Abstract
In the present study we used the water-soluble short chain phosphatidylcholine analogue L-alpha-dibutyryl-glycero-3-phosphatidylcholine (diC4PC) to investigate the mechanism involved in the canalicular secretion of phospholipids in rat liver. Uptake of 14C-labeled di-C4PC was studied in isolated microsomes as well as in basolateral (sinusoidal) and canalicular plasma membrane vesicles. Saturable uptake of diC4PC into an osmotically active space was observed in microsomes and canalicular membrane vesicles. In contrast, diC4PC uptake into basolateral membrane vesicles could be accounted for by cross-contamination with endoplasmic reticulum and canalicular membrane vesicles. Whereas the Km values for diC4PC uptake (37 degrees C) were similar in microsomes (7.4 +/- 2.6 mM) and canalicular membrane vesicles (8.2 +/- 2.0 mM), the Vmax values were approximately 2-fold higher in canalicular membrane vesicles (29.6 +/- 2.7 nmol/mg of protein x min) than in microsomes (16.7 +/- 2.1 nmol/mg of protein x min). Furthermore, Pronase treatment of the membrane vesicles reduced diC4PC uptake by 34-54% in both subfractions, whereas the D-[14C]glucose-accessible water space was only reduced by approximately 20%. These data provide direct evidence for the presence of a protein-mediated phosphatidylcholine translocating activity in the canalicular membrane of rat hepatocytes. This canalicular "flippase" has kinetic properties similar to those described previously in microsomes and provides a potential pathway for the translocation of bile salt dissolvable biliary phospholipids to the exoplasmic leaflet of the canalicular membrane.
Highlights
C4PC uptake into basolateral membravneesicles could be accounted for by cross-contamination with endoplasmic reticulum and canalicular membranevesicles
The canalicular secretion of both of these biliary lipids is closely coupled [3],and they are predominantly carriedin small unilamellarvesicles in primary hepatic bile fluid [4]. increasing bile salt secretion markedly stimulatesbiliary lipid output, it has been shown that bile salts are pumped out intboile canaliculi before the secretion of biliary phospholipid and cholesterol
The solubilized microdomains would occur by fusion of intracellular biliary lipid precursor vesicles with the canalicular membrane
Summary
Grade and obtained from either Sigma, Merck Incontrast,basolateral LPM vesicles exhibited a lower diC4PCuptakeactivi;ythan microsomes and canalicular membranes (Fig. 1) Most likely this low uptake activity thawed (37 "C water bath), diluted 15 with 50 mM Tris, adjusted to p H 7.4 with HCI, and preincubated for 10min at 37 "C in afinal volume of 100 pl. Kinetic experiments in control and Pronase-pretreatedvesicles demonstrated a Pronase-induced reduction of the V,, values by 65 and 72% in microsomes and canalicular LPMvesicles, respectively, whereas the apparent K , values remained the same These Pronase-induced reductions of diC4PC uptake cannot be explained by disintegration of the membrane vesicles alone,since the~-[‘~C]glucose-accessibw~aeter spacewas reduced by only about 20% in both membrane subfractions (data not shown).
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