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.     

Temperature effects on the inotropic and chronotropic responses to adrenaline in the frog heart

Summary The contractile force and frequency of the spontaneously beating auricles ofRana tempeoraria were recorded as a function of temperature. Tracings of the tension development, its integrated and derived functions showed that the isolated auricles of warmadapted winter frogs responded to temperature with changes in inotropy but not in the tension generated in one minute.Adrenaline, previously shown to act via the in the auricles of this frog, increased both the force and the frequency of the contractions between 5 and 25°C. The affinity for adrenaline was highest at 5°C for all the parameters examined. The maximal effect (efficacy) of adrenaline for T_max, T_max and the tension-time integral was highest at 5°C while the efficacy of adrenaline on the heart rate reached its maximum at 25°C. At 5°C the tension generated in one minute was doubled by the maximally effective dose of adrenaline (1.4 × 10^–6 M). These results strongly suggest that adrenaline, being the main sympathetic neurotransmitter in the frog, has an important role in adjusting the heart to maximal performance at the low temperatures.     

Positive inotropic effect of Murraya koenigii (Linn.) Spreng extract on an isolated perfused frog heart

Ethanolic extract of fresh leaves of M. koenigii (MKEE) showed a dose dependent positive inotropic effect on isolated frog heart. The responses to MKEE (62.5-1000 microg) were not affected in either way by theophylline, imidazole, propranolol and sildenafil. The change in potassium and sodium concentration did not alter MKEE-induced positive inotropic effect. Lignocaine did not alter the responses to MKEE significantly. Responses to MKEE were significantly inhibited when calcium concentration was reduced to half (from 1.58 to 0.79 mM) and were significantly potentiated when calcium concentration was doubled (from 1.58 to 3.16 mM). Verapamil was found to inhibit the responses significantly. The results suggest that M. koenigii induced positive inotropic effect possibly by increasing availability of calcium from extra cellular sites.     

Diltiazem potentiates the negative inotropic action of nimodipine in heart

In Langendorff perfused rat hearts, nimodipine enhances coronary flow and inhibits contractility. The binding of [3H]nimodipine (160 Ci/mmol) to sarcolemma isolated from dog heart revealed a KD of 0.2 nM. d-cis-Diltiazem, but not 1-cis-diltiazem, a less active stereoisomer, stimulated [3H]nimodipine (0.17 nM) binding to sarcolemmal membranes (ED50 for diltiazem = 1.1 microM). In the presence of 10 microM d-cis-diltiazem, [3H]nimodipine binding sites were doubled, but there was no change in the apparent affinity. Perfused rat hearts were treated with 250 nM d-cis-diltiazem. The negative inotropic response to nimodipine was dramatically potentiated (I50, from 1.1 to 0.033 microM). The pharmacological and binding effects were observed only at 37 degrees C. It is possible that diltiazem in some way converts low affinity to high affinity sites.     

Analysis of the negative inotropic effect of acetylcholine on frog atrial fibres

Voltage-clamp experiments have been performed on frog atrial preparations in order to study the mechanism of the inotropic effect of acetylcholine (ACh) at various concentrations. The amplitude of the slow inward current (Is) is reduced even at low ACh concentrations; such low concentrations have little or no effect on potassium permeability. Dose-effect relationships for Is inhibition (Is/Is max) by ACh show a half amplitude dose (K0.5 around 8 X 10(-8) M ACh. The reduction of Is is attributed largely to a decrease of the maximal conductance of the slow channel (gs). Steady-state activation and inactivation parameters are not affected by ACh. Experiments in a Na-free solution (Na replaced by Li ions) or in a Ca-free solution (with EGTA) indicate that the "slow sodium current" is more sensitive to ACh than the "slow Ca current", although these two currents both seem to flow through the slow channel. The decrease of the phasic component of contraction observed in the presence of ACh is very well correlated with the decrease of Is (K0.5 = 8 X 10(-8) M ACh), while the increase of the tonic tension may be related to the outward potassium current induced by high concentrations of ACh. The significant difference between the half amplitude dose (K0.5) observed in the dose effect curves with ACh for Is inhibition (K0.5 = 8 X 10(-8) M) and for ACh-induced extra-current (K0.5 - 10(-6) M) may indicate the presence of two muscarinic receptors.     

Isoproterenol-induced desensitization to the positive inotropic effect of isoproterenol in ventricular strips isolated from carp heart (Cyprinus carpio)

Whether the positive inotropic effect of isoproterenol in ventricular strips of carp heart is altered by previous exposure to the agonist was studied. Isoproterenol produced a concentration-dependent positive inotropic effect in these preparations which was competitively antagonized by propranolol. Isoproterenol dose-response curves were shifted significantly downward and to the right after previous treatment with and removal of isoproterenol. The desensitization could be demonstrated after 5 min incubation and near maximal desensitization was observed after 30 min exposure to 1000 nM isoproterenol. The responsiveness was only partially recovered after 180 min incubation in drug-free medium. Isoproterenol produces a rapidly developing desensitization to inotropic effects of the beta-adrenergic agonist. This phenomenon may be mediated by alterations in beta-adrenergic receptors, adenylate cyclase and/or the beta receptor-adenylate cyclase coupling mechanism.     

Chronotropic and inotropic effects of atrial peptides on the isolated systemic heart ofOctopus vulgaris

Summary The chronotropic and inotropic effects of four atrial peptides (cardiodilatin 1–16, atrial natriuretic factor 8–33 and atriopeptin I and III) on the isolated systemic heart ofOctopus vulgaris were studied.Using a preparation that produces a physiological stroke volume at physiological input pressures, it was found that ANF, atriopeptin I and atriopeptin III exerted both negative chronotropic and inotropic effects. In contrast, cardiodilatin produced a positive inotropic effect.A dose-response curve of ANF is reported, showing a threshold concentration of about 10^–12 M.The pharmacological and physiological implications of these results are discussed in relation to some characteristics of the cephalopod systemic heart.     

Catestatin (chromogranin A344-364) is a novel cardiosuppressive agent: inhibition of isoproterenol and endothelin signaling in the frog heart

The catecholamine release-inhibitory catestatin [Cts; human chromogranin (Cg) A(352-372), bovine CgA(344-364)] is a vasoreactive and anti-hypertensive peptide derived from CgA. Using the isolated avascular frog heart as a bioassay, in which the interactions between the endocardial endothelium and the subjacent myocardium can be studied without the confounding effects of the vascular endothelium, we tested the direct cardiotropic effects of bovine Cts and its interaction with beta-adrenergic (isoproterenol, ISO) and endothelin-1 (ET-1) signaling. Cts dose-dependently decreased stroke volume and stroke work, with a threshold concentration of 11 nM, approaching the in vivo level of the peptide. Cts reduced contractility by inhibiting phosphorylation of phospholamban (PLN). Furthermore, the Cts effect was abolished by pretreatment with either nitric oxide synthase (N(G)-monomethyl-l-arginine) or guanylate cyclase (ODQ) inhibitors, or an ET(B) receptor (ET(BR)) antagonist (BQ-788). Cts also noncompetitively inhibited the positive inotropic action of ISO. In addition, Cts inhibited the positive inotropic effect of ET-1, mediated by ET(A) receptors, and did not alter the negative inotropic ET-1 influence mediated by ET(BR). Cts action through ET(BR) was further suggested when, in the presence of BQ-788, Cts failed to inhibit the positive inotropism of both ISO and ET-1 stimulation and PLN phosphorylation. We concluded that the cardiotropic actions of Cts, including the beta-adrenergic and ET-1 antagonistic effects, support a novel role of this peptide as an autocrine-paracrine modulator of cardiac function, particularly when the stressed heart becomes a preferential target of both adrenergic and ET-1 stimuli.     

Cleavage of chromogranin A N-terminal domain by plasmin provides a new mechanism for regulating cell adhesion

It has been proposed that chromogranin A (CgA), a protein secreted by many normal and neoplastic neuroendocrine cells, can play a role as a positive or a negative modulator of cell adhesion. The mechanisms that regulate these extracellular functions of CgA are unknown. We show here that plasmin can regulate the anti/pro-adhesive activity of CgA by proteolytic cleavage of the N-terminal domain. Limited proteolytic processing decreased its anti-adhesive activity and induced pro-adhesive effects in fibronectin or serum-dependent fibroblast adhesion assays. Cleavage of Lys(77)-Lys(78) dibasic site in CgA(1-115) was relatively rapid and associated with an increase of pro-adhesive effect. In contrast, antibodies against the region 53-90 enhanced the anti-adhesive activity of CgA and CgA(1-115). Structure-activity relationship studies showed that the conserved region 47-64 (RILSILRHQNLLKELQDL) is critical for both pro- and anti-adhesive activity. These findings suggest that CgA might work on one hand as a negative modulator of cell adhesion and on the other hand as a precursor of positive modulators, the latter requiring proteolytic processing for activation. Given the importance of plasminogen activation in tissue invasion and remodeling, the interplay between CgA and plasmin could provide a novel mechanism for regulating fibroblast adhesion and function in neuroendocrine tumors.     

Localization and characterization of the N-terminal fragment of atrial natriuretic factor (ANF) precursor in the frog heart

The localization of the N-terminal fragment of the atrial natriuretic factor (ANF) precursor in the heart of the frog Rana ridibunda was examined by the indirect immunofluorescence and the immunogold techniques using an antiserum directed against synthetic rat ANF (Asp11-Ala37). At the optic level, positive material was found in most atrial myocytes. Staining of consecutive sections of frog heart with antibodies against N-terminal and C-terminal regions of the proANF molecule showed that both peptides are contained in the same cardiocytes. In the rat atrium, antibodies against the N-terminal ANF region induced a more intense labeling than in the frog atrium. Electron microscopic studies indicated that all secretory granules present in frog atrial cardiocytes contain N-terminal ANF-like immunoreactive material. The positive material localized in frog atrium was characterized by gel filtration and radioimmunological detection. Serial dilutions of frog atrial extracts exhibited displacement curves which were parallel to that obtained with synthetic human ANF (Asn1-Asp30). Sephadex G-50 gel chromatography of the immunoreactive material showed that the N-terminal ANF-like immunoreactivity eluted in a single peak corresponding to high molecular weight material. These results indicate that the N-terminal fragment of frog proANF is immunologically and biochemically related to the homologous mammalian peptide.     

Dilatory and inotropic effects of corticotropin-releasing factor (CRF) on the isolated heart. Effects on atrial natriuretic peptide (ANP) release.

The effects of CRF administration on cardiac performance, coronary flow and ANP release were investigated in the rat heart. Isolated hearts were perfused at a constant filling pressure according to working heart model with a Krebs-Henseleit solution containing glucose and insulin, saturated with a gas mixture containing 95% O2 and 5% CO2. Administration of CRF via a cannula into the left atrium elicited a prolonged increase in the coronary flow rate and a transient increase in the aortic pressure resulting in an overall increase in the pressure-volume work. The oxygen consumption, after the administration of CRF, increased in accordance with the cardiac effort. No changes were observed in the spontaneous heart rate. Furthermore, administration of CRF induced a short-term increase of ANP release into the coronary perfusate. Our experiments suggest that administration of CRF produces a prolonged dilatory effect on the coronary arteries while producing a transient positive inotropic effect and a transient increase of ANP release on the isolated rat heart.     

Frequency-dependent effects in an alternating electric field on an isolated frog heart

An isolated frog heart placed in a special chamber with Ringer solution (pH 7.2) was stimulated by electric field at a frequency of 0.3-4.0 Hz with graphite electrodes immersed in the solution. Unusual resonance phenomena were observed during a progressive increase or decrease of the stimulation frequency: the amplitude of the mechanical responses of the preparation rose at multiple frequencies of stimulation, whereas the frequency of the responses remained unchanged.     

The inotropic effect of digoxin on an isolated rat heart in hypercapnia and (or) hypoxia

The explanation for the increased frequency of troubles with digoxin therapy in patients with chronic pulmonary diseases is debated. The reported effects of hypoxia in vivo on myocardial levels of digoxin are contradictory, and there have been few studies on the effects of hypercapnia. In the past, it has been shown in rat myocardial tissue at rest in vitro that hypoxia decreased and hypercapnic acidosis increased the digoxin uptake. We performed a new study in vitro in an isolated beating rat heart perfused at constant flow (37 degrees C) and stimulated at a constant frequency (6 Hz). The performances were recorded with an intraventricular balloon equipped with a tip-manometer catheter. The action of digoxin was studied by recording systolic pressure (PS) and diastolic pressure (PD), the left ventricular developed pressure (LVDP = PS - PD), the (dP/dt)max, and the ratio (dP/dt)max/PS. First, the heart was perfused for 30 min with a modified Tyrode's solution perfusate aerated with carbogen (pH = 7.40; PCO2 = 37 mmHg; PO2 = 530 mmHg) (1 mmHg = 133.32 Pa). Various parameters of contractions were recorded (initial control values). Then the heart was perfused for 15 min with Tyrode's solution aerated either with a hypoxic gas mixture (pH = 7.41; PCO2 = 36 mmHg; PO2 = 122 mmHg), a hypercapnic gas mixture (pH = 7.08; PCO2 75 mmHg; PO2 = 485 mmHg), or a hypoxic-hypercapnic gas mixture (pH = 7.09; PCO2 = 73 mmHg; PO2 = 124 mmHg). Control hearts were continuously perfused with Tyrode's solution aerated with carbogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory influence of chromogranin A N-terminal fragment (vasostatin-1) on the spontaneous contractions of rat proximal colon

Very little is known about the role played by CGA and its fragments in the gastrointestinal physiology. We have studied the role of CGA N-terminal fragments in the regulation of intestinal smooth muscle contractility by measuring the influence of recombinant CGA 1-78 (VS-1) and synthetic CGA 7-57 peptides on the spontaneous mechanical activity of rat proximal colon in vitro. The mechanical activity was recorded as changes in the intraluminal pressure. VS-1 (0.1-30 nM) and CGA 7-57 (10-300 nM) produced concentration-dependent inhibitory effects, characterized by a progressive decrease in the mean amplitude of circular muscle spontaneous contractions, without affecting the resting tone. The response to VS-1 was antagonised by anti-CGA monoclonal antibodies (mAb5A8, B4E11, 7D1 or 4D5) but not by an irrelevant antibody, indicating that the effect was specific. The inhibitory responses to VS-1 and to CGA 7-57 were significantly reduced by pre-treatment of the preparations with N(omega)-nitro-l-arginine methyl ester (l-NAME) (300 microM), 1H-(1,2,4) oxadiazolo-(4,3-a) quinoxalin-1-one (ODQ) (10 microM), apamin (0.1 microM) or tetrodotoxin (TTX) (1 microM). The results suggest that VS-1 plays an inhibitory modulatory role on spontaneous contractions rat colon circular muscle, through mechanisms involving in part neural release of nitric oxide.     

Reduction in transmembrane Ca influx induced by the negative inotropic action of nicardipine in frog ventricular muscle

The effects of nicardipine, a new 1,4-dihydropyridine derivative, on electrical and mechanical properties of frog ventricular muscle were examined. Nicardipine (3 X 10(-7) M) reduced the twitch tension, and this reduction was frequency dependent, and considerable, in case of high frequencies. The resting potential was not affected by nicardipine (3 X 10(-7) M), but the plateau height of the action potential was decreased and the duration of the action potential was shortened. The suppression of this plateau height was frequency dependent. The nicardipine-induced suppression of tension and action potential could be almost completely antagonized by raising the concentration of [Ca]o or by applying isoprenaline (8 X 10(-7) M). These results suggest that the negative inotropic action of nicardipine is induced mainly by a reduction in the transmembrane Ca influx.