Brain Research 882 2000 256–265 www.elsevier.com locate bres
Interactive report
21
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
21 21
Present study revealed the stimulatory effects of d 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
21 21
administration of CdCl or 0-Ca
bath solutions. DPDPE-mediated [Ca ] elevation was abolished by pertussis toxin PTX
2 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 were
i 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 indicated
i 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
21
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
21 21
Ca VOC channel [1,12,18,19,26,33], to suppress basal
channel [7,20], to elevate [Ca ]
in NG108-15 cells
i 21
adenosine 39:59-cyclic monophosphosphatecAMP 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
s
Published on the World Wide Web on 25 September 2000.
seems to be involved in the latter case. Which mode of
Corresponding author. Tel.: 186-27-8754-3104; fax: 186-27-8754-
activities do opiates exerts depends on various parameters,
4375. E-mail address
: lychen163.net L. Chen.
such as experimental conditions, age, and receptor types or
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 0 4 - 8
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
21
opioid on [Ca ] in a rather simple and homogenous
The cell-bearing coverslip were rinsed three times with
i
preparation. buffer before loading and then incubated with fura-2 AM
SH-SY5Y cell is a kind of human neuroblastoma cell 2–5 mM 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, CaCl
2.5, MgCl 1, 4-2-
2 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.
21
increase inositol 1,4,5-trisphosphate IP accumulations Ca
-free buffer was the same as the above except that
3
[31]. It also mobilizes calcium store in the presence of MgCl
was substituted for CaCl , and EGTA ethylene
2 2
ongoing muscarinic receptor activation [2]. glycol-bis[b-aminoethyl ether]-N,N,N9,N9-tetraacetic acid
Microfluorometric technique was used to detect the 100 mM was added to the buffer.
21
[Ca ] in single SH-SY5Y cells. When the cells were
i 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
21
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. The
i
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
