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

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 Ca(2)(+) caused by ET-1(1-31) were significantly inhibited by BQ123, an ET(A) receptor antagonist, but not by BQ788, an ET(B) receptor antagonist, suggesting that ET-1(1-31) mediates chemotaxis through an ET(A) or ET(A)-like receptor.

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.

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.

Plasma levels of free and sulfoconjugated catecholamines in patients with atherosclerosis.

We have studied the relationship between free and sulfoconjugated catecholamines (CAs) in the plasma of patients with various cardiovascular diseases and have described the physiological significance of sulfoconjugated CAs in plasma. In the present study, we measured free and sulfoconjugated dopamine (DA) and noradrenaline (NA) in the plasma of patients with atherosclerosis (AS). Results showed that the plasma levels of free DA and NA in patients with atherosclerosis were higher than those in control subjects. Moreover, it was also observed that the plasma levels of conjugated DA and NA in patients had a tendency to be higher than the levels in control subjects. These results suggest the involvement of free CAs in atherosclerosis and that elevated free CAs may be converted to sulfoconjugated forms in patients with atherosclerosis.

Mechanism of endothelin-1-(1-31)-induced calcium signaling in human coronary artery smooth muscle cells.

We have found that human chymase produces a 31-amino acid endothelin [ET-1-(1-31)] from the 38-amino acid precursor (Big ET-1). We examined the mechanism of synthetic ET-1-(1-31)-induced intracellular Ca2+ signaling in cultured human coronary artery smooth muscle cells. ET-1-(1-31) increased the intracellular free Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (10(-14)-10(-10) M). This ET-1-(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon, Bowman-Birk inhibitor, and thiorphan. 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) at 10(-12) M did not cause Ca2+ influx, whereas 10(-7) M ET-1-(1-31) evoked marked Ca2+ influx, which was inhibited by nifedipine. ET-1-(1-31) also increased inositol trisphosphate formation. These results suggest that the ET-1-(1-31)-induced [Ca2+]i increase at relatively low concentrations is attributable to the release of Ca2+ from inositol trisphosphate-sensitive intracellular stores, whereas Ca2+ influx into the cells evoked by high concentration of ET-1-(1-31) probably occurs through voltage-dependent Ca2+ channels. We concluded that the physiological activity of ET-1-(1-31) may be attributable to Ca2+ mobilization from intracellular stores rather than influx of Ca2+ from extracellular space.

Comparison of the effects of endothelin-1, -2 and -3 (1-31) on changes in [Ca2+]i in human coronary artery smooth muscle cells.

We have previously found that human chymase selectively cleaves big endothelins (ETs) at the Tyr31-Gly32 bond to produce 31-amino-acid endothelins, ETs (1-31). In the present study, we investigated the effects of ETs (1-31) on changes in intracellular free Ca2+ ([Ca2+]i) in cultured human coronary artery smooth muscle cells (HCASMCs) using confocal laser microscopy. ETs (1-31) increased [Ca2+]i in a concentration-dependent manner. Phosphoramidon did not inhibit the increases in [Ca2+]i caused by ETs (1-31). The [Ca2+]i increases induced by ETs (1-31) were compared to those of ETs (1-21) and big ETs. ET-1 (1-21) was about 10-times more potent than big ET-1 or ET-1 (1-31), whereas big ET-2 was 10-times less potent than ET-2 (1-21) or ET-2 (1-31). ETs (1-31) may induce [Ca2+]i increase through ET(A)-type or ET(A)-type-like receptors. The 10(-12) M ET (1-31)-induced increases in [Ca2+]i were not affected by removal of extracellular Ca2+, but were inhibited by thapsigargin. These results suggested that ET-1, -2 and -3 (1-31) showed similar potencies in increasing [Ca2+]i and mechanisms of ET (1-31)-induced increases in [Ca2+]i may be similar among the three ETs (1-31).

Endothelin-1-(1-31), a novel vasoactive peptide, increases [Ca2+]i in human coronary artery smooth muscle cells.

We have previously found that human chymase cleaves big endothelins at the Tyr31-Gly32 bond and produces 31-amino acid long endothelins-(1-31), without any further degradation products. In this study, we investigated the effect of synthetic endothelin-1-(1-31) on the intracellular free Ca2+ concentration ([Ca2+]i) in cultured human coronary artery smooth muscle cells. Endothelin-1-(1-31) increased [Ca2+]i in a concentration-dependent manner (10(-14) to 10(-10) M). This endothelin-1-(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon (N-(alpha-Rhamnopyranosyloxyhydroxyphosphinyl)-L-Leucyl-L-Tryptoph an), an inhibitor of endothelin-converting enzyme. It was, however, inhibited by 10(-10) M BQ123 (Cyclo-(-D-Trp-D-Asp(ONa)-Pro-D-Val-Leu-)), an endothelin ET(A) receptor antagonist, but not by 10(-10) M BQ788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-yMeLeu-D-Trp(COOM e)-D-Nle-ONa), an endothelin ET(B) receptor antagonist. These results suggest that endothelin-1-(1-31) by itself exhibits vasoactive properties probably through endothelin ET(A) receptors. Since human chymase has been reported to play a role in atherosclerosis, endothelin-1-(1-31) may be one of the candidate substances for its cause.