Effect of gestational age and hypoxia on activity of ribonucleic acid polymerase in fetal guinea pig brain.

OBJECTIVE: The aim of this study was to determine the effect of gestational age and hypoxia on the activity of ribonucleic acid polymerase in fetal guinea pig brain. STUDY DESIGN: Fetal cerebral cortical neuronal nuclei were isolated at 40, 50, and 60 days (term) of gestation to determine the effect of gestational age on the activity of ribonucleic acid polymerase I, II, and III. Pregnant guinea pigs at 60 days' gestation were randomly assigned to a normoxic or hypoxic group to determine the effect of hypoxia on ribonucleic acid polymerase activity. The fetal neuronal nuclei were pooled from 6 pregnant animals in each group. In the normoxic group the pregnant guinea pigs were exposed to room air before delivery. In the hypoxic group delivery occurred after the pregnant guinea pig had been exposed to 7% oxygen for 60 minutes. The fetuses were delivered by cesarean, and the fetal cerebral cortical neuronal nuclei were isolated immediately. Ribonucleic acid polymerase activity was determined with nuclei suspended in a buffer containing adenosine triphosphate, guanosine triphosphate, cytidine triphosphate, and tritiated uridine triphosphate. Dactinomycin (actinomycin D) and polydeoxyadenylic-thymidylic acid were used to determine the activity of bound and free ribonucleic acid polymerase. alpha-Amanitin was used to determine the activity of ribonucleic acid polymerase II. RESULTS: The activity of total (bound and free) ribonucleic acid polymerase I and III increased from 85.4 +/- 9.4 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour at 40 days' gestation to 233.3 +/- 82.1 fmol at 50 days and to 343.4 +/- 231.6 fmol at 60 days (P =.02). Total ribonucleic acid polymerase II activity increased from 19.9 +/- 6.0 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour at 40 days to 123.8 +/- 53.0 fmol at 50 days and to 200.9 +/- 77.8 fmol at 60 days (P <.01). In the term fetal guinea pig brain the activity of bound ribonucleic acid polymerase I and III decreased from 116.8 +/- 107.2 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour under normoxic conditions to 92.8 +/- 76.0 fmol in hypoxic fetal brain, a decrease of 20.5%. Free ribonucleic acid polymerase I and III activity decreased from 199.2 +/- 115.2 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour in normoxic fetal brain to 132.0 +/- 66.4 fmol in hypoxic fetal brain, a decrease of 33.8%. Free ribonucleic acid polymerase II activity decreased from 62.4 +/- 70.4 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour in normoxic fetuses to 13.6 +/- 9.6 fmol in hypoxic fetal brain, a decrease of 78.2%. In contrast, however, in term fetal guinea pig brain, bound ribonucleic acid polymerase II activity increased from 8.0 +/- 10.4 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour under normoxic conditions to 35.2 +/- 8.8 fmol in hypoxic fetal brain, an increase of 340% (P <.01). CONCLUSION: The activity of ribonucleic acid polymerases I, II, and III increases throughout the latter half of gestation, from 40 to 60 days, in the fetal guinea pig brain. Hypoxia in utero is associated with a decrease in ribonucleic acid polymerase I and III activity. Although hypoxia is associated with a decrease in free ribonucleic acid polymerase II activity, we observed a marked increase in bound ribonucleic acid polymerase II activity, which may represent a hypoxia-induced alteration of gene expression.

Kainate receptor modification in the fetal guinea pig brain during hypoxia.

The present study tests the hypothesis that hypoxia alters the high-affinity kainate receptors in fetal guinea pig brain. Experiments were conducted in normoxic and hypoxic guinea pig fetus at preterm (45 days of gestation) and term (60 days of gestation). Hypoxia in the guinea pig fetus was induced by exposure to maternal hypoxia (FiO2 = 7%) for 60 min. Brain tissue hypoxia in the fetus was documented biochemically by decreased levels of ATP and phosphocreatine. [3H]-Kainate binding characteristics (Bmax = number of receptors, Kd = dissociation constant) were used as indices of kainate receptor modification. P2 membrane fractions were prepared from the cortex of normoxic and hypoxic fetuses and were washed six times prior to performing the binding assays. [3H]kainate binding was performed at 0 degrees C for 30 min in a 500 microliters medium containing 50 mM Tris-HCl buffer, 0.1 mM EDTA (pH 7.4), 300 micrograms protein and varying concentrations of radiolabelled kainate ranging from 1 to 200 nM. Non-specific binding was determined in the presence of 1.0 mM glutamate. During brain development from 45 to 60 days gestation, Bmax value increased from 330 +/- 16 to 417 +/- 10 fmoles/mg protein; however, the Kd was unchanged (8.2 +/- 0.4 vs 8.8 +/- 0.5 nM, respectively). During hypoxia at 60 days, the Kd value significantly increased as compared to normoxic control (15.5 +/- 0.7 vs 8.8 +/- 0.5 nM, respectively), whereas the Bmax was not affected (435 +/- 12 vs 417 +/- 10 fmol/mg protein, respectively). At 45 days, hypoxia also increased the Kd (11.9 +/- 0.6 vs 8.2 +/- 0.4 nM) without affecting the Bmax (290 +/- 15 vs 330 +/- 16 fmol/mg protein, respectively). The results show that the number of kainate receptors increase during gestation without change in affinity and demonstrate that hypoxia modifies the high-affinity kainate receptor sites at both ages; however the effect is much stronger at 60 days (term). The decreased affinity of the site could decrease the kainate receptor-mediated fast kinetics of desensitization and provide a longer period for increased Na(+)-influx, leading to increased accumulation of intracellular Ca2+ by reversal of the Na(+)-Ca2+ exchange mechanism. In addition, Kd values for kainate-type glutamate receptor sites are 30-40 fold lower (i.e. higher affinity) than those for NMDA-displaceable glutamate sites. The higher affinity suggests that the activation of the kainate-type glutamate receptor during hypoxia could precede initiation of NMDA receptor-mediated excitotoxic mechanisms. We propose that hypoxia-induced modification of the high affinity kainate receptor in the fetus is a potential mechanism of neuroexcitotoxicity.