Heme oxygenase and nitric oxide synthase in the placenta of the guinea-pig during gestation.

Nitric oxide (NO) and carbon monoxide (CO) are novel gaseous chemical messengers that play key roles in cell function and cell-cell communication in many organ systems, including the cardiovascular system. Although the presence of NO synthase (NOS) in the placenta and its role in the regulation of fetoplacental and uteroplacental blood flow are well established, little is known about placental expression and activity of heme oxygenase (HO), the enzyme that catalyses the oxidation of heme to CO, biliverdin and iron, during gestation. The objectives of this study were to elucidate the localization of HO-1 and HO-2 isoforms relative to NOS III protein, and to determine the enzymatic activity of HO in the placenta of the guinea-pig during gestation. Placentae were obtained from pregnant guinea-pigs at gestational day (GD) 34, 50, 62 and full term (term, about GD 68). Immunohistochemical localization of HO-1, HO-2 and NOS III protein was conducted using selective polyclonal antibodies. HO activity was determined by using a gas chromatographic method to measure the rate of formation of CO from heme. Faint staining for HO-1 was observed in the adventitial layer of larger fetal blood vessels of the placenta at GD 34. The intensity of this staining was higher at GD 50 and GD 62, and decreased at full term. Similar areas in serial sections of placentae obtained at these selected times during gestation exhibited lower staining intensity when incubated with anti-HO-2 antiserum. Placental HO activity was significantly increased (P<0.05) at GD 62 compared with GD 34, GD 50 and full term. NOS III (endothelial constitutive NOS) staining was highest at GD 34, decreasing thereafter, and was localized mostly to trophoblast lining maternal channels. The data demonstrate that, in the guinea-pig, placental HO and NOS differ in tissue localization during the second half of gestation, with expression of HO protein and its catalytic activity being higher during near-term pregnancy. In a preliminary immunohistochemical investigation of the full-term human placenta, HO-1 protein was localized primarily in the adventitial region of fetal blood vessels of stem chorionic villi. In view of the vasodilator action of CO and NO, the HO and NOS systems may play key roles in the regulation of placental haemodynamics.

Increased cerebral extracellular adenosine and decreased PGE2 during ethanol-induced inhibition of FBM.

Adenosine and PGE2 are neuromodulators, both of which inhibit fetal breathing movements (FBM). Although circulating PGE2 has been implicated as a mediator of ethanol-induced inhibition of FBM in the late-gestation ovine fetus, a role for adenosine has not been examined. The objective of this study was to determine the effect of maternal ethanol infusion on ovine fetal cerebral extracellular fluid adenosine and PGE2 concentrations by using in utero microdialysis and to relate any changes to ethanol-induced inhibition of FBM. Dialysate samples were obtained from the fetal parietal cortex over 70 h after surgery to determine steady-state extracellular fluid adenosine and PGE2 concentrations. On each of postoperative days 3 and 4, after a 2-h baseline period, ewes received a 1-h infusion of ethanol (1 g/kg maternal body wt) or an equivalent volume of saline, and the fetus was monitored for a further 11 h with 30-min dialysate samples collected throughout. Immediately after surgery, dialysate PGE2 and adenosine concentrations were 3.7 +/- 0.7 and 296 +/- 127 nM, respectively. PGE2 did not change over the 70 h, whereas adenosine decreased to 59 +/- 14 nM (P < 0.05) at 4 h and then remained unchanged. Ethanol decreased dialysate PGE2 concentration for 2 h (3.3 +/- 0.3 to 1.9 +/- 0.4 nM; P < 0.05) and increased adenosine concentration for 6 h (87 +/- 13 to a maximum of 252 +/- 59 nM, P < 0.05). Ethanol decreased FBM incidence from 47 +/- 7 to 16 +/- 5% (P < 0.01) for 8 h. Saline infusion did not change dialysate adenosine or PGE2 concentrations or FBM incidence. These data are consistent with the hypothesis that fetal cerebral adenosine, and not PGE2, is the primary mediator of ethanol-induced inhibition of FBM at 123 days of gestation in sheep.

Hippocampal nitric oxide synthase in the fetal guinea pig: effects of chronic prenatal ethanol exposure.

The effects of chronic maternal administration of ethanol on nitric oxide synthase (NOS) activity and the numbers of NOS containing neurons, and CA1 and CA3 pyramidal neurons in the hippocampus of the near term fetal guinea pig at gestational day (GD) 62 were investigated. Pregnant guinea pigs received oral administration of 4 g ethanol/kg maternal body weight (n = 5), isocaloric sucrose/pair feeding (n = 5) or water (n = 5), or no treatment (NT; n = 5) from GD 2 to GD 61. NOS activity in the 25,000 x g supernatant of hippocampal homogenate was determined using a radiometric assay. The numbers of NOS containing neurons, and CA1 and CA3 pyramidal neurons were determined using NADPH diaphorase histochemistry and cresyl violet staining, respectively. The chronic ethanol regimen produced a maternal blood ethanol concentration of 193 +/- 13 mg/dl at 1 h after the second divided dose on GD 57. Chronic ethanol exposure produced fetal body, brain, and hippocampal growth restriction and decreased fetal hippocampal NOS activity compared with the isocaloric sucrose/pair feeding, water, and NT experimental groups, but did not affect the number of NOS containing and CA1 or CA3 pyramidal neurons. These data demonstrate that, in the near term fetus, chronic maternal administration of ethanol suppresses hippocampal NOS activity and consequent formation of NO, without loss of NOS containing neurons and prior to loss of CA1 pyramidal neurons that occurs in the adult.

Ontogeny of nitric oxide synthase I and III protein expression and enzymatic activity in the guinea pig hippocampus.

60. NOS enzymatic activity increased throughout prenatal and postnatal life, and attained highest activity in the adult. The developmental profile of NOS III protein expression was similar to that for NOS enzymatic activity. There was differential expression of NOS I protein, which was low in the GD 50 fetus and increased rapidly during fetal development to attain adult level by GD 62. These data suggest that the guinea pig is a reliable animal model in which to investigate the roles of NO in normal hippocampal development and in mediating neuronal injury in this brain region.

Nitrate and nitrite anion concentration in the intact cerebral cortex of preterm and nearterm fetal sheep: indirect index of in vivo nitric oxide formation.

Pregnant sheep with a microdialysis probe implanted in the fetal cerebral cortex were used to determine if nitrate and nitrite anions (nitrate/nitrite) could be quantitated in the microdialysate as an indirect index of in vivo nitric oxide formation. Pregnant ewes (term, about 147 days) were surgically instrumented at gestational day (GD) 90 (n = 3; preterm) and GD 121 (n = 3; nearterm). Three days later, following an overnight probe equilibration period, five dialysate samples were collected continuously on ice at 1-h intervals (infusion rate of 1 (microl/min). The nitrate/nitrite concentration was determined by reducing a 10-microl aliquot of each dialysate fraction with hot acidic vanadium followed by chemiluminescence quantitation of the nitric oxide product. The lower limit of quantitative sensitivity of the method is 25 picomoles. Nitrate/nitrite concentration was 16.6+/-7.3 microM for the preterm fetus and 19.7+/-1.9 microM for the nearterm fetus. The data demonstrate that nitrate/nitrite, as an index of in vivo nitric oxide formation, can be quantitated in microdialysate samples collected from the intact fetal sheep cerebral cortex.

Ethanol neurobehavioral teratogenesis and the role of the hippocampal glutamate-N-methyl-D-aspartate receptor-nitric oxide synthase system.

The purpose of this review is to evaluate a proposed mechanism for ethanol neurobehavioral teratogenesis in the hippocampus, involving suppression of the glutamate-N-methyl-D-aspartate (NMDA) receptor-nitric oxide synthase (NOS) system. It is postulated that suppression of this signal transduction system in the fetus by chronic maternal consumption of ethanol plays a key role in hippocampal dysmorphology and dysfunction in postnatal life. This mechanism is evaluated critically based on the current literature and our research findings. In view of the apparent time course for loss of CA1 pyramidal cells in the hippocampus produced by chronic prenatal ethanol exposure that manifests in early postnatal life, it is proposed that therapeutic intervention, which targets the glutamate-NMDA receptor-NOS system, may prevent or lessen the magnitude of postnatal hippocampal dysfunction.

Effect of chronic prenatal ethanol exposure on nitric oxide synthase I and III proteins in the hippocampus of the near-term fetal guinea pig.

Chronic prenatal ethanol exposure suppresses nitric oxide synthase (NOS) enzymatic activity, in the hippocampus of the near-term fetal guinea pig at gestational day (GD) 62. The objective of this study was to determine if this decrease in NOS activity is the result of decreased NOS I and NOS III protein expression. Pregnant guinea pigs received oral administration of 4 g ethanol/kg maternal body weight/day (n = 8), isocaloric-sucrose/pair feeding (n = 8), or water (n = 8) from GD 2 to GD 61. The NOS I and NOS III protein expression and localization in the hippocampus were determined using Western blot analysis and immunohistochemistry, respectively. The chronic ethanol regimen produced fetal body, brain, and hippocampal growth restriction compared with the isocaloric-sucrose/pair fed and water groups but did not affect the expression or localization of NOS I and NOS III proteins in the hippocampus. The decrease in NOS enzymatic activity induced by chronic prenatal ethanol exposure may be the result of posttranslational modification of NOS I and/or NOS III protein in the hippocampus of the near-term fetal guinea pig.

Effect of chronic maternal ethanol administration on nitric oxide synthase activity in the hippocampus of the mature fetal guinea pig.

Nitric oxide is a novel messenger that is involved in neuronal cell-cell communication and seems to play a neurotrophic role in normal brain development. Chronic prenatal ethanol exposure can produce central nervous system (CNS) teratogenesis, in which one of the target sites is the hippocampus. The main objective of our study was to test the following hypothesis: chronic maternal administration of an ethanol dosage regimen that produces CNS teratogenesis decreases nitric oxide synthase (NOS) activity in the fetal hippocampus. The ontogeny of NOS activity in the hippocampus of the developing guinea pig was further elucidated at two prenatal and two postnatal ages. The effects of chronic maternal oral administration of 4 g of ethanol/kg maternal body weight/day, isocaloric sucrose and pair feeding, or water [given as two equally divided doses 2 hr apart from gestational day (GD) 2 to GD 61] on body, brain, and hippocampal weights and hippocampal NOS activity were determined in the mature fetal guinea pig at GD 62 (term, about GD 68). NOS activity in the 25,000 x g supernatant fraction of hippocampal homogenate was measured using an optimized radiometric assay, based on the oxidation of L-[14C]arginine to L-[14C]citrulline. For the chronic ethanol regimen, the maternal blood ethanol concentration at 1 hr after the second divided dose on GD 57 was 157 +/- 45 mg/dl. Chronic maternal administration of ethanol decreased fetal body, brain, and hippocampal weights, compared with the isocaloric-sucrose/pair-fed and water treatment groups. The rate of L-[14C]citrulline formation and NOS activity in the fetal hippocampus were decreased in the ethanol treatment group, compared with the isocaloric-sucrose/ pair-fed and water treatment groups. There was no difference in the rate of L-[14C]citrulline formation, NOS activity, and fetal hippocampal and body weights between the isocaloric-sucrose/pair-fed and water treatment groups; however, fetal brain weight was decreased in the isocaloric-sucrose group, compared with the water group. Data of this study support the research hypothesis by demonstrating that chronic maternal administration of ethanol decreases fetal hippocampal NOS activity that is correlated with restricted growth of this brain region.

Cyclic AMP prevents an increase in GAP-43 but promotes neurite growth in cultured adult rat dorsal root ganglion neurons.

High expression of the growth-associated protein GAP-43 in neurons is correlated with developmental and regenerative axon growth. It has been postulated that during development and after injury, GAP-43 expression is elevated due to the unavailability of a target-derived repressive signal, but that GAP-43 expression then declines upon target contact. Here we examine the cyclic AMP second messenger signaling pathway to determine if it might mediate retrograde transmission of a signal which represses GAP-43 expression and inhibits growth. Cultures of adult rat dorsal root ganglia were chronically exposed to membrane-permeable analogs of cyclic AMP and activators of adenyl cyclase. These treatments caused GAP-43 protein levels to decrease in a dose-dependent manner, although neuronal survival was not affected. GAP-43 mRNA was also decreases by cyclic AMP. GAP-43 protein levels were not repressed by neurotrophins, cytokines, or other agents. Surprisingly, cyclic AMP caused an increase in the rate of neurite outgrowth, even though the neurons were partially depleted of GAP-43. Growth stimulation was quickly inducible and reversible, could occur in the presence of transcription inhibitors, and did not entail alterations in branching pattern. These findings suggest that axon growth involving high levels of GAP-43 is distinct from the growth stimulation which is rapidly induced by cyclic AMP.

Ethanol-induced changes in prostaglandin E concentration in the intact cerebral cortex of preterm and near-term fetal sheep.

Ethanol-induced changes in fetal prostaglandin E (PGE) concentration may play a role in the toxic effects of prenatal ethanol exposure. Using the novel technique of in utero microdialysis, the present study tested the hypothesis that acute ethanol exposure changes PGE concentration in the intact cerebral cortex of preterm (93 +/- 1 days of gestation) and near-term (124 +/- 1 days of gestation; term, approximately 147 days) fetal sheep. Fetal sheep were surgically instrumented with a microdialysis probe placed in the parasagittal parietal cortex. Three days later, the effects of maternal infusion of 1 g of ethanol/kg maternal body weight on preterm (n = 6) and near-term (n = 7) fetal cerebral cortical and plasma PGE concentrations were determined. In the preterm fetal cerebral cortex, PGE concentration was increased after ethanol infusion in all six animals studied. The median peak increase was 160% with a 95% confidence interval of 115 to 784%. There was considerable variation in the time of occurrence, magnitude, and duration of this increase. In the near-term fetal cerebral cortex, an increase in PGE concentration was observed after ethanol infusion in 5 of the 7 animals studied, whereas a decrease in PGE concentration was observed in the other two animals. Overall, ethanol did not increase significantly near-term fetal cerebral cortical PGE concentration. For both age groups, ethanol infusion had no effect on fetal plasma PGE concentration. These data indicate that ethanol can affect PGE production in the fetal cerebral cortex and that this effect seems to be gestational-age-dependent.