Regulation of sgk by aldosterone and its effects on the epithelial Na+ channel

2000 ◽  
Vol 278 (4) ◽  
pp. F613-F619 ◽  
Author(s):  
Alexander Shigaev ◽  
Carol Asher ◽  
Hedva Latter ◽  
Haim Garty ◽  
Eitan Reuveny
Keyword(s):  
De Novo ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Na Channel ◽  
Xenopus Laevis Oocytes ◽  
Fourfold Increase ◽  
Tight Epithelia ◽  
High Level

Aldosterone is the major corticosteroid regulating Na+ absorption in tight epithelia and acts primarily by activating the epithelial Na+ channel (ENaC) through unknown induced proteins. Recently, it has been reported that aldosterone induces the serum- and glucocorticoid-dependent kinase sgk and that coexpressing ENaC with this kinase in Xenopus laevis oocytes increases the amiloride-sensitive Na+current (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Proc Natl Acad Sci USA 96: 2514–2519, 1999). The present study was done to further characterize regulation of sgk by aldosterone in native mammalian epithelia and to examine its effect on ENaC. With both in vivo and in vitro protocols, an almost fivefold increase in the abundance of sgk mRNA has been demonstrated in rat kidney and colon but not in lung. Induction of sgk by aldosterone was detected in kidney cortex and medulla, whereas the papilla expressed a constitutively high level of the kinase. The increase in sgkmRNA was detected as early as 30 min after the hormonal application and was independent of de novo protein synthesis. The observed aldosterone dose-response relationships suggest that the response is mediated, at least in part, by occupancy of the mineralocorticoid receptor. Coexpressing sgk and ENaC in Xenopus oocytes evoked a fourfold increase in the amiloride-blockable Na+ channel activity. A point mutation in the β-subunit known to impair regulation of the channel by Nedd4 (Y618A) had no significant effect on the response to sgk.

scholarly journals Distribution and vulnerability of transcriptional outputs across the genome in Myc-amplified medulloblastoma cells

2021 ◽  
Author(s):  
Rui Yang ◽  
Wenzhe Wang ◽  
Meichen Dong ◽  
Kristen Roso ◽  
Paula Greer ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Tumor Cells ◽  
Target Genes ◽  
De Novo ◽  
Inhibitory Effect ◽  
Nucleotide Synthesis ◽  
High Level ◽  
The Impact

Myc plays a central role in tumorigenesis by orchestrating the expression of genes essential to numerous cellular processes1-4. While it is well established that Myc functions by binding to its target genes to regulate their transcription5, the distribution of the transcriptional output across the human genome in Myc-amplified cancer cells, and the susceptibility of such transcriptional outputs to therapeutic interferences remain to be fully elucidated. Here, we analyze the distribution of transcriptional outputs in Myc-amplified medulloblastoma (MB) cells by profiling nascent total RNAs within a temporal context. This profiling reveals that a major portion of transcriptional action in these cells was directed at the genes fundamental to cellular infrastructure, including rRNAs and particularly those in the mitochondrial genome (mtDNA). Notably, even when Myc protein was depleted by as much as 80%, the impact on transcriptional outputs across the genome was limited, with notable reduction mostly only in genes involved in ribosomal biosynthesis, genes residing in mtDNA or encoding mitochondria-localized proteins, and those encoding histones. In contrast to the limited direct impact of Myc depletion, we found that the global transcriptional outputs were highly dependent on the activity of Inosine Monophosphate Dehydrogenases (IMPDHs), rate limiting enzymes for de novo guanine nucleotide synthesis and whose expression in tumor cells was positively correlated with Myc expression. Blockage of IMPDHs attenuated the global transcriptional outputs with a particularly strong inhibitory effect on infrastructure genes, which was accompanied by the abrogation of MB cells proliferation in vitro and in vivo. Together, our findings reveal a real time action of Myc as a transcriptional factor in tumor cells, provide new insight into the pathogenic mechanism underlying Myc-driven tumorigenesis, and support IMPDHs as a therapeutic vulnerability in cancer cells empowered by a high level of Myc oncoprotein.

scholarly journals Aldosterone rapidly activates p-PKC delta and GPR30 but suppresses p-PKC epsilon protein levels in rat kidney

2019 ◽  
Vol 53 (3) ◽  
pp. 154-164 ◽  
Author(s):  
Somchit Eiam-Ong ◽  
Mookda Chaipipat ◽  
Krissanapong Manotham ◽  
Somchai Eiam-Ong
Keyword(s):  
Rat Kidney ◽  
Kidney Cortex ◽  
Protein Abundance ◽  
Protein Levels ◽  
Pkc Delta ◽  
Pkc Epsilon ◽  
Male Wistar Rats ◽  
Pkc Alpha

AbstractObjectives. Aldosterone rapidly enhances protein kinase C (PKC) alpha and beta1 proteins in the rat kidney. The G protein-coupled receptor 30 (GPR30)-mediated PKC pathway is involved in the inhibition of the potassium channel in HEK-239 cells. GPR30 mediates rapid actions of aldosterone in vitro. There are no reports available regarding the aldosterone action on other PKC isoforms and GPR30 proteins in vivo. The aim of the present study was to examine rapid actions of aldosterone on protein levels of phosphorylated PKC (p-PKC) delta, p-PKC epsilon, and GPR30 simultaneously in the rat kidney.Methods. Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (150 µg/kg body weight). After 30 minutes, abundance and immunoreactivity of p-PKC delta, p-PKC epsilon, and GPR30 were determined by Western blot analysis and immunohisto-chemistry, respectively.Results. Aldosterone administration significantly increased the renal protein abundance of p-PKC delta by 80% (p<0.01) and decreased p-PKC epsilon protein by 50% (p<0.05). Aldosterone injection enhanced protein immunoreactivity of p-PKC delta but suppressed p-PKC epsilon protein intensity in both kidney cortex and medulla. Protein abundance of GPR30 was elevated by aldosterone treatment (p<0.05), whereas the immunoreactivity was obviously changed in the kidney cortex and inner medulla. Aldosterone translocated p-PKC delta and GPR30 proteins to the brush border membrane of proximal convoluted tubules.Conclusions. This is the first in vivo study simultaneously demonstrating that aldosterone administration rapidly elevates protein abundance of p-PKC delta and GPR30, while p-PKC epsilon protein is suppressed in rat kidney. The stimulation of p-PKC delta protein levels by aldosterone may be involved in the activation of GPR30.

Psoralen photoactivation promotes morphological and functional changes in fibroblasts in vitro reminiscent of cellular senescence

1998 ◽  
Vol 111 (6) ◽  
pp. 759-767
Author(s):  
G. Herrmann ◽  
P. Brenneisen ◽  
M. Wlaschek ◽  
J. Wenk ◽  
K. Faisst ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Molecular Data ◽  
Cellular Aging ◽  
Premature Aging ◽  
Functional Changes ◽  
Uva Irradiation ◽  
High Level

Premature aging of the skin is a prominent side effect of psoralen photoactivation, a treatment used widely for various skin disorders. The molecular mechanisms underlying premature aging upon psoralen photoactivation are as yet unknown. Here we show that treatment of fibroblasts with 8-methoxypsoralen (8-MOP) and subsequent ultraviolet A (UVA) irradiation resulted in a permanent switch of mitotic to stably postmitotic fibroblasts which acquired a high level of de novo expression of SA-beta-galactosidase, a marker for fibroblast senescence in vitro and in vivo. A single exposure of fibroblasts to 8-MOP/UVA resulted in a 5.8-fold up-regulation of two matrix-degrading enzymes, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), over a period of &gt;120 days, while TIMP-1, the major inhibitor of MMP-1 and MMP-3, was only slightly induced. This imbalance between matrix-degrading metalloproteases and their inhibitor may lead to connective tissue damage, a hallmark of premature aging. Superoxide anion and hydrogen peroxide, but not singlet oxygen, were identified as important intermediates in the downstream signaling pathway leading to these complex fibroblast responses upon psoralen photoactivation. Collectively, the end phenotype induced upon psoralen photoactivation shares several criteria of senescent cells. In the absence of detailed molecular data on what constitutes normal aging, it is difficult to decide whether the changes reported here reflect mechanisms underlying normal cellular aging/senescence or rather produce a mimic of cellular aging/senescence by quite different pathways.

scholarly journals BIOL-10. DISTRIBUTION AND VULNERABILITY OF TRANSCRIPTIONAL OUTPUTS ACROSS THE GENOME IN MYC-AMPLIFIED MEDULLOBLASTOMA CELLS

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i5-i5
Author(s):  
Rui Yang ◽  
Wenzhe Wang ◽  
Meichen Dong ◽  
Kristen Roso ◽  
Xuhui Bao ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Target Genes ◽  
De Novo ◽  
Inhibitory Effect ◽  
Nucleotide Synthesis ◽  
Expression Of Genes ◽  
High Level ◽  
The Impact

Abstract Myc plays a central role in tumorigenesis by orchestrating the expression of genes essential to numerous cellular processes. While it is well established that Myc functions by binding to its target genes to regulate their transcription, the distribution of the transcriptional output across human genome in Myc-amplified cancer cells, and the susceptibility of such transcriptional outputs to therapeutic interferences remain to be fully elucidated. Here, we analyze the distribution of transcriptional outputs in Myc-amplified medulloblastoma (MB) cells by profiling nascent total RNAs within a temporal context. This profiling reveals a major portion of transcriptional action in these cells was directed at the genes fundamental to cellular infrastructures, including rRNAs and particularly those in the mitochondrial genome (mtDNA). Notably, even when Myc protein was depleted by as much as 80%, the impact on transcriptional outputs across the genome was limited, with notable reduction mostly in genes of involved in ribosomal biosynthesis, genes residing in mtDNA or encoding mitochondria-localized proteins, and those encoding histones. In contrast to the limited direct impact of Myc depletion, we found that the global transcriptional outputs were highly dependent on the activity of Inosine Monophosphate Dehydrogenases (IMPDHs), rate limiting enzymes for de novo guanine nucleotide synthesis and whose expression in tumor cells was positively correlated with Myc’s expression. Blockage of IMPDHs attenuated the global transcriptional outputs with a particularly strong inhibitory effect on the aforementioned infrastructure genes, which was accompanied by the abrogation of MB cell’s proliferation in vitro and in vivo. Together, our findings reveal a real time action of Myc as a transcriptional factor in tumor cells, gain new insight into the pathogenic mechanism underlying Myc-driven tumorigenesis, and support IMPDHs as a therapeutic vulnerability in MB cells empowered by a high level of Myc oncoprotein.

scholarly journals Accumulation of Sulfate by Mitochondria of Rat Kidney Cortex

1962 ◽  
Vol 45 (4) ◽  
pp. 757-775 ◽  
Author(s):  
Robert W. Winters ◽  
Adelaide M. Delluva ◽  
Ingrith J. Deyrup ◽  
Robert E. Davies
Keyword(s):  
Rat Kidney ◽  
Kidney Cortex ◽  
Ph Optimum ◽  
Rat Kidney Cortex ◽  
Sulfate Ions ◽  
Straight Line ◽  
Freundlich Adsorption Isotherm ◽  
Ambient Concentrations

Twice washed mitochondria from rat kidney cortex can accumulate sulfate ions from low (10-7 M) ambient concentrations to create virtual gradients of several hundred to one. This sulfate is subsequently released. The activation energy for the uptake is 12,000 calories per mole; for release it is about 30,000 calories per mole. Variations in the sulfate concentration of the medium show that there is a straight line Freundlich adsorption isotherm over a million-fold range of concentration of sulfate in the medium. There are 9 x 104 sites at 10-5 M and 9 x 105 sites at 10-3 M sulfate per average single mitochondrion. Preincubation at 30°C rapidly destroys the ability to accumulate sulfate. Partial protection occurs if oxidative phosphorylation is proceeding during the preincubation. The concentration of the endogenous inorganic sulfate of twice washed mitochondria is 4.2 x 10-4 moles per liter of mitochondrial pellet water; 99.85 per cent of this endogenous sulfate is inexchangeable with external sulfate in vitro. It is all exchangeable in vivo. The pH optimum for accumulation of radiosulfate from dilute external sulfate concentrations is 5.5. These observations show that there is a delicate and specific mechanism in mitochondria from kidney cortex which accumulates sulfate. The chemical nature of the accumulated sulfate is unknown.

scholarly journals 3-Mercaptopicolinic acid, an inhibitor of gluconeogenesis

1974 ◽  
Vol 138 (3) ◽  
pp. 387-394 ◽  
Author(s):  
N. W. DiTullio ◽  
C. E. Berkoff ◽  
B. Blank ◽  
V. Kostos ◽  
E. J. Stack ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Diabetic Rats ◽  
Kidney Cortex ◽  
Hepatic Glycogen ◽  
Hypoglycaemic Effect ◽  
Rat Kidney Cortex ◽  
Urea Production ◽  
Rat Livers

1. 3-Mercaptopicolinic acid (SK&F 34288) inhibited gluconeogenesis in vitro, with lactate as substrate, in rat kidney-cortex and liver slices. 2. In perfused rat livers, gluconeogenesis was inhibited when lactate, pyruvate or alanine served as substrate, but not with fructose, suggesting pyruvate carboxylase or phosphoenolpyruvate carboxylase as the site of inhibition. No significant effects were evident in O2 consumption, hepatic glycogen, urea production, or [lactate]/[pyruvate] ratios. 3. A hypoglycaemic effect was evident in vivo in starved and alloxan-diabetic rats, starved guinea pigs and starved mice, but not in 4h-post-absorptive rats. 4. In the starved rat the hypoglycaemia was accompanied by an increase in blood lactate. 5. A trace dose of [14C]lactate in vivo was initially oxidized to a lesser extent in inhibitor-treated rats, but during 90min the total CO2 evolved was slightly greater. The total amount of the tracer oxidized was not significantly different from that in the controls.

Interaction with grp58 increases activity of the thiazide-sensitive Na-Cl cotransporter

2002 ◽  
Vol 282 (3) ◽  
pp. F424-F430 ◽  
Author(s):  
Bruce Wyse ◽  
Nawb Ali ◽  
David H. Ellison
Keyword(s):  
Rat Kidney ◽  
Glucose Deprivation ◽  
Kidney Cortex ◽  
Xenopus Laevis Oocytes ◽  
Rat Kidney Cortex ◽  
Sodium Uptake ◽  
Sodium Chloride Cotransporter ◽  
Important Interaction ◽  
Hybrid Screening

The thiazide-sensitive sodium-chloride cotransporter (NCC) is expressed by distal convoluted tubule cells of the mammalian kidney. We used yeast two-hybrid screening to identify that glucose-regulated protein 58 (grp58), a protein induced by glucose deprivation, binds to the COOH terminus of the NCC. Immunoprecipitation of rat kidney cortex homogenates using a guinea pig anti-NCC antibody confirmed that grp58 associates with the NCC in vivo. Northern blots indicated that grp58 is highly expressed in rat kidney cortex. Immunofluorescence showed that grp58 protein abundance in kidney is highest in epithelial cells of the distal nephron, where it colocalizes with NCC near the apical membrane. To determine whether this interaction has a functional significance, NCC and grp58 cRNA were coexpressed in Xenopus laevis oocytes. In oocytes overexpressing grp58, sodium uptake was increased compared with control. Because oocytes express endogenous grp58, antisense experiments were performed to evaluate whether endogenous grp58 affected NCC activity in oocytes. Sodium uptake was lower in oocytes injected with both antisense grp58 cRNA and sense NCC compared with sense NCC oocytes. Western blot analysis did not show any effect of grp58 expression on processing of the NCC. These data indicate a novel, functionally important interaction between grp58 and the NCC in rat kidney cortex.

Regulatory Factors Associated with Synthesis of the Osmolyte Glycine Betaine in the Halophilic MethanoarchaeonMethanohalophilus portucalensis

1999 ◽  
Vol 65 (2) ◽  
pp. 828-833 ◽  
Author(s):  
Mei-Chin Lai ◽  
Daw-Renn Yang ◽  
Ming-Jen Chuang
Keyword(s):  
Glycine Betaine ◽  
De Novo ◽  
Compatible Solutes ◽  
In Vitro Assays ◽  
Intracellular Accumulation ◽  
De Novo Synthesis ◽  
Factors Associated ◽  
High Level

ABSTRACT The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize de novo and accumulate β-glutamine, N ɛ-acetyl-β-lysine, and glycine betaine (betaine) as compatible solutes (osmolytes) when grown at elevated salt concentrations. Both in vivo and in vitro betaine formation assays in this study confirmed previous nuclear magnetic resonance 13C-labelling studies showing that the de novo synthesis of betaine proceeded from glycine, sarcosine, and dimethylglycine to form betaine through threefold methylation. Exogenous sarcosine (1 mM) effectively suppressed the intracellular accumulation of betaine, and a higher level of sarcosine accumulation was accompanied by a lower level of betaine synthesis. Exogenous dimethylglycine has an effect similar to that of betaine addition, which increased the intracellular pool of betaine and suppressed the levels of N ɛ-acetyl-β-lysine and β-glutamine. Both in vivo and in vitro betaine formation assays with glycine as the substrate showed only sarcosine and betaine, but no dimethylglycine. Dimethylglycine was detected only when it was added as a substrate in in vitro assays. A high level of potassium (400 mM and above) was necessary for betaine formation in vitro. Interestingly, no methylamines were detected without the addition of KCl. Also, high levels of NaCl and LiCl (800 mM) favored sarcosine accumulation, while a lower level (400 mM) favored betaine synthesis. The above observations indicate that a high sarcosine level suppressed multiple methylation while dimethylglycine was rapidly converted to betaine. Also, high levels of potassium led to greater amounts of betaine, while lower levels of potassium led to greater amounts of sarcosine. This finding suggests that the intracellular levels of both sarcosine and potassium are associated with the regulation of betaine synthesis inM. portucalensis.

Effect on kidney S35O4 uptake of compounds related to SO4 transport and metabolism

1964 ◽  
Vol 207 (1) ◽  
pp. 84-88 ◽  
Author(s):  
Ingrith J. Deyrup
Keyword(s):  
Amino Acids ◽  
Rat Kidney ◽  
Point Of View ◽  
Transport Systems ◽  
Kidney Cortex ◽  
Cellular Transport ◽  
Rat Kidney Cortex ◽  
Aryl Sulfatase

Experiments have been carried out to test the effects on S35O4 accumulation by rat kidney cortex slices in vitro of 1) compounds known to affect renal SO4 reabsorption (thiosulfate, amino acids); 2) compounds secreted by the kidney, or known to affect specific cellular transport systems (including tetraethylammonium ions, guanidine, creatinine, carinamide, probenecid, phloretin, diethylstilbestrol, ethylenediaminetetraacetate sodium); and 3) compounds related to SO4 metabolism (aryl sulfatase substrates). Under the conditions of the experiment, net S35O4 uptake was depressed by thiosulfate, certain amino acids, carinamide, phloretin, diethylstilbestrol, and aryl sulfatase substrates. It was enhanced by ethylenediaminetetraacetate sodium. Other compounds were without effect. These results are discussed from the point of view of the possible relationship between SO4 accumulation in vitro and transport in vivo.

scholarly journals FURTHER OBSERVATIONS ON UPTAKE OF S35-LABELLED SULFATE BY RENAL TISSUE IN VITRO

1956 ◽  
Vol 39 (6) ◽  
pp. 893-908 ◽  
Author(s):  
Ingrith J. Deyrup
Keyword(s):  
Rat Kidney ◽  
Mammalian Species ◽  
Renal Tissue ◽  
Metabolic Inhibitors ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Lower Vertebrates ◽  
The One

Additional studies have been made of the accumulation of S35 by renal cortical tissue incubated in media containing radiosulfate. This process was found to occur in several mammalian species in addition to the rat, but was not observed as a significant occurrence in three species of lower vertebrates. In the case of rat renal tissue, S35 uptake was found to be sensitive to the pH and osmolar concentration of the medium. The character of the anions present in conjunction with K+ affected it as well. Various factors known to be related to in vitro accumulative processes, as well as to renal sulfate reabsorption by the intact dog, were tested on rat kidney cortex to assess the effect on radiosulfate uptake. In general, all substances tested (amino acids, metabolic intermediates, ATP, metabolic inhibitors, competitive inhibitors for PAH accumulation in vitro) were found to lessen S35 uptake, or to be without effect upon it. The one striking exception was phlorhizin, which enhanced markedly S35 uptake in vitro, as it does sulfate reabsorption in vivo. Some implications of these findings have been discussed.

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