MECHANISMS OF LIVER CELL INJURY

Project: Research project

Project Details

Description

Our OVERALL OBJECTIVE is to define the critical cellular mechanisms
culminating in lethal liver cell injury. We will test the novel GENERAL
HYPOTHESIS that NON-LYSOSOMAL PROTEOLYSIS IS AN ESSENTIAL MECHANISM
MEDIATING LETHAL CELL INJURY DURING ANOXIA, OXIDATIVE STRESS AND BILE
SALT CYTOTOXICITY. We will employ current and complementary biochemical
and cell biological techniques to ascertain the regulatory and
mechanistic aspects of proteolysis during lethal liver cell injury. The
proposal has two SPECIFIC AIMS. FIRST, we will determine the cellular
localization, regulation and identity of protease activity during lethal
liver cell injury by directly testing: a) the HYPOTHESIS that proteolysis
occurs during cell injury in all liver cell types, and occurs
predominantly by nonlysosomal proteases, b) the HYPOTHESIS that
proteolysis during cell injury is regulated by cellular ATP, cytosolic
pH, vesicular pH and/or cytosolic free calcium; and c) the HYPOTHESIS
that specific identifiable classes of proteases are responsible for the
proteolysis occurring during cell injury. SECOND, we will determine the
mechanistic role of proteolysis as a cause of lethal liver cell injury by
directly testing: a) the HYPOTHESIS that proteolytic activity precedes
the onset of cell death; and b) the HYPOTHESIS that inhibition of
proteolytic activity delays the onset of lethal cell injury. The
studies will employ homogeneous cultures and suspensions of isolated rat
liver hepatocytes, sinusoidal endothelial cells and intrahepatic bile
duct epithelial cells. In dispersed cell suspensions, total, lysosomal
and non-lysosomal proteolysis, rates of cell killing, ATP depletion, and
protease class specific proteolysis will be measured under basal
conditions and during cell injury. Employing single cultured cells,
fluorescent probes and multiparameter digitized video microscopy,
cytosolic pH, vesicular pH, cytosolic free calcium, and protease class
specific proteolysis will be quantitated using commercially available and
novel synthetic fluorescent probes. Our LONG-TERM GOALS are to provide
new fundamental information regarding proteolysis as a mechanism of
lethal injury of liver cells during clinically relevant models of cell
injury. Interventional strategies must be predicated on an understanding
of the cellular mechanisms which lead to cell death. Successful
completion of this proposal could identify a protease responsible for
lethal liver cell injury. The results may lead to the identification of
treatment modalities effective in the presentation of liver tissue during
anoxia, oxidative stress, and cholestasis with retention of toxic bile
salts.
StatusFinished
Effective start/end date8/1/893/31/20

ASJC

  • Medicine(all)