This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mahata, M. Articles by O'Connor, D. T. Search for Related Content PubMed PubMed Citation Articles by Mahata, M. Articles by O'Connor, D. T. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL NICOTINE NICOTINE TARTRATE SODIUM

(Hypertension. 1998;32:907-916.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Proadrenomedullin N-Terminal 20 Peptide

Minimal Active Region to Regulate Nicotinic Receptors

Manjula Mahata; Sushil K. Mahata; Robert J. Parmer; ; Daniel T. O'Connor

From the Department of Medicine and Center for Molecular Genetics, University of California at San Diego, and San Diego Veterans Affairs Healthcare System, San Diego, Calif.

Correspondence to Daniel T. O'Connor, MD, Department of Medicine and Center for Molecular Genetics (9111H), University of California at San Diego, 3350 La Jolla Village Dr, San Diego, CA 92161. E-mail doconnor{at}ucsd.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—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 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 -helical. PAMP also blocked (EC₅₀ 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.

Key Words: chromogranin • adrenal gland • chromaffin granule • adrenomedullin • catecholamines

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Adrenomedullin is a novel hypotensive peptide originally isolated from human pheochromocytoma as a cAMP-elevating agent.¹ The 52–amino acid human adrenomedullin shares sequence homology with the calcitonin gene–related peptide and pancreatic amylin family.¹ 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.^{1 2 3} 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 (Gly⁴²Lys⁴³Arg⁴⁴), after which PAMP-[1-20] is posttranslationally amidated at its carboxy terminus in response to the Gly signal for the enzyme peptidylglycine -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^{1 2 3} (Table 1). PAMP-[1-20] is found in plasma, adrenal medulla, right atrium, kidney, and brain^{4 5} and exerts hypotensive activity in the rat and cat.^{1 6 7 8 9 10 11 12} Plasma levels of PAMP-[1-20] are elevated in human disease states such as essential hypertension,¹³ renal failure,¹⁴ and congestive heart failure,¹⁵ as well as in the spontaneously hypertensive rat.¹⁶ While adrenomedullin decreases vascular resistance by a direct action on vascular smooth muscle or by releasing nitric oxide from the endothelium,^{17 18 19 20} 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,^{5 21} direct or tone-dependent vasodilation in the hindquarters vascular bed of the rat,⁹ or cAMP-mediated vasodilator activity in the mesenteric and hindlimb vascular beds of the cat.^{10 22} 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.⁷ Synthetic human PAMP-[12-20] is also a vasodepressor in the rat and cat.⁸

View this table: [in this window] [in a new window]   Table 1. PAMP: Interspecies (Human,1 Rat,2 Pig3) Sequence Conservation

In bovine chromaffin cells, PAMP-[1-20] has been reported to inhibit nicotinic agonist–induced catecholamine secretion²³ and synthesis²⁴ and nicotinic agonist–induced Na⁺ and Ca²⁺ influx.^{23 25} However, the precise mechanism or site of PAMP-[1-20] action remains in doubt, with different reports implicating the nicotinic cholinergic receptor^{23 25} or an alternative mechanism involving a G protein–coupled receptor inhibiting voltage-gated calcium currents.²⁶

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 (IC₅₀ 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.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cell Culture
Rat PC12 pheochromocytoma cells²⁷ (at passage 8, obtained from Dr David Schubert, Salk Institute, La Jolla, Calif) were grown at 37°C/6% CO₂, 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).²⁸

In some experiments, cells were pretreated with pertussis toxin (100 ng/mL, 16 hours).²⁹

Secretagogue-Stimulated Release of Norepinephrine
Secretion of norepinephrine was monitored as previously described.²⁸ PC12 cells were plated on poly-D-lysine–coated polystyrene dishes (Falcon Labware), labeled for 3 hours with 1 µCi [³H]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 CaCl₂, 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 [³H]norepinephrine by liquid scintillation counting, and results were expressed as percent secretion: [amount released/(amount released+amount in cell lysate)]x100. Net secretion is secretagogue-stimulated release minus basal release, where basal norepinephrine release is typically 5.8±0.36% of cell total [³H]norepinephrine released over 30 minutes (n=10 separate secretion assays).

Synthetic Peptides
PAMP-[1-20]or its analogues were synthesized at 10 to 100 µmol scale by the solid-phase method³⁰ 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 A₂₈₀ (aromatic rings) or A₂₁₄ (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.

Neuronal Differentiation of PC12 Cells With Nerve Growth Factor
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.

²²Na⁺ Uptake by PC12 Cells
²²Na⁺ uptake was studied as described by Amy and Kirshner³¹ 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 CaCl₂, and 5 mmol/L HEPES adjusted to pH 7.4 with NaOH. To measure ²²Na⁺ influx, this medium was then supplemented with 5 mmol/L ouabain (to prevent active extrusion of newly taken-up ²²Na⁺ from cells) and 1.5 µCi/mL of ²²NaCl. 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 ²²Na⁺ 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.

⁴⁵Ca²⁺ Uptake by PC12 Cells
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 ⁴⁵Ca²⁺ (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 LaCl₃; the nonselective calcium channel blocker La³⁺ 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.

Structure Predictions
Secondary structure of PAMP was first predicted by the empirical statistical Chou-Fasman³³ or Robson-Garnier³⁴ 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,³⁵ 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 Bank³⁶ (PDB). Remote homologues were detected by the method of prediction-based threading³⁷ on the EMBL Web server.³⁸ -Helical structures were created in the graphic program Fold-It (light) (version 4.0.7; Reference 39³⁹ and jean-claude.jesior@imag.fr), 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.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Catecholamine Release–Inhibitory Actions of PAMP
Both PAMP-[1-20]-amide (carboxy terminus amidated, as in the natural form of PAMP) and PAMP-[1-20]-acid (with a free carboxy terminus) exerted dose-dependent inhibition of nicotine-stimulated catecholamine secretion from PC12 cells. IC₅₀ values for PAMP-[1-20]-amide and PAMP-[1-20]-acid were 0.35 µmol/L and 2.9 µmol/L, respectively (Figure 1A).

View larger version (27K): [in this window] [in a new window]   Figure 1. 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.

PAMP-[1-20]-amide activity was heat stable (no loss of inhibition after heating to 100°C for 10 minutes; data not shown) and at least partially reversible (Figure 1B): after 1 hour of incubation with PAMP-[1-20]-amide, two 10-minute washouts resulted in loss of 50% of the catecholamine release inhibition on PC12 cells.

PAMP as a Noncompetitive Nicotinic Cholinergic Antagonist
To test whether PAMP exerted its nicotinic antagonist action by a competitive or noncompetitive means, we stimulated PC12 cells with a spectrum of nicotine doses (10 to 1000 µmol/L), alone or with PAMP-[1-20]-amide (0.1 to 10 µmol/L). Nicotine failed to overcome inhibition by PAMP-[1-20]-amide at any agonist dose (Figure 2), and nicotine was not able to reverse the blocking effects of even submaximal inhibitory concentrations of PAMP. This result functionally establishes PAMP as a noncompetitive nicotinic antagonist (Figure 2).

View larger version (30K): [in this window] [in a new window]   Figure 2. 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).

PAMP Biological Activity Resides Toward Its C-Terminus
To define active residues within the PAMP-[1-20] sequence, we synthesized several PAMP analogues by N-terminal truncations (PAMP-[4-20]-amide, PAMP-[7-20]-amide, PAMP-[10-20]-amide, PAMP-[13-20]-amide, and PAMP-[16-20]-amide) or C-terminal truncations (PAMP-[1-17], PAMP-[1-14], PAMP-[1-11], PAMP-[1-8], and PAMP-[1-5]) and tested their effects on nicotine-induced catecholamine secretion from PC12 cells. Initial N-terminal truncation analogues did not impair PAMP activity; indeed, PAMP-[10-20]-amide was even more active (IC₅₀ 0.045 µmol/L) than wild-type PAMP-[1-20]-amide (Figures 3A and 5). Although PAMP-[13-20]-amide was still active (IC₅₀ 0.53 µmol/L for inhibition of nicotine-induced catecholamine secretion), PAMP-[16-20]-amide lost detectable activity (IC₅₀ >10 µmol/L). Any truncation from the C-terminus severely reduced catecholamine release–inhibitory activity: removal of even the C-terminal amide resulted in some loss of activity (to IC₅₀ 2.9 µmol/L), while truncation of 3 C-terminal amino acids (in PAMP-[1-17]) completely inactivated (IC₅₀ >10 µmol/L) the peptide (Figure 3B).

View larger version (25K): [in this window] [in a new window]   Figure 3. 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.

View larger version (23K): [in this window] [in a new window]   Figure 5. 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.

PAMP Inhibition Specific for Nicotinic Cholinergic Stimulation of Catecholamine Secretion
In addition to stimulation of the physiological (nicotinic cholinergic) secretory pathway (by nicotine, 60 µmol/L), we tested several secretagogues that act at stages in the pathway later than the nicotinic receptor (Table 2): membrane depolarization (55 mmol/L KCl) to open voltage-gated calcium channels, an alkaline earth (BaCl₂, 2 mmol/L; the effects of this secretagogue require participation of calcium channels⁴⁰), a calcium ionophore (A23187, 1 µmol/L), or alkalinization of the chromaffin vesicle core (chloroquine, 1 mmol/L). PAMP-[1-20]-amide suppressed norepinephrine release only when triggered by nicotine (Table 2) and not when secretion was caused by agents acting later (ie, distal to the nicotinic cholinergic receptor) in the secretory pathway (Table 2).

View this table: [in this window] [in a new window]   Table 2. PAMP Effects on Catecholamine Secretion From PC12 Cells: Dependence on Secretory Stimulation Pathway

Stimulation of P_2x purinergic receptors causes catecholamine secretion from PC12 cells.⁴¹ Since P_2x receptors and nicotinic cholinergic receptors are members of the same superfamily of extracellular ligand-gated cation channels,⁴¹ we tested the effect of PAMP on the function of this receptor, as well. Although the somewhat selective P_2x antagonist reactive blue 2⁴¹ substantially inhibited ATP-stimulated catecholamine release (from 52.1±0.09% to 20.6±1.49% release), PAMP-[1-20] was without effect on this ligand-receptor system (Table 2).

PAMP Effect on Neurite-Bearing PC12 Cells
PAMP-[10-20]-amide (RKKWNKWALSR-amide) inhibited catecholamine release from both normal PC12 (IC₅₀ 0.045 µmol/L) and NGF-treated PC12 cells (IC₅₀ 0.083 µmol/L) (Figure 4), with similar potencies.

View larger version (24K): [in this window] [in a new window]   Figure 4. 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.

PAMP and Nicotinic Cholinergic Signal Transduction: Na⁺ and Ca²⁺ Cation Fluxes
PAMP-[10-20]-amide blocked nicotine-induced uptake of ²²Na⁺ into PC12 cells, and the blockade was dose dependent (Figure 5A). The IC₅₀ value for inhibition of ²²Na⁺ uptake by PAMP-[10-20]-amide was 0.09 µmol/L (Figure 5A) compared with 0.045 µmol/L for inhibition of catecholamine release (Figure 5A).

To test the specificity of PAMP action on nicotinic signaling pathways, we studied the effects of PAMP-[1-20]-amide on ⁴⁵Ca²⁺ uptake into PC12 cells induced by nicotine (60 µmol/L) or by membrane depolarization (55 mmol/L KCl), which opens voltage-gated calcium channels. While PAMP-[1-20]-amide completely abolished nicotine-induced uptake of ⁴⁵Ca²⁺, it failed to block ⁴⁵Ca²⁺ uptake when voltage-gated calcium channels were directly opened by membrane depolarization (Figure 5B).

Testing Involvement of Inhibitory G Proteins in the PAMP Effect on PC12 Cells
To test whether the effect of PAMP on nicotine-induced norepinephrine release might be mediated by inhibitory G proteins, we pretreated PC12 cells with the G_i/G_o inactivator pertussis toxin (100 ng/mL, 16 hours²⁹) and then tested the effects of PAMP-[1-20]-amide on nicotine-induced catecholamine release. No effect of pertussis toxin on the PAMP-[1-20]-amide antisecretory effect was noted (data not shown). Similar studies were performed in NGF (100 ng/mL for 7 days)–treated PC12 cells, and we found no difference between pertussis toxin–pretreated or control PC12 cells in inhibition of catecholamine release by PAMP-[1-20]-amide (data not shown).

Role of PAMP Truncation Peptides in Inhibition of Catecholamine Secretion by PAMP-[1-20]-amide
We tested whether any of these functionally inactive PAMP truncation peptides antagonize the action of full-length PAMP-[1-20]-amide. None of the inactive PAMP analogues (PAMP-[16-20]-amide, PAMP-[1-17], PAMP-[1-14], PAMP-[1-11], PAMP-[1-8], or PAMP-[1-5]) reversed the action of PAMP-[1-20]-amide on nicotinic cholinergic–stimulated catecholamine release (Figure 6).

View larger version (28K): [in this window] [in a new window]   Figure 6. 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.

PAMP Inhibition of Nicotinic Cholinergic Desensitization of Catecholamine Release
Repeated or prolonged exposure of chromaffin cells to nicotinic cholinergic agonists results in diminution of subsequent catecholamine secretion responses to nicotinic rechallenge.⁴² To determine whether PAMP influences nicotinic desensitization, we exposed PC12 cells to nicotine (10 µmol/L) for 10 minutes, either alone or in combination with ascending doses (0.1 to 10 µmol/L) of PAMP-[1-20]-amide or substance P (as a positive control⁴²), removed nicotine and PAMP-[1-20]-amide by washing twice, and then rechallenged with nicotine (10 µmol/L) for 10 minutes. In dose-dependent fashion, both PAMP-[1-20]-amide (EC₅₀ 0.27 µmol/L) and substance P (EC₅₀ 0.21 µmol/L) blocked the desensitizing effect of prior nicotinic exposure on subsequent nicotinic-stimulated [³H]norepinephrine release (Figure 7A).

View larger version (29K): [in this window] [in a new window]   Figure 7. 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.

PAMP Blockade of Nicotinic Desensitization of ²²Na⁺ Uptake, the First Step in Nicotinic Signal Transduction
To test whether PAMP blockade of desensitization of catecholamine release is achieved at the most proximal step in nicotinic cholinergic signaling, we studied the effect of PAMP on ²²Na⁺ uptake during nicotinic rechallenge. In the initial incubation (incubation I), PC12 cells were exposed to nicotine (10 µmol/L; 10 minutes), nicotine plus PAMP- [10-20]-amide (10 µmol/L), or buffer alone. After washout, the same groups were exposed to nicotine (10 µmol/L; 10 minutes) for measurement of cellular ²²Na⁺ uptake. Prior exposure to nicotine alone caused 82.1% diminution of ²²Na⁺ uptake on reexposure to nicotine, but prior coincubation with PAMP-[10-20]-amide (10 µmol/L) completely reversed this effect (returning to 100.1% of secretion achieved without prior nicotine exposure; Figure 7B).

PAMP Structure
Both the Chou-Fasman³³ and Robson-Garnier³⁴ algorithms predicted an -helical conformation for PAMP. The -helix displayed no amphiphilicity on hydrophobic moment plot.³⁵

Twenty known-structure remote homologues of PAMP were detected by the threading algorithm,³⁷ with up to 30% (range, 15% to 30%) pairwise sequence identity with PAMP and with Z alignment scores up to 1.83 (range, 1.34 to 1.83). The homologous regions were found in the following PDB entries: 1bct, 4 gpb, 3 mdd, 1oct, 7ccp, 1hlb, 1 mgn, 2hmz, 1trk, 3ccp, 2cyp, 1ses, 1tph, 2spo, 2 mge, 1hle (4 regions), and 2ccy. Each of these homologous regions was predominantly -helical in the homologue of known structure, especially toward the C-terminus of each segment. An -helical conformation of the PAMP sequence was therefore created and subjected to energy minimization; after 261 iterations in the MM2 force field, the root mean square gradient (slope) minimized at 0.088, indicating a local energy minimum. The resulting -helix is displayed in side (Figure 8A) and axial (Figure 8B) views.

View larger version (38K): [in this window] [in a new window]   Figure 8. Structure of PAMP. After empirical statistical algorithms and homology search suggested PAMP to be an -helix, the PAMP sequence was created as an -helix and subjected to energy minimization in an MM2 (molecular mechanics) force field. Top, side view. Bottom, axial view.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present findings reveal that PAMP-[1-20]-amide is a potent inhibitor (IC₅₀ 0.35 µmol/L) of catecholamine secretion from PC12 pheochromocytoma cells, in keeping with an earlier report²³ in bovine chromaffin cells (IC₅₀ 1.6 µmol/L). Our results also indicate that this inhibition is specific for the nicotinic cholinergic pathway, since inhibition did not occur after secretory stimulation by a variety of maneuvers that bypass the nicotinic receptor (Table 2), including membrane depolarization, calcium ionophore, or alkalinization of secretory vesicles. Purinergic P_2x ionotropic receptors, which cause secretion in response to ATP,⁴¹ were not inhibited by PAMP-[1-20]-amide (Table 2). Since P_2x and nicotinic receptors are members of the same superfamily of extracellular ligand-gated cation channels,⁴¹ the specificity of PAMP-[1-20]-amide inhibitory effects within this receptor family is apparent.

Similar potency of PAMP-[10-20]-amide in both normal PC12 (IC₅₀ 0.045 µmol/L) and neurite-bearing PC12 cells (IC₅₀ 0.083 µmol/L) suggests its activity in noradrenergic neurites as well as chromaffin cells (Figure 4).

As predicted for a nicotinic antagonist, PAMP-[10-20]-amide blocked nicotinic-stimulated Na⁺ entry (IC₅₀ 0.09 µmol/L) into the cytosol, and PAMP-[10-20]-amide potency for blockade of Na⁺ entry (Figure 5A) was comparable to its potency for blockade of catecholamine secretion (IC₅₀ 0.045 µmol/L; Figure 5A); thus, nicotinic cholinergic antagonism is an entirely sufficient mechanism to account for the PAMP antisecretory effect in this setting.

In bovine chromaffin cells, Katoh et al²³ and Niina et al²⁴ found that PAMP-[1-20]-amide inhibited not only carbachol-induced ²²Na⁺ influx (IC₅₀ 2.5 µmol/L) but also carbachol- or nicotine-induced ⁴⁵Ca²⁺ influx through voltage-dependent Ca²⁺ channels. We also found that PAMP-[1-20]-amide blocked influx of ⁴⁵Ca²⁺ from the extracellular space into the cytosol (Figure 5B), but only when such entry was triggered by nicotinic stimulation and not when triggered by membrane depolarization (Figure 5B), which opens calcium channels at a signal transduction step distal to nicotinic stimulation.⁴⁰ In NGF-differentiated PC12 cells, Takano et al²⁶ described a very different action of PAMP on Ca²⁺ influx: PAMP-[1-20]-amide inhibited divalent cation inward currents through N-type Ca²⁺ channels, and this inhibition could be abolished by pretreatment with pertussis toxin, suggesting an action of PAMP on a receptor utilizing a pertussis toxin–sensitive G protein.²⁶ Therefore, we also examined whether pertussis toxin–sensitive G proteins mediate PAMP antisecretory activity in either normal PC12 cells, which express mainly L-type Ca²⁺ channels,⁴³ or in NGF-treated PC12 cells, which express N-type Ca²⁺ channels.⁴³ The catecholamine release–inhibitory action of PAMP-[1-20]-amide was not influenced by pertussis toxin in either normal (data not shown) or NGF-treated (data not shown) PC12 cells. Thus, in this PC12 cell system, the PAMP effect to inhibit catecholamine release can be entirely accounted for by actions on the nicotinic receptor.

The inability of nicotine to overcome PAMP-[1-20]-amide secretory inhibition even at very high agonist doses (Figure 2) indicates noncompetitive nicotinic inhibition, although we have not yet established the precise site at which PAMP interacts with the nicotinic receptor. In other studies, we recently identified a novel fragment of chromogranin A, catestatin (bovine chromogranin A_344-364), which also behaves as a noncompetitive nicotinic antagonist.⁴⁴

To define the minimal active region within the PAMP sequence, we synthesized PAMP analogues by N-terminal (PAMP-[4-20]-amide, PAMP-[7-20]-amide, PAMP-[10-20]-amide, PAMP-[13-20]-amide, PAMP-[16-20]-amide) or C-terminal (PAMP-[1-17], PAMP-[1-14], PAMP-[1-11], PAMP-[1-8], PAMP-[1-5]) truncations. Although truncation of even 3 C-terminal amino acids (PAMP-[1-17]) abolished PAMP antisecretory activity, truncation from the N-terminus (up to 9 residues) failed to diminish the activity and even augmented its potency (PAMP-[10-20]-amide; IC₅₀ 0.12 µmol/L); thus, residues crucial for PAMP activity reside toward its C-terminus (Figures 3A and B), while N-terminal residues 1 through 9 (ARLDVASEF) may actually be modestly inhibitory to the overall nicotinic cholinergic actions of PAMP. Even the 8–amino acid peptide PAMP-[13-20]-amide (WNKWALSR-amide) retained substantial potency (IC₅₀ 0.53 µmol/L; Figure 3A). The enhanced potency of PAMP-[10-20]-amide (IC₅₀ 0.045 µmol/L; RKKWNKWALSR-amide; Figure 5A) may be of importance in vivo, since PAMP[9-20] has been identified in the porcine adrenal medulla and exhibits a hypotensive effect on intravenous injection in the rat.⁷ In addition, the synthetic C-terminal PAMP fragment PAMP-[12-20]-amide (KWNKWALSR-amide) is also a vasodepressor in the rat and cat.⁸ Of note, the 12 functionally crucial amino acids at the C-terminus of PAMP are completely conserved in all mammalian species thus far studied (rat, pig, and human; Table 1).

Two different lines of evidence (empirical statistical algorithms^{33 34} and knowledge-based homology modeling³⁷) predicted an -helical structure for PAMP (Figure 8). The antisecretory activity of PAMP is retained in PAMP-[13-20] (WNKWALSR-amide; IC₅₀ 0.045 µmol/L; Figure 5). Since each hydrogen-bonding turn of an -helix comprises 3.5 amino acid residues, it is conceivable that an active -helical conformation might not be sustainable with <2 turns of helix, although peptides of up to 15 to 20 residues in length are typically required for exhibition of a preference for adopting stable helical structures in solution.⁴⁵

Both substance P⁴² and PAMP (Figure 7A) require C-terminal amidation for their effects, and each blocks nicotinic desensitization of both catecholamine release (Figure 7A) and ²²Na⁺ uptake (Figure 7B). Both of these peptides are quite basic in amino acid composition (PAMP-[1-20], pI 11.14; PAMP-[10-20], pI 12.49; substance P, pI 11.14), although they otherwise do not share common primary structure (for substance P: RPKPQQFFGLM-amide).

Proadrenomedullin fragments may have complex effects on blood pressure. Even the vascular actions of adrenomedullin are multiple: while adrenomedullin itself (adrenomedullin_1–52) is a vasodilator, the adrenomedullin fragments adrenomedullin_1–25, adrenomedullin_16–21, adrenomedullin_15–22, and adrenomedullin_16–31 actually have pressor effects, in species-specific fashion,²² and the pressor effects are abrogated by sympatholytic maneuvers such as -adrenergic blockade,^{46 47} catecholamine storage depletion,^{46 47} or adrenalectomy.⁴⁶ Since the pressor effect of adrenomedullin_15–22 is not abrogated by the nicotinic cholinergic antagonist hexamethonium,⁴⁶ this response is also unlikely to be influenced by the nicotinic antagonist activity of PAMP (the present report).

In conclusion, PAMP not only inhibits nicotinic cholinergic–stimulated chromaffin cell catecholamine release but also antagonizes the desensitization of catecholamine release induced by nicotine. These 2 effects of PAMP are selective for the physiological (nicotinic cholinergic agonist) stimulus to chromaffin cell secretion. At the nicotinic receptor, the inhibition is noncompetitive with agonist. The inhibition specifically impairs nicotinic cationic (Ca²⁺ and Na⁺) signal transduction. Since PAMP is coreleased with catecholamines,²³ it may therefore contribute to a novel, autocrine homeostatic (negative-feedback) mechanism controlling catecholamine release from chromaffin cells (Figure 9). We speculate that PAMP blockade of nicotinic desensitization of catecholamine release may be advantageous to an organism, particularly during circumstances of heightened sympathetic outflow, a setting in which this action of PAMP might sustain catecholamine release.

View larger version (37K): [in this window] [in a new window]   Figure 9. 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.

	Acknowledgments

 
This work was supported by grants from the Department of Veterans Affairs, National Institutes of Health (HL55583 to Dr O'Connor and DA11311 to Dr Mahata), and the American Heart Association.

Received April 22, 1998; first decision May 18, 1998; accepted June 26, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Kitamura K, Sakata J, Kangawa K, Kojima M, Matsuo H, Eto T. Cloning and characterization of cDNA encoding a precursor for human adrenomedullin [published correction appears in Biochem Biophys Res Commun. 1994;202:643]. Biochem Biophys Res Commun. 1993;194:720–725.
2. Sakata J, Shimokubo T, Kitamura K, Nakamura S, Kangawa K, Matsuo H, Eto T. Molecular cloning and biological activities of rat adrenomedullin, a hypotensive peptide. Biochem Biophys Res Commun. 1993;195:921–927.[Medline] [Order article via Infotrieve]
3. Kitamura K, Kangawa K, Kojima M, Ichiki Y, Matsuo H, Eto T. Complete amino acid sequence of porcine adrenomedullin and cloning of cDNA encoding its precursor [published correction appears in FEBS Lett. 1994;348:220]. FEBS Lett. 1994;338:306–310.
4. Washimine H, Kitamura K, Ichiki Y, Yamamoto Y, Kangawa K, Matsuo H, Eto T. Immunoreactive proadrenomedullin N-terminal 20 peptide in human tissue, plasma and urine. Biochem Biophys Res Commun. 1994;202:1081–1087.[Medline] [Order article via Infotrieve]
5. Ishizaka Y, Ishizaka Y, Tanaka M, Kitamura K, Kangawa K, Minamino N, Matsuo H, Eto T. Adrenomedullin stimulates cyclic AMP formation in rat vascular smooth muscle cells. Biochem Biophys Res Commun. 1994;200:642–646.[Medline] [Order article via Infotrieve]
6. Kitamura K, Kangawa K, Ishiyama Y, Washimine H, Ichiki Y, Kawamoto M, Minamino N, Matsuo H, Eto T. Identification and hypotensive activity of proadrenomedullin N-terminal 20 peptide (PAMP). FEBS Lett. 1994;351:35–37.[Medline] [Order article via Infotrieve]
7. Kuwasako K, Kitamura K, Ishiyama Y, Washimine H, Kato J, Kangawa K, Eto T. Purification and characterization of PAMP-12 (PAMP[9–20]) in porcine adrenal medulla as a major endogenous biologically active peptide. FEBS Lett. 1997;414:105–110.[Medline] [Order article via Infotrieve]
8. Fry RC, Champion HC, Lawrence TC, Murphy WA, Coy DH, Kadowitz PJ. Proadrenomedullin NH2-terminal peptide (PAMP)(12–20) has vasodepressor activity in the rat and cat. Life Sci. 1997;60:L161–L167.
9. Champion HC, Czapla MA, Friedman DE, Lambert DG, Murphy WA, Coy DH, Kadowitz PJ. Tone-dependent vasodilator responses to proadrenomedullin NH2-terminal 20 peptide in the hindquarters vascular bed of the rat. Peptides. 1997;18:513–519.[Medline] [Order article via Infotrieve]
10. Champion HC, Murphy WA, Coy DH, Kadowitz PJ. Proadrenomedullin NH2-terminal 20 peptide has direct vasodilator activity in the cat. Am J Physiol. 1997;272(pt 2):R1047–R1054.
11. Shimosawa T, Fujita T. Hypotensive effect of a newly identified peptide, proadrenomedullin N-terminal 20 peptide. Hypertension. 1996;28:325–329.[Abstract/Free Full Text]
12. Shimosawa T, Ando K, Fujita T. A newly identified peptide, proadrenomedullin N-terminal 20 peptide, induces hypotensive action via pertussis toxin–sensitive mechanisms. Hypertension. 1997;30:1009–1014.[Abstract/Free Full Text]
13. Kohno M, Hanehira T, Kano H, Horio T, Yokokawa K, Ikeda M, Minami M, Yasunari K, Yoshikawa J. Plasma adrenomedullin concentrations in essential hypertension. Hypertension. 1996;27:102–107.[Abstract/Free Full Text]
14. Eto T, Washimine H, Kato J, Kitamura K, Yamamoto Y. Adrenomedullin and proadrenomedullin N-terminal 20 peptide in impaired renal function. Kidney Int Suppl. 1996;55:S148–S149.[Medline] [Order article via Infotrieve]
15. Etoh T, Kato J, Washimine H, Imamura T, Kitamura K, Koiwaya Y, Kangawa K, Eto T. Plasma proadrenomedullin N-terminal 20 peptide (PAMP) in patients with congestive heart failure. Horm Metab Res. 1997;29:46–47.[Medline] [Order article via Infotrieve]
16. Inatsu H, Sakata J, Shimokubo T, Kitani M, Nishizono M, Washimine H, Kitamura K, Kangawa K, Matsuo H, Eto T. Distribution and characterization of rat immunoreactive proadrenomedullin N-terminal 20 peptide (PAMP) and the augmented cardiac PAMP in spontaneously hypertensive rat. Biochem Mol Biol Int. 1996;38:365–372.[Medline] [Order article via Infotrieve]
17. Vari RC, Adkins SD, Samson WK. Renal effects of adrenomedullin in the rat. Proc Soc Exp Biol Med. 1996;211:178–183.[Abstract]
18. Feng CJ, Kang B, Kaye AD, Kadowitz PJ, Nossaman BD. L-NAME modulates responses to adrenomedullin in the hindquarters vascular bed of the rat. Life Sci. 1994;55:L433–L438.
19. Nossaman BD, Feng CJ, Kaye AD, DeWitt B, Coy DH, Murphy WA, Kadowitz PJ. Pulmonary vasodilator responses to adrenomedullin are reduced by NOS inhibitors in rats but not in cats. Am J Physiol. 1996;270(pt 1):L782–L789.
20. Majid DS, Kadowitz PJ, Coy DH, Navar LG. Renal responses to intra-arterial administration of adrenomedullin in dogs. Am J Physiol. 1996;270(pt 2):F200–F205.
21. Shimosawa T, Ito Y, Ando K, Kitamura K, Kangawa K, Fujita T. Proadrenomedullin NH(2)-terminal 20 peptide, a new product of the adrenomedullin gene, inhibits norepinephrine overflow from nerve endings. J Clin Invest. 1995;96:1672–1676.
22. Champion HC, Erickson CC, Simoneaux ML, Bivalacqua TJ, Murphy WA, Coy DH, Kadowitz PJ. Proadrenomedullin NH2-terminal 20 peptide has cAMP-mediated vasodilator activity in the mesenteric vascular bed of the cat. Peptides. 1996;17:1379–1387.[Medline] [Order article via Infotrieve]
23. Katoh F, Kitamura K, Niina H, Yamamoto R, Washimine H, Kangawa K, Yamamoto Y, Kobayashi H, Eto T, Wada A. Proadrenomedullin N-terminal 20 peptide (PAMP), an endogenous anticholinergic peptide: its exocytotic secretion and inhibition of catecholamine secretion in adrenal medulla. J Neurochem. 1995;64:459–461.[Medline] [Order article via Infotrieve]
24. Niina H, Kobayashi H, Kitamura K, Katoh F, Eto T, Wada A. Inhibition of catecholamine synthesis by proadrenomedullin N-terminal 20 peptide in cultured bovine adrenal medullary cells. Eur J Pharmacol. 1995;286:95–97.[Medline] [Order article via Infotrieve]
25. Nagatomo T, Shibuya I, Kabashima N, Harayama N, Ueta Y, Toyohira Y, Uezono Y, Yanagihara N, Izumi F, Wada A, Yamashita H. Proadrenomedullin N-terminal 20 peptide (PAMP) reduces inward currents and Ca2+ rises induced by nicotine in bovine adrenal medullary cells. Life Sci. 1996;59:1723–1730.[Medline] [Order article via Infotrieve]
26. Takano K, Yamashita N, Fujita T. Proadrenomedullin NH2-terminal 20 peptide inhibits the voltage-gated Ca2+ channel current through a pertussis toxin-sensitive G protein in rat pheochromocytoma-derived PC 12 cells. J Clin Invest. 1996;98:14–17.[Medline] [Order article via Infotrieve]
27. Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976;73:2424–2428.[Abstract/Free Full Text]
28. Mahata M, Mahata SK, Parmer RJ, O'Connor DT. Vesicular monoamine transport inhibitors: novel action at calcium channels to prevent catecholamine secretion. Hypertension. 1996;28:414–420.[Abstract/Free Full Text]
29. Milligan G. Techniques used in the identification and analysis of function of pertussis toxin-sensitive guanine nucleotide binding proteins. Biochem J. 1988;255:1–13.[Medline] [Order article via Infotrieve]
30. Merrifield RB. Solid phase peptide synthesis, I: the synthesis of a tetrapeptide. J Am Chem Soc.. 1963;85:2149–2154.
31. Amy C, Kirshner N. 22Na+ uptake and catecholamine secretion by primary cultures of adrenal medulla cells. J Neurochem. 1982;39:132–142.[Medline] [Order article via Infotrieve]
32. Pandol SJ, Schoeffield MS, Fimmel CJ, Muallem S. The agonist-sensitive calcium pool in the pancreatic acinar cell: activation of plasma membrane Ca2+ influx mechanism. J Biol Chem. 1987;262:16963–16968.[Abstract/Free Full Text]
33. Chou PY, Fasman GD. Empirical predictions of protein conformations. Ann Rev Biochem. 1978;47:251–276.[Medline] [Order article via Infotrieve]
34. Garnier J, Osguthorpe DJ, Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978;120:97–120.[Medline] [Order article via Infotrieve]
35. Eisenberg D, Weiss RM, Terwilliger TC. The hydrophobic moment plot detects periodicity in protein hydrophobicity. Proc Natl Acad Sci U S A. 1984;81:140–144.[Abstract/Free Full Text]
36. Protein Data Bank. Available at: http://www.pdb.bnl.gov. Accessed September 1998.
37. Rost B, Schneider R, Sander C. Protein fold recognition by prediction-based threading. J Mol Biol. 1997;270:471–480.[Medline] [Order article via Infotrieve]
38. EMBL. Predict-protein web server. Available at: http://www.embl-heidelberg.de/predictprotein. Accessed September 1998.
39. Jesior J-C, Filhol A, Tranqui D. Fold-It (light): an interactive program for Macintosh computers to analyze and display Protein Data Bank coordinate files. J Appl Cryst. 1994;27:1075.
40. Kirshner N. Sodium and calcium channels in cultured bovine adrenal medulla cells. In: Rosenheck K, Lelkes PI, eds. Stimulus-Secretion Coupling in Chromaffin Cells. Vol 2. Boca Raton, Fla: CRC Press; 1987:71–86.
41. Inoue K, Nakazawa K, Ohara-Imaizumi M, Obama T, Fujimori K, Takanaka A. Antagonism by reactive blue 2 but not by brilliant blue G of extracellular ATP-evoked responses in PC12 phaeochromocytoma cells. Br J Pharmacol. 1991;102:851–854.[Medline] [Order article via Infotrieve]
42. Boksa P, Livett BG. Desensitization to nicotinic cholinergic agonists and K+, agents that stimulate catecholamine secretion, in isolated adrenal chromaffin cells. J Neurochem. 1984;42:607–617.[Medline] [Order article via Infotrieve]
43. Rausch DM, Lewis DL, Barker JL, Eiden LE. Functional expression of dihydropyridine-insensitive calcium channels during PC12 cell differentiation by nerve growth factor (NGF), oncogenic ras, or src tyrosine kinase. Cell Mol Neurobiol. 1990;10:237–255.[Medline] [Order article via Infotrieve]
44. Mahata SK, O'Connor DT, Mahata M, Yoo SH, Taupenot L, Wu H, Gill BM, Parmer RJ. Novel autocrine feedback control of catecholamine release: a discrete chromogranin a fragment is a noncompetitive nicotinic cholinergic antagonist. J Clin Invest. 1997;100:1623–1633.[Medline] [Order article via Infotrieve]
45. Grant GA. Synthetic Peptides: A User's Guide. New York, NY: WH Freeman and Co; 1992.
46. Champion HC, Fry RC, Murphy WA, Coy DH, Kadowitz PJ. Catecholamine release mediates pressor effects of adrenomedullin-(15–22) in the rat. Hypertension. 1996;28:1041–1046.[Abstract/Free Full Text]
47. Watanabe TX, Itahara Y, Inui T, Yoshizawa-Kumagaye K, Nakajima K, Sakakibara S. Vasopressor activities of N-terminal fragments of adrenomedullin in anesthetized rat. Biochem Biophys Res Commun. 1996;219:59–63.[Medline] [Order article via Infotrieve]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mahata, M. Articles by O'Connor, D. T. Search for Related Content PubMed PubMed Citation Articles by Mahata, M. Articles by O'Connor, D. T. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL NICOTINE NICOTINE TARTRATE