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

Abstract 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 Mg 2+ , 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 Mg 2+ : 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 Mg 2+ (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 Mg 2+ . 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 Mg 2+ and MgATP complex may play an important role in the Na + -ATPase physiological regulatory mechanisms.