Desensitization of Catecholamine Release
THE NOVEL CATECHOLAMINE RELEASE-INHIBITORY PEPTIDE CATESTATIN (CHROMOGRANIN A344-364) ACTS AT THE RECEPTOR TO PREVENT NICOTINIC CHOLINERGIC TOLERANCE*

Sushil K. MahataDagger , Manjula Mahata, Robert J. Parmer, and Daniel T. O'Connor

From the Department of Medicine and Center for Molecular Genetics, University of California, and San Diego Veterans Administration Healthcare System, San Diego, California 92161

    ABSTRACT
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Abstract
Introduction
References

Nicotinic cholinergic receptors undergo desensitization upon repeated or prolonged exposure to agonist. We investigated the effects of a novel chromogranin A catecholamine release-inhibitory fragment, catestatin (chromogranin A344-364), on agonist-induced desensitization of catecholamine release from pheochromocytoma cells. In a dose-dependent fashion, the nicotinic antagonist catestatin blocked agonist desensitization of both catecholamine release (IC50 ~ 0.24 µM) and 22Na+ uptake (IC50 ~ 0.31 µM), the initial step in nicotinic cationic signal transduction; both secretion inhibition and blockade of desensitization were noncompetitive with agonist. Desensitizing effects of the nicotinic agonists nicotine and epibatidine were blocked. This antagonist action was specific to desensitization by nicotinic agonists, since catestatin did not block desensitization of catecholamine release induced by agents which bypass the nicotinic receptor. Hill plots with slopes near unity suggested noncooperativity for catestatin effects on both nicotinic responses (secretory antagonism and blockade of desensitization). Human, bovine, and rat catestatins (as well as substance P) had similar potencies. IC50 values for secretion inhibition and blockade of desensitization paralleled each other (r = 0.76, n = 10 antagonists, p = 0.01) for several noncompetitive nicotinic antagonists. Peptide nicotinic antagonists (catestatins, substance P) were far more potent inhibitors of both secretion (p = 0.019) and desensitization (p = 0.005) than nonpeptide antagonists (trimethaphan, hexamethonium, procaine, phencyclidine, cocaine, or clonidine), and the peptides displayed enhanced selectivity to block desensitization versus secretion (p = 0.003). We conclude that catestatin is a highly potent, dose-dependent, noncompetitive, noncooperative, specific inhibitor of nicotinic desensitization, an effect which may have implications for control of catecholamine release.

    INTRODUCTION
Top
Abstract
Introduction
References

Nicotinic cholinergic receptors are extracellular ligand-gated pentameric cation channels activated by the endogenous neurotransmitter acetylcholine or its exogenous analogs such as nicotine (1, 2). The nicotinic family consists of several distinct but related receptor subtypes. 10 different nicotinic acetylcholine receptor subunits (alpha 2- alpha 8 and beta 2-beta 4) have been identified in vertebrate neurons (2, 3), and PC12 pheochromocytoma cells express alpha 3, alpha 5, alpha 7, beta 2, and beta 4 nicotinic subunits (4, 5).

Desensitization (sometimes referred to tolerance, refractoriness, subsensitivity, or down-regulation) denotes loss of cell or tissue responses after repeated or prolonged application of a stimulus. Nicotinic receptor desensitization by agonist has been known for at least 40 years, since Katz and Thesleff (6) first characterized the phenomenon at the neuromuscular junction. Nicotinic desensitization occurs at both muscle-type and neuronal-type nicotinic receptors (7, 8). Desensitization takes place in cell lines that express nicotinic functions, such as nicotine-induced release of norepinephrine from adrenal chromaffin cells (9), or agonist-induced Na+ (10, 11) or other cation (12) fluxes in PC12 pheochromocytoma cells. Cell lines transfected with defined subunits of nicotinic receptors (such as alpha 2-beta 2 (13) or alpha 4-beta 2 (14)) also desensitize upon exposure to agonist. Recently, nicotinic receptor desensitization has been shown to occur in midbrain dopamine neurons (15).

Desensitization of the nicotinic acetylcholine receptor may be modulated by a variety of factors. Noncovalent modulators include the noncompetitive nicotinic blockers (7), calcium (7), the thymic peptide hormones thymopoietin and thymopentin (7), substance P (9), and calcitonin gene-related peptide (7). Covalent modifications of receptor structure, such as receptor phosphorylation (16), may also play a role.

The neuropeptide substance P modifies the nicotinic response of chromaffin cells by two distinct actions: (i) substance P inhibits the secretion of catecholamines evoked by nicotinic agonists (17), and (ii) substance P protects against desensitization of this nicotinic response (17). These effects of substance P are selective for the nicotinic receptor ionophore complex, rather than tachykinin receptors (18).

The catestatin peptide fragment of the catecholamine secretory vesicle protein chromogranin A (bovine chromogranin A344-364) is a potent inhibitor of exocytotic catecholamine secretion from PC12 and chromaffin cells (19, 20). This peptide acts as a noncompetitive nicotinic cholinergic antagonist, with characteristic inhibitory effects on nicotinic cationic (Na+, Ca2+) signal transduction (19, 20). Indeed, catestatin is more potent than substance P in inhibiting nicotine-induced secretion of catecholamines (19).

Since catestatin and substance P have similar actions on catecholamine release, we investigated whether catestatin also affects nicotinic cholinergic desensitization of catecholamine release from chromaffin cells. Our results indicate that catestatin specifically and potently inhibits such desensitization.

    EXPERIMENTAL PROCEDURES

Cell Culture-- Rat PC12 pheochromocytoma cells (Ref. 21, at passage 8, obtained from Dr. David Schubert, Salk Institute, La Jolla, CA) were grown at 37 °C, 6% CO2, in 10-cm plates or 6-well plates, in Dulbecco's modified Eagle's/high glucose medium supplemented with 5% heat-inactivated fetal bovine serum, 10% heat-inactivated horse serum,, and 1% penicillin/streptomycin (100% stocks were 10,000 units/ml penicillin G and 10,000 µg/ml streptomycin sulfate; Life Technologies, Inc.), as described previously (19).

Secretagogue-stimulated Release of Norepinephrine-- Secretion of norepinephrine was monitored as described previously (19). PC12 cells were plated on poly-D-lysine-coated 6-well polystyrene dishes (Falcon Labware, Oxnard, CA), labeled for 3 h with 1 µCi of L-[3H]norepinephrine (71.7 Ci/mmol, NEN Life Science Products Inc., Boston, MA) in 1 ml of PC12 growth medium, washed twice with release buffer (150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM HEPES, pH 7), and then incubated at 37 °C for 30 min in release buffer with or without secretagogues, such as nicotine (0.1 to 1000 µM), or cell membrane depolarization (55 mM KCl). Release buffer for experiments involving KCl as secretagogue had NaCl reduced to 100 mM, to maintain isotonicity. After 30 min, secretion was terminated by aspirating the release buffer, and lysing cells into 150 mM NaCl, 5 mM KCl, 10 mM HEPES, pH 7, 0.1% (v/v) Triton X-100. Release medium and cell lysates were assayed for L-[3H]norepinephrine by liquid scintillation counting, and results were expressed as % secretion: (amount released/(amount released + amount in cell lysate)) × 100. Net secretion is secretagogue-stimulated release minus basal release.

Desensitization of Catecholamine Release, and Its Blockade by Catestatins, Substance P, or Other Nicotinic Cholinergic Antagonists-- Cells were preloaded with L-[3H]norepinephrine and exposed to nicotinic agonists either alone or in combination with nicotinic antagonists for periods of 10-60 min (incubation I). Cells were then washed twice (6 min each) in secretion buffer as described above and rechallenged with nicotinic agonists for a period of 10 min (incubation II) after which the cells were harvested for measurement of norepinephrine release from cells and medium.

Desensitization of 22Na+ Uptake by PC12 Cells and Its Blockade by Catestatin-- 22Na+ uptake was performed as described by Mahata et al. (19). Before experiments, PC12 cells were washed twice with 50 mM Na+-sucrose media containing 50 mM NaCl, 187 mM sucrose, 5 mM KCl, 2 mM CaCl2 and 5 mM HEPES adjusted to pH 7.4 with NaOH. To measure 22Na+ influx, this medium was then supplemented with 5 mM ouabain (to prevent active extrusion of newly taken-up 22Na+ from cells by membrane Na+/K+-ATPase) and 1.5 µCi/ml 22NaCl (specific activity: 840.21 mCi/mg, NEN Life Scinece Products Inc.). Incubation was carried out at 22 °C for 5 min, in the presence or absence of secretagogues, and then the cells were washed within 10 s with three changes (1 ml each) of 50 mM Na+-sucrose media containing 5 mM ouabain. The cells were lysed (see above) and 22Na+ in the cell lysate was measured in a gamma -counter (LKB 1274 RIAGAMMA, Wallac Inc., Gaithersburg, MD). The data were expressed as disintegrations/min/well.

Synthetic Peptides-- Chromogranin A fragment peptides such as the catestatins (bovine chromogranin A344-364, RSMRLSFRARGYGFRGPGLQL; human chromogranin A352-372, SSMKLSFRARAYGFRGPGPQL; and rat chromogranin A367-387, RSMKLSFRARAYGFRDPGPQL) were synthesized at 10-100 µmol scale by the solid-phase method (22) using t-boc or Fmoc protection chemistry, and then purified to >95% homogeneity by reversed phase high pressure liquid chromatography on C-18 silica columns, monitoring A280 (aromatic rings) or A214 (peptide bonds). Authenticity and purity of peptides were verified by re-chromatography, as well as electrospray-ionization or MALDI mass spectrometry, or amino acid composition. Substance P (RPKPQQFFGLM-amide; 40) was obtained from Peninsula Laboratories (Belmont, CA).

Statistics-- Results are recorded as the mean value ± one S.E. Mean value comparisons were made by t test or by nonparametric test (Wilcoxon or Mann Whitney), when data were not normally distributed. Regressions were done by Pearson product moment correlations, or by Spearman rank order correlations, when data were not normally distributed. Data were analyzed and plotted with the programs InStat (GraphPad Software, San Diego, CA), Kaleidagraph (Abelbeck Software, Reading, PA), or CricketGraph (Cricket Software, Malvern, PA).

    RESULTS

Nicotinic Cholinergic Desensitization: Effects on Norepinephrine Release-- PC12 cells were prelabeled with [3H]norepinephrine and exposed in an initial incubation (incubation I) to several doses (0 to 1000 µM) of nicotine for 10 min. The medium was collected for measurement of norepinephrine release in incubation I. Cells were then washed twice (6 min each) and rechallenged with a single dose of nicotine (10 µM) during a subsequent incubation (incubation II) for 10 min, after which both medium (supernatant) and cells were harvested for measurement of norepinephrine release. In the initial period (incubation I), nicotine released norepinephrine in a dose-dependent fashion, with the usual biphasic dose-response curve (Fig. 1A). Pre-exposure of PC12 cells to nicotine decreased subsequent norepinephrine release by 57.4 to 69.9% upon agonist rechallenge (Fig. 1B), and the apparent desensitization was nearly maximal for even the smallest initial exposure (incubation I) dose of nicotine, 10 µM (Fig. 1, A and B).


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Fig. 1.   Nicotinic cholinergic effects on catecholamine release: initial stimulation of secretion, with subsequent desensitization upon second exposure to agonist. A, nicotine effect to release catecholamines. PC12 cells were prelabeled with L-[3H]norepinephrine and then treated with ascending log doses of nicotine for 10 min (incubation I). Supernatants were collected for measurement of norepinephrine secretion. The data are expressed as net L-[3H]norepinephrine secretion (i.e. minus basal (nonstimulated) secretion). Basal release in incubation I was 437 ± 19 cpm/well (n = 3). B, nicotinic desensitization of catecholamine release. PC12 cells were washed twice (6 min each) after collection of supernatants in incubation I and then re-exposed to ascending log doses of nicotine for 10 min (incubation II). Supernatants were collected for measurement of L-[3H]norepinephrine secretion. Basal release in incubation II was 388 ± 12 cpm/well (n = 3).

Noncompetitive Mechanism of Peptide Antagonists at Nicotinic Cholinergic Receptors: Inhibition of Catecholamine Release from PC12 Cells-- Catestatin (bovine chromogranin A344-364) inhibits nicotine-induced release of norepinephrine, with an IC50 of ~200-300 nM (19). To demonstrate whether this nicotinic inhibition is noncompetitive, we treated PC12 cells with log10-ascending doses of nicotine (10 to 1000 µM) either alone or with ascending doses of catestatin or substance P (0.1 to 10 µM) for 30 min, after which cells were harvested for measurement of norepinephrine release. Even very high doses of nicotine (100-1000 µM) could not overcome catestatin or substance P inhibition of norepinephrine release (Fig. 2), suggesting noncompetitive inhibition.


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Fig. 2.   Noncompetitive nicotinic cholinergic inhibition by peptide antagonists: inhibition of catecholamine release from PC12 cells. Noncompetitive inhibition of catecholamine release by catestatin or substance P. L-[3H]Norepinephrine preloaded cells were treated with: A, logarithmically ascending doses (10-1000 µM) of nicotine; B, 10 µM nicotine; C, 100 µM nicotine; or D, 1 mM nicotine either alone or in combination with logarithmically ascending doses (0.1-10 µM) of bovine catestatin or human substance P for 30 min, before measurement of norepinephrine secretion.

When the effects of catestatin versus substance P are studied at several agonist concentrations (Fig. 2), catestatin had consistently greater potency (lower IC50) than substance P, especially at higher agonist concentrations (~11.6-fold lower IC50 for catestatin at 1 mM nicotine). The competitive nicotinic antagonist alpha -bungarotoxin (50 nM) did not affect nicotine (60 µM)-stimulated catecholamine release from PC12 cells: nicotine alone released 18.6 ± 0.62% of cell norepinephrine stores, while addition of alpha -bungarotoxin to nicotine still yielded 18.4 ± 1.1% release.

Peptide Nicotinic Antagonists (Including Catestatin) Block Agonist-induced Desensitization of Catecholamine Release in a Dose-dependent, Noncompetitive Effect-- Nicotinic antagonists may alter agonist desensitization responses (23). Is nicotinic agonist-induced desensitization of norepinephrine release (Fig. 1B) influenced by noncompetitive nicotinic antagonists such as catestatin? PC12 cells were exposed to nicotinic agonists (nicotine (10 µM) or epibatidine (1 µM)) for 10 min either alone or in combination with log10-ascending doses (0.1-10 µM) of catestatin or substance P, then washed twice (6 min each), and finally rechallenged with nicotine (10 µM, 10 min) for measurement of norepinephrine release. Pretreatment with catestatin or substance P caused dose-dependent blockade of nicotinic desensitization of norepinephrine secretion (Fig. 3), whether that desensitization was elicited by nicotine (Fig. 3A) or epibatidine (Fig. 3B). Even very high doses of nicotinic agonist pre-exposure (10-1000 µM nicotine) failed to overcome the effects of peptide antagonists (catestatin or substance P; 10 µM) to block prior agonist desensitization of secretion (Fig. 3C), suggesting that this action of the peptides is also noncompetitive with respect to agonist.


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Fig. 3.   Effect of catestatin or substance P to block nicotinic cholinergic desensitization of catecholamine release: a dose-dependent, noncompetitive effect. A, catestatin or substance P protection against desensitization of catecholamine release induced by prior exposure to the agonist nicotine (10 µM). L-[3H]Norepinephrine preloaded cells were treated with nicotinic cholinergic agonist (nicotine, 10 µM) either alone or in combination with logarithmically ascending doses (0.1-10 µM) of bovine catestatin or substance P for 10 min (incubation I), washed twice (6 min each), and rechallenged with nicotine (10 µM) for 10 min (incubation II) before measurement of norepinephrine secretion. Control cells received nicotine only in incubation II. B, catestatin or substance P blockade of desensitization of catecholamine release induced by prior exposure to the agonist epibatidine (1 µM). Same conditions as A, but initial agonist challenge in incubation I with the nicotinic agonist epibatidine (1 µM). C, nicotinic antagonist peptide blockade of prior agonist desensitization is noncompetitive with regard to agonist. L-[3H]Norepinephrine preloaded cells were treated with ascending doses of nicotinic cholinergic agonist (nicotine, 10-1000 µM) either alone or in combination with desensitization blocking doses (10 µM; see A) of bovine catestatin or substance P for 10 min (incubation I), washed twice (6 min each), and rechallenged with ascending doses of nicotine (10-1000 µM) for 10 min (incubation II) before measurement of norepinephrine secretion. Control cells received nicotine only in incubation II.

Relative Potencies of Peptide Nicotinic Antagonists (Catestatins or Substance P) to Block Agonist-induced Secretion Versus Desensitization of Catecholamine Release-- For secretion, PC12 cells were treated with nicotine (60 µM), either alone or in combination with log10-ascending doses (0.1-10 µM) of catestatins or substance P. Each peptide inhibited nicotine-stimulated catecholamine release, in a dose-dependent fashion. IC50 values for inhibition of nicotinic-stimulated secretion were: human catestatin, ~0.31 µM; bovine catestatin, ~0.3 µM; rat catestatin, ~1.2 µM; or substance P, ~1.0 µM.

For nicotinic desensitization, PC12 cells were pre-exposed to nicotine (10 µM, 10 min), either alone or in combination with log10-ascending doses (0.1-10 µM) of catestatins or substance P in incubation I, then washed twice (6 min each), and re-exposed to nicotine (10 µM, 10 min) in incubation II. Each peptide blocked desensitization in a dose-dependent fashion. IC50 values for blockade of nicotinic desensitization of release were: human catestatin, ~0.22 µM; bovine catestatin, ~0.24 µM; rat catestatin, ~0.62 µM; or substance P, ~0.25 µM.

Lack of Cooperativity in Catestatin's Effect to Inhibit Secretion or Desensitization: Hill Plots-- Hill plots (Fig. 4) were done for the fractional effect of catestatin to inhibit nicotinic-stimulated catecholamine secretion (Fig. 4A) versus desensitization of secretion (Fig. 4B). In each case, the plots were linear over a wide range of catestatin concentrations (2 orders of magnitude), and the Hill slopes were near unity (slope = 0.878 for secretion, slope = 0.958 for desensitization). Thus, in each case the catestatin effect seems to be noncooperative.


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Fig. 4.   Catestatin effects to inhibit secretion or block desensitization: Hill plots. Log10 of bovine catestatin dose (µM) is plotted against log10 of its fractional effect to inhibit nicotinic agonist-induced secretion (panel A) or to block nicotinic agonist-induced desensitization (panel B). v, % inhibition by any given dose of catestatin. V, maximal % inhibition at the highest dose of catestatin used (10 µM). A, catestatin inhibition of nicotinic agonist-stimulated secretion. L-[3H]Norepinephrine preloaded cells were treated with 60 µM nicotine either alone or in combination with ascending doses (0.01-10 µM) of bovine catestatin for 30 min, before measurement of norepinephrine secretion. n = 6 catestatin concentrations, r = 0.988, p < 0.0001, slope = 0.878. B, catestatin blockade of nicotinic agonist-induced desensitization. L-[3H]Norepinephrine preloaded cells were treated with 10 µM nicotine either alone or in combination with logarithmically ascending doses (0.01-10 µM) of bovine catestatin for 10 min (incubation I), washed twice (6 min each), and then secretion was rechallenged with nicotine (10 µM) for 10 min (incubation II) before measurement of norepinephrine secretion. Control cells received nicotine only in incubation II. n = 5 catestatin concentrations, r = 0.989, p < 0.0001, slope = 0.958.

Specificity of Catestatin to Block Nicotinic Desensitization of Norepinephrine Release-- After PC12 cell exposure to the secretagogues nicotine (10 µM), KCl (55 mM), or ATP (100 µM), either alone or with catestatin (10 µM) for 10 min, cells were washed twice (6 min each), and then re-exposed to nicotine (10 µM), KCl (55 mM), or ATP (100 µM) for 10 min, before harvesting for measurement of [3H]norepinephrine release (Fig. 5). Catestatin blocked apparent desensitization of norepinephrine release only when such desensitization was induced by nicotine (Fig. 5A), and not by stimuli which bypass the nicotinic receptor in triggering release of norepinephrine, such as 55 mM KCl or 100 µM ATP (Fig. 5, B and C).


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Fig. 5.   Specificity of catestatin effect to block only nicotinic desensitization of norepinephrine release. A, catestatin blockade of desensitization of catecholamine release induced by prior nicotine. L-[3H]Norepinephrine preloaded cells were treated with nicotine (60 µM) either alone or in combination with bovine catestatin (10 µM) for 10 min (incubation I), washed twice (6 min each), and then secretion was rechallenged with nicotine (10 µM) for 10 min (incubation II) before measurement of norepinephrine secretion. Control cells received nicotine only in incubation II. B, lack of catestatin blockade of desensitization of catecholamine release induced by prior membrane depolarization with KCl. L-[3H]Norepinephrine preloaded cells were treated with KCl (55 mM) either alone or in combination with bovine catestatin (10 µM) for 10 min (incubation I), washed twice (6 min each), and then secretion was rechallenged with KCl (55 mM) for 10 min (incubation II) before measurement of norepinephrine secretion. Control cells received KCl only in incubation II. C, lack of catestatin blockade of desensitization of catecholamine release induced by prior exposure to the P2x receptor agonist ATP. L-[3H]Norepinephrine preloaded cells were treated with ATP (100 µM) either alone or in combination with bovine catestatin (10 µM) for 10 min (incubation I), washed twice (6 min each), and then secretion was rechallenged with ATP (100 µM) for 10 min (incubation II) before measurement of norepinephrine secretion. Control cells received nicotine only in incubation II.

Catestatin Blockade of Nicotinic Desensitization of Extracellular 22Na+ Uptake as the Initial Step in Nicotinic Signal Transduction-- PC12 cells were exposed to nicotine (10 µM; 10 min), nicotine plus catestatin (10 µM), or vehicle (mock) in incubation I. After washing (6 min twice), the same cell groups were treated again with nicotine (10 µM; 10 min) in the presence of 22Na+. Pre-exposure to nicotine alone caused 82.1% diminution of subsequent 22Na+ uptake, but initial co-incubation with catestatin dose dependently reversed this nicotine effect (Fig. 6). Catestatin IC50 values for blockade of nicotinic desensitization were indistinguishable for 22Na+ uptake (IC50 = 0.31 µM; Fig. 6) or catecholamine release (IC50 = 0.24 µM).


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Fig. 6.   Catestatin blockade of desensitization of extracellular Na+ uptake, the first step in nicotinic cholinergic signal transduction. PC12 cells were treated with nicotine (10 µM) either alone or in combination with bovine catestatin (10 µM) versus vehicle (mock) in incubation I, then washed twice (6 min each), and finally treated with nicotine (10 µM) for 10 min in incubation II in the presence of extracellular 22Na+. Results of 22Na+ uptake into cells are shown.

Relative Potencies of Nonpeptide Nicotinic Antagonists to Block Agonist-induced Secretion Versus Desensitization of Catecholamine Release-- To see whether the effects of peptide (catestatin and substance P) nicotinic antagonists is comparable to that exerted by nonpeptide nicotinic antagonists we repeated the experiments described above using several nonpeptide nicotinic antagonists and found that the IC50 values for inhibition of nicotinic-stimulated secretion were (Fig. 7A): trimethaphan, ~8.3 µM; procaine, ~8.45 µM; phencyclidine, ~0.81 µM; cocaine, ~4.83 µM; clonidine, 19.4 µM; or hexamethonium, ~40 µM. IC50 values for blockade of nicotinic desensitization of release were (Fig. 7B): trimethaphan, ~18.0 µM; procaine, ~14.4 µM; phencyclidine, >10 µM; cocaine, ~23.1 µM; clonidine, ~4.91; or hexamethonium, >1000 µM.


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Fig. 7.   Relative potencies of nonpeptide noncompetitive nicotinic antagonists to block agonist-induced secretion versus desensitization of catecholamine release. A, inhibition of catecholamine release: [3H]-L-norepinephrine preloaded cells were treated with nicotine (60 µM) either alone or in combination with logarithmically ascending doses (0.1 to 1000 µM) of antagonist for 30 min before measurement of norepinephrine secretion. B, blockade of desensitization of catecholamine release: L-[3H]norepinephrine preloaded cells were treated with nicotine (10 µM) either alone or in combination with logarithmically ascending doses (0.1-10 µM) of antagonist for 10 min (incubation I), washed twice (6 min each), and then secretion was rechallenged with nicotine (10 µM) for 10 min (incubation II) before measurement of norepinephrine secretion. Control cells received nicotine only in incubation II.

Parallel Potencies for Nicotinic Antagonists to Block Agonist-induced Secretion Versus Desensitization of Catecholamine Release: Contrasting Effects of Peptide Versus Nonpeptide Antagonists-- Nicotinic antagonist IC50 values for inhibition of nicotinic-stimulated secretion correlated with their IC50 values for blockade of desensitization (r = 0.76, n = 10 antagonists, p = 0.01; Spearman rank-order correlation).

Of note, peptide antagonists as a group had generally lower IC50 values for both secretion and desensitization than did nonpeptide antagonists. When peptide versus nonpeptide antagonists were so stratified (Table I), peptide antagonists had not only higher molecular weights (p < 0.0001), and lower secretion IC50 values (p = 0.019) and desensitization IC50 values (p = 0.005), but also increased IC50 ratios of secretion/desensitization (p = 0.03). Thus, as compared with nonpeptides, peptide antagonists seemed to have a preferential effect to block desensitization (Table I).

                              
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Table I
Nicotinic antagonist effects on secretion versus desensitization: effects of peptides (catestatins or substance P) versus nonpeptides (trimethaphan, procaine, phencyclidine, cocaine, clonidine, or hexamethonium)
Values are mean ± 1 S.E.

Reversibility of Nicotinic Antagonist Blockade of Catecholamine Secretion-- Potential reversibility of secretory inhibition for both peptide (catestatin and substance P) and nonpeptide (trimethaphan, procaine, phencyclidine, cocaine, clonidine, and hexamethonium) nicotinic antagonists was studied by preincubation with a secretory inhibitory dose of antagonist, followed by extensive washing of the cells, with subsequent secretory stimulation by the agonist nicotine (100 µM) (Fig. 8).


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Fig. 8.   Test of reversibility of noncompetitive nicotinic antagonist effects to block agonist-stimulated catecholamine release. L-[3H]Norepinephrine preloaded PC12 cells were treated for 15 min with several nicotinic antagonists, at a concentration previously shown to exert substantial secretory inhibition by each antagonist (Fig. 7). Cells were then washed twice (2 min/wash) with secretion buffer, and secretion was then stimulated by the nicotinic agonist nicotine (100 µM). After 30 min, medium and cells were harvested for measurement of net % secretion of norepinephrine.

Secretion inhibitory effects of the nonpeptide antagonists phencyclidine and cocaine persisted even after washout, while effects of the other nonpeptide antagonists, as well as the peptide antagonists, seemed to be readily reversible (Fig. 8).

    DISCUSSION

In these experiments in pheochromocytoma cells, the chromogranin A fragment catestatin not only blocked nicotinic-stimulated catecholamine release (Fig. 2), but also antagonized the effect of prior nicotinic agonist exposure to desensitize the effect of a subsequent nicotinic challenge to induce secretion (Fig. 3). Both secretory blockade and desensitization blockade were noncompetitive with agonist (Figs. 2 and 3C), dose-dependent (Fig. 3) and noncooperative (Fig. 4).

Noncompetitive nicotinic cholinergic antagonists may modulate agonist desensitization of the receptor (24). In muscle-type nicotinic receptors, some noncompetitive nicotinic antagonists, particularly local anesthetics and some antibiotics, seem to exert their antagonist effects at least in part by acute desensitization of the receptor accompanied by an increase in agonist affinity; such effects have been reported for both the Torpedo californica receptor (25) and for the muscle-type receptor on mammalian cells (24).

By contrast, in PC12 pheochromocytoma cells we found that noncompetitive peptide (19) (Fig. 2) neuronal nicotinic antagonists seemed to block nicotinic desensitization (Fig. 3). This blockade of desensitization seemed to be specific, in that the nicotinic antagonist catestatin did not affect subsequent secretory responses to other chromaffin cell secretagogues (Fig. 5), such as membrane depolarization by KCl, or ATP-triggered opening of P2x cation channels (19, 26). Furthermore, in dose-dependent fashion, catestatin blocked nicotinic desensitization of the earliest event in nicotinic signal transduction, extracellular Na+ entry into the cytosol (Fig. 6), and its potencies were virtually identical for blockade of desensitization of nicotinic-stimulated catecholamine release (IC50 = 0.24 µM) and 22Na+ influx (IC50 = 0.31 µM; Fig. 6). Thus, a noncompetitive neuronal nicotinic antagonist seems to block desensitization in close parallel to its blockade of agonist-induced cation flux through the nicotinic pore. These results indicate that catestatin blockade of desensitization of catecholamine release is highly selective for the nicotinic receptor ionophore complex, a finding in line with extensive reports for substance P effects on chromaffin cells (9).

Several species' forms of catestatin (bovine, human, and rat) exhibited similar effects to block nicotinic desensitization of catecholamine release, consistent with the sequence conservation of the catestatin region of chromogranin A across mammalian species (19).

The ability of noncompetitive nicotinic antagonists to block desensitization paralleled their antagonism of nicotinic-stimulated secretion (r = 0.76, n = 10 antagonists, p = 0.01). Intriguingly, peptide and nonpeptide antagonists seemed to group into two potency families: indeed, there was no overlap at all for potency to block desensitization (Table I). Peptide antagonists as a group had superior potency to block both secretion (p = 0.019) and desensitization (p = 0.005), as well as higher secretion/desensitization ratios (p = 0.03) (Table I). Small, nonpeptide nicotinic antagonists may exert their actions by lodging deep within the nicotinic cation pore, bounded by the pore's membrane-spanning alpha -helical M2 domains (23). By contrast, catestatin is far too large to effectively enter the membrane-spanning portion of the cation pore; instead, it is likely to block nicotinic signaling by occluding the ~25-Å cation pore vestibule at its extracellular orifice (27). Thus, peptide and nonpeptide nicotinic antagonists differ not only in size, therefore likely receptor site of action to exert noncompetitive nicotinic blockade, but also in the potency of such action. It is conceivable that the superior potencies of peptide antagonists (Fig. 3, A and B; Table I) arise from more effective occlusion of the nicotinic cation pore as a result of vestibule occlusion by the larger antagonists.

Two of the nonpeptide nicotinic antagonists (phencyclidine and cocaine) also differed from the peptide antagonists in that the smaller molecules displayed relatively irreversible secretory inhibition (Fig. 8). Such smaller antagonists, with consequent increased ability to penetrate the nicotinic cation pore, may be more difficult to remove from the pore with a simple buffer wash. Other properties of such small antagonists, such as hydrophobicity or charge, may also be important for relative irreversibility.

Nicotinic receptor subunit composition seems to determine both activation and desensitization responses to agonists. Expressing neuronal nicotinic receptors in Xenopus oocytes, Fenster et al. (28) showed that receptors containing alpha 4 subunits were more sensitive than alpha 3-containing receptors to both activation and desensitization by nicotine. Fenster et al. (28) therefore suggested that the alpha  subunit type largely determines the apparent agonist affinity of active and desensitized states of a nicotinic receptor, while the beta  subunit influences the time course of development of desensitization; other studies (29, 30) suggest that the alpha  subunit also can influence the desensitization time course. Because homomeric alpha 7 receptors also desensitize, it is likely that the beta  subunit plays a modulatory rather than a permissive role in desensitization (28). alpha 3, alpha 5, alpha 7, beta 2, and beta 4 nicotinic subunit expression is detectable in PC12 cells (4, 5, 31), although the precise subunit stoichiometry of any given nicotinic heteropentamer may be difficult to establish. Lack of effect of alpha -bungarotoxin on nicotinic-stimulated PC12 cell catecholamine release weighs against a role for alpha 7 subunits in the secretion we observed (23).

Although detailed physical models of agonist mechanisms of both channel gating and desensitization have yet to emerge, electron diffraction images of the nicotinic acetylcholine receptor trapped in a channel-open conformation by rapid freezing suggest tilting and twisting of the M2 transmembrane alpha -helices accompanied by structural alterations near the neurotransmitter-binding site (32). During desensitization, photoactivatable probe incorporation studies suggest alterations in both the neurotransmitter-binding site and the pore-lining M2 amino acid side chains (33, 34). Fourier transform infrared difference spectroscopy and 1H/2H exchange difference spectroscopy (35) suggest that desensitization causes conformational changes principally in the agonist binding site.

Peptide antagonist fractional dose dependence for secretory inhibition and blockade of desensitization were parallel, as judged by Hill plots (Fig. 4), and in each case the Hill slopes were near unity. This suggests that peptide antagonist effects are noncooperative, and that such desensitization-blocking peptide antagonists binds to the same site, at the same affinity, on the nicotinic receptor to exert both noncompetitive antagonism and inhibition of desensitization.

The catestatin data (Figs. 2-6, Table I) are of principal interest because this peptide is formed endogenously from chromogranin A, a protein stored and released with catecholamines under nicotinic cholinergic stimulation (19). Thus, chromaffin cells and sympathetic axons may thereby generate an endogenous antagonist of nicotinic cholinergic desensitization. We have estimated that the concentration of just released chromogranin A in the local extracellular vicinity of the chromaffin cell may be as high as ~400 µM (19). Chromogranin A circulates in humans at concentrations ranging from ~1 nM, in healthy controls, to ~1 µM, in disease states such as pheochromocytoma (36), other neuroendocrine neoplasia (37), or renal failure (38). Since we found that even 100 nM catestatin caused substantial blockade of nicotinic desensitization, the potential physiologic consequences of such a mechanism cannot be ignored.

We speculate that catestatin 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 catestatin might sustain catecholamine release. Indeed, substance P may augment catecholamine release by protection against nicotinic desensitization during prolonged stress in an organism (39).

    FOOTNOTES

* This work was supported by the Department of Veterans Affairs, National Institutes of Health Grants HL55583 (to D. T. O'C.) and DA11311 (to S. K. M.), and the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept of Medicine and Center for Molecular Genetics, University of California, San Diego (9111H), 3350 La Jolla Village Dr., San Diego, CA 92161. Tel.: 619-552-8585 (ext. 2634); Fax: 619-552-7549; E-mail: smahata{at}ucsd.edu.

    REFERENCES
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Abstract
Introduction
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