Endosomal Acidification and Activation of NADPH Oxidase Isoforms Are Upstream Events in Hyperosmolarity-induced Hepatocyte Apoptosis

Abstract

Hyperosmotic exposure of rat hepatocytes induced a rapid oxidative stress (ROS) response as an upstream signal for proapoptotic CD95 activation. This study shows that hyperosmotic ROS formation involves a rapid ceramide-and protein kinase C (PKC)-dependent serine phosphorylation of p47 phox and subsequent activation of NADPH oxidase isoforms. Hyperosmotic p47 phox phosphorylation and ROS formation were sensitive to inhibition of sphingomyelinases and were strongly blunted after knockdown of acidic sphingomyelinase (ASM) or of p47 association, EGFR-catalyzed CD95-tyrosine phosphorylation, and translocation of the EGFR-CD95 complex to the plasma membrane, where formation of the deathinducing signaling complex occurs. These proapoptotic responses were not only sensitive to inhibitors of sphingomyelinase, PKC, or NADPH oxidases but also to ASM knockdown, bafilomycin, and DIDS, i.e. maneuvers largely preventing hyperosmolarity-induced endosomal acidification and/or ceramide formation. In hepatocytes from p47 phox knock-out mice, hyperosmolarity failed to activate the CD95 system. The data suggest that hyperosmolarity induces endosomal acidification as an important upstream event for CD95 activation through stimulation of ASM-dependent ceramide formation and activation of NADPH oxidase isoforms.

Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: a crucial role for nuclear p47phox

“…Qin et al [26] have shown that Angiotensin II induced apoptosis in H9c2 cells via an increase in cytoplasmic and apparent nuclear p47 phox expression as determined by western blot and immunocytochemistry, which resulted in intracellular ROS production; however, the effect on other subunits such as NOX2 was not determined. Reinehr et al [27] have shown in p47 phox knock-out mice that hepatocytes no longer produced ROS. Furthermore, Heymes et al [28] found an increase in translocation of p47 phox to the sarcolemmal membrane of cardiomyocytes in failing human hearts compared to non-failing hearts, which coincided with increased membrane and intracellular gp91 phox expression, analyzed by immunohistochemistry, which could suggest that both p47 phox and gp91 phox are necessary for cardiomyocyte damage.…”

Ionic determinants of pH of acidic compartments under hypertonic conditions in trout hepatocytes

“…Bafilomycin A1 reduced the hypertonicity-induced pH L decrease by about 50%. Previous studies in rat hepatocytes (Schreiber et al, 1994;Reinehr et al, 2006) have shown similar pH L acidification in response to exposure to hypertonic conditions. This acidification was also sensitive to V-ATPase inhibition by bafilomycin A1 (Reinehr et al, 2006), but not by concanamycin A (Schreiber et al, 1996).…”

“…Previous studies in rat hepatocytes (Schreiber et al, 1994;Reinehr et al, 2006) have shown similar pH L acidification in response to exposure to hypertonic conditions. This acidification was also sensitive to V-ATPase inhibition by bafilomycin A1 (Reinehr et al, 2006), but not by concanamycin A (Schreiber et al, 1996). In the study of Reinehr and colleagues, the effect of hypertonicity on pH L was removed at a bafilomycin A1 concentration of only 0.1 μmol l -1 (Reinehr et al, 2006).…”

“…However, other reports showed no effect of Cl -removal, and it was concluded that Cl -is not essential in maintaining steady-state pH L (Schreiber et al, 1996;Wu et al, 2000;Wu et al, 2001). Under hypertonic conditions, removal of extracellular Cl -or inhibition of Cl -transport using DIDS has been reported to eliminate the pH L acidification induced by hypertonicity in rat hepatocytes (Schreiber et al, 1996;Reinehr et al, 2006). In our study, hypertonicity-induced acidification was not inhibited by inhibition of Cl -uptake using Cl --free medium or the anion inhibitor SITS.…”

“…Interestingly, intracellular vesicular compartments have been reported to participate in cell volume-sensitive pathways, such as lysosomal proteolysis or the putative swelling-induced insertion/retrieval of bile acid-transporter molecules into/from the canalicular membrane in response to cell swelling/shrinkage (Schreiber et al, 1994). And recently, hypertonicity-induced endosomal acidification has been suggested to be an important upstream event signalling oxidative stress and triggering proapoptotic state in hepatocytes (Reinehr et al, 2006).…”