Antenatal maternal hypoxic stress: adaptations of the placental renin-angiotensin system in the mouse.

UNLABELLED: The stress of Antenatal Maternal Hypoxia (AMH) can lead to a number of physiological and pathological changes in both mother and fetus, changes which can be linked to alterations in placental morphology and gene regulation. Recently, in the Brown Norway rat "model" of placental insufficiency, we reported alterations in placental renin-angiotensin system (RAS) genes. Moreover, AMH can lead to reduced oxygen availability to the fetus, similar to a state of placental insufficiency. Thus, in pregnant mice dams we tested the hypothesis that antenatal maternal hypoxic stress leads to alterations in the placental RAS. These alterations may, in part, account for the phenotypic changes in both pregnant mice dams as well as fetus and adult offspring. METHODS: Pregnant FVB/NJ mice dams were either maintained as controls, or exposed to 10.5% O(2) for 48 h from 15.5 to 17.5 day post coitum. We then measured placental mRNA and protein expression of several RAS genes (n = 4 to 5; P < 0.05 was considered significant). RESULT: In murine placenta: (1) angiotensinogen (AGT) mRNA was undetectable; however, AGT protein was detectable and increased significantly with AMH. (2) In AMH, although renin mRNA was reduced protein expression increased, in association with decreased microRNA (miRNA) 199b, which can lead to increased renin translation. (3) Also in AMH placenta, angiotensin converting enzyme (ACE) -1 mRNA was unaltered; however, protein expression increased significantly, in association with decreased miRNA 27a, which can result in increased ACE-1 translation. (4) In AMH placenta, ACE-2 mRNA was reduced significantly, whereas protein expression was significantly greater, in association with reduced miRNA 429. (5) In AMH placenta, angiotensin II type (AT) -1a receptor mRNA expression was unaltered while AT-1b receptor mRNA was undetectable in both groups. Moreover, AT-1 receptor protein expression was unchanged in response to AMH. (6) AT-2 receptor mRNA and proteins were undetectable in both groups. CONCLUSION: The normal murine placenta possesses several components of RAS, and in response to AMH several of these elements undergo important changes. In addition, differential expression of RAS mRNA, miRNA and protein, indicate post-transcriptional regulatory mechanisms involved with hypoxic stress, and necessitate further investigation.

Placental gene expression in a rat 'model' of placental insufficiency.

OBJECTIVE: Placental insufficiency is a major factor associated with pregnancy complications such as miscarriages, intrauterine growth restriction and pre-eclampsia. Recent studies have identified the Brown Norway (BN) rat as a natural 'model' of placental insufficiency associated with decreased trophoblast remodeling of maternal uterine arteries. HYPOTHESIS: Genetic pathways involved in angiogenesis and immune cell regulation are dysregulated in the placenta of BN rats. METHODS: Global gene expression in placentas from BN rats were compared with that from Sprague-Dawley (SD) controls at 17.5 days of gestation using the Affimetrix Rat 1.0 microarray chip, and results confirmed with real-time PCR and immunoblotting. RESULTS: We found significant differences in 272 genes with 108 being up-regulated and 164 down-regulated in BN placentas compared to SD placentas. BN placentas overexpressed genes involved in the renin-angiotensin system (RAS) such as Ace, Ace2, Agtr1a, Nox4, and Ephx2, while key genes involved in angiogenesis, such as Mmp1, Mmp10, Fgfbp1, Esr1, Itga2, Rgs5, and Ccnb1 were down-regulated. We also observed increased expression of Timd2, Itm2a, Irak3, and Csf1r, and decreased expression of Slpi, Ncam1, and Igsf3 in BN placentas. In addition, we observed lower placental weights in BN males compared to BN females, together with increased expression of Cyp1a1 in BN males, as compared to BN females. CONCLUSIONS: The present study demonstrates differential expression of genes involved in blood pressure, angiogenesis and immune cell regulation in BN placenta, and suggests that the RAS may be involved in the pathogenesis of placental dysfunction observed in BN rats.

Placental gene expression responses to maternal protein restriction in the mouse.

OBJECTIVE: Maternal protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. We tested the hypothesis that, by the use of microarray technology, we could identify specific genes and cellular pathways in the developing placenta that are responsive to maternal protein deprivation, and propose a potential mechanism for observed gene expression changes. METHODS: We fed pregnant FVB/NJ mice from day post-coitum 10.5 (DPC10.5) to DPC17.5, an isocaloric diet containing 50% less protein than normal chow. We used the Affymetrix Mouse 430A_2.0 array to measure gene expression changes in the placenta. We functionally annotated the regulated genes, and examined over-represented functional categories and performed pathway analysis. For selected genes, we confirmed the microarray results by use of qPCR. RESULTS: We observed 244 probe sets, corresponding to 235 genes, regulated by protein restriction (p<0.001), with ninety-one genes being up-regulated, and 153 down-regulated. Up-regulated genes included those involved in the p53 pathway, apoptosis, negative regulators of cell growth, negative regulators of cell metabolism and genes related to epigenetic control. Down-regulated genes included those involved in nucleotide metabolism. CONCLUSIONS: Microarray analysis has allowed us to describe the genetic response to maternal protein deprivation in the mouse placenta. We observed that negative regulators of cell growth and metabolism in conjunction with genes involved in epigenesis were up-regulated, suggesting that protein deprivation may contribute to growth restriction and long-term epigenetic changes in stressed tissues and organs. The challenge will be to understand the cellular and molecular mechanisms of these gene expression responses.

Upregulation of eNOS in pregnant ovine uterine arteries by chronic hypoxia.

We tested the hypothesis that chronic high-altitude (3,820 m) hypoxia during pregnancy was associated with the upregulation of endothelial nitric oxide (NO) synthase (eNOS) protein and mRNA in ovine uterine artery endothelium and enhanced endothelium-dependent relaxation. In pregnant sheep, norepinephrine-induced dose-dependent contractions were increased by removal of the endothelium in both control and hypoxic uterine arteries. The increment was significantly higher in hypoxic tissues. The calcium ionophore A23187-induced relaxation of the uterine artery was significantly enhanced in hypoxic compared with control tissues. However, sodium nitroprusside- and 8-bromoguanosine 3',5'-cyclic monophosphate-induced relaxations were not changed. Accordingly, chronic hypoxia significantly increased basal and A23187-induced NO release. Chronic hypoxia increased eNOS protein and mRNA levels in the endothelium from uterine but not femoral or renal arteries. In nonpregnant animals, chronic hypoxia increased eNOS mRNA in uterine artery endothelium but had no effects on eNOS protein, NO release, or endothelium-dependent relaxation. Chronic hypoxia selectively augments pregnancy-associated upregulation of eNOS gene expression and endothelium-dependent relaxation of the uterine artery.

Cerebral artery K(ATP)- and K(Ca)-channel activity and contractility: changes with development.

The present study was designed to test the hypothesis that in cerebral arteries of the fetus, ATP-sensitive (K(ATP)) and Ca(2+)-activated K(+) channels (K(Ca)) play an important role in the regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) and that this differs significantly from that of the adult. In main branch middle cerebral arteries (MCA) from near-term fetal ( approximately 140 days) and nonpregnant adult sheep, simultaneously we measured norepinephrine (NE)-induced responses of vascular tension and [Ca(2+)](i) in the absence and presence of selective K(+)-channel openers/blockers. In fetal MCA, in a dose-dependent manner, both the K(ATP)-channel opener pinacidil and the K(Ca)-channel opener NS 1619 significantly inhibited NE-induced tension [negative logarithm of the half-maximal inhibitory concentration (pIC(50)) = 5.0 +/- 0.1 and 8.2 +/- 0.1, respectively], with a modest decrease of [Ca(2+)](i). In the adult MCA, in contrast, both pinacidil and NS 1619 produced a significant tension decrease (pIC(50) = 5.1 +/- 0.1 and 7.6 +/- 0.1, respectively) with no change in [Ca(2+)](i). In addition, the K(Ca)-channel blocker iberiotoxin (10(-7) to 10(-6) M) resulted in increased tension and [Ca(2+)](i) in both adult and fetal MCA, although the K(ATP)-channel blocker glibenclamide (10(-7) to 3 x 10(-5) M) failed to do so. Of interest, administration of 10(-7) M iberiotoxin totally eliminated vascular contraction and increase in [Ca(2+)](i) seen in response to 10(-5) M ryanodine. In precontracted fetal cerebral arteries, activation of the K(ATP) and K(Ca) channels significantly decreased both tension and [Ca(2+)](i), suggesting that both K(+) channels play an important role in regulating L-type channel Ca(2+) flux and therefore vascular tone in these vessels. In the adult, K(ATP) and the K(Ca) channels also appear to play an important role in this regard; however, in the adult vessel, activation of these channels with resultant vasorelaxation can occur with no significant change in [Ca(2+)](i). These channels show differing responses to inhibition, e.g., K(Ca)-channel inhibition, resulting in increased tension and [Ca(2+)](i), whereas K(ATP)-channel inhibition showed no such effect. In addition, the K(Ca) channel appears to be coupled to the sarcoplasmic reticulum ryanodine receptor. Thus differences in plasma membrane K(+)-channel activity may account, in part, for the differences in the regulation of contractility of fetal and adult cerebral arteries.

Dual role of PKC in modulating pharmacomechanical coupling in fetal and adult cerebral arteries.

This study tested the hypothesis that protein kinase C (PKC) has dual regulation on norepinephrine (NE)-mediated inositol 1,4, 5-trisphosphate [Ins (1,4,5)P(3)] pathway and vasoconstriction in cerebral arteries from near-term fetal ( approximately 140 gestational days) and adult sheep. Basal PKC activity values (%membrane bound) in fetal and adult cerebral arteries were 38 +/- 4% and 32 +/- 4%, respectively. In vessels of both age groups, the PKC isoforms alpha, beta(I), beta(II), and delta were relatively abundant. In contrast, compared with the adult, cerebral arteries of the fetus had low levels of PKC-epsilon. In response to 10(-4) M phorbol 12,13-dibutyrate (PDBu; PKC agonist), PKC activity in both fetal and adult cerebral arteries increased 40-50%. After NE stimulation, PKC activation with PDBu exerted negative feedback on Ins(1,4,5)P(3) and intracellular Ca(2+) concentration ([Ca(2+)](i)) in arteries of both age groups. In turn, PKC inhibition with staurosporine resulted in augmented NE-induced Ins(1,4,5)P(3) and [Ca(2+)](i) responses in adult, but not fetal, cerebral arteries. In adult tissues, PKC stimulation by PDBu increased vascular tone, but not [Ca(2+)](i). In contrast, in the fetal artery, PKC stimulation was associated with an increase in both tone and [Ca(2+)](i). In the presence of zero extracellular [Ca(2+)], these PDBu-induced responses were absent in the fetal vessel, whereas they remained unchanged in the adult. We conclude that, although basal PKC activity was similar in fetal and adult cerebral arteries, PKC's role in NE-mediated pharmacomechanical coupling differed significantly in the two age groups. In both fetal and adult cerebral arteries, PKC modulation of NE-induced signal transduction responses would appear to play a significant role in the regulation of vascular tone. The mechanisms differ in the two age groups, however, and this probably relates, in part, to the relative lack of PKC-epsilon in fetal vessels.

Cerebral artery sarcoplasmic reticulum Ca(2+) stores and contractility: changes with development.

To test the hypothesis that sarcoplasmic reticulum (SR) Ca(2+) stores play a key role in norepinephrine (NE)-induced contraction of fetal and adult cerebral arteries and that Ca(2+) stores change with development, we performed the following study. In main branch middle cerebral arteries (MCA) from near-term fetal ( approximately 140 days) and nonpregnant adult sheep, we measured NE-induced contraction and intracellular Ca(2+) concentration ([Ca(2+)](i)) in the absence and presence of different blockers. In adult MCA, after thapsigargin (10(-6) M), the NE-induced responses of tension and [Ca(2+)](i) were 37 +/- 5 and 47 +/- 7%, respectively, of control values (P < 0.01 for each). In the fetal artery, in contrast, this treatment resulted in no significant changes from control. When this was repeated in the absence of extracellular Ca(2+), adult MCA increases in tension and [Ca(2+)](i) were 32 +/- 5 and 13 +/- 3%, respectively, of control. Fetal cerebral arteries, however, showed essentially no response. Ryanodine (RYN, 3 x 10(-6) to 10(-5) M) resulted in increases in tension and [Ca(2+)](i) in both fetal and adult MCA similar to that seen with NE. For both adult and fetal MCA, the increased tension and [Ca(2+)](i) responses to RYN were essentially eliminated in the presence of zero extracellular Ca(2+). These findings provide evidence that in fetal MCA, in contrast to those in the adult, SR Ca(2+) stores are of less importance in NE-induced contraction, with such contraction being almost wholly dependent on Ca(2+) flux via plasma membrane L-type Ca(2+) channels. In addition, they suggest that in both adult and fetal MCA, the RYN receptor is coupled to the plasma membrane Ca(2+)-activated K(+) channel and/or L-type Ca(2+) channel.

Elizabeth M. Ramsey and the evolution of ideas of uteroplacental blood flow and placental gas exchange.

During the past century a number of investigators have contributed to an understanding of the regulation of uteroplacental blood flow and its influence on placental respiratory gas exchange and fetal oxygenation. Among these, Elizabeth M. Ramsey is noteworthy for her contributions to embryology and placental development. In addition, with several colleagues she performed pioneering cineangiographic studies on the patterns of blood flow in the primate placenta.

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.

Reproductive physician-scientists for the twenty-first century.

As we enter a new century, departments of obstetrics and gynecology in American medical schools face a number of challenges. A primary concern is the relative dearth of physician-scientists to explore basic mechanisms of cell function and relate these to the diseases of women. The question arises, what can we do to revitalize the spirit of investigation in many of our academic departments? This report reviews three programs designed to develop academic investigators: the Reproductive Scientist Development Program, the American Gynecological and Obstetrical Society/American Association of Obstetricians and Gynecologists Foundation Fellowship Program, and the Women's Reproductive Health Research Career Development Centers. On the basis of a decade of experience with the first two of these programs, the prospects for the third are promising. Clearly, the opportunities for obstetrician-gynecologist basic and clinical scientists are considerable. The question remains: Will leaders in the specialty work together to meet the challenge?

Role of Ca(2+) channels in NE-induced increase in [Ca(2+)](i) and tension in fetal and adult cerebral arteries.

In vascular smooth muscle, elevation of agonist-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) occurs via both Ca(2+) release from intracellular stores and Ca(2+) influx across the plasma membrane. In the cerebral vasculature of the fetus and adult the relative roles of these mechanisms have not been defined. To test the hypothesis that plasma membrane L-type and receptor-operated Ca(2+) channels play a key role in NE-induced vasoconstriction via alterations in plasma membrane Ca(2+) flux and that this may change with developmental age, we performed the following study. In main branch middle cerebral arteries (MCA) from near-term fetal ( approximately 140 days) and nonpregnant adult sheep, we quantified NE-induced responses of vascular tension and [Ca(2+)](i) (by use of fura 2) under standard conditions in response to several Ca(2+) channel blockers and in response to zero extracellular Ca(2+). In fetal and adult MCA, maximal NE-induced tensions (g) were 0.91 +/- 0.12 (n = 10) and 1.61 +/- 0.13 (n = 12), respectively. The pD(2) values for NE-induced tension were both 6.0 +/- 0.1, whereas the fetal and adult maximum responses (%K(max)) were 107 +/- 16 and 119 +/- 7, respectively. The fetal and adult pD(2) values for NE-induced increase of [Ca(2+)](i) were 6.2 +/- 0.1 and 6.4 +/- 0.1, respectively, whereas maximum [Ca(2+)](i) responses were 81 +/- 9 and 103 +/- 15% of K(max), respectively. After 10(-5) M NE-induced contraction, nifedipine resulted in dose-dependent decrease in vessel tone and [Ca(2+)](i) with pIC(50) values for fetal and adult tensions of 7.3 +/- 0.1 and 6.6 +/- 0.1, respectively (P < 0.01; n = 4 each), whereas pIC(50) for [Ca(2+)](i) responses were 7.2 +/- 0.1 and 6.9 +/- 0.1, respectively. The pIC(50) values for tension for diltiazem and verapamil were somewhat lower but showed a similar relationship. The receptor-operated Ca(2+) channel blocker 2-nitro-4 carboxyphenyl-N,N-diphenyl carbamate showed little effect on NE-induced vessel contractility or [Ca(2+)](i). In the absence of extracellular Ca(2+) for 2 min, 10(-5) M NE resulted in markedly attenuated responses of adult MCA tension and [Ca(2+)](i) to 39 +/- 7 and 73 +/- 8% of control values (n = 4). For fetal MCA, exposure to extracellular Ca(2+) concentration resulted in essentially no contractile or [Ca(2+)](i) response (n = 4). Similar blunting of NE-induced tension and [Ca(2+)](i) was seen in response to 10(-3) M lanthanum ion. These findings provide evidence to suggest that especially in fetal, but also in adult, ovine MCA, Ca(2+) flux via L-type calcium channels plays a key role in NE-induced contraction. In contrast, Ca(2+) flux via receptor-operated Ca(2+) channels is of less importance. This developmental difference in the role of cerebrovascular plasma membrane Ca(2+) channels may be an important association with increased Ca(2+) sensitivity of the fetal vessels.

Effect of chronic hypoxia on alpha-1 adrenoceptor-mediated inositol 1,4,5-trisphosphate signaling in ovine uterine artery.

The present study examined the effect of chronic hypoxia on coupling efficiency of alpha-1 adrenoceptors to inositol 1,4,5-trisphosphate (InsP3) signaling in ovine uterine artery. Chronic hypoxia did not change the time course of InsP3 formation, but significantly decreased the potency (pD2: 6.17 +/- 0.09 --> 5.26 +/- 0.12) and the maximal response (220.7 +/- 21.7 --> 147.7 +/- 15.3 pmol/mg protein) of norepinephrine-induced InsP3 synthesis. The coupling efficiency of alpha-1 adrenoceptors to InsP3 synthesis (picomoles InsP3 per femtomoles receptor) was decreased 45% by chronic hypoxia. In addition, simultaneous measurement of norepinephrine-induced contractions and InsP3 synthesis indicated that for a given amount of InsP3 generated, the contractile force of the uterine artery was significantly less in chronically hypoxic than in control tissues (0. 27 +/- 0.01 versus 0.35 +/- 0.02 g tension/pmol InsP3). InsP3 receptors were characterized using radioligand binding techniques. Although the density of InsP3 receptors was not changed by chronic hypoxia (Bmax: 325 +/- 35 --> 378 +/- 18 fmol/mg protein), the dissociation constant (Kd) of InsP3 to its receptors was significantly increased (Kd: 5.20 +/- 0.40 --> 7.81 +/- 0.34 nM). Analysis of InsP3 receptor occupancy-tension development relationship indicated no difference in intrinsic ability of the InsP3-receptor complex in eliciting contractions between the control and hypoxic tissues. Our results suggest that chronic hypoxia attenuates coupling efficiency of alpha-1 adrenoceptors to InsP3 synthesis in the uterine artery. In addition, the tissue contractile sensitivity to InsP3 is reduced, which is mediated predominantly by a decrease in InsP3 binding affinity to InsP3 receptors.

Training of academic staff in obstetrics and gynaecology in the USA.

As we enter a new century and millennium, departments of obstetrics and gynaecology in American medical schools face a number of challenges. Of primary concern among these is the relative dearth of physician-scientists to explore and connect the basic mechanisms and clinical aspects of the diseases of women. This report reviews some aspects of three programmes designed to educate such investigators: The Reproductive Scientist Development Program, The American Gynecological and Obstetrical Society--American Association of Obstetricians and Gynecologists Foundation Fellowship Program and the Women's Reproductive Health Research Career Development Centers. Based on a decade of experience with the first two of these programmes, the prospects for the relatively new third programme are promising. Nonetheless, the need for and opportunities for obstetrician-gynaecologist reproductive scientists far exceed the supply.

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.

High altitude, hypoxic-induced modulation of noradrenergic-mediated responses in fetal and adult cerebral arteries.

In response to high altitude long-term hypoxemia, the cerebral arteries of adult and fetal sheep show decreased contractile responses to norepinephrine and other agonists. In this review, we examine some of the presynaptic and postsynaptic contractile mechanisms that might account for these changes. In addition, we examine cerebral vessel relaxation and the role of pregnancy in altering these responses. In general, high altitude hypoxia is associated with augmented or "upregulation" of presynaptic functions. In contrast, postsynaptic functions tend to be significantly depressed or "downregulated." The results emphasize the role of high altitude, long-term hypoxemia in modulating adrenergic-mediated signal transduction in the cerebral vasculature. They specifically highlight the profound differences in acclimatization responses between common carotid and intracranial arteries, as well as the significant differences between responses in the fetus and adult.

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.

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.