Purification and characterization of PAMP-12 (PAMP[9-20]) in porcine adrenal medulla as a major endogenous biologically active peptide.

Proadrenomedullin N-terminal 20 peptide (PAMP-20) is a potent hypotensive peptide processed from the adrenomedullin (AM) precursor. We developed a specific radioimmunoassay which recognizes the C-terminal region of PAMP-20. Using this radioimmunoassay, the distribution of immunoreactive (ir-) PAMP was determined in porcine tissues. High concentrations of ir-PAMP were observed in the adrenal medulla and in the atrium, and these values were comparable to the corresponding concentrations of ir-AM. The concentration of ir-PAMP was almost the same as that of ir-AM in the kidney, while ir-PAMP was significantly lower than ir-AM in the ventricle, lung, and aorta. Reversed-phase high performance liquid chromatography in each porcine tissue sample revealed that two major peaks of ir-PAMP existed: one emerged at a position identical to that of authentic porcine PAMP-20; the other unknown peak was eluted earlier. The unknown peptide was purified to homogeneity from porcine adrenal medulla, and its complete amino acid sequence was determined. This peptide was found to be PAMP[9-20] with a C-terminal amide structure, and was named PAMP-12. Intravenous injections of PAMP-12 in anesthetized rats showed a significant hypotensive effect in a dose-dependent fashion, and the effect was comparable to that of PAMP-20. These data indicate that PAMP-12, a major component of ir-PAMP, is processed from the AM precursor, as is PAMP-20, and may participate in cardiovascular control.

Adrenomedullin and proadrenomedullin N-terminal 20 peptide in impaired renal function.

Proadrenomedullin N-terminal 20 peptide (PAMP) is a novel hypotensive peptide found in the N-terminal portion of the prohormone of adrenomedullin (AM), a vasodilator peptide. In this study, we examined the pathophysiological roles of the two peptides. Plasma concentrations of both peptides in patients with impaired renal function were measured and compared to those of the control subjects. Plasma AM concentrations in the study patients were significantly (P < 0.01) higher than in the controls with a serum creatinine of < 1 mg/dl (2.94 +/- 0.18 fmol/ml), and higher concentrations in those patients with a serum creatinine of > or = 2 mg/dl (Group II; 14.8 +/- 1.9 fmol/ml) were observed as compared to those at the 1 to 2 mg/dl level (Group I; 10.3 +/- 1.2 fmol/ml). Similarly, plasma PAMP concentrations tended to be higher in the Group I patients (0.82 +/- 0.05 fmol/ml) and significantly (P < 0.01) increased in the Group II patients (1.42 +/- 0.17 fmol/ml) when compared to the controls (0.53 +/- 0.04 fmol/ml). A significantly (P < 0.05) positive correlation was noted between the plasma AM and PAMP in the study patients. These findings suggest a potential role for these biologically active peptides in the regulation of blood pressure in impaired renal function.

Distribution and characterization of rat immunoreactive proadrenomedullin N-terminal 20 peptide (PAMP) and the augmented cardiac PAMP in spontaneously hypertensive rat.

Proadrenomedullin N-terminal 20 peptide (PAMP) is a novel hypotensive peptide processed from an adrenomedullin precursor. In this study, high concentrations of immunoreactive PAMP (ir-PAMP) were detected in rat cardiac atrium and adrenal gland by the radioimmunoassay (RIA) for rat PAMP. The mean plasma concentration of rat ir-PAMP was 3.8 +/- 0.3 fmol/ml. Analysis in atrium, adrenal gland and plasma with high performance liquid chromatographies showed that most ir-PAMP emerged as one major peak at the position exactly identical to that of the authentic rat PAMP. We further investigated the tissue and plasma concentrations of rat ir-PAMP in spontaneously hypertensive rat (SHR) to elucidate the role of PAMP in hypertension. The ir-PAMP concentration in heart tissue of SHR was found to be increased compared with that of the control rat. Especially, the atrial concentration of ir-PAMP of SHR (5.66 +/- 0.78 fmol/mg wet tissue) was significantly higher than that of the control (3.29 +/- 0.22 fmol/mg wet tissue). Cardiac PAMP might have a role for the protection from systemic hypertension.

Plasma concentration of human adrenomedullin in patients on hemodialysis.

To investigate a possible pathophysiological role of human adrenomedullin (AM), we measured the plasma concentration of immunoreactive-AM (ir-AM) in 38 patients with end-stage renal disease (ESRD) on hemodialysis (HD) and 38 healthy subjects (age and sex matched). In addition, plasma ir-AM was characterized by a reverse-phase high performance liquid chromatography. The mean value (+/- SEM) of plasma AM in the patients before HD (10.1 +/- 0.67 fmol/ml) was markedly higher than that in the control group (2.9 +/- 0.13 fmol/ml, p < 0.001), but plasma AM levels were not altered by HD. There was a significant correlation between plasma AM levels and mean blood pressure (MBP) in a group of subjects including both patients before HD and healthy subjects (p < 0.01). In chromatographic study, the major peak of ir-AM in the plasma from patients on HD, as well as healthy subjects, emerged at an elution time identical to that of synthetic AM, indicating that the active form of AM was present in the circulating blood. The secretion of AM seemed to be increased in response to the conditions elicited by ESRD such as hypervolemia and/or hypertension, and reduced renal excretion of the peptide may also contribute to its high plasma level.

Immunohistochemical identification of adrenomedullin in human, rat, and porcine tissue.

The histological localization was investigated of adrenomedullin (AM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma. The immunohistological distribution was examined of AM in human, rat, and porcine tissues using a polyclonal antibody to a fragment comprising C-terminal amino acids 40-52 of human adrenomedullin [AM(40-52)NH2]. Almost all of the human pheochromocytoma and normal adrenal medullary cells of all three species were immunostained and found to be intensely positive for AM. Furthermore, AM-immunoreactive cells were present in the pancreatic islets, gastrointestinal neuroendocrine system, anterior pituitary, and choroid plexus with some degree of interspecies heterogeneity. These findings indicate that AM-immunoreactive cells are widely distributed in the endocrine and neuroendocrine system, suggesting that AM plays some important role in the control of systemic and local circulation and also of humoral secretion.

Proadrenomedullin N-terminal 20 peptide (PAMP), an endogenous anticholinergic peptide: its exocytotic secretion and inhibition of catecholamine secretion in adrenal medulla.

In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca(2+)-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC50 = 50.1 microM) and catecholamines (EC50 = 63.0 microM), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP [1-20]NH2 inhibited carbachol-induced 22Na+ influx via nicotinic receptors (IC50 = 2.5 microM in a noncompetitive manner and thereby reduced carbachol-induced 45Ca2+ influx via voltage-dependent Ca2+ channels (IC50 = 1.0 microM) and catecholamine secretion (IC50 = 1.6 microM). It did not alter high K(+)-induced 45Ca2+ influx via voltage-dependent Ca2+ channels or veratridine-induced 22Na+ influx via voltage-dependent Na+ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca(2+)-dependent exocytosis in response to nicotinic receptor stimulation.

Identification and hypotensive activity of proadrenomedullin N-terminal 20 peptide (PAMP).

Proadrenomedullin N-terminal 20 peptide (PAMP) is a candidate for a novel biologically active peptide processed from an adrenomedullin precursor. Using a radioimmunoassay for human PAMP, major and minor immunoreactive PAMPs were purified from porcine adrenal medulla and complete amino acid sequences were determined. The major immunoreactive peptide was PAMP itself with an amidated carboxy terminus. The minor one was determined to be PAMP[5-20]. An intravenous bolus injection of human PAMP in anesthetized rats caused a rapid and strong hypotensive effect in a dose dependent manner. The present data indicate that PAMP is an endogenous biologically active peptide which is processed from adrenomedullin precursor.

Immunoreactive proadrenomedullin N-terminal 20 peptide in human tissue, plasma and urine.

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.

Horseshoe kidney and membranous glomerulonephritis with cold activation of complement.

A 48-year-old woman was admitted to our hospital because of proteinuria associated with persistent hypocomplementemia. Intravenous pyelography indicated the presence of horseshoe kidney without other abnormalities. Hypocomplementemia was caused by cold activation of complement. There were some findings suggestive of chronic liver disease (positive HCV antibody, hypergammaglobulinemia, low cholinesterase, etc.). Percutaneous renal biopsy showed the features of multiple evolutional phases of membranous glomerulonephritis.