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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activation of the renin-angiotensin system by sodium deficiency is associated with reciprocal changes in the expression of angiotensin II receptors in adrenal glomerulosa and vascular smooth muscle cells. The effects of dietary sodium changes on the expression of brain angiotensin receptor subtype 1 (AT1) mRNAs were examined in rats maintained on normal, low, and high sodium intake for 3 weeks. Plasma aldosterone and renin activity were elevated in rats maintained on a low salt diet compared with normal rats and were reduced in rats maintained on a high salt diet. These results are consistent with previous findings on the effects of altered dietary sodium on the renin-angiotensin system. The expression of AT1A and AT1B receptor subtype mRNAs was determined by quantitative reverse transcriptase-polymerase chain reaction during changes in sodium intake. The results revealed that sodium deprivation enhanced the expression of AT1B receptors in decorticated brains by 164% compared with high sodium intake. Conversely, high sodium diet increased the expression of AT1A receptors by 155% in the brain compared with low sodium intake. These data suggest that AT1A and AT1B receptors play reciprocal roles in central mechanisms for the control of fluid homeostasis. Further analysis of the molecular biology of angiotensin II receptor regulation in the brain may provide new insights into the interplay between the renin-angiotensin system and blood pressure regulation and also into the role of angiotensin II in the pathogenesis of essential hypertension.
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PMID:Regulation of angiotensin II receptors in rat brain during dietary sodium changes. 750 98

Mineralocorticoid receptors (MRs) are nonselective in vitro, binding corticosterone, cortisol, and aldosterone with similar affinity. In the distal nephron in vivo, MRs are selectively activated by aldosterone despite much higher glucocorticoid levels. This has been suggested to reflect the action of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which catalyzes rapid inactivation of corticosterone to 11-dehydrocorticosterone (cortisol to cortisone). However, cellular models of this effect have not been reported, and a recent study suggested that properties intrinsic to MR contribute to aldosterone selectivity. We have screened clonal mammalian cell lines for 11 beta-HSD activity. Pig kidney epithelial LLC-PK1 cells expressed by far the greatest 11 beta-HSD activity. In cell homogenates, this was NAD-dependent, with Km for corticosterone of 34.4 nM and cortisol of 89.7 nM. Intact LLC-PK1 cells showed similar apparent Km for corticosterone (13.9 nM) and cortisol (79.4 nM); only 11 beta-dehydrogenation was detected. These biochemical data indicate the expression of the type 2 isoform, 11 beta-HSD2. Using primers to conserved regions of 11 beta-HSD2, a reverse transcriptase-polymerase chain reaction product was obtained from LLC-PK1 cell RNA. Sequence analysis revealed close homology to previously cloned 11 beta-HSD2 cDNAs from several species. LLC-PK1 cell 11 beta-HSD activity was inhibited by carbenoxolone (IC50 approximately 10(-8) M) and high concentrations of estradiol or progesterone (10(-7) and 10(-6) M), but was induced at lower estradiol concentrations (10(-8) and 10(-9) M). To examine whether the 11 beta-HSD2 activity in LLC-PK1 cells regulates corticosterone access to MR, cells were transfected with the corticosteroid-inducible mouse mammary tumor virus long terminal repeat-luciferase reporter construct. Cell transfection by a lipofection method did not alter 11 beta-HSD activity in LLC-PK1 cells. LLC-PK1 cells expressed low levels of MR (13.9 fmol/mg protein, dissociation constant (Kd) 0.3 x 10(-9) M for aldosterone) and glucocorticoid receptors (GR; 18.5 fmol/mg protein, Kd 0.3 x 10(-9) M for dexamethasone). Transfection with mouse mammary tumor virus long terminal repeat-luciferase reporter construct alone suggested that the endogenous levels of MR and GR were insufficient to affect transcription. However, cotransfection of LLC-PK1 cells with pRShMR, an MR expression plasmid, allowed at least 50-fold induction of luciferase with 10(-8) M aldosterone; the ED50 0.3 x 10(-9) M closely reflects the in vitro affinity of MR for aldosterone. Corticosterone only weakly induced luciferase (maximum of 6-fold induction).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:LLC-PK1 cells model 11 beta-hydroxysteroid dehydrogenase type 2 regulation of glucocorticoid access to renal mineralocorticoid receptors. 758 9

The inactivation of physiological glucocorticoids by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) confers mineralocorticoid specificity to certain aldosterone target tissues. However, 11 beta-HSD activity in a human mineralocorticoid-responsive tissue has never been characterized. The present studies describe the features of 11 beta-HSD in the cultured human colonic epithelial cell line, T84. The 11 beta-HSD activity of T84 cells resided in the microsomal fraction and showed a marked preference for NAD rather than NADP as cofactor. NAD or NADP (200 microM) increased the conversion of corticosterone to 11-dehydrocorticosterone by 24.1 +/- 2.1 and 0.5 +/- 0.7 pmol.mg protein-1.20 min-1, respectively, indicating a > 40-fold preference for NAD vs. NADP. The Michaelis constant values for corticosterone and cortisol were 11.3 +/- 1.5 and 79.8 +/- 10 nM, respectively. The T84 11 beta-HSD was inhibited by 11-dehydrocorticosterone in a noncompetitive fashion [inhibition constant (Ki) = 180 +/- 9.6 nM] and by carbenoxolone in a competitive fashion (Ki = 17.4 +/- 1.3 nM). The expression of mineralocorticoid receptors in these cells was demonstrated by reverse transcriptase-polymerase chain reaction of mRNA isolated from T84 cells and by [3H]aldosterone binding studies. The coexpression of this NAD-dependent isoform of 11 beta-HSD and mineralocorticoid receptors is consistent with the view that the NAD-dependent isoform is responsible for the specificity of mineralocorticoid responses.
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PMID:NAD-dependent 11 beta-hydroxysteroid dehydrogenase in cultured human colonic epithelial cells. 761 67

Mineralocorticoid resistance (pseudohypoaldosteronism) is a rare condition first described in 1958 and associated with failure to thrive, salt wasting, and dehydration in infancy. In the index case it has previously been shown that binding of aldosterone to mineralocorticoid receptors in peripheral blood lymphocytes is absent; here, we report results of the molecular characterization of the mineralocorticoid receptor in this patient. Genomic DNA extracted from peripheral blood lymphocytes was subjected to Southern blot analysis after digestion with various restriction enzymes. There was no evidence of a major gene rearrangement or deletion. Oligonucleotide primers were designed on the basis of the published human complementary DNA sequence to cover the entire open reading frame of the mineralocorticoid receptor (MR). Total messenger ribonucleic acid (RNA) from lymphocytes was subjected to reverse transcription and amplification using the reverse transcriptase-polymerase chain reaction; the resulting fragments were then purified, subcloned, and sequenced. The patient showed no abnormality in the complementary DNA sequence corresponding to the open reading frame of the MR molecule compared with the published sequence. In addition, semiquantitative assessment of the patient's MR messenger RNA based on the reverse transcriptase-polymerase chain reaction technique suggested that he was producing MR RNA in roughly normal quantities. The mechanism of mineralocorticoid resistance in this case, therefore, remains uncertain, and the possibility must be considered that the underlying abnormality is not in the MR gene, but in an independent gene acting through yet to be characterized processes.
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PMID:Pseudohypoaldosteronism: molecular characterization of the mineralocorticoid receptor. 802 37

The 12-lipoxygenase (LO) pathway of arachidonic acid plays an important role in angiotensin II (AII)-mediated aldosterone synthesis. Several distinct isoforms of 12-LO have been cloned. However, in humans only the platelet form of 12-LO has been reported to be present. Western immunoblotting analysis in cultured human adrenal glomerulosa cells using polyclonal antibodies to porcine leukocyte 12-LO enzyme or peptide showed a specific 72-kilodalton band, which is identical to the molecular size of the porcine leukocyte form of 12-LO. In addition, AII (10(-7)) increased the intensity of the 72-kilodalton band nearly 2-fold over basal. In situ hybridization analysis indicated a strong positive reaction with the porcine leukocyte type of 12-LO antisense riboprobe in the zona glomerulosa of the adrenal cortex. The 12-LO probe also recognized a near 4-kilobase messenger RNA (mRNA) from human glomerulosa cells in Northern blots. Since the leukocyte type of 12-LO is highly homologous to human 15-LO, a reverse transcriptase and polymerase chain reaction was used to analyze the specific type of 12-LO mRNA in human cells. The mRNA for a porcine leukocyte type of 12-LO was detected in human adrenal glomerulosa cells, and the level of 12-LO transcripts was increased approximately 60-fold by AII (10(-7) M). The leukocyte type of 12-LO also was detected in human monocyte-like U937 cells, but not in IM-9 lymphocytes or human erythroleukemia cells. These results suggest that human adrenal glomerulosa cells and human monocyte-like U937 cells express a 12-LO which has immunological and molecular biological characteristics similar to the porcine leukocyte 12-LO.
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PMID:Evidence that a leukocyte type of 12-lipoxygenase is expressed and regulated by angiotensin II in human adrenal glomerulosa cells. 827 71

Specific regulatory mechanisms of aldosterone-stimulated Na+ reabsorption through the apical amiloride-sensitive channel are unknown. In this study, we examined the effects of aldosterone on Na+ channel gamma-subunit mRNA levels in cultured rabbit cortical collecting duct cells. With the use of reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA isolated from aldosterone-treated cells and degenerate primers, a 446-base pair (bp) PCR product was amplified and further characterized by nested PCR and sequencing. The nested PCR yielded a predicted 164-bp product. Sequencing of the 446-bp PCR product revealed 83% nucleotide and 91% amino acid identity to the rat colonic Na+ channel gamma-subunit. The relative abundance of Na+ channel mRNA was determined by quantitative PCR after a 24-h aldosterone treatment. The results demonstrate that Na+ channel gamma-subunit mRNA levels were significantly higher (2.6 +/- 0.42) in aldosterone-treated cultures vs. the controls. This increase, however, is less than the aldosterone-induced increase (3.2 +/- 2.0) in the amiloride-sensitive short-circuit current. These results indicate that Na+ channel gamma-subunit mRNA levels are increased by aldosterone and that this increase is likely to be responsible, at least in part, for the aldosterone-induced Na+ current in the kidney.
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PMID:Aldosterone regulation of sodium channel gamma-subunit mRNA in cortical collecting duct cells. 876 73

The purpose of the present study was to determine whether the renal inner medulla expresses mRNA for the rat epithelial Na+ channel (rENaC) and, if so, to define its regulatory properties using a low-Na+ diet model. We detected alpha, beta and gamma subunit mRNA in rat renal inner medulla using reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for rENaC alpha, beta and gamma subunits. Moreover, we have developed a specific probe for the alpha subunit using RT-PCR with rENaC alpha-subunit-specific primers. The resulting cDNA was verified by sequencing and was then used in Northern blot analysis of distal colon, whole kidney and inner medulla. The probe for the rENaC alpha subunit hybridized not only to distal colon RNA but also to inner medulla RNA derived from rats fed a normal diet. Furthermore, we examined the effect of a low-Na+ diet on alpha, beta and gamma subunit mRNA expression of rENaC using full-length cDNA as a probe. A marked elevation of rENaC alpha subunit mRNA abundance in the inner medulla was observed in response to a high plasma aldosterone concentration induced by dietary Na+ deprivation. On the other hand, neither beta nor gamma subunit mRNA expression was enhanced by a low-Na+ diet. From these results, it is suggested that rENaC is responsible for Na+ transport in the renal inner medulla and that is probably regulated via transcriptional control of the alpha subunit of ENaC.
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PMID:A low-Na+ diet enhances expression of mRNA for epithelial Na+ channel in rat renal inner medulla. 930 9

Increasing evidence suggests that mineralo- and glucocorticoids modulate cardiovascular homeostasis via the effects of circulating components generated within the adrenals but also through local synthesis. The aim of this study was to assess the existence of such a steroidogenic system in heart. Using the quantitative reverse transcriptase-polymerase chain reaction, the terminal enzymes of corticosterone and aldosterone synthesis (11beta-hydroxylase and aldosterone synthase, respectively) were detected in the rat heart. This pathway was shown to be physiologically active, since production of aldosterone, corticosterone, and their precursor, deoxycorticosterone, was detected in both the homogenate and perfusate of isolated rat hearts using radioimmunoassay after Celite column chromatography. Perfusion of angiotensin II or adrenocorticotropin for 3 h increased aldosterone and corticosterone production and decreased deoxycorticosterone, suggesting that aldosterone and corticosterone are formed within the isolated heart from a locally present substrate. Chronic regulation of this intracardiac system was then examined. As in adrenals cardiac 11beta-hydroxylase and aldosterone-synthase mRNAs were independently regulated by 1 week's treatment with either low sodium and high potassium diet (which increased aldosterone synthase mRNA level only), angiotensin II (which raised level of both mRNAs), or adrenocorticotropin (which stimulated the 11beta-hydroxylase gene exclusively). Changes in cardiac steroid levels during treatment were not directly related to their plasma levels suggesting independent regulating mechanisms. This study, therefore, provides the first evidence for the existence of an endocrine cardiac steroidogenic system in rat heart and emphasizes its potential physiological and pathological relevance.
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PMID:Myocardial production of aldosterone and corticosterone in the rat. Physiological regulation. 947 30

This study tests the hypothesis that aldosterone induces cardiac fibrosis through an increase of cardiac angiotensin II (Ang II) AT1 receptor levels, thereby potentiating the fibrotic effect of Ang II by determining the effects of spironolactone and losartan on cardiac fibrosis, AT1 density, and gene expression in aldosterone-salt-treated rats. Fibrosis was quantified by slot blots of collagen I and III mRNA levels and videomorphometry of Sirius red-stained collagen. AT1 receptor density was determined by (125I-Sar1-Ile8)-Ang II competition binding, and AT1 mRNA levels were analyzed by quantitative reverse transcriptase polymerase chain reaction. One month of aldosterone-salt treatment induced a decrease in plasma Ang II and an increase in blood pressure, left ventricular hypertrophy, and ventricular fibrosis. Spironolactone (20 mg/kg per day) and losartan spironolactone (10 mg/kg per day) had no effect on the first 3 parameters. Losartan was as effective as spironolactone in preventing ventricular collagen mRNA increase and fibrosis. Ventricular density of AT1 receptors increased 2-fold and was accompanied by a 3-fold increase in the corresponding mRNA in aldosterone-salt compared with sham-operated rats. Both spironolactone and losartan prevented the elevation of ventricular AT1 density and that of right ventricular AT1 mRNA levels. These results demonstrate that the mechanism by which aldosterone-salt induces cardiac fibrosis involves Ang II acting through AT1 receptors. They also suggest that the cardiac AT1 receptor is a target for aldosterone.
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PMID:Angiotensin AT1 receptor subtype as a cardiac target of aldosterone: role in aldosterone-salt-induced fibrosis. 1020 34

NCI-H295, a human adrenocarcinoma cell line, has been proposed as a model system to define the role of the renin-angiotensin system in the regulation of aldosterone production in humans. Because the precise cellular localization of the components of the renin-angiotensin system in human adrenal cortical cells remains unclear, we investigated their localization in this defined cell system. NCI-H295 cells expressed both angiotensinogen and renin as shown by reverse transcriptase polymerase chain reaction and immunohistochemistry. Human angiotensin-converting enzyme (ACE) was not detectable by immunocytochemistry, ACE binding, or reverse transcriptase polymerase chain reaction. However, 3.5 mmol/L K+ stimulated the formation of both angiotensin I and angiotensin II 1. 9- and 2.5-fold, respectively, and increased aldosterone release 3. 0-fold. The K+-induced stimulation of aldosterone release was decreased by captopril and enalaprilat (24% and 26%, respectively) and by the angiotensin type 1 (AT1)-receptor antagonist losartan (28%). Angiotensin II-induced stimulation of aldosterone release was abolished by losartan treatment. Specific [125I]Sar1-angiotensin II binding was detected by receptor autoradiography. The binding of [125I]Sar1-angiotensin II was completely displaced by the AT1 antagonist losartan but not by the AT2 receptor ligand PD 123319, confirming the expression of angiotensin II AT1 receptors in NCI-H295 cells. Our results demonstrate that NCI-H295 cells express most of the components of the renin-angiotensin system. Our failure to detect ACE, however, suggests that the production of angiotensin II in NCI-H295 cells may be ACE independent. NCI-H295 cells are able to produce angiotensin II, and K+ increases aldosterone secretion in part through an angiotensin-mediated pathway. The production of angiotensin II in NCI-H295 cells demonstrates that this human cell line can be useful to characterize the role of locally produced angiotensin II in the regulation of aldosterone release.
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PMID:Local renin-angiotensin system is involved in K+-induced aldosterone secretion from human adrenocortical NCI-H295 cells. 1020 42


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