Abstract
Inhibition of autophagic proteolysis by hypoosmotic or amino acid-induced hepatocyte swelling requires osmosignaling toward p38MAPK; however, the upstream osmosensing and signaling events are unknown. These were studied in the intact perfused rat liver with a preserved in situ environment of hepatocytes. It was found that hypoosmotic hepatocyte swelling led to an activation of Src (but not FAK), Erks, and p38MAPK, which was prevented by the integrin inhibitory hexapeptide GRGDSP, but not its inactive analogue GRGESP. Src inhibition by PP-2 prevented hypoosmotic MAP kinase activation, indicating that the integrin/Src system is located upstream in the osmosignaling toward p38MAPK and Erks. Inhibition of the integrin/Src system by the RGD motif-containing peptide or PP-2 also prevented the inhibition of proteolysis and the decrease in autophagic vacuole volume, which is otherwise observed in response to hypoosmotic or glutamine/glycine-induced hepatocyte swelling. These inhibitors, however, did not affect swelling-independent proteolysis inhibition by phenylalanine. In line with a role of p38MAPK in triggering the volume regulatory decrease (RVD), PP-2 and the RGD peptide blunted RVD in response to hypoosmotic cell swelling. The data identify integrins and Src as upstream events in the osmosignaling toward MAP kinases, proteolysis, and RVD. They further point to a role of integrins as osmo- and mechanosensors in the intact liver, which may provide a link between cell volume and cell function.
Highlights
Inhibition of autophagic proteolysis by hypoosmotic critical for creating volume-regulatory ion fluxes in response to or amino acid-induced hepatocyte swelling requires osmosignaling toward p38MAPK; the upstream osmosensing and signaling events are unknown
It was found that hypoosmotic hepatocyte swelling led to an activation of Src, Erks, and p38MAPK, which was prevented by the integrin inhibitory hexapeptide GRGDSP, but not its inactive analogue GRGESP
Inhibition of the integrin/Src system by the RGD motif-containing peptide or PP-2 prevented the inhibition of proteolysis and the decrease in autophagic vacuole volume, which is otherwise observed in response to hypoosmotic or glutamine/glycine-induced hepatocyte swelling
Summary
Liver Perfusion—Livers from male Wistar rats (160 –230 g), fed a standard diet, were perfused in an open non-recirculating manner at a flow rate of 3.5– 4.5 ml/min/g. For immune complex assay and Western blot determinations, liver lobes from perfused liver were excised at the respective time points (0, 2, 5, 10, 20, and 30 min after installation of hypoosmotic perfusion conditions), dounced with an Ultraturrax (Janke & Kunkel, Staufen, Germany) at 0 °C in lysis buffer containing 20 mmol/liter Tris-HCl (pH 7.4), 140 mmol/liter NaCl, 10 mmol/liter NaF, 10 mmol/liter sodium pyrophosphate, 1% Triton X-100, 1 mmol/liter EDTA, 1 mmol/liter EGTA, 1 mmol/liter sodium vanadate, 20 mmol/liter -glycerophosphate, and protease inhibitor mixture. Electron Microscopy—For electron microscopic morphometry, fixation of the liver lobes was performed as described previously [11] by perfusion of glutaraldehyde (3%) in Krebs-Henseleit medium for 30 s.
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