CYP2E1 overexpression inhibits microsomal Ca2+-ATPase activity in HepG2 cells
Introduction
Calcium homeostasis is essential for cell survival, and changes in intracellular calcium are pivotal to signal transduction (Grover et al., 2003). In mammalian cells, a 10,000× concentration gradient across the plasma membrane is achieved by permanent active cytoplasmic Ca2+ extrusion, through the Na+/Ca2+ exchanger, by using the Na+ gradient generated by Na+/K+ ATPase, and is fine-tuned by the plasma membrane Ca2+-ATPase (PMCA) activity. In addition, most of the intracellular calcium is sequestered in the luminal side of the endoplasmic reticulum, by active pumping from the cytoplasm promoted by the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), creating another similar transmembrane gradient (Souza dos Santos et al., 2007).
Several reports have described that reactive oxygen species (ROS) and reactive nitrogen species (RNS) can inhibit SERCA, leading to alterations in calcium homeostasis. Oxidative damage to SERCA was reported in sarcoplasmic reticulum vesicles treated in vitro with Fe2+/H2O2/ascorbate (Castilho et al., 1996), hypoxanthine/xanthine oxidase (Barnes et al., 2000), hydrogen peroxide (Grover et al., 1997), and peroxynitrite (Gutierrez-Martin et al., 2004). Partial inactivation of SERCA caused by oxidative stress has been associated with several pathological conditions such as biological aging (Viner et al., 1999), obesity (Li et al., 2006), heart failure (Lokuta et al., 2005), and high intensity exercise (Matsunaga et al., 2003).
An active source of cellular ROS is cytochrome P450. The cytochrome P450 enzymes are a superfamily of hemeproteins that serve as terminal oxidases in the mixed function oxidase system for metabolizing various endogenous substrates such as steroids and fatty acids, and xenobiotics including drugs, toxins and carcinogens (Caro and Cederbaum, 2004). Oxygen activation by P450, necessary for the enzyme catalytic function, can also result in the production of ROS. Small amounts of the superoxide anion radical (O2−) can be produced from decay of the oxygenated P450 complex, while hydrogen peroxide (H2O2) can form from either dismutation of O2− or decay of the peroxy P450 complex (Puntarulo and Cederbaum, 1998). Among the different P450 isoforms, CYP2E1 has a high capacity to reduce dioxygen to reactive oxy radicals (Caro and Cederbaum, 2004).
In this work, we evaluated the possibility that CYP2E1-generated reactive oxygen species may inhibit microsomal Ca2+-ATPase activity in intact liver cells.
Section snippets
Chemicals
Phosphate-buffered saline (PBS) was from Roche (Newark, NJ). G418 was from Invitrogen (Carlsbad, CA). Protein concentration was measured using the Bio-Rad DC protein assay (Hercules, CA). Most of the other chemicals used were from Sigma Chemical Co. (St. Louis, MO). Antibodies for immunoblots were from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture
Two human hepatoma HepG2 cell sublines described in Chen and Cederbaum (1998) were used as models in this study: (1) E47 cells, which constitutively
Microsomal Ca2+-ATPase activity in CYP2E1 overexpressing cells
Previous work has shown that E47 cells overexpress active microsomal CYP2E1 and generate higher levels of reactive oxygen species with respect to control C34 cells, which do not express significantly any cytochrome P450 (Caro and Cederbaum, 2004). In this work, we measured the microsomal Ca2+-ATPase activity in E47 versus C34 cells. The selected assay for microsomal Ca2+-ATPase activity measures inorganic phosphate (Pi) released by the ATPase activity in one-step, without removing proteins from
Discussion
Microsomal ATPase activity in HepG2 cells showed two components: (1) a Ca2+-independent activity (around 10% of the total), which did not show significant differences between C34 and E47 cells; and (2) a Ca2+-dependent activity (around 90% of the total), which showed a significant 55% decrease in E47 with respect to C34 cells. This suggests that CYP2E1 overexpression inhibits microsomal Ca2+-dependent ATPase activity in intact HepG2 cells. The ATPase activity of rat liver microsomes also showed
Conflict of interest
The authors declare that there are no conflicts of interest.
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