Expression of nitric oxide synthase isoforms and detection of nitric oxide in rat placenta

Nitric oxide (NO) production in therat placenta was monitored and quantified by electron paramagneticresonance (EPR) spectroscopy with hemoglobin and anFe-N-(dithiocarboxy)sarcosine (DTCS) complex as NO-trappingreagents. Expression of nitric oxide synthase (NOS) isoformswas also examined by quantitative RT-PCR analysis. The EPR spectrum ofthe placenta with hemoglobin trapping showed a three-line hyperfinestructure (g = 2.008 and a = 1.66-mT). The EPR signal was diminished after the placenta was homogenized or the NOSinhibitor L-NAME was administered to pregnant rats.Therefore, the specific signal was definitely identified as beingderived from endogenous NO spin-trapped by hemoglobin, and the EPRspectrum showed that the NO adduct existed as a pentacoordinate -NOheme species. The EPR spectrum of the placenta with Fe-DTCS trapping showed a triplet signal (g = 2.038) derived from anNO-Fe-DTCS complex. The height of the triplet signal did not varysignificantly with gestational stage during the last few days ofgestation. At the gestational stages examined, the level of NOS II mRNAexpression was significantly higher than that of NOS III mRNA. NOS IIexpression in term (day 21.5) placenta was significantlyincreased compared with that in preterm (day 19.5) placenta(P < 0.01, n = 4 or 5). These resultssuggest that NOS II is the predominant producer of NO in the placentaand that NOS II-generated NO plays significant roles in the maintenanceof placental functions immediately before birth.