Steroid-sensitivity of agonist binding to pituitary cell line histamine H3 receptors

eJi:l European Journal of Pharmacology Molecular PharmacologySection 267 (1994) 343-348

ELSEVIER

molecular pharmacology

Steroid-sensitivity of agonist binding to pituitary cell line histamine H 3 receptors Robert E. West, Jr. *, Joyce Myers, Adam Zweig, Marvin I. Siegel, Robert W. Egan, Mike A. Clark Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ, USA

(Received: 28 January 1994; accepted 8 February 1994)

Abstract

Histamine H 3 receptors have been identified in rat and guinea-pig pituitary glands and in the mouse pituitary tumor cell line, AtT-20. Histamine H 3 receptor agonists are reported to stimulate adrenocorticotropic hormone (ACTH) release from AtT-20 cells, an effect blocked by histamine H 3 but n o t n I o r H 2 receptor antagonists. To determine whether negative feedback regulation of the histamine H 3 receptor-mediated effect might occur, we tested the effects of steroid treatment upon binding of the agonist [3H]N~-methylhistamine to AtT-20 cell membranes. Consistent with feedback regulation, steroid treatment of the cells reduced [3H]N~-methylhistamine binding. The effect was dose-dependent and was greatest for glucocorticoids among the steroids tested. As the duration of steroid treatment increased, the amount of [3H]N"-methylhistamine binding decreased, to 15% of control at 36 h. However, the effect was not specific for histamine H 3 receptors. Somatostatin inhibits ACTH release from these cells and its binding was similarly reduced by steroid treatment. Because steroids have been reported to modulate levels of guanine nucleotide-binding proteins, the lack of receptor specificity could reflect an indirect effect of steroids upon agonist binding and, in fact, we show that [3H]N~-methylhistamine binding to these cells, like somatostatin, is pertussis toxin-sensitive. However, steroid treatment does not alter the apparent levels of pertussis toxin substrate in these cells. Whether steroid treatment affects histamine H 3 receptors of these cells directly or through some more subtle effect upon the guanine nucleotide-binding proteins to which they couple, the result is a negative feedback loop that attenuates [3H]N~-methylhistamine binding to these cells. Key words: Histamine H 3 receptor; AtT-20 cell; [3H]N~-Methylhistamine binding; ACTH (Adrenocorticotropic hormone)

release; Steroid effect

I. Introduction

A high-affinity histamine receptor that provides feedback control of histamine synthesis (Arrang et al., 1987a,b) and release (Arrang et al., 1983, 1987a; van der W e f t et al., 1987; Bado et al., 1992) from nerve terminals has been discovered and designated H 3. The histamine H 3 receptor has subsequently been shown to regulate the release of other neurotransmitters as well (lshikawa and Sperelakis, 1987; Trzeciakowski, 1988; Schlieker et al., 1988, 1992; T a m u r a et al., 1988; Ichinose and Barnes, 1989a,b; Fink et al., 1990; Menkveld and Timmerman, 1990; Luo et al., 1991; Hey et al., 1992; Koss and Hey, 1992; Molderings et al., 1992;

* Corresponding author. Tel.: 908-298-7241; Fax: 908-298-7175. 0922-4106/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0922-4106(94)00027-V

Taylor and Kilpatrick, 1992). Adrenocorticotropic hormone (ACTH) release in response to histamine administration is a long-recognized p h e n o m e n o n (reviewed by Hough, 1988), although the underlying mechanism is unresolved. Recently, H 3 receptors have been identified on the mouse pituitary cell line, AtT-20, and histamine H 3 receptor-specific stimulation of A C T H release from these cells has been demonstrated (Clark et al., 1992). The function of A C T H is to stimulate glucocorticoid release from the adrenal cortex and glucocorticoids have been shown to affect histamine turnover in rat brain and synaptosomes (MazurkiewiczKwilecki, 1983; Walejtys-Roda et al., 1989). We, therefore, tested glucoeorticoid effects upon the binding of [3H]N"-methylhistamine, a specific agonist ligand for histamine H 3 receptors (Korte et al., 1990; West et al., 1990) to determine whether their negative feedback on

344

R.E. West et al. / European Journal of Pharmacology - Molecular Pharmacology Section 267 (1994) 343-348

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histamine might occur at the receptor level in this system.

2. Materials and Methods

2.1. Materials [3H]N"-Methylhistamine (80 Ci/mmol) and [1251Tyrl]somatostatin (2000 Ci/mmol) were from Dupont NEN. Steroids were synthesized by the Schering-Plough Medicinal Chemistry Department. AtT-20 cells were from the American Type Culture Collection, Bethesda, MD. 2.2. Cell culture and treatment AtT-20 cells were grown in 25 mM Hepes-buffered Dulbecco's modified minimal essential medium/20% fetal calf serum in a 95% air/5% CO 2 atmosphere. Experiments were performed with cells at approximately 80% confluence. Steroids were applied in dimethylsulfoxide (final concentration of 0.1%) except for betamethasone which was aqueous. 2.3. Radioligand binding assays For membrane preparation, ceils were disrupted with a Polytron (Brinkmann) in ice-cold 50 mM TrisHC1, pH 7.5, and the homogenate was centrifuged 10 rain at 1000 × g. The supernatant was centrifuged 10 min at 50,000 × g and the membrane pellet was rinsed once by resuspension in buffer. For binding assays, triplicate determinations of total and nonspecific binding were made for four to eight concentrations (0.18-9 nM) of the appropriate radioligand incubated with 0.3 mg of membrane protein, determined by a bicinchoninic acid assay (Pierce) (Smith et al., 1985), in 0.5

ml of 50 mM Tris-HC1, pH 7.5, at 30°C for 30 min. Nonspecific binding was determined in the presence of 10 -6 M N"-methylhistamine (Calbiochem) for histamine H 3 assays and 10 -6 M [Tyrll]somatostatin (Peninsula Laboratories) for somatostatin assays. Assays were terminated by filtration over glass-fiber filters (Whatman, GF/C), presoaked for H 3 binding in 0.3% polyethylenimine (Bruns et al., 1983), followed by two 4-ml Tris-HC1 washes and liquid scintillation counting of filters at 40% efficiency for histamine H 3 receptor binding or gamma-counting for somatostatin binding. For saturation binding assays, binding constants were determined by linear regression of Scatchard plots of the data. 2. 4. ADP-ribosylation reactions Two/xg of pertussis toxin (List Biological Laboratories) was activated for 20 rain at 30°C in 20 /zl of 50 mM glycine, pH 8/25 mM dithiothreitol containing 0.5 mg of ovalbumin/ml. To this, reagents were added for a final volume of 100/~1 of 50 mM sodium phosphate, pH 7.5/10 mM thymidine/1 mM GTP/1 mM ATP/20 /zM [32p]NAD (3/zCi) containing 20/xg of membrane protein and the ADP-ribosylation reaction was incubated 1 h at 30°C. The reaction mix was precipitated with 10% trichloroacetic acid, resuspended, and electrophoresed on a 12% polyacrylamide gel (Laemmli, 1980). The dried gel exposed Kodak X-omat AR film overnight at room temperature. 2.5. Data analysis Values are expressed as means + standard errors of the mean except as noted. A paired, two-tailed t-test 13) r-

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was used to determine the degree to which treatments resulted in binding significantly different from that of controls. A paired test was used because each and every experiment included a control and treatment, and while the differences between these were generally dramatic, there were substantial differences in the levels of control binding from week to week.

3. Results

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Fig. 4. Saturation binding isotherm of [=25I-Tyrl]somatostatin binding to membranes from untreated and betamethasone (10-6M) pretreated AtT-20 cells. One of two such experiments is shown. Symbols: e, control, o, treated. Inset: Scatchard Plot.

histamine binding to 64% of control (22 + 3 f m o l / m g of protein for control, 14 _+ 1 f m o l / m g for membranes from treated cells; n = 6, P < 0.01). The affinity of the remaining binding was also reduced (control: K D = 1.0 _+ 0.1 nM, treated: K D = 1.5 _+ 0.2 nM; P < 0.01) (Fig. 1). Over a 36-h time course of betamethasone (10 -6 M) treatment, [3 H]N~_methylhistamine binding steadily decreased, to 15% of control at 36 h (Fig. 2). At 24 h, the effect of betamethasone was dose-dependent from 10 -1° to 10 -7 M with the ICs0 equal to 2 nM (Fig. 3). A number of steroids were tested for their ability to inhibit [3H]N"-methylhistamine binding. At the concentration tested (10 - 6 M ) , the effect was relatively

3.1. Steroid effects on [ 3 H ] N ~ - m e t h y l h i s t a m i n e binding

Betamethasone treatment (10 - 6 M) of AtT-20 cells for 24 h reduced maximum levels of [3H]N~-methyl-

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Percent inhibition ±S.E.M. 58 + 9 57 + 17 50 +_15 28 + 12 11 + 8 15 _+9 0 0

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R.E. West et aL / European Journal of Pharmacology MolecularPharmacology Section 267 (1994) 343-348

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specific for glucocorticoids (Table 1) although progesterone also significantly inhibited binding, a finding simililar to that with glucocorticoid-sensitive /3-adrenergic receptors (Nakada et al., 1987).

[3H]N~-methylhistamine binding had no effect on the levels of pertussis toxin substrate.

3.2. Betamethasone effects on [12SI-Tyr1]somatostatin binding

4. Discussion

To determine whether the glucocorticoid inhibition of binding was restricted to stimuli of ACTH release, the effect of betamethasone treatment upon binding of an inhibitor of ACTH release was assayed (Fig. 4). Similarly to [3H]N~-methylhistamine, the maximum binding of [~25I-Tyr1]somatostatin was reduced by betamethasone treatment (control: Bmax = 684 + 50 f m o l / m g of protein, treated: Bma x = 347 + 0 fmol/mg). The K o for binding to control membranes was 1.3 + 0.5 nM and for membranes from treated cells 0.6 + 0.1 nM (n = 2, values are means + range of duplicate determinations from the mean).

3.3. Pertussis toxin-sensitivity of radioligand binding Somatostatin receptors are known to be coupled to a pertussis toxin-sensitive G protein in these cells (Reisine and Guild, 1985) and glucocorticoids have been shown to regulate levels of G proteins in brain (Saito et al., 1989), so we tested [3H]N~-methylhistamine binding for its sensitivity t o pertussis toxin treatment of cells (Fig. 5). Pertussis toxin pretreatment of cells reduced the density of [3H]N"-methylhistamine binding to 60% of control (P < 0.01) and what binding remained showed reduced affinity (P < 0.05) (control: K D = 1.3 _+ 0.2 nM, Bmax = 36 _+ 7 f m o l / m g of protein; treated: K D = 3.3 _+ 0.5 nM, Bmax = 22 _+ 7 f m o l / m g of protein; n = 3. Paired values for individual experiments were as follows for controls and toxin treatments: KD: 1.1 and 2.6, 1.5 and 3.1, 1.4 and 4.1; Bmax: 25 and 13, 39 and 20, 43 and 33.) We next determined whether levels of pertussis toxin substrate in these cells were altered by betamethasone treatment. Fig. 6 is an autoradiogram of the 41 kDa pertussis toxin substrate in membranes from a betamethasone treatment time course and dose-response that were subsequently [32p]ADP-ribosylated. It is clear from these experiments that doses of betamethasone that reduced

Inasmuch as N'~-methylhistamine is reported to stimulate ACTH release from pituitary tumor-derived AtT-20 cells, ACTH stimulates glucocorticoid release from adrenal cortex, and glucocorticoids reduce [3H]N~-methylhistamine binding to AtT-20 cells, a negative feedback loop occurs. Its physiologic significance is unclear, however, because the reduction in binding is not limited to stimuli of ACTH release. Binding of somatostatin, which inhibits ACTH release (Richardson and Schonbrunn, 1981), is also reduced. The extreme reduction of histamine H 3 agonist binding to 15% of control by 36 h of betamethasone treatment is consistent with a functionally significant effect of steroids to suppress histamine H 3 receptor-mediated stimulation of ACTH release, but it remains to be explained why an inhibitor of ACTH release should be similarly affected by steroid treatment. Perhaps, because glucocorticoids directly and powerfully inhibit ACTH synthesis and release, the role of other modulators is diminished in their presence and thus the reduction in binding of stimulatory and inhibitory modulators is a reflection rather than the cause of their diminished role. Because binding to these receptors is currently characterized with radiolabeled agonists, changes in radioligand binding may reflect receptor changes or, because these are both G protein-coupled receptors, alterations in the G proteins to which the receptors couple. Steroids have been reported to modulate levels of G proteins (Saito et al., 1989) but, based on the experiment of Fig. 6, they have no effect upon levels of pertussis toxin substrate in AtT-20 cells. In complex with their receptors, glucocorticoids modulate gene transcription by binding to DNA sequences called glucocorticoid responsive elements to stimulate or inhibit transcription of particular genes (Beato, 1989). Among G protein-coupled receptors there are examples of glucocorticoids increasing (Lai et al., 1982; Nakada et al., 1987; F~ve et al., 1990; Biron et al., 1989) and

R.E. West et aL / European Journal of Pharmacology - Molecular Pharmacology Section 267 (1994) 343-348

decreasing (Sakue and Hoffmann, 1991; F~ve et al., 1992) receptor densities. So it is probable that the glucocorticoid-induced reduction of agonist binding to histamine H 3 receptors in AtT-20 cells is due to reduced transcription of the gene for this receptor. An issue of interest that these data raise is the common pertussis toxin-sensitivity of the histamine H3 and somatostatin receptors, because the former is reported to stimulate (Clark et al., 1992) and the latter to inhibit (Richardson and Schonbrunn, 1981) ACTH release from AtT-20 cells. The pertussis toxin-sensitive G proteins comprise G i a l , G i a 2 , Gia3, Gout, and Go~2 (Simon et al., 1991). The somatostatin receptor of these cells appears to couple to Gi,~l, Gi, 3 and Goa (Law et al., 1991). Presumably the histamine H 3 receptor couples to other of the pertussis toxin-sensitive G proteins in order to produce an effect opposite to that of somatostatin. Which of these is involved in histamine H 3 receptor-mediated signaling in AtT-20 cells has yet to be determined.

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