ANALYTICAL SCIENCES JANUARY 2A07, V OL. 23

2AA7 @ The Japan Society for Analytical Chemistry

81

Real-Time Detection of Dopamine Released from a Nerve ModelCell by an Enz,yme -Catalyied Lumine§cence Method and ltsApplication to Drug Assessment

Iliroaki §m¡os¿n¿'t and Feifei WaNc

Field. of Life, I$ormation and System Scierues, Graútate Scttool of Scimce and Engineering

IJnivercity of Toywta 3 DA Gofuha Toyuna 9 j O85 5 5' Japan

A real-time observatioa of neurctraasmiüer release from a nerve cell is a useful method for not only- neuroscieoce

researctr, but also .rroriog of the influence of chemicals, including drugs, on *te human nervou§ sy§tem' In this stud¡ a

more simple and sensitive-method for real-time monñoring of do,pamine release from a nerve modsl ce¡l was developed"

Higtüy sensitive detection of dopamine was performed by using tyr¿mine oxidase for dopamine oxida{9n whilh'was

rolowe¿ by a luminol luminesc-ence reactiou. rhis enryme-catalyzed luminescence method was applied to observe

dopamine ,elease from the pC12 cell as a nerve model cell upon stimulation with acetylcholine and an acetylcholine

rpcepúor agonist The resuln demonstrated that the real-time moniodng of the activation of rhe PC12 cell was e4sily

perfórmeO-Uy ttris method, This method possessed many advantage§, such as high sensitivity, rapid measurement and no

prerrr.rn*ifo, cells. It might be applied to drug scr;ning and the as§es§ment of harmful influences of food addiüves

and pesticides on the nerves.

(Received october 3{), 20ffi; Accepd November 6 2&K; Published January 1g'?Ñl\

Introduction

Easier and more sensitive methods for the biological assessment

of chemical compounds, such as drugs, food additives and

environmental pollutants, to the human nervous system have

been increasingly required. The real-time monioring of nerve-

cell acüvation, or the release of neurotra¡smitters, is very

effectivs for these purposes. Real-time mea§urement§ of nerve-

cell activation have been conventionally performed by using

fluorescence microscopy with a calcium indicatol.r'z However,

in this meüoü the indicator reagent should be loaded into the

cell bodies, and any excess of the reagent should be removed by

washing each time. Although HPLC analysis is also the usual

method to investigate neurotransmitter release by nerve-cell

activation,3l ÍIPLC analysis is complicated, and takes much

tirne. Therefore, the development of easier, real-time

observation methods is further requested.

In üis study, we aimed to develop a novel method for the

real-time detection of dopamine, a typical neuroransmitter,

released from the rat pheochromocytoma (PC12) cell as a

dopaminergic neuron model.!' Acetylcholine (ACh) and 1,1-

dimettryt-2}-phenylpiperazinium iodide (DMPP) were used as

stimulation agents for the PCl2 cell. It was well-known that

these compounds induce an increase of cytosolic Ca2n through

depolarization-induced activation of voltage-sensitive Ca?"

channels, and the release of Caa from the cytosolic vesicles'

and following neurotransmitter release ftom PCl2 cells.Ho

In this paper, a modified enzyme-luminescence detection

method for doPaminc is int¡oduccd as a novcl real-time

r To whom correspondence should be addressed.

E-mail: hshinoha@eng.u-oyama.ac jp

observation method to observe dopamine release from PCl2' In

this method, dopamine is firsüy oxidized to produce IIrO, by

tyramine oúdase (TOD). The produced H:Oz reacts ,wiÚ¡

tuminol to gerierate chemiluminescence in the presence ofhorseradish percxidase (IIRP)'

- The appücationr of

lactoperoxidase (trJOD)tt or tyr<xinaset2 for dopamine

oxidation was previously reported, however these euz¡nneg had

insufficient activity to detect dopamine in the examination

experiments" Therefore, in ttris study, ryramineoxidase (TOD'gC t.¿.¡.¿ t¡om Ar-throbacter sp.) was fir§t te§ted to oxidize

dopamine iastead of LFOD or tyrosinasé, because üe structure-

of its original substrate, tyramine is similar to thal ofdopamine.ri Furthenr¡ore, the ToD-catalyzed luminescence

¿eLction of dopamine was applied to detect dopamine deased

from PC12 cells upon ACh stimulhtion. Finally, we tried to

apply this method for drug assessment.

Experimental

Reagents and clwmicalsLuminol (5-amino2'3'ttihydro-l$'phthalazinedigne)'

tyrosinase from mushroom, hydrogen peroxide, and acetylcholine

b¡omide were obtained from Wako Pure Chemical Industries,

Ltd. Dopamine hydrochloride and lactoperoxidase (LPOD)

from bovine milk was obtained from Sigma' Peroxidase (POD)

wa.§ obtained from TOYOBO Co., Ltd. Tyrarnineoxidase

(fOD) from Anhrobacter sp' was obtained from ASAHI

KASEI PI{ARMA. DMPP (1,1 -dimethyl4-phenylpiperazihiumiodide) was obtained from ICN Biomedicals' Inc'

Cell culturePC12 cells (RC80009) were purchased from the cell bank of

Doparnine rele*edñom FC12

PCf8cdls

Fig. 1 Schematic illusnation of the principle of real-time detectionof dopamine released from PCl2 cells by drug stimulation with aluminescence detectable piate reader.

RIKEN BioResouce Center. Cells were grown in Dulbecco'smodified Eagle medium (DMEM) supplemented with 10% (v/v)horse serum (GIBCO), 57o (vlv) fetal bovine serum (ICNBiomedicals, Inc.), and 1% penicillin-streptomycin (GIBCO) at37'C in a humidified atmosphere containing 5%, COz. On theday before an experiment, cells were harvested with trypsin-EDTA and re-plated in a 9Gwell plate at a density of 0.5 x IffcellVml, and incubated for Z h to allow aüachment for a cellassay.

Characterizption of tle enzynes for doparnine oidationA comparison of three enzJme (TOD, tyrosinase, POD)

activities for dopamine oxidation was performed by a

colorimetric assay. The assay mixture contained 1 mM phenol,

0.5 mM 4-aminoanüpyrine, 0.5 mg/ml horseradish peroxidase,

10 pM - 2 mM dopamine, 0.1 M phosphate buffer (pH 7.4) at a

final volume of 3 ml. The reaction was started by adding üeenzyme in a final concentration of 0.1 mgiml. The absorbance

of üe solution was measured at 500 nm for 5 min with the v-560 spectrophotometer (JASCO Co.). The enzyme activity was

expressed as üe amount of dopamine oxidized per min and mg

of protein.

Deuction of dopamine with the e¡tzyme-luminescetrce method

The detection of dopamine was performed with a

iuminescence detectable plate reade¡ FLUOstar OIrIIMA(BMG LABTECH). Before a measurement, the medium was

replaced with [,ocke's solution Gü{ 7.4, 200 pl), then 0.25

mg/ml POD (30 td), 1.0 mg/mi TOD (30 ¡rl) and I mM luminol(30 pl) were added to each well. A dopamine solution of 10 plwas injected into the wells of the 96-well plate using an

automatic reagent injector to start the enzymatic reaction.

Lurninescence was detected by an equipped photomultiplier.

Enzync-catalyzed hnnine scence detection of dopunine released

fronPC|Z cellsPCl2 cells were plated in the 96-well plate at a density of

0.5 x ltr cellVml, aod incubarcd ovemight to Promotc ccll

adhesion. Just before a measurement, the Srowth medium was

removed from each well and cells were washed twice withLocke's solution. After washing, Locke's solution (200 U$,

ANALYTICAL SCIENCES JANUARY 2Ñ7, Y OL. 23

Table I Characterization of three enzymes for dopamineoxidation

Activityfu mgt KJ¡uM.

Tyr4mineoxidase CfOD)Tyrosinaselactoperoxidase &POD)

0.25 mg/¡ril POD (30 pl), l'0 mg/ml TOD (30 pi) and I mM

luminol (30¡tl) were added to each weil. The 96-well plate was

set in tl¡e plate reader and cell excitation was induced by the

injection of Ach or DMFP with the equipped auto-injector. The

principle of the real-time detection of dopamine released from

PCl2 cells by drug injection with the luminescence-detectable

plate reader is schematically shown in Fig. l.HPLC-ECD analysis was atso carried out to determine

dopamine released from the PCl2 cells by drug stimulation in

Locke's solution. Ion-pair chromarography with an ODS

capiltary column was used to separate dopamine after

deproteinization by a perchlorate pretreatment and

centrifugation. An LC-100 pump, ail I-C-165 on line valve, a

DA-5 chromatograph interface, and an LC-4C amperometric

detector were purchased from BAS.

Results and Discussion

Enzyme se lection for d.opamine oxidartonWe aimed firstly to find a good enzyme to detect dopamine

released from nerve model cells (PCl2) in real-time by enzyme-

catalyzed luminescence mea§urements. Table I rePresents the

three enzyme activities upon dopamine oxidation. TOD had the

highest activity (more than 6 times for tyrosinase and more than

2(tr times for LPOD) for dopamine oxidation, while there was

little difference in the K, value of three enzymes. Theretbre' itwas considered that TOD is much more efficient tbr dopamine

oxidation ar¡d HzO: production as compared with the previous

two enzymes.

Detection of doparuine with the enzyme-lwninescence mcthodTOD and POD were combined to detect dopamine by rhe

luminol luminescence method. When dopamine was injected

into the measurement soiution, luminescence was quickly

geúerated and reached the peak within a few seconds, as shown

in Fig. 2(A). The luminescence peak intensity increased withincreasing dopamine concenration within I UM. The detection

timit of dopamine was l0 nM, and its seositivity was

comparable to that of the ÍIPLC method. Figure 2(B) shows the

calibration cun¡es for dopamine with our TOD + POD method

and the LPOD + POD method. The comparison indicates that

the TOD + POD method is more sensitive to detect dopamine'

Real-time detection of dopamine rcleased from PCl2 cells by

the eniyme- catalyzed lurnine scenc e rnetho dPrior to the detection of dopamine released from PCl2 cells

by the developed enzyme-catalyzed lumine§cence method'

HPLC detection of catecholamine release<I from PCl2 cells was

performed. The HPLC anaJysis demonsuated that PCl2 cells

release only dopamine by ACh stimulation after one day ofculturing, though norepinephrine was also released after more

than n¡vo days. Next, the real-time observation of dopamine

release from PC12 cells after one day of culturing was

examined by the deveiopcd enzyme-cataiyzed luminescence

oñtr-:-'ry 5l3994

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ANALYTICAL SCIENCES JANUARY 2N7, YOL, 23

DM

510Time / sec,

0 t00 200 3m 4fl) 500

[DopamineJ /dr{

Fig. 2 Detection of dopamine by the enzyrre-luminescencerrrthod. (A) Tine course of luminescence after dopamine injection.(B) Dependence of the luminescence intensity on the dopamine

concenrratio¡. (a) TOD + POD method; (¡)) LPOD + POD ñpthod.

method. As shown in Fig. 3(A), the luminescence peak was

observed quickly within a few seconds after the injection ofACb. There was good reproducibility for three wells. The

dependence of the luminescence peak intensity on the

co¡centration of ACh was safurated at 100 pM with a detection

li¡nit of 5 !rM, as shorvn in Fig. 3(B). The EGo for ACh was 6ó

¡rM by this method and it was almost the sarne as the ECso (70

pM) by our HPLC analysis.Mo¡eover, we tried to apply this method to drug as§essment

for FCl2 cells. Luminescence geoer¿¡tion was quickly observed

by the injecüon of DMPP (a typical ACh receplor agonist) as

similar as ACt stimulation. The luminescence peak intensity

depended on the concentration of DMPP, a§ §hown in Fig. 4'

The EC5qby this meüod forDMPP was 20 pM and the ECsoby

our ÍIPIf analysis was 30 ¡tM. This result demonstrated that

this method can be used for dn¡g assessment.

Conclusions

It was demonstrated that our modified enzymeluminescencemethod is very useful for rcal-time observations of dopamine

release from PCl2 ceüs, as a doperminergic neuron model, by

acetylcholine and DMPP stimulation. The dependence of the

cell response on the drug concentration by this method

coresponded well to the dependence investigated by an HPLCanalysis. These results support that this method can be applied

to drug assessment for dopaminergic neuron§ or their model

10 20 30

Time/ sec.

0 100 2a0 300 400 500

lAchl I FtM

Fig. 3 Real-time dercdiou of dopamine ¡eleased ftom PCl2 cells.(A) Luminescence observation indicating dopamine ¡etrease fm¡nPCl2 cells by ACh stimulation The responses of th¡ee wells are

overlaid. The ACh concentration was 100 t¿M. (B) Dependence ofttre luminesence intessity on the ACh concenfation.

0 100 200 300 400 500

IDMPPI / l¡I,t

Fig. 4 Dependence of the luminescence intensity on the DMPP

conentration.

cells. This method has many advantages, such as highsensitivily for dopamine, rapid measurement (high througi¡pu$,and no pretreatment for cells (simple procedure). In addition,

the meüod mighr be usefui tbr the screening of chemical

compounds that have a harmful influence on the human nerve

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system. As our next direcüon, we would like o develop ttrismethod for the real-time imaging of dopamine rclease f¡omdopaminergic neurons, or rheir model cells. This fuure studymay help to find dopaminergic neurons in brain slice andprimary culturcd cells without any staini¡g. AIso it may beusefi¡l for monitoring the activity of focused dopaminergicneurons.

Acknowledgements

This work was supported by a grant of Toyama-Medical BioCluster ProjecL MEXT in Japan.

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