Related Experiment Videos
Osmotic regulation of the Na+/myo-inositol cotransporter and postinduction normalization
M A Yorek1, J A Dunlap, W L Lowe
1Department of Internal Medicine, Diabetes Endocrinology Research Center, and Veterans Affairs Medical Center, University of Iowa, Iowa City, Iowa, USA. myorek@ucva,gov
Background:
In renal cells, hyperosmolarity has been shown to induce the accumulation of myo-inositol, via the Na+/myo-inositol cotransporter (SMIT). Previously we showed that SMIT mRNA in the kidney is localized in the medullary thick ascending limb of Henle (TALH). Here we used renal cells derived from the rabbit outer medullary TALH to examine the regulation of myo-inositol transport by hyperosmolarity. In addition, using both cultured renal and endothelial cells, we examined the normalization of SMIT activity and mRNA levels following induction by hyperosmolarity.
Methods:
TALH cells were exposed to isotonic or hyperosmotic medium, and then SMIT mRNA levels and myo-inositol accumulation were determined. To examine postinduction normalization, cultured endothelial and renal cells were first exposed to hyperosmotic medium and then to isotonic medium containing actinomycin D or cycloheximide. Afterwards, SMIT mRNA levels and myo-inositol accumulation were determined.
Results:
Hyperosmolarity increased SMIT mRNA levels and myo-inositol accumulation in TALH cells. The hyperosmolarity-induced increase in myo-inositol uptake by TALH cells was characterized by an increase in the Vmax for the high-affinity myo-inositol transport system, with no change in the Km. This increase was blocked by actinomycin D or cycloheximide. Examination of postinduction normalization showed that returning hyperosmotic-treated cells to isotonic medium caused a rapid reversion of SMIT mRNA levels, followed by a return of myo-inositol accumulation to basal values. However, the addition of cycloheximide or actinomycin D partially to totally prevented the reversal in SMIT mRNA levels and activity.
Conclusions:
These results suggest that RNA and protein synthesis is required for the hyperosmotic induction of SMIT mRNA levels and myo-inositol accumulation by TALH cells. Furthermore, normalization of SMIT mRNA levels and myo-inositol accumulation following hyperosmotic induction requires RNA transcription and protein synthesis.