Cardiac steroids (CSs) are specific inhibitors of Na ؉ , K ؉ -ATPase activity. Although the presence of CS-like compounds in animal tissues has been established, their physiological role is not evident. In the present study, treatment of human NT2 cells with physiological concentrations (nanomolar) of CSs caused the accumulation of large vesicles adjacent to the nucleus. Experiments using N-(3-triethylammonium propyl)-4-(dibutilamino)styryl-pyrodinum dibromide, transferrin, low-density lipoprotein, and selected anti-transferrin receptor and Rab protein antibodies revealed that CSs induced changes in endocytosis-dependent membrane traffic. Our data indicate that the CS-induced accumulation of cytoplasmic membrane components is a result of inhibited recycling within the late endocytic pathway. Furthermore, our results support the notion that the CS-induced changes in membrane traffic is mediated by the Na ؉ , K ؉ -ATPase. These phenomena were apparent in NT2 cells at nanomolar concentrations of CSs and were observed also in other human cell lines, pointing to the generality of this phenomenon. Based on these observations, we propose that the endogenous CS-like compounds are physiological regulators of recycling of endocytosed membrane proteins and cargo. INTRODUCTION Naϩ , K ϩ -ATPase is an enzyme present in the plasma membrane of most eukaryotic cells that hydrolyzes ATP and uses the free energy thus generated to drive potassium into the cell and sodium out of the cell, against their electrochemical gradients. This enzyme is the major determinant of the Na ϩ and K ϩ electrochemical gradient. As such, it has an important role in regulating cell volume, the plasma membrane electrical potential, cytoplasmic pH, and Ca 2ϩ levels through the Na ϩ /H ϩ and Na ϩ /Ca 2ϩ exchangers, respectively, and in driving a variety of secondary transport processes (Lingrel, 1992;Blanco and Mercer, 1998;Mobasheri et al., 2000;Scheiner-Bobis, 2002). After the discovery, Ͼ40 years ago, of the Na ϩ , K ϩ -ATPase, it was found that plant and amphibian steroids (digitalis, cardenolides, and bufadienolides), collectively termed here as cardiac steroids (CSs), bind to a specific site on the enzyme and inhibit ATP hydrolysis and ion transport (Kelly and Smith, 1996). In the past decade, several groups have identified CS-like compounds in animal tissues. Ouabain was demonstrated in human plasma and adrenal (Hamlyn et al., 1991;Sich et al., 1996), digoxin was shown to be present in human urine (Goto et al., 1990), a ouabain isomer was found in bovine hypothalamus (Tymiak et al., 1993), 19-norbufalin and its peptide derivative were identified in cataractous human lenses (Lichtstein et al., 1993), and dihydropyrone-substituted bufadienolide in human placenta (Hilton et al., 1996). In addition, immunoreactivity of marinobufogenin-like (Bagrov et al., 1995) and proscillaridin A-like compounds (Sich et al., 1996), which also are members of the bufadienolide family, have been demonstrated in human plasma. The structural similarity between the differ...
Plasma membrane Na(+)-K(+)-ATPase, which drives potassium into and sodium out of the cell, has important roles in numerous physiological processes. Cardiac steroids (CS), such as ouabain and bufalin, specifically interact with the pump and affect ionic homeostasis, signal transduction, and endocytosed membrane traffic. CS-like compounds are present in mammalian tissues, synthesized in the adrenal gland, and considered to be new family of steroid hormones. In this study, the mechanism of Na(+)-K(+)-ATPase involvement in the regulation of endocytosis is explored. We show that the effects of various CS on changes in endosomal pH are mediated by the pump and correspond to their effects on endosomal membrane traffic. In addition, it was found that CS-induced changes in endocytosed membrane traffic were dependent on alterations in [Na(+)] and [H(+)] in the endosome. Furthermore, we show that various CS differentially regulate endosomal pH and membrane traffic. The results suggest that these differences are due to specific binding characteristics. Based on our observations, we propose that Na(+)-K(+)-ATPase is a key player in the regulation of endosomal pH and endocytosed membrane traffic. Furthermore, our results raise the possibility that CS-like hormones regulate differentially intracellular membrane traffic.
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