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 radioligand-binding analysis using 125I-[Tyr0]-dogfish (df)CNP. None of the modifications of the CNP molecule that occur at the time of iodination (addition of a Tyr residue at the N-terminus, introduction of iodine into Tyr0 or oxidation of Met17) affect the affinity of dfCNP for the receptors. Neither did oxidation of Met17 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 Kd of 24.0 pM and Bmax of 59.9 fmol/mg protein, and the other has low affinity (Kd > 1 nM) and high capacity (Bmax > 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. Affinity-labelling 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.