RESUMO
Cardiac steroids (CS), an important class of naturally occurring compounds, are synthesized in plants and animals. The only established receptor for CS is the ubiquitous Na(+),K(+)-ATPase, a major plasma membrane transporter. The binding of CS to Na(+),K(+)-ATPase causes the inhibition of Na(+) and K(+) transport and elicits cell-specific activation of several intracellular signaling mechanisms. It is well documented that the interaction of CS with Na(+),K(+)-ATPase is responsible for numerous changes in basic cellular physiological properties, such as electrical plasma membrane potential, cell volume, intracellular [Ca(2+)] and pH, endocytosed membrane traffic, and the transport of other solutes. In the present study we show that CS induces the formation of dark structures adjacent to the nucleus in human NT2 and ACHN cells. These structures, which are not surrounded by membranes, are clusters of glycogen and a distorted microtubule network. Formation of these clusters results from a relocation of glycogen and microtubules in the cells, two processes that are independent of one another. The molecular mechanisms underlying the formation of the clusters are mediated by the Na(+),K(+)-ATPase, ERK1/2 signaling pathway, and an additional unknown factor. Similar glycogen clusters are induced by hypoxia, suggesting that the CS-induced structural change, described in this study, may be part of a new type of cellular stress response.
Assuntos
Bufanolídeos/farmacologia , Glicogênio/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Adenocarcinoma , Cardiotônicos/farmacologia , Hipóxia Celular/fisiologia , Digoxigenina/farmacologia , Humanos , Neoplasias Renais , Sistema de Sinalização das MAP Quinases/fisiologia , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Células-Tronco Neurais/citologia , Nocodazol/farmacologia , Ouabaína/análogos & derivados , Ouabaína/farmacologia , Potássio/farmacologia , RNA Interferente Pequeno/genética , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Estresse Fisiológico/fisiologia , Moduladores de Tubulina/farmacologia , Células Tumorais CultivadasRESUMO
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.