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Interactive report

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Different stimulatory opioid effects on intracellular Ca

in SH-SY5Y

1

cells

*

Liangyi Chen , Shoubin Zou, Xuelin Lou, Hua-Guang Kang

Institute of Biophysics and Biochemistry, Huazhong University of Science and Technology, Wuhan 430074, PR China Accepted 7 September 2000

Abstract

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Present study revealed the stimulatory effects ofd opioid receptor on intracellular Ca concentration ([Ca ] ) in SH-SY5Y cells.i 21

Fura-2 based single cell fluorescence ratio (F345 / F380) was used to monitor the fluctuation of [Ca ] . Application of the selective_i

2,5

delta-opioid receptor agonist alone, [D-Pen ]-enkephalin (DPDPE), hardly had any effects on cells cultivated for 3–10 days. However, after the cells had been pre-stimulated with cholinoceptor agonist, carbachol, variable calcium elevation was found in 59% of the cultures. The response was naltridole-reversible and dose-dependent, and was abolished completely by thapsigargin (TG) treatment but not by

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administration of CdCl₂ or 0-Ca bath solutions. DPDPE-mediated [Ca ] elevation was abolished by pertussis toxin (PTX)_i

pretreatment but not cholera toxin (CTX), indicating coupling via G proteins of G / G subfamily. In 17.5% of the responding cells,i o

biphase response was found which may be due to both the stimulatory and the inhibitory effects of opioid. On the other hand, in acutely

21

dissociated cells, DPPDE alone induced [Ca ] increase in 50% of the cultures. The probability and the amplitude of the elevation werei 21

decreased considerably by application of nifedipine or 0-Ca bath solution and was little affected by application of TG. DPDPE

21

activated [Ca ] increase via a PTX-insensitive and CTX-sensitive pathway suggesting coupling through G subunit. All these indicatedi s 21

the opioid modulated the intracellular Ca regulation system through different pathways. SH-SY5Y cell line might be a suitable model for the investigation of the complex mechanism which underlies opioid function.  2000 Elsevier Science B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters, and receptors

Topic: Opioids: Anatomy, physiology, and behaviour

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Keywords: DPDPE; Intracellular Ca concentration; SH-SY5Y cells; Stimulatory effects of opioid

1. Introduction reduction of cellular excitability produced by opioid

through G or G subunit.i o

Opioid receptor, as a kind of GTP binding protein- However, from the middle of the 1970s, experiments coupled receptor, modulates various cellular functions. The began to show opioid might also take excitatory roles as best-documented predominant effects of opioid are inhib- well. For example, opiate were found to increase cAMP itory: to block calcium entry through voltage-operated production [8,25], to enhance both L- and N-typed VOC

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Ca (VOC) channel [1,12,18,19,26,33], to suppress basal channel [7,20], to elevate [Ca ]i in NG108-15 cells

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adenosine 39:59-cyclic monophosphosphate(cAMP) forma- (either through mobilization of Ca store or through tion [6,34], and to promote hyperpolarization by activation membrane depolarization) [10,11,30,36], to prolong action

1

of K channel [32], etc. All these inhibitory actions potential duration of DRG neuron [3,27–29], and finally to underlie the decrease in neurotransmitter release and enhance transmitter release from SK-N-SH and NMB cells [15,16,23]. Some of the stimulatory effects are pertussis toxin (PTX) sensitive while others are cholera toxin (CTX) sensitive [3,28,29]. A CTX-sensitive G subunit

1_{Published on the World Wide Web on 25 September 2000.} s

seems to be involved in the latter case. Which mode of *Corresponding author. Tel.: 186-27-8754-3104; fax: 1

86-27-8754-activities do opiates exerts depends on various parameters, 4375.

E-mail address: lychen@163.net (L. Chen). such as experimental conditions, age, and receptor types or

L. Chen et al. / Brain Research 882 (2000) 256 –265 257

drugs concentration. Therefore, the aim of present study 2.3. Fura-2 loading was to study both the excitatory and inhibitory effects of

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opioid on [Ca ] in a rather simple and homogenousi The cell-bearing coverslip were rinsed three times with

preparation. buffer before loading and then incubated with fura-2 /AM

SH-SY5Y cell is a kind of human neuroblastoma cell (2–5mM) for 30 min at 378C. The buffer solution contains line, expressing both m and d opioid receptor [3,13,14]. (mM): NaCl 140, KCl 2.0, CaCl2 2.5, MgCl2 1, 4-(2-Opioid was reported to negatively couple to adenylyl hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)

21

cyclase [21], to inhibit N-type Ca channel [26], to 10, glucose 10, surcose 40 and BSA 0.05%, pH 7.3.

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increase inositol (1,4,5)-trisphosphate (IP ) accumulations3 Ca -free buffer was the same as the above except that [31]. It also mobilizes calcium store in the presence of MgCl2 was substituted for CaCl , and EGTA (ethylene2

ongoing muscarinic receptor activation [2]. glycol-bis[b-aminoethyl ether]-N,N,N9,N9-tetraacetic acid) Microfluorometric technique was used to detect the (100mM) was added to the buffer.

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[Ca ] in single SH-SY5Y cells. When the cells werei

21

applied with d opioid receptor agonist, DPDPE, two _{2.4. Intracellular Ca measurements} different kinds of responses were found. For cells

culti-21

vated for 3–10 days, DPDPE hardly triggered any [Ca ]i _{The setup of fluorescence measurement was as follows:}

response alone. However, by pre-stimulating the cells with _{the fluorescent microscopy is constructed around a}

Ax-21

carbachol, it could elevate intracellular Ca level with _{iovert 100 (Zeiss, Germany); excitation light at 345 nm or}

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much higher probability through activation of Ca store. _{380 nm is provided by a monochromator system (T.I.L.L.} On the contrary, DPDPE alone was enough to elevate _{Photonics, Germany) which is controlled by MacQuadra}

21

[Ca ] in acutely dissociated cells (within 48 h). Thei _{650 computer (Apple Co., USA). The fluorescence signals} 21

response was attributed to the extracellular Ca entry _{are collected by a photomultiplier and converted to voltage}

21

instead of Ca store. PTX but not CTX inhibit the former _{values, then digitized and sent to the computer. X-CHART} responses but failed to block the former ones, while the _{(Heka, Germany) is used to calculate and monitor the}

21

reverse was true for the latter ones. These indicated that _{intracellular [Ca ] based on the equation provided by}

i

opioid may exert the same excitatory effects in the same _{Grynkiewicz et al. [9]. The fluorescence ratio of F345 /} cells via different modulation system. _{F380 was used in the experiments to reflect the fluctuation}

21 of [Ca ] .i

2. Method

3. Results

2.1. Chemicals and drugs

3.1. Response in cells passaged for 3 –10 days

Foetal bovine serum, HEPES, EGTA, carbamylcholine

21

2,5

3.1.1. DPDPE elevate [Ca ] only in the presence of

chloride(carbachol), [D-Pen ]-enkephalin(DPDPE), thap- i

carbachol sigargin, pertussis toxin and cholera toxin are purchased

For cells cultivated for 3–10 days, only 9% responded from Sigma, RPMI 1640 from Gibco, fura-2 /AM was from

to the application of selective d opioid agonist DPDPE Molecular Probe. Other ordinary drugs are from local

alone (n5130), while others showed no alternation in chemical drug companies.

fluorescence ratio, as Fig. 1a showed. However, pre-stimu-lated cells with cholinoceptor agonist, carbachol, increased

2.2. Cell culture the probability (59%, n5110) of the cells responding to

DPDPE application (Fig. 1b). Naltrindole, the d receptor The SH-SY5Y cells used here were obtained from antagonist, blocked this effect suggesting that it was Institute of Neuroscience, Beijing Medical University. mediated bydreceptor. To avoid receptor desensitization, They were cultured in RPMI 1640 supplemented with DPDPE were applied for 60 s at intervals of at least 5 min

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penicillin (100 iu ml ), streptomycin (100mg ml ) and [35]. Prolonged application of DPDPE was also tested and foetal bovine serum (15%) in a humidified incubator with no more pronounced increase in the amplitude was found 5% CO2 and 378C in temperature. They were passaged (data not shown).

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every week and were undifferentiated. Cells fresh disso- The response of [Ca ] to DPDPE was highly variable.i

ciated (within 48 h) or 3–10 days after passage were used Therefore, to investigate the maximum effectiveness of in these experiments, and they were planted onto glass DPDPE, different concentration of DPDPE were adminis-coverslips. Cells pretreated with PTX or CTX were trated to the same cell to decide DPDPE induced con-incubated overnight (12–14 h) in culture medium con- centration-dependent fluorescence ratio increase, where the

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Fig. 1. DPDPE elevated in the presence of carbachol. (A) No obvious fluorescence ratio increased in most of the cells tested (91% of 130) whendopioid receptor agonist, DPDPE (1mM) was applied alone. (B) After the cells been stimulated with cholinoceptor receptor agonist, carbachol (CCH, 50mM), DPDPE (1mM) elevated ratio in 59% of cells tested (n5110). The elevation was abolished bydreceptor antagonist, naltridole (1.5mM). Arrows in the

21

figures indicated puffing drugs for 60 s since prolonged time of drug application would not lead to more pronounced alternation of [Ca ] .i

21

nM in the presence of 50mM carbachol (n56) which was the DPDPE-triggered [Ca ] elevation may not be attribu-i 21

in good accordance of the results reported before [2]. ted to extracellular Ca entry.

21 21 Thapsigargin (TG) is an irreversible Ca / Mg

-AT-21

3.1.2. The effects of DPDPE attributed mainly to Ca Pase inhibitor that is located in the sarcoplasmic reticulum. store and were PTX-sensitive Pretreated the cells with it prevents the refilling of the

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Application of Ca channel antagonist, CdCl2 in the Ca store. Exposed the cell with 100 nM TG induced a bath solution, decreased the probability of responding cells gradual increase in fluorescence ratio. Further

administra-21

from 59% to 40% but not to none. Furthermore, in 0 mM tion of opioid elicit no change of [Ca ] (ni 520) even in

21

Ca bath solution, DPDPE still evoked rapid increase of the presence of carbachol (Fig. 3b).

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L. Chen et al. / Brain Research 882 (2000) 256 –265 259

Fig. 2. Concentration dependence of DPDPE-induced ratio increases in single SH-SY5Y cell. (A) Different concentrations of DPDPE were used to stimulate the same cell. Each stimulus was applied for 60 s at intervals of at least 5 minutes to avoid receptor desensitization. CCH (50mM) was always

Hill

presented in the bath solution. (B) The concentration-dependent curve was fitted based on the hill equation y5ymax/(11(K /x)d ) according to the data (n56). The EC50 for DPDPE was about 17 nM (P,0.05).

50), while CTX hardly had any effects on it (Fig. 4). That 3.2. Response in cells acutely dissociated indicated that the effect was mediated via Gi or Go

21

subunit. 3.2.1. DPDPE directly evoked [Ca ] increase_i

On the other hand, 1mM DPDPE alone applied for 20s 3.1.3. Stimulatory and inhibitory effects of opioid in the was enough to induce drastic fluorescence ratio increase in same cell 50% of the acutely dissociated cells (n5120), as a typical Of all the cells which responded to the DPDPE stimulus, figure shown in Fig. 6a. No biphase response was found in 17.5% (n570) showed biphase response. Fig. 5 showed a all the cells tested. Prolonged application of DPDPE (from

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typical response. The [Ca ]i first decreased then in- 20 to 100 s) led to augmentation in [Ca ] , as Fig. 6bi

creased after DPDPE application The decay phase was shows.

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attributed to extracellular Ca entry since replacing the

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normal buffer with Ca -free buffer (shown in Fig. 5), or 3.2.2. The effects of DPDPE attributed mainly to

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application of CdCl2 in the buffer (data not show) extracellular Ca entry and were PTX-insensitive

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Fig. 3. DPDPE mobilized calcium store in the presence of CCH. (A) In 0 mM Ca bath solution, DPDPE (1mM) still could elevated [Ca ] in thei

prsence of CCH in 55% of the cells (n550) tested. They were also inhibited by application of 1.5mM naltridole. (B) Calcium store inhibitor, thapsgargin

21

(GG, 100 nM) elevated [Ca ] and further blocked the simulatory effects of DPDPE completely (ni 510).

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or removal of Ca from bath solution considerably (8% for n530), as data summarized in Fig. 8. This hinted decreased even blocked the response evoked by DPDPE. that the effect might be act via a G subunit other than Gs i

The frequency of DPDPE evoked response was reduced to or G .o

25% (n530) in nifedipine-containing bath solution and

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even to 10% in Ca -free buffers (n540) (Fig. 8). Also as

Fig. 7a illustrated, the DPDPE triggered response was 4. Discussion

nearly completely abolished by switching the bath solution

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from normal buffer to Ca -free buffer. However, the Sarne and Gafni suggested that three factors might amplitude of it was hardly affected by administration of determine the stimulatory effects of opioid. They are: (1)

21

L. Chen et al. / Brain Research 882 (2000) 256 –265 261

Fig. 4. The frequency of DPDPE evoked responses in different preparation. Summary of results obtained in SH-SY5Y cells. DPDPE alone hardly triggered

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any responses. However, pre-stimulation with CCH increased the frequency greatly. To inhibit the extracellular Ca entry with CdCl or remove the Ca2 21

from bath solution hardly decreased the percentage of responding cells. Application of TG to deplete Ca store blocked the effects of DPDPE completely. Pretreated the cells with PTX inhibit the DPDPE evoked response to a great extent, while the reverse was true for CTX pretreatment. This suggested that the effect was mediated through PTX-sensitive G protein.

acutely dissociated cells that had nearly the same position in their division cycle. Both methods enhanced the excitat-ory effects of DPDPE, although the modulation mecha-nisms were different.

The use of a single-cell microfluorometric technique

21

enabled us to detect the intracelluar Ca changes within a single cell. Similar to the work conducted by Conner and Henderson [2], we found that DPDPE application alone

21

hardly triggered any [Ca ] augmentation in cells culti-i

vated for 3–10 days. However, when synergism with stimulatory agent as carbachol, the probability of DPDPE

21 triggered response increased. The elevation of [Ca ]i

21 caused by opioid was attributed mainly to the Ca store,

21

as 0 Ca bath solution would not affect it and administra-tion of TG blocked it completely. The mechanism might Fig. 5. Dual phase of DPDPE-induced ratio increase in single SH-SY5Y _{be that DPDPE antagonize long time carbachol treatment} cell. Biphase curves were found in 17.5% of the DPDPE evoked

induced desensitization of muscarinic receptor, then lead to response. The initial decay phase after DPDPE administration were found ₂₁

21 _{mobilization of Ca store, as discussed in [2]. PTX but}

due to extracellular factors, since removal the Ca from bath solution

21

Fig. 6. DPDPE alone evoked ratio rise in acutely dissociated SH-SY5Y cells. (A) DPDPE (1mM) applied alone could elevate [Ca ] in 50% of acutelyi

dissociated cells tested (n5120). Arrows indicated puffing DPDPE for 20 s. (B) Prolonged administration of DPDPE (1 mM) led to enhanced and prolonged elevation of fluorescence ratio (n55).

suggesting that it be mediated via a PTX-sensitive G ori stimulatory effect was different from former one.

Extracel-21

lular Ca entry, but not the intracellular store played a G protein.o

dominant role this time. TG treatment had little effect on The 17.5% of the culture that exhibited biphase curve

21

the Ca elevation caused by DPDPE. Prolonged applica-after DPDPE stimulus was not reported before. The first

21

21

tion of DPDPE lead to enhanced elevation of [Ca ] .

decay phase of [Ca ] curve was owed to extracellulari i

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Ca influx, while the second climbing phase owed to Previously had been reported that opiate elevated [Ca ]i 21

activation of Ca store. Carbachol had already found to through DHP-sensitive VOC channel in differentiate

45 21

elicited a muscarinic acetylcholine receptors (mAChR)- NG108-15 cells[2;10;20], and basal Ca uptake is at

21

mediated extracellular Ca entry in SK-N-SH human least partly mediated by VOC channel in NMB cells [24]. neuroblastoma [22], and induced a inward store-operated The existence of L-type calcium channel could not be

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current in gland cells [17]. Thus the possible explanation excluded by the fact that no Ca current was detected in may be that carbachol played an important role in the patch clamp experiment (data not shown). Addition of

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regulation of [Ca ]i after application, while DPDPE nifedipine into the bath solution both reduced the prob-exerted inhibitory effects on it. It also directly illustrated ability of the responding cells the amplitude of the how opioid coupled to different modulation system con- DPDPE-triggered elevation. Therefore, the increase of

21

temporarily to exerted totally different action on the [Ca ] was at least partially mediated by L type channel.i

intracellular calcium level in the same cell. However, the incomplete blockade by nifedipine compared

21

However, in 50% of acutely dissociated cells tested, to 0-Ca buffer suggested that opiate must evoke some

21

L. Chen et al. / Brain Research 882 (2000) 256 –265 263

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Fig. 7. The stimulatory effect of DPDPE was attributed to extracellular Ca entry. (A) After removal the Ca from bath solution, DPDPE (1mM) hardly

21

triggered fluorescence increase anymore (10% from 40 cells tested). A typical figure was shown here. (B) To deplete the Ca store with TG would not inhibit the stimulatory ability of DPDPE. All arrows indicated puffing DPDPE for 20 s.

VOC channel current. Receptor mediated channel may stimulatory effects may be canceled under normal situa-account for this although further work need to be done. tion. Elevation of GM1 level will lead to the conversion of The excitatory potency of DPDPE was enhanced rather opioid receptor from G –G coupled mode to G coupledi o s

than inhibited by PTX pretreatment, while the reverse was mode, and increase the efficacy of the G -couple receptors

true for CTX application. All these indicated a G me-s to the AC-cAMP transducer system, thus attenuated the diated mechanism existed in acutely dissociated SH-SY5Y inhibitory potency of opioid and uncover the excitatory cells, which is totally different from the previous recorded effects behind them. That might be the explanation why

in normal cultures. PTX enhanced rather than block the excitatory effects of

The different DPDPE-triggered excitatory responses of DPDPE. It might also be the GM1 level that decided the opioid might relate to the endogenous GM1 ganglioside dual activities of opioid in the same cell line although levels on the cell membrane. Gangliosides are abundantly confirmation of the assumption will need other mean of distributed on the surface of most neurons. Recently, a detection.

specific cAMP-PKA-dependent glycolipid, GM1 gan- In conclusion, opioid is a kind of substance that exerts glioside was found to play a important role in the complex regulation activities. Both the stimulatory and the excitatory opioid receptor functions [3–5]. Based on the inhibitory effects of opioid were found and much emphasis result they obtained, Crain and Shen brought forward the had been put on the mechanism of them and how the idea that intercoversion of opioid receptor between inhib- interconversion between the two modes taken place. itory and excitatory modes depends on GM1 level. Opioid However, from our experiment, the function of opioid may was supposed to couple to both G and G simultaneouslyi s be even more complicated than we had thought. DPDPE

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Fig. 8. The frequency of DPDPE evoked responses in acutely dissociated SH-SY5Y cells. DPDPE alone triggered responses in a large population of the

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acutely dissociated SH-SY5Y cells. The excitatory effect was abolished by nifedipine or 0-Ca bath solution, but not by application of TG. PTX pretreatment would not block this effect. Instead, they increased the population that responded to DPDPE stimulus. Reversely, CTX pretreatment abolished the effect of DPDPE suggesting coupling of G protein.s

tion of intracellular calcium in SH-SY5Y human neuroblastoma same time to exert opposite effects, and it increased the

21 cells, Br. J. Pharmacol. 117 (1996) 333–340.

basal Ca level also via different mechanisms and

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