Abstract
In thyroidectomized rats, a single injection of L-2,,5,2'-triiodothyronine (T3) (50mug/100 g body weight) elicited at 45% increase in (Na+ + k+)-dependent adenosine triphosphatase (NaK-ATPase) activity of the membrane-rich fraction of renal cortex at the optimal time of response, 48 h after injection. Three successive doses of T3 (50 mug/100 g body weight), given on alternate days, increased NaK-ATPase by 67% in the renal cortex but had no significant effect on the outer medulla or the papilla. Moreover, T3 had no effect on Mg2+-dependent adenosine trisphatase (MgATPase) in cortex, cedulla, or papilla. Three doses of T3 (50 mug/100 g body weight) given on alternate days to thyroidectomized rats elecited a 134, 79, and 46% increase in Vmax for ATP, Na4, and K+, respectively. There were no changes in the Km for ATP or the K1/2 values for Na+ and K+. Two methods were used to estimate the effect of T3 on the number of NaK-ATPase units (assumed to represent the number of Na+ pump sites); rat renal plasma membrane fractions were incubated with [gamma-32P]ATP, Mg2+, and Na+; the 32P-labeled membrane protein yeild was quantitatively dependent on Na+ and was hydrolyzed on addition of K+. There was a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of phosphorylated intermediate formed, in renal cortical membrane fractions from thyroidectomized rats given T3 or the diluent. There was also a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of [3H]ouabain specifically bound (Na+-, Mg2+-, APT-dependent) to the NaK-ATPase preparation. Injection of T3 resulted in a 70% increase in NaK-ATPase activity, a 79% increase in formation of the phosphorylated intermediate, and a 65% increase in the [H]ouabain specifically bound to the NaK-ATPase system. The T3-dependent increases in Vmax for ATP, Na+, and K+ and the proportionate increases in the phosphorylated intermediate and in the amount of [3H]ouabain bound indicate that T3 increases the number of NaK-ATPase units and that this increase accounts for the increase in NaK-ATPase activity.
Highlights
In thyroidectomized rats, a single injection of L-3,5,3’-triiodothyronine (T,) (50 Erg/100 g body weight) elicited a 45% increase in (Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) activity of the membrane-rich fraction of renal cortex at the optimal time of response, 48 h after injection
There was a linear correlation between the specific activity of NaK-ATPase (V,) and the amount of phosphorylated intermediate formed, in renal cortical membrane fractions from thyroidectomized rats given T, or the diluent
Activity in Renal Cortex, Medulla, and Papilla-To define the time course of the dependence of NaK-ATPase activity on thyroid status, thyroidectomized rats were injected with the diluent (5 x 10-l M NaOH) or T, (250 pg/lOO g body weight) and the kidneys removed at 12, 24, 48, 72, or 120 h after injection
Summary
Sprague-Dawley rats (body weights 180 to 220 g) maintained on Purina chow ad lib&urn were used in all experiments. The K,,, and V,,,, of the NaK-ATPase system for Nat were determined by adding -25 pg of membrane protein (L fraction of the renal cortex) to 1.0 ml of a medium that contained 0.02 M KCl, 0.003 M. The phosphorylated intermediate was generated in a l.O-ml reaction mixture that contained 0.1 M NaCl, 0.1 M TrislHCl, 0.003 M, MgCl,, 0.001 M EDTA, 1.5 x 1Om6 M lr-“ZP]ATP (1.5 Ci/mmol), pH 7.4, and 90 to 200 +g of membrane protein (L fraction of renal cortex) in Eppendorf vials. The L fraction of rat renal cortex (final concentration = 25 pg of protein/ml) was incubated in 1.0 ml of 0.1 M NaCl, 0.005 M MgCl,, 0.05 M Tris/HCl, and 9.5 x 10-O M [“Hlouabain (1.3 Ci/mmol), pH 7.4. England Nuclear and [y-32PlATP (1.5 Cilmmol) from Amersham/ Searle
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