α2-Adrenoceptor agonists stimulate high-affinity GTPase activity in a receptor subtype-selective manner

doi:10.1016/S0014-2999(99)00306-4

European Journal of Pharmacology

Volume 374, Issue 1, 11 June 1999, Pages 137-146

https://doi.org/10.1016/S0014-2999(99)00306-4 Get rights and content

Abstract

Transfected Chinese hamster ovary cells expressing human α_2A-, α_2B- and α_2C-adrenoceptor subtypes were used to monitor α₂-adrenoceptor-stimulated GTP hydrolysis. Incubation with 100 μM (−)-adrenaline resulted in stimulation of pertussis toxin-sensitive GTPase by 380% after activation of the α_2A-subtype, by 320% after activation of the α_2B-subtype and by 110% after activation of the α_2C-subtype. The agonists dexmedetomidine, UK14,304 (5-bromo-6-[2-imidazoline-2-ylamino]quinoxaline) and oxymetazoline showed subtype-dependent efficacy. Dexmedetomidine was a full agonist at the α_2B-subtype and a partial agonist at the α_2A- and the α_2C-subtypes. UK14,304 was a full agonist at the α_2A-subtype and a partial agonist at the other two. Oxymetazoline showed strong partial agonism at the α_2B-subtype (63% of adrenaline), but did not significantly activate the α_2A- and the α_2C-subtypes. These results agreed with cAMP accumulation experiments carried out with cell lines endogenously expressing the α_2A-subtype (human erythroleukemia, HEL) or the α_2B-subtype (neuroblastoma-glioma, NG108-15). The GTPase assay may thus provide a valuable tool for the identification of subtype-selective α₂-adrenoceptor agonists.

Introduction

Activated G-protein coupled receptors promote the exchange of GTP for GDP in the activated α-subunit of the receptor-associated G-protein. The newly bound GTP is subsequently hydrolysed by the GTPase activity intrinsic to the G-protein α-subunit. Guanine nucleotide exchange and GTP hydrolysis are the first detectable biochemical responses following activation of α₂-adrenoceptors. In principle, receptor-stimulated GTPase activity can be quite conveniently monitored by measuring the release of $^{32} P$ from [γ- $^{32} P$ ]GTP, but the agonist-mediated increase over basal GTP hydrolysis is often quite small compared to downstream responses in the cellular signaling pathways. Enzymatic GTP hydrolysis is not restricted to transmembrane signaling events; there are actually numerous possible sources of basal GTPase activity. However, since a considerable part of the total GTPase activity in cell membranes is eliminated after pretreatment with pertussis toxin (Gierschik et al., 1994), it appears that either receptor activity (ligand-dependent or -independent), or spontaneous receptor-independent G_i/o-protein activity, or both, actually contribute in a major way.

Physiological signaling pathways linked to the three α₂-adrenoceptor subtypes (α_2A, α_2B and α_2C) are mostly mediated by pertussis toxin sensitive G-proteins (G_i1, G_i2, G_i3 and G_o) but α₂-adrenoceptors have also been shown to couple to G_s, G_z, and G_q (Conklin et al., 1992; Eason et al., 1992; Wong et al., 1992; Chabre et al., 1994). Consequently, α₂-adrenoceptors can regulate several different types of effector enzymes, including adenylyl cyclases and phospholipases (A₂, C and D), as well as ion channels (K⁺ and Ca²⁺). The subtype-specific coupling characteristics of each α₂-adrenoceptor subtype have not yet been fully established.

Drug discovery projects aimed at the identification of novel receptor ligands usually use competitive radioligand binding assays for the initial characterisation of compounds. Since binding assays do not reliably reveal functional properties, they have to be followed up by functional experiments. Second messenger-based assays are commonly used for this purpose. However, especially when recombinant cell lines expressing high densities of receptors are used, maximal second messenger responses can often be evoked at relatively low degrees of receptor activation, and partial agonists can behave as full agonists (Adham et al., 1993; Pohjanoksa et al., 1997). Direct measurement of receptor-mediated activation of G-proteins would be expected to avoid this problem. The aim of the present study was therefore to develop a convenient assay to monitor α₂-adrenoceptor-stimulated GTPase activity, in order to enable the pharmacological characterisation of α₂-adrenoceptor agonists and their possible subtype-selectivity. For this purpose, we used Chinese hamster ovary (CHO) cells stably transfected to separately express the α_2A-, α_2B- and α_2C-adrenoceptor subtypes. The characterisation of the adenylyl cyclase regulation by α₂-adrenoceptor subtypes in transfected CHO cells has already been reported (Pohjanoksa et al., 1997). In order to further validate the GTPase results we now also determined α₂-adrenoceptor-mediated changes in cAMP production in two cell lines endogenously expressing low levels of two α₂-adrenoceptor subtypes: human erytholeukemia (HEL) cells expressing the α_2A-subtype and neuroblastoma-glioma (NG108-15) cells expressing the α_2B-subtype.

Section snippets

Materials

[γ- $^{32} P$ ]GTP, [ $^{3} H$ ]adenine and [ $^{3} H$ ]RX821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline) were from Amersham (Buckinghamshire, UK) and [ $^{3} H$ ]rauwolscine was from DuPont NEN (Boston, MA). (−)-Adrenaline, App(NH)p (5′-adenylylimidodiphosphate), ATP, charcoal, creatine phosphate, creatine phosphokinase, dithiothreitol, forskolin, IBMX (3-isobuthyl-1-methyl-xanthine), (−)-noradrenaline, oxymetazoline, ouabain, propranolol, quinacrine, pertussis toxin and G418 (Geneticin®) were from Sigma (St.

GTPase activity in CHO cell membranes

The basal and (−)-adrenaline stimulated release of $^{32} P$ from [γ- $^{32} P$ ]GTP in membranes from CHO cells transfected to express the three human α₂-adrenoceptor subtypes α_2A, α_2B and α_2C at densities of 1.88±0.40 pmol/mg, 2.40±0.65 pmol/mg and 2.04±0.42 pmol/mg total cellular protein is shown in Fig. 1. All tubes contained 0.1 nM [γ- $^{32} P$ ]GTP together with different amounts of unlabelled GTP. Increasing concentrations of unlabelled GTP reduced the hydrolysis of [γ- $^{32} P$ ]GTP and abolished the stimulatory

Discussion

A receptor-mediated increase in high-affinity GTPase activity is among the first detectable biochemical events that follow activation of G-protein coupled receptors and is in this respect a potentially useful indicator for the pharmacological characterisation of agonists. Kim et al. (1994)have shown that the effector enzyme, adenylyl cyclase, is quantitatively the limiting factor in the coupling of receptors via G_s-proteins to activation of adenylyl cyclase. This limiting step is avoided by

Acknowledgements

Anna-Mari Pekuri, Tuire Olli-Lähdesmäki, Sami Virolainen, Marjo Pihlavisto and Anne Marjamäki are gratefully acknowledged for assistance. The study was financially supported by the Technology Development Centre of Finland (TEKES).

References (48)

K Aktories et al.
Epinephrine inhibits adenylate cyclase and stimulates a GTPase in human platelet membranes via α-adrenoceptors
FEBS Lett.
(1981)
M.M Bradford
A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)
D.R Brandt et al.
Catecholamine-stimulated GTPase cycle
J. Biol. Chem.
(1986)
O Chabre et al.
Coupling of the α_2A-adrenergic receptor to multiple G-proteins
J. Biol. Chem.
(1994)
B.R Conklin et al.
Recombinant G_qα: mutational activation and coupling to receptors and phospholipase C
J. Biol. Chem.
(1992)
M.G Eason et al.
Simultaneous coupling of α₂-adrenergic receptors to two G-proteins with opposing effects
J. Biol. Chem.
(1992)
P Gierschik et al.
Receptor-stimulated hydrolysis of guanosine 5′-triphosphate in membrane preparations
Meth. Enzymol.
(1994)
M Halme et al.
Recombinant human α₂-adrenoceptor subtypes: comparison of [ $^{3} H$ ]rauwolscine, [ $^{3} H$ ]atipamezole and [ $^{3} H$ ]RX821002 as radioligands
Biochim. Biophys. Acta
(1995)
H Hasegawa et al.
Two isoforms of the prostaglandin E receptor EP3 subtype different in agonist-independent constitutive activity
J. Biol. Chem.
(1996)
T Higashijima et al.
The effect of GTP and Mg²⁺ on the GTPase activity and the fluorescent properties of G_o
J. Biol. Chem.
(1987)

J.F Jasper et al.

Ligand efficacy and potency at recombinant α₂-adrenergic receptors

Biochemical Pharmacology

(1998)

S Lazareno et al.

Pharmacological characterization of guanine nucleotide exchange reactions in membranes from CHO cells stably transfected with human muscarinic receptors M1–M4

Life Sciences

(1993)

P Leff

The two-state model of receptor activation

Trends Pharmacol. Sci.

(1995)

E.E MacNulty et al.

α₂-C10 adrenergic receptors expressed in rat 1 fibroblasts can regulate both adenylyl cyclase and phospholipase D-mediated hydrolysis of phosphatidylcholine by interacting with pertussis toxin-sensitive guanine nucleotide-binding proteins

J. Biol. Chem.

(1992)

A Marjamäki et al.

Use of recombinant human α₂-adrenoceptors to characterize subtype selectivity of antagonist binding

Eur. J. Pharmacol.

(1993)

M.C Michel et al.

α₂-Adrenergic receptor stimulation mobilizes intracellular Ca²⁺ in human erytholeukemia cells

J. Biol. Chem.

(1989)

I.F Musgrave et al.

α_2A-adrenoceptors mediate activation of non-selective cation channels via G_i-proteins in human erythroleukaemia (HEL) cells

Biochem. Pharmacol.

(1995)

Y Odagaki et al.

Pharmacological characterization of epinephrine-stimulated GTPase activity in human platelet membranes

Biochem. Pharmacol.

(1993)

J.M Peltonen et al.

Subtype-specific stimulation of [ $^{35} S$ ]GTPγS binding by recombinant α₂-adrenoceptors

Eur. J. Pharmacol.

(1998)

K Pohjanoksa et al.

α₂-Adrenoceptor regulation of adenylyl cyclase in CHO cells: dependence on receptor density, receptor subtype and present activity of adenylyl cyclase

Eur. J. Pharmacol.

(1997)

Y Salomon et al.

A highly sensitive adenylate cyclase assay

Anal. Biochem.

(1974)

M Von Zastrow et al.

Subtype-specific differences in the intracellular sorting of G protein-coupled receptors

J. Biol. Chem.

(1993)

A.G Williams et al.

Identification of the pertussis toxin-sensitive G proteins in platelets, megakaryocytes, and human erytholeukemia cells

Blood

(1990)

N Adham et al.

Receptor reserve masks partial agonist activity of drugs in a cloned rat 5-hydroxytryptamine_1B receptor expression system

Mol. Pharmacol.

(1993)

Cited by (23)

Age-related effects of dexmedetomidine on myocardial contraction and coronary circulation in isolated guinea pig hearts
2016, Journal of Pharmacological Sciences
Citation Excerpt :
The three receptors bind most α2 adrenergic drugs, including dexmedetomidine with similar affinities, but with different efficacies. It was reported that dexmedetomidine was a full agonist at the α2B subtype and a partial agonist at the α2C subtype and the α2A/D subtype (25). In the present study, dexmedetomidine almost completely inhibited the increase in LVP induced by EFS from hearts of guinea pigs of all ages.
Dexmedetomidine is a selective α₂ adrenergic agonist. Although dexmedetomidine is widely used for sedation and analgesia, it frequently produces hypotension and bradycardia. The present study aimed to evaluate the effects of dexmedetomidine on cardiac function and coronary circulation using Langendorff-perfused guinea pig hearts. Coronary perfusion pressure (CPP) and left ventricular pressure (LVP) were continuously monitored, and electric field stimulation (EFS) was applied to stimulate sympathetic nerve terminals. Dexmedetomidine almost completely inhibited the EFS-induced increase in LVP at all ages. The effect of dexmedetomidine on coronary artery resistance varied according to postnatal age, i.e., dexmedetomidine had little effect on CPP in young hearts (<4 weeks) but increased CPP by 10 mmHg at 4–8 weeks and by 15 mmHg at >8 weeks. The increase in CPP in adult hearts was inhibited by imiloxan, an α_2B antagonist, and prazosin, an α₁ antagonist. The results suggest that dexmedetomidine acts on α₂ adrenergic receptors at sympathetic nerve terminals to suppress the release of norepinephrine. In addition, the findings suggest that dexmedetomidine directly affects α₁ adrenoceptors and/or α_2B adrenoceptors on coronary smooth muscles to increase CPP. The age-related changes in α adrenoceptor subtypes may be linked to the cardiodepressant effects of dexmedetomidine.
Actions of the prototypical 5-HT<inf>1A</inf> receptor agonist 8-OH-DPAT at human α<inf>2</inf>-adrenoceptors: (+)8-OH-DPAT, but not (-)8-OH-DPAT is an α<inf>2B</inf> subtype preferential agonist
2010, European Journal of Pharmacology
8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] is the prototypical agonist at serotonin 5-HT_1A receptors; however, activity at other targets contributes to the functional effects of the compound as well. We examined the properties of 8-OH-DPAT and its enantiomers at recombinant human (h)α₂-adrenoceptor subtypes, using a panel of radioligand binding and functional tests. In competition binding experiments using [³H]-RX821002, about 10-fold selectivity of (+)8-OH-DPAT for the hα_2B subtype (pK_i about 7) over hα_2A- and hα_2C-adrenoceptors was observed. In contrast, the S(−) enantiomer of 8-OH-DPAT showed similar weak affinities for the three receptor subtypes (pK_is < 6). The binding affinity of (+)8-OH-DPAT at the hα_2B- and the hα_2A-adrenoceptor was found sensitive to GTPγS, a receptor/G protein-uncoupling agent, indicating agonist properties of the drug. Furthermore, using [³⁵S]GTPγS binding determination at CHO-hα_2B or CHO-hα_2A cell membranes and G protein coupled inwardly rectifying potassium (GIRK) current recordings in Xenopus oocytes expressing hα_2B, partial agonist activity of (+)8-OH-DPAT at the respective receptors was confirmed in these two different functional assays. Potency of (+)8-OH-DPAT for stimulation of [³⁵S]GTPγS incorporation was lower at the hα_2A- than at the hα_2B-adrenoceptor, consistent with binding affinities. Thus, (+)8-OH-DPAT and, as a consequence, racemic (±)8-OH-DPAT are partial agonists at hα₂-adrenoceptors with selectivity for the hα_2B subtype, a property that might contribute to the effects of the compound described in native systems.
Comparative pharmacology of adrenergic α<inf>2C</inf> receptors coupled to Ca<sup>2+</sup> signaling through different Gα proteins
2009, Neurochemistry International
Adrenergic α₁, α₂ and β receptors are members of the G-protein-coupled receptor families (GPCRs) mediating physiological responses to adrenaline (epinephrine) and noradrenaline (norepinephrine). Since GPCRs are major targets for potential therapeutic agents, development of robust, reliable and cost effective functional screening methods for these receptors is in the focus of pharmacological research. For this reason, the aim of the present study was to develop an intracellular calcium assay for investigating the pharmacology of the α_2C type of adrenergic receptors (α_2C-AR). Although activation of α_2C-AR is not linked to calcium mobilization, co-expression of these receptors with the chimeric Gα_qi5 protein, containing the five carboxyl-terminal amino acids from G_i, or promiscuosus Gα₁₆ protein can divert receptor signaling to the G_q pathway generating Ca²⁺ release from intracellular stores.
In order to assess the functional potency of α₂-AR agonists and antagonists, we established a fluorometric Ca²⁺ assay using cell lines stably and constitutively co-expressing α_2C-AR and Gα_qi5 or Gα₁₆ proteins (Gα_qi5/α_2C and Gα₁₆/α_2C). As part of the pharmacological characterization, we measured the changes in cytoplasmic Ca²⁺ levels due to activation of the chimeric Gα_qi5 or Gα₁₆ coupled recombinant α_2C receptors as a function of increasing concentration of several agonists (noradrenaline, brimonidine, oxymetazoline, clonidine, moxonidine) and antagonists (MK912, yohimbine). The binding affinities of α₂-AR agonist and antagonists and the inhibition of the forskolin-stimulated cAMP accumulation in α_2C-AR expressing cells were also measured. These results confirmed that the Gα_qi5/α_2C and Gα₁₆/α_2C recombinant systems can be useful for modelling the native G_i-coupled system. Our results indicate that a plate-reader based fluorometric Ca²⁺ assay may be suitable in high-throughput screening for α_2C-AR ligands as well.
Chapter 2 CCR5 Pharmacology Methodologies and Associated Applications
2009, Methods in Enzymology
Citation Excerpt :
MIP‐1β–dependent stimulation of GTP can be examined using a radiolabeled nonhydrolyzable form of GTP (γS‐GTP) exactly as described by (Haworth et al., 2007) to characterize the inverse agonism mechanism that is measurable in recombinant systems which underpins the functional antagonism of antiviral CCR5 inhibitors (Dorr et al., 2005b; Strizki et al., 2001). GTP binding to CCR5 is measured based on previously described methods (Jansson et al., 1999; Labrecque et al., 2005). These can also be adapted to a time‐resolved fluorometric assay, using a Europium (Eu3+) labeled γS‐GTP to avoid use of radioactivity.
The G protein–coupled chemokine (C‐C motif) receptor, CCR5, was originally characterized as a protein responding functionally to a number of CC chemokines. As with chemokine receptors in general, studies indicate that CCR5 plays a role in inflammatory responses to infection, although its exact role in normal immune function is not completely defined. The vast majority of research into CCR5 has been focused on its role as an essential and predominant coreceptor for HIV‐1 entry into host immune cells. Discovery of this role was prompted by the elucidation that individuals homozygous for a 32 bp deletion in the CCR5 gene do not express the receptor at the cell surface, and as a consequence, are remarkably resistant to HIV‐1 infection, and apparently possess no other clear phenotype. Multiple studies followed with the ultimate aim of identifying drugs that functionally and physically blocked CCR5 to prevent HIV‐1 entry, and thus provide a completely new approach to treating infection and AIDS, the world's biggest infectious disease killer. To this end, functional antagonists with potent anti–HIV‐1 activity have been discovered, as best exemplified by maraviroc, the first new oral drug for the treatment of HIV‐1 infection in 10 years. In this chapter, the specific methods used to characterize CCR5 primary pharmacology and apply the data generated to enable drug discovery, notably maraviroc, for the treatment of HIV infection and potentially inflammatory‐based indications, are described.
Role of adrenoceptor-linked signaling pathways in the regulation of CYP1A1 gene expression
2005, Biochemical Pharmacology
Citation Excerpt :
Atipamezole is a selective α2-AR antagonist, which penetrates rapidly into the brain and stimulates the release of central and plasma noradrenaline levels [23,41]. On the other hand, dexmedetomidine, a highly selective agonist of α2-adrenoceptors [42], suppresses brain and plasma noradrenaline levels in a dose-dependent manner [23,43,44]. Only subacute treatment with atipamezole or dexmedetomidine has been found to result in a sufficient increase or inhibition of hypothalamic noradrenaline release, respectively [23,43,45].
Alpha2-adrenoceptor agents as well as stress affect the activity of several hepatic monoxygenases including those related to CYP1A enzymes. This study was therefore designed to assess the role of central and/or peripheral catecholamines and, in particular, of adrenoceptors in the regulation of B(α)P-induced cytochrome CYP1A1 expression. In order to discriminate the role of central from that of peripheral catecholamines in the regulation of CYP1A1 induction, the effect of central and peripheral catecholamine depletion using reserpine versus only peripheral catecholamine depletion using guanethidine was assessed. By using selected agonists and antagonists, the role of alpha and beta-adrenoceptors in the regulation of CYP1A1 induction was evaluated. The results showed that the central catecholaminergic system has a negative regulatory effect on 7-ethoxyresorufin O-deethylase (EROD) inducibility by benzo(α)pyrene (B(α)P), and that this may be mediated via α1-, α2- and β-adrenoceptors. Specifically, stimulation of α2-adrenoceptors with dexmedetomidine and blockade of α1- or β-adrenoceptors with prazosin or propranolol respectively, resulted in a further increase of EROD inducibility. Adrenoceptors were found to be involved in the regulation of the CYP1A1 gene at mRNA level. Both, reduced noradrenaline release in central nervous system induced with dexmedetomidine and central catecholamine depletion, as well as blockade of central α1-adrenoceptors induced with prazosin, all were associated with up-regulation of CYP1A1 expression. In contrast, stimulation of central beta-adrenoceptors with isoprenaline resulted in a down-regulation of CYP1A1 expression. Our observations indicate that drugs, which stimulate or block adrenoceptors and catecholamine release may lead to complications in drug therapy and modulate the toxicity or carcinogenicity of drugs that are substrates for the CYP1A1.
Ligand modulation of [<sup>35</sup>S]GTPγS binding at human α<inf>2A</inf>, α<inf>2B</inf> and α<inf>2C</inf> adrenoceptors
2002, Cellular Signalling
Affinities and efficacies of chemically diverse ligands—some of them used as clinical agents—were examined, employing [³H]RX821,002 and [³⁵S]GTPγS binding assays, respectively, at human (h) cloned, hα_2A, hα_2B and hα_2C adrenoceptors (AR) expressed in Chinese hamster ovary (CHO) cells. As compared to noradrenaline (NA, efficacy defined as 100%), the majority of the 13 agonists tested generally behaved as partial agonists. Amongst 18 antagonists, pK_B and pK_i values, which were highly correlated for each α₂-AR subtype, failed to reveal any strikingly selective agents. Inverse agonist properties were not detected for any antagonist, consistent with a lack of constitutive activity suggested by the monophasic inhibition of [³⁵S]GTPγS binding by GTPγS. These data should facilitate interpretation of experimental and clinical actions of adrenergic agonists. Moreover, they emphasize the continuing need for α₂-AR subtype-selective antagonists in order to define further the roles and therapeutic relevance of hα_2A-, hα_2B-, and hα_2C-AR.

View all citing articles on Scopus

¹: Present address: Åland Central Hospital, FIN-22100 Mariehamn, Finland.

²: Present address: Quality Assurance Unit, University of Turku, Tykistökatu 4 A, FIN-20520 Turku, Finland.

³: Present address: Juvantia Pharma, Tykistökatu 6 A, FIN-20520 Turku, Finland.

View full text

α2-Adrenoceptor agonists stimulate high-affinity GTPase activity in a receptor subtype-selective manner

Abstract

Introduction

Section snippets

Materials

GTPase activity in CHO cell membranes

Discussion

Acknowledgements

FEBS Lett.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Meth. Enzymol.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Biochemical Pharmacology

Life Sciences

Trends Pharmacol. Sci.

J. Biol. Chem.

Eur. J. Pharmacol.

J. Biol. Chem.

Biochem. Pharmacol.

Biochem. Pharmacol.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Anal. Biochem.

J. Biol. Chem.

Blood

Receptor reserve masks partial agonist activity of drugs in a cloned rat 5-hydroxytryptamine1B receptor expression system

Mol. Pharmacol.

α₂-Adrenoceptor agonists stimulate high-affinity GTPase activity in a receptor subtype-selective manner

Receptor reserve masks partial agonist activity of drugs in a cloned rat 5-hydroxytryptamine_1B receptor expression system