Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The DHD/K12/PROb rat colonic epithelial cell line, which was originally derived from a chemically induced adenocarcinoma, expresses functional glucocorticoid receptors (GR) and has been reported to be growth inhibited by glucocorticoid agonists. In the present study the potential mechanisms underlying corticosteroid-mediated autoregulation of GR mRNA levels in this colonic cell line were investigated. The GR mRNA levels in the various treatment groups were quantitated via the ribonuclease protection assay using a specific 32P-cRNA probe. Time-course experiments demonstrated that in contrast to several other cell lines that are also growth inhibited by glucocorticoids, treatment of confluent monolayers of PROb cells with the pure GR agonist RU 28362 (1 microM) elicits a rapid and significant (65%) down-regulation of GR mRNA levels that is sustained for at least 36 h. This down-regulation, which is also elicited to a lesser extent by weaker GR agonists including corticosterone and aldosterone, is blocked by the GR antagonist RU 38486. The protein synthesis inhibitor cycloheximide was utilized to demonstrate that the initial phase (6 h) of agonist-mediated down-regulation occurs independently of ongoing protein synthesis, although new protein synthesis, perhaps of the GR protein itself, is required to maintain this down-regulation. Although agonist-mediated down-regulation in these cells probably occurs primarily at the level of GR gene transcription, inhibition of ongoing RNA synthesis with actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) demonstrated that during the initial phase (1 h) of this down-regulation, but not following maximal (18 h) down-regulation, RU 28362 treatment also significantly reduces the stability of the GR mRNA.
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PMID:Potential mechanisms underlying autoregulation of glucocorticoid receptor mRNA levels in the DHD/K12/PROb rat colonic adenocarcinoma cell line. 749 1

The effects of different steroids on the expression of angiotensin AT1 receptors by the human hepatoma cell line, PLC-PRF-5 was studied. Dexamethasone and aldosterone decreased the specific binding of [3H]angiotensin II to intact PLC-PRF-5 cells by 57 +/- 4% and 54 +/- 2%, respectively, compared to control, untreated cells. EC50 values for dexamethasone, cortisol and aldosterone were 1.8 +/- 0.6, 40 +/- 6, and 310 +/- 20 nM, respectively, suggesting that these effects were mediated via a glucocorticoid receptor. Scatchard analysis revealed that dexamethasone decreased the number of angiotensin AT1 receptors expressed (50 +/- 4% relative to control) with no change in receptor affinity. Treating cells with dexamethasone in the presence of either an angiotensin converting enzyme inhibitor or an angiotensin II receptor antagonist did not prevent the reduction in angiotensin AT1 receptor expression, ruling out a mechanism involving a dexamethasone induced increase in endogenous angiotensin II production. A ribonuclease protection assay established that the steady state level of angiotensin AT1 receptor mRNA in dexamethasone treated cells was reduced to 34.7 +/- 8.4% of untreated cells. The decrease in the number of angiotensin AT1 receptors expressed on the cell surface after treatment with dexamethasone therefore seems likely to reflect the decreased steady state level of the mRNA coding for this receptor.
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PMID:Glucocorticoids regulate the expression of angiotensin AT1 receptors, in the human hepatoma cell line, PLC-PRF-5. 777 81

Glucocorticoids and mineralocorticoids exert profound effects on electrolyte balance in the rat distal colon by binding with high affinity and specificity to intracellular receptor proteins, termed glucocorticoid (GR) and mineralocorticoid (MR) receptors, respectively. Hormonal regulation of GR and MR expression represents an important mechanism for maintaining appropriate tissue sensitivity to these two classes of adrenocorticosteroids. In the present study the corticosteroid regulation of the expression of these two rat colonic receptors has been evaluated at the protein and mRNA levels. Preliminary experiments demonstrated that colonic MR and GR binding levels are significantly increased (approximately 60%) after adrenalectomy (14 days). Experiments were therefore performed to test the hypothesis that these increases in binding levels correlate with increased GR and MR mRNA and protein levels. Receptor mRNA levels were quantitated via ribonuclease protection assays using [32P]cRNA probes specific for either GR or MR mRNA, and GR and MR protein levels were quantitated via Western immunoblotting using the anti-GR BuGR2 and anti-MR hMRsN antibodies. Fourteen days after adrenalectomy, significant increases in GR mRNA (2.1-fold) and protein levels (1.6-fold) were detected. In contrast, neither MR mRNA nor protein levels were up-regulated by removal of endogenous corticosteroids. Additionally, GR and MR mRNA levels were measured after ip injection of adrenalectomized rats with pharmacological doses of either the pure glucocorticoid agonist RU 28362 or the mineralocorticoid agonist aldosterone in combination with the glucocorticoid antagonist RU 38486 (blocks aldosterone binding to the GR). Multiple ip injections of RU 28362 resulted in a significant decrease (80%) in GR mRNA levels without affecting MR mRNA levels. In contrast, multiple ip injections of aldosterone (plus RU 38486) had no effect on either MR or GR mRNA levels. A single ip injection of RU 28362 resulted in a detectable decrease (30%) in GR mRNA levels and again had no effect on MR mRNA levels. Although a single ip injection of aldosterone did not down-regulate MR mRNA levels, it did elicit a significant decrease (45%) in GR mRNA levels. Taken collectively, these data demonstrate that rat colonic GR and MR mRNA and protein levels are differentially up-regulated after removal of endogenous corticosteroids. Additionally, these data demonstrate that in response to its cognate ligand, the GR is capable of homologously down-regulating its own mRNA and protein levels. However, in response to its cognate ligand, the MR appears incapable of homologously down-regulating its own mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:In contrast to glucocorticoid receptors, mineralocorticoid receptors are not autoregulated in rat distal colon epithelia. 811 55

K+ homeostasis depends on K+ absorption in digestive and renal epithelia. Recently, a cDNA encoding for a putative K(+)-adenosinetriphosphatase (ATPase) alpha-subunit has been characterized. We studied its expression by ribonuclease protection assay and in situ hybridization in the distal colon and the kidney of rats in various physiological states. In the distal colon of control rats, high expression of the colonic putative K(+)-ATPase mRNA was restricted to the surface epithelial cells. A low-K+ diet did not modify this expression, adrenalectomy decreased it, and aldosterone or dexamethasone treatment for 2 days restored normal levels. In the kidney of control rats, levels of K(+)-ATPase mRNA were very low. A low-K+ diet revealed a clear mRNA expression, which is consistent with a recent report [J.A. Kraut, F. Starr, G. Sachs, and M. Reuben. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F581-F587, 1995]. This expression was restricted to the outer medullary collecting duct, presumably in principal cells. Changes in corticosteroid status did not influence the renal expression. Our results, together with previous studies on K+ absorption and K(+)-ATPase activity, suggest that more than a single molecular form of K(+)-ATPase is likely to be responsible for the regulation of K+ absorption in the colon and distal nephron.
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PMID:Differential regulation of putative K(+)-ATPase by low-K+ diet and corticosteroids in rat distal colon and kidney. 877 35

In humans the last steps in the synthesis of aldosterone and cortisol rely on the activity of two cytochrome P450 genes termed CYP11B2 (aldosterone synthase; P450aldo) and CYP11B1 (11 beta hydroxylase; P450cl1). The mechanisms which lead to differential expression of these two genes within the adrenal cortex are not well-defined. The human adrenocortical cell line. H295R, was utilized in this study to examine the intracellular second messenger pathways regulating expression of P450aldo and P450c11. using specific ribonuclease protection assays. Treatment of H295R cells with angiotensin II or potassium (K+) caused a time-dependent induction in the level of P450aldo transcripts. While K+ treatment was more specific for the induction of P450aldo mRNA, treatment with angiotensin II increased levels of both P450aldo and P450c11 transcripts. To define the second messenger systems which influence transcript levels for these enzymes, the effects of agonists of the protein kinase A, protein kinase C, and calcium pathways were tested on the expression of P450aldo and P450c11. Activation of the protein kinase A pathway by the agonists, dibutyryl cAMP or forskolin, preferentially increased the P450c11 transcript to a greater degree than P450aldo. Interestingly, activation of the protein kinase C pathway by tetradecanoylphorbol acetate (TPA) did not alter transcripts for either P450aldo or P450c11. The calcium channel agonist BAYK 8644 mimicked the effects of K+ by increasing the transcript for P450aldo. However, the calcium channel blocker nifedipine attenuated the stimulatory effects of angiotensin II and K+ on the levels of P450aldo. However, the calcium channel blocker nifedipine attenuated the stimulatory effects of angiotensin II and K+ on the levels of P450aldo transcripts without affecting the stimulatory effect of dbcAMP. This study demonstrates that the protein kinase A pathway preferentially induces P450c11 mRNA over that of P450aldo. In addition, pharmacologic agents that affect calcium levels provide evidence for an additional regulatory mechanism in modulating the expression of P450aldo. This is of importance since the major physiologic regulators of aldosterone secretion, angiotensin II and K+ are able to increase intracellular calcium but have little effect on intracellular cAMP levels.
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PMID:Differential regulation of 11 beta-hydroxylase and aldosterone synthase in human adrenocortical H295R cells. 886 69

Hormones can regulate the expression of their own receptor. We have examined whether adrenalectomy (ADX) and hormone replacement by physiological doses of aldosterone or dexamethasone could modulate the expression of glucocorticoid receptor (GR) or mineralocorticoid receptor (MR) at the mRNA level in the rat kidney, distal colon, and heart. Adult rats were adrenalectomized and received or did not receive an infusion of aldosterone (5 micrograms.100 g-1.day-1) or dexamethasone (10 micrograms.100 g-1.day-1). No significant change in steady-state levels of both MR and GR mRNA was detectable by using ribonuclease (RNase) protection assay (RPA) after either ADX or hormone replacement. Because the kidney is heterogeneous with regard to MR expression, RPA was adapted for measurements on microdissected nephron segments. GR mRNA is expressed at comparable levels all along the nephron, whereas MR mRNA is restricted to the distal nephron. No effect of ADX or GR and MR mRNA levels was detected in any nephron segment that was either aldosterone sensitive or insensitive. In situ hybridization confirmed the absence of corticosteroid-dependent modulation of MR mRNA in all kidney cell types. We conclude that variations of corticosteroid status do not affect MR and GR mRNA steady-state levels in heart, colon, and kidney and thus do not participate to the functional adaptations that are known to depend on hormonal status.
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PMID:Corticosteroid receptor mRNA expression is unaffected by corticosteroids in rat kidney, heart, and colon. 896 34

We compared two models of cardiac fibrosis in which collagen synthesis is controlled at different levels. Regulation is pretranslational in aldosterone-salt-induced hypertension in young rats and posttranslational in 24-month-old rats. However, little is known about the role of matrix metalloproteinases (MMP) in fibrosis development. Ventricular MMP activities were studied by zymography, and MMP-2 and MMP-1 mRNA levels were determined using slot-blot and ribonuclease protection assay, respectively. After 1 month of aldosterone-salt treatment, proMMP-2, MMP-2, and proMMP-1 collagenolytic activities and their gene expression were unchanged compared with sham-operated rats. After 2 months, total MMP-2 activity was increased by 40% with parallel stimulation of its gene expression. These changes were localized by in situ zymography within the media of coronary vessels. These results suggest that MMP play a prominent role in vascular remodeling during the first steps of hypertension. During aging, however, there were 40% and 45% decreases in MMP-2 and proMMP-1 activity, respectively, with a corresponding down-regulation of MMP-2 mRNA. These observations suggest that depression of the degradative pathway is partly responsible for age-associated fibrosis. Thus, MMP have differing involvements in the cardiac remodeling associated with hypertension or aging.
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PMID:Differential regulation of matrix metalloproteinases associated with aging and hypertension in the rat heart. 916 91

Distal colon and renal cortical collecting ducts are major effectors of aldosterone-dependent Na homeostasis. Na is absorbed by entry through an apical amiloride-sensitive Na channel and extruded by Na-K-ATPase at the basolateral membrane. Using a ribonuclease protection assay, we studied, in vivo, aldosterone regulation of alpha-, beta-, gamma-subunits of the rat epithelial Na channel (rENaC) and alpha 1- and beta 1-subunits of Na-K-ATPase. In the kidney, Na-K-ATPase mRNAs were also assayed over discrete tubular segments by in situ hybridization. In rat colon, all three rENaC mRNAs were decreased by adrenalectomy, with a major effect on beta- and gamma-subunits, and were restored with 7 days, but not 2 days, of aldosterone treatment; in the kidney, however, only alpha-transcripts varied. Na-K-ATPase alpha 1- and beta 1-subunit mRNAs in both organs were not (in the case of the beta 1-subunit) or were mildly (in the case of the alpha 1-subunit) affected after adrenalectomy. Our conclusions are as follows: 1) Transcripts of rENaC and Na-K-ATPase subunits are not coordinately regulated by aldosterone in vivo; i.e., modulation involves mainly the Na channel, not Na-K-ATPase; the effect is not of comparable magnitude on each subunit mRNA and differs between tissues. 2) The delay of the aldosterone effect on transcripts is much longer than that required to restore normal Na transport in adrenalectomized rats, indicating that rENaC and Na-K-ATPase subunit transcript levels may depend on unidentified early aldosterone-induced proteins.
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PMID:Noncoordinate regulation of epithelial Na channel and Na pump subunit mRNAs in kidney and colon by aldosterone. 917 38

The main objective of this study was to find out if the reported changes in the aldosterone-suppressant activity of atrial natriuretic peptide (ANP) during different hormonal states in rats are due to a modulation of ANP receptors. In zona glomerulosa cells, ribonuclease protection assay detected mRNAs for guanylate cyclase (GC)-coupled ANP GC-A and GC-B receptors, and for ANP C receptors, which are not coupled to GC. Western analysis using polyclonal anti-GC-A and anti-GC-B receptor antibodies revealed the presence of GC-A but not GC-B receptor proteins in zona glomerulosa cells. Pregnancy (days 7, 16 and 21), oestradiol-17 beta and progesterone decreased mRNAs for all the three ANP receptors in zona glomerulosa cells. Pregnancy decreased GC-A receptor proteins in zona glomerulosa cells, but these recovered to virgin values on day 2 postpartum. ANP receptor mRNAs in zona glomerulosa cells increased by postpartum day 2, but did not reach the values found in virgin rats. Zona fasciculata mainly contained GC-A receptor mRNA. It is concluded that ANP receptors in rat adrenal zona glomerulosa are modulated by pregnancy, oestrogen and progesterone; a decrease in ANP GC-A receptors during pregnancy might explain the accompanying decrease in the aldosterone-suppressant effects of ANP.
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PMID:Downregulation of adrenal atrial natriuretic peptide receptor mRNAs and proteins by pregnancy in rats. 948 97

Aldosterone acts via mineralocorticoid receptors (MRs) to control salt and water flux in epithelial organs such as the kidney and colon to maintain circulatory homeostasis. Inappropriate glucocorticoid-mediated activation of MRs in aldosterone-target tissues is prevented by the glucocorticoid-metabolizing enzyme 11beta-hydroxysteroid dehydrogenase type 2 (HSD2). We have studied HSD2 expression in the mouse at the level of gene transcription and further analyzed, with HSD1, its pattern of tissue-restricted gene expression. The mouse HSD2 gene, including upstream regulatory regions, has been cloned, and its transcription start site has been mapped in colon and kidney. A 2.5 kb upstream region has been sequenced, and its proximal promoter region has been analyzed. We have compared the relative expression of HSD1 and HSD2 in a variety of tissues from male mice using ribonuclease protection analysis. HSD1 was expressed in liver, kidney, adrenal, lung, spleen, thymus, fat, small intestine, stomach, heart, skin, and epididymis. HSD2 was expressed in kidney, colon, small intestine, stomach, and epididymus. No expression of either HSD1 and HSD2 was detected in bladder, testis, seminal vesicles, vas deferens, prostate, or skeletal muscle. Finally, to investigate the specific roles of HSD2 in vivo, we have created "floxed" HSD2 alleles using gene targeting in mouse embryonic stem cells with the aim to create tissue-specific ablation of HSD2 in mice via Cre recombinase mediated gene targeting.
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PMID:Expression of the 11beta-hydroxysteroid dehydrogenase 2 gene in the mouse. 1076 59


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