A cytochemical procedure for determination of Na+,K+-ATPase activity in MDCK cells

Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) is an integral protein usually located in the basolateral membrane [1, 2] that actively pumps Na+ out and K+ in to the cell, through ATP hydrolysis [3]. The activity of Na+,K+-ATPase is essential for the existence of water and solute transport by epithelial cells [4,5]. Measurement of Na+ ,K+-ATPase activity is usually done by biochemical assays. Cytochemical methods [6] allow cellular enzyme localization and may offer an alternative approach for determination of Na+,K+-ATPase activity in disrupted cells. We have used a cytochemical method, based on measurements of inorganic phosphate generated during ATP hydrolysis by ATPases during the incubation period and precipitated by a lead ammonium citrate acetate complex (LACA) as lead phosphate [Pb3(PO4)2]. The colorless lead phosphate was converted into a brown precipitate of lead sulphide (PbS) by treatment with ammonium sulphide. We chose to evaluate Na+,K+-ATPase activity in MDCK (Madin-Darby canine kidney) cells because they retain many differentiated properties characteristic of distal tubule epithelia [4, 7]. In these cells, apical to basolateral salt and water transport was shown to be inhibited by ouabain and amiloride [7–10]. We show that this method is a simple, convenient, and could be easily performed to measure Na+,K+-ATPase activity and its modulation under physiological conditions