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(Journal of Leukocyte Biology. 2001;70:306-312.)
© 2001 by Society for Leukocyte Biology

A novel bioactive 31-amino acid endothelin-1 is a potent chemotactic peptide for human neutrophils and monocytes

Ping Cui{dagger}, Kenji Tani*, Hiroko Kitamura{dagger}, Yuushi Okumura{dagger}, Mihiro Yano{dagger}, Daisuke Inui{ddagger}, Toshiaki Tamaki{ddagger}, Saburo Sone* and Hiroshi Kido{dagger}

{dagger} Division of Enzyme Chemistry, Institute for Enzyme Research,
* Third Department of Internal Medicine, and
{ddagger} Department of Pharmacology, School of Medicine, The University of Tokushima, Tokushima, Japan

Correspondence: Hiroshi Kido, Division of Enzyme Chemistry, Institute for Enzyme Research, The University of Tokushima, Kuramoto-cho 3-18-15, Tokushima 770-8503, Japan. E-mail: kido{at}ier.tokushima-u.ac.jp


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ABSTRACT
 
Endothelin (ET)-1(1-31) is a novel 31-amino acid-length peptide derived from big ET-1 by chymase or other chymotrypsin-type proteases and is a major ET derivative in human neutrophils. In this study, we revealed that ET-1(1-31), but not big ET, exhibited chemotactic activities toward human neutrophils and monocytes as an inflammatory mediator, although the effects were less potent than those of formyl-methionyl-leucyl-phenylalanine or interleukin-8. However, the chemotactic effects of ET-1(1-31) were much greater than those of the 21-amino acid ET-1, ET-1(1-21). Checkerboard analyses revealed that the effects are chemotactic rather than chemokinetic. The effects of ET-1(1-31) are not mediated by interleukin-8 or monocyte chemoattractant protein-1. The chemotactic effects and an increase in intracellular-free Ca2+ caused by ET-1(1-31) were significantly inhibited by BQ123, an ETA receptor antagonist, but not by BQ788, an ETB receptor antagonist, suggesting that ET-1(1-31) mediates chemotaxis through an ETA or ETA-like receptor.

Key Words: chemotaxis • endothelin-1(1-31) • endothelin-1(1-21) • Ca2+ mobilization


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INTRODUCTION
 
The endothelins (ETs), a family of 21-residue peptides [ETs(1-21)], were first isolated from the culture medium of porcine endothelial cells and shown to be vasoconstrictors [1 , 2 ]. Three forms of the peptides have been characterized and designated as ET-1, -2, and -3. Recent studies indicated that ETs are found in various cell types [3 ] and have a variety of physiological and pathological functions, not only as smooth-muscle constrictors [4 , 5 ] but also as inflammatory mediators in the progression of inflammatory processes [6 7 8 ] and ischemia damage [9 ]. Inflammatory cytokines and interactions between neutrophils and endothelial cells stimulate the production of ETs [10 11 12 ]. Furthermore, ET-1(1-21) exhibits chemotactic activities toward neutrophils [8 , 13 ] and monocytes [14 ], although the cell-migration indexes are fairly low in comparison with demonstrated effects of chemoattractant factors, such as interleukin-8 (IL-8), monocyte chemoattractant protein (MCP)-1, and formyl-methionyl-leucyl-phenylalanine (fMLP).

Recently, the bioactive ET peptide family has expanded: novel, smooth-muscle-constricting, 31-amino acid endothelins [ET-1, -2, and, -3(1-31)], which are generated from big ETs through specific cleavage of the Tyr31-Gly32 bond by human chymase or other chymotrypsin-type proteases, have been found by our group [15 , 16 ] and Hanson et al. [17 ]. The smooth-muscle-constricting activity of ETs(1-31) is not the consequence of conversion to the corresponding ETs(1-21) by phosphoramidon-sensitive ET-converting enzymes or metalloendopeptidases [15 , 18 , 19 ]. These novel ETs as well as ETs(1-21) in human neutrophils and lungs were separated by reverse-phase high-performance liquid chromatography, and their levels were determined recently by means of a specific sandwich-type, enzyme-linked immunosorbent assay (ELISA) [20 ]. Among the ET derivatives in human neutrophils, ET-1(1-31) is the predominant bioactive peptide [20 ]. In addition, heavy immunoreactive deposits of monospecific antibodies against the C-terminal seven residues of ET-1(1-31) were detected in the cells (unpublished results). This suggests that ET-1(1-31) in neutrophils has some pivotal functions in the inflammatory reactions in an autocrine and/or paracrine manner. Although pharmacological analyses of the effects of ETs(1-31) on vascular and tracheal smooth-muscle constriction [15 , 21 ] and calcuim signaling [19 , 22 ] have been performed, characterization of ET-1(1-31) as a chemoattractant peptide for human leukocytes has not been performed so far.

To better understand the contribution of ET-1(1-31) to inflammation and pathophysiological vascular events, in this study we first analyzed the chemotactic effects of ET-1(1-31) on human neutrophils and monocytes by means of the 48-well microchemotaxis chamber technique. ET-1(1-31) exhibited chemotactic rather than chemokinetic activity toward neutrophils and monocytes. The chemotactic effects of ET-1(1-31) on these cells were considerably greater than those of ET-1(1-21), although the effects were less potent than those of fMLP and IL-8.


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MATERIALS AND METHODS
 
Reagents
Human ET-1(1-21), ET-1(1-31), and phosphoramidon were purchased from the Peptide Institute (Osaka, Japan). BQ123 [cyclo-(-D-Trp-D-Asp(ONa)-Pro-D-Val-Leu)] and BQ788 [N-cis-2,6-dimethylpiperidinocarbonyl-L-{gamma}-MeLeu-D-Trp(COOMe)-D-NIe-Ona] were obtained from Calbiochem Novabiochem (La Jolla, CA), and fMLP was from Sigma Chemical Co. (St. Louis, MO). ELISA kits for human IL-8 and human MCP-1 were purchased from Biosource International (Camarillo, CA), and one for ET-1(1-31) was from Immuno-biological Laboratories (Gumma, Japan). Anti-human IL-8 neutralizing antibody was from R&D System Inc. (Abingdon, UK). Human recombinant IL-8 was from PeproTechEC (London, England). All other reagents were commercial products of the highest grade available.

Isolation and culture of human leukocytes
Peripheral blood neutrophils were isolated under sterile conditions from the buffy coats of healthy volunteers with Lymphoprep (Nycomed Pharmas, Oslo, Norway), according to the manufacturer’s protocol. The purity of the isolated neutrophil fraction, as determined by morphologic examination, was greater than 96%. Human monocytes were isolated from normal donors with an isoosmotic Percoll gradient as demonstrated [23 ]. The purified monocytes exhibited more than 89% purity. The viability of neutrophils and monocytes isolated was >=98%, as determined by means of trypan blue exclusion.

Chemotaxis assays
Cell migration was evaluated by the 48-well microchemotaxis chamber (Neuro Probe, Cabin John, MD) technique [24 , 25 ]. Because ETs and phosphoramidon have hydrophobic properties, these reagents were dissolved in dimethyl sulfoxide (DMSO) as stock solutions of 10-7–10-2 M and then diluted with sterilized RPMI 1640 medium containing 1% bovine serum albumin (BSA) to yield a 1/100 working concentration. DMSO at concentrations of less than 1% had no effect on migration. A 27 µl aliquot of a chemotactic solution was placed in the lower compartment, and 50 µl of a cell suspension (1x106 neutrophils/ml or 3x106 monocytes/ml) was placed in the upper compartment of the chamber. The two compartments were separated by a polycarbonate filter with a pore size of 3 µm for neutrophils and 5 µm for monocytes. The chamber was incubated at 37°C under humidified air containing 5% CO2 for 30 min for neutrophil migration or 1 h for monocyte migration. After the incubation, the filter was removed, fixed, and stained with a Diff-Quik solution (International Reagents, Kobe, Japan). The numbers of migrating cells were determined in five random high-power fields (HPF; x400). Checkerboard analysis was performed to determine whether a stimulant was chemotactic or chemokinetic, as demonstrated [13 , 25 ]. All experiments were repeated at least three times with cells from different donors.

Determination of the levels of cytokines released from and associated with cells
Major chemotactic cytokines, such as IL-8 and MCP-1, released from and associated with human leukocytes were evaluated after treatment of the cells with ET-1(1-31). Neutrophils (3x106) and monocytes (1.8x106) in RPMI 1640 containing 1% BSA were incubated with various concentrations of ET-1(1-31) at 37°C under 5% CO2 for 30 min for neutrophils or 1 h for monocytes. Culture supernatants were then collected, centrifuged (400 g for 5 min at 4°C) to remove cells, and stored at -80°C. When cell-associated cytokines were analyzed, 0.3 ml ice-cold, phosphate-buffered saline (PBS) was added to the culture wells, and then the cells were gently scraped off and combined with the cell pellet obtained on centrifugation of the culture medium. The combined cell suspension was then centrifuged, and the cell pellet was lysed with 300 µl ice-cold lysis buffer [0.5 mM ethylenediaminetetraacetate (EDTA), 0.5 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA), 0.2 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 1 µM pepstatin A, and 1 µM aprotinin in PBS] and then stored at -80°C. Just before the assaying of IL-8 and MCP-1, the frozen supernatants and cell suspensions were thawed on ice. The cell suspensions were then centrifuged at 15,000 g for 10 min at 4°C. (This allowed most of the cells to rupture.) These supernatants were analyzed as to the levels of IL-8 and MCP-1 using specific ELISA kits. The manufacturer’s limits of detection are 15.6 pg/ml for IL-8 and 31.2 pg/ml for MCP-1.

Immuno-depletion of ET-1(1-31) and IL-8 in conditioned medium
The chemotactic activity in the conditioned medium after treatment with ET-1(1-31) was evaluated by passage through an anti-ET immunoglobulin G (IgG)-coupled Sepharose 4B column (ET-affinity gel, Immuno-biological Laboratories), which adsorbs various ET-1 derivatives. After incubation for 30 min of 3 x 106 cells with 4 x 10-7 M ET-1(1-31), the cell suspension was collected and centrifuged at 400 g for 5 min at 4°C. The supernatant was applied to 1 ml of an ET-affinity gel at 4°C to adsorb and deplete ET-1(1-31) in the sample. The flow-through fraction was collected and then lyophilized, which was then dissolved in 100 µl water. As a control, the supernatant was also applied to a nonimmunized IgG-coupled Sepharose 4B gel. The level of ET-1(1-31) in the medium was measured with a specific ELISA kit for ET-1(1-31). The manufacturer’s limit of detection is 0.16 pg/well.

The chemotactic activity of the conditioned medium after treatment of 1 x 10-6 neutrophils with 10-6 M ET-1(1-31) for 30 min was also evaluated by addition of neutralizing antibody against IL-8. Anti-IL-8 neutralizing antibody was added to the conditioned medium at a concentration of 30 µg/ml, enough of a dose to inhibit biological effects of IL-8 [26 ], and was incubated for an additional 30 min at 37°C, and then chemotactic activity of the medium was analyzed.

Intracellular [Ca2+] measurement
Human neutrophils were loaded with fura-2/AM according to the method of Hafstrom et al. [27 ] with some modifications. Isolated neutrophils were resuspended in Hanks’ balanced salt solution (HBSS) containing 0.5% BSA and then loaded with 1 µM fura-2/AM (Dojin, Kumamoto, Japan) at 37°C for 20 min with continuous shaking. The loaded cells were washed twice and then gently resuspended in HBSS (with 1.3 mM Ca2+ but without BSA and phenol red). The cells were stored at 22°C and protected from light until analysis. The cells were then warmed at 37°C with continuous stirring. The excitation wavelength was set at 340 nm, and the emission one was set at 510 nm. After a stable baseline had been established, the agonists were added, and then the emitted fluorescence was recorded. The inhibitory effects of ET antagonists, such as BQ123 and BQ788, were analyzed by preincubation with 1 and 10 µM of these antagonists for 20 min before stimulation with ET-1(1-31). At the end of each assay, maximum (Fmax) and minimum fluorescence (Fmin) were determined after the addition of 0.1% Triton X-100 and 10 mM EGTA, respectively. An intracellular-free Ca2+ concentration ([Ca2+]i) was calculated using standard equations [28 ]. The effects of ET derivatives and their antagonists on human monocytes, which attach to culture dishes coated with poly-L-lysine, were also analyzed by confocal laser microscopy, with Fluo-3/AM as the dye, as described [19 , 22 ]. After 1 min, the same cells were stimulated by addition of 10 µM ionomycin in each experiment to estimate Fmax.

Statistical analysis
Statistical significance was determined by means of the unpaired t-test for comparisons between the stimulated groups and the control groups using Statview 4.0 software. A value of P < 0.05 was accepted as significant.


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RESULTS
 
ET-1(1-31) exhibits considerably greater chemotactic activity toward neutrophils and monocytes than ET-1(1-21)
ET-1(1-21) exhibits weak but significant chemoattractant activity toward human neutrophils [8 , 13 ] and monocytes [14 ], and neutrophils possess binding sites for ET-1(1-21) [29 , 30 ]. As a result of these findings, the roles of ET peptides in inflammatory reactions and atherogenesis have attracted attention recently. Because a novel ET peptide, ET-1(1-31), is the predominant bioactive ET peptide in human neutrophils, in this study the chemotactic activities of ET-1(1-31) toward human neutrophils and monocytes were measured in comparison with the effects of ET-1(1-21). Increasing concentrations of ET-1(1-31) over 10-9 M in the lower microchemotaxis chamber lead to an increase in the number of neutrophil (Fig. 1A ) and monocyte migrations (Fig. 1B) . In contrast to the random migration in RPMI 1640 medium, ET-1(1-31) at 10-6 M caused fourfold to fivefold and 5.5- to tenfold higher migration of neutrophils and monocytes, respectively, although these effects were less potent than those of 10-7 M fMLP (a potent chemotactic factor for neutrophils and monocytes) and 50 ng/ml IL-8 (a chemotactic factor for neutrophils) as described below. In contrast, ET-1(1-21) caused only 1.4- and 1.2-fold higher migration of neutrophils and monocytes, respectively, in comparison with random migration, the levels being consistent with those demonstrated [8 , 13 ]. However, big ET-1 did not have any chemotactic effect on neutrophil migration (Fig. 1A) . To confirm that the biological function of ET-1(1-31) is not the consequence of conversion to ET-1(1-21) by phosphoramidon-sensitive ET-converting enzymes [31 , 32 ], the effect of phosphoramidon on the chemotactic activity of ET-1(1-31) was analyzed. Phosphoramidon at 10-4 M had no effect on neutrophil migration stimulated by ET-1(1-31) (Fig. 1A) , consistent with the effect of phosphoramidon on smooth-muscle constriction caused by ET-1(1-31) [15 , 18 ].



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  		Figure 1. The effects of various ET-1 peptides on the migration of human neutrophils and monocytes. (A) Human neutrophils were stimulated with increasing concentrations of ET-1(1-31) ({square}), ET-1(1-21) ({blacksquare}), big ET-1 ({blacktriangleup}), ET-1(1-31) plus 10-4 M phosphoramidon ({triangleup}), and big ET-1 plus 10-4 M phosphoramidon ({circ}). The chemotactic effect of fMLP (•) was also analyzed. The random cell migration in RPMI 1640 medium gave 50 ± 0.8 HPF. (B) Human monocytes were stimulated with increasing concentrations of ET-1(1-31) ({blacktriangleup}) and ET-1(1-21) ({blacksquare}). The effects of 10-7 M fMLP ({square}) and RPMI 1640 medium ({triangleup}) were also examined. The values are the means ± SD for five independent experiments. *, Significantly different from the medium control value (P<0.05).

To determine whether the stimulatory effect of ET-1(1-31) is chemotactic or chemokinetic, we carried out a checkerboard assay, with different ET-1(1-31) concentrations in the upper, lower, or both compartments. Neutrophil (Table 1 ) and monocyte (Table 2 ) migration increased with increasing concentrations of ET-1(1-31) in the lower compartment. However, the neutrophil and monocyte migration were not significant when the concentration of ET-1(1-31) was the same in the upper and lower compartments. These results indicate that the effects of ET-1(1-31) on neutrophil and monocyte migration are chemotactic rather than chemokinetic.


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  		Table 1. Checkerboard Analysis of Neutrophil Migration by ET-1(1-31)


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  		Table 2. Checkerboard Analysis of Monocyte Migration by ET-1(1-31)

The chemotactic effect of ET-1(1-31) is not the consequence of induced IL-8 and MCP-1
Various chemokines that enhance the motility and directional migration of neutrophils and monocytes were studied, and IL-8 and MCP-1 are the major potent chemokines of neutrophils [33 ] and monocytes [34 , 35 ], respectively. The question then arose as to the mechanism underlying the chemotactic effects of ET-1(1-31). For example, are the chemotactic effects the consequence of neutrophil chemotactic factor IL-8 and monocyte chemotactic factor MCP-1 induced by ET-1(1-31), or is the migration the direct effect of ET-1(1-31)? To determine the IL-8 released and produced during incubation for 30 min of neutrophils with ET-1(1-31), the levels of IL-8 in culture media and those in neutrophils were analyzed. As shown in Figure 2 , the levels of released, cell-associated, and total IL-8 increased in a dose-dependent manner after treatment with ET-1(1-31) at concentrations over 10-8 M. However, IL-8 induced in the incubation period was too low to induce neutrophil chemotaxis. From the data in Figure 2 , the maximum level of IL-8 released in the presence of 1 x 106 neutrophils is calculated to be about 65 pg/ml. The levels of IL-8 with the concentration of 0.1 ng/ml were not enough to induce neutrophil migration, as shown in Figure 3A . We next analyzed the effect of immuno-depletion of IL-8 in the conditioned medium by anti-IL-8-neutralizing antibody. The conditioned medium after treatment of neutrophils with 10-6 M ET-1(1-31) was treated with anti-IL-8-neutralizing antibody at 30 µg/ml at 37°C for 30 min, enough to inhibit the biological effects of IL-8 [26 ]. However, the neutralizing antibody did not show any inhibitory effect on neutrophil migration. The antibody by itself did not exhibit any effects on neutrophils.



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  		Figure 2. Changes in the levels of IL-8 and MCP-1 in culture media and cells after treatment with ET-1(1-31). Human neutrophils (A) and monocytes (B) were stimulated with increasing concentrations of ET-1(1-31) for 30 min and 1 h, respectively. Then the levels of IL-8 and MCP-1 released into the culture media ({triangleup}) and cell-associated (•), and the total amounts ({blacktriangleup}) were determined by ELISA as described in Materials and Methods. The values are the means ± SD for three independent experiments. *, Significantly different from the medium control value (P<0.05).



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  		Figure 3. Effects of immuno-depletion of IL-8 and ET-1(1-31) in conditioned medium on the activity of neutrophil migration. The chemotactic activity in the conditioned medium after treatment with ET-1(1-31) was evaluated by immuno-depletion of IL-8 (A) and ET-1(1-31) (B). (A) Neutrophils (1x106 cells) were stimulated with 10-6 M ET-1(1-31) for 30 min, and then the conditioned medium was prepared by removal of cell pellets. Anti-IL-8-neutralizing antibody was added to the conditioned medium at 30 µg/ml and incubated for additional 30 min, and then chemotactic activity of the medium was analyzed. The chemotactic activities of 0.1 and 50 ng/ml IL-8 were also analyzed as positive controls. (B) Neutrophils (3x106 cells) were stimulated with 4 x 10-7 M ET-1(1-31) for 30 min, and then the conditioned medium was applied to an ET-affinity gel or a nonimmunized, IgG-coupled Sepharose 4B column as described in Materials and Methods. The chemotactic activities of the original, conditioned medium and RPMI 1640 control medium were also analyzed. The values are the means ± SD for five independent experiments. *, Significantly different from the medium control value (P<0.05).

The levels of MCP-1 in culture media and those in cells after treatment of human monocytes with increasing concentrations of ET-1(1-31) remained unchanged during the incubation period of 1 h, and the values were also low enough to induce monocyte migration (Fig. 2B) .

We then analyzed the possibility of direct effect of ET-1(1-31) on neutrophil migration. The conditioned medium after incubation of 3 x 106 cells with 4 x 10-7 M ET-1(1-31) for 30 min was applied to an ET-affinity gel to deplete the ET in the medium. The level of ET-1(1-31) in the flow-through fraction determined by ELISA was 1.13 x 10-9 M, indicating that 99.7% of ET-1(1-31) was removed from the conditioned medium. As shown in Figure 3B , the flow-through fraction of the conditioned medium did not exhibit any effect on neutrophil migration, suggesting that ET-1(1-31) by itself induces chemotactic migration of neutrophils directly. On the contrary, the flow-through fraction from a nonimmunized, IgG-coupled Sepharose 4B column as a control showed that the level of ET-1(1-31) in the treated medium did not decrease (4–4.2x10-7 M), and the medium exhibited a significant chemotactic effect.

Effect of ET-receptor antagonists on neutrophil and monocyte migration induced by ET-1(1-31)
The functions of ETs are mediated by at least two distinct subtypes of receptors, the ETA and ETB receptors [36 37 38 ]. To determine whether the chemotactic effects of ET-1(1-31) on neutrophils and monocytes are mediated by the ETA or ETB receptor, these cells were pretreated with BQ123, a selective ETA-receptor antagonist, or BQ788, an ETB-receptor antagonist, for 1 h at 4°C, and then chemotactic activities were analyzed after stimulation of the cells by 10-6 M ET-1(1-31) (Fig. 4 ). BQ123, but not BQ788, inhibited chemotaxis of neutrophils and monocytes caused by ET-1(1-31) in a dose-dependent manner, and 10-5 M BQ123 suppressed the chemotactic effects on neutrophils and monocytes by 73% and 63%, respectively. Neither BQ123 nor BQ788 attenuated chemotactic activities toward neutrophils and monocytes by fMLP.



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  		Figure 4. Effects of ET-receptor antagonists on neutrophil (A) and monocyte (B) migration induced by ET-1(1-31). Neutrophils (1x106 cells) and monocytes (3x106 cells) were treated previously with 10-6 and 10-5 M BQ123 or BQ788 at 4°C for 1 h, and then cell migration was measured after addition of 10-6 M ET-1(1-31) as described in Materials and Methods. The values are the means ± SD for five independent experiments. * and **, Significantly different (P<0.05) from the medium control value and the 10-6 M ET-1(1-31) value, respectively.

Effect of ET-1(1-31) on mobilization of intracellular-free calcium
In the intracellular-signaling events evoked by ET-1(1-31), a transient increase in the [Ca2+]i has been observed in smooth-muscle cells [19 , 22 ]. To determine whether the chemotactic effects of ET-1(1-31) on neutrophils and monocytes also involve an increase in [Ca2+]i, human neutrophils were treated with ET-1(1-31) and the Ca2+ indicator fura-2/AM in the presence or absence of ET-receptor antagonists. An increase in the level of [Ca2+]i caused by 10-6 M ET-1(1-31) was observed after stimulation for 10 s. It reached a plateau after 30–35 s and then began to decrease slowly after 50–60 s, although the level of [Ca2+]i induced by fMLP reached a plateau after 15 s, continuing until 50 s (unpublished results). The levels of [Ca2+]i in neutrophils treated with 1% DMSO as a solvent remained unchanged during the incubation period.

The levels of [Ca2+]i after treatment of neutrophils with 10-6 M ET-1(1-31) in the presence or the absence of ET antagonists for 30 and 60 s are shown in Figure 5 . ET-1(1-31) at 10-6 M evoked an increase in [Ca2+]i of about threefold over [Ca2+]i before stimulation or in the presence of 1% DMSO, the maximum level about 67% of [Ca2+]i induced by 10-7 M fMLP. However, little increase in [Ca2+]i was observed in the cells treated with 10-6 M ET-1(1-21). The increase in the level of [Ca2+]i caused by ET-1(1-31) was inhibited by BQ123 in a dose-dependent manner but not inhibited at all by BQ788. BQ123 at 10-5 M suppressed the increase in the level of [Ca2+]i by ET-1(1-31) by 50% at 30 s and 42% at 60 s.



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  		Figure 5. Effects of ET-1(1-21) and ET-1(1-31), with or without ET-receptor antagonists, on the level of [Ca2+]i in human neutrophils. Human neutrophils were stimulated with 10-6 M ET-1(1-31) or ET-1(1-21) or 1% DMSO as a control. The cells were also stimulated with 10-6 M ET-1(1-31) in the presence of 10-6 and 10-5 M BQ123 or BQ788. The levels of [Ca2+]i after incubation for 30 s (A) and 60 s (B) were analyzed as described in Materials and Methods. The levels of [Ca2+]i induced by 10-7 M fMLP were also examined. The values are the means ± SD for five independent experiments. * and **, Significantly different (P<0.05) from the medium control value and the 10-6 M ET-1(1-31) value, respectively.

An increase in the level of [Ca2+]i in human monocytes after treatment with 10-6 M ET-1(1-31) was also observed, with similar results as those in neutrophils except for the effect of BQ123, which suppressed the increase in [Ca2+]i induced by ET-1(1-31) more efficiently than that of BQ123 in neutrophils (Fig. 6 ). Under these conditions, BQ123 at 10-5 M suppressed the increase in [Ca2+]i of monocytes by 92%.



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  		Figure 6. Effects of ET-1(1-21) and ET-1(1-31), with or without ET-receptor antagonists, on the level of [Ca2+]i in human monocytes. The effects of 10-6 M ET-1(1-21) and ET-1(1-31), in the presence or absence of ET-receptor antagonists BQ123 and BQ788 on the level of [Ca2+]i in human monocytes, were analyzed as described in Materials and Methods. Values are expressed as % difference of Fmax and Fmin as described [19 , 22 ]. The values are the means ± SD for five independent experiments. * and **, Significantly different (P<0.05) from the medium control value and the 10-6 M ET-1(1-31) value, respectively.


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DISCUSSION
 
In the present study, we found that a novel 31-amino acid ET-1, a predominant ET peptide in human neutrophils, exhibited more efficient chemotactic activity toward human neutrophils and monocytes than ET-1(1-21) and big ET-1 in vitro. ET-1(1-31) exhibited significant chemotactic effects toward human neutrophils and monocytes at concentrations over 10-8 M, consistent with those needed for smooth-muscle constriction in rat tracheae [15 ] and porcine coronary arteries [21 ]. At inflammatory loci or during clot formation [39 ], various mediators including ET-1(1-31) will be released from neutrophils, and ET-1(1-31) might cause other neutrophils and monocytes to migrate in vivo in a paracrine or autocrine manner. Based on these findings, we propose the pathophysiological role of ET-1(1-31) in neutrophils in the initiation or progression of inflammatory reactions and ischemia damage, which are characterized by the local accumulation of neutrophils and the adhesion of neutrophils to endothelial cells. In the secretory granules of neutrophils, there is a chymotrypsin-type serine protease, cathepsin G, which exhibits the most potent chemotactic activity for monocytes and neutrophils and chemokinetic activity for T lymphocytes [40 ]. Although cathepsin G converts big ET-1 to ET-1(1-31) transiently and also degrades ET-1(1-31) in vitro [15 ], studies about the interaction between cathepsin G and ET-1(1-31) in vivo remain to be solved.

The chemotactic effect of ET-1(1-31) is not the consequence of conversion to ET-1(1-21) by phosphoramidon-sensitive, ET-converting enzymes, as shown in Figure 1 , and is not mediated by an increase in the production of IL-8 in neutrophils or by the production of MCP-1 in monocytes during a short incubation period, as shown in Figures 2 and 3 . In addition, chemotactic activity of the conditioned medium after treatment of neutrophils with ET-1(1-31) was almost completely suppressed by immuno-depletion of ET-1(1-31) in the medium, suggesting that ET-1(1-31) induces chemotactic migration directly. However, the possibility of the involvement of some chemokine(s) other than IL-8 and MCP-1 in neutrophils and monocytes has not been ruled out completely. Furthermore, the possibility remains in vivo that ET-1(1-31) induces IL-8 at doses enough for a chemotactic response after long-time incubation for over 48 h, because ET-1(1-21) markedly stimulates IL-8 production in human brain-derived endothelial cells after incubation for 48 h [9 ]. These possibilities are now under investigation.

The chemotactic effects of ET-1(1-31) on human neutrophils and monocytes involve an increase in the level of [Ca2+]i (Figs. 5 and 6) . The difference in the effects on [Ca2+]i of human neutrophils and monocytes between ET-1(1-21) and ET-1(1-31) is consistent with that in the effects on the chemotaxis of these cells. BQ123 significantly inhibited the chemotactic effects of ET-1(1-31) on neutrophils and monocytes as well as the increase in [Ca2+]i by ET-1(1-31) in neutrophils and monocytes, but BQ788 did not. These results support the previous findings of the effects of these ET-receptor antagonists on the increase in [Ca2+]i caused by ET-1(1-31) in vascular smooth-muscle cells [19 , 22 ]. Based on these findings, we speculate that ET-1(1-31) by itself has a chemotactic effect, probably on a ETA or ETA-like receptor. Further studies on the effects of ET-1(1-31) on the chemotactic responses in vivo and identification of an ET-1(1-31) receptor are now under investigation.


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ACKNOWLEDGEMENTS
 
This study was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture (09670130) of Japan.

Received February 12, 2001; revised May 4, 2001; accepted May 4, 2001.


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