Adrenocorticotrophic hormone (ACTH) stimulation of sheep fetal adrenal cortex can occur without increased expression of ACTH receptor (ACTH-R) mRNA

2002 ◽  
Vol 14 (1) ◽  
pp. 1 ◽  
Author(s):  
A. M. Carter ◽  
Y. M. Petersen ◽  
M. Towstoless ◽  
D. Andreasen ◽  
B. L. Jensen
Keyword(s):  
Adrenal Gland ◽  
Mrna Expression ◽  
Ribonuclease Protection Assay ◽  
Acth Stimulation ◽  
Cortical Cells ◽  
Fetal Plasma ◽  
Chain Cleavage ◽  
Ribonuclease Protection ◽  
Sheep Fetus

In the present study, it was hypothesized that the adrenocorticotrophin hormone receptor (ACTH-R) would be up-regulated in the adrenal gland of the sheep fetus following infusion of physiological amounts of ACTH, as shown for adrenal cortical cells in culture. In chronically catheterized sheep, an intravenous infusion of ACTH1–24 was given to 6 fetuses for 24 h at a rate of 0.5 g h–1, starting on Day 126 or 127 of gestation (term ~147 days). Four control fetuses received an infusion of vehicle (saline). Total RNA was extracted from the fetal adrenal glands by the guanidinium thiocyanate method. Expression of specific mRNAs was determined by ribonuclease protection assay using cRNA probes directed against: ACTH-R; the steroid enzymes side-chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17 α-hydroxylase (P450c17) and 21β-hydroxylase (P450c21); and β-actin. Ratios of mRNA expression to β-actin mRNA expression (arbitrary units) were calculated to correct for differences in RNA quality between samples. The concentration (mean SEM) of immunoreactive cortisol in fetal plasma was greater after ACTH infusion than after vehicle infusion (47 3 v. 13 2 ng mL–1 respectively; (P<0.001). Adrenal expression of P450scc and P450c21 mRNA increased after ACTH infusion (P<0.05), whereas expression of P450c17 and 3β-HSD mRNA was unchanged. There was no difference in ACTH-R mRNA expression between ACTH- and vehicle-infused fetuses (254 48 v. 305 76 arbitrary units respectively). It was concluded that ACTH is able to increase plasma cortisol concentrations in the sheep fetus by up-regulating cortisol synthesis in the adrenal gland, but that in vivo this does not require up-regulation of ACTH-R mRNA.

Differential expression, abundance, and regulation of Na+-phosphate cotransporter genes in murine kidney

1998 ◽  
Vol 275 (4) ◽  
pp. F527-F534 ◽  
Author(s):  
Harriet S. Tenenhouse ◽  
Stéphane Roy ◽  
Josée Martel ◽  
Claude Gauthier
Keyword(s):  
Mrna Expression ◽  
Leukemia Virus ◽  
Mouse Kidney ◽  
Ribonuclease Protection Assay ◽  
Type I ◽  
Outer Cortex ◽  
Gh Treatment ◽  
Gibbon Ape Leukemia Virus ◽  
Ribonuclease Protection ◽  
Renal Expression

Three classes of high-affinity Na+-Picotransporters are expressed in mammalian kidney. These include Npt1 (type I), Npt2 (type II), and the cellular receptors for gibbon ape leukemia virus (Glvr-1) and amphotropic murine retrovirus (Ram-1) (type III). We defined the tissue distribution as well as the relative renal abundance of Npt1, Npt2, Glvr-1, and Ram-1 mRNAs and examined the effects of low-Pi diet, the Hyp mutation, and growth hormone (GH) on their renal expression by ribonuclease protection assay. In normal mouse kidney, Npt1, Npt2, Glvr-1, and Ram-1 accounted for 15 ± 1.0, 84 ± 1.0, 0.5 ± 0.2, and 0.5 ± 0.3% of total Na+-Picotransporter mRNAs, respectively. Evidence was obtained for low-abundance Npt1 mRNA expression in liver and Npt2 mRNA expression in intestine, whereas Glvr-1 and Ram-1 mRNAs were also detected in bone, intestine, heart, and liver. Npt2 mRNA was localized to proximal tubules in the renal outer cortex, whereas Glvr-1 transcripts were detected throughout the kidney by in situ hybridization. The Hyp mutation elicited a significant reduction in renal Npt1 and Npt2 mRNAs (78 ± 8 and 57 ± 3% of normal, respectively), whereas neither low-Pi diet nor GH influenced the renal abundance of Npt1 and Npt2 transcripts. Renal Glvr-1 mRNA expression was significantly increased in Hyp mice and GH-treated mice (145 ± 6 and 165 ± 5% of control, respectively), whereas the renal abundance of Ram-1 transcript was unaffected by either the Hyp mutation, low-Pi diet, or GH treatment. In summary, we demonstrate that Npt2 is the predominant Na+-Picotransporter in mouse kidney, that Npt2 and Glvr-1 have distinct patterns of renal expression, and that the Hyp mutation modulates the renal expression of Npt1, Npt2, and Glvr-1 mRNAs. Our results suggest that increased renal Glvr-1 mRNA may contribute to GH stimulation of renal Na+-Picotransport.

Prolactin and the expression of suppressor of cytokine signaling-3 in the sheep adrenal gland before birth

2006 ◽  
Vol 291 (5) ◽  
pp. R1399-R1405 ◽  
Author(s):  
S. Gentili ◽  
J. S. Schwartz ◽  
M. J. Waters ◽  
I. C. McMillen
Keyword(s):  
Adrenal Gland ◽  
Mrna Expression ◽  
Tissue Growth ◽  
Late Gestation ◽  
Cytokine Signaling ◽  
Suppressor Of Cytokine Signaling ◽  
Subsequent Increase ◽  
Fetal Sheep

The fetal pituitary-adrenal axis plays a key role in the fetal response to intrauterine stress and in the timing of parturition. The fetal sheep adrenal gland is relatively refractory to stimulation in midgestation (90–120 days) before the prepartum activation, which occurs around 135 days gestation (term = 147 ± 3 days). The mechanisms underlying the switch from adrenal quiescence to activation are unclear. Therefore, we have investigated the expression of suppressor of cytokine signaling-3 (SOCS-3), a putative inhibitor of tissue growth in the fetal sheep adrenal between 50 and 145 days gestation and in the adrenal of the growth-restricted fetal sheep in late gestation. SOCS-3 is activated by a range of cytokines, including prolactin (PRL), and we have, therefore, determined whether PRL administered in vivo or in vitro stimulates SOCS-3 mRNA expression in the fetal adrenal in late gestation. There was a decrease ( P < 0.005) in SOCS-3 expression in the fetal adrenal between 54 and 133 days and between 141 and 144 days gestation. Infusion of the dopaminergic agonist, bromocriptine, which suppressed fetal PRL concentrations but did not decrease adrenal SOCS-3 mRNA expression. PRL administration, however, significantly increased adrenal SOCS-3 mRNA expression ( P < 0.05). Similarly, there was an increase ( P < 0.05) in SOCS-3 mRNA expression in adrenocortical cells in vitro after exposure to PRL (50 ng/ml). Placental and fetal growth restriction had no effect on SOCS-3 expression in the adrenal during late gestation. In summary, the decrease in the expression of the inhibitor SOCS-3 after 133 days gestation may be permissive for a subsequent increase in fetal adrenal growth before birth. We conclude that factors other than PRL act to maintain adrenal SOCS-3 mRNA expression before 133 days gestation but that acute elevations of PRL can act to upregulate adrenal SOCS-3 expression in the sheep fetus during late gestation.

scholarly journals Construction of a ribozyme directed against human interleukin-6 mRNA: evaluation of its catalytic activity in vitro and in vivo

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3758-3765 ◽  
Author(s):  
M Mahieu ◽  
R Deschuyteneer ◽  
D Forget ◽  
P Vandenbussche ◽  
J Content
Keyword(s):  
Interleukin 6 ◽  
Ribonuclease Protection Assay ◽  
Mammalian Expression ◽  
Cell Clones ◽  
Ribonuclease Protection ◽  
Amniotic Cells ◽  
Necrosis Factor

We have designed a ribozyme (Rz) that cleaves human interleukin-6 (IL- 6) mRNA in vivo. This Rz was tested in vitro, and was found to give expected size fragments. It was then incorporated into a mammalian expression vector containing the constitutive cytomegalovirus (CMV) immediate early promoter and transfected into human U amniotic cells (UAC). Cell clones that stably express this catalytic RNA have been obtained. Some of them displayed a marked reduction of tumor necrosis factor (TNF)-induced IL-6 production. Their reduced ability to express IL-6 was related to the amount of Rz they produced and to the extent of IL-6 mRNA cleavage as observed by a ribonuclease protection assay. These data provide a method to study further the role of IL-6 production in various biologic situations, and suggest the feasibility of developing Rzs directed against various cytokines to study their biologic role and mechanism of action.

scholarly journals Tissue distribution of a menthyl-conjugated oligodeoxyribonucleotide antisense to PAI-1 mRNA.

2005 ◽  
Vol 52 (4) ◽  
pp. 849-856
Author(s):  
Janusz Szemraj ◽  
Khalid N I Al-Nedawi ◽  
Ewa Chabielska ◽  
Wlodzimierz Buczko ◽  
Zofia Pawlowska
Keyword(s):  
Rat Kidney ◽  
Inhibitory Effect ◽  
Organ Distribution ◽  
Ribonuclease Protection Assay ◽  
Ribonuclease Protection ◽  
And Control ◽  
Kidney Liver ◽  
Pai 1

The inhibitory effect of numerous analogues of PO-16, an hexadecadeoxyribonucleotide antisense to sequences -22 to -17 of PAI-1 mRNA coding for a fragment of the signal peptide, on the expression of PAI-1 in endothelial cells, and physiological consequences of the subsequently reduced PAI-1 activity tested in vitro and in vivo, were described in our previous studies. Of particular interest was PO-16 5'-O-conjugated with menthyl phosphorothioate (MPO-16R). In this work, tissue localisation of MPO-16R labelled with [(35)S] phosphorothioate at the 3'-end, was determined. [(35)S]MPO-16R and control [(35)S]MPO-16R-SENSE oligonucleotides were administered intravenously into 22 rats and organ distribution of the labelled bioconjugates was assessed after 24 and 48 h. For this purpose, tissue sections were subjected to autoradiography, and quantitated by liquid scintillation after solubilisation. Overall clearance of radioactivity was already seen after 24 h, with the radioactivity recovered mainly in the kidney and liver. A smaller fraction of radioactivity was also retained in the spleen and heart. The kidney concentration of the labelled probe was higher than that of liver by 50%. The distribution of PAI-1 mRNA in untreated rat kidney, liver, spleen and heart established by two independent techniques: Ribonuclease Protection Assay and Real-Time PCR, shows the same pattern as that observed for [(35)S]MPO-16R antisense.

scholarly journals Intrafetal Insulin-Like Growth Factor-I Infusion Stimulates Adrenal Growth But Not Steroidogenesis in the Sheep Fetus during Late Gestation

Endocrinology ◽  
2007 ◽  
Vol 148 (11) ◽  
pp. 5424-5432 ◽  
Author(s):  
J. T. Ross ◽  
I. C. McMillen ◽  
F. Lok ◽  
A. G. Thiel ◽  
J. A. Owens ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Adrenal Cortex ◽  
Plasma Cortisol ◽  
Zona Glomerulosa ◽  
Late Gestation ◽  
Zona Fasciculata ◽  
Synthetic Enzymes ◽  
Fetal Plasma ◽  
Igf I ◽  
Sheep Fetus

We investigated the effects of an intrafetal infusion of IGF-I on adrenal growth and expression of the adrenal steroidogenic and catecholamine-synthetic enzyme mRNAs in the sheep fetus during late gestation. Fetal sheep were infused for 10 d with either IGF-I (26 μg/kg·h; n = 14) or saline (n = 10) between 120 and 130 d gestation, and adrenal glands were collected for morphological analysis and determination of the mRNA expression of steroidogenic and catecholamine-synthetic enzymes. Fetal body weight was not altered by IGF-I infusion; however, adrenal weight was significantly increased by 145% after IGF-I infusion. The density of cell nuclei within the fetal adrenal cortex (the zona glomerulosa and zona fasciculata), and within the adrenaline synthesizing zone of the adrenal medulla, was significantly less in the IGF-I-infused fetuses compared with the saline-infused group. Thus, based on cell-density measurements, there was a significant increase in cell size in the zona glomerulosa and zona fasciculata of the adrenal cortex and in the adrenaline-synthesizing zone of the adrenal medulla. There was no effect of IGF-I infusion on the adrenal mRNA expression of the steroidogenic or catecholamine-synthetic enzymes or on fetal plasma cortisol concentrations. In summary, infusion of IGF-I in late gestation resulted in a marked hypertrophy of the steroidogenic and adrenaline-containing cells of the fetal adrenal in the absence of changes in the mRNA levels of adrenal steroidogenic or catecholamine-synthetic enzymes or in fetal plasma cortisol concentrations. Thus, IGF-I infusion results in a dissociation of adrenal growth and function during late gestation.

scholarly journals Differential Expression of Individual UT-A Urea Transporter Isoforms in Rat Kidney

2000 ◽  
Vol 11 (11) ◽  
pp. 1980-1986
Author(s):  
SERENA M. BAGNASCO ◽  
TAO PENG ◽  
YUSHI NAKAYAMA ◽  
JEFF M. SANDS
Keyword(s):  
Mrna Expression ◽  
Differential Expression ◽  
Rat Kidney ◽  
Northern Hybridization ◽  
Ribonuclease Protection Assay ◽  
Urea Transporter ◽  
Mrna Isoforms ◽  
Rapid Amplification ◽  
The One ◽  
Ribonuclease Protection

Abstract. The rat renal urea transporter UT-A includes four mRNA isoforms: UT-A1, UT-A2, UT-A3, and UT-A4. This study detected by rapid amplification of cDNA ends (RACE), primer extension, and ribonuclease protection assay (RPA) a single transcription start site for UT-A1, UT-A3, and UT-A4, distinct from the one for UT-A2 and identified by 3′-RACE new transcripts of UT-A1, UT-A2, and UT-A3, characterized by alternative 3′ untranslated sequences (UTR). Expression of an alternative 3′UTR resulted in UT-A1 and UT-A2 transcripts that are approximately 400 bp shorter than the original cDNA. These mRNA isoforms (UT-A1b and UT-A2b) were present in low abundance in the inner medulla. Expression of an alternative 3′UTR for UT-A3 resulted in a 3.5 kb transcript (UT-A3b), which is 1.5 kb longer than the original UT-A3 cDNA. UT-A3b mRNA was easily detected by Northern hybridization in the inner medulla. This study examined whether different states of hydration induce homogeneous changes in mRNA expression of individual UT-A isoforms in the kidney. Analysis of UT-A1, UT-A1b, UT-A2, UT-A2b, UT-A3, and UT-A3b mRNA expression in rat kidney revealed that water deprivation markedly increases the relative abundance of UT-A2, UT-A2b, UT-A3, and UT-A3b mRNA in renal inner medulla, whereas UT-A1 and UT-A1b remain almost unchanged. The conclusion is that differential expression of individual UT-A mRNA isoforms occurs in the kidney and probably involves multiple regulatory mechanisms.

scholarly journals Ultrastructural and biochemical evidence for a steroid-containing secretory organelle in the perfused cat adrenal gland.

1977 ◽  
Vol 72 (1) ◽  
pp. 209-215 ◽  
Author(s):  
R T Gemmell ◽  
S G Laychock ◽  
R P Rubin
Keyword(s):  
Adrenal Gland ◽  
Cortical Cell ◽  
Underlying Mechanism ◽  
Acth Stimulation ◽  
Cortical Cells ◽  
Fourfold Increase ◽  
Dense Granules ◽  
Golgi Region ◽  
Close Proximity

A correlative study of the ultrastructural and biochemical effects of ACTH on fasciculata cells was carried out on the isolated cat adrenal gland perfused in situ with Locke's solution. The outstanding morphologic feature of cortical cells exposed to microunit ACTH concentrations for 40 min was the abundance of electron-dense granules (0.2-0.4 mum). These organelles were observed in small groups in close proximity to the Golgi region and to the cell membrane. Morphometric and biochemical analysis of control and ACTH-treated glands demonstrated that ACTH stimulation was associated with a fourfold increase in the number of these granules and a comparable increase in the corticosteroid content of the gland. By contrast, ACTH failed to augment cortical lysosomal enzyme activity. These findings, which link steroid release to the appearance of intracellular granules, extend further the parallels between the mechanism of release of newly synthesized steroid and the release of preformed hormones stored in secretory organelles. These results also lend support to the concept that a process related to exocytosis may be the underlying mechanism for extruding steroid from the cortical cell.

Cobalt exerts opposite effects on erythropoietin gene expression in rat hepatocytes in vivo and in vitro

1995 ◽  
Vol 269 (5) ◽  
pp. R995-R1001
Author(s):  
T. Gopfert ◽  
K. U. Eckardt ◽  
B. Gess ◽  
A. Kurtz
Keyword(s):  
Gene Expression ◽  
Oxygen Sensor ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Ribonuclease Protection Assay ◽  
Mrna Levels ◽  
Adult Rats ◽  
Ribonuclease Protection

This study investigates the effects of hypoxia and of cobalt on erythropoietin (EPO) gene expression in hepatocytes in vivo and in vitro in neonatal, juvenile, and adult rats. With the use of the ribonuclease protection assay to quantify RNA, both hypoxia (0.1% CO or 9% O2) and cobalt (60 mg/kg) elicit production of increased amounts of EPO mRNA in neonatal and juvenile rat liver in vivo. In vitro hepatocyte EPO gene expression could be reproducibly stimulated by hypoxia (3% O2) but not by cobaltous chloride (50-150 microM) within 2-20 h. Conversely, cobalt substantially attenuated the rise of EPO mRNA levels in response to hypoxia. This inhibitory effect of cobalt was mimicked by zinc but not by other metals. CO attenuated the rise of EPO mRNA levels in vitro in response to hypoxia; this inhibitory effect coincided with an inhibition of total RNA synthesis as determined by [3H]uridine incorporation. The lack of specific inhibitory effects of CO and of specific stimulatory effects of cobalt on hepatocyte EPO gene expression in vitro suggests that a specific heme oxygen sensor may be less important than in hepatoma cells.

SOME ASPECTS OF THE CONTROL OF DEHYDROEPIANDROSTERONE SYNTHESIS IN THE HUMAN ADRENAL GLAND IN VITRO

1972 ◽  
Vol 55 (2) ◽  
pp. 311-321 ◽  
Author(s):  
VIBEKE JENSEN ◽  
PAMELA CARSON ◽  
N. DESHPANDE
Keyword(s):  
Adrenal Gland ◽  
Competitive Inhibition ◽  
Tissue Concentration ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Maximum Velocity ◽  
Chain Cleavage ◽  
17 Hydroxyprogesterone ◽  
Reduced Nicotinamide Adenine Dinucleotide

SUMMARY The control of dehydroepiandrosterone (DHA) synthesis in the human adrenal gland was studied by investigating the biosynthesis of its immediate precursor, 17-hydroxypregnenolone (3β,17α-dihydroxy-pregn-5-en-20-one) and the side-chain cleavage. Both conversions take place in the microsomal fraction and there is an obligatory requirement for the co-factor, reduced nicotinamide adenine dinucleotide phosphate (NADPH). Under optimal conditions, 17-hydroxylation is controlled only by the availability of the substrate and the co-factor. By using the same kinetic approach it was observed that the synthesis of DHA is controlled by the availability of substrate and co-factor. However, the reaction was inhibited by the oxidized form (NADP+) and when the ratio of NADP+:NADPH reached 1, the maximum velocity was halved. Two major metabolites of 17-hydroxypregnenolone, namely DHA and 17-hydroxyprogesterone, are non-competitive inhibitors of the reaction. The inhibition constants are of the magnitude of the tissue concentration of these steroids in the adrenal gland which suggests that non-competitive inhibition takes place in vivo.

scholarly journals Distribution, Neuronal Colocalization, and 17β-E2 Modulation of Small Conductance Calcium-Activated K+ Channel (SK3) mRNA in the Guinea Pig Brain

Endocrinology ◽  
2002 ◽  
Vol 143 (3) ◽  
pp. 1097-1107 ◽  
Author(s):  
Martha A. Bosch ◽  
Martin J. Kelly ◽  
Oline K. Rønnekleiv
Keyword(s):  
In Situ Hybridization ◽  
Guinea Pig ◽  
Mrna Expression ◽  
Negative Feedback ◽  
Ribonuclease Protection Assay ◽  
Protection Assay ◽  
Ribonuclease Protection ◽  
Vasopressin Neurons ◽  
Small Conductance

Abstract Molecular cloning has revealed the existence of three distinct small conductance (SK1–3) Ca2+-activated K+ channels. Because SK channels underlie the afterhyperpolarization (AHP) that is critical for sculpturing phasic firing in hypothalamic neurons, we investigated the distribution of these channels in the female guinea pig. Both SK1 and SK3 cDNA fragments were cloned using PCR, and ribonuclease protection assay as well as in situ hybridization analysis illustrated that the SK3 channel was the predominant subtype expressed in the guinea pig hypothalamus. Combined in situ hybridization and fluorescence immunocytochemistry revealed that SK3 mRNA was expressed in GnRH, dopamine, and vasopressin neurons, and all of these neurons exhibited an AHP current. Moreover, SK3 mRNA was found in other brain areas, including the septum, bed nucleus, amygdala, thalamus, midbrain, and hippocampus. Using quantitative ribonuclease protection assay, the rank order of SK3 mRNA expression was septum ≥ midbrain &gt; rostral thalamus ≥ rostral basal hypothalamus ≥ caudal thalamus ≥ preoptic area ≫ caudal basal hypothalamus ≥ hippocampus. Moreover, 17β-E2 treatment, which reduces plasma LH during the negative feedback phase, significantly increased SK3 mRNA levels in the rostral basal hypothalamus (P &lt; 0.05; n = 6). Therefore, these findings suggest that estrogen increases the mRNA expression of SK3 channels, which may represent a mechanism by which estrogen regulates hypothalamic neuronal excitability during negative feedback.

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