Characterization of a very-high-affinity ouabain binding site in term fetal guinea pig brain Na⁺,K⁺-ATPase

OBJECTIVE: To characterize the very-high-affinity ouabain binding site in fetal brain and determine its sensitivity to hypoxia. METHODS: Studies were performed on six normoxic and six hypoxic guinea pig fetuses at term and in six adult guinea pigs. Fetuses were delivered after the pregnant female had been exposed to 21% or 7% oxygen for 1 hour. Brain cell membranes were prepared and ouabain binding studies were performed. Ouabain binding was determined in the presence and absence of erythrosin B, a known inhibitor of high-affinity ouabain binding sites. RESULTS: Normoxic term fetal brain membrane had a Bmax (receptor number) of 84.2 ± 13.6 pmol/mg protein, which decreased to 5.9 ± 3.8 pmol/mg protein (93.0% decrease, P < .001) in the presence of erythrosin B. Normoxic fetal brain had a dissociation constant (Kd) (receptor affinity) of 24.6 ± 4.5 nmol/L, which was unchanged in the presence of erythrosin B (Kd = 20.7 ± 15.4 nmol/L, P = nonsignificant [NS]). Hypoxic term fetal brain had a Bmax of 74.7 ± 8.3 pmol/mg protein, which decreased to 7.1 ± 3.9 pmol/mg protein (90.5% decrease, P < .001) in the presence of erythrosin B. Hypoxic fetal brain had a Kd of 22.9 ± 1.9 nmol/L, which was unchanged in the presence of erythrosin B (Kd = 24.5 ± 9.9 nmol/L, P = NS). The adult control guinea pig brain had a Bmax of 104.1 ± 13.3 pmol/mg protein, which decreased to 44.9 ± 10.5 pmol/mg protein (P < .001) in the presence of erythrosin B, and a Kd of 214.3 ± 31.3 nmol/L, which remained unchanged in the presence of erythrosin B (Kd was 165.4 ± 36.0 nmol/L, P = NS). CONCLUSION: Fetal brain has a unique very-high-affinity ouabain binding site that is absent in adult brain and is sensitive to erythrosin B and resistant to hypoxia. We speculate that the presence of a Na⁺,K⁺ -ATPase molecule with a very-high-affinity site may be advantageous to the fetal brain during early maturation as well as during hypoxia or ischemia.