Kaname Saida

Differential activities of two distinct endothelin family peptides on ileum and coronary artery

A synthesized mouse vasoactive intestinal contractor peptide, which belongs to a novel member of the endothelin family, induced a prolonged contraction in mouse ileum as well as porcine coronary artery in vitro. Studies comparing the effects of vasoactive intestinal contractor and endothelin on different tissues revealed that the maximum ileum contraction of vasoactive intestinal contractor was much higher than that of endothelin in both guinea pig and mouse systems, but that the vasoconstriction activity of vasoactive intestinal contractor was weaker than that of endothelin in porcine artery. These resuts show that vasoactive intestinal contractor might be a novel gastrointestinal hormone.

Effects of vasoactive intestinal contractor (VIC) and endothelin on intracellular calcium level in neuroblastoma NG108‐15 cells

Effects on [Ca2+]i levels of endothelin‐1 (ET) and vasoactive intestinal contractor peptide (VIC), which is a novel member of the endothelin family, were examined in fura 2‐loaded neuroblastoma NG108‐15 cells. VIC was found to be a very effective stimulus for intracellular Ca2+ mobilization and to be more potent than ET. Intracellular calcium response to sequential addition of two stimulants exhibited the homologous desensitization of either ET or VIC, but no heterologous desensitization between ET and VIC. This indicates evidence suggesting that these two peptides act through distinct receptors.

RAPID DETECTION OF MYCOPLASMA CONTAMINATION IN CELL CULTURES USING SYBR GREEN-BASED REAL-TIME POLYMERASE CHAIN REACTION

SummaryWe have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluated the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, in suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.

cDNA cloning, sequence analysis and tissue distribution of a precursor for vasoactive intestinal contractor (VIC)

A full-length cDNA encoding preprovasoactive intestinal contractor (PPVIC) has been cloned. From the deduced 160 amino acid PPVIC, the mature VIC is predicted to be produced via a 37 residue intermediate, big VIC. The PPVIC also contains a VIC-like peptide of 16 amino acids structurally related to to the amino-terminal residues of VIC and flanked by pairs of dibasic amino acids, putative processing sites. RNA blot hybridization with PPVIC cDNA confirmed the PPVIC gene to be expressed in the small and large intestinal tract in a tissue specific manner.

Endothelin-2/vasoactive intestinal contractor: regulation of expression via reactive oxygen species induced by CoCl2, 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.

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 100000-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.

Increased gene expression and production of murine endothelin receptors after birth

We developed the real-time PCR quantification of endothelin-A (ET-A) and endothelin-B (ET-B) receptor genes and present their relative expression levels in various adult tissues and during development in mouse using the 2(-Delta Delta C(T)) method. ET-A and ET-B receptors were detected in all tissues examined. Gene expression of ET-A and ET-B receptors increases during the later stages of embryonic development in lung, heart, liver, kidney, and skin and reaches a maximum on the first one or two days after birth. The results, in agreement with our data on endothelin (ET) ligands, suggest that the ET system may be involved in the emergence and maintenance of functions vital after birth in these organs. These findings were corroborated through observation of the correlation between the gene expression and (poly)peptide production of the ET system in normal skin before and after parturition.

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.

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.

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.