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)

The aim of the present study was to determine whether mRNA for the three endothelin peptides (endothelin-1, endothelin-2, and endothelin-3) and the two known receptor subtypes (ETA and ETB) was present in human endometrium at different stages of the menstrual cycle (menstrual, early and mid-proliferative, and early, mid-, and late secretory). Endometrium was obtained from women undergoing surgery for benign disease, and total RNA was extracted using a guanidinium isothiocyanate method. mRNA for endothelin peptide and receptor was detected using the reverse transcriptase-polymerase chain reaction with nested oligonucleotide primers. mRNA for endothelin-1, endothelin-2, and endothelin-3 was demonstrated throughout the menstrual cycle, and three splice variants of mRNA encoding endothelin-3 were found in all samples. The ratio of ETA to ETB receptor mRNA was found to change throughout the menstrual cycle. In the proliferative phase, amplified cDNA product was almost exclusively confined to the ETA receptor, whereas an increase in the amplified product of the ETB receptor cDNA was seen in the secretory and menstrual phases. These studies show that mRNA for endothelin-1, endothelin-2, and endothelin-3 is present in human endometrium at all stages of the menstrual cycle and suggest that different physiological actions of the endothelin peptides may be mediated through changes in the ratio of the ETA and ETB receptor subtypes.
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PMID:Presence of messenger ribonucleic acid for endothelin-1, endothelin-2, and endothelin-3 in human endometrium and a change in the ratio of ETA and ETB receptor subtype across the menstrual cycle. 146 62

Our aim was to examine the hypothesis that vascular smooth-muscle cells (VSMCs) express only ETA mRNA and endothelial cells express only ETB mRNA and to determine which ET mRNA isoforms are expressed in these cell cultures. Using the reverse transcriptase polymerase chain reaction, we were able to detect ETB, ET-1 and splice variant ET-2 mRNA in cultured human umbilical vein endothelial cells (HUVECs) and ETA and splice variant ET-2 mRNA in cultured aortic smooth-muscle cells. The presence of ET-2 mRNA in cultured VSMCs has not been previously reported. These results agree with the hypothesis that ET-1 may be released from vascular endothelial cells to act predominantly on ETA receptors on VSMCs to stimulate contraction of the underlying smooth-muscle cells, and that endothelium-derived relaxing factor release may be mediated predominantly via the ETB receptors on HUVECs. The role of ET-2 expression from HUVECs and VSMCs is less clear.
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PMID:Endothelin-2 mRNA splice variants detected by RT-PCR in cultured human vascular smooth muscle and endothelial cells. 750 38

The thyroid gland is a highly vascular tissue, and its blood flow changes dramatically in various pathological conditions. Although the mechanisms regulating these changes in vascularity and blood flow are not well understood, candidate mediators include endothelin-1 (ET-1) and nitric oxide (NO). In the present study, we used a reverse transcriptase-polymerase chain reaction assay to determine which components of these vasoregulatory pathways are present in the thyroid and to analyze changes in gene expression in an experimental model of goiter formation and involution. Expression of messenger RNAs (mRNAs) encoding ET-1, ET receptors (ETA and ETB), ET-converting enzyme, and the three nitric oxide synthase (NOS) isoforms (NOS I, NOS II, and NOS III) was readily detected in the rat thyroid. After goiter formation was induced by thiouracil and a low iodine diet, there was increased expression of the genes encoding ET-related proteins (ET-1, 3.2-fold; ETA, 2.9-fold; ETB, 3.5-fold) as well as two of the three NOS isoforms (NOS I, 2.7-fold; NOS III, 4.9-fold). During iodide-induced involution, the ET-related mRNA levels remained elevated, whereas those of the two NOS isoforms returned to basal values. ET-converting enzyme, NOS II, and thyroglobulin mRNAs were minimally affected in this model, providing evidence for selective regulation of these genes. To assess whether NO plays a role in vascular changes during goiter formation, animals were treated with a NOS inhibitor, N-nitro-L-arginine methyl ester (NAME). NOS activity in the thyroid was inhibited by more than 75% after treatment with NAME. Thyroid hormone and TSH levels were unchanged. Although NAME had little effect on overall thyroid size, vascular expansion during goiter formation was decreased by 36%. We conclude that the thyroid gland expresses a complex network of vasoactive genes whose expression is regulated dynamically during thyroid goiter formation and involution. NO production and probably other locally produced vasoactive substances are involved in changes in thyroid vascularization.
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PMID:Expression of nitric oxide synthase isoforms in the thyroid gland: evidence for a role of nitric oxide in vascular control during goiter formation. 758 72

1. We have identified the endothelin receptors present in the media of human main stem renal artery and vein and characterized the subtypes mediating vasoconstriction in these blood vessels in vitro. 2. Messenger RNA encoding both ETA and ETB receptors was identified in the smooth muscle layer of human renal artery and vein by reverse transcriptase-polymerase chain reaction assay. In cryostat-cut cross-sections of both vessels autoradiographical visualisation suggested a majority of ETA receptors. Intense binding was obtained to the non-selective ligand [125I]-ET-1 and the ETA-selective [125I]-PD151242 but only weak labelling of sites by the ETB-selective [125I]-BQ3020. 3. ET-1 potently constricted renal artery and vein preparations with EC50 values of 4.06 nM and 1.00 nM, respectively. Sarafotoxin 6b was approximately ten times less potent than ET-1 with EC50 values of 36.3 nM and 13.8 nM respectively. In the renal artery, ET-3 and sarafotoxin 6c showed little or no activity up to 300 nM. Responses to these peptides were more variable in the renal vein. Preparations from three individuals did not respond to ET-3 but in three further cases, although ET-3 was much less potent than ET-1, full dose-response curves were obtained. S6c elicited dose-related contractions in vein preparations from 5/6 individuals and although more potent than ET-1, the maximum response was 30-60% of that obtained to ET-1. 4. ET-1-induced vasoconstriction of renal artery and vein was antagonized by the ETA-selective, BQ123 (3-10 microM). The dose-response curves to ET-1 were displaced in a parallel rightward fashion with no attenuation of the maximum responses. pA2 values were estimated to be 6.8 +/- 0.1 and 6.8 +/- 0.4 for artery and vein respectively.5. These data suggest that mRNA encoding both ETA and ETB receptors is present in the media of human main stem renal artery and vein. However, autoradiographical studies indicate that the majority of ET receptors expressed are of the ETA subtype. The relative potencies of ET-1 and ET-3 as vasoconstrictors of renal blood vessels in vitro is consistent with this being an ETA-mediated response,and therefore whilst responses to S6c indicate that constrictor ETB receptors may be present in renal veins from some individuals these are likely to be of less importance in these blood vessels.
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PMID:Vasoconstrictor endothelin receptors characterized in human renal artery and vein in vitro. 781 31

Recent studies have revealed that endothelin-1 (ET-1) may be produced by human cancer cell lines and have suggested that in vivo the peptide might play a modulatory role in the growth of stromal cells surrounding tumor cells and/or in the growth of the cancer cells themselves, through paracrine or autocrine mechanisms. Therefore, we investigated whether ET-1 and ET receptors could be expressed in the human gastric cancer cell line HGT-1. By applying the reverse transcriptase polymerase chain reaction (RT-PCR) to total RNA extracted from the cells, using oligonucleotides synthesized from the sequence of the prepro-ET-1 mRNA, we have amplified a cDNA at the expected size (453 bp), which hybridized with a labeled ET-1-specific probe. In addition, RT-PCR was carried out to test whether HGT-1 cells expressed mRNA for ETA and/or ETB receptor subtypes. The amplified products of cDNA were at the size predicted for the ETA receptor (368 bp), whereas no ETB receptor mRNA could be detected.
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PMID:Endothelin-1 and ETA receptor subtype are expressed in the gastric HGT-1 cell line. 858 61

Using reverse transcriptase-polymerase chain reaction, products corresponding to mRNA encoding endothelin-A and -B (ETA and ETB) receptors were demonstrated in human coronary arteries and veins with intact endothelium and in endothelium-denuded human coronary arteries. Vasomotor responses were studied on isolated segments of human epicardial coronary arteries and veins at resting tension and after precontraction with U46619. In both arteries and veins, endothelin-1 (ET) induced strong and potent contractions, and preincubation with different concentrations of the non-selective ETA/ETB receptor antagonist PD 145065 caused a rightward shift of the concentration-response curves without significantly changing maximum responses (pA2 value 6.7 arteries, 7.4 veins). The ETB receptor agonist IRL 1620 induced no contraction of arteries or veins at resting tension, but induced weak relaxation of all arteries and most precontracted veins, the relaxation being endothelium-dependent in arteries. ET at low concentrations induced weak relaxations of most precontracted arteries, but no veins. In conclusion, mRNA encoding ETA and ETB receptors is present in human coronary arteries and veins, ETA receptors mediating contraction and ETB receptors mediating relaxation. In arteries, mRNA for both receptor types was detected in the media, but ETB receptor-mediated relaxation was endothelium-dependent.
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PMID:Endothelin-A and -B receptors in human coronary arteries and veins. 883 23

1. The pharmacology and mRNA expression of endothelin (ET) receptors in human omental arteries were characterized by use of functional contractile assays and the reverse transcriptase-polymerase chain reaction (RT-PCR). 2. In freshly obtained segments of human omental arteries, ET-1 and ET-3 induced concentration-dependent contractions which were normalized to the response produced by 60 mM K+. ET-1 produced a maximum contraction (Emax) amounting to 151 +/- 17% of the K+ response. The pEC50 for this agonist was 8.64 +/- 0.17. The effect of ET-3 was less pronounced (Emax: 71 +/- 22% and pEC50: 6.69 +/- 0.17) than that of ET-1. The ET receptors involved were characterized with FR139317 (a selective ETA receptor antagonist), PD 145065 (a mixed ETA and ETB receptor antagonist) and BQ 788 (an ETB receptor antagonist). A high concentration of these antagonists (10 microM) abolished the contractile responses to ET-3, and produced a parallel rightward shift of the ET-1 concentration-response curve without changing the maximal effect. FR139317 and PD 145065 were equally effective while BQ 788 was much less effective. This is consistent with ETA receptors mediating contraction in human omental arteries. 3. Arterial segments cultured for 5 days in serum-free Dulbecco's medium at 37 degrees C under sterile and humidified conditions retained contractility although responses to 60 mM K+ were somewhat reduced. ET-3 was significantly more potent in the cultured arteries (pEC50: 8.56 +/- 0.15) and achieved a greater maximum effect (Emax: 116 +/- 19%). Responses were not antagonised by FR139317 but were competitively blocked by PD 145065 and BQ 788 with the latter antagonist being the more potent. In contrast Emax (179 +/- 17%) and pEC50 (8.66 +/- 0.23) values for ET-1 were not significantly different from those obtained with fresh arteries. PD 145065 still demonstrated a rightward shift of the ET-1-induced concentration-response curve, whereas FR139317 and BQ 788 caused non-significant shifts. These findings suggest that functional ETB receptors contribute significantly to the endothelin contractile response in cultured arteries. 4. Two-site analysis of the ET-1 induced concentration-response curve from cultured arteries suggests that ETB receptors, at the high potency component, and ETA receptors, at the low potency component, contribute both to the contractile response in relative proportion of 70% and 30%, respectively. Further analysis suggested that the ETA receptor would be capable of evoking at least 75% of the ET-1 contraction in the absence of ETB receptors, although with a lower potency as compared to fresh arteries. 5. Electrophoresis of RT-PCR products from the smooth muscle layer of freshly obtained human arteries indicated the presence of mRNA for both ETA and ETB receptors. Arteries cultured for 1 and 5 days demonstrated an increase of mRNA for the ETB receptor as compared to the ETA receptor. The identities of the PCR products were verified by restriction enzyme digestion. 6. In freshly obtained human omental arteries, the contractile effects of endothelins appear to be mediated predominantly by the ETA receptor subtype, with a negligible contribution by ETB receptors. Cultured arterial segments, however, exhibited a substantial ETB receptor mediated contractile response and an increase in ETB receptor mRNA content, consistent with an upregulation of functional ETB receptors. These in vitro data suggest plasticity in the smooth muscle cell expression of contractile ETB receptors.
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PMID:Plasticity of contractile endothelin-B receptors in human arteries after organ culture. 893 19

These studies tested the hypothesis that the cerebral vasospasm that follows subarachnoid hemorrhage (SAH) is due to alterations in endothelin (ET) and ET receptor expression. Eight monkeys underwent cerebral angiography and induction of SAH. Angiography was repeated 7 days later to confirm the presence of cerebral vasospasm, and animals were killed. RNA was isolated from right (vasospastic) and left (control) side middle cerebral arteries and surrounding cerebral cortex. The levels of prepro (PP) ET-1 (ppET-1) and ppET-3 and ETA and ETB receptor MRNAs were determined using a quantitative reverse transcriptase polymerase chain reaction-based assay. ET-1 peptide was also measured in CSF at baseline and after 7 days. Specific agonist binding to ETA and ETB receptors in both middle cerebral arteries and in surrounding brain cortex was measured in three animals by autoradiographic binding assays. Levels of ETB receptor mRNA were 3.4 +/- 2.2-fold higher in the right than in the left cerebral arteries (p < 0.01). There were no significant differences in the levels of ppET-1, ppET-3, or ETA receptor mRNA in cerebral arteries. ET-1 peptide was not elevated in CSF. Levels of ETA and ETB receptor mRNAs were 2.6 +/ 1.1- and 2.1 +/ 1.3-fold higher, respectively, in the right than in the left cerebral cortex, while the level of ppET-3 mRNA was 2.1 +/- 1.0-fold lower. There were no differences in ppET-1 mRNA levels between right and left cerebral cortex. Binding to ETA and ETB receptors in cerebral arteries and cortex did not differ significantly between right and left sides. These results do not support the hypothesis that overexpression of ET-1 is principal cause of vasospasm, but rather they suggest that SAH causes complex changes in the ET system that together are responsible for the cellular response to SAH.
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PMID:Increased expression of endothelin B receptor mRNA following subarachnoid hemorrhage in monkeys. 896 9

We have characterized the interaction of endothelin (ET) with cultured neonatal rat ventricular myocytes. Binding studies indicate a single population of ETA receptors [53,000 sites/cell, apparent dissociation constant (Kd) for ET-1 approximately 0.07 nM]. Analysis of mRNA levels for ET receptors using 35 cycles of reverse transcriptase-polymerase chain reaction demonstrates the presence of only ETA-receptor message. Studies with ET-1 and a variety of congeners and antagonists indicate that ETA receptors couple to both the stimulation of phosphoinositide turnover and the inhibition of adenylyl cyclase. In myocytes transfected with an atrial natriuretic factor (ANF) promoter linked to a luciferase reporter gene, ET-1 stimulates luciferase expression through an ETA receptor. These data indicate that the ETA receptor is the exclusive receptor on neonatal ventricular myocytes and that this receptor couples to both phosphoinositide hydrolysis and adenylyl cyclase. ET-1 also induces a threefold increase in mitogen-activated protein kinase (MAPK) activity, an effect that is not sensitive to pertussis toxin (PTx). By contrast, ET-stimulated ANF-luciferase expression is partially inhibited by treatment of cells with PTx, suggesting that both PTx-sensitive (Gi) and PTx-insensitive (Gq) pathways mediate the effects of ET-1 on ANF gene expression in neonatal myocytes and that hormonal regulation of ANF expression may utilize pathways in addition to the activation of MAPK.
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PMID:Endothelin ETA receptor regulates signaling and ANF gene expression via multiple G protein-linked pathways. 903 31

The biochemical and molecular endothelin-1 (ET-1) responses to high dose morphine sulfate infusion were studied in conscious newborn piglets (n = 6) that received a loading dose of 100 micrograms/kg over 5 min followed by a continuous i.v. infusion dose of 100 micrograms.kg-1.h-1 for 4 h. The control group (n = 6) received equivalent volume loading and infusion doses of 5% dextrose. Blood samples were drawn serially from the femoral artery and sagittal sinus vein before (0), during (30 min, 1, 2, 3, and 4 h), and post (1 and 2 h) infusion. Five micrograms of total RNA obtained from brainstem tissue homogenates was analyzed by reverse transcriptase--polymerase chain reaction (RT-PCR). The amounts of mRNA encoding ET-1, and endothelin receptor subtypes ETA and ETB, were semiquantitated using densitometric scanning. Morphine infusion resulted in elevated respiratory rate and mean arterial blood pressure, with no effect on arterial pH, Po2, and O2 saturation. Compared with the control group, morphine induced significant elevations in plasma ET-1 levels following the bolus dose (systemic: 13.2 +/- 3.6 vs. 8.6 +/- 2.2 pg/mL, p < 0.05; sagittal sinus vein: 13.7 +/- 3.4 vs. 8.2 +/- 0.9 pg/mL, p < 0.01). These effects lasted up to 2 h after discontinuation of morphine infusion (systemic: 14.5 +/- 3.4 to 18.7 +/- 5.7 pg/mL vs. 7.5 +/- 0.8 to 9.4 +/- 3.2 pg/mL, p < 0.05 to p < 0.01; sagittal sinus vein: 14.8 +/- 2.7 to 17.6 +/- 2.8 pg/mL vs. 7.5 +/- 1.4 to 9.4 +/- 3.4 pg/mL, p < 0.05 to p < 0.01). The RT-PCR assay showed a twofold (p < 0.02) upregulation in ET-1 and a threefold (p < 0.007) upregulation in ETA receptor mRNA expression in the brainstem of morphine-treated animals. In contrast, there was a threefold (p < 0.0001) downregulation of the ETB receptor mRNA expression. The rapid and sustained elevations in systemic arterial and sagittal sinus venous ET-1 levels suggest a role for ET-1 in the morphine-induced excitatory responses observed in newborn piglets. Upregulation of ETA receptors and downregulation of ETB receptors in the brainstem with high doses of morphine may indicate possible effects on cerebral vascular tone.
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PMID:Biochemical and molecular endothelin responses to morphine sulfate infusion in conscious newborn piglets. 979 54


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