Alterations of cell volume regulation in the development of hepatocyte necrosis

Intracellular Na⁺ accumulation has been shown to contribute to hepatocyte death caused by anoxia or oxidative stress. In this study we have investigated the mechanism by which Na⁺ overload can contribute to the development of cytotoxicity. ATP depletion in isolated hepatocytes exposed to menadione-induced oxidative stress or to KCN was followed by Na⁺ accumulation, loss of intracellular K⁺, and cell swelling. Hepatocyte swelling occurred in two phases: a small amplitude swelling (about 15% of the initial size) with preservation of plasma membrane integrity and a terminal large amplitude swelling associated with cell death. Inhibition of Na⁺ accumulation by the use of a Na⁺-free medium prevented K⁺ loss, cell swelling, and cytotoxicity. Conversely, blocking K⁺ efflux by the addition of BaCl₂ did not influence Na⁺ increase and small amplitude swelling, but greatly stimulated large amplitude swelling and cytotoxicity. Menadione or KCN killing of hepatocytes was also enhanced by inducing cell swelling in an hypotonic medium. However, increasing the osmolarity of the incubation medium did not protect against large amplitude swelling and cytotoxicity, since stimulated Na⁺ accumulation and K⁺ efflux. Altogether these results indicate that the impairment of volume regulation in response to the osmotic load caused by Na⁺ accumulation is critical for the development of cell necrosis induced by mitochondrial inhibition or oxidative stress.