Characterization of a Very-High-Affinity Ouabain Binding Site in Term Fetal Guinea Pig Brain Na+,K+-ATPase

Abstract

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 expoed 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 thatis 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.

Keywords

Guinea pig brain Na+K+-ATPase erythrosin B hypoxia

Get full access to this article

View all access options for this article.

References

1. Geering K . Subunit assembly and functional maturation of Na+,K+-ATPase. J Membr Biol 1990;115:109-121.

2. Lingrel JB . Na+,K+-ATPase: isoform structure, function, and expression. Journal of Bioenerg Biomemb 1992;24:263-270.

3. Silbergeld EK . Erythrosin B is a specific inhibitor of high affinity 3H-ouabain binding and iron transport in rat brain. Neuropharmacology 1980;20:87-89.

4. Graham EM , Mishra OP , Delivoria-Papadopoulos M . Brain cell membrane Na+,K+-ATPase modification following hypoxia in the guinea pig fetus. Neurosci Lett 1993;153:93-97.

5. Lowry OH , Rosebrough NJ , Farr AL , Randall RJ . Protein measurement with the folin phenol reagent. J Biol. Chem 1951;193:265-275.

6. Franck G , Grisar T , Moonen G . Glial and neuronal Na+-K+ pump. In: Federoff SL , Hertz L , eds. Advances in Cellular Neurobiology, Vol. 4, New York: Academic Press, 1983:133-159.

7. Shull GE , Greeb J , Lingrel JB . Molecular cloning of three distinct forms of the Na+,K+-ATPase α-subunit from rat brain. Biochemistry 1986;25:8125-8132.

8. Albers RW , Siegel GJ , Stahl WL . Membrane transport. In: Siegel GJ , Agranoff BW , Albers RW , Molinoff PB , eds. Basic Neurochemistry, 4th edition. New York: Raven Press, 1989:49-70.

9. Hamlyn JM , Blaustein MP , Bova S , et al. Identification and characterization of a ouabain-like compound from human plasma. Proc Natl Acad Sci USA 1991;88:6259-6263.

10.

10. Mathews WR , DuCharme DW , Hamlyn JM , et al. Mass spectral characterization of an endogenous digitalislike factor from human plasma. Hypertension 1991;17:930-935.

11.

11. Sweadner KJ . Two molecular forms of (Na++K+)-stimulated ATPase in brain. J Biol Chem 1979;254:6060-6067.

12.

12. Maki AA , Baskin DG , Stahl WL . 3H-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography. J Histochem Cytochem 1992;40:771-779.

13.

13. Graham EM , Mishra OP , Delivoria-Papadopoulos M . Effect of in utero hypoxia on the ouabain/strophanthidin binding site of the fetal guinea pig brain cell membrane Na+,K+-ATPase. Neurosci Lett 1995;185:159-162.

14.

14. Silbergeld EK , Anderson SM , Morris SJ . Interactions of erythrosin B (U.S. F,D&C Red 3) with rat cortical membranes. Life Sci 1982;31:957-969.

15.

15. Swann AC . Brain (NA+,K+)-ATPase: biphasic interaction with erythrosin B. Biochem Pharmacol 1982;31:2185-2190.