Catestatin: a multifunctional peptide from chromogranin A

Abstract

In 1997, we identified a novel peptide, catestatin (CST: bovine chromogranin A [CHGA](344-364): RSMRLSFRARGYGFRGPGLQL; human CHGA(352-372): SSMKLSFRARGYGFRGPGPQL), which is a potent inhibitor of nicotinic-cholinergic-stimulated catecholamine secretion. CST shows characteristic inhibitory effects on nicotinic cationic (Na(+), Ca(2+)) signal transduction, which are specific to the neuronal nicotinic receptor. Utilizing systematic polymorphism discovery at the human CHGA locus we discovered three human variants of CST: G(364)S, P(370)L, and R(374)Q that showed differential potencies towards the inhibition of catecholamine secretion. In humans, CHGA is elevated and its processing to CST is diminished in hypertension. Diminished CST is observed not only in hypertensive individuals but also in the early-normotensive offspring of patients with hypertension, suggesting that an early deficiency of CST might play a pathogenic role in the subsequent development of the disease. Consistent with human findings, prevention of endogenous CST expression by targeted ablation (knockout) of the mouse Chga locus (Chga-KO) resulted in severe hypertension that can be "rescued" specifically by replacement of the CST peptide. CST acts directly on the heart to inhibit the inotropic and lusitropic properties of the rodent heart and also acts as a potent vasodilator in rats and humans. While the G(364)S CST variant caused profound changes in human autonomic activity and seemed to reduce the risk of developing hypertension, CST replacement rescued Chga-KO mice from dampened baroreflex sensitivity. In addition, CST has been shown to induce chemotaxis and acts as an antimicrobial as well as an antimalarial peptide. The present review summarizes these multiple actions of CST.

Figure 1

Identification of CST in immunoprecipitated bovine adrenal medullary chromaffin granules. Aliquots (200 ml) of the low molecular weight chromaffin granule peptides (devoid of full length CHGA) separated by gel filtration were immunoprecipitated (20 ml of anti-bovine CST antiserum) and then subjected to MALDI mass spectrometry (1–2 ml). (A) Immunoprecipitation by preimmune serum. (B) Immunoprecipitation by rabbit anti-bovine CST antiserum. (C) Immunoprecipitation by rabbit anti-bovine CST antiserum, followed by adsorption and elution from a C-18 (Sep-Pak) cartridge. (D) Immunoprecipitation by rabbit anti-bovine CST after M³⁴⁶ oxidation by 10 μM H₂O₂. (Reproduced with permission from The American Society for Biochemistry & Molecular Biology).

Figure 2

Identification of crucial amino acids in the active core of CST for inhibition of catecholamine release. PC12 cells prelabeled with [³H]L-norepinephrine were incubated with 60 μM nicotine, with or without logarithmically ascending doses (0.01 to 10 μM) of bovine CST for 30 min. (A). 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 bCHGAR_344A, R³⁴⁴ is substituted by A. Alanine substitutions are also shown in bold letters. (B). Graphic comparison of crucial amino acids in the active CST core (bovine CHGA_344-358) sequence for blockade of nicotinic cholinergic-stimulated catecholamine release. Control (100%) release is that in the presence of nicotine (60 μM) stimulation alone. Results are shown as the mean ± SEM. IC₅₀ values of each peptide for inhibition of secretion are given in parentheses. bCHGA, Bovine chromogranin A. (Reproduced with permission from The Endocrine Society).

Figure 3

Altered efficacy of nicotinic inhibition by naturally occurring human CST variant peptides. PC12 cells prelabeled with [³H]L-norepinephrine were incubated with 60 μM nicotine, with or without bovine CST (0.01 to 10 μM) for 30 min. Control (100%) release is that in the presence of nicotine (60 μM) stimulation alone. Results are shown as the mean ± SEM. (Reproduced with permission from The University of Chicago Press).

Figure 4

CST effects on catecholamine release ex vivo from the superfused rat adrenal gland and in vivo from mouse adrenal gland. (A). Catecholamine secretion from superfused rat adrenal glands was induced by electrical stimulation of the splanchnic nerve (10 Hz, 30 sec), acetylcholine (10 μM, 2 min), and nicotine (10 mM, 2 min), and compared to basal secretion. 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. *, P < 0.03; **, P < 0.003; ***, P < 0.0001 (stimulation with CST or without [no CST]). (Reproduced with permission from The Endocrine Society). (B). Catecholamine release by nicotinic-cholinergic stimulation and blockade by nicotinic-cholinergic antagonists, including CST. The sympathoadrenal system was activated by the nicotinic-cholinergic agonist nicotine (2.5 mg/kg intraperitoneally) for comparison with vehicle alone (mock). Animals were pretreated 30 min prior to nicotine or vehicle alone (mock) or with nicotinic cholinergic antagonists (either the classical antagonist chlorisondamine 5 mg/kg intraperitoneally, or the novel peptide antagonist CST, 2.5 mg/kg intraperitoneally) to achieve an extracellular target concentration of ~4 μM. In each experiment, n = 6 males were studied, at age 60–70 days. 30 min after nicotine or vehicle, animals were anesthetized (ketamine, 60 mg/kg of body weight; xylazine, 6.4 mg/kg of body weight; acepromazine, 1.2 mg/kg of body weight), and blood was collected for determination of plasma catecholamines. Results are shown as mean ± S.E. (Reproduced with permission from The American Society for Biochemistry & Molecular Biology).

Figure 5

CST inhibition of desensitization of catecholamine release. L-[³H]-norepinephrine preloaded cells were treated with the nicotinic cholinergic agonist nicotine (30 μM) either alone or in combination with logarithmically ascending doses (0.01 to 10 μM) of bovine CST analogs or substance P (0.1 to 10 μM) 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. (Reproduced with permission from The American Society for Biochemistry & Molecular Biology).

Figure 6

CST effect on BP in vivo in rat. Typical BP response during 7.5 V 20 Hz stimulation of the sympathetic nervous system of a pithed rat, before and after treatment with intravenous CST (0.3 μmol), without (A), or with (B) prior adrenergic blockade by propranolol (2 mg/kg) and phenoxybenzamine (20 mg/kg).

Figure 7

Vasodilation by exogenous CST infusion into the human dorsal hand vein: Stratification by sex. CST exhibited dose-dependent vasodilation (p=0.019) in phenylephrine-induced venoconstriction (~70%), with the effect most prominent in female subjects (p=0.024; covariate: age). The F value (>1) indicates that the means are significantly different from one another. Assuming maximal venodilation with the highest concentration of CST (~5000 nM), the EC₅₀ (semi-maximal effective concentration) for females was ~30 nM.

Figure 8

CST reversal of heightened SBP and dampened baroreflex sensitivity in Chga-KO mice. (A). Rescue from elevated SBP by exogenous CST: exaggerated SBP fall in Chga-KO mice. SBP was monitored by telemetry before and after administration of CST (2.5mg/kg body weight, IP) at time 0 in wild type (WT; n=4) and Chga-KO (n=4) mice. Results were analyzed by 2-way, repeated-measures ANOVA, evaluating the effects of time (p<0.001), mouse strain (p=0.009), or strain/peptide interaction (p<0.001). (B). Baroreceptor slope after treatment with phenylephrine (PE: 0.005 μg/g bw iv) or sodium nitroprusside (SNP: 0.05 μg/g bw iv) in unconscious WT and Chga-KO mice, or after supplementation of CST (4 μg/g bw iv) in KO mice. Slopes in line drawings are presented from one representative animal per group. The slope values presented at the top of the Figure are the mean values ± one SEM (msec/mmHg; n=8 animals/group). “Set point” refers to the initial/resting/starting point for each animal (for SBP, in mmHg, and R-R interval, in msec), prior to administration of drugs.

Figure 9

Model showing the autocrine-paracrine homeostatic regulation (negative-feedback) of catecholamine secretion by CST and its regulation of cardiovascular parameters including BP, cardiac contractility and vasodilation. Binding of nicotine (acetylcholine surrogate) to the nicotinic-cholinergic receptor induces extracellular Na⁺ influx resulting in depolarization of the cell membrane that causes influx of calcium through voltage-gated calcium channels. Influx of calcium induces both catecholamine release by exocytosis (all-or-none secretion) and Chga gene transcription through a pathway involving activation of protein kinase C (PKC) and mitogen activated protein kinase (MAPK). CST formed in and secreted from chromaffin granules inhibits subsequent catecholamine secretion, decreases BP and cardiac contractility, and induces vasodilation. NE, norepinephrine; ERK, extracellular signal-regulated kinase; CRE, cAMP-response element; CREB, cAMP-response element-binding protein; p-CREB: phosphorylated CREB.