Critical factors in the radioimmunoassay of endothelin-1, endothelin-3, and big endothelin-1 in human plasma

The possible diagnostic or prognostic significance of changes in circulating level of endothelins in a variety of pathological conditions is currently of interest. Unfortunately, no consensus regarding optimization of sensitivity and extraction procedures for the reliable radioimmunoassay of endothelin-1 (ET-1), big endothelin-1 (BigET-1), and endothelin-3 (ET-3) currently exists. The object of the present study was to evaluate aspects of currently used extraction and assay procedures that limit accurate determination of ET in human plasma and define criteria to reduce variability. Critical parameters include the selectivity of commercial antibodies and the ability to remove interfering material after Sep-Pak absorption by selective washing with 24% ethanol in 4% acetic acid or methylene chloride in 0.1% trifluoroacetic acid. Assay sensitivity and specificity in the physiological range is improved by optimizing total binding parameters for the antibodies to give approximately 15-20% binding of radiolabeled peptide. With these modifications normal plasma values for ET-1, BigET-1, and ET-3 averaged 1.7 +/- 0.06, 2.5 +/- 0.3, and 5.8 +/- 0.2 pg/ml, respectively. These data suggest that such modifications may help to resolve many of the earlier difficulties concerning the role of ET under normal and pathological conditions.     

Comparison of the hemodynamic effects of endothelin-1 and big endothelin-1 in the rat

We compared the hemodynamic effects of continuous i.v. infusion of endothelin-1 and big endothelin-1 in anesthetized rats. Big endothelin-1 was fivefold less potent than endothelin-1 in decreasing cardiac output, heart rate, and stroke volume. However, big endothelin-1 produced a significantly larger increase in mean arterial pressure compared to endothelin-1 at doses that produced identical decreases in cardiac output. These findings support the hypothesis that the hypertensive effects of big endothelin-1 and endothelin-1 are produced by differential effects on systemic vascular resistances.     

Synthesis and degradation of endothelin-1

The endothelin-converting enzyme (ECE) is the main enzyme responsible for the genesis of the potent pressor peptide endothelin-1 (ET-1). It is suggested that the ECE is pivotal in the genesis of ET-1, considering that the knockout of both genes generates the same lethal developments during the embryonic stage. Several isoforms of the ECE have been disclosed, namely ECE-1, ECE-2, and ECE-3. Within each of the first two groups, several sub-isoforms derived through splicing of single genes have also been identified. In this review, the characteristics of each sub-isoform for ECE-1 and 2 will be discussed. It is important to mention that the ECE is, however, not the sole enzyme involved in the genesis of endothelins. Indeed, other moieties, such as chymase and matrix metalloproteinase II, have been suggested to be involved in the production of ET intermediates, such as ET-1 (1-31) and ET-1 (1-32), respectively. Other enzymes, such as the neutral endopeptidase 24-11, is curiously not only involved in the degradation and inactivation of ET-1, but is also responsible for the final production of the peptide via the hydrolysis of ET-1 (1-31). In this review, we will attempt to summarize, through the above-mentioned characteristics, the current wisdom on the role of these different enzymes in the genesis and termination of effect of the most potent pressor peptide reported to date.     

Phosphoramidon inhibits the conversion of intracisternally administered big endothelin-1 to endothelin-1

It is suggested that endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). We examined the effects of intracisternal administration of big ET-1 on the cerebral arteries in the absence or presence of pretreatment with phosphoramidon, an inhibitor of ET converting enzyme, in anesthetized dogs. After intracisternal administration of big ET-1 (10 micrograms/dog), the caliber of the basilar artery on the angiogram was decreased to about 59% of the control. This was accompanied by a marked increase in immunoreactive ET in the cerebrospinal fluid. Systemic arterial pressure was markedly elevated following big ET-1 injection. All changes induced by big ET-1 were effectively prevented with phosphoramidon. These data suggest that intracisternally administered big ET-1 is converted to ET-1 and that the generated ET-1 produces cerebral vasospasm and hypertension. A phosphoramidon-sensitive metalloproteinase appears to contribute to this conversion.     

miRNA-1 regulates endothelin-1 in diabetes

Aims

MicroRNAs (miRNAs) play important roles in several biological processes. In this study, we investigated the role of miR-1, an endothelin-1 (ET-1) targeting miRNA, in endothelial cells (ECs) and tissues of diabetic animals. ET-1 is known to be of pathogenetic significance in several chronic diabetic complications.

Main methods

PCR array was used to identify alterations of miRNA expression in ECs exposed to glucose. miR-1 expression was validated by TaqMan real-time PCR assay. Human retinal ECs (HRECs) and human umbilical vein ECs (HUVECs) exposed to various glucose levels with or without miR-1 mimic transfection, and tissues from streptozotocin-induced diabetic animals after two months of follow-up, were examined for miR-1 expression, as well as ET-1 and fibronectin (FN) mRNA and protein levels.

Key findings

Array analyses showed glucose-induced alterations of 125 miRNAs (out of 381) in ECs exposed to 25 mM glucose compared to 5 mM glucose. Fifty-one miRNAs were upregulated and 74 were downregulated. 25 mM glucose decreased miR-1 expression and increased ET-1 mRNA and protein levels. miR-1 mimic transfection prevented HG-induced ET-1 upregulation. Furthermore, glucose induced upregulation of FN, which is mediated partly by ET-1, was also prevented by such transfection.Diabetic animals showed decreased miR-1 expression in the retina, heart and kidneys. In parallel, ET-1 mRNA expressions were increased in these tissues of diabetic animals, in association with upregulation of FN.

Significance

These results indicate a novel glucose-induced mechanism of tissue damage, in which miR-1 regulates ET-1 expressions in diabetes. Identifying such mechanisms may lead to RNA based treatment for diabetic complications.     

Fluorescence studies of endothelin-1

Fluorescence measurements and singlet singlet energy transfer experiments on endothelin-1 provide information on the conformation of this peptide in dilute aqueous solution. The tyrosine fluorescence quantum yield in the absence of transfer (in [Phe²¹]endothelin-1) is relatively large (Φ_tyr = 0.39), indicating the side-chain is oriented away from fluorescence quenching groups such as the two disulfide bonds of the peptide. The fluorescence emission maximum (λ = 351 nm) and quantum yield (Φ_trp = 0.099) of tryptophan in endothelin-I suggests that this residue is fully accessible to the solvent and that the indole ring is not located near the fluorescence quenching histidinium moiety or the disulfide bonds.

Singlet-singlet fluorescence energy transfer measurements of the Tyr¹³/Trp²¹ intramolecular distance by both donor fluorescence quenching and relative enhancement of acceptor fluorescence yield a distance of about 12.8 ± 0.6 Å. Molecular modeling of a fully extended C-terminal hexapeptide indicates a Tyr¹³/Trp²¹ distance of about 25 Å. Thus, the C-terminal residues must bend back towards the bicyclic portion of the molecule.     

Effects of endothelin-1 and conversion of big endothelin-1 in the isolated perfused rabbit lung.

We examined the effects of endothelin-1 (ET-1) on pulmonary hemodynamic and transvascular fluid filtration and the conversion of big endothelin-1 (big ET-1), a precursor of ET-1, in isolated perfused rabbit lungs at constant vascular and airway pressures. Furthermore we examined whether ET-1 contributes to cyclooxygenase metabolism. The perfusate flow decreased significantly after bolus administration of 1 or 0.1 nmol of ET-1. Lung weight did not increase throughout the experimental period. Big ET-1- (1 nmol) induced decrease in the flow was slow in developing, although the maximum response was comparable to that induced by the same dose of ET-1. The concentration of bit ET-1 in the perfusate progressively decreased, while that of ET-1 increased in a time-dependent manner. Phosphoramidon, an inhibitor of metalloproteinase, suppressed the pressor effect of big ET-1 (P less than 0.01) and the increase in the concentration of ET-1 in the perfusate (P less than 0.05). The present findings provide the first evidence suggesting that the potent vasocontractile effect of big ET-1 in pulmonary circulation can be attributed to the production of ET-1 by the conversion from big ET-1 in the vascular bed. ET-1-induced perfusate flow changes were not affected by indomethacin, and the concentration of 6-ketoprostaglandin F1 alpha, a metabolite of prostacyclin, did not increase after ET-1 administration.     

Different affinities and selectivities of endothelin-1 and endothelin-3 binding to various rat tissues

Inhibition of [125I]endothelin-1 ([125I]ET-1) binding to membrane fractions from various rat tissues by ET-1 and endothelin-3 (ET-3) was investigated. Brain tissue demonstrated a 37-fold higher affinity for ET-1 compared to lung, while a greater than 1000-fold difference in affinity for ET-3 was observed in these two tissues. Furthermore, the ratio of the IC50 value of ET-3 to that of ET-1 in each tissue varied from approximately 2 in brain, kidney and liver to greater than 100 in heart and spleen. These experiments show that the tissues examined have different affinities as well as different selectivities for ET-1 and ET-3.     

Arrhythmogenic action of endothelin-1(1-31) through conversion to endothelin-1(1-21)

Endothelin (ET)-1(1-21) is known to play an important role in the pathogenesis of acute ischemic arrhythmia. In the present study, we attempted to determine whether administration of ET-1(1-31) would result in arrhythmia in perfused isolated rat hearts. Forty-eight Sprague-Dawley rats weighing approximately 250-350 g were randomized into 6 groups. Heart was isolated and perfused in a Langendorff mode. The effects of ET-1(1-31) on arrhythmia, heart rate, coronary flow, and heart function were analyzed. Perfusion with 1 nM ET-1(1-31) resulted in frequent ventricular ectopic beats (VEBs) and ventricular tachycardia (VT). Overall VEB was 128.0 (approximately 66.0-1015.0), and the arrhythmia score (AS) was 2.18 +/- 0.87; both were significantly higher than those of the control group (P < 0.01). Pretreatment with perfusion of 10 nM of the ETA-receptor antagonist BQ(123) markedly attenuated the occurrence of VEB and VT induced by ET-1(1-31). AS in 10 nM BQ123 group was significantly lower than that in 1 nM ET-1(1-31) group (P < 0.01). The arrhythmia induced by 1 nM ET-1(1-31) was partially but significantly reduced by phosphoramidon (1 microM), a neutral endopeptidase/ET-converting enzyme inhibitor. ET-1(1-31) per se caused arrhythmia in perfused isolated rat hearts. This arrhythmogenic action is in part mediated by ET(A) receptor and may be attributed mainly to the conversion of ET-1(1-31) to ET-1(1-21.).     

Increased expression of endothelin-1 and inducible nitric oxide synthase isoform II in aging arteries in vivo: implications for atherosclerosis

We here report that aging increases expression of endothelin-1 and NO synthases in the vasculature and kidney of normotensive rats in vivo. Expression of preproendothelin-1 mRNA was quantified by RT-PCR and in situ hybridization, and endothelin-1 protein was determined by radioimmunoassay/HPLC. Vascular mRNA expression of NO synthase isoforms II and III was analyzed by RT-PCR. In young animals, vascular endothelin-1 protein was differentially expressed (aorta < renal artery < carotid artery) and increased with aging in all vascular beds (P < 0.05). In the intact aorta of aged rats, mRNA expression of preproendothelin-1, "inducible" NO synthase II, and endothelial cell NO synthase III gene was up-regulated (P < 0.05). Moreover, preproendothelin-1 mRNA expression increased in glomeruli and tubulointerstitial cells (P < 0.05). To our knowledge this is the first study demonstrating local vascular up-regulation of the trophic factor endothelin under physiological conditions. Activation of vascular endothelin and NO synthases may be important, pressure-independent factors contributing to structural and functional abnormalities of age-dependent diseases, including atherosclerosis.     

Phosphoramidon inhibits the intracellular conversion of big endothelin-1 to endothelin-1 in cultured endothelial cells

Effects of various protease inhibitors on the conversion of big endothelin (ET)-1 to ET-1 in cultured endothelial cells were analyzed. A metal protease inhibitor, phosphoramidon, decreases the amount of ET-1 and increase that of big ET-1 released. This effect is dose-dependent and not nonspecific. When the contents of ET-1 and big ET-1 in the cells after culturing in the medium with or without phosphoramidon were measured, the ratio of ET-1: big ET-1 in the cells was 3.3 : 1 and phosphoramidon inverted the ratio in the cells to 1 : 3.5. These data strongly suggest that a phosphoramidon-sensitive protease converts big ET-1 to mature ET-1 intracellularly.     

Articular chondrocyte passage number: influence on adhesion, migration, cytoskeletal organisation and phenotype in response to nano- and micro-metric topography

The isolation and culture of articular chondrocytes is a prerequisite of their use in tissue engineering, but prolonged culture and passaging is associated with de-differentiation. In this paper we studied the influence of nanometric and micrometric grooves (85 nm to 8 microm in depth and 2 microm to 20 microm in width) on 1st and 2nd passage ovine chondrocytes since our earlier findings indicate that primary cells are not affected by such features. 1st and 2nd passage chondrocytes cultured on grooved substrata showed a polarisation of cell shape parallel to the groove long axis and F-actin condensations were evident at groove ridge boundaries. An increase in cell migration with increasing groove depth was observed. Both passages of chondrocytes maintained type II collagen expression, but to a lesser degree in 2nd. This study demonstrates that passage number alters the response of chondrocytes to micrometric and nanometric topography, and could be important in ex vivo cartilage engineering.     

Lipid phosphatase SHIP-1 regulates chondrocyte hypertrophy and skeletal development

SH2-containing inositol-5′-phosphatase-1 (SHIP-1) controls the phosphatidylinositol-3′-kinase (PI3K) initiated signaling pathway by limiting cell membrane recruitment and activation of Akt. Despite the fact that many of the growth factors important to cartilage development and functions are able to activate the PI3K signal transduction pathway, little is known about the role of PI3K signaling in chondrocyte biology and its contribution to mammalian skeletogenesis. Here, we report that the lipid phosphatase SHIP-1 regulates chondrocyte hypertrophy and skeletal development through its expression in osteochondroprogenitor cells. Global SHIP-1 knockout led to accelerated chondrocyte hypertrophy and premature formation of the secondary ossification center in the bones of postnatal mice. Drastically higher vascularization and greater number of c-kit + progenitors associated with sinusoids in the bone marrow also indicated more advanced chondrocyte hypertrophic differentiation in SHIP-1 knockout mice than in wild-type mice. In corroboration with the in vivo phenotype, SHIP-1 deficient PDGFRα + Sca-1 + osteochondroprogenitor cells exhibited rapid differentiation into hypertrophic chondrocytes under chondrogenic culture conditions in vitro. Furthermore, SHIP-1 deficiency inhibited hypoxia-induced cellular activation of Akt and extracellular-signal-regulated kinase (Erk) and suppressed hypoxia-induced cell proliferation. These results suggest that SHIP-1 is required for hypoxia-induced growth signaling under physiological hypoxia in the bone marrow. In conclusion, the lipid phosphatase SHIP-1 regulates skeletal development by modulating chondrogenesis and the hypoxia response of the osteochondroprogenitors during endochondral bone formation.     

PTHrP modulates chondrocyte differentiation through AP-1 and CREB signaling

During the process of differentiation, chondrocytes integrate a complex array of signals from local or systemic factors like parathyroid hormone-related peptide (PTHrP), Indian hedgehog, bone morphogenetic proteins and transforming growth factor beta. While PTHrP is known to be a critical regulator of chondrocyte proliferation and differentiation, the signaling pathways through which this factor acts remain to be elucidated. Here we show that both cAMP response element-binding protein (CREB) and AP-1 activation are critical to PTHrP signaling in chondrocytes. PTHrP treatment leads to rapid CREB phosphorylation and activation, while CREB DNA binding activity is constitutive. In contrast, PTHrP induces AP-1 DNA binding activity through induction of c-Fos protein expression. PTHrP activates CRE and TRE reporter constructs primarily through PKA-mediated signaling events. Both signaling pathways were found to be important mediators of PTHrP effects on chondrocyte phenotype. Alone, PTHrP suppresses maturation and stimulates proliferation of the chondrocyte cultures. However, in the presence of dominant negative inhibitors of CREB and c-Fos, these PTHrP effects were suppressed, and chondrocyte maturation was accelerated. Moreover, in combination, the effects of dominant negative c-Fos and CREB are synergistic, suggesting interaction between these signaling pathways during chondrocyte differentiation.     

Sphingosine-1-phosphate stimulates rat primary chondrocyte proliferation

Rat primary chondrocytes express the sphingosine-1-phosphate (S1P) receptor, S1P(2), S1P(3), S1P(4), but not S1P(1). When chondrocytes were stimulated with S1P or phytosphingosine-1-phosphate (PhS1P, an S1P(1)- and S1P(4)-selective agonist), phospholipase C-mediated cytosolic calcium increase was dramatically induced. S1P and PhS1P also stimulated two kinds of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) and p38 kinase in chondrocytes. In terms of the two phospholipids-mediated functional modulation of chondrocytes, S1P and PhS1P stimulated cellular proliferation. The two phospholipids-induced chondrocyte proliferations were almost completely blocked by PD98059 but not by SB203580, suggesting that ERK but not p38 kinase is essentially required for the proliferation. Pertussis toxin almost completely inhibited the two phospholipids-induced cellular proliferation and ERK activation, indicating the crucial role of G(i) protein. This study demonstrates the physiological role of two important phospholipids (S1P and PhS1P) on the modulation of rat primary chondrocyte proliferation, and the crucial role played by ERK in the process.     

Receptor kinetics differ for endothelin-1 and endothelin-2 binding to Swiss 3T3 fibroblasts

The equilibrium binding, kinetics of ligand-receptor interactions, and biological activity of endothelin-1 and -2 have been studied in Swiss 3T3 fibroblasts. Scatchard analyses of saturation binding data for ET-1 and -2, performed at 4 degrees C to prevent internalization of the occupied receptor, revealed similar affinity constants and numbers of binding sites for endothelin-1 and -2. Experiments designed to determine ligand-induced effects on 45Ca efflux demonstrated no qualitative or quantitative differences between the two endothelin isoforms. In contrast, kinetic studies resulted in different rates of dissociation for the two isoforms and different extents of dissociation. Specifically, only 40% of the bound [125I]endothelin-1 was dissociated at 4 h following the addition of excess unlabeled ligand, whereas 85-90% of the bound [125I]endothelin-2 was dissociated under the same conditions. Endothelin-1 and -2 also differed in the percent of specific cell-associated ligand bound after a 2 h incubation at 37 degrees C following an initial equilibration at 4 degrees C. The differences in dissociation rates and association or internalization rates at 37 degrees C are the first data that differentiate between the two isoforms. It is suggested that isoform-specific differences in the rate of dissociation from cell surface endothelin receptors influence the level of cell-associated endothelin and may be important in determining physiologic responses in vivo.