Abstract
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 approximately 0.24 microM) and 22Na+ uptake (IC50 approximately 0.31 microM), 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.
Highlights
THE NOVEL CATECHOLAMINE RELEASE-INHIBITORY PEPTIDE CATESTATIN (CHROMOGRANIN A344–364) ACTS AT THE RECEPTOR TO PREVENT NICOTINIC CHOLINERGIC TOLERANCE*
Noncompetitive Mechanism of Peptide Antagonists at Nicotinic Cholinergic Receptors: Inhibition of Catecholamine Release from PC12 Cells—Catestatin inhibits nicotine-induced release of norepinephrine, Chromogranin A and Desensitization of Catecholamine Release with an IC50 of ϳ200 –300 nM [19]. To demonstrate whether this nicotinic inhibition is noncompetitive, we treated PC12 cells with log10-ascending doses of nicotine (10 to 1000 M) either alone or with ascending doses of catestatin or substance P (0.1 to 10 M) for 30 min, after which cells were harvested for measurement of norepinephrine release
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). In these experiments in pheochromocytoma cells, the chromogranin A fragment catestatin blocked nicotinicstimulated catecholamine release (Fig. 2), and antagonized the effect of prior nicotinic agonist exposure to desensitize the effect of a subsequent nicotinic challenge to induce secretion (Fig. 3)
Summary
THE NOVEL CATECHOLAMINE RELEASE-INHIBITORY PEPTIDE CATESTATIN (CHROMOGRANIN A344–364) ACTS AT THE RECEPTOR TO PREVENT NICOTINIC CHOLINERGIC TOLERANCE*. 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 (Naϩ, Ca2ϩ) signal transduction [19, 20]. Our results indicate that catestatin and potently inhibits such desensitization
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