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Endocrinology Vol. 141, No. 9 3397-3402
Copyright © 2000 by The Endocrine Society

ARTICLES

Adenosine Is an Agonist of the Growth Hormone Secretagogue Receptor

Søren Tullin, Birgit Sehested Hansen, Michael Ankersen, Jette Møller, Karen Arevad von Cappelen and Lars Thim

Health Care Discovery, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark

Address all correspondence and requests for reprints to: Søren Tullin, Health Care Discovery, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark. E-mail: stu{at}novo.dk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Growth hormone secretagogues (GHSs) are synthetic compounds that induce GH release in several species, including man. The aim of the current study was to identify hypothalamic GHS receptor (GHS-R) agonists. This led to the discovery of adenosine as a GHS-R agonist. We demonstrate that adenosine as well as the A1 adenosine receptor agonist N6-R-phenylisopropyladenosine (R-PIA) induce calcium responses, with EC50 values of 50 nM and 0.5 nM, respectively, in cells which express recombinant human GHS-R. However, neither compound induces a calcium response in nontransfected cells. Binding experiments show that adenosine and the GHS compound MK-0677 bind to membranes from GHS-R expressing cells with nearly identical Bmax values (2.6 ± 0.1·10-10 mol/mg protein for adenosine and 2.0 ± 0.3·10-10 mol/mg protein for MK-0677). However, no binding to membranes from nontransfected cells could be detected. Furthermore, we show that the IC50values for inhibition of the adenosine, R-PIA, and GHS induced calcium responses by the GHS-R antagonist [D-Arg1, D-Phe5, D-Trp7,9, D-Leu11]-substance P are similar. These findings strongly suggest that adenosine and R-PIA are agonists of the GHS-R. Interestingly, neither adenosine nor R-PIA were able to induce GH release from rat pituitary cells in vitro. The implications of the latter finding is discussed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RELEASE OF GH from the anterior pituitary gland is regulated by at least two hypothalamic hormones the GH-releasing hormone (GHRH) and somatostatin (1). Moreover, a new class of GH secretagogues (GHSs) based on small synthetic peptides that induce GH release in vivo (2), has emerged. Examples of such GHSs include the D-amino acid containing peptide GHRP-6 (His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) and its nonpeptide mimetic MK-0677 (3, 4). It was not until 1996 that complementary DNA (cDNA) for a receptor with high affinity for GHRP-6 and MK-0677 was cloned from pig pituitary gland (5). This receptor, denoted the GHS receptor (GHS-R), was shown to be G{alpha}11 coupled with 7 transmembrane (7TM) spanning domains. The human, chimpanzee, bovine, rat, and mouse genes have also been cloned. These were shown to be 93–98% identical to the porcine receptor at the amino acid level (5). Recently, a GHS-R agonist (ghrelin), which induce GH release in vivo and in vitro, was isolated from stomach (6).

In situ hybridization studies have demonstrated high expression of GHS-R in the arcuate nucleus of the hypothalamus, the CA2 and CA3 regions of the hippocampus, substantia nigra, ventral tegmental area, dorsal, and median raphe nuclei (5, 7). Furthermore, a low and diffuse expression of GHS-R has been detected in the pituitary gland (5, 7). Recent reports have also described high affinity binding sites for hexarelin with low affinity for MK-0677 in the pituitary gland, the brain (8, 9), and the heart (10, 11). These are presumably nonGHS-R binding sites because GHS-R has high affinity for MK-0677.

Apart from inducing GH release, many GHSs stimulate feeding (12, 13, 14). Furthermore, intracerebroventricular (icv) injection of some GHSs induce feeding without affecting GH release (14). As GHS-R and neuropeptide Y (NPY) are expressed in the same hypothalamic neurons (15) and because c-Fos is induced in NPY neurons following GHS administration (16), it is a possibility that the (hypothalamic) GHS-R mediates the orexigenic effects of the GHSs.

The aim of the current study was to identify hypothalamic GHS-R agonist(s). Because GHSs induce an increase in the intracellular Ca2+ concentration in cells that express recombinant GHS-R (5), we used a Fura-2-based calcium assay in conjunction with BHK cells that express human GHS-R to screen a fractionated rat hypothalamic extract. This approach led to the discovery of adenosine as a high affinity agonist for GHS-R.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue extraction
Hypothalamus tissue (2.68 g in total) was removed from eighteen 250 g female Wistar rats and frozen on dry ice. This was followed by homogenization for 3 min at 4 C in 25 ml of ethanol/0.7 M HCl (3/1, vol/vol), using an Ultra-Turrax type TP18/10 homogenizer (IKA, Staufen, Germany) equipped with a 10N head, and stirring at 4 C for 18 h. After centrifugation at 18,000 rpm for 30 min, the supernatant was concentrated to 20% of the original volume by vacuum centrifugation. The concentrated supernatant was lyophilized and extracted with 20 ml 2 M acetic acid. After centrifugation at 18000 rpm for 30 min, the supernatant was lyophilized. The lyophilized powder was redissolved in 50 ml PBS-buffer (50 mM sodium phosphate, 100 mM NaCl, pH 7.2) containing a mixture of protease inhibitors (Complete, Roche Molecular Biochemicals, Mannheim, Germany).

HPLC purification
A volume of 250 µl of the hypothalamic extract was injected onto a 4.6 x 250 mm Vydac 218TP54 reverseD-phase C18 HPLC column (The Separation Group, Hesperia, CA), which had been equilibrated at 30 C at a flow rate of 1.0 ml/min of 0.1% (vol/vol) TFA. The concentration of acetonitrile in the eluting solvent was increased from 0% to 30% (vol/vol) over 30 min and the UV absorption was measured at 214 nm. Fractions corresponding to individual UV-peaks were collected and analyzed in the calcium imaging assay. All UV-peaks in the chromatogram were scanned from 190–600 nm.

Transfection
Lipofectamine (Life Technologies, Inc., Rockville, MD) was used for transfection of HEK293 and BHK cells with a GHS-R (human) expression vector (based on pcDNA3.1 from Invitrogen, Carlsbad, CA). The transfected cells are referred to as stably or transiently transfected BHK/GHS-R cells or transiently transfected HEK293/GHS-R cells. GHS-R expressing cells were cultured in Lab-Tek chambered coverglasses (Nalge Nunc International, Naperville, IL).

Calcium imaging
Before the experiment cells were loaded with the Ca2+ sensitive dye, Fura-2-AM (Molecular Probes, Inc., Eugene, OR), according to standard procedures. The chambers were placed on a temperature-regulated microscope stage and kept at 37 C. Fluorescence images were acquired using the MetaFluor software package (Universal Imaging Corp., West Chester, PA) together with a Carl Zeiss Axiovert 100S inverted microscope (Carl Zeiss, Oberkochen, Germany) and a Princeton MicroMAX-5–1300Y CCD camera (Princeton Instruments, Trenton, NJ). The microscope was also equipped with a 530 nm ± 15 nm emission filter, a 500 nm dichroic mirror (DELTA Light and Optics, Lyngby, Denmark) and a filterwheel (LUDL electronic products, Hawthorne, NY) harboring 340 nm ± 10 nm and 380 nm ± 10 nm excitation filters (DELTA Light and Optics). Image pairs were acquired every 3 sec. After acquisition of 12–14 images, the cells were stimulated with HPLC fractions, adenosine, R-PIA, or MK-0677. In each experiment, the Fura-2 ratio was followed in 50 cells. A normalized ratio was generated for each cell by dividing the Fura-2 ratio at time t with the ratio at time zero. The data shown represent the average normalized Fura-2 ratio for 50 cells in a typical experiment. The ordinate in dose-response experiments represent the percent increase in Fura-2 ratio at the peak of the response (e.g. ~60% for the fraction 57 curve shown in Fig. 1Go). Dose-response curves were generated using the nonlinear regression feature of the GraphPad Software, Inc. Prism software package (GraphPad Software, Inc., San Diego, CA) and potencies (EC50 values) were calculated from these curves. All experiments were repeated at least three times.



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  		Figure 1. Reverse phase HPLC (Vydac 218TP54 column) of a crude hypothalamic extract. A, Entire chromatogram. B, Details of the chromatogram from 6 to 9 min corresponding to the inset. C, UV-absorption spectrum of HPLC fraction no. 57.

 
Receptor binding assay
Ligand binding to receptor was assayed as previously described (17). Briefly, crude membranes from stably transfected BHK/GHS-R cells were suspended at 0.25 mg protein/ml in homogenization buffer (2.5 mM Tris-base, 2.5 mM EDTA, 10 mM MgCl2 and 30 µg/ml bacitracin). In a microtiter plate 10 µl membrane suspension was combined with either 35S-MK-0677 (Amersham Pharmacia Biotech, Essex, UK) or 2-3H-adensoine (Amersham Pharmacia Biotech) as well as binding buffer (2.5 mM Tris-base, 2.5 mM EDTA, and 10 mM MgCl2) to a total volume of 250 µl. Nonspecific binding was determined by adding 10 µM MK-0677 or 10 µM adenosine to the assay. The mixture was subsequently incubated at 30 C for 60 min, followed by application of the samples to GF/B filters (Whatman, Kent, UK), which had been pretreated with 0.5% polyethylenimine for 60 min. The filters were subsequently washed in 0.9% NaCl and counted using an Optiphase ‘HiSafe 3' counter (Wallac, Inc. Turku, Finland). Specific binding was determined as the difference between total binding and nonspecific binding (binding in the presence of 10 µM unlabelled ligand). Binding curves were generated using either the nonlinear regression or Scatchard plot feature of the GraphPad Software, Inc. (San Diego, CA) Prism software package. KD and Bmax were derived from the Scatchard plots.

GH release assay
Rat pituitary cells were isolated and stimulated as previously described (17). Moreover, the adenosine deaminase inhibitor erythro-9-(2-hydroxyl-3-nonyl) adenine (EHNA) was included in some experiments (also during the preincubation).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A crude hypothalamic extract was subjected to HPLC fractionation (Fig. 1Go) and fractions corresponding to the individual UV-peaks were collected. Individual fractions were subsequently lyophilized and redissolved in assay buffer. When applied to Fura-2 loaded GHS-R expressing BHK cells (BHK/GHS-R 1A cells), only fraction 57 (Fig. 1BGo) induced a significant response whereas the neighboring fractions were inactive (Fig. 2Go). As the UV absorption spectrum of the material in fraction 57 (Fig. 1CGo) was identical to the UV absorption spectrum of adenosine, this compound was analyzed in the same HPLC setup that was used for fractionation of the hypothalamic extract. Analysis of a mixture of adenosine and the hypothalamus extract showed coelution of adenosine and fraction 57 (data not shown).



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  		Figure 2. Effects of HPLC fractions 56–58 on the intracellular calcium concentration in GHS-R expressing cells. Stably transfected and Fura-2 loaded BHK/GHS-R cells were stimulated at 35 sec with HPLC fraction 56 (closed circles), fraction 57 (open triangles), or fraction 58 (open circles). The data shown are representative of at least three independent experiments.

 
When applied to Fura-2-loaded BHK/GHS-R cells, adenosine induced a response with a potency (EC50) of approximately 50 nM (Fig. 3AGo). In contrast, 1 µM adenosine or lower concentrations had no effect on the untransfected BHK cells, whereas 5 µM adenosine induced a weak response (Fig. 3AGo). We also tested the effect of the A1 adenosine receptor agonist N6-R-phenylisopropyladenosine (R-PIA). This compound induced a response with an EC50 of 0.5 nM when applied to Fura-2 loaded BHK/GHS-R cells (Fig. 3BGo). Furthermore, 100 µM of the A1 adenosine receptor antagonist caffeine was unable to inhibit the response to 0.5 nM R-PIA (data not shown). As observed for adenosine, R-PIA had no effect on the untransfected BHK cells (Fig. 3BGo). When applied to Fura-2 loaded BHK/GHS-R cells MK-0677 induced a response with an EC50 of approximately 0.7 nM (Fig. 3CGo). Moreover, MK-0677 had no effect on the untransfected BHK cells.



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  		Figure 3. Concentration-response for the effects of adenosine (A), R-PIA (B), and MK-0677 (C) on Fura-2 loaded and stably transfected BHK/GHS-R cells. The ordinate represent the percent increase in Fura-2 ratio at the peak of the response (e.g. ~60% for the fraction 57 curve shown in Fig. 2Go). Results are expressed as mean ± SEM (n = 3–4).

 
We also studied the effects of adenosine and MK-0677 on the intracellular calcium concentration in transiently transfected cells. Both 200 nM adenosine and 10 nM MK-0677 induced a calcium response in transiently transfected HEK293 and BHK cells (Fig. 4Go). The weaker response to MK-0677 and adenosine of the transiently transfected BHK/GHS-R cells was the result of a low transfection efficiency, verified by parallel transfection with a green fluorescent protein expression vector (data not shown), and consequently a lower fraction of responding cells. As for untransfected BHK cells, the untransfected HEK293 cells did not respond to 1 µM adenosine nor 100 nM MK-0677 (data not shown).



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  		Figure 4. Effects of adenosine and MK-0677 on the intracellular calcium concentration in transiently transfected GHS-R expressing cells. Transiently transfected and Fura-2 loaded BHK/GHS-R cells (circles) or HEK293/GHS-R cells (triangles) were stimulated at 35 sec with 10 nM MK-0677 (open symbols) or 200 nM adenosine (closed symbols). The data shown are representative of at least three independent experiments.

 
Pretreatment of stably transfected and Fura-2 loaded BHK/GHS-R cells for 30 sec with 1 µM of the GHS-R antagonist [D-Arg1, D-Phe5, D-Trp7,9, D-Leu11]-substance P completely inhibited the response to 100 nM adenosine, 1 nM R-PIA and 5 nM MK-0677 (Fig. 5Go). In contrast, pretreatment with 0.1 µM of the antagonist had little if any effect on the R-PIA induced response and no effect on the adenosine and MK-0677 induced responses (Fig. 5AGo). At 0.33 µM, the GHS-R antagonist had an intermediary effect on the adenosine and MK-0677 induced responses, whereas the R-PIA response was completely inhibited (Fig. 5Go). It should be noticed that the GHS-R antagonist alone had little if any effect on the intracellular calcium concentration in BHK/GHS-R cells, i.e. 1 µM of the GHS-R antagonist induced a -3% ± 4% change in the Fura-2 ratio (n = 8).



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  		Figure 5. Effect of the GHS-R antagonist [D-Arg1, D-Phe5, D-Trp7,9, D-Leu11]-substance P on the calcium responses induced by adenosine, R-PIA, and MK-0677. Fura-2 loaded BHK/GHS-R cells were pretreated for 30 sec with the indicated concentrations of GHS-R antagonist followed by stimulation with 100 nM adenosine (open bars), 5 nM MK-0677 (hatched bars) or 1 nM R-PIA (closed bars). The ordinate represent the percent increase in Fura-2 ratio at the peak of the response (e.g. ~60% for the fraction 57 curve shown in Fig. 1Go). Results are expressed as mean ± SEM (n = 3–5).

 
In contrast to the effect of the GHS-R antagonist, the nonselective adenosine receptor antagonist theophylline (at 200 µM) had no effect on the adenosine or MK-0677 induced calcium responses in BHK/GHS-R cells (data not shown).

Binding experiments showed specific binding of 3H-adenosine (KD = 90 ± 10 nM and Bmax= 2.6 ± 0.1·10-10 mol/mg protein) to membranes from stably transfected BHK/GHS-R cells (Fig. 6AGo). In contrast, no binding of 3H-adenosine to membranes from untransfected BHK cells could be detected (Fig. 6AGo). Binding of 35S-MK-0677 to membranes from BHK/GHS-R cells gave a KD-value of 0.4 ± 0.1 nM and a Bmax of 2.0 ± 0.3·10-10 mol/mg protein, whereas no binding to membranes from untransfected BHK cells could be detected (Fig. 6BGo).



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  		Figure 6. Binding of 3H-adenosine and 35S-MK-0677 to membranes from stably transfected BHK/GHS-R cells and nontransfected BHK cells. A, Binding of 3H-adenosine to membranes from stably transfected BHK/GHS-R cells (closed diamonds) or wild-type BHK cells (open circles). Nonspecific binding was determined using 10 µM adenosine. Results are expressed as mean ± SEM (n = 4). B, Binding of 35S-MK-0677 to membranes from stably transfected BHK/GHS-R cells (closed diamonds) or nontransfected BHK cells (open circles). Nonspecific binding was determined using 10 µM MK-0677. Results are expressed as mean ± SEM (n = 5). Insets show Scatchard plots of the BHK/GHS-R binding data.

 
We were unable to demonstrate any GH-releasing effects of adenosine and R-PIA when they were applied to primary rat pituitary cells (Fig. 7AGo). The lack of response to adenosine was not due to degradation of the compound because culture supernatants from the incubations with 1 µM and 10 µM adenosine, sampled at the end of the incubation, induced a significant calcium response when applied to Fura-2 loaded BHK/GHS-R cells (data not shown). Furthermore, in the presence of 25 µM of the adenosine deaminase inhibitor EHNA, adenosine still had no effect on the GH release by the primary rat pituitary cells (Fig. 7AGo). We also tested whether adenosine was able to interfere with the GH releasing effects of GHRH or GHRP-6. This showed that adenosine in a concentration range from 0.1 nM to 10 µM had no effect on GH release induced by 10 nM GHRP-6 or 1 nM GHRH (Fig. 4BGo).



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  		Figure 7. Effect of MK-0677, adenosine and R-Pia on GH release by primary rat pituitary cells. A, Concentration-response, measured in a GH release assay using primary rat pituitary cells, for MK-0677 (closed squares), R-PIA (open diamonds) as well as adenosine with (closed inverted triangles) or without (closed triangles) EHNA. Results are expressed as mean ± SEM (n = 3–5). B, Concentration-response curves for adenosine measured in a GH release assay using primary rat pituitary cells. The cells were incubated with either adenosine alone (open inverted triangles), adenosine plus 10 nM GHRP-6 (closed squares), or adenosine plus 1 nM GHRH (closed triangles). Results are expressed as mean ± SEM (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings provide evidence that adenosine is an endogenous ligand of the GH secretagogue receptor (GHS-R). Both adenosine and the GH secretagogue MK-0677 induced an increase in the intracellular calcium concentration in BHK and HEK293 cells, which expressed recombinant human GHS-R. However, the two compounds had no effect on the intracellular calcium concentration in untransfected BHK or HEK293 cells. Furthermore, both compounds were able to bind to membranes from GHS-R expressing BHK cells, whereas no binding was observed to membranes from untransfected BHK cells. The KD values for binding of MK-0677 and adenosine to GHS-R were 0.2 nM and 90 nM, respectively. These values are comparable with the EC50 values for the calcium responses in GHS-R expressing BHK cells (about 50 nM for adenosine and approximately 0.7 nM for MK-0677). Because the affinities of adenosine receptors for adenosine range from 100 nM in case of the A2A receptor to about 10 µM in case of the A2B receptor (18), GHS-R has a comparatively high affinity for adenosine. The binding experiments, furthermore, showed that the number of binding sites for adenosine and MK-0677 was nearly identical (Bmax = 2.6 ± 0.1·10-10 mol/mg protein for adenosine and Bmax = 2.0 ± 0.3·10-10 mol/mg protein for MK-0677). It should, finally, be noticed that the IC50values for inhibition of the adenosine, R-PIA, and MK-0677 induced calcium responses by the GHS-R antagonist [D-Arg1, D-Phe5, D-Trp7,9, D-Leu11]-substance P were similar. These findings strongly suggest that adenosine and R-PIA are agonists of the GHS-R. This conclusion correlates with recent findings by R. G. Smith and co-workers, which identified adenosine as an agonist of the GHS-R (R. G. Smith, personal communication).

To date, four different mammalian adenosine receptor subtypes (denoted A1, A2A, A2B, and A3) have been identified. The homology between subtypes is relatively low, with e.g. 41% identity and 50% similarity at amino acid level between the human A2B (GenBank Accession No. M97759) and A3 receptors (GenBank Accession No. l20463). For comparison, the human GHS-R (GenBank Accession No. U60179) and its most homologous adenosine receptors (the A2b and A3 receptors) exhibit approximately 28% identity and 38% similarity. Overall homology considerations therefore neither support nor argue against the possibility that GHS-R is a novel adenosine receptor subtype.

We also tested whether adenosine had an effect on GH secretion by primary rat somatotrophs. In conclusion, adenosine neither stimulated GH secretion on its own nor did it affect GHRP-6 or GHRH-stimulated GH release. The fact that adenosine is an agonist on GHS-R and yet has no effect on GH release could indicate that GHS-R is not mediating the GH-releasing effects of the GHSs on primary somatotrophs. Accordingly, a yet undiscovered (pituitary) GHS receptor should mediate the GH releasing effects of ghrelin and GHSs. As a different possibility, alternative splicing, posttranslation modification(s) or two binding sites on GHS-R with coupling to two separate signaling pathways could result in two functionally distinct GHS-R versions. One form (e.g. the pituitarian) could then be activated by ghrelin and GHSs, whereas the other (e.g. the hypothalamic) could be activated by adenosine, GHSs, and perhaps ghrelin. As support for the existence of another GHS receptor recent reports have described binding sites with high affinity for the GHS hexarelin and low affinity for MK-0677 in the pituitary gland and the brain (8, 9). These are presumably nonGHS-R binding sites because GHS-R has high affinity for MK-0677.

The A1 adenosine receptor agonist R-PIA [Kd ~ 5 nM on the A1 receptor (19)] is a very potent GHS-R agonist with an EC50 ~ 0.5 nM. Moreover, the A1 receptor antagonist caffeine is unable to block the effect of R-PIA on GHS-R expressing cells. Interestingly, R-PIA in doses from 0.1 µg and upwards induce feeding in rats upon icv administration and again caffeine is unable to block or inhibit the response (20). These findings together with results showing increased feeding upon icv injection of GHSs (13, 14) could indicate that the hypothalamic GHS-R mediates the feeding responses induced by adenosine, R-PIA, and GHSs.


   Acknowledgments
 
We are grateful to Anne-Mette Petersen and Anni Demandt for excellent technical assistance and to Dr. Esper Boel and Dr. Nils Billestrup for critical comments.

Received January 19, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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