The In Vitro Regulatory Mechanism Of Na, K-ATPase Induced By Low Potassium

Na,K-ATPase, or sodium pump, is a heterodimer membrane protein that is composed of α and β subunits. The basic function of the Na,K-ATPase is to maintain the high Na and K gradients across the plasma membrane of animal cells. It has an important role in regulating the cell volumn, cytoplasmic PH and Ca2+ levels through the Na+/H+ and Na/Ca exchangers, respectively, and in driving a variety of secondary transport processes such as Na+-dependent glucose and amide acid transport. In present study , low K+ transiently increased the activity of reactive oxygen species (ROS) in MDCK cells. Inhibition of ROS activity by catalase, N-acetylcysteine or diphenylene iodonium abrogated low K+-induced increases in the α-subunit promoter activity, α1- and β1-subunit polypeptide abundance, and binding sites of Na,K-ATPase, indicating the role of ROS in the effect of low K+. However, brief pulse with 1000 μM H2O2 or with low K+ and then incubation with control medium up to 24 hours failed to increase the Na,K-ATPase binding sites and protein abundance, in spite of the increased promoter activity. Extension of low K+ treatment to 12 hours did not increase the binding sites or protein abundance either. Among ROS species, H2O2 was specifically involved in the up-regulation of Na,K-ATPase induced by low K. 96 base pairs of upstream of Na,K-ATPase α subunit promoter was the key cis-element in transcriptional regulation of promoter activity. Sp1 was alsoinvolved in the regulation of Na,K-ATPase α subunit. This study also took advantage of the fact that confluent MDCK cells can survive in a serum-free medium for several days to examine whether the up-regulation of Na,K-ATPase by low K+ required serum. It was found that serum was essential for low K+ to induce an increase in the Na,K-ATPase binding sites as quantified by ouabain binding assays. Further analysis identified that transferrin, not EGF or IGF1, could simulate the effect of serum. Moreover, transferrin was also required for low K+ -induced increases in α1-subunit promoter activity, (1- and (1-subunit protein abundance of the Na,K-ATPase. In the presence of transferrin, low K+ enhanced cellular uptake of iron approximately by 70%. Inhibition of intracellular iron activity by deferoxamine (40 (M) abrogated the effect of low K+ on the Na,K-ATPase. Like deferoxamine, catalase (100 U/ml) also ablated the effect of low K+. In conclusion, low K+ increases the activity of ROS that induce de novo synthesis of the Na,K-ATPase. However, an additional translational or post-translational regulatory mechanism also mediates the effect of low K+, the stimulation of the Na,K-ATPase by low K+ is dependent on transferrin. The effect of transferrin is mediated by increased iron transport and reactive oxygen species activity.