We have investigated the processing of the non-exchangeable fluorescent
phospholipid analogue
phosphatidyl(N-sulphorhodamine B sulphonyl)ethanolamine (
N-Rh-PE) by rat liver cells. In the hepatocyte couplet system,
N-Rh-PE was incorporated into the plasma membrane at 2 degrees C and readily internalized upon warming to 37 degrees C. Fluorescence was initially found to be concentrated in vesicles clustered throughout the cell, but subsequently it started to accumulate in pericanalicular vesicles, tentatively identified as lysosomes, and in the bile canalicular lumen. Analysis of cells and media by t.l.c. revealed the slow formation of at least two metabolites. After
intravenous injection into bile-
fistula rats of [9,10-3H-oleoyl]
N-Rh-PE incorporated in small
unilamellar liposomes, the initial rates of elimination from plasma of 3H and
rhodamine label were virtually identical. However, biliary secretion of the 3H label (5.5% of dose at 2 h) was much slower than that of the
rhodamine label (49.3% at 2 h). The
rhodamine label in bile was
chloroform-soluble, but not identical to the native molecule, and was resistant to
phospholipase A2 and alkaline hydrolysis. To gain insight in the mechanism of the rapid bile secretion of this metabolite, we compared the processing of
N-Rh-PE, its deacylated form [glycerophospho(N-
sulphorhodamine B sulphonyl)
ethanolamine; Gly-N-Rh] and the
rhodamine label itself (
sulphorhodamine B sulphonyl
chloride; SRho).
Intravenous injection of
chloroform-soluble
N-Rh-PE and of
methanol/water-soluble Gly-N-Rh complexed with
albumin both resulted in rapid bile secretion of
chloroform-soluble fluorescent compounds (60.2% and 86.3% respectively at 2 h), which showed behaviour identical to that of the metabolite of liposomal
N-Rh-PE on t.l.c.
Methanol/water-soluble SRho was also rapidly secreted into bile (89.5% at 2 h) without being metabolized. Bile secretion of the
chloroform-soluble metabolite of
N-Rh-PE and of SRho was markedly impaired (-31% and -52% respectively) in GY Wistar rats, which express a genetic defect in the hepatobiliary transport of organic
anions. Our data show that the rat hepatocyte is capable of modifying the structure of
N-Rh-PE, a process which proceeds considerably faster in vivo than in vitro. The
chloroform-soluble metabolite is subsequently rapidly removed via the bile. The canalicular organic
anion transporting system, which is deficient in GY rats, appears to be involved in the excretion of this apolar product of hepatic metabolism.