Localization of Receptors for Natriuretic Peptide and Endothelin in the Duct of the Epididymis of the Freshwater Turtle

Abstract

The presence of receptor subtypes for natriuretic peptides (NPs) and endothelin (ET) in the epididymis of the freshwater turtle, Amyda japonica, was examined by quantitative in vitro autoradiography using iodinated mammalian-type atrial NP (125I-ANP(1–28)), phylogenically conserved C-type NP (125I-[Tyr0]-CNP(1–22)), and ET-1 (125I-ET-1) as radiolabeled ligands. To characterize NP receptor (NPR) subtypes, we also performed an activation of particulate guanylyl cyclase (GC) in membranes of the epididymis by NPs. Specific 125I-ANP(1–28) and 125I-[Tyr0]-CNP(1–22) bindings were localized in surrounding smooth muscle cell layer of the duct of the epididymis with an apparent dissociation constant (Kd) of 0.84 ± 0.15 and 1.74 ± 0.39 nM and a maximal binding capacity (Bmax) of 0.47 ± 0.11 and 0.08 ± 0.01 fmol/mm2, respectively. Bindings of 125I-ANP(1–28) and 125I-[Tyr0]-CNP(1–22) to these sites were also displaced by des[Gln18,Ser19,Gly20, Leu21,Gly22]ANF(4–23), a specific ligand of the NP clearance receptor. Production of 3′,5′-cyclic guanosine monophosphate by particulate GC in membranes of the epididymis was stimulated by ANP(1–28), BNP(1–26), and CNP(1–22). Receptor subtypes for ET in the epididymis were characterized by competition with BQ 123 and BQ 788 as specific antagonists for ET receptors, type A (ETA) and type B (ETB) subtypes, respectively. Specific 125I-ET-1 bindings were localized in the smooth muscle cell layer of the duct of the epididymis with Kd and Bmax of 0.21 ± 0.03 nM and 0.52 ± 0.05 fmol/mm2, respectively. These specific bindings were potently inhibited in a dose-dependent manner by BQ 123, whereas BQ 788 (10 μM) was not in competing for specific 125I-ET-1 bindings in this structure. Therefore, these results indicate that specific NP and ET receptors are localized in surrounding smooth muscle cells of the duct of the epididymis of the freshwater turtle. It is also suggested that biological and clearance NPR-like subtypes coexist in these cells, and the predominant ET receptor subtype in this tissue is the ETA-like receptor. The localization of specific receptors for NPs and ET in the epididymis may be involved in the control of the transport of sperm in the freshwater turtle.