Abstract 259 Poster Session III, Monday, 5/3 (poster 118)

Previous studies have shown that calbindin-D28k, a calcium-binding protein localized in high concentrations in restricted neuronal populations, controls intracellular calcium levels. The present study investigates calbindin-D28k concentration in subcellular fractions of cerebellar cells in the guinea pig fetus following hypoxia. Studies were conducted on 14 pregnant guinea pigs at term (63 days). Brain tissue from fetuses of normoxic (N×, n=4) animals were compared to fetuses obtained from pregnant guinea pigs exposed to hypoxia (Hx) but allowed to recover for 0, 24, or 72 hr in utero (H×0, n=4; H×24, n=4; H×72, n=2). Maternal hypoxia was induced by decreasing FiO2 from 21% to 7% for 1 hr. Pregnant animals were subsequently allowed to recover in room air for the specified period. Subcellular fractions (nuclei, cytosol and membranes) were prepared from the cerebellum. Fetal brain tissue hypoxia was documented biochemically by measuring levels of ATP and phosphocreatine. Calbindin-D28k was separated using 12% SDS-PAGE and detected using a monoclonal calbindin-D28k antibody (CL-300, Sigma). The protein bands were analyzed by imaging densitometry (GS 700, BioRad). The density of the protein was expressed as absorbance units (OD × mm2). During hypoxia the brain tissue ATP level decreased by 76% from 4.21±0.53 to 1.21±0.3 µmoles/g brain, and PCr decreased by 73% from 3.82±0.43 to 1.03±0.33 µmoles/g brain. In the cytosolic fraction calbindin-D28k immunoreactivity increased during hypoxia (22.84 ± 2.35 H×0 vs 19.33 ± 0.97 N× OD × mm2, p<0.05) but returned to a level close to that of the normoxic group after 24hr (20 ± 1.20 OD × mm2, p=NS) and decreased below control values after 72hr to 15.95 ± 0.49 OD × mm2 (p=0.06 vs H×0). In the nuclear fraction calbindin-D28k decreased significantly following hypoxia (8.66 ± 0.26 H× 0 vs 11.01 ± 0.06 N× OD × mm2, p<0.05), returned to control levels in H×24 (11.05 ± 1.62 OD × mm2), and subsequently increased in H×72 (12.97 ± 0.42 vs 11.01 ± 0.06 N× OD × mm2, p<0.05). No significant changes were observed in the membrane fraction (12.87 ± 1.83 N×, 14.85 ± 2 H×0, 13.37 ± 2.1 H×24, 14.89 ± 0.67 H×72 OD × mm2). The data show that cytosolic calbindin-D28k concentration increases transiently during hypoxia, returns back to normal at 24hr and then decreases at 72hr. The nuclear calbindin-D28k initially decreases during hypoxia followed by a continuous increase during the 24-72 hrs of reoxygenation. We conclude that hypoxia alters the subcellular sorting of calbindin-D28k in the cerebellum of the guinea pig fetus. We speculate that while the increase in cytosolic calbindin-D28k may represent a physiological cellular response to hypoxia preventing the increase of intracellular Ca++, the early-immediate decrease of nuclear calbindin-D28k may reflect oversaturation of this Ca++ buffering protein and may lead to a transient increase in intranuclear Ca++ concentration leading to expression of proapoptotic genes.

(Funded by NIH-HD-20337)