We have previously found that the infusion of a placental extract inhibits spontaneous fetal breathing in sheep. The action was primarily found in the 1-10 kD subfraction and was tissue specific. Because prostaglandins function as a placentally-derived respiratory inhibitor before birth, it is possible that they are the active principle contained in the placental extract. Inprotocol 1, we hypothesized that if prostaglandins were the active factors in the placental extract, then inhibition of placental prostaglandins with Indomethacin prior to the removal of the placenta, should significantly decrease the activity of the extract. Therefore, after continuous breathing had been induced by the administration of 3mg/kg of Indomethacin into the fetal circulation, the ewe was sacrificed and the placenta removed. Infusion of the 1-10 kD subfraction obtained from the Indomethacin treated placentas inhibited breathing in 66% (19/29) of the experiments compared to 14% (3/22) with Krebs solution (control; p = 0.0002). This inhibition was not different than the one observed with the untreated placental extract (80%; 16/20; p = 0.3). Breathing output (∫ EMGdixfrequency) significantly decreased with administration of the Indomethacin treated and untreated placental extracts, from 416 ± 4 to 158 ± 5 arbitrary units (p = 0.0001) and from 476 ± 9 to 183± 15 (p = 0.001), respectively. No significant effect was observed with the Krebs solution. In protocol 2, we determined the dose response of the 1-10 kD extract, reconstituted to different concentrations and dilutions after lyophilization. Concentrating the infusates one, two, and four fold inhibited breathing in 75, 84 and 92% of the experiments respectively. On the contrary, this activity was significantly decreased in the infusates diluted ×1/2 (47%), ×1/4 (19%),×1/8 (16%), and ×1/16 (10%). Regression analysis of changes in breathing activity with various concentrations of the extracts showed a significant correlation (R2 = 0.36; p = 0.0001), the activity decreasing with dilution of the extract. These findings suggest that: i) the inhibition of breathing observed with the placental extract is not related to prostaglandins, and ii) this inhibition was dependent upon the concentration of the placental extract infused into the fetus. Our studies using the placental extract suggest that a non prostanoid mediator, likely a peptide, is responsible for the inhibition of fetal breathing. (Supported by MRC & Child. Hosp. Res. Found.)
