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

Abstract

Only C-type natriuretic peptide (CNP) has been identified in primitive elasmobranch fish. CNP is the most conserved molecule in the natriuretic peptide family, suggesting that it is the ancestral type. As a first step to investigating the ancestral type of natriuretic peptide receptors, CNP receptors were characterised in an elasmobranch (dogfish, Triakis scyllia) by radioligandbinding analysis using 17 decrease the ability of CNP to stimulate cGMP production. In the tissues examined, CNP receptors were densest in the gill cells followed by the intestine, interrenal gland and rectal gland, all of which are involved in osmoregulation in elasmobranchs. CNP-stimulated guanylate cyclase (GC) activity was highest in the interrenal gland, intestine, brain and rectal gland, followed by the gill cells. Since the gill cells seem to contain both GC-coupled and uncoupled receptors, this tissue was used to characterise dogfish CNP receptors. Scatchard analysis of the saturation isotherm revealed two classes of binding site: one has a K d of 24·0 p and B max of 59·9 fmol/mg protein, and the other has low affinity (K d >1n) and high capacity (B max >200 fmol/mg protein). The higher-affinity binding sites may represent GC-uncoupled receptors, because C-ANF, a specific ligand for GC-uncoupled receptors, almost completely displaced CNP binding. Affinitylabelling experiments showed that dogfish receptors have molecular masses of about 90, 170 and 340 kDa, and CNP binding to the former two receptors is inhibited by C-ANF. After reduction with 2-mercaptoethanol, most 170 kDa labelling was shifted to 90 kDa. It is concluded that GC-uncoupled receptors in the dogfish gill have higher molecular mass than those of mammals and eel (about 65 kDa), and are present mostly as monomers even in non-reducing conditions. However, a small population of GC-coupled receptors is also present, as demonstrated by an increase in cGMP production.

CNP is the only Natriuretic Peptide in an Elasmobranch Fish, Triakis scyllia

“…Mature CNP-22 stimulates NaCl secretion from the Squalus rectal gland (Silva et al, 1999). However, it is not known whether circulating proCNP is cleaved to CNP-22 before acting on target tissues, since dogfish guanylyl cyclase-coupled receptor, probably NPR-B (Aller et al, 1999), has low ligand selectivity in Triakis (Sakaguchi and Takei, 1998). CNP is highly conserved in elasmobranchs; this high degree of conservation extends not only to mature CNP-22 that is located at the C-terminus, but also to the whole proCNP sequence as shown in Fig.…”

Cardiovascular cGMP-Generating Systems in Physiological and Pathological Conditions

“…Interestingly, these receptors resemble the mammalian receptors in terms of both structure and signal transduction cascade. Three types of NPRs have been identified in nonmammalian vertebrates, two are similar to NPR-A and NPR-B and signal through cGMP, possibly involving also G proteins [77,79,80,81], the other appears to be a NPR-Clike receptors [see 74 for references].…”