Neurochemical Research, VoL 17, No. 3, 1992, pp. 215-222
Muscarinic Binding Sites in a Catecholaminergic Human Neuroblastoma Cell Line Giuseppe Sorrentino 1,4, Indrapal N. Singh 1, Alphonse Hubsch 2, Julian N. Kanfer ~,3, Serge Mykita 2, and Raphael Massarelli 2 (Accepted July 29, 1991)
Tyrosine hydroxylase (TH) a characteristic enzyme activity for the catecholaminergic clonal cell line LA-N-1 and choline acetyltransferase (CHAT) a characteristic enzyme activity for the cholinergic clonal cell line LA-N-2 were previously shown to be increased in these cells exposed to 10 -5 M retinoic acid (R_A) as differentiating agent. An investigation of the receptor characteristics suggests a complementarity between the two cell lines. The binding of QNB, a muscarinic ligand, was undetectable with the LA-N-2 cells but was present in the LA-N-1 cells and possessed a kD of 1.8 nM and 2.2 nM and a Bmax of 0.56 and 0.68 for control and RA grown cells respectively. There was a gradual increase in QNB binding to LA-N-1 cells from 2 days in vitro (DIV) until 6 DIV in both control and RA grown cells. An ICso of 2.5 x 10 -8 M and 0.9 x 10 -8 M for atropine inhibition was obtained for the control and RA grown cells respectively. The corresponding values for carbachol inhibition were 7 x 10 -z M and 3 x 10 -e M respectively. The inhibition by the agonist oxotremorine is comparable to that of carbachol and 1 mM pilocarpine inhibited the binding by 21%. QNB binding showed a low affinity for pirenzepine and for AF-DX-116 but was inhibited with a rather high affinity by 4-DAMP (IC5o:110 p~M) thus suggesting the presence of an M3 receptor. Acetylcholine (100 p~M) plus eserine (50 ~M) and BW284c55 (1 IxM), an acetylcholinesterase inhibitor, reduced the binding of QNB by approximately 25%. Nicotine (1 raM) caused a 36% reduction of binding and hemicholinium-3 (HC-3) (1 txM), an inhibitor of choline uptake, inhibited the binding by 53%. There was a down regulation of QNB binding observed with cells grown for 24 hours with either the antagonist atropine or the agonists carbachol or oxotremorine. Low amounts of c~-bungarotoxin (c~-BgTx) binding sites were barely detectable in both LA-N-1 and LA-N-2 cells. The LA-N-1 muscarinic receptor is coupled to polyphosphoinositide hydrolysis without increased cyclic AMP formation further suggesting its being an M~ receptor. KEY WORDS: LA-N-1 Cells; muscarinic receptor; human neuroblastomaclones; polyphosphoinositidebreakdown; cAMP. INTRODUCTION
proliferative capacity of these cells (1). Thus the treatment of glial ceils and primary neuronal cultures, or their corresponding established cell lines with cholinergic agonists such as carbachol (CCh) induces D N A synthesis only when the receptors linked to PI hydrolysis are ac-
The activation of muscarinic acetylcholine receptors (mAChRs) coupled to the hydrolysis of polyphosphoinositides (PI) in brain cells appears to influence the
a Address reprint requests to: Dr. J. N. Kanfer, Department of Biochemistry and Molecular Biology, University of Manitoba, Faculty
of Medicine, 770 Bannatyne Avenue, Winnipeg, Manitoba R3E 0W3, Canada. 4 Present address of Dr. G. Sorrentino: Istituto di Scienze Neurologiche, P Facoltfi di Medicina, Via Pansini 5, 80131 Napoli, Italy.
1 Department of Biochemistry and Molecular Biology, The University of Manitoba, Winnipeg, Manitoba R3E 0W3, Canada. z Centre de Neurochimie-Cronenbourg, BP20 CR, 67037 Strasbourg Cedex, France.
215 0364-3190/92/0300-0215506.50/09 1992PlenumPublishingCorporation
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Sorrentino, Singh, Ilubsch, Kanfer, Mykita, and Massarelli
tivated (2). Acetylcholine (ACh) stimulated PI hydrolysis by brain tissue is maximum at birth (3). It has been shown that CCh stimulates the PI hydrolysis in primary astroglial cells (4) and PI hydrolysis appears to be correlated with cell proliferation (5). Several different types of mAChRs designated M1 to M4 and perhaps M5 have been described to be present in mammalian tissues. In the CNS these receptors appear to be coupled to a number of biochemical effector systems resulting in a stimulation of PI turnover or an inhibition of cAMP production and activation of cGMP production (6). These responses have been observed with a variety of tissues, primary cell cultures and several cell lines of neuronal or of astroglial origin (7,8). It has been shown that the different receptors have different primary structures (9-11), different ligand binding properties (1,10,12,) and are expressed in tissue specific patterns
(12-14). We have previously investigated the developmental profile of several enzymatic activities in the human neuroblastoma clones LA-N-1 and "LA-N-2. Choline acetyltransferase (CHAT) activity of the LA-N-2 cells, a cholinergic clonal ceil line, increased gradually until 8 days in vitro (DIV) and then declined (15). In contrast acetylcholinesterase (ACHE) activity decreased at 8 DIV and was maintained at a low level. The activity of tyrosine hydroxylase (TH) in LA-N-1 cells, a catecholaminergic clone, gradually increased until 4 DIV and remained constant. The presence of 10 -5 M retinoic acid in the growth medium increased both of these synthetic enzymatic activities about two to three fold (15). It seemed useful based upon these observations to characterize the neurotransmitter receptor profile of these neuroblastoma cell lines under conditions of enhanced differentiation and to correlate the properties of receptor activation with effector-coupling.
Los Angeles, and their growth and maintenance as previously described (15). Binding of QNB. Ceils were detached and harvested by centrifugation at 500 g for 10 rain, resuspended in Leibovitz medium (L15) without serum and incubated for 30 rain at 37~ The cells were then centrifuged at 750 g for 10 min, the pellet was homogenized in a minimum volume of either 10 mM phosphate at pH 7.4 or 50 mM phosphate at pH 7.4. The incubation mixture, in a final volume of 100 Ixl, contained 0.3 mg of homogenate protein, varying concentrations of [3H]quinuclidinylhenzilate (QNB, specific activity: 41.5 Ci/mmol, Amersham) containing 210,000 dpms and 2 to 10 Ixl of 1 mM atropine sulfate and shaken at 37~ or 25~ for varying periods of time. Unless indicated otherwise the final routine concentration of [3H] QNB was 15 nM, that of atropine was 10 rzM and the time of incubation was 30 rain at 37~ The incubation was stopped by chilling the tubes in an ice bath followed by centrifugation at 10,000g for 5 min. The supematant was removed and the pellet resuspended in cold 10 mM phosphate at pH 7.4 and centrifuged again at 10,000 g for 2 rain. After removal of the supernatant the pellet was digested with 100 Ixl of 1 N NaOH, transferred to scintillation vials and the radioactivity determined with 10 ml of Scintiverse. Non specific binding was determined by measuring the radioactivity with homogenates incubated in the presence of 10 ~M atropine sulfate. Agonists and antagonists were included at the indicated concentrations during the incubations as indicated. Binding of Alpha-Bungarotoxin (a-BgTx). The binding of [tzsl] a-BgTx (specific activity: 200 Ci/mmol, Amersham) was carried out in incubations containing 0.1--0.75 mg of protein, 100 mM NaC1, 50 mM phosphate buffer at pH 7.4, and 5, 10 and 30 nM [lz5I] cx-BgTx at 37~ for 60 rain. Non specific binding was estimated with incubations containing 10 ~M d-tubocurarine. The assay procedure was the same as that employed for QNB binding.
Regulation of Muscarinic Binding by Agonists and Antagonists. To investigate the regulation of the muscarinic binding sites the LAN-1 cells were maintained in culture medium containing either the
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EXPERIMENTAL PROCEDURE Drugs and Other Chemicals. All radioisotopes and cAMP RIA (radioimmunoassay) kit were obtained from Amersham. Carbachol, 3quinuclidinyl benzilate (QNB), atropine, pirenzepine, d-tubocurarine, oxotremorine, scopolamine, nicotine, hemicholinium-3, aeetylcholine iodide, acetylcholinesterase inhibitor BW 284C55, pilocarpine, eserine, and retinoic acid were obtained from Sigma (St. Louis, MO, U.S.A.); and cell culture L-15 medium and heat-inactivated fetal calf serum were obtained from Flow Labs. AF-DX-116 [ll,2-(diethylamino)methyl-l-piperidinyl-acetyl-5, ll-dihydro-6H-pyrido-2, 3-b, 1,4 benzodiazepine-6-one and 4-DAMP (diphenylacetoxy-4-methylpiperidine methiodide) were obtained as gifts from Boehfinger Ingelheim (Canada) Ltd., Burlington, Ontario. Cell Cultures. The human neuroblastoma cell lines, LA-N-1 and LA-N-2 were obtained from Dr. R. Seeger, University of California,
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Muscarinic Binding Sites in Human Neuroblastoma Cell Line
217
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LOG (4-DAMP)
C non-specific antagonist atropine (10 ~M), or the M1 antagonist pirenzepine (i0 ~M) (I6) or the strong agonist carbachol (1 raM) or the weak agonist oxotremorine (1 raM) for 4 or 24 hrs. At that time the cells were collected and [3H]QNB binding was estimated as described above.
Cyclic Adenosine 3',5' Monophosphate (cAMP) Quantitations. The cells were washed three times with 5 ml of phosphate saline buffer (PBS), 10 IxM, pH 7.4, extracted with 200 ILl of 1 N HCI04, scraped, transferred to Eppendorf tubes, sonicated and centrifuged in the cold. The supernatant was removed, neutralized with 3 M K~CO~, centrifuged at 5,000g for 15 min and the cAMP content measured according to the instructions accompanying the Amersham kit.
Quantitation of the Products of Pl's Hydrolysis. Control and RAtreated LA-N-1 cells were incubated for 24 hrs with 44 ~M [3H]myoinositoI (1 p,Ci/ml, specific activity: 14.2 Ci/mmol, Amersham). At
the end of the incubation the cultures were washed with PBS containing 10 rnM LiC1 and the cells were homogenized in 2 ml of water. The radioactive water soluble inositol containing compounds were separated on a Dowex (1 x 8) formate form column employing the methodology described by Brown et al. (17). Proteins were estimated using the classical procedure of Lowry et al. (18). Statistical analysis. Statistical significance was assessed using student's t-test.
RESULTS
QNB Binding. Scatchard plots o f the binding of [3H]QNB s h o w e d saturable b i n d i n g in L A - N - 1 cells with
218
Sorrentino, Singh, Hubsch, Kanfer, Mykita, and Massarelli
an apparent dissociation constant (KD) of 1.8 nM and a Bmax of 0.56 (Figure 1). The LA-N-1 cells had a slightly increased KD of 2.2 nM and a Bm,x of 0.68 (Figure 1) when grown in the presence of 10 -5 M retinoic acid (4 DIV). There was no detectable binding of [3H]QNB to the LA-N-2 cells. Using [125I] c~-BgTx as a ligand (30 nM), a binding capacity of 20.2 and 26.6 fmol/mg protein respectively for LA-N-1 and LA-N-2 control cells and 30.5 and 82.0 fmol/mg protein respectively for LAN-1 and LA-N-2 RA treated cells. Effects of Agonists and Antagonists. To further demonstrate the presence of muscarinic receptors in the LA-N-1 ceils the effect of varying concentrations of the antagonist atropine was analysed (Figure 2a). Hill plots of the dose response curve for atropine yielded an ICso of 2.5 x 10 -8 M for control cells and 0.9 x 10 -8 M for the retinoic acid treated celIs and a coefficient below 1.0. Similarly pirenzepine had a very low affinity for the receptor in both control and retinoic acid treated cells (Figure 2b). The agonist carbachol was also examined (Figure 3a) and a Hill plot yielded an ICso of 7 x 10 -2 M for control cells and 3 x 10 -2 M for RA treated cells. The Hill coefficient was 1.0 for control cells and 1.8 for RA grown cells. Oxotremorine showed a greater competition for the binding of [3H]QNB than carbachol (Figure 3b). Incubation of membranes with 0.05-10 t~M AF-DX-116 [11,2-(diethylamino)methyl- 1-piperidinylacetyl-5, 11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepine-6-one;], a selective inhibitor of M2 receptor binding, did not affect the binding of QNB (results not shown) while 4-DAMP (diphenylacetoxy-4-methylpiperidine methiodide) a compound with selectivity for M1 and M3 receptors (19) inhibited the binding with an apparent affinity of 110 I~M (Figure 2c). Survey of Agonists and Antagonists. In an attempt to characterize the type of muscarinic receptor, the effect of several agonists and antagonists on the [3H]QNB binding was examined. In general, the magnitude of the effects of these agents were consistently greater with the retinoic acid treated cells as compared to untreated cells. Pilocarpine caused an inhibition; eserine and acetylcholine together produced an inhibition. In the retinoic acid grown cells the specific acetylcholinesterase inhibitor BW 284c55 was a more potent inhibitor of [3H]QNB binding than eserine. A decreased [3H]QNB binding was also obtained with the nicotinic receptor agonist nicotine and hemicholinium-3, an inhibitor of choline uptake (Table I). The muscarinic receptor antagonists atropine and scopolamine, at 10 IxM concentration, caused complete inhibition (data not shown). These results are usual for muscarinic receptors.
Developmental Profile of [3H]QNB Binding Sites.
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It was previously demonstrated with LA-N-1 and LAN-2 cells that the levels of activities of several enzymes increased during growth in culture (15) and with growth in the presence of retinoic acid. It seemed of interest to quantify the [3H]QNB binding capacity as a function of time in cultures of LA-N-1 cells. The
Muscarinic Binding Sites in Human Neuroblastoma Cell Line
219
Table I. Agonists and Antagonists: Effect of Various Drugs on QNB Binding in LA-N-1 Cells Control
Retinoic Acid (10-~M)
fmol/mg protein/hr
% inhibition
fmol/mg protein/hr
% inhibition
470.0 • 15.3 372.6 • 19.3"** 422.0 _ 47.0
0 20.8 10.2
733.4 • 34.5 354.0 • 30.1"** 466.0 • 16.1'**
51.7 36.4
300.0 - 66.1"* 344.3 • 77
36.1 26.7
378.6 • 50.5*** 354.7 • 23.7***
48.4 51.6
509.1 ___ 22.5 442.4 • 6.5** 321.4 • 30.2***
0 12.9 36.7
540 • 51.5 348.2 ~ 25.8*** 179.8 • 20.0***
0 53.5 66.7
237.2 • 164.7 •
53.3 65.6
121.6 - 10.0"** 56.8 --- 4.0***
77.5 89.5
Experiment I No addition Pilocarpine 1 mM Eserine 50 v.M Eserine 50 IxM plus ACh 100 IzM BW 284c55 1 ~M
Experiment 11 No addition Nicotine 10 ~M 1 mM Hemicholinium-3 1 ~M 10 IxM
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developmental profile pattern showed that [3H]QNB binding increased two fold at 6 DIV as compared to 0.5 DIV and that retinoic acid treatment does not change the binding capacity of the cells as compared to controls (Figure 4). Regulation of [3H]QNBBinding. The regulation of the muscarinic binding properties was investigated by the quantitation of the [3H]QNB binding capacity of the LA-N-1 cells grown in the presence of agonists and antagonists for either 4 or 24 hrs (Tables II and III). The results indicate that exposure of the control cells with pirenzepine, particularly for 4 hrs of incubation (Table II) apparently upregulated [3H]QNB binding. Exposure to atropine, carbachol and especially oxotremorine resulted in down regulation of [3H]QNB binding to varying ctegrees. The magnitude of these effects was lower in the RA treated cultures. No statistically significant difference was observed with cultures exposed to these agents for 24 hours except for the differentiated cells with carbachol and oxotremorine which resulted in down regulation of [3H]QNB binding (Table III). Second MessengersActivation. In an attempt to determine the nature of the coupling of the muscarinic binding in the LA-N-1 neurons to second messengers, the breakdown of phosphoinositides and the concentration of cAMP were measured after stimulation with cholinergic agonists. After 28 hrs of incubation with 44 I~M [3H]myoinositol the prelabeled LA-N-1 neurons were challenged
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DIV Fig. 4. Developmental profile of [3H]QNB binding in LA-N-1 cell membranes. At various days in vitro (DIV) control cells ( o - - o ) or RA-(10-SM) treated cells (o----.--~)were tested for [aH]QNB binding. RA was added from day 1. Each point represents the average of 4-12 determinations _ SD.
220
Sorrentino, Singh, Hubsch, Kanfer, Mykita, and Massarelli Table II. Regulationof QNB Binding in LA-N-1 Cells Grown in the Presence of Agonists and Antagonistsfor 4 Hours Control fmol/mg protein/hr No addition Atropine 10 ~M Pirenzepine 10 I~M Carbachol 1 mM Oxotremorine 1raM
504.4 354.5 760.7 233.8 184.6
• • • • •
Retinoic Acid (10-5M) % change
23.8 31.4'* 80.6** 18.1"** 24.0***
-30 +50 -54 -64
fmol/mg protein/hr 452.3 • 17.1 420.4 • 26.8 496.2 • 17.6 ND 400.5*
% change -7 + 10 -12
LA-N-1 cell cultures grown in the absence or presence of 10-5 M RA (4 DIV) were incubated with the agonists and antagonistsfor 4 hrs. Each value corresponds to the average of three determinations. * Average of two determinations;ND: not determined; 9* P < 0.05, *** P < 0.02.
Table III. Regulationof MuscarinicQNB Binding in LA-N-1 Cells Exposed to Agonists and Antagonistsfor 24 Hours i
A.
Control Retinoic Acid (i0-sMi fmol/mg protein/hr fmol/mgprotein/hr No addition Atropine 10 I~M % change Pirenzepine 10 I~M % change Carbachol 1 mM % change Oxotremorine 1 mM % change
CONTROL
i
528.2 -+- 202.0 268.6 --_ 136 - 49 511.4 • 421.6 - 3.2 449.5 • 100.8 - 15 322.6 • 215 - 39
570.1 • 34.0 362.0 • 172 - 36 377.7 • 105 - 34 445 • 50** - 22 321.7 • 105"** -44
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LA-N-1 cell culturesgrown in the absenceor presence of i0 -5 M RA (4 DIV) were incubatedwith the agonists and antagonistsfor 24 hrs. Each value is the average of three determinations.M mean of the three separate experiments • SD. ** P < 0.1; *** P < 0.005.
with 1 m M CCh, in the presence or absence of 12 IJ,M atropine for 20 rain at 37~ CCh greatly stimulated the appearance of phosphoinositides metabolites and this effect was completely abolished by the presence of atropine (Figure 5a). Retinoic acid treated cells showed a lesser CCh provoked PI breakdown, as compared to untreated ceils, however the effects of atropine were the same (Figure 5b). Experiments were undertaken to measure cAMP content after atropine, CCh or oxotremorine treatment. There were only slight but statistically significant changes caused by exposure to oxotremorine resulting in a decrease of cAMP levels in the untreated cells and increased levels in the retinoic acid treated cells (Table IV). These changes were however, not blocked by the presence of atropine. The magnitude of these changes were smaller in the CCh and atropine treated cultures. The RA treated cells had a 2.5-3 fold higher basal cAMP content than the untreated cells (Table IV).
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fraction number Fig. 5. Effect of carbachol on polyphosphoinositidebreakdown products in LA-N-1 neuroblasts. Cells were preincubatedwith [aH]myoinositol and incubatedwith lmM CCh (o----o), with 1 mM CCh plus 10 ~M atropine (e-----o) or no additions (A--A). PI degradation products were isolated on Dowex columns accordingto Brown et al. (17). A. Control cells. B. RA-(10-SM) treated cells. Each point represents the average of values obtainedfrom three petri dishes. DISCUSSION Five forms of muscarinic receptors have been characterized in the last few years based upon pharmacolog-
Muscarinic Binding Sites in Human Neuroblastoma Cell Line
221
Table IV. Effect of Agonists and Antagonists on the cAMP Content of the LA-N-1 Cells cAMP (pmol/mg protein)
% Control Carbachol 0.1 mM Carbachol 0.1 mM + Atropine 10 IxM Atropine 10 IxM Oxotremorine 0.1 mM Oxotremorine 0.1 mM + Atropine 10 ~M i
Control
change
3.44 - 0.06 3.32 --- 0.09
-3.5
% Retinoic Acid (10-5M) 8.30 --- 0.25 8.70 - 0.54
change +4.8
3.37 -+ 0.09 3.02 --- 0.09*** 3.11 • 0.08**
-2.0 -12.3 -9.6
8.55 -+ 0.27 8.47 --- 0.32 9.15 • 0.33**
+3.0 +2.0 +10.2
3.00 -+ 0.05***
-13.0
9.00 - 0.17"*
+8.4
i
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Membrane preparations of the cells grown in the absence or presence of 10 -5 M RA (4 DIV) were incubated in the presence of the agonist and/or of the antagonist during 30 rain. cAMP content was measured with the Amersham RIA Kit as explained in the Experimental Procedure section. Results expressed as mean • SD of four determinations. ** P < 0.01; *** P < 0.001.
ical differentiation. The use of pirenzepine, a drug selective for M1 receptors, AF-DX-116 which is selective for M2 receptors and 4-DAMP which is selective for M1 and M3 receptors have provided tools to distinguish these subtypes and to define their effector coupling properties. Muscarinic ligand binding may result in an increase of polyphosphoinositide degradation coupled to M1, M2 and M3 receptors and in a decreased cAMP synthesis coupled to M2 and M4 receptors (20). Several human neuroblastoma cell lines have been established in continuous culture. These cell lines are capable of synthesizing neurotransmitters, are morphologically similar to neuronal cells and can be induced to differentiate biochemically and morphologically upon treatment with various drugs (21). Retinoic acid causes inhibition of growth and enhanced morphological differentiation of human neuroblastoma cells in culture (2227). Muscarinic binding has been investigated with the catecholaminergic SK-N-SH neuroblasts, their derived neuronal clone SH-SY-5Y and the melanocyte clone SHEP1 (28,29). The latter showed a high affinity for 4DAMP and low affinities for pirenzepine and AF-DX116 suggesting the presence of M3 receptors. The present results demonstrate that the LA-N-1 clone, which is catecholaminergic, displays muscarinic receptor binding characteristics in contrast to clone LAN-2 which is cholinergic and does not display muscarinic binding but may display few nicotinic binding sites. The dissociation constant of [3H]QNB binding in these LA-N-1 ceils is similar to this type of binding in the nervous tissue. The differentiation of the neuroblasts induced by RA does not change the [3H]QNB binding when compared to the untreated cells. The effects of CCh, atropine, pirenzepine and oxotremorine upon
[ 3 H ] Q N B binding with the RA treated cells was different to that of untreated cells. Small displacements of the inhibition curves might indicate changes in affinity because of the morphological changes produced by RA in the number and extension of neurites rather than upon induction of receptors or their modification. The most striking observation concerned the coupling of the carbachol binding to the provoked degradation of polyphosphoinositides (Figure 5). A reduced formation of PI metabolites was observed in RA grown cells as compared to control cells (Figure 5) (see also 30,31). The other notable difference was the greater basal level of cAMP in the RA grown cells (Table IV). The present observations demonstrate that the LAN-1 cells do not seem to express M1 receptor properties based upon the low affinity of the pirenzepine binding. Such low affinity binding of pirenzepine suggested the possible presence of an M2 receptor but this usually inhibits cAMP formation, does not usually cause PI hydrolysis and is inhibited by AF-DX-116. The possibility remains that the LA-N-1 muscarinic receptor type might be related to an M3 receptor. It has been shown, in SKN-SH human neuroblasts, that the M3 receptor is coupled to PI breakdown (32). The inhibition by 4-DAMP and the lack of effect by AF-DX-116 support the possibility that LA-N-1 neuroblasts possess a muscarinic receptor of the M3 type similar to the SK-N-SH neuroblasts.
ACKNOWLEDGMENTS Supported by grants from the Medical Research Council of Canada, NATO and CNRS (Centre National de la Recherche Scientifique, France).
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Sorrentino, Singh, Hubsch, Kanfer, Mykita, and M a s s a r e l l i
REFERENCES 1. Ashkenazi, A., Peralta, E. G., Winslow, J. W., Ramachandran J., and Capon, D. J. 1989. Functionally distinct G proteins selectively couple different receptors to PI hydrolysis in the same cell. Cell 56:487-493. 2. Ashkenazi, A., Ramachandran J., and Capon, D. J. 1989. Acetylcholine analogue stimulates DNA synthesis in brain-derived cells via a specific muscarinic receptor subtypes. Nature 340:146150. 3. Balduini, W., Murphy, S. D., and Costa, L. G. 1987. Developmental changes in muscarinic receptor-stimulated phosphoinositide metabolism in rat brain. J. Pharmacol. Exper. Therap. 241:421-427. 4. Pierce, B., Cambray-Deakin, M., Morrow, C., Grimble, J., and Murphy, S. 1985. Activation of muscarinic and of alphal-adrenergic receptors on astrocytes results in the accumulation of inositol phosphates. J. Neurochem. 45:1534-1540. 5. Berridge, M. J. 1987. Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu. Rev. Biochem. 56:159193. 6. Hulme, E. C., Birdsall, N. J. M., and Buckley, N. J. Muscarinic receptor subtypes. Ann. Rev. Pharmacol. Toxicol. 30:633-673, 1990. 7. Pinkas-Kramarski, R., Edelman, R., and Stein, R. 1990. Indications for selective coupling to phosphoinositide hydrolysis or to adenylate cyclase inhibition by endogenous muscarinic receptor subtypes M3 and M4 but not M2 in tumor cell lines. Neurosci. Lett. 108:335-340. 8. Lambert, D. G., and Nahorskf, S. R. 1990. Muscarinic receptor mediated changes in intracellular Ca2§ and inositol 1,4,5-trisphosphate mass in a human neuroblastoma cell line, SH-SY5Y. Biochem. J. 265:555-562. 9. Kubo, T., Fukuda, K., Mikami, A., Maeda, A., Takahashi, H., Mishina, M., Haga, T., Haga, K., Ichiyama, A., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T., and Numa, S. 1986. Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature 323:411--416. 10. Peralta, E. G., Winslow, J. W., Peterson, G. L., Smith, D. H., Ashkenazi, A., Ramachandran, J., Schimerlik, M. I., and Capon, D. J. 1987 a. Primary structure and biochemical properties of an M2 muscarinic receptor. Science 236:600--605. 11. Bonner, T. I., Buckley, N. J., Young, A. C., and Brann, M. R. 1987. Identification of a family of muscarinic acetylcholine receptor genes. Science 237:527-532. 12. Bonner, T. I., Young, A. C., Brann, M. R., and Buckley, N. J. 1988. Cloning and expression of human and rat m5 muscarinic acetylcholine receptor genes. Neuron 1:403-410. 13. Peralta, E. G., Ashkenazi, A., Winslow, J. W., Smith, D. H., Ramachandran, J., and Capon, D. J. 1987 b. Distinct primary structure, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholinereceptors. EMBO J. 6:3923-3929. 14. Maeda, A., Kubo, T., Mishina, M., and Numa, S. 1988. Tissue distribution of mRNAs encoding muscarinic acetylcholine receptor subtypes. FEBS Lett. 239:339-342. 15. Singh, I., Sorrentino, G., McCartney, D. G., Massarelli, R., and Kanfer, J. N. 1990. Enzymatic activities during differentiation of the human neuroblastoma cells LA-N-1 and LA-N-2. J. Neurosci. Res. 25:476--485. 16. Roeske, W. R., Wang, J. X., Gulya, K., Korc, M., Sera, M.,
17. 18. 19. 20. 21. 22.
23. 24.
25.
26.
27.
28. 29.
30.
31. 32.
Ackerman, M., Mei, L., Vickroy, T., Watson, M., and Yamamura, H. I. 1987. Biochemical, functional and anatomic evidence for putative muscarinic receptor subtypes using selective antagonists [3H] AFDX-116 and [3H]pirenzepine. Cell. Mol. Basis Cholinergic Funct., Ed. Dowdal, M. J. and Hawthorne, J. N.: Chichester (England): Horwood, U.K., pp. 64-71. Brown, E., Kendall, D. A., and Nahorski, S. R. 1984. Inositol phospholipid hydrolysis in rat cerebral cortical slices: I. receptor characterization. J. Neurochem. 42:1379-1387. Lowry, O. H., Rosebrough, J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Barlow, R. B., and Shepherd, M. K. 1989. A further search for selective antagonists at M2-muscarinic receptors. Br. J. Pharmacol. 89:837-843. Sokolovsky, M. 1989. Muscarinic cholinergic receptors and their interaction with drugs. Adv. Drug Res. 18:431-509. Spengler, B. A., Ross, R. A., and Biedler, J. L. 1986. Differential drug sensitivity of human neuroblastoma cells. Cancer Treatment Rep. 70:959-965. Seeger, R. C., Rayner, S. A., Bannerjee, A., Chung, H., Lang, W. E., Neustein, H. B., and Benedict, W. F. 1977. Morphology, growth, chromosomal pattern and fibrinolytic activity of two new human neuroblastoma cell lines. Cancer Res. 37:1364-1371. Sidell, N. 1982. Retinoic acid-induced growth inhibition and morphologic differentiation of human neuroblastoma cells in vitro. J. Natn. Cancer Inst. 68:589-596. Spinelli, W., Sonnenfeld, K. H., and Ishii, O. N. 1982. Effects of phorbol esters tumor promoters and nerve growth factor on neurite outgrowth in cultured neuroblastoma cells. Cancer Res. 42:5067-5073. Recio-Pinto, E., and Ishii, O. N. 1984. Neurite outgrowth in cultured human neuroblastoma cells was induced by insulin, insulin-like growth factor I and nerve growth factor. Brain Res. 302:323-334. Rupniak, M. T., Rein, G., Powell, J. F., Ryder, T. H., Carson, S., Povey, S., and Hill, B. T. 1984. Characterization of a new human neuroblastoma cell line which differentiate in response to cyclic adenosine 3', 5'- monophosphate. Cancer Res. 44:26002607. Sidell, N., Sarafian, T., Kelly, M., Tsuchida, T., and Haussler, M. 1986. Retinoic acid-induced differentiation of human neuroblastoma: a cell variant system showing two distinct responses. Expl. Cell Biol. 54:287-300. Clementi, F., Cabrini, D., Gotti, C., and Sher, E. 1986. Pharmacological characterization of cholinergic receptors in a human neuroblastoma cell line. J. Neurochem. 47:291-297. Lambert, D. G., Ghataorre, A. S., and Nahorski, S. R. 1989. Muscarinic receptor binding characteristics of human neuroblastoma SK-N-SH and its clone SH-SY5Y and SH-EP1. Eur. J. Pharmacol. 165:71-77. Lanciotti, M., Lomgone, P., and Cornaglia-Ferraris Ponzoni, M. 1989. Retinoic acid inhibits phosphatidylinositol turnover only in RA-sensitive while not in RA-resistant human neuroblastoma cell lines. Biochem. Biophys. Res. Comm. 161:284-289. Ponzoni, M., and Lanciotti, M. 1990. Retinoic acid rapidly decreases phosphatidylinositol turnover during neuroblastoma cell differentiation. J. Neurochem. 54:540-546. Fisher, S. K., and Heacock, A. M. 1988. A putative M3 muscarinic cholinergic receptor of high molecular weight couples to phosphoinositides hydrolysis in human SK-N-SH neuroblastoma cells. J. Neurochem. 50:984-987.
Muscarinic Binding Sites in a Catecholaminergic Human Neuroblastoma Cell Line Giuseppe Sorrentino 1,4, Indrapal N. Singh 1, Alphonse Hubsch 2, Julian N. Kanfer ~,3, Serge Mykita 2, and Raphael Massarelli 2 (Accepted July 29, 1991)
Tyrosine hydroxylase (TH) a characteristic enzyme activity for the catecholaminergic clonal cell line LA-N-1 and choline acetyltransferase (CHAT) a characteristic enzyme activity for the cholinergic clonal cell line LA-N-2 were previously shown to be increased in these cells exposed to 10 -5 M retinoic acid (R_A) as differentiating agent. An investigation of the receptor characteristics suggests a complementarity between the two cell lines. The binding of QNB, a muscarinic ligand, was undetectable with the LA-N-2 cells but was present in the LA-N-1 cells and possessed a kD of 1.8 nM and 2.2 nM and a Bmax of 0.56 and 0.68 for control and RA grown cells respectively. There was a gradual increase in QNB binding to LA-N-1 cells from 2 days in vitro (DIV) until 6 DIV in both control and RA grown cells. An ICso of 2.5 x 10 -8 M and 0.9 x 10 -8 M for atropine inhibition was obtained for the control and RA grown cells respectively. The corresponding values for carbachol inhibition were 7 x 10 -z M and 3 x 10 -e M respectively. The inhibition by the agonist oxotremorine is comparable to that of carbachol and 1 mM pilocarpine inhibited the binding by 21%. QNB binding showed a low affinity for pirenzepine and for AF-DX-116 but was inhibited with a rather high affinity by 4-DAMP (IC5o:110 p~M) thus suggesting the presence of an M3 receptor. Acetylcholine (100 p~M) plus eserine (50 ~M) and BW284c55 (1 IxM), an acetylcholinesterase inhibitor, reduced the binding of QNB by approximately 25%. Nicotine (1 raM) caused a 36% reduction of binding and hemicholinium-3 (HC-3) (1 txM), an inhibitor of choline uptake, inhibited the binding by 53%. There was a down regulation of QNB binding observed with cells grown for 24 hours with either the antagonist atropine or the agonists carbachol or oxotremorine. Low amounts of c~-bungarotoxin (c~-BgTx) binding sites were barely detectable in both LA-N-1 and LA-N-2 cells. The LA-N-1 muscarinic receptor is coupled to polyphosphoinositide hydrolysis without increased cyclic AMP formation further suggesting its being an M~ receptor. KEY WORDS: LA-N-1 Cells; muscarinic receptor; human neuroblastomaclones; polyphosphoinositidebreakdown; cAMP. INTRODUCTION
proliferative capacity of these cells (1). Thus the treatment of glial ceils and primary neuronal cultures, or their corresponding established cell lines with cholinergic agonists such as carbachol (CCh) induces D N A synthesis only when the receptors linked to PI hydrolysis are ac-
The activation of muscarinic acetylcholine receptors (mAChRs) coupled to the hydrolysis of polyphosphoinositides (PI) in brain cells appears to influence the
a Address reprint requests to: Dr. J. N. Kanfer, Department of Biochemistry and Molecular Biology, University of Manitoba, Faculty
of Medicine, 770 Bannatyne Avenue, Winnipeg, Manitoba R3E 0W3, Canada. 4 Present address of Dr. G. Sorrentino: Istituto di Scienze Neurologiche, P Facoltfi di Medicina, Via Pansini 5, 80131 Napoli, Italy.
1 Department of Biochemistry and Molecular Biology, The University of Manitoba, Winnipeg, Manitoba R3E 0W3, Canada. z Centre de Neurochimie-Cronenbourg, BP20 CR, 67037 Strasbourg Cedex, France.
215 0364-3190/92/0300-0215506.50/09 1992PlenumPublishingCorporation
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Sorrentino, Singh, Ilubsch, Kanfer, Mykita, and Massarelli
tivated (2). Acetylcholine (ACh) stimulated PI hydrolysis by brain tissue is maximum at birth (3). It has been shown that CCh stimulates the PI hydrolysis in primary astroglial cells (4) and PI hydrolysis appears to be correlated with cell proliferation (5). Several different types of mAChRs designated M1 to M4 and perhaps M5 have been described to be present in mammalian tissues. In the CNS these receptors appear to be coupled to a number of biochemical effector systems resulting in a stimulation of PI turnover or an inhibition of cAMP production and activation of cGMP production (6). These responses have been observed with a variety of tissues, primary cell cultures and several cell lines of neuronal or of astroglial origin (7,8). It has been shown that the different receptors have different primary structures (9-11), different ligand binding properties (1,10,12,) and are expressed in tissue specific patterns
(12-14). We have previously investigated the developmental profile of several enzymatic activities in the human neuroblastoma clones LA-N-1 and "LA-N-2. Choline acetyltransferase (CHAT) activity of the LA-N-2 cells, a cholinergic clonal ceil line, increased gradually until 8 days in vitro (DIV) and then declined (15). In contrast acetylcholinesterase (ACHE) activity decreased at 8 DIV and was maintained at a low level. The activity of tyrosine hydroxylase (TH) in LA-N-1 cells, a catecholaminergic clone, gradually increased until 4 DIV and remained constant. The presence of 10 -5 M retinoic acid in the growth medium increased both of these synthetic enzymatic activities about two to three fold (15). It seemed useful based upon these observations to characterize the neurotransmitter receptor profile of these neuroblastoma cell lines under conditions of enhanced differentiation and to correlate the properties of receptor activation with effector-coupling.
Los Angeles, and their growth and maintenance as previously described (15). Binding of QNB. Ceils were detached and harvested by centrifugation at 500 g for 10 rain, resuspended in Leibovitz medium (L15) without serum and incubated for 30 rain at 37~ The cells were then centrifuged at 750 g for 10 min, the pellet was homogenized in a minimum volume of either 10 mM phosphate at pH 7.4 or 50 mM phosphate at pH 7.4. The incubation mixture, in a final volume of 100 Ixl, contained 0.3 mg of homogenate protein, varying concentrations of [3H]quinuclidinylhenzilate (QNB, specific activity: 41.5 Ci/mmol, Amersham) containing 210,000 dpms and 2 to 10 Ixl of 1 mM atropine sulfate and shaken at 37~ or 25~ for varying periods of time. Unless indicated otherwise the final routine concentration of [3H] QNB was 15 nM, that of atropine was 10 rzM and the time of incubation was 30 rain at 37~ The incubation was stopped by chilling the tubes in an ice bath followed by centrifugation at 10,000g for 5 min. The supematant was removed and the pellet resuspended in cold 10 mM phosphate at pH 7.4 and centrifuged again at 10,000 g for 2 rain. After removal of the supernatant the pellet was digested with 100 Ixl of 1 N NaOH, transferred to scintillation vials and the radioactivity determined with 10 ml of Scintiverse. Non specific binding was determined by measuring the radioactivity with homogenates incubated in the presence of 10 ~M atropine sulfate. Agonists and antagonists were included at the indicated concentrations during the incubations as indicated. Binding of Alpha-Bungarotoxin (a-BgTx). The binding of [tzsl] a-BgTx (specific activity: 200 Ci/mmol, Amersham) was carried out in incubations containing 0.1--0.75 mg of protein, 100 mM NaC1, 50 mM phosphate buffer at pH 7.4, and 5, 10 and 30 nM [lz5I] cx-BgTx at 37~ for 60 rain. Non specific binding was estimated with incubations containing 10 ~M d-tubocurarine. The assay procedure was the same as that employed for QNB binding.
Regulation of Muscarinic Binding by Agonists and Antagonists. To investigate the regulation of the muscarinic binding sites the LAN-1 cells were maintained in culture medium containing either the
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EXPERIMENTAL PROCEDURE Drugs and Other Chemicals. All radioisotopes and cAMP RIA (radioimmunoassay) kit were obtained from Amersham. Carbachol, 3quinuclidinyl benzilate (QNB), atropine, pirenzepine, d-tubocurarine, oxotremorine, scopolamine, nicotine, hemicholinium-3, aeetylcholine iodide, acetylcholinesterase inhibitor BW 284C55, pilocarpine, eserine, and retinoic acid were obtained from Sigma (St. Louis, MO, U.S.A.); and cell culture L-15 medium and heat-inactivated fetal calf serum were obtained from Flow Labs. AF-DX-116 [ll,2-(diethylamino)methyl-l-piperidinyl-acetyl-5, ll-dihydro-6H-pyrido-2, 3-b, 1,4 benzodiazepine-6-one and 4-DAMP (diphenylacetoxy-4-methylpiperidine methiodide) were obtained as gifts from Boehfinger Ingelheim (Canada) Ltd., Burlington, Ontario. Cell Cultures. The human neuroblastoma cell lines, LA-N-1 and LA-N-2 were obtained from Dr. R. Seeger, University of California,
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Muscarinic Binding Sites in Human Neuroblastoma Cell Line
217
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LOG (4-DAMP)
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Cyclic Adenosine 3',5' Monophosphate (cAMP) Quantitations. The cells were washed three times with 5 ml of phosphate saline buffer (PBS), 10 IxM, pH 7.4, extracted with 200 ILl of 1 N HCI04, scraped, transferred to Eppendorf tubes, sonicated and centrifuged in the cold. The supernatant was removed, neutralized with 3 M K~CO~, centrifuged at 5,000g for 15 min and the cAMP content measured according to the instructions accompanying the Amersham kit.
Quantitation of the Products of Pl's Hydrolysis. Control and RAtreated LA-N-1 cells were incubated for 24 hrs with 44 ~M [3H]myoinositoI (1 p,Ci/ml, specific activity: 14.2 Ci/mmol, Amersham). At
the end of the incubation the cultures were washed with PBS containing 10 rnM LiC1 and the cells were homogenized in 2 ml of water. The radioactive water soluble inositol containing compounds were separated on a Dowex (1 x 8) formate form column employing the methodology described by Brown et al. (17). Proteins were estimated using the classical procedure of Lowry et al. (18). Statistical analysis. Statistical significance was assessed using student's t-test.
RESULTS
QNB Binding. Scatchard plots o f the binding of [3H]QNB s h o w e d saturable b i n d i n g in L A - N - 1 cells with
218
Sorrentino, Singh, Hubsch, Kanfer, Mykita, and Massarelli
an apparent dissociation constant (KD) of 1.8 nM and a Bmax of 0.56 (Figure 1). The LA-N-1 cells had a slightly increased KD of 2.2 nM and a Bm,x of 0.68 (Figure 1) when grown in the presence of 10 -5 M retinoic acid (4 DIV). There was no detectable binding of [3H]QNB to the LA-N-2 cells. Using [125I] c~-BgTx as a ligand (30 nM), a binding capacity of 20.2 and 26.6 fmol/mg protein respectively for LA-N-1 and LA-N-2 control cells and 30.5 and 82.0 fmol/mg protein respectively for LAN-1 and LA-N-2 RA treated cells. Effects of Agonists and Antagonists. To further demonstrate the presence of muscarinic receptors in the LA-N-1 ceils the effect of varying concentrations of the antagonist atropine was analysed (Figure 2a). Hill plots of the dose response curve for atropine yielded an ICso of 2.5 x 10 -8 M for control cells and 0.9 x 10 -8 M for the retinoic acid treated celIs and a coefficient below 1.0. Similarly pirenzepine had a very low affinity for the receptor in both control and retinoic acid treated cells (Figure 2b). The agonist carbachol was also examined (Figure 3a) and a Hill plot yielded an ICso of 7 x 10 -2 M for control cells and 3 x 10 -2 M for RA treated cells. The Hill coefficient was 1.0 for control cells and 1.8 for RA grown cells. Oxotremorine showed a greater competition for the binding of [3H]QNB than carbachol (Figure 3b). Incubation of membranes with 0.05-10 t~M AF-DX-116 [11,2-(diethylamino)methyl- 1-piperidinylacetyl-5, 11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepine-6-one;], a selective inhibitor of M2 receptor binding, did not affect the binding of QNB (results not shown) while 4-DAMP (diphenylacetoxy-4-methylpiperidine methiodide) a compound with selectivity for M1 and M3 receptors (19) inhibited the binding with an apparent affinity of 110 I~M (Figure 2c). Survey of Agonists and Antagonists. In an attempt to characterize the type of muscarinic receptor, the effect of several agonists and antagonists on the [3H]QNB binding was examined. In general, the magnitude of the effects of these agents were consistently greater with the retinoic acid treated cells as compared to untreated cells. Pilocarpine caused an inhibition; eserine and acetylcholine together produced an inhibition. In the retinoic acid grown cells the specific acetylcholinesterase inhibitor BW 284c55 was a more potent inhibitor of [3H]QNB binding than eserine. A decreased [3H]QNB binding was also obtained with the nicotinic receptor agonist nicotine and hemicholinium-3, an inhibitor of choline uptake (Table I). The muscarinic receptor antagonists atropine and scopolamine, at 10 IxM concentration, caused complete inhibition (data not shown). These results are usual for muscarinic receptors.
Developmental Profile of [3H]QNB Binding Sites.
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It was previously demonstrated with LA-N-1 and LAN-2 cells that the levels of activities of several enzymes increased during growth in culture (15) and with growth in the presence of retinoic acid. It seemed of interest to quantify the [3H]QNB binding capacity as a function of time in cultures of LA-N-1 cells. The
Muscarinic Binding Sites in Human Neuroblastoma Cell Line
219
Table I. Agonists and Antagonists: Effect of Various Drugs on QNB Binding in LA-N-1 Cells Control
Retinoic Acid (10-~M)
fmol/mg protein/hr
% inhibition
fmol/mg protein/hr
% inhibition
470.0 • 15.3 372.6 • 19.3"** 422.0 _ 47.0
0 20.8 10.2
733.4 • 34.5 354.0 • 30.1"** 466.0 • 16.1'**
51.7 36.4
300.0 - 66.1"* 344.3 • 77
36.1 26.7
378.6 • 50.5*** 354.7 • 23.7***
48.4 51.6
509.1 ___ 22.5 442.4 • 6.5** 321.4 • 30.2***
0 12.9 36.7
540 • 51.5 348.2 ~ 25.8*** 179.8 • 20.0***
0 53.5 66.7
237.2 • 164.7 •
53.3 65.6
121.6 - 10.0"** 56.8 --- 4.0***
77.5 89.5
Experiment I No addition Pilocarpine 1 mM Eserine 50 v.M Eserine 50 IxM plus ACh 100 IzM BW 284c55 1 ~M
Experiment 11 No addition Nicotine 10 ~M 1 mM Hemicholinium-3 1 ~M 10 IxM
8.5*** 8.2***
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LA-N-1 cells grown in the absence or presence of 10 -s M RA (4 DIV) were incubated with [3H]QNB andthe various compounds for 30 min specified in Experimental Procedure section. Each value represents the average of three determinations _ SD. ** P < 0.005; *** P < 0.001.
developmental profile pattern showed that [3H]QNB binding increased two fold at 6 DIV as compared to 0.5 DIV and that retinoic acid treatment does not change the binding capacity of the cells as compared to controls (Figure 4). Regulation of [3H]QNBBinding. The regulation of the muscarinic binding properties was investigated by the quantitation of the [3H]QNB binding capacity of the LA-N-1 cells grown in the presence of agonists and antagonists for either 4 or 24 hrs (Tables II and III). The results indicate that exposure of the control cells with pirenzepine, particularly for 4 hrs of incubation (Table II) apparently upregulated [3H]QNB binding. Exposure to atropine, carbachol and especially oxotremorine resulted in down regulation of [3H]QNB binding to varying ctegrees. The magnitude of these effects was lower in the RA treated cultures. No statistically significant difference was observed with cultures exposed to these agents for 24 hours except for the differentiated cells with carbachol and oxotremorine which resulted in down regulation of [3H]QNB binding (Table III). Second MessengersActivation. In an attempt to determine the nature of the coupling of the muscarinic binding in the LA-N-1 neurons to second messengers, the breakdown of phosphoinositides and the concentration of cAMP were measured after stimulation with cholinergic agonists. After 28 hrs of incubation with 44 I~M [3H]myoinositol the prelabeled LA-N-1 neurons were challenged
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DIV Fig. 4. Developmental profile of [3H]QNB binding in LA-N-1 cell membranes. At various days in vitro (DIV) control cells ( o - - o ) or RA-(10-SM) treated cells (o----.--~)were tested for [aH]QNB binding. RA was added from day 1. Each point represents the average of 4-12 determinations _ SD.
220
Sorrentino, Singh, Hubsch, Kanfer, Mykita, and Massarelli Table II. Regulationof QNB Binding in LA-N-1 Cells Grown in the Presence of Agonists and Antagonistsfor 4 Hours Control fmol/mg protein/hr No addition Atropine 10 ~M Pirenzepine 10 I~M Carbachol 1 mM Oxotremorine 1raM
504.4 354.5 760.7 233.8 184.6
• • • • •
Retinoic Acid (10-5M) % change
23.8 31.4'* 80.6** 18.1"** 24.0***
-30 +50 -54 -64
fmol/mg protein/hr 452.3 • 17.1 420.4 • 26.8 496.2 • 17.6 ND 400.5*
% change -7 + 10 -12
LA-N-1 cell cultures grown in the absence or presence of 10-5 M RA (4 DIV) were incubated with the agonists and antagonistsfor 4 hrs. Each value corresponds to the average of three determinations. * Average of two determinations;ND: not determined; 9* P < 0.05, *** P < 0.02.
Table III. Regulationof MuscarinicQNB Binding in LA-N-1 Cells Exposed to Agonists and Antagonistsfor 24 Hours i
A.
Control Retinoic Acid (i0-sMi fmol/mg protein/hr fmol/mgprotein/hr No addition Atropine 10 I~M % change Pirenzepine 10 I~M % change Carbachol 1 mM % change Oxotremorine 1 mM % change
CONTROL
i
528.2 -+- 202.0 268.6 --_ 136 - 49 511.4 • 421.6 - 3.2 449.5 • 100.8 - 15 322.6 • 215 - 39
570.1 • 34.0 362.0 • 172 - 36 377.7 • 105 - 34 445 • 50** - 22 321.7 • 105"** -44
2000-
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ii
LA-N-1 cell culturesgrown in the absenceor presence of i0 -5 M RA (4 DIV) were incubatedwith the agonists and antagonistsfor 24 hrs. Each value is the average of three determinations.M mean of the three separate experiments • SD. ** P < 0.1; *** P < 0.005.
with 1 m M CCh, in the presence or absence of 12 IJ,M atropine for 20 rain at 37~ CCh greatly stimulated the appearance of phosphoinositides metabolites and this effect was completely abolished by the presence of atropine (Figure 5a). Retinoic acid treated cells showed a lesser CCh provoked PI breakdown, as compared to untreated ceils, however the effects of atropine were the same (Figure 5b). Experiments were undertaken to measure cAMP content after atropine, CCh or oxotremorine treatment. There were only slight but statistically significant changes caused by exposure to oxotremorine resulting in a decrease of cAMP levels in the untreated cells and increased levels in the retinoic acid treated cells (Table IV). These changes were however, not blocked by the presence of atropine. The magnitude of these changes were smaller in the CCh and atropine treated cultures. The RA treated cells had a 2.5-3 fold higher basal cAMP content than the untreated cells (Table IV).
E•,
20 fraction number
30
RETINOIC ACID 2000-
.~ 1500 I o ,ooo E
"~ 500o
i o
GP ,
?3 o
'b_o~ ,~.d \ ~b
"o-o 2~
i
30
fraction number Fig. 5. Effect of carbachol on polyphosphoinositidebreakdown products in LA-N-1 neuroblasts. Cells were preincubatedwith [aH]myoinositol and incubatedwith lmM CCh (o----o), with 1 mM CCh plus 10 ~M atropine (e-----o) or no additions (A--A). PI degradation products were isolated on Dowex columns accordingto Brown et al. (17). A. Control cells. B. RA-(10-SM) treated cells. Each point represents the average of values obtainedfrom three petri dishes. DISCUSSION Five forms of muscarinic receptors have been characterized in the last few years based upon pharmacolog-
Muscarinic Binding Sites in Human Neuroblastoma Cell Line
221
Table IV. Effect of Agonists and Antagonists on the cAMP Content of the LA-N-1 Cells cAMP (pmol/mg protein)
% Control Carbachol 0.1 mM Carbachol 0.1 mM + Atropine 10 IxM Atropine 10 IxM Oxotremorine 0.1 mM Oxotremorine 0.1 mM + Atropine 10 ~M i
Control
change
3.44 - 0.06 3.32 --- 0.09
-3.5
% Retinoic Acid (10-5M) 8.30 --- 0.25 8.70 - 0.54
change +4.8
3.37 -+ 0.09 3.02 --- 0.09*** 3.11 • 0.08**
-2.0 -12.3 -9.6
8.55 -+ 0.27 8.47 --- 0.32 9.15 • 0.33**
+3.0 +2.0 +10.2
3.00 -+ 0.05***
-13.0
9.00 - 0.17"*
+8.4
i
ii
Membrane preparations of the cells grown in the absence or presence of 10 -5 M RA (4 DIV) were incubated in the presence of the agonist and/or of the antagonist during 30 rain. cAMP content was measured with the Amersham RIA Kit as explained in the Experimental Procedure section. Results expressed as mean • SD of four determinations. ** P < 0.01; *** P < 0.001.
ical differentiation. The use of pirenzepine, a drug selective for M1 receptors, AF-DX-116 which is selective for M2 receptors and 4-DAMP which is selective for M1 and M3 receptors have provided tools to distinguish these subtypes and to define their effector coupling properties. Muscarinic ligand binding may result in an increase of polyphosphoinositide degradation coupled to M1, M2 and M3 receptors and in a decreased cAMP synthesis coupled to M2 and M4 receptors (20). Several human neuroblastoma cell lines have been established in continuous culture. These cell lines are capable of synthesizing neurotransmitters, are morphologically similar to neuronal cells and can be induced to differentiate biochemically and morphologically upon treatment with various drugs (21). Retinoic acid causes inhibition of growth and enhanced morphological differentiation of human neuroblastoma cells in culture (2227). Muscarinic binding has been investigated with the catecholaminergic SK-N-SH neuroblasts, their derived neuronal clone SH-SY-5Y and the melanocyte clone SHEP1 (28,29). The latter showed a high affinity for 4DAMP and low affinities for pirenzepine and AF-DX116 suggesting the presence of M3 receptors. The present results demonstrate that the LA-N-1 clone, which is catecholaminergic, displays muscarinic receptor binding characteristics in contrast to clone LAN-2 which is cholinergic and does not display muscarinic binding but may display few nicotinic binding sites. The dissociation constant of [3H]QNB binding in these LA-N-1 ceils is similar to this type of binding in the nervous tissue. The differentiation of the neuroblasts induced by RA does not change the [3H]QNB binding when compared to the untreated cells. The effects of CCh, atropine, pirenzepine and oxotremorine upon
[ 3 H ] Q N B binding with the RA treated cells was different to that of untreated cells. Small displacements of the inhibition curves might indicate changes in affinity because of the morphological changes produced by RA in the number and extension of neurites rather than upon induction of receptors or their modification. The most striking observation concerned the coupling of the carbachol binding to the provoked degradation of polyphosphoinositides (Figure 5). A reduced formation of PI metabolites was observed in RA grown cells as compared to control cells (Figure 5) (see also 30,31). The other notable difference was the greater basal level of cAMP in the RA grown cells (Table IV). The present observations demonstrate that the LAN-1 cells do not seem to express M1 receptor properties based upon the low affinity of the pirenzepine binding. Such low affinity binding of pirenzepine suggested the possible presence of an M2 receptor but this usually inhibits cAMP formation, does not usually cause PI hydrolysis and is inhibited by AF-DX-116. The possibility remains that the LA-N-1 muscarinic receptor type might be related to an M3 receptor. It has been shown, in SKN-SH human neuroblasts, that the M3 receptor is coupled to PI breakdown (32). The inhibition by 4-DAMP and the lack of effect by AF-DX-116 support the possibility that LA-N-1 neuroblasts possess a muscarinic receptor of the M3 type similar to the SK-N-SH neuroblasts.
ACKNOWLEDGMENTS Supported by grants from the Medical Research Council of Canada, NATO and CNRS (Centre National de la Recherche Scientifique, France).
222
Sorrentino, Singh, Hubsch, Kanfer, Mykita, and M a s s a r e l l i
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