Kenji Kangawa

C-Type natriuretic peptide (CNP): A new member of natriuretic peptide family identified in porcine brain

Two types of natriuretic peptide, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), very similar to each other in structure and in pharmacological effect, are known to be present in mammalian heart and brain. In our present survey for unidentified peptides in porcine brain extracts, we found a new peptide of 22 amino acid residues, eliciting a potent relaxant activity on chick rectum. The amino acid sequence determined for the peptide shows remarkable similarity to those of ANP and BNP, especially in the 17-residue sequences flanked by two cysteine residues. The peptide shows a pharmacological spectrum similar to ANP and BNP. Thus, the peptide was designated C-type natriuretic peptide (CNP), the third member to join the natriuretic peptide family. In contrast to ANP and BNP, CNP terminates in the second cysteine residue, lacking a further C-terminal extension.

Cloning and sequence analysis of a cDNA encoding a precursor for rat C‐type natriuretic peptide (CNP)

Recent identification of a C‐type natriuretic peptide (CNP) in porcine brain strongly suggested that a third member of the natriuretic peptide family still remains to be identified in other species of mammals. A cDNA encoding a precursor for rat CNP was cloned from a rat brain cDNA library and sequenced. The precursor was a 126‐residue peptide, carrying a 23‐residue signal sequence at the N‐terminus and the known porcine CNP‐53 sequence at the C‐terminus. By RNA blot analysis, rat CNP mRNA was found to be expressed exclusively in the brain, implying that CNP may function in the central nervous system as a neuropeptide.

Gene and precursor structures of human C-type natriuretic peptide

We have isolated the gene for human C-type natriuretic peptide (CNP) from a human genomic library using a cloned porcine CNP genomic DNA fragment as probe. Human CNP gene consists of at least two coding blocks and an intron, and encodes a 126-residue CNP precursor (human prepro-CNP). From a comparison of the amino acid sequences of porcine and rat prepro-CNPs, human prepro-CNP is found to be processed to generate 22-and 53-residue peptides (human CNP-22 and human CNP-53, respectively) as major endogenous CNPs in human. Interestingly, human CNP-53 has two amino acid substitutions as compared to the porcine and rat CNP-53s, whereas human CNP-22 is identical to the porcine and rat CNP-22s. Intravenous injection of human CNP-53 into anesthetized rats induces diuretic-natriuretic and hypotensive activities with same potencies as porcine CNP-53 does, although these activities were considerably lower (about 1/100) than those of human alpha-ANP.

DISTRIBUTION AND CHARACTERIZATION OF IMMUNOREACTIVE PORCINE C-TYPE NATRIURETIC PEPTIDE

C-type natriuretic peptide (CNP) is a new member of the natriuretic peptide family recently identified in porcine brain (1). We raised an antiserum against porcine CNP and set up a radioimmunoassay (RIA) for CNP. Using this RIA system, distribution of immunoreactive (ir-) CNP in porcine tissue was measured and compared with that of ir-atrial natriuretic peptide (ANP) and ir-brain natriuretic peptide (BNP). Tissue concentration of ir-CNP in brain was the highest of the three natriuretic peptides at about 0.79 pmol/g wet wt. CNP was present in medulla-pons in high concentration, with a significant concentration detected in cerebellum. In contrast, ir-CNP was not detected in peripheral tissue, including heart, in a significant concentration. These data demonstrated sharp contrasts in the distribution of the three natriuretic peptides, suggesting that CNP is a natriuretic peptide functioning in the central nervous system.

Characterization of immunoreactive human C-type natriuretic peptide in brain and heart.

Amino acid sequence of human C-type natriuretic peptide (CNP) has recently been deduced to be identical to those of porcine and rat CNPs in the bioactive unit of C-terminal 22 residues (CNP-22) (1). Thus, tissue concentrations and molecular forms of immunoreactive (ir-) CNP in human brain and heart were determined or characterized using a radioimmunoassay established for porcine CNP. In human brain (hypothalamus and medullapons), ir-CNP was detected at a concentration of 1.04 pmol/g, being about 25 times or 70 times higher than ir-atrial (A-type) natriuretic peptide (ANP) or ir-brain (B-type) natriuretic peptide (BNP). CNP was present mainly as CNP-53, with CNP-22 as well as 13K CNP (presumed to be pro-CNP) as minor components. In heart, 1 approximately 5 pmol/g of ir-CNP was detected in both atrium and ventricle, but this ir-CNP was shown to be derived from crossreactivity of ANP. These results demonstrated that human CNP functions exclusively in the central nervous system in contrast to ANP and BNP which mainly function in the circulation system.

Novel natriuretic peptide, CNP, potently stimulates cyclic GMP production in rat cultured vascular smooth muscle cells.

The newly identified peptide C-type natriuretic peptide (CNP) caused only a slight elevation of cGMP in rat renal glomeruli. In contrast, CNP potently increased cGMP levels in cultured rat vascular smooth muscle cells (VSMC) and stimulated guanylate cyclase activity in the particulate fraction of the cells. The extent of maximum activation of the enzyme induced by CNP was 4-fold higher than that by human atrial natriuretic peptide (alpha-hANP) while CNP was 4- and 16-fold weaker than alpha-hANP in binding affinity for the putative receptors on VSMC and vasorelaxant activity for rat aorta, respectively. These results indicate that CNP is a potent stimulator of cGMP formation in VSMC but not in glomeruli and pharmacological feature of CNP is distinct from that of ANP.

Characterization of immunoreactive brain natriuretic peptide in human cardiac atrium.

Based on cDNA sequence data for human brain natriuretic peptide (BNP) precursor (1), a radioimmunoassay (RIA) system highly specific to human BNP (hBNP) was developed and used to characterize immunoreactive (ir-) hBNP in cardiac atrium. Gel filtration of ir-hBNP in atrium indicated that ir-hBNP was mainly comprised of two molecular forms of 13-15K and 4K. In reverse phase high performance liquid chromatography (HPLC), the low molecular weight (MW) ir-hBNP emerged as a single peak at an elution time identical to that of synthetic hBNP-32. The high MW ir-hBNP was also eluted as a single peak. On the other hand, tissue concentrations of ir-hBNP in cardiac atria were found to be 9.98-593.22 pmol/g in 13 specimens, being about 1/150 the concentration of ir-human atrial natriuretic peptide (hANP). These results demonstrate that hBNP is present as a peptide in human heart, suggesting that hBNP is secreted from heart and functions together with hANP as a hormone.

Isolation and identification of C-type natriuretic peptide in chicken brain

C-type natriuretic peptide (CNP) has recently been identified in porcine brain as a third member of the mammalian natriuretic peptide family (1). Using a radioimmunoassay system for porcine CNP, we found a significant concentration of immunoreactive (ir-) CNP in chicken brain, from which a new peptide was isolated. By microsequence analysis, the amino acid sequence of the peptide was determined to be Gly-Leu-Ser-Arg-Ser-Cys-Phe- Gly-Val-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys. Based on its high homology to porcine CNP, the peptide was designated chicken C-type natriuretic peptide. Chicken CNP also elicits pharmacological effects highly similar to porcine CNP, suggesting that CNP functions as a neuropeptide in the chicken central nervous system.

Gene and precursor structure of porcine C-type natriuretic peptide

Recently we isolated from porcine brain two related peptides, a 22-residue peptide (CNP-22) and its N-terminally elongated peptide (CNP-53; 53-residue), which belong to the third type of mammalian natriuretic peptide designated C-type natriuretic peptide family (CNP) (1,2). To elucidate the structure of their precursor form, we have now isolated the gene for this porcine CNP and prepared its cDNA from COS-1 cells transfected with the gene. Nucleotide sequence analyses have revealed that the gene consists of a least two exons and an intron and encodes the 126-residue CNP precursor (porcine prepro-CNP), in which a putative signal peptide and the CNP-53 sequence are located at the N- and C-terminus, respectively. The C-terminal cysteine codon of CNP-53 is directly followed by a termination codon, indicating that the C-terminus of porcine CNP is generated per se.

Cloning and sequence analysis of complementary DNA encoding a precursor for chicken natriuretic peptide

Chicken α‐natriuretic peptide (α‐chNP) has been identified in chicken heart, which showed higher homology to brain natriuretic peptide (BNP) than to atrial natriuretic peptide (ANP) [1]. Complementary DNA (cDNA) clone encoding a chNP precursor (pre‐chNP) precursor (pre‐chNP) was isolated from cardiac cDNA library and sequenced. Pre‐chNP was 140‐residue signal peptide at the N‐terminus and α‐chNP at the C‐terminus, and did not exhibit high homology to poreine BNP except for the C‐terminal region. However, a characteristic AT‐rich nucleotide sequence commonly found in mammalian BNPs was also present in the 3′‐untranslated region. Thus, chNP is concluded to be classified into the BNP‐type