Characterisation of natriuretic peptide receptors in eel gill

Abstract

A radioligand-binding assay was established for eel atrial natriuretic peptide (eANP), and ANP receptors were characterised in the eel gill. ANP binding to the gill membrane fraction was saturable with increasing ligand concentrations and was specific to ANP peptides, i.e. eANP, ventricular natriuretic peptide (VNP) and C-type natriuretic peptide (CNP). A Scatchard analysis revealed a single class of high-affinity receptors with a Kd of 59.2 pM and a Bmax of 67.9 fmol/mg protein. The Kd value is within the range of plasma ANP concentration of the eel. Kd and Bmax did not differ between freshwater (FW)- and seawater (SW)-adapted fish. The gill receptors exhibited similar affinity for eANP, eVNP and eCNP, and ANP binding was almost completely displaced by C-ANF, a specific ligand for guanylate cyclase-uncoupled receptors. The presence of this type of receptor (natriuretic peptide receptor (NPR)-C and NPR-D) was also indicated in eel gill by affinity labelling. cGMP production was stimulated by the addition of eCNP but not by eANP and eVNP, and this was observed only in FW eels. Thus, most ANP receptors in eel gill are NPR-C and NPR-D, but a small number of the NPR-B type are also present in FW eels. In addition to the gill, specific ANP receptors were detected in the red body of the swim bladder, the brain, digestive tracts, kidney, head kidney and urinary bladder, in most of which ANP action has been reported. The density of ANP receptors decreased in most tissues after adaptation to SW except the gill, brain, atrium and spleen.

Characterisation of C-type natriuretic peptide receptors in the gill of dogfish Triakis scyllia

“…The function of dense GCuncoupled receptors in the dogfish gill may be to regulate plasma CNP titre, as has been suggested for the trout (Olson & Duff 1993) and eel . Most of the GC-uncoupled receptors may be homodimeric NPR-C, as observed in mammalian lung (Shimonaka et al 1987) and teleost gill (Olson & Duff 1993, Donald et al 1994, Mishina & Takei 1997, because most 170 kDa receptors are converted to 90 kDa after reduction with 2-mercaptoethanol. The presence of tetrameric NPR-D was not excluded in the dogfish gill, but its population is minor as shown in eel gill (Mishina & Takei 1997).…”

“…They weighed 0·8-2·5 kg at the time of experimentation. Membrane fractions were prepared from various tissues as described previously (Mishina & Takei 1997). The dogfish were anaesthetised by immersion in 1% tricaine methanesulphonate for 10 min and the heart was exposed.…”

“…The membrane fractions thus prepared were stored in liquid nitrogen until use. ]-dfCNP were not metabolised after incubation under these conditions (Mishina & Takei 1997). After incubation, 1 ml ice-cold buffer B was added to each tube to terminate the reaction.…”

“…GC activity was determined as described previously (Mishina & Takei 1997). Membrane fraction prepared from each tissue (30 µg) was incubated at 25 C for 10 min in 80 µl 50 m Tris-HCl buffer, pH 7·6, containing 1 mg/ml BSA, 0·5 m isobutyl-1-methylxanthine, 15 m phosphocreatine and 31·25 U/ml creatine kinase.…”

Some Factors Affecting Drinking Behavior and Their Interactions in Seawater-Acclimated Eels, Anguilla japonica

“…A radioligand-binding assay showed that ANP receptors exist in the gills, but the predominant receptors are clearancetype NPR-C in eels (Sakaguchi et al, 1993) and toadfish (Donald et al, 1994). Further, Mishina and Takei (1997) showed that the NPR-A type is not detectable in the gills of SW eels as shown by the absence of cGMP accumulation after addition of ANP to isolated gill cells. These results provide correlative support for the absence of ANP effects on the gills of SW eels.…”