Regulation of the glucocorticoid receptor in fetal rat lung during development

The effect of the synthetic glucocorticoid betamethasone on the regulation of the glucocorticoid receptor mRNA and on receptor protein was studied in fetal rat lung during development. Using a glucocorticoid receptor cRNA probe, glucocorticoid receptor mRNA was examined by Northern blot hybridization and by solution hybridization. A monoclonal antibody against the glucocorticoid receptor was used to study regulation of the receptor protein by the Western immunoblotting technique. In fetal rat lungs, of 16-21 days of gestation, as well as in adult lungs, betamethasone treatment resulted in a significant decrease of glucocorticoid receptor mRNA to 50-65% of the control level. In contrast, betamethasone treatment did not down-regulate the receptor protein in rat lungs of 16-19 days of gestation, whereas a decrease of glucocorticoid receptor protein to 40-60% of control was seen in lungs of 21 days of gestation, in postnatal and adult lung. These results provide data for a change in regulation in vivo of the glucocorticoid receptor by its homologous ligand in fetal rat lung during development.     

Phenytoin induced oxidative stress in pre- and postnatal rat development - effect of vitamin E on selective biochemical variables

A pre- and postnatal study was carried out to investigate the effect of high dose (500 mg/kg) of the natural antioxidant vitamin E (VIT E) on biochemical variables in the model of chronic intrauterine hypoxia. Chronic hypoxia was induced by administration of the anticonvulsant phenytoin (PHT) during pregnancy. Rats were orally treated with PHT (150 mg/kg) from day 7 to 18 of gestation and VIT E prior to PHT orally on the same days. The activity of the lysosomal enzyme N-acetyl-ss-D-glucosaminidase (NAGA) and the level of glutathione (GSH) were used as markers of tissue damage. In the prenatal study PHT-induced embryofoetal toxicity was associated with an increase in NAGA activity and decrease of GSH level in maternal serum and heart and with an increase in NAGA activity in the placenta. Administration of VIT E did not inhibit the above given changes. PHT increased the activity of NAGA and decreased the level of GSH in foetal organs (liver, lungs, brain). VIT E did not reverse these changes. In the postnatal study, we did not find any significant differences in NAGA activity in the organs of 1-day-old pups. An increase of liver GSH level was found in PHT and VIT E+PHT groups of pups and in the group VIT E+PHT in the lungs. In conclusion, supplementation with a high-dose of VIT E failed to protect maternal, foetal and new-born rat organs from PHT induced changes of selective biochemical variables.     

SNAT4 isoform of system A amino acid transporter is expressed in human placenta

The system A amino acid transporter is encoded by three members of the Slc38 gene family, giving rise to three subtypes: Na+-coupled neutral amino acid transporter (SNAT)1, SNAT2, and SNAT4. SNAT2 is expressed ubiquitously in mammalian tissues; SNAT1 is predominantly expressed in heart, brain, and placenta; and SNAT4 is reported to be expressed solely by the liver. In the placenta, system A has an essential role in the supply of neutral amino acids needed for fetal growth. In the present study, we examined expression and localization of SNAT1, SNAT2, and SNAT4 in human placenta during gestation. Real-time quantitative PCR was used to examine steady-state levels of system A subtype mRNA in early (6-10 wk) and late (10-13 wk) first-trimester and full-term (38-40 wk) placentas. We detected mRNA for all three isoforms from early gestation onward. There were no differences in SNAT1 and SNAT2 mRNA expression with gestation. However, SNAT4 mRNA expression was significantly higher early in the first trimester compared with the full-term placenta (P < 0.01). We next investigated SNAT4 protein expression in human placenta. In contrast to the observation for gene expression, Western blot analysis revealed that SNAT4 protein expression was significantly higher at term compared with the first trimester (P < 0.05). Immunohistochemistry and Western blot analysis showed that SNAT4 is localized to the microvillous and basal plasma membranes of the syncytiotrophoblast, suggesting a role for this isoform of system A in amino acid transport across the placenta. This study therefore provides the first evidence of SNAT4 mRNA and protein expression in the human placenta, both at the first trimester and at full term.     

Effects of prenatal hypoxia on expression of amyloid precursor protein and metallopeptidases in the rat brain

Summary In the present study we have investigated the effect of prenatal hypoxia on expression of amyloid precursor protein (APP) and some metallopeptidases, which regulate β-amyloid peptide (Aβ) levels (neprilysin (NEP) and endothelin-converting enzyme (ECE-1)) in the cortex of rats during different periods of postnatal development. We have found that the level of APP in the sensorimotor cortex (SMC) of rats, analysed by Western blotting, increases from days 1 to 5 of postnatal development and then steadily decreases with age, with the most dramatic decline in the period from day 180 to 600. In the cortex of rats subjected to prenatal hypoxia on day 13.5 of embryogenesis, the postnatal levels of APP were higher than in the control. Secretion of the soluble form of APP (sAPP) by α-secretase was found to be the most active on day 30 of postnatal development and there was a significant decrease in the production of sAPP after prenatal hypoxia. NEP was found to be expressed in the cortex of rats only at the early stages of postnatal development and it was barely detectable in adult rats. The decline of NEP levels during ageing might contribute to accumulation of Aβ in later life in humans. Prenatal hypoxia resulted in a significant decrease of NEP expression on day 10, but its level was recovered when animals were preconditioned to mild hypoxia. A similar phenomenon was observed when the expression of ECE-1 was analysed. Overall, prenatal hypoxia leads to significant changes in the levels of APP and expression of metallopeptidases involved in amyloid metabolism during all postnatal life and preconditioning to hypoxia appeared to be neuroprotective.     

Autoradiographic study of the ontogeny of the imipramine receptors in the cerebral cortex of rats

Localization of 3H-imipramine binding sites in the cerebral cortex of rats was studied on the 19th day of prenatal development and on the 3d and 14th days of postnatal ontogenesis. Imipramine was injected into rats on days 17, 18 and 19 of gestation. The maximal imipramine receptor density was found in cortex layers IV-V. The therapeutic doses of imipramine provoked marked stimulation of the formation of imipramine receptors on the 3d day of postnatal development. However, on the 14th day the 3H-imipramine binding level was stabilized.     

Newborn Hypoxia/Anoxia Inhibits Cardiomyocyte Proliferation and Decreases Cardiomyocyte Endowment in the Developing Heart: Role of Endothelin-1

In the developing heart, cardiomyocytes undergo terminal differentiation during a critical window around birth. Hypoxia is a major stress to preterm infants, yet its effect on the development and maturation of the heart remains unknown. We tested the hypothesis in a rat model that newborn anoxia accelerates cardiomyocyte terminal differentiation and results in reduced cardiomyocyte endowment in the developing heart via an endothelin-1-dependent mechanism. Newborn rats were exposed to anoxia twice daily from postnatal day 1 to 3, and hearts were isolated and studied at postnatal day 4 (P4), 7 (P7), and 14 (P14). Anoxia significantly increased HIF-1α protein expression and pre-proET-1 mRNA abundance in P4 neonatal hearts. Cardiomyocyte proliferation was significantly decreased by anoxia in P4 and P7, resulting in a significant reduction of cardiomyocyte number per heart weight in the P14 neonates. Furthermore, the expression of cyclin D2 was significantly decreased due to anoxia, while p27 expression was increased. Anoxia has no significant effect on cardiomyocyte binucleation or myocyte size. Consistently, prenatal hypoxia significantly decreased cardiomyocyte proliferation but had no effect on binucleation in the fetal heart. Newborn administration of PD156707, an ET_A-receptor antagonist, significantly increased cardiomyocyte proliferation at P4 and cell size at P7, resulting in an increase in the heart to body weight ratio in P7 neonates. In addition, PD156707 abrogated the anoxia-mediated effects. The results suggest that hypoxia and anoxia via activation of endothelin-1 at the critical window of heart development inhibits cardiomyocyte proliferation and decreases myocyte endowment in the developing heart, which may negatively impact cardiac function later in life.     

Effects of dietary protein restriction on nephron number in the mouse

In rats, maternal protein restriction reduces nephron endowment and often leads to adult hypertension. Sex differences in these responses have been identified. The molecular and genetic bases of these phenomena can best be identified in a mouse model, but effects of maternal protein restriction on kidney development have not been examined in mice. Therefore, we determined how combined prenatal and postnatal protein restriction in mice affects organ weight, glomerular number and dimensions, and renal expression of angiotensin receptor mRNA, in both male and female offspring. C57/BL6/129sv mice received either a normal (20% wt/wt; NP) or low (9% wt/wt; LP) protein diet during gestation and postnatal life. Offspring were examined at postnatal day 30. Protein restriction retarded growth of the kidney, liver, spleen, heart, and brain. All organs except the brain weighed less in female than male offspring. Protein restriction increased normalized (to body weight) brain weight, with females having relatively heavier brains than males. The effects of protein restriction were not sex dependent, except that normalized liver weight was reduced in males but increased in females. Glomerular volume, but not number, was greater in female than in male mice. Maternal protein restriction reduced nephron endowment similarly in male and female mice. Renal expression of AT(1A) receptor mRNA was approximately sixfold greater in female than male NP mice, but similar in male LP and female LP mice. We conclude that maternal protein restriction reduces nephron endowment in mice. This effect provides a basis for future studies of developmental programming in the mouse.     

MicroRNA-29a inhibited epididymal epithelial cell proliferation by targeting nuclear autoantigenic sperm protein (NASP)

Cell proliferation often decreases gradually during postnatal development of some organs. However, the underlying molecular mechanisms remain unclear. Epididymis, playing important roles in sperm maturation, is a typical organ of this type, which displays a decreased proliferation during postnatal development and even ceased at the adult stage. Here, epididymis was employed as a model to explore the underlying mechanisms. We profiled the microRNA and mRNA expression of newborn (1 day) and adult (90 day) rat epididymis by microarray analysis, and found that the level of miR-29a was dramatically up-regulated during postnatal development of rat epididymis. Subsequent investigations demonstrated that overexpression of miR-29a inhibited the proliferation of epididymal epithelial cells in vitro. The nuclear autoantigenic sperm protein (NASP), a novel target of miR-29a, was significantly down-regulated during postnatal development of rat epididymis. Further analysis showed that silence of NASP mimicked the anti-proliferation effect of miR-29a, whereas overexpression of this protein attenuated the effect of miR-29a. As in rat epididymis, miR-29a was up-regulated and Nasp was down-regulated during postnatal development of mouse epididymis, heart, liver, and lung. Moreover, miR-29a can also inhibit the proliferation of cancer cells by targeting Nasp. Thus, an increase of miR-29a, and hence decrease of Nasp, may contribute to inhibit cell proliferation during postnatal organ development.     

Plasma corticosterone concentrations in the perinatal rat

1. Plasma corticosterone concentrations were determined in the foetal rat during the gestational period from day 18·5 to term and in postnatal rats over the first few hours after delivery. 2. The plasma corticosterone concentrations in foetal rats are as high as six times maternal values at day 19 of gestation and are approximately equal to maternal values from day 20 to term. 3. In postnatal rats the plasma corticosterone concentrations rise 3·5-fold on average within 5hr. of delivery. 4. The results are discussed in relation to the function of adrenal steroids in postnatal liver development.     

Fetal exposure to a maternal low protein diet impairs nephrogenesis and promotes hypertension in the rat

Epidemiological evidence suggests that hypertension and coronary heart disease are programmed by exposure to a poor diet during intrauterine life. It has been proposed that the prenatal environment may exert an adverse effect on the development of the kidney and hence later control of blood pressure. These assertions are supported by animal experiments. In the rat, fetal exposure to a maternal low protein diet is associated with disproportionate patterns of fetal growth and later elevation of blood pressure. Pregnant female rats were fed control (18% casein) or low protein diets throughout pregnancy, or during specific periods. Nephron number was determined at day 20 gestation, full term and 4 weeks of age. Exposure to low protein throughout gestation, or in mid-late gestation increased total nephron number at day 20. By term nephron number was reduced, relative to controls, in rats that were undernourished between days 8-14 or 15-22 gestation. At 4 weeks postnatally rats exposed to low protein throughout fetal life had a reduced (13%) nephron complement and blood pressures 13 mmHg above control animals. Lower renal size and elevated blood pressure persisted to 19 weeks of age, at which time glomerular filtration rate was normal. The data are consistent with the hypothesis that maternal undernutrition may programme the renal nephron number and hence impact upon adult blood pressure and the development of renal disease.     

Insulin receptors in developing rat liver: Acquisition of down regulation in the immediate postnatal period

Alterations in the high and low affinity insulin receptor concentrations in developing rat liver were investigated. The number of high affinity receptors in partially purified plasma membranes from fetal rats increased from Days 19 through 22 of gestation, with no further increase in binding during the postnatal period. Fetuses of diabetic rats had approximately three times as many high affinity insulin receptors as age-matched fetuses of normal rats; however, by 1 day after birth the receptor number decreased to the normal level. Neither the number of low affinity receptors nor the affinity of insulin binding to high or low affinity receptors changed during development or between offspring of normal and diabetic rats. These changes in the number of high affinity hepatic insulin receptors from prenatal animals did not correlate with the concentration of plasma insulin. When suckling pups were rendered diabetic the changes in the number of high affinity insulin receptors correlated with alterations in plasma insulin concentrations. The number of high affinity sites/microgram DNA in hepatocytes from Day 18 fetal rats was not altered when cells were cultured for 48 h in medium containing 0, 250, or 5000 μU/ml of added insulin. When cultured hepatocytes derived from 1-day-old and adult rats were maintained in medium with added insulin concentrations of 250 or 5000 μU/ml the number of high affinity receptors/microgram DNA decreased as compared to the number of high affinity receptors in hepatocytes cultured in medium with no added insulin. This decrease in receptor number was accompanied by an increase in the affinity of insulin binding to its high affinity receptors. The data show that (i) only the high affinity insulin receptor number increases in rat liver during the prenatal period, (ii) fetuses of diabetic rats show a greater increase in high affinity receptors than do fetuses of normal animals, and (iii) the phenomenon of down regulation for high affinity insulin receptors is not observed in fetal rat liver, but is acquired in the immediate postnatal period.     

Ontogeny of cytosolic proteins capable of modulating sarcoplasmic reticulum calcium transport in heart muscle

In a previous study we described the inhibitory action of a cytosolic protein fraction from heart muscle on ATP-dependent Ca² uptake by sarcoplasmic reticulum (SR); further, this inhibition was shown to be blocked by an inhibitor antagonist, also derived from the cytosol (Narayanan et al. Biochim Biophys Acta 735: 53–66, 1983). The present study investigated the ontogenetic expression of the activities of Ca² transport inhibitor and inhibitor antagonist in heart cytosol during fetal and postnatal development of the rat. The SR Ca² transport inhibitor activity was undetectable in the cytosol of fetal (15- or 20-days gestation) rat heart but was manifested in the cytosol as early as one day after birth and increased progressively thereafter to reach almost adult levels within the first two weeks of postnatal development. The activity of the SR Ca² transport inhibitor antagonist was barely detectable in the near-term (20 days gestation) fetus but increased substantially during early postnatal development, in parallel with the rise in activity of the inhibitor. The ontogenetic appearance and increase in the activities of the Ca² transport inhibitor and its antagonist correlated well with the concurrent appearance and increase in the amounts of two polypeptides of apparent molecular weights 43 kDa and 64 kDa, which we have tentatively identified as the inhibitor and inhibitor antagonist, respectively. The co-ordinated expression of both the inhibitor and inhibitor antagonist activities in the cytosol during the early postnatal period parallels the morphogenesis and functional maturation of SR in cardiac muscle suggesting likely involvement of these cytosolic proteins in the physiological regulation of SR function.     

Downregulation of hippocampal NMDA receptor expression by prenatal exposure to dioxin.

Gestational exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) has been implicated as causative to disparities between ethnic groups with respect to learning disabilities. Dioxin is an extremely toxic environmental pollutant that bioaccumulates in maternal adipose tissue, and is transferred to the developing organism during gestation and lactation. Long-term cognitive deficits have been reported following prenatal exposure to dioxin. N-methyl-D-aspartate (NMDA) receptors in the central nervous system (CNS) have been well known to play an important role in the activity-dependent synaptic plasticity underlying learning and memory and in CNS development including brain cell differentiation. Here, the effects of prenatal exposure to dioxin on the developmental expression profiles of rat hippocampal NMDA receptor subtype 1 mRNA and protein was examined. F-344 rats were exposed to 0 and 700 ng of dioxin/kg on gestational day 15. Real-time PCR and Western blot analysis clearly revealed that dioxin significantly downregulated NMDAR1 mRNA and protein expression during the first postnatal month. The study provides support to the hypothesis that NMDA receptors are important targets for dioxin-induced neurotoxicity in F1 preweaning pups. The results also support the concept that prenatal exposure to dioxin may contribute to the pathogenesis of diseases in the adult.     

Expression of autotaxin and lysophosphatidic acid receptors 1 and 3 in the developing rat lung and in response to hyperoxia

Abstract

We sought to evaluate lysophosphatidic acid (LPA) signaling improvement in lung development by assessing the expression of autotaxin and LPA receptor 1 and 3 (LPAR1 and LPAR3) in the neonatal rat lung during normal perinatal development and in response to hyperoxia. In the developmental study, rats were sacrificed on days 17, 19, and 21 of gestation; on postnatal days 1, 4, and 7; and at adulthood (postnatal 9 weeks). In the hyperoxia study, 42 postnatal 4-day-old rat pups were divided into seven groups and exposed to either 85% O₂ for 24, 72, or 120 h or room air for 0, 24, 72, or 120 h. The rats were then euthanized after 0, 24, 72, and 120 h of exposure. Immunofluorescence demonstrated that autotaxin, LPAR1, and LPAR3 proteins were broadly colocalized in airway epithelial cells, but mainly distributed in vascular endothelial and mesenchymal cells during the first postnatal week. The expression of autotaxin, LPAR1, and LPAR3 were increased during late gestation and then decreased after birth. Autotaxin expression and enzymatic activity were significantly increased at 72 and 120 h after exposure to hyperoxia. LPAR1 and LPAR3 expression was also increased after 120 h of hyperoxic exposure. These findings suggest that LPA-associated molecules were upregulated at birth and induced by hyperoxia in the developing rat lung. Therefore, the LPA pathway may be involved in normal lung development, including vascular development, as well as wound-healing processes of injured neonatal lung tissue, which is at risk of neonatal hyperoxic acute lung injury.     

Developmental changes in rat hepatic casein kinases 1 and 2

Cytosolic histone kinase and casein kinase activities varied considerably in the late fetal and postnatal periods of liver development. Both activities showed a maximum at day 21 of gestation and decreased at birth to values close to those of adult rats. The changes in total casein kinase activity were due to variations of casein kinase 1 and casein kinase 2. Similarly the activities of both the cyclic-AMP-dependent protein (histone) kinase and the cyclic-AMP-independent histone kinase varied during development. Besides the changes in total activity, the affinity of casein kinases 1 and 2 for casein also varied in fetal and postnatal development. The Km values of casein kinase 2 increased from day 18, reached a maximum at day 20 of gestation and then started to decrease until one day after birth. In contrast the Km values of casein kinase 1 decreased from day 18, reached its lowest value at day 21 of gestation and attained values similar to those in the adult at the day of birth. Changes in this parameter were also observed when insulin (3 IU/kg) was administered by intraperitoneal injection to one-day-old rats. The Km values of casein kinase 1 decreased while those of casein kinase 2 increased after administration of this hormone. On the other hand, the Km values for ATP of casein kinases 1 and 2 as well as their apparent molecular masses and sensitivity to heparin and GTP did not significantly change during ontogeny of rat liver.     

The use of fluorescent antimyosin and DNA labeling for the estimation of the myoblast and myocyte population of primary rat heart cell cultures

Cell division in heart muscle cells progressively ceases during the development of the rat heart, leading to an adult stage with muscle cells incapable of cell division. We have quantitatively determined the number of dividing and nondividing heart muscle cells in cultures derived from different stages of the developing rat heart with the use of ³HTdR continuous labeling and fluorescent antimyosin staining. The cultures were derived from 14 and 17 day postcoital (dPC) rat embryos and from 1 and 4 day postnatal (dPN) rats. The percent nondividing cells increased with development and the age of the postnatal rat. The percent nondividing cells in 14 dPC equalled 21%, 17 dPC equalled 25%, 1 dPN equalled 44%, and 4 dPN equalled 60%. This method for the quantitative determination of dividing and nondividing cells in the developing rat heart provides a model that is useful for the study of the mechanism of the loss of cell division capacity.     

Impact of forskolin and amino acid depletion upon System A activity and SNAT expression in BeWo cells

Amino acid transport System A (SysA) plays an important role in mediating the transplacental transfer of neutral amino acids from mother to fetus. Given that prior work has demonstrated that SysA activity is regulated, both over gestation and in response to dietary restriction during pregnancy, we examined the response of SysA activity and sodium-dependent neutral amino acid transporter (SNAT; responsible for SysA activity) expression to cAMP analogues and amino acid deprivation in BeWo cells, an accepted model of placental syncytia. SysA activity was unaffected by forskolin, a cAMP agonist, at 48 and 72 h. Amino acid depletion was associated with an up-regulation of SysA activity, largely mediated through an enhancement of SNAT2 (Slc38a2) expression at both the protein and mRNA level. SNAT1 (Slc38a1) expression did not change in response to amino acid depletion, while SNAT4 (Slc38a4) could not be detected. In summary, SysA activity in BeWo cells responds to amino acid depletion through the differential regulation of SNAT subtypes.     

Pre- and postnatal stimulation of pulmonary surfactant protein D by in vivo dexamethasone treatment of rats.

Fetal (days 18 and 20 of gestation), neonatal (days 0, 2 and 4 of neonate) and adult rats were injected with dexamethasone (1 mg/kg) in vivo and 24 hours later the effect on the contents of surfactant protein D (SP-D) in the rat lungs were examined in comparison with surfactant protein A, disaturated phosphatidylcholine and phosphatidylglycerol. In vivo dexamethasone treatment resulted in significant increases of SP-D content as the other 3 components of surfactant in both fetuses and neonates, but not in adults. Responsiveness to glucocorticoid treatment on SP-D content was maximum on day 1 of neonate (2.7 times control value). The contents of surfactant components examined tend to respond better to steroid in postnatal rats. These data demonstrated that glucocorticoid treatment in vivo for short durations exhibits the stimulatory effect on the contents of SP-D in the fetal and neonatal rat lungs.