Beta3-Adrenoceptor in the eel (Anguilla anguilla) heart:negative inotropy and NO-cGMP-dependent mechanism

Abstract

Neuroendocrine regulation of cardiac function involves a population of three types of β-adrenoceptors (ARs). In various mammalian species,β 1- 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 theβ 3-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 G_i/o proteins inhibitor pertussis toxin (PTx; 0.01 nmol l^-1), suggesting a G_i/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 ofβ 3-like adrenoceptors in the eel Anguilla anguilla heart identifying, for the first time in a working fish heart, theβ 3-AR-dependent negative inotropy discovered in mammals.