0013-7227/90/1276-3240$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 127, No. 6 Printed in U.S.A.

DEXAMETHASONE ACTION ON RAT THYMIC ATRIAL NATRIURETIC PEPTIDE ANGELIKA M. VOLLMAR AND RUDIGER SCHULZ Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Kbniginstr. 16, 8000 Munich 22, FRG SUMMARY: The rat thymus gland expresses the ANP gene and synthesizes the ANP-precursor, suggesting a role for this peptide within the immune system. This study provides further evidence for this notion, as expression of ANP mRNA in the thymus is altered in response to administration of the glucocorticoid dexamethasone to rats (1.5 mg/kg). This drug causes involution of the thymic gland and at day 4 after steroid exposure a striking increase (up to 30 fold) in mRNA coding for ANP was observed. The increase of the ANP precursor was found to be 4-fold as compared to saline treated controls. During regeneration of the thymus the mRNA and precursor levels normalize, reaching control values the day 18 after dexamethasone. The strong stimulation of the thymus ANP system subsequent to the glucocorticoid may suggest a critical function of this peptide in mechanisms leading to cellular depletion or during the regeneration of the gland after exposure to the steroid. INTRODUCTION gland (1-2 g) was extracted for total RNA using the The recent discovery of atrial natriuretic peptide LiCl/Urea technique and mRNA was enriched by (ANP) precursor in lymphoid nodules of the gut (1), submitting 1 mg of total RNA to oligo-dT-cellulose the spleen (2), and the thymus (3) links this peptide chromatography. 30 ug from each preparation was to the immune system. It is, thus, assumed that denatured in 1 M glyoxal, applied to gel changes in the function of the mammalian immune system electrophoresis (1.2 % agarose gel) and blotted onto a are associated with an altered expression of ANP. The Nytran 13 N membrane (Schleicher and Schlill, FRG) by glucocorticoids are powerful pharmacological tools capillary forces. The ANP-specific cRNA hybridization, affecting a number of biochemical mechanisms of the probe (obtained from K. Bloch and J. Seidman, Boston) lymphoid system. These drugs decrease the synthesis of was prepared as described (3) using 100 uCi -P-UTP proteins in lymphocytes (4). They lower RNA polymerase (400 Ci/mmol, Amersham) and the SP6-polymerase activity (5) and inhibit ATP production (6). The promotor system. Membranes were hybridized and washed present study was designed, therefore, to investigate as reported (3) prior to exposure to X-ray film (4 the effect of dexamethasone on ANP in rat thymus, an days). Filters were rehybridized using a B-actin probe organ known to be highly sensitive to the immune (2 hrs exposure time). The autoradiographic signals depressive action of glucocorticoids (7,8). We report were semiquantitatively evaluated by a scanning here, that a single administration of dexamethasone densitometry. substantially elevates the amount of mRNA coding for Quantification of immunoreactive ANP 1-126 (IR-ANP): ANP in the thymus which in turn increased the Extraction, characterization and quantification of synthesis of ANP precursor material (ANP 1-126). thymic IR-ANP has been performed as described in MATERIALS AND METHODS detail elsewhere (1-3). Briefly, frozen thymus (1-2 g) Rats: Male' Sprague Dawley rats (100-120 g) were was extracted with 0.1 N HC1 (95°C) and submitted to injected once subcutaneously with dexamethasone (1.5 Amberlite XAD 4 resin (Serva, FRG). The retained mg/kg; Fortecortin, Bayer, Leverkusen, FRG). On day 4, material was eluted by 80 % acetonitrile in 0.1 % 10 and 18 after treatment the animals were decapitated trifluoroacetic acid and the lyophilized extracts and the thymi quickly removed, weighed, and underwent gel filtration (Sephadex G-50). immediately frozen on dry ice. Control animals Quantification of ANP was performed by submitting received saline injection. aliquots of the fractions to a RIA procedure as Northern blot hybridization: RNA blot hybridization previously described (1,2). Further characterization of IR-ANP was conducted by RP-HPLC (1-3). The recovery has been conducted as described (3). Briefly, thymus rate, as determined by addition of synthetic rat ANP Received in Iowa City, Iowa, Septermber 11, 1990 (2-126) to the thymus prior to homogenization, was in the range of 28-36 %. Values reported have been

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RAPID COMMUNICATIONS corrected for the individual loss. RESULTS Effect of dexamethasone on the thymus gland Dexamethasone induced thymic involution as documented by a decrease in thymic weight of 73 % (n=50; 0.577g _+ 0.082 vs. 0.146g _+ 0.036) and in thymic cell number of 94 % (n=10; 6.1 ± 0.8 x 10 8 vs. 0.38 + 0.05 x 10 8 cells/thymus) at day 4 after exposure to the steroid. Glands removed at day 10 were found in the process of recovery as indicated by an increased thymus weight (31 % below controls, 0.398g +_ 0.043, n=50) and cell number (28 % of controls, 1.7 _+ 0.2 x 10 8 cells/ thymus, n=5). At day 18 after injection glands displayed a tissue weight 17 % below controls (n=5, 0.478g +_ 0.039) whereas the cell number was 26 % below (n=5, 4.5 + 0.6 x 10 8 cells/thymus). Histological examination of the different stages was in line with these results (data not shown). Thus, the thymic cortex and the size of lobules at day 4 after dexamethasone treatment was strongly reduced, whereas at day 10 reconstruction of cortical areas and repopulation by small thymocytes was apparent. At day 18 we observed no distinct difference from control tissue. Effect of dexamethasone on ANP mRNA The thymus of rats receiving a single dexamethasone injection displays increased amounts of mRNA coding for ANP. As demonstrated in Figure 1, a 22 to 30-fold elevated level is manifest the 4th day after dexamethasone (n=3). During regeneration of the tissue (10th day) ANP mRNA decreased again exhibiting only a 1.5 to 2-fold elevated level (n=3). At day 18 approximately control level were measured (n=3). ANP mRNA in control thymus glands amounts to less than 0.1% of the atrial ANP transcript (right lane). RNA hybridization with a rat R-actin probe has been performed to adjust measurements for equal amounts of loaded mRNA. CN

d4

d1O

d18

atria

Figure 1: mRNA coding for ANP in rat thymus treated with dexamethasone. ANP and B-actin mRNA from thymic

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tissue collected at day (d) 4,10, and 18 after single steroid treatment as well as from saline treated animals (control), is shown (30 ug mRNA/lane). Atrial mRNA coding for ANP (0.5 ug total RNA) was used as control tissue. IR-ANP content in thymus after dexamethasone treatment IR-ANP of glands from treated and untreated rats has been submitted to RP-HPLC, revealing mainly the presence of the precursor molecule ANP 1-126 (data not shown). The control tissue glands contain 45.2 +_ 12.8 fmol IR-ANP per mg protein (n=3), whereas dexamethasone exposed thymic tissue (4 days after treatment) displays a 4 fold increase (191.5 +_ 30.2 fmol/mg protein, n=4). Recovery was observed at day 18 after treatment exhibiting 53.8 _+ 10.6 fmol IR-ANP (n=3) per mg protein (controls: 64.3 +_ 15.8 fmol/mg protein). DISCUSSION The data reported here disclose a substantial effect of dexamethasone on ANP expression in the rat thymus. Since the glucocorticoids cause an involution of the gland (7-13), one may predict that their catabolic effect on protein biosynthesis, e.g. on lymphocytes (4,11), causes a decrease of the ANP level in the thymus. However, the mRNA coding for ANP was found to increase 30-fold and ANP 1-126 displays 4-fold elevated levels as consequence of a single dexamethasone treatment. These effects are reversible as the gland recovers. Taking into consideration that glucocorticoid induced lysis of thymocytes accounts for the involution of the gland (7,8) the IR-ANP detected here may be linked to those cells not lysed and thus resistant to the action of dexamethasone. It is not clear yet, whether these cells belong to those thymocytes synthesizing ANP (3). It is furthermore uncertain, whether the glucocorticoid resistant cells, which are believed to represent mature, immunocompetent thymocytes as well as thymic precursor cells (10-14), are the only source of ANP mRNA and ANP 1-126. In addition, one should consider the fact that during involution and regeneration of the thymus a variety of phenotypic and functional changes of thymocytes occur. For instance, subpopulations of cells emerge, such as cytolytic T-lymphocyte precursors (13) and large blast cells (9,10,12), which eventually disappear upon complete thymic regeneration. Furthermore not only lymphocytes are affected by glucocorticoids, also thymic epithelial

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RAPID COMMUNICATIONS cells respond to hydrocortisone with an increase of certain subpopulations of cells (14). Due to the complex action of dexamethasone the cell types responsible for the substantial increase of ANP mRNA in the thymus are not defined yet. However, glucocorticoids are known to stimulate the ANP system in other cells such as atrial and ventricular myocytes and pneumocytes in vivo as well as in vitro in a time dependent way, but the effects observed were of minor degree (2-3 fold) on ANP mRNA and IR-ANP, respectively (15-17). Interestingly, it has been shown that glucocorticoids stimulate ANP processing and secretion by atrial cultures (17). It would be worthwhile to search also for this effect on thymic cells. The surprisingly strong effect of dexamethasone on the ANP in the thymus, communicated here, may be unique as compared to other tissues. It may thus reflect a specific action of dexamethasone on the immune system and we suggest that ANP exerts a critical function in the regeneration thymic gland subsequent to steroid exposure. REFERENCES 1. Vollmar AM, Friedrich A, Sinowatz F, Schulz R 1988 Presence of atrial natriuretic peptide like material in guinea pig intestine. Peptides 9:965-971. 2. Vollmar AM, Friedrich A, Schulz R 1989 Immunoreactive atrial natriuretic peptide in the guinea pig spleen. Life Sci 45:1293-1297. 3. Vollmar AM, Schulz R 1990 Atrial natriuretic peptide is synthesized in the human thymus. Endocrinology 126:2277-2281. 4. Morita Y, Munck A 1964 Effects of glucocorticoids in vivo and in vitro on net glucose uptake and ami no acid incorporation in rat thymus cells. Biochem Biophys Acta 93:150-157. 5. Bell PA, Borthwick NM 1975 Glucocorticoid effects on DNA-dependent RNA polymerase activity of rat thymocytes. J steroid Biochem 7:1147-1150. 6. Nordeen SK, Young DA 1976 Glucocorticoid action on rat thymic lymphocytes: experiments utilizing adenosine to support cellular metabolism lead to a reassessment of catabolic hormone actions. J Biol Chem 251:7295-7303. 7. Compton MM, Caron LAM, Cidlowski JA 1987 Glucocorticoid action on the immune system. J steroid Biochem 27:201-208.

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8. Munck A, Crabtree GR 1981 Glucocorticoid-induced lymphocyte death. In: Cell death in biology and pathology (Eds: Bowen ID and Locksin A) Chapman and Hall New York, pp. 329-359. 9. Weissman I 1972 Thymus cell maturation. Studies on the origin of cortisone-resistant thymic lymphocytes. J Exp Med 137:504-510. 10. Blomgren H, Andersson B 1971 Characteristics of immunocompetent cells in the mouse thymus: cell population changes during cortisone-induced atrophy and subsequent regeneration. Cell Immunol 1:545-560. 11. Rothenberg E 1980 Expression of differentiation antigens in subpopulations of mouse thymocytes: regulation at the level of de Novo synthesis. Cell 20:1-9. 12. Boersma W, Betel I, van der Westen G 1979 Thymic regeneration after dexamethasone treatment as a model for subpopulation development. Eur J Immunol 9:45-52. 13. Ceredig R, Cummings DE 1983 Phenotypic and functional properties of murine thymocytes III, kinetic analysis of the recovery of intrathymic cytolytic T lymphocyte precursor after in vivo administration of hydrocortisone acetate. J Immunol 130:33-37. 14. Savino W, Cirne-Lima E0, Soares JFT, Leite-de-Moraes MC, Ono IPC, Dardenne M 1988 Hydrocortisone increases the number of KL 1 cells, a discrete thymic epithelial cell subset characterized by high molecular weight cytokeratin expression. Endocrinology 123:2557-2564. 15. Gardner DG, Hane S, Trachewsky D, Schenk D, Baxter JD 1986 Atrial natriuretic peptide mRNA is regulated by glucocorticoids in vivo. Biochem Biophys Res Commun 139:1047-1054. 16. Matsubara H, Mori Y, Umeda Y, Oikawa S, Nakazato H, Inada M 1988 Atrial natriuretic peptide gene expression and its secretion by pneumocytes derived from neonatal rat lungs. Biochem Biophys Res Commun 156:619-627. 17. Shields PP, Dixon JE, Glembotski CC 1988 The secretion of atrial natriuretic factor (99-126) by cultured cardiac myocytes is regulated by glucocorticoids. J Biol Chem 263:12619-12628.

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Dexamethasone action on rat thymic atrial natriuretic peptide.

The rat thymus gland expresses the ANP gene and synthesizes the ANP-precursor, suggesting a role for this peptide within the immune system. This study...
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