In vitro macro- and micro-autoradiographic localization of atrial natriuretic peptide in the rat kidney

We investigated the receptor localization of atrial natriuretic peptide (ANP) in the rat kidney, by in vitro macro- and micro-autoradiography (ARG) of [125I]-ANP using nonfixed frozen sections. First, we examined the optimum conditions for ARG with respect to the effects of polyethylenimine (PEI), thickness of section, incubation time and degradation of [125I]-ANP. Saturation experiments of [125I]-ANP using rat kidney sections revealed the presence of high affinity binding sites of [125I]-ANP in the renal cortex (Kd = 0.52 nM). Macro-autoradiograms showed that the dense grains representing specific binding sites of [125I]-ANP were distributed in the cortex in a punctate pattern. Using micro-autoradiography, the localization of the dense grains on the emulsion was compared with the staining pattern of the same section subjected to double staining. In the renal cortex, the dense grains were observed on the glomerulus, blood vessels and proximal tubules. Dense grains were also observed in the mesangium area of glomeruli, the inside wall of blood vessels (especially endothelium), and the inside wall of proximal tubules (possibly brush border). These results suggested that the physiological action of ANP in the kidney is mediated by its receptors. Also, ARG was useful for accurately detecting the action sites of ANP.