A Transfected m l Muscarinic Acetylcholine Receptor Stimulates. Adenylate Cyclase via Phosphatidylinositol Hydrolysis*. (Received for publication, March 17, ...
THEJOURNAL O F BIOLOGICAL CHEMISTRY
Vol. 264, No. 34, Issue of December 5, pp. 20356-20362,1989 Printed in U.S.A.
A Transfected m l Muscarinic Acetylcholine Receptor Stimulates Adenylate Cyclasevia Phosphatidylinositol Hydrolysis* (Received for publication, March 17, 1989)
Christian C. Felder, Robert Y. KantermanS, Alice L. Ma, and JuliusAxelrod From the Laboratoryof Cell Biology, Section on Pharmacology, National Institute of Mental Health, Bethesda, Maryland20892 and the $Howard Hughes Medical Institute, National Institutes of Health, Research Scholars Program, Bethesda, Maryland 20892
The m l muscarinic acetylcholine receptor gene was systems are activated upon receptor occupation. a-l-adrenerA9 L cells. gic stimulation of both phospholipase AZ and phospholipase transfected into and stably expressed in The muscarinic receptor agonist, carbachol, stimulated C in FRTL5 cells and MDCK cells occurs through separate re- G proteins (1,2). A dissociation of bradykinin-induced phosinositolphosphate generation,arachidonicacid lease, and cAMP accumulation in these cells. Carbachol pholipase Az and phospholipase C was shown in Swiss 3T3 stimulated arachidonic acid and inositol phosphate re- cells and MDCK cells (3, 4). Dopamine-1-stimulated adenyllease with similar potencies, while cAMP generation ate cyclase and phospholipase C activity has been described required a higher concentration. Studies were perin renal membrane preparations ( 5 ) . Muscarinic agonists formed todetermine if the carbachol-stimulated cAMP decreased adenylate cyclase activity and increased phosphoaccumulation was due to direct coupling of the m l muscarinic receptor to adenylate cyclase via a GTP lipase C activity inhuman astrocytoma cells (6) and increased binding protein or mediated by other second messen- both of these responses in SK-N-SH neuroblastoma cells (7). It cannot be ascertained from these studies if the added gers. Carbachol failed to stimulate adenylate cyclase agonist is stimulating oneor more as yet unidentified subtypes activity in A9L cell membranes, whereas prostaglandin E2 did, suggesting indirect stimulation. The phor- of the receptor to induce multiple intracellular signals. The bo1 ester, phorbol 12-myristate13-acetate (PMA), cloning, transfection, and functional expression of single stimulated arachidonic acid release yet inhibited cAMP receptor genes into host cells has allowed the study of the accumulation in response to carbachol. PMA also in- coupling of a single receptor subtype to second messenger hibited inositol phosphate release in response to car- systems without the presence of multiple receptor subtypes bachol, suggesting that activation of phospholipase C from the same receptor family (8-10). From these model might be involved in cAMP accumulation. PMA did not systems, it may be possible to determine if one receptor is cholera toxin-, or forskolin- coupling to multiple G proteins and effector enzymes to induce inhibit prostaglandin E2-, stimulated cAMP accumulation. The phospholipase A2 pleiotropic responses or if a second messenger generated from inhibitor eicosatetraenoic acid and the cyclooxygenase a single receptor-transducer interactionis activating asecond inhibitors indomethacin and naproxen had no effect effector on enzyme, independent of the receptor or G protein. carbachol-stimulated cAMP accumulation. CarbacholIn this paper, a model system was utilized in which the stimulated cAMP accumulationwasinhibitedwith gene for the mlsubtype of a family of five muscarinic acetylTMB-8, an inhibitor of intracellular calcium release, choline receptors has been transfected into and stably exand W7,a calmodulin antagonist. These observations pressed in the A9 L fibroblast to study the coupling of a single suggest that carbachol-stimulatedcAMP accumulation receptor subtype to multiple transduction pathways. In the does not occur through direct m l muscarinic receptor coupling or through the release of arachidonic acid and A9 L cell, the ml receptor generates several second messenits metabolites, but is mediated through the activation gers including arachidonic acid release, inositol phosphate of phospholipase C. The generationof cytosolic calcium generation, and cAMP accumulation (10,ll). Themuscarinic agonist carbachol stimulated cAMPaccumulation, which may via inositol 1,4,5-trisphosphate and subsequent activation of calmodulin by m l muscarinic receptor stim- be the result of direct coupling of the receptor to adenylate cyclase through a G protein, or secondary to stimulation of ulation of phospholipase C appears to generate the phospholipase C or phospholipase Az. Our data indicate that accumulation of CAMP. the carbachol-stimulated cAMP accumulation in these cells is a consequence of phospholipase C activation. Hormones and neurotransmitters generate intracellular signals through a sequence involving binding to cell surface receptors which are coupled to guanine nucleotide regulatory proteins (G proteins), which then stimulate or inhibit effector enzymes. Initially, it was thought that a receptor coupled to only a single transduction pathway to stimulate one intracellular second messenger. Recently, a number of more complex systems have been described in which multiple transduction * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
EXPERIMENTAL PROCEDURES
Materials [5,6,8,9,11,12,14,15-3H]Arachidonic acid and [32P]adenosine5’-triphosphate,tetra(triethy1ammonium) salt was purchased from Du Pont-New England Nuclear and [3H]inositol from American Radiolabeled Chemicals Inc. Phorbol 12-myristate 13-acetate (PMA)’ and
’
The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; IP3, inositol 1,4,5-trisphosphate; IP2, inositol 1,4-bisphosphate; IP, inositol 1-monophosphate; IBMX, 3-isobutyl-1-methylxanthine; TMB-8, 3,4,5-trimethoxybenzoic acid8-(diethylamino)octylester; W7, N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide hydrochloride; W5, N-(6-aminohexyl)-l-naphthalenesulfonamide hydro-
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Muscarinic Receptor Stimulated Accumulation CAMP calmodulin (bovine brain) were purchased from Calbiochem. A9 L cells were obtained from the American Type Culture Collection and are a fibroblast-like subclone of the cell line ATCC CCL 1. Reagents for the determination of CAMP byRIAwas supplied by Dr. Gary Brooker (Georgetown University, Washington D.C.). 4-(3-Butoxy-4methoxybenzyl)-2-imidazolidinone(RO 20-1724) and 1-methyl-3480butylxanthine (IBMX)was purchased from Biomol (Plymouth Meeting, PA). All other reagents were obtained from Sigma. Cell Culture ofA9 L Cells Transfected with and Stably Expressing the Cloned ml Muscarinic Receptor A9 L cells which were transfected with and stably expressing the m l subtype of the muscarinic acetylcholine receptor were generously supplied by Dr. Mark Brann and Dr. Noel Buckley(National Institute of Neurological Disorders and Stroke). A9 L cell clones were selected that were expressing receptor densities similar to densities found in commonly studied cells and membranes (100-200 fmol/mg of protein) (11).The level of carbachol-stimulated arachidonic acid release, inositol phosphate release, and cAMP accumulation was consistent throughout the duration of the study. All experiments were performed in 24-well Costar plastic culture plates (Becton Dickinson, Oxnard, CA) with A9 L cells grown in adherent monolayers at a density of greater than 90% confluence as previously described (10). Measurement of cAMP Accumulation in Whole Cells and Adenylate Cyclase Activity in Cell Membrane Preparations Whole Cells-A9 L cells were grown to confluence, and thegrowth media were replaced with 250 pl of serum-free media containing 20 mM HEPES and 1 mM IBMX with or without the experimental agents to be tested. The reaction was stopped after 5 min with 250 PI of an ice cold solution containing 0.1 N HC1 and 1 mM CaCI2. The accumulation of cAMP was measured by RIA as previously described (12). In the absence of IBMX, no cAMP accumulation was detected. Following carbachol stimulation, no cAMP was detected in the media without lysing the cells, indicating that little if any transport of cAMP occurred under the assay conditions described above. Cell Membrane Preparation-A9 L cells were grown to confluence, and growth media were replaced with serum-free media containing 20 mM HEPES. The cells were scraped off the surface of the culture flask and centrifuged at 1000 X g in a Beckman TJ-6 centrifuge for 15 min. The pellet was resuspended in serum-free media containing 20 mM HEPES and 1 mM IBMX. The pellet was sonicated for 30 s with a Kontes Cell Disrupter (Kontes Equipment Co., Vineland, NJ) set a t a frequency of 4 and power of 5. Complete disruption of the cells was verified by loss of adenylate cyclase activity in the absence of added ATP and an ATP regeneration system, as well as the loss of PGE,, stimulated cAMP accumulation in the absence of GTP, ATP, and an ATP regeneration system. Membranes were then centrifuged at 24,000 X g for 30 min, and the pellet was resuspended in ATP regeneration buffer containing 10 mM HEPES, 140 mM NaCI, 3 mMMgC12, 1 mM IBMX, 0.3 mM ATP, 6.7 mM phosphocreatine, 30 units/ml creatinephosphokinase, 1pM GTP. ThecAMP produced over 5 min at 30 "C with and without added experimental agents was analyzed by RIA as described above for whole cells. Measurement of [3HHrachidonicAcid Release A9 L cells were incubated with arachidonic acid (0.25 pCi/well) to isotopic equilibrium (18-24 h). Prior to the addition of experimental agents, the cells were washed twice with 1 mlof serum-free media supplemented with 20 mM HEPES and 0.2% bovine serum albumin (fatty acid-free). The experimental agents were added in a final volume of 1 ml, and the reaction was allowed to proceed for 15 min unless otherwise noted. The reaction was stopped by removing the incubation media, and released [3H]arachidonic acid was measured with a liquid scintillation spectrophotometer. Measurement of [3H]inositolPhosphate Release A9 L cells were incubated with [3H]inositol (0.5 pCi/well) to isotopic equilibrium (18-24 h). Prior to the addition of experimental chloride; ETYA, eicosatetraenoic acid; NDGA, nordihydroguaiaretic acid HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid; RO 20EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; G,, stimula1724, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone; tory guanyl nucleotide binding regulatory protein; RIA, radioimmunoassay; PG, prostaglandin.
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agents, the cells were washed twice with 1 mlof serum-free media supplemented with 20 mM HEPES and10 mM LiC12.The experimental agents were added in a final volume of 0.5 ml, and the reaction wasallowed to proceed for 15 min unless otherwise noted. The reaction was stopped by the addition of 0.5 mlof an ice cold stop solution containing 1 M KOH, 18 mM NazB407,3.8 mM EDTA, and 7.6 mM NaOH. The stop solution was immediately neutralized with the addition of 0.5 ml of a solution containing 7.5% HCI. Released inositol phosphates were separated by anion exchange chromatography as previously described (13). Measurement of Protein Kinase C Activity in A9 Cell Membranes and Cytosol A9 cells were removed from the flask with brief trypsin (0.01%) treatment, washed once in growth media, and resuspended in serumfree media buffered with 20 mM HEPES (pH 7.4). A9 cells were incubated with lo" M PMA in a final volume of 250 pl for the times indicated in the figure legend at 37 "C. The reaction was stopped by diluting the reaction mixture with 10 mlofice cold phosphatebuffered saline solution. Protein kinase C was assayed by the method of Kraft andAnderson (14) with modifications by Zatz et al. (15). A9 cells in suspension followingbrief trypsin treatment had similar carbachol-stimulated arachidonic acid release, inositol phosphate generation, and increase in intracellular calcium as cells grown in monolayers in plastic culture dishes (data not shown). Proteins were measured by the method of Bradford (Bio-Rad protein assay). Depletion of Calmodulin from A9 Cell Membranes Calmodulin depletion was attempted using a modification of the procedures of Brostrom et al. (30) and MacNeil et al. (31) as follows. Cells were washed twice with incubation buffer containing 10 mM HEPES, 140 mMNaC1, 3 mMMgC1, and then scraped from the surface of the culture flask. The cells were centrifuged at 1,000 X g in a Beckman TJ-6 centrifuge for 15 min, and the pellet was resuspended in incubation buffer containing LaC13 (20 p ~ 200 , p ~ or, 2,000pM) or EGTA (1 mM, 3 mM, 5 mM). The cells were then sonicated for 15 s with a Kontes cell disrupter as described for the membrane preparation above. The membranes were incubated with either the Lac13 or EGTA at 4 "C for 15 min, then washed three times with incubation buffer without the added EGTA or LaCL by centrifugation at 10,000 X g for 10 min in a Microfuge. The final membrane preparation was resuspended in ATP regeneration buffer as described above for the RIA of CAMP. The membranes retained PGE2-, cholera toxin-, and forskolin-stimulated adenylate cyclase activity over the range of EGTA or Lack tested, while higher concentrations were inhibitory. RESULTS
Tramfected and Stably Expressed ml Muscarinic Receptor Gene Generates Multiple Second Messengers-In A9 L fibroblasts transfected with and stably expressing the m l muscarinic receptor, carbachol stimulated inositol phosphate release (EC50IP = 6 g ~ IP2 , = 14 g ~ IP, , = 15 PM) and arachidonic acid release (EC50 = 7 p ~ with ) similar potencies when assayed under identical assay conditions (Fig. 1). Higher concentrations of carbachol were required to stimulate cAMP M) accumulation (EC50= 41 g M ) (Fig. 1). Carbachol caused an immediate rapid increase in cAMP accumulation in whole cells and adenylate cyclase activity in membrane preparations that peaked at 5 min (data not shown). Carbachol-stimulated inositol phosphate generation was also immediate and rapid, but was sustained over 30 min (the last timepoint tested). Carbachol-stimulated arachidonic acid release was not as rapid initially but was sustained for 30 min (the lasttime point tested) (datanot shown). The Muscarinic ml Receptor Is Not Directly Linked to cAMP Accumulation through a GTP Binding Protein-Carbachol-stimulated cAMP accumulation could occur through linkage of the m l receptor to adenylate cyclase via a GTP binding protein (G& Carbachol did not increase adenylate cyclase activity in A9 L cell membrane preparations (Fig. 2). Carbachol added with GTP did not increase adenylate cyclase
Muscarinic Receptor Stimulated
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FIG.1. Carbachol-stimulated arachidonicacid release, inositol phosphate release, and cAMP accumulation in A9 L cells expressing the m l receptor. Experiments were performed as described under “ExperimentalProcedures”and were undersimilar assay conditions. Data is the mean ? S.E. of threeexperiments performed in triplicate. Thecurue represented by the closed triangles is the mean f S.E. of the responses for all three second messenger systems performed in triplicate in the presence of atropine andwere not significantly different from each other or basal levels ( p < 0.01 analysis of variance, Newman Keuls Test). Atropine was added 15 min prior to the addition of carbachol.
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FIG.3. PMA enhanced basal and carbachol-stimulated arachidonic acid release yet inhibitedcarbachol-stimulated cAMP accumulation inA 9 L cells expressing the m l receptor. A , arachidonic acid release; B, cAMP accumulation. Arachidonic acid release and cAMP accumulation were measured as described under “Experimental Procedures” and were under similar assay conditions. PMA was preincubated with cells for 1 h. The data are the mean If: S.E. of three experimentsperformed in triplicate.
5 15a
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lated cAMP accumulationwas tested (Fig. 3, A and B). PMA increased both basal and carbachol-stimulated PGEz formaFIG.2. Carbachol didnot stimulate adenylatecyclase activ- tion over a similar concentration range to arachidonic acid ity, but GTP enhanced PGEz-stimulated adenylate cyclase release (data not shown). In contrast, PMA inhibited caractivity in A9 L cell membranes prepared from cells express- bachol-stimulated cAMP accumulation (ICso = 20 nM), but ing the m l receptor. Adenylate cyclase activity was measured in had no effect on basal cAMP levels (Fig. 3B). From these A9 L cell membranes in the presence of an ATP regeneration system as described under “Experimental Procedures.”Data are the mean f observations, it is unlikely that arachidonicacid or its metabS.E. of triplicate measurements and are representative of three ex- olites are involved in carbachol-stimulated cAMP accumulation. periments performed in triplicate.Concentrations used were M carbachol, M PGE,, and M GTP. Several inhibitors of the production of arachidonic acid metabolites were tested for their ability to block carbacholactivity more than GTP alone. To test for the presence of stimulated cAMP accumulation.Nordihydroguaiareticacid functional G,, adenylate cyclase activity was stimulated with (NDGA), a lipoxygenase inhibitor, did block carbachol-stima concentration-dependent PGEz which has been shown to couple to adenylate cyclase ulatedcAMPaccumulationin = 53 p ~ with ) complete inhibition at 500 p M through G, (16). PGE, alone increased adenylate cyclase manner (Fig. 4A), but also inhibited carbachol-stimulatedrelease of activity in membrane preparations, and, when added with = 40 pM IP, GTP, there was a marked increase in activity (Fig. 2). These inositol phosphates with similar potencies observations indicate that PGEz receptor-G,-mediated acti- 24 pM IP2, 22 p M IP3) with complete inhibition at 100 p M vation of adenylate cyclase can occur in A9 L cells and that (Fig. 4B). This demonstrates the lack of specificity of this carbachol-stimulated adenylate cyclase activity may not be putative lipoxygenase inhibitor since it inhibited the release of arachidonic acid and inositol phosphates with equal podirectly coupled through a G T P binding protein. Carbachol-stimulated CAMPAccumulation Is Not Mediated tency. NDGA did not alter the binding characteristicsof the by the Products of Arachidonic Acid Metabolism-PMA has m l receptor as defined with N-methyl[3H]scopolamine and acid (ETYA), a been shown to stimulate both basal and carbachol-stimulated-atropine (data not shown). Eicosatetraenoic arachidonic acid release in A9 L cells (10).Similar stimulation potent 12-lipoxygenase inhibitor and partial 5-lipoxygenase, of arachidonicacidrelease was observed under the assay cyclooxygenase, and phospholipase Az inhibitor, had noeffect conditions used in these experiments (Fig. 3A). To determine on carbachol-stimulated cAMP accumulation up to 500 pM, if the increase in cAMP accumulationwas due to the release yet inhibited carbachol-stimulated arachidonic acidrelease . and indomethacin,cyclooxygenase (IC50= 50 p ~ )Naproxen of arachidonic acid, the effect of PMA on carbachol-stimuBASAL
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FIG.5. PMA inhibited the release of inositol phosphates in A9 L cells expressing the m l receptor. PMA inhibition of carbachol-stimulated inositol 1,4,5-trisphosphate (IPa), inositol 1,4-bisphosphate (IPZ), and inositol 1-monophosphate (IP) release. The data is the mean f S.E. of three experiments performed in triplicate. PMA was added 1h before the addition of carbachol.
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FIG.4. Inhibition of carbachol-stimulated cAMP accumulation and inositol 1,4,5-trisphosphate (IPS)release by NDGA in A9 L cells expressing the m l receptor. A, cyclic AMP accumulation. B , IP, release. Cyclic AMP accumulation and IPa release were measured as described under "Experimental Procedures." The data are the mean f S.E. of three experiments performed in triplicate. NDGA was added 15 min before the addition of carbachol. Similar results were observed for the release of IPz and IP(data not shown). inhibitors, had no effect on carbachol-stimulated cAMP accumulation up to 500 p ~ Indomethacin . and naproxen inhibited carbachol-stimulated PGEz release with equal potency . addition of arachidonic acid M) had (ICBo= 5 p ~ )The no effect on basal cAMP accumulation. In view of the above observations, it is unlikely that PGEZ or any otherarachidonic acid metabolite mediates the carbachol-stimulated cAMP accumulation observed in these cells. PMA Inhibits Inositol Phosphate Release and CAMPAccumulation with Equal Potency-PMA was used to determine if the phospholipase C pathway was involved in carbacholstimulated cAMP accumulation. PMA has been shown to inhibit inositol phosphate release in the A9 L cell (10). The effect of PMA on carbachol-stimulated inositol phosphate release and cAMP accumulation was measured under identical assay conditions. PMA inhibitedcarbachol-stimulated inositol phosphate release (Fig. 5) IP = 10 nM, IP2 = 14 nM, IP3 = 18 nM) and cAMP accumulation (Fig. 3 B ) (ICbo= 20 nM) with similar potencies. These findings suggest an indirect stimulation of cAMP accumulation as a result of m l muscarinic receptor-coupled phospholipase C activation. The effects of PMA described above may be mediated by protein kinase C. Brief incubation of cells with PMA has been shown to cause an apparent activation and translocation of protein kinase C from the cytoplasm to the membrane, while extended incubation with PMA desensitizes protein kinase C (17). Similar results were observed in A9 cells transfected with the m l receptor (Fig. 6). PMA stimulated proteinkinase C activity in A9 cell membranes up to 30 min which was
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FIG.6. The phorbol ester PMA stimulated protein kinase C activity in A9 membranes and decreased activity in the cytoplasm. PMA was incubated with A9 cells in suspension for the times indicated as described under "Experimental Procedures." Data are representative of three experiments performed in duplicate.
followed by a desensitization that was maximal by 2 h of incubation. PMA caused a decrease in cytosolic protein kinase C activity that was further decreased by 2 h of incubation. When PMA was preincubated with the A9 L cells for various times, maximal inhibition of cAMP accumulation and inositol phosphate release was observed between 30 min and 3 h. After prolonged incubation (4-18 h), the effect was abolished, indicatingadesensitization of protein kinase C (data not shown). This is consistent with the view that the effects of PMA are exerted throughthe activation of protein kinase C. Protein kinase C could regulate cAMP generation at the receptor protein (18), G protein (19), or adenylate cyclase directly (20). To determine whether protein kinase C acted directly on cAMP generation, the effect of PMA was tested on PGE2-, cholera toxin (a direct activator of GJ, and forskolin (a direct activator of adenylatecyclase)-stimulated cAMP accumulation. PMA, at a concentration that had maximal inhibitory effects on carbachol-stimulated cAMP accumulation and inositol phosphate release, did not inhibit PGE2, cholera toxin, or forskolin-stimulated cAMP accumulation (data not shown). In addition, carbachol-stimulated cAMP accumulation was additive with maximal PGE2-, cholera toxin-, andforskolin-stimulated cAMP accumulation (Fig. 7). These observations suggest that PMA is not inhibitingcAMP
~
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CAMPAccumulation
Muscarinic Receptor Stimulated
I +CARBACHOL
FIG. 7. Maximal carbachol-stimulated cAMP accumulation was additive withmaximal PGE2, cholera toxin-,or forskolinstimulated cAMP accumulation in A9 L cells expressing the m l receptor. Cells were preincubated in growth media with cholera toxin (100 ng/ml) ( C T x ) for 4 h.Cyclic AMP accumulation was measured over 5 min after exchanging the growth media with serumM), cholera toxin (100 ng/ml), or free media containing PGEz M), with or without carbachol M) as indicated. forskolin Data are the mean +- S.E. of three experiments performed in triplicate.
30
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ill 10 D 5
log [TMB-E3 or W7]
vestigate the role of calmodulin in this pathway (22). W7 inhibited carbachol-stimulated cAMP accumulation with an IC50 of 17 pM that was maximal at 100 p~ (Fig. 8). W7 had no effect on basal or carbachol-stimulated inositol phosphate release up to 50 pM. An inactive analog of W7, N-(6-aminohexyl)-1-naphthalenesulfonamidehydrochloride (W5), had no effect on carbachol-stimulatedcAMP accumulation or inositol phosphate release (data not shown). The inhibition of carbachol-stimulated cAMP accumulation with W7 suggests a role for calmodulin in this pathway. In an attempt to clarify the role of calmodulin in adenylate cyclase activation, cell membranes were incubated with calmodulin (10-6-10-9 M) over a range of calcium concentrations (10-10-10-3 M). Membranes were subjected to a calmodulin depletion procedure involving treatment with either EGTA or Lac13 (see “Experimental Procedures”). Addition of calmodulin to untreated orcalmodulin-depleted membranes did not significantly stimulateadenylate cyclase activity. Calmodulin did not activate adenylate cyclase activity in whole cell homogenates. Muscarinic receptor-mediated stimulation of calcium/calmodulin-sensitive phosphodiesterases have been shown in 132N1 human astrocytoma cells (6, 23). The phosphodiesterase inhibitors, 3-methyl-1-isobutylxanthine(IBMX) and 4(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO 20-1724) were added from a concentration of 100 nM up to saturating M and 5 X lo-* M, respectively, to concentrations of 5 X test if muscarinic receptor-mediated inhibition of phosphodiesterase was responsible for cAMP accumulation. In the M IBMX, cAMP accumulation was presence of 5 X increased 4-fold. Inthe presence of IBMX (5 X M) and M), cAMP accumulation was stimulated carbachol (1 X 9-fold. Inthe presence of 5 X M RO 20-1724, CAMP accumulation was increased 50% and was stimulated 9-fold M). with the addition of carbachol (1 X DISCUSSION
When the product of a single transfected receptor gene is FIG. 8. TMB-8, an inhibitor of intracellular calcium reexpressed in a host cell, multiple signal transduction pathways lease, and W7,a calmodulin inhibitor, inhibited carbacholstimulated cAMP accumulation in A 9 L cells expressing the can be activated (8-11). The ml muscarinic acetylcholine m l receptor. Cells were preincubated with increasing concentrareceptor gene, when transfected into and stably expressed in tions of TMB-8 or W7 for 15 min and basal and carbachol-stimulated A9 L cells, stimulated inositol phosphate and arachidonic acid cAMP accumulation measured as described under“Experimental release with similar potencies IP = 6 pM, IP, = 14 pM, Procedures.” Data are the mean f S.E.of three experiments perIPS= 15 p ~arachidonic , acid = 7 p ~ and ) stimulated cAMP formed in triplicate (CC = carbachol). accumulation at a higher concentration (ECIO= 41 pM). Our data indicate that the carbachol-stimulated cAMP accumugeneration directly and reinforces the idea that itsinteraction lation is not due to direct coupling of the m l receptor to with carbachol-stimulated cAMP generation is mediated by adenylate cyclase through aGTP binding protein. In addition, its effects (presumably throughproteinkinase C) on the carbachol-stimulated cAMP accumulation is nota consephospholipase C pathway. quence of arachidonic acid release and subsequent production Calcium and Calmodulin Antagonists Inhibit Carbacholof prostaglandins. It is carbachol-stimulated inositol phosstimulated CAMPAccumulation-All the above data suggest phate release that appears to activate the increase in cAMP that the phospholipase C pathway is involved in cAMP generation. Activation of phospholipase C causes the release of througha mechanism involving intracellular calcium and inositol 1,4,5-trisphosphate (IPS)which induces the release of calmodulin (Fig. 9). The role of calcium and calmodulin in carbachol-stimulated intracellular calcium (13). Several agents that modify calcium cAMP accumulation was investigated with TMB-8, an inhibmetabolism were tested to determine the role of calcium in itor of intracellular calcium release, and W7, a calmodulin carbachol-stimulated cAMP accumulation. TMB-8, an inhibitor of intracellular calcium release (21), blocked carbachol- antagonist. TMB-8 blocked carbachol-stimulated CAMP acstimulated CAMPaccumulation with an ICs0 of2 p~ that was cumulation at a concentration which did not inhibit inositol maximal at 100 p~ (Fig. 8).T M B S did not inhibit carbachol- phosphate release, suggesting that this agent blocks CAMP stimulated inositol phosphate release up to 2 p~ and required generation at a step beyond phospholipase C. W7, but not the inactive analog W5, blocked carbachol-stimulated cAMP acgreater than 100 KM for complete inhibition. Calmodulin has been shown to stimulate cAMPgeneration. cumulation at a concentration that did not inhibit inositol The calmodulin antagonist N-(6-aminohexyl)-5-chloro-l- phosphate release, suggesting a role for calmodulin in CAMP naphthalenesulfonamide hydrochloride (W7) was used to in- generation. To furtherclarify the role of calmodulin in aden-
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Muscarinic Receptor Stimulated CAMPAccumulation
U
.
muscarinic m l receptor
, FIG. 9. A model of the proposed pathways involved in m l muscarinic receptor activation of adenylate cyclase. m l muscarinic receptoractivated phospholipase C releases intracellular inositol 1,4,5-trisphosphate(IP,) which then activates adenylate cyclase through a rise in intracellular calcium in concert with calmodulin. The activated adenylate cyclase may be distinct from the population stimulated by PGEz. The inhibition of inositol phosphate release by protein kinase C may occur at a step beyond the m l muscarinic receptor.
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t t
Kinase C
ylate cyclase stimulation, calmodulin was added directly to A9 cell membranes. Addition of calmodulin to A9 cell membranes failed to stimulate adenylate cyclase activity above carbachol-stimulated activity. Endogenous calmodulin may have been present in the membrane preparations preventing further activation. Therefore, two methods of calmodulin depletion were attempted which have successfully depleted membrane-bound calmodulin from a variety of cell types (for review, see Ref. 29). Again, calmodulin failed to stimulate adenylate cyclase activity. It is possible that endogenous calmodulin is not easily removed from A9 cell membranes and would therefore block the effect of added calmodulin. It is also possible that several factors, such as protein kinases, are required in conjunction with calmodulin for adenylate cyclase activation that were removed during the membrane preparation. Calmodulin failed to stimulate adenylate cyclase activity in whole cell homogenates suggesting that even if essential factors are present, the cell must remain intact for muscarinic receptor-stimulated adenylate cyclase to function. Experiments with W7 and W5 offer preliminary evidence for a role of calmodulin in adenylate cyclase activation. The phorbol ester PMA has been shown to stimulate protein kinase C, which is involved in the regulation of a number of cell processes including the stimulation or inhibition of adenylate cyclase and phospholipase C activity (24-27). In the A9 cell, PMA stimulated membrane-bound proteinkinase C with a concomitant decrease in cytosolic protein kinase C activity consistent with anapparent translocation of the enzyme from cytosol to membrane. Similar results were seen with the addition of carbachol M), andthe stimulation and desensitization occurred with a similar time course.' PMA inhibited carbachol-stimulatedinositol phosphate release and cAMP accumulation with equal potencies. In contrast, PMA stimulated arachidonic acid release. These observations suggest a linkage between carbachol-stimulated inositol phosphate generation and cAMP accumulation, but obviates a role for arachidonic acid and itsmetabolites in this pathway. The inhibition of carbachol-stimulated inositol phosphate release and cAMP accumulation by PMA could occur at multiple sites by means of protein kinase C-mediated phosphorylation.Thesepotentialsites could be GTP binding proteins, adenylatecyclase, the mlmuscarinic receptor, phosC. C. Felder, R. Y. Kanterman, A. L. Ma, and J. Axelrod, preliminary observations.
pholipase C, or enzymes involved in the inositol phospholipid cascade. PMA treatment did not inhibit cholera toxin- or forskolin-stimulated cAMPaccumulation, ruling out a protein kinase C-mediated inhibition ofG, or adenylate cyclase, respectively. Since PMA simultaneously activated carbacholstimulated arachidonic acid release and inhibited carbacholstimulated inositol phosphate generation, it is unlikely that direct phosphorylation of the m l muscarinic receptor is involved. Thus, themost likely phosphorylation sites are phospholipase C, its associated GTP binding protein, or enzymes involved in the inositol phospholipid cascade (Fig. 9). PGEz stimulated cAMP accumulation inboth untransfected cells and in cells expressing the mlmuscarinic receptor. PMA hadno effect on PGEz-stimulatedcAMP accumulation, but inhibited carbachol-stimulated cAMP accumulation. In membrane preparations, PGE, stimulated adenylate cyclase activity while carbachol failed to generate CAMP. Furthermore, PGE2- and carbachol-stimulated cAMP accumulation were additive. These observations indicate that PGE, stimulates adenylate cyclase by a different mechanism than carbachol-stimulated cAMPgeneration. This could involve multiple active sites on adenylate cyclase or two populations of this enzyme. Other molecular mechanisms have been proposed for the indirect stimulation of adenylate cyclase, not involving Glinked receptors. Verghese et al. (28) have suggested that chemoattractant-elicited stimulation of adenylate cyclase in human polymorphonuclear leukocytes requires extracellular calcium and is regulated by cAMP phosphodiesterases. These mechanisms do not appear to be acting in the A9 L cell since both verapamil (a voltage-sensitive calcium channel blocker) and EGTA (a calcium-chelating agent) had little effect on both basal and carbachol-stimulated cAMP accumulation (data not shown). Muscarinic receptor-induced attenuation of cAMP accumulation has been shown to occur through the stimulation of a calcium/calmodulin-sensitive phosphodiesterase in 1321N1 human astrocytoma cells (6, 23). It is possible that in the A9 cell the m l muscarinic receptor could be coupled to the inhibition of phosphodiesterase activity, resulting in an apparent stimulation of cAMP levels. In A9 cells, carbachol stimulated cAMP accumulation severalfold over levels achieved in the presence of saturating concentrations of the phosphodiesterase inhibitors IBMX and RO 201724. These data suggest that cAMP phosphodiesterases inhibited by these two compounds do not play a role in the
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regulation of CAMP levels, but does not ruletheout possibility of other calcium/calmodulin-sensitive phosphodiesterases being involved. These results offer an example of the complexity of interactions between transmembrane signalling systems activated by a receptor expressed from a single gene. Our results indicate that the generation of cAMP is not directly coupled to t h e m l muscarinicreceptor,butis a consequence of m l receptor-st.imulated phospholipase C . Cyclic A M P has been previously thought of as a second messenger, but in some situations may be better described as a third or even fourth messenger. REFERENCES 1. Burch, R. M., Luini, A., and Axelrod, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 83,7201-7205 2. Slivka, R. S., and Insel, P. A. (1988) J. Biol. Chem. 2 6 2 , 42004207 3. Burch, R. M., and Axelrod, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,6374-6378 4. Slivka, R. S., and Insel, P. A. (1988) J. Biol. Chem. 2 6 3 , 1464014647 5. Felder, C.C., Jose, P. A., and Axelrod, J. (1989) J. Pharmacol. Exp. Ther. 2 4 8 , 171-175 6. Meeker, R.B., and Harden, T. K. (1982) Mol. Pharrnacol. 2 2 , 310-319 7. Baumgold, J., and Fishman, P. H. (1988) Biochem. Biophys. Res. Comrnun. 154,1137-1143 8. Stein, R., Pinkas-Kramarski, R., and Sokolovsky, M. (1988) EMBO J. 7,3031-3035 9. Peralta, E. G., Ashkenazi, A., Winslow, J. W., Ramachandran, J., andCapon, D. J. (1988) Nature 334,434-437 10. Conklin, B. R., Brann, M. R., Buckley, N. J., Ma, A. L., Bonner, T. I., and Axelrod, J. (1988) Proc. Natl. Acad. Sci.U.S. A. 8 5 , 8698-8702
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11. Brann, M. R., Buckley, N. J., Jones, S. V. P., and Bonner, T. I. (1987) Mol. Pharmacol. 3 2 , 450-455 12. Brooker, G., Harper, J. F., and Teraski, W. L. (1979) Adv. Cyclic Nucleotide Res. 10, 1-33 13. Berridge, M. J., Downes, C., and Hanley, M. R. (1982) Biochem. J. 206,587-595 14. Kraft, A. S., and Anderson, W. B. (1983) Nature 3 0 1 , 621-623 15. Zatz,M., Mahan, L. C., and Reisine, T. (1987) J. Neurochem. 48,106-110 16. Stadel, J. M., DeLean, A., and Lefkowitz,R. J. (1982) Adu. Enzymol. Relat. AreasMol. Biol. 5 3 , 1-43 17. Nishizuka, Y. (1984) Nature 308,693-698 18. Garte, S., and Belman, S. J. (1980) Nature 2 8 4 , 171-173 19. Simpson, I. A., and Pfeuffer, T. (1980) Eur. J. Biochem. 111, 111-116 20. Bell, J. D., Buxton, I. L. O., and Bruton, L. L. (1985) J. Biol. Chem. 260,2625-2628 21. Chiou, C. Y., and Malagodi, M. H. (1975) Br. J. Pharmucol. 5 3 , 279-285 22. Chafouleas, J. G., Bolton, W. E., Hidaka, H., Boyd, A. E., and Means, A. R. (1982) Cell 2 8 , 41-50 23. Tanner, L. I., Harden, T. K., Wels, J. N., and Martin, M.W. (1986) Mol. Pharmacol. 29,455-460 24. Nikula, H., Naor, Z., Parvinen, M., and Huhtaniemi, I. (1987) Mol. Cell. Endocrinol. 4 9 , 39-49 25. Kawai, Y., and Clark, M. R. (1985) Endocrinology 1 1 6 , 23202326 26. Cronin, M.J., and Canonico, P. L. (1985) Biochem. Biophys. Res. Commun. 129,404-410 27. Mukhopadhyay, A. K., Bohnet, H. G., and Leidenberger, F. A. (1984) Biochem. Biophys. Res. Commun. 1 1 9 , 1062-1067 28. Verghese, M. W., Fox, K, McPhail, L.C., and Snyderman, R. (1985) J. Biol. Chem. 260,6769-6775 29. MacNeil, S., Lakey, T., and Tomlinson, S. (1985) Cell Calcium 6,213-226 30. Brostrom, M.A., Brotman, L. A., and Brostrom, C. 0.(1982) Bwchim. Bwphys. Acta7 2 1 , 227-235 31. Walker, S. W., MacNeil, S., Senior, H. J., Bleehen, S. S., and Tomlinson, S. (1984) Biochem. J. 219,941-946
