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 p38MAPK 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 p38MAPK activation. Immune complex assays from perfused liver showed that hypo-osmotic activation of p38MAPK 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 p38MAPK 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 p38MAPK is required for this process and occurs upstream of the putative microtubule regulation site.