cDNA cloning and sequence analysis of preproendothelin-1 (PPET-1) from salmon, Oncorhynchus keta

The presence of endothelin (ET)-like immunoreactivity and the cardiovascular effects of mammalian ET-1 in fish have been reported. To identify ET-related peptides in fish, we screened the cDNA library of the salmon (Oncorhynchus keta) stomach by means of rapid amplification of cDNA ends, and we cloned cDNAs encoding an ET-related peptide. The salmon ET-related sequence of 21 amino acids is identical to the trout ET-1 peptide recently purified from kidney specimens of Oncorhynchus mykiss. The deduced amino acid sequence of salmon pre-proET-1 (PPET-1) comprises 244 amino acids, including a putative signal sequence and mature ET-1, as well as big ET-1 and ET-1-like sequences. This precursor, the first reported PPET-1 sequence for Salmoniformes, Teleostei, has low homology with the sequences of human, mouse, frog (Xenopus laevis), and zebrafish (Danio rerio) PPET-1 (26%, 29%, 24%, and 39%, respectively).     

The human preproendothelin-1 gene. Complete nucleotide sequence and regulation of expression

Endothelin-1 is a 21-amino acid potent vasoconstrictor peptide produced by vascular endothelial cells. We have cloned the whole length of the human preproendothelin-1 (PPET-1) gene and the corresponding cDNA and determined the complete nucleotide sequences. The 2026-nucleotide human mRNA for PPET-1 (excluding the polY(A) tail) is encoded in five exons distributed over 6836 base pairs of the genome. The 5'-flanking region of the gene contains (i) octanucleotide sequences for the phorbol ester-responsive elements, also known as the binding elements for FOS.JUN complex; (ii) consensus motifs for the binding site of nuclear factor 1, which may mediate the induction described previously of PPET-1 mRNA by transforming growth factor-beta; (iii) hexanucleotide sequences for the acute phase reactant regulatory elements that may be involved in the induction of endothelin-1 under acute physical stress in vivo. Further, the 3'-nontranslated sequence of human PPET-1 mRNA contains three AUUUA motifs, which may mediate selective translation-dependent destabilization of the mRNA. Northern blot analysis in cultured endothelial cells from human umbilical veins shows that PPET-1 mRNA is in fact rapidly induced by the active phorbol ester 12-O-tetradecanoylphorbol 13-acetate within 10 min. Analysis of mRNA life span by using actinomycin D demonstrates that PPET-1 mRNA has a short intracellular half-life of about 15 min and is superinduced by cycloheximide. This superinduction is found to be due to the stabilization of the mRNA by cycloheximide, as in the case of other known AUUUA-containing mRNAs. These findings suggest that the regulation of expression of PPET-1 mRNA may be mediated in part by these sequence elements.     

Rapid quantification of murine endothelin-1 and vasoactive intestinal contractor gene expression levels by a real-time PCR system

A rapid quantitative analysis method for murine endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC) gene expression levels was established using a real-time polymerase chain reaction (PCR). We designed primer pairs and TaqMan probes specific for murine prepro-ET-1 (PPET-1) and prepro-VIC (PPVIC) genes, based on the cDNA sequence region common to both mouse and rat. The dynamic range for detection in this system spanned 100000-fold of the starting molecule. The gene expression levels of PPET-1 and PPVIC were estimated as gene expression rates normalized by the expression of the house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase. To examine the reproducibility of this assay system, we calculated the intra-assay and interassay coefficients of variation of the gene expression rate, which ranged from 16.2 to 55.0% and from 24.2 to 56. 5%, respectively. Using this system, we examined gene expression levels of PPET-1 and PPVIC in mouse tissues. PPET-1 gene expression was found in all tissues at relatively high levels, whereas high levels of PPVIC gene expression were observed only in stomach, intestine, uterus, and ovary. The gene expression patterns agreed well with those determined by RNase protection assay and conventional PCR. These results show that this new rapid method is accurate and reproducible.     

Cloning and expression of rat preproendothelin-3 cDNA.

We report here the cloning and expression of a rat full-length cDNA encoding preproendothelin-3 (preproET-3). The predicted rat preproET-3 consisted of 167 amino acid residues. As in other ET-family peptides, the mature rat ET-3 was predicted to be produced through unusual processing from a 41-residue intermediate, the big ET-3 in rat. Transient transfection of COS-7 cells with the cloned preproET-3 cDNA resulted in the production of mature ET-3 and this production was inhibited by phosphoramidon, a metaloprotease inhibitor. This suggested that a phosphoramidon sensitive mechanism was involved in the production of ET-3 in the transfected COS-7 cells. Northern blot analysis showed that an approximately 3.0-kb rat preproET-3 mRNA was expressed in rat tissues, including the eye ball, submandibular gland, brain, kidney, jejunum, stomach and spleen. A 2.0-kb and a 3.3-kb mRNA were also detected in the eye ball and small intestine, respectively. The distinct distribution of rat preproET-3 mRNA from that of preproET-1 mRNA suggested that ET-1 and ET-3 played different roles.     

Early activation of cardiac and renal endothelin systems in experimental heart failure

We investigated the time course of the expression of cardiac and renal endothelin systems in tachycardia-induced heart failure in dogs. Eleven beagles underwent rapid pacing at a progressively increased rate over a period of 5 wk, with a weekly clinical examination, echocardiography, measurement of circulating and urinary endothelin-1 (ET-1), and myocardial and renal tissue biopsies. Real-time quantitative PCR was used for determinations of tissue prepro-ET-1 (ppET-1), ET-1-converting enzyme (ECE-1), and ETA and ETB receptor mRNA. Cardiac and renal tissue ET-1 contents were evaluated by immunostaining and measured by radioimmunoassay at autopsy. Rapid pacing caused a progressive increase in end-systolic and end-diastolic ventricular volumes (P < 0.05) from week 2 together with a decrease in ejection fraction and in mean velocity of circumferential shortening (P < 0.05) from week 1. These changes were tightly correlated to myocardial ppET-1 and renal ETA receptor mRNA and less so to myocardial ECE-1 mRNA, and they occurred before any increase in plasma and urinary ET-1 (P < 0.05 from week 4) and clinical signs of heart failure. Renal ppET-1 did not change. Both cardiac and renal ET-1 peptide contents were increased at autopsy. We conclude that tachycardia-induced heart failure in dogs is characterized by an early activation of the cardiac and renal tissue endothelin systems, which occurs before any changes in circulating and urinary ET-1 and is closely related to altered ventricular function.     

A transformed murine Leydig cell line expresses the ETA receptor subtype

We recently demonstrated that transformed murine Leydig cells (MA-10) responded to endothelin-1 (ET-1) via increased steroidogenesis. This study addresses the endothelin receptor subtype present on this cell line and whether or not the cells produce ET-1. The expression of the preproendothelin-1 (PPET-1) gene was investigated by Northern blot analysis, and PPET-1 mRNA was found to be <0.2% of that present in pulmonary endothelial cells. The medium from MA-10 cells, maintained under serum-free conditions, was analyzed by radio-immunoassay to determine immunoreactive-ET-1 production and ET-1 levels were found to be below the sensitivity of the assay (<10 pg/ml). The data from competitive binding experiments with [¹²⁵I]ET-1 and unlabeled ET-1, ET-3 and receptor subtype selective ligands yielded a single class of high affinity binding sites with ET_A receptor subtype characteristics. The results of this study demonstrate that MA-10 cells possess the ET_A receptor subtype but do not produce significant quantities of ET-1 under basal conditions.     

Analysis of responses to endothelins 1, 2, and 3 and sarafotoxin 6b in airways of the cat.

Airway responses to endothelin (ET) 1, ET-2, ET-3, and sarafotoxin 6b (S6b) were investigated in paralyzed, anesthetized, mechanically ventilated cats. Intravenous injections of ET-1 (0.1-1 nmol/kg) increased transpulmonary pressure (Ptp) and lung resistance (RL) and decreased dynamic compliance (Cdyn) in a dose-related manner. Airway responses to ET-1 were decreased significantly by sodium meclofenamate, a cyclooxygenase inhibitor, and by SKF 96148, a thromboxane receptor blocking agent. In terms of relative bronchoconstrictor activity, the thromboxane mimic, U-46619, was threefold more potent than ET-1 on a molar basis in increasing Ptp. ET-1 and ET-3 had similar bronchoconstrictor activity, and these peptides were less potent than ET-2 and S6b. Bronchoconstrictor responses to ET-2, ET-3, and S6b were also decreased significantly by meclofenamate and by thromboxane receptor blocking agents. The ET-1 precursor ET-1-(1-38) (big ET-1) caused a significant slowly developing increase in Ptp, RL, and aortic pressure (PAO) and a decrease in Cdyn, whereas a monocyclic ET-1 analogue and ET-1-(16-21) hexapeptide fragment had little or no activity in the airways. The present data indicate that ET-1, ET-2, ET-3, and S6b have significant bronchoconstrictor activity in the cat and that responses are dependent in part on the release of arachidonic acid and the formation of thromboxane A2. These data also suggest that big ET-1 is converted into a mature peptide in the cat and that ET-1-(16-21) hexapeptide fragment and a monocyclic ET-1 analogue have little if any bronchoconstrictor activity in the anesthetized cat.     

Regulation of ACE2 in cardiac myocytes and fibroblasts

Angiotensin-converting enzyme 2 (ACE2) preferentially forms angiotensin-(1-7) [ANG-(1-7)] from ANG II. We showed that cardiac ACE2 is elevated following treatment of coronary artery-ligated rats with AT1 receptor blockers (ARBs). Cardiac myocytes and fibroblasts were isolated from neonatal rats to determine the molecular mechanisms for the ACE2 upregulation by ARB treatment. ANG II significantly reduced ACE2 activity and downregulated ACE2 mRNA in cardiac myocytes, effects blocked by the ARB losartan, indicating that ANG II regulates ACE2. ANG II also reduced ACE2 mRNA in cardiac fibroblasts; however, no enzyme activity was detected, reflecting the limited expression of ACE2 in these cells. Endothelin-1 (ET-1) also significantly reduced myocyte ACE2 mRNA. The reduction in ACE2 mRNA by ANG II or ET-1 was blocked by inhibitors of mitogen-activated protein kinase kinase 1, suggesting that ANG II or ET-1 activates extracellular signal-regulated kinase (ERK) 1/ERK2 to reduce ACE2. Although ACE2 mRNA was not affected by ANG-(1-7), both the ANG II- and ET-1-mediated reductions in ACE2 mRNA were blocked by the heptapeptide. The ANG-(1-7) modulatory effect was prevented by the ANG-(1-7) receptor antagonist [D-Ala7]-ANG-(1-7), indicating that the ANG-(1-7) response was mediated by a specific AT(1-7) receptor. Myocyte treatment with atrial natriuretic peptide (ANP) also reversed the ACE2 mRNA downregulation by ANG II or ET-1, whereas treatment with ANP alone was ineffective. These results indicate that multiple hypertrophic and anti-hypertropic peptides regulate ACE2 production in myocytes, suggesting that ACE2 expression in the heart is dependent upon the compliment and concentration of regulatory molecules.     

cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA.

We report the cloning of a full-length cDNA encoding rat preproendothelin-1 (preproET-1). The predicted rat preproET-1 consists of 202 amino acid residues and highly similar to human, porcine and bovine preproET-1, respectively. The deduced 21-residue sequence of mature rat ET-1 is identical to human, porcine, canine and bovine ET-1. As in other mammalian species, the mature ET-1 is predicted to be produced from a 39-residue big ET-1 in the rat. Northern blot analysis showed that a single 2.3-kb preproET-1 mRNA is expressed not only in vascular endothelial cells but also in other rat tissues, including the lung, brain, uterus, stomach, heart, adrenal gland and kidney. These findings suggest that ET-1 may play roles as a local mediator in multiple organs both within and outside the cardiovascular system in the rat.     

Thromboxane blockade reduces blood pressure and progression of renal failure independent of endothelin-1 in uremic rats

This study was designed to investigate the role of eicosanoids, thromboxane A2 (TXA2) and prostacyclin (PGI2) as well as their relationship with endothelin-1 (ET-1) in the pathogenesis of renal parenchymal hypertension. Uremic rats were prepared by renal mass ablation and compared with sham-operated controls. The stable metabolites of TXA2 (TXB2) and PGI2 (6-keto-PGF1alpha) and immunoreactive ET-1 concentrations were measured by specific RIAs in biological fluids and in vascular and renal tissues. To investigate the functional role of TXA2 in the progression of hypertension and renal failure, a group of uremic rats were treated with ridogrel (25 mg/kg/day), a TXA2 synthase inhibitor and receptor antagonist. Renal preproET-1 expression was assessed by Northern blot analysis. Systolic blood pressure (SBP), serum creatinine and proteinuria were found to be higher in uremic rats as compared to sham-operated controls (P < 0.01). TXB2 and ET-1 concentrations were increased in blood vessels, the renal cortex and in urine (P < 0.05). 6-keto-PGF1alpha concentrations were also increased in blood vessels and the renal cortex but decreased in urine (P < 0.05). Ridogrel significantly lowered SBP and proteinuria (P < 0.05) and blunted the increase of serum creatinine. Treatment with ridogrel resulted in a marked fall in vascular, renal and urine TXA2 concentrations, while ET-1 and 6-keto-PGF1alpha concentrations remained unchanged. The preproET-1 expression was higher in uremic rats than in the controls and was unaffected by ridogrel. These results suggest that TXA2 is involved in the pathogenesis of hypertension and renal failure progression in rats with subtotal 5/6 nephrectomy and that this effect is independent of the ET-1 system.     

The lithium tolerance of the Arabidopsis cat2 mutant reveals a cross-talk between oxidative stress and ethylene

In order to investigate the effects of a permanent increase in cellular H(2)O(2) on cation homeostasis we have studied a T-DNA insertion mutant of the Arabidopsis CATALASE 2 gene. This mutant (cat2-1) exhibits 20% of wild-type leaf catalase activity and accumulates more H(2)O(2) than the wild type under normal growth conditions. In addition to reduced size, a pale green color and great reduction in secondary roots, the cat2-1 mutant exhibited increased sensitivity to H(2)O(2), NaCl, norspermidine, high light and cold stress. On the other hand, the germination of the cat2-1 mutant is more tolerant to lithium than the wild type. This novel phenotype cannot be explained by changes in lithium transport. Actually, the uptake of lithium (and of other toxic cations such as sodium and norspermidine) is increased in the cat2-1 mutant while K(+) levels were decreased. The lithium tolerance of this mutant seems to result both from insensitivity to the inhibitory ethylene induced by this cation and a reduced capability for ethylene production. Accordingly, induction by ethylene of responsive genes such as PR4 and EBP/ERF72 is decreased in cat2-1. Mutants insensitive to ethylene such as etr1-1 and ein3-3 are lithium tolerant, and inhibition of ethylene biosynthesis with 2-aminoisobutyrate protects against lithium toxicity. Microarray analysis of gene expression indicates that the expression of genes related to cation transport and ethylene synthesis and perception was not altered in the cat2-1 mutant, suggesting that H(2)O(2) modulates these processes at the protein level. These results uncover a cross-talk between oxidative stress, cation homeostasis and ethylene.     

Cytokine-induced release of endothelin-1 from porcine renal epithelial cell line.

To elucidate the mechanism by which endothelin-1 (ET-1) is released from renal epithelial cells, we have investigated the effects of several compounds on release of ET-1-like immunoreactivity (LI) from LLCPK1 cell line. Thrombin, transforming growth factor-beta, cytokines (tumor necrotizing factor-alpha, interleukin-1 beta), and phorbol ester stimulated ET-1-LI release in a time- and dose-dependent manner. The cytokine-induced ET-1-LI release was not affected by indomethacin. Northern blot analysis using cDNA for porcine preproET-1 as a probe revealed a single major band corresponding to the size of ET-1 mRNA in LLCPK1. These data indicate that the preproET-1 gene is also expressed in renal epithelial cells and the release of ET-1 from renal cells is regulated by the similar mechanism to that from endothelial cells.     

Time-dependent expression of renal vaso-regulatory molecules in LPS-induced endotoxemia in rat

To elucidate roles of microvascular factors in the pathogenesis of renal complications during endotoxemia, that is characterized by renal vasoconstriction and systemic hypotension/generalized non-renal vasodilation, we profile the expression pattern and time-course of three key vaso-regulators, namely endothelin (ET)-1, nitric oxide (NO), and angiotensin II (Ang II). We hypothesize that disruption of the overall balance between vasodilatation and vasoconstriction in the kidney, during the early phase of sepsis, contribute to its (kidney) predisposition to acute renal failure. Adult male Wistar rats were rendered endotoxemic at different time points (1, 3, 6 and 10 h) by a single i.p. injection of lipopolysaccharide (LPS) (15 mg/kg) dissolved in saline. Control group was injected vehicle only (saline). Both systolic and diastolic blood pressures significantly decreased at different time points after LPS administration. Surprisingly, renal histopathological evaluation showed no remarkable changes in LPS-induced endotoxemia. However, overall, levels of the vaso-regulators and, where applicable, their respective receptors were upregulated: (1) plasma ET-1 increased 25-fold and peaked, as renal ET-1 mRNA, at 3 h; renal ET-1 protein and its receptors, ET type A (ET(A)) receptor (vasoconstrictive) and ET type B (ET(B)) receptor (vasodilatatory) increased in a time-dependent fashion, (2) Ang II increased by 53% compared to control, peaking at 6 h. However, while levels of Ang II type 1 (AT1) receptor increased over time after LPS injection, those of Ang II type 2 (AT2) receptor were downregulated, (3) data of NO system (NO-NOS), the key vasodilator, were the most intriguing. Whereas levels of renal NO increased time-dependently following LPS administration, with a 2240-fold increase in renal iNOS expression, levels of eNOS, were almost unchanged. In conclusion, the present study overall reveals intriguing and complex dynamics between levels of vasoconstrictors and vasodilators during the early phase of LPS-induced endotoxemia. These shifts in molecular expressions are likely triggered by compensatory mechanisms aimed at counteracting the undesirable and dominant effects of one group of vaso-regulatory moiety over the other.     

Nucleotide sequence analysis of the cat gene of Proteus mirabilis: comparison with the type I (Tn9) cat gene.

In Proteus mirabilis PM13 chloramphenicol resistance is mediated by the cat gene, a single copy of which is present in both resistant and sensitive isolates and which reverts at a high frequency. RNA measurements show an about 8.5-fold increase in cat-specific mRNA in cells expressing the resistance phenotype as compared with those which are sensitive to chloramphenicol. DNA sequence analysis has revealed a high degree of homology between the P. mirabilis cat gene and the type I cat variant (Tn9), 76% at the amino acid level and 73% when nucleotides in the coding sequence are compared. Sequence homology between the strain PM13 cat variant and Tn9 cat was not apparent however in the 5' and 3' flanking regions. Segments of near identity were seen when the upstream sequence of the cat of P. mirabilis was compared with the 5' regions of the Salmonella typhimurium flagellin genes H1 and H2, which are alternately expressed by a flip-flop control mechanism involving an invertible promoter and a trans-acting product.     

Endothelin-1 in chronic renal failure and hypertension

Investigation into the role of endothelin-1 (ET-1) in renal function has revealed two major direct actions leading to the control of extracellular volume and blood pressure. These are the regulation of renal hemodynamics and glomerular filtration rate and the modulation of sodium and water excretion. In the rat remnant kidney model of chronic renal failure, ET-1 production is increased in blood vessels and renal tissues. These changes are related to an increase in preproET-1 expression and correlate with the rise in blood pressure, the development of cardiovascular hypertrophy, and the degree of renal insufficiency and injury. Selective ETA receptor blockade prevents the progression of hypertension and the vascular and renal damage, supporting a role for ET-1 in chronic renal failure progression. The increase in ET-1 production can be associated with other local mediators, including angiotensin II, transforming growth factor-beta1 and nitric oxide, the local production of which is also altered in chronic renal failure. In human patients with essential hypertension, atherosclerosis, and nephrosclerosis, plasma ET-1 levels are increased compared with patients with uncomplicated essential hypertension. Similarly, plasma ET-1 concentrations are markedly increased in patients with end-stage renal disease undergoing dialysis, and this correlates with blood pressure, suggesting that ET-1 may contribute to hypertension in these patients. The treatment of anemia in patients with renal failure with human recombinant erythropoietin increases blood pressure by accentuating the underlying endothelial dysfunction and the elevated vascular ET-1 production. Overall, these results support a role for ET-1 in hypertension and the end-organ damage associated with chronic renal failure. ETA receptor blockade may then represent a potential target for the management of hypertension and cardiovascular and renal protection.     

Endothelin isoforms and the response to myocardial stretch

Myocardial stretch elicits a biphasic increase in developed force with a first rapid force response and a second slow force response (SFR). The rapid phase is due to an increase in myofilament Ca(2+) responsiveness; the SFR, analyzed here, is ascribed to a progressive increase in Ca(2+) transients. Experiments were performed in cat papillary muscles to further elucidate the signaling pathway underlying the SFR. Although the SFR was diminished by BQ-123, a similar endothelin (ET)-1-induced increase in force was not affected: 23 +/- 2 vs. 23 +/- 3% (not significant). Instead, BQ-123 suppressed the contractile effects of ET-2 or ET-3 (21 +/- 2 and 25 +/- 3% vs. -1 +/- 1 and -7 +/- 3% respectively, P < 0.05), suggesting that ET-2 or ET-3, but not ET-1, was involved in the SFR. Each isoform activated the Na(+)/H(+) exchanger (NHE-1), increasing intracellular Na(+) concentration by 2.0 +/- 0.1, 2.3 +/- 0.1, and 2.1 +/- 0.4 mmol/l for ET-1, ET-2, and ET-3, respectively (P < 0.05). The NHE-1 inhibitor HOE-642 prevented the increases in force and intracellular Na(+) concentration induced by all the ET isoforms, but only ET-2 and ET-3 effects were sensitive to BQ-123. Real-time RT-PCR measurements of prepro-ET-1, -ET-2, and -ET-3 were performed before and 5, 15, and 30 min after stretch. No changes in ET-1 or ET-2, but an increase of approximately 60% in ET-3, mRNA after 15 min of stretch were detected. Stretch-induced ET-3 mRNA upregulation and its mechanical counterpart were suppressed by AT(1) receptor blockade with losartan. These data suggest a role for AT(1)-mediated ET-3 released in the early activation of NHE-1 that follows myocardial stretch.