Uridine catabolism in Kupffer cells, endothelial cells, and hepatocytes

Abstract

1. Kupffer cells, endothelial cells, and hepatocytes were separated by centrifugal elutriation. The rate of uracil formation from [2-I4C]uridine, the first step in uridine catabolism, was monitored in suspensions of the three different liver cell types. Kupffer cells demonstrated the highest rate of uridine phosphorolysis. 3 5 min after the addition of the nucleoside the label in uracil amounted to 51%, 13%, and 19% of total radioactivity in the medium of Kupffer cells, endothelial cells, and hepatocytes, respectively. If corrected for Kupffer cell contamination, hepatocyte suspensions demonstrated similar activities as endothelial cells.2. In contrast to non-parenchymal cells, hepatocytes continuously cleared uracil from the incubation medium. The lack of uracil consumption by Kupffer cells and endothelial cells points to uracil as the end-product of uridine catabolism in these cells.3. Kupffer cells and endothelial cells did not produce radioactive C 0 2 upon incubation in the presence of [2-14C]uridine. Hepatocytes, however, were able to degrade uridine into C 0 2 , ,!I-alanine, and ammonia as demonstrated by active formation of volatile radioactivity from the labeled nucleoside.4. There was almost no detectable formation of thymine from thymidine or of cytosine, uracil, or uridine from cytidine by any of the different cell types tested. These results are in line with low thymidine phosphorolysis and cytidine deamination in rat liver.5. Our studies suggest a co-operation of Kupffer cells, endothelial cells, and hepatocytes in the breakdown of uridine from portal vein blood with uridine phosphorolysis predominantly occurring in Kupffer cells and with uracil catabolism restricted to parenchymal liver cells.Uridine is present in blood plasma of different species in relatively constant micromolar concentrations [l -51 providing a source for nucleotide synthesis on the salvage pathway [5,6]. The pyrimidine nucleoside level in serum has been found independent from dietary RNA supply [3] or food restriction [l] and revealed no diurnal variations [l]. These observations suggested a regulatory mechanism for monitoring blood plasma uridine. A single-pass exchange of uridine in the isolated perfused rat liver points to the liver as the key site in maintaining constant levels of uridine in the circulation [7, 81. More than 90% of infused uridine is degraded in a single passage while constant amounts of the nucleoside are released into the hepatic vein blood [7,8]. Nonphysiological concentrations of circulating uridine are depleted up to at least 25 pmol/l [8]. The clearance of uridine from portal vein blood by degradation is a prerequisite for monitoring serum uridine levels Correspondence to A. Holstege,

Succinate is a paracrine signal for liver damage

“…Hepatocytes and cholagiocytes were isolated by collagenase perfusion, according to protocols described previously (6;7). Kupffer cells were isolated by collagenase perfusion followed by centrifugal elutriation (8) and their purity was confirmed by immunostaining with commercially obtained antibodies (Serotec; Dusseldorf, Germany) for the Kupffer cell marker F4/80 (9), while sinusoidal endothelial cells were isolated by collagenase perfusion followed by Percoll gradient centrifugation (10) and their purity was confirmed by staining with commercially obtained antibodies for endothelial nitric oxide synthase (eNOS) (BD Biosciences; San Jose, CA).…”

The role of nonparenchymal and parenchymal liver cells in the catabolism of extracellular purines

“…Isolation and Separation of Different Liver Cell Populations. The cell isolation and separation procedure is based on a method developed by Kirn et al (35) and was performed essentially as described (34). After in situ perfusion of the liver with collagenase, livers were excised and cut into small pieces, followed by further collagenase treatment of the fragments.…”

Potent combination therapy for human breast tumors with high doses of 5-fluorouracil: remission and lack of host toxicity

“…Indeed, more than 90% of the circulating uridine is catabolized in a single pass through the liver by the activity of hepatic uridine phosphorylase (UP, EC 2.4.2.3), while constant amounts of uridine are synthesized de novo and released into the hepatic vein blood [28,29]. Less than 2% of the uridine metabolized by the liver is salvaged and recovered in the uracil nucleotide pool in tissues of whole animals [27,[30][31][32], perfused rat liver [6,13], or isolated liver cells [31]. The remainder is rapidly catabolized to products beyond uracil in the pyrimidine catabolic pathway [13,28,33].…”