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Volume 419, Issue 2-3 p. 171-174
Full-length article
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Modulation of forskolin-mediated adenylyl cyclase activation by constitutively active Gs-coupled receptors

Astrid E Alewijnse

Astrid E Alewijnse

Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

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Martine J Smit

Martine J Smit

Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

Present address: Department of Pharmacology, Mount Sinai School of Medicine, New York, NY 10029, USA. Search for more papers by this author
M Sol Rodriguez Pena

M Sol Rodriguez Pena

Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

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Dennis Verzijl

Dennis Verzijl

Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

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Henk Timmerman

Henk Timmerman

Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

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Rob Leurs

Corresponding Author

Rob Leurs

Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

Corresponding author. Fax: +31 (20) 4447610. E-mail: [email protected]Search for more papers by this author
First published: 14 January 1998
Citations: 35

Abstract

In transfected CHO cells constitutively active histamine H2 receptors not only increase the basal cAMP level, but also enhance forskolin-induced cAMP production. The increased forskolin response was inhibited by inverse H2 agonists with potencies similar to those determined at basal levels. The modulation of the forskolin response was also observed after H2 receptor expression in HEK-293 and Sf9 cells or TSH receptor expression in COS-7 cells. The enhancement of forskolin-induced cAMP production seems to be a general characteristic of constitutively active Gs-coupled receptors and can be very useful to study inverse agonism at wild-type receptors.

1 Introduction

Classical models of G-protein-coupled receptors (GPCRs) require agonist occupation of the receptor to activate signal transduction pathways. Nowadays, it is well documented that GPCRs can be spontaneously active and this agonist-independent receptor activity is often referred to as constitutive receptor activity [1-5]. Inverse agonists reduce the constitutive GPCR activity, whereas neutral antagonists do not affect the basal GPCR activity. Constitutive activity was first shown for mutant GPCRs and has proved to be the mechanistic basis for several genetic diseases [6]. Moreover, a detailed understanding of constitutive GPCR activity will give better insights of agonist-induced GPCR activation [5]. Because of these important implications the phenomenon of constitutive GPCR activity and inverse agonism currently receives considerable attention [1-5].

To study inverse agonism GPCRs should possess a detectable level of constitutive activity. Constitutive activity of wild-type receptors is generally low and inverse agonism is therefore often studied using constitutively active mutant (CAM) GPCRs. However, as a result of introduced mutations, the pharmacological behaviour of ligands can be seriously affected [7, 8]. For example, as a result of a single point mutation in TM4 of the δ-opioid and μ-opioid receptors antagonists became surprisingly full agonists [8]. Moreover, the β-blocker propranolol was reported to change from an inverse agonist at the wild-type β2-adrenergic receptor [9]to a neutral antagonist at a constitutively active β2-adrenergic receptor mutant [10]. In view of these findings the use of wild-type receptors in the study of inverse agonism is preferred.

Recently, the use of highly sensitive reporter gene technology has been introduced to observe constitutive activity of wild-type Gq-coupled receptors [11]. Similarly, modifications of the extracellular sodium concentration have been shown to increase the constitutive activity of bradykinin B2 [12], TSH [13]and LH receptor [13]. Moreover, overexpression of Gαq in NIH-3T3 cells results in a detectable constitutive activity of the coexpressed, wild-type muscarinic m3 receptor [14]. However, this latter approach is not always feasible. Overexpression of Gαs in NG108-15 cells did not affect the constitutive activity of the β2-adrenergic receptor [15], probably because Gαs is not the limiting factor in the signal transduction.

Activation of Gs-coupled receptors and reconstitution of adenylyl cyclase isoenzymes with purified Gαs subunits are known to have a synergistic effect on the forskolin-induced adenylyl cyclase activation [16, 17]. Consequently, constitutively active Gs-coupled receptors can be anticipated to potentiate forskolin responses by increasing the levels of free Gαs subunits. We previously noticed that stable expression of the rat H2 receptor in CHO cells not only enhanced the basal cAMP level but also potentiated the forskolin-induced cAMP production [18]. In this study, we provide evidence that the enhancement of the forskolin response is indeed the consequence of the constitutive activity of the wild-type H2 receptor. We show that modulation of the forskolin response offers a useful method to investigate constitutive GPCR activity in several cell lines and inverse agonism at wild-type Gs-coupled receptors under normal experimental conditions.

2 Materials and methods

2.1 Chemicals

Histamine dihydrochloride, isobutylmethylxanthine (IBMX), aminopotentidine, cyclic AMP (cAMP), DEAE-dextran, chicken egg albumin and forskolin were obtained from Sigma Chemical Company (USA). [2,8-3H]cAMP (40 Ci/mmol) was obtained from Amersham. VUF 8299 was taken from laboratory stock [19]. Gifts of cimetidine (SmithKline Beecham, United Kingdom), ranitidine dihydrochloride (Glaxo, United Kingdom) and famotidine (Merck Sharp and Dohme, The Netherlands) are greatly acknowledged.

2.2 Cell culture and transfection

Chinese Hamster ovary (CHO) cells deficient in dihydrofolate reductase (CHOdhfr), H2 receptor expressing CHO (CHOH2) and human embryonic kidney (HEK-293H2) cells, African green monkey kidney (COS-7) cells and Sf9 insect cells were grown as described previously [20-22].

COS-7 cells were transiently transfected with the expression vectors pSVL, pSVL-TSHI486F, pSVL-TSHF631L and pSVL-TSH (5 μg DNA/106 cells) using DEAE-dextran [23]. Sf9 cells were infected with a recombinant baculovirus containing the epitope-tagged H2 receptor as described by Beukers et al. [22].

2.3 Biochemical measurements

The determination of H2 receptor binding with the H2 antagonist [125I]iodoaminopotentidine ([125I]APT) was performed as described by Leurs et al. [24]. The cAMP production in CHO, HEK-293, COS-7 (48 h after transfection) and Sf9 cells was determined as described previously [18]. Protein concentrations were determined according to Bradford using bovine serum albumin as a standard [25].

2.4 Statistical analysis

All data shown are expressed as mean±standard error (mean±S.E.M.) of at least three independent experiments. Statistical analysis was carried out by Student's t-test. P-values <0.05 were considered to indicate a significant difference.

3 Results

In CHOdhfr cells, stably expressing the H2 receptor [24]at a density of 1079±313 fmol/mg protein (mean±S.E.M., n=6) (CHOH2 cells), the basal cAMP level was about 2-fold higher (170±37 pmol cAMP/mg protein, mean±S.E.M., n=6) than in CHOdhfr cells (78±13 pmol cAMP/mg protein, mean±S.E.M., n=6, P<0.05). Forskolin dose-dependently increased the cAMP formation in both CHOdhfr and CHOH2 cells (Fig. 1 ). At each tested concentration the forskolin-induced cAMP production in CHOH2 cells was significantly higher than in CHOdhfr cells. The absolute difference in cAMP production was maximal (1600 pmol cAMP/mg protein) at a concentration of 1 μM of forskolin. A potentiation of the forskolin response was also seen at higher concentrations of forskolin, but due to the increased response in the CHOdhfr cells the relative increase was lower at these concentrations (Fig. 1).

figure image
Forskolin-induced increase in cAMP production in untransfected (open circles) and CHOH2 cells (filled circles). The data shown represent the mean±S.E.M. of six experiments. The asterisk indicates a significant difference (P<0.05) compared to CHOdhfr cells.

The inverse agonist cimetidine dose-dependently reduced the elevated basal cAMP level of CHOH2 cells to 52±3% (mean±S.E.M., n=12) (Table 1 ). Cimetidine also inhibited the forskolin-induced cAMP production in CHOH2 cells, whereas in CHOdhfr cells cimetidine did not exhibit any negative intrinsic activity (Fig. 2 A). VUF8299, a cimetidine analogue with strongly reduced H2 receptor affinity [19], showed up to 10 μM no negative intrinsic activity in CHOH2 cells (Fig. 2B). The pIC50 values for cimetidine, as determined by inhibition of either the basal cAMP level or forskolin-induced (1 or 10 μM) cAMP formation, were not significantly different (Table 1). Moreover, these values correspond with the observed pKi value of 6.2±0.2 (mean±S.E.M., n=3), as determined from [125I]APT displacement studies. The percentage maximal inhibition by the inverse agonist decreased at higher concentrations of forskolin (Table 1), as expected from the data in Fig. 1. Besides cimetidine, ranitidine and famotidine were also tested for their effect on the forskolin response in CHOH2 cells. The pIC50 values for these inverse agonists, determined by inhibition of the forskolin response, also correspond well with the values determined by inhibition of the basal cAMP level (Table 2 ). The negative intrinsic activity (αinv) of cimetidine determined by inhibition of the forskolin response in CHOH2 cells was significantly lower than that of famotidine and ranitidine (Table 2). At the basal cAMP level no significant differences in intrinsic activity of the inverse agonists were found (Table 2).

figure image
A: Effect of cimetidine on forskolin-induced (10 μM) cAMP production in CHOdhfr (open circles) and CHOH2 cells (filled circles). The data shown represent the mean±S.E.M. of respectively four or eight experiments. B: Effect of cimetidine (filled circles) and the inactive analogue, VUF 8299 (open circles), on forskolin-induced (10 μM) cAMP production in CHOH2 cells. The data shown represent the mean±S.E.M. of respectively eight or six experiments.
Table Table 1. The pIC50 and the maximal inhibitory response of cimetidine in CHOH2 cells determined on the basal cAMP level and forskolin-induced cAMP formation
Conditions pIC50 % inhibition n
Basal 6.3±0.2 48±3 12
1 μM forskolin 6.3±0.1 41±2* 4
10 μM forskolin 6.0±0.2 36±2* 8
  • The pIC50 and the maximal inhibition were determined from the inhibition curve of cimetidine.
  • * indicates a significant difference (P<0.05) from the % inhibition of the basal cAMP level.
  • n, number of experiments.
Table Table 2. pIC50 and αinv of different inverse agonists in CHOH2 cells determined on the basal cAMP level and forskolin-induced cAMP formation
Drug Basal Forskolin (1 μM)
pIC50 αinv pIC50 αinv
Cimetidine 6.3±0.2 0.88±0.04 6.3±0.1 0.83±0.02*
Famotidine 6.7±0.2 0.88±0.05 6.7±0.3 0.93±0.04
Ranitidine 6.8±0.2 1.00±0.03 6.7±0.3 1.00±0.02
  • pIC50 and the αinv were determined from the inhibition curve of the indicated drug on either the basal cAMP level or the forskolin-induced cAMP production. The data shown represent the mean±S.E.M. of three independent experiments performed in duplicate.
  • * indicates a significant difference (P<0.05) from αinv of ranitidine which caused a maximal inhibition in this cell line.

The modulation of the forskolin response by constitutively active H2 receptors was not restricted to CHO cells. Stable expression of the H2 receptor in HEK-293 cells at a density of 1594±17 fmol/mg protein [21]resulted in an approximately 10-fold increase in the cAMP production induced by 10 μM forskolin (HEK-293 cells: 805±245 pmol/mg protein (mean±S.E.M., n=6), HEK-293H2 cells: 7471±998 pmol/mg protein (mean±S.E.M., n=6)). The increase in the basal cAMP level was approximately 15-fold (HEK-293 cells: 33±5 pmol/mg protein (mean±S.E.M., n=6), HEK-293H2 cells: 572±83 pmol/mg protein (mean±S.E.M., n=6)). The increase in the forskolin response by the constitutive activity of the H2 receptor in HEK-293H2 cells was inhibited by cimetidine (pIC50=6.0±0.1, mean±S.E.M., n=3). Furthermore, in previous studies we showed that the wild-type H2 receptor can also be functionally expressed in insect Sf9 cells [22]. Infection of Sf9 cells with recombinant baculovirus resulted in high expression levels of H2 receptor proteins (2.7 pmol/mg protein). Expression of the H2 receptor did not significantly elevate the basal cAMP level in Sf9 cells [22]. Yet, the response to 100 μM forskolin was enhanced after H2 receptor expression (53±14 pmol cAMP/106 cells, mean±S.E.M., n=3) compared to mock-infected Sf9 cells (15±4 pmol cAMP/106 cells, mean±S.E.M., n=3). Cimetidine showed no significant effect on the basal cAMP production in these cells (Fig. 3 ). However, a small, but significant, inhibition of the forskolin-induced (100 μM) cAMP production by cimetidine was observed (Fig. 3).

figure image
Effect of cimetidine (hatched bars) on basal and forskolin-induced cAMP production in Sf9 cells expressing the H2 receptor. Sf9 cells infected with recombinant baculovirus containing an epitope-tagged H2 receptor were exposed to 100 μM cimetidine, in the presence or absence of 100 μM forskolin. Basal cAMP level in Sf9 cells expressing the H2 receptor: 10.5±3.9 pmol/106 cells. The data shown represent the mean±S.E.M. of four experiments. The single asterisk indicates a significant difference (P<0.05).

Finally, we transiently expressed the thyrotropin (TSH) receptor and the constitutively activated Ile486Phe and Phe631Leu TSH receptor mutants [26]in COS-7 cells. Expression of the wild-type receptor resulted in a small, but significant elevation of the basal cAMP level compared to mock- (pSVL) transfected COS-7 cells (Fig. 4 ). As reported previously [26], the two clinically important CAM TSH receptors both caused a marked elevation of the basal cAMP level. In the presence of forskolin the constitutive activity of the wild-type TSH receptor became quite prominent and easy to detect in comparison to the minor increase in the basal cAMP level (Fig. 4). The constitutive activity of the two CAM receptors was further magnified (Fig. 4).

figure image
Basal (white bars) and forskolin-induced (10 μM) cAMP production (hatched bars) in COS-7 cells transiently transfected with pSVL (C), pSVL-TSH (WT), pSVL-TSHI486F (I486F) and pSVL-TSHF631L (F631L). The data shown represent the data of a typical experiment, from three independent experiments, each performed in triplicate.

4 Discussion

In this study we show that the modulation of forskolin response by constitutively active Gs-coupled receptors offers a simple way to investigate inverse agonism at wild-type Gs-coupled receptors. Stable expression of the H2 receptor in CHO cells both enhanced basal and forskolin-induced cAMP production, probably by increasing the level of free Gαs subunits. The increase in forskolin-induced cAMP production was maximal at a concentration of 1 μM. At higher forskolin concentrations the relative increase in forskolin-induced cAMP production decreased due to the increased response in CHOdhfr cells. If the increase in forskolin-induced cAMP production is a direct effect of the constitutive activity of the H2 receptor, inverse agonists should, as shown at the basal cAMP level [18], be able to inhibit the H2 receptor-mediated increase of the forskolin-induced cAMP production. Indeed, a dose-dependent inhibition of the forskolin-induced cAMP production by cimetidine was observed. As expected, the maximal inhibition by cimetidine in CHOH2 cells was slightly higher at a forskolin concentration of 1 μM than at a concentration of 10 μM. The pIC50 values of cimetidine determined from the inhibition of the forskolin responses were not significantly different from the values determined from the inhibition of the basal cAMP level (Table 1). In addition, investigation of the inverse agonistic properties of famotidine and ranitidine in CHOH2 cells revealed no significant differences in inverse agonistic behaviour determined in either the absence or presence of forskolin (Table 2). Modulation of the forskolin response by constitutively active H2 receptors thus represents a useful tool to study inverse agonism at the H2 receptor. The method seems quite sensitive as in CHOH2 cells cimetidine could be classified as a partial inverse agonist at forskolin-stimulated cAMP levels. At the basal cAMP level no differences in negative intrinsic activity (αinv) between cimetidine, famotidine and ranitidine could be detected. This is probably due to the fact that the forskolin-induced cAMP levels are about 20-fold higher than basal cAMP levels and small differences can consequently be detected easier.

The increase of the forskolin response by H2 receptor expression is not limited to a particular cell type. Compared to CHOH2 cells the constitutive activity of the H2 receptor was more pronounced in HEK-293 cells. Stable expression of the H2 receptor in this cell line resulted in a strongly enhanced basal and forskolin-induced cAMP production, that was inhibited by the inverse agonist cimetidine with comparable potency as observed in CHOH2 cells. In insect Sf9 cells the H2 receptor is not very efficiently coupled to adenylyl cyclase activation [22]. Consequently, in these cells no inverse agonism by cimetidine could be detected at the basal cAMP level. Nevertheless, this inverse agonist was able to decrease the forskolin-induced cAMP production in Sf9 cells expressing the H2 receptor. These data further support the high sensitivity of the described method to measure inverse agonism.

Importantly, transient expression of the Gs-coupled TSH receptor and constitutively active TSH mutant receptors [26]in COS-7 cells also modulated the forskolin response. A clear constitutive activity of the wild-type TSH receptor was observed after evaluation of the forskolin responses. Our method to study inverse agonism at the H2 receptor may thus be of general use in the study of inverse agonism at Gs-coupled receptors. Further evidence supporting this suggestion is available in literature [27-29]. Takuma et al. [29]showed e.g. that propranolol reduces the forskolin response in rat parotid acinar cells endogenously expressing the β1 receptor. This inhibition was not attributed to inverse agonism as this phenomenon was relatively new at that moment. From experimental data obtained with heterologous expression systems and/or CAM receptors, the modulation of the forskolin response by constitutively active Gs-coupled receptors can often be deduced [30, 31]. In line with our findings, constitutively active Gi-coupled receptors also affect forskolin response [32, 33]. As expected, the forskolin response is modulated in a way opposite to that of Gs-coupled receptors and consequently the elevation of the forskolin response by inverse agonists can be regarded as a related assay for inverse agonism at Gi-coupled receptors [32, 33].

In conclusion, we observed that constitutively active Gs-coupled receptors (H2 and TSH receptor) not only enhance basal cAMP production but also increase the forskolin response in a variety of cell lines (CHO, HEK-293, COS-7 and Sf9 cells). Inverse agonists are able to reduce the increased forskolin response. Thorough investigation of the inverse agonistic properties of cimetidine, famotidine and ranitidine in CHOH2 cells revealed no significant differences in inverse agonistic potency determined in either the absence or presence of forskolin. Inhibition of forskolin responses may thus be very useful to investigate inverse agonism at wild-type receptors and eliminate the need of using CAM receptors.

Acknowledgements

The authors thank Dr. G. Vassart (Université Libre de Bruxelles, Belgium) for the kind gift of cDNAs encoding the wild-type and CAM TSH receptors and Dr. M. Beukers (Leiden/Amsterdam Center for Drug Research, Leiden) for help with the baculovirus expression.