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 (IC 50 ≈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 (22 Na+,45 Ca 2+) signal transduction was disrupted by this peptide, and potencies for inhibition of 22 Na+ 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 α-helical. PAMP also blocked (EC 50 ≈270 nmol/L) nicotinic cholinergic agonist desensitization of catecholamine release, as well as desensitization of nicotinic signal transduction (22 Na+ 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 is a novel hypotensive peptide originally isolated from human pheochromocytoma as a cAMP-elevating agent. The 52–amino acid human adrenomedullin shares sequence homology with the calcitonin gene–related peptide and pancreatic amylin family. cDNA sequences of rat, pig, and human forms reveal that preproadrenomedullin consists of 185 amino acids, with 3 sites of paired basic amino acids as targets for prohormone-processing proteolytic cleavage. Excision of the signal peptide between amino acids Thr21 and Ala22 in preproadrenomedullin yields a proadrenomedullin propeptide of 164 amino acids.
Proadrenomedullin N-terminal 20 peptide (PAMP-[1-20]; ARLDVASEFRKKWNKWALSR-amide) is liberated from proadrenomedullin by proteolytic cleavage at the first group of basic amino acids (Gly 42 Lys 43 Arg 44), after which PAMP-[1-20] is posttranslationally amidated at its carboxy terminus in response to the Gly signal for the enzyme peptidylglycine α-amidating monooxygenase. cDNA sequences of rat, pig, and human forms show 80% sequence identity of the PAMP-[1-20] region (Table 1), and 12 amino acids at the C-terminus are conserved in all 3 species (Table 1). PAMP-[1-20] is found in plasma, adrenal medulla, right atrium, kidney, and brain and exerts hypotensive activity in the rat and cat. Plasma levels of PAMP-[1-20] are elevated in human disease states such as essential hypertension, renal failure, and congestive heart failure, as well as in the spontaneously hypertensive rat. While adrenomedullin decreases vascular resistance by a direct action on vascular smooth muscle or by releasing nitric oxide from the endothelium, PAMP may induce vasodilation by a variety of site- or species-dependent mechanisms: inhibition of norepinephrine release from adrenergic nerve endings in the mesenteric vascular bed of the rat, direct or tone-dependent vasodilation in the hindquarters vascular bed of the rat, or cAMP-mediated vasodilator activity in the mesenteric and hindlimb vascular beds of the cat. Recently, PAMP-[9-20] (a carboxy-terminal fragment of PAMP) has been identified in porcine adrenal medulla and causes hypotension on intravenous injection in the rat. Synthetic human PAMP-[12-20] is also a vasodepressor in the rat and cat.
In bovine chromaffin cells, PAMP-[1-20] has been reported to inhibit nicotinic agonist–induced catecholamine secretion and synthesis and nicotinic agonist–induced Na+ and Ca 2+ influx. However, the precise mechanism or site of PAMP-[1-20] action remains in doubt, with different reports implicating the nicotinic cholinergic receptor or an alternative mechanism involving a G protein–coupled receptor inhibiting voltage-gated calcium currents.
We examined the effects of PAMP-[1-20] on catecholamine secretion from rat pheochromocytoma PC12 cells, determining the crucial active residues in its sequence. We also studied its actions on signal transduction mechanisms for catecholamine release and uncovered a novel effect on nicotinic cholinergic desensitization. Our results reveal that PAMP-[1-20] inhibits catecholamine secretion (IC 50 ≈350 nmol/L), acting in a noncompetitive manner specifically at the nicotinic cholinergic receptor; in addition, PAMP-[1-20] inhibits the desensitization of catecholamine release evoked by nicotinic agonists. Peptide deletion studies establish that the carboxy-terminal domain of PAMP-[1-20] is crucial to its antisecretory activity.
Rat PC12 pheochromocytoma cells (at passage 8, obtained from Dr David Schubert, Salk Institute, La Jolla, Calif) were grown at 37°C/6% CO 2, in 10-cm plates or 6-well plates, in DME/high-glucose medium supplemented with 5% fetal bovine serum, 10% horse serum, 100 U/mL penicillin, and 1% penicillin/streptomycin (100% stocks were 10 000 U/mL penicillin G and 10 000 μg/mL streptomycin sulfate; Life Technologies, Inc).
In some experiments, cells were pretreated with pertussis toxin (100 ng/mL, 16 hours).
Secretion of norepinephrine was monitored as previously described. PC12 cells were plated on poly-d-lysine–coated polystyrene dishes (Falcon Labware), labeled for 3 hours with 1 μCi [3 H]l-norepinephrine (71.7 Ci/mmol, DuPont/NEN) in 1 mL of PC12 growth medium, washed twice with release buffer (150 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L CaCl 2, 10 mmol/L HEPES, pH 7), and then incubated at 37°C for 30 minutes in release buffer with or without secretagogues, such as nicotine (60 μmol/L), or cell membrane depolarization (55 mmol/L KCl). Release buffer for experiments involving KCl as secretagogue had NaCl reduced to 100 mmol/L to maintain isotonicity. After 30 minutes, secretion was terminated by aspirating the release buffer and lysing cells into 150 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L HEPES, pH 7, 0.1% (vol/vol) Triton X-100. Release medium and cell lysates were assayed for [3 H]norepinephrine by liquid scintillation counting, and results were expressed as percent secretion: [amount released/(amount released+amount in cell lysate)]×100. Net secretion is secretagogue-stimulated release minus basal release, where basal norepinephrine release is typically 5.8±0.36% of cell total [3 H]norepinephrine released over 30 minutes (n=10 separate secretion assays).
PAMP-[1-20]or its analogues were synthesized at 10 to 100 μmol scale by the solid-phase method with t-boc or f-moc protection chemistry and then purified to >95% homogeneity by reversed-phase high-pressure liquid chromatography on C-18 silica columns, with monitoring of A 280 (aromatic rings) or A 214 (peptide bonds). Authenticity and purity of peptides were verified by rechromatography, as well as by electrospray-ionization or matrix-assisted laser desorption ionization (MALDI) mass spectrometry or amino acid composition. For some experiments, small-scale (1 μmol) peptide syntheses were accomplished by pin technology (Chiron Mimotopes), after which peptides were cleaved from the resin and washed.
PC12 cells were split to 50% confluence and treated with nerve growth factor (NGF) (2.5S, murine, natural, 100 ng/mL; GIBCO-BRL). The medium was changed every other day with NGF addition to the new medium. After 7 days of treatment, the neurite-bearing cells were used for secretion studies, as described above.
22 Na+ uptake was studied as described by Amy and Kirshner with minor modifications. Before experiments, PC12 cells were washed twice with 50 mmol/L Na+-sucrose media containing 50 mmol/L NaCl, 187 mmol/L sucrose, 5 mmol/L KCl, 2 mmol/L CaCl 2, and 5 mmol/L HEPES adjusted to pH 7.4 with NaOH. To measure 22 Na+ influx, this medium was then supplemented with 5 mmol/L ouabain (to prevent active extrusion of newly taken-up 22 Na+ from cells) and 1.5 μCi/mL of 22 NaCl. Incubation was performed at 22°C for 5 minutes, in the presence or absence of secretagogues, and then the cells were washed within 10 seconds with 3 changes (1 mL each) of 50 mmol/L Na+-sucrose media with 5 mmol/L ouabain. The cells were lysed (see above), and 22 Na+ in the cell lysate was measured in a gamma counter (LKB 1274 RIAGAMMA, Wallac Inc). The data were expressed as disintegrations per minute per well.
Cells were seeded onto poly-d-lysine–coated 6-well polystyrene culture dishes 2 days before assay. Cells were rinsed with 1 mL of release buffer and preincubated with 1 mL of release buffer for 30 minutes at 37°C. Then the cells were incubated for 5 minutes at 37°C in 1 mL of release buffer (without unlabeled calcium) containing 2 μCi of 45 Ca 2+ (14.95 mCi/mg, DuPont/NEN). The drugs tested were present in the release buffer during the 1-minute incubation period. Calcium uptake was stopped abruptly after 1 minute by inversion of the plate to decant all 6 wells simultaneously, followed promptly by addition of 2 mL of ice-cold release buffer containing 1 mmol/L LaCl 3; the nonselective calcium channel blocker La 3+ terminates further uptake of extracellular labeled calcium.32 The culture dishes were then rinsed twice with ice-cold release buffer. To cells in each well, 1 mL of cell lysis buffer was added and collected for liquid scintillation counting. The data were expressed as disintegrations per minute per well.
Secondary structure of PAMP was first predicted by the empirical statistical Chou-Fasman or Robson-Garnier algorithms with the use of the program MacVector (version 5.0.2; Oxford Molecular Group PLC). Secondary structure predictions for amphiphilicity, in which hydrophobic moment plots were used, were also done on MacVector.
Homology modeling allowed prediction of 3-dimensional structure on the basis of known (x-ray crystallographic– or nuclear magnetic resonance–derived) structures of remote homologues in the Protein Data Bank (PDB). Remote homologues were detected by the method of prediction-based threading on the EMBL Web server. α-Helical structures were created in the graphic program Fold-It (light) (version 4.0.7; Reference 39 and admin@frankenthalerfoundation.org), and the PDB format (x, y, z coordinates) fileswere imported into the program CS Chem3D Pro (version 3.2; CambridgeSoft) for energy minimization by molecular mechanics with the use of the MM2 force field and the method of steepest descent. The MM2 force field includes both covalent (bond stretching, bending, and torsion) and noncovalent (Van der Waals, charge-charge, charge-dipole, and dipole-dipole) terms.
Both PAMP-[1-20]-amide (carboxy terminus amidated, as in the nat
