Proadrenomedullin (1-20) (rat)

Proadrenomedullin N-terminal 20 peptide (PAMP-[1-20]; ARLDVASEFRKKWNKWALSR-amide) is a potent hypotensive and catecholamine release-inhibitory peptide released from chromaffin cells. We studied the mechanism of PAMP action and how its function is linked to structure. We tested human PAMP-[1-20] on catecholamine secretion in PC12 pheochromocytoma cells and found it to be a potent, dose-dependent (IC50 approximately 350 nmol/L) secretory inhibitor. Inhibition was specific for nicotinic cholinergic stimulation since PAMP-[1-20] failed to inhibit release by agents that bypass the nicotinic receptor. Nicotinic cationic (22Na+,45Ca2+) signal transduction was disrupted by this peptide, and potencies for inhibition of 22Na+ uptake and catecholamine secretion were comparable. Even high-dose nicotine failed to overcome the inhibition, suggesting noncompetitive nicotinic antagonism. N- and C-terminal PAMP truncation peptides indicated a role for the C-terminal amide and refined the minimal active region to the C-terminal 8 amino acids (WNKWALSR-amide), a region likely to be alpha-helical. PAMP also blocked (EC50 approximately 270 nmol/L) nicotinic cholinergic agonist desensitization of catecholamine release, as well as desensitization of nicotinic signal transduction (22Na+ uptake). Thus, PAMP may exert both inhibitory and facilitatory effects on nicotinic signaling, depending on the prior state of nicotinic stimulation. PAMP may therefore contribute to a novel, autocrine, homeostatic (negative-feedback) mechanism controlling catecholamine release.

Adrenomedullin (AM), a potent and novel vasodilator 52-residue peptide originally isolated from pheochromocytoma, is processed from a precursor molecule (preproAM) in which another unique 20-residue sequence, termed proadrenomedullin N-terminal 20 peptide (PAMP), exists. Using [125I Tyr0] rat PAMP as a radioligand, we have examined PAMP binding sites in various rat tissues and cultured vascular smooth muscle cells (VSMC) from rat aorta. Specific binding sites for rat PAMP, although very low, were widely distributed in various rat tissues examined. The relatively more abundant sites were present in aorta and adrenal glands, followed by lung, kidney, brain, spleen, and heart. An equilibrium binding study using cultured rat VSMC revealed the presence of a single class of high-affinity [dissociation constant (Kd): 3.5 x 10(-8) M] binding sites for rat PAMP with a maximal binding capacity of 4.5 x 10(6) sites per cell. Binding studies revealed that synthetic rat PAMP(1-19)-NH2 was about 10-fold less potent, and rat PAMP(1-20)-OH and human PAMP were about 20-fold less potent than rat PAMP(1-20)-NH2. SDS-polyacylamide gel electrophoresis after affinity-labeling of membranes from various rat tissues (aorta, adrenal glands, lung) and VSMC revealed a distinct labeled band with the apparent molecular mass of 90 kDa, which was diminished by excess unlabeled rat PAMP. A nonhydrolyzable GTP analog (GTP-gammaS) dose-dependently reduced binding of [125I] rat PAMP to VSMC membranes, while ATP-gammaS had no effect. Neither cyclic AMP nor inositol-1,4,5-triphosphate formation was affected by rat PAMP in rat VSMC. The present study demonstrates for the first time that PAMP receptors are widely distributed in various rat tissues, among which aorta and adrenal glands have the most abundant sites. Our data suggest that PAMP receptors are functionally coupled to G-proteins, although its signal transduction remains obscure. The present study also shows that amidation of C-terminal residue of PAMP is critical for receptor binding. The physiological function of PAMP remains undetermined.

Proadrenomedullin N-terminal 20 peptide (PAMP) is generated from post-transcriptional enzymatic processing of a 185-amino acid precursor for adrenomedullin (AM), a potent vasodilator peptide. We have reported that AM is secreted from cultured neonatal rat cardiac myocytes and fibroblasts, and that secreted AM modulates the growth of these cells; however, it is unknown whether or not the cardiac cells produce PAMP. In this study, we examined the production of PAMP in cultured neonatal rat cardiac myocytes and fibroblasts. Both the cardiac myocytes and fibroblasts cultured with serum-free media secreted PAMP time-dependently at rates of 5.7+/-0.9 fmol/10(5) cells/40 h and 8.4+/-0.7 fmol/5x10(4) cells/48 h (mean+/-SD), respectively. Reverse-phase high performance liquid chromatography showed that immunoreactive PAMP secreted from these cells was identical to PAMP[1-20], a whole active molecule. PAMP and AM secretions were significantly (P<0.01) stimulated by 10(-6) mol/L angiotensin II (Ang II) and 10% fetal bovine serum (FBS) in myocytes and fibroblasts, whereas the ratio of PAMP to AM secretion in the myocytes was smaller than that of the fibroblasts. These results suggest that PAMP is secreted along with AM from rat cardiac myocytes and fibroblasts, and the secretion is augmented by the growth-promoting stimuli of Ang II and FBS for these cells.