Nothing is known about the effects of nitric oxide (NO) on cardiac performance in fish. Using an in vitro working heart preparation that generates physiological values of output pressure, cardiac output and ventricular work and power, we assessed the effects of NO on the cardiac performance of the eel Anguilla anguilla. We examined basal cardiac performance (at constant preload, afterload and heart rate), the effects of cholinergic stimulation and the Frank–Starling response (preload-induced increases in cardiac output at constant afterload and heart rate). The NO synthase (NOS) inhibitors NG -monomethyl-L-arginine (L-NMMA) and L-N5 (1-iminoethyl)ornithine (L-NIO), the guanylate cyclase inhibitor 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ) and Triton X-100, a detergent that damages the endocardial endothelium, all increased stroke volume (VS) and stroke work (WS). In contrast, the endogenous NOS substrate L-arginine, tested before and after treatment with haemoglobin, the NO donor 3-morpholinosydnonimine, tested with and without the superoxide scavenger superoxide dismutase, and the stable cGMP analogue 8-bromoguanosine 3’, 5’ -cyclic monophosphate (8-Br-cGMP) decreased VS and WS. Acetylcholine chloride produced a biphasic effect. At nanomolar concentrations, in 34 % of the preparations, it induced a NO–cGMP-dependent positive inotropism that required the integrity of the endocardial endothelium. Pretreatment with Triton X-100 or with NO–cGMP pathway inhibitors (L-NMMA, L-NIO, NG -nitro-L-arginine methyl ester, Methylene Blue and ODQ) abolished the positive effect of acetylcholine. In contrast, at micromolar concentrations, acetylcholine produced a negative effect that involved neither the endocardial endothelium nor the NO–cGMP pathway. Pre-treatment with L-arginine (10− 6 mol l− 1) was without effect, whereas L-NIO (10− 5 mol l− 1) significantly reduced the Frank–Starling response. Taken together, these three experimental approaches provide evidence that NO modulates cardiac performance in the eel heart.

SUMMARY: Neuroendocrine regulation of cardiac function involves a population of three types of β-adrenoceptors (ARs). In various mammalian species,β1- and β2-AR stimulation produces an increase in contractility;whereas β3-AR activation mediates negative inotropic effects. At the moment, nothing is known about the physiological role of β3-AR in fish.Using an isolated working heart preparation, we show that a β3-AR selective agonist BRL37344 (0.1-100 nmol l-1) elicits a dose-dependent negative inotropism in the freshwater eel Anguilla anguilla. This effect was insensitive to the β1/β2-AR inhibitor nadolol (10 μmol l-1), but was blocked by theβ3-AR-specific antagonist SR59230 (10 nmol l-1). The analysis of the percentage of stroke work (SW) variations, in terms of EC50 values, induced by BRL37344 alone (10 nmol l-1), and in presence of SR59230 (10 nmol l-1), indicated a competitive antagonism of SR59230. In addition to the classic positive inotropism, the non-specific β agonist isoproterenol (100 nmol l-1) induced, in 30% of the preparations, a negative inotropic effect that was abrogated by pre-treatment with SR59230, pointing to a β3-mediated pathway. The BRL37344-induced negative inotropic effect was abolished by exposure to a Gi/o proteins inhibitor pertussis toxin (PTx; 0.01 nmol l-1), suggesting a Gi/o-dependent mechanism. Using L-N5(l-imino-ethyl)ornithine (L-NIO; 10 μmol l-1), as a nitric oxide (NO) synthase (NOS) blocker and haemoglobin (Hb; 1 μmol l-1), as a NO scavenger, we demonstrated that NO signalling is involved in the BRL37344-induced response. Pre-treatment with either an inhibitor of soluble guanylate cyclase (GC) 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ; 10 μmol l-1), or an inhibitor of the cGMP-activated protein kinase (PKG) KT5823 (100 nmol l-1), abolished the β3-dependent negative inotropism,indicating the cGMP-PKG component as a crucial target of NO signalling. Taken together, our findings provide functional evidence for the presence ofβ3-like adrenoceptors in the eel Anguilla anguilla heart identifying, for the first time in a working fish heart, theβ3-AR-dependent negative inotropy discovered in mammals.

Due to their uncontrolled use, plastics has become an environmental concern, not only for their varying dimension but also for the potential release of substances such as phthalates (PAEs) and non-phthalates (NPPs) into the water. Phthalates are the most common plasticizers of concern, but non-phthalate plasticizers such as di (2-ethylhexyl) terephthalate (DEHT) have also been lately found in the marine environment. Mytilus galloprovincialis is a well-known bioindicator of aquatic environments due to its ability to accumulate a wide variety of xenobiotics, including plasticizers. Hence, aim of this study was to evaluate the potential bioaccumulation and effects of the NPP DEHT on M. galloprovincialis. To this purpose, following exposure to DEHT at 1 mg/l (DEHT1) and 100 mg/l (DEHT100), its accumulation in tissues and its effects on total lipids and fatty acid (FA) composition, protein content, cell viability, ability to recover volume and changes in biomarkers of oxidative stress were assessed. Mussels were able to bioaccumulate DEHT in their tissues, with a statistically significant increase compared to the control organisms. Differences in FA composition were observed after exposure, since C16:0, C18:0, C20:5ω-3 and C22:6ω-3 were significantly decreased from control to exposed groups. As a result, total SFA, MUFA and PUFA were affected in DEHT-exposed groups. Also, total protein varied following DEHT exposure, and significantly decreased in the DEHT100-group. Considering the physiological responses, both DEHT-exposed groups lost their ability to return to the original volume of digestive gland (DG) cells. On the other hand, oxidative biomarkers in the gills and DG were not significantly affected by the DEHT exposure. Overall, this study showed for the first time that DEHT exposure differentially affect mussels, in their lipid and protein metabolism, as well as cellular parameters.