title: Localization of nitric oxide synthase in human trophoblast cells: role of nitric oxide in trophoblast proliferation and differentiation.
abstract: OBJECTIVE
  There are conflicting reports about the isoform of nitric oxide synthase (NOS) present in trophoblast cells. In this study, we have examined the presence of different NOS isoforms in trophoblast cells. In addition, the role of nitric oxide (NO) in trophoblast function has also been studied by investigating the possible role of nitric oxide in trophoblast proliferation and differentiation.
  
  
  METHODS
  NOS isoforms in primary-term trophoblast and JEG-3 cells were identified by immunocytochemistry. The intracellular localization of this enzyme was determined by confocal laser scanning microscopy. Trophoblast proliferation was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasolium bromide (MTT) conversion assay and cellular differentiation was monitored by human chorionic gonodotropin (hCG) and progesterone secretion, measured by radioimmunoassay.
  
  
  RESULTS
  The immunoreactive NOS was present in human trophoblast cells of normal term placenta and JEG-3 cells (a choriocarcinoma cell line) maintained in culture. Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase activity overlapped with the immunostaining of NOS. Specific antibodies against the different isoforms of NOS detected the presence of neuronal-type NOS (nNOS) only. The other two isoforms, i.e., eNOS (endothelial) and iNOS (macrophage specific) were completely absent. The nNOS was localized in cell cytoplasm. In culture, JEG-3 cells normally undergo proliferation and cytotrophoblast cells in primary culture differentiate to form hormone-secreting syncytial cells. Sodium nitroprusside (SNP), a nitric oxide donor, when added to the culture, significantly increased proliferation of JEG-3 cells and inhibited the differentiation of cytotrophoblast cells. The arrest by SNP in the formation of syncytial cells was further evidenced by the low secretion profile of hCG and progesterone.
  
  
  CONCLUSIONS
  Our findings suggest for the first time the presence of nNOS in the human trophoblast cells and a previously unrecognized role of NO in trophoblast proliferation and differentiation.
authors.affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi.
authors.forename: M
authors.initials: M
authors.lastname: Sanyal
authors.forename: T C
authors.initials: TC
authors.lastname: Nag
authors.forename: C
authors.initials: C
authors.lastname: Das
mesh_terms: D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D008058:Dihydrolipoamide Dehydrogenase; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D018613:Microscopy, Confocal; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D011247:Pregnancy; D013194:Staining and Labeling; D014327:Trophoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D014407:Tumor Cells, Cultured
chemical_list: D007527:Isoenzymes; D009599:Nitroprusside; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D008058:Dihydrolipoamide Dehydrogenase