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Endocrinology, doi:10.1210/en.2007-1397
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Endocrinology Vol. 149, No. 5 2391-2402
Copyright © 2008 by The Endocrine Society

β1-Adrenoceptors in Rat Anterior Pituitary May Be Constitutively Active. Inverse Agonism of CGP 20712A on Basal 3',5'-Cyclic Adenosine 5'-Monophosphate Levels

Kristel Janssens, Magaly Boussemaere, Stefan Wagner, Klaus Kopka and Carl Denef

Laboratory of Cell Pharmacology (K.J., M.B., C.D.), University of Leuven, Medical School, Gasthuisberg, B-3000 Leuven, Belgium; Department of Nuclear Medicine (S.W., K.K.), University Hospital of the University of Münster, D-48129 Münster, Germany; and European Institute of Molecular Imaging (S.W., K.K.), D-48149 Münster, Germany

Address all correspondence and requests for reprints to: Professor Carl Denef, Laboratory of Cell Pharmacology, University of Leuven, Medical School, Campus Gasthuisberg (O & N), B-3000 Leuven, Belgium. E-mail: Carl.Denef{at}med.kuleuven.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Catecholamines directly stimulate GH, ACTH, and prolactin secretion from rat anterior pituitary through the β2-adrenoceptor (AR). We recently showed that gonadotrophs express the β1-AR and that glucocorticoids drastically increase its mRNA expression level. The present investigation explores whether β1-ARs are functionally coupled to adenylate cyclase. In anterior pituitary cell aggregates, the highly selective β1-AR antagonists CGP 20712A and ICI 89,406–8a attenuated isoproterenol-stimulated cAMP accumulation, but no agonist action of norepinephrine could be detected. Remarkably, CGP 20712A inhibited basal cAMP levels by its own for at least 50%, an action that tended to be more effective in dexamethasone-supplemented medium. The latter effect was abolished by the β-AR antagonist carvedilol, but not by other β-AR antagonists. Pretreatment with pertussis toxin abolished the action of CGP 20712A on basal cAMP. CGP 20712A also attenuated isoproterenol-induced cAMP accumulation in the gonadotroph cell lines {alpha}T3–1 and LβT2, but not in the somatotroph precursor cell line GHFT and the folliculo-stellate cell line TtT/GF. However, in LβT2 cells CGP 20712A did not inhibit basal cAMP levels by its own. The present data suggest that β1-AR in the anterior pituitary is positively coupled to adenylyl cyclase but is constitutively active in a pertussis toxin-sensitive manner. CGP 20712A may act as an inverse agonist with approximately 50% negative intrinsic activity, suggesting that the β1-AR significantly contributes to basal adenylate cyclase activity in the pituitary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STRESS ACTIVATES THE hypothalamic-pituitary-adrenal axis and the output of the catecholamines norepinephrine by the autonomic nervous system and epinephrine from the adrenal medulla. Corticosteroids and catecholamines interact at the level of the pituitary, and affect the secretion and gene expression of ACTH (1, 2, 3, 4, 5, 6, 7), {alpha}-MSH (1, 2, 3, 4, 5, 6, 7), LH (8, 9, 10), TSH, prolactin (PRL) (11, 12, 13, 14, 15), and GH (1, 16, 17, 18, 19, 20, 21, 22, 23, 24). Catecholamines exert their action through {alpha}- and β-adrenoceptors (ARs) that are distinguished on the basis of differences in affinity for certain agonists and antagonists, second messenger responses and homology in amino acid and gene sequences (25), and belong to the large superfamily of G protein-coupled receptors (GPCRs) (26). The primary transduction mechanism of β-ARs is activation of adenylate cyclase through Gs{alpha} (27), but all β-ARs may also couple to Gi/o{alpha} (28, 29, 30, 31, 32, 33), and coupling can switch from Gs{alpha} to Gi/o{alpha} (34, 35).

It has been demonstrated before that the predominant ARs in the rat pituitary are the {alpha}1- (2, 17, 36) and β2-AR (4, 11, 12, 19, 37, 38, 39), whereas in porcine pituitary the β1-AR appears to be the predominant β-AR (38). The β2-AR in the rat mediates stimulation of GH (16, 18, 20, 38), {alpha}-MSH (5), and PRL (11, 12) release, whereas stimulation of TSH secretion is mediated by the {alpha}1-AR (36). In bovine folliculo-stellate cells, both β1- and β2-ARs seem to be present (40), and catecholamines modulate IL-6 release (41). In a companion paper, we reported β1-AR mRNA expression in the rat anterior pituitary and that expression is highly dependent on glucocorticoids (42). β1-AR immunostaining of intact pituitary and quantitative RT-PCR and immunoblot analysis of various cell lines indicated that expression was confined to the gonadotrophs. However, glucocorticoid dependency was not located in the gonadotrophs, but in a nonidentified cell type. Although β-ARs can be expressed at the protein level, their trafficking and targeting to the cell plasma membrane and their functional coupling needs to be demonstrated to allow conclusions about their functional activity and biological role. The present investigation was intended to answer the question whether or not in the anterior pituitary and in pituitary derived cell lines the β1-AR is functionally coupled to the adenylate cyclase system, the canonical signal transduction system of all β-ARs, and whether or not glucocorticoids potentiate this coupling. Therefore, we measured alterations in cAMP accumulation levels to several selective and nonselective β-AR agonists and antagonists. We present evidence that anterior pituitary cells express functional β1-ARs but that the receptor is constitutively active through coupling to Gi/o{alpha}. We demonstrate that CGP 20712A acts as an inverse β1-AR agonist, of which the action is abrogated by carvedilol, but not by various other β1-AR antagonists. Furthermore, it was found that the activity of CGP 20712A tended to be more effective in the presence of glucocorticoids. Functional β1-ARs, but without constitutive activity, were found in the gonadotrophic cell lines {alpha}T3–1 and LβT2 as well, but not in the somatotrophic precursor cell line GHFT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormones and drugs
Xamoterol, salmeterol, CGP 20712A, and ICI 118,551 were purchased from Tocris Biosciences (Bristol, UK), and isoproterenol (ISO), norepinephrine, betaxolol, and propranolol from Sigma-Aldrich (Steinheim, Germany). Carvedilol was obtained from Roche Diagnostics (Indianapolis, IN) and domperidone from Janssen Pharmaceutica (Beerse Belgium). ICI 89,406–5a (43) and ICI 89,406–8a (44, 45) were kindly donated by Dr. K. Kopka (University Hospital of Münster, Münster, Germany). ISO and norepinephrine were dissolved at 1 mM in 1% ascorbic acid, CGP 20712A, ICI 118,551, betaxolol, propranolol, and xamoterol were diluted from a 1 mM stock in H2O, domperidone and salmeterol were stored at 1 mM in ethyl alcohol, ICI 89,406–5a was dissolved in 50% ethyl alcohol (500 µM), ICI 89,406–8a (500 µM) in 10% dimethylsulfoxide (DMSO), and carvedilol was stored at 90 mM in DMSO. T3 and dexamethasone (DEX) were purchased from Serva (Heidelberg, Germany). T3 was diluted into the culture medium from a 0.5 µM stock solution made in 0.9% NaCl. DEX was diluted from a 0.8 mM stock solution in ethanol. All control culture dishes were treated with the corresponding vehicles. Final dilutions were: ascorbic acid 1:100, ethanol 1:10,000, and DMSO 1:10,000. Pertussis toxin (PTx) was purchased from Sigma-Aldrich and used at a dose of 100 ng/ml diluted from a 50 ng/µl stock solution in water.

Animals
Male Wistar rats (11–12 wk old) were purchased from Elevage Janvier (Bio-Services, Uden, The Netherlands). They were kept in the animal house facilities (University of Leuven, Leuven, Belgium) during maximum 7 d under constant temperature, humidity, and day-night cycle, and had free access to food and water. After CO2 anesthesia rats were killed by decapitation. All experiments were conducted in accordance with the Guidelines for Care and Use of Experimental Animals, and were approved by the University Ethical Committee.

Aggregate cell culture and cell line culture
Aggregate cell cultures of anterior pituitary lobes were prepared by methods described previously (46) and in the companion paper (42). After cell dispersion the cell pellet was resuspended in serum-free DMEM-F12 medium as previously described (46, 47). This medium was supplemented with 50 pM T3, and according to experimental setup, with 80 nM DEX. Cells (2 x 106/2 ml culture medium) were allowed to re-associate in 35-mm nontreated culture dishes (Iwaki, Scitech division, Chiba, Japan) on a gyratory shaker (AppliTek, Nazareth, Belgium) at 63 rpm in a humidified 1.5–1.9% CO2-air incubator at 37 C. Culture medium was renewed on d-2 culture.

GHFT cells, LβT2 cells, {alpha}T3–1 cells (all from P. Mellon, University of California, San Diego, CA), and AtT20 cells (ATCC; LGC Promochem, Teddington, UK) were cultured in Advanced DMEM-F12 mixture (Invitrogen Corp., Carlsbad, CA) supplemented with GlutaMAX, streptomycin, and penicillin (Invitrogen), and 10% fetal calf serum (FCS) (Cambrex Bio Science, Verviers, Belgium). GH3 cells (ATCC; LGC Promochem) and TtT/GF cells (Riken Cell Bank, Tsukuba, Ibaraki, Japan) were cultured in Advanced DMEM-F12 supplemented with GlutaMAX, streptomycin, penicillin, and 12.5% horse serum (Invitrogen) –2.5% FCS and 10% horse serum –2.5% FCS, respectively. Cells were seeded in 75 cm2 culture flasks and were trypsinized (TrypLE; Invitrogen) twice a week. Cultures were kept in a humidified 5% CO2-air incubator at 37 C.

cAMP measurement
Assays of cAMP accumulation in pituitary cell aggregates in response to agonists and antagonists were performed on d 5 in culture. Each experimental group consisted of three dishes with each 2 x 106 cells, except for studies shown in Figs. 1AGo and 2AGo, for which aggregates were redistributed on d-5 culture so that each experimental group consisted of three dishes with 106 cells per dish. For testing agonists and antagonists on cAMP accumulation, the culture medium was replaced by the same medium containing 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), and cells were preincubated for 10 min. According to the experimental design, one or two antagonists were included in the preincubation medium. During the subsequent 30 min, cells were incubated in the same medium with agonists and antagonists. In case the effect of antagonists on their own was tested, aggregates were incubated during 40 min (10 min preincubation + 30 min incubation). Aggregates were then collected in 200 µl 0.1 M HCl and sonicated for 10 sec on ice. Cell fragments were spun down at 600 x g for 10 min, and the cAMP levels in the supernatant were quantified in duplicate by an enzymatic immunoassay from Assay Designs (Ann Arbor, MI).


Figure 1
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  		FIG. 1. Effect of the selective β1-AR antagonist CGP 20712A (CGP) and of the selective β2-AR antagonist ICI 118,551 (ICI) (A and B), and of the β1-AR antagonist ICI 89,406–8a (C) on ISO-stimulated intracellular cAMP accumulation in rat pituitary cell aggregates. Antagonists were added 10 min before and during stimulation with ISO (30 min). Values were expressed as percentage of controls, with controls (only vehicles added) set to 100% (A), or as absolute cAMP levels per dish (B). In the controls, levels of intracellular cAMP in 80 nM DEX were approximately 66% of levels found in steroid-free medium (P = 0.001). Three-way GLM ANOVA (A and B) was performed on ln-transformed data: ***/**, significantly different from control in corresponding medium (hormone free or DEX supplemented) with P = 0.001/0.01; and °°°, significantly different from 10 nM ISO in corresponding medium with P = 0.001. Two-way GLM ANOVA (C) was performed on log-transformed data: ***/**, significantly different from control; and °, significantly different from 10 nM ISO in the absence of ICI 89,406–8a with P = 0.05.

 

Figure 2
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  		FIG. 2. A, Effect of the β1-AR partial agonist xamoterol (xamo) (three doses) and of the β2-AR agonist salmeterol (salme) (50 nM) on cAMP accumulation in rat pituitary cell aggregates. ISO (100 nM) was taken as an internal estimate of approximate full intrinsic activity. Levels of basal cAMP were comparable in DEX (80 nM) and DEX-free conditions. Data are expressed as percentage of controls (no substances added), and controls were set to 100%. Three-way GLM ANOVA was performed on log-transformed data: ***, significantly different from control in corresponding medium (hormone free or DEX supplemented) with P = 0.001; and °°°/°°, significantly different from 100 nM ISO in corresponding medium with P = 0.001/0.01. B, Lack of effect of norepinephrine (norepi) on cAMP accumulation in pituitary cell aggregates under conditions of β2-AR blockade by 100 nM ICI 118,551 (ICI) and D2-R blockade by 10 nM (in case of 1 and 10 µM norepinephrine) or 100 nM (in case of 100 µM norepinephrine) domperidone. Three-way GLM ANOVA on raw data revealed no statistically significant differences.

 
For the cell line studies, the cells were treated in the culture flasks with 80 nM DEX or vehicle (diluted ethanol) for 3–4 d, after which they were seeded in 24-well plates at a density of 3 x 105 cells per well and kept in fresh culture medium. The following day the cells were exposed to agonists and/or antagonists in the same way as the aggregates. At the end of the incubation period, the cells were lysed in 200 µl 0.1 M HCl to analyze intracellular cAMP levels.

Statistical analysis
Values were expressed as the mean ± SEM of at least three independent experiments, each experimental group being done in triplicate for each experiment. Most data were log or ln transformed because of heterogeneity of variance. Data of experimental groups were compared by two-way or three-way generalized linear model (GLM) ANOVA [factors: medium-condition, a(nta)gonists and cultures] with the Tukey-Kramer comparison test according to the experimental design. The statistical package used was NCSS (Statistical Solutions Ltd., Cork, Ireland).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Selective β1- and β2-AR antagonists inhibit ISO-stimulated cAMP accumulation in anterior pituitary cell aggregates
We first tested whether the stimulation of cAMP accumulation by the nonselective β-AR full agonist ISO could be blocked by selective β1-AR and β2-AR antagonists. Figure 1AGo shows the results with cAMP values expressed as percent change over basal levels in DEX and steroid-free condition. In both culture conditions, ISO (10 nM) caused a 2-fold increase in intracellular cAMP accumulation. The response was completely blocked by 100 nM of the β1-AR selective antagonist CGP 20712A [a dose ~100 x above the dissociation constant for the β1-AR (48)] in aggregates supplemented with DEX, but blockade appeared incomplete in aggregates in steroid-free condition. The β2-AR antagonist ICI 118,551 completely blocked the cAMP response to ISO, regardless of the presence of DEX. Interestingly, combination of both antagonists led to a decrease in cAMP below basal levels.

Figure 1BGo shows the same data but expressed as absolute cAMP values. Although DEX attenuated absolute levels of cAMP (Table 1Go), the effects of the β1- and β2-AR antagonists remained essentially the same.


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  		TABLE 1. Basal cAMP accumulation in pituitary cell lines (3 x 105 cells) and aggregates (106 cells) cultured in hormone-free or DEX-supplemented medium

 
An even more selective and potent β1-AR blocker, ICI 89,406–8a [5 nM, a dose ~100 fold above the dissociation constant for the β1-AR (44, 45)] abolished cAMP accumulation stimulated by 10 nM ISO (Fig. 1CGo). The same dose of ICI 89,406–8a did not abolish stimulation by a 10 or 100-fold higher dose of ISO, consistent with competition between both agents for the β1-AR.

The present data are consistent with the coupling of both β1- and β2-ARs to adenylate cyclase, although the action of the antagonists on their own needs to be tested before a final conclusion can be reached. This was done in a separate experiment (see below).

β1-AR agonists but not β2-AR agonists fail to stimulate cAMP levels in anterior pituitary cell aggregates
The partial β1-AR agonist xamoterol (20 nM to 2 µM doses) was unable to elevate levels of cAMP, whereas 50 nM salmeterol (β2-AR selective) caused a 2-fold increase, regardless of the presence of DEX (Fig. 2AGo). The 2-µM dose can be considered a receptor-saturating dose because the EC50 concentration of xamoterol in cardiac myocytes is approximately 10 nM (49). Because xamoterol is a partial agonist for the β1-AR with an intrinsic activity of 10–30% (49), we studied the effect of the full agonist norepinephrine under conditions that prevent any action of norepinephrine through the β2-AR and the type 2 dopamine receptor (D2-R), which would result in an inhibitory effect on cAMP accumulation (12). Under these conditions norepinephrine did not affect cAMP levels, even at a dose of 100 µM (Fig. 2BGo). The apparent difference in cAMP response to ISO between steroid-free and DEX-supplemented aggregates was statistically not significant; there was also no difference at a 10 nM ISO dose (Fig. 1Go).

The selective β1-AR antagonist CGP 20712A inhibits basal cAMP accumulation in anterior pituitary cell aggregates
The internal inconsistency between the results obtained with the β1-AR antagonist CGP 20712A and those obtained with β1-AR agonists together with the finding that combined treatment with β1-AR and β2-AR antagonists decreased cAMP accumulation for 50% below basal levels prompted us to test the hypothesis that CGP 20712A may have an intrinsic inhibitory action on cAMP accumulation not related to the β1-AR or might act as an inverse agonist as already demonstrated in artificial expression systems (50). CGP 20712A significantly depressed basal cAMP levels in anterior pituitary cell aggregates in both DEX and non-DEX conditions but tended to be more effective in DEX-supplemented cultures, albeit the difference reached only significance in the CGP plus ICI group (P = 0.001) (Fig. 3AGo). The β2-AR antagonist ICI 118,551 was inactive in this respect.


Figure 3
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  		FIG. 3. A, Effect of 100 nM CGP 20712A (CGP) and 100 nM ICI 118,551 (ICI) (alone and in combination) on basal cAMP levels in pituitary cell aggregates. No differences in absolute basal cAMP levels in the control condition were seen between the absence and presence of DEX in the culture medium. Data are expressed as percentage of basal cAMP levels with the latter controls for each condition set to 100%. The data were log transformed and three-way GLM ANOVA: ***, significantly different from control in corresponding medium (hormone free or DEX supplemented) with P = 0.001; and ###, significantly different from corresponding hormone-free condition with P = 0.001. B, Influence of PTx pretreatment on the inhibition of basal cAMP accumulation by CGP 20712A (CGP). Experiments were run in DEX-supplemented (80 nM) medium in the absence and presence of ICI 118,551 (ICI). Cell aggregates were exposed to 100 ng/ml PTx during the last 16 h of the 5-d culture period after which the cAMP responses were tested. Basal cAMP values were not affected by PTx and set to 100%. Data were log transformed and three-way GLM-ANOVA: ***, significantly different from control in corresponding medium (untreated or PTx treated) with P = 0.001.

 
PTx abolishes the inhibitory effect of CGP 20712A on basal cAMP accumulation
The inhibition of basal cAMP accumulation by CGP 20712A may be based on two possible actions, i.e. an inverse agonist effect on the β1-AR by which the antagonist exerts negative intrinsic activity on a constitutively active β1-AR, or an agonist effect via another, negatively coupled GPCR. Coupling to Gi/o-proteins was tested by studying the effect of pretreatment of anterior pituitary cell aggregates with 100 ng/ml PTx for 16 h. The latter treatment completely abolished the inhibitory effect of CGP 20712A on basal cAMP accumulation, an effect not influenced by ICI 118,551 (Fig. 3BGo). PTx pretreatment did not affect basal cAMP levels on its own (data not shown).

Carvedilol abolishes the inhibition of cAMP accumulation by CGP 20712A
To test the specificity of CGP 20712A on basal cAMP accumulation and to evaluate whether the effect might be due to a partial β1-AR agonist action on β1-ARs negatively coupled via a Gi/o-protein, we checked whether other β-AR antagonists also affect basal cAMP accumulation and/or could block the effect of CGP 20712A. Propranolol, betaxolol (Fig. 4AGo), carvedilol, and ICI 89,406–5a (Fig. 4BGo) were ineffective in reducing cAMP levels on their own. In contrast, ICI 89,406–8a (Fig. 4CGo) slightly (~20%) decreased basal cAMP accumulation, although it was borderline statistically significant. Neither propranolol nor betaxolol (Fig. 4AGo), ICI 89,406–5a (Fig. 4BGo), and ICI 89,406–8a (Fig. 4CGo) could block the effect of CGP 20712A, excluding a partial agonist action of the latter on Gi/o-protein-coupled β-AR. However, the β-AR antagonist carvedilol completely abolished the CGP 20712A effect (Fig. 4BGo). The selective interaction of CGP 20712A and carvedilol is consistent with the hypothesis that the former is an inverse agonist and the latter a "neutral antagonist" of the β1-AR.


Figure 4
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  		FIG. 4. Effect of 100 nM CGP 20712A (CGP) alone and in combination with 100 nM propranolol (prop), 100 nM betaxolol (Bet) (A), 30 nM ICI 89,406–5a (5a), or 900 nM carvedilol (Carve) (B) and of 5 nM ICI 89,406–8a (8a) (C) on cAMP accumulation in pituitary cell aggregates. These experiments were only performed in DEX-supplemented (80 nM) medium. All data were expressed as percentage of control (only vehicles added), which was set to 100%. Three-way GLM ANOVA on log-transformed data: ***/*, significantly different from control with P = 0.001/0.052.

 
Selective β1- and β2-AR antagonists decrease ISO-stimulated cAMP accumulation in gonadotroph cell lines
To localize putatively the functional coupling of β1-AR to adenylate cyclase in a particular cell type, we tested the action of β1-AR and β2-AR blockers on the ISO-stimulated cAMP accumulation in the GH3, GHFT, LβT2, {alpha}T3–1, and TtT/GF cells. No change in cAMP levels in response to 10 nM ISO was observed in GH3 cells cultured in either DEX or steroid-free condition (data not shown), consistent with the absence of β-AR binding sites in this cell line (51). Both GHFT and TtT/GF cells accumulated cAMP in response to ISO to a level 20 and three times above control, respectively (Fig. 5Go, A and B). CGP 20712A did not affect ISO-induced cAMP accumulation, whereas ICI 118,551 completely abolished this response, regardless of DEX supplementation. In {alpha}T3–1 cells (Fig. 5CGo), ISO induced a 29-fold increase in cAMP level in the absence and an 81-fold increase in the presence of DEX during culture. Both CGP 20712A and ICI 118,551 strongly attenuated ISO-induced cAMP accumulation, and adding both antagonists together completely blocked the cAMP response, regardless of whether DEX was present or not. In LβT2 cells (Fig. 5DGo), ISO induced a 7-fold increase of cAMP level in DEX-supplemented and a 5-fold increase in steroid-free condition. CGP 20712A partially blocked the response to ISO, statistical significance being reached only in the DEX condition. ICI 118,551 significantly attenuated the response in both the absence and presence of DEX. In both steroid and steroid-free conditions, combined administration of the two β-AR antagonists completely abolished the response. On the basis of these results, it appears that the contribution of the β1-AR to adrenergic stimulation of cAMP accumulation is largest in the {alpha}T3–1 cell line, in which it is also larger than the contribution of the β2-AR.


Figure 5
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  		FIG. 5. Effect of CGP 20712A (CGP) and ICI 118,551 (ICI) on 10 nM ISO-stimulated cAMP accumulation in GHFT (A), TtT/GF (B), {alpha}T3–1 (C), and LβT2 (D) cells. In the absence of ISO and antagonists, there was no difference in basal cAMP accumulation between hormone-free and DEX-supplemented condition for the GHFT, {alpha}T3–1, and TtT/GF cells, whereas basal cAMP levels in LβT2 cells were three times higher in steroid-free cultures than in DEX-treated cultures. Hormone-free and DEX-treated controls were all set to 100%. ln-transformed data were analyzed with three-way GLM ANOVA: ***/**/*, significantly different from control in corresponding medium (hormone free or DEX supplemented) with P = 0.001/0.01/0.05; ###/#, significant difference between corresponding DEX-supplemented and hormone-free conditions with P = 0.001/0.05; and °°°/°°, significantly different from 10 nM ISO in corresponding medium with P = 0.001/0.01.

 
To ensure that the effect of CGP 20712A and ICI 118,551 on ISO-stimulated cAMP accumulation was due to competitive antagonism, the effect of the latter compounds on their own should also be tested. This was done for CGP 20712A in the LβT2 cells, and no effect was seen (see Fig. 8Go). Because no effect of ICI 118,551 on its own was found in the pituitary aggregates, we did not check for this action in cell lines. Moreover, because a combination of the two antagonists did not decrease cAMP levels below the basal control value (as was the case in the aggregates), an effect of either one on its own was not expected.


Figure 8
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  		FIG. 8. Lack of effect of propranolol (Prop) and betaxolol (Bet) on the inhibition of basal cAMP accumulation by CGP 20712A (CGP) (A) and effect of ICI 89,406–8a on ISO-stimulated cAMP accumulation (B) in LβT2 cells. Data are expressed as percentage of untreated control (A), and as percentage of control values in untreated and ICI 89,406–8a-treated cultures (B), with controls set at 100%. Three-way GLM ANOVA on ln- (A) and log- (B) transformed data: ***, significantly different from control in corresponding medium (untreated or ICI 89,406–8a-treated) with P = 0.001; and °°°, significantly different from 10 nM ISO in the absence of ICI 89,406–8a with P = 0.001.

 
As shown in Table 1Go, basal cAMP formation was quite different among cell lines. Levels were lowest in {alpha}T3–1 and TtT/GF cells, whereas GHFT and LβT2 cells showed higher levels. DEX had no effect on cAMP levels except in LβT2 cells, in which it significantly depressed basal levels. Basal cAMP levels in several cell lines were considerably higher than in pituitary aggregates. However, note that the exact number of cells at testing is not known.

β1-AR agonists stimulate cAMP levels in the L βT2 and TtT/GF cell lines
As expected, GH3 cells did not show any cAMP responses to either β1- or β2-AR agonists (data not shown; n = 3). In GHFT cells the β2-AR agonist salmeterol dose dependently stimulated cAMP levels, regardless of glucocorticoid treatment, whereas the β1-AR partial agonist xamoterol (used only at the receptor-saturating dose) was inactive (Fig. 6AGo). The {alpha}T3–1 cells did not show β1-AR-mediated cAMP responses to xamoterol, whereas salmeterol induced a clear effect that was enhanced in DEX-supplemented condition (Fig. 6BGo). The only cell line showing a cAMP response to xamoterol was the LβT2 cell line (Fig. 6CGo), but the effect was small. In view of the fact that the dissociation constant of the β1-AR agonist xamoterol for the β2-AR in the rat is close to the concentration we used (48), we retested the effect of xamoterol on the LβT2 cells in the presence of the β2-AR blocker ICI 118,551. In this situation xamoterol was still stimulating cAMP levels, confirming an action through the β1-AR (data not shown; n = 3).


Figure 6
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  		FIG. 6. Effect of the selective β1-AR agonist xamoterol (xamo) and the β2-AR agonist salmeterol (salme) on cAMP accumulation in GHFT (A), {alpha}T3–1 (B), and LβT2 (C) cells. The effect of 100 nM ISO was tested to have an indication of full intrinsic activity. Data are expressed as percentage of control levels in hormone-free and DEX-supplemented cultures. Three-way GLM ANOVA was performed on log-transformed data: ***/**, significantly different from control in corresponding medium (hormone free or DEX supplemented) with P = 0.001 / 0.01; ###/##/#, significant difference between corresponding DEX-supplemented and hormone-free conditions with P = 0.001/0.01/0.05; and °°°/°°, significantly different from 100 nM ISO in corresponding medium with P = 0.001/0.01.

 
In both the steroid-free and DEX-supplemented media, the LβT2 cells showed a clear cAMP response to norepinephrine under conditions of masking effects via β2-AR and D2-R obtained by coincubation with 100 nM ICI 118,551 and 10 nM domperidone (Fig. 7AGo). However, a difference in responsiveness between DEX and steroid-free medium was difficult to assess because the lowest concentration of norepinephrine tested already evoked a maximal response. Under β2-AR masking with 100 nM ICI 118,551, TtT/GF cells showed a cAMP response to norepinephrine in a dose-dependent manner. Importantly, this effect was only seen in medium supplemented with DEX (Fig. 7BGo).


Figure 7
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  		FIG. 7. Effects of different doses of norepinephrine (norepi) on cAMP accumulation in LβT2 cells (A) and in TtT/GF cells (B) tested under conditions of β2-AR blockade by 100 nM ICI 118,551 (ICI) and D2-R blockade by 10 nM (in case of 1 and 10 µM norepinephrine) or 100 nM (in case of 100 µM norepinephrine) domperidone. Data are expressed as percentage of control values in hormone-free and DEX-supplemented cultures (only vehicles added), which were set to 100%. Three-way GLM ANOVA was performed on log- (LβT2) and ln- (TtT/GF) transformed data: ***, significantly different from control in corresponding medium (hormone free or DEX supplemented) with P = 0.001; and #, significant difference between corresponding DEX-supplemented and hormone-free condition with P = 0.05.

 
CGP 20712A antagonizes ISO-stimulated but fails to inhibit basal cAMP accumulation in LβT2 cells
In contrast to what was seen in aggregates, CGP 20712A failed to affect basal cAMP levels in LβT2 cells (Fig. 8AGo). This was also seen when CGP 20712A was coadministered with propranolol or betaxolol. ICI 89,406–8a also failed to suppress basal cAMP levels in LβT2 cells (data not shown) but blocked ISO-stimulated cAMP levels (Fig. 8BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows that pharmacological interference with selective β1-AR ligands modulates intracellular cAMP accumulation in aggregate cell culture of the anterior pituitary consistent with functional coupling to adenylate cyclase, but several data indicate mediation through a noncanonical transduction pathway. ISO-stimulated cAMP accumulation was abolished by the highly selective β1-AR antagonists CGP 20712A and ICI 89,406–8a, which is indicative for competitive antagonism of the action of ISO at both the β1-AR and β2-AR. However, the partial β1-AR agonist xamoterol as well as the full agonist norepinephrine (tested under conditions precluding interference via β2-AR and D2-R obtained by coincubation with the selective β2-AR antagonist ICI 118,551 and the D2-R blocker domperidone) failed to stimulate cAMP accumulation, even at a dose as high as 2 and 100 µM, respectively. These findings suggest that the effect of CGP 20712A on ISO-induced cAMP formation might be based on another mechanism than pharmacological competitive antagonism of the β1-AR. Indeed, in the absence of an agonist, CGP 20712A also depressed basal cAMP accumulation by more than 50%. Negative intrinsic activity on basal cAMP levels, although considerably smaller, was found with ICI 89,406–8a as well, but other β-AR antagonists, such as propranolol (nonselective), betaxolol (β1-AR preferring), ICI 89,406–5a (β1-AR selective), and carvedilol (nonselective), had no such effect. A plausible explanation for these findings is that the β1-AR is constitutively active, and that CGP 20712A and ICI 89,406–8a act as inverse agonists on this receptor, thereby decreasing agonist-stimulated as well as basal cAMP accumulation. It has been shown that GPCRs can exist in an active state if "empty" or "un-liganded," i.e. coupled to a G protein and stimulating adenylate cyclase in the absence of an agonist, a process that is called constitutive activity (50). Many GPCR antagonists have the potential to inhibit this constitutive activity of the receptor and, therefore, are called "inverse agonists," hereby decreasing cAMP levels below basal levels. These substances counteract the effect of a classical agonist, but their action is primarily due to an allosteric effect leading to a molecular conformation unable to interact with the G protein. Antagonists that do not influence the spontaneous activity of the receptor but antagonize the action of a classical agonist by displacement of its binding, are known as "neutral antagonists" (50). Although constitutive activity is often seen in situations in which receptors are present at high density, such as in artificial expression systems (51), or after certain amino acid mutations in the receptor (52), constitutive activity has also been found for certain receptors (H3-R, 5-HT1A-R, 5-HT2C-R, GABAA-R) in certain cell types under normal in vivo conditions (53, 54, 55, 56). Constitutively active β1-ARs have been demonstrated in guinea pig and human ventricular myocytes (57, 58), in transfected COS-7 cells, and in transgenic mice with heart-specific overexpression of human β1-ARs (59). CGP 20712A has exerted inverse agonism in other systems (59, 60). Assuming that CGP 20712A is acting as an inverse agonist in our pituitary system, its action in the presence of an agonist would lead to the same result as if it would act as a pure antagonist, i.e. lowering cAMP accumulation. An important further and direct argument for our interpretation was that the inhibitory action of CGP 20712A on basal cAMP formation was abolished by carvedilol, known as a neutral antagonist (59), whereas several other β-AR blockers (propranolol, betaxolol, and ICI 89,406–5a) did not. An important finding was that the inhibition of cAMP levels by CGP 20712A was abolished by PTx that is known to block signal transduction via Gi/o{alpha} proteins (61). Coupling of a constitutively active β1-AR has, so far, been shown only for Gs{alpha} proteins, but, at least in cardiac myocytes, the constitutively active form of the β2-AR weakly couples to Gi/o{alpha} (29, 62). Taking all data together, it seems reasonable to assume that in the pituitary cell aggregate system, the β1-AR is constitutively coupled to Gi/o{alpha}, and that CGP 20712A and ICI 89,406–8a act as inverse agonists. The presumptive constitutive activity of the β1-AR could explain the failure to find an agonist effect of norepinephrine and xamoterol on cAMP accumulation because constitutive activity is so high that a cAMP increase over what is already generated by the active receptor is difficult to detect. Although stimulation of cAMP by an activated GPCR is often mediated by Gs{alpha}, adenylate cyclase may also be activated via coupling to Gi/o{alpha}, of which the β{gamma}-subunit can act in a stimulatory fashion on adenylate cyclase (63).

We challenged the hypothesis of a constitutively active β1-AR by examining two other possible explanations. One possibility is that CGP 20712A acts as an agonist at another Gi/o{alpha}-coupled GPCR. A strong argument against this explanation is the finding that the β-AR antagonist carvedilol abolished the inhibitory action of CGP 20712A on basal cAMP accumulation. That both CGP 20712A and carvedilol would act at the same nonspecific (non-β-adrenergic) target seems highly unlikely. Moreover, ICI 89,406–8a [a β1-AR antagonist even more selective than CGP 20712A (45)] also caused some depression of basal cAMP levels. Again, it is unlikely that two compounds with high selectivity for the β1-AR and displaying a considerable structural difference would bind to the same nonspecific target. Another possible explanation for the intrinsic action of CGP 20712A could be that the compound acts as a competitive antagonist on β1-ARs that are tonically activated by an endogenous adrenergic agonist, acting in a tonic paracrine or autocrine manner and through PTx-sensitive β1-AR coupling. The action of CGP 20712A on its own could then easily be explained by interference of the latter compounds with that paracrine/autocrine loop. The lack of effect of exogenous β1-AR agonists (norepinephrine and xamoterol) on cAMP levels could be explained by assuming a high activity of the endogenous adrenergic system, making a cAMP increase over what is already produced by the endogenous ligand difficult to detect. Several nonneuronal tissues such as mesangial cells (64), T lymphocytes (65), and fetal cardiomyocytes (66) synthesize norepinephrine that appears to act in an autocrine/paracrine loop. However, although the enzymatic machinery for dopamine production may be present in the anterior pituitary (67, 68), there is no evidence for the presence of norepinephrine or epinephrine (69). When the anterior pituitary is disconnected from the hypothalamus by transplantation under the kidney capsule, some norepinephrine was found in the transplant, but the source remained unknown (70). Although it remains possible that in cultured pituitary cell aggregates the cellular machinery for catecholamine biosynthesis is recruited, it remains difficult to explain why only CGP 20712A and ICI 89,406–8a and not propranolol, betaxolol, ICI 89,406–5a, and carvedilol would antagonize that endogenous agonist. Moreover, the finding that carvedilol abolished the action of CGP 20712A cannot be reconciled with the hypothesis of an endogenous ligand tonically activating the β1-AR. Thus, the latter hypothesis reasonably has to be rejected. However, it should be reminded that a growing number of GPCRs is being reported that appear promiscuous in their ligand selectivity and can bind substances with markedly differing structures (71). Therefore, it cannot be excluded that the β1-AR is tonically activated by an unconventional substance in the aggregates.

In a companion paper, we found that the β1-AR is located in gonadotrophs (42). To gain more information as to the cellular localization of β1-AR coupling, the effect of β1-AR agonists on cAMP accumulation and of β1-AR antagonists on ISO-stimulated cAMP accumulation was studied in the gonadotrophic cell lines {alpha}T3–1 and LβT2, and compared with that in GHFT, GH3, and TtT/GF cells. No effect of ISO, β1- or β2-AR agonists was found in GH3 cells, consistent with previous findings that they do not contain β-ARs (51). CGP 20712A blocked ISO-stimulated cAMP formation in the two gonadotroph cell lines, but not in the GHFT and TtT/GF cells, supporting coupling of the β1-AR to adenylate cyclase in cells of the gonadotroph lineage. This was confirmed by a stimulatory action of norepinephrine and xamoterol on cAMP accumulation in the LβT2 cells. The observed effects of the agonists were not due to interference with β2-ARs because the agonist actions were studied in the presence of the β2-AR antagonist ICI 118,551. Thus, the data are consistent with the presence of β1-AR immunoreactivity in gonadotrophs of the intact pituitary, as demonstrated in a companion paper (42).

Importantly, in LβT2 cells there was no evidence for constitutive activity of the β1-AR because CGP 20712A did not affect basal cAMP accumulation in the latter cells. Thus, β1-ARs in LβT2 cells are functionally coupled to adenylate cyclase in a canonical manner. By combining the pituitary aggregates with the cell line data, a picture emerges that the gonadotrophs express a functional β1-AR and that constitutive activity is either expressed cell autonomously in the normal pituitary but not in the gonadotrophic cell lines or that it needs a normal pituitary microenvironment. In the latter case, the constitutive activity may be transduced by another cell type to the gonadotrophs, or it may depend on the three-dimensional nature of the aggregate cell cultures, which may create a dense association between cells. However, the aggregation phenomenon by itself is not likely to be involved in the constitutive activity because LβT2 cells in "monolayer culture" are in fact growing as small aggregate-like associations with cells superposing each other. Under our trypsinization conditions, these aggregates are not fully dispersed (indicating their tight association), and the aggregate-like associations are detected within 1-d replating (at the time of testing the cAMP responses). Whether constitutive activity (and the inverse agonist activity of CGP 20712A) is cell autonomous or depends on paracrine/autocrine interactions in the pituitary is a most attractive issue for future research. However, at present, cell separation and recombination experiments are unfeasible due to the limited yield of cells in gonadotroph-enriched fractions and their low basal cAMP levels.

Previous work showed a strong up-regulation of β1-AR mRNA levels by glucocorticoids in pituitary aggregates (42). In the present study, no obvious potentiation of ISO-stimulated cAMP accumulation by DEX was found. The antagonism of ISO-stimulated cAMP accumulation by CGP 20712A was somewhat more pronounced in DEX-treated aggregates (Fig. 1Go), but the difference did not reach statistical significance. This is not surprising because even when more β1-AR is expressed in the DEX-supplemented aggregates, it cannot be seen under stimulation with ISO because the unliganded β1-AR is already effectively coupled to adenylate cyclase. For this reason there was also no effect of norepinephrine on cAMP levels when the β2-AR was pharmacologically blocked. However, the inverse agonism appeared to be more pronounced in DEX-supplemented culture conditions.

Consistent with previous observations that DEX did not increase β1-AR expression in pituitary cell lines (42), no effect of DEX on the cAMP response to ISO, xamoterol, and norepinephrine was observed in LβT2 cells, and the blunting of the cAMP response to ISO by the β1-AR antagonist was of a similar magnitude in DEX-free as in DEX-supplemented cultures. However, in the folliculo-stellate TtT/GF cell line, norepinephrine (under β2-AR selective blockade) stimulated cAMP accumulation only when DEX was present in the culture medium. However, the significance of the DEX effect remains questionable because β1-AR mRNA levels found in the TtT/GF cells are extremely low (42).

The role of constitutive activity in GPCRs is poorly understood in general, except for a few cases such as for the melanocortin-4 receptor, for which a physiological agonist ({alpha}-MSH) and a physiological inverse agonist (agouti-related peptide) exist (72, 73). The physiological significance of β1-AR constitutive activity could be related to the close morphological association of β1-AR-expressing gonadotrophs with "cup-shaped" lactotrophs shown in the companion paper (42). It has been demonstrated by a number of investigators that the normal PRL-secreting activity of lactotrophs requires normal activity of gonadotrophs (reviewed in Ref. 74). In that context the constitutive activity of the β1-AR could be a mechanism to keep a basal trophic activity of gonadotrophs upon lactotrophs. For example, β1-AR may indirectly have a role in maintaining basal PRL release. Because the proportion of gonadotrophs in the pituitary is less than 10%, and lactotrophs are more numerous and have a relatively high basal cAMP formation rate (75), one has to consider that the 50% decrease in cAMP formation by CGP 20712A represents an effect in gonadotrophs that is amplified by a gonadotroph-to-lactotroph communication mechanism. The latter may be mediated by extracellular molecules or even by intracellular cAMP through gap junctions. Within gonadotrophs basal cAMP may also be important for basal expression of the GnRH receptor, gonadotropin and follistatin production (76, 77, 78, 79). Whereas maintenance of gonadotroph responsiveness to GnRH in terms of LH and FSH production critically depends on the pulsatile nature of GnRH delivery to the tissue (80), it seems biologically meaningful that a mechanism exists capable of preserving sustained basal production of these molecules, independent of GnRH pulse frequency. Such a role could reside in constitutive formation of cAMP by the β1-AR. Another possibility is that β1-AR-generated cAMP is functionally related to the intrinsic pulsatility in basal gonadotropin release within the pituitary (81, 82), just like it is positively related to pulsatile GnRH release within GnRH neurons (83). Regardless of the exact functions the β1-AR may have, its constitutive activity may be affected during stress because glucocorticoids tended to enhance it. Finally, because single amino acid mutations have caused or altered constitutive activity in certain GPCRs (84), the present observations may be relevant for the pathogenesis of pituitary disease, such as progression of GH or PRL cell adenomas that are dependent on cAMP levels (85). Crucial further questions are whether β1-AR-generated cAMP is targeted to the canonical protein kinase A cascade or to other targets such as R-ras-Rap1, particularly because R-ras and Rap1 are known to play an important role in cell adhesion through integrin activation (86, 87), a possible hypothesis to consider in relation to the association between gonadotrophs and lactotrophs. The β2-AR has already been shown to signal through the Rap1 pathway (86, 87).

The present data confirmed canonical β2-AR coupling to adenylate cyclase in pituitary cell aggregates, and report for the first time the functional expression of β2-AR in GHFT, {alpha}T3–1, LβT2, and TtT/GF cells. The selective β2-AR antagonist ICI 118,551 blocked ISO-stimulated cAMP formation, and the selective β2-AR agonist salmeterol stimulated cAMP accumulation. These findings are consistent with the broad distribution of the β2-AR in the anterior pituitary. In the {alpha}T3–1 cells, the β2-AR coupling to adenylate cyclase appeared to be potentiated by DEX because the cAMP response to salmeterol was higher in DEX-supplemented than in DEX-free condition.

In conclusion, we propose that β1-AR is functionally expressed in pituitary gonadotrophs, and that the nonliganded receptor is constitutively active, accounting directly or indirectly for a large part of basal cAMP production in the tissue in culture and that the constitutive activity signals through a PTx-sensitive G protein. The latter activity may be dependent on the microenvironment of the normal pituitary because no such activity was observed in the gonadotrophic cell line LβT2. To our knowledge, the β1-AR in gonadotrophs may be the first example of a constitutively active GPCR in the normal pituitary, although most recently, constitutively active somatostatin receptors were demonstrated in the AtT20 and TtT/GF pituitary cell lines (88).


   Acknowledgments
 
We thank Roche Diagnostics (Indianapolis, IN) for the kind donation of carvedilol and K. Rillaerts and Y. Van Goethem for skillful technical assistance.


   Footnotes
 
This work was supported by grants from the Flemish Ministry of Science Policy (Concerted Research Actions) and the Fund for Scientific Research Flanders (Belgium) (Fonds voor Wetenschappelijk Onderzoek).

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 17, 2008

Abbreviations: AR, Adrenoceptor; DEX, dexamethasone; DMSO, dimethylsulfoxide; FCS, fetal calf serum; GLM, generalized linear model; GPCR, G protein-coupled receptor; ISO, isoproterenol; PRL, prolactin; PTx, pertussis toxin; D2-R, type 2 dopamine receptor.

Received October 11, 2007.

Accepted for publication January 7, 2008.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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K. Janssens, O. Krylyshkina, N. Hersmus, H. Vankelecom, and C. Denef
{beta}1-Adrenoceptor Expression in Rat Anterior Pituitary Gonadotrophs and in Mouse {alpha}T3-1 and L{beta}T2 Gonadotrophic Cell Lines
Endocrinology, May 1, 2008; 149(5): 2313 - 2324.
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