Primary Structure and Function of the Catecholamine Release Inhibitory Peptide Catestatin (Chromogranin A344-364): Identification of Amino Acid Residues Crucial for Activity

Sushil K. Mahata, Manjula Mahata, Arun R. Wakade and Daniel T. O’Connor

Department of Medicine and Center for Molecular Genetics (S.K.M., M.M., D.T.O.) University of California, and Veterans Affairs San Diego Healthcare System San Diego, California 92161
Department of Pharmacology (A.R.W.) Wayne State University School of Medicine Detroit, Michigan 48201


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The novel chromogranin A fragment catestatin (bovine chromogranin A344-364; RSMRLSFRARGYGFRGPGLQL) is a potent inhibitor of catecholamine release (IC50, ~0.2–0.3 µM) by acting as a nicotinic cholinergic antagonist. To define the minimal active region within catestatin, we tested the potencies of synthetic serial three-residue deletion (amino-terminal, carboxyl-terminal, or bidirectional) fragments to inhibit nicotine-stimulated catecholamine secretion from PC12 pheochromocytoma cells. The results revealed that a completely active core sequence of catestatin was constituted by chromogranin A344-358. Nicotinic cationic signal transduction was affected by catestatin fragments in a manner similar to that for secretion (confirming the functional importance of the amino-terminus). To identify crucial residues within the active core, we tested serial single amino acid truncations or single residue substitutions by alanine on nicotine-induced catecholamine secretion and desensitization. Nicotinic inhibition by the active catestatin core was diminished by even single amino acid deletions. Selective alanine substitution mutagenesis of the active core revealed important roles for Met346, Leu348, Phe350, Arg351, Arg353, Gly354, Tyr355, Phe357, and Arg358 on catecholamine secretion, whereas crucial roles to inhibit desensitization of catecholamine release were noted for Arg344, Met346, Leu348, Ser349, Phe350, Arg353, Gly354, Tyr355, Gly356, and Arg358. We conclude that a small, 15-amino acid core of catestatin (chromogranin A344-358) is sufficient to exert the peptide’s typical inhibitory effects on nicotinic cholinergic-stimulated catecholamine secretion, signal transduction, and desensitization. These studies refine the biologically active domains of catestatin and suggest that the pharmacophores for inhibition of nicotinic secretion and desensitization may not be identical.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Catecholamine secretion from adrenal medullary chromaffin cells is influenced by cholinergic (acetylcholine and its analog nicotine) and peptidergic [pituitary adenylyl cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP)] stimuli conducted by preganglionic sympathetic neurons that are located in the intermediolateral cell column of the thoracic spinal cord and extend out to the adrenal medulla through thoracic splanchnic nerves (1, 2, 3, 4, 5). It has been reported that cholinergic and peptidergic transmitters control catecholamine secretion at different levels of neuronal activity, in that acetylcholine stimulates the secretion of norepinephrine and epinephrine, whereas PACAP and VIP predominantly stimulate the secretion of epinephrine (6).

We recently identified a novel peptide, catestatin (bovine chromogranin A344-364), which is a potent inhibitor of nicotinic cholinergic-stimulated catecholamine release from chromaffin and pheochromocytoma cells (7, 8, 9). This peptide is formed within and secreted from chromaffin granules (7, 10) and acts as a nicotinic cholinergic antagonist, with characteristic inhibitory effects on nicotinic cationic (Na+, Ca2+) signal transduction (7). Like substance P (2, 11) catestatin also blocks nicotinic agonist-induced desensitization of catecholamine release (9). This blockade of desensitization appears to be specific to neuronal nicotinic receptors, in that catestatin fails to inhibit desensitization caused by secretagogues, which bypass nicotinic receptors such as membrane depolarization, or ATP-triggered opening of P2x cation channels (9).

To identify the important amino acid residues in the catestatin sequence, we first synthesized serial three-amino acid truncation peptides (amino-terminal, carboxyl-terminal, or bidirectional) and tested their potencies at inhibition of catecholamine release. In a second round of syntheses, we examined the roles of individual amino acid residues. In so doing, we were able to define an active catestatin core as well as the roles of crucial amino acid residues for inhibition of nicotinic cholinergic-stimulated catecholamine secretion, signal transduction, and agonist desensitization.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of the Minimal, Active Core Region in the Catestatin (Bovine Chromogranin A344-364) Sequence: Effects on Catecholamine Release
Catestatin and Analog Potency to Inhibit Catecholamine Release
To test the efficacy of a catestatin peptide, [3H]norepinephrine-prelabeled cells were treated with nicotine (60 µM), either alone or in combination with 12 ascending doses (from 0.01–10 µM) of the peptide, for 30 min. Control (100%) net norepinephrine release was that in the presence of nicotine stimulation alone, without catestatin. The IC50 values of full-length and two catestatin analogs are presented in Fig. 1AGo.



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Figure 1. Identification of the Minimal Active Region in Catestatin (Bovine Chromogranin A344-364)

L-[3H]Norepinephrine-preloaded PC12 cells were treated with nicotine (60 µM) either alone or in combination with ascending doses (0.01–10 µM) of wild-type catestatin (bovine chromogranin A344-364) or its analogs (amino-terminal, carboxyl-terminal, or bidirectional truncations) for 30 min for measurement of norepinephrine secretion. Control (100%) net norepinephrine release was that in the presence of nicotine stimulation alone, without catestatin. The IC50 value of a catestatin peptide was then interpolated as the peptide dose that inhibited 50% of nicotine-stimulated secretion. Results are shown as the mean ± SEM. A, Examples of the dose-dependent effects of wild-type catestatin or catestatin analogs to inhibit nicotinic cholinergic-stimulated norepinephrine release. B, Effect of amino-terminal truncations. C, Effect of carboxyl-terminal truncations. D, Effect of bidirectional truncations. bCgA, Bovine chromogranin A. *, P < 0.05; **, P < 0.01 (vs. wild type).

 
Amino-Terminal Truncations
The catecholamine release inhibitory potency was already affected (~3-fold loss of potency; P < 0.05) after deletion of even 3 residues from the amino-terminus; deletion of 6 residues decreased potency approximately 14-fold (P < 0.01), and further deletion of 9–12 residues abolished measurable potency (IC50, >10 µM; Fig. 1BGo).

Carboxyl-Terminal Truncations
The catecholamine release inhibitory potency was preserved after deletion of 6 carboxyl-terminal residues (from IC50 of 0.41 ± 0.022 µM to IC50 of 0.18 ± 0.021 µM). Further deletions of 9–12 residues resulted in a progressive loss of potency (P < 0.01), while deletion of 15 residues abolished potency (IC50, >10 µM; Fig. 1CGo).

Bidirectional Truncations
Deletion of three residues from each terminus caused an approximately 6.6-fold loss of potency (P < 0.05). Deletion of six residues resulted in barely detectable potency (IC50, ~10 µM; P < 0.01), and further deletion abolished the potency (IC50, >10 µM; Fig. 1DGo).

Roles of Individual Amino Acids in the Active Catestatin Core Sequence (Bovine Chromogranin A344-358): Effects on Catecholamine Release
As the results of serial 3-amino acid deletions (Fig. 1Go) suggested an active 15 amino acid catestatin core (bovine chromogranin A344-358; RSMRLSFRARGYGFR), further deletion and substitution mutagenesis were conducted on this active core.

Amino-Terminal Residues
Deletion of even the first residue (Arg344) from the amino-terminus caused a 16-fold loss in catecholamine release inhibition (P < 0.01). Deletion of the next two residues (Ser345 and Met346) caused no further falls (~13- to 16-fold; P < 0.01) in potency, whereas deletion of the next four residues abolished potency altogether (IC50, >10 µM; Fig. 2AGo).



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Figure 2. Identification of Crucial Amino Acids in the Active Core of Catestatin (Bovine Chromogranin A344-358) for Inhibition of Catecholamine Release

PC12 cells were preloaded with L-[3H]norepinephrine and then treated with nicotine (60 µM) either alone or in combination with ascending doses (0.01–10 µM) of catestatin analogs (amino-terminal or carboxyl-terminal truncations, alanine-scanning mutants, change of each arginine to lysine, change of levo to dextro forms of the amino acids, or reversal of the peptide sequence) vs. wild-type active catestatin core (bovine chromogranin A344-358) for 30 min for measurement of norepinephrine secretion. Results are shown as the mean ± SEM. A, Effect of amino-terminal truncations. B, Effect of carboxyl-terminal truncations. C, Effect of alanine substitution of individual amino acids. Amino acids preceding the numbers 344–358 represent the particular amino acid (and its position) substituted by alanine. For example, in bovine chromogranin AR344A, Arg344 is substituted by Ala. Alanine substitutions are also shown in bold letters. D, Qualitative changes in multiple residues: effect of conversion of all arginine residues to lysines (lysines in bold), and conversion of all amino acids from levorotatory L to dextrorotatory D isomers (D-amino acids shown in lower case). bCgA, Bovine chromogranin A. *, P < 0.05; **, P < 0.01 (vs. wild type).

 
Carboxyl-Terminal Residues
Deletion of one carboxyl-terminal residue (Arg358) from the core resulted in an approximately 5-fold decrement in potency (Fig. 2BGo; P < 0.05). Deletion of the next four residues caused an approximately 7- to 10-fold loss of potency (P < 0.01). Potency was completely lost (IC50, >10 µM) upon deletion past five residues (Arg353 or further; Fig. 2B).

Interior Amino Acids: Alanine Substitution (Alanine-Scanning) Mutants
To test the roles of individual residue side-chains, we systematically replaced each residue in the 15-amino acid catestatin core with L-alanine (methyl-glycine), the smallest amino acid that retains chirality. Alanine substitution of bovine chromogranin A residues 344 (R344A), 345 (S345A), 347 (R347A), 349 (S349A), and 356 (G356A) had little effect on potency (P > 0.05). Alanine substitution of residues 346 (M346A), 354 (G354A), or 355 (Y355A) caused moderate (~4- to 5-fold; P < 0.01) loss of potency. Substitution of residues 348 (L348A), 350 (F350A), 351 (R351A), 353 (R353A), 357 (F357A), or 358 (R358A) caused more substantial (~8- to 21-fold; P < 0.01) loss of potency (Fig. 2CGo).

Qualitative Residue Changes: Arginine->Lysine and Levo (L)->Dextro (D)
Converting each arginine to lysine in chromogranin A344-358 caused an approximately 6-fold (P < 0.01) loss of antisecretory potency (Fig. 2DGo). Converting chromogranin A344-358 from all L (levorotatory) to all D (dextrorotatory) amino acid isomers resulted in approximately 9-fold (P < 0.01) loss of antisecretory potency (Fig. 2DGo).

Nicotinic Cholinergic Receptor Cationic Signal Transduction: Role of Catestatin Domains
We have previously shown that catestatin (bovine chromogranin A344-364) dose dependently blocks nicotine-induced uptake of 22Na+ into PC12 cells, the initial cationic stage in nicotinic cholinergic signal transduction; the IC50 of approximately 0.25 µM paralleled that for blockade of catecholamine release (IC50, ~0.20 µM) (7). Here we tested the potencies of several catestatin analogs at a 10-µM dose on nicotine-induced uptake of 22Na+; not only full-length catestatin (bovine chromogranin A344-364; secretion IC50, 0.31 ± 0.032 µM), but also two of its active analogs [bovine chromogranin A344-358 (secretion IC50, 0.22 ± 0.023 µM) and chromogranin A347-358 (secretion IC50, 3.64 ± 0.213 µM)] substantially (>80%; P < 0.01) inhibited nicotine-induced 22Na+ uptake (Fig. 3Go). However, an inactive catestatin analog (bovine chromogranin A348-358; secretion IC50, >10 µM) was relatively ineffective (~20%, although still P < 0.01) in inhibiting 22Na+ influx triggered by nicotine (Fig. 3Go).



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Figure 3. Effects of Catestatin Analogs on Nicotinic Cationic Signal Transduction (Nicotine-Induced Uptake of 22Na+) into PC12 Cells

PC12 cells were treated with 22Na+ plus nicotine (60 µM), in the presence or absence of bovine catestatin analogs (chromogranin A344-364, chromogranin A344-358, chromogranin A347-358, or chromogranin A348-358; 10 µM), for 5 min. Afterward the medium was removed, and cells were lysed for measurement of 22Na+ uptake. Control (100%) net 22Na+ uptake is that in the presence of nicotinic (60 µM) stimulation alone, without catestatin analog. Results are shown as the mean ± SEM. bCgA, Bovine chromogranin A. **, P < 0.01 vs. wild type.

 
Blockade of Nicotinic Cholinergic Desensitization of Catecholamine Release: Roles of Individual Amino Acids in the Active Catestatin Core (Bovine Chromogranin A344-358)
Desensitization, a loss of cell or tissue response after repeated or prolonged application of a stimulus, occurs in PC12 cells after prior exposure to nicotinic cholinergic agonists, and the desensitization may be blocked by catestatin (9). The phenomenon is illustrated in Fig. 4AGo: preexposure of PC12 cells to nicotine (60 µM; incubation I) decreased subsequent catecholamine release in response to rechallenge with nicotine (incubation II). Such desensitization could be prevented by inclusion of the nicotinic antagonist catestatin during agonist preexposure (Fig. 4AGo).



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Figure 4. Identification of Crucial Amino Acids in the Active Catestatin Core (Bovine Chromogranin A344-358) Sequence for Blockade of Nicotinic Cholinergic Desensitization of Catecholamine Release

L-[3H]Norepinephrine-preloaded cells were treated with nicotine (60 µM) either alone or in combination with ascending doses (0.01–10 µM) of wild-type catestatin (chromogranin A344-358) or catestatin analogs (amino-terminal or carboxyl-terminal truncations, or alanine-scanning mutants) for 10 min (incubation I) and washed twice (6 min each), then norepinephrine secretion was activated by rechallenge with nicotine (60 µM) for 10 min (incubation II). Control cells received nicotine only in incubation II. Results are shown as the mean ± SEM. A, Effect of catestatin, active 15-mer (bovine chromogranin A344-358) catestatin, or an inactive catestatin analogs (bovine chromogranin A344-352) on desensitization of catecholamine release. B, Effect of amino-terminal catestatin truncation peptides to block nicotinic desensitization of catecholamine release. C, Effect of catestatin carboxyl-terminal truncation peptides to block nicotinic desensitization of catecholamine release. D, Effect of alanine substitution on individual amino acid in catestatin (chromogranin A344-358) on inhibition of desensitization of catecholamine release. Amino acids preceding the numbers 344–358 represent the identity and position of the amino acid substituted by alanine. For example, in bovine chromogranin R344A, Arg344 is substituted by Ala. Alanine substitutions are also shown in bold letters. bCgA, Bovine chromogranin A. *, P <0.05; **, P < 0.01 (vs. wild type).

 
In these experiments, ascending doses (0.01–10 µM) of catestatin (bovine chromogranin A344-364) or the catestatin active core (bovine chromogranin A344-358) were coincubated with nicotine (60 µM) for 10 min in incubation I and washed twice to remove nicotine (6 min each), then catecholamine secretion was stimulated by rechallenge with nicotine (60 µM) for 10 min in incubation II. Prior exposure to nicotine caused an approximately 58% decrease in catecholamine release upon reexposure to nicotine. Both catestatin (bovine chromogranin A344-364; IC50, 0.54 ± 0.034 µM) and the active catestatin core (bovine chromogranin A344-358; IC50, 0.50 ± 0.022 µM) dose dependently inhibited this desensitization of catecholamine release, whereas a small, inactive catestatin fragment (bovine chromogranin A344-352; secretion IC50, >10 µM; Fig. 2BGo) also failed to inhibit desensitization (IC50, >10 µM; Fig. 4AGo).

Individual Amino-Terminal Residues
To test the role of amino-terminal amino acids in inhibition of desensitization, catestatin amino-terminal truncations were tested as described above; deletion of even the first amino-terminal residue from the catestatin core (Arg344) caused an approximately 4.8-fold loss of inhibition (P < 0.01). Deletion of the next two residues (Ser345 and Met346) caused modest decrease (~6- to 7.5-fold in potency). Deletion of four to seven additional residues (Arg347 through Phe350) resulted in complete loss of detectable potency (Fig. 4BGo).

Individual Carboxyl-Terminal Residues
Deletion of even the first carboxyl-terminal residue (Arg358) caused an approximately 9-fold decrease in inhibition of desensitization (P < 0.01). Deletion of the next four residues (Phe357 through Gly354) resulted in further loss (~14-fold) of potency. Further deletions (past Arg353) eliminated detectable potency (Fig. 4CGo).

Individual Interior Amino Acids: Alanine Substitution (Alanine-Scanning) Mutants
Alanine substitution of residues 345 (S345A), 347 (R347A), 351 (R351A), and 357 (F357A) had little effect on the ability of the peptide to inhibit desensitization (P > 0.05). Alanine substitution of residue 344 (R344A), 350 (F350A), 353 (R353A), 354 (G354A), 355 (Y355A), 356 (G356A), or 358 (R358A) caused modest (~2.2- to ~4.4-fold) loss of inhibition of desensitization (P < 0.01 to P < 0.05). Substitution of residue 346 (M346A), 348 (L348A), or 349 (S349A) caused severe (~4.8- to 7-fold) loss of potency (P < 0.01; Fig. 4DGo).

Comparative Potencies for Inhibition of Secretion vs. Desensitization: Differences in Crucial Individual Amino Acid Residues
In Fig. 5Go, we present detailed dose-response curves for the effects of alanine substitution mutants of catestatin on secretion (Fig. 5AGo) vs. desensitization (Fig. 5BGo). Rightward shifts of dose-response curves for crucial mutants are apparent in each case. Also apparent is a biphasic effect of catestatin peptides to inhibit desensitization, with a decline in activity at very high peptide concentrations.



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Figure 5. Graphic Comparison of Individual Amino Acids in the Active Catestatin Core (Bovine Chromogranin A344-358) Sequence Crucial for Blockade of Nicotinic Cholinergic-Stimulated Catecholamine Release vs. Desensitization of Catecholamine Release

Ascending 0.01- to 10-µM doses of catestatin (or catestatin analogs by alanine substitution) were used in each case. Catecholamine secretion studies were conducted as described in Fig. 2CGo, whereas desensitization studies were accomplished as noted in Fig. 4DGo. Results are shown as the mean ± SEM. A, Effects of catestatin analogs on secretion. B, Effects of catestatin analogs on desensitization. C, Direct comparison of IC50 values of each peptide for inhibition of secretion vs. desensitization. bCgA, Bovine chromogranin A. Comparing secretion vs. desensitization IC50 values: *, P < 0.02 to P < 0.03; **, P < 0.0014 to P < 0.006; ***, P < 0.0005 to P < 0.0001.

 
The relative importance of each individual amino acid in the active catestatin core, as determined by alanine scanning mutagenesis, for inhibition of nicotinic cholinergic-stimulated catecholamine secretion (Fig. 2CGo) vs. blockade of desensitization (Fig. 4DGo) is plotted in composite Fig. 5CGo.

Catestatin Effects in Vivo
In the in situ perfused rat adrenal gland (Fig. 6Go), catecholamine release was triggered by both splanchnic nerve stimulation (10 Hz, 30 sec) and nicotinic cholinergic agonists (acetylcholine or nicotine). In each case, catestatin inhibited secretion, although the inhibition was most efficient (>80%) for secretion caused by nicotine itself. Of note, neural stimulation can mobilize both acetylcholine and PACAP as ganglionic transmitters (6), and acetylcholine may trigger catecholamine secretion by both muscarinic and nicotinic pathways in the rodent (12); by contrast, catestatin is selective for the nicotinic cholinergic mechanism (7).



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Figure 6. Catestatin Effects on Catecholamine Release in Vivo: Results in the in Situ Perfused Rat Adrenal Gland

Perfused left rat adrenal glands were exposed to three stimuli to cause exocytotic release of catecholamines, electrical stimulation of the splanchnic nerve (10 Hz, 30 sec), acetylcholine (10 µM, 2 min), and nicotine (10 µM, 2 min), or to no stimulation. Perfusates were collected for 2 min for catecholamine assay. Experiments were conducted on 3 different days, and the results were averaged (mean ± SEM) after subtraction of basal (unstimulated) release. bCgA, Bovine chromogranin A. *, P < 0.03; **, P < 0.003; ***, P < 0.0001 (stimulation with or without catestatin).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study, we examined the effects of wild-type, full-length catestatin (bovine chromogranin A344-364) and several mutants on nicotinic cholinergic secretory responses of pheochromocytoma cells. Catestatin is formed within and secreted from chromaffin granules (7, 10) and is known to inhibit catecholamine release from chromaffin cells and noradrenergic neurons by acting as a nicotinic cholinergic antagonist both in cultured cells (7, 8) and in the intact adrenal gland (Fig. 6Go). Catestatin may therefore constitute an endogenous autocrine feedback regulator of sympathoadrenal activity. Finally, catestatin is also a potent, specific inhibitor of nicotinic agonist-induced desensitization (9). Here we attempted to identify active domains within the catestatin sequence that exert such actions on the nicotinic cholinergic system.

Serial three-amino acid deletions (Fig. 1Go) defined an active core (chromogranin A344-358) required for inhibition of nicotine-stimulated catecholamine release. Serial single amino acid deletions of the active core (Fig. 2Go) revealed the particular importance of amino-terminal residues; removal of even Arg344 alone reduced potency by about 12-fold. Within the interior of chromogranin A344-358, alanine substitution mutagenesis revealed crucial roles for Leu348, Phe350, Arg351, Arg353, and Arg358. Of note, both hydrophobic (Leu348, Phe350) and charged (Arg351, Arg353, Arg358) residues were important. Although removal of residue Arg344 caused a substantial (~16-fold; P < 0.01) decrement in potency (Fig. 2AGo), substitution to Ala344 (in mutant R344A; Fig. 2CGo) did not significantly (P > 0.05) affect potency, suggesting that although the presence of a residue at position 344 is important, its qualitative nature may be less important. By contrast, removal of Arg358 caused only an approximately 5-fold (P < 0.05) decrease in potency (Fig. 2BGo), whereas substitution to Ala358 (in mutant R358A; Fig. 2CGo) caused a more substantial, approximately 21-fold (P < 0.01) decrement in potency, suggesting that the precise nature of the residue’s guanidino side-chain at position 358 is important.

Earlier studies with substance P analogs showed that both the carboxyl- and amino-termini are required for inhibition of nicotine-evoked secretion (13), binding to chromaffin cell membranes (14), and inhibition of agonist-stimulated Na+ influx in pheochromocytoma cells (15).

We also tested the effects of multiple or qualitative changes in catestatin amino acids (Fig. 2DGo). Although both Arg and Lys residues carry a positive charge, conversion of each Arg to Lys decreased potency about 6-fold (P < 0.01); the guanidino nitrogens of Arg carry especially high pKa (-log10 Ka where Ka = dissociation constant) values (pKa 12.48) (16), perhaps in part accounting for the strong maximum of positive electrostatic potential at the tip of the catestatin loop (17), which may be crucial for the docking of catestatin at the electronegative vestibule of the nicotinic receptor cation pore (17). The nicotinic antagonist potency of catestatin was also diminished about 9-fold (P < 0.01) upon inversion of chirality of each amino acid (all L->D isomers), suggesting that charge alone does not determine catestatin’s potency; instead, particular side chain spatial orientations seem to be important (Fig. 2DGo).

The initial step in nicotinic cholinergic signal transduction is movement of extracellular Na+ into the intracellular space through the nicotinic receptor, which is an extracellular ligand-gated cation pore (18). We have previously shown that catestatin blocks such nicotinic signaling in parallel with its blockade of nicotine-stimulated secretion (7). In the present experiments, we have shown that although active catestatin analogs or fragments (e.g. bovine chromogranin A344-358) block nicotinic signal transduction, fragments that have no effect on secretion (e.g. bovine chromogranin A348-358; secretion IC50, >10 µM) are also relatively ineffective in blocking signal transduction (Fig. 3Go). Thus, blockade of nicotinic signaling seems to be a prerequisite for the secretory potency of catestatin peptides.

Nicotinic agonist-induced catecholamine release displays desensitization after repeated exposure to agonist (9), and catestatin is a potent inhibitor of this desensitization response (9). Here we show that although active catestatin fragments (e.g. bovine chromogranin A344-358; secretion IC50, 0.22 µM) also inhibit desensitization (IC50, 0.50 µM), inactive catestatin fragments (e.g. bovine chromogranin A344-352; secretion IC50, >10 µM), by contrast, also fail to block desensitization (IC50, >10 µM).

We have recently shown that nicotinic antagonist catestatin inhibits agonist-induced desensitization of catecholamine release. This blockade of desensitization was dose dependent (IC50, 0.54 µM; Fig. 4AGo), noncompetitive with agonist, and noncooperative (9). In the present study, the catestatin active core (bovine chromogranin A344-358) also displayed dose-dependent inhibition (IC50, 0.50 µM; Fig. 4AGo) of desensitization of catecholamine release. Previous studies revealed that the carboxyl-terminus of substance P is important for the blockade of nicotinic agonistinduced desensitization (13).

Within the catestatin core (chromogranin A344-358), different amino acid residues seemed to be crucial for the peptide’s actions to block nicotinic cholinergic agonist-stimulated catecholamine release (Fig. 2Go) compared with agonist-induced desensitization of release (Fig. 4Go). At the amino-terminus, deletion of Arg344 caused an approximately 16-fold decrement in potency for secretion inhibition (Fig. 2AGo), whereas for desensitization (Fig. 4BGo), loss of Arg344 caused only about 4.8-fold loss of potency. At the carboxyl-terminus, deletion of Arg358 caused only about a 5.1-fold decline in potency for secretion inhibition (Fig. 2BGo), but a 9.3-fold fall in potency for desensitization (Fig. 4CGo). Alanine substitution mutagenesis also suggested differences. For secretion inhibition (Fig. 2CGo), residues Met346, Leu348, Phe350, Arg351, Arg353, Gly354, Tyr355, Phe357, and Arg358 were crucial (P < 0.01), whereas for blockade of desensitization (Fig. 4DGo), residues Arg344, Met346, Leu348, Ser349, Phe350, Arg353, Gly354, Tyr355, Gly356, and Arg358 were most important (P < 0.01 to P < 0.05). Composite Fig. 5CGo (on the effects of alanine scanning mutants on inhibition of secretion vs. desensitization) illustrates how particular regions of the active catestatin core were differentially important for these two functions; alanine substitutions toward the amino-terminus (especially R344A, M346A, and S349A) had a tendency to inactivate desensitization blockade, whereas alanine substitutions toward the carboxyl-terminus (especially R358A, F357A, R353A, R351A, and F340A) were particularly apt to affect secretion inhibition.

Although we observed dissociation (Fig. 5CGo) in catestatin domains important for inhibition of nicotine-stimulated secretion (Fig. 2Go) vs. those involved in inhibiting nicotinic desensitization (Fig. 4Go), the significance of these differences is not completely apparent; however, the nicotinic heteropentamer itself may occupy different structural conformations during agonist-stimulated secretion compared with desensitization (19).

In summary, we provide substantial information about the relationship between primary structure and function of the catecholamine release inhibitory peptide catestatin (chromogranin A344-364). A core of 15 amino acids (chromogranin A344-358) seems to be sufficient to exert catestatin’s typical effects on catecholamine release (Figs. 1Go and 2Go), nicotinic cationic signal transduction (Fig. 3Go), and blockade of nicotinic agonist-induced desensitization (Fig. 4BGo). However, different amino acids within the core seem to be especially crucial for differentiating the catecholamine release inhibitory and desensitization inhibitory functions of the peptide.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
Rat PC12 pheochromocytoma cells (passage 8) (20) were used in this study; they were obtained from David Schubert, The Salk Institute (La Jolla, CA). Cells were grown at 37 C in 6% CO2 in 10-cm dishes or six-well plates in DMEM/high glucose medium supplemented with 5% heat-inactivated FBS, 10% heat-inactivated horse serum, and 1% penicillin/streptomycin (100% stocks were 10,000 U/ml penicillin G and 10,000 µg/ml streptomycin sulfate; Life Technologies, Inc., Gaithersburg, MD), as previously described (21).

Secretagogue-Stimulated Release of Norepinephrine
Norepinephrine secretion from PC12 cells was monitored as previously described (21). Cells were plated on poly-D-lysine-coated six-well polystyrene dishes (Falcon Labware, Oxnard, CA), labeled for 3 h with 1 µCi L-[3H]norepinephrine (71.7 Ci/mmol; NEN Life Science Products, Boston, MA) in 1 ml PC12 growth medium, washed twice with release buffer (150 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 10 mM HEPES pH 7), and then incubated at 37 C for 30 min in release buffer with or without secretagogues, such as nicotine (0.1–1000 µM), or cell membrane depolarization (55 mM KCl). 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), and 0.1% (vol/vol) Triton X-100. Release medium and cell lysates were assayed for L-[3H]norepinephrine by liquid scintillation counting, and results were expressed as the percent secretion: [amount released/(amount released + amount in cell lysate)] x 100. Net secretion is secretagogue-stimulated release minus basal release.

Desensitization of Catecholamine Release and Its Blockade by Catestatin
Desensitization of catecholamine release was performed as described previously (9). Briefly, cells were preloaded with L-[3H]norepinephrine and then exposed to nicotine (60 µM) for 10 min (incubation I). Cells were washed twice (6 min each) in secretion buffer, as described above, and rechallenged with nicotine (60 µM) for 10 min (incubation II), after which the cells were harvested for measurement of norepinephrine release. Control cells received nicotine (60 µM) only in incubation II.

Synthetic Peptides
Catestatin 21-mer (bovine chromogranin A344-364; RSMRLSFRARGYGFRGPGLQL), catestatin core 15-mer (bovine chromogranin A344-358; RSMRLSFRARGYGFR), or their truncation or substitution analogs were synthesized at 10–100 µmol scale by the solid phase method (22) using t-boc (tertiary butoxycarbonyl) or f-moc (9-fluorenylmethoxycarbonyl) protection chemistry and then were purified to more than 95% homogeneity by reverse phase HPLC on C18 silica columns, monitoring A280 (aromatic rings) or A214 (peptide bonds). The authenticity and purity of wild-type peptides were verified by rechromatography. Peptides were characterized by ion spray mass spectrometry on a Perkin-Elmer Corp. Sciex API III instrument (Norwalk, CT); in each case, the molecular mass of the appropriate target peptide was identified. For some experiments, small (1 µmol) scale peptide syntheses were accomplished by the pin technology (Chiron Corp., San Diego, CA), after which peptides were cleaved from the resin and washed.

Catestatin Effects in Vivo
The in situ perfused rat adrenal gland preparation was used, as previously described (6, 23). The three stimuli used to cause exocytotic release of catecholamines were electrical stimulation of the splanchnic nerve (10 Hz, 30 sec), acetylcholine (10 µM, 2 min), and nicotine (10 µM, 2 min). This degree of electrical field stimulation selectively activates splanchnic nerves, but not chromaffin cells (6). Between stimulations, secretion was allowed to return to basal values over 1 h. Incised left adrenal glands were perfused through a left renal vein cannula (continuous with the left adrenal vein) at 37 C with a Krebs-bicarbonate solution at 700 µL/gland·2 min. Two-minute adrenal effluent samples were collected from the incised glands onto an ice bath. Catecholamines were measured by spectrofluorometry (23). Experiments were conducted on 3 different days, and the results were averaged (mean ± 1 SEM).

Statistics
Curve fitting was accomplished in the program Kaleidagraph (Synergy Software, Reading, PA), using the Stineman function, which applies a geometric weight ±10% of the data range, to arrive a smoothed curve. The IC50 value of a peptide was interpolated as the concentration that achieved 50% inhibition of nicotinic-stimulated catecholamine release or desensitization. Experiments were performed in triplicate, with data (including IC50 values) reported as the mean ± 1 SEM. When only two conditions (e.g. control and experimental) were compared, the data were evaluated by unpaired t tests. When multiple conditions (e.g. several peptides) were compared, one-way ANOVA was used, followed by Dunnett’s multiple comparison post-hoc test, if appropriate. Statistical significance was concluded at P < 0.05. Statistics were computed with the program InStat (GraphPad Software, Inc., San Diego, CA).


    FOOTNOTES
 
Address requests for reprints to: Sushil K. Mahata, Ph.D., Department of Medicine, University of California (9111H), 3350 La Jolla Village Drive, San Diego, California 92161. E-mail: smahata{at}ucsd.edu

This work was supported by the Department of Veterans Affairs and the NIH (DA-11311 to S.K.M., and HL-55583 and HL-58120 to D.T.O.C.).

Received for publication November 18, 1999. Revision received June 1, 2000. Accepted for publication June 16, 2000.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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