Arachidonic Acid Release in Cell Lines Transfected with Muscarinic Receptors

Cell. Signal. Vol. 11, No. 6, pp. 405–413, 1999 Copyright  1999 Elsevier Science Inc.

ISSN 0898-6568/99 $–see front matter PII S0898-6568(99)00006-6

Arachidonic Acid Release in Cell Lines Transfected with Muscarinic Receptors: A Simple Functional Assay to Determine Response of Agonists Frank P. Bymaster,* David O. Calligaro and Julie F. Falcone Lilly Neuroscience Research, Lilly Corporate Center, Indianapolis, IN 46285, USA

ABSTRACT. Muscarinic agonists stimulated arachidonic acid release from 10- to 32-fold in Chinese hamster ovary (CHO) cells transfected with muscarinic M1, M3 and M5 receptor subtypes. Muscarinic agonists liberated arachidonic acid from the cAMP-coupled M2 and M4 cells only in the presence of ATP. Partial agonists were less efficacious at liberating arachidonic acid than full agonists. The ability of muscarinic agonists to liberate arachidonic acid and stimulate phosphoinositide hydrolysis in the same CHO M1, M3 and M5 cells was well correlated; however, partial agonists were more efficacious at stimulating phosphoinositide hydrolysis than arachidonic acid release. The efficacy and potency of 13 muscarinic agonists to liberate arachidonic acid was characterised. Influx of external calcium was required for arachidonic acid release even after initiation of agonistinduced release. It is concluded that arachidonic acid release is a simple assay suitable for evaluation of muscarinic agonists, antagonists and the flux of external calcium into cells. cell signal 11;6:405–413, 1999.  1999 Elsevier Science Inc. KEY WORDS. Muscarinic receptors, Muscarinic agonist, Arachidonic acid release, Phosphoinositide hydrolysis, Functional assay, Calcium channels, Cell lines

INTRODUCTION Five structurally related, but functionally distinct, subtypes of muscarinic receptors (M1–M5) have been identified, and the M1, M3 and M5 subtypes are preferentially coupled to stimulation of phospholipase C, whereas the M2 and M4 receptor subtypes are negatively coupled to adenylyl cyclase [1]. Additionally, the release of another second messenger, arachidonic acid (AA), was shown to be mediated by muscarinic receptors in a number of tissues and cell lines [2–5]. For example, activation of M1 and M3 receptors in A9 L cells [3, 6] and the M5 receptor in Chinese hamster ovary k1 (CHO) cells [7] stimulated marked liberation of AA. Muscarinic agonists did not liberate AA from the cAMP-linked M2 and M4 cell lines [3] but were able to produce marked release of AA in the presence of ATP [8]. In addition to its role as a first or second messenger, another important role of AA is serving as the precursor of biologically active compounds such as prostaglandins and * Author to whom all correspondence should be addressed. Tel: 11-317276-9444; Fax: 11-317-276-5546; E-mail: [email protected] Abbreviations: AA–arachidonic acid; ATP–adenosine triphosphate; BSA–bovine serum albumin; CHO–Chinese hamster ovary; DMEM– Dulbecco’s modified Eagle’s medium; dpm–disintegrations per minute; EC50– concentration of agonist required to produce one-half of the maximal effect; EDTA–ethylenediamine tetraacetic acid; EGTA–ethylene glycolbis[b-aminoethylether]-N,N,N9,N9-tetraacetic acid; PI–phosphoinositide; IC50–concentration of antagonist required to block 50% of the effect; MEM–modified Eagle’s medium; S.E.M.–standard error of the mean. Received 27 August 1998; and accepted 9 November 1998.

leukotrienes [2]. Although AA may be liberated from phospholipids by multiple pathways, the major source of AA release in A9 L and CHO cells was shown to be due to action of the enzyme phospholipase A2 [3, 7]. Activation of phospholipase A2 results in the formation of AA and lysophospholipids from a variety of phospholipids. Phospholipase A2 activity is calcium dependent and can therefore be altered by changes in intracellular Ca21 levels from external sources [3, 9] and by activation of protein kinase C [7]. There is much interest in synthesising muscarinic agonists for reversing cognitive deficits in Alzheimer’s disease [10, 11], and suitable second messenger assays are needed to determine functional effects and selectivity of agonists. Responses to receptor activation in A9 L cells, including activation of phosphoinositol (PI) hydrolysis, calcium mobilisation, membrane hyperpolarisation, cell proliferation and AA release, were shown for the full agonist carbachol and the partial agonist pilocarpine [6]. Pilocarpine was found to have about one-half the potency of carbachol in PI hydrolysis, Ca21 mobilisation and AA release, suggesting that these assays would be suitable for determining agonist efficacy. Determination of AA release is quite simple and requires no laborious steps such as chromatographic separation or radioimmunoassays, suggesting that it might be ideal for ready determination of the functional effects of agonists [12, 13]. We report here that the efficacy and potency of muscarinic agonists can be determined by utilising the release of AA from M1, M3 and M5 cell lines. The ability of muscarinic ag-

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onists to liberate AA and increase phosphoinositide hydrolysis also was compared. MATERIALS AND METHODS Cell Culture Chinese hamster ovary cells (CHO-k1) and A9-L cells transfected with human muscarinic receptor subtypes [14] were obtained from Dr. Mark R. Brann (University of Vermont). Untransfected CHO-k1 cells were obtained from American Type Culture Collection (Rockville, MD). The growth medium used was Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum, 10 mM MEM non-essential amino acid solution, penicillin-streptomycin [penicillin G sodium (10,000 units) and 10,000 mg streptomycin sulfate, 1 mL/100 mL media], fungizone (250 mg amphotericin B, 1 mL/100 mL media). Geneticin (100 mg/mL) was added to the media for muscarinic receptor selection. Cells were maintained in monolayer culture in a humidified incubator at 378C and 95% O2/5% CO2. Arachidonic Acid Release Arachidonic acid release was determined according to our published method [6]. Briefly, cells were harvested by using Trypsin (0.25%)-EDTA (1 mM) and centrifuged at 1000 rpm for 7 min in a Beckman countertop centrifuge. A cell count was performed on the suspension, and cells were plated in 24-well culture plates at a density of 50,000 cells per well in growth media and returned to the incubator. When wells appeared to be approximately 70% confluent, growth media containing 0.0625 mCi/0.5 mL [3H]-AA was added to each well, and the plates were incubated generally for 18–24 h. Immediately prior to the addition of the experimental agents, the wells were washed twice with 1 mL of DMEM containing 2 mg/mL fatty acid-free bovine serum albumin (BSA). Test compounds were dissolved in water and diluted in DMEM medium containing 2 mg/mL of fatty acid-free BSA. Experimental agents were added in a 0.5-mL volume in triplicate and incubated for the appropriate length of time. Muscarinic antagonists were added 30 min prior to the addition of agonists, and the plates were incubated for an additional 45 min. To induce release in cAMPcoupled CHO M2 and M4 cell lines, adenosine-5-triphosphate (ATP) was added to all wells. Oxotremorine-M (10 mM) was used to determine maximal liberation of AA. Calcium mechanism studies were run in DMEM media containing calcium and in calcium-free MEM (GIBCO). After incubation, aliquots of media from each well were placed into scintillation vials containing Beckman Ready Solv HP (Beckman, Fullerton, CA), and radioactivity was determined by liquid scintillation spectrometry. The total radioactivity incorporated into cells was measured by solubilising the cells in 3% sodium dodecyl sulfate (SDS) and determining total radioactivity in the solution. PI Hydrolysis For phosphatidylinositol hydrolysis determination, CHO M3 cells were plated as described previously in DMEM

F. P. Bymaster et al.

growth media and were prelabelled with 1 mCi per well of [3H]-myoinositol (2–10 Ci/mmol). Plates were incubated for at least 16 h to allow incorporation of [3H]-myoinositol into phospholipid. Prior to the addition of the experimental agents, medium in the wells was removed and washed twice with a DMEM assay medium containing 10 mM lithium chloride, 10 mM myoinositol and 10 mM HEPES. Dilutions of the experimental agents were made in the assay media, were added in a 0.5-mL volume in triplicate to the wells and were incubated at 378C for 60 min in 95% O2/5% CO2. The reaction was terminated by aspirating off the media and then adding 1 mL of ice-cold 10 mM LiCl and 10 mM HEPES in water to each well. Plates were then placed on ice and allowed to sit for 30 min, and PI hydrolysis was determined by using a previously described method [15]. Briefly, assay samples were removed from each well, and the wells were washed with 0.5 ml H20. Samples and wash were added to the columns (Sep-Pak, Waters, Milford, MA) followed by the addition of 10 mL of H20 and then 10 mL of 5 mM sodium borate. [3H]Inositol monophosphate was eluted into scintillation vials with the use of 4 mL of a reagent containing 0.1 M ammonium formate, 0.01 M formic acid and 5 mM sodium borate. Beckman Ready Solv HP was added to all vials, and radioactivity was determined by liquid scintillation spectrometry. Data Calculations The EC50 and IC50 values of compounds were determined in at least three separate experiments by using Allfit [16]. Inhibition constants (Ki) values were calculated by using the Cheng-Prusoff equation [17]. The levels of significance were calculated by using Student’s t-test. Materials [3H]Arachidonic acid (100 Ci/mmol) and [3H]myoinositol (2–10 Ci/mmol) were obtained from New England Nuclear (Boston, MA). S-Aceclidine, R-aceclidine, A23187 and RS-86 were provided by the Eli Lilly Research Laboratories (Indianapolis, IN). Other muscarinic agents were obtained from either Sigma Chemical Co. (St., Louis, MO) or Research Biochemicals International (Natick, MA). The following reagents were obtained from GIBCO (Grand Island, NY): foetal bovine serum , MEM non-essential amino acid solution, penicillin-streptomycin, fungizone, geneticin, trypsin-EDTA and DMEM containing high glucose with L-glutamine, pyridoxine hydrochloride and no sodium pyruvate. Fatty acid-free BSA and HEPES were obtained from Sigma Chemical Co. RESULTS AA Incorporation in Cells The incorporation of [3H]AA (about 0.065 mCi) into CHO M5 cells increased from 30 min to 16 h, and about 62% of the radioactivity was incorporated into the cells by 16 h

Muscarinic Agonist-Induced Arachidonic Acid Release

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FIGURE 1. Incorporation of [3H]AA into CHO cells transfected

with muscarinic M5 receptors and agonist-induced AA release. [3H]AA (0.065 mCi) was incubated with CHO cells transfected with the muscarinic M5 receptor subtype from 15 min to 16 h; the cells were washed and then incubated with or without oxotremorine-M (10 mM) as described in the Materials and Methods section. AA incorporated into the cells and AA released (basal) was determined as described in the Materials and Methods section. Each data point represents the mean dpm 6 S.E.M. from a representative experiment in triplicate.

FIGURE 2. Concentration-dependent release by oxotremorine-M of [3H]AA from CHO cells transfected with the M1, M3 and M5 receptor subtypes and A9 L cells transfected with the muscarinic M1 receptor subtype. The release of [3H]AA from CHO M1, M3, M5 and A9 L M1 cell lines by oxotremorine-M (from 10 nM to 100,000 nM) was determined as described in the Materials and Methods section. Basal release was amount of release with no agonist added. Each data point represents the mean dpm 6 S.E.M. of triplicate samples.

(Fig. 1). The amount of [3H]-radioactivity released by oxotremorine-M (10 mM) was about the same percentage at each preloading time point. Sixteen hours for incorporation was used in all subsequent experiments to maximise the amount of [3H]AA released. AA Release by Muscarinic Agonists in M1, M3 and M5 Cell Lines The full agonist oxotremorine-M concentration dependently stimulated release of [3H]AA from A9 L cells transfected with the M1 receptor or CHO cells transfected with M1, M3 or M5 muscarinic receptor subtypes (Fig. 2). Basal release of [3H]AA was 1591, 875, 1080 and 559 dpm, respectively, for A9 L M1, CHO M1, CHO M3 and CHO M5 cell lines. Oxotremorine-M (10 mM) increased release of AA to 16-, 23-, 33- and 33-fold above basal levels, respectively, in these cell lines. In untransfected CHO-k1 cells, AA was incorporated into the cells, but the release of AA was 1011 6 122 and 1009 6 33 dpm in the absence and presence of oxotremorine-M (10 mM), respectively (data not shown). FIGURE 3. Release of [3H]AA from CHO cells transfected with

AA Release in Muscarinic M2 and M4 Cell Lines Oxotremorine-M (10 mM) did not appreciably stimulate release of [3H]AA alone from M2 or M4 CHO cells (Fig. 3) but, in combination with ATP, increased liberation of [3H]AA. For example, the release of [3H]AA from CHO M2 cells was increased to 9-fold of the basal level by the combi-

the muscarinic M2 and M4 receptor subtypes by oxotremorine-M (OXO-M) and ATP. The release of [3H]AA from CHO M2 and M4 cell lines by control (basal), oxotremorine-M (10 mM), ATP (5 mM) or the combination of ATP and oxotremorine-M was determined as described in the Materials and Methods section. Each data point represents the mean dpm 6 S.E.M. of triplicate samples. *P , 0.05 versus basal release, #P , 0.05 versus ATP and oxotremorine-M alone.

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FIGURE 4. Time course of release of [3H]AA from CHO cells

transfected with the muscarinic M5 receptor subtype. The release of [3H]AA liberated by control (basal), atropine (ATR, 1 mM), oxotremorine-M (OXO-M, 10 mM) or combination of atropine (1 mM) 1 oxotremorine-M (10 mM) treatments was determined from 3 to 120 min. Each data point represents the mean dpm 6 S.E.M. of triplicate samples.

nation of ATP and oxotremorine-M, whereas ATP alone increased AA release 3-fold above basal. Oxotremorine-M and ATP increased AA release 1.06- and 1.07-fold above basal level in M4 cells, respectively, but the combination increased AA release to 3.5-fold above basal. Time- and Receptor-Dependent Release of AA Oxotremorine-M (10 mM) liberated [3H]AA from CHO M5 cells from 1 to 120 min in a time-dependent manner (Fig. 4). The muscarinic antagonist atropine (1 mM) did not alter [3H]AA release from basal levels on its own but, in combination with oxotremorine-M, totally blocked agonist-stimulated release. Basal release of [3H]AA did not appreciably change during the test period.

F. P. Bymaster et al.

FIGURE 5. Concentration-dependent stimulation of [3H]AA release and PI hydrolysis from CHO M3 cells by the muscarinic full agonist carbachol and the partial agonist pilocarpine. The stimulation of [3H]AA release and PI hydrolysis by the concentrations (0.1 nM to 100,000 nM) of the muscarinic agonists was determined three times in triplicate, as described in the Materials and Methods section. Each data point represents the mean % 6 S.E.M. of the maximal effect of carbachol.

The maximal effect of nine agonists to stimulate AA release and phosphoinositide hydrolysis in the CHO M3 cell line was compared with a full agonist (Fig. 6). The ability of the agonist to stimulate AA liberation and phosphoinositide hydrolysis was highly correlated (r 5 0.861, P , 0.01), but partial agonists were less effective at stimulating AA release than phosphoinositide hydrolysis. However, the concentration of agonist required to stimulate AA release and phosphoinositide hydrolysis by 50% of the maximum of that compound (EC50) was similar (Fig. 7) and highly correlated (r 5 0.973, P , 0.001). Similar results were found with the M5 cell line (data not shown). Efficacy and Potency of Muscarinic Agonists to Liberate AA

Stimulation of AA Release and PI Hydrolysis by Muscarinic Agonists The effect of carbachol and pilocarpine on stimulation of AA release and inositol phosphate hydrolysis was compared in the CHO M3 cell line (Fig. 5). Carbachol concentration dependently stimulated AA release and formation of inositol-monophosphates as much as 11- and 9-fold above basal, respectively. Pilocarpine stimulated AA release and formation of inositol-monophosphate as much as 4- and 7-fold above basal, respectively. The concentration-dependent curves for carbachol on stimulation of AA release and phosphoinositide hydrolysis were nearly coincidental. However, the partial agonist pilocarpine more efficaciously stimulated phosphoinositide hydrolysis than AA release.

The ability of 13 muscarinic agonists to liberate [3H]AA from CHO M1, CHO M3 and CHO M5 cell lines was compared (Table 1). Oxotremorine-M, carbachol, cis-dioxolane and muscarine produced the maximal effect in all three cell lines, and S-aceclidine produced the maximal effect in the CHO M1 cell line. The natural neurotransmitter acetylcholine produced the maximal effect in the M1 and M3 cell lines but produced only 87% of the maximal effect in the M5 cell line. Oxotremorine, arecoline, pilocarpine, R-aceclidine, bethanecol, MCN-A-343 and RS-86 were partial agonists in all three cell lines. S-Aceclidine was more efficacious in liberating [3H]AA from each cell line than its enantiomer R-aceclidine. Pilocarpine, MCN-A-343 and RS-86 were substantially more efficacious in the CHO M1 cell lines

Muscarinic Agonist-Induced Arachidonic Acid Release

FIGURE 6. The correlation of the maximal (MAX.) effect of

muscarinic agonists on [3H]AA release (x axis) and on [3H]PI hydrolysis (y axis) from cell lines transfected with muscarinic M3 receptor subtype. The percent maximal induction of [3H]AA release and the percent maximal stimulation of [3H]PI hydrolysis for each agonist were compared with the maximal effect obtainable with a full agonist. The agonists in the correlation are designated by the following numbers in the graph: 1, carbachol; 2, oxotremorine; 3, oxotremorine-M; 4, pilocarpine; 5, arecoline; 6, MCN-A-343; 7, RS-86; 8, R-aceclidine; 9, S-aceclidine. Data are from at least three experiments in triplicate. The correlation coefficient (r) was 0.861, and the P value was ,0.01.

than in CHO M3 or CHO M5. RS-86 was particularly inefficacious in the CHO M5 cell line. The potency of the agonists to liberate [3H]AA varied greatly (Table 1). Oxotremorine-M, oxotremorine, cis-dioxolane and acetylcholine were among the most potent agonists, and bethanecol was the least potent. Acetylcholine was considerably more potent at M3 receptors than at M1 or M5 receptor subtypes. Blockade of AA Release by a Muscarinic Antagonist The ability of the relatively selective M1 antagonist pirenzepine to block oxotremorine-M-stimulated release of [3H]AA from CHO M1, CHO M3 and CHO M5 cell lines was investigated (Fig. 8). Pirenzepine antagonised the oxotremorine-M-induced release of [3H]AA in a concentration-dependent manner and with IC50 values of 152 6 21, 725 6 125 and 3915 6 600 nM for CHO M1, CHO M3 and CHO M5 cell lines, respectively. The calculated Ki values for pirenzepine were 13, 96 and 487 nM for M1, M3 and M5 receptors, respectively. Pirenzepine alone did not appreciably alter basal AA release. Role of External Calcium in AA Release External calcium has been shown to play a key role in AA release [3], and the effect of various techniques for increasing or blocking Ca21 entry into CHO M5 cells was investigated. The divalent ionophore A23187 (10 mM) and the muscarinic agonist oxotremorine-M (10 mM) stimulated AA

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FIGURE 7. The correlation of the potency of muscarinic agonists on stimulation of [3H]AA release (x axis) and PI hydrolysis (y axis) from CHO M3 cells. The log concentration required to produce one-half of the maximal effect (EC50) on [3H]AA release and [3H]PI hydrolysis for each agonist was calculated. The agonists in the correlation are designated by the following numbers in the graph: 1, carbachol; 2, oxotremorine; 3, oxotremorine-M; 4, pilocarpine; 5, arecoline; 6, MCN-A-343; 7, RS-86; 8, R-aceclidine; 9, S-aceclidine. Data are from at least three experiments in triplicate. The correlation coefficient (r) was 0.973, and the P value was ,0.001.

release 15- and 16-fold above basal release, respectively (Fig. 9). The combination of oxotremorine-M and A23187 was additive, and [3H]AA release was increased to 28-fold above basal release. In contrast, there was no substantial muscarinic agonist-induced [3H]AA release in Ca21-free medium or in medium containing the calcium chelator EGTA. The liberation of [3H]AA by oxotremorine-M was reduced to 40% by the non-selective Ca21-channel-blocker cadmium chloride (1 mM). Cadmium alone did not alter basal release of AA (data not shown). Cessation of AA Release by a Muscarinic Antagonist or Calcium Chelation Blockade of agonist-induced AA release from CHO M5 cells after initiation of the liberation process was attempted by utilising either receptor blockade with atropine or calcium chelation with EGTA. In separate samples, EGTA (10 mM) was added at 20 min or atropine (1 mM) at 30 min after initiation of [3H]AA release with oxotremorine-M (10 mM), and AA release was determined at the end of a 90min incubation (Fig. 10). Further [3H]AA liberation from CHO M5 cells was halted upon addition of antagonist or calcium chelator. Addition of atropine or EGTA at other time points produced similar results (data not shown). DISCUSSION In this study, muscarinic agonists produced large increases in AA release in CHO M1, M3 and M5 and A9 L M1 cell

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F. P. Bymaster et al. TABLE 1. Potency and percent maximal effect of muscarinic agonists on [3H]AA release from

CHO M1, M3, and M5 cell lines

Compound Carbachol Muscarine Oxotremorine-M cis-Dioxolane Acetylcholine S-aceclidine Bethanecol Oxotremorine Arecoline R-aceclidine Pilocarpine MCN-A-343 RS-86

M1: EC50, nM (% max.)

M3: EC50, nM (% max.)

M5: EC50, nM (% max.)

1507 6 483 (104 6 2) 377 6 21 (106 6 6) 95 6 16 (100) 92 6 24 (96 6 6) 110 6 37 (99 6 5) 1230 6 65 (107 6 14) 7712 6 738 (69 6 7) 64 6 17 (70 6 4) 672 6 175 (69 6 0) 2107 6 542 (64 6 4) 1473 6 212 (71 6 5) 1258 6 77 (83 6 7) 1164 6 65 (82 6 10)

329 6 61 (107 6 3) 393 6 126 (109 6 3) 153 6 40 (100) 66 6 23 (106 6 3) 563 (95 6 4) 1027 6 174 (82 6 4) 7195 6 930 (77 6 3) 70 6 15 (68 6 4) 308 6 72 (77 6 4) 4793 6 961 (43 6 5) 1772 6 251 (42 6 4) 8358 6 472 (19 6 4) 796 6 94 (47 6 5)

570 6 135 (103 6 6) 1130 6 265 (102 6 11) 142 6 20 (100) 245 6 90 (94 6 3) 430 6 130 (87 6 2) 1750 6 485 (67 6 8) 6900 6 2100 (64 6 5) 89 6 2 (61 6 4) 1930 6 500 (60 6 4) 7585 6 2710 (39 6 6) 3860 6 1900 (36 6 3) 3480 6 790 (31 6 5) — (16 6 2)

Note: CHO cells were transfected with muscarinic M1, M3 or M5 receptors. The EC50 value 6 S.E.M. is the concentration of agonist required to produce one-half of its maximal effect. Maximal liberation of AA release (% max.) for each agonist was determined from at least six concentrations in triplicate (as high as 100 mM) and are the mean 6 S.E.M. of values from at least three separate experiments.

lines. The liberation of the second-messenger AA may be determined directly and simply by determining the radioactivity in the medium as an indication of receptor stimulation. High levels of radioactivity (80–100,000 dpm) can be incorporated into cells by incubation with [3H]AA (0.065 mCi) overnight for 16 to 24 h. Incubation for longer periods reduces the amount of radioactivity releasable by muscarinic agonists (unpublished observation). After incorporation of [3H]AA into the cells, unincorporated radioactivity can be removed by washing the cells in fatty acid-free BSA. The cells are then incubated with agonists in medium containing fatty acid-free BSA to trap liberated AA. Basal release of AA or AA release in the presence of high concentrations of antagonists did not change appreciably for at least 2 h of incubation. For determination of AA release, the radioactivity in an aliquot of medium was simply counted by using liquid scintillation spectrometry. We found that, under normal incubation conditions, CHO and A9 L cells adhere well enough to microtitre plates to not require centrifugation prior to aliquot removal. Because of the simplicity of determining functional receptor stimulation in this assay, this method is amenable to high throughput automation [18]. Arachidonic acid may be released by neuronal receptor subtypes that couple to external calcium

channels in cell lines containing phospholipase A2 such as CHO or A9 L [7]. The liberation of AA was increased from 15- to 32-fold above basal levels in the various cell lines by addition of muscarinic agonists. Liberation of AA release was mediated by muscarinic receptors, as shown by lack of agonist-induced release in untransfected cells and total blockade of release by muscarinic antagonists. Furthermore, muscarinic agonistinduced AA release was concentration and time dependent. Partial agonists such as pilocarpine liberated AA to a lesser extent than full agonists, consistent with previous reports [6, 13]. Muscarinic agonists effectively released AA from the PIcoupled muscarinic receptor subtypes but did not liberate AA from the cAMP-coupled M2 and M4 receptor subtypes, consistent with previous reports [3, 8]. However, addition of ATP increased AA release about 2-fold above the level of ATP alone (this paper and [8]). Receptor-mediated stimulation of phosphoinositide hydrolysis has generally been used to evaluate functional effects of muscarinic agonists in M1, M3 and M5 cell lines [19, 20], but changes in the second-messenger product 1,4,5inositol trisphosphate are transient and difficult to determine. However, stimulation of phosphoinositide hydrolysis may be estimated by measuring formation of inositol-mono-

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FIGURE 8. Antagonism by pirenzepine of oxotremorine-M-induced

release of [3H]AA from CHO cell lines transfected with M1, M3 and M5 muscarinic receptor subtypes. The cells were incubated with various concentrations of the muscarinic antagonist pirenzepine for 30 min prior to addition of oxotremorine-M (1 mM). The liberation of AA was determined 45 min after addition of oxotremorine-M. Each data point represents the percent release 6 S.E.M. of triplicate samples from three separate experiments compared with that obtained with oxotremorine-M alone.

phosphates in the presence of the inositol-1-phosphatase inhibitor lithium. Inositol-monophosphate accumulation may then be determined chromatographically. The dose-dependent stimulation of phosphoinositide hydrolysis and AA liberation by carbachol were compared in CHO M3 and M5 cell lines. The maximal stimulation of AA release and PI hydrolysis in M3 cell lines was well correlated, but partial agonists were more effective in stimulating PI hydrolysis than AA release. Similar results were found in the M5 cell lines. Similar efficacy of partial agonists to stimulate phosphoinositide hydrolysis and release AA was found in M3 cell lines [13]; however, the density of muscarinic receptors in the cell lines used in that study were considerably lower than that in the present study. In addition, the potency of muscarinic agonists to stimulate AA release or PI hydrolysis was well correlated in the present study. These data suggest that the receptor reserve or coupling efficiency of AA release (or both) is less than that for phosphoinositide hydrolysis in the cell lines used in this study, which have relatively high expression of receptors with Bmax values of 2549, 1072 and 1727 fmol/mg protein for CHO M1, CHO M3 and CHO M5 cell lines, respectively (Bymaster, unpublished observation). The stimulation of AA release by a number of muscarinic agonists was compared in M1, M3 and M5 cell lines. The agonists produced a wide range of maximal AA liberation compared with the full agonist oxotremorine-M. No agonists examined demonstrated striking selectivity for a particular subtype, in agreement with similar comparisons using PI hydrolysis [19, 20]. Oxotremorine-M, carbachol, muscarine

FIGURE 9. Effect of calcium-containing medium, calcium-free medium, EGTA, cadmium and A23187 on [3H]AA release in CHO M5 cells. The liberation of AA by control (basal), oxotremorine-M (10 mM)-, calcium ionophore A23187 (10 mM)- and A23187 (10 mM) 1 oxotremorine-M (10 mM)treated samples was determined after a 45-min incubation as described in the Materials and Methods section. The release of AA induced by oxotremorine-M (10 mM) was also determined in triplicate in calcium-free medium, medium containing EGTA (10 mM) or cadmium Cl (1 mM). Each data point represents the percent release 6 S.E.M. of various treatments compared with that obtained with oxotremorine-M (10 mM) alone. Basal release was not altered appreciably by EGTA, Ca21-free medium or cadmium. *P , 0.05 versus basal release; #P , 0.05 versus ATP or oxotremorine-M alone.

and cis-dioxolane produced the maximal effect in all three cell lines. However, several agonists show distinct differences on stimulation of AA release between the receptor subtypes. For example, MCN-A-343 and pilocarpine were more effective at liberating AA release in cells with M1 receptors than M3 or M5 receptors, consistent with results utilising phosphoinositide hydrolysis [19, 20] and AA release in M1 and M3 cell lines [13]. S-Aceclidine was more effective at stimulating AA release from cells with any of the three receptors than the less-active enantiomer R-aceclidine [21]. RS-86 was much less effective at liberating AA release from cells with M5 receptors than M1 or M3 receptors, consistent with previous data utilising phosphoinositide hydrolysis [20]. Muscarinic agonists also varied widely in potency. For example, oxotremorine-M and cis-dioxolane had EC50 values in the range of 100 nM, whereas bethanecol had EC50 values of about 7 mM, consistent with previous reports on potency for stimulating phosphoinositide hydrolysis [20]. Acetylcholine was 22 and 86 times more potent at M3 receptors than at M1 or M5 receptors, respectively. The muscarinic antagonist pirenzepine blocked liberation of AA induced by oxotremorine-M in a concentration-dependent fashion in

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FIGURE 10. Effect of addition of EGTA (10 mM) added at 20

min (1) or atropine (1 mM) added at 30 min (2) on oxotremorine-M (10 mM)-induced release of [3H]AA from CHO M5 cells. The release of AA from the samples containing EGTA or atropine was determined at 90 min. Each data point (triplicates) represents the percent release 6 S.E.M. (when not covered by the data point) of that obtained with oxotremorine-M at 90 min.

the cell lines. However, pirenzepine more potently blocked agonist-induced release of AA from M1 cell lines than M3 or M5 cells, consistent with its higher affinity for the M1 receptor in binding assays [14]. Thus, antagonism of muscarinic agonist-induced release of AA may be used to determine the selectivity of antagonists for muscarinic receptors. In this study, AA release was demonstrated to require constant receptor occupancy and continual availability of external calcium; the requirement of external calcium was previously demonstrated [3, 9]. After initiation of AA release by muscarinic agonists, the liberation of AA could be rapidly terminated by either occupancy of the receptor with muscarinic antagonists or addition of the calcium chelator EGTA. This is in agreement with previous findings suggesting that receptor occupancy by muscarinic agonists mediates opening of a voltage-insensitive Ca21 channel, and Ca21 influx activates phospholipase A2 [7]. Furthermore, these data suggest that this is a dynamic receptor-mediated process that is not readily down-regulated. The role of external calcium on AA release was further investigated. The divalent ionophore A23187, which functions as a carrier of external calcium into the cell [22], has been shown to stimulate release of AA and potentiate dopamine-induced AA release from CHO cells transfected with dopamine D2 receptors [23]. In CHO cells transfected with M5 receptors, A23187 increased AA liberation. Further, A23187 additively increased AA release induced by oxotremorine-M alone. On the other hand, deletion of calcium from the medium or chelation of calcium with EGTA completely abolished agonist-induced AA release. The non-selective calcium-channel-blocker cadmium partly in-

F. P. Bymaster et al.

hibited muscarinic agonist-induced AA release. These data demonstrate the Ca21 requirement for the process that liberates AA and that the Ca21 flux produced by muscarinic agonists is not sufficient to saturate the enzyme involved, presumably phospholipase A2 [7]. The liberation of arachidonic acid from cells transfected with PI-coupled muscarinic receptors would be a sensitive technique for studying calcium influx and calcium channels coupled to muscarinic receptors. In summary, these data indicate that muscarinic agonistinduced AA release from CHO or A9 L cell lines transfected with M1, M3 or M5 muscarinic receptor subtypes is a simple method for determining the response of muscarinic agonists at the respective subtypes. In addition, partial agonist effects could be demonstrated with this assay. Muscarinic agonist-induced liberation of AA was reproducible and increased from 10- to 30-fold above basal levels. The liberation of AA may be induced from M2 and M4 cell lines by muscarinic agonists in the presence of ATP, but the amount of AA released is much smaller than with phosphoinositide-coupled receptors. Antagonist blockade of liberation of AA induced by muscarinic agonists may be used to determine selectivity of muscarinic antagonists. Because of the simplicity of the assay, it is amenable to development as a high throughput assay [18]. Mechanistically, muscarinic agonist-induced AA release from PI-coupled cell lines is a dynamic process that requires constant receptor occupancy and influx of external calcium. References 1. Peralta E. G., Ashkenazi A., Winslow J. W., Ramachandran J. and Capon D. J. (1988) Nature (Lond.) 334, 434–437. 2. Axelrod J., Burch R. M. and Jelsema C. L. (1988) Trends Neurosci. 11, 117–123. 3. Conklin B. R., Brann M. R., Buckley N. J., Ma A. L., Bonner T. I. and Axelrod J. (1988) Proc. Natl. Acad. Sci. USA 85, 8698–8702. 4. Kanterman R. Y., Ma A. L., Briley E. M., Axelrod J. and Felder C. C. (1990) Neurosci. Lett. 118, 235–237. 5. Tence M., Cordier J., Premont J. and Glowinski J. (1994) J. Pharmacol. Exp. Ther. 269, 646–653. 6. Baumgold J., Dyer K., Falcone J. F. and Bymaster F. P. (1995) Cell. Signal. 7, 39–43. 7. Felder C. C., Deter P., Kinsella J., Tamura K., Kanterman R. Y. and Axelrod J. (1990) J. Pharmacol. Exp. Ther. 255, 1140–1147. 8. Felder C. C., Williams H. L. and Axelrod J. (1991) Proc. Natl. Acad. Sci. USA 88, 6477–6480. 9. Brooks R. C., McCarthy K. D., Lapetina E. G. and Morell P. (1989) J. Biol. Chem. 264, 20147–20153. 10. Sauerberg, P., Olesen P. H., Nielsen S., Treppendahl S., Sheardown M. J., Honore T., Mitch C. H., Ward J. S., Pike A. J., Bymaster F. P., Sawyer B. D. and Shannon H. E. (1992) J. Med. Chem. 35, 2274–2283. 11. Bymaster F. P., Whitesitt C. A., Shannon H. E., DeLapp N., Ward J. S., Calligaro D. O., Shipley L. A., Buelke-Sam J. L., Bodick N. C., Farde L., Sheardown M. J., Olesen P. H., Hansen K. T., Suzdak P. D., Swedberg M. D. B., Sauerberg P. and Mitch C. H. (1997) Drug Dev. Res. 40, 158–170. 12. Lahti R. A., Figur L. M., Piercey M. F., Ruppel P. L. and Evans D. L. (1992) Mol. Pharmacol. 42, 432–438.

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