Autoradiographic localization and characterization of angiotensin II binding sites in the spleen of rats and mice

Pepttde~. Vol 8, pp 737-742 ~ Pergamon Journals Ltd, 1987 Printed m the U S A

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Autoradiographic Localization and Characterization of Angiotensin II Binding Sites in the Spleen of Rats and Mice EERO CASTRI~N, MASAKI KURIHARA

A N D J U A N M. S A A V E D R A 1

Unit on Preclinical Neuropharmacology, Section on Clinical Pharmacology, Laboratory o f Climcal Science National Institute o f Mental Health 9000 Rockville Pike, Building 10, R o o m 2D46, Bethesda, M D 20892 R e c e i v e d 17 F e b r u a r y 1987 CASTRt~N, E , M. KURIHARA AND J M. SAAVEDRA. Autoradlographw locahzanon and characterzzatton of angtotensm 11 binding ~ttes in the spleen of rat~ and rowe PEPTIDES 8(4) 737-742, 1987.--Specific binding sites for anglotensm II (Ang II) were locahzed m the red pulp of the spleen of rats and mice by quantitative autoradlography using lzsI-Sar~-Ang II as a hgand. In the rat, the binding was saturable and speofic, and the rank order for Ang II denvat,ves as competitors of 125I-Sarl-Ang II binding correlates well with their affimty for Ang II receptors m other tissues. Kinetic analysis m the rat spleen revealed a single class of binding sites with a KD of 1.11 nM and a Bmax value of 81 6 fmol/mg protein Ang II binding sites were also Iocahzed on isolated rat spleen cells with slmdar affimty but with much lower Bma\, 9 75 fmol/mg protein Ang II receptors were not detected m thymus sections from rats or mice, or on Isolated rat thymocytes. The binding sites described here m,ght represent a functmnal Ang II receptor w,th a role m the regulation of splenic volume and blood flow and in the modulation of the lymphocyte function. Spleen

Thymus

Anglotensm II

Receptor autoradlography

obtained from Zlvic Miller, Alliston Park, PA, and male mice (BALB/c) from Charles River, Vermonton, MA. The animals were housed under constant temperature (20-+ I°C) with lights on from 06.00 hr to 18.00 hr and were given free access to food and water. Rats and mice were killed by decapitation at the age of four and six weeks, respectively, between 09.00 and 12.00 hr. The spleen and thymus were removed and frozen by immersion in lso-pentane at -30°C. Within 24 hr of death, 16/zm thick tissue sections were cut in a cryostat at - 16°C, thaw-mounted onto gelatin-coated glass slides, and stored overnight under vacuum at 4°C. The glass slides containing the tissue sections were preincubated in l0 mM phosphate buffer (pH 7.4) containing 120 mM NaC1, 5 mM Na2EDTA, 0.1 mM bacitracin, and 0.2% bovine serum albumin for fifteen minutes at room temperature. The slides with rat tissues were then transferred into the same buffer containing lzsI-Sarl-Ang II concentrations between 0.1 and 4.4 nM [9,10]. Tissue sections from mice tissues were incubated under the same conditions with 2 nM 125I-Sar~-Ang II. After one hour incubation at room temperature, the slides were washed four t~mes, one minute for each wash, in ice-cold 50 mM Tris-HCi (pH 7.56), dipped into ice-cold distilled water, dried under a cold stream of air, and exposed to [3H]-Ultrofilm (LKB Instruments, Rockville, MD) for seven days. Non-specific binding was determined by in-

A N G I O T E N S I N II (Ang II) IS involved In the regulation of blood pressure and body fluid homeostasis by direct actions on cell surface receptors present in vasculature, adrenal gland, kidney, and certain parts of the central nervous system [12]. In addition, Ang I! has been shown to have effects on the components of the immune system [6, 17, 18, 23]. Binding sites for Ang II have been localized on human peripheral lymphocytes [17] and on murine granuloma macrophages [20,22]. Ang II has also been shown to suppress human mononuclear cell reactivity by stimulating suppressor T-lymphocytes [18,19]. These data suggest that Ang II may have a role in the regulation of immune response and inflammation. Several neuropeptides have recently been proposed to play a role in the regulation of the immune response [23]. We have recently showed the presence of binding s~tes for substance P and atrial natriuretic peptide in the rat spleen and thymus with quantitative autoradiography [ 11,16]. These observations have prompted us to investigate the presence of Ang II binding sites in the lymphoid organs of laboratory rodents commonly utilized in immunological studies. METHOD Male Sprague-Dawley rats (specific pathogen free) were ~Requests for repnnts should be addressed to J. M. Saavedra

737

CASTRI~N, KURIHARA AND SAAVEDRA

738

SPLEEN

A

b

B

b

C

FIG

I

THYMUS

A U T O R A D I O G R A P H I C L O C A L I Z A T I O N A N D A N G II cubatmg adjacent tissue sections in the presence of 5 ~ M unlabeled Ang II. To determine the ability of related peptides to compete with Ang II binding, tassue sections were incubated with 2 nM of 12~I-Sar~-Ang II in the presence of I / z M of Ang I, Ang II, Ang III, Sar~-Ang II, Sari-AlaS-Ang II, and the nonrelated peptides atrial natrluretic peptade and substance P. Single-cell suspensions of spleen and thymus from individual rats were prepared by teasing apart organs anto RPMI 1640 medium (GIBCO Laboratories, NY) supplemented with L-glutamine, 50 units/ml of penicillin, 50 p.g/ml of streptomycin and 10% fetal calf serum, followed by filtration through a nylon mesh. The erythrocytes were lysed by treatlng the cells for 10 minutes in ice-cold 10 mM Tris-buffered 0.85% ammonium chloride [7]. After three consecutive washes, precipitated cells were transferred into Eppendorf tubes which contained M-1 embedding matrix (Llpshaw Mfg. Co., Detroit, MI) (1 1, v/v), centrifuged at 3000 rpm (4°C) for 15 minutes and frozen in iso-pentane at -30°C. The resuiting pellets were removed from the tubes, mounted onto cryostat specimen holders, and cut into 16 ~tm-thlck sections in a cryostat at -16°C. Blood collected from rats after decapitation was centrifuged, the plasma discarded, and a sample of the red blood cell-rich pellet was transferred into an Eppendorf tube together with the embedding matrix. After centrifugation, the tubes were frozen and the pellets were removed, mounted on the specamen holders, and cut anto 16/~m sections in a cryostat at - 16°C Ang II binding sites were quantified with computerized densltometry and comparison made with ~25I standards [9,10]. A complete set of 125I standards was carried through the whole exposure and development procedure for each film. After determination of the standard curve (In optical density × 100 vs. In disintegrations per mln of standards), the optical densities corresponding to the tassue images were determined and interpolated in the straight line to obtain the corresponding dpm bound to the tissue [21]. To determine the kinetic parameters, Scatchard plots were generated, and the presence of only one class of binding sites was confirmed by using the L I G A N D program [14]. Results are expressed as mean value -+SEM. Statistical significances were determined by Student's t-test, and p-values less than 0.05 were considered statistically significant. RESULTS High density of Ang II binding sites was localized throughout the red pulp of rat spleen (Fig. l) Binding was saturable and specific, since it could be displaced by a high concentration of unlabeled Ang II. The non-specific binding was less than 20% of the total binding. Scatchard analysis revealed a single class of binding sites with a KD value of 1.11-+0.11 nM and Bm~ of 81.6-+4.67 fmol/mg protein (n=8) (Fig. 2). The whate pulp of rat spleen was devoid of specific Ang II binding sates (Fig. l).

739 The relative potency of Ang II derivatives in competing with I25I-Sari-Ang II binding m rat spleen sections was Sar~-Ang II > Sarl-AlaS-Ang II > Ang II > Ang III > Ang I (Fig. 3) The unrelated peptides, atrial natriuretic peptide and substance P did not inhibit binding significantly at I/zM concentration (Fig. 3). In the rat spleen cell preparataons, Ang II bound to a single class of binding sites with binding affinity similar to that of the red pulp of the spleen sections (KD 0.7--+0.28 nM, n=4). The binding capacity, however, was considerably lower in the cell preparation than in the whole spleen red pulp (Bm.~, 9.75-+0.75 fmol/mg protein, n=4) (Fig 2). Binding sites for Ang II were also localized in the red pulp of mouse spleen. The binding concentration in mouse spleen sections incubated with 2 nM ~25I-Sar~-Ang II was 50.8 fmol/mg protein, and the competition properties of angxotensm analogs were similar to those observed in the rat tissue sections (data not shown). Again, white pulp was devoid of Ang II binding sites. We could not detect binding of Ang II to the thymus of either the rat (Fig. 1) or the mouse. In addatlon, the red blood cells from peripheral circulation were devoid of Ang II bindlng sites (data not shown). DISCUSSION Our results are the first demonstration of specific Ang II binding sites discretely localized in the red pulp of rat and mouse spleen The white pulp of the spleen was completely devoid of Ang II binding sites. The kinetic analysis in the red pulp of the rat spleen sections revealed a single class of binding sites with an affinity comparable to that of Ang II receptors in anterior pituitary and adrenal gland [10]. The ablhty of anglotensin I1 analogs to compete with ~2~I-SarlAng II binding correlates well with the affinities of these compounds to Ang II receptors in other tissues [13], which suggests that these binding sites might have physiological significance. The spleen consists of two different structures, the white and the red pulp. The immunological activity of the spleen is mainly located in the white pulp, whereas the red pulp is an important storage of intact red blood cells, and has metabolic activity [1, 2, 8, 15]. Smooth muscle cells in the capsule and trabeculae of the stroma of the red pulp, probably together with reticular cells, which have been shown to have contractile properties [1,8], receive noradrenergic innervation [5], and regulate the volume of the spleen [15]. The stimulation of the sympathetic nerve produces contraction of these elements, which leads to depletion of intact red blood cells from the spleen into the circulation. In addition to a response to norepinephrlne, smooth muscle cells in the splenic stroma are also responsive to Ang II, which causes a marked vasoconstriction and a reduction in the volume of the spleen [4,15]. This action o f A n g II appears to be at least partially a direct one, not caused indirectly by catecholamine release, because Ang II effects cannot be abolished by either denerration or pharmacological blockade of catecholamine recep-

FACING PAGE FIG. I. Autoradlographlc localization of anglotensm II binding sites m rat spleen and thymus (A) typical sections of spleen and thymus stained with Hematoxyhn and Eosm, (B) adjacent sections incubated with 1 8 nM (103400 cpm/50 p.1) ~2zI-Sar~-Ang11, opposed against [3H]-Ultrofilm for 7 days, (C) adjacent sections to (B) incubated with 1 8 nM ~25I-Sarl-AngII and 5/LM unlabeled anglotenstn I1 to illustrate non-specific binding (a) White pulp of the spleen, (b) red pulp of the spleen, (c) thymlc cortex, (d) thymlc medulla

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l~sI-Sarl-Ang II (nM) FIG. 2 Saturation curves and Scatchard analysis (inserts) of specific ~2~I-Sarlanglotensm II binding to the red pulp of rat spleen (upper panel) and isolated spleen cells (lower panel). Tissue sections or sections of spleen cell-nch pellet from single ammals were Incubated with ~s[-Saff-Ang [I eoneentraUons ranging from 0.1 nM to 4 4 nM (see the Method secnon). Each curve represents one typical experiment which was repeated with 4 0solated cells) or 8 (tissue sections) d~fferent rats. The umt of the abscissa of the Scatchard curves (B/F) is (fmol/mg protem)/nM

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FIG 3 The ability of anglotensln analogs and two peptldes not related to anglotensln to compete with 12~I-Sarl-angtotensm II binding in the red pulp of the rat spleen sections. Each column represents mean data (_+SEM) obtained from 6 rats analyzed individually. *p
tors [3,4]. On the other hand, Ang II may interact with adrenergic innervat]on, since the blockade of adrenergic receptors increases the effects of Ang II on the spleen [4]. Our results suggest that the majority of the splenic Ang II binding sites are located in the stroma, because the concentration of the binding sites on the isolated spleen cells was almost one order of magnitude less than that in the rat spleen

741 sections, and, in addition, red blood cells did not bind Ang II. The binding sites in the splenic stroma might mediate the actions of Ang II on the blood flow and volume regulation of the spleen [8], which is consistent with a physiological role for circulating Ang II as a major regulator of blood volume and pressure. The presence of Ang II binding s~tes has been reported on human peripheral blood lymphocytes [17] and murine granuloma macrophages [20,22], and Ang II has an inhibitory action on human leukocytes by a stimulation of the suppressor T-cells [18,19]. We have detected specific binding sites for Ang II in isolated rat spleen cells. These binding sites may be located on lymphocytes or macrophages and they might be comparable to those described in human lymphocytes [17]. The physiological function, if any, of these binding sites is yet unclear, but they might mediate the effects of Ang II as reported for human lymphocytes [18,19]. We did not find specific binding sites for Ang II on either thymlc sections or isolated thymocytes. Since T-lymphocyte maturation takes place in thymus, the presence of binding s~tes on isolated spleen cells but not on thymocytes indicates that Ang II binding sites on isolated spleen cells might be predominantly located on the other subsets, such as B lymphocytes or macrophages. It is also possible that Ang II binding sites are expressed only in the matured T-cells released into the circulation and not In the maturing lymphocytes m the thymus, or that the effects of Ang II on the function of the lymphocytes described in humans [18,19l may be absent in rodents. In conclusion, our demonstration of specific Ang II binding sites In the red pulp of rat and mouse spleen and on isolated rat spleen cells supports the role suggested for angmtensin in the regulation of splenic volume and blood flow and in the modulation of the lymphocyte function. ACKNOWLEDGEMENTS The authors wish to thank Drs S. Gutkind, D. McKenna and A J Nazarah for their suggestions to this manuscript E C. has been partially supported by E Aaltonen Foundation

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8 Hartwig, H. and H. G. Hartwlg Structural characteristics of the mammalian spleen indicating storage and release of red blood cells Aspects of evolutionary and environmental demands Expenentta 41: 159-163, 1985 9. Israel, A , F M. A. Correa, M. Nlwa and J. M. Saavedra Quantitative determination of anglotensln II binding sites in the rat brain and pituitary gland by autoradlography Brain Re~ 322: 341-345, 1984. 10. Israel, A., L Plunkett and J M. Saavedra. Quantitative autroadiographlc characterization of receptors for angiotensin II and other neuropeptides in individual brain nuclei and peripheral tissues from single rats. Cell Mol Neurobtol 5: 211-222, 1985. 11. Kunhara, M., K. Shlgematsu and J. M Saavedra Localization of atrial natnuretlc peptlde ANP-(99-126) binding sites in the rat thymus and spleen with quantitative autoradlography. Regul Pept 15: 341-346, 1986. 12. Mendelsohn, F A O. Localization and properties of anglotensln receptors. J Hypertens 3: 307-316, 1985

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13. Mendelsohn, F. A. O., R. Qmnon, J. M Saavedra, G Aqudera and K J Cart Autoradmgraphlc locahzatmn of angmtensm II receptors in rat brain. Pro~ Natl At ad S ~ USA 81: 1575-1579, 1984 14 Munson, P. J. LIgand. A computerized analysis ofhgand binding data In Method.~ m Enzymology. Vol 92, lmmuno~ hernt~ al Te~ hntque~, Part E, edited by J J Langone and H. van Vunakls New York. Academic Press, 1983, pp 543-576 15 Reflly, F. D Innervatlon and vascular pharmacodynamlcs of the mammahan spleen Expertentta 41: 187-192, 1985 16. Shlgematsu, K , J Saavedra and M Kunhara Specific substance P binding sites m rat thymus and spleen m v~tro autoradmgraphlc study Regul Pep 16: 147-156, 1986 17 Shlmada, K. and Y Yazakl. Binding sites for angmtensm I1 m human mononuclear leucocytes J Btochem 84: 1013-1015, 1978 18 Simon, M R , D E E n g e l a n d J V Wemstock Anglotensm I1 suppression of human mononuclear cell reactivity ~s a~socmted with enhanced OKT8 + lymphocyte tymldlne mcorporatmn / lmmunopharmat ol 8: 28%297, 1986.

CASTRI~N, K U R I H A R A A N D S A A V E D R A

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