Modulation of ouabain-insensitive Na+-ATPase activity in the renal proximal tubule by Mg2+, MgATP and furosemide

In addition to the (Na+ + K+)ATPase another P-ATPase, the ouabain-insensitive Na+-ATPase has been observed in several tissues. In the present paper, the effects of ligands, such as Mg2+ MgATP and furosemide on the Na+-ATPase and its modulation by pH were studied in the proximal renal tubule of pig. The principal kinetics parameters of the Na+-ATPase at pH 7.0 are: (a) K-0.5 for Na+ = 8.9 +/- 2.2 mM; (b) K-0.5 for MgATP = 1. 8 0.4 mM; (c) two sites for free Mg2+: one stimulatory (K-0.5 = 0.20 +/- 0.06 mM) and other inhibitory (I-0.5 = 1.1 +/- 0.4mM); and (d)I-0.5 for furosemide = 1.1 +/- 0.2 mM. Acidification of the reaction medium to pH 6.2 decreases the apparent affinity for Na+ (K-0.5 = 19.5 +/- 0.4) and MgATP (K-0.5 = 3.4 +/- 0.3 mM) but increases the apparent affinity for furosemide (0. 18 +/- 0.02 mM) and Mg2+ (0.05 +/- 0.02 mM). Alkalization of the reaction medium to pH 7.8 decreases the apparent affinity for Na+ (K-0.5 = 18.7 1.5 mM) and furosemide (I-0.5 = 3.04 +/- 0.57 mM) but does not change the apparent affinity to MgATP and Mg2+. The data presented in this paper indicate that the modulation of the Na+-ATPase by pH is the result of different modifications in several steps of its catalytical cycle. Furthermore, they suggest that changes in the concentration of natural ligands such as Mg2+ and MgATP complex may play an important role in the Na+-ATPase physiological regulatory mechanisms. (C) 2002 Elsevier Science Ltd. All rights reserved.