A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family. Molecular cloning, expression, and biological activity.

A new peptide family (endothelin (ET] consisting of three members in mammals appears to be present in mice according to genomic Southern blot analysis. Two ET-related genes were identified by cloning and sequence analysis of a mouse genome. One encoded a peptide identical to porcine and human vasoconstrictor peptide ET, and the other encoded a novel peptide differing from ET in 3 amino acid residues, with 4 cysteines in the same positions as in ET. This novel peptide was synthesized and confirmed to have in vivo pressor activity similar to that of ET. Northern blot analysis, however, indicated the gene of this novel peptide to be expressed only in the intestine, and not in other tissues or cell lines, or endothelial cells. Furthermore, the peptide evoked a strong contractile response in the guinea pig ileum. This peptide may thus be reasonably classified as a gastrointestinal peptide, vasoactive intestinal contractor.

* This work was supported by project grants of Basic Technology for Future Industry and Research and Development for the elucidation of Biological Functions from the Ministry of International Trade and Industry of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "uduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bunk with accession number(s) M26497andM26498.
Recently, a novel family of peptides was identified (9-12). Some of them, such as peptide YY, pancreatic polypeptide, and neuropeptide Y (9), were found to have unique physiological functions in different tissues. To discover new peptides homologous to ET, an ET family gene from the mouse genome was examined. Cloning of two mouse genes, their tissuespecific expression, and their physiological roles are discussed.

MATERIALS AND METHODS
Isolation and Sequencing of Mouse ET and VZC Genes-A mouse genomic library was constructed by insertion of BALB/c mouse liver genomic DNA partially digested with Sau3AI into the BamHI sites of Charon 30 (kindly provided by N. Takahashi, Nagoya University, Japan). Approximately 2 X lo6 plaques from the library were screened by hybridization with a 32P-labeled EcoRI-EcoRI 1.1-kb fragment of human E T cDNA (7). Restriction fragments of the genomic inserts were suhcloned into plasmid pUC 18 and sequenced by the dideoxy chain termination method (13). All DNA sequences were confirmed by sequencing both strands. The standard recombinant DNA procedures used are as described (14, 15).
Synthesis of Mouse VIC-Mouse putative VIC was assembled with an Applied Biosystems model 430.4 peptide synthesizer according to the primary structure predicted from the nucleotide sequence. The purity of the final product was determined by analytical HPLC and amino acid analysis. The disulfide bond arrangement of synthetic VIC was the same as that of ET, based on HPLC profiles (6, 8).
Assay of in Viuu Pressor Effect in Conscious Rats-Male Wistar rats (350-400 g) were used. After synthetic E T or mouse VIC had been injected intravenously, blood pressure was measured by the tailcuff method (17).
Nurthern Blot Analysis-Poly(A)' RNAs were prepared (18) from various mouse tissues and cell lines (brain, kidney, lung, liver, spleen, stomach, intestine, whole body, mouse sarcoma Engelbreth-Holm-Swarm tumor, granulocyte WEHI-3, T-cell BW5147, mast-cell P815, macrophage 57'74, B-cell myeloma P3X63Ag8, fibroblast L929, and NIH 3T3) and cultured bovine and human endothelial cells. The methods for endothelial cell preparation were the same as described previously (19). Five fig of poly(A)+ RNA from cells and tissues was subjected to electrophoresis in a 1% agarose gel and transferred to nitrocellulose filters (20). Hybridization was conducted as described in the Southern blot analysis procedure. The 32P-labeled probes are shown in Fig. 1A. The filters were subsequently washed at 50 "C in a solution containing 1 X SSC, 0.1% SDS.

RESULTS AND DISCUSSION
On using a human ET cDNA fragment (EcoRI-EcoRI, 1.1 kb (7)) for analysis of a Southern blot of a BALB/c mouse genomic DNA at low stringency, a thick band, possibly indicating the mouse ET gene, and several faint bands, possibly genes homologous to ET, were observed (data not shown). The same probe detected 20 hybridization-positive clones from 2 X lo6 screenings with a BALB/c mouse liver genomic DNA library. These clones were subdivided into two groups, mET and mVIC, by restriction enzyme mapping (Fig. 1A). small horizontal lines in Fig. 1A were also hybridized with synthetic oligonucleotides (7) derived from porcine ET. In Southern blotting of BALB/c mouse genomic DNA at high stringency (Fig. 1B), an mET probe hybridized with a 19.4kb BamHI band and an mVIC probe hybridized with a 1.8-kb BumHI band. This and the mapping data in Fig. 1A indicate mET and mVIC to be located at different. genomic loci.

Fragments of the mET and mVIC probe regions indicated by
Sequence analysis of mET and mVIC indicated the genes of two putative peptides, ET and VIC, to be present in the mouse genome. Mouse E T was identical to porcine and human ET, while VIC was a novel peptide homologous to ET, demonstrating the possible presence of an ET family in the mouse (Fig. 2). The nucleotide and amino acid sequences of this E T family from several species were compared (Fig. 2). ET and its flanking amino acid residues were completely identical in mouse, pig, and human. But mouse VIC had 3 amino acid residues differing from those of ET, at amino acid positions 9, 11, and 12; that is, Ser, Leu, and Met changed to Asn, Trp, and Leu, respectively. These substitutions were very conservative (from polar to polar and non-polar to non-polar). The positions of the 4 cysteine residues and a dibasic pair of amino acids, Arg-Arg, directly preceding the mature peptides and recognized by processing endopeptidases (21), were also conserved. Trp-Val, possibly recognized by the "ET-converting enzyme" (6), was also conserved.
In Southern blotting of mouse genomic DNA at low stringency (Fig. 1B), a faint band between the 19.4-and 1.8-kb bands showed a gene homologous to the ET and VIC genes. In Southern blotting of rat genomic DNA hybridized with mET and mVIC probes, the hybridization-positive bands2 differing in size from rat E T (8) suggest that the ET and VIC genes in the mouse may also be present in the rat. Rat E T ( 8 ) (designated as rut EHP in Fig. 2) has 6 amino acids differing from those of E T or VIC, the substitutions were less conservative, and the recognition site of the ET-converting enzyme changed from Trp-Val to Trp-Ile. These facts indicate rat E T to possibly be a third member of the ET family, an "ET homologous peptide" (EHP), whose expression has yet to be confirmed (8). Thus, the gene corresponding to the faint band between the 19.4-and 1.8-kb bands in Fig. 1B suggest the presence of E H P in the mouse. Since our discovery of the mouse E T family,3 human ET family genes have been re-p~r t e d ,~ although none of them is identical to mouse VIC. We observed2 by Southern blot analysis the presence of E T family genes in Xenopus as well. The presence of the ET family (ET, VIC, and EHP) in vertebrates may thus be a possibility. The amino acid sequences of ET, VIC, and EHP differ mainly in their amino-terminal halves, and their conserved carboxylteminal halves may be essential for binding to receptors, as observed for the ligand binding site of IL-2 receptors (22,23).
The structural homology between ET and VIC prompted us to synthesize VIC and examine its pressor activity. Intravenous injection of VIC exerted as characteristically long lasting an in vivo pressor effect in conscious Wistar rats (Fig.  3) as ET (6). VIC (1 nmol/kg) caused a rise of 22 mm Hg (1 mm Hg = 1.333 x 10' pascal, pascal = m" kg s-') in blood pressure, and the profile of in vivo pressor activities was essentially the same as that for E T (6). Thus, possibly VIC may be a novel vasoconstrictor peptide with a potency nearly equal to that of ET.
To examine the tissue-specific expression of the ET and VIC genes, poly(A)+ RNAs isolated from various mouse tissues, mouse cell lines, and cultured bovine and human endothelial cells were analyzed by Northern blotting (Fig. 4). An mET probe (Fig. 1A) hybridized with a 2.3-kb transcript in bovine and human endothelial cells as expected, but this probe failed to detect any transcripts in other cells or tissues. In contrast, an mVIC probe (Fig. lA) hybridized with a 1.4-kb transcript in the intestine but not in other tissues, cell lines, or endothelial cells (Fig. 4). It is thus evident that the expression of ET and VIC genes is regulated in a tissue-specific manner.
In consideration of the tissue-specific expressions of the ET and VIC genes, the possibility that VIC may have contractile effect on the intestine was examined. I n vitro isotonic contraction activity of VIC in guinea pig ileum was confirmed and calculated as 29.8 If: 6.7% (at M VIC, n = 3), this value being the percentage of contraction induced by M acetylcholine. Based on the above results, VIC may be classified as a gastrointestinal peptide in consideration of its tissue-specific expression and activity. The biological characterization of VIC has been presented in greater detail (24). N. Ishida, unpublished data. "Saida, K., Mitsui, Y., Nishimatsu, S., and Ishida, N. (1988) Scientific Meeting ofthe Molecular Biology Society of Japan, Decemher 20, 1988, p. 220, Tokyo, Japan. Yanagisawa, M., Inoue, A., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K., andMasaki, T. (1989) (6), human (7), and rat (8) E T (designated here as rat "ET homologous peptide" (EHP)) family are compared. Open boxes show regions of the mature peptide (21 amino acid residues). Nucleotides and amino acids differing from those of porcine E T are underlined. The putative introns for mouse and rat genes are typed in small letters. These sequences fit well with the consensus sequence for the 5' end of the intron (25). Nucleotide and amino acid numbers are indicated above the structures. In conclusion, we have demonstrated the existence of a gene for a novel peptide, the VIC, and ET and EHP genes as well, suggesting the existence of a new peptide (endothelin) family. In consideration of the findings that the VIC gene is expressed in mouse intestine but not in endothelial cells, and that VIC induces strong contraction of the guinea pig ileum, VIC may possibly function as a gastrointestinal peptide. The production of a specific antibody against VIC which does not cross- react with E T or EHP is now being carried out to identify VIC-producing cells in the intestine and study the physiological and pathological roles of VIC.