Cell hydration controls autophagosome formation in rat liver in a microtubule-dependent way downstream from p38(MAPK) activation

Autophagic proteolysis in rat liver is under the control of the cellular hy dration state. Because the morphological site of swelling-dependent proteol ysis regulation has not yet been identified, the formation of autophagosome s was investigated with transmission electron microscopy in slices from per fused livers. In livers from fed rats, hypo-osmotic exposure (185 mosmol/l) led within 30 min to a decrease in fractional cytoplasmic autophagosome vo lume that was sensitive to colchicine and p38(MAPK) inhibition. Similarly, the decrease in autophagosome volume, but not the increase in cell volume c aused by insulin or glutamine/ glycine, was strongly inhibited by colchicin e and SE 203580, an inhibition of p38(MAPK) activation. Immune complex assa ys from perfused liver showed that hypo-osmotic activation of p38(MAPK) was not inhibited by colchicine. Further, experiments using confocal laser mic roscopy in cultivated hepatocytes incubated with mouse-derived anti-(alpha -tubulin) showed that microtubular structures were not influenced by the in hibition of p38(MAPK) by SE 203580. It is concluded that the sequestration of autophagic vacuoles is a major site of proteolysis regulation by cell hy dration. Swelling-induced activation of p38(MAPK) is required for this proc ess and occurs upstream of the putative microtubule regulation site.