Project Details
Description
Bile is the principal pathway for cholesterol elimination from the body,
and abnormalities in bile secretion may result in gallstone formation,
cholestasis, biliary cirrhosis, hypercholesterolemia and fat
malabsorption. The applicant has proposed physicochemical and molecular
biological experiments to delineate the hepatocellular mechanisms whereby
bile salts promote biliary secretion of cholesterol together with highly
specific phosphatidylcholines. Preliminary studies in model systems
suggest that bile salts in submicellar concentrations promote transfer
of biliary phosphatidylcholine molecules from smooth endoplasmic
reticulum to canalicular plasma membranes via a specific
phosphatidylcholine transfer protein (PC-TP). To test the hypothesis that
PC-TP plays a key role phosphatidylcholine selection and transport in
vivo, bile salt activation of this protein will be examined employing
native smooth endoplasmic reticulum and canalicular plasma membrane
vesicles. Potential regulation of molecular expression of hepatic PC-TP
by bile salts will be explored by measuring steady state cytosolic
protein and mRNA levels as well as gene transcription rates. A putative
transfer protein for biliary phosphatidylcholines necessitates
independent cytosolic transfer of biliary cholesterol, hypothesized
herein to be via hepatic sterol carrier protein 2 (SCP2). The influence
of submicellar bile salts on inter-membrane ferrying of cholesterol
molecules by this protein will be studied in vitro. As with PC-TP,
molecular regulation of SCP2 by bile salts as well as potential
overexpression in cholesterol gallstone disease will be investigated.
Both phosphatidylcholine and cholesterol molecules translocate across
canalicular plasma membranes for biliary secretion. Whereas this may
occur passively for cholesterol, a specific bilayer translocase is
required for phosphatidylcholine. In preliminary experiments, the
principal investigator has demonstrated functional activity in hepatocyte
membranes of an ATP-independent long-chain phosphatidylcholine
translocase that is distinct from the ATP-dependent phosphatidylcholine
translocating activity of the multidrug resistance gene (mdr2) product,
P-glycoprotein. The responsible protein will be isolated from canalicular
plasma membrane vesicles by immobilized artificial membrane affinity
chromatography and then characterized physical-chemically and
biochemically. Following secretion into bile canaliculi, bile salts are
believed to promote biliary phosphatidylcholine and cholesterol secretion
as vesicles. The hypothesis that vesiculation of phosphatidylcholine and
cholesterol molecules results from physical-chemical partitioning of
dilute bile salts into exoplasmic leaflets of canalicular membranes will
be tested using an automated Langmuir-Pockels surface balance and
quasielastic light scattering spectroscopy. These studies should, in
part, elucidate biliary lipid secretion at a fundamental cellular level
and potentially lead to early interventions in cholelithiasis and
cholestasis as well as new strategies for management of
hypercholesterolemia.
Status | Finished |
---|---|
Effective start/end date | 2/1/96 → 1/31/02 |
Funding
- National Institute of Diabetes and Digestive and Kidney Diseases: $146,745.00
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