Evidence for atrial natriuretic peptide-(5-28) production by macrophages of the rat spleen: An immunochemical and nonradioactive in situ hybridization approach

Atrial natriuretic peptide (ANP), a 28-amino acid peptide, is produced and secreted by cardiac atriocytes to modulate cardiovascular functions. Recently, biologically active receptors for ANP have been demonstrated in the spleen; we report here the production of ANP-(5-28) and its 15-kDa (K) mol wt (Mr) presumptive precursors by macrophages of rat splenic tissues. Splenic, hypothalamic, and heart tissues were collected from adult male Sprague-Dawley rats and acid ex-tracted for ANP assay. The splenic content of immunoreactive (ir) ANP (mean P SEM, 428 P 68 pg/tissue; n = 7) was approx-imately a fifth of that found in the hypothalamus and about 4 orders of magnitude lower than that in the heart of the same animals. The Sephadex G-50 column profile of splenic extracts revealed two immunoreactive peaks; the major peak eluted in positions consistent with 15K Mr, while a minor peak coeluted with synthetic rat ANP-(1-28) of 3K Mr. HPLC analysis of the 3K Mr species showed a single peak of immunoreactivity, which eluted with a retention time similar to that of ANP-(5-28). In rat splenic sections, immunoperoxidase localization of ir-ANP revealed positive cells sparsely distributed in marginal sinuses and the red pulp of the tissue; employing a double staining technique, S22, a surface marker for macrophages, was colocal-ized on the same splenocytes. Furthermore, colorimetric in situ hybridization with antisense oligonucleotide probes labeled with digoxigenin, identified specific signals for pro-ANP mRNA in splenocytes of tissue sections. In monolayer cultures of vehicle-treated splenocytes, approximately 87% of the adherent cells stained positive for S22; this marker was colocalized with ir-ANP in approximately 15% of the cells. Twenty-four-hour treatment with lipopolysaccharide (50 μg/ml), a bacterial endotoxin, tripled the proportion of adherent cells (32 P 4%; P < 0.01) staining positive for ir-ANP over that in control cultures (mean P SEM, 11 P 3%; 10⁴ cells/sample; n = 5). Furthermore, an equivalent dose of lipopolysaccharide, but not Concanavalin-A (50 μg/ml), quadrupled ir-ANP content compared to that in vehicle-treated cultures (<5 pg/well). Thus, our findings suggest that ANP-(5-28) is produced by a small population of splenic macrophages and raise the possibility that the peptide may play a signalling role at the tissue level.