Chromogranin A-derived peptides pancreastatin and catestatin: emerging therapeutic target for diabetes.

Chromogranin A (ChgA) is an acidic pro-protein found in neuroendocrine organs, pheochromocytoma chromaffin granules, and tumor cells. Proteolytic processing of ChgA gives rise to an array of biologically active peptides such as pancreastatin (PST), vasostatin, WE14, catestatin (CST), and serpinin, which have diverse roles in regulating cardiovascular functions and metabolism, as well as inflammation. Intricate tissue-specific role of ChgA-derived peptide activity in preclinical rodent models of metabolic syndrome reveals complex effects on carbohydrate and lipid metabolism. Indeed, ChgA-derived peptides, PST and CST, play a pivotal role in metabolic syndrome such as obesity, insulin resistance, and diabetes mellitus. Additionally, supplementation of specific peptide in ChgA-KO mice have an opposing effect on physiological functions, such as PST supplementation reduces insulin sensitivity and enhances inflammatory response. In contrast, CST supplementation enhances insulin sensitivity and reduces inflammatory response. In this review, we focus on the tissue-specific role of PST and CST as therapeutic targets in regulating carbohydrate and lipid metabolism, along with the associated risk factors.

Chromogranin A-derived peptide CGA47-66 protects against septic brain injury by reducing blood-brain barrier damage through the PI3K/AKT pathway.

CGA47-66 (Chromofungin, CHR), is a peptide derived from the N-terminus of chromogranin A (CgA), has been proven to inhibit the lipopolysaccharide (LPS)-induced brain injury. However, the underlying mechanism is still unknown. We found that CGA47-66 exerted a protective effect on cognitive impairment by inhibiting the destruction of the blood-brain barrier (BBB) in the LPS-induced sepsis mice model. In addition, the hCMEC/D3 cell line was used to establish an in vitro BBB model. Under LPS stimulation, CGA47-66 could significantly alleviate the hyperpermeability of the BBB, the destruction of tight junction proteins, and the rearrangement of F-actin. To investigate the underlying mechanism, we used LY294002, a PI3K inhibitor, which partially reduced the protective effect of CGA47-66 on the integrity of BBB. Indicating that the PI3K/AKT pathway plays a vital role in the brain-protective function of CGA47-66, which might be a potential therapeutic target for septic brain injury.

Catestatin enhances ATP-induced activation of glial cells mediated by purinergic receptor P2X4.

The activation of glial cells and its possible mechanism play an extremely important role in understanding the pathophysiological process of some clinical diseases, and catestatin (CST) is involved in regulating this activation. In this project, we found that CST could enhance the activation of satellite glial cells (SGCs) and microglial cells and that the expression of P2X4 was increased; the co-expression of the P2X4 receptor with glial fibrillary acidic protein (GFAP) and the P2X4 receptor with CD11b was also increased significantly in glial cells of the ATP + CST group, and TNF-α and IL-1ß also showed a rising trend; the expression of phosphorylated ERK1/2 was also increased in the ATP + CST group. In summary, we conclude that CST could enhance ATP-induced activation of SGCs and microglial cells mediated by the P2X4 receptor and that the ERK1/2 signaling pathway may be involved in this activation process.

The Catestatin-Derived Peptides Are New Actors to Fight the Development of Oral Candidosis.

Resistance to antifungal therapy of Candida albicans and non-albicans Candida strains, frequently associated with oral candidosis, is on the rise. In this context, host-defense peptides have emerged as new promising candidates to overcome antifungal resistance. Thus, the aim of this study was to assess the effectiveness against Candida species of different Catestatin-derived peptides, as well as the combined effect with serum albumin. Among Catestatin-derived peptides, the most active against sensitive and resistant strains of C. albicans, C. tropicalis and C. glabrata was the D-isomer of Cateslytin (D-bCtl) whereas the efficiency of the L-isomer (L-bCtl) significantly decreases against C. glabrata strains. Images obtained by transmission electron microscopy clearly demonstrated fungal membrane lysis and the leakage of the intracellular material induced by the L-bCtl and D-bCtl peptides. The possible synergistic effect of albumin on Catestatin-derived peptides activity was investigated too. Our finding showed that bovine serum albumin (BSA) when combined with the L- isomer of Catestatin (L-bCts) had a synergistic effect against Candida albicans especially at low concentrations of BSA; however, no synergistic effect was detected when BSA interacted with L-bCtl, suggesting the importance of the C-terminal end of L-bCts (GPGLQL) for the interaction with BSA. In this context in vitro D-bCtl, as well as the combination of BSA with L-bCts are potential candidates for the development of new antifungal drugs for the treatment of oral candidosis due to Candida and non-Candida albicans, without detrimental side effects.

The antifungal peptide CGA-N12 inhibits cell wall synthesis of Candida tropicalis by interacting with KRE9.

CGA-N12, an antifungal peptide derived from chromogranin A, has specific antagonistic activity against Candida spp., especially against Candida tropicalis, by inducing cell apoptosis. However, the effect of CGA-N12 on the Candida cell wall is unknown. The Candida protein KRE9, which possesses ß-1,6-glucanase activity, was screened by affinity chromatography after binding to CGA-N12. In this study, the effect of CGA-N12 on KRE9 and the interaction between CGA-N12 and KRE9 was studied to clarify the effect of CGA-N12 on C. tropicalis cell wall synthesis. The effect of CGA-N12 on recombinant KRE9 ß-1,6-glucanase activity was investigated by analyzing the consumption of glucose. The results showed that CGA-N12 inhibited the activity of KRE9. After C. tropicalis was treated with CGA-N12, the structure of the C. tropicalis cell wall was damaged. The interaction between CGA-N12 and KRE9 was analyzed by isothermal titration calorimetry (ITC). The results showed that their interaction process was involved an endothermic reaction, and the interaction force was mainly hydrophobic with a few electrostatic forces. The results of the fluorescence resonance energy transfer (FRET) assay showed that the distance between CGA-N12 and KRE9 was 7 ∼ 10 nm during their interaction. Therefore, we concluded that the target of CGA-N12 in the C. tropicalis cell membrane is KRE9, and that CGA-N12 weakly binds to KRE9 within a 7 ∼ 10 nm distance and inhibits KRE9 activity.

Catestatin reverses the hypertrophic effects of norepinephrine in H9c2 cardiac myoblasts by modulating the adrenergic signaling.

Catestatin (CST) is a catecholamine release-inhibitory peptide secreted from the adrenergic neurons and the adrenal glands. It regulates the cardiovascular functions and it is associated with cardiovascular diseases. Though its mechanisms of actions are not known, there are evidences of cross-talk between the adrenergic and CST signaling. We hypothesized that CST moderates the adrenergic overdrive and studied its effects on norepinephrine-mediated hypertrophic responses in H9c2 cardiac myoblasts. CST alone regulated the expression of a number of fetal genes that are induced during hypertrophy. When cells were pre-treated CST, it blunted the modulation of those genes by norepinephrine. Norepinephrine (2 µM) treatment also increased cell size and enhanced the level of Troponin T in the sarcomere. These effects were attenuated by the treatment with CST. CST attenuated the immediate generation of ROS and the increase in glutathione peroxidase activity induced by norepinephrine treatment. Expression of fosB and AP-1 promoter-reporter constructs was used as the endpoint readout for the interaction between the CST and adrenergic signals at the gene level. It showed that CST largely attenuates the stimulatory effects of norepinephrine and other mitogenic signals through the modulation of the gene regulatory modules in a characteristic manner. Depending upon the dose, the signaling by CST appears to be disparate, and at 10-25 nM doses, it primarily moderated the signaling by the ß1/2-adrenoceptors. This study, for the first time, provides insights into the modulation of adrenergic signaling in the heart by CST.

The chromogranin A-derived antifungal peptide CGA-N9 induces apoptosis in Candida tropicalis.

CGA-N9, a peptide derived from human chromogranin A (CGA), was found to have antimicrobial activity in our previous investigation, but its mechanism of action remains unclear. Herein, the mechanism of action of CGA-N9 was investigated. We found that CGA-N9 induced the depolarization of the cell membrane and uptake of calcium ions into the cytosol and mitochondria. With the disruption of the mitochondrial membrane potential, the generation of intracellular reactive oxygen species (ROS) increased. Accordingly, we assessed apoptotic processes in Candida tropicalis cells post-treatment with CGA-N9 and found cytochrome c leakage, chromatin condensation and DNA degradation. The interaction of CGA-N9 with DNA in vitro showed that CGA-N9 did not degrade DNA but bound to DNA via an electrostatic interaction. In conclusion, CGA-N9 exhibits antifungal activity by inducing apoptosis in C. tropicalis.

Catestatin regulates vesicular quanta through modulation of cholinergic and peptidergic (PACAPergic) stimulation in PC12 cells.

We have previously shown that the chromogranin A (CgA)-derived peptide catestatin (CST: hCgA352-372) inhibits nicotine-induced secretion of catecholamines from the adrenal medulla and chromaffin cells. In the present study, we seek to determine whether CST regulates dense core (DC) vesicle (DCV) quanta (catecholamine and chromogranin/secretogranin proteins) during acute (0.5-h treatment) or chronic (24-h treatment) cholinergic (nicotine) or peptidergic (PACAP, pituitary adenylyl cyclase activating polypeptide) stimulation of PC12 cells. In acute experiments, we found that both nicotine (60 µM) and PACAP (0.1 µM) decreased intracellular norepinephrine (NE) content and increased 3H-NE secretion, with both effects markedly inhibited by co-treatment with CST (2 µM). In chronic experiments, we found that nicotine and PACAP both reduced DCV and DC diameters and that this effect was likewise prevented by CST. Nicotine or CST alone increased expression of CgA protein and together elicited an additional increase in CgA protein, implying that nicotine and CST utilize separate signaling pathways to activate CgA expression. In contrast, PACAP increased expression of CgB and SgII proteins, with a further potentiation by CST. CST augmented the expression of tyrosine hydroxylase (TH) but did not increase intracellular NE levels, presumably due to its inability to cause post-translational activation of TH through serine phosphorylation. Co-treatment of CST with nicotine or PACAP increased quantal size, plausibly due to increased synthesis of CgA, CgB and SgII by CST. We conclude that CST regulates DCV quanta by acutely inhibiting catecholamine secretion and chronically increasing expression of CgA after nicotinic stimulation and CgB and SgII after PACAPergic stimulation.

Catestatin Enhances Neuropathic Pain Mediated by P2X4 Receptor of Dorsal Root Ganglia in a Rat Model of Chronic Constriction Injury.

BACKGROUND/AIMS: Neuropathic pain (NPP) is the consequence of a number of central nervous system injuries or diseases. Previous studies have shown that NPP is mediated by P2X4 receptors that are expressed on satellite glial cells (SGCs) of dorsal root ganglia (DRG). Catestatin (CST), a neuroendocrine multifunctional peptide, may be involved in the pathogenesis of NPP. Here, we studied the mechanism through which CST affects NPP. METHODS: We made rat models of chronic constriction injury (CCI) that simulate neuropathic pain. Rat behavioral changes were estimated by measuring the degree of hyperalgesia as assessed by the mechanical withdrawal threshold (MWT) and the thermal withdrawal latency (TWL). P2X4 mRNA expression was detected by quantitative real-time reverse transcription-polymerase chain reaction. P2X4 protein level and related signal pathways were assessed by western blot. Additionally, double-labeled immunofluorescence was employed to visualize the correspondence between the P2X4 receptor and glial fibrillary acidic protein. An enzyme-linked immunosorbent assay was performed to determine the concentration of CST and inflammatory factors. RESULTS: CST led to lower MWT and TWL and increased P2X4 mRNA and protein expression on the SGCs of model rats. Further, CST upregulated the expression of phosphor-p38 and phosphor-ERK 1/2 on the SGCs of CCI rats. However, the expression level of phosphor-JNK and phosphor-p65 did not obviously change. CONCLUSION: Taken together, CST might boost NPP by enhancing the sensitivity of P2X4 receptors in the DRG of rats, which would provide us a novel perspective and research direction to explore new therapeutic targets for NPP.

Striking Oxygen Sensitivity of the Peptidylglycine α-Amidating Monooxygenase (PAM) in Neuroendocrine Cells.

Interactions between biological pathways and molecular oxygen require robust mechanisms for detecting and responding to changes in cellular oxygen availability, to support oxygen homeostasis. Peptidylglycine α-amidating monooxygenase (PAM) catalyzes a two-step reaction resulting in the C-terminal amidation of peptides, a process important for their stability and biological activity. Here we show that in human, mouse, and insect cells, peptide amidation is exquisitely sensitive to hypoxia. Different amidation events on chromogranin A, and on peptides processed from proopiomelanocortin, manifest similar striking sensitivity to hypoxia in a range of neuroendocrine cells, being progressively inhibited from mild (7% O2) to severe (1% O2) hypoxia. In developing Drosophila melanogaster larvae, FMRF amidation in thoracic ventral (Tv) neurons is strikingly suppressed by hypoxia. Our findings have thus defined a novel monooxygenase-based oxygen sensing mechanism that has the capacity to signal changes in oxygen availability to peptidergic pathways.

Vasostatin-1 Stops Structural Remodeling and Improves Calcium Handling via the eNOS-NO-PKG Pathway in Rat Hearts Subjected to Chronic ß-Adrenergic Receptor Activation.

PURPOSE: Chronically elevated catecholamine levels activate cardiac ß-adrenergic receptors, which play a vital role in the pathogenesis of heart failure. Evidence suggests that vasostatin-1 (VS-1) exerts anti-adrenergic effects on isolated and perfused hearts in vitro. Whether VS-1 ameliorates hypertrophy/remodeling by inducing the chronic activation of ß-adrenergic receptors is unknown. The present study aims to test the efficacy of using VS-1 to treat the advanced hypertrophy/remodeling that result from chronic ß-adrenergic receptor activation and to determine the cellular and molecular mechanisms that underlie this response. METHODS AND RESULT: Rats were subjected to infusion with either isoprenaline (ISO, 5 mg/kg/d), ISO plus VS-1 (30 mg/kg/d) or placebo for 2 weeks. VS-1 suppressed chamber dilation, myocyte hypertrophy and fibrosis and improved in vivo heart function in the rats subjected to ISO infusion. VS-1 increased phosphorylated nitric oxide synthase levels and induced the activation of protein kinase G. VS-1 also deactivated multiple hypertrophy signaling pathways that were triggered by the chronic activation of ß-adrenergic receptors, such as the phosphoinositide-3 kinase (PI3K)/Akt and Ca2+/calmodulin-dependent kinase (CaMK-II) pathways. Myocytes isolated from ISO + VS-1 hearts displayed higher Ca2+ transients, shorter Ca2+ decays, higher sarcoplasmic reticulum Ca2+ levels and higher L-type Ca2+ current densities than the ISO rat hearts. VS-1 treatment restored the protein expression of sarcoplasmic reticulum Ca2+ uptake ATPase, phospholamban and Cav1.2, indicating improved calcium handling. CONCLUSIONS: Chronic VS-1 treatment inhibited the progression of hypertrophy, fibrosis, and chamber remodeling, and improved cardiac function in a rat model of ISO infusion. In addition, Ca2+ handling and its molecular modulation were also improved by VS-1. The beneficial effects of VS-1 on cardiac remodeling may be mediated by the enhanced activation of the eNOS-cGMP-PKG pathway.

Molecular interactions of the physiological anti-hypertensive peptide catestatin with the neuronal nicotinic acetylcholine receptor.

Catestatin (CST), a chromogranin-A-derived peptide, is a potent endogenous inhibitor of the neuronal nicotinic acetylcholine receptor (nAChR). It exerts an anti-hypertensive effect by acting as a 'physiological brake' on transmitter release into the circulation. However, the mechanism of interaction of CST with nAChR is only partially understood. To unravel molecular interactions of the wild-type human CST (CST-WT) as well as its naturally occurring variants (CST-364S and CST-370L, which have Gly→Ser and Pro→Leu substitutions, respectively) with the human α3ß4 nAChR, we generated a homology-modeled human α3ß4 nAChR structure and solution structures of CST peptides. Docking and molecular dynamics simulations showed that ~90% of interacting residues were within 15 N-terminal residues of CST peptides. The rank order of binding affinity of these peptides with nAChR was: CST-370L>CST-WT>CST-364S; the extent of occlusion of the receptor pore by these peptides was also in the same order. In corroboration with computational predictions, circular dichroism analysis revealed significant differences in global structures of CST peptides (e.g. the order of α-helical content was: CST-370L>CST-WT>CST-364S). Consistently, CST peptides blocked various stages of nAChR signal transduction, such as nicotine- or acetylcholine-evoked inward current, rise in intracellular Ca(2+) and catecholamine secretion in or from neuron-differentiated PC12 cells, in the same rank order. Taken together, this study shows molecular interactions between human CST peptides and human α3ß4 nAChR, and demonstrates that alterations in the CST secondary structure lead to the gain of potency for CST-370L and loss of potency for CST-364S. These findings have implications for understanding the nicotinic cholinergic signaling in humans.

Chromogranin A is a T cell antigen in human type 1 diabetes.

Chromogranin A (ChgA) is a beta cell secretory granule protein and a peptide of ChgA, WE14, was recently identified as a ligand for diabetogenic CD4 T cell clones derived from the NOD mouse. In this study we compared responses of human CD4 T cells from recent onset type 1 diabetic (T1D) and control subjects to WE14 and to an enzymatically modified version of this peptide. T cell responders to antigens were detected in PBMCs from study subjects by an indirect CD4 ELISPOT assay for IFN-γ. T1D patients (n = 27) were recent onset patients within one year of diagnosis, typed for HLA-DQ8. Controls (n = 31) were either 1st degree relatives with no antibodies or from the HLA-matched general population cohort of DAISY/TEDDY. A second cohort of patients (n = 11) and control subjects (n = 11) was tested at lower peptide concentrations. We found that WE14 is recognized by T cells from diabetic subjects vs. controls in a dose dependent manner. Treatment of WE14 with transglutaminase increased reactivity to the peptide in some patients. This work suggests that ChgA is an important target antigen in human T1D subjects and that post-translational modification may play a role in its reactivity and relationship to disease.

The protective effects of Chromofungin in oligomeric amyloid ß42 (Aß42)-induced toxicity in neurons in Alzheimer's disease.

Oligomeric Aß42 is considered to play a harmful role in the pathophysiology of Alzheimer's disease (AD). Prolonged exposure to oligomeric Aß42 could induce neuronal damage including cellular senescence. Amelioration of Aß42-induced cellular senescence has been considered as a promising strategy for the treatment of AD. Chromofungin, a chromogranin A-derived peptide, has displayed various biological functions in different types of cells and tissues. However, the effects of Chromofungin on oligomeric Aß42-induced cellular senescence have not been previously reported. In the current study, we report a novel function of Chromofungin by showing that treatment with Chromofungin could ameliorate Aß42-induced neurotoxicity in M17 neuronal cells. The Cell Counting Kit-8 (CCK-8) assay and the lactate dehydrogenase (LDH) release experiments revealed that 0.5 and 1 mM are the optimal concentrations of Chromofungin for cell culture in M17 cells. Challenging with oligomeric Aß42 (5 µM) for 7 and 14 days led to a significant decrease in telomerase activity, which was rescued by Chromofungin dose-dependently. Additionally, the senescence-associated ß-galactosidase (SA-ß-gal) staining assay demonstrated that Chromofungin mitigated oligomeric Aß42-induced cellular senescence. Correspondingly, treatment with Chromofungin reversed the gene expression of human telomerase reverse transcriptase (hTERT), telomeric repeat-binding factor 2 (TERF2), and p21 against oligomeric Aß42 in M17 neurons. Interestingly, Chromofungin attenuated oligomeric Aß42-induced oxidative stress (OS) in M17 cells by reducing the production of intracellular reactive oxygen species (ROS) but increasing the levels of intracellular superoxide dismutase (SOD). Importantly, the presence of Chromofungin reduced the expression of cyclooxygenase2 (COX-2) as well as the generation of prostaglandin E2 (PGE2). Transduction with Ad-COX-2 impaired the effects of Chromofungin on telomerase activity and the profile of cellular senescence. Our findings suggest that Chromofungin might act as a potential agent for the treatment of AD.

Functional genetic variants of the catecholamine-release-inhibitory peptide catestatin in an Indian population: allele-specific effects on metabolic traits.

Catestatin (CST), a chromogranin A (CHGA)-derived peptide, is a potent inhibitor of catecholamine release from adrenal chromaffin cells and postganglionic sympathetic axons. We re-sequenced the CST region of CHGA in an Indian population (n = 1010) and detected two amino acid substitution variants: G364S and G367V. Synthesized CST variant peptides (viz. CST-Ser-364 and CST-Val-367) were significantly less potent than the wild type peptide (CST-WT) to inhibit nicotine-stimulated catecholamine secretion from PC12 cells. Consistently, the rank-order of blockade of nicotinic acetylcholine receptor (nAChR)-stimulated inward current and intracellular Ca(2+) rise by these peptides in PC12 cells was: CST-WT > CST-Ser-364 > CST-Val-367. Structural analysis by CD spectroscopy coupled with molecular dynamics simulations revealed the following order of α-helical content: CST-WT > CST-Ser-364 > CST-Val-367; docking of CST peptides onto a major human nAChR subtype and molecular dynamics simulations also predicted the above rank order for their binding affinity with nAChR and the extent of occlusion of the receptor pore, providing a mechanistic basis for differential potencies. The G364S polymorphism was in strong linkage disequilibrium with several common CHGA genetic variations. Interestingly, the Ser-364 allele (detected in ∼15% subjects) was strongly associated with profound reduction (up to ∼2.1-fold) in plasma norepinephrine/epinephrine levels consistent with the diminished nAChR desensitization-blocking effect of CST-Ser-364 as compared with CST-WT. Additionally, the Ser-364 allele showed strong associations with elevated levels of plasma triglyceride and glucose levels. In conclusion, a common CHGA variant in an Indian population influences several biochemical parameters relevant to cardiovascular/metabolic disorders.

Catestatin (chromogranin A(352-372)) and novel effects on mobilization of fat from adipose tissue through regulation of adrenergic and leptin signaling.

Chromogranin A knock-out (Chga-KO) mice display increased adiposity despite high levels of circulating catecholamines and leptin. Consistent with diet-induced obese mice, desensitization of leptin receptors caused by hyperleptinemia is believed to contribute to the obese phenotype of these KO mice. In contrast, obesity in ob/ob mice is caused by leptin deficiency. To characterize the metabolic phenotype, Chga-KO mice were treated with the CHGA-derived peptide catestatin (CST) that is deficient in these mice. CST treatment reduced fat depot size and increased lipolysis and fatty acid oxidation. In liver, CST enhanced oxidation of fatty acids as well as their assimilation into lipids, effects that are attributable to the up-regulation of genes promoting fatty acid oxidation (Cpt1α, Pparα, Acox, and Ucp2) and incorporation into lipids (Gpat and CD36). CST did not affect basal or isoproterenol-stimulated cAMP production in adipocytes but inhibited phospholipase C activation by the α-adrenergic receptor (AR) agonist phenylephrine, suggesting inhibition of α-AR signaling by CST. Indeed, CST mimicked the lipolytic effect of the α-AR blocker phentolamine on adipocytes. Moreover, CST reversed the hyperleptinemia of Chga-KO mice and improved leptin signaling as determined by phosphorylation of AMPK and Stat3. CST also improved peripheral leptin sensitivity in diet-induced obese mice. In ob/ob mice, CST enhanced leptin-induced signaling in adipose tissue. In conclusion, our results implicate CST in a novel pathway that promotes lipolysis and fatty acid oxidation by blocking α-AR signaling as well as by enhancing leptin receptor signaling.

Catestatin reduces myocardial ischaemia/reperfusion injury: involvement of PI3K/Akt, PKCs, mitochondrial KATP channels and ROS signalling.

Catestatin (CST) limits myocardial ischaemia/reperfusion (I/R) injury with unknown mechanisms. Clearly phosphoinositide-3-kinase (PI3K), protein kinase C (PKC) isoforms, including intra-mitochondrial PKCε, mitochondrial KATP (mitoKATP) channels and subsequent reactive oxygen species (ROS)-signalling play important roles in postconditioning cardioprotection, preventing mitochondrial permeability transition pore (mPTP) opening. Therefore, we studied the role of these extra- and intra-mitochondrial factors in CST-induced protection. Isolated rat hearts and H9c2 cells underwent I/R and oxidative stress, respectively. In isolated hearts CST (75nM, CST-Post) given in early-reperfusion significantly reduced infarct size, limited post-ischaemic contracture, and improved recovery of developed left ventricular pressure. PI3K inhibitor, LY-294002 (LY), large spectrum PKC inhibitor, Chelerythrine (CHE), specific PKCε inhibitor (εV1-2), mitoKATP channel blocker, 5-Hydroxydecanoate (5HD) or ROS scavenger, 2-mercaptopropionylglycine (MPG) abolished the infarct-sparing effect of CST. Notably the CST-induced contracture limitation was maintained during co-infusion of 5HD, MPG or εV1-2, but it was lost during co-infusion of LY or CHE. In H9c2 cells challenged with H2O2, mitochondrial depolarization (an index of mPTP opening studied with JC1-probe) was drastically limited by CST (75nM). Our results suggest that the protective signalling pathway activated by CST includes mitoKATP channels, ROS signalling and prevention of mPTP opening, with a central role for upstream PI3K/Akt and PKCs. In fact, all inhibitors completely abolished CST-infarct-sparing effect. Since CST-anti-contracture effect cannot be explained by intra-mitochondrial mechanisms (PKCε activation and mitoKATP channel opening) or ROS signalling, it is proposed that these downstream signals are part of a reverberant loop which re-activates upstream PKCs, which therefore play a pivotal role in CST-induced protection.

The novel chromogranin A-derived serpinin and pyroglutaminated serpinin peptides are positive cardiac ß-adrenergic-like inotropes.

Three forms of serpinin peptides, serpinin (Ala26Leu), pyroglutaminated (pGlu)-serpinin (pGlu23Leu), and serpinin-Arg-Arg-Gly (Ala29Gly), are derived from cleavage at pairs of basic residues in the highly conserved C terminus of chromogranin A (CgA). Serpinin induces PN-1 expression in neuroendocrine cells to up-regulate granule biogenesis via a cAMP-protein kinase A-Sp1 pathway, while pGlu-serpinin inhibits cell death. The aim of this study was to test the hypothesis that serpinin peptides are produced in the heart and act as novel ß-adrenergic-like cardiac modulators. We detected serpinin peptides in the rat heart by HPLC and ELISA methods. The peptides included predominantly Ala29Gly and pGlu-serpinin and a small amount of serpinin. Using the Langendorff perfused rat heart to evaluate the hemodynamic changes, we found that serpinin and pGlu-serpinin exert dose-dependent positive inotropic and lusitropic effects at 11-165 nM, within the first 5 min after administration. The pGlu-serpinin-induced contractility is more potent than that of serpinin, starting from 1 nM. Using the isolated rat papillary muscle preparation to measure contractility in terms of tension development and muscle length, we further corroborated the pGlu-serpinin-induced positive inotropism. Ala29Gly was unable to affect myocardial performance. Both pGlu-serpinin and serpinin act through a ß1-adrenergic receptor/adenylate cyclase/cAMP/PKA pathway, indicating that, contrary to the ß-blocking profile of the other CgA-derived cardiosuppressive peptides, vasostatin-1 and catestatin, these two C-terminal peptides act as ß-adrenergic-like agonists. In cardiac tissue extracts, pGlu-serpinin increased intracellular cAMP levels and phosphorylation of phospholamban (PLN)Ser16, ERK1/2, and GSK-3ß. Serpinin and pGlu-serpinin peptides emerge as novel ß-adrenergic inotropic and lusitropic modulators, suggesting that CgA and the other derived cardioactive peptides can play a key role in how the myocardium orchestrates its complex response to sympathochromaffin stimulation.

Development of a pharmacophore model for the catecholamine release-inhibitory peptide catestatin: virtual screening and functional testing identify novel small molecule therapeutics of hypertension.

The endogenous catecholamine release-inhibitory peptide catestatin (CST) regulates events leading to hypertension and cardiovascular disease. Earlier we studied the structure of CST by NMR, molecular modeling, and amino acid scanning mutagenesis. That structure has now been exploited for elucidation of interface pharmacophores that mediate binding of CST to its target, with consequent secretory inhibition. Designed pharmacophore models allowed screening of 3D structural domains. Selected compounds were tested on both cultured catecholaminergic cells and an in vivo model of hypertension; in each case, the candidates showed substantial mimicry of native CST actions, with preserved or enhanced potency and specificity. The approach and compounds have thus enabled rational design of novel drug candidates for treatment of hypertension or autonomic dysfunction.

Reduced serum levels of vasostatin-2, an anti-inflammatory peptide derived from chromogranin A, are associated with the presence and severity of coronary artery disease.

AIMS: We here investigated the endothelial effects of the chromogranin A-derived peptide vasostatin-2 and its relation to coronary artery disease (CAD). METHODS AND RESULTS: We assessed the impact of recombinant vasostatin-1 and vasostatin-2 on tumour necrosis factor-alpha (TNFα)-, angiotensin II-, and oxidized low-density lipoprotein (oxLDL)-induced expression of adhesion molecules in human arterial endothelial cells. Vasostatin-1 and vasostatin-2 levels were examined in coronary endarterectomy specimens (n= 23), atherosclerotic aortas (n= 16), non-significant-atherosclerotic internal mammary arteries (n= 30), and non-atherosclerotic aortas (n= 10), as well as in peripheral blood mononuclear cells (PBMCs) from severe CAD patients (n= 50) and healthy volunteers (n= 21). Serum levels of vasostatin-2 were analysed in 968 consecutive patients undergoing coronary angiography. Vasostatin-1 and vasostatin-2 concentration-dependently inhibited TNFα-, angiotensin II-, and oxLDL-induced expression of adhesion molecules; and attenuated TNFα-induced adhesion of U937 monocytes to endothelial cells. Vasostatin-2 levels were significantly decreased in endarterectomy samples and atherosclerotic aortas compared with non-atherosclerotic internal mammary arteries and aortas, as well as in PBMCs of severe CAD patients compared with healthy controls (all P< 0.05). Serum vasostatin-2 levels were significantly lower in CAD patients (diameter stenosis ≥ 50%, n= 554) than in controls (normal arteries or diameter stenosis <30%, n= 281) (P< 0.001). Its concentrations correlated with the number of diseased coronary arteries and Syntax score in CAD patients (all P< 0.05). At multivariable regression analysis, decreased vasostatin-2 levels remained associated with CAD when other variables were taken into account. CONCLUSION: Vasostatin-2 has anti-inflammatory properties and is decreased in atherosclerotic plaque specimens and in PBMC of CAD patients. Decreased serum vasostatin-2 levels are associated with the presence and severity of CAD.