Hypertonic activation of the renal betaine/GABA transporter is microtubuledependent

Background Epithelial cells in the renal inner medulla accumulate osmolytes such as betaine to maintain normal cell volume during prolonged extracellu lar hypertonic stress. Betaine accumulation is the result of activation of transcription of the BGT1 transporter gene followed by increased betaine tr ansport. Methods. We studied the possible role of microtubules in this adaptive mech anism using renal cells in culture. Results. In cultured renal cell lines [Madin-Darby canine kidney (MDCK) and mouse inner medullary collecting duct (mIMCD-3)], up-regulation of BGT1 ac tivity was maximal after 24 to 30 hours in growth medium made hypertonic (5 10 mOsm/kg) by the addition of sucrose or NaCl. Up-regulation was reversed within 24 to 36 hours after returning cells to isotonic medium. Both cycloh eximide (20 mu mol/L) and nocodazole (20 mu mol/L) blocked the hypertonic u p-regulation of BGT1. Nocodazole was partially effective even when added 16 to 20 hours after the switch to hypertonic medium. Recovery from nocodazol e action was rapid, and there was full activation of BGT1 transport within three to six hours after nocodazole removal, suggesting rapid trafficking t o the cell surface once microtubules repolymerized. Hypertonic activation o f BGT1 transport was detected in an isolated membrane fraction and was bloc ked by cycloheximide but not by nocodazole. Confocal microscopy confirmed t he increased abundance of BGT1 proteins in the plasma membrane of hypertoni c cells and showed that BGT1 remained intracellular during nocodazole treat ment. Conclusions. Hypertonic activation of BGT1 in renal cells requires de novo protein synthesis and microtubule-dependent trafficking of additional trans porters to the cell surface. The apparent resistance of membrane BGT1 to no codazole blockade is likely due to the presence in the membrane fraction of an increased intracellular pool of active BGT1 transporters.