Shoichi Ohkubo

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.

Identification and functional characterization of a novel subtype of neuromedin U receptor.

Neuromedin U is a bioactive peptide isolated originally from the porcine spinal cord. We recently identified neuromedin U as the cognate ligand for the orphan G protein-coupled receptor FM-3. In this study, we isolated cDNA coding for a novel G protein-coupled receptor, TGR-1, which was highly homologous with FM-3. We found that neuromedin U specifically and clearly elevated the extracellular acidification rates, arachidonic acid metabolite release, and intracellular Ca2+ mobilization in Chinese hamster ovary cells expressing TGR-1. Radiolabeled neuromedin U specifically bound with high affinity to membrane fractions prepared from these cells. These results show that TGR-1, like FM-3, is a specific and functional receptor for neuromedin U. We analyzed TGR-1 mRNA tissue distribution in rats using quantitative reverse transcription-polymerase chain reaction and found it to considerably differ from that of FM-3 mRNA. TGR-1 mRNA was primarily expressed in the uterus, suggesting that TGR-1 mediates the contractile activity of neuromedin U in this tissue. The identification of specific and functional receptor subtypes for neuromedin U will facilitate the study of their physiological roles and the search for their specific agonists and antagonists.

Characterization of murine PACAP mRNA

A murine PACAP precursor cDNA was isolated by screening a brain cDNA library. The amino acid sequence of the precursor was highly similar (from 81% to 93% similarity) to its rat, human, and ovine counterparts. The primary structure of murine PACAP is identical with those from sheep, humans, and rats, indicating that the mature PACAP is well conserved among mammals. Northern blot analysis revealed that the approximately 2.4 kb transcript for the PACAP precursor is expressed in murine brain. The verification that murine PACAP is identical to its human counterpart provides a rationale for physiological and pathophysiological studies of PACAP in mice.

One of the endothelin gene family, endothelin 3 gene, is expressed in the placenta

A cDNA encoding human endothelin 3 (ET‐3) precursor was cloned from a cDNA library from the placenta, and its nucleotide and deduced amino acid sequences were determined. This ET‐3 cDNA was found to contain 2.3 kb pairs and encode prepro‐ET‐3 protein consisting of 224 amino acid residues. The putative big‐ET‐3 seems to consist of 42 amino acid residues. Two of the intron insertion sites were determined with information from nucleotide sequences of the cloned genomic ET‐3 gene. This is the first direct evidence that the ET‐3 gene is transcribed and expressed in the placenta.

Specific expression of human endothelin-2 (ET-2) gene in a renal adenocarcinoma cell line Molecular cloning of cDNA encoding the precursor of ET-2 and its characterization

Analysis of culture medium of human renal adenocarcinoma cells ACHN by RP‐HPLC suggested that the cells specifically secreted human endothelin‐2 (ET‐2). cDNAs encoding human ET‐2 precursor were cloned from a cDNA library constructed with mRNA derived from the ACHN cells, and the nucleotide and deduced amino acid sequences were determined. The ET‐2 cDNA was revealed to contain 1.3 kb and encode prepro‐ET‐2 protein consisting of 178 amino acid residues. The ET‐like sequence found in the prepro‐ET‐1 and ‐ET‐3 was conserved in this prepro‐ET‐2. The Northern blot analysis of mRNA suggested that the transcript of ET‐2 gene was 1.4 kb. This is the first direct evidence that human ET‐2 gene was expressed specifically in tumor cells.

Expression of human pituitary adenylate cyclase activating polypeptide (PACAP) cDNA in CHO cells and characterization of the products

cDNA encoding human PACAP precursor was expressed in non‐neuroendocrine Chinese hamster ovary cells, CHO‐K1. The cells were transfected with expression vector (pTS705) containing the human PACAP cDNA by electroporation. A cell line which produced more than 80 ng/ml of immunoreactive PACAP (ir‐PACAP) into the conditioned medium was established. RP‐HPLC analysis of culture medium of this established cell line exhibited the presence of two types of PACAP, i.e. PACAP38 and PACAP27. At the same time, it was also revealed that immunoreactive PACAP‐related peptide (ir‐PRP) was secreted into the cultured medium. The ir‐PACAPs were confirmed to have biological activities such as induction of cAMP and neurite outgrowth in rat pheochromocytoma PC12h cells.

Expression of endothelin-2 (ET-2) gene in a human renal adenocarcinoma cell line : purification and cDNA cloning of ET-2

It has been found that human renal adenocarcinoma ACHN cells synthesize and secrete immunoreactive endothelin (ir-ET) in the culture medium. Partial characterization of this material with reverse-phase high-performance liquid chromatography (RP-HPLC) suggested that ACHN cells synthesized only human endothelin-2 (ET-2). Isolation and characterization of this ir-ET-2 has revealed that this peptide has almost the same amino acid sequence and molecular weight as that of human ET-2 deduced from the nucleotide sequence of cloned human ET-2 gene. To delineate the precise structure of human ET-2 precursor, ET-2 cDNAs were cloned from a cDNA library constructed with mRNA derived from the ACHN cells, and the nucleotide and deduced amino acid sequences were determined. The ET-2 cDNA that has the longest open reading frame encodes prepro-ET-2 protein, consisting of 178 amino acid residues. The ET-like sequence found in the prepro-ET-1 and -ET-3 was also conserved in this prepro-ET-2. The Northern blot analysis of mRNA revealed that the transcript of the human ET-2 gene was 1.4 kb.

Epidermal growth factor (EGF) decreased endothelin-2 (ET-2) production in human renal adenocarcinoma cells.

Production of immunoreactive (ir‐) endothelin‐2 (ET‐2) in renal adenocarcinoma cells, ACHN, was reduced by transforming growth factor‐β, basic fibroblast growth factor, transforming growth factor‐α and, most strikingly, by epidermal growth factor (EGF). These growth factors did not show such inhibitory effects on the secretion of ir‐ET‐1 in ET‐1‐producing cells, indicating that the production of ET‐2 and ET‐1 is regulated differently by the growth factors. EGF specifically reduced the secretion of not only ir‐ET‐2 but also ir‐big ET‐2 with only a small decrease in total protein synthesis. Northern blot analysis indicated that EGF controls the ET‐2‐production at the transcription levels of ET‐2 gene.