Tomoh Masaki

Hemodynamic shear stress stimulates endothelin production by cultured endothelial cells

We have examined the effect of shear stress on the production of endothelin by cultured porcine endothelial cells. Low shear stress stimulated the expression of endothelin mRNA in polygonal endothelial cells with a peak time of 2 to 4 hours and also increased the release of immunoreactive endothelin into the culture medium. The expression of endothelin mRNA in the ellipsoidal endothelial cells under higher shear stress was not different from that of the control level. Our results suggest a possible role for hemodynamic shear stress in the regulation of endothelin production in vascular endothelial cells.

Transforming growth factor-β stimulates the expression of endothelin mRNA by vascular endothelial cells

Endothelin is a potent vasoconstrictor peptide produced by vascular endothelial cells. Incubation of the serum-deprived confluent porcine aortic endothelial cells with 10-300 pM TGF-beta 1, resulted in a several fold increase in endothelin mRNA levels with a peak time of 2 h. An enzyme-linked immunosorbent assay revealed that the levels of endothelin in endothelial cell conditioned media was also increased by TGF-beta 1. These results suggest that TGF-beta 1, secreted by activated platelets, is involved not only in wound healing, but in the regulation of local vascular tone by stimulating endothelin production in the endothelial cells.

Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: Identity of human and porcine endothelin

A cDNA encoding a human endothelium‐derived vasoconstrictor peptide, endothelin, was isolated from a human placenta cDNA library. The nucleotide sequence of this cDNA clone showed that the primary structure of the human preproendothelin has 212 amino acid residues and is highly homologous to porcine preproendothelin, and that human endothelin is identical with porcine endothelin.

Endothelin is a potent secretagogue for atrial natriuretic peptide in cultured rat atrial myocytes.

Using cultured neonatal rat atrial cardiocytes, we have studied the effect of synthetic porcine endothelin (pET), a novel potent vasoconstrictor isolated from endothelial cells, on the release of immunoreactive (IR) rat atrial natriuretic peptide (rANP). pET stimulated IR-rANP secretion in a dose-dependent manner (10(-10)-10(-7) M) with an approximate half-maximally stimulatory dose of 2 x 10(-10) M. The pET-induced IR-rANP secretion was attenuated by Ca2+-channel blocker nicardipine, but no further stimulation was induced when combined with a Ca2+-channel agonist BAY-K 8644. pET in combination with tetradecanoyl-phorbol-acetate resulted in a synergistic effect on IR-rANP secretion. These data suggest that ET may play as an endogenous secretagogue for rANP by modulating Ca2+ influx through the voltage-dependent Ca2+-channels in atrial cardiocytes.

Cloning and functional expression of human cDNA for the ETB endothelin receptor

We report the cloning of human cDNA encoding an ETB (non-isopeptide-selective) subtype of the endothelin receptor. The predicted amino acid sequence of the human ETB endothelin receptor was 87.8% and 62.9% identical with the previously cloned rat ETB and ETA receptors, respectively. COS cells transiently transfected with the cloned cDNA expressed specific, high-affinity binding sites for endothelin isopeptides and responded to the peptides with a transient increase of [Ca2+]i; endothelin-1 and endothelin-3 exhibited approximately equal potencies both in displacing 125I-labeled endothelin-1 binding and in eliciting [Ca2+]i transients. The ETB receptor mRNAs were expressed in various human tissues and also in the intact porcine aortic intimal cells ex vivo.

A novel peptide vasoconstrictor, endothelin, is produced by vascular endothelium and modulates smooth muscle Ca2+ channels

Since the discovery of endothelium-dependent vasodilation, vascular endothelium has been recognized as an important functional unit contributing to the regulation of vascular tonus. Recent purification, structure determination and molecular cloning of a novel peptidergic vasoconstrictor, endothelin, from the culture supernatant of porcine aortic endothelial cells has further substantiated this concept. Starting from a large quantity of serum-fee endothelial cell-conditioned medium, endothelin was purified to homogeneity after three steps of high performance liquid chromatography by collecting active fractions and constricting porcine coronary artery strips. A peptide sequence analysis showed that endothelin was a previously unknown 21-residue peptide with two intrachain disulphide bonds. The EC50 of endothelin for contraction of porcine coronary artery was 4.0 x 10-10 mol/l, indicating that endothelin is one of the most potent vasoconstrictors known. The pharmacological and structural properties of endothelin suggest that the peptide acts by activating voltage-dependent Ca2+ channels in vascular smooth muscle cells. Cloning and sequence analysis of complementary (c)DNA encoding porcine and human endothelin precursors showed that endothelin was produced de novo in endothelial cells from an approximately 200-residue prepropeptide through an unusual proteolytic processing by a putative `endothelin-converting enzyme‘. Northern blot analysis showed that preproendothelin messenger (m)RNA was expressed not only in cultured endothelial cells but also in fresh intimal tissue from porcine aorta, and that mRNA can be induced markedly in response to several vasoactive agents in cultured endothelial cells, suggesting the existence of a novel endothelium-mediated cardiovascular control system.

Interleukin 1 increases the production of endothelin-1 by cultured endothelial cells

We examined the effect of human recombinant interleukin 1 (IL-1) on the production of endothelin-1 by cultured porcine endothelial cells. The induction of endothelin-1 mRNA began within 1 hr of exposure to IL-1, showed twin peaks at 4 and 24 hr, and declined thereafter. Enzyme-linked immunosorbent assay revealed that the amount of endothelin-1 peptide in conditioned media was also increased by IL-1 in a dose- and time-dependent manner. Our results suggested that IL-1, a macrophage-derived cytokine, may affect the contraction and proliferation of vascular smooth muscle cells by stimulating the production of endothelin by endothelial cells.

cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA

We report the cloning of a full-length cDNA encoding rat preproendothelin-1 (preproET-1). The predicted rat preproET-1 consists of 202 amino acid residues and highly similar to human, porcine and bovine preproET-1, respectively. The deduced 21-residue sequence of mature rat ET-1 is identical to human, porcine, canine and bovine ET-1. As in other mammalian species, the mature ET-1 is predicted to be produced from a 39-residue big ET-1 in the rat. Northern blot analysis showed that a single 2.3-kb preproET-1 mRNA is expressed not only in vascular endothelial cells but also in other rat tissues, including the lung, brain, uterus, stomach, heart, adrenal gland and kidney. These findings suggest that ET-1 may play roles as a local mediator in multiple organs both within and outside the cardiovascular system in the rat.

Binding and receptor down-regulation of a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells

Binding of a novel endothelium‐derived vasoconstrictor endothelin (ET) and the regulation of its receptor were studied in cultured rat vascular smooth muscle cells. 125I‐labeled‐ET bound to the cells was resistant to acid extraction and the majority of the acid‐resistant compartment was extractable with chloroform/methanol with minimal degradation. Autoradiographic studies using electron microscopy revealed that the grains were predominantly localized in the plasma membranes, but some were adjacent to and within the lysosome. Pretreatment with ET resulted in a substantial reduction of ET receptor number without changing its binding affinity. ET‐induced increase in cytosolic free Ca2+ levels ([Ca2+]i] was absent or attenuated in the ET‐pretreated cells. These data suggest that tight association of ET with its receptor is due to a strong interaction of its hydrophobic domain with the membrane lipids and/or its internalization within cells and that down‐regulation of ET receptor is functionally linked to decreased ET‐induced [Ca2+]i response.

Structure-activity relationships of endothelin: importance of the C-terminal moiety

The vasoconstrictor activities of various forms of derivatives of endothelin (ET) were characterized in vitro by measuring the contraction of porcine coronary artery strips. The removal of the C-terminal Trp21 reduced the molar potency of the peptide by nearly 3 orders of magnitude. The removal of amino acid residues from the C-terminus of ET(1-20) further attenuated the activity. Replacement of Trp21 with D-Trp, reduction and carboxamidomethylation of the four Cys residues, or cleavage at Lys9 by lysyl endopeptidase all lowered the potency approximately 200 fold. While both native ET and [D-Trp21]ET induced a very slow and sustained vasoconstriction, the other derivatives of ET listed above showed a much more rapid kinetics of vasoconstriction. These results indicate that the C-terminal Trp of ET is especially important for the potent and extremely long-lasting vasoconstrictor activity characteristic to ET.