A subclone of the human colon adenocarci-

S _nnuay A panel of human colonic adenocarcinoma cell lines was examined both for expression of mRNAs of the nitric oxide synthase (NOS) gene family and for evidence of enzymic activity based on citrulline and nitrite (NO,-) formation. Reverse transcription-polymerase chain reaction (RT-PCR). revealed that all lines (SW480. SW620. DLD-1 and WiDr) expressed mRNA for the Ca-+-dependent endothelial (e)NOS. while SW480 cells also expressed the Ca2'-dependent neuronal (n)NOS. The mRNA for the Ca'+-independent inducible (i)NOS was expressed both by cytokine-stimulated and by unstimulated SW480. SW620 and DLD-1 cells, but none was seen at any time in the WiDr cells. There was, however, little correlation between mRNA expression and enzynic activity based on citruUline and NO,-formation. Thus none of the cell lines exhibited measurable Ca'-dependent NOS activity, while Ca2'-independent NOS activity was seen in all but the WiDr cells. Furthermore. DLD-1 cells generated citrulline with resultant NO.-formation only after stimulation with lipopolysacchanrde (LPS) and or cytokines. while SW480 and SW620 did so constitutively. Thus RT-PCR studies indicate that tumour cells of similar epithelial origin display a diverse pattern of NOS gene family expression. and parallel biochemical studies clearly indicate that such expression does not always result in measurable enzymic activity leading to the generation of NO.

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The aim of this study is to investigate the changes in expression and activity of inducible nitric oxide synthase (iNOS) in the developing murine embryo and mouse and human placenta. Using reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, and in-situ hybridization (ISH) we identified iNOS mRNA in mouse placenta at 9.5, 12, 14, 16, 18 and 20 days post coitum. Northern blot analysis demonstrated that the quantity of murine iNOS transcript was expressed at a stable level between days 12-20 although the level of calcium-independent NOS activity declined with advancing gestation. RT-PCR detected iNOS-specific mRNA in murine embryonic stem cells, but not in embryos at later stages (4-cell or blastocyst). ISH failed to show iNOS-specific mRNA in either murine placenta or the underlying myometrium on day 7, but did so in the trophoblast by day 9.5. Later in gestation, extensive labelling was observed in both spongiotrophoblast and trophoblast giant cells. iNOS mRNA was also detected both in immature human placentae (16-18 weeks) and at term, predominantly in syncytiotrophoblasts and placental artery smooth muscle. In conclusion, iNOS is constitutively expressed in mouse and human placenta at a time and in a location that suggests a role in placentation.

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