Application of Developmental Biotechnology to the Investigation on the Clinical Implication of Natriuretic Peptide Family

Natriuretic peptide family, which possesses potent diuretic, natriuretic and vasorelaxing properties, is now recognized to be composed of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). We have elucidated that ANP and BNP are the cardiac hormone mainly secreted from the atrium, and the ventricle, respectively. We have also discovered that CNP first recognized as the neuropeptide is produced in endothelial cells and considered to be an autocrine/paracrine regulator in the vascular wall. The aim of this study is to elucidate clinical significance of natriuretic peptide family using molecular biology and developmental biotechnology technique and seek for the clinical application of natriuretic peptide family for cardiovascular disorders.To elucidate the significance of natriuretic peptide family in chronic regulation of blood pressure and body fluid homeostasis, we isolated mouse BNP gene and cDNA and tried to generate BNP gene overexpressing transgenic mice. Mouse BNP gene contains three exons and two introns. Typical TATAAA sequence exists about 100 base pairs upstream of the translation initiation site. By microinjecting the expression vector which contained cloned mouse BNP gene linked to human serum amyloid P promoter into mouse male pronucleus of fertilized egg, we succeed in obtaining several F1 transgenic mice with various copy numbers of BNP gene (15∼50 copies). Northern blot analysis revealed that mouse BNP mRNA was abundantly expressed in the liver in the BNP transgenic mice. The BNP mRNA concentration in the liver was ten-times higher than that in the ventricle. We established the radioimmunoassay specific to mouse BNP and examined the plasma level of BNP in the transgenic mice. The plasma BNP concentration in the transgenic mice was 2-15pmol/ml, which was at least 10 to 100 times higher than the control mice (less than 0.16pmol/ml). Blood pressure of the transgenic mice determined by the direct measurement was 106±1mmHg, which was significantly lower than that of the control mice (126±2mmHg). These findings indicate that BNP can chronically exert biological action to modulate blood pressure and body fluid homeostasis, and support the long term effectiveness of natriuretic peptide family for clinical application. The significance of natriuretic peptide family is now under investigation, using the transgenic mice. We also succeeded in cloning mouse CNP gene. The mouse CNP gene is composed of at least two exons and one intron. The 5'-flanking region contains an array of cis-acting regulatory elemnts, including TATA box, Y box, GC box and CRE-like sequence, and a dinucleotide CA repeat (microsatellite). CNP mRNA expression was observed widely in mouse tissues, suggesting the role of CNP as a local regulator. On the basis of the polymerase chain reaction-analyzed microsatellite length polymorphisms among recombinant inbred strains of mice, the CNP gene (Nppc) was assigned to mouse chromosome 1. This is well contrast to the fact that the genes of ANP and BNP, which act as the cardiac hormone, are tightly linked on chromosome 4. CNP gene over-expressing transgenic mice and CNP knock-out mice have been now under development for the investigation of the significance of CNP in cardiovascular homeostasis.