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Biomedical subjects

J F Padbury

Publications and source records attributed to J F Padbury.

At least 19 recordsLinked to original sources
PubMed2001

A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the beta1-adrenergic receptor gene.

The effects of glucocorticoids on expression of the beta1-adrenergic receptor (beta1AR) gene have been varied. To study the mechanism underling hormonal regulation of the beta1AR, transient transfection of progressively deleted ovine beta1AR promoter fragments was used to identify a 43-bp region (-1274 to -1232 from the translation start site) that contains a novel glucocorticoid regulatory unit (GRU) and confers glucocorticoid responsiveness. Using DNase I footprinting and electrophoretic mobility shift assays (EMSA), we demonstrated the GRU was composed of a palindrome, 5'-TAATTA-3', which is a core binding motif for the homeodomain proteins, an E-box (5'-CACGTG-3'), binding site for the Myc/Max family proteins, and an overlapping glucocorticoid response element (GRE) half-site (5'-TGTTCT-3'). EMSA demonstrated that the GRE half-site is critical for GRU-protein interactions, which also require binding of proteins to the E-box and the homeodomain region. Co-transfection of a plasmid expressing a c-myc antisense construct significantly reduced glucocorticoid responsiveness of the ovine beta1AR promoter. Furthermore, expression of proteins binding to the GRU was shown to be developmentally regulated, being high in embryonic, reduced in newborn and not detectable in adult heart. We conclude that the ovine beta1AR promoter contains a novel, functional GRU and that glucocorticoid receptor (GR) and the Myc/Max family proteins are involved in the cell-specific nuclear factor binding and transactivation via this element. The results suggest an alternative pathway through which glucocorticoids may exert their effects on genes lacking a full consensus GRE.

Animals↗
PubMed2001

Molecular cloning of the rat TA1/LAT-1/CD98 light chain gene promoter.

The rat LAT-1 (L-amino acid transporter-1) gene is a CD98 light chain highly expressed in cancer and development. As an initial study of the molecular basis underlying regulation of its expression, we cloned 2 kb of the LAT-1 5' flanking region. Inverse RACE and primer extension methods were used to define the transcription initiation site at 80 bp upstream from the translational start site. Functional studies carried out in normal hepatic cells using constructs containing progressive 5' deletion from region -1958 to -185 showed 3-5-fold beta-galactosidase activities over control. The presence of an activator site(s) between -52 and -185 was indicated by low activities conferred by the construct spanning this region.

Animals↗
PubMed2001

The interactive effects of endotoxin with prenatal glucocorticoids on short-term lung function in sheep.

OBJECTIVE: Previously we have shown that neonatal lung function in sheep after preterm birth is profoundly enhanced by intra-amniotic injection of endotoxin, with a magnitude at least equal to that induced by maternal betamethasone administration. This study investigated the effects of betamethasone on lung maturation and growth in the presence of inflammation by treating sheep with both maternal intramuscular betamethasone and intra-amniotic endotoxin injections. STUDY DESIGN: Time-mated pregnant ewes at 118 days' gestation were allocated at random to receive maternal intramuscular or intra-amniotic saline solution injection (n = 10), maternal intramuscular betamethasone injection (0.5 mg/kg; n = 7), intra-amniotic endotoxin injection (20 mg Escherichia coli B055;B5; n = 11) by ultrasonographic guidance, or both betamethasone and endotoxin injections (n = 7). The lambs were delivered abdominally at 125 days' gestation (term is 150 days' gestation), and the neonates were ventilated for 40 minutes before postmortem examination. RESULTS: Combined treatment with betamethasone and endotoxin resulted in significantly greater improvements in neonatal lung function than occurred after treatment with either agent alone, and this effect was not accompanied by a further increase in surfactant levels. The reduction in birth weight that is seen after maternal betamethasone treatment was not seen when this treatment was combined with endotoxin. Endotoxin treatment resulted in inflammatory responses in cord blood and alveolar wash, and these responses were not inhibited by betamethasone treatment. There were no pregnancy losses. CONCLUSION: Both intra-amniotic endotoxin injection and maternal intramuscular betamethasone injection promoted fetal lung maturation. When these treatments were combined, there were additive effects on short-term postnatal lung function but not on surfactant levels. Endotoxin negated the growth restriction in sheep caused by maternal betamethasone treatment. These findings provide evidence that the lung maturation induced by glucocorticoids and that induced by endotoxin are mediated by different mechanisms.

Acute-Phase Reaction↗
PubMed2001

Beta-adrenergic receptors (betaAR) regulate cardiomyocyte proliferation during early postnatal life.

Cardiomyocyte development switches from hyperplasmic to hypertrophic growth between postnatal days 3 and 4 in rats. The mechanisms responsible for this transition have been controversial. beta-Adrenergic receptor (betaAR) activation of mitogenic responses in vitro has been reported. We hypothesized that tonic activation of the betaAR signaling regulates cell division in neonatal cardiomyocytes via effects on signaling kinases known to be important in cell cycle regulation. The purpose of the current study was to elucidate the roles of betaAR in rat cardiomyocyte growth in vivo. We demonstrated that betaAR blockade induced a significant reduction in cardiomyocyte proliferation as measured by the BrdU labeling index. Blockade of betaAR did not affect p38 or p44/42 MAPK activities. We further demonstrated that betaAR blockade induced a prompt deactivation of the p70 ribosomal protein S6 kinase (p70 S6K). To confirm these results, we measured p70 S6K activity directly. Basal activity of p70 S6K in neonatal cardiomyocytes was fourfold higher than that of insulin-treated adult rat liver. The activity of p70 S6K was reduced by 60% within 1 min after betaAR blockade. We conclude that the betaAR are involved in regulation of neonatal cardiomyocyte proliferation and that this mitogenic control may be mediated via the p70 S6K pathway.

Adrenergic beta-Antagonists↗
PubMed2001

Inhibition of placental 11beta-hydroxysteroid dehydrogenase type 2 by catecholamines via alpha-adrenergic signaling.

The placenta expresses high levels of 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) that converts cortisol into inactive 11-keto metabolites and effectively protects the developing fetus from maternal cortisol during pregnancy. Impairment of this glucocorticoid barrier has adverse effects on fetal outcomes. A similar spectrum of adverse fetal effects is induced by antenatal stress during pregnancy. To examine the hypothesis that physiological stress may regulate placental 11betaHSD2 gene expression, we examined the effects of the catecholamines norepinephrine (NE) and epinephrine (E) on 11betaHSD2 expression in human trophoblastic cells. With the use of Northern blotting and semiquantitative RT-PCR, we determined that NE and E rapidly downregulate 11betaHSD2 steady-state mRNA levels in early- and late-gestation human trophoblasts and BeWo trophoblastic cells. Experiments using different adrenoceptor subtype-selective agonists and antagonists demonstrated that this catecholamine suppression of 11betaHSD2 mRNA expression is mediated via both alpha(1)- and alpha(2)-adrenoceptors and is independent of beta-adrenergic stimulation. To examine transcriptional regulation, BeWo cells were transiently transfected with a reporter construct in which an 11betaHSD2 human promoter sequence was inserted upstream of the luciferase gene. Treatment with 10(-7) M NE decreased luciferase activity by ~60% (n = 3, P < 0.01). These results suggest the NE/E-mediated decrease in placental 11betaHSD2 gene expression is an instance of alpha-adrenoceptor-specific rapid transcriptional inhibition of an adrenergic target gene. This molecular mechanism may be involved in the deleterious effects of antenatal physiological stress on fetoplacental growth and development.

Adrenergic alpha-Antagonists↗
PubMed2001

Liganded and unliganded steroid receptor modulation of beta 1 adrenergic receptor gene transcription.

The classical model of gene regulation by hormones involves a hormone-bound receptor interacting with a DNA response element to increase or decrease gene transcription. Steroid hormone regulation more commonly involves atypical cis-elements, co-receptors, accessory proteins, and unique modes of interaction on different genes. The thyroid hormone and retinoic acid receptors belong to the super family of steroid nuclear receptors and may modify gene expression even in the absence of ligand binding. In these studies, we characterized thyroid receptor- and retinoic acid receptor-mediated regulation of beta1 adrenergic receptor (beta1AR) gene expression. Using cloned fragments of the ovine beta1AR in a luciferase reporter vector, we examined the effects of thyroid receptor and retinoic acid receptor, alone and in combination with T3 or retinoic acid on beta1AR expression. We examined expression in SK-N-SH neuroblastoma cells, CV-1 fibroblasts, and, in neonatal rat, primary cardiomyocytes. We demonstrated that even in the absence of ligand binding, thyroid receptor and retinoic acid receptor can significantly increase beta1AR transcription activity. This effect is important in the developmental transition in beta1AR expression during fetal and postnatal life.

Animals↗
PubMed2000

Cloning and sequence analysis of the rat norepinephrine transporter promoter.

We isolated a 2.5-kb fragment of the promoter for the rat norepinephrine transporter (NET) gene. The transcription start site was identified approximately 200 base pairs upstream from the translation start site. Several potential regulatory elements were identified by sequence analysis. The structure of the rat NET promoter was compared to mouse and human. Expression studies in placental and neuroblastoma cells suggested the presence of a 'repressor' element more than 500 base pairs upstream from the transcription start site. These studies provide the basis for examination of transcriptional regulation of this gene and for understanding its temporal and tissue-specific modes of regulation.

5' Untranslated Regions↗
PubMed2000

Antenatal glucocorticoids alter premature newborn lamb neuroendocrine and endocrine responses to hypoxia.

Glucocorticoids are administered for preterm labor to improve postnatal adaptation. We assessed the effect of antenatal betamethasone (Beta) treatment on preterm newborn lamb neuroendocrine [catecholamine, arginine vasopressin (AVP)] and endocrine [triiodothyronine (T(3)), ANG II, and atrial natriuretic factor (ANF)] adaptive responses following delivery and a hypoxic challenge. Beta treatment included direct fetal injection at 0.2 (F(0.2); n = 8) or 0.5 (F(0.5); n = 7) mg/kg estimated fetal body weight or maternal injection with 0.2 (n = 8) or 0.5 mg/kg (M(0.5); n = 8). Control animals received fetal and maternal intramuscular injections of saline (n = 8). After 24 h, lambs were delivered by cesarean section, surfactant treated, and ventilated for 4 h. Relative to the control lambs, 3 h after delivery, there was a marked suppression of plasma cortisol, epinephrine, norepinephrine, and ANG II levels and elevated plasma T(3) and ANF levels, systolic blood pressure, and left ventricular contractility (dP/dt; F(0.5) and M(0.5)) values in F(0.5) and both maternal Beta-treated groups. However, Beta treatment augmented the cardiac output, cortisol, norepinephrine, AVP, and ANF responses to 20 min of hypoxia (PO(2) = 25-30 mmHg). We concluded that short-term (24 h) antenatal glucocorticoid exposure 1) alters preterm newborn postnatal blood pressure regulation in the face of marked depression of plasma cortisol, catecholamine, and ANG II levels and 2) augments the postnatal neuroendocrine and endocrine responses to a hypoxic challenge.

Angiotensin II↗
PubMed2000

Endotoxin-induced lung maturation in preterm lambs is not mediated by cortisol.

Antenatal exposure to glucocorticoids, amnionitis, intraamniotic interleukin (IL)-1alpha, or endotoxin can improve postnatal lung function after preterm delivery. The relationship between early lung maturation and the dose and duration of a proinflammatory stimulus has not been evaluated. The effects of proinflammatory stimuli on fetal plasma cortisol also have not been evaluated. We hypothesized that intraamniotic endotoxin would induce early lung maturation in fetal sheep without increasing fetal cortisol. Intraamniotic injections of 1, 4, 20, or 100 mg of Escherichia coli 055:beta5 endotoxin caused 2-fold increases in compliance, 4- to 5-fold increases in lung gas volumes, and 20-fold increases in alveolar saturated phosphatidylcholine (Sat PC) when given 7 d before preterm delivery at 125 d gestation. Animals treated with 20 mg endotoxin for treatment to delivery intervals of 5 h to 15 d had no significant elevations in cord plasma cortisol levels. Increases in Sat PC in lung tissue and alveolar washes were detected 2 d after endotoxin treatment and lung function improved 4 d after endotoxin treatment. Two doses of endotoxin given 3 and 7 d or 7 and 15 d before treatment resulted in lung maturation responses equivalent to single dose comparison groups without elevations in cortisol. Early lung maturation induced by intraamniotic endotoxin in fetal sheep occurred without an increase in fetal plasma cortisol, indicating that endotoxin promoted lung maturation by a mechanism independent of cortisol.

Amnion↗
PubMed2000

The use of inhaled glucocorticosteroids and recovery from adrenal suppression after systemic steroid use in a VLBW premature infant with BPD: case report and literature discussion.

Despite development of many prevention and treatment modalities for bronchopulmonary dysplasia (BPD), a form of chronic respiratory insufficiency in premature infants recovering from respiratory distress syndrome, BPD remains a treatment challenge and a significant cause of long-term morbidity. A ventilator-dependent very low birth weight infant in our newborn special care unit was receiving multiple courses of systemic dexamethasone for severe respiratory failure. The infant demonstrated adrenal suppression manifested by a baseline cortisol concentration below reported levels in infants of similar birth weight and postnatal age. We hypothesized that he had developed adrenal insufficiency as a result of the prolonged systemic steroid administration used to treat his respiratory problems. We further hypothesized that inhaled beclomethasone therapy would aid in the infant's recovery phase during relative adrenal insufficiency--and so substituted inhaled for systemic steroids. Inhaled corticosteroid treatment improved the clinical respiratory course and postnatal growth of this premature infant with BPD without inhibiting his recovery from adrenal insufficiency.

Administration, Inhalation↗
PubMed1999

Placental biogenic amine transporters: in vivo function, regulation and pathobiological significance.

The biogenic amine transporters are part of a large family of plasma membrane transporters. These carriers mediate the re-uptake of neurotransmitters from the synaptic cleft and plasma compartments. Re-uptake process is inhibited by drugs like cocaine, fluoxetine and tricyclic antidepressants. There are specific transporters for norepinephrine, epinephrine, dopamine and serotonin. The placenta expresses the norepinephrine and serotonin transporters, which is unusual as they are otherwise expressed predominantly in neuronal tissue. Fetal catecholamine clearance rate is higher than under any other physiological conditions and is mediated in large measure by the placental transporters. The high intrauterine catecholamine secretion and clearance rates are part of the unique fetal neuroendocrine milieu. They condition the fetus to a high capacity for catecholamine secretion in the early postnatal period when elevated sympathoadrenal system activity is vital for postnatal survival. Because of the prominent catecholamine clearance rate, the fetus is vulnerable to the adverse effects of re-uptake inhibitors. Understanding the mechanisms of expression and regulation of placental biogenic amine transporters is important to the pathobiology of fetal conditions associated with elevated catecholamine levels or intrauterine exposure to uptake inhibitors like cocaine.

Biogenic Monoamines↗
PubMed1998

Antenatal thyrotropin-releasing hormone to prevent lung disease in preterm infants. North American Thyrotropin-Releasing Hormone Study Group.

BACKGROUND: Pulmonary disease is common in preterm infants, despite antenatal glucocorticoid therapy. The addition of antenatal thyrotropin-releasing hormone therapy has been reported to decrease pulmonary morbidity in these infants. METHODS: We enrolled 996 women at 13 North American centers who were in preterm labor at <30 weeks' gestation in a double-blind, placebo-controlled, randomized trial of antenatal thyrotropin-releasing hormone, given intravenously in four doses of 400 microg each at eight-hour intervals. The primary outcome was chronic lung disease or death of the infant on or before the 28th day after delivery, and secondary outcomes were respiratory distress syndrome and chronic lung disease or death at 36 weeks' postmenstrual age. Complete data were available for 981 women and their 1134 live-born infants. The 769 infants born at < or = 32 weeks' gestation were defined as the group at risk. RESULTS: There were no significant differences between the at-risk treatment and placebo groups in mean (+/-SD) birth weight (1109+/-354 vs. 1097+/-355 g), gestational age (27.9+/-2.1 vs. 27.9+/-2.1 weeks), sex, or race. The frequencies of respiratory distress syndrome (66 percent vs. 65 percent), death at 28 days (11 percent vs. 11 percent), chronic lung disease or death at 28 days (45 percent vs. 42 percent) and at 36 weeks (32 percent vs. 34 percent), and other neonatal complications as well as the severity of lung disease were not significantly different in the at-risk treatment and placebo groups. Similarly, there were no differences in outcome between the treatment and placebo groups for the infants born at >32 weeks' gestation. CONCLUSIONS: In preterm infants at risk for lung disease, antenatal administration of thyrotropin-releasing hormone and glucocorticoid is no more beneficial than glucocorticoid alone.

Adult↗
PubMed1998

Expression of a pulmonary endothelial norepinephrine transporter.

To determine whether pulmonary endothelial cells express the plasma membrane norepinephrine transporter (NET), an ovine-specific riboprobe was prepared from a partial NET cDNA clone isolated by PCR from a placental cDNA library. Northern hybridization detected a predominant 5.8 kb and a weaker transcript at 3.6 kb in RNAs isolated from near-term ovine fetal pulmonary endothelial cells. These data should be useful for future studies examining the molecular mechanisms underlying pulmonary endothelial NET expression and regulation.

Animals↗
PubMed1998

An inverted cAMP response element mediates the cAMP induction of the ovine beta 1-adrenergic receptor gene.

We identified an inverted, functional cAMP response element (CRE) located at--1599 bp relative to the translation start site within the ovine beta 1-adrenergic receptor (beta 1 AR) gene promoter. In transfection studies with SK-N-MC cells, a 40-bp oligonucleotide containing the potential CRE, beta 1 AR-CRE, conferred a 3- to 4-fold increase in luciferase activity mediated by cAMP. The induction was mimicked by co-transfecting the cells with a vector overexpressing the alpha-catalytic subunit of the cAMP-dependent protein kinase (PKA) without treatment, and was blocked by overexpressing a PKA inhibitor (PKI). In electrophoretic mobility shift assays, a discrete binding pattern was shown in cell nuclear extract probed with the 40 bp beta 1 AR-CRE. The binding was shown to be specific and supershifted by addition of a CRE binding protein (CREB-1) antibody. These data demonstrate that cAMP mediates the induction of beta 1 AR gene expression by interacting with an inverted CRE within the promoter region. This is the first reported functional CRE among all beta 1 AR genes.

Animals↗
PubMed1997

A comparative study of cardiovascular, endocrine and behavioural effects of betamethasone and dexamethasone administration to fetal sheep.

1. Chronically instrumented, late-gestation fetal sheep were prepared to: (1) characterize cardiovascular, endocrine and behavioural effects of fetal treatment with clinical doses of betamethasone and dexamethasone; (2) define specific differences, if any, in the actions of betamethasone and dexamethasone of measured fetal responses; and (3) assess the contribution of changes in peripheral vascular resistance to the glucocorticoid-induced hypertension. 2. Following baseline, either saline (n = 9), betamethasone (n = 9), or dexamethasone (n = 6) was infused for 48 h in fetal sheep commencing at 125 days of gestation. A pronounced increase in fetal blood pressure occurred following both betamethasone and dexamethasone treatment. The nature and magnitude of this increase was similar following treatment with either glucocorticoid. 3. To address possible mechanisms contributing to the glucocorticoid-induced fetal hypertension, fetal plasma catecholamine levels and changes in fetal femoral haemodynamics were assessed following fetal glucocorticoid treatment. A fall in fetal plasma noradrenaline and adrenaline concentrations occurred during betamethasone and dexamethasone treatment. In contrast, a progressive femoral vasoconstriction occurred during betamethasone treatment. 4. A modest fall in the incidence of fetal breathing movements occurred during fetal treatment with either betamethasone or dexamethasone. The magnitude of this reduction was similar with treatment of either glucocorticoid. The fall in fetal breathing during betamethasone and dexamethasone treatment was not associated with a fall in the incidence of fetal low voltage electrocortical activity. 5. Our results indicate that prenatal betamethasone and dexamethasone treatment of late-gestation fetal sheep, in doses similar to those employed clinically, is associated with fetal cardiovascular, endocrine and behavioural effects. Both betamethasone and dexamethasone induce similar increases in fetal blood pressure and similar falls in the incidence of fetal breathing movements. The pronounced betamethasone-induced fetal hypertension is associated with an increase in fetal femoral vascular resistance.

Animals↗