Localization of Atrial Natriuretic Factor Receptors in the Mesenteric Arterial Bed

Abstract

Although receptors for atrial natriuretic factor (ANF) and angiotensin II (Ang II) have been reported in rat mesenteric arteries, both peptides induce weak biological responses. Endothelin-1 (ET-1) evokes a potent vasoconstriction in the mesenteric artery. To identify the tissue localization of ANF, Ang II, and ET-1 receptors, radioligand binding experiments with ¹²⁵I-ANF, ¹²⁵I–[Sar¹,Ile⁸]Ang II, and ¹²⁵I–ET-1 were performed in defatted mesenteric arteries and in the surrounding adipose tissue. ¹²⁵I-ANF binding assays in adipose tissue showed a single class of high-affinity binding sites (B_max, 420±16 fmol/mg protein; K_d, 343±16 pmol/L). In vascular membranes, most ¹²⁵I-ANF binding was nonspecific. The majority of receptors present in adipose tissue recognized ANF, C-type natriuretic peptide (CNP), and des-[Gln¹⁸,Ser¹⁹,Gly²⁰,Leu²¹,Gly²²]ANF-(4-23) (C-ANF) with close affinities, with C-ANF competing for >98% of the binding sites. In adipocytes, ANF and CNP stimulated cGMP generation. cGMP production by mesenteric arteries was stimulated by sodium nitroprusside but not by ANF or CNP. Autoradiographic localization of ¹²⁵I-ANF and ¹²⁵I–ET-1 showed that in the case of ANF, most specific binding occurred in adipocytes, whereas for ET-1, specific binding was present in both adipose tissue and mesenteric arteries. Cross-linking of ¹²⁵I-ANF followed by SDS-PAGE revealed two receptor species of 130 and 70 kD in adipose membranes and none in vascular tissue. Both were completely displaced by ANF, CNP, and C-ANF. ¹²⁵I–[Sar¹,Ile⁸]Ang II binding assays in adipose tissue exhibited a single class of binding sites (B_max, 211±4 fmol/mg protein; K_d, 520±10 pmol/L). In mesenteric arteries, ¹²⁵I–[Sar¹,Ile⁸]Ang II saturation assays showed little specific binding. The profile of ligands competing for ¹²⁵I–[Sar¹,Ile⁸]Ang II was consistent with an AT₁ receptor subtype. ¹²⁵I–ET-1 binding assays demonstrated high-affinity binding sites in both mesenteric arteries and adipose tissue (B_max, 179±21 and 312±7 fmol/mg protein, respectively; K_d, 215±45 and 180±111 pmol/L, respectively), the majority corresponding to BQ-123–sensitive sites. Thus, ANF and Ang II binding sites were either absent or barely expressed in the mesenteric artery. Binding sites for ET-1 were abundantly expressed in the mesenteric artery. Adipose tissue expressed high-affinity binding sites for ANF, Ang II, and ET-1, but their biological role, if any, remains to be defined.