Specific sandwich-type enzyme immunoassays for smooth muscle constricting novel 31-amino acid endothelins

We established highly sensitive and specific sandwich-enzyme immunoassays (EIAs) for three newly discovered bioactive 31-amino acid endothelins [ETs(1-31)], which can detect as little as 0.16 pg/well of ET-1(1-31), 0.39 pg/well of ET-2(1-31), and 0.16 pg/well of ET-3(1-31). The EIAs showed no crossreactivity with 21-amino acid endothelins [ETs(1-21)] or big ETs at the usual assay concentrations below 1-5 ng/ml. In reversed-phase HPLC, immunoreactive ETs(1-31) in the granulocytes of normal human subjects eluted at the exact positions of authentic ETs(1-31), except for the presence of one additional unknown immunoreactive ET-1(1-31). The results also indicate that ETs(1-31) exist in the granulocytes at levels higher than or similar to those of ETs(1-21). This study is the first to establish EIAs for novel bioactive ETs(1-31). These assays can be utilized to assess the pathophysiological roles of ETs(1-31).     

Effect of endothelin-1(1-31) on p38 mitogen-activated protein kinase in cultured human mesangial cells

It was reported that human chymase cleaves big endothelins (ETs) at the Tyr31-Gly32 bond and produces 31-amino acid ETs(1-31). In this study, we investigated the effect of ET-1(1-31) on p38 mitogen-activated protein kinase (p38-MAPK) activity in human mesangial cells (HMCs). By measuring the kinase activity, we demonstrated that ET-1 (1-31) activated the p38-MAPK dose-dependently (10(-9) M to 10(-7) M), which was inhibited by SB203580. The p38-MAPK activation induced by ET-1(1-31) peaked at 10 minutes. BQ123 almost abolished ET-1(1-31)-induced p38-MAPK activation, whereas BQ788 failed to inhibit it. These findings suggest that the stimulatory effect of ET-1(1-31) on p38-MAPK activation is mediated through ET(A) or ET(A)-like receptor. In conclusion, ET-1(1-31) induced increase in p38-MAPK activation in cultured HMCs.     

Mechanisms transducing the aldosterone secretagogue signal of endothelins in the human adrenal cortex

Evidence has been provided that the 21-amino acid hypertensive peptide endothelin (ET)-1 exerts a potent secretagogue effect on human adrenocortical zona glomerulosa (ZG), acting through two receptor subtypes, called ET(A) and ET(B), the signaling mechanism(s) of which has (have) not yet been investigated. Collagenase dispersed human ZG cells were obtained from normal adrenals of patients undergoing nephrectomy/adrenalectomy for renal cancer. The selective ET(A)- and ET(B)-receptor activation was obtained by exposing dispersed cells to ET-1 plus the ET(B)-receptor antagonist BQ-788 and to the ET(B)-receptor agonist BQ-3020, respectively. The phospholipase (PL) C inhibitor U-73122 abolished ET(A) receptor-mediated secretory response, but only partially prevented the ET(B) receptor-mediated one. The phosphatidylinositol 3-kinase inhibitor wortmannin, the calmodulin inhibitor W-7 and the protein kinase (PK) C inhibitor calphostin-C significantly blunted the secretory responses ensuing from the activation of both receptor subtypes. When added together, calphostin-C and wortmannin or W-7 abolished ET(A)-mediated secretory response, but only decreased ET(B)-mediated one. The ET(B) receptor-, but not the ET(A) receptor-mediated aldosterone response was partially reversed by the cyclooxygenase (COX) inhibitor indomethacin, which when added together with U-73122 abolished it. ET(A)-receptor activation raised inositol triphosphate (IP(3)) production from dispersed ZG cells, while ET(B)-receptor stimulation enhanced both IP(3) and prostaglandin-E(2) production. Collectively, our findings indicate that ETs stimulate aldosterone secretion from human ZG cells, acting through ET(A) receptors exclusively coupled to PLC/PKC-dependent pathway and ET(B) receptors coupled to both PLC/PKC- and COX-dependent cascades.     

Expression of endothelins and their receptors promotes an invasive phenotype of breast tumor cells but is insufficient to induce invasion in benign cells

There is increased staining of endothelins (ET-1, -2, and -3) and receptors (ET-RA and -RB) in invasive breast tumors compared to nonneoplastic tissue, and ETs stimulate MCF-7 cell invasion in vitro. We analyzed ETstimulation of benign and transformed mammary epithelial cells, and whether expression of ETs is sufficient to induce invasiveness. In breast cancer patient serum, ET-1 was increased in those patients with lymph node metastases compared to those with no lymph node involvement; ETs, however, had no mitogenic effect on breast tumor cell lines in vitro. The benign mammary epithelial cell line, hTERT-HME1, and the poorly invasive breast tumor cell line MCF-7 secreted low levels of ET-1, while the invasive cell lines SKBR3 and MDAMB231 secreted high levels. Expression of the ETs and receptors by the cell lines broadly correlated with their in vitro invasiveness; overexpression of ETs in MCF-7 cells increased basal invasion. ET-mediated invasion involved both receptors and a calcium influx to induce a pertussis toxin-sensitive MAPK pathway. MMP-14 activity was induced via ET-RA in an autocrine manner. In contrast to transformed cells, ET stimulation or overexpression did not induce an invasive phenotype in benign cells. Benign cells do not respond to ETs, and ET expression is not sufficient to induce invasion; however, the level of ET production by tumor cells correlates with their invasiveness, and increasing expression of the ET axis promotes breast tumor cell invasion via both receptors, while MMP-14 is induced via ET-RA.     

Effects of endothelins on signal transduction and proliferation in human melanocytes

We demonstrate here that human melanocytes could be regulated by endothelin (ET) derivatives, potent vasoconstrictive peptides synthesized by endothelial cells, to stimulate their proliferation and melanization via a receptor-mediated signal transduction pathway. Receptor-binding assay using [125I]ET indicated that unlabeled ET-1 or ET-2 competitively inhibited each binding of labeled ETs to melanocytes with a concentration for half-maximal inhibition (IC50) of 0.7 or 0.9 nM, respectively. The dissociation constant (Kd) and the number of sites of the specific bindings of ET-1 and those of ET-2 were almost the same (Kd: 1.81 nM, binding sites: 7.0-8.0 x 10(4) per cell). Upon incubation with cultured cells, the mass contents of inositol 1,4,5-trisphosphate and intracellular calcium level were substantially increased by 10 nM ET-1, ET-2, and ET-3, but not by big-ET with maximal response at 80-130-s postincubation. The addition of ET-1 and ET-2 at 1-50 nM concentrations caused human melanocytes to significantly stimulate DNA [( 3H]thymidine incorporation) and melanin synthesis (3H2O release and [14C] thiouracil incorporation). Furthermore, ETs exhibited an additive stimulatory effect on basic fibroblast growth factor-stimulated DNA synthesis. In a long-term serum-free culture system, the strongest stimulation of growth by 10 nM ET-1 or ET-2 was observed in the presence of 10 nM cholera toxin and 0.2% bovine pituitary extract, resulting in a 4.5-fold increase in cell number for 12 culture days. These findings strongly suggest involvement of ET in the mechanism regulating proliferation and melanization of human melanocytes.     

High-yield production of recombinant endothelin-1.

A fusion protein (pETB-42P), which encodes the 42-amino acid leader peptide and the 38-amino acid peptide of human big endothelin (ET)-1, was synthesized in Escherichia coli, isolated as inclusion bodies, and purified by DEAE-chromatography. Trypsin digestion of the purified pETB-42P gave big ET-1(1-37) in a yield of 70%; then pepsin digestion of the purified big ET-1(1-37) gave ET-1(1-21) in a yield of 74% (overall yield: 52%). Sequential trypsin and pepsin digestions of the purified fusion protein in the same reaction vessel also allowed recovery of ET-1 in a yield of 60%. One milligram of ET-1 or 2.0 mg of big ET-1(1-37) was obtained from 1.8 liters of culture broth. Recombinant ET-1 thus obtained was identical to authentic ET-1 in terms of amino acid sequence and vasoconstrictor potency, and recombinant big ET-1(1-37) had almost the same in vitro and in vivo biological activities as big ET-1(1-38).     

Role of endothelins in septic,cardiogenic, and hemorrhagic shock

Shock is a condition where blood flow is inadequate for tissue needs. In all forms of shock, the concentrations of endothelins (ETs) are elevated, and they are especially high in septic shock. The rise in ETs plasma levels may initially have some positive homeostatic effects, for ETs can help restore normal vascular tone. However, high levels of ETs compromise the appropriate matching of flow to tissue needs and contribute to the pathophysiology of shock. Attempts at regulating the effects of ETs by the use of pharmacological blockers is made complicated by important interactions between the ETA and ETB receptors and potentially different effects on different tissues. We conclude that antagonism of ET receptors is unlikely to be helpful for cardiogenic or hemorrhagic shock. Furthermore, selective blockade is unlikely to be helpful. However, moderate doses of a mixed ET receptor antagonist may be of use for the management of septic patients.     

Effects of endothelins on hepatic stellate cell synthesis of endothelin-1 during hepatic wound healing

Endothelin-1 production is increased after liver injury and the subsequent wounding response. Further, endothelin-1 has prominent effects on hepatic stellate cells (key effectors of the hepatic wounding response), including on collagen synthesis, proliferation, and expression of smooth muscle proteins. We tested the hypothesis that endothelins (ETs) may regulate endothelin-1 production during hepatic wounding, and have investigated potential mechanisms underlying this process. Studies were performed on isolated stellate cells from normal and injured livers; in addition, potential autocrine effects of ET were assessed in vivo using an ET receptor antagonist in a model of liver injury. In stellate cells isolated from either normal or injured rat livers, ET receptor stimulation with endothelin-3 or sarafotoxin S6C (preferential ET(B) agonists) caused a dose-dependent increase in endothelin-1 production. Additionally, administration of a mixed ET antagonist in vivo during injury led to reduced stellate cell production of endothelin-1. The mechanism by which ETs stimulated endothelin-1 in this system appeared to be through upregulation of ET converting enzyme-1 (which converts precursor ET to mature peptide), rather than by modulation of precursor endothelin-1. We conclude that during liver injury and wound healing, stellate cell endothelin-1 production is, at least partially, stimulated by ETs via autocrine mechanisms that occur at the level of ET converting enzyme-1.     

Symbiotic Characteristics of Rhizobium leguminosarum bv. trifolii Isolates Which Represent Major and Minor Nodule-Occupying Chromosomal Types of Field-Grown Subclover (Trifolium subterraneum L.)

The symbiotic effectiveness and nodulation competitiveness of Rhizobium leguminosarum bv. trifolii soil isolates were evaluated under nonsoil greenhouse conditions. The isolates which we used represented both major and minor nodule-occupying chromosomal types (electrophoretic types [ETs]) recovered from field-grown subclover (Trifolium subterraneum L.). Isolates representing four ETs (ETs 2, 3, 7, and 8) that were highly successful field nodule occupants fixed between 2- and 10-fold less nitrogen and produced lower herbage dry weights and first-harvest herbage protein concentrations than isolates that were minor nodule occupants of field-grown plants. Despite their equivalent levels of abundance in nodules on field-grown subclover plants, ET 2 and 3 isolates exhibited different competitive nodulation potentials under nonsoil greenhouse conditions. ET 3 isolates generally occupied more subclover nodules than isolates belonging to other ETs when the isolates were mixed in 1:1 inoculant ratios and inoculated onto seedlings. In contrast, ET 2 isolates were less successful at nodulating under these conditions. In many cases, ET 2 isolates required a numerical advantage of at least 6:1 to 11:1 to occupy significantly more nodules than their competitors. We identified highly effective isolates that were as competitive as the ET 3 isolates despite representing serotypes that were rarely recovered from nodules of field-grown plants. When one of the suboptimally effective isolates (ET2-1) competed with an effective and competitive isolate (ET31-5) at several different inoculant ratios, the percentages of nodules occupied by the former increased as its numerical advantage increased. Although subclover yields declined as nodule occupancy by ET2-1 increased, surprisingly, this occurred at inoculant ratios at which large percentages of nodules were still occupied by ET31-5.     

Localization of the 31-amino-acid endothelin-1 in hamster tissue

Endothelin (ET)-1(1-31) is a novel vasoconstrictor peptide produced by human mast cell chymase, which selectively cleaves big ET-1 at the Try(31)-Gly(32) bond. We investigated the localization of ET-1(1-31) in various hamster tissues by immunohistochemistry and compared it to the distribution of ET-1(1-21). We found that the localization and amount of ET-1(1-31) were different from those of ET-1(1-21) in each tissue. ET-1(1-31)-like immunoreactivities (IR) in the heart, lung, and adrenal gland were observed in the same areas as ET-1(1-21) but were significantly weaker, suggesting that ET-1(1-31) might play a role only in mast cell/chymase-related pathological conditions in these tissues. In the liver, ET-1(1-31)-like IR was strongly detected in Kupffer cells where ET-1(1-21)-like IR was seen more weakly. In the kidney, ET-1(1-31)-like IR was slightly higher than ET-1(1-21). These results suggest that ET-1(1-31) might have physiological roles distinct from those of ET-1(1-21) in some hamster tissues.     

Concomitant secretion of big endothelin and its C-terminal fragment from human and bovine endothelial cells

A specific radioimmunoassay (RIA) for the carboxyl-terminal fragment (CTF) of big porcine endothelin (pET), an intermediate form of pET, was established to characterize big ET-like and its CTF-like immunoreactivity (LI) secreted from cultured bovine and human endothelial cells (EC). The antibody used crossreacted equally with big pET(1-39) and its CTF(22-39), but not with pET(1-21). Serial dilution curves of the culture media from bovine and human EC were parallel to that of standard CTF. Reverse-phase HPLC coupled with RIAs for big ET and ET of the culture media from bovine and human EC revealed essentially the same elution profiles: two major CTF-LI components, one corresponding to big pET(1-39) and the other to its CTF(22-39), in addition to one major ET-LI component corresponding to pET(1-21). The amounts of CTF-LI were almost equal to that of ET-LI on a molar basis. These data suggest that big ET is processed by a putative ET converting enzyme to yield its CTF and the mature ET(1-21) in EC.     

Activation of particulate guanylyl cyclase by endothelins in cultured SV-40 transformed cat iris sphincter smooth muscle cells

We investigated the effects of endothelins (ETs) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ET-3 increased cGMP formation in a concentration-dependent manner (EC50 = 98nM), which was 2.5 times higher than that of ET-1. The ET(B)receptor agonists sarafotoxin-S6c and IRL 1620 also increased cGMP production, mimicking the effects of the ETs. The ET(B) receptor antagonist BQ 788, but not the ET(A) receptor antagonist BQ610, dose-dependently blocked ET-3-stimulated cGMP formation (IC50=10nM). The phorbol ester, Phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylyl cyclase in smooth muscle, dose-dependently inhibited ET-3-stimulated cGMP accumulation (IC50=66nM). LY83583 and ODQ, inhibitors of soluble guanylyl cyclases, as well as inhibitors of the nitric oxide cascade and of intracellular Ca2+ elevation had no appreciable effect on ET-3-induced cGMP production. ET-3 markedly inhibited carbachol-induced intracellular Ca2+ mobilization. We conclude that ET-3 increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ET(B) receptor subtype and subsequent stimulation of the membrane-bound guanylyl cyclase. Elevation of cGMP by ET and the subsequent inhibition of muscarinic stimulation of intracellular Ca2+ mobilization by the cyclic nucleotide could serve to modulate the contractile effects of Ca2+-mobilizing agonists in the iris sphincter smooth muscle.     

Modulation and roles of the endothelins in the pathophysiology of pulmonary embolism

Recent research on the endothelins (ETs) and their pathways in acute pulmonary embolism (APE) has led to significant advances in the understanding of this disease. ETs are potent vasoconstrictors and bronchoconstrictors found abundantly in the lung and can be released by stimuli such as endothelial injury, hypoxia, or thrombin, a key product in the coagulation cascade. Many studies using different approaches and methods of inducing pulmonary embolization, both in vitro and in vivo in various species, have mostly shown that ETs play an important role in the pathophysiology of APE. These results were obtained by comparing the hemodynamic data in the presence or absence of various ETs inhibitors, but also by assessing the modulation of the ET-related elements of this system by molecular, cell biology, and pharmacological methods. Based on the current understanding, a mechanism involving the ET pathway in the pathophysiology of APE is proposed for the reader's considerations. We postulate that ETs are primary mediators in APE based on the following: (i) their source from pulmonary endothelial cells where the primary injury takes place; (ii) their direct vasconstrictive, bronchoconstrictive, and promitogenic effects via distinct ET receptors; and (iii) their indirect effects associated with the secondary release of thromboxane and other mediators, which are released from inflammatory cells and platelets, which together can potentiate the overall hemodynamic response, most specifically the pulmonary vascular bed. Such combined effects of ETs on bronchomotor and vasomotor tone in the lung can adversely affect ventilation perfusion matching and lead to severe hypoxemia without causing significant changes in the chest X-ray of these patients. Thus, we may consider ET inhibitors as future current therapeutic agents in patients with PE.     

Cultured bovine endothelial cells convert big endothelin isopeptides to mature endothelin isopeptides

The incubation of big endothelin-1 (big ET-1), big ET-2 or big ET-3 with cultured bovine endothelial cells (ECs) resulted in their conversions to mature endothelins (ETs). These conversions apparently exhibited Michaelis-Menten kinetics as a function of each big ET isopeptide. The conversions of big ETs were abolished by phosphoramidon. These results indicate that vascular endothelium can convert exogenous big ET-1 to mature ET-1 through a phosphoramidon-sensitive metalloprotease, and that this enzyme has also high affinities for big ET-2 and big ET-3.     

Effect of endothelin-1(1-31) on intracellular free calcium in cultured human mesangial cells

We found that human chymase selectively produces 31-amino-acid length endothelins (1-31) (ETs(1-31)). We investigated the effect of synthetic ET-1(1-31) on intracellular free Ca2+ concentration ([Ca2+]i) in cultured human mesangial cells. ET-1(1-31) increased [Ca2+]i in a concentration-dependent manner to a similar extent as ET-1. The ET-1 (1-31)-induced [Ca2+]i increase was not influenced by removal of extracellular Ca2+ but was inhibited by thapsigargin. ET-1(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon. It was inhibited by BQ123, but not by BQ788. These results suggest that ET-1(1-31) by itself exhibits bioactive properties probably through endothelin ET(A) or ET(A)-like receptors. Since human chymase has been reported to exist in the kidney, ET-1(1-31) may be a candidate substance for mesangium-relevant diseases.     

A solid-phase enzyme immunoassay of thromboxane B2

A solid-phase enzyme immunoassay for thromboxane B2 was developed using a conjugate of thromboxane B2 and beta-galactosidase. Anti-thromboxane B2 IgG was bound to a polystyrene tube, and the enzyme-labeled and unlabeled thromboxane B2 were allowed to react in a competitive manner with the immobilized antibody. Then, the specifically bound beta-galactosidase was assayed fluorimetrically, and the enzyme activity was correlated with the amount of unlabeled thromboxane B2. By using a calibration curve, thromboxane B2 was determined in the range of 20 fmol-14 pmol. 2,3-Dinor- and 2,3,4,5-tetranor-thromboxane B2 cross-reacted with thromboxane B2 to the extents of 18.6% and 0.4%, respectively. Most prostaglandins and their metabolites tested showed cross-reactivities of less than 1%. In application of the method to human blood and urine, an octadecylsilyl silica column was utilized for extraction and concentration of thromboxane B2. The crude extract contained a substance(s) which disturbed the enzyme immunoassay and gave an apparently high value of thromboxane B2, and the interfering substance was separated from thromboxane B2 by reverse-phase HPLC. Various amounts of authentic thromboxane B2 added to the purified material from human plasma could be determined by the enzyme immunoassay with a recovery of about 80% and the results correlated well with the values obtained by radioimmunoassay (r = 0.979). When the extract from human urine was analyzed by reverse-phase HPLC, the 2,3-dinor metabolite rather than thromboxane B2 was the predominant compound detected by the enzyme immunoassay.     

Pepsin, an aspartic protease, converts porcine big endothelin to 21-residue endothelin

Porcine big endothelin (big ET-39) at 1 nM, a concentration with no influence on contractile activity in isolated rat aorta, induced a slow-onset and sustained contraction by the pre-incubation with pepsin. When the incubation mixture of big ET-39 with pepsin was analyzed by high-performance liquid chromatography on an octadecyl silica column, two major products of pepsin hydrolysis were obtained; their amino acid sequences were identical with those of 21-residue endothelin (ET-21) and a C-terminal peptide of big ET-39, big ET (22-39), respectively. On the other hand, no degradation of ET-21 was observed by pepsin treatment. These results indicate that pepsin specifically cleaves a Trp21-Val22 bond in the big ET-39 molecule, producing ET-21 and big ET (22-39). Thus, the possibility that pepsin-like aspartic protease may participate in the conversion of big ET-39 to ET-21 in vivo warrants further attention.