CNP is the only natriuretic peptide in an elasmobranch fish, Triakis scyllia

The natriuretic peptide (NP) family consists of endocrine ANP/BNP/VNP in the heart and paracrine CNP in the brain in vertebrates ranging from teleosts to mammals. In elasmobranchs, however, only CNP has been identified thus far in the heart and brain. To delineate the molecular evolution of this hormone family, it is essential to determine whether CNP is the only NP in this primitive fish group. In the present study, PCR cloning of all types of piscine NP was performed from the heart and brain of a dogfish, Triakis scyllia, using degenerate primers that amplified eel ANP, VNP and CNP. However, only CNP cDNA with an identical sequence was cloned from the heart and brain. Southern blot analysis showed that the CNP gene is a single copy gene, showing that endocrine CNP from the heart and paracrine CNP in the brain originate from the same gene. Since expression of the CNP gene was so high as demonstrated by Northern blot analysis, the abundantly expressed CNP mRNA could have interfered the amplification of other NP mRNAs expressed in small amounts. Therefore, a method was developed to cleave the cloned CNP mRNA specifically at the 3'-untranslated region with RNase H. After removal of the cloned CNP mRNA by this technique, no other NP cDNAs could be cloned, but small amounts of CNP cDNAs with shorter 3' sequence were amplified. These results strongly suggest that only CNP is present in elasmobranchs. Thus, it is likely that CNP is an ancestral form of the NP family and endocrine ANP/BNP/VNP have appeared later in the vertebrate evolution.