To evaluate the mechanisms involved in macrophage proliferation and activation, we studied the regulation of the nucleoside transport systems. In murine bone marrow-derived macrophages, the nucleosides required for DNA and RNA synthesis are recruited from the extracellular medium. M-CSF induced macrophage proliferation and DNA and RNA synthesis, whereas interferon gamma (IFN-gamma) led to activation, blocked proliferation, and induced only RNA synthesis. Macrophages express at least the concentrative systems N1 and N2 (CNT2 and CNT1 genes, respectively) and the equilibrative systems es and ei (ENT1 and ENT2 genes, respectively). Incubation with M-CSF only up-regulated the equilibrative system es. Inhibition of this transport system blocked M-CSF-dependent proliferation. Treatment with IFN-gamma only induced the concentrative N1 and N2 systems. IFN-gamma also down-regulated the increased expression of the es equilibrative system induced by M-CSF. Thus, macrophage proliferation and activation require selective regulation of nucleoside transporters and may respond to specific requirements for DNA and RNA synthesis. This report also shows that the nucleoside transporters are critical for macrophage proliferation and activation.
Primary cultures of rat-liver parenchymal cells show carrier-mediated nucleoside uptake by a mechanism that mainly involves concentrative, Na ؉ -dependent transport activity. In contrast, the hepatoma cell line FAO shows high nucleoside transport activity, although it is mostly accounted for by Na ؉ -independent transport processes. This is associated with a low amount of sodium purine nucleoside transporter (SPNT) mRNA. SPNT encodes a purinepreferring transporter expressed in liver parenchymal cells. To analyze whether SPNT expression is modulated during cell proliferation, SPNT mRNA levels were determined in the early phase of liver growth after partial hepatectomy and in synchronized FAO cells that had been induced to proliferate. SPNT mRNA amounts increased as early as 2 hours after partial hepatectomy. FAO cells induced to proliferate after serum refeeding show an increase in SPNT mRNA levels, which is followed by an increase in Na ؉ -dependent nucleoside uptake and occurs before the peak of 3 H-thymidine incorporation into DNA. FAO cells also express significant equilibrative nucleoside transport activity, which may be accounted for by the expression of the nitrobenzylthioinosine (NBTI)-sensitive and -insensitive isoforms, rat equilibrative nucleoside transporter 1 (rENT1) and rENT2, respectively. Interestingly, rENT2 mRNA levels follow a similar pattern to that described for SPNT when FAO cells are induced to proliferate, whereas rENT1 appears to be constitutively expressed. Liver parenchymal cells show low and negligible mRNA levels for rENT1 and rENT2 transporters, respectively, although most of the equilibrative transport activity found in hepatocytes is NBTI-resistant. It is concluded that: 1) SPNT expression is regulated both in vivo and in vitro in a way that appears to be dependent on cell cycle progression; 2) SPNT expression may be a feature of differentiated hepatocytes; and 3) equilibrative transporters are differentially regulated, rENT2 expression being cell cycle-dependent. This is consistent with its putative role as a growth factor-induced delayed early response gene. (HEPATOLOGY 1998;28:1504-1511.)Nucleosides and nucleoside analogues have a wide range of potent physiological and pharmacological properties. Purines, essentially adenosine, play a multifactorial role in liver physiology by modulating key metabolic pathways of the hepatocyte 1-5 and influencing, among other functions, hepatic arterial pressure-flow autoregulation, 6 vasodilation, 7 and superoxide anion generation. 8
In murine bone marrow macrophages, lipopolysaccharide (LPS) induces apoptosis through the autocrine production of tumor necrosis factor-␣ (TNF-␣), as demonstrated by the fact that macrophages from TNF-␣ receptor I knock-out mice did not undergo early apoptosis. In these conditions LPS up-regulated the two concentrative high affinity nucleoside transporters here shown to be expressed in murine bone marrow macrophages, concentrative nucleoside transporter (CNT) 1 and 2, in a rapid manner that is nevertheless consistent with the de novo synthesis of carrier proteins. This effect was not dependent on the presence of macrophage colony-stimulating factor, although LPS blocked the macrophage colony-stimulating factor-mediated up-regulation of the equilibrative nucleoside transport system es. TNF-␣ mimicked the regulatory response of nucleoside transporters triggered by LPS, but macrophages isolated from TNF-␣ receptor I knock-out mice similarly up-regulated nucleoside transport after LPS treatment. Although NO is produced by macrophages after LPS treatment, NO is not involved in these regulatory responses because LPS up-regulated CNT1 and CNT2 transport activity and expression in macrophages from inducible nitric oxide synthase and cationic amino acid transporter (CAT) 2 knock-out mice, both of which lack inducible nitric oxide synthesis. These data indicate that the early proapoptotic responses of macrophages, involving the up-regulation of CNT transporters, follow redundant regulatory pathways in which TNF-␣-dependent-and -independent mechanisms are involved. These observations also support a role for CNT transporters in determining extracellular nucleoside availability and modulating macrophage apoptosis.
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