Previous studies have shown that
hypoxia results in increased Ca2+ influx in neuronal nuclei and generation of
nitric oxide (NO)
free radicals in the cerebral cortical tissue of newborn piglets. The present study tests the hypothesis that
hypoxia results in modification of the
inositol triphosphate (
IP3) receptor characteristics in neuronal nuclei and that the
hypoxia-induced modification of the
IP3 receptor is NO mediated. Studies were performed in piglets, 3-5 days old, divided into normoxic (n = 5), hypoxic (n = 5), and
NO synthase (NOS) inhibitor N-nitro-
L-arginine (NNLA)-treated hypoxic (n = 5) groups. The NNLA-treated hypoxic group received an infusion of NNLA (40 mg/kg) over 1 hr prior to hypoxic exposure. The
hypoxia was induced by lowering the FiO2 to 0.05-0.07 for 1 hr. Brain tissue
hypoxia was documented biochemically by determining
ATP and
phosphocreatine (PCr) levels. Neuronal nuclei were isolated from the cerebral cortical tissue, and
IP3 receptor binding was performed in a medium containing 50 mM
HEPES (pH 8.0), 2 mM
EDTA, 3H-IP3 (7.5-100 nM), and 100 microg
nuclear protein. Nonspecific binding was determined in the presence of 10 microM unlabelled IP3. The
IP3 receptor characteristics Bmax (number of receptor sites) and Kd (dissociation constant) were determined. In normoxic, hypoxic, and NNLA-hypoxic groups,
ATP levels were 4.46 +/- 0.35, 1.52 +/- 0.10 (P <.05 vs. normoxic), and 1.96 +/- 0.33 micromoles/g brain, respectively (P <.05 vs. normoxic). PCr levels were 3.75 +/- 0.35, 0.87 +/- 0.09 (P <.05 vs. normoxic), and 1.31 +/- 0.10 micromoles/g brain, respectively (P <.05 vs. normoxic).
IP3 receptor binding characteristics in normoxic nuclear membranes showed that the Bmax value was 150.0 +/- 14.1 pmoles/mg
protein compared with 239.3 +/- 13.6 pmoles/mg
protein in the hypoxic group (P <.05). In the NNLA-treated hypoxic group, the Bmax value was 159.0 +/- 42.6 pmoles/mg
protein (P <.05 vs. hypoxic a, P = NS vs. normoxic). Similarly, the Kd was 25.2 +/- 0.28 nM in the normoxic group, 44.6 +/- 5.4 nM in the hypoxic group (P <.05), and 28.1 +/- 6.4 nM in the NNLA-treated hypoxic group. (P <.05 vs. hypoxic and P = NS vs. normoxic). The results show that
hypoxia results in increased Bmax and Kd values for the
IP3 receptor. Furthermore, the data demonstrate that administration of NNLA prior to
hypoxia prevents the
hypoxia-induced modification of the
IP3 receptor in neuronal nuclei of newborn piglets. Because NNLA inhibits NOS and prevents generation of NO, we conclude that the mechanism of
hypoxia-induced modification of the neuronal nuclear membrane
IP3 receptor is NO mediated. We propose that NO-mediated modification of the
IP3 receptor during
hypoxia may lead to increased intranuclear Ca2+, resulting in altered transcription of apoptotic genes and activation of cascades of
hypoxia-induced programmed neuronal death.