Immunoreactive Proadrenomedullin N-Terminal 20 Peptide in Human Tissue, Plasma and Urine

doi:10.1006/bbrc.1994.2039

Biochemical and Biophysical Research Communications

Volume 202, Issue 2, 29 July 1994, Pages 1081-1087

https://doi.org/10.1006/bbrc.1994.2039 Get rights and content

Abstract

Proadrenomedullin N-terminal 20 peptide (PAMP) is a candidate for a novel biologically active peptide processed from proadrenomedullin. This study clearly demonstrates the existence of PAMP in vivo that had been deduced from analysis of cDNA. To identify PAMP in vivo, we established a radioimmunoassay for PAMP and characterized immunoreactivities in human tissue, plasma and urine. Half maximal inhibition of the assay was observed at 10 fmol/tube. A high concentration of immunoreactive PAMP was found in adrenal medulla (18.4 ± 8.95 fmol/mg, mean ± S.D.) and pheochromocytoma tissue (12.3 ± 9.82 fmol/mg) where the concentrations are comparable to that of adrenomedullin. As determined by three different kinds of chromatography, most of the immunoreactive peptide in pheochromocytoma was eluted at a position exactly identical to that of synthetic PAMP. Further, considerable concentration of immunoreactive PAMP was found in human plasma and urine. The present data indicate that PAMP as well as adrenomedullin is processed from an adrenomedullin precursor.

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Cited by (103)

Proadrenomedullin N-terminal 20 peptide (PAMP) and its C-terminal 12-residue peptide, PAMP(9–20): Cell selectivity and antimicrobial mechanism
2020, Biochemical and Biophysical Research Communications
Citation Excerpt :
Although AM and PAMP both have hypotensive effects in the cardiovascular system, they also exert diverse and distinct effects on endocrine physiology, innate immunity, cytoskeletal biology, and receptor signaling pathways [2]. Similar to AM, PAMP is potently expressed and secreted by adrenal chromaffin cells but is also widely found throughout the body, including the plasma and urine [3]. Kuwasako et al. identified the C-terminal 12-residues of PAMP [PAMP(9–20)] in porcine adrenal medulla as a major endogenous active peptide of the AM precursor and demonstrated that its hypotensive activity is comparable to that of PAMP [4].
Proadrenomedullin N-terminal 20 peptide (PAMP) is a regulatory peptide that is found in various cell types. It is involved in many biological activities and is rich in basic and hydrophobic amino acids, a common feature of antimicrobial peptides (AMPs). In this study, the cell selectivity and antimicrobial mechanism of PAMP and its C-terminal peptide, PAMP(9–20), were investigated. PAMP and PAMP(9–20) displayed potent antimicrobial activity (minimum inhibitory concentration: 4–32 μM) against standard bacterial strains, but showed no hemolytic activity even at the highest tested concentration of 256 μM. PAMP(9–20) showed 2- to 4-fold increase in antimicrobial activity against gram-negative bacteria compared to PAMP. Cytoplasmic membrane depolarization, leakage of calcein dye from membrane mimic liposomes, SYTOX Green uptake, membrane permeabilization, and flow cytometry studies indicated that the major target of PAMP and PAMP(9–20) is not the microbial cell membrane. Interestingly, laser-scanning confocal microscopy demonstrated that FITC-labeled PAMP and PAMP(9–20) enter the cytoplasm of Escherichia coli similar to buforin-2, and gel retardation assay indicated that PAMP and PAMP(9–20) effectively bind to bacterial DNA. These results suggest that the intracellular target mechanism is responsible for the antimicrobial action of PAMP and PAMP(9–20). Collectively, PAMP and PAMP(9–20) could be considered promising candidates for the development of new antimicrobial agents.
Genetic loss of proadrenomedullin N-terminal 20 peptide (PAMP) in mice is compatible with survival
2019, Peptides
Citation Excerpt :
Far fewer studies have addressed the role of PAMP in physiology and pathophysiology. Like AM, PAMP is potently expressed and secreted by adrenal chromaffin cells but is also found widely throughout the body, including in plasma and urine [5]. PAMP is hydrolyzed by neprilysin, and like other neprilysin targets, exerts a hypotensive effect [6].
Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are small peptides derived from a common precursor, pre-proadrenomedullin. Although AM and PAMP share hypotensive effects in the cardiovascular system, the peptides also exert diverse and distinct effects on endocrine physiology, innate immunity, cytoskeletal biology and receptor signaling pathways. Tremendous knowledge has been gleaned from the study of several genetic animal models of AM deletion or overexpression, some of which also simultaneously delete the coding region for PAMP peptide. However, deletion of PAMP without concurrent deletion of AM in an animal model is not currently available for the study of PAMP function. Here, we present the generation of Adm^Δ^PAMP/^Δ^PAMP and Adm^Δ^PAMP/− mice, which lack the coding sequence for PAMP while preserving the coding sequence for AM. Adm^Δ^PAMP/^Δ^PAMP mice survive to adulthood without any obvious abnormalities and are fertile, though Adm^Δ^PAMP/− females have small litters. Interestingly, these animals express lower levels of Adm mRNA and AM peptide than wild type animals, but these levels are still compatible with survival. Importantly, despite reduced levels, the spatiotemporal expression of AM peptide within the hearts of Adm^Δ^PAMP/− mice remains similar to wild type animals. Adm^Δ^PAMP/^Δ^PAMP mice are now a publicly available tool for future investigations of PAMP function.
Adrenomedullin is a novel adipokine: Adrenomedullin in adipocytes and adipose tissues
2007, Peptides
Adrenomedullin (AM) is a multifunctional regulatory peptide that is produced and secreted by various types of cells. The production and the secretion of AM have been demonstrated in cultured adipocytes and adipose tissues. Inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and lipopolysaccharide are strong stimulators for AM expression in adipocytes. Furthermore, AM expression in the adipose tissue is increased in obesity, and plasma concentrations of AM are increased in obese subjects. One possible (patho)physiological role of AM secreted by adipose tissue may be actions against complications of the metabolic syndrome characterized by obesity, type 2 diabetic mellitus and hypertension, via its antioxidant and potent vasodilator effects. These findings indicate that AM is a new member of the adipokine family.
Peripheral adrenomedullin inhibits gastric emptying through CGRP<inf>8-37</inf>-sensitive receptors and prostaglandins pathways in rats
2006, Peptides
The effects of intravenous (iv) adrenomedullin (AM) on gastric emptying were investigated in conscious rats. AM induced a maximal 50% inhibition of gastric emptying at a dose of 1.2 nmol/kg. AM was about two-fold less potent than α-calcitonin gene-related peptide (α-CGRP), which induced a similar 50% maximal inhibition of gastric emptying at 0.6 nmol/kg. Delayed gastric emptying induced by i.v. AM and α-CGRP was prevented by peripheral injection of the selective CGRP₁ antagonist, CGRP_8–37, and by pretreatment with indomethacin, while not altered by blockade of the sympathetic nervous system with propranolol. These data indicate that peripheral AM inhibits gastric emptying through the interaction with CGRP_8–37-sensitive receptors, likely CGRP₁ receptors, and the recruitment of prostaglandin-dependent mechanisms.
Expression of the adrenomedullin gene in adipose tissue
2005, Regulatory Peptides
Adrenomedullin (AM) is a potent vasodilating peptide originally isolated from human pheochromocytoma cells. This report concerns the expression and secretion of AM from adipose tissue. Northern blot analysis demonstrated marked expression of AM mRNA in mouse adipose tissue. Expression levels in adipose tissues were 2.5–3.2 times higher than in the kidney. AM mRNA level in mature adipocytes was 7.3 times higher than in the stroma–vascular fraction of adipose tissue. In mature adipocyte culture, time-dependent increase of AM peptide concentration in the culture medium was detected. AM expression was also detected in human subcutaneous adipose tissue. Adipose AM expression significantly increased in obesity mouse model, high-fat diet fed mice and ob/ob mice. These results suggest that adipose tissue, especially mature adipocytes, is major source of AM in the body, and that adipocyte-derived AM plays a pathophysiological role in obesity.
Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides
2005, Biochemical and Biophysical Research Communications
Citation Excerpt :
Our data showed that MrgX2 responded to PAMP-12 with an EC50 value of 20–50 nM (Figs. 1 and 2). Under physiological conditions, immunoreactive-PAMP was reported to be present at 222.3 ± 27.2 fmol/mg in the wet tissue of the porcine adrenal medulla [4] and 18.4 fmol/mg in the wet tissue of the human adrenal medulla [6], which indicates that a 10–100 nM order of PAMP would exist in the tissue. The expression of MrgX2 in the adrenal chromaffin cells (Fig. 4B) suggests that PAMP would act in the adrenal medulla locally and that MrgX2 would be a previously proposed receptor that regulates catecholamine secretion and production from the adrenal medulla by PAMP in an autocrine or paracrine fashion under physiological conditions.
Proadrenomedullin N-terminal 20 peptide (PAMP[1–20]/PAMP-20) and its truncated analog, PAMP[9–20]/PAMP-12, are endogenous peptides that elicit hypotension through inhibiting catecholamine secretion from sympathetic nerve endings and adrenal chromaffin cells. Although the binding sites for PAMP are widely distributed, the nature of its receptor has been elusive. In an effort to identify potential PAMP receptor(s), we found that a human G-protein-coupled receptor, MrgX2, was specifically activated by PAMP. Although a previous study revealed that MrgX2 was a receptor for cortistatin, a neuropeptide involved in sleep regulation and locomotor activity, our present data indicated that the rank order of the agonistic effect against MrgX2 was “PAMP-12 ≥ cortistatin > PAMP-20”. These activities were confirmed by the inhibition of the forskolin-elevated cAMP accumulation, Ca²⁺ mobilization, and [³⁵S]guanosine 5′-(γ-thio)triphosphate binding assays. These findings suggest that MrgX2 couples with not only G_αq but also G_αi, consistent with previous reports on the pharmacological profile of PAMP signaling. Furthermore, by immunostaining, we found that MrgX2 was expressed in the adrenal chromaffin cells as well as the dorsal root ganglia. From these results, we concluded that MrgX2 is a potential human PAMP-12 receptor that regulates catecholamine secretion from adrenal glands. The present discovery will eventually lead to a better understanding of the pathophysiological role of proadrenomedullin peptides.

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Regular ArticleImmunoreactive Proadrenomedullin N-Terminal 20 Peptide in Human Tissue, Plasma and Urine

Abstract

Regular Article
Immunoreactive Proadrenomedullin N-Terminal 20 Peptide in Human Tissue, Plasma and Urine