Naoto Minamino

Brain natriuretic peptide is a novel cardiac hormone

Using a radioimmunoassay for brain natriuretic peptide (BNP), we have measured levels of BNP-like immunoreactivity (-LI) in extract of the porcine heart, in perfusate from the isolated porcine heart and in porcine plasma. BNP-LI was detected in the extract of the atrium, though no detectable amount of BNP-LI (more than 1 ng/g) was present in the ventricle. The BNP-LI level in the porcine atrium was 148.7 +/- 23.3 ng/g. BNP-LI was also detected in the perfusate from the heart. Basal secretory rate of BNP was 3.18 +/- 0.76 ng/min. Moreover, BNP-LI was detected in porcine plasma at the concentration of 4.2 +/- 1.3 pg/ml. Gel filtration studies showed that BNP is present in the atrium as a large molecule and is secreted into the circulation as a small molecule. The percentage of BNP-LI to atrial natriuretic peptide (ANP)-LI was almost the same among the extract, the perfusate and the plasma (2-3 percent). These results indicate that BNP is synthesized in and is secreted into the circulation from the heat in a similar fashion as ANP.

Isolation and sequence determination of rat cardiac natriuretic peptide

We have isolated a cardiac natriuretic peptide of 5K daltons from the rat atrium and determined its amino acid sequence. The 5K cardiac natriuretic peptide was elucidated to be a 45-amino acid peptide with the sequence of S-Q-D-S-A-F-R-I-Q-E-R-L-R-N-S-K-M-A-H-S-S-S-C-F-G-Q-K-I-D-R-I-G-A-V-S-R- L-G-C-D - G-L-R-L-F by sequencing the native peptide and its lysyl endopeptidase digests. The sequence of this peptide was identical to the amino acid sequence [51-95] of the rat brain natriuretic peptide (BNP) precursor deduced from the cDNA sequence. The 5K cardiac natriuretic peptide, or BNP[51-95], was identified as the major storage and secretory form derived from the BNP precursor in the rat heart.

Radioimmunoassay for brain natriuretic peptide (BNP) detection of BNP in canine brain

We established a highly sensitive and specific radioimmunoassay (RIA) for BNP. Our RIA detected BNP-like immunoreactivity (-LI) in the porcine and canine brains but did not detect BNP-LI in the human, monkey or rat brain. The widespread distribution of BNP-LI was demonstrated both in the porcine and canine brains, with the highest concentration in the medulla oblongata. In contrast, the highest concentration of ANP-LI determined simultaneously was in the midbrain and the olfactory bulb. High performance-gel permeation chromatography coupled with RIA revealed that the major component of BNP-LI was eluted at the position of synthetic BNP with a small molecular weight (3K). These results indicate that the RIA for BNP serves as a useful tool to investigate physiological roles of BNP.

Global metabolomic analysis of heart tissue in a hamster model for dilated cardiomyopathy

Dilated cardiomyopathy (DCM), a common cause of heart failure, is characterized by cardiac dilation and reduced left ventricular ejection fraction, but the underlying mechanisms remain unclear. To investigate the mechanistic basis, we performed global metabolomic analysis of myocardial tissues from the left ventricles of J2N-k cardiomyopathic hamsters. This model exhibits symptoms similar to those of human DCM, owing to the deletion of the δ-sarcoglycan gene. Charged and lipid metabolites were measured by capillary electrophoresis mass spectrometry (MS) and liquid chromatography MS(/MS), respectively, and J2N-k hamsters were compared with J2N-n healthy controls at 4 (presymptomatic phase) and 16weeks (symptomatic) of age. Disturbances in membrane phospholipid homeostasis were initiated during the presymptomatic phase. Significantly different levels of charged metabolites, occurring mainly in the symptomatic phase, were mapped to primary metabolic pathways. Reduced levels of metabolites in glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, together with large decreases in major triacylglycerol levels, suggested that decreased energy production leads to cardiac contractile dysfunction in the symptomatic phase. A mild reduction in glutathione and a compensatory increase in ophthalmate levels suggest increased oxidative stress in diseased tissues, which was confirmed by histochemical staining. Increased levels of 4 eicosanoids, including prostaglandin (PG) E2 and 6-keto-PGF1α, in the symptomatic phase suggested activation of the protective response pathways. These results provide mechanistic insights into DCM pathogenesis and may help identify new targets for therapeutic intervention and diagnosis.

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554–577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

Peptidomics-based discovery of an antimicrobial peptide derived from insulin-like growth factor-binding protein 5.

Antimicrobial peptides (AMPs) are effector molecules that are able to kill or inactivate microbial pathogens. However, most AMPs harbor multiple basic amino acids that hamper current proteomic identification. In our peptidomic survey of endogenous peptides, we identified a novel intramolecular disulfide-linked 22-residue amidated peptide. This peptide, designated AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5), showed antimicrobial activity against six of the eight microorganisms tested at concentrations comparable to or lower than those for well-characterized AMPs cathelicidin and β-defensin-2. AMP-IBP5 is identical at the amino acid level between human, mouse, rat, pig, and cow. Natural occurrence of this peptide as the originally isolated form was demonstrated in the rat brain and intestine, using mass spectrometric characterization of major immunoreactivity. The peptide is flanked N-terminally by a single arginine and C-terminally by a common amidation signal, indicating that insulin-like growth factor-binding protein 5 (IGFBP-5) undergoes specific cleavage by a defined set of processing proteases. Furthermore, the intramolecular linkage C199-C210 reveals itself as a correct disulfide pairing in the precursor protein, the finding not inferred from closely related family members IGFBP-4 and -6. In principle, neither conventional proteomics nor bioinformatics would achieve the identification of this AMP. Our study exemplifies the impact of peptidomics to study naturally occurring peptides.

Proteomic Analysis of Proteins Eliminated by Low‐Density Lipoprotein Apheresis

Low‐density lipoprotein apheresis (LDL‐A) treatment has been shown to decrease serum LDL cholesterol levels and prevent cardiovascular events in homozygous patients with familial hypercholesterolemia. Recently, LDL‐A treatment has been suggested to have beneficial effects beyond the removal of LDL particles. In this study, to clarify the preventive effects of LDL‐A treatment on atherosclerosis, the waste fluid from the adsorption columns was analyzed. The waste fluid of LDL adsorption columns was analyzed by two‐dimensional electrophoresis followed by mass spectrometry. Serum concentrations of the newly identified proteins before and after LDL‐A treatment were measured by enzyme‐linked immunosorbent assay. We identified 48 kinds of proteins in the waste fluid of LDL adsorption columns, including coagulation factors, thrombogenic factors, complement factors, inflammatory factors and adhesion molecules. In addition to the proteins that were reported to be removed by LDL‐A treatment, we newly identified several proteins that have some significant roles in the development of atherosclerosis, including vitronectin and apolipoprotein C‐III (Apo C‐III). The serum levels of vitronectin and Apo C‐III decreased by 82.4% and 54.8%, respectively, after a single LDL‐A treatment. While Apo C‐III was removed with very low‐density lipoprotein (VLDL) and LDL, vitronectin was removed without association with lipoproteins. The removal of proteins observed in the waste fluid has a certain impact on their serum levels, and this may be related to the efficacy of LDL‐A treatment. Proteomic analysis of the waste fluid of LDL adsorption columns may provide a rational means of assessing the effects of LDL‐A treatment.

Hexarelin Treatment in Male Ghrelin Knockout Mice after Myocardial Infarction

Both ghrelin and the synthetic analog hexarelin are reported to possess cardioprotective actions that are mainly exerted through different receptors. However, their effects on acute myocardial infarction have not been compared in vivo. This study aimed to clarify whether hexarelin treatment can compensate for ghrelin deficiency in ghrelin-knockout mice and to compare the effects of hexarelin (400 nmol/kg/d, sc) and equimolar ghrelin treatment after myocardial infarction. Myocardial infarction was produced by left coronary artery ligation in male ghrelin-knockout mice, which then received ghrelin, hexarelin, or vehicle treatment for 2 weeks. The mortality within 2 weeks was significantly lower in the hexarelin group (6.7%) and ghrelin group (14.3%) than in the vehicle group (50%) (P < .05). A comparison of cardiac function 2 weeks after infarction showed that in the ghrelin and hexarelin treatment groups, cardiac output was greater, whereas systolic function, represented by ejection fraction, and diastolic function, represented by dP/dt min (peak rate of pressure decline), were significantly superior compared with the vehicle group (P < .05). Hexarelin treatment was more effective than ghrelin treatment, as indicated by the ejection fraction, dP/dt max (peak rate of pressure rise), and dP/dt min. Telemetry recording and heart rate variability analysis demonstrated that sympathetic nervous activity was clearly suppressed in the hexarelin and ghrelin groups relative to the vehicle group. Our data demonstrated that hexarelin treatment can result in better heart function than ghrelin treatment 2 weeks after myocardial infarction in ghrelin-knockout mice, although both hormones have similar effects on heart rate variability and mortality.

Molecular Characterization and Biological Function of Neuroendocrine Regulatory Peptide-3 in the Rat

Neuroendocrine regulatory peptide (NERP)-3, derived from the neurosecretory protein VGF (non-aconymic), is a new biologically active peptide identified through peptidomic analysis of the peptides secreted by an endocrine cell line. Using a specific antibody recognizing the C-terminal region of NERP-3, immunoreactive (ir)-NERP-3 was identified in acid extracts of rat brain and gut as a 30-residue NERP-3 with N-terminal pyroglutamylation. Assessed by radioimmunoassay, ir-NERP-3 was more abundant in the brain, including the posterior pituitary (PP), than in the gut. Immunohistochemistry demonstrated that ir-NERP-3 was significantly increased in the suprachiasmatic nucleus, the magnocellular division of the paraventricular nucleus, and the external layer of the median eminence, but not in the supraoptic nucleus, after dehydration. The immunoreactivity was, however, markedly decreased in all of these locations after chronic salt loading. Intracerebroventricular administration of NERP-3 in conscious rats induced Fos expression in a subset of arginine vasopressin (AVP)-containing neurons in the supraoptic nucleus and the magnocellular division of the paraventricular nucleus. On in vitro isolated rat PP preparations, NERP-3 caused a significant AVP release in a dose-related manner, suggesting that NERP-3 in the PP could be an autocrine activator of AVP release. Taken together, the present results suggest that NERP-3 in the hypothalamo-neurohypophyseal system may be involved in the regulation of body fluid balance.

Endothelium-Derived C-Type Natriuretic Peptide Contributes to Blood Pressure Regulation by Maintaining Endothelial Integrity.

We previously reported the secretion of C-type natriuretic peptide (CNP) from vascular endothelial cells and proposed the existence of a vascular natriuretic peptide system composed of endothelial CNP and smooth muscle guanylyl cyclase-B (GC-B), the CNP receptor, and involved in the regulation of vascular tone, remodeling, and regeneration. In this study, we assessed the functional significance of this system in the regulation of blood pressure in vivo using vascular endothelial cell–specific CNP knockout and vascular smooth muscle cell–specific GC-B knockout mice. These mice showed neither the skeletal abnormality nor the early mortality observed in systemic CNP or GC-B knockout mice. Endothelial cell–specific CNP knockout mice exhibited significantly increased blood pressures and an enhanced acute hypertensive response to nitric oxide synthetase inhibition. Acetylcholine-induced, endothelium-dependent vasorelaxation was impaired in rings of mesenteric artery isolated from endothelial cell–specific CNP knockout mice. In addition, endothelin-1 gene expression was enhanced in pulmonary vascular endothelial cells from endothelial cell–specific CNP knockout mice, which also showed significantly higher plasma endothelin-1 concentrations and a greater reduction in blood pressure in response to an endothelin receptor antagonist than their control littermates. By contrast, vascular smooth muscle cell–specific GC-B knockout mice exhibited blood pressures similar to control mice, and acetylcholine-induced vasorelaxation was preserved in their isolated mesenteric arteries. Nonetheless, CNP-induced acute vasorelaxation was nearly completely abolished in mesenteric arteries from vascular smooth muscle cell–specific GC-B knockout mice. These results demonstrate that endothelium-derived CNP contributes to the chronic regulation of vascular tone and systemic blood pressure by maintaining endothelial function independently of vascular smooth muscle GC-B.