Leukotriene uptake by hepatocytes and hepatoma cells

The uptake of tritiated cysteinyl leukotrienes (LTC 4 , LTD 4 , LTE 4 ) and LTB 4 was investigated in freshly isolated rat hepatocytes and different hepatoma cell lines under initial‐rate conditions. Leukotriene uptake by hepatocytes was independent of an Na + gradient and a K + diffusion potential across the hepatocyte membranes as established in experiments with isolated hepatocytes and plasma membrane vesicles. Kinetic experiments with isolated hepatocytes indicated a low‐ K m system and a non‐saturable system for the uptake of cysteinyl leukotrienes as well as LTB 4 under the conditions used. AS‐30D hepatoma cells and human Hep G2 hepatoma cells were deficient in the uptake of cysteinyl leukotrienes, but showed significant accumulation of LTB 4 . Moreover, only LTB 4 was metabolized in Hep G2 hepatoma cells. Competition studies on the uptake of LTE 4 and LTB 4 (10 nM each) indicated inhibition by the organic anions bromosulfophthalein, S ‐decyl glutathione, 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonate, probenecid, docosanedioate, and hexadecanedioate (100 μM each), but not by taurocholate, the amphiphilic cations verapamil and N ‐propyl ajmaline, and the neutral glycoside ouabain. Cholate and the glycoside digitoxin were inhibitors of LTB 4 uptake only. Bromosulfophthalein, the strongest inhibitor of leukotriene uptake by hepatocytes, did not inhibit LTB 4 uptake by Hep G2 hepatoma cells under the same experimental conditions. Leukotriene‐binding proteins were analyzed by comparative photoaffinity labeling of human hepatocytes and Hep G2 hepatoma cells using [ 3 H]LTE 4 and [ 3 H]LTB 4 as the photolabile ligands. Predominant leukotriene‐binding proteins with apparent molecular masses in the ranges of 48–58 kDa and 38–40 kDa were labeled by both leukotrienes in the particulate and in the cytosolic fraction of hepatocytes, respectively. In contrast, no labeling was obtained with [ 3 H]LTE 4 in Hep G2 cells. With [ 3 H]LTB 4 a protein with a molecular mass of about 48 kDa was predominantly labeled in the particulate fraction of the hepatoma cells, whereas in the cytosolic fraction a labeled protein in the range of 40 kDa was detected. Our results provide evidence for the existence of distinct uptake systems for cysteinyl leukotrienes and LTB 4 at the sinusoidal membrane of hepatocytes; however, some of the inhibitors tested interfere with both transport systems. Only LTB 4 , but not cysteinyl leukotrienes, is taken up and metabolized by the transformed hepatoma cells.