Ouabain Suppresses CD63 Loading into Extracellular Vesicles <i>via</i> Na<sup>+</sup>/K<sup>+</sup>-ATPase-Dependent Localization to Autophagosomes

Extracellular vesicles (EVs), including exosomes, mediate intercellular communication by transferring lipids, proteins, and nucleic acids. However, the mechanisms determining selective cargo loading into EVs remain poorly understood. Here, we identify the cardiac glycoside ouabain as a selective inhibitor of CD63 loading into EVs. Using a luciferase-based high-throughput assay with CD9- and CD63-tagged reporter cells, ouabain was found to specifically suppress CD63 loading into EVs without affecting CD9 loading into EVs. Ouabain, Na⁺/K⁺-ATPase inhibitor, did not suppress EV secretion but markedly decreased CD63 incorporation. Other cardiac glycoside with strong Na⁺/K⁺-ATPase inhibitory activity, such as bufalin, exhibited similar effects, whereas weak inhibitors did not. Ouabain induced the internalization of Na⁺/K⁺-ATPase (ATP1A1) with CD63, resulting in the disappearance of CD63 from the plasma membrane. Furthermore, ouabain activated autophagy and promoted the colocalization of CD63 with autophagosomes, thereby selectively inhibiting the loading of CD63 into EVs. These effects required both Na⁺/K⁺-ATPase-dependent endocytosis and autophagy, as rapamycin-induced autophagy alone did not remove surface CD63. Our findings reveal a previously unrecognized mechanism in which cardiac glycoside regulates EV cargo composition by coupling Na⁺/K⁺-ATPase-mediated endocytosis with autophagy. Given that endogenous and therapeutic cardiac glycosides are implicated in cardiovascular and cancer biology, this mechanism may broadly influence EV-mediated intercellular communication and represent a potential target for modulating EV functions.