Natriuretic peptide binding sites in the gills of the pouched lamprey Geotria Australis

Abstract

Iodinated atrial natriuretic peptide (ANP) binding sites were examined in the gills and ventral aorta of the adult upstream-migrating lamprey Geotria australis using tissue section autoradiography, in vitro competition analysis and affinity cross-linking, while guanylate cyclase assays were performed on gill membranes of both adult and juvenile lampreys. A partial natriuretic peptide (NP) receptor sequence was amplified using reverse transcription/polymerase chain reaction (RT-PCR). The results indicated that there was specific NP binding to the aortic endothelium and to pillar cell regions in the axial plate and secondary lamellae. In competition studies, 50 % of NP binding was abolished by 4 nmoll-1 rat ANP, 35 nmoll-1 porcine C-type NP (CNP) and 45 nmoll-1 C-ANF (a truncated ANP). Affinity cross-linking followed by SDS-PAGE demonstrated two binding sites at 205 and 65kDa under non-reducing conditions and at 85 and 65kDa under reducing conditions. Guanylate cyclase assays demonstrated that, while no NP-stimulated GC activity occurred in adult lampreys, NP-stimulated enhancement of cyclic GMP accumulation was found in juveniles in fresh water and more particularly in salt water. RT-PCR amplified a 471 base pair fragment with 68 % amino acid sequence homology to the eel natriuretic peptide receptor D (NPR-D). This study suggests that NP binding sites in the adult gill and aorta are of an NPR-C/D type, whereas an additional GC-coupled site exists in juveniles.