Neurohypophyseal Hormone Receptors in the Rat ...

00l3-7227/90/1265-2703$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society

Vol. 126, No. 5 Printed in U.S.A.

Neurohypophyseal Hormone Receptors in the Rat Thymus, Spleen, and Lymphocytes JACK ELANDS, ANNELIES RESINK, AND E. RONALD DE KLOET Rudolf Magnus Institute, Medical Faculty, University of Utrecht, 3521 GD Utrecht, The Netherlands

ABSTRACT. Receptor sites for the neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin (OT) have been identified and characterized in some tissues involved in immune function in the rat. Novel radioiodinated ligands for the detection of neurohypophyseal hormone receptors, with a high specific radioactivity and affinity, enabled the selective detection of OT receptors in the thymus and vasopressin (VP) receptors in the spleen. OT receptors were detected in thymic membrane preparations and on thymocytes, which had a ligand selectivity similar to that of uterine OT receptors. AVP receptors of the Vi pressor

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HE NEUROHYPOPHYSEAL hormones vasopressin (VP) and oxytocin (OT) exert multiple actions on peripherally and centrally regulated functions (1-3). In cultured rat thymic lymphocytes mitotic activity (4) and DNA synthesis (5) have been reported to be stimulated by OT and lysine VP (LVP). Some of these findings with OT and a synthetic VP analog [desamino,8-D-arginine]vasopressin (dDAVP) have been confirmed in rat thymic organotypic cultures (6). OT has also been shown to stimulate glucose oxidation in rat thymocytes (7). Recently, arginine VP (AVP) immunoreactivity was detected in rat and mice thymic extracts (8), and OT, AVP, and neurophysin immunoreactivity in human thymic epithelium (9-11). OT appeared to be far more abundant than AVP. Furthermore, the presence of AVP and OT mRNA has been reported in human thymic epithelium (11). The thymic nurse cell, a cell type thought to be involved in T-cell maturation (12,13), has been demonstrated to be a source of this OT, AVP, and neurophysin in mice (14). Various researchers report the presence of receptors for neurohypophyseal hormones or their synthetic analogs on whole cells or membrane preparations of lymphocytes, spleen, and splenic lymphocytes. Human peripheral blood lymphocytes possess binding sites for [125I]AVP (15) and [125I]dDAVP (16). Human mononuclear phagocytes are reported to have Received October 20,1989. Address requests for reprints to: Dr. J. Elands, Rudolf Magnus Institute, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands.

type were present in a splenic membrane preparation. Specific AVP-binding sites, probably of the V! type, were also present on splenic lymphocytes. Binding sites for AVP and OT could not be detected on mononuclear cells in peripheral blood of the rat. This study demonstrates that the use of the newly developed radioiodinated AVP and OT receptor ligands, with high specific radioactivity and affinity, enables the selective characterization of receptor sites for the neurohypophyseal hormones, even in the thymus, where previously no binding sites could be detected. (Endocrinology 126: 2703-2710,1990)

specific [125I] AVP-binding sites resembling a Vx-like type (17), which mediate prostaglandin E production (18). Moreover, AVP has been shown to act synergistically with CRF in human peripheral blood mononuclear cells (PBMC) (19, 20). AVP receptors have also been detected on preparations of the rat spleen (21), and receptors were shown to be of the Vx type. Specific AVP-binding sites have been detected with [3H]AVP on cultured mouse splenic lymphocytes (22). These AVP-binding sites are probably involved in the production of r-interferon (2325). Accordingly, evidence has accumulated to suggest that neurohypophyseal hormones are involved in immune processes and that the tissues of the immune system contain receptors for these neurohypophyseal peptides. The present study was designed to establish the identity of the receptor types for neurohypophyseal hormones in the thymus, spleen, splenic lymphocytes, and PBMC of the Wistar rat. Such a study is feasible, since we recently have developed two radioiodinated antagonists, an OT antagonist (OTA) (26) and an AVP antagonist (AVA) (27). Both antagonists have a high specific radioactivity and a high affinity for their receptors in the rat (Kd = 0.05 nM for [1251]OTA to uterine OT receptors and Kd = 0.28 nM for [125I]AVA to liver AVP W1 receptors). Moreover, [125I]OTA is very selective for OT receptors in the rat; its affinity is about 330-440 times lower for AVP receptors. Using these ligands we have shown that the immune system has a remarkably differentiated pattern of AVP and OT receptors. 2703

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Materials and Methods Preparation of r5I]d(CHJb[Tyr(Me)*Thr\Tyr-NH2*]OVTand P2bI]d(CH?)b[Tyr(Me)2Tyr-NH2*A VP The OT-[l-(/3-mercapto-/3,/3-cyclopentamethylene propionic acid, 2-O-methyltyrosine, 4-threonine, 8-ornithine, 9-tyrosylamide]vasotocin (OTA) and AVP antagonist [l-(/3-mercapto-/3,/3-cyclopentamethylene propionic acid, 2-O-methyltyrosine, 9-tyrosylamide]VP arginine vasopressin (AVA) were synthesized as described by Elands et al. (26, 27) and were iodinated as described previously (27) with a few minor modifications. Briefly, 40 nmol OTA or 4 nmol AVA (antagonists were dissolved in methanol) were incubated in 75 /A lmM KH2PO4, pH 7.5, 10 Mg chloramine-T (Merck, Darmstadt, West Germany) and 1 mCi [125I]Na (NEZ-033A, New England Nuclear, Boston, MA) in an Eppendorf minivial. After 1.5 min the mixture was removed from the vial and injected immediately onto a Waters C18 /uBondapak column (Waters, Milford, MA). The reaction products were separated with a 40-min linear gradient of 40-80% solvent B (0.067% trifluoroacetic acid in 60% acetonitrile) in solvent A (0.067% trifluoroacetic acid). The flow rate was 1 ml/min. The organic solvents from the ligand containing eluate were removed under N2 at room temperature. A second identical HPLC step on a separate reverse phase Waters C18 ^Bondapak column was carried out to purify the 125I-labeled ligands after the addition of 1 mg BSA (Sigma, fraction V). The organic solvents were again removed under N2. The specific radioactivity of the 125I-labeled ligands was 2000 Ci/mmol. Male Wistar rats (200-300 g) were obtained from the Centraal Proefdieren Bedrijf (TNO, Zeist, The Netherlands) or our own breeding colony. Some rats received three consecutive injections of dexamethasone (10 ng in 100 /A physiological saline/100 g BW on days 1, 3, and 5) or physiological saline (100 MI/100 g BW).

Thymic and splenic membranes The thymus and spleen were dissected and homogenized in ice-cold 10 mM Tris-HCl (pH 7.4) buffer with a Polytron (Kinematica, Kriens, Switzerland) at setting 6 for three periods of 20 sec. The homogenate was spun down at 1,000 X g for 10 min. The resulting supernatant was centrifuged at 14,000 X g for 30 min. The pellet was washed in 10 mM Tris-HCl (pH 7.4), resuspended in 15 ml sucrose (0.32 M) in 10 mM Tris-HCl (pH 7.4), and layered onto 15 ml sucrose (1.12 M) in 10 mM TrisHCl (pH 7.4). After centrifugation for 2 h at 100,000 x g in a SW 28 swing-out bucket rotor (Beckman ultracentrifuge, Palo Alto, CA), the membranes were collected at the 0.32-1.12 M interface. The membranes were dispersed in 50 mM Tris-HCl (pH 7.4), 5 mM (AVA assays) or 10 mM MgCl2 (OTA assays), washed, and resuspended in the same medium. PBMC Male rats were decapitated, and the trunk blood was collected in heparinized tubes. The blood was diluted 7-fold in Minimum Essential Medium (MEM; Gibco, Grand Island, NY), filtered through surgical gauze, and layered onto Percoll (70% in MEM)

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(28). The lymphocytes were collected at the interface after centrifugation for 30 min at 1000 X g, washed twice in PBS, and resuspended in MEM containing 1 mg/ml BSA (MEMBSA) to give 107 cell/ml. When indicated, membranes were prepared from this fraction with a Potter-Elvehjem homogenizer (four strokes). Membranes prepared from 5 X 108 cells were used per assay. Thymocytes Thymi were cut into small pieces and gently homogenized in PBS at room temperature in a Potter-Elvehjem homogenizer (four strokes) with a large clearance. The homogenate was filtered through two layers of surgical gauze, and the filtrate was centrifuged at 500 x g. The resulting pellet was resuspended in PBS and again filtered through surgical gauze. The thymocytes in the filtrate were washed twice with PBS and resuspended in MEM-BSA to give 107 cells/ml. Splenocytes Spleens were gently rubbed through a stainless steel gauze in ice-cold MEM. The crude cell suspension obtained was washed twice with PBS and once with MEM. After filtration through a four-layer gauze, the cell suspension was layered onto 70% Percoll and prepared as described above for the peripheral blood lymphocyte preparation. The remaining stromal tissue was washed twice with PBS, and membranes were prepared as described above for thymic and splenic membranes. Binding assays Membranes were incubated in 50 mM Tris-HCl (pH 7.4), containing 5 mM (incubations with [125I]AVA) or 10 mM (incubations with [125I]OTA) MgCl2, 1 mg/ml BSA, and various concentrations of labeled and unlabeled peptides in a total volume of 80 n\. The incubations (30 C) lasted 40 min, unless otherwise indicated. Nonspecific binding was determined in the presence of a 1000-fold excess of AVP (for [125I]AVA) or OT (for [125I]OTA). Nonspecific binding determined in this way gave similar values as those obtained with a 250-fold excess of nonradiolabeled monoiodinated AVA or OTA (see also Refs. 26 and 27). The incubation was terminated by the addition of 4 ml ice-cold filtration buffer (10 mM Tris-HCl, pH 7.4, and 1 mM MgCl2). Bound and free radioactivity were separated by filtration through Whatman GF/C filters (Clifton, NJ), presoaked in 10 mg/ml BSA (for at least 2 h); 16 ml filtration buffer were used, and filtration lasted approximately 15 sec. The radioactivity retained on the filters was counted. In dissociation experiments after incubation (40 min) with [125I]OTA (0.04-0.05 nM) or [125I]AVA (0.20-0.34 nM), as described for the other binding experiments, a 100-fold dilution was made by the addition of 8 ml incubation buffer. The dissociation was stopped by filtration of the incubation buffer over Whatman GF/C filters, as described above. Suspensions of peripheral blood cells, thymocytes, and splenocytes (107 cells/ml) were incubated for 30 min in 1 ml MEM containing 1 mg/ml BSA, 2 mM MgCl2, and different concentrations of labeled and unlabeled peptides. Nonspecific binding was determined with a 1000-fold excess of unlabeled AVP for [125I]AVA and a 1000-


NEUROHYPOPHYSEAL HORMONE RECEPTORS fold excess of OT for [125I]OTA (26, 27). After 30 min, 9 ml icecold PBS containing 5 mM MgCl2 (PBS-Mg) were added, and the cell suspension was centrifuged at 500 X g for 10 min. After resuspension in 9 ml ice-cold PBS-Mg, the suspension was again centrifuged at 500 x g for 10 min. The cell pellet was resuspended in 2 ml PBS-Mg and transferred to a counting vial, and the radioactivity was determined. We checked whether the 125I-labeled ligands were degraded during the incubations by taking an aliquot before and after incubation and separating it on HPLC, as described for the iodination procedure. No degradation could be detected by HPLC in any of the membrane or cell preparations. Binding constants for the unlabeled peptides were determined in competition binding experiments in the presence of either 0.330.49 nM [125I]AVA or 0.04-0.11 mM [125I]OTA and different concentrations of the unlabeled peptides. Data analysis Estimates of the dissociation constants (Kd) and the maximal number of binding sites (Bmnx) were derived from linear regression of the Scatchard analysis of results obtained from concentration dependence experiments at equilibrium. The slope index and K values of competition experiments for [125I]ligand binding were calculated by fitting the experimental data to the expected linear relationship log[(Bo/B) - l][([T]/Kd*I) + 1] = log[I] - log(Ki), in which Bo represents specific binding in the absence and B the specific binding in the presence of unlabeled competitor, [*I] is the concentration of[125I]ligand, and KdT is the dissociation constant at equilibrium of [125I]ligand. The results of groups of homologous and heterologous competition binding experiments were analyzed by a nonlinear regression analysis with the PC version of the computer program Ligand (29) (version 3.0). The data were fitted to oneand two-site models. Association and dissociation experiments were analyzed with the program Kinetic (30,31) and were fitted to mono- and biexponential models. Selection of the best fit was based on an F test (29). All values are expressed as the mean ± SD. Peptides The following OT and AVP analogs were kindly donated by Dr. M. Manning (Toledo, OH): d(CH2)5[Tyr(Me)2Tyr-NH29] AVP, d(CH2)6[Tyr(Me)2Thr4,Tyr-NH29]OVT (for iodination), d(CH2)5[Tyr(Me)2]AVP, [Thr4,Gly7]OT, and des,Gly-(NH2)9d(CH2)5 [Tyr(Me)2Thr]OVT. AVP, OT, and dDAVP were obtained from Organon (Oss, The Netherlands). The biological activities of these peptides are indicated in Table 1.

Results pMjjOTA and ^IJAVA

binding to thymus and spleen

[125I]AVA and [125I]OTA specifically bound to thymic and [125I]AVA to splenic membrane preparations. Binding of [125I]OTA to thymic and [125I]AVA to splenic membrane preparations was time dependent, reversible (Fig. 1), saturable, and of high affinity. For the thymic

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membranes, Scatchard analysis revealed a dissociation constant for [125I]OTA of 0.06 ± 0.02 nM (n = 5; Fig. 2A), the association rate constants (KJ for [125I]OTA binding were 6.1 and 8.3 X 107 M"1 min (determined in two separate experiments), while the dissociation rate constants (K_x) were 4.6 and 5.7 X 10~3 min"1 (determined in two separate experiments). The calculated dissociation constant (0.07 nM) thus appeared to be in good agreement with the estimated Kd value. The binding of [125I]AVA to splenic membranes occurred with a somewhat lower affinity; Scatchard analysis (Fig. 2B) gave a dissociation constant of 0.13 ± 0.07 nM (n = 4). In the spleen, the K : values were 0.8 and 1.4 X 108 M"1 min"1 (determined in two separate experiments), while the K_i values were 7.2 and 11.8 X 10~3 min"1 (determined in two separate experiments), resulting in a calculated dissociation constant of 0.09 nM, which was, again, in good agreement with the value determined from the Scatchard analyses. Binding reached equilibrium within 40 min, with a half-time of 8 min for the thymic membranes and 6 min for the splenic membranes. Both association and dissociation were adequately described by a monoexponential model (P values for a biexponential model: thymus, P = 1.000; spleen, P = 0.386). The maximal binding capacity was 42 ± 5 fmol [125I]OTA/mg protein for the thymic membranes (n = 5) and 103 ± 26 fmol [125I]AVA/ mg protein for the splenic membranes (n = 4). PBMC Neither [125I]OTA nor [125I]AVA bound specifically to intact PBMC or to PBMC membrane preparations. [125I] AVP, prepared as described for [125I]OTA, did not bind to any of the rat PBMC preparations. Ligand specificity of thymic and splenic binding sites To determine the nature of the neurohypophyseal hormone receptors that bound [125I]OTA and [125I]AVA, competition binding experiments were carried out with a series of AVP and OT analogs. OT appeared to have a high affinity (K; = 1.6 ± 0.9 nM; n = 3) for the thymic [125I]OTA-binding sites (Fig. 3), as was the case for the potent oxytocic agonist [Thr4,Gly7]OT and antagonist des,Gly-(NH2)9-d(CH2)5-[Tyr(Me)2Thr4]OVT [K; = 1.0 ± 0.4 (n = 2) and 0.5 ± 0.2 nM (n = 3), respectively]. AVP also displayed a high affinity (Kj = 2.18 ± 0.02 nM; n = 4), while one of the most selective Vx receptor ligands presently known, the antagonist d(CH2)5.[Tyr(Me)2] AVP, had a considerably lower affinity for the thymic binding sites (K; = 29 ± 13 nM; n = 3). A reversed situation was observed in the spleen (Fig. 3). Both AVP and its potent antagonistic analog d(CH2)5[Tyr(Me)2] AVP had a high affinity for the [125I]AVA-binding sites [K; = 1.5 ± 0.6 (n = 3) and 0.3 ± 0.1 nM (n = 2),


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TABLE 1. Biological activities of AVP, OT, and labeled and nonlabeled analogs Peptide

Antivasopressor

Antiantidiuretic

dDAVP" [Thr'.Gly^OT" d(CH2)6[Tyr(Me)2]AVP< d(CH2)5[Tyr(Me)2|1-Tyr-NH29]AVP« d(CH2)5[Tyr(Me)2Thr4|rTyr-NH29]OVT'> Des,Gly(NH2)9d(CH2)5[Tyr(Me)2Thr4]OVT;

369 ± 6 4.3 ± 0.12 0.39 ± 0.02