br Conflict of interest br Acknowledgements We
Conflict of interest
Acknowledgements We would like to thank Editage (www.editage.jp) for English language editing. This work was supported by Grants-in-Aid for Scientific Research, Japan Society for the Promotion of Science, Grant Number 24592576.
Co-existence of β- and β-adrenergic receptor subtypes, with a predominance and importance of the β-subtype, has been established in cardiac tissues of many species including humans (), cats and guinea pigs () and in rat atria (). Altered function and H 89 of the cardiac β-adrenergic receptor pathway have been reported in heart failure in humans (), whereas the role of the β-adrenergic receptor subtype is still being debated. Despite the fact that turkeys have been frequently used as an animal model to investigate human dilated cardiomyopathy (DCM) (), β-adrenergic receptors have not been well characterised in the different cardiac chambers of turkeys. Moreover, in turkey farms there is considerable economic loss due to premature animal death (presumably caused by multifactorial diseases related to the cardiovascular system). The aim of this investigation was to characterise the β-adrenergic receptor density and subtype distribution in adult turkey heart chambers (right and left ventricles, right and left atria). Heart samples were collected from 16-week old British United Turkey (BUT) BIG 6 female turkey poults which had been slaughtered at a local abattoir. Hearts were quickly transported to the laboratory and immediately dissected into four chambers. Samples were frozen at −80 °C prior to use. For the radioligand binding studies, membranes were prepared from all cardiac chambers (400 mg) as previously described (). The β-adrenergic receptor density and the relative amount of β- and β-adrenergic receptor subtypes were determined in cardiac membrane preparations by saturation and displacement binding studies using (−)-[I]-iodocyanopindolol (ICYP) and in the presence or absence of increasing concentrations (10–10 M) of the β-receptor selective (CGP 20712A) or β-receptor selective (ICI 118.551) antagonists as well as nonselective β-agonists (isoproterenol, epinephrine and norepinephrine) as previously described (). All binding data were analysed using the iterative, non-linear curve fitting GraphPad Prism software (GraphPad Software). Comparisons were made between chambers using analysis of variance (ANOVA) with post-hoc Bonferroni test, and < 0.05 indicated the level of significance. Specific ICYP binding was saturable and of high affinity (). There was no statistical difference between β-adrenergic receptor density (B, ~40 fmol/mg protein) and dissociation constant (K; ~30 pM) in all cardiac chambers of adult turkeys. In all chambers, ICYP was displaced by ICI 118.551 and CGP 20712A in a concentration-dependent steep monophasic manner. The K values for CGP 20712A and ICI 118.551 are shown in . The agonist displacement curves for the right atria and ventricles were biphasic (), with the rank order of potency as follows: isoproterenol > norepinephrine ≥ epinephrine. Our data strongly suggest the exclusive presence of the β-adrenergic receptor subtype in adult turkey heart chambers. The fact that the concentration–inhibition curves of CGP 20712A and ICI 118.551 were steeply monophasic implies that both antagonists displaced ICYP from one binding site. The selective β-adrenergic receptor antagonist CGP 20712A was highly potent in discriminating the β-receptor subtype, in accordance with the drug's selectivity for the subtype found in other pharmacological studies (K range, 10–100 nM; ), but it was less potent in discriminating the β-receptor subtype (K range, >4000–10,000; ). In contrast, ICI 118.551 was almost 25-fold less potent in discriminating the β-adrenergic receptors in turkey chambers when compared to other reported data (K range, <5 nM; ).