CONFOCAL ANALYSIS OF HEPATOCELLULAR LONG-CHAIN FATTY-ACID UPTAKE

Transmembrane transport and cytosolic accumulation of fatty acids were investigated using confocal laser scanning microscopy (cLSM). A Zeiss LSM 310 system was used to determine the uptake of the fluorescent fa tty acid derivative nitrobenz-2-oxa-1,3-diazol-4-yl)amino]octadecanoic acid (12-NBD stearate) (C-18) in single rat hepatocytes. Uptake was a saturable process with a Michaelis-Menten constant value of 68 nM. In itial uptake velocity was dependent on extracellular presence of album in and beta-lactoglobulin. Absence of albumin reduced uptake to 32 +/- 16% (P < 0.01) of control values. In the presence of unlabeled steara te, uptake of 12-NBD stearate was lowered to 49 +/- 12% (P < 0.01). Io n substitution experiments showed no sodium dependency of uptake. Incr ease in membrane potential led to a pronounced accumulation of the fat ty acid derivative within the plasma membrane and in the adjacent cyto plasmic compartment, whereas membrane depolarization had no effect on uptake rates. In separate experiments line scans through representativ e hepatocytes were analyzed to generate ''x-t'' plots. 12-NBD stearate showed a fluorescence pattern with prominent staining of the area of the plasma membrane and the adjacent cytoplasm, dependent on the prese nce of extracellular albumin. For the hepatocellular cytosolic accumul ation process of 12-NBD stearate a diffusion constant of 22.2 +/- 6.2 x 10(-9) cm(2) is was calculated. In contrast to the long-chain fatty acid derivative 12-NBD stearate, short (C-5)- and medium (C-11)-chain fatty acids revealed no membrane interaction with hepatocytes. Erythro cytes also lacked a membrane interaction process for 12-NBD stearate. In conclusion, it was demonstrated that cLSM is capable of directly ev aluating the cellular fatty acid uptake process at a subcellular level .