Modulation of ouabain-insensitive Na(+)-ATPase activity in the renal proximal tubule by Mg(2+), 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±0.4 mM); 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.