Vasodilatory effects of homologous adrenomedullin 2 and adrenomedullin 5 on isolated blood vessels of two species of eel

Abstract

In mammals, adrenomedullin (AM) is a potent vasodilator through signalling pathways that involve the endothelium. In teleost fishes, a family of five AMs are present (AM1/4, AM2/3 and AM5) with four homologous AMs (AM1, AM2/3 and AM5) recently cloned from the Japanese eel, Anguilla japonica. Both AM2 and AM5 have been shown to be strong in vivo vasodepressors in eel, but the mechanism of action of homologous AMs on isolated blood vessels has not been examined in teleost fish. In this study, both eel AM2 and AM5 caused a marked vasodilation of the dorsal aorta. However, only AM5 consistently dilated the small gonadal artery in contrast to AM2 that had no effect in most preparations. Neither AM2 nor AM5 had any effect when applied to the first afferent branchial artery; in contrast, eel ANP always caused a large vasodilation of the branchial artery. In the dorsal aorta, indomethacin significantly reduced the AM2 vasodilation, but had no effect on the AM5 vasodilation. In contrast, removal of the endothelium significantly enhanced the AM5 vasodilation only. In the gonadal artery, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) significantly reduced the AM5 vasodilation suggesting a role for soluble guanylyl cyclase in the dilation, but l-NNA and removal of the endothelium had no effect. The results of this study indicate that AM2 and AM5 have distinct vasodilatory effects that may be due to the peptides signalling via different receptors to regulate vascular tone in eel.

Introduction

Mammalian adrenomedullin (AM) was first isolated from human pheochromocytoma cells and was classified as a member of the calcitonin gene-related peptide (CGRP) family (Kitamura et al., 1993). In pufferfish (Ogoshi et al., 2003) and medaka (Ogoshi et al., 2006), comparative genomic analyses have shown that AM is highly diversified and consists of five paralogous AMs termed AM1 to 5; multiple AMs have been found in other teleost species including zebrafish and trout (Takei et al., 2004a). In medaka and pufferfish, the five AMs can be divided into three groups, AM1/4, AM2/3 and AM5, based on deduced precursor sequences, with AM1 considered the orthologue of the original mammalian AM (Ogoshi et al., 2006, Takei et al., 2010). In Japanese eel, Anguilla japonica, four AMs (AM1, AM2, AM3 and AM5) have been cloned and sequenced (Nobata et al., 2008). Following the discovery of five AMs in teleost fishes, genomic analysis revealed that AM2 (also known as intermedin) and AM5 are also found in mammals, reptiles and amphibians (AM2: Roh et al., 2004, Takei et al., 2004a, Takei et al., 2004b; AM5: Takei et al., 2008, Takei et al., 2010, Takei et al., 2013).

It is known that members of the CGRP family, and more specifically mammalian AM, bind to calcitonin receptor-like receptors (CLRs; McLatchie et al., 1998). The CLRs on their own offer little binding affinity for mammalian AM and must associate with one of three receptor activity-modifying proteins (RAMPs) to become functional. In mammals, it has been shown that AM binds to a CLR in association with RAMP2 (called the AM₁ receptor) or RAMP3 (called the AM₂ receptor). A recent study has shown that mammalian AM2 demonstrates binding affinity to both AM₁ and AM₂ receptors, but preferentially activates the AM₂ receptor (Hong et al., 2011). In addition, mammalian AM2 has also been shown to interact with the CGRP receptor (CLR/RAMP1) but with much less affinity (Kuwasako et al., 2011). As the discovery of AM5 in mammals is relatively new, the receptor(s) are as yet unknown, but it has been suggested that mammalian AM5 may act through a receptor other than a CLR/RAMP complex (Takei et al., 2008, Takei et al., 2010, Takei et al., 2013, Rademaker et al., 2012). In teleost fish, AM receptors have been characterised in pufferfish, Takifugu obscurus. Three CLRs and five RAMPs were cloned and it was found that AM2 and AM5 could bind to the CLR1/RAMP3 combination or AM₂ receptor (Nag et al., 2006).

It has been well documented that mammalian AM is a key regulator of the cardiovascular system of mammals by mediating hypotension via central and peripheral mechanisms (Brain and Grant, 2004). In addition, both AM2 and AM5 have vasodepressor effects, but these are not as potent as AM (Fujisawa et al., 2004, Pan et al., 2005, Takei et al., 2010). AM is a dilator of isolated blood vessels but the signalling systems involved in vasodilation are variable and dependent on the type of blood vessel (Brain and Grant, 2004). A considerable number of studies have provided evidence that AM acts indirectly via the calcium-dependent activation of endothelial nitric oxide synthase (NOS3) in endothelial cells (Hayakawa et al., 1999, Ross and Yallampalli, 2006) and the subsequent release of NO, as AM vasodilation is attenuated by inhibitors of soluble GC (sGC) and NOS. In contrast, other studies have shown that AM mediates endothelium-independent vasodilation involving cAMP and K channels (Roh et al., 2004, Bayram et al., 2010).

Previously, mammalian AM was found to cause hypotension in trout, Oncorhynchus mykiss but not in the cod, Gadus morhua (Aota, 1995), and a weak dilation of the trout coeliac artery (Aota, 1995). In A. japonica, injection of homologous AM1 (AjAM1), AM2 (AjAM2) and AM5 (AjAM5) in vivo caused varying degrees of hypotension of both the branchial and systemic circulations (Nobata et al., 2008), with the vasodepressor effects of both AjAM2 and AjAM5 being significantly more potent than that of AjAM1. However, the effects of homologous AMs on isolated blood vessels from the systemic and branchial vasculature of teleost fish have not been determined. Therefore, the aim of this study was to assess the effects of AjAM2 and AjAM5 on eel blood vessels to determine if the endothelium, sGC or prostaglandins are involved in their vascular effects. AM1 was not examined because of its weak hypotensive effect in A. japonica (Nobata et al., 2008).