Cryptic Na+, K+–Atpase Activity in Rat Liver Canalicular Plasma Membranes: Evidence for Its Basolateral Origin

Abstract

Controversy exists concerning the localization of the enzyme Na+,K(+)-ATPase to canalicular membranes in hepatocytes. Most studies find enzyme activity only at the basolateral plasma membrane domain of the hepatocyte. However, Na+,K(+)-ATPase activity has been detected recently in a canalicular membrane fraction prepared by Mg++ precipitation, suggesting that differences in membrane domain fluidity account for these discrepancies. To reinvestigate this question, we used free-flow electrophoresis to further purify canalicular liver plasma membranes originally separated by sucrose density centrifugation. With this technique, canalicular membranes devoid of Na+,K(+)-ATPase activity by routine assay were separated into six subfractions. More than 80% of the activities of canalicular marker enzymes was recovered in two subfractions closest to the anode, which were totally devoid of Na+,K(+)-ATPase activity. However, Na+,K(+)-ATPase activity could now be detected in the four other fractions that contained only small amounts of canalicular marker enzymes. The basolateral marker enzyme, glucagon-stimulated adenyl cyclase, comigrated with this cryptic Na+,K(+)-ATPase activity. Furthermore, addition of 6 mumol/L [12-(2-methoxyethoxy)-ethyl-8-(cis-2-n-octylcyclopropyl)-octanoate ], a membrane-fluidizing agent, to the original canalicular membrane preparation and to all subfractions did not stimulate or unmask latent Na+,K(+)-ATPase activity. Finally, when canalicular membranes isolated by Mg++ precipitation were subjected to free-flow electrophoresis, they could not be separated from the more positively charged Na+,K(+)-ATPase-containing fractions, probably because of alterations in surface charge. Together these findings suggest that Na+,K(+)-ATPase is a basolateral enzyme, that represents a small contaminant when present in canalicular liver plasma membranes and that methodological differences may account for previous discrepancies.

Mechanisms and regulation of bile secretion

“…Basolateral membrane vesicles from human liver show levels of Na'-K' ATPase activity similar to those found in the rat (16), confirming the functional importance of this transporter in human hepatocytes. Characterization and basolateral location of this exchanger was described more than 10 yr ago (€9, but ongoing controversy exists concerning whether the enzyme is also present on the canalicular membrane (10, [17][18][19][20]. Recent studies using free-flow electrophoresis show that highly purified canalicular membranes are devoid of Na+-K' ATPase activity even after the fluidity of these normally stiff membranes is increased (17).…”

Signaling pathway involved in the inhibitory effect of FTY720P on the Na+/K+ ATPase in HepG2 cells

“…The hepatocytes' functions are fulfilled and accomplished through the concerted action of various transporters like the Na (Graf and Häussinger 1996), which rely directly or indirectly on the sodium gradient established by the Na + /K + ATPase, known also as the Na + /K + pump. The ATPase is located in the basolateral membrane (Sellinger et al 1990) and maintains the ionic constancy of the intracellular milieu which is needed for proper cellular activities. Sphingosine 1-phosphate (S1P) was shown previously to reduce the activity of the Na + /K + ATPase in HepG2 cells (Dakroub and Kreydiyyeh 2012), a human liver cancer cell line, and a similar inhibitory effect was demonstrated for its analogue, FTY720P (Al Alam and Kreydiyyeh 2016), which has been approved lately for the treatment of multiple sclerosis because of its immunomodulatory properties.…”

A Quantitative Immunocytochemical Study of Na+, K+-ATPase in Rat Hepatocytes After STZ-induced Diabetes and Dietary Fish Oil Supplementation

“…There was a discrepancy between the bile canalicular localization of the ␣ 1 -subunit and the basolateral localization found in a number of biochemical and cytochemical studies (Blitzer and Boyer 1978;Boyer et al 1983;Gorvel et al 1983;Takemura et al 1984;Yamamoto et al 1984;Leffert et al 1985;Sztul et al 1987;Chamlian et al 1988;Hara et al 1988;Benkoël et al 1995;Simon et al 1995). This controversy could be explained by variations in membrane lipid fluidity (Sutherland et al 1988), mobility of Na ϩ ,K ϩ -ATPase from basolateral membranes to apical membranes as described in renal epithelium (Molitoris 1993;Paller 1994), and the possibility that anti-␣ 1 -subunit antibody could recognize similar epitopes in Ca ϩϩ -ATPase (Lingrel et al 1990;Sellinger et al 1990;Lemas et al 1994).…”