Effects of acute hepatic ischemia on the electrophoretic patterns of the polypeptides and phosphopolypeptides of the microsomal membrane fraction of rat liver

Abstract

The purpose of this study was to establish when alterations of the electrophoretic patterns of the polypeptides and phosphopolypeptides of the microsomal membrane fraction of the livers of rats become observable after initiation of acute hepatic ischemia. Ischemia was initiated by clamping the vascular supply to the left and median lobes of the livers of adult male rats. The animals were killed at various times thereafter (up to 6 h, and in certain instances, 24 h) and microsomal membrane fractions were prepared from each. The patterns of the polypeptides and phosphopolypeptides of these fractions were analysed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, using staining with Coomassie blue to analyse the polypeptides and radioautography to analyse 32P-labelled phosphopolypeptides. Alterations of the polypeptide pattern were apparent in the fractions from animals killed at 4 h and after; prior to this time point, subtle alterations, at most, could be distinguished. Effects of acute ischemia on the pattern of phosphopolypeptides of the microsomal membrane fraction were studied after phosphorylation in vivo (produced by intraperitoneal injection of [32P]phosphoric acid) and in vitro (using [gamma-32P]ATP as phosphate donor). No marked changes in the phosphopolypeptide pattern produced by phosphorylation in vivo were observed until 6 h after clamping, by which time a diminution of the radioactivity in the majority of the phosphopolypeptides was evident. However, noteworthy alterations of the pattern of phosphopolypeptides produced by phosphorylation in vitro were observable in the membrane fractions from animals subjected to 2 h of ischemia. Overall the study provides a base line delineating the time sequence during which alterations of the electrophoretic patterns of the polypeptides and phosphopolypeptides of rat liver microsomal membranes become evident following the onset of acute hepatic ischemia and reveals that gross alterations of the polypeptide patterns of these membranes and of certain other subcellular fractions are not an early occurrence following this severe type of injury. The possible utility of the application of phosphorylation in vitro for detecting early alterations in membrane structure following cell injury is suggested.