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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2010 Jan 28;159(4):820–830. doi: 10.1111/j.1476-5381.2009.00584.x

Lack of effect of the α2C-adrenoceptor Del322-325 polymorphism on inhibition of cyclic AMP production in HEK293 cells

MD Montgomery 1, DB Bylund 1
PMCID: PMC2829208  PMID: 20128806

Abstract

Background and purpose:

The α2C-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human α2C polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells.

Experimental approach:

Human α2C wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200–2320 fmol·mg−1 protein) was measured following agonist treatment.

Key results:

Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325.

Conclusions and implications:

The α2C WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the α2C-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.

Keywords: adrenoceptor, α2C, Del322-325, polymorphism, signalling, heart failure, adenylyl cyclase

Introduction

The α2-adrenoceptors are activated by the endogenous hormone adrenaline as well as the neurotransmitter noradrenaline. Upon ligand binding, these G protein-coupled receptors (GPCRs) primarily activate Gi/Go proteins, initiating downstream signalling pathways such as inhibition of adenylyl cyclase and voltage-gated Ca2+ channels, and activation of receptor-operated K+ channels and the mitogen-activated protein (MAP) kinase cascade (Limbird, 1988; Kobilka, 1992).

The physiological functions of the three α2-adrenoceptor subtypes, α2A, α2B and α2C, have been studied in receptor knockout mice and include regulatory roles in the cardiovascular, endocrine, ocular and peripheral and central nervous systems (Kable et al., 2000). The α2C-adrenoceptor has been found to be important in locomotion (Sallinen et al., 1998b), stress responses (Sallinen et al., 1999) and the startle reflex (Sallinen et al., 1998a). This receptor also regulates the release of noradrenaline and adrenaline from chromaffin cells via a negative feedback mechanism (Gilsbach et al., 2007). Both α2A- and α2C-adrenoceptors are involved in the inhibition of neurotransmitter release from presynaptic sympathetic nerve terminals (Hein et al., 1999; Trendelenburg et al., 2003). These receptors serve as autoreceptors for both the central and peripheral nervous systems, with α2A mediating inhibition of noradrenaline release at higher stimulation frequencies and α2C mediating inhibition at lower stimulation frequencies (Hein et al., 1999). Characterization of α2A and α2C single- and double-knockout mice in the absence and presence of cardiac pressure overload (Hein et al., 1999; Brede et al., 2002) revealed the importance of α2A- and α2C-adrenoceptors for the correct regulation of catecholamine release in vivo and suggests that loss of these receptors, or their function, may be associated with heart failure. Similar studies in transgenic mice expressing only one functional copy of the α2C-adrenoceptor gene showed that, in the peripheral sympathetic nervous system, heterozygous expression of the α2C-adrenoceptor was sufficient to inhibit catecholamine release (Gilsbach et al., 2007). These mice did, however, have decreased left ventricular contractility and relaxation after transverse aortic constriction compared with wild-type (WT) animals, suggesting that they are at greater risk for developing heart failure. In the adrenal glands of heterozygous α2C mice, there was an increased release of catecholamines under basal conditions and a roughly 50% reduction in the ability to inhibit catecholamine release, suggesting that in the adrenal glands the α2C-adrenoceptor operates without a receptor reserve (Gilsbach et al., 2007).

These studies with transgenic mice have revealed the critical role of α2-adrenoceptors in the regulation of hormone and neurotransmitter release. Changes in receptor expression or function, as can occur with genetic polymorphisms, may result in physiological effects leading to various disorders. A variety of characteristics, such as agonist or antagonist binding, protein coupling, and receptor trafficking and regulation can be affected by receptor polymorphisms (Johnson and Lima, 2003; Small et al., 2003). Therefore, polymorphisms may play a role in predisposition for disease, progression of disease, response to pharmacological treatment and the inter-individual variation observed among patients (Buscher et al., 1999, Small et al., 2003; Eichelbaum and Evans, 2006).

The human α2C-adrenoceptor has a twelve nucleic acid deletion polymorphism (Del322-325) within its coding region that results in the loss of four amino acids (Gly-Ala-Gly-Pro) in the third intracellular loop (Small et al., 2000). When expressed in CHO cells, the Del322-325 receptor was reported to have decreased inhibition of membrane adenylyl cyclase activity compared with the WT receptor (Small et al., 2000). The maximum inhibition of adenylyl cyclase activity with brimonidine, also known as UK-14 304, in WT and Del322-325 receptors was approximately 68% and 31%, respectively, in cells expressing 1500–1600 fmol·mg−1, resulting in a roughly 50% reduction in signalling with the Del322-325 receptor. The 50% reduction was observed with other full and partial agonists; however, EC50 values remained similar. In cells expressing lower receptor levels (500–600 fmol·mg−1), the reduction in signalling with the Del322-325 receptor was even greater (10% maximal inhibition versus 73% for WT with adrenaline). The activation of MAP kinase and accumulation of inositol phosphate were also impaired with Del322-325. Competition binding studies with Del322-325 revealed a slight decrease in high-affinity binding of agonist compared with the WT receptor (Small et al., 2000). Because binding of a G protein to the GPCR is required for the receptor to be in its high-affinity state (Maudsley et al., 2005), the decreased high affinity binding of α2 agonists and the reduced signalling were proposed to be a result of decreased coupling of the receptor to its G protein.

To better understand the mechanism behind the reported decrease in signalling of the α2C-Del322-325 receptor in CHO cells, we attempted to replicate this reduced signalling in HEK293 cells. Because different cell lines are known to have different protein expression levels and different effector molecules present, it was important to show similar effects in a cell line other than CHO cells. Therefore, we compared the inhibition of cyclic AMP production by α2C WT and Del322-325 receptors stably expressed in HEK293 cells at various expression levels.

Methods

Cell culture

HEK293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 0.1 µg·mL−1 gentamicin and 5 µg·mL−1 amphotericin B, supplemented with 10% fetal bovine serum (FBS). Cells were grown in 5% CO2 at 37°C.

Creating WT construct and sequencing

The cDNA of the human α2C-adrenoceptor from OriGene contained a 12-nucleotide deletion in the DNA coding for the third intracellular loop and was determined to be that of the previously identified α2C-Del322-325 adrenoceptor polymorphism. A non-conserved C to T mutation at bp 1015, resulting in a serine rather than proline at position 339, was also identified in the cDNA. The α2C cDNA from Missouri S&T cDNA Resource Center was also shown to be the Del322-325 variant; however, the C to T mutation was not present. Site-directed mutatgenesis was performed on the OriGene α2C-Del322-325 adrenoceptor cDNA to convert it to the WT α2C-adrenoceptor.

To create the human WT cDNA, the Quickchange II XL kit (Stratagene, La Jolla, CA, USA) was used to add the 12 nucleotide insert, GGGGGCGGGGCC (nucleotides 963-974; accession number AF280399), into the human α2C-Del322-325 cDNA. Two 44-nucleotide primers were used: 5′-GCGGTGCGGACGGGCAGGGGGCGGGGCCGGGGGCGGCTGAGTCG-3′ (forward) and 5′-CGACTCAGCCGCCCCCGGCCCCGCCCCCTGCCCGTCCGCACCGC-3′ (reverse). For a 50 µL reaction, 20 ng of α2C-Del322-325 template and 125 ng of primers were used. The temperature programme was similar to the Stratagene protocol: 95°C for 1 min (1 cycle), 95°C for 50 s, 60°C for 50 s, 68°C for 15 min (18 cycles), followed by 68°C for 15 min (1 cycle). Dpn I treatment of the reaction product was carried out for 3 h. For conversion of the non-conserved C-T mutation at position 1015 back to the WT C nucleotide, the Quickchange II XL kit was again used. Two 25-nucleotide primers were used: 5′-GACCGCCTCCAGGTCCCCGGGGCCC-3′ (forward) and 5′-GGGCCCCGGGGACCTGGAGGCGGTC-3′ (reverse). For a 50 µL reaction, 10 ng of vector template and 125 ng of primers were used. The temperature programme was similar to the Stratagene protocol: 95°C for 2 min (1 cycle), 95°C for 50 s, 55°C for 50 s, 68°C for 15 min (18 cycles), followed by 68°C for 15 min (1 cycle).

The modified insert was excised from the OriGene pCMV6-XL4 vector using EcoR I and Xba I and ligated into pcDNA3.1(+). DNA sequencing of the WT and Del322-325 constructs was performed using a Perkin-Elmer Applied Biosystems Model 377 DNA Sequencer (Foster City, CA, USA). Sequencing was performed in the forward and reverse directions with the T7 (forward) and BGHR (reverse) primers. The following forward primers were also used: 5′-GCGCCACAGAACCTCTTC-3′, 5′-TTCCCGCCGCTGGTCTCG-3′, 5′-CAGCGCAGCGGCCGAGAG-3′ and 5′-GAGCAGCAGGCGGCGATG-3′. The sequence of our α2C-Del322-325 adrenoceptor variant was identical to the sequence published in GenBank (accession number AAG28077). Our WT α2C-adrenoceptor sequence was also identical to the WT sequence in GenBank (accession number NP_000674).

Transfection

Human α2C WT and Del322-325 (Missouri S&T cDNA Resource Center, Rolla, MO, USA) adrenoceptor cDNAs were transformed into competent bacterial cells for amplification and then purified by Qiagen Maxi DNA kit (Valencia, CA, USA). Each cDNA was stably transfected into HEK293 cells using the calcium phosphate method. Briefly, 1 µg DNA was added to a 1:10 solution of calcium chloride and nanopure water and mixed. This solution was added dropwise to an equal volume of 2X HEPES buffer with constant mixing. The combined solution was incubated at room temperature for 20 min. The calcium phosphate/DNA solution was added dropwise to HEK293 cells that were approximately 70% confluent with gentle mixing, and the cells were then incubated for 6 h in 5% CO2 at 37°C. Cells were then washed twice and DMEM containing 0.1 µg·mL−1 gentamicin, 5 µg·mL−1 amphotericin B and 10% FBS was added. Stable receptor transfections were selected for and maintained in medium containing 200 µg·mL−1 G418. Individual clones of α2C WT and Del322-325 were isolated by limiting dilution and maintained in selection media (DMEM containing 0.1 µg·mL−1 gentamicin, 5 µg·mL−1 amphotericin B, 10% FBS and 200 µg·mL−1 G418).

Membrane preparation

Cells were grown to confluence in 150 mm dishes. The medium was aspirated and 20 mL ice-cold phosphate-buffered saline was added to each dish. Cells were scraped from the dish and transferred to a 50 mL conical tube. Cells were centrifuged at 1400 g for 10 min. The supernatant was discarded and the cells resuspended in 1.5 mL ice-cold 50 mM sodium phosphate (NaPO4) buffer. Cells were homogenized for 15 s with an Ultra Turrax (A. Daigger & Company, Vernon Hills, IL, USA) at setting 3. The solution was transferred to Sorvall tubes and cells were centrifuged at 44 300 g for 10 min. The pellets were resuspended in 1.5 mL ice-cold 50 mM NaPO4buffer and homogenized for 15 s at setting 3. Membranes were centrifuged at 44 300 g for 10 min. The supernatant was discarded and cell membrane pellets were stored at −80°C.

Saturation binding

Saturation binding assays were performed as described previously (Deupree et al., 1996) with some modification. Briefly, 970 µL of cell membranes of clones stably expressing α2C WT or Del322-325 receptors in 25 mM buffer, pH 7.4 (either NaPO4 or Tris-HCl, 10 mM MgSO4 and 0.5 mM EDTA) were incubated without (total binding) or with (non-specific binding) 10 µL noradrenaline (0.2 mM final concentration) and 20 µL of various concentrations of [3H]RX821002 for 1 h with mixing at 30 min. Six concentrations of [3H]RX821002 were used over a 100-fold range. Reaction mixtures were filtered through GF/B glass-fibre filter strips using a 48-well Brandel Cell Harvester (Gaithersburg, MD, USA) to terminate the reactions. The tubes and filters were rinsed three times with 5 mL ice-cold 50 mM Tris HCl pH 8.0. Filters were then placed in scintillation vials with 5 mL Econo-safe liquid scintillation cocktail and quantified in a liquid scintillation counter the next day. Duplicate tubes were used for total and non-specific binding, with 0.2 mM noradrenaline used to determine non-specific binding. Specific binding was calculated as total binding minus non-specific binding. The specific activity of [3H]RX821002 was 49 Ci·mmol−1 for experiments using NaPO4 buffer and 60 Ci·mmol−1 for experiments using Tris buffer. Kd and Bmax values were determined using non-linear regression analysis with Prism 4 software (GraphPad, San Diego, CA, USA). Kd values were compared using an unpaired t-test. The total amount of protein present in the binding assays was determined by protein assays using the BCA protein assay kit (Pierce, Rockford, IL, USA). The amount of membrane protein per sample used for binding varied between clones, with high-expressing clones requiring less protein than low-expressing clones in order to bind less than 10% of the total added radioligand. For the saturation binding assays, between 0.034 and 0.114 mg protein per assay tube were used. Saturation binding assays were performed on non-transfected HEK293 cells to determine receptor expression level. These cells were found to contain 8 ± 2 fmol·mg−1 receptor (n= 3).

Competition binding

Competition binding assays were performed as described previously (Deupree et al., 1996) with some modification. Stably transfected clones, one WT clone (Bmax= 1460 fmol·mg−1 protein) and one Del322-325 clone (Bmax= 2320 fmol·mg−1 protein), were used for the standard competition binding experiments (one-site binding). Cell membranes containing α2C WT or Del322-325 receptors in 25 mM NaPO4buffer, pH 7.4 were incubated with various concentrations of agonist in the presence of [3H]RX821002 (specific activity = 49 Ci·mmol−1) for 1 h with mixing at 30 min (final volume of 1.0 mL). Agonists used were noradrenaline, clonidine and brimonidine (UK-14 304). Eleven drug concentrations (one to two per log unit) and one blank (water) were used, giving a total of 12 conditions, and experiments were performed in duplicate. The concentration of [3H]RX821002 used was 0.8 ×Kd. Data were analysed with Prism 4 (GraphPad) software using a one-site competition curve to determine IC50 values and a sigmoid analysis with a variable slope to determine Hill slopes. The Ki values were calculated from the IC50 values by the Cheng–Prusoff equation (Cheng and Prusoff, 1973). Mean pKi values were calculated from pKi values obtained from individual experiments.

For experiments designed to detect high- and low-affinity agonist binding, competition binding assays were performed as described previously (Small et al., 2000) with some modifications. Stably transfected clones, one WT clone (Bmax= 170 fmol·mg−1 protein) and one Del322-325 clone (Bmax= 140 fmol·mg−1 protein), were used for these experiments. Cell membranes containing α2C WT or Del322-325 receptors in 25 mM Tris-HCl, pH 7.4, 10 mM MgSO4, 0.5 mM EDTA were incubated with [3H]RX821002 (specific activity = 60 Ci·mmol−1) and 22 concentrations of agonist in the absence or presence of 100 µM Gpp(NH)p for 1 h with mixing at 30 min (final volume of 1.0 mL). Agonists used were noradrenaline, clonidine and brimonidine, with experiments performed in duplicate. Two blank (water) and 22 drug concentrations were used, for a total of 24 points. Drug concentrations ranged from 10 pM to 300 µM. Two drug concentrations per log unit were used at low (<3 nM) and high (>30 µM) ends of the curve, while four drug concentrations per log unit were used for the rest of the curve (10 nM to 10 µM). The concentration of [3H]RX821002 used was approximately 0.8 ×Kd (see Table 1). Data were analysed with Prism 5 software (GraphPad) using a global one- or two-site competition curve. For the global fit analysis, Prism 5 uses the Kd and the concentration of the radioligand to calculate shared pKi and RH (percentage of receptors in the high-affinity state) values and compares the fit of the data to one-site or two-site models using the extra sum-of-squares F test.

Table 1.

Affinity of [3H]RX821002 for human α2C WT and Del322-325 receptors in NaPO4 and Tris buffers

Wild type
 Del322-325
 P
Kd±SEM (pM) n Kd±SEM (pM) n
NaPO4 buffer 263 ± 8 26 281 ± 8 19 0.51
Tris buffer 1560 ± 140 7 1151 ± 101 4 0.36
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Saturation binding assays were performed on HEK293 cell membranes of clones stably expressing various levels of WT or Del322-325 receptors. Experiments were performed in either 25 mM NaPO4 buffer, pH 7.4 or 25 mM Tris-HCl, pH 7.4, 10 mM MgSO4, 0.5 mM EDTA buffer. Data were analysed with GraphPad Prism software using nonlinear regression analysis, from which Kd values were determined. Kdvalues from all experiments were compared using an unpaired t-test.

Inhibition of cyclic AMP

The inhibition of forskolin-stimulated cyclic AMP production in intact cells was measured as described previously (Shimizu et al., 1969; Johnson and Toews, 1990) with some modification. HEK293 cells stably expressing either α2C WT or Del322-325 receptors were grown in poly-D-lysine-coated 6-well plates. When the cells were approximately 80% confluent, each well was rinsed once with HEPES-buffered DMEM and then 1 mL medium containing 2 µCi·mL−1[3H]adenine was added. The plates were then incubated at 37°C in a non-CO2 incubator for 1 h, after which each well was rinsed once with HEPES-buffered DMEM. The medium was aspirated and 1 mL of medium containing 30 µM forskolin alone or with various concentrations of clonidine or brimonidine (but without a phosphodiesterase inhibitor) was added to each well. Plates were incubated at 37°C in a non-CO2 incubator for 5 min, after which the stimulation solution was aspirated and 1 mL 5% trichloracetic acid/1 mM cyclic AMP was added to stop the reaction. The solution from each well was passed over Dowex and alumina columns to isolate the [3H]ATP and [3H]cyclic AMP as described previously (Johnson and Toews, 1990) and the [3H]ATP and [3H]cyclic AMP were quantified. Experiments were performed in triplicate for each condition. Data were expressed as percent conversion of [3H]ATP to [3H]cyclic AMP and analysed with Prism 5 (GraphPad) using sigmoidal dose-response curves to obtain pEC50 values. Data were normalized to forskolin-stimulated cyclic AMP production and percent inhibition was calculated as 100-(bottom of curve). Mean pEC50 and mean percent inhibition values were derived from sigmoidal dose-response curves of the combined data from all experiments. Binding studies with non-transfected HEK293 cells indicated a very low level of endogenous α2-adrenoceptors (<10 fmol·mg−1 protein). We were also able to detect inconsistent inhibition of forskolin-stimulated cyclic AMP production by brimonidine, confirming the presence of a low density of α2 receptors in non-transfected cells.

Statistical analysis

For binding, Kd and pKi values were analysed using an unpaired t-test, with significance at P < 0.05. For functional studies, linear regression analysis was used to determine if the slope of each line was different from zero and whether the slopes for WT and Del322-325 receptors were different, with significance at P < 0.05. Data are reported as means ± SEM.

Materials

HEK293 cells were purchased from ATCC (Manassas, VA, USA) (catalogue # CRL-1573). The human α2C-Del322-325 adrenoceptor cDNA was purchased from OriGene (Rockville, MD; catalog number TC124091) and Missouri S&T cDNA Resource Center (catalog number AR0A2C0000). The OriGene construct was in the pCMV6-XL4 vector and subcloned into EcoR I and Sal I sites. The Missouri S&T cDNA Resource Center construct was in pcDNA3.1(+) and subcloned into BamH I and Xho I sites. DMEM was purchased from Gibco, Invitrogen (Carlsbad, CA, USA). Quickchange II XL kits were from Stratagene. Qiagen Maxi DNA kits were purchased from Qiagen. GF/B glass-fiber filter strips were from Whatman (Clifton, NJ, USA). Noradrenaline and guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate [Gpp(NH)p] were purchased from Sigma-Aldrich (St. Louis, MO, USA). Brimonidine and clonidine were purchased from Research Biochemicals International (Natick, MA, USA). The BCA Protein Assay kits were purchased from Pierce. Prism versions 4 and 5 software from GraphPad.

Results

α2C WT and Del322-325 receptor clones

HEK293 cells stably transfected with α2C WT or Del322-325 receptors were characterized by saturation binding using membrane preparations to determine expression levels (Bmax) and Kd values. The affinity of [3H]RX821002 for each receptor type was determined in NaPO4 and Tris buffers (Table 1). Kd values for WT and Del322-325 did not differ in either buffer; however, Kd values were higher in Tris buffer than in NaPO4. The expression levels of three WT and five Del322-325 clones were determined in NaPO4 buffer (Table 2). WT Bmax values ranged from 470 to 1460 fmol·mg−1 protein and Del322-325 Bmax ranged from 200 to 2320 fmol·mg−1 protein.

Table 2.

Potency and efficacy values of brimonidine and clonidine for the inhibition of cyclic AMP production by α2C WT and Del322-325 receptors

Wild type
 Del322-325
Expression level
 Inhibition of cyclic AMP
 Expression level
 Inhibition of cyclic AMP
Bmax (fmol·mg−1) n pEC50±SEM % inhibition±SEM n Bmax (fmol·mg−1) n pEC50±SEM % Inhibition±SEM n
Brimonidine
470 [467, 464]a 2 9.17 ± 0.10 87 ± 3 3 200 [200, 203]a 2 8.40 ± 0.07 89 ± 3 3
530 ± 50 5 8.45 ± 0.04 93 ± 1 3 670 [706, 637]a 2 9.32 ± 0.21 78 ± 4 3
1460 ± 104 6 9.37 ± 0.06 96 ± 2 3 1100 [914, 1278]a 2 9.13 ± 0.05 101 ± 2 4
1380 ± 292 5 9.44 [9.44, 9.513]a 100 [97, 100]a 2
2320 [2343, 2299]a 2 9.14 ± 0.10 98 ± 3 4
Clonidine
470 [467, 464]a 2 8.49 ± 0.09 84 ± 3 3 200 [200, 203]a 2 7.86 ± 0.07 82 ± 2 3
530 ± 50 5 8.13 ± 0.10 87 ± 3 3 670 [706, 637]a 2 9.09 ± 0.21 72 ± 4 3
1460 ± 104 6 8.66 ± 0.06 95 ± 2 3 1100 [914, 1278]a 2 8.43 ± 0.08 101 ± 3 4
1380 ± 292 5 8.74 [8.74. 8.99]a 99 [98, 100]a 2
2320 [2343, 2299]a 2 8.58 ± 0.11 97 ± 3 4
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Inhibition of cyclic AMP production was determined at multiple receptor expression levels for each receptor type. HEK293 cells stably transfected with either WT or Del322-325 were treated with forskolin alone or in the presence of various concentrations of agonists, with each condition performed in triplicate. Data were normalized to forskolin-stimulated cyclic AMP production and analysed by non-linear regression. Mean pEC50 and mean % inhibition values, which were calculated as 100-(bottom of curve), were derived from sigmoidal dose-response curves of the combined data from all experiments.

a

At n= 2, SEM is not reported. Instead, values from individual experiments are shown in brackets.

Binding characteristics of α2C WT and Del322-325 receptors

The binding characteristics of α2C WT and Del322-325 receptors stably expressed in HEK293 cells were investigated by radioligand competition binding in cell membranes. The affinities of three α2-adrenoceptor agonists, noradrenaline, brimonidine and clonidine, for both WT and Del322-325 receptors were determined (Table 3). pKi values for noradrenaline were not significantly different between WT and Del322-325 receptors. Ki values of brimonidine and clonidine were approximately twofold higher for Del322-325 compared with WT, with statistically significant differences in pKi values.

Table 3.

Receptor binding affinities of adrenoceptor agonists for α2C WT and Del322-325

Wild type
 Del322-325
Ki (nM) pKi±SEM Hill slope Ki (nM) pKi±SEM Hill slope
Noradrenaline 843 6.07 ± 0.10 −0.72 654 6.18 ± 0.06 −0.80
Brimonidine 340 6.47 ± 0.03 −0.85 561 6.25*± 0.06 −0.87
Clonidine 85 7.07 ± 0.05 −0.87 186 6.73*± 0.06 −0.99
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Competition binding assays were performed on WT (Bmax= 1460 fmol·mg−1) and Del322-325 (Bmax= 2320 fmol·mg−1) receptors stably transfected into HEK293 cells using the radioligand [3H]RX821002. pKi values were derived using the Cheng–Prusoff equation with IC50 values from one-site competition curves with fixed slopes and were used to calculate mean pKi. Hill slopes were obtained using a sigmoid analysis with variable slope. Data are from three experiments, each in duplicate.

*

P < 0.05 compared with WT using an unpaired t-test.

Additional binding studies were performed with the same ligands under conditions favourable for detecting binding to high- and low-affinity sites of the receptor. A two-site inhibition curve was preferred for all three ligands in the absence of Gpp(NH)p (Table 4). In the presence of Gpp(NH)p, only the brimonidine curve was shifted to a one-site inhibition for both WT (Figure 1A) and Del322-325 (Figure 1B). Gpp(NH)p had a minimal effect on the clonidine inhibition;, however, it increased KiH and KiL values (lowered affinity) for noradrenaline. This trend was observed with both WT and Del322-325 receptors. Under these conditions, noradrenaline, brimonidine and clonidine had similar binding affinities for the high- and low-affinity sites of the WT and Del322-325 receptors.

Table 4.

High- and low-affinity receptor binding of adrenoceptor agonists for α2C WT and Del322-325

Wild type
KiH (nM) pKiH±SEM (nM) RH±SEM (%) KiL (nM) pKiL±SEM (nM) P value F
Noradrenaline 83 7.08 ± 0.13 47 ± 6 912 6.04 ± 0.14 <0.0001 13.93
Noradrenaline + Gpp(NH)p 166 6.78 ± 0.21 49 ± 3 1202 5.92 ± 0.23 0.0011 4.85
Brimonidine 13 7.88 ± 0.21 17 ± 4 513 6.29 ± 0.05 <0.0001 17.01
Brimonidine + Gpp(NH)p (one-site) (one-site) 0 447 6.35 ± 0.02 0.1686 1.64
Clonidine 25 7.61 ± 0.33 16 ± 1 407 6.39 ± 0.07 0.0001 6.16
Clonidine + Gpp(NH)p 95 7.02 ± 0.35 21 ± 8 447 6.35 ± 0.13 0.0057 3.82
Del322-325
KiH (nM) pKiH±SEM (nM) RH±SEM (%) KiL (nM) pKiL±SEM (nM) P-value F
Noradrenaline 49 7.31 ± 0.16 43 ± 4 575 6.24 ± 0.13 <0.0001 11.41
Noradrenaline + Gpp(NH)p 138 6.86 ± 0.22 48 ± 3 871 6.06 ± .21 0.0010 4.92
Brimonidine 26 7.58 ± 0.18 33 ± 4 490 6.31 ± 0.09 <0.0001 15.47
Brimonidine + Gpp(NH)p (one-site) (one-site) 0 380 6.42 ± 0.03 0.0945 2.03
Clonidine 13 7.87 ± 0.34 14 ± 3 309 6.51 ± 0.06 <0.0001 6.96
Clonidine + Gpp(NH)p 10 7.98 ± 0.52 5 ± 5 282 6.55 ± 0.05 0.0078 3.62
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RH, percentage of receptors in the high affinity state.

Competition binding assays were performed on HEK293 cell membranes from clones stably expressing WT (Bmax= 170 fmol·mg−1) or Del322-325 (Bmax= 140 fmol·mg−1) receptors. In the absence or presence of Gpp(NH)p, α2-adrenoceptor agonists competed with [3H]RX821002 under conditions suitable for determining binding to high- and low-affinity receptor binding sites. Three experiments were performed for each condition at each receptor type; and each experiment was performed in duplicate. The experiments were first analysed individually comparing one- and two-site fits. For the WT receptor, in the absence of Gpp(NH)p, in 7 out of 9 experiments the data best fit a two-site model, whereas in the presence of Gpp(NH)p 7 of 9 experiments fit best to the one-site model. Similarly, for the Del322-325 receptor in the absence of Gpp(NH)p, in 8 out of 9 experiments, the data fit best to a two-site model, whereas in the presence of Gpp(NH)p, 6 of 9 experiments fit best to the one-site model. Data from all three experiments for each condition were then analysed with GraphPad Prism 5 software using a comparison analysis to determine a global one-site versus global two-site inhibition fit. The mean, shared pKi value was obtained from the global fit analysis. Shared pKi values were transformed to mean Ki. The mean RH values were obtained by averaging RH from each individual experiments analysed by global two-site inhibition. P values and F-scores were taken from the Prism global one-site versus two-site inhibition analysis.

Figure 1.

Figure 1

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High- and low-affinity competition binding of brimonidine at α2C WT (A) and Del322-325 (B) receptors in the absence or presence of Gpp(NH)p. These curves are from single experiments and are representative of the results from the global fit analysis. Experiments were performed in duplicate and data are plotted as means ± SEMs.

Inhibition of cyclic AMP production by α2C WT and Del322-325 receptors

The inhibition of cyclic AMP production in intact HEK293 cells was studied as a measure of functional response of stably transfected α2C WT and Del322-325 receptors. Clones with various levels of receptor expression were used to study the effects of the deletion polymorphism on inhibition by brimonidine (highly efficacious) and clonidine (a less efficacious agonist). In cells expressing high levels of either WT or Del322-325 receptors (1460 and 1100 fmol·mg−1, respectively), both agonists were able to completely inhibit the forskolin-stimulated production of cyclic AMP (Figure 2). The highest concentration used for the brimonidine curves was 1 µM due to a reduced inhibitory effect of brimonidine on cyclic AMP production at concentrations exceeding 1 µM (data not shown), which may reflect a change in coupling of some of the receptors from Gi to Gs proteins (Eason et al., 1992). For brimonidine (Figure 2A) and clonidine (Figure 2B), the functional responses of WT and Del322-325 were essentially identical. These data indicate that WT and Del322-325 receptors have similar efficacies (percentage inhibition) and potencies (pEC50 values) for the inhibition of production of cyclic AMP in HEK293 cells.

Figure 2.

Figure 2

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Inhibition of cyclic AMP production by the α2 agonists brimonidine (A) and clonidine (B) acting at α2C WT and Del322-325 adrenoceptors stably expressed in HEK293 cells. The data are normalized to the maximal forskolin stimulation and the values plotted are the means ± SEM from three to four experiments. WT Bmax= 1460 fmol·mg−1 protein; Del322-325 Bmax= 1100 fmol·mg−1 protein.

Additional WT and Del322-325 clones with various expression levels were also studied. Table 2 shows expression levels for all WT and Del322-325 clones, which range from 200 to 2320 fmol·mg−1; % inhibition and pEC50 values for brimonidine and clonidine were found to be similar for WT and Del322-325 clones of similar receptor expression levels. The α2C receptor-mediated inhibition of cyclic AMP production was measured in the presence of forskolin-stimulated cyclic AMP production. As expected, the conversion of [3H]ATP to [3H]cyclic AMP in basal conditions was extremely low and variable; however, in the presence of 30 µM forskolin, % conversion values were approximately 10- to 100-fold higher and showed less variation (Table 5).

Table 5.

Basal and forskolin-stimulated conversion of [3H]ATP to [3H]cyclic AMP

Bmax (fmol·mg−1) n % conversion (%±SEM)
 n
Basal Forskolin stimulation
WT
470 [467, 464]a 2 0.13 ± 0.03 1.28 ± 0.19 6
530 ± 50 5 0.10 ± 0.02 0.91 ± 0.05 6
1460 ± 104 6 0.02 ± 0.02 0.68 ± 0.16 6
Del322-325
200 [200, 203]a 2 0.13 ± 0.03 1.07 ± 0.17 6
670 [706, 637]a 2 0.11 ± 0.03 0.83 ± 0.10 6
1100 [914, 1278]a 2 0.12 ± 0.03 2.43 ± 0.44 8
1380 ± 292 5 0.08 ± 0.03 1.89 ± 0.67 4
2320 [2343, 2299]a 2 0.04 ± 0.01 1.42 ± 0.31 8
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To measure α2C-mediated inhibition of cyclic AMP production in clones with various expression levels of WT or Del322-325 receptors, cyclic AMP production was stimulated with forskolin. Basal levels of cyclic AMP production were measured in the absence of forskolin or α2agonist. Stimulation with 30 µM forskolin is shown in the absence of α2 agonist. Percent conversion values represent the % of [3H]ATP converted to [3H]cyclic AMP. Means ± SEMs are reported.

a

At n= 2, SEM is not reported. Instead, values from individual experiments are shown in brackets.

To test for similarities or differences across the entire range of expression levels, the efficacy and potency values for brimonidine and clonidine were plotted against the log Bmax for all clones and the data analysed by linear regression (Figure 3 and Table 6). For brimonidine, a comparison of the slopes of the lines for WT and Del322-325 receptors showed no significant differences in either the % inhibition (Figure 3A) or the pEC50 values (Figure 3C). Similar results were found for the slopes of the lines for % inhibition (Figure 3B) and pEC50 (Figure 3D) with clonidine in WT and Del322-325 receptors. The relatively large error values in slope parameter meant that we could not detect a statistically significant small change in slope. This novel way of expressing the functional data over a range of receptor expression levels shows that there is no difference in the inhibition of cyclic AMP production by the Del322-325 receptor compared with the WT receptor when expressed in HEK293 cells.

Figure 3.

Figure 3

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Comparison of the functional responses of α2C WT and Del322-325 adrenoceptors at various receptor expression levels in HEK293 cells. Efficacy and potency values for the inhibition of cyclic AMP production by brimonidine (A and C, respectively) and clonidine (B and D, respectively) are plotted against the log Bmax values of multiple WT and Del322-325 clones. Plotted values are the means ± SEM. The slopes of the lines for WT and Del322-325 were determined by linear regression analysis and did not differ between the two receptors for either agonist.

Table 6.

Slope values for linear regression analysis of efficacy and potency versus log Bmax

% inhibition
 pEC50
WT Del322-325 P WT Del322-325 P
Brimonidine 14.0 ± 9.6 13.2 ± 11.7 0.98 1.04 ± 1.46 0.78 ± 0.37 0.83
Clonidine 20.8 ± 3.3 20.0 ± 13.9 0.98 0.67 ± 0.74 0.67 ± 0.54 1.00
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Inhibition of cyclic AMP production by multiple WT and Del322-325 clones of various receptor densities were performed using brimonidine and clonidine. The relationship of average efficacy or potency to log Bmax was analysed by linear regression and the slope values of WT and Del322-325 clones were compared by linear regression analysis using Prism 5.

Discussion and conclusions

This study used intact HEK293 cells to investigate a functional response mediated by the α2C-Del322-325 adrenoceptor, which had previously been reported to exhibit reduced signalling compared with the WT receptor when expressed in CHO cells. Ligand binding studies showed that the Del322-325 receptor had similar binding affinities (within about twofold) as compared with the WT receptor for three classic α2-adrenoceptor agonists: noradrenaline, brimonidine and clonidine. More detailed competition binding experiments in Tris and Mg++ buffer revealed high- and low-affinity agonist binding sites. For brimonidine, Gpp(NH)p converted the two-site competition binding to a one-site model; however, with noradrenaline, the percentage of receptors in the high-affinity state remained unchanged in the presence of Gpp(NH)p. It is unclear why such differences were observed, but it may be due to different ligand-specific receptor conformations stabilized by brimonidine and the endogenous ligand noradrenaline (Urban et al., 2007). Although Gpp(NH)p was not always able to completely convert the binding to a single site, the WT and Del322-325 receptors behaved identically.

Receptor function was studied with an intact cell assay in which the inhibition of cyclic AMP production was measured. In this system, brimonidine and clonidine had similar efficacies and potencies in clones expressing either WT or Del322-325 receptors. To rule out any differences due to different expression levels, multiple clones of WT and Del322-325 receptors at different densities (Bmax values) were tested in the functional assay. The relationships between efficacy or potency and receptor density were assessed by linear regression and the slopes of the lines for WT and Del322-325 were compared. If signalling were reduced with the Del322-325 polymorphism, the magnitude of this reduction might be amplified at lower receptor densities, resulting in a steeper (i.e. larger value) slope for the Del322-325 receptor. The slopes of the regression lines for both efficacy and potency, however, were not different between WT and Del322-325 receptors with both brimonidine and clonidine. These data strongly indicate that there is no difference in WT and Del322-325 function across the range of expression levels tested. Therefore, in intact HEK293 cells, the α2C-Del322-325 polymorphism does not affect the signalling of the receptor to adenylyl cyclase.

There are multiple differences between our study and that of Small et al. (2000), in which a difference in coupling of Del322-325 was observed. One difference is the cell line used, with the previous study using CHO cells and the current study using HEK293 cells. Due to variations observed from one cell line to another (Chaudhry and Granneman, 1994), validation of the previous findings in a second cell line is necessary before further study of the implications of this polymorphism in vivo. Another difference is that the decrease in Del322-325 inhibition of adenylyl cyclase activity in CHO cells was determined in membrane preparations, whereas our results in intact HEK293 cells showed no decrease in the inhibition of cyclic AMP production compared with the WT. Whereas the Small study reported a roughly 50–60% maximal inhibition of adenylyl cyclase activity, the current study achieved >90% inhibition of forskolin-stimulated cyclic AMP production. These differences in efficacy are due to measuring adenylyl cyclase enzyme activity in membrane preparations versus cyclic AMP production in intact cells. For example, in CHO cells expressing 770 fmol·mg−1 of the human α2C-adrenoceptor, it has been shown that the maximum inhibition of adenylyl cyclase activity in membranes is 58, 61 and 47% for noradrenaline, dexmedetomidine and clonidine, respectively, whereas in intact cells the maximum inhibition of cyclic AMP production is 92, 89 and 91% respectively (Pohjanoksa et al., 1997).

Additionally, receptor expression levels differed between the two studies. Small et al. (2000) performed functional assays on cells ranging from roughly 500–1600 fmol·mg−1 receptor, claiming that the 500 fmol·mg−1 level was more similar to that found physiologically. In our study, we tested functional response in cells with a much broader range of receptor expression level, from 200 to 2320 fmol·mg−1. The density of α2-adrenoceptors in the human brain has been reported as approximately 70–140 fmol·mg−1 in the frontal cortex, 105 fmol·mg−1 in the cerebellum and 62 fmol·mg−1 in the caudate (Petrash and Bylund, 1986; Sastre and Garcia-Sevilla, 1994). By contrast, mouse brain and rat cortex express α2-adrenoceptors at 200–300 fmol·mg−1 (MacMillan et al., 1996; Bucheler et al., 2002) and 200 fmol·mg−1 (Wallace et al., 1994) respectively. Our studies include lower, more physiological levels.

These are not the only conflicting studies reported for the α2C-Del322-325 polymorphism. Many studies have investigated the association of this polymorphism with certain clinical conditions, such as heart failure, with inconsistent results. Rosskopf and Michel (2008) present a comprehensive review on the association (or lack thereof) of the α2C-Del322-325 polymorphism with various cardiovascular phenotypes. The rationale for potential cardiovascular effects is that α2A- and α2C-adrenoceptors work as autoreceptors inhibiting the release of noradrenaline in the peripheral nervous system. If this inhibitory effect was reduced, as could occur with loss-of-function receptor polymorphisms, the excess release of noradrenaline would cause increased sympathetic stimulation of the β1-adrenoceptors of cardiac myocytes. Stimulation of the β1- and β2-adrenoceptors results in increased inotropic and chronotropic effects on the heart (Bristow, 1998). Chronic stimulation of β-adrenoceptors leads to the uncoupling of receptors from their G proteins and the down-regulation of β1-adrenoceptors (Hajjar and MacRae, 2002). Therefore, it is suggested that the progression of heart failure is related to a sustained increase in sympathetic drive.

Although some clinical studies have found an association of the α2C-Del322-325 polymorphism with increased levels of circulating catecholamines, increased risk of heart failure (Rosskopf and Michel, 2008), more progressive heart failure (Brede et al., 2002) and better responses to β-blocker therapy (Regitz-Zagrosek et al., 2006), other studies do not support a role for the Del322-325 variant in cardiovascular diseases. Clonidine and other α2-adrenoceptors agonists are used in the treatment of hypertension to reduce the sympathetic tone, thereby lowering blood pressure and heart rate. While clonidine effectively lowers blood pressure in Caucasian hypertensive patients, it is not as effective in controlling blood pressure in African Americans (Materson et al., 1993; Materson and Reda, 1994). The allele frequency of the Del322-325 polymorphism is relatively low in the Caucasian population (0.04); however, it is much more prevalent among the African American population (0.38) (Small et al., 2000). Kurnik et al. (2008) looked at the association of the Del322-325 polymorphism with variation in pharmacological responses of Caucasian and African American hypertensive patients treated with dexmedetomidine, an α2-selective agonist. Sympathetic tone decreased similarly in Caucasians and African Americans and there was no association of the Del322-325 variant with dexmedetomidine response. There was also no association of hypertension with the Del322-325 variant, either alone or in combination with a poorly expressed α2A variant (Li et al., 2006). Another study of black South Africans found all control and heart failure patients genotyped to be homozygous for the Del322-325 variant. In patients who were also homozygous for the β1-Arg389 variant and not undergoing β-blocker treatment, there was no association with heart failure phenotype, severity or progression of the disease (Woodiwiss et al., 2008).

The reason for the conflicting results from these clinical studies may be partially explained by the haplotype expressed in the subsets of patients studied. It has been shown that the Del322-325 variant is found in nine different haplotypes of the α2C gene, with one of these haplotypes found in approximately 48% of Del322-325 carriers (Small et al., 2004). Therefore, other factors may contribute to an association, or lack thereof, of the Del322-325 polymorphism and heart failure. For example, if the Del322-325 variant is found in a haplotype resulting in low receptor expression levels, this could amplify the phenotype resulting in an association with heart failure. However, our studies failed to show different coupling between WT and Del322-325 even at lower expression levels.

Although the Del322-325 polymorphism has no rodent homologue, information obtained from α2C genetically modified mice may give some indications of what occurs physiologically with a loss-of-function variant of this receptor. Recently, mice expressing just one functional copy of the α2C-adrenoceptor have been created (Gilsbach et al., 2007). These mice show that the loss of one copy of the α2C-adrenoceptor is sufficient to impair the inhibition of catecholamine secretion from the adrenal gland. This differs from catecholamine release in the peripheral nervous system, which was unaffected by loss of one receptor copy. In the rat brain, only 20% of the receptors are required for a full response (Adler et al., 1987; Agneter et al., 1997) whereas in adrenal chromaffin cells of mice, which have approximately 30 fmol·mg−1 receptors (Lymperopoulos et al., 2007), no receptor reserve is available. The reduced inhibition of adrenaline release from the adrenal cells of heterozygous mice was shown to result in tachycardia, and these mice developed heart failure after aortic banding (Gilsbach et al., 2007). Therefore, the adrenal cells would be much more sensitive to the effects of an α2C loss-of-function receptor polymorphism and these heterozygous mice could potentially be a good model for such a polymorphism.

Overall, our failure to show reduced function in HEK293 cells compared with the decreased function reported previously in CHO cells adds to the complexities of the phenotypic consequences of the α2C-adrenoceptor Del322-325 genetic polymorphism. It is possible that further studies on the molecular basis for the different results in HEK293 and CHO cells might shed light on the marked differences seen between our studies and those of Small et al. (2000). Previous studies on the β3-adrenoceptor have revealed such cell type-specific differences, where the receptor is desensitized in HEK293 cells but not in CHO cells (Chaudhry and Granneman, 1994; Vrydag et al., 2009). Unexplained variation in receptor sub-cellular localization, dimerization and downstream signalling components could contribute to the variability observed in vitro. Similarly, the inconsistent cardiovascular effects observed with the Del322-325 polymorphism may be due to the presence of additional polymorphisms in other downstream signalling components. Signalling of the α2C-adrenoceptor is linked to multiple downstream effector proteins. Although inhibition of adenylyl cyclase is a classical signalling mechanism of α2-adrenoceptors, they are also involved in the inhibition of voltage-gated Ca++ channels (Limbird, 1988). Activation of receptor-operate K+ channels and MAP kinase is also an α2 receptor signalling mechanism (Limbird, 1988; Kobilka, 1992). The Gi/o-α subunit is most likely responsible for these signalling events. However, for the α2C receptor, it has been reported that the Gi/o-βγ is responsible for a Ca2+ mobilization response (Kukkonen et al., 1998). In addition, there is some evidence that α2-adrenoceptors can couple to the stimulatory Gs protein, leading to stimulation of cyclic AMP production (Eason et al., 1992). Therefore, although the α2C-Del322-325 adrenoceptor polymorphism does not impact the inhibition of cyclic AMP production in HEK293 cells, it is possible that other signalling events are affected by this variant.

Acknowledgments

The authors thank Dr Christopher Taylor, Dr Ronald Rimerman, Nancy Schulte, Amy Lussetto and Jeff Anderson for their technical support. This work was funded by an unrestricted gift from Procter and Gamble, Inc.

Glossary

Abbreviations:

AMP

adenosine monophosphate

CHO

Chinese hamster ovary

Del322-325

deletion 322-325

DMEM

Dulbecco's modified Eagle's medium

FBS

fetal bovine serum

GPCR

G protein-coupled receptor

HEK293

human embryonic kidney 293

MAP

mitogen-activated protein

WT

wild type

Conflicts of interest

There are no conflicts of interest.

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