Abstract 394
Poster Session III, Monday, 5/3 (poster 167)
We have previously shown that the apparent number of NMDA receptors (Bmax) increases during development in the sheep, and that treatment with dexamethasone (DX) for 48 hr had no effect on number of receptors in the sheep fetus at 60% or 80% of gestation, but decreased the Bmax in 5-day-old lambs. The present study tests the hypothesis that the dexamethasone-mediated decrease in Bmax is due to altered expression of NMDA receptor subunit proteins (NR1, NR2A, NR2B). Studies were performed in 6 fetuses at 124 d (term=154 d) and 7 five-day-old lambs. Fetal treatment consisted of DX, 6 mg IM (n=3), or saline (n=3) q 12 hr × 4 doses to pregnant ewes at 122 d gestation. Lambs received DX, 0.01 mg/kg/dose (n=2) or 0.25 mg/kg/dose (n=2), or an equal volume of saline (n=3) IM q 12 hrs × 4 doses on postnatal days 3 and 4. Postnatal doses of DX were chosen to match fetal exposure to maternal steroids and doses used in preterm infants. Animals were studied 12-18 hr after the last dose of DX. The P2 membrane fraction was prepared from cerebral cortex and immunoprecipitated with antibodies directed specifically against each of the subunits. Immune complexes were incubated with protein A Sepharose, washed, suspended in Laemmli sample buffer, and heated at 90°C for 3-5 min. Sepharose was pelleted by centrifugation and the supernatant analyzed by SDS-PAGE. Proteins were transferred to nitrocellulose membranes and reacted with horseradish peroxidase-labeled secondary antibodies. After washing, membranes were incubated with chemiluminescent substrate and exposed to X-ray film. Protein bands were analyzed using imaging densitometry. No differences were found between lambs treated with high-dose and low-dose DX; therefore, data from all treated lambs were analyzed together. In saline-treated animals, the amount of NR1 protein was greater in 5-day-old lambs than in 124-day fetuses. In contrast, NR2A and NR2B protein decreased 40% in lambs compared to fetal values. In utero treatment with dexamethasone decreased the level of NR2A protein by 20% compared to age-matched saline-treated fetuses. However, DX treatment had no effect on levels of NR1 or NR2B protein in fetal lambs. In 5-day-old lambs, DX had no effect on any subunit. Thus expression of NMDA receptor subunit proteins changed with development in the sheep in a pattern similar to that seen in other species. Treatment with dexamethasone decreased NR2A expression in the fetus but had no other effects on the NMDA receptor proteins studied. We speculate that the apparent decrease in NMDA receptor number previously observed in newborn lambs after steroid treatment is not due to decreased expression of receptor subunits, but may be due to nonspecific effects on cell membrane structure that alter receptor configuration and binding characteristics. The decrease in NR2A in the fetus to newborn levels may represent steroid-induced acceleration of the normal maturational process.
