Calcium-dependent nitric oxide synthesis is potently stimulated by tetrahydrobiopterin in human primordial placenta

Homogenized first trimester human placenta exhibits both Ca(2+)-dependent (90-95 per cent) and Ca(2+)-independent (5-10 per cent) nitric oxide (NO)-synthesizing activities. Addition of tetrahydrobiopterin (BH4) to homogenates containing Ca2+ in maximally activating concentrations (> 0.5 microM) results in a further 2-2.5-fold activation of NO synthesis, with half-maximal stimulation observed at 26 +/- 8.2 microM BH4 (mean +/- SEM, n = 4). Chelation of Ca2+ in the medium abolishes the stimulatory effect, indicating that only a Ca2(+)-dependent NO-synthase (NOS) isoform is activated by BH4. Based on our previous findings, we suggest that this isoform is the endothelial or Type III NOS. Importantly, BH4 has no significant effect on the Ca2(+)-dependency of NOS activity, the apparent Km values for Ca2+ are comparable in the absence (1.8 +/- 0.4 microM, mean +/- SEM, n = 6) or presence (2.5 +/- 0.6 microM, mean +/- SEM, n = 6) of 50 microM BH4. The BH4 content of these placentae is 207.4 +/- 86.7 pmol/g wet tissue (mean +/- s.d., n = 9), therefore, BH4 added to the homogenate does not simply restore the concentrations that occur endogenously. The results provide the first evidence that in the early human placenta, a constitutively expressed CA 2(+)-dependent NOS isoform is stimulated by exogenous BH4, raising the possibility that BH4 is an important regulator of NOS activity in this tissue. This novel aspect of the NO-generating pathway may have implications in the aetiology and treatment of pregnancy-induced hypertension and pre-eclampsia.