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

Abstract

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.

Proadrenomedullin N-Terminal 20 Peptide (PAMP), Acting Through PAMP(12–20)-Sensitive Receptors, Inhibits Ca 2+ -Dependent, Agonist-Stimulated Secretion of Human Adrenal Glands

“…This observation makes PAMP (12)(13)(14)(15)(16)(17)(18)(19)(20) a good candidate as a PAMP receptorselective antagonist, a contention supported by the present finding that PAMP (12)(13)(14)(15)(16)(17)(18)(19)(20), although ineffective per se, partially suppressed the PAMP inhibitory action on the secretory response of ZG and AM to Ca 2ϩ -dependent agonists. Because the PAMP(9 -20) fragment is the major endogenous PAMP peptide in porcine AM, 25 we hypothesized that posttranslational processing of the prepro-ADM molecule gives rise not only to PAMP but also its fragments. Along with our present findings concerning PAMP (12)(13)(14)(15)(16)(17)(18)(19)(20), this raises the interesting possibility that adrenal PAMP receptors may be agonistically and antagonistically modulated by endogenous ligands.…”

Pathophysiological Function of Adrenomedullin and Proadrenomedullin N-Terminal Peptides in Adrenal Chromaffin Cells

“…This finding led us to seek novel PAMP20-related peptides in the adrenal medulla. By using an antibody recognizing the C-terminal structure of PAMP20, it was found that a high level of PAMP12, a 12 amino acid peptide identical to PAMP20 at amino acids 9-20, was present in pigs and cows (24,25). In addition, a considerable level of PAMP16, a peptide identical to PAMP20 at amino acids 5-20, was identified in the bovine adrenal medulla (24).…”

Proadrenomedullin N-Terminal 20 Peptide

“…10,22 Recently, PAMP- [9][10][11][12][13][14][15][16][17][18][19][20] (a carboxy-terminal fragment of PAMP) has been identified in porcine adrenal medulla and causes hypotension on intravenous injection in the rat. 7 Synthetic human PAMP- [12][13][14][15][16][17][18][19][20] is also a vasodepressor in the rat and cat. 8 In bovine chromaffin cells, PAMP- [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] has been reported to inhibit nicotinic agonist-induced catecholamine secretion 23 and synthesis 24 and nicotinic agonist-induced Na ϩ and Ca 2ϩ influx.…”

“…7 Synthetic human PAMP- [12][13][14][15][16][17][18][19][20] is also a vasodepressor in the rat and cat. 8 In bovine chromaffin cells, PAMP- [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] has been reported to inhibit nicotinic agonist-induced catecholamine secretion 23 and synthesis 24 and nicotinic agonist-induced Na ϩ and Ca 2ϩ influx. 23,25 However, the precise mechanism or site of PAMP- [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]…”

“…Our results reveal that PAMP- [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] inhibits catecholamine secretion (IC 50 Ϸ350 nmol/L), acting in a noncompetitive manner specifically at the nicotinic cholinergic receptor; in addition, PAMP- [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] inhibits the desensitization of catecholamine release evoked by nicotinic agonists. Peptide deletion studies establish that the carboxy-terminal domain of PAMP- [1][2][3][4][5][6][7][8][9][10]…”