Carbon monoxide: effects on oxygenation of the fetus in utero.

The partial pressure of oxygen in fetal blood decreases in proportion to the carboxyhemoglobin concentrations in fetal and maternal blood. Because fetal oxygen tensions normally equal 20 to 30 percent of the values for adults, this reduction can result in severe hypoxia of vital tissues. Decreases in oxygen tension may be a factor in the lower birth weights of infants born to women who smoke or are exposed to severe air pollution.

Respiratory function of the placenta as determined with carbon monoxide in sheep and dogs.

A technique is described for studying the respiratory function of the placenta using carbon monoxide, a gas whose exchange across the placenta between the maternal and fetal circulations is limited by diffusion rather than blood flow. During the steady state before the introduction of CO, the normal concentration of carboxyhemoglobin in the ewe, [COHb](M), is approximately 0.90%, and that in the fetus is 2.9%, the ratio [COHb](F)/[COHb](M) being 3.2. In dogs the corresponding values are 1.9%, 4.8%, and 2.4%. After the introduction of CO into the mother animal, CO diffused across the placenta slowly with an equilibration half-time of approximately 2 hours. The average carbon monoxide diffusing capacity (D(Pco)) of the placenta during maternal to fetal exchange was 0.54 ml per (minute x mm Hg x kg fetal weight) (SD +/- 0.13) in sheep and 0.57 ml per (minute x mm Hg x kg) (SD +/- 0.18) in dogs. The fetal to maternal placental diffusing capacity in two sheep was 0.54 ml per (minute x mm Hg x kg). Calculations considering the relative rates of reaction of O(2) and CO with red cell hemoglobin and the relative rates of diffusion of the two gases suggest that the true D(Po2) should be about 1.2 to 2 times greater than the D(Pco) or 0.65 to 1.1 per (minute x mm Hg x kg). This is about 5 times greater than the reported value of D(Po2) calculated from measurements of P(O2) in the mixed uterine and umbilical venous blood. With a diffusing capacity of this magnitude the maternal and fetal placental end capillary P(O2) would approach equilibrium, becoming too small to measure, and the calculation of D(Po2) would be unreliable. We suggest that the apparent end capillary P(o2) gradients of 15 to 20 mm Hg, obtained from sampling uterine and umbilical venous blood, result from a combination of uneven distribution of maternal and fetal placental blood flow and from placental oxygen consumption.

Uneven distribution of maternal and fetal placental blood flow, as demonstrated using macroaggregates, and its response to hypoxia.

A technique is described for studying the distribution of blood flow to the maternal and fetal placental vessels in sheep and dogs with radioactive labeled macroaggregates of albumin. When the maternal animal breathed room air the distribution of maternal placental blood flow was uneven among the cotyledons as well as within a given cotyledon. Fetal blood flow was also distributed nonuniformly among and within the cotyledons. The relation of maternal to fetal placental blood flow was also markedly uneven (coefficient of correlation, tau = 0.066). After the animal was made hypoxic by breathing 10-12% O(2) the distribution of maternal, fetal, and maternal/fetal placental flows became more uniform. The coefficient of correlation of maternal to fetal flow was high (tau = 0.53, P < 0.01). While the maternal animal breathed room air, after ligation of a major branch of the umbilical artery the distribution of maternal, fetal, and maternal/fetal flows in the remaining two-thirds to three-fourths of the placenta became more uniform. The correlation coefficient for maternal to fetal flow was high (tau = 0.35, P < 0.01).It appears that under normal circumstances with uneven distribution of blood flows there is a considerable portion of the placenta that does not receive blood flow in optimum quantities to promote efficient O(2) exchange. Failure to consider the influence of nonuniform maternal flow/fetal flow will result in overestimation of mean maternal-fetal oxygen tension gradients, and thus underestimation of the placental diffusing capacity for oxygen. In response to maternal hypoxia or compromise of the fetal placental circulation the distribution of maternal, fetal, and maternal/fetal flows becomes more uniform, thereby increasing the efficiency of placental O(2) exchange.

Amino acid transport in placental slices. Mechanism of increased accumulation by prolonged incubation.

The accumulation of alpha-aminoisobutyric acid by placental slices is increased dramatically upon prior incubation of the slices in amino acid-free, buffered saline. This increase is inhibited by inhibitors of protein synthesis and is accompanied by an increased V for the transport process. While alternative explanations are discussed, these data suggest that the incubation effect may be mediated through an increase in the number of available transport sites which are synthesized during the incubation period. Incubation with an amino acid mixture diminishes the increase as well as general protein synthesis, suggesting that a reduced availability of amino acids may initiate compensatory changes in the synthesis of cellular transport proteins.

Regulation of basal adrenocorticotropin and cortisol secretion by arginine vasopressin in the fetal sheep during late gestation.

This study tested the hypothesis that arginine vasopressin (AVP) is involved in the regulation of basal ACTH secretion in the ovine fetus near term. In five fetuses challenged with AVP (1 microgram/ml, iv bolus) plasma ACTH concentrations increased to an 8-fold peak within 10 min of the preceding baseline (55 +/- 6 to 403 +/- 241 pg/ml). Cortisol in fetal circulation subsequently increased 2-fold (11 +/- 1 to 28 +/- 5 ng/ml) within 15 min of the AVP injection. The AVP-induced rise in plasma ACTH and cortisol concentrations was blocked when the fetus was pretreated with the AVP V1 receptor antagonist d(Ch2)5Tyr(Me)AVP. In a total of seven studies, antagonist (10 micrograms/kg estimated BW, iv bolus) was administered to three fetuses, aged 137-147 days gestation, followed 40 min later by the exogenous AVP challenge, as described above. After AVP antagonist treatment, basal ACTH and cortisol concentrations were not significantly different from the preinjection baseline levels (P greater than 0.05, by analysis of variance). Moreover, plasma ACTH and cortisol remained unchanged after the AVP challenge. To further define the role of endogenous AVP in basal ACTH and cortisol secretion, the AVP antagonist was administered (five studies in two fetuses) at 30-min intervals for a total of three injections per fetus. This extended AVP antagonist regimen also failed to alter fetal circulating concentrations of ACTH or cortisol (P greater than 0.05). Cortisol in the maternal circulation was not affected by any of the fetal AVP or AVP antagonist treatments. Lambs were born at 146 +/- 2 days gestation (n = 5), within the range for the normal duration of pregnancy. These data do not support the hypotheses that AVP is involved in the regulation of basal ACTH secretion in the fetal sheep during the 10 days preceding parturition. Rather, the ability of AVP antagonist to block the AVP-induced rise in plasma ACTH and cortisol in the fetus suggests that basal and stimulated ACTH secretion are under separate regulatory mechanisms.

Fibroblast growth factor enhances human chorionic gonadotropin synthesis independent of mitogenic stimulation in Jar choriocarcinoma cells.

The Jar choriocarcinoma cell line was used as an in vitro placental cell model to determine the effects of polypeptide growth factors on hCG beta secretion. Epidermal and fibroblast growth factor (FGF) treatment of serum-free cultures stimulated hCG beta secretion 2.5- and 4.0-fold over basal serum-free control levels within a 15-h incubation period. Insulin-like growth factor I, nerve growth factor, and transforming growth factor-beta had no significant effect on hCG beta secretion. FGF at concentrations as low as 0.125 ng/ml significantly elevated medium hCG beta levels without increasing cell number or total cellular protein. FGF stimulation of secretion was not detectable until 2 h of treatment. Intracellular hCG beta remained constant (23%) relative to total hCG beta (cell plus medium) as total hCG beta increased 3-fold, suggesting that FGF stimulated de novo hCG beta synthesis. Insulin significantly augmented the FGF-induced hCG beta stimulation without stimulating hCG beta production itself.

Fibroblast growth factor enhances the transcription and stability of human chorionic gonadotropin beta-subunit messenger ribonucleic acid in Jar choriocarcinoma cells.

In previous studies, we found that basic fibroblast growth factor (bFGF) significantly stimulated the secretion of hCG beta in the Jar choriocarcinoma cell line. In the present study, the effect of bFGF on the steady state hCG beta mRNA level in this cell line was determined. Application of Northern analyses with total RNA isolated from bFGF-stimulated Jar cells revealed that, in a time-dependent manner, the steady state hCG beta mRNA level increased progressively, reaching 4-fold of the control value within 4 h after exposure to bFGF. The observed accumulation was due in part to increased transcription (2.4-fold relative to that in control cultures), as determined by nuclear transcription studies. In addition, bFGF increased the stability of the hCG beta message; the message half-life was increased from approximately 3 h (in control cultures) to greater than 6 h (in bFGF-treated cultures). These data demonstrate that bFGF stimulates hCG beta mRNA accumulation in a complex manner regulated through both transcriptional and posttranscriptional mechanisms.

Dissociation of pulsatile cortisol and adrenocorticotropin secretion in fetal sheep during late gestation.

In the fetal sheep, plasma cortisol concentrations gradually increase in the last weeks of gestation and abruptly rise during the final 48-72 h preceding birth. To determine if these changes in mean circulating cortisol concentrations result from increased pulsatile secretion and are driven by changes in ACTH pulses, blood samples from five chronically catheterized fetuses were collected every 5 min for 2 h at 133 days gestation and every 4 days thereafter until delivery at 146 +/- 2 days. Volume was replaced after each blood sample and erythrocytes were returned every 20 min. Plasma cortisol and ACTH secretion were pulsatile in fetuses at all ages. Cortisol pulse frequency increased significantly with gestation from a mean of 2.2 pulses/2 h at 133 days to 4.8 pulses/2 h at 146 days. The interpulse interval (mean +/- SE) decreased between 133 and 146 days from 54 +/- 11 min to 23 +/- 3 min, respectively. Cortisol pulse amplitude increased significantly from 10 +/- 2 ng/ml at 133 days to 44 +/- 13 ng/ml at 146 days. In contrast to cortisol, ACTH pulse frequency (3 +/- 0.6 pulses/2 h) and amplitude (21 +/- 3 pg/ml) were similar at 133 days and 146 days. The coincidence of cortisol and ACTH pulses did not change between 133 and 146 days. Furthermore, the number of coincident pulses failed to exceed random associations (hypergeometric probability analysis) and could have occurred by chance alone (P values ranged from 0.11-0.63). A point by point comparison of cortisol and ACTH concentrations in fetal circulation indicate that only 36% of the variance in cortisol concentrations could be explained by variance in ACTH (cross-correlation analysis). These data suggest that fetal cortisol and ACTH secretion are pulsatile and that, as gestation advances, increases in constitutive cortisol pulse amplitude and frequency may not be predominantly driven by pulsatile changes in ACTH in the ovine fetal circulation near term.

Effect of chronic hypoxia on adrenoceptor responses of ovine foetal umbilical vessels.

1. The effects of chronic hypoxia on alpha1-adrenoceptor-mediated contractions were investigated in foetal umbilical vessels obtained from near-term (approximately 140 day gestation) pregnant sheep maintained near sea level ( 300 m) and at high altitude (3820 m) from 30 day gestation. 2. Chronic hypoxia significantly decreased contractile sensitivity of the umbilical vein to noradrenaline (pD2: 6.22+/-0.19 vs 5.67+/-0.09) and reduced the maximum response by 43%. Noradrenaline-induced contraction of the umbilical artery was abolished. In contrast, contractions to KCI were not affected by chronic hypoxia. 3. In umbilical vein, the apparent dissociation constant (KA) of noradrenaline to alpha1-adrenoceptors was increased from 0.54+/-0.06 microM in control animals to 1.35+/-0.14 microM in chronically hypoxic animals. In accordance, radioligand binding of agonist showed high and low affinity binding sites for noradrenaline in both normoxic and chronically hypoxic tissues. Addition of GTPgammaS (100 microM) abolished apparent high affinity binding sites. Whereas proportional binding sites were not changed by chronic hypoxia, the apparent high affinity of noradrenaline was significantly decreased (pKi: 7.80+/-0.17 vs 7.20+/-0.16). 4. Chronic hypoxia significantly decreased alpha1-adrenoceptor density (fmol mg protein(-1)) in umbilical vein (24.6+/-3.2 vs 12.3+/-3.1) and the artery (7.1+/-0.4 vs 3.1+/-0.9) with no change in [3H]-prazosin binding affinity. There was a linear correlation of the maximum contractions to noradrenaline and alpha1-adrenoceptor density. 5. We conclude that chronically hypoxic-induced depression in contractions of ovine foetal umbilical vessels to noradrenaline is mediated predominantly by decreases in alpha1-adrenoceptor density and the agonist binding affinity.

Ovine placentome morphology: effect of high altitude, long-term hypoxia.

The effect of high altitude, long-term hypoxaemia on placentome morphology in the sheep was examined using singleton and twin pregnant ewes. Normoxic twins had lower fetal and placental weights (3.7+/-0.2 kg and 215+/-26 g, respectively) than normoxic singleton fetuses (4.3+/-0.2 kg and 336+/-17 g, respectively). Fetal and placental weights were similar in normoxic singleton and high altitude (3820 m) hypoxic singleton fetuses (4.3+/-0.2 and 4.4+/-0.4 kg, 336+/-17 and 342+/-62 g, respectively). The distribution of placentome types was classified into four major categories (A-D) and for normoxic singletons was as follows: A=76+/-4, B=22+/-3, C=1+/-2, and D=1+/-1. Normoxic twins tended to have more type B (type A=63+/-10, B=33+/-8, C=2+/-1, and D=2+/-1). High altitude hypoxic singletons had significantly fewer type A (33+/-4) and more type B (50+/-3), C (10+/-7), D (7+/-1) placentomes than normoxic singletons. In addition, in the sea-level control group, five animals were found to be spontaneously hypoxic with a placentome distribution similar to that of the high altitude hypoxic fetuses. In conclusion, both high altitude, long-term hypoxia and low altitude spontaneous hypoxia lead to a significant change in placentome distribution with less type A and increases in types B, C and D. Physiologically, the change in the several placentome types with high altitude hypoxia suggests an acclimatization response to optimize transplacental exchange efficiency.

Term ovine placental vasculature: comparison of sea level and high altitude conditions by corrosion cast and histomorphometry.

The placental vascular architecture differs significantly at high altitude from that at sea level in the human and guinea-pig. Four sheep between 137 and 140 days of gestation, kept near sea level throughout gestation, were used as a normoxic control group for comparison of the placental vasculature with 10 other ewes, kept at high altitude (3820 m above sea level; Barcroft Laboratory, White Mountain Research Station, CA, USA). Placentomes from both groups were prepared for histology and scanning electron microscopy of vascular corrosion casts. Singular perfusion of fetal placentae, as well as combined maternal/fetal injection was performed. The influence of long-term hypoxaemia was determined by qualitative and semi-quantitative evaluation of corrosion casts and histological sections. The fetal vessel casts show a distinct difference in the arrangement of vessels of all sizes in response to long-term hypoxaemia. In the control group, stem arteries and veins are straight and parallel. In contrast, this is much less evident in the hypoxaemic group because arterioles and venules branch off the stem vessels more frequently and in an irregular manner. This leads to a capillary bed that is much more dense due to increased branching and capillary coiling. These observations are confirmed by histomorphometry. In the fetal vessels of high altitude sheep placentomes, we observed a decreased number of vascular cross sections (21.6 +/- 4.7 SEM versus 27.7 +/- 4.0 SEM; P = 0.02). However, the average luminal size per cross section (77.9 +/- 10.5 microns2 SEM versus 59.4 +/- 7.4 microns2 SEM; P = 0.004) was increased at high altitude and the percentage of lumina of the total area (5.7 +/- 0.5 SEM versus 5.3 +/- 0.3 SEM; P = 0.09) indicated a trend towards an increase. In maternal vessels of high altitude placentomes, the number of vessel cross sections (6.5 +/- 0.7 SEM versus 6.0 +/- 0.5 SEM; P = 0.2) remained unchanged, whereas the average luminal size (1108 +/- 122 microns2 SEM versus 844 +/- 77 microns2 SEM; P < 0.001) and the percentage of lumina out of the total area (20.9 +/- 1.8 SEM versus 17.5 +/- 1.7 SEM; P < 0.001) were increased. The interhaemal distance appeared to be slightly but not significantly increased at high altitude. These findings indicate that at high altitude the sheep placenta develops an increased materno-fetal absorptive surface to help guarantee substance exchange.

Regional anatomy of the term human placenta.

We have studied regional anatomical variability in four term placentae, comparing both whole placental regions and intralobar zones and plates. In addition, we have emphasized the need for careful selection of the area to be sampled, rather than strictly randomized sampling of the whole placenta. A unique contribution is our quantitative data for a number of structures of the several intralobar zones and plates. The data confirm the hypothesis that the area best suited to physiological exchange is the central region parabasal plate. The relative homogeneity of the intralobar zones in this area make it a representative area for sampling for various placental studies.

Placental anatomy and diffusing capacity in guinea pigs following long-term maternal hypoxia.

To determine the relation of placental structure to placental diffusing capacity (DPCO), we exposed Hartley guinea pigs to 12 or 14 per cent O2 from day 15 of gestation to near term (64 days). At that time we measured DPCO and fetal body and placental weights. In addition, we used stereological techniques to measure placental parameters important to diffusing capacity. We also used a mathematical model with results from the stereological measurements to predict the diffusing capacity. In the first hypoxic group (E1), measured DPCO decreased 10.1 +/- 3.7 per cent, while that predicted was 2.4 per cent less than control. Total vascular volume decreased 6.6 +/- 3.6 per cent, while tissue volume and mean diffusion distance increased 10.2 +/- 5.6 per cent and 12.9 +/- 7.0 per cent, respectively. In the pair-fed animals, measured DPCO decreased 22.6 +/- 4.6 per cent, while that predicted was 20.0 per cent less than control. There were no significant stereological differences in this group. In the second (E2) hypoxic group, measured DPCO increased 27.2 +/- 7.4 per cent, while that predicted increased 38.2 per cent. For this same group, total vascular volume increased 11.7 +/- 3.0 per cent, and tissue volume and mean diffusion distance decreased 18.2 +/- 4.6 per cent and 17.8 +/- 3.8 per cent, respectively. These results demonstrate the dependence of placental diffusing capacity upon placental structure.

Physiologic assessment of fetal compromise: biomarkers of toxic exposure.

Understanding the physiologic and endocrinologic basis of fetal development is a major goal of perinatal biology. During the past decade a number of technological developments have allowed more precise evaluation of the fetus in utero and diagnosis of abnormalities. Despite these methodological achievements, however, there are no specific biological markers currently available to indicate that exposure to a given xenobiotic is associated with a cellular, subcellular, or pharmacodynamic event. This paper evaluates the following issues: What are some of the unique physiologic and endocrinologic features of the fetal milieu intérieur? What problems are peculiar to fetal assessment? Of what value are techniques such as ultrasonography, amniocentesis, chorionic villus sampling, fetoscopy, and fetal blood and tissue sampling for obtaining appropriate biomarkers? What are some examples of validated biomarkers and their applicability? What promising biomarkers are on the horizon? What are some of the promising techniques such as the evaluation of fetal body movements, breathing activity, electronic heart rate monitoring, and nuclear magnetic resonance? How may molecular probes be of value as biological markers of fetal compromise? What are some of the major research gaps and needs, and how should research priorities be set? Some of these topics are addressed. Moreover, the more general role(s) that various diagnostic methods and biological markers can have in an understanding of the regulation of fetal growth and differentiation and the role of xenobiotics in affecting the normal course of events are discussed.

Plasma testosterone surge and luteinizing hormone beta (LH-beta) following parturition: lack of association in the male rat.

Studies examining the role of luteinizing hormone (LH) in the initiation of the postnatal surge of testosterone in the male rat have produced ambiguous results. We examined the pattern of postnatal LH secretion in the newborn male rat, coincident with plasma testosterone levels, using a specific monoclonal antibody for LH-beta. In some males, we attempted to block LH secretion and the postnatal testosterone surge by injecting males with a gonadotropin-releasing hormone (GnRH) antagonist, an LH antibody or progesterone immediately after delivery by cesarean section on day 22. Following injection, animals were immediately sacrificed (time 0) or housed in a humidified incubator maintained at 30 degrees C until sacrifice at 60, 120, 240, 360 or 480 min after delivery. Plasma from individual animals was measured subsequently for LH-beta and testosterone by radioimmunoassay. Results revealed a postnatal surge of testosterone which peaked at 2 h after delivery in males from all treatment groups. This testosterone surge was not accompanied by a postnatal rise in plasma LH-beta in any group. Administration of the GnRH antagonist or the ethanol vehicle produced a transient drop of approximately 25% in LH-beta levels at 60 min but did not decrease the postnatal testosterone surge in the same animals. Additional studies in untreated males and females born by cesarean section or natural birth also failed to reveal a postnatal rise in plasma LH-beta during the first 3 h after birth. Plasma levels in both sexes were significantly lower in animals delivered by cesarean section compared to natural birth.(ABSTRACT TRUNCATED AT 250 WORDS)

Free radical-induced elevation of ornithine decarboxylase activity in developing rat brain slices.

OBJECTIVE: In developing brain, we have previously shown both in vivo [L.D. Longo, S. Packianathan, J.A. McQueary, R.B. Stagg, C.V. Byus and C.D. Cain, Acute hypoxia increases ornithine decarboxylase activity and polyamine concentrations in fetal rat brain, Proc. Natl. Acad. Sci. USA, Vol. 90 (1993) 692-696] and in vitro [S. Packianathan, C.D. Cain, B.H. Liwnicz and L.D. Longo, Ornithine decarboxylase activity in vitro in response to acute hypoxia: a novel use of newborn rat brain slices, Brain Res., Vol. 688 (1995) 61-71] that acute hypoxia is associated with a significant increase in ornithine decarboxylase (ODC) activity and polyamine concentrations. We tested the hypothesis that oxygen free radicals induce an increase in ODC activity similar to that of hypoxia and that both this and the hypoxia-induced response are inhibited by free radical scavengers. MATERIALS AND METHODS: Slices of cerebrum, 300-500 microm thick, were made from P3 newborn Sprague-Dawley rat pups and equilibrated for 1 h in artificial cerebrospinal fluid continuously bubbled with 95% O2/5% CO2. Free radical-induced ODC activity response was measured beginning after a 1-h recovery period. Experiments were performed on slices treated with 5 X 10(-7) M xanthine (X) + 10 mU/ml xanthine oxidase (XO), with or without the free radical scavengers superoxide dismutase (SOD; 100 U/ml), catalase (CAT; 700 U/ml) or glutathione peroxidase (GPX; 3 U/ml). We also quantified slice malonaldehyde concentrations in response to hypoxia (21% O2/5% CO2/74% N2). RESULTS: Under control conditions, ODC activity was stable during the 2-h post-recovery period. In response to X/XO treatment, ODC activity increased 2.3-fold at 1.5 h post-recovery. In examining ODC activity as a function of xanthine dose, we noted that ODC activity increased in response to 2.5 X 10(-7) M xanthine; however, it decreased in response to 7.5 X 10(-7) M or higher concentrations. Free radical-induced ODC activity was significantly decreased by addition of the free radical scavengers, SOD, CAT or GPX. In addition, the hypoxic-induced increases in ODC activity and malonaldehyde concentration was also eliminated by the addition of SOD with CAT. CONCLUSIONS: (1) Oxygen free radicals, particularly hydroxyl radical (OH.), appear to trigger an induction of ODC activity in newborn rat cerebrum slices. (2) Oxygen free radicals also appear to mediate the hypoxic-induced increase in ODC activity. (3) Any consequent increase in polyamine synthesis may have profound effects on neurogenesis and neurodifferentiation in the developing brain.

In situ and immunocytochemical localization of E-FABP mRNA and protein during neuronal migration and differentiation in the rat brain.

The present study compares the temporal-spatial expression and tissue localization of the rat epidermal type fatty acid binding protein (E-FABP) (DA11/C-FABP/S-FABP/LEBP/KLBP) in the developing rat central nervous system (CNS). In situ hybridization (ISH) and immunocytochemistry (ICC) studies demonstrate that mRNA E-FABP and protein are expressed at high levels during neurogenesis, neuronal migration, and terminal differentiation. Migrating pyramidal cells in the cerebral cortex, Purkinje cells and deep nuclear neurons in the cerebellum, and neurons in the olfactory bulb and retina exhibited a strong E-FABP-like immunoreactivity (E-FABP-LI) throughout the entire process of differentiation and migration. The levels of E-FABP mRNA and protein were dramatically higher in prenatal and early postnatal neurons, as compared to adult neurons. The E-FABP antibody immunoreacted with growing neurites, and nuclear and cytoplasmic regions of neurons. The intracellular multiregional pattern of localization of E-FABP and its differential temporal expression during development, are consistent with its proposed role in transporting long chain free fatty acids and/or other hydrophobic ligands during neuronal differentiation and axon growth.

Ornithine decarboxylase activity in vitro in response to acute hypoxia: a novel use of newborn rat brain slices.

In fetal as well as newborn rats, acute hypoxic exposure results in significantly elevated brain ornithine decarboxylase (ODC) activity, polyamine concentrations, and ODC mRNA. The interpretations of these in vivo hypoxic-induced changes, however, are complicated by maternal confounding effects. To test the hypothesis that acute hypoxia will also increase ODC activity in vitro, we developed a brain slice preparation which eliminates such maternal effects. Sections of whole cerebrum, approximately 300-500 microns thick, were made from 3- to 4-day old Sprague-Dawley rat pups. The slices were equilibrated for 1 h in artificial cerebrospinal fluid (ACSF) continuously bubbled with 95% O2/5% CO2, prior to induction of hypoxia. We induced hypoxia by changing the oxygen concentration to 40%, 30%, 21%, 15%, 10%, or 0% O2, all with 5% CO2 and balance N2. In the normoxic control brain slices, low but stable basal ODC activity persisted for up to 5 h post-sacrifice. Slices in ACSF treated with bovine serum albumin (BSA), or both BSA and fetal bovine serum (FBS), however, showed stable ODC activity values 2- to 3-fold higher than slices in ACSF alone, for up to 5 h. In response to acute hypoxia (i.e., 15, 21, and 30% O2), ODC activity was elevated 1.5- to 2-fold above control values between 1 and 2 h after initiation of hypoxia. Qualitative light and electron microscopic examination of the neonatal brain slices following 2 h hypoxic exposure suggested that the great majority of cells did not show severe hypoxic damage or necrosis. It was concluded that: (1) in neonatal rat brain slices in vitro, stable ODC activity values approximating the whole brain ODC activity seen at sacrifice, can be maintained for several hours; (2) the in vivo hypoxic-induced increase in ODC activity can be approximated in vitro; (3) the neonatal rat brain slice preparation may be an alternative to other methods for studying hypoxic-induced ODC enzyme kinetics, or other brain enzymes, without maternal confounding effects; and (4) ODC activity may be an indicator of active metabolism within the newborn brain slice both in normoxia and hypoxia.

Noradrenaline-mediated contractions of ovine uterine artery: role of inositol 1,4,5-trisphosphate.

To elucidate the role of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) as a second messenger through which noradrenaline regulates contractions of the uterine artery, we present here studies designed to characterize simultaneously the noradrenaline-mediated contractions and Ins(1,4,5)P3 formation in isolated uterine arteries from near-term pregnant sheep. Noradrenaline stimulated a rapid increase of Ins(1,4,5)P3 formation with the peak at 30 second. Simultaneous measurement of noradrenaline-induced contractile responses and Ins(1,4,5)P3 formation revealed a significant linear correlation between these two events. In accordance with the contractile results, the noradrenaline-mediated inositol phosphate accumulation was blocked by prazosin (0.1 microM), but not by yohimbine (0.1 microM). Pre-treatment of tissues with pertussis toxin (200 ng/ml, 3 h) failed to block noradrenaline-induced inositol phosphate accumulation. We conclude that, in the uterine artery of late pregnancy, the alpha 1-adrenoceptor-elicited contraction, at least the initial phasic component, is predominantly mediated by the formation of Ins(1,4,5)P3, leading to release of Ca2+ from intracellular stores.