Physiological levels of chromogranin A prevent doxorubicin-induced cardiotoxicity without impairing its anticancer activity.

The clinical use of doxorubicin (Doxo), a widely used anticancer chemotherapeutic drug, is limited by dose-dependent cardiotoxicity. We have investigated whether chromogranin A (CgA), a cardioregulatory protein released in the blood by the neuroendocrine system and by the heart itself, may contribute to regulation of the cardiotoxic and antitumor activities of Doxo. The effects of a physiologic dose of full-length recombinant CgA on Doxo-induced cardiotoxicity and antitumor activity were investigated in rats using in vivo and ex vivo models and in murine models of melanoma, fibrosarcoma, lymphoma, and lung cancer, respectively. The effect of Doxo on circulating levels of CgA was also investigated. In vivo and ex vivo mechanistic studies showed that CgA can prevent Doxo-induced heart inflammation, oxidative stress, apoptosis, fibrosis, and ischemic injury. On the other hand, CgA did not impair the anticancer activity of Doxo in all the murine models investigated. Furthermore, we observed that Doxo can reduce the intracardiac expression and release of CgA in the blood (i.e., an important cardioprotective agent). These findings suggest that administration of low-dose CgA to patients with low levels of endogenous CgA might represent a novel approach to prevent Doxo-induced adverse events without impairing antitumor effects.-Rocca, C., Scavello, F., Colombo, B., Gasparri, A. M., Dallatomasina, A., Granieri, M. C., Amelio, D., Pasqua, T., Cerra, M. C., Tota, B., Corti, A., Angelone, T. Physiological levels of chromogranin A prevent doxorubicin-induced cardiotoxicity without impairing its anticancer activity.

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.

Phosphodiesterase type-2 and NO-dependent S-nitrosylation mediate the cardioinhibition of the antihypertensive catestatin.

The chromogranin A (CHGA)-derived peptide catestatin (CST: hCHGA(352-372)) is a noncompetitive catecholamine-release inhibitor that exerts vasodilator, antihypertensive, and cardiosuppressive actions. We have shown that CST directly influences the basal performance of the vertebrate heart where CST dose dependently induced a nitric oxide-cGMP-dependent cardiosuppression and counteracted the effects of adrenergic stimulation through a noncompetitive antagonism. Here, we sought to determine the specific intracardiac signaling activated by CST in the rat heart. Physiological analyses performed on isolated, Langendorff-perfused cardiac preparations revealed that CST-induced negative inotropism and lusitropism involve ß(2)/ß(3)-adrenergic receptors (ß(2)/ß(3)-AR), showing a higher affinity for ß(2)-AR. Interaction with ß(2)-AR activated phosphatidylinositol 3-kinase/endothelial nitric oxide synthase (eNOS), increased cGMP levels, and induced activation of phosphodiesterases type 2 (PDE2), which was found to be involved in the antiadrenergic action of CST as evidenced by the decreased cAMP levels. CST-dependent negative cardiomodulation was abolished by functional denudation of the endothelium with Triton. CST also increased the eNOS expression in cardiac tissue and human umbilical vein endothelial cells. cells, confirming the involvement of the vascular endothelium. In ventricular extracts, CST increased S-nitrosylation of both phospholamban and ß-arrestin, suggesting an additional mechanism for intracellular calcium modulation and ß-adrenergic responsiveness. We conclude that PDE2 and S-nitrosylation play crucial roles in the CST regulation of cardiac function. Our results are of importance in relation to the putative application of CST as a cardioprotective agent against stress, including excessive sympathochromaffin overactivation.

Nitrite is a positive modulator of the Frank-Starling response in the vertebrate heart.

Evidence from both mammalian and nonmammalian vertebrates indicates that intracardiac nitric oxide (NO) facilitates myocardial relaxation, ventricular diastolic distensibility, and, consequently, the Frank-Starling response, i.e., the preload-induced increase of cardiac output. Since nitrite ion (NO(2)(-)), the major storage pool of bioactive NO, recently emerged as a cardioprotective endogenous modulator, we explored its influence on the Frank-Starling response in eel, frog, and rat hearts, used as paradigms of fish, amphibians, and mammals, respectively. We demonstrated that, like NO, exogenous nitrite improves the Frank-Starling response in all species, as indicated by an increase of stroke volume and stroke work (eel and frog) and of left ventricular (LV) pressure and LVdP/dt max (rat), used as indexes of inotropism. Unlike in frog and rat, in eel, the positive influence of nitrite appeared to be dependent on NO synthase inhibition. In all species, the effect was sensitive to NO scavengers, independent on nitroxyl anion, and mediated by a cGMP/PKG-dependent pathway. Moreover, the nitrite treatment increased S-nitrosylation of lower-molecular-weight proteins in cytosolic and membrane fractions. These results suggest that nitrite acts as a physiological source of NO, modulating through different species-specific mechanisms, the stretch-induced intrinsic regulation of the vertebrate heart.

Integrating nitric oxide, nitrite and hydrogen sulfide signaling in the physiological adaptations to hypoxia: A comparative approach.

Hydrogen sulfide (H(2)S), nitric oxide (NO) and nitrite (NO(2)(-)) are formed in vivo and are of crucial importance in the tissue response to hypoxia, particularly in the cardiovascular system, where these signaling molecules are involved in a multitude of processes including the regulation of vascular tone, cellular metabolic function and cytoprotection. This report summarizes current advances on the mechanisms by which these signaling pathways act and may have evolved in animals with different tolerance to hypoxia, as presented and discussed during the scientific sessions of the annual meeting of the Society for Experimental Biology in 2011 in Glasgow. It also highlights the need and potential for a comparative approach of study and collaborative effort to identify potential link(s) between the signaling pathways involving NO, nitrite and H(2)S in the whole-body responses to hypoxia.

The evolutionary functions of cardiac NOS/NO in vertebrates tracked by fish and amphibian paradigms.

During early ectotherm vertebrate evolution the heart was redesigned as a high pressure pump adapted to perfuse larger body sizes. To compensate the consequent higher organ complexity and heterogeneity (ventricular myoarchitecture and blood supply), conceivably the three principal cardiac cell components, the endocardium, the contractile myocardium and the epicardium recruited and diversified the cardiac NOS system for functioning not only as a major modulator, but also as a spatio-temporal integrator of heart function. In the context of NOS isoform evolution, we will use fish and amphibian paradigms to illustrate major aspects of cardiac spatial and temporal integration achieved by the NOS/NO systems. This may reveal a primordial cardiac NOS/NO function, allocating it in a wider biological framework than so far envisioned.

Role of vasostatin-1 C-terminal region in fibroblast cell adhesion.

Fibroblast adhesion can be modulated by proteins released by neuroendocrine cells and neurons, such as chromogranin A (CgA) and its N-terminal fragment vasostatin-1 (VS-1, CgA(1-78)). We have investigated the mechanisms of the interaction of VS-1 with fibroblasts and of its pro-adhesive activity and have found that the proadhesive activity of VS-1 relies on its interaction with the fibroblast membrane via a phospholipid-binding amphipathic alpha-helix located within residues 47-66, as well as on the interaction of the adjacent C-terminal region 67-78, which is structurally similar to ezrin-radixin-moesin-binding phosphoprotein 50 (a membrane-cytoskeleton adapter protein), with other cellular components critical for the regulation of cell cytoskeleton.

The chromogranin A1-373 fragment reveals how a single change in the protein sequence exerts strong cardioregulatory effects by engaging neuropilin-1.

AIM: Chromogranin A (CgA), a 439-residue long protein, is an important cardiovascular regulator and a precursor of various bioactive fragments. Under stressful/pathological conditions, CgA cleavage generates the CgA1-373 proangiogenic fragment. The present work investigated the possibility that human CgA1-373 influences the mammalian cardiac performance, evaluating the role of its C-terminal sequence. METHODS: Haemodynamic assessment was performed on an ex vivo Langendorff rat heart model, while mechanistic studies were performed using perfused hearts, H9c2 cardiomyocytes and in silico. RESULTS: On the ex vivo heart, CgA1-373 elicited direct dose-dependent negative inotropism and vasodilation, while CgA1-372 , a fragment lacking the C-terminal R373 residue, was ineffective. Antibodies against the PGPQLR373 C-terminal sequence abrogated the CgA1-373 -dependent cardiac and coronary modulation. Ex vivo studies showed that CgA1-373 -dependent effects were mediated by endothelium, neuropilin-1 (NRP1) receptor, Akt/NO/Erk1,2 pathways, nitric oxide (NO) production and S-nitrosylation. In vitro experiments on H9c2 cardiomyocytes indicated that CgA1-373 also induced eNOS activation directly on the cardiomyocyte component by NRP1 targeting and NO involvement and provided beneficial action against isoproterenol-induced hypertrophy, by reducing the increase in cell surface area and brain natriuretic peptide (BNP) release. Molecular docking and all-atom molecular dynamics simulations strongly supported the hypothesis that the C-terminal R373 residue of CgA1-373 directly interacts with NRP1. CONCLUSION: These results suggest that CgA1-373 is a new cardioregulatory hormone and that the removal of R373 represents a critical switch for turning "off" its cardioregulatory activity.

Nitrite exerts potent negative inotropy in the isolated heart via eNOS-independent nitric oxide generation and cGMP-PKG pathway activation.

The ubiquitous anion nitrite (NO(2)(-)) has recently emerged as an endocrine storage form of nitric oxide (NO) and a signalling molecule that mediates a number of biological responses. Although the role of NO in regulating cardiac function has been investigated in depth, the physiological signalling effects of nitrite on cardiac function have only recently been explored. We now show that remarkably low concentrations of nitrite (1 nM) significantly modulate cardiac contractility in isolated and perfused Langendorff rat heart. In particular, nitrite exhibits potent negative inotropic and lusitropic activities as evidenced by a decrease in left ventricular pressure and relaxation, respectively. Furthermore, we demonstrate that the nitrite-dependent effects are mediated by NO formation but independent of NO synthase (NOS) activity. Specifically, nitrite infusion in the Langendorff system produces NO and cGMP/PKG-dependent negative inotropism, as evidenced by the formation of cellular iron-nitrosyl complexes and inhibition of biological effect by NO scavengers and by PKG inhibitors. These data are consistent with the hypothesis that nitrite represents an eNOS-independent source of NO in the heart which modulates cardiac contractility through the NO-cGMP/PKG pathway. The observed high potency of nitrite supports a physiological function of nitrite as a source of cardiomyocyte NO and a fundamental signalling molecule in the heart.

Catestatin improves post-ischemic left ventricular function and decreases ischemia/reperfusion injury in heart.

The Chromogranin A (CgA)-derived anti-hypertensive peptide catestatin (CST) antagonizes catecholamine secretion, and is a negative myocardial inotrope acting via a nitric oxide-dependent mechanism. It is not known whether CST contributes to ischemia/reperfusion injury or is a component of a cardioprotective response to limit injury. Here, we tested whether CST by virtue of its negative inotropic activity improves post-ischemic cardiac function and cardiomyocyte survival. Three groups of isolated perfused hearts from adult Wistar rats underwent 30-min ischemia and 120-min reperfusion (I/R, Group 1), or were post-conditioned by brief ischemic episodes (PostC, 5-cycles of 10-s I/R at the beginning of 120-min reperfusion, Group 2), or with exogenous CST (75 nM for 20 min, CST-Post, Group-3) at the onset of reperfusion. Perfusion pressure and left ventricular pressure (LVP) were monitored. Infarct size was evaluated with nitroblue-tetrazolium staining. The CST (5 nM) effects were also tested in simulated ischemia/reperfusion experiments on cardiomyocytes isolated from young-adult rats, evaluating cell survival with propidium iodide labeling. Infarct size was 61 ± 6% of risk area in hearts subjected to I/R only. PostC reduced infarct size to 34 ± 5%. Infarct size in CST-Post was 36 ± 3% of risk area (P < 0.05 respect to I/R). CST-Post reduced post-ischemic rise of diastolic LVP, an index of contracture, and significantly improved post-ischemic recovery of developed LVP. In isolated cardiomyocytes, CST increased the cell viability rate by about 65% after simulated ischemia/reperfusion. These results suggest a novel cardioprotective role for CST, which appears mainly due to a direct reduction of post-ischemic myocardial damages and dysfunction, rather than to an involvement of adrenergic terminals and/or endothelium.

Catecholamines, cardiac natriuretic peptides and chromogranin A: evolution and physiopathology of a 'whip-brake' system of the endocrine heart.

In the past 50 years, extensive evidence has shown the ability of vertebrate cardiac non-neuronal cells to synthesize and release catecholamines (CA). This formed the mindset behind the search for the intrinsic endocrine heart properties, culminating in 1981 with the discovery of the natriuretic peptides (NP). CA and NP, co-existing in the endocrine secretion granules and acting as major cardiovascular regulators in health and disease, have become of great biomedical relevance for their potent diagnostic and therapeutic use. The concept of the endocrine heart was later enriched by the identification of a growing number of cardiac hormonal substances involved in organ modulation under normal and stress-induced conditions. Recently, chromogranin A (CgA), a major constituent of the secretory granules, and its derived cardio-suppressive and antiadrenergic peptides, vasostatin-1 and catestatin, were shown as new players in this framework, functioning as cardiac counter-regulators in 'zero steady-state error' homeostasis, particularly under intense excitatory stimuli, e.g. CA-induced myocardial stress. Here, we present evidence for the hypothesis that is gaining support, particularly among human cardiologists. The actions of CA, NP and CgA, we argue, may be viewed as a hallmark of the cardiac capacity to organize 'whip-brake' connection-integration processes in spatio-temporal networks. The involvement of the nitric oxide synthase (NOS)/nitric oxide (NO) system in this configuration is discussed. The use of fish and amphibian paradigms will illustrate the ways that incipient endocrine-humoral agents have evolved as components of cardiac molecular loops and important intermediates during evolutionary transitions, or in a distinct phylogenetic lineage, or under stress challenges. This may help to grasp the old evolutionary roots of these intracardiac endocrine/paracrine networks and how they have evolved from relatively less complicated designs. The latter can also be used as an intellectual tool to disentangle the experimental complexity of the mammalian and human endocrine hearts, suggesting future investigational avenues.

The catecholamine release-inhibitory peptide catestatin (chromogranin A344-363) modulates myocardial function in fish.

Catestatin (CST), the 21-amino acid, cationic and hydrophobic peptide proteolytically derived from the ubiquitous chromogranin A (CgA), is an endogenous inhibitor of catecholamine release, a potent vasodilator in vivo and an anti-hypertensive agent in mammals, including humans. Recently, we discovered that CST also functions as an important negative modulator of heart performance in frog and rat. To gain an evolutionary perspective on CST cardiotropism in fish, we analysed the influence of bovine CST (CgA344₋364) on the eel heart, as well as the eventual species-specific mechanisms of its myocardial action. Experiments were carried out on fresh-water eels (Anguilla anguilla L.) using an electrically paced isolated working heart preparation. Stroke volume and stroke work were used as measures of ventricular performance. Under basal conditions, CST (from 11 nmol l⁻¹ to 165 nmol l⁻¹) caused a concentration-dependent negative inotropism, which was abolished by inhibitors of either ß1/ß2 (propranolol) or ß3 (SR59230) adrenergic receptors, or by G(i/o) protein (PTx) or nitric oxide synthase (L-NMMA), or guanylate cyclase (ODQ) blockers. This suggests a ß-adrenergic receptor-G(i/o) protein-NO-cGMP-dependent mechanism. By contrast, the CST-induced cardio-suppression was not influenced by atropine, unspecific muscarinic antagonist, thus excluding cholinergic receptor involvement. CST also counteracted the adrenergic (isoproterenol)-mediated positive inotropism. Under increased preload (i.e. Frank-Starling response) conditions, CST induced a significant increase of the Frank-Starling response, which was blocked by L-NMMA and thapsigargin, but independent from guanylate cyclase. In conclusion, this is the first report in fish that CST modulates myocardial performance under basal, as well as under increased preload, conditions and counteracts the adrenergic-mediated positive inotropism, which strikingly supports the evolutionary significance and establishes the cardioactive role of this peptide.

Morphological and physiological study of the cardiac NOS/NO system in the Antarctic (Hb-/Mb-) icefish Chaenocephalus aceratus and in the red-blooded Trematomus bernacchii.

The nitric oxide synthase (NOS)/nitric oxide (NO) system integrates cellular biochemical machinery and energetics. In heart microenvironment, dynamic NO behaviour depends upon the presence of superoxide anions, haemoglobin (Hb), and myoglobin (Mb), being hemoproteins are major players disarming NO bioactivity. The Antarctic icefish, which lack Hb and, in some species, also cardiac Mb, represent a unique model for exploring Hb and Mb impact on NOS/NO function. We report in the (Hb(-)/Mb(-)) icefish Chaenocephalus aceratus the presence of cardiac NOSs activity (NADPH-diaphorase) and endothelial NOS (eNOS)/inducible NOS (iNOS) zonal immuno-localization in the myocardium. eNOS is localized on endocardium and, to a lesser extent, in myocardiocytes, while iNOS is localized exclusively in myocardiocytes. Confronting eNOS and iNOS expression in Trematomus bernacchii (Hb(+)/Mb(+)), C. hamatus (Hb(-)/Mb(+)) and C. aceratus (Hb(-)/Mb(-)) is evident a lower expression in the Mb-less icefish. NO signaling was analyzed using isolated working heart preparations. In T. bernacchii, L-arginine and exogenous (SIN-1) NO donor dose-dependently decreased stroke volume, indicating decreased inotropism. L-arginine-induced inotropism was NOSs-dependent, being abolished by NOSs-inhibitor NG-monomethyl-L-arginine (L-NMMA). A SIN-1-induced negative inotropism was found in presence of SOD. NOS inhibition by L-N5-N-iminoethyl-L-ornithine (L-NIO) and L-NMMA confirmed the NO-mediated negative inotropic influence on cardiac performance. In contrast, in C. aceratus, L-arginine elicited a positive inotropism. SIN-1 induced a negative inotropism, which disappeared in presence of SOD, indicating peroxynitrite involvement. Cardiac performance was unaffected by L-NIO and L-NIL. NO signaling acted via a cGMP-independent mechanism. This high conservation degree of NOS localization pattern and signaling highlights its importance for cardiac biology.

The antihypertensive chromogranin a peptide catestatin acts as a novel endocrine/paracrine modulator of cardiac inotropism and lusitropism.

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 beta2-adrenergic receptors, Gi/o protein, nitric oxide or cGMP, indicating involvement of beta2-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 beta-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.

Nitric oxide modulates the frog heart ventricle morphodynamics.

The aim of this work was to investigate in the avascular heart of the frog Rana esculenta the influence of nitric oxide (NO) on ventricular systolic and diastolic functions by using a novel image analysis technique. The external volume variations of the whole ventricle were monitored during the heart cycle by video acquisition(visible light) and analysed by an appropriately developed software with a specific formula for irregular convex solids. The system, which measures the rate of volume changes and the ejection fraction, directly determined the volumetric behaviour of the working frog heart after stimulation or inhibition of NOS-NOcGMP pathway. End-diastolic volume (EDVext), end-systolic volume (ESVext), contraction and relaxation velocities (dV/dtsys and dV/dtdia, respectively), stroke volume (SV) and ejection fraction (EF), were measured before and after perfusion with NOS substrate (L-arginine), NO donor (SIN-1), cGMP analogue (8-Br-cGMP),NOS inhibitors (NG-monomethyl-L-arginine, L-NMMA; L-N(5)-(1-iminoethyl)-ornithine, L-NIO; 7-Nitroindazole,7-NI) and guanylyl cyclase inhibitor (ODQ). The results showed that NO reduces ventricular systolicfunction improving diastolic filling, while NOS inhibition increases contractility impairing ventricular filling capacity. The presence of activated eNOS (p-eNOS) was morphologically documented, further supporting that the mechanical activity of the ventricular pump in frog is influenced by a tonic release of NOS-generated NO.

The morphological and functional significance of the NOS/NO system in the respiratory, osmoregulatory, and contractile organs of the African lungfish.

This review aims to summarize the changes of the NOS/NO system which occur in the lungs, gills, kidney, heart, and myotomal muscle of air breathing fish of the genus Protopterus, i.e. P. dolloi and P. annectens, in relation to the switch from freshwater to aestivation, and vice-versa. The modifications of NOS and its partners Akt and Hsp-90, and HIF-1α, detected by immunohistochemical and molecular biology methods, are discussed together with the apoptosis rate, evaluated by TUNEL. We hypothesize that these molecular components are key elements of the stress-induced signal transduction/integration networks which allow the lungfish to overcome the dramatic environmental challenges experienced at the beginning, during, and at the end of the dry season.

Crucial role of cytoskeleton reorganization in the negative inotropic effect of chromogranin A-derived peptides in eel and frog hearts.

Vasostatins (VSs), i.e. the main biologically active peptides generated by the proteolytic processing of chromogranin A (CGA) N-terminus, exert negative inotropism in vertebrate hearts. Here, using isolated working eel (Anguilla anguilla) and frog (Rana esculenta) heart preparations, we have studied the role of the cytoskeleton in the VSs-mediated inotropic response. In both eel and frog hearts, VSs-mediated-negative inotropy was abolished by treatment with inhibitors of cytoskeleton reorganization, such as cytochalasin-D (eel: 10 nM; frog: 1 nM), an inhibitor of actin polymerisation, wortmannin (0.01 nM), an inhibitor of PI3-kinase (PI3-K)/protein kinase B (Akt) signal-transduction cascade, butanedione 2-monoxime (BDM) (eel: 100 nM; frog: 10 nM), an antagonist of myosin ATPase, and N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide (W7) (eel: 100 nM; frog: 1 nM), a calcium-calmodulin antagonist. These results demonstrate that changes in cytoskeletal dynamics play a crucial role in the negative inotropic influence of VSs on eel and frog hearts.

Granin-derived peptides.

The granin family comprises altogether 7 different proteins originating from the diffuse neuroendocrine system and elements of the central and peripheral nervous systems. The family is dominated by three uniquely acidic members, namely chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII). Since the late 1980s it has become evident that these proteins are proteolytically processed, intragranularly and/or extracellularly into a range of biologically active peptides; a number of them with regulatory properties of physiological and/or pathophysiological significance. The aim of this comprehensive overview is to provide an up-to-date insight into the distribution and properties of the well established granin-derived peptides and their putative roles in homeostatic regulations. Hence, focus is directed to peptides derived from the three main granins, e.g. to the chromogranin A derived vasostatins, betagranins, pancreastatin and catestatins, the chromogranin B-derived secretolytin and the secretogranin II-derived secretoneurin (SN). In addition, the distribution and properties of the chromogranin A-derived peptides prochromacin, chromofungin, WE14, parastatin, GE-25 and serpinins, the CgB-peptide PE-11 and the SgII-peptides EM66 and manserin will also be commented on. Finally, the opposing effects of the CgA-derived vasostatin-I and catestatin and the SgII-derived peptide SN on the integrity of the vasculature, myocardial contractility, angiogenesis in wound healing, inflammatory conditions and tumors will be discussed.

Characterization of natural vasostatin-containing peptides in rat heart.

Chromogranin A (CGA) is a protein that is stored and released together with neurotransmitters and hormones in the nervous, endocrine and diffuse neuroendocrine systems. As human vasostatins I and II [CGA(1-76) and CGA(1-113), respectively] have been reported to affect vessel motility and exert concentration-dependent cardiosuppressive effects on isolated whole heart preparations of eel, frog and rat (i.e. negative inotropism and antiadrenergic activity), we investigated the presence of vasostatin-containing peptides in rat heart. Rat heart extracts were purified by RP-HPLC, and the resulting fractions analyzed for the presence of CGA N-terminal fragments using dot-blot analysis. CGA-immunoreactive fractions were submitted to western blot and MS analysis using the TOF/TOF technique. Four endogenous N-terminal CGA-derived peptides [CGA(4-113), CGA(1-124), CGA(1-135) and CGA(1-199)] containing the vasostatin sequence were characterized. The following post-translational modifications of these fragments were identified: phosphorylation at Ser96, O-glycosylation (trisaccharide, NAcGal-Gal-NeuAc) at Thr126, and oxidation at three methionine residues. This first identification of CGA-derived peptides containing the vasostatin motif in rat heart supports their role in cardiac physiology by an autocrine/paracrine mechanism.