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Aims: The function, regulation and gene expression of the endothelin (ET) system in the intestine is not well understood. We investigated the dependence on feeding schedule and biological clock of the regulation of ET-1 gene expression in mouse colon., Main Methods: Mice were fed freely, fasted for 48 h and re-fed after fasting., Key Findings: Where indicated ET-1 gene expression was highest in the colon compared with other tissues examined in fasted mice. Fasting increased the level, while maintaining the rhythmicity, of ET-1 gene expression in epithelial colonic tissue. Re-feeding, however, decreased ET-1 gene expression and suppressed rhythmic oscillation, and the rhythmicity also changed for gene expression for circadian clocks, period-1 and period-2 (Per1 and Per2). Furthermore, the decrease in ET-1 gene expression induced by re-feeding was blocked by pre-treatment with hexamethonium and atropine. The daily change in ET-1 gene expression in colon, which depends on feeding schedule via the autonomic nervous system, is synchronized with peripheral circadian oscillators under conditions of free feeding and fasting but not re-feeding. The decrease in ET-1 gene expression in the proximal colon induced by re-feeding occurs via the nervous system., Significance: ET-1 plays an important physiological role, which is dependent on feeding behavior., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The present investigation was designed to evaluate the renal microvascular endothelial cell responses following exposure to preformed antibodies against human leukocyte antigens (HLA) in the recipient. We hypothesize that activation of endothelial cell genes has a pivotal role in renal allograft survival. In this study, we used cultured human umbilical cord vein endothelial cells (HUVEC), human microvascular glomerular endothelial cells (HMGEC), activated with and without IFN-γ and TNF-α, and pre-transplant blood group O patient sera containing multispecific HLA class I and class II antibodies. Molecular HLA typing revealed the HMGEC haplotype to be HLA-A*01, HLA-A*68, HLA-B*14, HLA-B*35, HLA-C*04, HLA-C*08, HLA-DRβ1*13, and HLA-DRβ1*15. Flow cytometry was used for phenotypic characterization of both inactivated and activated HUVECs and HMGECs with IFN-γ and TNF-α. HUVECs were positive for HLA-ABC, HLA-DR/DQ, von Willebrand factor, endoglin, PECAM, ICAM, MCAM, integrin beta-3, thrombomodulin, E-selectin, VCAM-1, and tissue factor, and negative for alpha smooth muscle actin and P-selectin antibodies. HMGECs were positive for HLA-ABC, HLA-DR/DQ, von Willebrand factor, endoglin, ICAM, MCAM, integrin beta-3, thrombomodulin, VCAM-1, and tissue factor; and negative for PECAM, E-selectin, P-selectin, and for blood group antigens A and B. 42 samples were analyzed by real time PCR and categorized into the following groups: the control group (HMGEC only, n=12), group 1 (HMGECs incubated with patient sera, n=15), and group 2 (HMGECs activated by TNF-α and IFN-γ and incubated with patient sera, n=15). Expression levels of the vasoconstriction genes endothelin 1 (EDN1), endothelin 2 (EDN2), and endothelin receptor type A (EDNRA) were up-regulated in both groups 1 and 2 compared to the control group. The thrombomodulin (THBD) gene was also up-regulated in both groups 1 and 2 compared to the control. Chemokine genes CCL5 and CX3CL1 were up-regulated in both groups 1 and 2 compared to the controls; whereas, CCL2 was up-regulated only in group 2. Cytokine activity genes colony stimulating factor 2 (CSF2), tumor necrosis factor (TNF), tumor necrosis factor (ligand) superfamilymember 10 (TNFSF10), interleukin 1 beta (IL1B), and interleukin 6 (IL6) were up-regulated in both groups 1 and 2 compared to the control; whereas, IL11 was up-regulated only in group 1 and IFNB1 in group 2. Adhesion molecule genes intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and integrin beta 3 (ITGB3) were up-regulated in both groups 1 and 2 compared to the control; whereas, CDH5 and COL18A1 were up-regulated only in group 2. Anti-apoptosis genes BCL2A1, CFLAR, and SPHK1 were up-regulated only in group 2 compared to controls. Apoptosis and caspase-activation genes CASP, RIPK1, and FAS were up-regulated only in group 2 compared to the control. Angiopoietin 1 (ANGPT1) and prostaglandin I2 (prostacyclin) synthase (PTGIS) were down-regulated in both groups 1 and 2 compared to the control group. Our results indicate that expression of the endothelin gene in endothelial cells may contribute to vasoconstriction of blood vessels in post-renal allograft transplantation. In addition, thrombomodulin, by reducing thrombogenic activity, and interleukin 11, through its cytoprotective effects, may have a role in transplant accommodation in the presence of pre-formed HLA antibodies. This study showed that activation of the vasoconstriction genes, thrombomodulin gene, chemokine genes, cytokine activity genes, adhesion genes, anti-apoptosis genes, and apoptosis and caspase-activation genes could have consequential effects on renal allograft survival., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aims: Endothelin-1 (ET-1) and endothelin-2 (ET-2; Trp(6)Leu(7)ET-1) are expressed by different cell types, but are considered to display identical pharmacological properties on endothelin receptors. We studied agonist-dependent aspects of endothelin(A) (ET(A))-receptor function and the importance of amino acids 6 and 7 of ET-1 and ET-2 in this respect., Main Methods: We used isolated rat mesenteric resistance arteries in wire myographs, in a setting that minimizes influences of endothelium and sensorimotor nerves, to study arterial smooth muscle ET(A)-receptor-mediated vasomotor responses, to ET-1, ET-2 and chimeras thereof (Trp(6)ET-1 and Leu(7)ET-1)., Key Findings: ET-1 and ET-2 cause arterial contractions with comparable sensitivities and maximal responses. BQ123 (ET(A)-antagonist) reduces sensitivity to ET-1 more potently than that to ET-2 (pK(B): 7.1 ± 0.2 versus 5.6 ± 0.4). However, 1 μM BQ123 relaxes maximal contractile responses to ET-2 more markedly than those to ET-1. Leu(7)ET-1 is a contractile agonist with lower potency and similar maximal effect compared to ET-1 and greater sensitivity to BQ123 than ET-2. Up to 256 nM Trp(6)ET-1 did not cause contraction and did not antagonize arterial responses to ET-1., Significance: Arterial smooth muscle ET(A)-receptor function displays agonist-dependent aspects. This involves roles of amino acids on position 6 and 7 of the endothelin sequence. Agonist-dependent pathologies may benefit from the design of specific, agonist-selective ET-receptor antagonists., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The endothelin system consists of three ligands (ET-1, ET-2 and ET-3) and at least two receptors (ETA and ETB). In mice ET-2 counterpart is a peptide originally called "vasoactive intestinal contractor" (VIC) for this reason, this peptide is frequently named ET-2/VIC. In intestinal villi, fibroblasts-like cells express endothelin's receptors and response to ET-1 and ET-3 peptides, changing their cellular shape. Several functions have been attributed to these peptides in the "architecture" maintenance of intestinal villi acting over sub-epithelial fibroblasts. Despite this, ET-2/VIC has not been analyzed in depth. In this work we show the intestine gene expression and immunolocalization of ET-1, ET-2 and the ETA and ETB receptors from duodenum to rectus and in the villus-crypt axis in mice, allowing a complete analysis of their functions. While ET-1 is expressed uniformly, ET-2 had a particular distribution, being higher at the bottom of the villi of duodenum, ileum and jejunum and reverting this pattern in the crypts of colon and rectus, where the higher expression was at the top. We postulated that ET-2 would act in a cooperative manner with ET-1, giving to the villus the straight enough to withstand mechanical stress., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We previously found that endothelin-2/vasoactive intestinal contractor (ET-2/VIC) greatly increased in mouse epidermis after birth. In the present study, we evaluated whether ET-2/VIC expression was associated with the calcium-induced differentiation of cultured mouse keratinocytes. The differentiation induction was revealed by morphological change, cornified envelope (CE) formation, and involucrin and transglutaminase 1 (TG 1) expressions. ET-2/VIC gene expression and peptide production subsequently increased in the induction of the differentiation. We also found that Y-27632, a Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) inhibitor, suppressed up-regulation of ET-2/VIC gene expression, the induction of morphological change, the CE formation, and TG 1 expression, but not involucrin expression. These results indicate new three findings, (1) ET-2/VIC expression increases and has potential as a differentiation marker, (2) ET-2/VIC expression is mediated by ROCK, and (3) the ROCK regulated TG 1 expression, on the calcium-induced differentiation of mouse keratinocytes.

Endothelin (ET) is a peptide composed of 21 amino acids, derived from a larger precursor, the big-endothelin, by action of the endothelin-converting enzyme (ECE) family; three isoforms of endothelin, named ET-1, ET-2 and ET-3, have been identified. Endothelin-1 is generated mainly by vascular endothelial cells and exerts various important biological actions, mediated by two receptor subtypes, ET-A and ET-B, belonging to the G protein-coupled family that have been identified in various human tissues such as the cardiac tissue. Endothelin-1 is a potent vasoconstrictive agent, has inotropic and mitogenic actions, modulates salt and water homeostasis and plays an important role in the maintenance of vascular tone and blood pressure in healthy subjects. Endothelin-1, as well as ET-A and ECE-1, also has an important role in cardiovascular development, as observed by the variety of abnormalities related to neural crest-derived tissues in mouse embryos deficient of a member of the ET-1/ECE-1/ET-A pathway. Various evidence indicates that endogenous endothelin-1 may contribute to the pathophysiology of conditions associated with sustained vasoconstriction, such as heart failure. In heart failure, elevated circulating levels of both endothelin-1 and big-endothelin-1 are observed; in failing hearts an activation of the endothelin system is found: tissue level of ET-1 is increased with respect to non-failing hearts as well as receptor density, due mainly to an upregulation of the ET-A subtype, the prevalent receptor subclass in cardiac tissue. Finally, studies in both humans and animal models of cardiovascular disease show that inhibition of the endothelin function (anti-endothelin strategy) is associated with an improvement of haemodynamic conditions; these observations indicate that endothelin receptor antagonists or endothelin-converting enzyme inhibitors may constitute a novel and potentially important class of agents for the treatment of this disease., (Copyright 2001 Academic Press.)