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Regulation of acetylcholine binding by ATP at the muscarinic M1 receptor in intact CHO cells
Brain Research 839 Ž1999. 94–99 www.elsevier.comrlocaterbres
Research report
Regulation of acetylcholine binding by ATP at the muscarinic M 1 receptor in intact CHO cells Marianne K.O. Grant, Arthur Christopoulos, Esam E. El-Fakahany
)
Department of Psychiatry, Pharmacology and Neuroscience, UniÕersity of Minnesota Medical School, Minneapolis, MN 55455, USA Accepted 8 June 1998
Abstract ATP may have a modulatory effect on cholinergic transmission, as it is known that ATP is released as a co-transmitter with acetylcholine from nerve terminals. The ability of ATP to influence the binding of acetylcholine to the M 1 muscarinic acetylcholine receptor expressed in intact CHO cells was investigated. In competition binding experiments, acetylcholine completely inhibited the binding of w3 Hx N-methylscopolamine, but yielded a shallow competition isotherm that was best described in terms of two affinity states. When these experiments were repeated in the presence of 1 mM ATP, the acetylcholine competition curve was better described in terms of a single, low-affinity state with a Hill slope not significantly different from unity. This modulatory effect of ATP was completely reversed by the addition of the P2 purinoceptor antagonist, suramin, to the assay medium. When the competition between the muscarinic receptor antagonist, atropine, and w3 Hx N-methylscopolamine was investigated, however, ATP was unable to modulate the binding of atropine, which was consistent with a one-site binding model in each instance. In contrast to the intact cell studies, ATP did not affect either affinity state of acetylcholine binding when studied in homogenate preparations. The results of the present study indicate that ATP, acting via endogenously expressed purinoceptors, is able to influence agonist binding to the M 1 muscarinic acetylcholine receptor via a cross-talk that requires the functional integrity of intact CHO cells. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Acetylcholine; ATP; Agonist binding; Cross-talk; Muscarinic receptor; Suramin
1. Introduction Muscarinic acetylcholine receptors belong to the super family of cell-surface proteins that mediate responses to extracellular neurotransmitters and agonists via coupling to heterotrimeric guanine nucleotide-binding proteins ŽG proteins. w7,21x. In common with most classes of G proteincoupled receptors, the distribution of the agonist– muscarinic receptor complex between G protein-coupled and uncoupled forms is often evident in competition binding assays, conducted on cellular homogenate preparations in the absence of guanine nucleotides, whereby the agonist binding isotherm is found to deviate from a simple hyperbolic relationship and displays multiple affinity states w24x. In many of these latter instances, the addition of guanine
)
Corresponding author. Neuroscience Research in Psychiatry, Box 392, Mayo Memorial Building, University of Minnesota Medical School, Minneapolis, MN 55455, USA. Fax: q1-612-624-8935; E-mail: [email protected]
nucleotides to the homogenate assay medium has been shown to convert multiphasic agonist binding curves to a single, lower-affinity state, presumably reflecting the existence of the G protein-uncoupled agonist–receptor complex w24x. In contrast, the addition of adenine nucleotides to homogenates containing receptors and their cognate G proteins under similar assay conditions exerts a minimal effect on agonist binding w3,15,20,28,31x. These findings have been generally interpreted in terms of the necessary allosteric linkage between agonist, receptor, G protein and guanine nucleotide binding in the process of signal transduction via these receptors w17x, whereas adenine nucleotides are presumed not to be required for the initiation of this event. Interestingly, studies conducted on intact cell preparations have found that adenine nucleotides, and in particular, ATP, can exert an effect on G protein-coupled receptor activation in a functional manner. For example, Hurley et al. w22x have shown that extracellular ATP is able to modify the mobilization of intracellular calcium stores that is mediated by both muscarinic and a 1-adrenoceptors in
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 7 2 0 - 5
M.K.O. Grant et al.r Brain Research 839 (1999) 94–99
rat submandibullar gland acini. Functional interactions between transfected muscarinic receptors or adrenoceptors and endogenous P2Y purinoceptors have also been demonstrated in recombinant expression systems w1,6,13x. Taken together, these findings, along with the fact that ATP is stored in autonomic nerve terminals and released as a co-transmitter with acetylcholine w5,18,30x, suggest that endogenously produced adenine nucleotides may be active participants in the process of receptor cross-talk in intact cells. Physiologically, this raises the possibility of multiple modes of regulation of neurotransmitter actions, including potential effects on the binding of extracellular agonists that are not detectable in cellular homogenate preparations because they require a functionally intact system. Thus, the aim of the present study was to reassess the interaction between ATP and the multiple affinity binding sites of acetylcholine at muscarinic receptors using a radioligand binding paradigm in intact Chinese hamster ovary ŽCHO. cells, stably expressing the human M 1 muscarinic acetylcholine receptor and an endogenous P2Y receptor.
2. Materials and methods
95
final pellet was then resuspended in HEPES buffer for immediate use. 2.4. Competition binding assays Intact CHO cells or cell homogenates were incubated with w3 Hx N-methylscopolamine Ž0.2 nM. and increasing concentrations of acetylcholine Ž0.1 mM–10 mM. in the absence or presence of ATP Ž1 mM. for 1 h at 378C. Non-specific binding was determined using 10 mM atropine. Additional experiments on intact cells were performed in the absence or presence of the P2 purinoceptor antagonist suramin Ž100 mgrml.. The reaction was terminated by filtration using a Brandell Cell Harvester. Whatman GFrC filters were used for intact cell studies; GFrB filters were used for homogenate studies. Filters were washed three times with 4 ml aliquots of ice-cold saline and dried before radioactivity Ždisintegrations per minute. was measured using liquid scintillation counting. Saturation binding parameters for w3 Hx N-methylscopolamine were determined previously w11,12x. 2.5. Data analysis
2.1. Materials w3 Hx N-Methylscopolamine Ž84.5 Cirmmol. was purchased from NEN Dupont ŽWilmington, DE.; Dulbecco’s modified Eagle’s medium was purchased from Gibco ŽGaithersburg, MD.; geneticin was obtained from Calbiochem ŽLa Jolla, CA.; bovine calf serum was supplied by Hyclone ŽLogan, UT.. All other reagents were purchased from Sigma ŽSt. Louis, MO.. 2.2. Cell culture CHO cells, stably expressing the human M 1 muscarinic acetylcholine receptor Žprovided by Dr. M. Brann, University of Vermont Medical School, Burlington, VT., were grown for 4 days at 378C in Dulbecco’s modified Eagle’s medium, supplemented with 10% bovine calf serum and 50 mgrml geneticin, in a humidified atmosphere consisting of 5% CO 2 and 95% air. Cells were harvested 4 days after subculture by trypsinization, followed by centrifugation Ž300 = g, 3 min. and re-suspension of the pellet in HEPES buffer Ž110 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2 , 1 mM MgSO4 , 25 mM glucose, 50 mM HEPES, 58 mM sucrose; pH 7.4; 340 mosM., repeated twice.
Competition binding isotherms were analyzed via nonlinear regression using PRISM 2.01 ŽGraphPad Software, San Diego, CA. in order to derive estimates of the Hill slope factor and the IC 50 Žmidpoint location or potency parameter.. Assuming simple competition, the data were refitted according to both one- and two-site mass-action binding models, and the better model was determined by an extra-sum-of-squares test using PRISM. IC 50 values were converted to K I values Žcompetitor–receptor dissociation equilibrium constant. according to the following equation w8x: KIs
IC 50 1 q Ž w D x rK D .
Where w D x and K D denote the concentration and dissociation constant of the radioligand, respectively. Results are expressed as mean " S.E.M. Statistical significance was determined by unpaired t-test. A probability Ž p . value of 0.05 was taken to indicate statistical significance.
3. Results
2.3. Membrane preparation CHO cells were harvested as described above and then homogenized twice in HEPES buffer using a polytron homogenizer for 10 s and placed on ice. The cells were centrifuged for 10 min at 1000 = g. The supernatant was collected and centrifuged at 30,000 = g for 30 min. The
3.1. Effect of ATP on the binding of acetylcholine in intact cell preparations Acetylcholine was able to inhibit completely the binding of 0.2 nM w3 Hx N-methylscopolamine at the M 1 muscarinic receptor expressed in intact cho cells in a concen-
M.K.O. Grant et al.r Brain Research 839 (1999) 94–99
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Fig. 1. Inhibition of the specific binding of 0.2 nM w3 Hx N-methylscopolamine at the human M 1 muscarinic acetylcholine receptor by acetylcholine in the absence Ž`. or presence Žv . of 1 mM ATP in intact CHO cells. Drugs were incubated with cells for 1 h at 378C in high ionic strength HEPES buffer Žsee Section 2. before termination by vacuum filtration. Non-specific binding was defined using 10 mM atropine. Points represent the mean"S.E.M. of five experiments conducted in triplicate.
Fig. 2. Inhibition of the specific binding of 0.2 nM w3 Hx N-methylscopolamine at the human M 1 muscarinic acetylcholine receptor by acetylcholine in the absence Ž`. or presence Žv . of 1 mM ATP in homogenates prepared from CHO cells. Points represent the mean" S.E.M. of four experiments conducted in duplicate. All other details are the same as in Fig. 1.
3.2. Lack of effect of ATP on the binding of acetylcholine in broken cell preparations tration-dependent manner ŽFig. 1.. Nonlinear regression analysis of the data found the competition to be best described in terms of two distinct affinity states for the agonist ŽTable 1.. When the agonist competition experiments were repeated in the presence of 1 mM ATP, a significant change Ž p - 0.05. in the binding profile of acetylcholine was observed ŽFig. 1., whereby the data were best described by a one-site binding model ŽTable 1.. On its own, ATP, at a concentration of 1 mM, had a minimal effect on the control binding of the radioligand Ž- 5% inhibition; data not shown..
Table 1 Competition binding parameters for acetylcholine at the human M 1 muscarinic acetylcholine receptor expressed in intact or homogenized CHO cell membranes, in the absence or presence of 1 mM ATP pK Ha Intact cells yATP 5.31"0.15 Ž78.1"7.5%. qATP Homogenates yATP 6.08"0.48 Ž44.2"8.0%. qATP 5.10"0.24 Ž45.3"10.4%.
pK Lb
pK Ic
ndH
ne
0.79"0.08
5
0.87"0.11
5
4.18"0.12
0.67"0.04
4
3.77"0.17
0.76"0.02
4
3.62"0.15 4.58"0.13
When acetylcholine competition binding was studied at the M 1 muscarinic receptor in homogenates obtained from CHO cells, two-site binding was observed ŽFig. 2; Table 1., as above. Importantly, and in contrast to the findings observed with the intact cells, when the homogenate experiments were repeated in the presence of 1 mM of ATP, no significant effect Ž p ) 0.05. on the binding parameters for acetylcholine was observed ŽFig. 2; Table 1.. 3.3. Lack of effect of ATP on the binding of atropine in intact cell preparations Further experiments were designed in order to ascertain whether the effect of ATP discriminates between agonist
a
Negative logarithm of the dissociation constant for the high-affinity agonist binding site; percentage of binding sites shown in parentheses. b Negative logarithm of the dissociation constant for the low-affinity agonist binding site. c Negative logarithm of the dissociation constant for binding to a single affinity site. d Hill slope factor. e Number of experiments.
Fig. 3. Inhibition of the specific binding of 0.2 nM w3 Hx N-methylscopolamine at the human M 1 muscarinic acetylcholine receptor by atropine in the absence Ž`. or presence Žv . of 1 mM ATP in intact CHO cells. Points represent the mean"S.E.M. of four experiments conducted in triplicate. All other details are the same as in Fig. 1.
M.K.O. Grant et al.r Brain Research 839 (1999) 94–99 Table 2 Competition binding parameters for acetylcholine at the human M 1 muscarinic acetylcholine receptor expressed in intact CHO cells, in the absence or presence of 1 mM ATP with or without 100 mgrml suramin. All other details are as for Table 1 pK H yATP and suramin qATP alone qATP and suramin
pK L
5.14"0.10 3.81"0.51 Ž82.2"8.9%. 5.31"0.10 4.08"0.18 Ž61.8"7.9%.
pK I
nH
n
0.81"0.06 10 4.86"0.13 0.86"0.10 0.68"0.03
6 6
and antagonist binding at the M 1 receptor expressed in intact CHO cells. Competition experiments were undertaken with the muscarinic receptor antagonist atropine as the unlabelled competitor. As shown in Fig. 3, atropine was able to inhibit completely the specific binding of w3 Hx N-methylscopolamine in a concentration-dependent manner. In contrast to the acetylcholine competition isotherms, however, the interaction between atropine and the radioligand was adequately described in terms of a one-site fit ŽFig. 3., with nonlinear regression analysis yielding a pK I value of 8.66 " 0.04 and a Hill slope of 1.05 " 0.03 Ž n s 4.. Additionally, the binding of atropine was not significantly affected Ž p ) 0.05. by the concomitant presence of 1 mM ATP ŽFig. 3., being characterized by a pK I of 8.77 " 0.03 and Hill slope of 1.04 " 0.10 Ž n s 4.. 3.4. Effect of suramin on the interaction between acetylcholine and ATP in intact cell preparations It is possible that activation of endogenous P2Y purinoceptors by ATP can alter the binding characteristics of agonists to the M 1 muscarinic receptor through a crosstalk between the two receptors. To test this hypothesis, acetylcholine competition experiments were undertaken in the absence and presence of both 1 mM ATP and 100 mgrml of the purinoceptor antagonist, suramin. As is shown in Table 2, the presence of suramin was able to abolish the effects of ATP on the acetylcholine competition curve, yielding a biphasic acetylcholine isotherm that was almost identical to that obtained under control conditions in the absence of ATP.
4. Discussion The majority of radioligand binding studies on G protein-coupled receptors have utilized broken cell preparations in order to allow the experimenter a greater degree of flexibility in the control of the assay environment w9x. A general dogma that has resulted from these types of experiments is that the existence of multiple affinity states for
97
agonist binding to a single receptor population is due to the presence of G protein-coupled and uncoupled forms of the receptor, and that guanine nucleotides, but not adenine nucleotides, are able to modulate this binding Žsee Section 1.. By contrast, relatively few studies have examined these types of interactions utilizing intact cells, due to the operation of additional physiologically relevant mechanisms, such as receptor desensitization and internalization, that could confound the interpretation of the observed agonist binding profiles w26x. It is not surprising, therefore, that an effect of adenine nucleotides on agonist binding in intact cells has not been reported until now. In the present study, we have demonstrated that ATP is able to abolish completely the high-affinity component of acetylcholine binding observed at the recombinant human M 1 muscarinic receptor expressed in intact CHO cells. It is unlikely that this effect of ATP on agonist binding was due to a direct interaction of the nucleotide with the muscarinic receptor protein for at least two reasons. First, the phenomenon was not observed when examined in homogenate preparations. Although the receptor environment may have been altered due to the homogenization procedure, the assay medium was kept the same for all experiments. The lack of effect of ATP on agonist binding to muscarinic receptors in homogenate preparations is in agreement with previous studies w3,15,20,28,31x and also argues against the possibility of an alteration of receptor binding properties due to any phosphorylating effects of the nucleotide on the receptor protein. Second, the inability of ATP to influence the binding of atropine ŽFig. 3. or the radioligand, w3 Hx Nmethylscopolamine, implies that the nucleotide does not interact with the classical binding site on the M 1 receptor that is shared by the antagonists and by agonists such as acetylcholine w34x. If a direct effect on the M 1 receptor were to account for the observed actions of ATP, this would imply the presence of an additional binding site on the receptor that is able to mediate an agonist-specific conformational change detectable only in intact cells. Although allosteric interactions at the muscarinic receptor family have been well-documented w10,25,32x, they are generally not compatible with such a scheme. The most likely explanation for our observations involves a form of cross-talk between the transfected M 1 receptors and endogenous P2Y purinoceptors known to be expressed in CHO cells w23x. In particular, the ability of the P2 purinoceptor antagonist, suramin, to reverse the effect of ATP on acetylcholine binding strongly supports a role for the activation of P2Y purinoceptors. It is also worth noting that although suramin itself has been shown to exert direct inhibitory effects on the coupling of receptors and G proteins through an interaction occurring at the cytosolic face of the cell membrane w2,33x, this phenomenon would not be relevant to the intact cell situation, as suramin is highly charged and cannot penetrate the cell membrane w27x. This cross-talk might be mediated by components within the signal transduction pathway utilized by both
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M.K.O. Grant et al.r Brain Research 839 (1999) 94–99
muscarinic and purinoceptors through which these two receptor types are able to exert heterologous effects on one another. Since both M 1 receptors and P2Y receptors couple preferentially to the Gqr11 family of G proteins w7,27x, it is possible that the observed differences in acetylcholine binding in the absence, compared to the presence of ATP may have been due to a competition of both receptor types for a common, limiting pool of G protein. Although theoretically possible, experimental evidence for such a phenomenon occurring in radioligand binding studies is scarce Žsee Ref. w19x.. Furthermore, multiphasic binding due to the formation of an agonist–receptor–G protein ternary complex is better detected in homogenate preparations than in intact cells w4x,w24x, and thus, any competition between different receptor types for a common pool of G protein would have been expected to be manifested in our homogenate experiments. As shown in Fig. 2, however, this was not the case, suggesting that the cross-talk between the two receptors involves intracellular mediators that are generated downstream from the level of G protein coupling. In contrast to the paucity of observations made using radioligand binding studies, there exists ample evidence for cross-talk between different types of G protein-coupled receptors occurring at the functional level, involving the convergence or divergence of various elements of intracellular signal transduction pathways beyond the level of the G protein Žfor review, see Ref. w29x.. Importantly, functional interactions between muscarinic receptors and purinoceptors have been reported. For instance, Carroll et al. w6x showed a marked enhancement of M 4 receptormediated phospholipase C signaling in CHO cells due to concurrent stimulation of P2Y purinoceptors by ATP. A similar observation was made by Dolezal et al. w13x with regard to P2Y and M 4 receptor-mediated calcium signaling. Recently, Fukushi w16x has demonstrated that P2Z purinoceptor-mediated activation of a phospholipase D and, subsequently, of a protein kinase C can lead to a heterologous desensitization of muscarinic receptors in rat parotid acinar cells. Hence, it is quite possible that the ability of different receptor types to activate intracellular mediators, especially receptor kinases, may result in a heterologous modification of not only intracellular signal transduction but also of agonist–receptor binding properties. Indeed, phosphorylation of cell-surface receptors represents one of the best-established mechanisms for the attenuation of agonist-mediated signaling w14x. Irrespective of the specific mechanism, however, the present findings suggest that extracellular ATP is able to modify acetylcholine binding to the M 1 receptor in intact cells under conditions more akin to those observed physiologically. Such regulation mechanisms might be operative in vivo, since ATP and acetylcholine are co-released by nerve terminals w5,18x. In general terms, the occurrence of a cross-talk between different receptor types manifested at
the binding level highlights an additional mechanism whereby different neurotransmitters may modulate each other’s actions in the maintenance of physiological homeostasis. Acknowledgements This work was supported by NIH grant NS25743. References w1x K.E. Akerman, J. Nasman, P.E. Lund, R. Shariatmadari, J.P. Kukkonen, Endogenous extracellular purine nucleotides redirect a 2adrenoceptor signaling, FEBS Lett. 430 Ž1998. 209–212. w2x W. Beindl, T. Mitterauer, M. Hohenegger, A.P. Ijzerman, C. Nanoff, M. Freissmuth, Inhibition of receptorrG protein coupling by suramin analogues, Mol. Pharmacol. 50 Ž1996. 415–423. w3x C.P. Berrie, N.J. Birdsall, A.S. Burgen, E.C. Hulme, Guanine nucleotides modulate muscarinic receptor binding in the heart, Biochem. Biophys. Res. Commun. 87 Ž1979. 1000–1005. w4x J. Bockaert, C. Brand, L. Journot, Do recombinant receptor assays provide affinity and potency estimates?, Ann. NY Acad. Sci. 812 Ž1997. 55–70. w5x G. Burnstock, Purinergic nerves, Pharmacol. Rev. 24 Ž1972. 509– 581. w6x R.C. Carroll, A.D. Morielli, E.G. Peralta, Coincidence detection at the level of phospholipase C activation mediated by the m4 muscarinic acetylcholine receptor, Curr. Biol. 5 Ž1995. 536–544. w7x M.P. Caulfield, Muscarinic receptors — characterization, coupling and function, Pharmacol. Ther. 58 Ž1993. 319–379. w8x Y.-C. Cheng, W.H. Prusoff, Relationship between the inhibition constant Ž K I . and the concentration of inhibitor which causes 50 per cent inhibition Ž I50 . of an enzymatic reaction, Biochem. Pharmacol. 22 Ž1973. 3099–3108. w9x A. Christopoulos, E.E. El-Fakahany, Qualitative and quantitative assessment of relative agonist efficacy, Biochem. Pharmacol. 58 Ž1999. 1–2. w10x A. Christopoulos, A. Lanzafame, F. Mitchelson, Allosteric interactions at muscarinic cholinoceptors, Clin. Exp. Pharmacol. Physiol. 25 Ž1998. 184–194. w11x A. Christopoulos, T.L. Pierce, J.L. Sorman, E.E. El-Fakahany, On the unique binding and activating properties of xanomeline at the M 1 muscarinic acetylcholine receptor, Mol. Pharmacol. 53 Ž1998. 1120–1130. w12x A. Christopoulos, J.L. Sorman, F. Mitchelson, E.E. El-Fakahany, Characterization of the subtype selectivity of the allosteric modulaX tor heptane-1,7-bis-Ždimethyl-3 -pthalimidopropyl. ammonium bromide ŽC 7 r3-phth. at cloned muscarinic acetylcholine receptors, Biochem. Pharmacol. 57 Ž1999. 171–179. w13x V. Dolezal, V. Lisa, S. Tucek, Differential effects of the M 1 –M 5 muscarinic acetylcholine receptor subtypes on intracellular calcium and on the incorporation of choline into membrane lipids in genetically modified Chinese hamster ovary cell lines, Br. Med. Bull. 42 Ž1997. 71–78. w14x S.S.G. Ferguson, L.S. Barak, J. Zhang, M.G. Caron, G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins, Can. J. Physiol. Pharmacol. 74 Ž1996. 1095–1110. w15x K.E. Flaim, G.W. Gessner, S.T. Crooke, J.R. Heys, J. Weinstock, Regulation of agonist and antagonist binding to striatal D-1 dopamine receptors: studies using the selective D-1 antagonist w3 HxSK&F R-83566, Life Sci. 38 Ž1986. 2087–2096. w16x Y. Fukushi, Heterologous desensitization of muscarinic receptors by P2Z purinoceptors in rat parotid acinar cells, Eur. J. Pharmacol. 364 Ž1999. 55–64.
M.K.O. Grant et al.r Brain Research 839 (1999) 94–99 w17x A.G. Gilman, G proteins: transducers of receptor-generated signals, Annu. Rev. Biochem. 56 Ž1987. 615–649. w18x J.L. Gordon, Extracellular ATP: effects, sources and fate, Biochem. J. 233 Ž1986. 309–319. w19x D. Graeser, R.R. Neubig, Compartmentation of receptors and guanine nucleotide-binding proteins in NG108-15 cells: lack of cross-talk in agonist binding among the a 2-adrenergic, muscarinic and opiate receptors, Mol. Pharmacol. 43 Ž1993. 434–443. w20x T.K. Harden, A.G. Scheer, M.M. Smith, Differential modification of the interaction of cardiac muscarinic cholinergic and beta-adrenergic receptors with a guanine nucleotide binding componentŽs., Mol. Pharmacol. 21 Ž1982. 570–580. w21x E.C. Hulme, N.J.M. Birdsall, N.J. Buckley, Muscarinic receptor subtypes, Annu. Rev. Pharmacol. Toxicol. 30 Ž1990. 633–673. w22x T.W. Hurley, D.D. Shoemaker, M.P. Ryan, Extracellular ATP prevents the release of stored Ca2q by autonomic agonists in rat submandibular gland acini, Am. J. Physiol. 265 Ž1993. C1472– C1478. w23x P.A. Iredale, S.J. Hill, Increases in intracellular calcium via activation of an endogenous P2 -purinoceptor in cultured CHO-K1 cells, Br. J. Pharmacol. 110 Ž1993. 1305–1310. w24x T.P. Kenakin, Drug receptor theory, in: T.P. Kenakin ŽEd.., Pharmacologic Analysis of Drug–Receptor Interaction, Lippincott-Raven, Philadelphia, PA, 1997, pp. 1–42. w25x N.H. Lee, E.E. El-Fakahany, Allosteric antagonists of the muscarinic acetylcholine receptor, Biochem. Pharmacol. 42 Ž1991. 199–205.
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w26x H.J. Motulsky, L.M. Mahan, P. Insel, Radioligand, agonists and membrane receptors on intact cells: data analysis in a bind, Trends Pharmacol. Sci. 6 Ž1985. 317–319. w27x V. Ralevic, G. Burnstock, Receptors for purines and pyrimidines, Pharmacol. Rev. 50 Ž1998. 413–492. w28x J.R. Schlegel, S.J. Peroutka, Nucleotide interactions with 5-HT1A binding sites directly labeled by w3 Hx-8-hydroxy-2-Ždi-n-propylamino.tetralin Žw3 Hx-8-OH-DPAT., Biochem. Pharmacol. 35 Ž1986. 1943–1949. w29x L.A. Selbie, S.J. Hill, G protein-coupled receptor cross-talk: the fine-tuning of multiple receptor-signaling pathways, Trends Pharmacol. Sci. 19 Ž1998. 87–93. w30x T.W. Stone, Physiological roles for adenosine and adenosine 5X-triphosphate in the nervous system, Neuroscience 6 Ž1981. 523–555. w31x B.I. Terman, S.R. Slivka, R.J. Hughes, P.A. Insel, a 1-adrenergic receptor-linked guanine nucleotide-binding protein in muscle and kidney epithelial cells, Mol. Pharmacol. 31 Ž1987. 12–20. w32x S. Tucek, J. Proska, Allosteric modulation of muscarinic acetylcholine receptors, Trends Pharmacol. Sci. 16 Ž1995. 205–212. w33x M. Waldhoer, E. Bofill-Cardona, G. Milligan, M. Freissmuth, C. Nanoff, Differential uncoupling of A 1 adenosine and D 2 dopamine receptors by suramin and didemethylated suramin ŽNF037., Mol. Pharmacol. 53 Ž1998. 808–818. w34x J. Wess, Mutational analysis of muscarinic acetylcholine receptors: structural basis of ligandrreceptorrG protein interactions, Life Sci. 53 Ž1993. 1447–1463.
Research report
Regulation of acetylcholine binding by ATP at the muscarinic M 1 receptor in intact CHO cells Marianne K.O. Grant, Arthur Christopoulos, Esam E. El-Fakahany
)
Department of Psychiatry, Pharmacology and Neuroscience, UniÕersity of Minnesota Medical School, Minneapolis, MN 55455, USA Accepted 8 June 1998
Abstract ATP may have a modulatory effect on cholinergic transmission, as it is known that ATP is released as a co-transmitter with acetylcholine from nerve terminals. The ability of ATP to influence the binding of acetylcholine to the M 1 muscarinic acetylcholine receptor expressed in intact CHO cells was investigated. In competition binding experiments, acetylcholine completely inhibited the binding of w3 Hx N-methylscopolamine, but yielded a shallow competition isotherm that was best described in terms of two affinity states. When these experiments were repeated in the presence of 1 mM ATP, the acetylcholine competition curve was better described in terms of a single, low-affinity state with a Hill slope not significantly different from unity. This modulatory effect of ATP was completely reversed by the addition of the P2 purinoceptor antagonist, suramin, to the assay medium. When the competition between the muscarinic receptor antagonist, atropine, and w3 Hx N-methylscopolamine was investigated, however, ATP was unable to modulate the binding of atropine, which was consistent with a one-site binding model in each instance. In contrast to the intact cell studies, ATP did not affect either affinity state of acetylcholine binding when studied in homogenate preparations. The results of the present study indicate that ATP, acting via endogenously expressed purinoceptors, is able to influence agonist binding to the M 1 muscarinic acetylcholine receptor via a cross-talk that requires the functional integrity of intact CHO cells. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Acetylcholine; ATP; Agonist binding; Cross-talk; Muscarinic receptor; Suramin
1. Introduction Muscarinic acetylcholine receptors belong to the super family of cell-surface proteins that mediate responses to extracellular neurotransmitters and agonists via coupling to heterotrimeric guanine nucleotide-binding proteins ŽG proteins. w7,21x. In common with most classes of G proteincoupled receptors, the distribution of the agonist– muscarinic receptor complex between G protein-coupled and uncoupled forms is often evident in competition binding assays, conducted on cellular homogenate preparations in the absence of guanine nucleotides, whereby the agonist binding isotherm is found to deviate from a simple hyperbolic relationship and displays multiple affinity states w24x. In many of these latter instances, the addition of guanine
)
Corresponding author. Neuroscience Research in Psychiatry, Box 392, Mayo Memorial Building, University of Minnesota Medical School, Minneapolis, MN 55455, USA. Fax: q1-612-624-8935; E-mail: [email protected]
nucleotides to the homogenate assay medium has been shown to convert multiphasic agonist binding curves to a single, lower-affinity state, presumably reflecting the existence of the G protein-uncoupled agonist–receptor complex w24x. In contrast, the addition of adenine nucleotides to homogenates containing receptors and their cognate G proteins under similar assay conditions exerts a minimal effect on agonist binding w3,15,20,28,31x. These findings have been generally interpreted in terms of the necessary allosteric linkage between agonist, receptor, G protein and guanine nucleotide binding in the process of signal transduction via these receptors w17x, whereas adenine nucleotides are presumed not to be required for the initiation of this event. Interestingly, studies conducted on intact cell preparations have found that adenine nucleotides, and in particular, ATP, can exert an effect on G protein-coupled receptor activation in a functional manner. For example, Hurley et al. w22x have shown that extracellular ATP is able to modify the mobilization of intracellular calcium stores that is mediated by both muscarinic and a 1-adrenoceptors in
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 7 2 0 - 5
M.K.O. Grant et al.r Brain Research 839 (1999) 94–99
rat submandibullar gland acini. Functional interactions between transfected muscarinic receptors or adrenoceptors and endogenous P2Y purinoceptors have also been demonstrated in recombinant expression systems w1,6,13x. Taken together, these findings, along with the fact that ATP is stored in autonomic nerve terminals and released as a co-transmitter with acetylcholine w5,18,30x, suggest that endogenously produced adenine nucleotides may be active participants in the process of receptor cross-talk in intact cells. Physiologically, this raises the possibility of multiple modes of regulation of neurotransmitter actions, including potential effects on the binding of extracellular agonists that are not detectable in cellular homogenate preparations because they require a functionally intact system. Thus, the aim of the present study was to reassess the interaction between ATP and the multiple affinity binding sites of acetylcholine at muscarinic receptors using a radioligand binding paradigm in intact Chinese hamster ovary ŽCHO. cells, stably expressing the human M 1 muscarinic acetylcholine receptor and an endogenous P2Y receptor.
2. Materials and methods
95
final pellet was then resuspended in HEPES buffer for immediate use. 2.4. Competition binding assays Intact CHO cells or cell homogenates were incubated with w3 Hx N-methylscopolamine Ž0.2 nM. and increasing concentrations of acetylcholine Ž0.1 mM–10 mM. in the absence or presence of ATP Ž1 mM. for 1 h at 378C. Non-specific binding was determined using 10 mM atropine. Additional experiments on intact cells were performed in the absence or presence of the P2 purinoceptor antagonist suramin Ž100 mgrml.. The reaction was terminated by filtration using a Brandell Cell Harvester. Whatman GFrC filters were used for intact cell studies; GFrB filters were used for homogenate studies. Filters were washed three times with 4 ml aliquots of ice-cold saline and dried before radioactivity Ždisintegrations per minute. was measured using liquid scintillation counting. Saturation binding parameters for w3 Hx N-methylscopolamine were determined previously w11,12x. 2.5. Data analysis
2.1. Materials w3 Hx N-Methylscopolamine Ž84.5 Cirmmol. was purchased from NEN Dupont ŽWilmington, DE.; Dulbecco’s modified Eagle’s medium was purchased from Gibco ŽGaithersburg, MD.; geneticin was obtained from Calbiochem ŽLa Jolla, CA.; bovine calf serum was supplied by Hyclone ŽLogan, UT.. All other reagents were purchased from Sigma ŽSt. Louis, MO.. 2.2. Cell culture CHO cells, stably expressing the human M 1 muscarinic acetylcholine receptor Žprovided by Dr. M. Brann, University of Vermont Medical School, Burlington, VT., were grown for 4 days at 378C in Dulbecco’s modified Eagle’s medium, supplemented with 10% bovine calf serum and 50 mgrml geneticin, in a humidified atmosphere consisting of 5% CO 2 and 95% air. Cells were harvested 4 days after subculture by trypsinization, followed by centrifugation Ž300 = g, 3 min. and re-suspension of the pellet in HEPES buffer Ž110 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2 , 1 mM MgSO4 , 25 mM glucose, 50 mM HEPES, 58 mM sucrose; pH 7.4; 340 mosM., repeated twice.
Competition binding isotherms were analyzed via nonlinear regression using PRISM 2.01 ŽGraphPad Software, San Diego, CA. in order to derive estimates of the Hill slope factor and the IC 50 Žmidpoint location or potency parameter.. Assuming simple competition, the data were refitted according to both one- and two-site mass-action binding models, and the better model was determined by an extra-sum-of-squares test using PRISM. IC 50 values were converted to K I values Žcompetitor–receptor dissociation equilibrium constant. according to the following equation w8x: KIs
IC 50 1 q Ž w D x rK D .
Where w D x and K D denote the concentration and dissociation constant of the radioligand, respectively. Results are expressed as mean " S.E.M. Statistical significance was determined by unpaired t-test. A probability Ž p . value of 0.05 was taken to indicate statistical significance.
3. Results
2.3. Membrane preparation CHO cells were harvested as described above and then homogenized twice in HEPES buffer using a polytron homogenizer for 10 s and placed on ice. The cells were centrifuged for 10 min at 1000 = g. The supernatant was collected and centrifuged at 30,000 = g for 30 min. The
3.1. Effect of ATP on the binding of acetylcholine in intact cell preparations Acetylcholine was able to inhibit completely the binding of 0.2 nM w3 Hx N-methylscopolamine at the M 1 muscarinic receptor expressed in intact cho cells in a concen-
M.K.O. Grant et al.r Brain Research 839 (1999) 94–99
96
Fig. 1. Inhibition of the specific binding of 0.2 nM w3 Hx N-methylscopolamine at the human M 1 muscarinic acetylcholine receptor by acetylcholine in the absence Ž`. or presence Žv . of 1 mM ATP in intact CHO cells. Drugs were incubated with cells for 1 h at 378C in high ionic strength HEPES buffer Žsee Section 2. before termination by vacuum filtration. Non-specific binding was defined using 10 mM atropine. Points represent the mean"S.E.M. of five experiments conducted in triplicate.
Fig. 2. Inhibition of the specific binding of 0.2 nM w3 Hx N-methylscopolamine at the human M 1 muscarinic acetylcholine receptor by acetylcholine in the absence Ž`. or presence Žv . of 1 mM ATP in homogenates prepared from CHO cells. Points represent the mean" S.E.M. of four experiments conducted in duplicate. All other details are the same as in Fig. 1.
3.2. Lack of effect of ATP on the binding of acetylcholine in broken cell preparations tration-dependent manner ŽFig. 1.. Nonlinear regression analysis of the data found the competition to be best described in terms of two distinct affinity states for the agonist ŽTable 1.. When the agonist competition experiments were repeated in the presence of 1 mM ATP, a significant change Ž p - 0.05. in the binding profile of acetylcholine was observed ŽFig. 1., whereby the data were best described by a one-site binding model ŽTable 1.. On its own, ATP, at a concentration of 1 mM, had a minimal effect on the control binding of the radioligand Ž- 5% inhibition; data not shown..
Table 1 Competition binding parameters for acetylcholine at the human M 1 muscarinic acetylcholine receptor expressed in intact or homogenized CHO cell membranes, in the absence or presence of 1 mM ATP pK Ha Intact cells yATP 5.31"0.15 Ž78.1"7.5%. qATP Homogenates yATP 6.08"0.48 Ž44.2"8.0%. qATP 5.10"0.24 Ž45.3"10.4%.
pK Lb
pK Ic
ndH
ne
0.79"0.08
5
0.87"0.11
5
4.18"0.12
0.67"0.04
4
3.77"0.17
0.76"0.02
4
3.62"0.15 4.58"0.13
When acetylcholine competition binding was studied at the M 1 muscarinic receptor in homogenates obtained from CHO cells, two-site binding was observed ŽFig. 2; Table 1., as above. Importantly, and in contrast to the findings observed with the intact cells, when the homogenate experiments were repeated in the presence of 1 mM of ATP, no significant effect Ž p ) 0.05. on the binding parameters for acetylcholine was observed ŽFig. 2; Table 1.. 3.3. Lack of effect of ATP on the binding of atropine in intact cell preparations Further experiments were designed in order to ascertain whether the effect of ATP discriminates between agonist
a
Negative logarithm of the dissociation constant for the high-affinity agonist binding site; percentage of binding sites shown in parentheses. b Negative logarithm of the dissociation constant for the low-affinity agonist binding site. c Negative logarithm of the dissociation constant for binding to a single affinity site. d Hill slope factor. e Number of experiments.
Fig. 3. Inhibition of the specific binding of 0.2 nM w3 Hx N-methylscopolamine at the human M 1 muscarinic acetylcholine receptor by atropine in the absence Ž`. or presence Žv . of 1 mM ATP in intact CHO cells. Points represent the mean"S.E.M. of four experiments conducted in triplicate. All other details are the same as in Fig. 1.
M.K.O. Grant et al.r Brain Research 839 (1999) 94–99 Table 2 Competition binding parameters for acetylcholine at the human M 1 muscarinic acetylcholine receptor expressed in intact CHO cells, in the absence or presence of 1 mM ATP with or without 100 mgrml suramin. All other details are as for Table 1 pK H yATP and suramin qATP alone qATP and suramin
pK L
5.14"0.10 3.81"0.51 Ž82.2"8.9%. 5.31"0.10 4.08"0.18 Ž61.8"7.9%.
pK I
nH
n
0.81"0.06 10 4.86"0.13 0.86"0.10 0.68"0.03
6 6
and antagonist binding at the M 1 receptor expressed in intact CHO cells. Competition experiments were undertaken with the muscarinic receptor antagonist atropine as the unlabelled competitor. As shown in Fig. 3, atropine was able to inhibit completely the specific binding of w3 Hx N-methylscopolamine in a concentration-dependent manner. In contrast to the acetylcholine competition isotherms, however, the interaction between atropine and the radioligand was adequately described in terms of a one-site fit ŽFig. 3., with nonlinear regression analysis yielding a pK I value of 8.66 " 0.04 and a Hill slope of 1.05 " 0.03 Ž n s 4.. Additionally, the binding of atropine was not significantly affected Ž p ) 0.05. by the concomitant presence of 1 mM ATP ŽFig. 3., being characterized by a pK I of 8.77 " 0.03 and Hill slope of 1.04 " 0.10 Ž n s 4.. 3.4. Effect of suramin on the interaction between acetylcholine and ATP in intact cell preparations It is possible that activation of endogenous P2Y purinoceptors by ATP can alter the binding characteristics of agonists to the M 1 muscarinic receptor through a crosstalk between the two receptors. To test this hypothesis, acetylcholine competition experiments were undertaken in the absence and presence of both 1 mM ATP and 100 mgrml of the purinoceptor antagonist, suramin. As is shown in Table 2, the presence of suramin was able to abolish the effects of ATP on the acetylcholine competition curve, yielding a biphasic acetylcholine isotherm that was almost identical to that obtained under control conditions in the absence of ATP.
4. Discussion The majority of radioligand binding studies on G protein-coupled receptors have utilized broken cell preparations in order to allow the experimenter a greater degree of flexibility in the control of the assay environment w9x. A general dogma that has resulted from these types of experiments is that the existence of multiple affinity states for
97
agonist binding to a single receptor population is due to the presence of G protein-coupled and uncoupled forms of the receptor, and that guanine nucleotides, but not adenine nucleotides, are able to modulate this binding Žsee Section 1.. By contrast, relatively few studies have examined these types of interactions utilizing intact cells, due to the operation of additional physiologically relevant mechanisms, such as receptor desensitization and internalization, that could confound the interpretation of the observed agonist binding profiles w26x. It is not surprising, therefore, that an effect of adenine nucleotides on agonist binding in intact cells has not been reported until now. In the present study, we have demonstrated that ATP is able to abolish completely the high-affinity component of acetylcholine binding observed at the recombinant human M 1 muscarinic receptor expressed in intact CHO cells. It is unlikely that this effect of ATP on agonist binding was due to a direct interaction of the nucleotide with the muscarinic receptor protein for at least two reasons. First, the phenomenon was not observed when examined in homogenate preparations. Although the receptor environment may have been altered due to the homogenization procedure, the assay medium was kept the same for all experiments. The lack of effect of ATP on agonist binding to muscarinic receptors in homogenate preparations is in agreement with previous studies w3,15,20,28,31x and also argues against the possibility of an alteration of receptor binding properties due to any phosphorylating effects of the nucleotide on the receptor protein. Second, the inability of ATP to influence the binding of atropine ŽFig. 3. or the radioligand, w3 Hx Nmethylscopolamine, implies that the nucleotide does not interact with the classical binding site on the M 1 receptor that is shared by the antagonists and by agonists such as acetylcholine w34x. If a direct effect on the M 1 receptor were to account for the observed actions of ATP, this would imply the presence of an additional binding site on the receptor that is able to mediate an agonist-specific conformational change detectable only in intact cells. Although allosteric interactions at the muscarinic receptor family have been well-documented w10,25,32x, they are generally not compatible with such a scheme. The most likely explanation for our observations involves a form of cross-talk between the transfected M 1 receptors and endogenous P2Y purinoceptors known to be expressed in CHO cells w23x. In particular, the ability of the P2 purinoceptor antagonist, suramin, to reverse the effect of ATP on acetylcholine binding strongly supports a role for the activation of P2Y purinoceptors. It is also worth noting that although suramin itself has been shown to exert direct inhibitory effects on the coupling of receptors and G proteins through an interaction occurring at the cytosolic face of the cell membrane w2,33x, this phenomenon would not be relevant to the intact cell situation, as suramin is highly charged and cannot penetrate the cell membrane w27x. This cross-talk might be mediated by components within the signal transduction pathway utilized by both
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M.K.O. Grant et al.r Brain Research 839 (1999) 94–99
muscarinic and purinoceptors through which these two receptor types are able to exert heterologous effects on one another. Since both M 1 receptors and P2Y receptors couple preferentially to the Gqr11 family of G proteins w7,27x, it is possible that the observed differences in acetylcholine binding in the absence, compared to the presence of ATP may have been due to a competition of both receptor types for a common, limiting pool of G protein. Although theoretically possible, experimental evidence for such a phenomenon occurring in radioligand binding studies is scarce Žsee Ref. w19x.. Furthermore, multiphasic binding due to the formation of an agonist–receptor–G protein ternary complex is better detected in homogenate preparations than in intact cells w4x,w24x, and thus, any competition between different receptor types for a common pool of G protein would have been expected to be manifested in our homogenate experiments. As shown in Fig. 2, however, this was not the case, suggesting that the cross-talk between the two receptors involves intracellular mediators that are generated downstream from the level of G protein coupling. In contrast to the paucity of observations made using radioligand binding studies, there exists ample evidence for cross-talk between different types of G protein-coupled receptors occurring at the functional level, involving the convergence or divergence of various elements of intracellular signal transduction pathways beyond the level of the G protein Žfor review, see Ref. w29x.. Importantly, functional interactions between muscarinic receptors and purinoceptors have been reported. For instance, Carroll et al. w6x showed a marked enhancement of M 4 receptormediated phospholipase C signaling in CHO cells due to concurrent stimulation of P2Y purinoceptors by ATP. A similar observation was made by Dolezal et al. w13x with regard to P2Y and M 4 receptor-mediated calcium signaling. Recently, Fukushi w16x has demonstrated that P2Z purinoceptor-mediated activation of a phospholipase D and, subsequently, of a protein kinase C can lead to a heterologous desensitization of muscarinic receptors in rat parotid acinar cells. Hence, it is quite possible that the ability of different receptor types to activate intracellular mediators, especially receptor kinases, may result in a heterologous modification of not only intracellular signal transduction but also of agonist–receptor binding properties. Indeed, phosphorylation of cell-surface receptors represents one of the best-established mechanisms for the attenuation of agonist-mediated signaling w14x. Irrespective of the specific mechanism, however, the present findings suggest that extracellular ATP is able to modify acetylcholine binding to the M 1 receptor in intact cells under conditions more akin to those observed physiologically. Such regulation mechanisms might be operative in vivo, since ATP and acetylcholine are co-released by nerve terminals w5,18x. In general terms, the occurrence of a cross-talk between different receptor types manifested at
the binding level highlights an additional mechanism whereby different neurotransmitters may modulate each other’s actions in the maintenance of physiological homeostasis. Acknowledgements This work was supported by NIH grant NS25743. References w1x K.E. Akerman, J. Nasman, P.E. Lund, R. Shariatmadari, J.P. Kukkonen, Endogenous extracellular purine nucleotides redirect a 2adrenoceptor signaling, FEBS Lett. 430 Ž1998. 209–212. w2x W. Beindl, T. Mitterauer, M. Hohenegger, A.P. Ijzerman, C. Nanoff, M. Freissmuth, Inhibition of receptorrG protein coupling by suramin analogues, Mol. Pharmacol. 50 Ž1996. 415–423. w3x C.P. Berrie, N.J. Birdsall, A.S. Burgen, E.C. Hulme, Guanine nucleotides modulate muscarinic receptor binding in the heart, Biochem. Biophys. Res. Commun. 87 Ž1979. 1000–1005. w4x J. Bockaert, C. Brand, L. Journot, Do recombinant receptor assays provide affinity and potency estimates?, Ann. NY Acad. Sci. 812 Ž1997. 55–70. w5x G. Burnstock, Purinergic nerves, Pharmacol. Rev. 24 Ž1972. 509– 581. w6x R.C. Carroll, A.D. Morielli, E.G. Peralta, Coincidence detection at the level of phospholipase C activation mediated by the m4 muscarinic acetylcholine receptor, Curr. Biol. 5 Ž1995. 536–544. w7x M.P. Caulfield, Muscarinic receptors — characterization, coupling and function, Pharmacol. Ther. 58 Ž1993. 319–379. w8x Y.-C. Cheng, W.H. Prusoff, Relationship between the inhibition constant Ž K I . and the concentration of inhibitor which causes 50 per cent inhibition Ž I50 . of an enzymatic reaction, Biochem. Pharmacol. 22 Ž1973. 3099–3108. w9x A. Christopoulos, E.E. El-Fakahany, Qualitative and quantitative assessment of relative agonist efficacy, Biochem. Pharmacol. 58 Ž1999. 1–2. w10x A. Christopoulos, A. Lanzafame, F. Mitchelson, Allosteric interactions at muscarinic cholinoceptors, Clin. Exp. Pharmacol. Physiol. 25 Ž1998. 184–194. w11x A. Christopoulos, T.L. Pierce, J.L. Sorman, E.E. El-Fakahany, On the unique binding and activating properties of xanomeline at the M 1 muscarinic acetylcholine receptor, Mol. Pharmacol. 53 Ž1998. 1120–1130. w12x A. Christopoulos, J.L. Sorman, F. Mitchelson, E.E. El-Fakahany, Characterization of the subtype selectivity of the allosteric modulaX tor heptane-1,7-bis-Ždimethyl-3 -pthalimidopropyl. ammonium bromide ŽC 7 r3-phth. at cloned muscarinic acetylcholine receptors, Biochem. Pharmacol. 57 Ž1999. 171–179. w13x V. Dolezal, V. Lisa, S. Tucek, Differential effects of the M 1 –M 5 muscarinic acetylcholine receptor subtypes on intracellular calcium and on the incorporation of choline into membrane lipids in genetically modified Chinese hamster ovary cell lines, Br. Med. Bull. 42 Ž1997. 71–78. w14x S.S.G. Ferguson, L.S. Barak, J. Zhang, M.G. Caron, G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins, Can. J. Physiol. Pharmacol. 74 Ž1996. 1095–1110. w15x K.E. Flaim, G.W. Gessner, S.T. Crooke, J.R. Heys, J. Weinstock, Regulation of agonist and antagonist binding to striatal D-1 dopamine receptors: studies using the selective D-1 antagonist w3 HxSK&F R-83566, Life Sci. 38 Ž1986. 2087–2096. w16x Y. Fukushi, Heterologous desensitization of muscarinic receptors by P2Z purinoceptors in rat parotid acinar cells, Eur. J. Pharmacol. 364 Ž1999. 55–64.
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