Natriuretic Peptide Receptors on Rat Thymocytes: Inhibition of Proliferation by Atria1 Natriuretic Peptide*

Because the thymus expresses the natriuretic peptides (NP) as well as their respective receptors, an involvement of NP in the physiology of this organ has been suggested. To evaluate functional aspects of NP in the thymus, we looked for thymic cells bearing NP receptors (Npr). Furthermore, the regulation of Npr expression by activation of cells and the influence of NP on the proliferation of thymocytes was studied. Expression of receptor messenger RNAs CmRNAs) was examined by PCR and Northern blot. Existence of functional Npr was confirmed by measurement of cGMP, the second messenger of NP. Proliferation of thymocytes upon concanavalin A (Con A) stimulation was analyzed by incorporation of [“Hlthymidine. We report here that thymocytes H ORMONES and neuropeptides are potent immunomodulators acting on various immune competent cells (for review see Refs. 1,2). It is well documented that the thymus, where bone marrow-derived precursor cells proliferate and differentiate to mature T-lymphocytes, is under endocrine control (3, 4). The regulation of these processes appears to be extremely complex (5). In addition to circulating hormones affecting these functions, complete neuropeptide systems exist involving synthesis of peptides and expression of their respective receptors by thymic cells (3). For example, GH, PRL, oxcytocin (OT), arginine-vasopressin (AVP), or p-endorphin are likely to control thymus physiology via paracrine and autocrine mechanisms in addition to the classical endocrine pathway (3, 6-9). Our previous work as well as data from others (10-13) implicate the natriuretic peptides (NP) in thymus regulation. This peptide family consists of atria1 natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) and affects regulation of cardiovascular homeostasis (14-16). ANP and BNP are produced predominantly by the heart atrium and ventricle and act mainly as circulating hormones (14-16). In contrast, CNP is the major NP in the central nervous system (15,16). However, it is also synthesized by endothelial cells and seems to function as a local vascular regulator (15, 16, 17). Most of the biological actions of NP are thought to be mediated by two guanylyl cyclase-linked receptor subtypes (Npra and Nprb) with different ligand selectivities (18). The Npra receptor is activated Received August 24, 1995. Address all correspondence and requests for reprints to: Dr. Angelika Vollmar, Institute of Pharmacology, Toxicology and Pharmacy, Universitv of Munich, Koniginstrasse 16, Munich, Germany D-80539. ‘* This work is supported by the Deutsche Forschungsgemeinschaft (Vo-376/h-1). express mRNAs for the three Npr, namely Npra, Nprb, and Nprc and that activation of Npra and Nprb increases cGMP levels. Stimulation of thymocytes with Con A (1 pg/ml, 48 h) resulted in an increase of mRNA coding for Npra, the receptor specific for atria1 natriuretic peptide (ANP) and brain natriuretic peptide. Nprb and Nprc receptor expression was not altered under these conditions. In agreement with these data only ANP, but not the C-type natriuretic peptide, elicited increased cGMP response in Con A-stimulated cells. ANP inhibited also the proliferation of Con A stimulated thymocytes, whereas C-type natriuretic peptide did not show this effect. These results suggest that ANP affects the complex mechanisms of thymocyte proliferation and differentiation. (Endocrinology 137:1706-1713, 1996) by ANP and BNP, whereas CNP is considered to be the specific ligand of the Nprb receptor (16, 18, 19). cGMP is thought to act as the second messenger (18,19). All three NP bind to the C-type receptor (Nprc). This type of receptor lacks guanylate cyclase activity and elicits clearance function of the NP (19, 20). We showed recently that all three NP are produced by the rat thymus (11). Expression of the thymic NP is regulated by different mechanisms because involution of this organ caused by dexamethasone or x-ray increases ANP but not BNP and CNP production (11,21,22). The detection of messenger RNA (mRNA) transcripts for all three NP receptors in the thymus (11) suggests the existence of a local NP system that may intrathymically modulate immune functions. The aim of the present study was to investigate NP receptor expression on thymic cells and functional aspects of NP/ NP-receptor interaction. Thymocytes have been examined for the corresponding Npr messenger RNAs (mRNAs) as well as for their guanylate cyclase response upon exposure to NP. In a second set of experiments, alterations of Npr expression by immunological stimuli as the mitogen Concavalin A (Con A) was evaluated. Materials and Methods Cell preparation and culture condition Rats (Sprague Dawley, male, 100 g) were decapitated, and the thymi quickly removed and either employed for RNA extraction or for isolation of thymocytes. Thymocytes were isolated as described (10) and purified by means of Ficoll (Pharmacia, Freiburg, Germany) gradient centrifugation. Identity and homogeneity of the isolated cell population were assessed by staining cells with a monoclonal mouse antirat thymocyte antigen Thy 1.1 antibody (Serotec Camon, Heidelberg, Germany) and a secondary fluorescein isothiocyanate-labeled antimouse IgG (Becton Dickinson, Heidelberg, Germany). Cells were then submit-