Hypoxia-induced modification of the inositol triphosphate receptor in neuronal nuclei of newborn piglets: role of nitric oxide.

Abstract

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.