Cloning of full-length preproendothelin-2 cDNA and its expression in dog.

Endothelin-2 (ET2) is a member of the endothelin family of 21-amino acid peptides with vasoconstrictive activity. We report here the molecular cloning of the canine full-length cDNA of the precursor form of ET2, prepro-ET2 (PPET2), from intestinal tissue by means of reverse transcription-polymerase chain reaction (RT-PCR) in conjunction with 5'- and 3'-rapid amplification of cDNA ends (RACE). Aside from the poly (A) tail the cDNA was found to be 1195 bp and included an open reading frame of 534 bp encoding a PPET2 polypeptide of 178 residues, in which the regions corresponding to bioactive mature ET2 peptide, an intermediate form big-ET2, and endothelin-like peptide are found. The organ distributions of PPET2 mRNA and a splicing variant were analyzed by RT-PCR. PPET2 transcript was detected in duodenum, colon, stomach, lung, liver, uterus, ovary, testis and kidney, but not in spleen. A splicing variant was found in none of the organs. Thus, based on the cloned cDNA sequence, we established a quantitative assay for dog PPET2 mRNA level using a real-time PCR system. Quantitative analysis by this method in various organs of the dog demonstrated that the dominant gene expression occurs in the intestine, with higher expression in large intestine than in small intestine.

Subcellular distribution of ryanodine receptors in the cardiac muscle of carp (Cyprinus carpio).

We examined the subcellular localization of ryanodine receptors (RyR) in the cardiac muscle of carp using biochemical, immunohistochemical, and electron microscopic methods and compared it with those of rats and guinea pigs. To achieve this goal, an anti-RyR antibody was newly raised against a synthetic peptide corresponding to an amino acid sequence that was conserved among all sequenced RyRs. Western blot analysis using this antibody detected a single RyR band following the SDS-PAGE of sarcoplasmic reticulum (SR) membranes from carp atrium and ventricle as well as from mammalian hearts and skeletal muscles. The carp heart band had slightly greater mobility than those of mammalian hearts. Although immunohistochemical staining showed evident striations corresponding to the Z lines in longitudinal sections of mammalian hearts, clusters of punctate staining, in contrast, were distributed ubiquitously throughout carp atrium and ventricle. Electron microscopic images of the carp myocardium showed that the SR was observed largely as the subsarcolemmal cisternae and the reticular SR, suggesting that the RyR is localized in the junctional and corbular SR.