Effect of in utero hypoxia on the ouabain/strophanthidin binding site of the fetal guinea pig brain cell membrane Na +,K +-ATPase

Abstract

This study investigates the effect of hypoxia on the high affinity strophanthidin/ouabain binding site of brain cell membrane Na +,K +-ATPase in 30, 45 and 60 day (term) fetal guinea pigs. Studies were performed on 30 fetuses randomized to either normoxic or hypoxic conditions. The hypoxic fetuses were exposed to maternal hypoxia (FiO 2 = 7%) for 60 min. Brain cell membrane fractions were prepared, and the rate of ATP hydrolysis was determined at varying concentrations of strophanthidin. In every experiment with 45 and 60 day brain preparations hypoxia caused a leftward shift in the IC 50, but this did not reach the level of statistical significance (4.0 × 10 −5 normoxic, 9.0 × 10 −6 hypoxic, P = 0.069, at 45 days; 9.5 × 10 −6 M normoxic, 8.5 × 10 −6 M hypoxic, P = 0.23, at 60 days). If hypoxia does cause a true left shift this would indicate greater sensitivity of the hypoxic brain to inhibitor. In addition, [ 3H]ouabain binding studies were performed. In the 30 day normoxic brain preparations, the K d was 24.7 ± 5.6 nM, and the B max was 0.26 ± 0.08 pmol/mg protein. At 45 days the ouabain binding sites showed no change in affinity following hypoxia ( K d = 14.6 ± 1.7 nM normoxic, 13.0 ± 0.8 nM hypoxic, P = NS); however, there was a significant decrease in receptor number following hypoxia ( B max = 22.1 ± 2.2 pmol/mg protein normoxic, 16.9 ± 0.3 pmol/mg protein hypoxic, P < 0.05). In 60 day hypoxic brain, ouabain binding sites showed a significant decrease in K d ( K d = 213.3 ± 39.0 nM normoxic, 161.1 ± 33.6 nM hypoxic, P < 0.05) reflecting an increased affinity, and a significant decrease in B max (154.7 ± 61.6 pmol/mg protein normoxic, 76.0 ± 28.5 pmol/mg protein, hypoxic, P = 0.02). At 45 days only B max is changed by hypoxia, indicating that following hypoxia, there are fewer enzyme molecules, but the remaining molecules are not affected. Term brain Na +,K +-ATPase molecules are even more sensitive to hypoxia, with hypoxia resulting in a decrease in both B max and K d. This suggests that a term not only are there fewer enzyme molecules following hypoxia, but that remaining molecules are modified as well.