Skip to main content
NCBI home page
Endocrinology logoLink to Endocrinology
. 2008 Jun 5;149(10):4780–4793. doi: 10.1210/en.2008-0318

The Antihypertensive Chromogranin A Peptide Catestatin Acts as a Novel Endocrine/Paracrine Modulator of Cardiac Inotropism and Lusitropism

Tommaso Angelone 1, Anna Maria Quintieri 1, Bhawanjit K Brar 1, Pauline T Limchaiyawat 1, Bruno Tota 1, Sushil K Mahata 1, Maria Carmela Cerra 1
PMCID: PMC2582908  PMID: 18535098

Abstract

Circulating levels of catestatin (Cts; human chromogranin A352–372) decrease in the plasma of patients with essential hypertension. Genetic ablation of the chromogranin A (Chga) gene in mice increases blood pressure and pretreatment of Chga-null mice with Cts prevents blood pressure elevation, indicating a direct role of Cts in preventing hypertension. This notable vasoreactivity prompted us to test the direct cardiovascular effects and mechanisms of action of wild-type (WT) Cts and naturally occurring human variants (G364S-Cts and P370L-Cts) on myocardial and coronary functions. The direct cardiovascular actions of WT-Cts and human variants were determined using the Langendorff-perfused rat heart. WT-Cts dose-dependently increased heart rate and coronary pressure and decreased left ventricular pressure, rate pressure product and both positive and negative LVdP/dt. WT-Cts not only inhibited phospholamban phosphorylation, but also the inotropic and lusitropic effects of WT-Cts were abolished by chemical inhibition of β2-adrenergic receptors, Gi/o protein, nitric oxide or cGMP, indicating involvement of β2-adrenergic receptors-Gi/o protein-nitric oxide-cGMP signaling mechanisms. In contrast, G364S-Cts did not affect basal cardiac performance but abolished isoproterenol-induced positive inotropism and lusitropism. P370L-Cts decreased rate pressure product and inhibited only isoproterenol-induced positive inotropism and lusitropism by 70%. Cts also inhibited endothelin-1-induced positive inotropism and coronary constriction. Taken together, the cardioinhibitory influence exerted on basal mechanical performance and the counterregulatory action against β-adrenergic and endothelin-1 stimulations point to Cts as a novel cardiac modulator, able to protect the heart against excessive sympathochromaffin overactivation, e.g. hypertensive cardiomyopathy.

CHROMOGRANIN A (CgA), an index member of the chromogranin/secretogranin protein family, is a secretory proprotein (1,2,3) that is endoproteolytically processed to give rise to several peptides of biological importance including the dysglycemic hormone pancreastatin (4), vasodilator vasostatin (5), and the catecholamine release inhibitory peptide catestatin [human CgA352–372, bovine CgA344–364; (6,7,8,9,10)]. CgA is costored with catecholamines, and its corelease documents exocytosis as the mechanism of physiologic catecholamine release in humans (11). Catestatin (Cts) and pancreastatin have been postulated as important counterregulatory hormones in zero steady-state error homeostasis [i.e. the perfect equilibrium generated by the balance between two counterregulatory hormones (12)], a role now extended also to vasostatin 1 (VS-1) in cardiac biology (Ref. 3 and references therein). The importance of CgA in cardiovascular homeostasis in man is documented by its increased plasma levels in various diseases, such as neuroendocrine tumors (13,14) and chronic heart failure (15) and its colocalization with brain natriuretic peptide and overexpression in human dilated and hypertrophic cardiomyopathy (16). Basal plasma levels of CgA correlate with sympathetic tone (17), showing a high heritability (18). Plasma level of Cts peptide (∼1.5 nm) decreases in patients with essential hypertension, the complex chronic disorder with a poorly understood pathogenesis, and also in normotensive subjects with a family history of hypertension and increased epinephrine secretion (19); this implicates that Cts is an inhibitor of chromaffin cell catecholamine secretion in vivo. Genetic ablation of the chromogranin A (Chga) gene results in high blood pressure in mice, which can be rescued by either pretreatment with Cts peptide or the introduction of the human CHGA gene in the Chga−/− background (20). Accordingly, decreased Cts levels may predict augmented adrenergic responses to stressors and increased risk of hypertension.

To understand human genetic variation at the level of the CHGA gene, the group of O’Connor and his colleagues at the University of California, San Diego (21), systematically searched for polymorphisms at the CHGA locus and reported three naturally occurring amino acid substitution variants within the region of Cts. Although two of the Cts variants (Pro370Leu and Arg374Gln) were reported to be relatively rare (minor allele frequencies 0.3–0.6%, respectively), one variant, Gly364Ser (S352SMKLSFRARGYS364FRGPGPQL372; position in the mature CgA protein) showed an allele frequency of about 3–4%. Boldface represents amino acid variation. These variants displayed differential potencies toward inhibition of nicotinic cholinergic agonist-evoked catecholamine secretion from sympathochromaffin cells in vitro with the following rank order of potency Pro370Leu>wild- type (WT)>Gly364Ser>Arg374Gln (11). In vivo, human carriers of the 364Ser allele had profound alterations in autonomic activity, in both the parasympathetic and sympathetic branches, and may be protected against the future development of hypertension, especially evident in males (22). On the basis of this remarkable vasoreactivity of Cts, we reasoned that its human variants could also act as cardiotropic agents exerting differential effects on the heart.

The working heart and the arterial system are such close functional complements that the analysis of their dynamic interaction represents an obligatory step in the integral understanding of cardiovascular homeostasis both under normal and abnormal (hypertension) conditions. There have been no studies investigating the direct action of Cts on isolated rat heart preparations, which being independent from extrinsic neuronal and endocrine influences is an ideal model for analyzing the direct cardiac effects of a substance. Using the Langendorff perfused rat heart as a mammalian cardiac paradigm, we show that Cts and its naturally occurring human variants directly influence both inotropic and lusitropic functions. In addition to signaling analyses addressed to determine the possible mechanisms of action of WT-Cts, we demonstrate that Cts inhibits the positive inotropic actions of both isoproterenol (ISO) and endothelin-1 (ET-1). The results provide a novel insight into the role of Cts as an endocrine/paracrine cardiac modulator and inhibitor of β-adrenergic and ET-1 actions on the heart, adding new aspects on the structure-function relationship of Cts variants and their anti-hypertensive potential.

Materials and Methods

Animals

Male Wistar rats (Morini, Bologna, Italy), weighing 180–250 g, were housed (three per cage) in a ventilated cage rack system under standard conditions. Animals had food and water access ad libitum. The investigation conforms to the Guide for the Care and Use of Laboratory Animals, according to National Institutes of Health publication 85-23 (revised 1996).

Drugs

WT-Cts, the pro370leu variant (P370L-Cts), and gly364ser variant of Cts (G364S-Cts) were synthesized by the solid-phase method, using 9-fluorenylmethoxy-carbonyl protection chemistry (7). Peptides were purified to greater than 95% homogeneity by preparative reverse-phase HPLC on C-18 silica column. Authenticity and purity of the peptides was further verified by analytical chromatography (reverse-phase HPLC) and electrospray-ionization or matrix-assisted laser desorption mass spectrometry. ISO; ET-1; the nitric oxide (NO) scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO); inhibitors of either nitric oxide synthase [NG-monomethyl-l-arginine (L-NMMA)] and (N5-(1-imino-3-butenyl)-l-ornithine) or guanylate cyclase (ODQ) or protein kinase G (KT5823) were purchased from Sigma Chemical Co. (St. Louis, MO). All drug-containing solutions were freshly prepared before experimentation.

Isolated heart preparation

Rats were anesthetized with ethyl carbamate (2 g/kg rat, ip), the hearts rapidly excised, and then transferred in ice-cold buffered Krebs-Henseleit solution (KHs). As previously described (23), the aorta was immediately cannulated with a glass cannula and connected to Langendorff apparatus to start perfusion at constant flow-rate (12 ml/min). Briefly, the apex of the left ventricle (LV) was pierced to avoid fluid accumulation. A water-filled latex balloon, connected to a pressure transducer (BLPR; WRI, Inc., Sarasota, FL), was inserted through mitral valve into the LV, allowing isovolumic contractions and continuous mechanical parameters recording. Another pressure transducer located just above the aorta recorded coronary pressure (CP). The perfusion solution consisted of a modified nonrecirculating KHs containing (in millimoles) NaCl 113, KCl 4.7, NaHCO3 25, MgSO4 1.2, CaCl2 1.8, KH2PO4 1.2, glucose 11, mannitol 1.1, Na-pyruvate 5 (pH 7.4; 37 C; 95% O2-5% CO2). Hemodynamic parameters were assessed using a PowerLab data acquisition system and analyzed using Chart software (both purchased by ADInstruments, Basile, Italy).

Basal conditions

Heart performance was evaluated from the LV pressure (LVP; in mm Hg), which is an index of contractile activity, the rate-pressure product [RPP; heart rate (HR) × LVP, in 104 mm Hg × beats/min], which is an index of cardiac work (24), the maximal value of the first derivative of LVP (25) (mm Hg per second), which is an index of the maximal rate of LV contraction, the time to peak tension of isometric twitch, which is an assessment of inotropism. Lusitropism was determined by calculating the maximal rate of LVP decline [(LVdP/dT)max; mm Hg/sec], the half-time relaxation (HTR) (seconds), which is the time required for tension to fall from the peak to 50% and T/−t ratio obtained by +(LVdP/dT)max/−(LVdP/dT)max (24,26). T/−t is the ratio between the maximal rate of LV contraction (+(LVdP/dT)max) and the maximal rate of LV relaxation (−(LVdP/dT)max). Mean CP was calculated by averaging values obtained during several cardiac cycles (23).

Cts-stimulated preparations

Repetitive exposure of each heart to a single concentration (33 nm) of WT-Cts revealed absence of desensitization (data not shown). Thus, concentration-response curves were generated by perfusing cardiac preparations with KHs supplemented with increasing concentrations of WT-Cts (from 11 to 200 nm) for 10 min.

Adrenergic and cholinergic receptors involvement

To obtain information on the involvement of β1/β2, α-adrenergic receptors (ARs) and cholinergic receptors on the inotropic and lusitropic effects induced by WT-Cts, cardiac preparations, stabilized for 20 min with KHs, were perfused with Nadolol (10 nm), phentolamine (100 nm), or atropine (10 nm) for 10 min and then washed out with KHs. After returning to control conditions, each heart was perfused with KHs containing a single concentration of Cts WT (110 nm) plus nadolol (10 nm), phentolamine (100 nm), or atropine (10 nm) for an additional 10 min.

Involvement of inhibitory G protein (Gi/o)

To evaluate the involvement of inhibitory G proteins in the cardiac action of WT-Cts, hearts were preincubated for 60 min with KHs enriched with pertussis toxin (PTx; 0.01 nm) and then exposed for 10 min to WT-Cts (110 nm). PTx catalyzes ADP-ribosylation of Gi/o α-subunit, uncoupling Gi membrane receptor interaction.

Cardiac signaling

Hearts were perfused with or without 110 nm WT-Cts as before. At the end of the experiment, hearts were snap frozen under liquid nitrogen and homogenized with 1 ml of ice-cold 0.2 m sucrose; Tris maleate (pH 7.0) buffer supplemented with 2 mm EDTA (pH 8.0); 1 mm sodium orthovanadate; 10 mm sodium pyrophosphate; 1 mm phenylmethylsulfonyl fluoride; 10 μg/ml leupeptin; 10 μg/ml aprotinin; and 0.1 mm 3-isobutyl-1-methylxanthine. Sarcoplasmic reticulum (SR) membrane fractions were isolated from cytosolic proteins as described before (27). Protein content was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA), and 100 μg of cytosolic protein were subjected to SDS-PAGE immunoblot analysis for phosphorylated ERK, total ERK (Santa Cruz Biotechnology, Santa Cruz, CA). Two hundred micrograms of cytosolic protein were used for immunoblot detection of phosphorylated protein kinase B (Akt)-Ser473 (P-Akt), total-Akt, phosphorylated glycogen synthase kinase-3 (P-GSK-3-Ser9) and actin (Cell Signaling Technology, Beverly, MA). For total phospholamban (T-PLN) and phosphorylated PLN [P-PLN-serine 16 (Ser16)] level assessment, 10 μg of SR membranes were subjected to electrophoresis and the immunoblots were probed with antimouse PLN antibody (Affinity Bioreagents, Golden, CO) and anti-P-PLN-Ser16 (Badrilla, Leeds, UK).

NO-pathway inhibitor stimulated preparations

Hearts were stabilized for 20 min with KHs and perfused with 110 nm WT-Cts for 10 min, and then the peptide was washed out with KHs. After returning to control conditions, each heart was perfused with KHs containing either the NO scavenger PTIO, the nonspecific NOS inhibitor L-NMMA, a soluble guanylate cyclase inhibitor (ODQ), or a protein kinase G blocker (KT5823). Subsequently the hearts were exposed to the specific signaling inhibitor plus 110 nm of WT-Cts.

Isoproterenol stimulated preparations

Cardiac preparations were stabilized for 20 min with KHs and then perfused with 5 nm ISO for 10 min. ISO was washed out with KHs. After returning to control conditions, each heart was perfused with KHs containing a single concentration of WT-Cts (110 nm), P370L-Cts (33 nm), or G364S-Cts (200 nm) with 5 nm ISO for a further 10 min.

To further describe the antagonistic action of WT-Cts (33 nm), P370L-Cts (110 nm), or G364S-Cts (200 nm) against ISO-dependent stimulation, dose-response curves were generated by perfusing the heart preparations with KHs enriched with increasing concentrations of ISO (0.1 nm to 1 μm) alone. These curves were then compared with those obtained by exposing other cardiac preparations to the same perfusion medium containing increasing concentrations of ISO (0.1 nm to 1 μm) plus a single concentration of WT-Cts (33 nm), P370L-Cts (110 nm), or G364S-Cts (200 nm).

ET-1-stimulated preparations

Hearts, stabilized for 20 min with KHs, were perfused for 10 min with ET-1 (1 nm) and then washed out with KHs. After returning to control conditions, each heart was perfused with KHs containing a single concentration of WT-Cts (33 nm), P370L-Cts (110 nm), or G364S-Cts (200 nm) plus 1 nm ET-1 for a further 10 min.

Statistics

Data are expressed as the mean ± sem. Curve fitting was accomplished in the program Kaleidagraph (Synergy Software, Reading, PA). Peptide EC50 and IC50 values were interpolated as the concentration that achieved 50% stimulation and inhibition, respectively. For analysis of phosphorylated proteins, levels were quantified using Bio-Rad QuantifyOne and volumes of phosphorylated proteins were divided by the levels of nonphosphorylated proteins to calculate fold activation. Stimulation of protein activity was expressed as fold increase over vehicle-treated control. The means and sem were calculated for each treatment. Multiple comparisons were made using either one-way ANOVA followed by Bonferroni’s post hoc test or two-way ANOVA. Statistical significance was concluded at P < 0.05. Statistics were computed with the program InStat (GraphPad Software, Inc., San Diego, CA).

Results

Basal conditions

After 20 min of stabilization, the following basal recordings were measured: LVP, 89 ± 3 mm Hg; HR, 280 ± 7 beats/min; RPP, 2.5 ± 0.1 104 mm Hg beats/min; CP, 63 ± 3 mm Hg; +(LVdP/dT)max, 2492 ± 129 mm Hg/sec; T/−t, 0.08 ± 0.01 sec; −(LVdP/dT)max, 1663 ± 70 mm Hg/sec; HTR, 0.05 ± 0.01 (sec); and T/−t or +(LVdP/dT)max/−LVdP/dT)max, 1.49 ± 1.84 mm Hg/sec. Endurance and stability of the preparation, analyzed by measuring performance variables every 10 min, showed that the heart preparation is stable for up to 180 min on the perfusion apparatus.

Inotropic and lusitropic actions of WT-Cts

To test whether WT-Cts alters basal cardiac parameters, heart preparations were exposed to increasing concentrations of WT-Cts to generate concentration-response curves. Exposure of single repeated doses of WT-Cts (33 nm) showed absence of desensitization (data not shown). WT-Cts effect on LVP reached its maximum at 5 min after administration, remaining stable for 15 min and then gradually decreased with time. Accordingly, cardiac parameters were measured at 10 min.

WT-Cts significantly increased HR at 33 nm (5.63%), reaching a maximum at 165 nm (8.82%; EC50 ∼12.5 nm; Fig. 1A). Myocardial parameters were markedly inhibited by WT-Cts: LVP by 28.39%, IC50 ∼69 nm) RPP by 27.86%, IC50 ∼69 nm; and +(LVdP/dt)max by 36.44%, IC50 ∼72 nm) (Fig. 1B). Among the lusitropic parameters, WT-Cts caused a concentration-dependent increment in T/−t (by 5.41%; EC50 ∼60 nm) and decrements in −(LVdP/dt)max (by 32.89%; IC50 ∼74 nm) and HTR (by 38.67%; IC50 ∼44 nm) (Fig. 1C). WT-Cts also caused a dose-dependent increment in CP with a maximum response (by 37.7%) at 200 nm (Fig. 1D).

Figure 1.

Figure 1

Open in a new tab

Dose-dependent response curves of WT-Cts (11–200 nm) on HR (A), myocardial parameters [LVP, RPP, and +(LVdP/dT)max] (B), lusitropic parameters [−(LVdP/dT)max and T/−t] (C), and CP (D) on Langendorff perfused rat heart preparation. For abbreviations and basal values, see Results. Percentage changes were evaluated as means ± sem of eight experiments. Significance of difference from control values was done by one-way ANOVA followed by Bonferroni’s post hoc test: α, P < 0.05; β, P < 0.01; γ, P < 0.001.

Catestatin signaling to cardiac modulation

The negative inotropic and lusitropic effects of WT-Cts were abolished by inhibition of β1/β2-ARs (by nadolol), reduced by α-AR antagonist (phentolamine), or remained unaffected by inhibition of cholinergic receptors (by atropine) (Fig. 2).

Figure 2.

Figure 2

Open in a new tab

Effects of WT-Cts (110 nm) before and after treatment with either nadolol (10 nm), phentolamine (100 nm), or atropine (10 nm) on LVP, +(LVdP/dT)max, and −(LVdP/dT)max on the isolated and Langendorff perfused rat heart. Significance between the control and WT-Cts-treated values were done by one-way ANOVA (P < 0.0001) followed by Bonferroni’s post hoc test (n = 5): P value α, Kreb’s buffer vs. WT-Cts; P value β, WT-Cts vs. inhibitors.

It is well established that Gi/o proteins are involved in the negative inotropism exerted by several cardiodepressive agents, including CgA-derived VS-1 (24). To evaluate Gi/o proteins involvement in Cts-dependent negative inotropism, hearts were perfused with the specific inhibitor PTx, in presence of WT-Cts (110 nm). PTx abolished WT-Cts-mediated negative inotropism [LVP and +(LVdP/dT)max] and lusitropism [−(LVdP/dT)max], indicating Gi involvement in Cts signaling (Fig. 3).

Figure 3.

Figure 3

Open in a new tab

Effects of WT-Cts (110 nm) alone and WT-Cts in presence of PTx on LVP, +(LVdP/dT)max, and −(LVdP/dT)max. Percentage changes were evaluated as means ± sem of five experiments. Significance between the control and WT-Cts-treated values were done by one-way ANOVA (P < 0.001) followed by Bonferroni’s post hoc test: Bonferroni test, P value α, buffer vs. WT-Cts; P value β, Wt-Cts vs. PTx.

The NO synthase (NOS)-NO-cGMP-cGMP-dependent protein kinase (PKG) cascade plays a key role in the control of contractile performance in mammals (28). Accordingly, we tested NO-cGMP-PKG involvement in WT-Cts-dependent cardiotropism by perfusing heart preparations with either PTIO (10 μm), L-NMMA (10 μm), ODQ (10 μm), or KT5823 (0.1 μm) in the presence of Cts. Antagonist concentrations were selected from preliminary dose-response curves that determined the minimum antagonist concentration that did not alter basal cardiac function. WT-Cts (110 nm)-induced reduction of negative inotropism [i.e. +(LVdP/dT)max] and lusitropism [i.e. −(LVdP/dT)max] was abolished by NO removal by PTIO, NOS inhibition by L-NMMA, and soluble guanylate cyclase blockade with ODQ. PKG inhibition by KT5823 failed to affect Cts-induced changes in cardiac performance (Fig. 4). In addition, preliminary experiments performed with selective NOS inhibitors have shown that the LVP reduction induced by WT-Cts (LVP = −18.19 ± 2.27) was abolished by N5-(1-Imino-3-butenyl)-L-ornithine (L-NIO), an endothelial NO synthase (eNOS) selective inhibitor (LVP = 5.04 ± 1.69), and reduced by Vinyl-L-NIO, an neuronal NOS selective inhibitor (LVP = −13.14 ± 2.25). This indicates that Cts signals specifically through eNOS.

Figure 4.

Figure 4

Open in a new tab

Effects of WT-Cts (110 nm) alone and WT-Cts in presence of L-NMMA, PTIO, or ODQ or KT5823 on LVP, +(LVdP/dT)max, and −(LVdP/dT)max. Significance between the control and WT-Cts-treated values were done by one-way ANOVA (P < 0.05) followed by Bonferroni’s post hoc test (n = 6): Bonferroni test, P value β, Cts vs. Cts plus L-NMMA.

The mechanism of action of Cts is summarized in Table 1.

Table 1.

Signaling pathways involved in the negative inotropic action of WT-Cts

Signaling pathways Negative inotropism
α-Adrenoceptors Reduced
β-Adrenoceptors Abolished
Muscarinic receptors Unchanged
Gi/o proteins Abolished
eNOS-NO-cGMP-PKG Abolished
Open in a new tab

Inotropism was evaluated in the presence of selective antagonists of α- and β-adrenergic and muscarinic receptors, Gi/o proteins, and the NO pathway. 

Intracellular Ca2+ coordinates cardiac contraction (sarcoplasmic reticulum, SR, intracytosol Ca2+ release via ryanodine receptors) and relaxation [SR Ca2+ reuptake through Ca2+ ATPase sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)-2a pump]. PLN, i.e. the 52-amino acid transmembrane SR phosphoprotein regulating Ca2+ ATPase SERCA2a, in its dephosphorylated state inhibits Ca2+ pump activity. PLN phosphorylation alters the PLN-SERCA2a interaction, relieving Ca2+ pump inhibition and enhancing relaxation rates and contractility. P-Akt, GSK-3, and ERK1/2 proteins also modulate cardiac function. Therefore, we checked whether WT-Cts signals through these proteins. WT-Cts inhibited phosphorylation of PLN at the PKA-specific site, Ser16 (by 49%; Fig. 5A), AKT at Ser473 (by 48%; Fig. 5B), ERK1/2 (by 54%; Fig. 5C), and GSK-3β at Ser9 (by 73%; Fig. 5D).

Figure 5.

Figure 5

Open in a new tab

Immunoblot analysis of total and P-PLN (A), AKT (B), ERK1/2 (C), and GSK-3β (actin instead of total) (D) in control and WT-Cts-treated hearts. Ten micrograms of SR membranes were used for detection of T-PLN and P-PLN. P-PLN was normalized to T-PLN. Then 200 μg of cytosolic protein was used for immunoblot analysis for P-Akt, T-Akt, P-GSK-3β, and actin; 100 μg of cytosolic protein was used for immunoblot analysis for phosphorylated ERK1/2 and total ERK1/2. The phosphorylated proteins were normalized either to their corresponding nonphosphorylated forms or actin. Control, n = 3; WT-Cts, 110 nm, n = 4.

Inotropic and lusitropic actions of naturally occurring human catestatin variants

G364S-Cts caused a significant increase in RPP, even at 33 nm (by 22.5%) (Fig. 6B). Other parameters tested were unaffected by treatment with this variant (Fig. 6, A–C).

Figure 6.

Figure 6

Open in a new tab

Concentration-dependent response curves of G364S-Cts (11–200 nm) on HR and myocardial parameters [LVP, RPP, and +(LVdP/dT)max] on −(LVdP/dT)max). Percentage changes were evaluated as means ± sem of eight experiments. Significance of difference from control values was done by one-way ANOVA (P < 0.05) followed by Bonferroni’s post hoc test.

P370L-Cts induced a negative inotropism from 110 to 200 nm, the maximum decrease in LVP (by 52.9%) and RPP (by 52.3%), being at concentration of 200 nm (Fig. 7B). This peptide also caused marked decrease in +(LVdP/dT)max starting from 110 nm, with a maximum decrease at 200 nm (by 52.3%; Fig. 7B) and −(LVdP/dT)max (by 79.2%; Fig. 7C). These effects were not accompanied by changes in HR (Fig. 7A) and CP (data not shown).

Figure 7.

Figure 7

Open in a new tab

Concentration-dependent response curves of P370L-Cts (11–200 nm) on HR and myocardial parameters [LVP, RPP, and +(LVdP/dT)max] on −(LVdP/dT)max. Percentage changes were evaluated as means ± sem of eight experiments. Significance of difference from control values was done by one-way ANOVA (P < 0.05) followed by Bonferroni’s post hoc test.

Inotropic and lusitropic effects of the three Cts variants are summarized in Table 2.

Table 2.

Effects of WT-Cts, P370L-Cts, and G364S-Cts on cardiac parameters under basal conditions

Cardiac parameters WT-Cts P370 liter-Cts G364S-Cts
Inotropism Negative Negative No effect
Lusitropism Negative No effect No effect
Activity: WT-Cts > P370L-Cts > G364S-Cts
Open in a new tab

Cts modulation of isoproterenol-induced cardiac changes

Cardiac preparations exposed to the β-adrenergic agonist ISO (5 nm) revealed positive inotropic and lusitropic responses that were associated with vasodilation. These modifications were indicated by an increase in LVP (26.7%), RPP, +(LVdP/dT)max (25.5%), −(LVdP/dT)max (38.5%), HTR (32.5%), and T/−t (7.8%) and a decrease in the CP (11.6%) (Fig. 8). As reported previously (29,30), ISO effects were significant up to 5 min from its initial application. Preliminary experiments performed by consecutive exposures of ISO (5 nm) on each heart showed absence of desensitization (data not shown). Because Cts variants display differential effects on nicotine-evoked catecholamine secretion (11), we tested the efficacies of the three human Cts peptides in counteracting ISO-mediated positive inotropism, lusitropism, and coronary dilation. Hearts were perfused with KHs containing ISO (5 nm) plus a single concentration of either WT-Cts (11, 33, or 110 nm), P370L-Cts (110 nm), or G364S-Cts (200 nm). All parameters were measured at 5 min after the drug application. All three peptides abolished the ISO stimulatory effect on LVP in the following rank order: WT-Cts greater than G364S-Cts greater than P370L-Cts (Fig. 8A). The ISO-induced coronary dilation was blocked by both WT-Cts (86%) and G364S-Cts (85%), remaining unchanged by P370L-Cts (Fig. 8B). In addition, Cts peptides counteracted the ISO-dependent positive inotropism. In fact, WT-Cts (by ∼90%) and G364S-Cts (by >100%) blocked +(LVdP/dT)max (Fig. 8C). Likewise, the ISO-induced positive lusitropism was blocked by WT-Cts (by ∼75%) and G364S-Cts (by ∼100%). P370L-Cts failed to modulate the lusitropic effect of ISO (Fig. 8D). The ISO-dependent decrease of T/−t was blocked only by G364S-Cts (Fig. 8E). All three peptides blocked ISO-induced HTR (Fig. 8F).

Figure 8.

Figure 8

Open in a new tab

Effects of ISO before and after treatment with WT-Cts, P370L-Cts, or G364S-Cts on LVP, +(LVdP/dT)max, CP, −(LVdP/dT)max, HTR, T/−t, or +(LVdP/dT)max/−(LVdP/dT)max. For abbreviations and basal values, see Results. Percentage changes were evaluated as means ± sem of six experiments for each group. Significance of difference from control and ISO-treated values were done one-way ANOVA (P < 0.05) followed by Bonferroni’s post hoc test. Bonferroni test: P value α, buffer vs. ISO; P value β, Iso vs. ISO plus Cts.

To characterize the inhibitory action of WT-Cts (11, 33, or 110 nm), G364S-Cts (200 nm), or P370L-Cts (110 nm) toward ISO-dependent stimulation of cardiac function, heart preparations were perfused with KHs containing increasing concentrations of ISO (0.1 nm to 1 μm) either alone or in combination with one of the three Cts variants.

ISO alone increased LVP significantly at concentrations ranging from 5 nm to 1 μm (Fig. 9). The EC50 values of the percent increase in LVP stimulation were determined from increasing concentrations of ISO alone or ISO plus WT-Cts (11, 33, and 110 nm), P370L-Cts (110 nm), or G364S-Cts (200 nm). WT-Cts at concentrations of 33 and 110 nm, G364S-Cts at a concentration of 200 nm, and P370L-Cts at a concentration of 165 nm significantly inhibited ISO-induced stimulation of LVP and displayed an interaction with ISO (Fig. 9). The EC50 values of LVP (in logM, molar concentration) of ISO alone were −8.67 ± 0.3 (r2 = 0.84), of ISO plus WT-Cts at concentrations of 11, 33, and 110 nm were −8.7 ± 0.35 (r2 = 0.85), −7.35 ± 0.46 (r2 = 0.73), and −7.2 ± 1.14 (r2 = 0.29), respectively. ISO plus P370L-Cts (165 nm) resulted in an EC50 value of LVP at −8.51 ± 0.46 (r2 = 0.76). Because increasing concentrations of ISO failed to overcome the antagonistic effect of Cts, the actions of Cts are considered as a noncompetitive type of antagonism (Fig. 9). The counteraction of adrenergic stimulation by Cts is summarized in Table 3.

Figure 9.

Figure 9

Open in a new tab

The concentration-response curves of ISO-mediated stimulation on LVP of ISO (10−10 to 10−6 m) alone and ISO (10−10 to 10−6 m) plus a single concentration of WT-Cts (11, 33, and 110 nm), P370L-Cts (165 nm), or G364S-Cts (200 nm). Contraction is expressed as a percentage of LVP [baseline = 0%, peak constriction by ISO and ISO plus WT-Cts = 100%]. The EC50 values (in log M, molar concentration) were: ISO alone, −8.67 ± 0.3 (r2 = 0.84); ISO plus WT-Cts (11, or 33, or 110 nm), −8.7 ± 0.35 (r2 = 0.85), −7.35 ± 0.46 (r2 = 0.73), −7.2 ± 1.14 (r2 = 0.29), respectively; plus P370L-Cts (165 nm), −8.51 ± 0.46 (r2 = 0.76); or plus G364S-Cts (200 nm), −8.61 ± 0.569 (r2 = 0.60). Comparison between groups and interaction between ISO vs. catestatin peptides were done by two-way ANOVA (n = 6): ISO dose, P < 0.0001 (WT-Cts at 11, 33, and 110 nm; G364S-Cts; and P370L-Cts); ISO vs. Cts, P < 0.0001 (WT-Cts at 33 and 110 nm; G364S-Cts, and P370L-Cts); interaction, P < 0.0001 (WT-Cts at 33 and 110 nm), P < 0.0019 (G364S-Cts), and P < 0.00016 (P370L-Cts).

Table 3.

Antiadrenergic actions of WT-Cts and its variants

Cardiac effects ISO alone ISO + WT-Cts ISO + P370L-Cts ISO + G364S-Cts
Inotropism Positive Abolished Abolished Abolished
Lusitropism Positive Abolished Reduced Abolished
Coronary pressure Vasodilation Abolished No effect abolished
EC50 −8.67 ± 0.3 −7.2 ± 1.14 −8.51 ± 0.46 −8.61 ± 0.57
Order of potency: WT-Cts > G364S-Cts > P370L-Cts
Open in a new tab

Cts blockade of ET-1 stimulated cardiac functions

Cts-induced vasodilation in vivo appears mediated, at least in part, by histamine release through activation of H1 receptors (31). However, it is unknown whether Cts is also able to relax preconstricted vessels in the isolated rat heart. As reported previously, exogenous ET-1 constricts coronary vessels in the isolated mammalian heart (32) and induces either positive (33) or negative inotropic effects, which are secondary to coronary constriction (34).

To verify Cts’s ability to counteract the ET-1-mediated inotropic and coronary effects, hearts were perfused with KHs containing ET-1 alone or in combination with one of the Cts variants.

Administration of ET-1 alone induced a dose-dependent biphasic effect on contractility. At a concentration of 1 nm ET-1 increased LVP and +(LVdP/dT)max, whereas at higher doses (10 nm), Cts decreased both parameters, thus inducing a negative inotropic effect. Cts peptides differentially affected the ET-1-induced inotropic effects. In fact, positive inotropism was blocked by WT-Cts (33 nm), P370L-Cts (110 nm), and G364S-Cts (200 nm) (Fig. 10). Moreover, ET-1 alone (1 and 10 nm) typically induced a significant increase in coronary constriction (34), which was blocked by all three human Cts variants (Fig. 10).

Figure 10.

Figure 10

Open in a new tab

ET-1 effects before and after treatment with WT-Cts, P376L-Cts, or G364S-Cts on LVP, +(LVdP/dT)max, and CP. Percentage changes were evaluated as means ± sem of six experiments for each group. Significance of difference from control and ET-1-treated values were done by one-way ANOVA followed by Bonferroni’s post hoc test (n = 6), P < 0.05. Bonferroni test, P value α, buffer vs. ET-1; P value β, ET-1 vs. Cts.

Discussion

Cardiovasoreactivity of Cts

The present study reveals that Cts, in addition to its relevant vasoreactivity shown in animal models (8) and man (11,19), elicits a relevant cardiosuppressive influence on the isolated Langendorff-perfused rat heart under both basal and chemically stimulated conditions. In fact, we find that WT-Cts increases HR and decreases LVP, RPP, +(LVdP/dt)max, −(LVdP/dt)max, and HTR. Thus, in addition to its important role in the control of blood pressure, Cts is emerging as a peptide that has direct cardiovascular actions. This suggests that the newly uncovered negative inotropism and lusitropism of WT-Cts may be important components of its hypotensive action. Although this finding fits well with the reported rescue of high blood pressure by Cts in Chga−/− mice, its effect on coronary vasoconstriction is difficult to explain at the moment. It remains to be elucidated whether it is either the result of a direct action of the peptide on the coronary tissue or as a consequence of an antiadrenergic effect exerted at the adrenergic innervation of the coronaries. We also report that human Cts variants are potent inhibitors of ISO and ET-1 mediated actions in the rat heart. In the Langendorff rat heart, the inotropic and lusitropic effects of the human recombinant VS-1 (hCgA1–76) are comparable with those of the synthetic rat rCgA1–64 fragment (encompassing most of the VS-1 sequence) (5). Conceivably, it can be inferred that also the effects of the heterologous human Cts variants are of physiological significance. On the whole the data suggest that Cts acts as an endocrine/paracrine cardiosuppressive modulator, being also of potential biomedical interest in those physiopathological conditions characterized by prolonged and excessive adrenergic activity, e.g. heart failure and hypertensive cardiomyopathy.

Signaling transduction mechanisms

Cts-induced contractile effects are mediated by β2-ARs-Gi/o Protein-eNOS-NO-cGMP-PKG mechanisms. The finding that WT-Cts-induced negative inotropic and lusitropic effects are either abolished or reduced by β2-AR and α-AR inhibition, respectively, and are blocked by PTx pretreatment strongly suggests that Cts is acting through interaction with inhibitory G protein-coupled receptor (Gi/o) and/or a number of signaling molecular components present in cell membrane microdomains (e.g. caveolae). So far, it is known that Cts interacts with nicotinic cholinergic receptors to inhibit catecholamine secretion from sympathochromaffin cells in vivo in mice (8) and in vitro in rat PC12 (8,9) and primary bovine chromaffin cells (6,7). Moreover, Cts vasodilatation in vivo is mediated by histamine release through H1 receptor and is seemingly unrelated to the autocrine inhibition of catecholamine release in the adrenal medulla in vitro (3). Our data suggest that Cts inotropic effects are mediated mainly by β2-adrenoceptors, partially by α-adrenoceptors, but not by cholinergic receptors. Further investigations are needed to determine whether this occurs by direct receptor interaction and/or via an allosteric modulation.

It is well known that the eNOS-NO-cGMP-PKG cascade plays a key role in mediating specific intracardiac signals involved in the control of contractile performance. For example, in rat ventricular myocytes, it has been shown that the NOS-produced NO, targets soluble GC, and thus PKG, negatively affect contractility through the reduction of L-type Ca2+ current (35,36) and phosphorylation of troponin I, thereby reducing troponin C affinity for calcium and depressing contractility (37). The finding that the inhibitor of PKG (KT5823) attenuates but does not abolishes Cts-induced inotropy suggests that NO influences Cts action with other mechanisms. Interestingly, WT-Cts significantly depressed the mean peak height calcium transient in isolated rat cardiac myocytes. Of note, we have recently shown that the N-terminal CgA1–76 fragment (VS-1) exerts its cardioinhibitory effects through a NO-cGMP-PKG mechanism in both rat and eel heart (25,38) and signals through a phosphoinositide 3 kinase-dependent NO release by endothelial cells to exert its antiadrenergic effect, rather than signaling through a direct action on cardiomyocytes (39).

Moreover, emerging evidences indicate that NO synthesized by eNOS attenuates cardiac β-adrenergic ventricular developed pressure in isolated hearts and in vivo (40). It has been reported that stimulation of β2AR activates eNOS through activation of Akt (41) and that phosphorylated-Akt, in turn, phosphorylates eNOS to generate NO (Fig. 11) (42). In addition, it has been shown that in endothelial cells Akt activation to β2AR stimulation is mediated by Gi (43).

Figure 11.

Figure 11

Open in a new tab

Schematic diagram showing the putative ET-1, ISO, or Cts signaling in endothelial and myocardial cells. AC, Adenylate cyclase; β-ARK, β-adrenergic receptor kinase; DHPR, dihydropyridine receptor; ETAR, ET receptor subtype A; IP3, inositol triphosphate; NCX, Na+/Ca2+ exchangers; PKA, protein kinase A; AKT, protein kinase B; PI-3-K, phosphoinositide 3 kinase; PLC, phospholipase C; PMCA, plasma membrane Ca2+-ATPases; RyR, ryanodine receptor; +, stimulation; −, inhibition; ±, no effect.

Further analysis of the intracellular effectors of Cts action revealed that in the presence of WT-Cts, P-PLN at residue serine 16 (P-PLN-Ser16) is reduced, suggesting PLN involvement in the Cts-induced negative inotropism and lusitropism. Indeed, both levels and degree of PLN phosphorylation critically modulate basal Ca2+ handling and contractility, elicited via P-PLN-induced increase of SR Ca2+ uptake by the SR Ca2+ pump (SERCA2a) and by enhancement of SR Ca2+ release via ryanodine receptor-2. As a result, intracellular Ca2+ transients increase, with consequences on inotropy and lusitropy (44,45). We also found that perfusion with WT-Cts is associated with decreased phosphorylation of ERK1/2, a member of the MAPK family, which is phosphorylated via MAPK kinase as well as Akt and GSK-3β, known markers of contractility and hypertrophy (46,47). However, due to the redundance of these signal-transduction cascades, activated by a number of important neuroendocrine (e.g. catecholamines) and paracrine (e.g. endothelins) agents (48), any inference regarding a more specific mechanistic involvement of Cts with these downstream signaling molecular components appears, at this stage of the study, premature.

Cardiac profile of Cts variants

Recent reports revealed that Gly/Ser heterozygotes display profound changes in autonomic activity including increased parasympathetic and decreased sympathetic indices, coupled with decrements in excretion of catecholamine (22). In addition, the Gly364Ser variant is associated with a decrease in diastolic blood pressure, and this antihypertensive effect is apparent in males only (22). In the present study, we found that G364S-Cts augments mechanical performance by increasing RPP (HR × LVP) without affecting HR and LVP (Fig. 6). In contrast, P370L-Cts caused decrements in RPP. Like WT-Cts, the P370L-Cts decreases LVP, +(LVdP/dt)max, and −(LVdP/dt)max (Fig. 7). Because P370L-Cts variation is rare (0.6%) and occurs in individuals from African ancestry, its association with cardiovascular function is yet to be established (21,49).

Inhibition of ISO and ET-1-induced actions

Notably, we found that human Cts variants act as potent inhibitors of ISO. Whereas the rank order of efficacies for counteracting positive inotropism are WT-Cts greater than G364S-Cts greater than P370L-Cts, the efficacies for counteracting ISO-induced lusitropic effect are in the following order: G364S-Cts greater than WT-Cts greater than P370L-Cts. The Cts variants also showed different coronary vasoactivity. The dose-dependent increments in ISO failed to overcome the inhibitory effects of Cts on positive inotropism, thus indicating that Cts inhibition is noncompetitive. This agrees with our previous report showing that Cts inhibition of nicotine-evoked catecholamine secretion is also noncompetitive (6,7,8,50). Of note, these findings indicate that Cts variants differ in their efficacies in counteracting the positive inotropic and lusitropic effects of ISO, thus providing a cardiotropic counterpart of the antihypertensive effects shown by the peptide. In particular, the Cts-induced negative lusitropism can be of importance under conditions of dilated cardiomyopathy. Whether Cts inhibits ISO-mediated signaling at the level of the β-adrenergic receptor or at specific downstream signaling events needs to be determined.

Moreover, Cts counteracts the positive inotropic, lusitropic, and coronary constrictor effects of ET-1. ET-1 exerts diverse and important cardiovascular actions including myocardial contractile and vasoactive coronary effects (34), promotion of vascular smooth muscle hypertrophy/mitogenesis, and regulation of angiogenesis (51). All Cts variants differ in their myocardial ET-1-inhibitory efficacies with the following rank order: P370L-Cts greater than G364S-Cts greater than WT-Cts. The Cts variants also counteract ET-1-induced coronary constriction with the following rank order: G364S-Cts greater than WT-Cts greater than P370L-Cts.

Although the coronary constriction evoked by Cts under basal conditions remains to be mechanistically explained, it is important that the peptide abolished the ISO-dependent vasodilation and, at the same time, acts as a vasodilator on ET-1 preconstricted coronaries, like the N-terminal CgA peptide vasostatin (3).

Figure 11 summarizes the putative pathways involved in Cts cardiotropism.

Cts in the context of the endocrine heart

The antiadrenergic and anti-ET-1 influences of Cts point to its role as a counterregulatory modulator in zero steady-state error homeostasis (14). The heart is the target of major stresses, which are associated with adrenosympathogenic overactivity (mainly catecholamines, aldosterone, and cortisol). This can induce a condition that is named sympathetic storm (52), characterized by fever, tachycardia, hypertension, posturing, and diaphoresis (exaggerated excitatory stimuli exerted by the potent autocrine-paracrine intracardiac agent endothelin can also contribute to, and potentiate, the stress-induced cardiotoxicity (Ref. 52 and references therein).

Taken together, our data strongly support the hypothesis that Cts, together with the other CgA fragments (i.e. the N-terminal CgA1–76, VS-1), may be a component of an endocrine/paracrine regulation of cardiac function (5). In fact, in the rat heart, CgA is stored in nonadrenergic myoendocrine atrial cells in association with atrial natriuretic peptide (53) and in both atrial and ventricular Purkinje fibers containing the calcium channel α1E subunit (54). CgA-derived fragments may also originate from cardiac sympathetic nerve termini (55). In the rat heart, intact CgA, four vasostatin-containing CgA peptides (i.e. CgA4-113, CgA1-124, CgA1-135, and CgA1-199) and larger-sized fragments containing the C terminus have been identified (56). Very recently Pieroni et al. (16) also demonstrated the presence of CgA in the human myocardium and its involvement in the neuroendocrine activation in patients with chronic heart failure. The possibility exists that locally derived Cts may regulate cardiac function because in the normal or stressed heart, a specific stimulus-induced proteolytic activation could generate CgA fragments. The presence of extracellular proteases both on cardiomyocyte cell membranes and in the extracellular matrix suggests that extracellular processing does occur (3). According to these observations, the heart may be affected by either circulating or locally produced Cts. It has already been established that Cts acts as an endogenous autocrine/paracrine regulator of catecholamine secretion from chromaffin cells in vitro (8) and adrenal medulla in vivo (10). The cardioactive property of Cts may also add a new piece of information to the expanding and fascinating area of brain-heart connections, as epitomized by various models of stress, including human neurogenic heart disease, in which the myocardium is excitotoxically damaged by abnormally high levels of catecholamines, either circulating or released by cardiac sympathetic nerve terminals (52).

Perspectives

In conclusion, because the heart and the arterial system interact as a closed-loop control system, the former being a target organ of prolonged and excessive adrenosympathogenic activity, such as hypertension with consequent hypertrophy and ischemic cardiomyopathy, it is of relevance that the recently discovered antihypertensive modulatory action of Cts now appears associated with a direct powerful counterexcitatory cardiac action. This cardiotropism of Cts may be inherently important for normal heart function but also suggests a unique inhibitory role of the peptide under abnormal cardiac conditions characterized by adrenosympathogenic overactivation. Until now, blockade of the β-adrenergic receptors is one of the most effective pharmacologic interventions in hypertensive patients. Future studies of administration of Cts to hypertensive animal models will determine whether the peptide has also any therapeutic potential for the treatment of hypertensive cardiomyopathy.

Footnotes

This work was supported by “Fondazione Cassa di Risparmio di Calabria e Lucania” research project “Cuore-cervello: nuovi orizzonti biomedici nello studio di neuropeptidi ad attività cardiovascolare” (to B.T., M.C.C., and T.A.); Ministero dell’lstruzione e dell’Università (to B.T. and M.C.C.); the “Dottorato di Fisiologia”; University of Turin; the “Istituto Nazionale di Ricerca Cardiovascolare” and “Compagnia San Paolo” (Italy) (to A.M.Q.); and Grants R01 DA011311 and P01 HL58120 from the National Institutes of Health and Department of Veterans Affairs (to S.K.M.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 5, 2008

See editorial p. 4778.

Abbreviations: AR, Adrenergic receptor; CgA, chromogranin A; CP, coronary pressure; Cts, catestatin; eNOS, endothelial NO synthase; ET-1, endothelin-1; Gi/o, inhibitory G protein; HR, heart rate; HTR, half- time relaxation; ISO, isoproterenol; KHs, Krebs-Henseleit solution; L-NMMA, NG-monomethyl-l-arginine; LV, left ventricle; LVP, LV pressure; NOS, NO synthase; P-Akt, phosphorylated protein kinase B (Akt)-Ser473; P-GSK-3-Ser9, phosphorylated glycogen synthase kinase-3; PKG, cGMP-dependent protein kinase; P-PLN, phosphorylated PLN; PTx, pertussis toxin; RPP, rate-pressure product; NO, nitric oxide; ODQ, guanylate cyclase; PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; Ser16, serine 16; SERCA, sarcoendoplasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; T-PLN, total phospholamban; VS-1, vasostatin 1; WT, wild type.

References

  1. Winkler H, Fischer-Colbrie R 1992 The chromogranins A and B: the first 25 years and future perspectives. Neuroscience 49:497–528 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Taupenot L, Harper KL, O'Connor DT 2003 Mechanisms of disease: the chromogranin-secretogranin family. N Engl J Med 348:1134–1149 [DOI] [PubMed] [Google Scholar]
  3. Helle KB, Corti A, Metz-Boutigue MH, Tota B 2007 The endocrine role for chromogranin A: a prohormone for peptides with regulatory properties. Cell Mol Life Sci 64:2863–2886 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Tatemoto K, Efendic S, Mutt V, Makk G, Feistner GJ, Barchas JD 1986 Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion. Nature 324:476–478 [DOI] [PubMed] [Google Scholar]
  5. Aardal S, Helle KB, Elsayed S, Reed RK, Serck-Hanssen G 1993 Vasostatins, comprising the N-terminal domain of chromogranin A, suppress tension in isolated human blood vessel segments. J Neuroendocrinol 5:405–412 [DOI] [PubMed] [Google Scholar]
  6. Mahata SK, O'Connor DT, Mahata M, Yoo SH, Taupenot L, Wu H, Gill BM, Parmer RJ 1997 Novel autocrine feedback control of catecholamine release. A discrete chromogranin A fragment is a noncompetitive nicotinic cholinergic antagonist. J Clin Invest 100:1623–1633 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Mahata SK, Mahata M, Wakade AR, O'Connor DT 2000 Primary structure and function of the catecholamine release inhibitory peptide catestatin (chromogranin A344–364): identification of amino acid residues crucial for activity. Mol Endocrinol 14:1525–1535 [DOI] [PubMed] [Google Scholar]
  8. Mahata SK, Mahapatra NR, Mahata M, Wang TC, Kennedy BP, Ziegler MG, O'Connor DT 2003 Catecholamine secretory vesicle stimulus-transcription coupling in vivo. Demonstration by a novel transgenic promoter/photoprotein reporter and inhibition of secretion and transcription by the chromogranin A fragment catestatin. J Biol Chem 278:32058–32067 [DOI] [PubMed] [Google Scholar]
  9. Mahata SK, Mahata M, Wen G, Wong WB, Mahapatra NR, Hamilton BA, O'Connor DT 2004 The catecholamine release-inhibitory “catestatin” fragment of chromogranin A: naturally occurring human variants with different potencies for multiple chromaffin cell nicotinic cholinergic responses. Mol Pharmacol 66:1180–1191 [DOI] [PubMed] [Google Scholar]
  10. Mahata SK 2004 Catestatin—the catecholamine release inhibitory peptide: a structural and functional overview. Curr Med Chem Immun Endocrinol Metab Agents 4:221–234 [Google Scholar]
  11. Takiyyuddin MA, Cervenka JH, Sullivan PA, Pandian MR, Parmer RJ, Barbosa JA, O'Connor DT 1990 Is physiologic sympathoadrenal catecholamine release exocytotic in humans? Circulation 81:185–195 [DOI] [PubMed] [Google Scholar]
  12. Koeslag JH, Saunders PT, Wessels JA 1999 The chromogranins and the counter-regulatory hormones: do they make homeostatic sense? J Physiol 517:643–649 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ceconi C, Ferrari R, Bachetti T, Opasich C, Volterrani M, Colombo B, Parrinello G, Corti A 2002 Chromogranin A in heart failure: a novel neurohumoral factor and a predictor for mortality. Eur Heart J 23:967–974 [DOI] [PubMed] [Google Scholar]
  14. Stridsberg M, Oberg K, Li Q, Engstrom U, Lundqvist G 1995 Measurements of chromogranin A, chromogranin B (secretogranin I), chromogranin C (secretogranin II) and pancreastatin in plasma and urine from patients with carcinoid tumours and endocrine pancreatic tumours. J Endocrinol 144:49–59 [DOI] [PubMed] [Google Scholar]
  15. Nobels FR, de Herder WW, Kwekkeboom DJ, Coopmans W, Mulder A, Bouillon R, Lamberts SW 1994 Serum chromogranin A in the differential diagnosis of Cushing’s syndrome. Eur J Endocrinol 131:589–593 [DOI] [PubMed] [Google Scholar]
  16. Pieroni M, Corti A, Tota B, Curnis F, Angelone T, Colombo B, Cerra MC, Bellocci F, Crea F, Maseri A 200 Myocardial production of chromogranin A in human heart: a new regulatory peptide of cardiac function. Eur Heart J 29:1117–1127 [DOI] [PubMed] [Google Scholar]
  17. Takiyyuddin MA, Baron AD, Cervenka JH, Barbosa JA, Neumann HP, Parmer RJ, Sullivan PA, O'Connor DT 1991 Suppression of chromogranin-A release from neuroendocrine sources in man: pharmacological studies. J Clin Endocrinol Metab 72:616–622 [DOI] [PubMed] [Google Scholar]
  18. Takiyyuddin MA, Parmer RJ, Kailasam MT, Cervenka JH, Kennedy B, Ziegler MG, Lin MC, Li J, Grim CE, Wright FA, O'Connor DT 1995 Chromogranin A in human hypertension. Influence of heredity. Hypertension 26:213–220 [DOI] [PubMed] [Google Scholar]
  19. O'Connor DT, Kailasam MT, Kennedy BP, Ziegler MG, Yanaihara N, Parmer RJ 2002 Early decline in the catecholamine release-inhibitory peptide catestatin in humans at genetic risk of hypertension. J Hypertens 20:1335–1345 [DOI] [PubMed] [Google Scholar]
  20. Mahapatra NR, O'Connor DT, Vaingankar SM, Hikim AP, Mahata M, Ray S, Staite E, Wu H, Gu Y, Dalton N, Kennedy BP, Ziegler MG, Ross J, Mahata SK 2005 Hypertension from targeted ablation of chromogranin A can be rescued by the human ortholog. J Clin Invest 115:1942–1952 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wen G, Mahata SK, Cadman P, Mahata M, Ghosh S, Mahapatra NR, Rao F, Stridsberg M, Smith DW, Mahboubi P, Schork NJ, O'Connor DT, Hamilton BA 2004 Both rare and common polymorphisms contribute functional variation at CHGA, a regulator of catecholamine physiology. Am J Hum Genet 74:197–207 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rao F, Wen G, Gayen JR, Das M, Vaingankar SM, Rana BK, Mahata M, Kennedy BP, Salem RM, Stridsberg M, Abel K, Smith DW, Eskin E, Schork NJ, Hamilton BA, Ziegler MG, Mahata SK, O'Connor DT 2007 Catecholamine release-inhibitory peptide catestatin [chromogranin A(352–372)]: naturally occurring amino acid variant Gly364Ser causes profound changes in human autonomic activity and alters risk for hypertension. Circulation 115:2271–2281 [DOI] [PubMed] [Google Scholar]
  23. Cerra MC, De Iuri L, Angelone T, Corti A, Tota B 2006 Recombinant N-terminal fragments of chromogranin-A modulate cardiac function of the Langendorff-perfused rat heart. Basic Res Cardiol 101:43–52 [DOI] [PubMed] [Google Scholar]
  24. Georget M, Mateo P, Vandecasteele G, Lipskaia L, Defer N, Hanoune J, Hoerter J, Lugnier C, Fischmeister R 2003 Cyclic AMP compartmentation due to increased cAMP-phosphodiesterase activity in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type 8 (AC8). FASEB J 17:1380–1391 [DOI] [PubMed] [Google Scholar]
  25. Cappello S, Angelone T, Tota B, Pagliaro P, Penna C, Rastaldo R, Corti A, Losano GA, Cerra MC 2007 Human recombinant chromogranin A-derived vasostatin-1 mimics preconditioning via an adenosine/nitric oxide signalling mechanism. Am J Physiol Heart Circ Physiol 293:H719–H727 [DOI] [PubMed] [Google Scholar]
  26. Vittone L, Mundina-Weilenmann C, Mattiazzi A, Cingolani H 1994 Physiologic and pharmacologic factors that affect myocardial relaxation. J Pharmacol Toxicol Methods 32:7–18 [DOI] [PubMed] [Google Scholar]
  27. Brar BK, Jonassen AK, Egorina EM, Chen A, Negro A, Perrin MH, Mjos OD, Latchman DS, Lee KF, Vale W 2004 Urocortin-II and urocortin-III are cardioprotective against ischemia reperfusion injury: an essential endogenous cardioprotective role for corticotropin releasing factor receptor type 2 in the murine heart. Endocrinology 145:24–35 [DOI] [PubMed] [Google Scholar]
  28. Cerra MC, Pellegrino D 2007 Cardiovascular cGMP-generating systems in physiological and pathological conditions. Curr Med Chem 14:585–599 [DOI] [PubMed] [Google Scholar]
  29. Abbott D, Lodola A 1992 The effects of isoproterenol on the isolated perfused rat heart. Res Commun Chem Pathol Pharmacol 76:125–128 [PubMed] [Google Scholar]
  30. Asimakis GK, Inners-McBride K, Conti VR, Yang CJ 1994 Transient β adrenergic stimulation can precondition the rat heart against postischaemic contractile dysfunction. Cardiovasc Res 28:1726–1734 [DOI] [PubMed] [Google Scholar]
  31. Kennedy BP, Mahata SK, O'Connor DT, Ziegler MG 1998 Mechanism of cardiovascular actions of the chromogranin A fragment catestatin in vivo. Peptides 19:1241–1248 [DOI] [PubMed] [Google Scholar]
  32. Baydoun AR, Peers SH, Cirino G, Woodward B 1989 Effects of endothelin-1 on the rat isolated heart. J Cardiovasc Pharmacol 13:S193–S196 [DOI] [PubMed] [Google Scholar]
  33. Takanashi M, Endoh M 1991 Characterization of positive inotropic effect of endothelin on mammalian ventricular myocardium. Am J Physiol 261:H611–H619 [DOI] [PubMed] [Google Scholar]
  34. Brunner F, Bras-Silva C, Cerdeira AS, Leite-Moreira AF 2006 Cardiovascular endothelins: essential regulators of cardiovascular homeostasis. Pharmacol Ther 111:508–531 [DOI] [PubMed] [Google Scholar]
  35. Abi-Gerges N, Fischmeister R, Mery PF 2001 G protein-mediated inhibitory effect of a nitric oxide donor on the L-type Ca2+ current in rat ventricular myocytes. J Physiol 531:117–130 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mery PF, Lohmann SM, Walter U, Fischmeister R 1991 Ca2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. Proc Natl Acad Sci USA 88:1197–1201 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Hove-Madsen L, Mery PF, Jurevicius J, Skeberdis AV, Fischmeister R 1996 Regulation of myocardial calcium channels by cyclic AMP metabolism. Basic Res Cardiol 91:1–8 [DOI] [PubMed] [Google Scholar]
  38. Imbrogno S, Angelone T, Corti A, Adamo C, Helle KB, Tota B 2004 Influence of vasostatins, the chromogranin A-derived peptides, on the working heart of the eel (Anguilla anguilla): negative inotropy and mechanism of action. Gen Comp Endocrinol 139:20–28 [DOI] [PubMed] [Google Scholar]
  39. Gallo MP, Levi RC, Ramella R, Brero A, Boero O, Tota B, Alloatti G 2007 Endothelium-derived nitric oxide mediates the anti-adrenergic effect of human vasostatin-1 (CgA 1–76) in rat ventricular myocardium. Am J Physiol Heart Circ Physiol 292:H2906–H2912 [DOI] [PubMed] [Google Scholar]
  40. Barouch LA, Harrison RW, Skaf MW, Rosas GO, Cappola TP, Kobeissi ZA, Hobai IA, Lemmon CA, Burnett AL, O'Rourke B, Rodriguez ER, Huang PL, Lima JA, Berkowitz DE, Hare JM 2002 Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms. Nature 416:337–339 [DOI] [PubMed] [Google Scholar]
  41. Iaccarino G, Ciccarelli M, Sorriento D, Cipolletta E, Cerullo V, Iovino GL, Paudice A, Elia A, Santulli G, Campanile A, Arcucci O, Pastore L, Salvatore F, Condorelli G, Trimarco B 2004 AKT participates in endothelial dysfunction in hypertension. Circulation 109:2587–2593 [DOI] [PubMed] [Google Scholar]
  42. Liu S, Premont RT, Kontos CD, Zhu S, Rockey DC 2005 A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension. Nat Med 11:952–958 [DOI] [PubMed] [Google Scholar]
  43. Ciccarelli M, Cipolletta E, Santulli G, Campanile A, Pumiglia K, Cervero P, Pastore L, Astone D, Trimarco B, Iaccarino G 2007 Endothelial β2 adrenergic signaling to AKT: role of Gi and SRC. Cell Signal 19:1949–1955 [DOI] [PubMed] [Google Scholar]
  44. Viatchenko-Karpinski S, Gyorke S 2001 Modulation of the Ca(2+)-induced Ca(2+) release cascade by β-adrenergic stimulation in rat ventricular myocytes. J Physiol 533:837–848 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ginsburg KS, Bers DM 2004 Modulation of excitation-contraction coupling by isoproterenol in cardiomyocytes with controlled SR Ca2+ load and Ca2+ current trigger. J Physiol 556:463–480 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Lips DJ, Bueno OF, Wilkins BJ, Purcell NH, Kaiser RA, Lorenz JN, Voisin L, Saba-El-Leil MK, Meloche S, Pouyssegur, Pagès G, De Windt LJ, Doevendans PA, Molkentin JD 2004 MEK1-ERK2 signaling pathway protects myocardium from ischemic injury in vivo. Circulation 109:1938–1941 [DOI] [PubMed] [Google Scholar]
  47. Bueno OF, De Windt LJ, Tymitz KM, Witt SA, Kimball TR, Klevitsky R, Hewett TE, Jones SP, Lefer DJ, Peng CF, Kitsis RN, Molkentin JD 2000 The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J 19:6341–6350 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sugden PH 2001 Signalling pathways in cardiac myocyte hypertrophy. Ann Med 33:611–622 [PubMed] [Google Scholar]
  49. Biswas N, Vaingankar SM, Mahata M, Das M, Gayen JR, Taupenot L, Torpey JW, O'Connor DT, Mahata SK 2008 Proteolytic cleavage of human chromogranin A containing naturally occurring catestatin variants: differential processing at catestatin region by plasmin. Endocrinology 149:749–757 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Mahapatra NR, Mahata M, Mahata SK, O'Connor DT 2006 The chromogranin A fragment catestatin: specificity, potency and mechanism to inhibit exocytotic secretion of multiple catecholamine storage vesicle co-transmitters. J Hypertens 24:895–904 [DOI] [PubMed] [Google Scholar]
  51. Schiffrin EL, Deng LY, Larochelle P 1994 Effects of a β-blocker or a converting enzyme inhibitor on resistance arteries in essential hypertension. Hypertension 23:83–91 [DOI] [PubMed] [Google Scholar]
  52. Samuels MA 2007 The brain-heart connection. Circulation 116:77–84 [DOI] [PubMed] [Google Scholar]
  53. Steiner HJ, Weiler R, Ludescher C, Schmid KW, Winkler H 1990 Chromogranins A and B are co-localized with atrial natriuretic peptides in secretory granules of rat heart. J Histochem Cytochem 38:845–850 [DOI] [PubMed] [Google Scholar]
  54. Weiergraber M, Pereverzev A, Vajna R, Henry M, Schramm M, Nastainczyk W, Grabsch H, Schneider T 2000 Immunodetection of α1E voltage-gated Ca(2+) channel in chromogranin-positive muscle cells of rat heart, and in distal tubules of human kidney. J Histochem Cytochem 48:807–819 [DOI] [PubMed] [Google Scholar]
  55. Miserez B, Annaert W, Dillen L, Aunis D, De Potter W 1992 Chromogranin A processing in sympathetic neurons and release of chromogranin A fragments from sheep spleen. FEBS Lett 314:122–124 [DOI] [PubMed] [Google Scholar]
  56. Glattard E, Angelone T, Strub JM, Corti A, Aunis D, Tota B, Metz-Boutigue MH, Goumon Y 2006 Characterization of natural vasostatin-containing peptides in rat heart. FEBS J 273:3311–3321 [DOI] [PubMed] [Google Scholar]

Articles from Endocrinology are provided here courtesy of The Endocrine Society

RESOURCES