Proadrenomedullin N-Terminal 20 Peptide by Manjula Mahata, Sushil K. Mahata, Robert J. Parmer, and Daniel T. O’Connor Hypertension Volume 32(5):907-916 November 1, 1998 Copyright © American Heart Association, Inc. All rights reserved.

Potency of catecholamine secretion inhibition by human PAMP Potency of catecholamine secretion inhibition by human PAMP. A, PAMP dose-response curve. [3H]l-norepinephrine–prelabeled cells were incubated with 60 μmol/L nicotine, with or without different doses (0.01 to 10 μmol/L) of synthetic human PAMP-[1-20]-amide (with carboxy-terminal amidation, as in the natural version of PAMP; ARLDVASEFRKKWNKWALSR-amide) or PAMP-[1-20]-acid (with a free carboxy terminus) for 30 minutes. Potency of catecholamine secretion inhibition by human PAMP. A, PAMP dose-response curve. [3H]l-norepinephrine–prelabeled cells were incubated with 60 μmol/L nicotine, with or without different doses (0.01 to 10 μmol/L) of synthetic human PAMP-[1-20]-amide (with carboxy-terminal amidation, as in the natural version of PAMP; ARLDVASEFRKKWNKWALSR-amide) or PAMP-[1-20]-acid (with a free carboxy terminus) for 30 minutes. Control (100%) net norepinephrine release is that in the presence of nicotine (60 μmol/L) stimulation alone, without PAMP. Results are mean±SEM; n=3 replicates per condition. B, Reversibility of PAMP action. [3H]l-norepinephrine–prelabeled cells were preincubated with PAMP-[1-20]-amide (10 μmol/L) for 1 hour and washed twice (10 minutes each), then [3H] norepinephrine secretion was stimulated by 60 μmol/L nicotine. The results are expressed as percentage of net [3H]norepinephrine release. Results are mean±SEM; n=3 replicates per condition. Manjula Mahata et al. Hypertension. 1998;32:907-916 Copyright © American Heart Association, Inc. All rights reserved.

PAMP as a noncompetitive nicotinic cholinergic antagonist. PAMP as a noncompetitive nicotinic cholinergic antagonist. Inhibition of catecholamine release was provoked by a spectrum of concentrations of nicotinic cholinergic agonist. PC12 cells were labeled with [3H]l-norepinephrine, and secretion over a 30-minute time course was studied in response to different doses of nicotine (10 to 1000 μmol/L), either alone or in combination with different doses of human PAMP-[1-20]-amide (0.1 to 10 μmol/L), or a “positive control” neuronal nicotinic antagonist, the noncompetitive nicotinic antagonist (channel blocker) hexamethonium (100 μmol/L). Manjula Mahata et al. Hypertension. 1998;32:907-916 Copyright © American Heart Association, Inc. All rights reserved.

Identification of the minimal active region within the PAMP sequence Identification of the minimal active region within the PAMP sequence. [3H]l-norepinephrine–prelabeled cells were incubated with 60 μmol/L nicotine, with or without different doses of human PAMP-amide truncation peptides (wild-type PAMP-[1-20]-amide, PAMP-[4-20]-amide, PAMP-[7-20]-amide, PAMP-[10-20]-amide, PAMP-[13-20]-amide, PAMP-[16-20]-amide, PAMP-[1-20]-acid, PAMP-[1-17], PAMP-[1-14], PAMP-[1-11], PAMP-[1-8], or PAMP-[1-5]; at 0.01 to 10 μmol/L) for 30 minutes. Identification of the minimal active region within the PAMP sequence. [3H]l-norepinephrine–prelabeled cells were incubated with 60 μmol/L nicotine, with or without different doses of human PAMP-amide truncation peptides (wild-type PAMP-[1-20]-amide, PAMP-[4-20]-amide, PAMP-[7-20]-amide, PAMP-[10-20]-amide, PAMP-[13-20]-amide, PAMP-[16-20]-amide, PAMP-[1-20]-acid, PAMP-[1-17], PAMP-[1-14], PAMP-[1-11], PAMP-[1-8], or PAMP-[1-5]; at 0.01 to 10 μmol/L) for 30 minutes. Control (100%) net norepinephrine release is that in the presence of nicotine (60 μmol/L) stimulation alone, without PAMP. IC50 values for inhibition of catecholamine release by each peptide were then calculated with net norepinephrine release by nicotine alone used as control (100%). A, N-terminal PAMP truncation peptides. B, C-terminal PAMP truncation peptides. Manjula Mahata et al. Hypertension. 1998;32:907-916 Copyright © American Heart Association, Inc. All rights reserved.

PAMP-[10-20]-amide effect on neurite-bearing PC12 cells. PAMP-[10-20]-amide effect on neurite-bearing PC12 cells. PAMP-[10-20]-amide effect on neurite-bearing (NGF-differentiated) PC12 cells. The neurites were grown by treatment with NGF (2.5S form, 100 ng/mL, 7 days) or vehicle. Cells were then labeled with [3H]l-norepinephrine, and secretion over a 30-minute time course was studied in response to 60 μmol/L nicotine, either alone or in combination with different doses of PAMP-[10-20]-amide (RKKWNKWALSR-amide). Results are mean±SEM; n=3 replicates per condition. Manjula Mahata et al. Hypertension. 1998;32:907-916 Copyright © American Heart Association, Inc. All rights reserved.

PAMP and nicotinic cholinergic signal transduction: 22Na+ and Ca2+ cation fluxes. PAMP and nicotinic cholinergic signal transduction: 22Na+ and Ca2+ cation fluxes. A, PAMP-[10-20]-amide effect on nicotine-induced [3H]norepinephrine release or uptake of 22Na+ in PC12 cells. For catecholamine release, [3H]l-norepinephrine–prelabeled cells were incubated with 60 μmol/L nicotine, with or without different doses (0.01 to 10 μmol/L) of synthetic human PAMP-[10-20]-amide for 30 minutes, followed by measurement of [3H]norepinephrine release in cell and in media. For the 22Na+ uptake study, cells were treated with 22Na+ plus nicotine (60 μmol/L), in the presence or absence of human PAMP-amide (PAMP-[10-20]-amide; 0.1 to 10 μmol/L), for 5 minutes, followed by removal of the medium and cell lysis for measurement of 22Na+ uptake. Control (100%) net [3H]norepinephrine release of 22Na+ uptake is that in the presence of nicotine (60 μmol/L) stimulation alone, without PAMP-[10-20]-amide. Results are mean±SEM; n=3 replicates per condition. B, Effect of human PAMP-[1-20]-amide on secretagogue-induced uptake of 45Ca2+ from PC12 cells. Cells were treated with 45Ca2+ plus nicotine (60 μmol/L) or KCl (55 mmol/L), in the presence or absence of human PAMP-[1-20]-amide (10 μmol/L), for 1 minute, followed by removal of the medium and cell lysis for measurement of 45Ca2+ uptake. Results are mean±SEM; n=3 replicates per condition. Manjula Mahata et al. Hypertension. 1998;32:907-916 Copyright © American Heart Association, Inc. All rights reserved.

In a determination of whether inactive PAMP truncation peptides antagonize the effect of PAMP-20 on catecholamine secretion, [3H]l-norepinephrine–prelabeled cells were incubated with 60 μmol/L nicotine alone, in presence of full-length PAMP-[1-20]-amide (10 μmol/L) either alone or in combination with an inactive PAMP truncation peptide (PAMP-[16-20]-amide, PAMP-[1-17], PAMP-[1-14], PAMP- [1-11], PAMP-[1-8], or PAMP-[1-5]; each at 10 μmol/L). In a determination of whether inactive PAMP truncation peptides antagonize the effect of PAMP-20 on catecholamine secretion, [3H]l-norepinephrine–prelabeled cells were incubated with 60 μmol/L nicotine alone, in presence of full-length PAMP-[1-20]-amide (10 μmol/L) either alone or in combination with an inactive PAMP truncation peptide (PAMP-[16-20]-amide, PAMP-[1-17], PAMP-[1-14], PAMP- [1-11], PAMP-[1-8], or PAMP-[1-5]; each at 10 μmol/L). The results are expressed as percentage of net [3H]norepinephrine release. Results are mean±SEM; n=3 replicates per condition. Manjula Mahata et al. Hypertension. 1998;32:907-916 Copyright © American Heart Association, Inc. All rights reserved.

PAMP blockade of nicotinic cholinergic desensitization of catecholamine release, as well as blockade of nicotinic cholinergic–stimulated 22Na+ uptake. PAMP blockade of nicotinic cholinergic desensitization of catecholamine release, as well as blockade of nicotinic cholinergic–stimulated 22Na+ uptake. A, PAMP effects on desensitization of catecholamine release. [3H]l-norepinephrine–prelabeled cells were incubated with nicotine (10 μmol/L) either alone or in combination with PAMP-[1-20]-amide or substance P (0.1 to 10 μmol/L) for 10 minutes (incubation I) and washed twice (6 minutes each), then secretion was rechallenged with nicotine (10 μmol/L) for 10 minutes (incubation II) before measurement of [3H]l-norepinephrine secretion. The EC50 of PAMP-[1-20]-amide for inhibition of desensitization of catecholamine release is ≈0.27 μmol/L, while that of substance P is ≈0.21 μmol/L. Results are mean±SEM; n=3 replicates per condition. B, PAMP-[10-20]-amide effects on 22Na+ uptake. PC12 cells were treated with nicotine (10 μmol/L) either alone or in combination with PAMP-[10-20]-amide (10 μmol/L) or vehicle in incubation I, washed twice (6 minutes each), and then treated with nicotine (10 μmol/L) for 10 minutes in incubation II in the presence of extracellular 22Na+. Cells were then harvested for measurement of 22Na+ uptake. Results are mean±SEM; n=3 replicates per condition. Manjula Mahata et al. Hypertension. 1998;32:907-916 Copyright © American Heart Association, Inc. All rights reserved.

Structure of PAMP. After empirical statistical algorithms and homology search suggested PAMP to be an α-helix, the PAMP sequence was created as an α-helix and subjected to energy minimization in an MM2 (molecular mechanics) force field. Structure of PAMP. After empirical statistical algorithms and homology search suggested PAMP to be an α-helix, the PAMP sequence was created as an α-helix and subjected to energy minimization in an MM2 (molecular mechanics) force field. Top, side view. Bottom, axial view. Manjula Mahata et al. Hypertension. 1998;32:907-916 Copyright © American Heart Association, Inc. All rights reserved.

Model for the action of PAMP on catecholamine secretion from chromaffin cells. Model for the action of PAMP on catecholamine secretion from chromaffin cells. The physiological secretagogue for chromaffin cells is acetylcholine (ACh), which acts on neuronal-type nicotinic cholinergic receptors. Binding of acetylcholine to its receptor causes influx of sodium, which in turn causes depolarization of the cell membrane, resulting in influx of calcium through voltage-gated calcium channels. Antagonists at nicotinic receptors may be either competitive (with the agonist binding site) or noncompetitive (often cation channel blockers). M2 indicates cation channel domain of the nicotinic cholinergic receptor; Pro-AM, proadrenomedullin. Manjula Mahata et al. Hypertension. 1998;32:907-916 Copyright © American Heart Association, Inc. All rights reserved.