Takeshi Katafuchi

Novel fish-derived adrenomedullin in mammals: structure and possible function.

Adrenomedullin (AM) has been recognized as a member of the calcitonin (CT)/CT gene-related peptide (CGRP) family. However, an independent AM family consisting of five paralogous peptides exists in teleost fish. Among them, the peptide named AM1 is an ortholog of mammalian AM as determined by the linkage analysis of orthologous genes and the presence of proAM N-terminal 20 peptide (PAMP)-like sequence in the prosegment. Since the peptides named AM2 and 3 are distinct from other members with respect to the precursor sequence, tissue distribution of the transcripts, and exon-intron organization, we searched for their mammalian orthologs from genome databases, which resulted in an identification of AM2 in human, rat, and mouse. AM2 was expressed abundantly in the submaxillary gland, kidney, and some vascular and digestive tissues of mice. AM2 injected in vivo induced potent cardiovascular and renal effects in mice. In the heart and kidney of mice, AM2 was localized in endothelial cells of the coronary vessels and in glomeruli and vasa recta, respectively. AM2 increased cAMP accumulation in cells expressing human CT receptor-like receptor (CRLR) and one of receptor activity-modifying proteins (RAMPs), but it was no more potent than CGRP and AM. AM2 was also less potent than CT in cells expressing CT receptor and RAMP. There remains a possibility that a new AM2-specific receptor or an additional RAMP that enables CRLR to be an AM2-specific receptor, exists in mammals.

Regulation of adrenomedullin secretion from cultured cells

Characterization of immunoreactive adrenomedullin (AM) secreted from cultured human vascular smooth muscle cells and 7 other cells indicates that AM is synthesized and secreted from all cultured cells we surveyed. The secretion rate of AM measured ranges from 0.001-6.83 fmol/10(5) cells/h, and endothelial cells, vascular smooth muscle cells and fibroblasts generally secrete AM at high rates. Based on the results of regulation of AM secretion from vascular wall cells, fibroblasts, macrophages and other cells measured in this and previous studies, AM secretion is found to be generally stimulated by inflammatory cytokines, lipopolysaccharide (LPS) and hormones. Especially, vascular smooth muscle cells and fibroblasts elicited uniform and strong stimulatory responses of AM secretion to tumor necrosis factor (TNF), interleukin-1 (IL-1), LPS and glucocorticoid, but endothelial cells did not elicit such prominent responses. AM secretion of monocyte-macrophage was mainly regulated by the degree of differentiation into macrophage and activation by LPS and inflammatory cytokines including interferon-gamma. The other examined cells showed weaker responses to LPS and IL-1. Although cultured cells may have been transformed as compared with those in the tissue, these data indicate that AM is widely synthesized and secreted from most of the cells in the body and functions as a local factor regulating inflammation and related reactions in addition to as a potent vasodilator. The responses of AM secretion to LPS and inflammatory cytokines suggest that fibroblasts, vascular smooth muscle cells and macrophage are the major sources of AM in the septic shock.

Calcitonin Receptor-stimulating Peptide, a New Member of the Calcitonin Gene-related Peptide Family ITS ISOLATION FROM PORCINE BRAIN, STRUCTURE, TISSUE DISTRIBUTION, AND BIOLOGICAL ACTIVITY

We isolated a novel biologically active peptide, designated calcitonin receptor-stimulating peptide (CRSP), from the acid extract of the porcine brain by monitoring cAMP production in the porcine kidney cell line LLC-PK1. Determination of the amino acid sequence and cDNA analysis encoding a CRSP precursor showed that this peptide has ∼60% identity in the amino acid sequence with human calcitonin gene-related peptide type-α (αCGRP), type-β (βCGRP), and porcine CGRP. Northern blot analysis and radioimmunoassay demonstrated that CRSP is expressed mainly in the thyroid gland and the central nervous system, in which the calcitonin receptor was abundantly expressed. Synthetic CRSP elicited a potent stimulatory effect on the cAMP production in LLC-PK1 cells. Although it shows significant sequence similarity with CGRPs, this peptide did not elicit cAMP elevation in cells that endogenously expressed a CGRP receptor or an adrenomedullin receptor or were transfected with either of these recombinant receptors. Administration of CRSP into anesthetized rats did not alter the blood pressure but induced a transient decrease in the plasma calcium concentration. In fact, this peptide potently increased the intracellular cAMP concentration in COS-7 cells that expressed the recombinant calcitonin receptor. These unique properties indicate that CRSP is not a porcine counterpart of βCGRP and probably elicits its biological effects via the calcitonin receptor.

Adrenomedullin suppresses interleukin-1β-induced tumor necrosis factor-α production in Swiss 3T3 cells

We demonstrated that adrenomedullin (AM) inhibited interleukin‐1β‐induced tumor necrosis factor‐α (TNF‐α) secretion and gene transcription in Swiss 3T3 fibroblasts maximally to 23% and 18% of control, while the other peptides elevating intracellular cAMP levels elicited much weaker effects. AM rapidly reduced the gene transcript level of TNF‐α, inducing a maximal effect within 1 h. The inhibitory effect of AM was restored with an AM receptor antagonist as well as a cAMP‐dependent protein kinase inhibitor. These findings indicate that AM is a potent and quick suppressor of TNF‐α production in Swiss 3T3 cells acting through the cAMP protein kinase A pathway. As TNF‐α is a major inflammatory cytokine and stimulates AM production in fibroblasts, AM is deduced to be an autocrine or paracrine factor suppressing inflammation through the inhibition of TNF‐α production.

Cardiac fibroblasts are major production and target cells of adrenomedullin in the heart in vitro

OBJECTIVE Adrenomedullin (AM) is a potent vasodilator peptide. Plasma AM concentration is increased in patients with various heart diseases, and both myocytes (MCs) and non-myocytes (NMCs) secrete AM and express its receptors. These facts suggest that cardiac cells possess an autocrine/paracrine capability mediated by AM. METHODS MCs and NMCs were prepared from cardiac ventricles of neonatal rats. AM and endothelin-1 concentrations were measured by radioimmunoassays, and interleukin-6 level by a specific bioassay. Total nitrite/nitrate contents were measured with a fluorescence assay kit. RESULTS A basal secretion rate of AM from NMCs was 2.8-fold higher than that from MCs. Interleukin-1beta, tumor necrosis factor-alpha and lipopolysaccharide stimulated AM secretion from NMCs but not from MCs. AM stimulated interleukin-6 production in the presence of these cytokines or lipopolysaccharide, which was more prominent in NMCs. In the presence of interleukin-1beta, AM augmented nitric oxide synthesis 2.7-fold in NMCs, but slightly in MCs. NMCs secreted endothelin-1 at a rate nine times higher than MCs, and AM inhibited endothelin-1 secretion from NMCs. CONCLUSION This in vitro study suggests that AM in the heart is mainly produced in NMCs and exerts its effects through NMCs, especially under inflammatory conditions.

Role of Natriuretic Peptide Receptor Type C in Dahl Salt-Sensitive Hypertensive Rats

The natriuretic peptide system is suggested to be involved in the pathogenesis of salt-sensitive hypertension; a recent report indicated that disruption of the atrial natriuretic peptide precursor gene caused salt-sensitive hypertension. However, natriuretic peptide receptor (NPR)-A knockout mice did not show enhanced salt sensitivity of blood pressure. The aim of the present study was to investigate the role of NPR-C, the other receptor for atrial natriuretic peptide, in increased salt sensitivity of blood pressure. Dahl salt-sensitive (DS) and salt-resistant (DR) rats were placed on a 0.3% or 8% NaCl diet for 4 weeks. Blood pressure was elevated by salt loading only in DS rats. RNase protection assay demonstrated that NPR-C transcript level in the kidney was reduced by chronic salt loading in both DR and DS rats, whereas expression of NPR-A and NPR-B was not altered. The reduction of NPR-C mRNA in response to salt loading was enhanced in DS compared with DR rats. In situ hybridization indicated that the salt-induced NPR-C change was attributed mainly to suppressed expression of NPR-C in the podocytes. NPR-C gene expression was regulated by salt loading in a tissue-specific manner; the marked decrease in NPR-C mRNA by salt loading was seen only in the kidney. These data suggest that the exaggerated salt-induced reduction of NPR-C in the kidney of DS rats may play an important role in the pathogenesis of salt hypertension in this animal, possibly related to impaired renal sodium excretion.

Structure and biological properties of three calcitonin receptor-stimulating peptides, novel members of the calcitonin gene-related peptide family

In this review, we describe the structure and biological properties of calcitonin receptor-stimulating peptide-1 (CRSP-1), CRSP-2 and CRSP-3, the novel members of the CGRP family. CRSP-1, which has been identified in the pig, cow, dog, and horse, is a specific ligand for the calcitonin (CT) receptor, and porcine CRSP-1 elicits a 100-fold greater effect on a recombinant porcine CT receptor than porcine CT, although this peptide has high structural similarity with CGRP. CRSP-1 is expressed and synthesized mainly in the central nervous system (CNS), pituitary and thyroid gland. In an in vivo experiment, bolus administration of CRSP-1 into rats reduced the plasma calcium concentration, but did not alter blood pressure, indicating its action as a CT receptor agonist in the peripheral circulation. In the CNS, CRSP-1 is also deduced to be an endogenous agonist for the CT receptor. CRSP-2 has been identified in the pig and dog, and CRSP-3 has been identified only in the pig. They are expressed and synthesized mainly in the CNS and thyroid gland. However, their endogenous molecular forms, receptors, and biological activity remain unidentified.

Identification of second and third calcitonin receptor-stimulating peptides in porcine brain

We identified two cDNAs encoding new calcitonin receptor-stimulating peptides (CRSPs) in porcine hypothalamus cDNA library by cross-hybridization with the CRSP cDNA, and designated the second and third peptides as CRSP-2 and CRSP-3. The putative amino acid sequences of prepro-CRSP-2 and prepro-CRSP-3 showed higher identity with that of prepro-CRSP-1 than that of prepro-calcitonin gene-related peptide (CGRP), respectively, and these three CRSPs are considered to form a new family in the CGRP superfamily. RT-PCR analysis demonstrated that both CRSP-2 and CRSP-3 gene transcripts were expressed mainly in the central nervous system and thyroid gland. Synthetic CRSP-2 and CRSP-3 stimulated cAMP production very weakly in LLC-PK(1) cells compared with CRSP and calcitonin (CT). Furthermore, CRSP-2 and CRSP-3 did not elicit a cAMP elevation at all in the COS-7 cells expressing CT receptor or CT-like receptor with or without one of receptor activity-modifying proteins. These results suggest the presence of still unidentified action mechanisms and functions of the peptides in the CGRP superfamily.

Calcitonin receptor-stimulating peptide: Its evolutionary and functional relationship with calcitonin/calcitonin gene-related peptide based on gene structure☆

This review focuses on the evolutionary and functional relationship of calcitonin receptor-stimulating peptide (CRSP) with calcitonin (CT)/calcitonin gene-related peptide (CGRP) in mammals. CRSP shows high sequence identity with CGRP, but distinct biological properties. CRSP genes (CRSPs) have been identified in mammals such as pigs and dogs of the Laurasiatheria, but not in primates and rodents of the Euarchontoglires or in non-placental mammals. CRSPs have genomic organizations highly similar to those of CT/CGRP genes (CT/CGRPs), which are located along with CGRPs in a locus between CYP2R1 and INSC, while the other members of the CGRP superfamily, adrenomedullin and amylin, show genomic organizations and locations distinct from CT, CGRP, and CRSP. Thus, we categorized these three peptides into the CT/CGRP/CRSP family. Non-placental mammals having one and placental mammals having multiple CT/CGRP/CRSP family genes suggests that multiplicity of CT/CGRP started at an early stage of mammalian evolution. In the placental mammals, Laurasiatheria generally possesses multiple CRSPs and only one CT/CGRP, while Euarchontoglires possesses CT/CGRP and CGRPbeta but no CRSP, indicating an increase in the diversity and multiplicity of this family of genes in mammalian evolution. Phylogenetic analysis suggests that some CRSPs have been generated very recently in mammalian evolution. Taken together, the increase in the number and complexity of the CT/CGRP/CRSP family genes may have due to evolutionary pressure to facilitate adaptation during mammalian evolution. In this regard, it is important to elucidate the physiological roles of CT, CGRP and CRSP from the viewpoint of the CT/CGRP/CRSP family even in Euarchontoglires.

Human adrenal tumor cell line SW-13 contains a natriuretic peptide receptor system that responds preferentially to ANP among various natriuretic peptides

A new type of ANP receptor system which clearly distinguishes natriuretic peptides A and B (ANP and BNP) has been identified in the human adrenal tumor cell line SW-13 and characterized. SW-13 cells responded to nanomolar concentrations of ANP with large increases in cGMP levels but in the case of BNP, much higher concentrations were required to produce the same extent of response. This property is unique since the 140-kDa ANP receptors so far characterized do not discriminate between ANP and BNP. For comparison, various natriuretic peptide receptors were also re-characterized using the recently identified CNP.