title: Evidence for atrial natriuretic peptide-(5-28) production by macrophages of the rat spleen: an immunochemical and nonradioactive in situ hybridization approach.
abstract: 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 extracted for ANP assay. The splenic content of immunoreactive (ir) ANP (mean +/- SEM, 428 +/- 68 pg/tissue; n = 7) was approximately 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 colocalized 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 micrograms/ml), a bacterial endotoxin, tripled the proportion of adherent cells (32 +/- 4%; P less than 0.01) staining positive for ir-ANP over that in control cultures (mean +/- SEM, 11 +/- 3%; 10(4) cells/sample; n = 5). Furthermore, an equivalent dose of lipopolysaccharide, but not Concanavalin-A (50 micrograms/ml), quadrupled ir-ANP content compared to that in vehicle-treated cultures (less than 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.
authors.affiliation: Neuroendocrine Laboratory, Mental Health Research Institute of Victoria, Royal Park Hospital, Parkville, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Throsby
authors.forename: D
authors.initials: D
authors.lastname: Lee
authors.forename: W Q
authors.initials: WQ
authors.lastname: Huang
authors.forename: Z Y
authors.initials: ZY
authors.lastname: Yang
authors.forename: D L
authors.initials: DL
authors.lastname: Copolov
authors.forename: A T
authors.initials: AT
authors.lastname: Lim
mesh_terms: D000818:Animals; D009320:Atrial Natriuretic Factor / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D002478:Cells, Cultured; D002850:Chromatography, Gel; D007124:Immunoenzyme Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000096:biosynthesis*; D011498:Protein Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D013154:Spleen / Q000737:chemistry / Q000166:cytology*
chemical_list: D008070:Lipopolysaccharides; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D011498:Protein Precursors; D012333:RNA, Messenger; D009320:Atrial Natriuretic Factor; C054538:atrial natriuretic factor (5-28)