Recent studies demonstrate that the IP3 receptor, located on the inner nuclear membrane, controls the IP3 dependent increase in calcium in the nucleoplasm and influences gene transcription during hypoxia. In order to understand the mechanism of regulation of nuclear Ca++ concentration during hypoxia, the present study examines the effect of brain tissue hypoxia on the density and affinity of the IP3 receptor in cerebral cortical nuclear membranes of normoxic and hypoxic guinea pig fetuses. Nuclear membranes were prepared from normoxic (21% O2, n=4) and hypoxic (7% O2 for 1 hr, n=4) guinea pig fetuses. 3H-IP3 binding was performed in nuclear membranes and receptor characteristics Bmax (receptor number) and Kd (receptor affinity) determined. 3H-IP3 binding was performed in a medium containing 50 mM HEPES buffer, (pH 8.0), 2 mM EDTA,3 H-IP3 (7.5 to 100 nM) and 150 μ g membrane protein. Nonspecific binding was performed in the presence of 10μM unlabelled IP3. In normoxic nuclear membranes the Bmax and the Kd values were 34±4 fmoles/mg protein and 18±2 nM, respectively. In hypoxic nuclear membranes the Bmax and Kd values were 110±8 fmoles/mg protein and 15±2 nM, respectively. The data show that there is a 3-fold increase in the number of IP3 receptor sites in hypoxic nuclear membranes. However, the affinity of the receptor for 3H-IP3 is not altered by hypoxia. The data indicate that hypoxia-induced alteration of nuclear membranes leads to an increase in accessible IP3 receptor sites. We speculate that an increase in the number of accessible IP3 receptor sites may lead to an increase in intranuclear Ca++, and result in altered gene expression in the hypoxic brain.
(Funded by NIH#HD-20337 and MOD 6-FY94-0135)
