Dissociation of lipopolysaccharide-mediated induction of nitric oxide synthase and inhibition of DNA synthesis in RAW 264.7 macrophages and rat aortic smooth muscle cells

Abstract

1. The active component of endotoxin, lipopolysaccharide (LPS), inhibited basal DNA synthesis in both RAW 264.7 macrophages (IC₅₀ 0.05±0.03 μg ml⁻¹) and rat aortic smooth muscle cells (RASMC) (IC₅₀ 9.7±0.4 μg ml⁻¹).

2. In both cell types, serum differentially affected LPS-stimulated inhibition of DNA synthesis. In RAW 264.7 macrophages the presence of serum reduced the IC₅₀ for LPS-stimulated inhibition of DNA synthesis (1.4±0.85 ng ml⁻¹). However, in RASMC serum stimulated DNA synthesis and further increased the IC₅₀ value for LPS-stimulated inhibition of thymidine incorporation (57.3±7.8 μg ml⁻¹).

3. LPS also stimulated the induction of nitric oxide synthase (NOS) in RAW 264.7 macrophages with maximal expression at concentrations of 13 μg ml⁻¹. This was wholly dependent upon the presence of serum. In RASMC LPS alone, up to concentrations of 100 μg ml⁻¹, did not induce nitric oxide synthase and required co-incubation with the direct activator of adenylyl cyclase, forskolin. Under these conditions stimulated expression of NOS was inhibited by the presence of serum.

4. Incubation with the nitric oxide synthase inhibitors N^ω-nitro-L-arginine methyl ester (L-NAME) and L-canavanine did not reverse the inhibition of [³H]-thymidine incorporation in response to LPS but prevented the formation of nitrite in both cell types.

5. These results indicate that the effects of LPS upon cell growth are independent of the induction of the 130 kDa isoform of nitric oxide synthase and nitric oxide formation in both RAW 264.7 macrophages and RASMC.

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