Vasoactive Intestinal Contractor Research Articles

Endothelin-2, the forgotten isoform: emerging role in the cardiovascular system, ovarian development, immunology and cancer.

Endothelin-2 [ET-2; also known as vasoactive intestinal contractor (VIC), in rodents] differs from endothelin-1 (ET-1) by only two amino acids, and unlike the third isoform, endothelin-3 (ET-3), it has the same affinity as ET-1 for both ET(A) and ET(B) receptors. It is often assumed that ET-2 would mimic the actions of the more abundant ET-1 and current pharmacological interventions used to inhibit the ET system would also block the actions of ET-2. These assumptions have focused research on ET-1 with ET-2 studied in much less detail. Recent research suggests that our understanding of the ET family requires re-evaluation. Although ET-2 is very similar in structure as well as pharmacology to ET-1, and may co-exist in the same tissue compartments, there is converging evidence for an important and distinct ET-2 pathway. Specifically is has been demonstrated that ET-2 has a key role in ovarian physiology, with ET-2-mediated contraction proposed as a final signal facilitating ovulation. Furthermore, ET-2 may also have a pathophysiological role in heart failure, immunology and cancer. Comparison of ET-2 versus ET-1 mRNA expression suggests this may be accomplished at the level of gene expression but differences may also exist in peptide synthesis by enzymes such as endothelin converting enzymes (ECEs) and chymase, which may allow the two pathways to be distinguished pharmacologically and become separate drug targets. LINKED ARTICLES This article is part of a themed section on Endothelin. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.168.issue-1.

Endothelin system in intestinal villi: A possible role of endothelin-2/vasoactive intestinal contractor in the maintenance of intestinal architecture

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.

Characterization of CoCl2-induced reactive oxygen species (ROS): Inductions of neurite outgrowth and endothelin-2/vasoactive intestinal contractor in PC12 cells by CoCl2 are ROS dependent, but those by MnCl2 are not

CoCl(2) and MnCl(2) are hypoxic mimetic agents. We previously found that expression of ET-2/VIC, one of hypoxia-related factors, and the induction of neurite outgrowth in PC12 cells through ROS induced by CoCl(2). MnCl(2) also are known to induce neurite outgrowth in PC12 cells. However, it is unclear whether the mechanism of the effect induced by these metals is same. In the present study, we evaluated biological effects induced by MnCl(2) and compared with those induced by CoCl(2). Furthermore, we analyzed sources of CoCl(2)-induced ROS generation. MnCl(2) up-regulated ET-2/VIC gene expression and ET-2/VIC peptide production as CoCl(2) did, but not affect ET-1 gene expression, in the neurite outgrowth of PC12 cells. NAC did not at all inhibit the effects induced by MnCl(2). Furthermore, addition of MnCl(2) to the culture medium did not generate ROS as CoCl(2) did. These results indicate that ET-2/VIC expression is a common pathway in neurite outgrowth induced by CoCl(2) and MnCl(2), but the effects induced by CoCl(2) are ROS dependent, whereas the effects induced by MnCl(2) are ROS independent. Taken together, the mechanism for the effects by CoCl(2) was different from that by MnCl(2). The ROS, were not decomposed by catalase or SOD, were rapidly generated by reaction of CoCl(2) mainly with components of HS rather than with FBS or DMEM. Some ROS generated by reaction of CoCl(2) with components of HS may participate in the observed neurite outgrowth of PC12 cells.

Endothelin-2/vasoactive Intestinal Contractor via ROCK regulates transglutaminase 1 on differentiation of mouse keratinocytes

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.

Chromatic effects of endothelin family peptides in non-innervated fish,Synbranchus marmoratus, melanophores

The biological activity of endothelins (ETs) in non-innervated Synbranchus marmoratus melanophores was demonstrated. These peptides induced a dose-dependent pigment aggregation (lightening skin) in these cells. However, they presented EC50's (effective concentration required to produce 50% of response) 26, 106 and 35 times higher than, respectively, the melanin concentrating hormone (MCH) EC50, and exhibited a characteristic temporal and dose-dependent autodessensibilization of the aggregative effect on the melanophores of this fish. The receptor characterization suggested the presence of the ET(B) subtype, since BQ-788 (selective antagonist of ET(B)) but not BQ-485 (selective antagonist of ET(A)) blocked the aggregative effect of the hormones. Confirming these data, sarafotoxin (SRTX) S6c, a toxin selective for ET(B), induced maximal aggregation of pigment granules. S6c presented an EC50 6.8 times higher than the MCH EC50, and 3.9, 15.6 and 5.1 times lower than the EC50's ETs, respectively. The melanotropic effect of SRTX S6b and vasoactive intestinal contractor (VIC) were demonstrated for the first time in this work. SRTX S6b induced a dose-dependent pigment aggregation and presented an EC50 2.54 and 17.2 times higher than the S6c and MCH EC50's, respectively. Compared to the ETs it was 1.53, 6.19 and 2.03 times lower, respectively.

Endothelin-2/Vasoactive Intestinal Contractor: Regulation of Expression via Reactive Oxygen Species Induced by CoCl22, and Biological Activities Including Neurite Outgrowth in PC12 Cells

This paper reviews the local hormone endothelin-2 (ET-2), or vasoactive intestinal contractor (VIC), a member of the vasoconstrictor ET peptide family, where ET-2 is the human orthologous peptide of the murine VIC. While ET-2/VIC gene expression has been observed in some normal tissues, ET-2 recently has been reported to act as a tumor marker and as a hypoxia-induced autocrine survival factor in tumor cells. A recently published study reported that the hypoxic mimetic agent CoCl2 at 200 µM increased expression of the ET-2/VIC gene, decreased expression of the ET-1 gene, and induced intracellular reactive oxygen species (ROS) increase and neurite outgrowth in neuronal model PC12 cells. The ROS was generated by addition of CoCl2 to the culture medium, and the CoCl2-induced effects were completely inhibited by the antioxidant N-acetyl cysteine. Furthermore, interleukin-6 (IL-6) gene expression was up-regulated upon the differentiation induced by CoCl2. These results suggest that expression of ET-2/VIC and ET-1 mediated by CoCl2-induced ROS may be associated with neuronal differentiation through the regulation of IL-6 expression. CoCl2 acts as a pro-oxidant, as do Fe(II, III) and Cu(II). However, some biological activities have been reported for CoCl2 that have not been observed for other metal salts such as FeCl3, CuSO4, and NiCl2. The characteristic actions of CoCl2 may be associated with the differentiation of PC12 cells. Further elucidation of the mechanism of neurite outgrowth and regulation of ET-2/VIC expression by CoCl2 may lead to the development of treatments for neuronal disorders.

Cobalt chloride induces neurite outgrowth in rat pheochromocytoma PC-12 cells through regulation of endothelin-2/vasoactive intestinal contractor

We investigated whether endothelin-2/vasoactive intestinal contractor (ET-2/VIC) gene expression, upregulated by hypoxia in cancer cells, was associated with differentiation in neuronal cells. RT-PCR analysis, morphological observations, and immunostaining revealed that CoCl2, a hypoxic mimetic agent, at 200 microM increased expression of the ET-2/VIC gene, decreased expression of the ET-1 gene, and induced neurite outgrowth in PC-12 rat pheochromocytoma cells. These effects induced by 200 microM CoCl2 were completely inhibited by the antioxidant N-acetyl cysteine at 20 mM. In addition, CoCl2 increased the level of intracellular reactive oxygen species (ROS) at an early stage. Furthermore, interleukin (IL)-6 gene expression was upregulated upon the differentiation induced by CoCl2. These results suggest that expression of ET-2/VIC and ET-1 mediated by ROS may be associated with neuronal differentiation through the regulation of IL-6. When the cells were treated with 500 microM CoCl2 for 24 hr, however, ET-2/VIC gene expression disappeared, IL-6 gene expression was downregulated, and necrosis was subsequently induced in the PC-12 cells.

Molecular Cloning and Sequence Analysis of the Porcine Precursor of Endothelin-2

The amino acid sequences of two of the three endothelin (ET) family peptides, ET-1 and ET-3, are identical among mammals, whereas for the other family member, ET-2 or vasoactive intestinal contractor (VIC), the mouse and rat sequences differ from the human counterpart ET-2 by one amino acid residue. To examine more deeply the structural diversity among ET-2/VIC orthologs (EDN2), we screened porcine ET-2/VIC-like cDNAs using the 5' rapid amplification of cDNA ends (RACE) method with degenerate primers based on ET-2/VIC mature peptides. Sequence analysis of the cDNAs showed that ET-2 is present in pig. The full-length cDNA sequence, produced by combining 5' RACE and 3' RACE products, revealed the porcine precursor protein of ET-2 (PPET-2). Porcine PPET-2, made up of 214 amino acids, includes a 26-residue putative signal sequence, big ET-2, mature ET-2, and ET-2-like peptide. The percent sequence identity of porcine PPET-2 with human PPET-2, and rat or mouse precursor protein of VIC runs between approximately 70% and 74%. ET-2, although expressed in intestine, has no anti-microbial activity.

Isoform‐specific quantification of endothelins in HUVEC culture supernatants by on‐line high‐performance liquid chromatography/electrospray mass spectrometry

A method for the quantitative analysis of endothelin peptides in human umbilical vein endothelial cell (HUVEC) culture supernatants is reported. The analysis is isoform-specific and employs solid-phase extraction and subsequent HPLC fractionation followed by HPLC-ESIMS analysis. The peptide vasoactive-intestinal-contractor (VIC) was used as internal standard for the HPLC-ESIMS analysis. Linearity of calibration curves was from 50 fmol to 25 pmol. The limit of detection of the HPLC-ESIMS step using a buffer matrix was estimated at 50 fmol (S/N > 3). The overall limit of detection for supernatants of HUVEC was 500 fmol/mL. In HUVEC culture supernatants only ions of endothelin-1 (ET1) were observed. Basal levels were determined to be 1.8 +/- 0.3 pmol/mL. Quantitative results obtained for ET1 were in agreement with those obtained by using a standard addition method and by an ELISA method.

Vasoactive intestinal contractor/endothelin-2 gene expression in the murine central nervous system

Vasoactive intestinal contractor (VIC) is a member of the endothelin (ET) family. We have investigated the regional distribution of VIC/ET-2 and of ET-1 gene expression in the adult murine brain and pituitary gland. We used real-time quantitative reverse transcription-linked polymerase chain reaction. VIC/ET-2 gene expression was observed at high levels in the pituitary gland and medulla oblongata in both the mouse and rat. Moderate to low levels of expression were observed in other brain regions. On the contrary, ET-1 gene expression was quite low in the pituitary gland in comparison with the levels observed in the cerebral cortex, striatum, and midbrain. Cold injury to the mouse cerebral cortex caused a significant decrease in VIC/ET-2 gene expression in this structure, whilst expression of the ET-1 gene was increased. These results suggest that VIC/ET-2 may have certain physiological roles that differ from those of ET-1 in the brain and pituitary gland.

Expression of endothelin-1 and vasoactive intestinal contractor genes in mouse organs during the perinatal period.

In an attempt to understand the significance of endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC)/ET-2 peptides in organs during perinatal development, we performed quantitative analysis of ET-1 and VIC gene expression in mouse organs obtained from embryos at days 14 and 17 (E-14 and E-17) of pregnancy, neonates at days 0, 1, 3 and 7 after birth (N-0, -1, -3 and -7), and adult mice (10 weeks old). In intestine, VIC gene expression progressively increased between E-14 and N-1 (approximately 10-fold) and then remained constant into adulthood. ET-1 gene expression exhibited a one-step increase between E-17 and N-0, subsequently remaining constant. In lung, a sharp increase in ET-1 mRNA level (approximately 10-fold) was noticed between E-14 and N-0. The gene expression pattern of VIC, with a peak at N-0, was similar to that of ET-1 although the expression level of VIC was two to three orders of magnitudes lower than that of ET-1. Gene expression patterns of ET-1 and VIC remained nearly constant in brain, heart, liver and kidney throughout the period examined. Considering that the intestinal and pulmonary gene expression levels of both genes reached almost the same level as observed in adult soon after birth, we suggest that these peptides may be involved in the emergence and maintenance of intestinal and pulmonary functions vital after birth.

Effect of aging on gene expression rates of endothelin-1 and endothelin-2/vasoactive intestinal contractor in ethanol-induced gastric mucosal injury of the mouse.

To elucidate the physiological roles of endothelin-1 (ET-1) and endothelin-2 (ET-2)/vasoactive intestinal contractor (VIC) in gastric injury of mice, we measured the gene expression rates of ET-1 and ET-2/VIC in gastric injury induced by ethanol in young (8 weeks) and old (>33 weeks) mice. Mice that were fasted for 24 h were injected with absolute ethanol intragastrically and killed after 1 or 4 h of ethanol exposure. The size of the gastric lesions increased gradually after ethanol exposure and was at its greatest after 4 h, in both young and old mice. The gene expression of ET-1 tended to increase after 1 h and to decrease by 4 h of ethanol exposure in both young and old mice. However, the gene expression of ET-2/VIC in young mice increased significantly after 1 and 4 h of ethanol exposure, whereas the gene expression of ET-2/VIC in the old mice did not change after ethanol exposure. Based on these results, we conclude that aging influences the gene expression of ET-2/VIC but not lesion size or gene expression of ET-1 in ethanol-induced gastric injury in the mouse. We therefore suggest that regulation of gene expression of these two genes differs during the course of aging.

Rapid quantification of murine endothelin-1 and vasoactive intestinal contractor gene expression levels by a real-time PCR system

A rapid quantitative analysis method for murine endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC) gene expression levels was established using a real-time polymerase chain reaction (PCR). We designed primer pairs and TaqMan probes specific for murine prepro-ET-1 (PPET-1) and prepro-VIC (PPVIC) genes, based on the cDNA sequence region common to both mouse and rat. The dynamic range for detection in this system spanned 100 000-fold of the starting molecule. The gene expression levels of PPET-1 and PPVIC were estimated as gene expression rates normalized by the expression of the house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase. To examine the reproducibility of this assay system, we calculated the intra-assay and interassay coefficients of variation of the gene expression rate, which ranged from 16.2 to 55.0% and from 24.2 to 56.5%, respectively. Using this system, we examined gene expression levels of PPET-1 and PPVIC in mouse tissues. PPET-1 gene expression was found in all tissues at relatively high levels, whereas high levels of PPVIC gene expression were observed only in stomach, intestine, uterus, and ovary. The gene expression patterns agreed well with those determined by RNase protection assay and conventional PCR. These results show that this new rapid method is accurate and reproducible.

Fluctuating gene expression and localized cellular distribution of vasoactive intestinal contractor (VIC) in mouse uterus.

To understand the physiological roles of vasoactive intestinal contractor (VIC) and endothelin-2 (ET-2) in the uterus, we examined the expression levels of VIC mRNA by real-time quantitative reverse transcription-linked polymerase chain reaction (RT-PCR) and characterized the cellular distribution of VIC peptide and mRNA by immunostaining and in situ hybridization in mouse uterus. In pregnant mouse uterus, VIC mRNA expression changed considerably between Days 10.5 and 12.5 of pregnancy. The expression levels were significantly (p<0.05) higher (approximately fivefold) in the later stage of pregnancy (Days 12.5-17.5) than in the earlier stage (Days 7.5-10.5). In nonpregnant uterus, VIC mRNA expression was significantly (p <0.05) higher (approximately threefold) in proestrus and estrus than in diestrus. Immunohistochemical studies demonstrated the presence of VIC peptide in endometrial epithelial cells, myometrial cells, and vascular smooth muscle cells during the estrous cycle and pregnancy and after parturition. Notably, myometrial cells showed dominant immunostaining in proestrus and estrus, in the later pregnancy stage, and in the early postpartum period, analogous to the expression pattern of VIC mRNA. In situ hybridization confirmed localization of VIC mRNA in myometrial cells. These findings suggest that VIC may play an important role in the function of myometrial cells.

The Prepro Vasoactive Intestinal Contractor (VIC)/Endothelin-2 Gene (EDN2): Structure, Evolution, Production, and Embryonic Expression

Murine vasoactive intestinal contractor (VIC) and its human analog endothelin-2 (ET2) are potent vasoactive hormones composed of 21 amino acids. To study the structural characteristics of the VIC/ET2 gene (HGMW-approved symbol EDN2), we isolated the full length of the mouse VIC gene. Sequence analysis indicates that a biologically active mature VIC peptide is produced from a 175-residue precursor protein; preproVIC (PPVIC). Several remarkable similarities of the PPVIC gene to the human preproendothelin-1 gene strongly suggest that the two genes have arisen from a common progenitor by gene duplication. Transfection of ACHN adenocarcinoma cells with the cDNA resulted in the production of VIC peptide. VIC production was increased by the deletion of the 3′-untranslated region, which contains an AU-rich mRNA destabilizing sequence. Increased PPVIC gene expression during the late embryonic stage suggests an important function in development. This study provides the basis for disruption and regulation analysis of the gene, which may lead to a better understanding of VIC/ET2's physiological significance.

Quantitative Analysis of Endothelin-1 and Vasoactive Intestinal Contractor/Endothelin-2 Gene Expression in Rats by Real-Time Reverse Transcriptase Polymerase Chain Reaction

Summary: We established a real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) system for the analysis of rat endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC)/ET-2 gene expression. We used this technique to examine the expression levels in rat in 16 different organs. ET-1 gene expression was observed in all organs examined, while VIC mRNA was detected in some organs such as heart, lung, ovary, stomach, and intestine. Ovary and intestine express both ET-1 and VIC mRNA at high levels, suggesting the importance of both peptides in these organs. In addition, we examined the gene expression levels in intestinal epithelial and mesenchymal tissues from rat fetuses at 16.5 and 19.5 days postcoitus (E16.5 and E19.5). We observed distinct differences in the temporal gene expression patterns for ET-1 and VIC in fetal intestinal epithelial tissue. In fetal mesenchymal tissue the expression level of ET-1 is significantly higher than that of VIC, and the levels of both genes remain unchanged over the time period observed. These findings suggest distinct biological roles and gene regulation mechanisms for ET-1 and VIC in intestinal epithelial and mesenchymal tissues.

Quantitative analysis of endothelin-1 and vasoactive intestinal contractor/endothelin-2 gene expression in rats by real-time reverse transcriptase polymerase chain reaction.

We established a real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) system for the analysis of rat endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC)/ET-2 gene expression. We used this technique to examine the expression levels in rat in 16 different organs. ET-1 gene expression was observed in all organs examined, while VIC mRNA was detected in some organs such as heart, lung, ovary, stomach, and intestine. Ovary and intestine express both ET-1 and VIC mRNA at high levels, suggesting the importance of both peptides in these organs. In addition, we examined the gene expression levels in intestinal epithelial and mesenchymal tissues from rat fetuses at 16.5 and 19.5 days postcoitus (E16.5 and E19.5). We observed distinct differences in the temporal gene expression patterns for ET-1 and VIC in fetal intestinal epithelial tissue. In fetal mesenchymal tissue the expression level of ET-1 is significantly higher than that of VIC, and the levels of both genes remain unchanged over the time period observed. These findings suggest distinct biological roles and gene regulation mechanisms for ET-1 and VIC in intestinal epithelial and mesenchymal tissues.

The Endothelin-2/Vasoactive Intestinal Contractor Gene

In order to understand the physiological roles of vasoactive intestinal contractor (VIC)/endothelin-2 (ET-2), we examined the expression of this peptide by specific reverse transcriptase polymerase chain reaction (RT-PCR) analysis and found that PC12 rat pheochromocytoma cells express the VIC gene. The 5'-flanking 1.0 kilo base pair (kb) region of the mouse VIC gene is sufficient to express a secreted alkaline phosphatase (SEAP) reporter gene in transiently transfected PC12 cells. The 1.0 kb promoter region may contain cis-acting elements that determine the rate of the VIC gene transcription in PC12 cells.

Structure of mouse preproendothelin-3 and phylogenetic analysis of the endothelins.

Endothelin (ET)-related peptide hormones, ET-1, vasoactive intestinal contractor (VIC), ET-2 and ET-3, have multiple physiological roles including vasoconstriction. To reveal the structural diversity of the precursor proteins of the ET family, cDNAs, cross-hybridizing with ET-1 and VIC probes, were cloned from the mouse intestine library. The deduced protein is a 214-amino acid precursor of mouse ET-3, preproendothelin-3 (PPET-3), which is the counterpart of the hypothalamus-, not placenta-, derived human PPET-3. Sequence identities of PPET-3 amino acids of mouse with human and rat are 65% over 194 amino acids and 65% over 214 amino acids. Phylogenetic analysis of the precursor proteins for ET-1, VIC and ET-3 suggest that members of the ET family are distantly related and probably descended from a common ancestral gene.

The Endothelin-2/Vasoactive Intestinal Contractor Gene

Summary: In order to understand the physiological roles of vasoactive intestinal contractor (VIC)/endothelin-2 (ET-2), we examined the expression of this peptide by specific reverse transcriptase polymerase chain reaction (RT-PCR) analysis and found that PC12 rat pheochromocytoma cells express the VIC gene. The 5'-flanking 1.0 kilo base pair (kb) region of the mouse VIC gene is sufficient to express a secreted alkaline phosphatase (SEAP) reporter gene in transiently transfected PC12 cells. The 1.0 kb promoter region may contain cis-acting elements that determine the rate of the VIC gene transcription in PC12 cells.