Chymase inhibitor-sensitive synthesis of endothelin-1 (1-31) by recombinant mouse mast cell protease 4 and human chymase.

Important structural differences imply that human and mouse mast cell chymases may differ with respect to their enzymatic properties. We compared in this study the catalytic efficiencies of recombinant human chymase (rCMA1) and its functional murine homologue recombinant mouse mast cell protease-4 (rmMCP-4) toward a fluorogenic chymase substrate (Suc-Ala-Ala-Pro-Phe-7-amino-4-methylcoumarin (AMC) and by their ability to convert Big-endothelin (ET)-1 into ET-1 (1-31) using a LC/MS/MS system. Activities toward a fluorogenic substrate (Suc-Leu-Leu-Val-Tyr-AMC) and Big ET-1 were also measured in extracts from mouse peritoneal mast cells, LUVA human mast cell-like cells and human aortas. The specificity of these activities was assessed with the chymase inhibitor TY-51469 (2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonyl-phenyl]thiazole-4-carboxylic acid). For similar affinities, rmMCP-4 showed a higher activity toward the fluorogenic substrate and a higher ability to process Big ET-1 as compared to recombinant CMA1 (chymase activity (kcat/KM in µM(-1)s(-1)): 2.29 × 10(-4)vs. 6.41 × 10(-6); ET-1 (1-31) production: 2.19 × 10(-3)vs. 6.57 × 10(-5)), and both of these activities of mouse and human chymase were sensitive to TY-51469. Furthermore, extracts from mouse peritoneal mast cells, LUVA cells and human aorta homogenates contained processing activities toward the fluorogenic chymase substrate as well as Big ET-1, all of which were sensitive to TY-51469. Finally, the pressor responses to Big ET-1 but not to ET-1 were significantly reduced in conscious and free moving mMCP-4 KO mice when compared to wild type congeners. Our results suggest that both mouse and human chymases have potent ET-1 (1-31)-producing abilities, with the murine isoform being more efficient.

Cardiovascular effects of endothelin-11₋31 microinjected into the nucleus tractus solitarius of anesthetized rats.

Endothelin-11₋31 (ET-11₋31) is a 31-amino-acid vasoactive peptide that plays an important role in the regulation of cardiovascular function. However, the cardiovascular effects of central ET-11₋31 are still not fully understood. In this study, we assess the effects of ET-11₋31 within the nucleus tractus solitarius (NTS) of anesthetized rats and explore the underlying mechanisms of these effects. Bilateral microinjections of ET-11₋31 into the NTS produced dose-dependent hypotension and bradycardia, very similar to the effects of a unilateral microinjection of ET-11₋31 into the NTS. Bilateral microinjections of ET-11₋31 into the NTS significantly decreased baroreflex function in a time-dependent manner. The hypotensive and bradycardic effects induced by the microinjection of ET-11₋31 into the NTS were significantly decreased by the ETA receptor antagonist BQ123 and by kynurenic acid, but not by the ETB receptor antagonist BQ788. These results show that ET-11₋31 injected into the NTS produces hypotension and bradycardia, mediated by ETA receptors and, at least partly, by the glutamate receptor.

Pivotal role of mouse mast cell protease 4 in the conversion and pressor properties of Big-endothelin-1.

The serine protease chymase has been reported to generate intracardiac angiotensin-II (Ang-II) from Ang-I as well as an intermediate precursor of endothelin-1 (ET-1), ET-1 (1-31) from Big-ET-1. Although humans possess only one chymase, several murine isoforms are documented, each with its own specific catalytic activity. Among these, mouse mast cell protease 4 (mMCP-4) is the isoform most similar to the human chymase for its activity. The aim of this study was to characterize the capacity of mMCP-4 to convert Big-ET-1 into its bioactive metabolite, ET-1, in vitro and in vivo in the mouse model. Basal mean arterial pressure did not differ between wild-type (WT) and mMCP-4(-/-) mice. Systemic administration of Big-ET-1 triggered pressor responses and increased blood levels of immunoreactive (IR) ET-1 (1-31) and ET-1 that were reduced by more than 50% in mMCP-4 knockout (-/-) mice compared with WT controls. Residual responses to Big-ET-1 in mMCP-4(-/-) mice were insensitive to the enkephalinase/neutral endopeptidase inhibitor thiorphan and the specific chymase inhibitor TY-51469 {2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonylphenyl]thiazole-4-carboxylic acid}. Soluble fractions from the lungs, left cardiac ventricle, aorta, and kidneys of WT but not mMCP-4(-/-) mice generated ET-1 (1-31) from exogenous Big-ET-1 in a TY-51469-sensitive fashion as detected by high-performance liquid chromatography/ matrix-assisted laser desorption/ionization-mass spectrometry. Finally, pulmonary endogenous levels of IR-ET-1 were reduced by more than 40% in tissues derived from mMCP-4(-/-) mice compared with WT mice. Our results show that mMCP-4 plays a pivotal role in the dynamic conversion of systemic Big-ET-1 to ET-1 in the mouse model.

Fluorescence correlation spectroscopy in drug discovery: study of Alexa532-endothelin 1 binding to the endothelin ETA receptor to describe the pharmacological profile of natural products.

Fluorescence correlation spectroscopy and the newly synthesized Alexa532-ET1 were used to study the dynamics of the endothelin ET(A) receptor-ligand complex alone and under the influence of a semisynthetic selective antagonist and a fungal extract on living A10 cells. Dose-dependent increase of inositol phosphate production was seen for Alexa532-ET1, and its binding was reduced to 8% by the selective endothelin ET(A) antagonist BQ-123, confirming the specific binding of Alexa532-ET1 to the endothelin ET(A) receptor. Two different lateral mobilities of the receptor-ligand complexes within the cell membrane were found allowing the discrimination of different states for this complex. BQ-123 showed a strong binding affinity to the "inactive" receptor state characterized by the slow diffusion time constant. A similar effect was observed for the fungal extract, which completely displaced Alexa532-ET1 from its binding to the "inactive" receptor state. These findings suggest that both BQ-123 and the fungal extract act as inverse agonists.

Endothelin-1(1-31) induces spreading depolarization in rats.

BACKGROUND: The vasoconstrictor endothelin-1(1-21) (ET-1) seems to induce cerebral vasospasm after aneurismal subarachnoid hemorrhage (aSAH). Moreover, ET-1 causes spreading depolarization (SD) via vasoconstriction/ischemia. ET-1(1-31) is an alternate metabolic intermediate in the generation of ET-1. Our aim was to investigate whether endothelin-1(1-31) causes SD in a similar fashion to ET-1. METHOD: Increasing concentrations of either ET-1, ET-1(1-31) or vehicle were brain topically applied in 29 rats. Each concentration was superfused for one hour while regional cerebral blood flow (rCBF) and direct current electrocorticogram (DC-ECoG) were recorded. FINDINGS: In response to the highest concentration of 10(-6) M, all animals of both ET groups developed typical SD. At concentrations below 10(-6) M only ET-1 induced SD (n=14 of 19 rats). Thus, the efficacy of ET-1(1-31) to induce SD was significantly lower (P<0.001, two-tailed Fisher's Exact Test). CONCLUSIONS: Our findings suggest that ET-1(1-31) less potently induces SD compared to ET-1 which implicates that it is a less potent vasoconstrictor. Speculatively, it could be interesting to shift the metabolic pathway towards the alternate intermediate ET-1(1-31) after aSAH as an alternative strategy to ETA receptor inhibition. This could decrease ET-induced vasoconstriction and SD generation while a potentially beneficial basal ETA receptor activation is maintained.

Ablation of transient receptor potential vanilloid 1 abolishes endothelin-induced increases in afferent renal nerve activity: mechanisms and functional significance.

Endothelin 1 (ET-1) and its receptors, ETA and ETB, play important roles in regulating renal function and blood pressure, and these components are expressed in sensory nerves. Activation of transient receptor potential vanilloid (TRPV) 1 channels expressed in sensory nerves innervating the renal pelvis enhances afferent renal nerve activity (ARNA), diuresis, and natriuresis. We tested the hypothesis that ET-1 increases ARNA via activation of ETB, whereas ETA counterbalances ETB in wild-type (WT) but not TRPV1-null mutant mice. ET-1 alone or with BQ123, an ETA antagonist, perfused into the left renal pelvis increased ipsilateral ARNA in WT but not in TRPV1-null mutant mice, and ARNA increases were greater in the latter. [Ala1, 3,11,15]-endothelin 1, an ETB agonist, increased ARNA that was greater than that induced by ET-1 in WT mice only. [Ala1, 3,11,15]-endothelin 1-induced increases in ARNA were abolished by chelerythrine, a protein kinase C inhibitor, but not by H89, a protein kinase A inhibitor. Chelerythrine, H89, and BQ788, an ETB antagonist, did not affect ARNA triggered by capsaicin in WT mice. Substance P release from the renal pelvis was increased by [Ala1, 3,11,15]-endothelin 1 in WT mice only, and the increase was abolished by chelerythrine but not by H89. Chelerythrine, H89, and BQ788 did not affect capsaicin-induced substance P release. Our data show that ET1 increases ARNA via activation of ETB, whereas ETA counterbalances ETB in WT but not in TRPV1-null mutant mice, suggesting that TRPV1 mediates ETB-dependent increases in ARNA, diuresis, and natriuresis possibly via the protein kinase C pathway.

Endothelin-1 (1-31): from chymase-dependent synthesis to cardiovascular pathologies.

The mast cell-derived serine protease chymase is importantly involved not only in degradation, but in synthesis of bioactive peptides as well. Several studies suggest that chymase is the predominant enzyme in the production of angiotensin II (Ang II) from angiotensin-I in interstitial tissues. Interestingly, chymase has also been suggested to mature endothelin-1 (ET-1) from its precursor, big-ET-1 in vitro. The lack of availability of specific chymase inhibitors, beyond the chymotrypsin-like inhibitor chymostatin, currently hampers the investigation of the chymase/ET-1/Ang II paradigm in physiology and cardiovascular diseases. Nonetheless, the recent advent of highly selective chymase inhibitors is shedding new light on the role of this enzymatic pathway in the several inflammatory prone vascular diseases as summarized in the present review. Considering increasing evidence towards significant interactions between Ang II and ET-1 in cardiovascular diseases, the present review will address the role of chymase in the production of those two peptides. Whether chymase-dependent production of ET-1 plays an important role in cardiovascular pathologies will also be discussed.

Identification of ETA and ETB binding domains using ET-derived photoprobes.

Using the structure of ET-1 as a template, a series of photosensitive analogs were developed to investigate the binding domain of ETA and ETB receptors. Accordingly, a p-benzoyl-l-phenylalanine (Bpa) residue was introduced into the peptide chain following a pattern aiming at scanning N- to C-terminal portions of the molecule. Among the analogs, those containing a Bpa amino acid in position 7 ([L-Bpa7, Tyr(125I)13]hET-1) or 12 ([Nle7, L-Bpa12, Tyr(125I)13]hET-1) exhibited the capacity to activate both receptors, thus showing that residues Met-7 and Val-12 of ET-1 do not play a key role in the activation process. The binding capacity of the probes was also evaluated on transfected CHO cells overexpressing either ETA or ETB receptors. Subsequently, these photoprobes were used to label ETA and ETB receptors overexpressed in transfected CHO cells. Enzymatic digestions and chemical cleavages were then performed on ligand-receptor complexes and fragments produced by the lysis were analyzed to point out putative interaction areas on the receptors. Results showed that Phe147-Lys166, covering the second segment of EC I and the top part of TM III, contains a contact point for [Nle7, L-Bpa12, Tyr(125I)13]hET-1 on ETA receptors whereas Ile292-Trp319, spanning from the second half of the intracellular loop III up to the middle turns of TM VI, includes a residue that can interact with [L-Bpa7, Tyr(125I)13]hET-1. Moreover, upon binding of [Nle7, L-Bpa12, Tyr(125I)13]hET-1, it was observed that Thr263-Met266 (EC II) of the ETB receptor would come close with the ligand.

Effects of endothelin-11-31 on cell viability and [Ca2+]i in cultured neonatal rat cardiomyocytes

We previously found that endothelin-1(1-31) (ET-1(1-31)) exhibited a pro-arrhythmogenic effect in isolated rat hearts. In this study, we further investigated the effects of ET-1(1-31) on a cell viability and observed [Ca(2+)](i) in cultured cardiomyocytes. Cultured neonatal rat cardiomyocytes were treated with 0.1, 1, and 10 nM ET-1(1-31) for 24h in the presence or absence of ET(A) receptor antagonist (BQ(123)) or phosphoramidon, a NEP/ECE inhibitor. Cell injury was evaluated by supernatant lactate dehydrogenase (LDH) assay, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content. Cell viability was assessed by MTT assay. [Ca(2+)](i) was measured with Fluo-3/AM under a laser confocal microscope. 1) ET-1(1-31) dose-dependently increased LDH release and decreased cell viability. 2) LDH and MDA levels were significantly elevated and SOD activity decreased after administration of 1 nM ET-1(1-31) for 24h, and these changes were markedly attenuated by 1 uM BQ(123). 3) Exposure to 10 nM ET 1(1-31) caused a continuous increase in [Ca(2+)](i) to cultured beating cardiomyocytes and termination of [Ca(2+)](i) transient within 6 min, and this change was reversed by 1 uM BQ(123) and attenuated by 0.5 mM phosphoramidon. These results suggest that ET-1(1-31) could cause cell injury, and that the effect of ET-1(1-31) on [Ca(2+)](i) transients is mainly mediated by ET(A) receptor and partially attributed to the conversion of ET-1(1-31) to ET-1(1-21).

Big ET-1 processing into vasoactive peptides in arteries and veins.

The endothelin (ET) peptides are more potent in contracting veins than arteries. The precursor big ET-1 is metabolized by endothelin converting enzyme [ECE; to ET-1 (1-21)], matrix metalloproteases [MMPs; to ET-1 (1-32)] and chymase [to ET-1(1-31)]. We hypothesized that arteries and veins were differently dependent in conversion of big ET-1 to vasoconstrictors. Immunohistochemical, western, zymographic and isometric contractile assays in rat aorta and vena cava were used. Big ET-1 contracted aorta [60+/-17% phenylephrine contraction] but was more efficacious in vena cava [478+/-61% norepinephrine contraction]. ECE and its product ET-1(1-21) were detected in aorta and vena cava, and the ECE inhibitors phosphoramidon and CGS-26393 reduced big ET-1-induced contraction. ET-1 (1-32) contracted aorta and vena cava but inhibition of MMPs with minocycline or GM6001 did not reduce big ET-1-induced contraction; zymography confirmed active tissue MMPs. Aorta and vena cava contracted to the product of chymase, ET-1 (1-31). Chymase was detected in aorta and only weakly in vena cava. Inhibition of chymase (chymostatin, 100 muM) reduced arterial (19% control) but not venous constriction to big ET-1. These results suggest at least one potential significant difference - the role of chymase - in in vitro enzymatic processing of big ET-1 in arteries and veins.

Production of monocyte chemoattractant protein-1 and cytokine-induced neutrophil chemoattractant-1 in rat brain is stimulated by intracerebroventricular administration of an endothelin ETB receptor agonist.

The role of endothelin (ET)B receptors in chemokine production in the brain of rats was examined. Intracerebroventricular administration of 500 pmol/day of Ala(1,3,11,15)-ET-1, a selective ETB agonist, for 3 or 7 days increased monocyte chemoattractant protein (MCP)-1 and cytokine-induced neutrophil chemoattractant (CINC)-1 mRNA in the caudate-putamen and cerebrum, whereas it had no effects on regulated on activation normal T-cell expressed and secreted (RANTES), fractalkine and stromal cell-derived factor (SDF)-1alpha mRNA expression. Immunoreactive MCP-1 and CINC-1 in the caudate-putamen and the cerebrum were increased by the ETB agonist. Immunohistochemical observations on the Ala(1,3,11,15)-ET-1-infused rats showed that glial fibrillary acidic protein-positive astrocytes had immunoreactivity for MCP-1 and CINC-1. These findings indicate that the activation of brain ETB receptors causes the production of MCP-1 and CINC-1, and suggest a pathophysiological role for brain ETB receptors in nervous system damage.

Development of photolabile caged analogs of endothelin-1.

Photoactivable caged analogs of endothelin-1 (ET-1) were obtained after derivatization with the photolabile 4,5-dimethoxynitrobenzyl (DMNB) group. This was achieved by the incorporation of N-alpha-Fmoc caged building blocks of Lys, Asp, Glu and Tyr during the solid phase peptide synthesis step. The C-terminal carboxylic function was also derivatized. However, difficulties were encountered with the introduction of the Asp and Glu photoactivable building blocks. As a matter of fact, formation of an aminosuccinyl derivative, through cyclization of the Asp(ODMNB) residue, and the formation of a pyrrolidone ring from the Glu(ODMNB) residue were highly favored by the electronic properties of the photocleavable function. ET-1 analogs were also tested in the ET(A) and ET(B) paradigms and specific pharmacological profiles were obtained for each peptide.

Cardiovascular effects of centrally applied endothelin-1 1-31 and its relationship to endothelin-1 1-21 in rats.

Endothelin-1(1-31) (ET-1(1-31)) is a novel member of the endothelin family, which comprises 31 amino acids and derived from the selective hydrolysis of big ET-1 by chymase. Although ET-1(1-31) has been reported to be involved in biological effects via direct or indirect (converting to ET-1(1-21)) mechanisms, the cardiovascular effects of central ET-1(1-31) are not fully identified. The present study was designed to comparatively investigate the cardiovascular effects of intracerebroventricular (icv) application of ET-1(1-31) or ET-1(1-21) in anesthetized rats. Injection (icv) of ET-1(1-31) (500 pmol) produced a biphasic blood pressure response: an initial increase (from 118+/-8 to 138+/-14 mmHg, P<0.05) followed by a sustained decrease in BP (from 118+/-8 to 58+/-9 mmHg, P<0.05), which was very similar to BP response to icv injection of big ET-1 (500 pmol) or ET-1(1-21) (25 pmol)(.) The cardiovascular effects of icv injection of ET-1(1-31) or ET-1(1-21) were completely antagonized by ET(A) receptor antagonist BQ123 but not ET(B) receptor antagonist BQ788. Furthermore, pretreatment with ET converting enzyme inhibitor phosphoramidon (10 nmol) abolished the cardiovascular effects evoked by icv injection of ET-1(1-31) or big ET-1. In conclusion, the current data showed that central ET-1(1-31) produced the similar cardiovascular effects as those of central ET-1(1-21), and suggesting that the central cardiovascular effects of ET-1(1-31) resulted from it converting to ET-1(1-21) and then activating ET(A) receptors.

Enzymatic pathways involved in the generation of endothelin-1(1-31) from exogenous big endothelin-1 in the rabbit aorta.

We investigated whether blood vessels contribute to the production of ET-1(1-31) from exogenous big endothelin-1 (BigET-1) in the rabbit and assessed which enzymes are involved in this process. Vascular reactivity experiments, using standard muscle bath procedures, showed that BigET-1 induces contraction in endothelium-intact rabbit aortic rings. Preincubation of the rings with phosphoramidon, CGS35066 or thiorphan reduced BigET-1-induced contraction. Conversely, chymostatin did not affect BigET-1-induced contraction. Thiorphan and phosphoramidon, but not CGS35066 or chymostatin, reduced ET-1(1-31)-induced contraction. None of the enzymatic inhibitors affected the contraction afforded by ET-1.BQ123-, but not BQ788-, selective antagonists for ET(A) and ET(B) receptors, respectively, produced concentration-dependent rightward displacements of the ET-1(1-31) and ET-1 concentration-response curves. By the use of enzymatic assays, we found that the aorta, as well as the heart, lung, kidney and liver, possess a chymase-like activity. Enzyme immunoassays detected significant levels of Ir-ET-1(1-31) in bathing medium of aortas after the addition of BigET-1 (30 nM). Neither thiorphan nor chymostatin altered the levels of Ir-ET-1(1-31). Conversely, the levels of Ir-ET-1(1-31) were increased in the presence of phosphoramidon. This marked increase of the 31-amino-acid peptide was abolished when phosphoramidon and chymostatin were added simultaneously. The major new finding of the present work is that the rabbit aorta generates ET-1(1-31) from exogenously administered BigET-1. Additionally, by measuring the production of ET-1(1-31), we showed that a chymase-like enzyme is involved in this process when ECE and NEP are inhibited by phosphoramidon. Our results also suggest that ET-1(1-31) is an alternate intermediate in the production of ET-1 following BigET-1 administration. Finally, we showed that NEP is the predominant enzymatic pathway involved in the cleavage of ET-1(1-31) to a bioactive metabolite that will act on ET(A) receptors to induce contraction in the rabbit aorta.

Blood pressure responses of endothelin-1 1-31 within the rostral ventrolateral medulla through conversion to endothelin-1 1-21.

Endothelin-1 1-31 (ET-1 1-31), a novel member of the endothelin family comprising 31 amino acids and derived from the selective hydrolysis of big ET-1 by chymase, directly activates endothelin receptors or converts to ET-1 1-21 by ET converting enzyme (ECE). The cardiovascular effects of central ET-1 1-31 are not identified. The present study was designed to investigate the cardiovascular actions of ET-1 1-31 within the rostral ventrolateral medulla (RVLM) in anesthetized rats. Bilateral injection of ET-1 1-31 (0.5, 1, and 2 pmol for each side) into the rostral ventrolateral medulla produced an initial pressor and/or a long-lasting hypotensive action but did not affect HR. Unilateral microinjection of 2 and 4 pmol of ET-1 1-31 into the rostral ventrolateral medulla only produced a significant (P < 0.05) transient increase in blood pressure by an average of 13 and 12 mm Hg, respectively, whereas unilateral microinjection of 8 pmol of ET-1 1-31 produced a sustained fall in blood pressure (from 92 +/- 6 to 69 +/- 8 mm Hg, P < 0.05). The transient pressor effect of unilaterally injecting ET-1 1-31 (4 pmol) into the rostral ventrolateral medulla was completely abolished by pretreatment with either ETA receptor antagonist BQ123 (83 +/- 2 versus 84 +/- 5 mm Hg, P > 0.05) or ET converting enzyme inhibitor phosphoramidon (99 +/- 5 versus 99 +/- 7 mm Hg, P > 0.05) but not ETB receptor antagonist IRL1038 (89 +/- 6 versus 96 +/- 7 mm Hg, P < 0.05). In addition, prior injection of phosphoramidon also completely abolished the long-lasting hypotension of intra-RVLM ET-1 1-31 (8 pmol) but did not modify the depressor action of intra-RVLM ET-1 1-21 (from 100 +/- 6 to 76 +/- 8 mm Hg, P < 0.05). In conclusion, the current results suggest that the cardiovascular effects of intra-RVLM ET-1 1-31 might be the result of conversion of ET-1 1-31 to ET-1 1-21 through activation of ETA receptors.

Effect of chymase on intraocular pressure in rabbits.

Chymase is a chymotrypsin-like serine protease that is stored exclusively in the secretory granules of mast cells and converts big endothelins to endothelin-1 (1-31). The aim of this study was to evaluate the effect of chymase on intraocular pressure in rabbits. Chymase injection (3 and 10 mU) resulted in a trend toward increased intraocular pressure and a significant increase in intraocular pressure at a dose of 10 mU compared with the control. A specific chymase inhibitor, Suc-Val-Pro-Phe(P)(OPh)(2), attenuated the ocular hypertension induced by chymase. Endothelin-1 (1-31) also caused ocular hypertension, which was inhibited by a selective endothelin ET(A) receptor antagonist, cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123). Moreover, chymase-induced ocular hypertension was inhibited by BQ-123. These results suggest that chymase influences the regulation of intraocular pressure, and it is likely that the formation of endothelin-1 (1-31) and subsequent activation of endothelin ET(A) receptors are involved in the development of ocular hypertension induced by chymase.

Endothelins stimulate the expression of neurotrophin-3 in rat brain and rat cultured astrocytes.

Endothelins play a role in the regulation of astrocytic functions in brain pathologies such as hyperplasia and neurotrophic factor production. The present study examined the effects of endothelins on production of neurotrophin-3, a member of the neurotrophin family of neurotrophic factors, in cultured astrocytes and rat brain. Quantitative reverse transcription-PCR analysis of mRNA copy numbers showed that cultured astrocytes expressed comparable numbers of neurotrophin-3 and neurotrophin-4/5 mRNA copies to nerve growth factor and brain-derived neurotrophic factor. Endothelin-1 (100 nM) and Ala1,3,11,15-endothelin-1 (an endothelinB receptor agonist, 100 nM) caused a transient increase in neurotrophin-3 mRNA levels, but not in neurotrophin-4/5 levels, in cultured astrocytes. The increases in mRNA levels were accompanied with that in extracellular release of neurotrophin-3. The effects of endothelin-1 on neurotrophin-3 mRNA levels were reduced by BQ788, an endothelinB receptor antagonist. I.c.v. administration of 500 pmol/day Ala1,3,11,15-endothelin-1 increased mRNA and peptide levels of neurotrophin-3 in rat caudate putamen and cerebrum. On the other hand, neurotrophin-3 production in hippocampus was not affected by Ala1,3,11,15-endothelin-1. Immunohistochemical examination of Ala1,3,11,15-endothelin-1-infused rats showed that neurotrophin-3 was mainly expressed in glial fibrillary acidic protein-positive astrocytes in caudate putamen and cerebrum. endothelin-induced increases in neurotrophin-3 expression in cultured astrocytes were inhibited by chelation of intracellular Ca2+ and PD98095 (an ERK inhibitor). These results suggest that endothelin is an extracellular signal that stimulates astrocytic neurotrophin-3 production in brain pathologies.

Endothelin-1 (1-31) is an intermediate in the production of endothelin-1 after big endothelin-1 administration in vivo.

The precursor of endothelin-1, big endothelin-1, can be hydrolyzed by chymase to generate endothelin-1 (1-31) in vitro. In the present study, we explored the processes involved in the production of endothelin-1 (1-31) as well as its pharmacodynamic characteristics in the rabbit in vivo. Endothelin-1 (1-31) (1 nmol/kg, injected into the left cardiac ventricle) induced a monophasic increase of mean arterial blood pressure similarly to big endothelin-1 (1-38), whereas endothelin-1 induces a biphasic response. Phosphoramidon, a dual neutral endopeptidase and endothelin-converting enzyme inhibitor, blocked both pressor responses to endothelin-1 (1-31) and big endothelin-1 but not those afforded by endothelin-1. Thiorphan, a neutral endopeptidase inhibitor, markedly inhibited the response to endothelin-1 (1-31) but only weakly reduced that of big endothelin-1. In contrast, CGS 35066, an endothelin-converting enzyme inhibitor, was significantly more efficient against the pressor response to big endothelin-1 than to endothelin-1 (1-31). Furthermore, injection of big endothelin-1 concomitantly with phosphoramidon induced an increase in endothelin-1 (1-31) plasma levels. Finally, intracardiac-administered endothelin-1 (1-31) induced an increase of endothelin-1 plasma levels, which are markedly reduced by phosphoramidon and thiorphan but not by CGS 35066. Our results thus demonstrate that endothelin-1 (1-31) is an alternate intermediate in the production of endothelin-1 after big endothelin-1 administration in the rabbit in vivo.

High-resolution X-ray structure of the unexpectedly stable dimer of the [Lys(-2)-Arg(-1)-des(17-21)]endothelin-1 peptide.

Previous structural studies on the [Lys((-2))-Arg((-1))]endothelin-1 peptide (KR-ET-1), 540-fold less potent than ET-1, strongly suggested the presence of an intramolecular Arg(-1)-Asp(8) (R(-1)-D(8)) salt bridge that was also observed in the shorter [Lys((-2))-Arg((-1))-des(17-21)]endothelin-1 derivative (KR-CSH-ET). In addition, for these two analogues, we have shown that the Lys-Arg dipeptide, which belongs to the prosequence, significantly improves the formation of the native disulfide bonds (>or=96% instead of approximately 70% for ET-1). In contrast to what was inferred from NMR data, molecular dynamics simulations suggested that such an intramolecular salt bridge would be unstable. The KR-CSH-ET peptide has now been crystallized at pH 5.0 and its high-resolution structure determined ab initio at 1.13 A using direct methods. Unexpectedly, KR-CSH-ET was shown to be a head-to-tail symmetric dimer, and the overall interface involves two intermolecular R(-1)-D(8) salt bridges, a two-stranded antiparallel beta-sheet, and hydrophobic contacts. Molecular dynamics simulations carried out on this dimer clearly showed that the two intermolecular salt bridges were in this case very stable. Sedimentation equilibrium experiments unambiguously confirmed that KR-ET-1 and KR-CSH-ET also exist as dimers in solution at pH 5.0. On the basis of the new dimeric structure, previous NMR data were reinterpreted. Structure calculations were performed using 484 intramolecular and 38 intermolecular NMR-derived constraints. The solution and the X-ray structures of the dimer are very similar (mean rmsd of 0.85 A). Since the KR dipeptide at the N-terminus of KR-CSH-ET is present in the prosequence, it can be hypothesized that similar intermolecular salt bridges could be involved in the in vivo formation of the native disulfide bonds of ET-1. Therefore, it appears to be likely that the prosequence does assist the ET-1 folding in a chaperone-like manner before successive cleavages that yield the bioactive ET-1 hormone.