Journal of Neurochemislry Raven Press, Ltd., New York 0 1992 International Society for Neurochemistry

Lan-1: A Human Neuroblastoma Cell Line With M, and M, Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+Channels Activated by Membrane Depolarization A. Fatatis, A. Bassi, M. R. Monsurrh, G. Sorrentino, *G. D. Mita, G. F. Di Renzo, and L. Annunziato Section of Pharmacology, Department of Science of Human Communication, 2nd School of Medicine, University of Naples “Federico II’l and *IIGB (CNR), Naples, Italy

Abstract: The LAN- 1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1- 1,000 p M ) caused a dose-dependent increase of the intracellular free Ca2+concentration ([Ca2+Ii).This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+reduced the magnitude of the peak phase to -70% but completely abolished the plateau phase. The muscarinic-activated Ca2+channel was gadolinium (Gd3+) blockable and nimodipine and w-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca”], . The CCh-induced [Ca2+], elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpipendine methiodide, two rather selective antagonists of MI and M, muscarinic receptor subtypes, respectively,whereas methoctramine, an M, antagonist, was ineffective. The coupling of MI and M, receptor activation with [Ca2+Iielevation does not seem to

be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca”], elicited by MI and M, muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitiveintracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+Iimobilization, and, finally, LAN- I cells appear to lack caffeinesensitive Ca2+stores, because the methylxanthine was unable to elicit intracellular Ca2+mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 p M ) , or after thapsigargin. Key Words: LAN- 1 cells-Neuroblastoma cells-MI, M,, and M3 muscarinic receptor subtypes-Intracellular Ca2+ concentration-Ca2+ channels. Fatatis A. et al. LAN- 1: A human neuroblastoma cell line with MI and M, muscarinic receptor subtypes coupled to intracellular Ca2+elevation and lacking Ca2+channels activated by membrane depolarization. J. Neurochern. 59, 1-9 ( 1992).

LAN-1 is a cell line derived from a human bone marrow metastasis of a neuroblastoma, producing vanillyl mandelic and homovanillic acid. These cells contain large amounts of tyrosine hydroxylase (1 5 times more than that found in the brain) and have multiple cytoplasmic dense cores (Seeger et al., 1977), such as those present in sympathetic neurons, pheochromocytomas, and other neuroblastoma cells. In these cells the existence of muscarinic receptors

involved in breakdown of polyphosphoinositides and in elevation of the intracellular free Ca2+concentration ([Ca”],) has been previously demonstrated (Pozzan et al., 1986; Fisher and Heacock, 1988; Lambert and Nahorski, 1990). The availability nf fluorescent dyes like fura-2 has allowed the measurement of variations in [Ca2+Iiboth in cell suspension and in singlecell preparations (Tsien and Poenie, 1986; Malgaroli et al., 1987).

Received July 3, 1991; revised manuscript received October 22, I99 I ;accepted December 2, 1991. Address correspondence and reprint requests to Prof. L. Annunziato at Section of Pharmacology, Department of Science of Human Communication, 2nd School of Medicine, University of Naples “Federico 11,” Via Pansini 5 , 8013 1 Napoli, Italy. Abbreviations used: 8-Br CAMP, 8-bromo cyclic A M P [Ca2’],,

intracellular free Ca2+concentration; CCh, carbachol: w-CgTX, wconotoxin: 4-DAMP, 4-diphenylacetoxy-N-methylpipendinemethiodide: G protein, guanine nucleotide-binding protein; IP,, inositol 1,4,5-trisphosphate; PK-C, protein kinase C: PTX. pertussis toxin: TMB-8, 8-(N,N-diethylamino)octyl 3.45-trimethoxybenzoate hydrochloride: TPA. phorbol L 2-myristate 13-acetate: TTX, tetrodotoxin.

1

2

A. FATATIS ET AL.

The purpose of the present study was to investigate the existence of muscarinic receptors coupled to an elevation of [Ca2+], in LAN-1 neuroblastoma cells and to identify which subtype of muscarinic receptors is involved. The effect of the cholinergic receptor agonist carbachol (CCh) in the presence of selective antagonists of the different muscarinic receptor subtypes M,, M,, and M3 (Doods et al., 1987; Bonner, 1989; Hulme et al., 1990) was investigated. In addition, the contribution of extracellular Ca2+ entrance to the [Ca2+],elevation following muscarinic receptor activation and the possible pharmacological sensitivity of the Ca2+channel to organic and inorganic blockers was evaluated. Because it was previously reported that veratridine induces a 22Na+uptake in LAN-1 cells (West et al., 1977), the hypothesis that Ca2+channels can be activated by membrane depolarization was verified. To clarify some of the transduction mechanisms that link muscarinic receptors to [Ca2+],modification, the possible involvement of a pertussis toxin (PTX)-sensitive guanine nucleotide-binding protein (G protein) or of the diacylglycerol-protein kinase C (PK-C) system was examined. Finally, the mobilization of intracellular Ca2+in LAN-1 cells from inositol 1,4,5-trisphosphate (IP,)-sensitive and -insensitive stores was explored using compounds that interfere with these two distinct compartments (Wakui et al., 1990).

MATERIALS AND METHODS Cell culture The LAN- I human neuroblastoma cell line was cultured as a monolayer in polystyrene dishes and maintained in Dulbecco's modified Eagle's medium (Flow Laboratories, Milan, Italy) containing 15% heat-inactivated fetal calf serum, 50 IU of penicillin/ml, 50 fig of streptomycin/ml, and 2.5 pg/ml of amphotericin B (Fungizone). Cells were grown in a humidified incubator at 37°C in a 5% CO, atmosphere and were fed twice a week. All experiments were performed with cells from passages 72-8 1.

[Ca*+],measurements Ftira-2 loading. Just before the experiment, LAN- 1 cells were detached by gently streaming the culture medium on the surface of the monolayers, centrifuged, and resuspended in a balanced salt solution of the following composition: 159 mMNaCl,5 mMKCI, 1.2 W M g C I , , 10 mMHEPES, 10 mM glucose, 1.2 mM CaCI,, and 0.2% bovine serum albumin (osmolality, 320 mOsmol/kg), pH adjusted to 7.4 with 1 M Tris. For single-cell experiments, LAN-1 cells were grown on 22-mm clean no. 1 glass coverslips, previously coated with collagen (Vitrogen) and poly-l-lysine (10 Kg/ ml), and transferred in a 35-mm-diameter plastic petri dish (Falcon). Cells were then incubated for 30 min at room temperature with 5 p A 4 fura-2 acetoxymethyl ester (Molecular Probes, Junction City, OR, U.S.A.). After the loading period, the medium was diluted with 2 volumes of the same balanced salt solution, incubated at room temperature for 10 min, and then washed twice before the experiment was performed.

J. Neurochern.. V d 59, No 1. 1992

CeN suspension experiments. [Ca2+],was measured in a 2-ml suspension of LAN-I cells at room temperature in a quartz cuvette equipped with a magnetic stirrer bar. Fura-2 fluorescence was monitored in a Perkin-Elmer model LS5B spectrofluorimeter. The excitation wavelength was 340 nm, with the emission at 490 nm. Calibration of the fluorescent signal was performed according to the method of Grynkiewicz et al. (1985). by means of the following calibration equation for a dye using intensity values at one wavelength:

[Ca2'li

K D

X

( F - Frnin)/(Frnax

-

F)

where K,, is 224 M,Fmin is the fluorescence of fura-2 after cell lysis induced by 0.1% Triton X-100 in the presence on 4 is the fluorescence of fura-2 m M external EGTA, and Fmax after its complete saturation with Ca2+(addition of 10 mM CaCI,). Single-cell experimem. Coverslips were mounted on a coverslip chamber for fluorescence measurements. All measurements were made at room temperature (22°C). Cytoplasmic fluorescence appeared uniform throughout the cells. Fura-2 fluorescence was imaged with an inverted Nikon Diaphot microscope using a Nikon 40X/ l .3 NA Fluor DL objective lens. The cells were illuminated with a xenon lamp (Osram, F.R.G.) with quartz collector lenses. A shutter and a filter wheel containing the two different interference filters (340 and 380 nm) were controlled by a computer. Emitted light was passed through a 400-nm dichroic mirror, filtered at 490 nm, and collected by a CCD camera connected with a light intensifier (Photonic Sciences Ltd., Sussex, U.K.). Images were digitized and averaged in an image processor (Joyce-Loebl Ltd., Dukesway Gateshead, U.K.) connected to a computer equipped with Tardis software. For the calibration of fluorescent signals, we used LAN-1 cells loaded with fura-2; R , and Rminare ratios at saturating and 0 [Ca"li, respectively, and were obtained by perfusing cells with a salt solution containing 15 mMCaCI, and 5 p M digitonin and subsequently with a Ca2+-freesalt solution containing 20 mMEGTA. The values of the obtained R,,, and Rmin,expressed as mean gray level, were used to calculate the calibration curve using the Tardis software. The [Ca2+],was determined according to the equation of Grynkiewicz et al. ( 1 985).

Materials Drugs were obtained as follows: CCh, atropine, tetrodotoxin (TTX), veratndine, staurosporine, poly-l-lysine, PTX, phorbol 12-myristate 13-acetate (TPA), and w-conotoxin (w-CgTX) were purchased from Sigma. Forskolin, Bay K 8644, and 8-bromo cyclic CAMP (8-Br CAMP)were purchased from Calbiochem. Thapsigargin was purchased from LC Service (Woburn, MA, U.S.A.).Nimodipine was obtained through the courtesy of Dr. Garthoff (Bayer, Wuppertal, F.R.G.) and verapamil through the courtesy of Dr. Kretzschmar and Dr. Scherrer (Knoll, Ludwigshafen, F.R.G.). Gadolinium (Gd3') and 8-(N,N-diethylamino)octyl 3,4,5-tnmethoxybenzoate hydrochloride (TMB-8) were purchased from Aldrich. 4-Diphenylacetoxy-N-methylpipendinemethiodide (4DAMP) and methoctramine were the generous gifts of Prof. Marchi (Department of Pharmacology, University of Genova, Genova, Italy). Pirenzepine was obtained through the courtesy of Dr. Ladinsky (De Angeli, Milano, Italy). Fura-2

M , AND M3 RECEPTORS AND [Ca2+IiIN LAN-1 CELLS acetoxymethyl ester and ryanodine were purchased from Calbiochem (San Diego, CA, U.S.A.).

[Ca++

3

li (nM)

RESULTS Effects of CCh stimulation on intracellular Ca" levels The muscarinic agonist CCh in concentrations ranging from 1 pMto 1 M c a u s e d a dose-dependent elevation of basal [Ca2+]levels (Fig. 1). This increase was characterized by an early phase that peaked in 10 s; this phenomenon was followed by a delayed decline, which reached a lower plateau phase in -60120 s, regardless of the level of the muscarinic agonist present. When extracellular Ca2+was removed, CCh still evoked a concentration-dependent increase in [Ca2+Ii;however, the magnitude of the peak phase was reduced to -70% (Fig. 2). The time required to reach the peak (10 s) was the same in both the presence and the absence of extracellular Ca2+.The removal of extracellular Ca2+completely abolished the plateau phase in a manner that converted the biphasic CCh response into a monophasic peak. Furthermore, when 1.2 mM Ca2+was added back to the medium, the plateau phase reappeared with the same lag phase (60 s) that had been detected when Ca2+was present from the beginning. [Ca++li

nM

225 91 -

t

1

p M CCh

519 78 -

$4-t

10 p M C C h 875 -

84 -

86-

t

CCh

552 385

92-

4 LJ

FIG. 2. Effect of 100 FM CCh on [Ca2'Ii in the presence (A) or in the absence (8) of extracellular Ca2+. In the experiments performed in the absence of extracellular Caz' (B), cells were preincubated for 5 min in a Ca*+-free,1 mM EGTA-containingmedium before CCh addition. CaCI, (1.2 mM) was added where indicated by the arrow.

In the single-cell experiments, a similar pattern of response was observed in three different LAN-1 cells (Fig. 3). Effects of inorganic and organic Ca2+channel blockers on the [CaZ+liincrease elicited by CCh Gd3+,an inorganic Ca2+entry blocker (Docherty, 1988), completely abolished the plateau phase of the [Ca2+Iielevation and reduced the magnitude of the peak phase to -60% when added 60 s before 100 pM CCh. It is interesting that if Gd3+was added after the occurrence of the peak phase and during the plateau phase, a fast return (45-60 s) of [Ca2+],to basal levels took place (Fig. 4). Among the organic Ca2+entry blockers, verapamil did not affect the [Ca2+],rise induced by 100 pLMCCh at a concentration of 1 pM, indeed, at a 30 pM concentration, it caused a 55% reduction of the peak and plateau phase. If verapamil(30 p M ) was added during [Ca++Ii

nM

t

100 p M C C h

lO00-l

855 -

95-

t

1 m M CCh FIG. 1. Concentration-dependenteffect of CCh on [Ca"], in fura2-loaded LAN-1 cells. Each trace is representativeof four experiments. The mean -e SEM basal value was 90 f 4 nM (n = 37). The mean -+ SEM peak value after CCh addition was as follows: 1 pM, 205 f 7 nM; 10 pM, 593 & 92 nM; 100 gM, 876 f 39 nM; and 1 mM, 834 f 40 nM. All peak values were statistically significant when compared with corresponding basal values (p < 0.01).

1

t

CCh

--

250

0

Seconds

FIG. 3. Effect of 100 pM CCh on [Ca"], in single-cell experiments with three different LAN-1 cells (traces A-C). Data points were taken every 4 s.

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A. FATATIS ET AL.

4

80

-

t

CCh

-

59594

m

et al., 1990), dose-dependently inhibited the [Ca2+Ii rise evoked by CCh. It is interesting that when pirenzepine ( 10 p M ) or 4-DAMP ( 1 p M ) was added during the CCh-induced plateau phase, the intracellular Ca2+ levels soon declined to basal values (Fig. 7). Moreover, the M, antagonist methoctramine, added before CCh stimulation at a 1 pA4 concentration, was not able to counteract the [Ca2+],rise (data not shown).

Gdl t

CCh

3 rnin

FIG. 4. Effect of Gd3+(30 1.M)addition on [Ca2+],,after (A) and before (B) exposure to 100 pM CCh. Data are from a single experiment typical of three others performed in suspended LAN-1 cells.

the plateau phase, [Ca2+Iideclined but did not reach the basal values. The dihydropyridine nimodipine (0.1-1 p M ) , another organic Ca2+entry blocker that has been demonstrated to interfere with the L-type Ca2+channel (Miller, 1987; Tsien et al., 1988), did not counteract the Ca2+influx after addition of CCh to LAN-1 cells; however, at a 1 pM concentration, only a small effect occurred either on the basal Ca2' level or on the plateau phase. This effect was probably due to some interference with the fura-2 signal (Fig. 5). Finally, o-CgTX, a marine toxin able to block the N-type Ca2+channel (McCleskey et al., 1987; Pin and Bockaert, 1990),did not interfere with the [Ca2+],rise when added at a 1.5 pLM concentration 30 min before CCh stimulation (data not shown). Effects of depolarizing stimuli on [Ca2+], in LAN-1 cells Elevated concentrations of K+, capable of depolarizing several excitable cells (Di Virgilio et al., 1987; Thayer et al., 1988),did not change [Ca2+Iiin LAN-1 neuroblastoma cells; similarly, veratridine (40 p M ) was unable to promote a [Ca2+],increase in LAN-1 cells. Furthermore, no K+-dependent [Ca2+Iiincrease was observed even though the cells were pretreated with the Ca2+channel activator Bay K 8644 (100 nM) or with 25 pM forskolin or 2 mM 8-Br CAMP. In addition, the selective Na+ channel blocker TTX did not prevent the [Ca2+Iiincrease evoked by the cholinergic agonist CCh. All the LAN-1 cells were responsive to the further addition of CCh (Fig. 6).

Effects of PTX, staurosporine, and TPA on the elevation of [Ca2+],evoked by CCh If LAN- 1 neuroblastoma cells were preincubated for 18 h in the presence of 20 ng/ml of PTX, the [Ca2+Iirise induced by CCh was completely unaffected. The inhibition of PK-C by adding staurosporine (20 and 100 nM)for 2 h before performing the experiment (Yanagihara et al., 1991) or the activation by 100 nM TPA for 20 min did not interfere with the mechanisms leading to the [Ca2+Iirise on CCh treatment (Fig. 8). Effect of compounds interfering with IP,-sensitive and -insensitive intracellular Ca2+stores TMB-8, a compound able to interfere with Ca2+ release from IP,-sensitive intracellular stores (Clapper and Lee, 1985; Schumaker and Sze, 1987; Palade et al., 1990)when used at 1 and 10 pMconcentrations in [Ca++li 880

nY

-

392 87 -

920a95 -

&&

t CCh

t CCh

t

t CCh

90 -

CCh 900 840 -

86 -

Cdh

Effects of putative antagonists of muscarinic receptor subtypes on CCh-induced elevation of [Ca2+liin LAN-1 cells Atropine, the muscarinic antagonist that does not discriminate among the different receptor subtypes, completely abolished [Ca2+Iielevation when added at a concentration of 1 pMbefore 100pMCCh (data not shown). Pirenzepine (0.1- 10 p M ) , a selective antagonist of the M, muscarinic receptor subtype, and 4DAMP ( 100 nM- 1 pM), a relatively selective M3 receptor antagonist (Fisher and Heacock, 1988; Hulme J. Neurochcm.. Val. 59, No. I , 1992

1BI

CCh

IDI

-

888 858 -

81

t CCh

t CCh

3 min

FIG. 5. Effect of the organic Ca2+entry blockers verapamil (VER; 1 or 30 1.M)and nimodipine (NIM; 0.1 or 1 pM), added to suspended LAN-1 cells before (A and C) and after (B and D) 100 f l CCh. Data are from a single experiment typical of two others.

M , AND M3 RECEPTORS AND [Ca’+/, IN LAN-1 CELLS [Ca+*Ii

[Ca*+Ii

nM

nM

1

CCh

975

FK

-

904

CCh

-

L

FIG. 6. Effect of different depolarizing conditions on [Ca”], in suspended LAN-1 cells. Cells were treated with 55 mM KCI and 40 pM veratridine. Before 55 mM KCI, cells were also pretreated with 25 pM forskolin, 2 mM 8-Br CAMP, or 100 nM Bay K 8644. All cells were subsequently stimulated with 100 pM CCh. Pretreatment with 1 pM TTX before 100 f l CCh addition was also performed.

55mM KCI

Veratridine 98

t

CLh

CCh

TTX

-

85

5

845

-

78

-

t CCh

__r t

t

BAY K 8644

CCh

I

3 rnin

experiments performed after extracellular Calf removal, inhibited the [Ca2+Iielevation induced by CCh by 66 and 80%, respectively. When LAN-1 neuroblastoma cells were treated with TMB-8 at a 100 puM dose before CCh addition, the [Ca2+],rise was completely prevented. Conversely, 10 pkfryanodine, a blocker of Ca2+-induced Ca2+release from IP,-insensitive intracellular [Ca++Ii 880

57.3

IAl

nM

-

stores (Thayer et al., 1987a, 1988),did not prevent the [Ca2+Iielevation evoked by muscarinic receptor activation when preincubated for 5 and 30 rnin (Fig. 9A). Analogously, in single-cell experiments, the methylxanthine caffeine, at a concentration of 40 mM, failed to elicit a [Ca”], rise in LAN-1 cells both under resting conditions and after CCh stimulation (Fig. 9B). Even though caffeine was added in conjunction with a subthreshold dose of CCh (300 nM),in the absence of extracellular Ca2+,no mobilization of Ca2+from intracellular stores was detected (Fig. IOA). Finally, 1 p k f thapsigargin, a compound that inhibits endoplasmic reticulum Ca2+-ATPase,caused a [Ca2+],elevation, which was followed by a sustained Ca2+level; the subsequent addition of 40 mMcaffeine [Ca++Ii

Pirenzepine

nM

(BI 1068-

I

108 Ckh

CCh

4-DAMP 3 min

FIG. 7. Dose-response curve of pirenzepine (A) and 4-DAMP (6) on the 100 pM CCh-induced [Ca2’li increase in suspended LAN-1 cells. Drugs were also added at the highest concentration during the plateau phase of the CCh-induced [Ca”], increase. Trace c, control.

-

-

t CCh

3 min

FIG. 8. Effect of TPA pretreatment on the CCh-induced [Ca*+], increase. Suspended LAN-1 cells were treated with 100 nM TPA 10 rnin before CCh addition. Data are from a single experiment typical of two others.

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A . FATATIS ET AL.

6

[Ca++Ili n Y

*Ay:A

7234 11327--

FIG. 9. Effect of TMB-8 (N), ryanodine, and caffeine on intracellular Ca2+ release from different stores. A Doseresponse curve of TMB-8 on the [Ca2+], increase evoked by 100 pM CCh (left) and effect of 10 pM ryanodine, added 30 rnin before 100 N CCh addition in a Ca2+-free medium, on [Ca2+],in suspended LAN-I cells (right). B: Effect of 10 mM caffeine addition to LAN-1 cells studied in a single cell. Extracellularmedia were changed as indicated. CCh was added as indicated by arrows. Trace c, control.

85

R yanodine

TYB-8

-

-4

t

t

CCh [Ca++Ii

3 min

CCh

nY

B45-

,

1O;Y-

Caffeine 92 -

+ca*'

-

-

Ca**free

t

t

1Om.M Caffeine

CCh

I

I

minutes

0

did not elicit a further increase of [Ca"], or any oscillatory pattern (Fig. IOB). DISCUSSION The results of the present study showed the existence of muscarinic receptors on LAN-1 neuroblastoma cells, whose activation leads to an increase of [Ca2+Ii.A mobilization of Caz+ from intracellular stores after CCh addition has already been reported in other neuroblastoma cell lines like SH-SYSY (Sher et al., 1988; Lambert and Nahorski, 1990), pheochromocytoma cells (Pozzan et al., 1986), and forebrain neurons (Reynolds and Miller, 1989). This phenomenon appears to be related to muscarinic receptor activation, which induces IP, formation, through the breakdown of polyphosphoinositides (Ashkenazi et al., 19896).The elevation of [Ca2+],elicited by muscarink activation in LAN- 1 cells is dependent on both Ca2+mobilization from intracellular stores and a Ca" influx from the extracellular space. Ca2+influx also contributes to the magnitude of the peak phase, because in the absence of extracellular Ca2+the peak phase was reduced to 70%.It is interesting that during the peak phase, Ca2+entrance from the extracellular space and Ca2+ release from the internal stores occurred in the same interval ( 10 s). This result supports the proposal that a common biochemical pathway promotes, at the same time, both extracellular Ca2+ entry and Caz+ release from intracellular stores. In this regard, it is noteworthy that IP, ,a second messenger produced after M, and M, muscarinic receptor stimulation (Ashkenazi et al., 1989~;Lambert and Nahorski, 1990) when added to the cytoplasmic site of chromaffin cells, opens Ca" channels, functioning J. Neurochem., Vol. 59, No. 1. 1992

19

as a Ca2+ channel opener (Mochizuki-Oda et al., 1991). In addition, the entrance of extracellular Ca2+ through the Ca2+channels, which follows muscarinic receptor stimulation, also exerts a predominant role nM

[Ca++li

-

700 -

A

and 0.3Faileine Y CCh

1 0COCphM I

1

85-

I

Ca++

0

[Ca++li

820

78

'12,

1

free

minutes

I 6

nM

El

-

-

Caffeine

0

minutes

22

FIG. 10. A Effect of simultaneous addition of 40 mM caffeine and a subthresholddose of CCh (0.3 pM) in the absence of extracellular Ca2+in singlecell experiments in LAN-1 cells. A stimulatory concentration of CCh (100 p M ) was added 3 min after CCh plus caffeine addition. B Effect of 40 mM caffeine on the 1 pM thapsigargin-induced[Ca"], increase. Caffeine was added at min 10 after thapsigargin addition.

M , AND M,, RECEPTORS AND [Ca2+IiIN LAN-1 CELLS on the maintenance of the plateau phase, because the removal of the bivalent cation or the blockade of the Ca2+ channel by Gd3+ completely abolished the plateau phase. Similarly, Lambert and Nahorski (1990) found that in SH-SYSY neuroblastoma cells Ca2+ channels activated by muscarinic stimulation are sensitive to nickel. Furthermore, muscarinic-activated Ca2+channels in LAN-1 cells appear to be insensitive to the organic Ca2+entry blockers nimodipine and w-CgTX, which are able to block the L-type and N-type Ca2+channels (Hurwitz, 1986; Thayer et al., 1987b; Pin and Bockaert, 1990). At variance with this observation, Reynolds and Miller ( 1989) found that in forebrain neurons, a Ca2+channel activated by muscarinic receptor stimulation is sensitive to the blockade of the dihydropyridine nimodipine. In addition, these brain Ca2+ channels seem to be voltage sensitive and are activated by a cascade of events that initially involve the blockade of the outward K+ channels, the activation of voltage-sensitive Na+ channels, and then voltage-sensitive Ca2+channel opening. In LAN- 1 cells this mechanism does not seem to be operative because TTX did not prevent the [Ca2+Iielevation induced by CCh. These findings indicate that LAN- 1 neuroblastoma cells possess muscarinic receptor-activated Ca2+channels, which are unaffected by compounds able to block L-type and N-type voltagesensitive Ca2+channels. These channels are only partially blocked by verapamil at a rather elevated concentration of 30 pM. A similar effect of organic Ca2+ channel entry blockers has already been reported in the CNS (Annunziato et al., 1986). Moreover, two depolarizing stimuli-the Na+ channel activator veratridine and high extracellular K+ concentrations-were unable to induce an increase of [Ca2+Ii.Furthermore, even when LAN-1 cells were pretreated with a selective Ca2+channel activator like Bay K 8644 or with compounds like forskolin or 8-Br CAMP, which through the increased availability of cyclic AMP facilitate Ca2+ channel opening (Hess et al., 1984; Levitan, 1988), depolarizing concentrations of K+ were unable to elicit a [Ca2+],increase. These results demonstrate that LAN1 cells do not show any Ca2+ influx on membrane depolarization; this suggests that in these cells voltagesensitive Ca2+channels could be absent or not activatable. Another fact that emerges from the present work is the existence of MI and M, muscarinic receptors coupled to [Ca2+],elevation in LAN- I neuroblastoma cells. In fact, pirenzepine and 4-DAMP, two rather selective receptor antagonists of the M, and M, subtype, respectively, dose-dependently counteracted the [Ca2+],increase induced by the cholinergic agonist CCh. However, it should be mentioned that if each of the two MI and M, antagonists was used individually, at the highest dose a complete blockade of the [Ca2+Ii increase was observed. This result could be inter-

7

preted either as a consequence of the blockade of a single population of receptors with mixed M, and M, properties or as a loss of receptor specificity of the antagonists when used at elevated concentrations. Furthermore, methoctramine, a putative receptor antagonist of the M, cardiac subtype (Hulme et al., 1990), failed to prevent CCh-induced [Ca2+],elevation. The receptor antagonist study also revealed that the occupation of the M, and M, receptors with the agonist is not only responsible for the occurrence of the peak phase, but also for the maintenance of the late plateau period. In fact, if pirenzepine or 4-DAMP was added during the plateau phase, [Ca2+Iisuddenly returned to basal levels. Because it has been widely demonstrated that, in several cell types, the stimulation of MI and M, receptors is coupled to the phosphatidylinositol bisphosphate-phospholipase C pathway, via G proteins, which can be either sensitive or insensitive to PTX (Ashkenazi et al., 1 9 8 9 ~;Downes, 1989; Hulme et al., 1990),LAN- 1 cells were preincubated in the presence of PTX before cholinergic receptor stimulation was performed. The results obtained suggested that LAN-1 cells have M I and M, receptors that are not modulated by a PTX-sensitive G protein. On the other hand, because the breakdown of polyphosphoinositides elicited by MI and M, receptor activation leads also to the formation of the PK-C activator diacylglycerol (Kaczmarek, 1987; Huang, 1989; Shearman et al., 1989), it was interesting to verify if PK-C is involved in the modulation of the muscarinic receptor-activated Ca2+ channel. The data showed that the inhibition of PK-C with staurosporine or its activation with TPA does not interfere with the CChinduced [Ca2+],increase, suggesting that the M I and M, receptor-mediated [Ca2+Iiincrease does not require an involvement of PK-C. It is now well demonstrated in several cell types that on M, and M, receptor activation, the polyphosphoinositide breakdown leads to Ca2+ mobilization from an IP,-sensitive intracellular store localized into the endoplasmic reticulum (Ashkenazi et al., 19896). Even in LAN-1 cells, the Ca2+release from internal stores induced by MI and M, receptor stimulation seems to be dependent on an IP,-sensitive pool because TMB-8, a compound that prevents Ca2+release from intracellular IP,-sensitive stores (Palade et al., 1990), dose-dependently prevented the CCh-induced [Ca2+Iielevation in a Ca2+-freemedium. In addition, ryanodine, a paralyzing alkaloid that prevents the release of the Ca2+from an intracellular store insensitive to IP,, failed to prevent the [Ca*+],elevation elicited by CCh. Consistent with this result, caffeine, a methylxanthine that induces intracellular Ca2+ release from this compartment in different cell types (Thayer et al., 1988; Wyskovsky et al., 1990), failed to evoke a [Ca2+Iielevation in LAN-1 cells, when used alone or in combination with 0.3 pM CCh or after thapsigargin addition. These results suggest that in J. Neurochem., Vol. 59, No. 1. 1992

A . FATATIS ET AL.

8

LAN- 1 cells, caffeine-sensitive stores cannot be depleted either by a subthreshold dose of CCh or by depletion of the IP3-sensitiveintracellular Ca2+stores induced by thapsigargin, in contrast with results obtained in rat parotid cells (Foskett and Wong, 199 I). Although at the moment there is no definite explanation for this insensitivity to caffeine, it is possible that LAN- 1 cells are deficient in the caffeine-sensitive intracellular Ca2+release. consistent with this observation, Palade et al. (1990) found that caffeine was unable to elicit Ca2+-inducedCa2+release in brain microsomes. Collectively, the results of the present study indicate that human neuroblastoma LAN- 1 cells have M, and M3 muscarinic receptor subtypes. The stimulation of these receptors is linked to intracellular Ca2+ mobilization from an IP,-sensitive internal store and to Ca2+ influx through a voltage-insensitive, Gd3+blockable channel. This increase of [Ca2+],does not appear to be mediated by a PTX-sensitive G protein or by the PK-C system. Finally, LAN-1 cells seem to lack Ca2+channels activated by membranedepolarization and caffeine-sensitive intracellular Ca2+stores. Acknowledgment: T h e authors wish to express their gratitude to Dr. J. Kanfer (Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Canada) and Dr. R. Massarelli (Centre de Neurochimie-Cronenbourg, Strasbourg, France) for providing the LAN- 1 clone and for helpful advice on setting u p the neuroblastoma cell culture, Prof. M. Marchi for 4-DAMP and methoctramine, and Dr. H. Ladinsky for pirenzepine. This work was supported by CNR grants 89.02549.04 to G. F. D i Renzo and 89.02397.04t o L. Annunziato and by C N R Target Project on Biotechnology and Bioinstrumentation.

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