid concentrations of Texas Red and daunorubicin were determined using a microfluorometric technique. Nanoliter droplets of secreted fluid were collected in optically flat glass capillaries and dye concentration was determined from fluorescence intensity measured by confocal laser scanning microscopy. Net transepithelial flux of each compound was then calculated as the product of its concentration in the secreted fluid and the fluid secretion rate. Net transepithelial flux of Texas Red increased when fluid secretion was stimulated by tyramine, cyclic AMP or hypoosmotic saline. Net flux decreased when fluid secretion rate of cAMP-stimulated tubules was reduced by elevating saline osmolality with sucrose. Net transepithelial flux of daunorubicin increased when fluid secretion was stimulated by cAMP. Significant increases in dye flux were seen only when the dyes were present at concentrations close to or greater than the concentration required for half maximal transport. Regression analyses showed that 57- 88% of the change in dye flux was attributable to the change in fluid secretion rate when tubules were stimulated with cAMP, cGMP, or tyramine. The results do not suggest that the effects of tyramine and cAMP are mediated through changes in transepithelial potential, nor do they indicate the direct effects of the stimulants on MRP2-like or p-glycoprotein-like transporters (e.g., via protein kinases). Instead, the results suggest that increases in fluid secretion rate minimize diffusive backflux of these dyes and, thus, facilitate higher rates of net transepithelial transport indirectly.
在黑腹果蝇的马氏管中，研究了促液分泌刺激物对多药耐药相关蛋白2（MRP2）底物德克萨斯红和P-糖蛋白底物柔红霉素的净跨上皮转运的影响。使用拉姆齐测定法测定液分泌率，使用微荧光测定技术测定德克萨斯红和柔红霉素的分泌液浓度。在光学平面玻璃毛细管中收集纳升分泌液滴，并通过共聚焦激光扫描显微镜测量的荧光强度确定染料浓度。然后将每种化合物的净跨上皮通量计算为其在分泌液中的浓度与液分泌率的乘积。当用酪胺、环磷酸腺苷（cAMP）或低渗盐水刺激液分泌时，德克萨斯红的净跨上皮通量增加。当用蔗糖提高盐水渗透压来降低cAMP刺激的小管的液分泌率时，净通量降低。当用cAMP刺激液分泌时，柔红霉素的净跨上皮通量增加。仅当染料浓度接近或高于半数最大转运所需浓度时，染料通量才会显著增加。回归分析表明，当用cAMP、环磷酸鸟苷（cGMP）或酪胺刺激小管时，染料通量变化的57%-88%可归因于液分泌率的变化。结果并不表明酪胺和cAMP的作用是通过跨上皮电位的变化介导的，也未表明刺激物对MRP2样或P-糖蛋白样转运体的直接作用（例如，通过蛋白激酶）。相反，结果表明液分泌率的增加使这些染料的扩散性反流最小化，从而间接促进更高的净跨上皮转运速率。
