The effect of temperature and substrate concentration on the kinetics of [6- 3H]uridine incorporation into RNA of toad bladder epithelial cells: The role of aldosterone

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1. The effects of temperature and uridine substrate concentration during incorporation of [6- 3H]uridine into RNA were studied in epithelial cells of the isolated urinary bladder of the toad, Bufo marinus, harvested after incubation. 2. Gross accumulation of newly synthesized RNA in the acid-insoluble fraction of whole epithelial cells was maximal at 33°C., even though net RNA synthesis, i.e. RNA specific activity, was significantly reduced compared to 24°C. Cold exposure at 8°C decreased accumulation of newly synthesized RNA when compared to warmer temperatures. 3. A uridine substrate concentration constant ( K′ m ), derived in a manner similar to the Michaelis-Menten constant ( K m ), was determined to be K′ m = 2.13 · 10 −5 mole/l at 24°C for non-hormonally treated urinary bladder. 4. The in vitro effect of the hormone, aldosterone, on RNA synthesis at 24°C was evaluated by quantitating the aldosterone-control difference of both RNA and RNA specific activity in paired samples of scraped epithelial cells. By minimizing dilution of the [6- 3H]uridine due to variations in the endogenous uridine RNA precursor pool size, through the technique of providing a constant excess of exogenous uridine with the radioisotope, aldosterone stimulation of RNA specific activity can be reproducibly demonstrated. 5. Aldosterone significantly increased RNA specific activity but not bulk RNA, per se, even after 20 h of hormone exposure. 6. The mean weight ratio of epithelial cell RNA/DNA determined in 116 different bladder parts derived from 30 toads, was 2.5 ± 0.32 S.E. 7. A 1.5-h exposure to aldosterone produced a significant increase (31.5 %) in the incorporation of [6- 3H]uridine into heterogenous RNA extracted from purified nuclei, but not in RNA extracted from either cytoplasm or whole cell. This primary nuclear response to aldosterone was followed by a significant increase in aldosterone-stimulated specific activity of whole cell RNA at 3 h (74 %) and 20 h (200 %).