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NEUROPHARMACOLOGY

Functional Characterization of Opioid Receptor Ligands by Aequorin Luminescence-Based Calcium Assay

Laboratory of Biomolecular Chemistry, Medical University of Lodz, Lodz, Poland (J.F., K.G., M.P., A.J.); Euroscreen s.a., Gosselies, Belgium (E.B.); and Laboratory for Developmental Physiology, Genomics, and Proteomics, Zoological Institute, Catholic University, Leuven, Belgium (J.P., J.V.B.)

Received December 20, 2005; accepted February 17, 2006.

	Abstract

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A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in intracellular calcium levels, was used to examine relative potency and efficacy of the µ-opioid agonists endomorphin-1, endomorphin-2, morphiceptin, and their position 3-substituted analogs, as well as the -agonist deltorphin-II. The results of the aequorin assay, performed on recombinant cell lines, were compared with those obtained in the functional assay on isolated tissue preparations (guinea pig ileum and mouse vas deferens). A range of nine opioid peptide ligands produced a similar rank order of potency for the µ- and -opioid receptor agonists in both functional assays. The highest potency at the µ-receptor was observed for endomorphin-1, endomorphin-2, and [D-1-Nal³]morphiceptin, whereas deltorphin-II was the most potent -receptor agonist. In the aequorin assay, the µ- and -agonist-triggered luminescence was inhibited by the opioid antagonists naloxone and naltrindole, respectively. We can conclude that the use of the aequorin assay for new µ- and -receptor-selective opioid analogs gives pharmacologically relevant data and allows high-throughput compound screening, which does not involve radioactivity or animal tissues. This is the first study that validates the application of this assay in the screening of opioid analogs.

In the present study, we used an alternative functional assay, which does not involve radioactivity or animal tissues and allows high-throughput screening of opioid peptide analogs. This assay is based on the recombinant mammalian cell line expressing an opioid receptor together with a luminescent reporter protein.

Opioid receptors belong to a large family of the G protein-coupled receptors (GPCR), which on stimulation by a ligand induce the activation of the phospholipase C, with the subsequent generation of the primary second messenger metabolites: diacylglycerol and inositol 1,4,5-trisphosphate, followed by a release of calcium from intracellular stores. These agonist-induced receptor-mediated changes in calcium levels can be quantitatively assessed and used to determine agonist potency and efficacy.

A number of groups (Stables et al., 1997; Ungrin et al., 1999; George et al., 2000; Torfs et al., 2002c; Niedernberg et al., 2003) have reported the use of the photoprotein aequorin (Shimomura et al., 1962) as a sensitive detector of increases in Ca²⁺ levels. Apoaequorin is a 21-kDa photoprotein, isolated from the jellyfish Aequorea victoria, which forms a bioluminescent complex when linked to the chromophore cofactor coelenterazine (Shimomura, 1995). The binding of Ca²⁺ to this complex results in an oxidation reaction of coelenterazine, followed by the production of apoaequorin, coelenteramide, CO₂, and light with a _max of 469 nm, which can be detected by conventional luminometry (Stables et al., 1997). Since its discovery in the 1960s, aequorin has been used as an intracellular calcium indicator. The first applications of aequorin involved its microinjection into cells. The cloning of its gene in 1985 opened the way to the stable expression of apoaequorin in different cell lines (Inouye et al., 1985). In a cell line expressing recombinant apoaequorin, reconstitution of aequorin can be simply obtained by the addition of coelenterazine to the medium (Brini et al., 1995).

Methods have been developed to use aequorin-based functional assay for agonist and antagonist screening of GPCR, ion channels, and tyrosine kinase receptors (Dupriez et al., 2002; Le Poul et al., 2002; Liu et al., 2003). For example, this assay was extensively used for characterization of human, as well as insect, neurokinin receptors (Torfs et al., 2002a,b; Poels et al., 2004; Janecka et al., 2005), a histamine receptor (Brini et al., 1995), and mouse -4, -2 nicotinic acetylcholine receptors (Karadsheh et al., 2004).

The aim of the present study was to characterize the relative agonist potency and efficacy of three structurally related µ-selective opioid peptides, endomorphin-1, endomorphin-2, and morphiceptin and their position 3-substituted analogs, as well as a -agonist, deltorphin-II, in the aequorin assay performed on the genetically engineered Chinese hamster ovary (CHO) cell lines CHO-MOR-Aeq and CHO-DOR-Aeq coexpressing, respectively, µ- or -opioid receptors and apoaequorin. A comparison with the data obtained previously from conventional opioid receptor binding and functional GPI/MVD assays was also performed.

	Materials and Methods

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Peptide Synthesis. Peptides were synthesized by standard solidphase procedures as described before (Fichna et al., 2004) using techniques for N-(9-fluorenyl)methoxycarbonyl-protected amino acids on 4-methylbenzhydrylamine Rink peptide resin (100–200 mesh, 0.8 mM/g; Novabiochem, San Diego, CA). Twenty percent piperidine in dimethylformamide was used for deprotection of N-(9-fluorenyl)-methoxycarbonyl groups, and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetra-methyluronium tetrafluoroborate was used to facilitate coupling. Simultaneous deprotection and cleavage from the resin were accomplished by treatment with trifluoroacetic acid (TFA)/triisopropylsilane/water (95:2.5:2.5) for 3 h at room temperature. Crude peptides were purified by reverse phase high-performance liquid chromatography on a Vydac C₁₈ column (1 x 25 cm) using the solvent system of 0.1% TFA in water (A)/80% acetonitrile in water containing 0.1% TFA (B) and a linear gradient. Calculated values for protonated molecular ions were in agreement with those obtained using fast atom bombardment mass spectrometry.

Cell Lines. The CHO cell lines CHO-MOR-Aeq and CHO-DOR-Aeq, permanently expressing both mitochondria-targeted apoaequorin and the mammalian µ and receptor, respectively, were obtained from Euroscreen. The cells were cultured at 37°C with 5% CO₂ in Ham's F-12 medium (BioWhittaker, Verviers, Belgium) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 400 µg/ml G418, and 250 µg/ml Zeocin (all of the cell medium components were purchased from Invitrogen, Carlsbad, CA). The cells were grown as monolayers and were trypsinized every 3 days.

Aequorin Charging Protocol. The CHO cells were grown until 90% confluent monolayers were obtained. Cells in mid-log phase were detached from culture flasks by flushing with phosphate-buffered saline/EDTA (5 mM EDTA), centrifuged (5 min, 150g), and resuspended in "bovine serum albumin (BSA) medium" (Dulbecco's modified Eagle's medium/F-12 with HEPES and 0.1% BSA, without phenol red) at a concentration of 5 x 10⁶ cells/ml. Cells were incubated at room temperature for 4 h in BSA medium supplemented with 5 µM Coelenterazine h (Molecular Probes, Leiden, The Netherlands). After coelenterazine loading, the cells were diluted 10 times in the same medium and incubated for 1 h.

Aequorin Assay. Test substances were dissolved in 50 µl of BSA medium and pipetted into the wells of the white 96-well plates. Light emission was recorded (EG&G Microplate Luminometer LB96V, Berthold, Germany) for 30 s immediately after injection of 50 µl of cell suspension (i.e., 25,000 cells) into each well. Cells were then lysed by a second injection of 50 µl of 0.3% Triton X-100, followed by a 15-s monitoring period. Luminescence data (peak integration) were calculated using Winglow software (PerkinElmer, Boston, MA), which was linked to the Microsoft Excel (Redmond, WA) program. Results are expressed as the fractional luminescence, i.e., the ratio of the agonist-generated signal and the total luminescence (agonist + lysed cells), thereby correcting for potential well-to-well variation in the number of injected cells (Knight et al., 2003).

Data Analysis. Statistical and curve-fitting analyses were performed using Prism 4.0 (GraphPad Software Inc., San Diego, CA). Three independent experiments for each assay were carried out in duplicate. All the values are expressed as mean ± S.E.M.

	Results

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Calcium Measurements and Concentration-Response Curves. Recombinant mammalian cell lines, CHO-MOR-Aeq and CHO-DOR-Aeq, expressing, respectively, the µ- or -opioid receptors and apoaequorin were used to study agonist-induced bioluminescent responses. Three structurally related µ-selective opioid peptides, endomorphin-1, endomorphin-2, and morphiceptin and their position 3-substituted analogs, as well as a -agonist, deltorphin-II, were chosen for the study. The concentration-response curves for the Ca²⁺ responses in the µ-receptor-expressing cells are depicted in Fig. 1. Calculated EC₅₀ values and maximal Ca²⁺ increases are listed in Table 1. A maximal increase in Ca²⁺ concentration at the µ-opioid receptor was obtained with endomorphin-1, endomorphin-2, morphiceptin, and analogs 4 and 5. For analogs 6 through 8, the maximal Ca²⁺ stimulation reached only approximately 75% of the level found with peptides 1 through 5, indicating that these analogs behaved as partial agonists at the µ-opioid receptor. Despite the lower maximal responses, activity of analogs 6 through 8 was detectable at concentrations as low as 10^–11 to 10^–12 M. Concentration-response curves for the Ca²⁺ response induced in the -opioid receptor-expressing cells are shown in Fig. 2. Deltorphin-II behaved as a potent -agonist, with the EC₅₀ value of 0.0017 ± 0.0001 nM and a maximal Ca²⁺ stimulation of 86% (100% = total luminescence as determined after cell lysis). All of the other tested peptides behaved as partial agonists, reaching 51 to 75% of the total luminescence level. Their EC₅₀ values were at least two or three orders of magnitude higher than the value obtained for deltorphin-II.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Concentration-response curves for the calcium increase induced by µ- and -selective opioid peptides in the CHO-MOR-Aeq cells. The data represent mean ± S.E.M. of three independent experiments carried out in duplicate.

View this table: [in this window] [in a new window]   TABLE 1 Comparison of EC50 values and maximal calcium increase for the µ- and -mediated intracellular calcium response induced by µ- and -selective opioid peptides

View larger version (32K): [in this window] [in a new window]   Fig. 2. Concentration-response curves for the calcium increase induced by µ- and -selective opioid peptides in the CHO-DOR-Aeq cells. The data represent mean ± S.E.M. of three independent experiments carried out in duplicate.

Effect of Opioid Antagonists on Agonist-Evoked Responses in CHO-MOR-Aeq and CHO-DOR-Aeq Cells. The opioid antagonists naloxone and naltrindole were used to assess their influence on the agonist-induced bioluminescent responses in the µ- and -opioid receptor-expressing cells, respectively. Naloxone produced a concentration-dependent rightward shift of concentration-response curves for the µ-selective peptides in CHO-MOR-Aeq cells. The exemplary results for endomorphin-1 and the most potent µ-agonist, analog 5, are shown in Fig. 3. The effect produced by deltorphin-II was blocked by pretreatment with the -antagonist naltrindole (Fig. 4).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Concentration-dependent effect of naloxone on the concentration-response curves for the calcium increase induced by endomorphin-1 1 (A) and [D-1-Nal3]morphiceptin 5 in the CHO-MOR-Aeq cells. The data represent mean ± S.E.M. of three independent experiments carried out in duplicate.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Concentration-dependent effect of naltrindole on the concentration-response curves for the calcium increase induced by deltorphin-II 9 (A) and [D-ClPhe3]morphiceptin 7 in the CHO-DOR-Aeq cells. The data represent mean ± S.E.M. of three independent experiments carried out in duplicate.

	Discussion

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Endogenous ligands with high affinity and selectivity toward the µ-opioid receptor endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) were isolated from mammalian brain in 1997 (Zadina et al., 1997). These tetrapeptide amides are structurally related to another µ-selective ligand, morphiceptin (Tyr-Pro-Phe-Pro-NH₂), obtained from the enzymatic digest of bovine casein (Chang et al., 1981, 1985).

Here we have selected endomorphins and morphiceptin, as well as their position 3-modified analogs, to study their relative potency and efficacy at the µ- and -opioid receptors expressed in CHO cells. For comparison, a highly -selective peptide, deltorphin-II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH₂), originating from amphibian skin (Kreil et al., 1989), has also been studied. We have used a functional assay based on agonist-evoked changes of intracellular Ca²⁺ levels in CHO cells stably transfected with the opioid receptor (µ or ) and apoaequorin cDNA. The light-emission responses obtained in the aequorin-based assay for all of the tested peptides were concentration-dependent, and they were inhibited by opioid antagonists.

The results from this study were compared with our previous data obtained in conventional functional assay (GPI/MVD) and with a traditional binding assay on rat brain membrane preparations (Table 2). In both functional assays, the most potent compounds at the µ-receptor were endogenous ligands, endomorphin-1, endomorphin-2, and morphiceptin analog 5 (Tyr-Pro-D-1-Nal-Pro-NH₂). These three peptides were almost equipotent in the GPI/MVD assay but showed a significant difference in potency in the calcium assay. Endomorphin-1 was the most potent and selective peptide of the series. Analog 5 was 7 times less potent, whereas the second endogenous ligand, endomorphin-2, was 180 times less potent than endomorphin-1. Morphiceptin was the only peptide showing much higher selectivity in the GPI/MVD assay than in the calcium assay. Analog 7 (Tyr-Pro-D-ClPhe-Pro-NH₂) was the only analog with higher potency for the -receptor over the µ-receptor in both tests.

View this table: [in this window] [in a new window]   TABLE 2 Competitive binding and GPI/MVD functional assay data obtained with µ- and -selective opioid peptides

Although the rank order of potencies of tested peptides was comparable in both functional assays, the EC₅₀ values observed in the bioluminescent calcium assay were several orders of magnitude lower than the IC₅₀ values observed in the GPI/MVD assay and in the radioligand binding assay on rat brain membrane preparations. This clearly illustrates the extraordinary sensitivity of the cell-based aequorin assay, which therefore can differentiate the potency of opioid analogs much better than the GPI/MVD assay. This may be because cell clones expressing a specific receptor type ensure much higher selectivity of this assay system than the traditional bioassays on complex tissue preparations.

In conclusion, the selected group of peptides, including well known ligands of high µ-affinity (endomorphin-1 and endomorphin-2), moderate µ-affinity (morphiceptin), high -affinity (deltorphin-II), and position 3-substituted analogs of endomorphins and morphiceptin, which in the binding experiments exhibited different degree of µ- or -affinity, were tested on the cell lines expressing µ- or -opioid receptor and a reporter protein, apoaequorin. The obtained results allowed us to differentiate all of the tested peptides in terms of their potency to a much greater degree than was possible using a functional assay on tissue preparations.

	Acknowledgements

 
We thank Dr. M. Detheux (Euroscreen) for advocating the use of the cell lines and Jozef Cieslak and Sofie Van Soest for excellent technical assistance.

	Footnotes

 
This work was supported by a grant for Bilateral Scientific Cooperation between Flanders and Poland (BIL03/18). In addition, the authors gratefully acknowledge the Belgian Interuniversity Attraction Poles Programme (IUAP/PAI P5/30, Belgian Science Policy) and the Fonds voor Wetenschappelijk Onderzoek—Vlaanderen (FWO) for financial support. J. Poels is supported by the FWO as a postdoctoral research associate.

doi:10.1124/jpet.105.099986.

ABBREVIATIONS: GPI, guinea pig ileum; MVD, mouse vas deferens; GPCR, G protein-coupled receptor(s); CHO, Chinese hamster ovary; TFA, trifluoroacetic acid; BSA, bovine serum albumin.

Address correspondence to: Dr. Anna Janecka, Laboratory of Biomolecular Chemistry, Medical University of Lodz, Mazowiecka 6/8, 92–215 Lodz, Poland. E-mail: ajanecka{at}zdn.am.lodz.pl

	References

Top Abstract Materials and Methods Results Discussion References

 

Brini M, Marsault R, Bastianutto C, Alvarez J, Pozzan T, and Rizzuto R (1995) Transfected aequorin in the measurement of cytosolic Ca²⁺ concentration ([Ca²⁺]c). A critical evaluation. J Biol Chem 270: 9896–9903.[Abstract/Free Full Text]

Chang KJ, Lillian A, Hazum E, Cuatrecasas P, and Chang J-K (1981) Morphiceptin (NH4-Tyr-Pro-Phe-Pro-CONH2): a potent and specific agonist for morphine (mu) receptors. Science (Wash DC) 212: 75–77.[Abstract/Free Full Text]

Chang KJ, Su YF, Brent DA, and Chang JK (1985) Isolation of a specific mu-opiate receptor peptide, morphiceptin, from an enzymatic digest of milk proteins. J Biol Chem 260: 9706–9712.[Abstract/Free Full Text]

Dupriez VJ, Maes K, Le Poul E, Burgeon E, and Detheux M (2002) Aequorin-based functional assays for G-protein-coupled receptors, ion channels and tyrosine kinase receptors. Receptors Channels 8: 319–330.[CrossRef][Medline]

Fichna J, Do-Rego JC, Costentin J, Chung NN, Schiller PW, Kosson P, and Janecka A (2004) Opioid receptor binding and in vivo antinociceptive activity of position 3-substituted morphiceptin analogs. Biochem Biophys Res Commun 320: 531–536.[CrossRef][Medline]

George SE, Schaeffer MT, Cully D, Beer MS, and McAllister G (2000) A high-throughput glow-type aequorin assay for measuring receptor-mediated changes in intracellular calcium levels. Anal Biochem 286: 231–237.[CrossRef][Medline]

Inouye S, Noguchi M, Sakaki Y, Tagaki Y, Miyata T, Iwanaga S, Miyata T, and Tsuji S (1985) Cloning and sequence analysis of cDNA for the luminescent protein aequorin. Proc Natl Acad Sci USA 82: 3154–3158.[Abstract/Free Full Text]

Janecka A, Poels J, Fichna J, Studzian K, and Vanden Broeck J (2005) Comparison of antagonist activity of spantide family at human neurokinin receptors measured by aequorin luminescence-based functional calcium assay. Regul Pept 131: 23–28.[CrossRef][Medline]

Karadsheh MS, Shah MS, Tang X, Macdonald RL, and Stitzel JA (2004) Functional characterization of mouse alpha4beta2 nicotinic acetylcholine receptors stably expressed in HEK293T cells. J Neurochem 91: 1138–1150.[CrossRef][Medline]

Knight PJK, Pfeifer TA, and Grigliatti TA (2003) A functional assay for G-protein-coupled receptors using stably transformed insect tissue culture cell lines. Anal Biochem 320: 88–103.[CrossRef][Medline]

Kreil G, Barra D, Simmaco M, Erspamer V, Falconieri-Erspamer G, Negri L, Severini C, Corsi R, and Melchiorri P (1989) Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for delta opioid receptors. Eur J Pharmacol 162: 123–128.[CrossRef][Medline]

Kruszynski R, Fichna J, do-Rego J-C, Chung NN, Schiller PW, Kosson P, Costentin J, Janecka A (2005) Novel endomorphin-2 analogs with µ-opioid receptor antagonist activity. J Pept Res 66: 125–131.[CrossRef][Medline]

Le Poul E, Hisada S, Mizuguchi Y, Dupriez VJ, Burgeon E, and Detheux M (2002) Adaptation of aequorin functional assay to high throughput screening. J Biomol Screen 7: 57–65.[Abstract/Free Full Text]

Liu AMF, Ho MKC, Wong CSS, Chan JHP, Pau AHM, and Wong YH (2003) G16/z chimeras efficiently link a wide range of G protein-coupled receptors to calcium mobilization. J Biomol Screen 8: 39–49.[Abstract/Free Full Text]

Niedernberg A, Tunaru S, Blaukat A, Harris B, and Kostenis E (2003) Comparative analysis of functional assays for characterization of agonist ligands at G protein-coupled receptors. J Biomol Screen 8: 500–510.[Abstract/Free Full Text]

Paterlini MG, Avitabile F, Ostrowski BG, Ferguson DM, and Protoghese PS (2000) Stereochemical requirements for receptor recognition of the mu-opioid peptide endomorphin-1. Biophys J 78: 590–599.[Medline]

Poels J, Van Loy T, Franssens V, Detheux M, Nachman RJ, Oonk HB, Ackerman KE, Vassart G, Parmentier M, De Loof A, et al. (2004) Substitution of conserved glycine residue by alanine in natural and synthetic neuropeptide ligands causes partial agonism at the stomoxytachykinin receptor. J Neurochem 90: 472–478.[CrossRef][Medline]

Sasaki Y, Sasaki A, Ariizumi T, Igari Y, Sato K, Kohara H, Niizuma H, and Ambo A (2004) 2',6'-Dimethylphenylalanine (Dmp) can mimic the N-terminal Tyr in opioid peptides. Biol Pharm Bull 27: 244–247.[CrossRef][Medline]

Shimomura O (1995) Luminescence of aequorin is triggered by the binding of two calcium ions. Biochem Biophys Res Commun 211: 359–363.[CrossRef][Medline]

Shimomura O, Johnson FH, and Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59: 223–239.[CrossRef][Medline]

Stables J, Green A, Marshall F, Fraser N, Knight E, Sautel M, Milligan G, Lee M, and Rees S (1997) A bioluminescent assay for agonist activity at potentially any G-protein-coupled receptor. Anal Biochem 252: 115–126.[CrossRef][Medline]

Torfs H, Akerman KE, Nachman RJ, Oonk HB, Detheux M, Poels J, Van Loy T, De Loof A, Meloen RH, Vassart G, et al. (2002a) Functional analysis of synthetic insectatachykinin analogs on recombinant neurokinin receptor expressing cell lines. Peptides 23: 1999–2005.[CrossRef][Medline]

Torfs H, Detheux M, Oonk HB, Akerman KE, Poels J, Van Loy T, De Loof A, Vassart G, Parmentier M, and Vanden Broeck J (2002b) Analysis of C-terminally substituted tachykinin-like peptide agonists by means of aequorin-based luminescent assays for human and insect neurokinin receptors. Biochem Pharmacol 63: 1675–1682.[CrossRef][Medline]

Torfs H, Poels J, Detheux M, Dupriez V, Van Loy T, Vercammen L, Vassart G, Parmentier M, and Vanden Broeck J (2002c) Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca²⁺-levels in Drosophila S2 cells. Invert Neurosci 4: 119–124.[CrossRef][Medline]

Ungrin MD, Singh LM, Stocco R, Sas DE, and Abramovitz M (1999) An automated aequorin luminescence-based functional calcium assay for G-protein-coupled receptors. Anal Biochem 272: 34–42.[CrossRef][Medline]

Yaksh TL, Malmberg AB, Ro S, Schiller PW, and Goodman M (1995) Characterization of the spinal antinociceptive activity of constrained peptidomimetic opioids. J Pharmacol Exp Ther 275: 63–72.[Abstract/Free Full Text]

Zadina JE, Hackler L, Ge LJ, and Kastin AJ (1997) A potent and selective endogenous agonist for the mu-opiate receptor. Nature (Lond) 386: 499–502.[CrossRef][Medline]

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This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.099986v1 317/3/1150    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fichna, J. Articles by Janecka, A. Search for Related Content PubMed PubMed Citation Articles by Fichna, J. Articles by Janecka, A.