Cell hydration controls autophagosome formation in rat liver in a microtubule-dependent way downstream from p38MAPK activation

Autophagic proteolysis in rat liver is under the control of the cellular hydration state. Because the morphological site of swelling-dependent proteolysis regulation has not yet been identified, the formation of autophagosomes was investigated with transmission electron microscopy in slices from perfused livers. In livers from fed rats, hypo-osmotic exposure (185mosmol/l) led within 30min to a decrease in fractional cytoplasmic autophagosome volume that was sensitive to colchicine and p38^MAPK inhibition. Similarly, the decrease in autophagosome volume, but not the increase in cell volume caused by insulin or glutamine/glycine, was strongly inhibited by colchicine and SB 203580, an inhibition of p38^MAPK activation. Immune complex assays from perfused liver showed that hypo-osmotic activation of p38^MAPK was not inhibited by colchicine. Further, experiments using confocal laser microscopy in cultivated hepatocytes incubated with mouse-derived anti-(α-tubulin) showed that microtubular structures were not influenced by the inhibition of p38^MAPK by SB 203580. It is concluded that the sequestration of autophagic vacuoles is a major site of proteolysis regulation by cell hydration. Swelling-induced activation of p38^MAPK is required for this process and occurs upstream of the putative microtubule regulation site.