GENERAL AND COMPARATIVE ENDOCRINOLOGY General and Comparative Endocrinology 139 (2004) 20–28 www.elsevier.com/locate/ygcen Influence of vasostatins, the chromogranin A-derived peptides, on the working heart of the eel (Anguilla anguilla): negative inotropy and mechanism of action Sandra Imbrognoa, Tommaso Angelonea,b, Angelo Cortic, Cristina Adamoa, Karen B. Helled, Bruno Totaa,e,* a Departments of Cellular Biology, University of Calabria, Via P. Bucci, Arcavacata di Rende 87030, CS, Italy b Departments of Pharmaco-Biology, University of Calabria, Arcavacata di Rende 87030, CS, Italy c Department of Biological and Technological Research, San Raffaele H Scientific Institute, Milan 20132, Italy d Department of Biomedicine, Division of Physiology, University of Bergen, Bergen 5020, Norway e Zoological Station ‘‘A. Dohrn’’, Naples 80121, Italy Received 25 May 2004; revised 6 July 2004; accepted 19 July 2004 Abstract We have studied the effects of exogenous human recombinant Vasostatin-1 (VS-1), Vasostatin-2 (VS-2) and the human Chromogranin A (CGA) 7–57 synthetic peptides on the mechanical performance of the isolated and perfused working eel (Anguilla anguilla) heart. Under basal conditions, the three peptides decreased stroke volume (SV) and stroke work (SW), thus exerting negative inotropism. The VS-1-mediated negative inotropism was abolished by exposure to inhibitors of either Gi/o protein (pertussis toxin; PTx) or M1 muscarinic receptors (Pirenzepine) or calcium (Lantanum and Diltiazem) and potassium (Ba2+, 4-aminopyridine, tetraethylammonium, glibenclamide) channels, while it required an intact endocardial endothelium (EE). Using NG-monomethyl-L -arginine (L -NMMA) as an inhibitor of nitric oxide (NO) synthase (NOS), and hemoglobin as a NO scavenger, we demonstrated the obligatory role of NO signaling in mediating the vasostatin response. Pretreatment with either a specific inhibitor of soluble guanylate cyclase (GC) 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ), or the inhibitor of the cGMP-activated protein kinase (PKG) KT5823, abolished the VS-1-mediated inotropism, indicating the cGMP-PKG component as a crucial target of NO signaling. Of note, VS-1 was effective in counteracting the adrenergic (Isoproterenol and Phenylephrine)-mediated positive inotropism. These findings provide the first evidence that vasostatins exert cardiotropic action in fish, thus suggesting their long evolutionary history as well as their species-specific mechanisms of action.
2004 Elsevier Inc. All rights reserved. Keywords: CGA-derived peptides; Eel heart; Nitric oxide; Calcium channels; Potassium channels; Catecholamines 1. Introduction Chromogranins represent a family of acidic and water-soluble proteins secreted by the diffuse neuroen* docrine system (Winkler and Fischer-Colbrie, 1992; and references therein). Chromogranin A (CGA),1 the first member of this family to be identified, occurs in the parathyroid (Cohn et al., 1982), thyroid C-cells Corresponding author. Fax: +39 0984 492906. E-mail address: tota@unical.it (B. Tota). 1 Abbreviations used: ACh, acetylcholine; 4-Aminopyr, 4-Aminopyridine; CGA, Chromogranin A; CO, cardiac output; EE, endocardial endothelium; GC, guanylate cyclase; Glib, Glibenclamide; Hb, haemoglobin; HR, heart rate; ISO, isoproterenol; L -NMMA, NG-monomethyl-L arginine; NO, nitric oxide; NOS, nitric oxide synthase; ODQ, 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one; PKG, cGMP-activated protein kinase; PTx, pertussis toxin; SV, stroke volume; SW, stroke work; TEA, tetraethilammonium chloride; VS-1, Vasostatin-1; VS-2, Vasostatin-2. 0016-6480/$ - see front matter
2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2004.07.008 S. Imbrogno et al. / General and Comparative Endocrinology 139 (2004) 20–28 (OÕConnor et al., 1983), endocrine pancreas (Ehrhart et al., 1986), adenohypophysis (Serck-Hanssen and OÕConnor, 1984) and adrenergic (Li et al., 1999) and non-adrenergic (Somogyi et al., 1984) neurones of the central nervous system. CGA is the precursor of several regulatory peptides (i.e. vasostatins, pancreastatin, catestatin, parastatin), generated by cell-, tissue- and species-specific proteolytic processes (Helle et al., 2001 and references therein), most of which act as powerful inhibitors of endocrine secretion. Recently, we have shown that recombinant human vasostatins (VS), corresponding to the chromogranin A (CGA) amino acids 1–76 (VS-1) and 1–113 (VS-2), respectively, exert cardio-suppressive actions on both the isolated and perfused frog and rat hearts and counteract the positive inotropism of b-adrenergic (i.e. isoproterenol; ISO) stimulation (Angelone et al., 2003; Corti et al., 2002). Therefore, in addition to the vaso-relaxing action of the natural bovine VS-1 on human vessels which have been pre-contracted by several agonists (e.g. catecholamines) (Aardal and Helle, 1992), these novel findings further support the hypothesis of VS acting as inhibitory regulators of the cardio-circulatory system. In a comparative perspective, using an isolated working eel (A. anguilla) heart preparation (Imbrogno et al., 2001), we have now demonstrated that exogenous human recombinant VS-1, VS-2 and the human synthetic CGA7–57 peptide exert inhibitory actions on mechanical cardiac performance and counteract the adrenergic-mediated positive inotropism. However, unlike in the frog heart, where vasostatin-induced negative inotropism involves neither the endocardial endothelium (EE) nor nitric oxide (NO)-guanylate cyclase (GC) system, nor G protein system (Corti et al., 2004), in the eel heart the same inotropic effects are mediated by G proteins and require an EE-NO-cGMP signal transduction mechanism. Taken together, these data suggest an early role of VS in vertebrates and, at the same time, emphasize the importance of species-specific mechanisms underlying the cardiac actions of these peptides. 21 hearts, isolated and connected to a perfusion apparatus as previously described (Imbrogno et al., 2001), received RingerÕs solution from an input reservoir and pumped against an afterload pressure given by the height of an output reservoir. The composition of the perfusate (in millimoles per litre) was: NaCl 115.17, KCl 2.03, KH2PO4 0.37, MgSO4 2.92, (NH4)2SO4 50, CaCl2 1.27, glucose 5.55, Na2HPO4 1.90; pH was adjusted to 7.7–7.9 by adding NaHCO3 (about 1 g/l) (Imbrogno et al., 2001); RingerÕs solution was equilibrated with a mixture of O2:CO2 at 99.5:0.5%. Experiments were carried out at room temperature (18–20 C). The controlled non-paced hearts operate at a frequency of about 50 bpm (see Imbrogno et al., 2001). Hearts were stimulated with an LE 12006 stimulator (frequency identical to that of control, non-paced hearts; pulse width fixed at 0.1 ms; voltage: 1.2 ± 0.1 V, means ± SEM). 2.2. Measurements and calculations Pressure was measured through T-tubes placed immediately before the input cannula and after the output cannula, and connected to two MP-20D pressure transducers (Micron Instruments, Simi Valley, CA, USA) in conjunction with a Unirecord 7050 (Ugo Basile, Comerio, Italy). Pressure measurements (input and output) were expressed in kilopascal (kPa) and corrected for resistances of cannula and of tubes length. Heart rate (HR) was calculated from pressure recording curves. Cardiac output (CO) was collected over 1 min and weighed; values were corrected for fluid density and expressed as volume measurements. The afterload (mean aortic pressure) was calculated as two-thirds diastolic pressure plus one-third maximum pressure. Stroke volume (SV; ml kg 1; CO/HR) was used as a measure of ventricular performance; changes in SV were considered to be inotropic effects. CO and SV were normalised per kilogram of wet body mass. Ventricular stroke work [SW; mJ g 1; (afterload–preload) · SV/ventricle mass] served as an index of systolic functionality. 2.3. Experimental protocols 2. Materials and methods 2.1. Isolated and perfused working heart preparations We used specimens of freshwater European eel (Anguilla anguilla L.), weighing 84.5 ± 3.6 g (means ± SEM, n = 84). Fish were provided by a local hatchery and kept at room temperature (18–20 C) without feeding for 5–7 days. In accordance with accepted standards of animal care, the experiments were organised to minimize stress and number of animals used. Experiments were performed from November to April. Each eel was anaesthetised in benzocaine (0.2 g/L) for about 15 min. The Basal conditions. Isolated perfused hearts were allowed to maintain a spontaneous rhythm for up to 15–20 min. In all experiments the control conditions were established at a mean output pressure of about 3 kPa, with a CO set to 10 ml/min/kg body mass by appropriately adjusting the filling pressure. These values are within the physiological range (for references see Imbrogno et al., 2001). Cardiac parameters were simultaneously measured during experiments. To analyse the inotropic effects distinct from the chronotropic actions of substances, the preparations were electrically paced. Hearts that did not stabilise within 20 min from the onset of perfusion were discarded. 22 S. Imbrogno et al. / General and Comparative Endocrinology 139 (2004) 20–28 Drug application. After the 15–20 min control period, paced hearts were perfused for 20 min with RingerÕs solution enriched with VS-1 or VS-2 or CGA7–57 at increasing concentrations to construct cumulative concentration–response curves. Heart preparations were used to test the effects of 33 nM VS-1 in the presence of adrenergic agonists (isoproterenol and phenylephrine) and antagonists (propanolol and phentolamine), cholinergic antagonists (atropine, pirenzepine and AF-DX 116), the NO scavenger haemoglobin, the nitric oxide synthase (NOS) inhibitor NGmonomethyl-L -arginine (L -NMMA)], the soluble guanylate cyclase (GC) specific inhibitor [1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ)], the protein kinase G (PKG) blocker (KT5823) and the inhibitors of calcium (lanthanum and diltiazem) and potassium (Ba2+, 4aminopyridine, tetraethylammonium and glibenclamide) channels. In the above-mentioned protocols the hearts were perfused for 20 min with RingerÕs solution enriched with the specific drug before the addition of VS-1. In another set of experiments the effects of VS-1 (33 nM) were tested after inhibition of G-proteins by pertussis toxin (PTx); in this case the hearts were pre-incubated for 60 min with PTx. The effect of VS-1 (33 nM) was also studied after inducing functional damage of the ventricular EE with the detergent Triton X-100; 0.1 ml of Triton X-100 at a concentration of 0.05% was injected through a needle inserted into the posterior ventral region of the ventricular wall to avoid damage to the atrium (for further details see Imbrogno et al., 2001). Since the performance of the in vitro eel heart is stable for about 2 h (see Imbrogno et al., 2001), our experiments were carried out within this period. 2.4. Statistics of Percentage changes were evaluated as means ± SEM percentage changes obtained from individual experiments. Because each heart acted as its own control, the statistical significance of differences withingroup was assessed using the paired StudentÕs t test (P < 0.05). Comparisons between groups were made using two-way analysis of variance (ANOVA). Significant differences were detected using DuncanÕs multiplerange test (P < 0.05). 2.5. Drugs and chemicals VS-1, VS-2 and the synthetic CGA7–57 peptide were produced and characterised as previously described (Corti et al., 1997). Haemoglobin, L -NMMA, ODQ, PTx, Triton X-100, ISO, phenylephrine, propanolol, phentolamine, atropine sulphate salt, pirenzepine, lanthanum, diltiazem, Ba2+, 4-aminopyridine, tetraethylammonium and glibenclamide were purchased from Sigma Chemical Company (St. Louis, MO, USA). KT5823 (used in a darkened perfusion apparatus to prevent degradation) was purchased from Calbiochem (Milan). AF-DX 116 was a generous gift from Boehringer Ingelheim (Biberach, Germany). All the solutions were prepared in double-distilled water (ODQ was prepared in ethanol); dilutions were made in RingerÕs solution immediately before use. 3. Results 3.1. The isolated and perfused working heart preparation The in vitro isolated and perfused whole heart preparation works at physiological loads and generates values of output pressure, CO, SV, SW and power that mimic the physiological values of the in vivo animal, as previously described (see Table 1, Imbrogno et al., 2001). Table 1 Absolute values for stroke volume (SV; ml kg 1) and stroke work (SW; mJ g 1) under control conditions and after the addition of VS-1 (11–110 nM), or VS-2 (7.5–75 nm) or CGA7–57 (16–160 nM) VS-1 SV SW Control 11 nM 33 nM 71 nM 110 nM 0.196 ± 0.04 0.88 ± 0.21 0.193 ± 0.04 0.873 ± 0.21 0.156 ± 0.02 0.744 ± 0.14 0.153 ± 0.05 0.73 ± 0.23 0.126 ± 0.01 0.70 ± 0.07 Control 7.5 nM 22 nM 49 nM 75 nM 0.15 ± 0.025 0.876 ± 0.25 0.13 ± 0.025 0.753 ± 0.27 0.116 ± 0.023 0.70 ± 0.24 0.103 ± 0.018 0.623 ± 0.21 0.103 ± 0.03 0.613 ± 0.27 VS-2 SV SW CGA 7–57 SV SW Control 16 nM 50 nM 100 nM 160 nM 0.18 ± 0.01 0.996 ± 0.12 0.162 ± 0.01 0.911 ± 0.15 0.162 ± 0.02 0.884 ± 0.14 0.147 ± 0.02 0.839 ± 0.17 0.157 ± 0.01 0.887 ± 0.13 S. Imbrogno et al. / General and Comparative Endocrinology 139 (2004) 20–28 23 Fig. 1. Cumulative dose–response curve for VS-1, VS-2 and CGA7–57 on stroke volume (SV) and stroke work (SW) in isolated and perfused paced eel hearts. Percentage changes were evaluated as means ± SEM (n = 3–4 experiments for each group). Significance of differences from control values (t test); *P < 0.05. 3.2. Effects of VS-1, VS-2 and CGA7–57 on basal mechanical performance Under basal (unstimulated) conditions cumulative concentration–response curves of the three CGA-derived peptides VS-1, VS-2 and CGA7–57 were generated (Fig. 1). VS-1 at and above 33 nM induced a significant reduction of SV and SW, consistent with negative inotropism, while VS-2 induced a negative inotropic effect significant at the concentration of 49 nM. The synthetic peptide CGA7–57 significantly reduced SW at and above 16 nM while the effect on SV was significant only at the higher concentrations (100 and 160 nM) tested. Since VS-1 appears more potent than the other two peptides tested, it was used at the minimum effective concentration (33 nM) to study the transduction mechanisms activated by this peptide. cific muscarinic antagonist, at a concentration of 1 lM blocked the VS-1-mediated negative inotropism. Specific muscarinic inhibitors such as pirenzepine (M1 specific antagonist, 10 lM and 10 nM) and AF-DX 116 (M2 specific antagonist, 100 nM) were used to discriminate the subtype of muscarinic receptors involved in the VS cardiac response. While VS-1 response was not influenced by AF-DX 116 treatment, it was completely blocked in the presence of pirenzepine (Fig. 2). To test whether adrenergic receptors were involved in the transduction mechanism of VS-1, the cardiac preparations were pretreated with b- (propanolol) and a(phentolamine) adrenergic antagonists prior exposure to VS-1. The blockage of VS-1 response indicates the involvement of the adrenergic receptors in the VS-1 action (SV and SW values for VS-1 in presence of propan- 3.3. G-protein interaction Located at the interface between receptor–response coupling, guanine nucleotide binding regulatory proteins (G-proteins) process many biological signals in intracellular language. To evaluate the involvement of G proteins in the negative inotropic action of VS-1 (33 nM), cardiac preparations were pre-treated with PTx (0.01 nM), which uncouples signal transduction between several families of receptors and Gi or Go proteins (Ai et al., 1998 and references therein). While PTx alone did not modify basal cardiac performance (data not shown) its pre-treatment abolished the inotropic effect of VS-1 (SV and SW values for PTx plus VS-1 were 0.9 ± 0.15% and 2.12 ± 1.22%, respectively). 3.4. Cholinergic and adrenergic receptors We analysed the involvement of cholinergic receptors in the VS-1 response. We found that atropine, an unspe- Fig. 2. Effects of VS-1 (33 nM) before and after treatment with Atropine (1 lM), Pirenzepine (10 lM; 10 nM) and AF-DX 116 (100 nM) on stroke volume (SV) and stroke work (SW) in isolated and perfused paced eel hearts. Percentage changes were evaluated as means ± SEM (n = 3–4 experiments for each drug). Significance of differences from control values (t test); *P < 0.05. Comparison between groups (ANOVA, DuncanÕs test); §P < 0.05. 24 S. Imbrogno et al. / General and Comparative Endocrinology 139 (2004) 20–28 olol and phentolamine were 1.5 ± 3% and 2.77 ± 3.09%, respectively). All these treatments alone did not modify basal cardiac performance (data not shown). 3.5. Involvement of EE-NO-cGMP-PKG signal transduction pathway To analyse whether the VS-1 response involves a NOcGMP pathway, the heart preparations were pre-treated with either haemoglobin (1 lM) (NO scavenger), or L NMMA (10 lM) (NOS inhibitor) or ODQ (10 lM) (guanylyl cyclase blocker). All these treatments abolished the effect of VS-1 demonstrating their dependence on a NOcGMP mechanism (Fig. 3). It is well known that cGMP could modulate cardiac contractility via activation of a cGMP-PKG pathway (Hove-Madsen et al., 1996) and we have shown that this is the case also in the eel heart (Imbrogno et al., 2003). By pretreating the cardiac preparation with a specific inhibitor of PKG (KT5823, 100 nM), no significant decrease in SV and SW values with VS-1 treatment was observed (Fig. 3). This result indicates the involvement of PKG in the VS-1 response. The heart of A. anguilla possesses a highly trabeculated ventricle with an extensive EE surface that, being an important source of NO, modulates cardiac performance (Imbrogno et al., 2001, 2003). The EE impairment caused by Triton X-100 (0.05%), a detergent that, at this concentration, damages, functionally but not structurally, the EE (Imbrogno et al., 2001), abolished the VS-1 (33 nM)-mediated inotropic effect, thereby implicating EE modulation in the transduction of VS-1 signaling (Fig. 3). 3.6. Calcium and potassium channels Several studies suggest that CGA is a high-capacity, low-affinity Ca2+-binding protein but it is not known to what extent VS-1 binds calcium (Helle et al., 2001 and references therein). In the bovine coronary artery, the inhibitory activity of CGA1–40 on the calcium-dependent vascular tone was sensitive to potassium channel blockers (Brekke et al., 2002). The importance of ICa in the teleost heart is acknowledged (Llach et al., 2004 and references therein; Vornanen et al., 2002). To ascertain whether in A. anguilla the cardiac effects of VS-1 are mediated by Ca2+, we used two calcium channel antagonists: lanthanum and diltiazem, the latter specific for the L-type calcium channels. Concentration–response curves for lanthanum (1–50 nM) and diltiazem (1– 50 nM) showed a negative inotropic effect, significant from the concentration of 25 nM on up (Fig. 4). Yet both treatments at the concentrations of 10, 25 and 50 nM, abolished the VS-1 negative inotropic effect, suggesting an involvement of calcium channels in this response (Fig. 4). The absence of phosphate in the ringer did not modify the effects of calcium channel blockers (data not shown). To evaluate putative involvement of K+ channels, we pre-treated the hearts with Ba2+, an antagonist of the inward rectifying K+ (KIR) channels, or 4-aminopyridine, an inhibitor of the voltage-sensitive-channels (KV), or TEA, an inhibitor of calcium activated-channels (KCa2+), or glibenclamide, an inhibitor of both the sarcolemmal and mitochondrial ATP-potassium channels (KATP) (Brekke et al., 2002). The cardio-suppressive effect induced by VS-1 was not influenced by Ba2+ or 4aminopyridine, while it was completely blocked by TEA and glibenclamide (Fig. 5). All these treatments per se did not modify basal cardiac performance (data not shown). 3.7. Cardioinhibitory activity of CGA N-terminal fragments on adrenergic-stimulated preparations Fig. 3. Effects of VS-1 (33 nM) before and after treatment with haemoglobin (Hb; 1 lM), NG-monomethyl-L -arginine (L -NMMA; 10 lM), 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ; 10 lM), KT5823 (100 nM) and Triton X-100 (0.05%) injections, on stroke volume (SV) and stroke work (SW) in isolated and perfused paced eel hearts. Percentage changes were evaluated as means ± SEM (n = 3–4 experiments for each drug). Significance of differences from control values (t test); *P < 0.05. Comparison between groups (ANOVA, DuncanÕs test); §P < 0.05. Since VS have been shown to counteract the adrenergic-mediated positive inotropism in frog (Corti et al., 2002) and rat (Angelone et al., 2003), we have studied in the eel heart the positive inotropic effect induced by b- (isoproterenol) and a- (phenylephrine) adrenergic agonists after pretreating the cardiac preparations with VS-1. The results indicate that also in the eel heart VS-1 conteracts the adrenergic-mediated positive inotropism (SV and SW values for ISO plus phenylephrine were 24.23 ± 7% and 27.06 ± 8.02%, respectively; SV and SW values for VS-1 in presence of ISO plus phenylephrine were 6 ± 1.4% and 4.2 ± 1.5%, respectively). To discriminate the subtype of adrenergic receptors involved, we studied the response to ISO before and after VS-1 treatment. The results clearly indicate an involve- S. Imbrogno et al. / General and Comparative Endocrinology 139 (2004) 20–28 25 Fig. 5. Effects of VS-1 (33 nM) before and after treatment with Ba2+ (30 lM), or 4-Aminopyridine (4-Aminopyr; 1 mM), or Tetraethilammonium chloride (TEA; 0.5 mM) or Glibenclamide (Glib; 10 lM) on stroke volume (SV) and stroke work (SW) in isolated and perfused paced eel hearts. Percentage changes were evaluated as means ± SEM (n = 3–4 experiments for each drug). Significance of differences from control values (t test); *P < 0.05. Comparison between groups (ANOVA, DuncanÕs test); §P < 0.05. Fig. 4. Top panel: dose–response curves for Lanthanum (1–50 nM) and Diltiazem (1–50 nM) on stroke volume (SV). Middle panel: effects of VS-1 (33 nM) before and after treatment with Lanthanum (10– 50 nM) on stroke volume (SV) and stroke work (SW). Bottom panel: effects of VS-1 (33 nM) before and after treatment with Diltiazem (10– 50 nM) on stroke volume (SV) and stroke work (SW). Percentage changes were evaluated as means ± SEM (n = 3–4 experiments for each drug). Significance of differences from control values (t test); *P < 0.05. Comparison between groups (ANOVA, DuncanÕs test); §P < 0.05. ment of b receptors (SV and SW values for ISO were 13 ± 1.5% and 14.2 ± 2%; SV and SW values for ISO plus VS-1 were 5.35 ± 1.6% and 3.83 ± 1.43%, respectively). 4. Discussion 4.1. Effects of VS-1, VS-2 and CGA7–57 on basal mechanical performance In the present study we show that on the isolated working heart of the eel A. anguilla VS-1, VS-2 and the human CGA7–57 synthetic peptide decrease SV and SW, thus exerting clear cardio-suppressive inotropic influence. VS-1 appears more potent than the other peptides as inhibitory inotropic agent. Similarly, using an in vitro isolated working heart of Rana esculenta as bioassay, Corti et al. (2002) showed that VS exert direct suppressive action on the mechanical performance of both non-stimulated and adrenergically stimulated cardiac preparations. The same authors observed that the negative inotropic effect of VS-1 was higher than that elicited by VS-2, and that the region 7–57 of VS-1 contains the structural determinants for this activity. It was suggested that the lower activity of VS-2 with respect to VS-1 could be due to conformational changes in this peptide (Corti et al., 2004). For this reason, only VS-1 was used in the following part of this study centred on transduction mechanisms activated by this peptide. The negative inotropy of VS reported by us in fish, frog and rat hearts strongly supports a ubiquitous cardio-depressive role of these peptides in vertebrates. 4.2. G protein interaction Despite their wide-ranging actions, there are very few studies on the signal-transduction mechanism that may be activated by CGA-derived peptides. For example, it has been suggested that pancreastatin, the CGA-derived peptide known as a counter-regulatory agent of insulin action, may affect cardiac function through interaction with GTP binding proteins (G proteins) (GonzalezYanes et al., 2001). The limited information available on VS peptides suggests the presence of species-related variations and, in the same species, different responses among a variety of tissues districts. For example, in the frog heart the 26 S. Imbrogno et al. / General and Comparative Endocrinology 139 (2004) 20–28 cardio-suppressive action exerted by vasostatins is independent from Gi protein-mediated signaling pathway (Corti et al., 2004). In contrast, we show here that on the eel heart the VS-1-induced negative inotropism is blocked by PTx, which uncouples signal transduction between several families of receptors and Gi or Go proteins (Ai et al., 1998 and references therein). Similarly, it has been suggested that VS-mediated inhibition of parathyroid cells activity requires G protein interaction with subsequent modulation of parathormone secretion (Angeletti et al., 2000). In the heart, the PTx sensitive G proteins, located within the caveolae together with cholinergic and adrenergic receptors, calcium channels, endothelial nitric oxide synthase (eNOS), are involved in various inhibitory transduction cascades triggered by both chemical and physical stimuli (Hare et al., 1998). Apart from specific receptor interactions still to be detected, VS could activate G proteins through spatially localised cell membrane perturbation caused by the interaction of the lipophylic portion of VS-1 with a lipidic bilayer domain. Indeed, such a mechanism was suggested to explain the antimicrobic action of some VS-derived fragments (Lugardon et al., 2002; MagetDana et al., 2002). 4.3. Cholinergic and adrenergic receptor systems In the isolated and perfused preparation the exogenous cholinergic and adrenergic stimuli are excluded. However, the possibility that intracardiac sources of these substances can be released from either intracardiac nerve terminals or chromaffin cells must be taken into consideration. In the same preparation we have found that exogenous acetylcholine (ACh) produces a biphasic inotropic response: a positive response mediated by M1 muscarinic receptors, mostly located on the EE, and a negative one mediated by M2 muscarinic receptors, mostly expressed in the myocardiocytes (Imbrogno et al., 2001). We observed that, in absence of exogenous acetylcholine, atropine, a non-specific muscarinic antagonist, blocked the VS-1 inotropic response, suggesting a cholinergic-mediated mechanism in A. anguilla heart. The use of specific muscarinic receptor inhibitors, i.e. pirenzepine (M1 specific antagonist) and AF-DX 116 (M2 specific antagonist) allowed to discriminate the subtype of receptors involved. In fact, while the VS-1 response was not influenced by AF-DX 116 treatment, it was completely blocked in presence of pirenzepine. Since in A. anguilla M1 receptors are mostly located on the EE (Imbrogno et al., 2001), our findings point to the EE as possible site where VS-1 signal-transduction cascade is generated (see below). Furthermore, in absence of exogenous adrenergic agonists, pre-treatment with b- (propanolol) and a- (phentolamine) adrenergic antagonists completely blocked the VS-1-mediated inotropism, thus uncovering an involvement of the adrenergic receptor system in the determinism of VS-1 effect. A preferential involvement of b-(either b2 or b3)adrenoceptors, suggested by our work in progress, needs to be analysed by further study. 4.4. Involvement of EE-NO-cGMP-PKG signal transduction pathway The heart of A. anguilla is characterised by a highly trabeculated ventricle lined by an extensive EE surface which is the only barrier between the cardiac lumen and the subjacent myocardium. In the working eel heart, the EE exerts a tonic mild negative inotropic influence through a NO-cGMP transduction pathway activated by both mechanical and chemical endoluminal stimuli (Imbrogno et al., 2001, 2003). Our findings that VS-1mediated negative inotropism is abolished by pre-treatment with both NO scavenger (Hb) and NOS (L -NMMA) or soluble GC (ODQ) inhibitors, are consistent with VS-1-induced stimulation of NO-cGMP pathway. In the eel heart, PKG is an important target of NO (Imbrogno et al., 2003). Since pre-treatment with the PKG inhibitor KT5823 abrogates the effect of VS-1, we can conclude that PKG is involved in the VS-1-mediated negative inotropism. In isolated mammalian ventricular cardiomyocytes, PKG exerts direct effect on calcium influx (Méry et al., 1991), and, through phosphorylation of troponin I, reduces the affinity of troponin C for calcium, thereby negatively regulating cardiac contractility (Hove-Madsen et al., 1996). The EE impairment caused by Triton X-100 (0.05%) abolished the VS-1-mediated inotropic effects, thereby implicating an EE-mediated mechanism in the transduction of VS-1 signaling. Of note, in the frog heart, Corti et al. (2004) have reported that the cardio-suppressive effect induced by VS-1 is EE-independent. In the bovine aorta, no interactions between vasostatins and vascular endothelium were detected (Mandalà et al., 2000). The results obtained by our laboratory regarding the different role of the EE in frog vs eel might be explained by EE species-specific differences (from ultrastructural to biochemical and molecular levels), which may affect peptide binding, internalisation, trans-endocardial transport, etc. For example, scavenger receptors have been characterised in the EE of the teleost heart (Seternes et al., 2001). Since the search for specific VS receptors has been so far elusive, it would be of interest to assess whether blood-borne endoluminal VS peptides could interact with scavenger receptors, thereby triggering an EE-mediated mechanism, which in turn affects myocardial inotropy. 4.5. Calcium and potassium channels Aardal and Helle (1992) in their study on isolated segments of human blood vessels observed that the S. Imbrogno et al. / General and Comparative Endocrinology 139 (2004) 20–28 vasoinhibitory activity induced by VS was independent from extracellular Ca2+ in the saphenous vein but not in the artery. In the eel heart, as previously shown in the frog heart (Corti et al., 2002), pre-treatments with Ca2+ channel antagonists lanthanum and diltiazem abolish VS-1-induced cardio-suppressive effect, suggesting an involvement of calcium influx. In the trout (Oncorhynchus mykiss) heart ICa appears to be a major pathway for activating contraction while the calcium channels blockade by Verapamil reduces substantially force development both in atrial and ventricular myocytes (Aho and Vornanen, 1999). Moreover, unlike the mammalian myocardium, in the cardiomyocytes of both eel and frog, in which the transverse tubular system is lacking, the immediate source of Ca2+ for tension development is the extracellular space (Llach et al., 2004; Vornanen et al., 2002; Morad and Cleemann, 1987). Recent evidence indicates that the action of CGA Nterminal fragments is abolished by pre-treatment with Ba2+, or 4-aminopyridine, or TEA, or glibenclamide (frog heart: Corti et al., 2004; bovine coronary arteries: Brekke et al., 2002). Interestingly, in the isolated working eel heart, the VS-1-mediated negative inotropism was not influenced by Ba2+ or 4-aminopyridine, while being abrogated by TEA and glibenclamide treatments. Although, the relevance of the transient outward K current in the teleost heart remains to be established, our data suggest that in the eel heart the modulation of calcium-dependent K+ channels and ATP-potassium channels may be prerequisites for peptide activity. 4.6. Cardioinhibitory activity of CGA N-terminal fragments on the adrenergically stimulated preparations In view of the postulated role of VS as homeostatic regulators of the cardiovascular system, possibly counteracting the excitatory adrenergic influences (Angelone et al., 2003; Corti et al., 2002; Helle et al., 2001), it is of interest that in the fish heart pre-treatment with VS-1 blocked the positive inotropism induced by b- and aadrenoceptor agonists. This cardiac ‘‘anti-drenergic’’ action of VS in an early vertebrate supports the hypothesis that these CGA N-terminal fragments can act not only as vasostatins but also as cardiostatins, i.e. hormones able to counterbalance the influence of physiological cardio-stimulating factors (Corti et al., 2004; Tota et al., 2003). Indeed, the heart of many teleosts, including the eel, are exposed to the stimulatory effects of circulating and intracardiac catecholamines, particularly under stress conditions (Imbrogno et al., 2003 for references), which may became potentially harmful in absence of local counter-regulatory mechanisms. The possibility that vasostatins could exert local cardio-inhibitory protection versus systemic and/or intracardiac cascades of excitatory stimuli targeting the heart, should stimulate further studies, particularly in view of the chal- 27 lenging concept of ‘‘zero steady-state error’’ homeostasis recently proposed for other CGA-derived peptides (Koeslag et al., 1999). In conclusion, the present data extend for the first time to a working fish heart the basal negative inotropy and the ‘‘anti-adrenergic’’ influence of vasostatins, previously reported by us on in vitro frog and rat hearts. In an evolutionary perspective, this suggests an early role of VS as cardio-circulatory inhibitory peptides (i.e. cardiostatins) in vertebrates and, at same time, highlights intriguing species-specific differences underlying the mechanisms of action of these CGA-derived fragments. To verify the occurrence of CGA-derived peptides in fish is a challenge for future studies. 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