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Pharmacological properties of cloned muscarinic receptors expressed in A9 L cells; comparison with in vitro models
of P/x7rmacology, 202 (1991) 15l-157 0 1991 Elsevier Science Publishers 6.V. All righls reserved 0014-2999/91/$03,50
European Journal ADONIS
151
0014299991005815
EJP 52022
H.W.G.M. Boddeke and M. Buttini Preclinicai Research, S~ndoz Pharma Ltd., CH-4002
Bask, Switzerland
Received 31 January 1991, revised MS received 8 May 1991, accepted 25 June 1991
The effects of a series of muscarinic agonists and antagonists at cloned ml and m3 muscarinic receptors expressed in mouse fibroblast A9 L cells have been compared with their effects in in vitro models of M, (rat superior cervical ganglion) and M, (guinea-pig ileum) muscarinic receptors. A good correlation existed between the potencies of rnuscarinic agonists at cloned ml muscarinic receptors and the M, sites in rat gangho: (r = 0.80) as well as at cloned m3 receptors and guinea-pig ileum M, receptors (r = 0.87). However, cross correlations of potencies in rat ganglion and cloned m3 receptors as well as in guinea-pig ileum and in cloned ml receptors also yielded relalively high correlation coefficients (0.71 and 0.91, respectively). Low correlation coefficients were found for the maximal responses of muscarinic agonists in rat ganglion and cloned ml receptors (0.53) and in guinea-pig ileum and cloned m3 receptors (0.36). A high correlation between pA, values of muscarinic antagonists at cloned ml receptors and in rat ganglion (r = 0.97) and between cloned m3 receptors and guinea-pig ileum (r = 0.98) was found. Cross correlation of pA z values in rat ganglion and cloned m3 receptors and in guinea-pig ileum and cloned ml receptors yielded correlation coefficients of 0.82 and 0.72, respectively. The data indicate that the cloned muscarinic receptor sites seem similar to the corresponding endogenous sites. The good correlations in corresponding but also non-corresponding receptor models reflect the relatively low selectivity of the majority of the compounds investigated. Muscarinic receptors (cloned); A,, L cells (mouse fibroblast); Muscarinic Mi receptors; Muscarinic M, receptors
1. Introduction
Five muscarinic receptors have been cloned and expressed in various cell lines (Kubo et al., 1986; Bonner ei al., 1987; Peralta et al., 1987). Biochemical studies have revealed that activation of ml, m3 and m5 muscarinic receptors expressed in A9 L cells leads to increased phosphatidylinositol (PI) metabolism. The G-protein mediating ml, m3 and m5 muscarinic receptor-induced PI metabolism is pertussis toxin-insensitive (Brann et al., 1988). Whole organ pharmacology has revealed three ‘native’ muscarinic receptors, Ml, M, and M, (Doods et al., 1987). Recently, a uniform nomenclature for muscarinic receptors, defined in whole organ experiments and in cloned receptors, has been devised (Birdsall et al., 1989). Activation of M, and M, muscarinic receptors in animal tissues induces an increase in PI metabolism via a pertussis toxin-insensitive G-protein (Brown et al., 1989; Horwitz et al., 1985; Salomon and Bolton, 1988). Since a clear analogy
Corrcspondcnce to: f1.W.G.M. Boddckc. Prcclinical Research. Sandoz Pharma Ltd., Cf+4OIf2, Basle, Switzerland.
exists between the second messenger systems of M, and M, muscarinic receptors in animal tissue and those expressed in A9 L cells, it seemed of interest to compare the responses of a series of muscarinic agonists and antagonists in these two models. For this purpose the increase in free intracellular calcium induced by ml or m3 receptor activation was measured using the calcium indicator fura-2/AM in A9 L cells. The rat superior cervical ganglion was used as an in vitro model for M, receptors (Brown et al., 1980) and the guinea-pig ileum was used as a model for M, muscarinic receptors (Lazareno and Roberts, 1989).
2. Materials and methods 2.1. Rat superior cewical ganglion Experiments on rat superior cervical ganglia were performed as described by Brown et al. (1980). Male rats (Sprague-Dawley 200-300 g) were anaesthetized with urethane (1.2 g/kg i-p.) and superior cervical ganglia were excised. Ganglia were desheathed, suspended vertically in a heated chamber (36°C) and
superfused (1 ml/min) with oxygenated Krebs solution of the following composition (in mM): NaCl 124.0, KCI NaH,PO,, 1.25, NaHCO, 5.0, CaCI, 2.0 and glucos$ 10.0. The preparations were allowed to equilibrate for 30-45 min. Muscarinic agonist-induced depolarizations were recorded differentially between the ganglion and its postganglionic trunk using calomel electrodes. The DC potentials were amplified by microvoltmeters and monitored on computer. After 4-6 min perfusion with agonist, a concentration-dependent maximal depolarization was obtained which was used as a measure of agonist effect.
yield the correct minimum fluorescence value Fmin: Fmin= F(q) + 0.27 X (F,,,,, - F(q) (Fisher and Snider, 1987; Capponi et al., 1989). The increase in cytosolic free calcium was calculated from the equation: [Ca”] = K, X ((F - F,i”)/(F,,, F)) (Grynkiewicz et al., 1985), where F is the measured fluorescence intensity and K, is the dissociation constant of fura- for Ca2+ and has the value 225 nM. Agonist activity was defined as the maximal increase in intracellular calcium concentration induced by a single concentration of agonist. 2.4. Agonist potencies
2.2. Guinea-pig ileum
Male guinea-pigs (300-350 g. Ivanwas) were killed by a blow to the head and a segment of the ileum was removed. A piece of longitudinal muscle strip was suspended vertically in an organ bath containing 20 ml oxygenated Krebs solution (36°C) at a resting tension of 7.5 mN. The preparation was allowed to equilibrate for 30 min. Muscarinic agonist-induced contractions were measured with force-displacement transducers and monitored on a computer. 2.3. Measurement of free intracellular calcium A9 L cells transfected with cloned ml or m3 muscarinic receptors, (obtained from NIH, Bethesda, Maryland. USA) were cultured in Dulbecco’s modified Eagle’s Medium supplemented with 5% fetal caif serum. The cells were cultured not longer than 12 passages. Cell suspensions with a density of approximately 10’ cells were seeded on glass coverslips and grown to confluent monolayers. The coverslips were washed in physiological salt solution (PSS; composition in mM: NaCl 145.0, KC1 5.0, CaClz 1.9, MgC12 1.0, HEPES 5.0 and glucose 10.0, pH 7.4). The cells were incubated at 37°C tor 30 min in PSS containing 5 PM fura- acetylmethoxyester and 0.02% w/v pluronic. The monolayers were washed and allowed to rest for 40 min at room temperature. The glass coverslips were placed in a quartz cuvette filled with 3 ml PSS and the cuvette was placed in a thermostated cuvette-holder (37°C) of an LS-5b Perkin-Elmer Luminescence Spectrophotometer. Continuous stirring was achieved by a magnetic microstirrer. Changes in fura- fluorescence upon agonist stimulation of the cells were measured using an excitation wavelength of 340 nm and an emission wavelength of 509 nm. Calibration was performed with 5 PM ionomycin to determine the maximal fluorescence (F,,,;,,) and 5 mM MnCI, to determine minimal fluorescence (F,i,). The value F(q) obtained after the addition of MnCl, represents the complete quenching of fura- fluorescence by MnCl, and has to be modified according to the following equation to
In rat superior cervical ganglia and guinea-pig ilea, concentration-response curves were made with single concentrations (six to eight) of agonist applied at 20-45 min intervals followed by a washout phase until the baseline was reached. In the transfected cells, a complete concentration-response curve was made by using one coverslip with single concentrations (6-8) of agonist. Between the measurements the cells were allowed to rest in PSS for 15-20 min. The potency of an agonist is express& as its pD, value, i.e. the -log of the molar concentration that induced a response that was 50% of the individual maximal effect. For the quantification of partial agonism, the response of carbachol was set to 100%. 2.5. Antagonist affinities Rat superior cervical ganglia zmd guinea-pig ilea were allowed to equilibrate for 30-60 min. Concentration-response curves of carbachol were obtained before and after addition of an antagonist. Only one concentration-response curve per coverslip could be made with the transfected cells, Therefore, control and antagonist-shifted concentration-response curves were made with different coverslips. EC,,, values were determined for the control and the antagonist-shifted concentration-response curves. Three to four concentrations of antagonist were tested in all preparations after a 60-min equilibration period. Each concentration of antagonist was tested 4-6 times and the ratios of agonist molar EC,,, values obtained in the absence or presence of antagonist were calculated. Schild plots were calculated using linear regression by the method of least squares. 2.6. Statistics All data are presented as means + S.E.M. Statistical significance (P < 0.05) was evaluated by means of an analysis of variance and Student’s t-test. Correlations were performed according to the method of least squares.
153 TABLE
1
The pD, values and maximal responses of a series of muscarinic agonists at cloned ml and m3 muscarinic receptors, rat superior cervical ganglion (i&J,) and guinea-pig ileum (MS). The data represent mean values+S.E.M. (MFMT = methylfurmethide; n.m. = not measured). C’ompound
m 1 receptors
ml receptors (maximal response %I
(PI%) Carbachol ~xotremorine BM-5 Arecoline Pilocarpine AF-102h AF30 RS-86 210-086 MFMT McN-A-343
5.7kO.l 5.720.1 6.1 +0.3 6.3kO.2 h.OkO.2 5.5iO.l 4.3+0.1 4.6 + 0.2 5.9kO.4 5.7*0.3 6.OLtO.2 5.1 kO.2
100 98k6.2 65 -14.2 15*2.1 65+ 1.2 69+ 1.4 2023.6 lOk2.1 58zt3.3 49+2.5 b8+ 4.6 56+&l
m3 receptors
7.2+0.2 7.1 kO.1 4.9+0.1 7.4&0.1 6.6&O.] 6.2 + 0.3 6.1 f0.3 6.0+0.1 6.7fO.2 7.0+0.1 7.2kO.l 6.0 + 0.2
m3 receptors (maximal response p/o)
(PI&) Carbachol
5.rkktJ.4
100
Muscarine Oxotremorine BM-5 Arecoline Pilocarpine AF-IMb AF-30 RS86
5.6&0.1 6.6,0.X 6.2+0.9 6.1 k0.6 5.3* 1.0 4.5 rt 0.7 4.7 f 0.6 6.2 rt 0.5
104 f 9.2 53 + 2.2 lo+ 1.4 322 1.6 5 rt 0.8 lOk2.7 21 rt4.6 29+ 1.1
210-086
5.7&0.4
MFMT McN-A-343
M, receptors (PI%)
M, receptors
M, receptors (maximal response R) 100 95 rt 2.7 92k4.9 107+7.1 309+3,.5 84 * 4.3 52 * 2.Y 91 +4.7
6.7+0.3 6.8k0.1 7.1 kO.1 6.6 2 0.2 hS+O.l 5.9 rt 0.2 4.9 + 0. I 4.8kO.2
97+x.2
6.1kO.4 6.3+ 0.I
I1 I f 7.6 I lS+h.Y
6.X f 0.3 < 4.0
61 f 3.3 n.m.
6.2 f 0.5 < 4.0
100 96 * 4.3 43 f 4.4 33k4.6 80 f 3.8 13Ok5.9 79 f 6.2 56?4.9 80 + 7.2 134+3*5 136~9.2 122k7.3
(PI&)
5X52.1
MI receptors (maximal response Q)
n.m.
--
200 180 160 depolarization
(9.) 60
140 : calcium
[nMl 120 t
9
8
7
6
5
agonist -1oq [Ml Fig I. (A) The mnct’ntmtion-rcsptlnse curve for the depolarization in rat superior cervical ganglion in (B) The concentration-response cuwc for the contraction in guinea-pig ileum induced by carhachol ( mean vidues f S.E.M. (n = 6.8).
aqonist
-log IMI ). The data rcpres
,.’ 7 *.
in DMSO; the final concentration exceed 0.1%.
~‘lf~~l~~fltflt~~ll.Wi
Carbamoylcholinc hydrochloride, E. Merck, DarmSt&t, FRG: DL-muscarinc chloride, Sigma St. Louis MO. USA; oxotremorine sesquifumarate, Sigma St. Louis MO. USA; BM-5 (N-methyl-N-( I-methyl-4-pyrr~~lidino-I-ye-~-but}~nyl)~lcctatnide oxalatef, gift Professor D. Jenden. UCLA University. USA; arecoline hydrohromide. Fluka AG, Buchs. Switzerland; pilocarpine hydrochloride. Sigma St. Louis MO. USA: AF-102b (2-methyl-spiro(l.C(-oxothiolan-4,3)-quiniclidine). Sandoz Ltd.. Switzerland: AF-30 (2-methyl-spiro (1.3-di~xolan-4,3~quiniclidine~, Sandoz Ltd.. Switzerland; RSXh (2-ethyl-&methyl-Wdinzaspiro-(4,Sbdccan- 13-dion hydrobromide), Sandoz Ltd., Switzerland: ~I~)-~~~6 f( - )[~-rneth}~i-Spiro-(1,3-dioxolan-4,4 ]-l-mcthylpiperidine hydrogenmaleinate). Sandoz Ltd.. Switzerland: methylfurmethic,c (S-m~:thylfurfuryltrimethyl amm~nium iodide). Sandoz Ltd.. Switzerland: h&N-A-343 ~4-13-chlorophen~~lcarhamoyloxy~-~-butinyltrimethyl ammonium chloride), RBI. Natick, MA. USA; atropine methylnitrate, Sigma. St. Louis MO, USA; pirenzepine, gift Boehringer Ingelheim. FRG; telenzepine dihydrochloride, gift Byk Gutden, D-Lomberg, FRG; AF-DX 116. gift Boehringer Ingelheim, FRG; methoctramine tetrahydrochloride, RBI. Natick MA. USA; para-fluoro-hexahydro-sila-difenidol hydrochloride, gift Professor G. Lambrecht, University of Frankfurt, FRG; 4DAMP methiodide. RBI, Natick MA, USA; fura-2/ acetylmethoxy ester, Sigma, St. Louis MO. USA; pluronic. Sigma, St. Louis MO, USA; ionomycin, Serva, Heidelberg, FRG. All drugs were dissolved in a saline (0.9%) solution. Stock solutions of fura- and ionomycin were prepared
TABLE
of DMSO did not
3. Results
The 12 agonists studied all induced a concentration-dependent depolarization of rat superior cervical ganglia. The corresponding pD, values are listed in table 1. Full agonist behaviour was observed for carbachol, which induced a maximal depolarization of 0.92 L4:0.08 mV (fig. l), musearine, pilocarpine, methylfurmethide, 210-086 and McN-A-343. The other compounds behaved as partial agonists, with maximal responses varying frum 33 _t 4.6 to 80 + 3.7% compared with carbachol (table 1). All antagonists investigated produced parallel shifts of the concentration-response curve of carbachol, as indicated by Schild analysis yielding slopes not significantly different from 1. The pA, values are listed in table 2. 3.2. Gttirren-pig ilertm In fig. I the concentration-response curve of carbachol the guinea-pig ileum is shown. The maximal contraction induced by carbaehol (43 + 4.2 mN1 was set to 100%. With the exception of McN-A-343, all the muscarinic agonists studied contracted guinea-pig ileum in a concentration-dependent manner (table 1). Pilocarpine and AF-102b behaved as partial agonists, with maximal responses of 84 + 5.6 and 52 t_ 3.4% of the maximal response of muscarine, respectively. All an-
2
The PA, values and the slopes of the Schild ;kts of a series d rnuscarinic antagonists :tt cloned ml and m3 muscatinic receptors. rat superior ~rtktl ~n~li~~n tM,) and ruined-pin ileum (M,). The data represent mean va1u~s-fS.E.M. Compcwnd
m I receptors (PA,)
ml receptors (slope)
MI receptors (PA,)
ML receptors (slope)
Attopine Pitenzepine Telenzcpine AF-DX 1Ih Methoctramine pF-HHSiD d-DAMP
X.6-f I!. 10 8. I -1:0.07 x.3+02-l h.-l+o.fX 7.hto.II 7.1 *(1.(15 Y. I rt 0.0x
I.ffX+o.ll 0.X6 f 0. I6 o.u3 4 0.09 O.XYrt (I.06 O.Yh2 0.13 1.07rt 0.0’~ O.YSI o.tw
Y.1+0.13 8.3 * o.aY 8.4~O.Il 6.3*0.17 7.3 5 0.0s 72 f 0.04 Y.0 * (I, I4
0.98+0.11 O.Yhf 0. I4 0.84+ 0.I3 I.05 + 0.08 0.94 f O,ll4 Il.88 rt 0.07 0.91 + 0.1 I
Compound
m3 reccplors (PA,)
m3 receptors (slope)
M ,, receptors (PA,)
M3 receptors Nope)
AIropine Pitenzepine Telenzepine AF-FX 116 Met~{~~tt~mine pF-WlSiD J-DAMP
X.Y t 0. I fi-7.0 f O.Oh 7. I -f 0.0x 6.1+11.13 h.Y~O.Ih 7.0 i: 0.w X.CJ + 0. I4
0.87 + o.tKl O.YZ* I). I 1 l.UY t_BNi 0.06 + 0. IO
8.Y rt 0.09 h.XZkO.13 h.7 f 0.0s 6. I + 0.07
1.11i: 0.0s
6.5+ 0.08
1.I& + 0.07 0.94 * 0. IO I .I12f. 0.08 0.x9*0.1 I I .07 * 0.04
--
l.l7~ll.l:!
7.7 * 0.00
O.Xh+ O.OH
9.5+ 0.I I)
1,(I1* 0.03 I. 12* 0.00
TABLE
3
The correlations of the potencies, the maximal responses and the antagonist affinities of musrarinic agonists and antagonists at cloned ml and m3 muscarinic receptors, rat superior cervical ganglion (MI) and guinea-pig ileum (M,). Correlations Correlation coefficient
Regression equation
(rf Agonist potencies
0.80
Y = 0.67X+ 2.97
0.87 0.91 0.71 0.85
Y Y Y Y
M1 M3
0.53 0.36
Y = @.60X + 52.54 Y = 0.18X + 87.72
MI Mz M, MI M J
0.97 0.98 0.72 0.82 0.73
Y ‘i Y Y Y
Cloned ml vs. in vitro M , Cloned m3 vs. in vitro M3 Cloned ml vs. in vitro M 3 Cloned m3 vs. in vitro Ml In vitro M t vs. in vitro MJ
= = = =
1.00X + 0.51 1.11x+0.003 0.52X + 3.78 1.47x+3.x1
Maximnlrespmtses Cloned ml vs. in vitro Cloned m3 vs. in vitro Antagmist rrffiniries Cloned ml vs. in vitro Cloned m3 vs. in vitro Cloned ml vs. in vitro Cloned m3 vs. in vitro In vitro M, vs. m vitro
= = = = =
1.09x - 0.68 1.20X - 1.56 1.00x - 0.50 0.80X + 1.91 o.Yzx+o.18
carinic receptors. Concentration-response curves of carbachol at both ml and m3 receptors are shown in fig. 2. Carbachol and muscarine showed full agonist behaviour at both ml and m3 receptors and induced maximal increases in intracellular calcium of 190 i_ 3 and 165 t 5 nM, respectively. Whereas McN-A-343 was a partial agonist at ml receptors, no activity for this compound could be detected at m3 receptors. PartiaI agonist behaviour at ml and m3 receptors was also found for the other muscarinic agonists studied. The pD, values and the m~imal responses for alf agonists investigated are given in table 1. All antagonists investigated shifted the concentration-response curve of carba~hol for its response at either ml or m3 receptors to the right in a parallel fashion and no depression of the maxima occurred. At both ml and m3 receptors the slopes of the Schild plots were not significantly different from unity. The pA, values of the antagonists investigated are given in table 2. 3.4. Correlatiori of in vitro systems and cloned muscnrinic receptors
tagonists studied shifted the concentration-response curve of carbachol in a parallel manner. The pA, values and the slopes of the Schild plots are given in table 2. 3.3. Muscarinic ml and m3 receptors in A9 L cells Concentration-dependent increases in cytosolic calcium were induced by addition of muscarinic agonists in A9 L cells transfected with either ml or m3 mus-
All correlation coefficients and the corresponding regression equations are presented in table 3. In rat superior cervical ganglion and cloned ml receptors, a good correlation between the potencies cf muscarinic agonists (r = 0.80) was found. A lower correlation coefficient (r = 0.53) was observed for the maximal responses of the muscarinic agonists in both systems. Correlation coefficients of 0.87 and 0.36 were found for pD2 values and maximal responses, respectively, at cloned m3 muscarinic receptors and guinea-pig ileum. Correlation of the pD, values at ganglion and cloned
80
contraption calcium [nMl
(F)
i
60
! 100
50 8
7
6
agonist
5 -log
4 [M]
3
agonist -log
[Ml
Fig 2. (A) The co,~centFution-rrsponsc ctnvc for the incrcasc in i~~tr~~ceiiularcalcium induced hy c~Ir~?~teht~l (e) and AF-1tQh in A9 L cells in intr;iccllular calcium induced hy translktcd with ml f&J muscarinic rcccptors. (B) The concentration-rosponsc curve for the incrcasc cal l>achol(e) and AF-lO2b in A0 L ~11s transfcctcd with m.7 (R) muscarinic rcccptorh. The diII;I rcprckent mean V~I~IU~~ S.E.M. h =: 6-s).
m3 receptors or at ileum and cloned ml receptors yielded correlation coefficients of 0.71 and 0.87, respectively. In addition, a good correlation between potencies in rat superior cervical ganglion and guineapig ileum (r = 0.85) was found. Excellent correlations (r = 0.97 and 0.98) were found for the pA, values of a series of muscarinic antagonists at rat superior cervical ganglion and cloned ml receptors and at guinea-pig ileum and cloned m3 receptors, respectively. Cross correlations of pA, values at rat superior cervical ganglion and cloned m3 receptors or guinea-pig ileum and cloned ml receptors yielded lower correlation coefficients of 0.83 and 0.72, respectively. Also a lower correlation coefficient was found (r = 0.73) for the correlation of pA, values in rat superior cervical ganglion and guinea-pig ileum.
4. Discussion Recently, five subtypes of muscarinic receptors have been cloned and expressed in an A9 L mouse fibroblast cell line (Bonner, 1989). Activation of cloned ml, m3 and m5 receptors in these cells induces an increase in PI turnover (Brann et al., 1988). An increase in phosphoinositide hydrolysis and thus generation of the second messenger IP, causes a rapid transient release of intracellular calcium in the cytoplasm of cells (Berridge, 1977). Indeed. an increase in free cytosolic calcium and calcium-dependent increases of potassium and chloride conductances have been demonstrated in A9 L cells after stimulation of ml or m3 muscarinic receptors (Jones et al., 1990). In the present study we describe a method which allows the activity of muscarinic compounds to be defined in these transfected A9 L cells in an easy and efficient manner. Well-attached monolayers of cells were cultured on glass coverslips and could be used several times to quantify agonist-induced changes in intracellular calcium. No desensitization was observed when up to six concentrations of muscarinic agonist were tested on one coverslip (data not shown). This enabled us to make a complete concentration-response curve with one monolayer of cells. Thus A9 L cells provide the basis for an efficient measurement of intracellular calcium. In order to establish the validity of this new test system, the pharmacological characteristics of the transfected cells were compared with those in vitro. As a model for M, muscarinic receptors, the slow depolarization evoked in rat superior cervical ganglion was used (Brown et al., 1980). An increase in PI turnover is believed to play an important role in this depolarization (Brown et al., 1989, Patterson and Voile, 1984). For muscarinic M, receptor activation, the contractile response in guineapig ileum (Lazareno and Roberts, 19S9), wiucS is also mediated by an increase in PI turnove , was \; {died.
The affinities of the antagonists compared well with those from radioligand binding studies in similarly liansfected cells (Buckley et al., 1989), which supports the use of transfected cell lines as described in this study. Moreover, the data found in the transfected cells, both for agonists and antagonists, are generally consistert with those found in rat superior cervical ganglion and guinea-pig ileum described in this study. Whereas agonist potencies as well as antagonist affinities in the transfected cell lines correlated well with the corresponding in vitro systems, relatively good correlations were also observed for the non-corresponding (ml vs. M, and m3 vs. M ,I systems (see table 3). This indicates that most of the compounds investigated lack receptor subtype selectivity. Nevertheless, selective antagonists like pirenzepine and telenzepine, as well as the agonist McN-A-343, displayed selectivity both in transfected cells and in vitro. For Instance, the M, selectivity of McN-A-343 found when using rat superior cervical ganglion and guinea-pig ileum was also observed in transfected cells. AF-102b and AF-30 were the only two agonists of which the results in animal tissue and at cloned muscarinic receptors were inconsistent. Whereas these compounds show a preference for M, over M, receptors in the in vitro models, no selectivity at the cloned muscarinic receptors was observed (see table 1). One possibility might be that the activity of both AF-102b and AF-30 does not reflect muscarinic receptor activation. However, this seems unlikely since the responses of the two agonists in rat superior cervical ganglion and guinea-pig ileum could be blocked completely by atropine (lo-’ M) (data not shown). At present we have no explanation for the lack of selectivity of AF-102b and AF-30 at the cloned muscarinic receptors. The potencies of the muscarinic agonists found in the cells transfected with ml muscarinic receptors were 4- to 63-fold lower than those found in rat superior cervical ganglion (table 1); 1.3- to 16-fold lower potencies at m3 receptors than in guinea-pig ileum were found. The potency of an agonist depends on the number of steps and thus amplification between receptor activation and the parameters measured (Kenakin, 1984). Thus the different parameters measured, i.e. calcium mobilization vs. depolarization and calcium mobilization vs. contraction, may be a factor in the potency differences of the agonists found in the different systems. Moreover, it may well be that differences in the type and number of G-proteins in trre various tissues exist which possibly affect agonist-receptor coupling. In addition, differences in receptor reserve may determine differences in agonist potencies. Some of the compounds that were full agonists in the in vitro models were partial agonists in the transfected ceils, again indicating differences in receptor reserve (table 1). Differences in receptor reserve would be expected
157
to underlie the relatively weak correlations obtained between the maximal responses in the cloned receptor systems and the in vitro models. A feature which may complicate the interpretation of agonist data determined in the rat superior cervical ganglion is the M, receptor-mediated hyperpolarization (Newberry et al., 1985; Newberry and Gilbert, 1989). This may explain the observation that compounds like pilocarpine, 220086, methylfurmethide and McN-A-343 produced higher maximal responses compared to carbachol in the rat superior cervical ganglion whereas they behaved as partial agonists in the cells transfected with ml receptors. The good correlation founci between the pD, values in the rat superior cervical ganglion and the A9 L cells transfected with ml receptors, however, suggests that this is not an important factor. In theory, cell lines expressing only one receptor subtype, as is the case in A9 L cells transfected with cloned muscarinic receptors, would be expected to detect the activity and selectivity of muscarinic drugs more reliably than animal tissues, which generally contain more than one muscarinic receptor subtype. The use of transfected cells as described in this paper provides an efficient method to determine potencies and affinities of muscarinic agonists and antagonists, respectively, and provides data generally consistent with that of existing in vitro models, The lack of well discriminating muscarinic ligands, however, makes it difficult to estimate the predicative value of cloned receptor systems for in vitro models.
References Berridge, M.J., 1987, Inositol Iriphosphate and diacylglycerol: IWO interacting second messengers, Ann. Rev. Biochem. 56, 159. Birdsall. N.J.M., N. Buckley, H.N. Doods, K. Fukuda, R. Giachetti, R. Hammer, H. Kilbinger, G. Lambrecht, E. Mutschler, N. Nathanson, A. North and R. Schwarz, 1989, Nomenclature for muscarinic receptors recommenCed by symposium, Trends Pharmacol. Sci. IO (SuppI. Subtypes of Muscarimc Receptors IV), VII. Brann, M.R., B. Conklin, N.M. Dean, R.M. Collins, T-1. Banner and N.J. Buckley, 1988, Cloned muscarinic receptors couple to different G-proteins and second messengers, Sot. Neurosci. Abstr. 14, 600. Brown, D.A.. A. Forward and S. Marsh, 1980. Antagonist discrimination between ganglionic and ileal muscarinic receptors, Br. J. Pharmacol. 71,362. Brown, D.A., N.V. Marrion and T.G. Smart, 1989, On the transduction mechanism for muscarine-induced inhibition of M-current in cultured rat sympathetic neurones, J. Physiol. 413, 469.
Banner,T.I., 1989,
The molecular basis of muscarinic receptor diversity, Trends Neurosci. 12. 148. Banner, T.f., A.C. Young, M.R. Brann and N.J. Euckley, 1987, Cloning and expression of the human and rat m5 muscariuic receptor genes, Neuron 1, 403. Buckley. N.J., T.1. Bonnet, C.M. Buckley and M.R. Brann, 1989, Antagonist binding properties of five cloned muscarinic receptors ir! CHO-Kl cells, Mol. Pharmacol. 35, 469. CaPponi, A.M..P.D. Lew, W. Schlegel and T. Pozzan, 1989, Use of intracellular calcium and membrane potential fluorescent indicators in neuroendocrine cells, in: Neuroendocrine Peptide Methodology, Selected Methods in Enzymology, ed. P.M. Conn (Academic Press, New York) p. 315. Doods H.N., M.N. Mathy, D. Davidesko, K.J. Van Charldorp, A. De Jonge and P.A. Van Zwieten. 1987, Selectivity of muscarinic agonists in radioligand and in vitro experiments for the putative Ml, Mz and M, receptors, J. Pharmacol. Exp. Ther. 242,257. Fisher. SK. and R.H. Snider, 1987, Differential receptor occupancy requirements for muscarinic cholinergic stimulation of inositol lipid hydrolysis in brain and in neuroblastomas, Mol. Pharmacol. 32,81. Glynkiewiecz, G., M. Poenie and R.Y. Tsien, 1985, A new generation of calcium indicators with greatly improved fluorescence properties, J. Biol. Chem. 260. 3440. Horwitz, J., S. Tsymbalow and R.L. Perlman, 1985, Muscarine stimulates the hydrolysis of inositol-containing phospholipids in the superior cervical ganglion, J. Pharmacol. Exp. Ther. 233, 235. Jones. S.V.P.. J.L. Barker, M.B. Goodman and M.R. Brann, 1990, Inositol triphosphate mediates cloned muscarinic receptoractivated conductances in transfected mouse fibroblast A9 L cells, J. Physiol. 412, 499. Kenakin. T.P., 1984, The classification of drugs and drug receptors in isolated tissues, Pharmacol. Rev. 63, 165. Kubo, T., K. Fukuda, A. Mikami, A. Maeda, H. Takahashi, M. Mishima. T. Haga, M. Haga, A. Ichiyama, K. Kangawa. M. Kojimd, H. Matsuo, T. Hirose and S. Numa, 19% Cloning. sequencing and expression of cosrplementary DNA encoding the muscarinic acetylcholine receptor, Nature 323, 41 I. Lazareno, S. and F.F. Roberts, 1989, Functional and binding studies with M,-subtype selective antagonists, Br. J. Pharmacol. 98, 309. Newberly, N.R. and M.J. Gilbert. 1989, Biphasic dose response curve to muscarine on the rat superior cervical ganglion, Eur. J. Pharmacol. 163, 237. Newberry, N.R., T. Priestley and G.N. Woodruff, 1985. Pharmacological distinction between two muscarinic responses on the isolated superior cervical ganglion of the rat, Eur. J. Pharmacol. 116. 191. Patterson, B.A. and R.L. Voile, 1984, Muscarinic receptors and [“Hlinositol incorporation in a rat sympathetic ganglion cell, J. Auton. Nerv, Syst. 10. 69. Peralta E.G., A. Ashkenazi, J.W. Winslow, D.H. Smith, J. Ra. machandran and D.J. Capon. 1987, Distinct primary structures. ligand binding and tissue specific expression of four human muscarinic acetylcholine receptors, EMBO J. 6. 3923. Sabmon, D.M.W. and T.B. Bolton. 19X8. Early events in inositol phosphate metabolism in longitudinal smooth muscle from guinea-pig intestine stimulated with carbachol. Biochem. 3. 245. 553.
European Journal ADONIS
151
0014299991005815
EJP 52022
H.W.G.M. Boddeke and M. Buttini Preclinicai Research, S~ndoz Pharma Ltd., CH-4002
Bask, Switzerland
Received 31 January 1991, revised MS received 8 May 1991, accepted 25 June 1991
The effects of a series of muscarinic agonists and antagonists at cloned ml and m3 muscarinic receptors expressed in mouse fibroblast A9 L cells have been compared with their effects in in vitro models of M, (rat superior cervical ganglion) and M, (guinea-pig ileum) muscarinic receptors. A good correlation existed between the potencies of rnuscarinic agonists at cloned ml muscarinic receptors and the M, sites in rat gangho: (r = 0.80) as well as at cloned m3 receptors and guinea-pig ileum M, receptors (r = 0.87). However, cross correlations of potencies in rat ganglion and cloned m3 receptors as well as in guinea-pig ileum and in cloned ml receptors also yielded relalively high correlation coefficients (0.71 and 0.91, respectively). Low correlation coefficients were found for the maximal responses of muscarinic agonists in rat ganglion and cloned ml receptors (0.53) and in guinea-pig ileum and cloned m3 receptors (0.36). A high correlation between pA, values of muscarinic antagonists at cloned ml receptors and in rat ganglion (r = 0.97) and between cloned m3 receptors and guinea-pig ileum (r = 0.98) was found. Cross correlation of pA z values in rat ganglion and cloned m3 receptors and in guinea-pig ileum and cloned ml receptors yielded correlation coefficients of 0.82 and 0.72, respectively. The data indicate that the cloned muscarinic receptor sites seem similar to the corresponding endogenous sites. The good correlations in corresponding but also non-corresponding receptor models reflect the relatively low selectivity of the majority of the compounds investigated. Muscarinic receptors (cloned); A,, L cells (mouse fibroblast); Muscarinic Mi receptors; Muscarinic M, receptors
1. Introduction
Five muscarinic receptors have been cloned and expressed in various cell lines (Kubo et al., 1986; Bonner ei al., 1987; Peralta et al., 1987). Biochemical studies have revealed that activation of ml, m3 and m5 muscarinic receptors expressed in A9 L cells leads to increased phosphatidylinositol (PI) metabolism. The G-protein mediating ml, m3 and m5 muscarinic receptor-induced PI metabolism is pertussis toxin-insensitive (Brann et al., 1988). Whole organ pharmacology has revealed three ‘native’ muscarinic receptors, Ml, M, and M, (Doods et al., 1987). Recently, a uniform nomenclature for muscarinic receptors, defined in whole organ experiments and in cloned receptors, has been devised (Birdsall et al., 1989). Activation of M, and M, muscarinic receptors in animal tissues induces an increase in PI metabolism via a pertussis toxin-insensitive G-protein (Brown et al., 1989; Horwitz et al., 1985; Salomon and Bolton, 1988). Since a clear analogy
Corrcspondcnce to: f1.W.G.M. Boddckc. Prcclinical Research. Sandoz Pharma Ltd., Cf+4OIf2, Basle, Switzerland.
exists between the second messenger systems of M, and M, muscarinic receptors in animal tissue and those expressed in A9 L cells, it seemed of interest to compare the responses of a series of muscarinic agonists and antagonists in these two models. For this purpose the increase in free intracellular calcium induced by ml or m3 receptor activation was measured using the calcium indicator fura-2/AM in A9 L cells. The rat superior cervical ganglion was used as an in vitro model for M, receptors (Brown et al., 1980) and the guinea-pig ileum was used as a model for M, muscarinic receptors (Lazareno and Roberts, 1989).
2. Materials and methods 2.1. Rat superior cewical ganglion Experiments on rat superior cervical ganglia were performed as described by Brown et al. (1980). Male rats (Sprague-Dawley 200-300 g) were anaesthetized with urethane (1.2 g/kg i-p.) and superior cervical ganglia were excised. Ganglia were desheathed, suspended vertically in a heated chamber (36°C) and
superfused (1 ml/min) with oxygenated Krebs solution of the following composition (in mM): NaCl 124.0, KCI NaH,PO,, 1.25, NaHCO, 5.0, CaCI, 2.0 and glucos$ 10.0. The preparations were allowed to equilibrate for 30-45 min. Muscarinic agonist-induced depolarizations were recorded differentially between the ganglion and its postganglionic trunk using calomel electrodes. The DC potentials were amplified by microvoltmeters and monitored on computer. After 4-6 min perfusion with agonist, a concentration-dependent maximal depolarization was obtained which was used as a measure of agonist effect.
yield the correct minimum fluorescence value Fmin: Fmin= F(q) + 0.27 X (F,,,,, - F(q) (Fisher and Snider, 1987; Capponi et al., 1989). The increase in cytosolic free calcium was calculated from the equation: [Ca”] = K, X ((F - F,i”)/(F,,, F)) (Grynkiewicz et al., 1985), where F is the measured fluorescence intensity and K, is the dissociation constant of fura- for Ca2+ and has the value 225 nM. Agonist activity was defined as the maximal increase in intracellular calcium concentration induced by a single concentration of agonist. 2.4. Agonist potencies
2.2. Guinea-pig ileum
Male guinea-pigs (300-350 g. Ivanwas) were killed by a blow to the head and a segment of the ileum was removed. A piece of longitudinal muscle strip was suspended vertically in an organ bath containing 20 ml oxygenated Krebs solution (36°C) at a resting tension of 7.5 mN. The preparation was allowed to equilibrate for 30 min. Muscarinic agonist-induced contractions were measured with force-displacement transducers and monitored on a computer. 2.3. Measurement of free intracellular calcium A9 L cells transfected with cloned ml or m3 muscarinic receptors, (obtained from NIH, Bethesda, Maryland. USA) were cultured in Dulbecco’s modified Eagle’s Medium supplemented with 5% fetal caif serum. The cells were cultured not longer than 12 passages. Cell suspensions with a density of approximately 10’ cells were seeded on glass coverslips and grown to confluent monolayers. The coverslips were washed in physiological salt solution (PSS; composition in mM: NaCl 145.0, KC1 5.0, CaClz 1.9, MgC12 1.0, HEPES 5.0 and glucose 10.0, pH 7.4). The cells were incubated at 37°C tor 30 min in PSS containing 5 PM fura- acetylmethoxyester and 0.02% w/v pluronic. The monolayers were washed and allowed to rest for 40 min at room temperature. The glass coverslips were placed in a quartz cuvette filled with 3 ml PSS and the cuvette was placed in a thermostated cuvette-holder (37°C) of an LS-5b Perkin-Elmer Luminescence Spectrophotometer. Continuous stirring was achieved by a magnetic microstirrer. Changes in fura- fluorescence upon agonist stimulation of the cells were measured using an excitation wavelength of 340 nm and an emission wavelength of 509 nm. Calibration was performed with 5 PM ionomycin to determine the maximal fluorescence (F,,,;,,) and 5 mM MnCI, to determine minimal fluorescence (F,i,). The value F(q) obtained after the addition of MnCl, represents the complete quenching of fura- fluorescence by MnCl, and has to be modified according to the following equation to
In rat superior cervical ganglia and guinea-pig ilea, concentration-response curves were made with single concentrations (six to eight) of agonist applied at 20-45 min intervals followed by a washout phase until the baseline was reached. In the transfected cells, a complete concentration-response curve was made by using one coverslip with single concentrations (6-8) of agonist. Between the measurements the cells were allowed to rest in PSS for 15-20 min. The potency of an agonist is express& as its pD, value, i.e. the -log of the molar concentration that induced a response that was 50% of the individual maximal effect. For the quantification of partial agonism, the response of carbachol was set to 100%. 2.5. Antagonist affinities Rat superior cervical ganglia zmd guinea-pig ilea were allowed to equilibrate for 30-60 min. Concentration-response curves of carbachol were obtained before and after addition of an antagonist. Only one concentration-response curve per coverslip could be made with the transfected cells, Therefore, control and antagonist-shifted concentration-response curves were made with different coverslips. EC,,, values were determined for the control and the antagonist-shifted concentration-response curves. Three to four concentrations of antagonist were tested in all preparations after a 60-min equilibration period. Each concentration of antagonist was tested 4-6 times and the ratios of agonist molar EC,,, values obtained in the absence or presence of antagonist were calculated. Schild plots were calculated using linear regression by the method of least squares. 2.6. Statistics All data are presented as means + S.E.M. Statistical significance (P < 0.05) was evaluated by means of an analysis of variance and Student’s t-test. Correlations were performed according to the method of least squares.
153 TABLE
1
The pD, values and maximal responses of a series of muscarinic agonists at cloned ml and m3 muscarinic receptors, rat superior cervical ganglion (i&J,) and guinea-pig ileum (MS). The data represent mean values+S.E.M. (MFMT = methylfurmethide; n.m. = not measured). C’ompound
m 1 receptors
ml receptors (maximal response %I
(PI%) Carbachol ~xotremorine BM-5 Arecoline Pilocarpine AF-102h AF30 RS-86 210-086 MFMT McN-A-343
5.7kO.l 5.720.1 6.1 +0.3 6.3kO.2 h.OkO.2 5.5iO.l 4.3+0.1 4.6 + 0.2 5.9kO.4 5.7*0.3 6.OLtO.2 5.1 kO.2
100 98k6.2 65 -14.2 15*2.1 65+ 1.2 69+ 1.4 2023.6 lOk2.1 58zt3.3 49+2.5 b8+ 4.6 56+&l
m3 receptors
7.2+0.2 7.1 kO.1 4.9+0.1 7.4&0.1 6.6&O.] 6.2 + 0.3 6.1 f0.3 6.0+0.1 6.7fO.2 7.0+0.1 7.2kO.l 6.0 + 0.2
m3 receptors (maximal response p/o)
(PI&) Carbachol
5.rkktJ.4
100
Muscarine Oxotremorine BM-5 Arecoline Pilocarpine AF-IMb AF-30 RS86
5.6&0.1 6.6,0.X 6.2+0.9 6.1 k0.6 5.3* 1.0 4.5 rt 0.7 4.7 f 0.6 6.2 rt 0.5
104 f 9.2 53 + 2.2 lo+ 1.4 322 1.6 5 rt 0.8 lOk2.7 21 rt4.6 29+ 1.1
210-086
5.7&0.4
MFMT McN-A-343
M, receptors (PI%)
M, receptors
M, receptors (maximal response R) 100 95 rt 2.7 92k4.9 107+7.1 309+3,.5 84 * 4.3 52 * 2.Y 91 +4.7
6.7+0.3 6.8k0.1 7.1 kO.1 6.6 2 0.2 hS+O.l 5.9 rt 0.2 4.9 + 0. I 4.8kO.2
97+x.2
6.1kO.4 6.3+ 0.I
I1 I f 7.6 I lS+h.Y
6.X f 0.3 < 4.0
61 f 3.3 n.m.
6.2 f 0.5 < 4.0
100 96 * 4.3 43 f 4.4 33k4.6 80 f 3.8 13Ok5.9 79 f 6.2 56?4.9 80 + 7.2 134+3*5 136~9.2 122k7.3
(PI&)
5X52.1
MI receptors (maximal response Q)
n.m.
--
200 180 160 depolarization
(9.) 60
140 : calcium
[nMl 120 t
9
8
7
6
5
agonist -1oq [Ml Fig I. (A) The mnct’ntmtion-rcsptlnse curve for the depolarization in rat superior cervical ganglion in (B) The concentration-response cuwc for the contraction in guinea-pig ileum induced by carhachol ( mean vidues f S.E.M. (n = 6.8).
aqonist
-log IMI ). The data rcpres
,.’ 7 *.
in DMSO; the final concentration exceed 0.1%.
~‘lf~~l~~fltflt~~ll.Wi
Carbamoylcholinc hydrochloride, E. Merck, DarmSt&t, FRG: DL-muscarinc chloride, Sigma St. Louis MO. USA; oxotremorine sesquifumarate, Sigma St. Louis MO. USA; BM-5 (N-methyl-N-( I-methyl-4-pyrr~~lidino-I-ye-~-but}~nyl)~lcctatnide oxalatef, gift Professor D. Jenden. UCLA University. USA; arecoline hydrohromide. Fluka AG, Buchs. Switzerland; pilocarpine hydrochloride. Sigma St. Louis MO. USA: AF-102b (2-methyl-spiro(l.C(-oxothiolan-4,3)-quiniclidine). Sandoz Ltd.. Switzerland: AF-30 (2-methyl-spiro (1.3-di~xolan-4,3~quiniclidine~, Sandoz Ltd.. Switzerland; RSXh (2-ethyl-&methyl-Wdinzaspiro-(4,Sbdccan- 13-dion hydrobromide), Sandoz Ltd., Switzerland: ~I~)-~~~6 f( - )[~-rneth}~i-Spiro-(1,3-dioxolan-4,4 ]-l-mcthylpiperidine hydrogenmaleinate). Sandoz Ltd.. Switzerland: methylfurmethic,c (S-m~:thylfurfuryltrimethyl amm~nium iodide). Sandoz Ltd.. Switzerland: h&N-A-343 ~4-13-chlorophen~~lcarhamoyloxy~-~-butinyltrimethyl ammonium chloride), RBI. Natick, MA. USA; atropine methylnitrate, Sigma. St. Louis MO, USA; pirenzepine, gift Boehringer Ingelheim. FRG; telenzepine dihydrochloride, gift Byk Gutden, D-Lomberg, FRG; AF-DX 116. gift Boehringer Ingelheim, FRG; methoctramine tetrahydrochloride, RBI. Natick MA. USA; para-fluoro-hexahydro-sila-difenidol hydrochloride, gift Professor G. Lambrecht, University of Frankfurt, FRG; 4DAMP methiodide. RBI, Natick MA, USA; fura-2/ acetylmethoxy ester, Sigma, St. Louis MO. USA; pluronic. Sigma, St. Louis MO, USA; ionomycin, Serva, Heidelberg, FRG. All drugs were dissolved in a saline (0.9%) solution. Stock solutions of fura- and ionomycin were prepared
TABLE
of DMSO did not
3. Results
The 12 agonists studied all induced a concentration-dependent depolarization of rat superior cervical ganglia. The corresponding pD, values are listed in table 1. Full agonist behaviour was observed for carbachol, which induced a maximal depolarization of 0.92 L4:0.08 mV (fig. l), musearine, pilocarpine, methylfurmethide, 210-086 and McN-A-343. The other compounds behaved as partial agonists, with maximal responses varying frum 33 _t 4.6 to 80 + 3.7% compared with carbachol (table 1). All antagonists investigated produced parallel shifts of the concentration-response curve of carbachol, as indicated by Schild analysis yielding slopes not significantly different from 1. The pA, values are listed in table 2. 3.2. Gttirren-pig ilertm In fig. I the concentration-response curve of carbachol the guinea-pig ileum is shown. The maximal contraction induced by carbaehol (43 + 4.2 mN1 was set to 100%. With the exception of McN-A-343, all the muscarinic agonists studied contracted guinea-pig ileum in a concentration-dependent manner (table 1). Pilocarpine and AF-102b behaved as partial agonists, with maximal responses of 84 + 5.6 and 52 t_ 3.4% of the maximal response of muscarine, respectively. All an-
2
The PA, values and the slopes of the Schild ;kts of a series d rnuscarinic antagonists :tt cloned ml and m3 muscatinic receptors. rat superior ~rtktl ~n~li~~n tM,) and ruined-pin ileum (M,). The data represent mean va1u~s-fS.E.M. Compcwnd
m I receptors (PA,)
ml receptors (slope)
MI receptors (PA,)
ML receptors (slope)
Attopine Pitenzepine Telenzcpine AF-DX 1Ih Methoctramine pF-HHSiD d-DAMP
X.6-f I!. 10 8. I -1:0.07 x.3+02-l h.-l+o.fX 7.hto.II 7.1 *(1.(15 Y. I rt 0.0x
I.ffX+o.ll 0.X6 f 0. I6 o.u3 4 0.09 O.XYrt (I.06 O.Yh2 0.13 1.07rt 0.0’~ O.YSI o.tw
Y.1+0.13 8.3 * o.aY 8.4~O.Il 6.3*0.17 7.3 5 0.0s 72 f 0.04 Y.0 * (I, I4
0.98+0.11 O.Yhf 0. I4 0.84+ 0.I3 I.05 + 0.08 0.94 f O,ll4 Il.88 rt 0.07 0.91 + 0.1 I
Compound
m3 reccplors (PA,)
m3 receptors (slope)
M ,, receptors (PA,)
M3 receptors Nope)
AIropine Pitenzepine Telenzepine AF-FX 116 Met~{~~tt~mine pF-WlSiD J-DAMP
X.Y t 0. I fi-7.0 f O.Oh 7. I -f 0.0x 6.1+11.13 h.Y~O.Ih 7.0 i: 0.w X.CJ + 0. I4
0.87 + o.tKl O.YZ* I). I 1 l.UY t_BNi 0.06 + 0. IO
8.Y rt 0.09 h.XZkO.13 h.7 f 0.0s 6. I + 0.07
1.11i: 0.0s
6.5+ 0.08
1.I& + 0.07 0.94 * 0. IO I .I12f. 0.08 0.x9*0.1 I I .07 * 0.04
--
l.l7~ll.l:!
7.7 * 0.00
O.Xh+ O.OH
9.5+ 0.I I)
1,(I1* 0.03 I. 12* 0.00
TABLE
3
The correlations of the potencies, the maximal responses and the antagonist affinities of musrarinic agonists and antagonists at cloned ml and m3 muscarinic receptors, rat superior cervical ganglion (MI) and guinea-pig ileum (M,). Correlations Correlation coefficient
Regression equation
(rf Agonist potencies
0.80
Y = 0.67X+ 2.97
0.87 0.91 0.71 0.85
Y Y Y Y
M1 M3
0.53 0.36
Y = @.60X + 52.54 Y = 0.18X + 87.72
MI Mz M, MI M J
0.97 0.98 0.72 0.82 0.73
Y ‘i Y Y Y
Cloned ml vs. in vitro M , Cloned m3 vs. in vitro M3 Cloned ml vs. in vitro M 3 Cloned m3 vs. in vitro Ml In vitro M t vs. in vitro MJ
= = = =
1.00X + 0.51 1.11x+0.003 0.52X + 3.78 1.47x+3.x1
Maximnlrespmtses Cloned ml vs. in vitro Cloned m3 vs. in vitro Antagmist rrffiniries Cloned ml vs. in vitro Cloned m3 vs. in vitro Cloned ml vs. in vitro Cloned m3 vs. in vitro In vitro M, vs. m vitro
= = = = =
1.09x - 0.68 1.20X - 1.56 1.00x - 0.50 0.80X + 1.91 o.Yzx+o.18
carinic receptors. Concentration-response curves of carbachol at both ml and m3 receptors are shown in fig. 2. Carbachol and muscarine showed full agonist behaviour at both ml and m3 receptors and induced maximal increases in intracellular calcium of 190 i_ 3 and 165 t 5 nM, respectively. Whereas McN-A-343 was a partial agonist at ml receptors, no activity for this compound could be detected at m3 receptors. PartiaI agonist behaviour at ml and m3 receptors was also found for the other muscarinic agonists studied. The pD, values and the m~imal responses for alf agonists investigated are given in table 1. All antagonists investigated shifted the concentration-response curve of carba~hol for its response at either ml or m3 receptors to the right in a parallel fashion and no depression of the maxima occurred. At both ml and m3 receptors the slopes of the Schild plots were not significantly different from unity. The pA, values of the antagonists investigated are given in table 2. 3.4. Correlatiori of in vitro systems and cloned muscnrinic receptors
tagonists studied shifted the concentration-response curve of carbachol in a parallel manner. The pA, values and the slopes of the Schild plots are given in table 2. 3.3. Muscarinic ml and m3 receptors in A9 L cells Concentration-dependent increases in cytosolic calcium were induced by addition of muscarinic agonists in A9 L cells transfected with either ml or m3 mus-
All correlation coefficients and the corresponding regression equations are presented in table 3. In rat superior cervical ganglion and cloned ml receptors, a good correlation between the potencies cf muscarinic agonists (r = 0.80) was found. A lower correlation coefficient (r = 0.53) was observed for the maximal responses of the muscarinic agonists in both systems. Correlation coefficients of 0.87 and 0.36 were found for pD2 values and maximal responses, respectively, at cloned m3 muscarinic receptors and guinea-pig ileum. Correlation of the pD, values at ganglion and cloned
80
contraption calcium [nMl
(F)
i
60
! 100
50 8
7
6
agonist
5 -log
4 [M]
3
agonist -log
[Ml
Fig 2. (A) The co,~centFution-rrsponsc ctnvc for the incrcasc in i~~tr~~ceiiularcalcium induced hy c~Ir~?~teht~l (e) and AF-1tQh in A9 L cells in intr;iccllular calcium induced hy translktcd with ml f&J muscarinic rcccptors. (B) The concentration-rosponsc curve for the incrcasc cal l>achol(e) and AF-lO2b in A0 L ~11s transfcctcd with m.7 (R) muscarinic rcccptorh. The diII;I rcprckent mean V~I~IU~~ S.E.M. h =: 6-s).
m3 receptors or at ileum and cloned ml receptors yielded correlation coefficients of 0.71 and 0.87, respectively. In addition, a good correlation between potencies in rat superior cervical ganglion and guineapig ileum (r = 0.85) was found. Excellent correlations (r = 0.97 and 0.98) were found for the pA, values of a series of muscarinic antagonists at rat superior cervical ganglion and cloned ml receptors and at guinea-pig ileum and cloned m3 receptors, respectively. Cross correlations of pA, values at rat superior cervical ganglion and cloned m3 receptors or guinea-pig ileum and cloned ml receptors yielded lower correlation coefficients of 0.83 and 0.72, respectively. Also a lower correlation coefficient was found (r = 0.73) for the correlation of pA, values in rat superior cervical ganglion and guinea-pig ileum.
4. Discussion Recently, five subtypes of muscarinic receptors have been cloned and expressed in an A9 L mouse fibroblast cell line (Bonner, 1989). Activation of cloned ml, m3 and m5 receptors in these cells induces an increase in PI turnover (Brann et al., 1988). An increase in phosphoinositide hydrolysis and thus generation of the second messenger IP, causes a rapid transient release of intracellular calcium in the cytoplasm of cells (Berridge, 1977). Indeed. an increase in free cytosolic calcium and calcium-dependent increases of potassium and chloride conductances have been demonstrated in A9 L cells after stimulation of ml or m3 muscarinic receptors (Jones et al., 1990). In the present study we describe a method which allows the activity of muscarinic compounds to be defined in these transfected A9 L cells in an easy and efficient manner. Well-attached monolayers of cells were cultured on glass coverslips and could be used several times to quantify agonist-induced changes in intracellular calcium. No desensitization was observed when up to six concentrations of muscarinic agonist were tested on one coverslip (data not shown). This enabled us to make a complete concentration-response curve with one monolayer of cells. Thus A9 L cells provide the basis for an efficient measurement of intracellular calcium. In order to establish the validity of this new test system, the pharmacological characteristics of the transfected cells were compared with those in vitro. As a model for M, muscarinic receptors, the slow depolarization evoked in rat superior cervical ganglion was used (Brown et al., 1980). An increase in PI turnover is believed to play an important role in this depolarization (Brown et al., 1989, Patterson and Voile, 1984). For muscarinic M, receptor activation, the contractile response in guineapig ileum (Lazareno and Roberts, 19S9), wiucS is also mediated by an increase in PI turnove , was \; {died.
The affinities of the antagonists compared well with those from radioligand binding studies in similarly liansfected cells (Buckley et al., 1989), which supports the use of transfected cell lines as described in this study. Moreover, the data found in the transfected cells, both for agonists and antagonists, are generally consistert with those found in rat superior cervical ganglion and guinea-pig ileum described in this study. Whereas agonist potencies as well as antagonist affinities in the transfected cell lines correlated well with the corresponding in vitro systems, relatively good correlations were also observed for the non-corresponding (ml vs. M, and m3 vs. M ,I systems (see table 3). This indicates that most of the compounds investigated lack receptor subtype selectivity. Nevertheless, selective antagonists like pirenzepine and telenzepine, as well as the agonist McN-A-343, displayed selectivity both in transfected cells and in vitro. For Instance, the M, selectivity of McN-A-343 found when using rat superior cervical ganglion and guinea-pig ileum was also observed in transfected cells. AF-102b and AF-30 were the only two agonists of which the results in animal tissue and at cloned muscarinic receptors were inconsistent. Whereas these compounds show a preference for M, over M, receptors in the in vitro models, no selectivity at the cloned muscarinic receptors was observed (see table 1). One possibility might be that the activity of both AF-102b and AF-30 does not reflect muscarinic receptor activation. However, this seems unlikely since the responses of the two agonists in rat superior cervical ganglion and guinea-pig ileum could be blocked completely by atropine (lo-’ M) (data not shown). At present we have no explanation for the lack of selectivity of AF-102b and AF-30 at the cloned muscarinic receptors. The potencies of the muscarinic agonists found in the cells transfected with ml muscarinic receptors were 4- to 63-fold lower than those found in rat superior cervical ganglion (table 1); 1.3- to 16-fold lower potencies at m3 receptors than in guinea-pig ileum were found. The potency of an agonist depends on the number of steps and thus amplification between receptor activation and the parameters measured (Kenakin, 1984). Thus the different parameters measured, i.e. calcium mobilization vs. depolarization and calcium mobilization vs. contraction, may be a factor in the potency differences of the agonists found in the different systems. Moreover, it may well be that differences in the type and number of G-proteins in trre various tissues exist which possibly affect agonist-receptor coupling. In addition, differences in receptor reserve may determine differences in agonist potencies. Some of the compounds that were full agonists in the in vitro models were partial agonists in the transfected ceils, again indicating differences in receptor reserve (table 1). Differences in receptor reserve would be expected
157
to underlie the relatively weak correlations obtained between the maximal responses in the cloned receptor systems and the in vitro models. A feature which may complicate the interpretation of agonist data determined in the rat superior cervical ganglion is the M, receptor-mediated hyperpolarization (Newberry et al., 1985; Newberry and Gilbert, 1989). This may explain the observation that compounds like pilocarpine, 220086, methylfurmethide and McN-A-343 produced higher maximal responses compared to carbachol in the rat superior cervical ganglion whereas they behaved as partial agonists in the cells transfected with ml receptors. The good correlation founci between the pD, values in the rat superior cervical ganglion and the A9 L cells transfected with ml receptors, however, suggests that this is not an important factor. In theory, cell lines expressing only one receptor subtype, as is the case in A9 L cells transfected with cloned muscarinic receptors, would be expected to detect the activity and selectivity of muscarinic drugs more reliably than animal tissues, which generally contain more than one muscarinic receptor subtype. The use of transfected cells as described in this paper provides an efficient method to determine potencies and affinities of muscarinic agonists and antagonists, respectively, and provides data generally consistent with that of existing in vitro models, The lack of well discriminating muscarinic ligands, however, makes it difficult to estimate the predicative value of cloned receptor systems for in vitro models.
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