June 2021

Medical Mass Spectrometry Vol. 5 No. 1

Research Paper

Dopamine enhances the glial endocannabinoid signaling
1

1

1

1

2,3

Hiroshi Ochi *, Yukari Hirata , Makoto Hamajima , Ichiro Isobe , Kazuo Igarashi
1

Department of Legal Medicine, Fujita Health University School of Medicine,1‒98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470‒1192, Japan
2
Association of Medicinal Analysis, 2‒3‒11‒608 Mayakaigandori, Nada-ku, Kobe, Hyogo 657‒0855, Japan3
Department of Legal Medicine, Kinki University School of Medicine,377‒2 Oonohigashi, Osakasayama, Osaka 589‒8511, Japan
Abstract

Endocannabinoid system (ECS) is widely distributed throughout the body and plays important roles in thephysiological functions. In this study, we investigated the eﬀect of some neurotransmitters for the expression state of
ECS-related genes of C6 cells. We recognized the signiﬁcant up-regulation of phospholipase c beta 4 (PLC β 4) anddown-regulation of monoacylglycerol lipase (MGLL) by dopamine (DA) treatment. The similar signiﬁcant expression
change of PLC β 4 and MGLL were observed by selective D1 receptor agonist treatment. Because PLC β 4 and MGLL wereinvolved in turnover of 2-arachidonylglycerol (2-AG), it was suggested that DA might aﬀect 2-AG productivity in C6 cells.
After pre-treatment with DA, C6 cells were treated with calcium ionophore to induce the 2-AG production. The amount of
2-AG signiﬁcantly increased in DA pre-treatment group. It is known that endocannabinoids act as retrograde messengers
and inhibit several synaptic signal transductions. In this study, we used C6 cells as a model of astrocyte. In the synapse, astrocyte forms the tripartite synapse with presynaptic neuron and postsynaptic neuron, and is known to inﬂuence the synaptic signal transductions. If astrocytes actually respond to increased DA in the same manner as C6 cells, we suppose that theincreased DA at synapse induces up-regulation of 2-AG production through activation of D1 receptors, and then the 2-AG
acts on the CB1 receptors existing on the presynapse and inhibit DA release from the presynaptic ends. In conclusion, ourﬁndings suggest that the glial 2-AG signaling may act as inhibitory mechanism of synaptic DA signaling.
Key words: dopamine, endocannabinoid system, phospholipase c beta 4, monoacylglycerol lipase, 2-arachidonylglycerol

Introduction

phosphatidylethanolamine phospholipase D (NAPE-PLD),

2-arachidonylglycerol (2-AG) and N-arachidonylethanol-

phospholipase c beta 4 (PLC β 4), diacylglycerol lipase.
The endocannabinoid system (ECS) consists of these endo
cannabinoids (2-AG and AEA), cannabinoid receptors

alpha (DAGL α ), fatty acid amide hydrolase (FAAH) andtype1 (CB1) and type2 (CB2), and enzymes involved inbiosynthesis or degradation of endocannabinoids (N-acyl

lipid which is the component of the cell membrane (AEA is
synthesized by NAPE-PLD and 2-AG is synthesized by

1‒3)

amide (AEA) are known as major endocannabinoids

4)

monoacylglycerol lipase (MGLL)) . Endocannabinoids areproduced with an activity dependent manner from phospho-

PLC β 4 and DAGL α ). Then endocannabinoids are released*Corresponding author

to the extracellular space and act on the receptors of target

Hiroshi Ochi

cells. After having acted, endocannabinoids are hydrolyzed(AEA is degraded by FAAH and 2-AG is degraded by

Department of Legal Medicine, Fujita Health University Schoolof Medicine, 1‒98 Dengakugakubo, Kutsukake-cho, Toyoake,
Aichi 470‒1192, Japan
Tel: ＋81‒562‒93‒2438, Fax: ＋81‒562‒92‒4580

MGLL). These ECS is widely distributed throughout theReceived August 11, 2020.

(CNS), endocannabinoids act as retrograde messengers andbody and plays important roles in the several physiological
5‒11)

functionsE-mail: hochi@fujita-hu.ac.jp

Accepted September 24, 2020.

. Especially, in the central nervous system
12‒14)

inhibit several synaptic transductions

Epub November 13, 2020.

.

The CNS is comprised of neuron and glial cells. The

DOI: 10.24508/mms.2021.06.001
2

June 2021

Medical Mass Spectrometry Vol. 5 No. 1

major function of neurons is signal transduction. In the
CNS, there are three types of glial cells: astrocyte, oligo-

was collected at every 24 h after treatment until 72 h. For
endocannabinoids analysis, C6 cells were pre-treated withdendrocyte and microglia. Oligodendrocytes form myelin

or without DA for 24, 48 or 72 h. Then the culture medium

around axons, and contribute to saltatory conduction ofaction potential. Microglias have macrophage-like function,

was changed to FBS and DA free DMEM. C6 cells weretreated with A23187 for 10 minutes, and then culture super-and play a role in the immune system in the CNS. It has
been thought that major function of astrocytes was regula-natant and cells were collected.

tion of synapse environment and energy supply to the neurons. However, it was revealed that various neurotransmit-

RNA isolation, PCR and quantitative real-time PCR(qRT-PCR)

ters are released from astrocytes, and act to the neurons toTotal RNA was extracted using TRIzol (Thermo Fisher
Scientiﬁc, MA, USA) and puriﬁed by chloroform/isopropa-

15)

regulate the neuronal functions .
We have been noted the expression state of ECS-related

nol method, followed by DNase treatment. cDNA was syn-genes in the glial cell as factors to prescribe a function of
ECS in the CNS. In this study, we hypothesized some neu-

thesized using Rever Tra Ace qPCR RT Master Mix

rotransmitters as the factor that aﬀect the expression stateof ECS-related genes in glial cell. We used C6 rat glioma

instruction. The PCR with Platinum PCR SuperMix(TOYOBO, Osaka, Japan) following the manufacturer s(Thermo Fisher Scientiﬁc, MA, USA) was performed. The
ampliﬁed products and 20-bp DNA ladder were electro-

cell line as a model of astrocytes to conﬁrm the eﬀect ofphoresed with agarose gel. The qRT-PCR with Platinum
SYBR Green qPCR SuperMIX-UDG (Thermo Fisher Sci-

neurotransmitters on the expression state of these genes.
Furthermore, we discussed the possibility of regulation ofsynaptic transduction by astrocyte-derived ECS.

entiﬁc, MA, USA) was performed using the ABI Prism
7900HT Real-Time PCR System (Thermo Fisher Scientiﬁc,Materials and Methods

MA, USA). The Expression levels of each ECS-related

Reagents and chemicals

gene were normalized against the level of hypoxanthine

Aldrich (MO, USA), and listed as follows: dopamine (DA),phosphoribosyltransferase (HPRT) in the same sample. The
qRT-PCR data were shown as the value of comparative Ct

glutamic acid (Glu), gamma aminobutyric acid (GABA),

method. The primers for PCR and qRT-PCR were as fol-

SKF38393 (SKF), quinpirole (QNP), calcium ionophore(A23187), N-arachidonoylethanolamide (AEA), 2-arachid-lows

All reagents and chemicals were purchased from Sigma-

16,17)

CB1:-CAAGCACGCCAACAACACA-3′5′onyl glycerol (2-AG), methyl heneicosanoate, methanol(MeOH), acetonitrile (MeCN), N,O-bis(trimethylsilyl)triﬂu-(forward)-TCTTAACGGTGCTCTTGATTGCA-3′
5′

oroacetamide＋trimethylchlorosilane (BSTFA＋TMCS). All
primary antibodies were purchased from Abcam plc (Cam-(reverse);-TTCCCCCTGATCCCCAACGACTA-3′
5′

CB2

bridge, UK) and secondary antibody was purchased from
Cell Signaling Technology (MA, USA).

(forward)-CTCTCCACTCCGCAGGGCATAAAT-3′
5′

Cell culture and treatment(reverse);-ATGACCATGTTGGCCATGG-3′
5′

MGLL

The C6 rat glioma cell line was purchased from American Type Culture Collection (VA, USA). Cells were cul-(forward)-CGTGCTGCAACAAATCTCTGAC-3′
5′tured in Dulbecco s Modiﬁed Eagle s Medium (DMEM,
Sigma-Aldrich, MO, USA) supplemented with 10% v/v(reverse);-GCTCTGAAACTCATTGACCGC-3′
5′

PLC β 4heat-inactivated fetal bovine serum (FBS, Nichirei, Tokyo,(forward)-ACGTAGCAATCTGCCATTCTCA-3′
5′

Japan) in a humidiﬁed incubator at 37°
C with a atmosphere
5% CO2. Cells were reseeded in the 6-well plates (for RNA
analysis) or 100 mm dishes (for endocannabinoids analysis)
4

FAAH

2

at a density 5×10 cells/cm . For RNA analysis, 24 h after(reverse);-CTGCCCAGCTGGTTTAAAAGA-3′
5′(forward)-AGGACGCATACTGTTGAGAAAGG-3′
5′reseeded, C6 cells were treated by several conditions. RNA
3

June 2021

NAPE-PLDDAGL αHPRT

Medical Mass Spectrometry Vol. 5 No. 1(reverse);-CTTTTGACCTCGCGGCTATT-3′
5′

formed in positive electron ionization mode. The GC/MS
conditions were as follows: column, DB-5MS (30 m×(forward)-CATAAACCACCTTGGCTCATAAGC-3′
5′0.25 mm, i.d. 0.25μm, Agilent Technologies, CA, USA);
column oven program, 120°
C for 3 min followed by an(reverse);-AAGAATGTCACTCTCGGGATGG-3′
5′

increase at 10°
C/min to 300°
C and keep 9 min; injector,(forward)-AGTGATGCAGACGCTGAGGAT-3′
5′200°
C, respectively; carrier gas and ﬂow rate, helium at(reverse);-GCAGTACAGCCCCAAAATGG-3′
5′monitoring mode using m/z values of 432, 103 and 129 for
2-AG, m/z values of 404, 85 and 116 for AEA and m/z val-

(forward)-AACAAAGTCTGGCCTGTATCCAA-3′
5′

ues of 340 for IS. Eight-point calibration curves were constructed for 2-AG and AEA in the concentration range of(reverse).

50‒1,000 ng/mL (r ＝0.99 and r ＝0.99, respectively). In

interface and ion source temperature, 260°
C, 260°C and
19)

1 mL/min . MS detection was carried out in selected ion

22

addition, the ﬁnal concentration of these endocannabinoids

Western blot assay

were standardized by total protein concentration of cells

C6 cells and rat brain samples were treated with cell lysis

derived from the same culture dish.

buﬀer to obtain total protein. The protein concentration wasdetermined by the Quant-iT Protein Assay kit and Qubit ﬂu-

Statistical analysis
All data were shown as the mean±standard error (SE). A
one-way analysis of variance followed by Tukey‒Kramer

orometer (Thermo Fisher Scientiﬁc, MA, USA). Twenty
micrograms of total proteins were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis andmethod was used to compare the signiﬁcance of diﬀerencetransferred to the polyvinylidene ﬂuoride membrane (Merck
Millipore, MA, USA). The membrane was incubated withamong all groups. Results were considered signiﬁcant at*p＜0.05 and **p＜0.01.

the primary antibody (1 : 1,000) at room temperature for 1 h
and with anti-rabbit secondary antibody (1 : 10,000) at room

Results

Expression change of ECS-related genes by neurotransmitter treatment

temperature for 1 h. The ECL detection reagent was used todetermine the expression levels of the proteins.

The expression state of ECS-related genes in C6 cells
were conﬁrmed by PCR method. As the result, the expres-

Solid phase extraction and derivatization of endocannabinoids

sion of CB1, MGLL, PLC β 4, FAAH, NAPE-PLD and

The endocannabinoids in collected culture supernatants
were extracted using Oasis HLB cartridge (Waters, MA,

DAGL α were detected, but the expression of CB2 was notclear in C6 cells (Fig. 1A). We examined the eﬀect of neu-

USA). The cartridge was conditioned with MeOH followed

rotransmitter for expression state of ECS-related genes. C6

by washing with water. After loading the sample, cartridge
was washed with 5% MeCN in water. The endocannabi-cells were treated with 100μM DA, Glu and GABA. Then
total RNA was collected at every 24 h after treatment until
72 h. As the results of qRT-PCR, signiﬁcant up-regulation

noids were eluted from the cartridge with MeCN. Methyl
eluted solution . The eluted solution was evaporated, andof PLC β 4 and signiﬁcant down-regulation of MGLL wereobserved by 100μM DA treatment (Fig. 1B). These expres-the residue was derivatized with BSTFA＋TMCS for 1 h at
60°
C. Derivatized samples were analysed by gas chroma-sion changes continued after DA treatment from 24 to 72 h.
Moreover, expression states of other ECS-related genes

tography/mass spectrometry (GC/MS).

were not changed by DA treatment (Fig. 1B). On the other
hand, expression states of all ECS-related genes were not

Quantiﬁcation of endocannabinoids by GC/MS

aﬀected by Glu and GABA treatment (Fig. 1B). In addition,
LDH assay was performed to conﬁrm inﬂuence on cell via-

heneicosanoate was added as an internal standard (IS) to
18)

GC/MS analysis was performed on GCMS-QP2010(Shimadzu, Kyoto, Japan) equipped with an AOC-20i

bility by neurotransmitter treatment, and signiﬁcantauto-injector (Shimadzu, Kyoto, Japan). Analysis was per-changes were not observed among all conditions (data not4

June 2021

Fig. 1.

Medical Mass Spectrometry Vol. 5 No. 1

Eﬀect of neurotransmitter for expression of ECS-related genes.

(A) The expression state of ECS-related genes in C6 cells were conﬁrmed by PCR methods. The ampliﬁed products and 20-bp
DNA ladder were electrophoresed with agarose gel. The each predicted amplicon size was as follows: CB1, 68 bp; CB2, 369 bp;
MGLL, 87 bp; PLC β 4, 54 bp; FAAH, 87 bp; NAPE-PLD, 53 bp; DAGL α , 63 bp; HPRT, 85 bp. (B) C6 cells were treated with 100
μM DA, Glu and GABA for 0, 24, 48 and 72 h. The expression levels of each ECS-related gene were normalized against the levelof HPRT in the same sample. The expression levels of each ECS-related genes in C6 cells treated for 0-h was set at 1.00. Data wereshown as folds of expression level at 0-h. All results were showed as mean±SE (n＝3). **P＜0.01, compared with control sample(cont.).

5

June 2021

Fig. 2.

Medical Mass Spectrometry Vol. 5 No. 1

DA dose dependence for expression change of PLC β 4 and MGLL.

C6 cells were treated with 0, 10, 25, 50, 75 and 100 μM DA for 0, 24, 48 and 72 h. The expression levels of PLC β 4 and MGLL
were normalized against the level of HPRT in the same sample. The expression levels of PLC β 4 and MGLL in C6 cells treated for
0-h was set at 1.00. Data were shown as folds of expression level at 0-h. All results were showed as mean±SE (n＝3). **P＜0.01,
compared with 0 μM DA.

DA receptor selectivity for expression change of PLC
β4 and MGLL

shown). From these results, next we performed moredetailed examination for expression change of PLC β 4 and25, 50, 75 and 100μM DA to reveal DA dose dependency

The DA receptors are classiﬁed in D1-like family andD2-like family. D1-like DA receptors consist of D1 and D5

for expression change of PLC β 4 and MGLL. C6 cells were

subtypes, whereas D2-like DA receptors consist of D2, D3

treated with DA for 0, 24, 48 and 72 h, and total RNA werecollected at each time point. For up-regulation of PLC β 4

and D4 subtypes. Next we investigated which DA receptor
subtypes were involved in up-regulation of PLC β 4 andexpression, clear DA dose dependency was observed (Fig.
2), and it was reconﬁrmed that up-regulation of PLC β 4 wasdown-regulation of MGLL. By western blotting analysis,

MGLL by DA treatment. C6 cells were treated with 0, 10,

expression of D1, D3 and D4 receptors were detected in C6
cells (Fig. 3A). By using the selective agonists for D1-like

continued until 72 h. Furthermore, clear DA dose depen-

DA receptor or D2-like DA receptor, we investigateddency and continuity of expression change were observedin down-regulation of MGLL (Fig. 2).

involvement of DA receptor subtypes in the expression
change of PLC β 4 and MGLL. SKF and QNP were used as
selective D1-like DA receptor agonist and selective D2-likeDA receptor agonist, respectively. C6 cells were treated6June 2021

Medical Mass Spectrometry Vol. 5 No. 1

with 100 μM SKF or QNP, and total RNA collected at every
24 h after treatment until 72 h. As the result, signiﬁcant
up-regulation of PLC β 4 and down-regulation of MGLL
were observed by 100μM SKF treatment (Fig. 3B), andthese expression changes continued until 72 h. On the other
hand, any signiﬁcant changes were not observed in PLC β 4
and MGLL expression by QNP treatment (Fig. 3B). In addition, LDH assay was performed to conﬁrm inﬂuence on cell
viability by SKF or QNP treatment, and signiﬁcant changes
were not observed among all conditions (data not shown).

Eﬀect of DA for 2-AG turnover in C6 cells

PLC β 4 and MGLL were enzymes involved in 2-AG

turnover. Therefore, it was suggested that production abilityof 2-AG in C6 cells changed by intense DA exposure. Next
we investigated whether 2-AG production and release in C6
cells were induced by DA treatment. After pre-treated with
100μM DA for 24, 48 or 72 h, C6 cells were treated withcalcium ionophore A23187 for 10 min, and then culture
supernatants and cells were collected. The collected culture
supernatants and cells were used for quantiﬁcation of endocannabinoids and protein, respectively. In addition, C6 cells
that were pre-treated with 0μM DA for 24, 48 or 72 h wereused for the control samples. Endocannabinoids wereextracted from collected culture supernatants by solid phaseextraction, and analyzed as trimethylsilyl (TMS) derivatives. In Fig. 4A, EI mass spectra from TMS derivatives ofstandard 2-AG and AEA were shown. In the control samples, 2-AG concentrations induced in culture supernatantsby A23187 treatment were 32.3±10.0, 34.4±1.1 and 38.8±
0.2 ng/mg protein at each time point (Fig. 4B). On the other
hand, 2-AG concentrations in the DA pre-treated samples
were 43.6±9.8, 49.9±1.0 and 91.7±7.8 ng/mg protein ateach time point (Fig. 4B). In the 24-h pre-treated samples,no signiﬁcant diﬀerence was observed in the 2-AG concenFig. 3.

tration between control and DA samples. However, in the48- and 72-h pre-treated samples, 2-AG amount were sig-

DA receptor selectivity for expression of PLC β 4
and MGLL mRNA.

niﬁcantly higher in the DA samples than control samples(P＜0.05 (at the 48-h samples) and P＜0.01 (at the 72-h

(A) The expression state of DA receptors in C6 cells
and Wistar rat whole brain (for positive control). Immunoblots were performed with whole cell extract (n＝2).
Antibodies to detect D1 (ab81296), D2 (ab85367), D3(ab155098), D4 (ab135978) and D5 (ab181623) wereused. (B) C6 cells were treated with 100 μM SKF or
QNP for 0, 24, 48 and 72 h. The expression levels of
PLC β 4 and MGLL were normalized against the level of
HPRT in the same sample. The expression levels of PLC
β 4 and MGLL in C6 cells treated for 0-h was set at 1.00.
Data were shown as folds of expression level at 0-h. All
results were showed as mean±SE (n＝3). **P＜0.01,
compared with control sample (cont.).

samples)). On the other hand, AEA was not detected in all
samples.

Discussion

The endocannabinoid system acts as the regulatory mechanism of synaptic signaling. Glial cells particularly astrocytes, also exist on synapses and form a tripartite synapse

7June 2021

Medical Mass Spectrometry Vol. 5 No. 1

diﬀerent, CB1 is mainly found in the central nervous system, whereas CB2 is mainly found in peripheral immune
cells. Furthermore, CB2 is reported slightly expressed in
microglia but not in astrocytes

20,21). Therefore, the absenceof CB2 expression in C6 cells as a model of astrocytes is
consistent with these facts.
We conﬁrmed that only highly-concentrated (100μM)
DA treatment induced up-regulation of PLC β 4 anddown-regulation of MGLL. Moreover, it was suggested thatthe expression changes of PLC β 4 and MGLL wereDA-speciﬁc, because these expression changes were foundto be dose-dependent on DA treatment. On the other hand,highly-concentrated (100 μM) Glu or GABA treatment did
not aﬀect the expression state of PLC β 4 and MGLL. In
another preliminary study, although C6 cell treated with 1
and 10 μM Glu or GABA for 0, 24, 48 and 72 h, there were
not signiﬁcant changes in the expression state of PLC β 4
and MGLL (data not shown). These results also supported
the DA-speciﬁcity for expression change of PLC β 4 andMGLL.
DA receptors are classiﬁed into D1-like receptor andD2-like receptor, and their actions conﬂict with each otherdue to the diﬀerence in the coupled G-proteins. Therefore,
in order to conﬁrm the receptor selectivity for expression
change of PLC β 4 and MGLL with DA treatment, SKF andQNP were used as selective D1-like receptor agonist andselective D2-like receptor agonist, respectively. The signiﬁFig. 4.

cant expression change of PLC β 4 and MGLL by SKF

Eﬀect of DA pre-treatment for 2-AG turnover.

treatment similar to that of DA treatment suggested that theactivation of D1-like receptors was involved in the expres-(A) The electron impact mass spectra obtained fromTMS derivatives of standard 2-AG and AEA. (B) Extracellular 2-AG amount induced by calcium ionophore
A23187 treatment. C6 cells were pre-treated with 0 or
100 μM DA for 24, 48 and 72 h. Then cells were treated with A23187 for 10 min. Culture supernatants werecollected for GC/MS analysis. Samples from pre-treatedwith 0 μM DA were used control (cont.). All results were
showed as mean±SE (n＝3). *P＜0.05 and **P＜0.01,
compared with cont.

sion change of these genes by DA treatment. We assumed
as follow that signal transduction mechanism that contributes to up-regulation of PLC β 4 via the activation of
D1-like receptor by DA treatment. The activation of D1-likereceptor induces activation of adenylate cyclase, because
this receptor is coupled with Gs. The concentration of intracellular cyclic AMP (cAMP) is risen by activated adenylate
cyclase, and as the result protein kinase A (PKA) is acti-with neurons. It is known that astrocytes play a role in theregulation of synapse functions. Therefore, we used C6
cells as a model of astrocytes to conﬁrm the eﬀect of neu-

vated. Activated PKA is known to phosphorylate various

rotransmitters on the expression state of ECS-related genes

enzymes and transcription factors. It is suggested that
up-regulation of PLC β 4 by DA treatment is involvement ofin these cells, and discussed the possibility of regulation ofsynaptic transduction by astrocyte-derived ECS. The

these cAMP dependent pathways. On the other hand,amount of MGLL mRNA was down-regulated by DA treat-

expression of ECS-related genes except CB2 in C6 cells
suggests that these cells have suﬃcient ECS function. It is

ment. Generally, it is thought that decrease of mRNAamount is derived from inhibition of transcription by tran-

known that the distribution of CB1 and CB2 is distinctly

scriptional control element or facilitation of mRNA degra8

June 2021

Medical Mass Spectrometry Vol. 5 No. 1

dation as posttranscriptional regulation. However, further
studies are required to reveal what kind of mechanism par-The brain reward system is one of the functions of CNS. In
this system, neurons are mainly projected from ventral teg-

ticipates in the down-regulation of MGLL mRNA in
DA-treated C6 cells. The enzymatic hydrolysis to arachi-mental area to nucleus accumbens or prefrontal cortex, anddonic acid and glycerol of 2-AG is performed mainly byother neurons. Generally, this nervous system is activatedDA acts as main neurotransmitter to communicate with

19)

25)

MGLL . Because it is reported that overexpression of
22)
MGLL is involved in decrease of 2-AG amount and sup-when desires are satisﬁed . Then the DA release isincreased and thereby the individual gets pleasure. How-pression of MGLL mRNA by RNAi results in enhancement23)of 2-AG signaling , MGLL is thought to play an important

ever, it is known that brain reward system is directly acti-role in control of 2-AG amount. Therefore, it is suggested
that 2-AG production is induced by the down-regulation of

stimulates the brain reward system for a long period and

MGLL mRNA by DA treatment.

addiction. The amount of DA around synapse is known to26)

vated by addictive drugs . Repeated use of these drugs
produces undesirable changes including dependence andBoth PLC β 4 and MGLL are enzymes involved in meta-

abnormally increase by the eﬀect of some addictive

bolic turnover of 2-AG. Consequently, next we investigated
the inﬂuence of DA on 2-AG metabolic turnover in C6

drugs . The abnormality of dopaminergic neuron iscells. Generally, it is known that endocannabinoid produc-thought that drug dependence and addiction are produced

tion and release are triggered by increase in intracellular

by abnormality of DA signaling.

27)

occured by repeated abuse of these drugs. As the result, it iscalcium ion . Therefore, A23187 was used as a calcium
ionophore to induce production of 2-AG by experimentallyIf the phenomena that we conﬁrmed in this study reappear in vivo, it seems that 2-AG production of glial cells

increase the concentration of intracellular calcium ion in C6

existing around synapse is signiﬁcantly induced by abnormality of DA signaling. In the CNS, it is known that 2-AG

19)

cells. Using this experimental model, we conﬁrmed
whether the expression changes in ECS-related genes

retrogradely release from postsynapse to presynapse, act on

induced by DA treatment were associated with the changes
in 2-AG productivity. As the result, it was revealed thatthe cannabinoid receptor exist in presynapse, and as the12‒14)

2-AG production of C6 cells were induced by DA pre-treat-. In the synapse,
astrocyte forms the tripartite synapse with presynaptic neu-

ment. Furthermore, the inﬂuence of DA was signiﬁcantly
increased in proportion to pre-treatment period. From theseron and postsynaptic neuron, and is known to inﬂuence thesynaptic signal transductions. Therefore, 2-AG derived

results, it is suggested that 2-AG production ability of C6
cells changes only when cells are exposed to highly-con-from glial cells may show inhibitory eﬀects for DA signal-

centrated DA for a long term. Because D1 receptor is cou-

glial cells competes with eﬀect of addictive drugs.

result, neurotransmission is suppressed

ing. In conclusion, it is suggested that ECS derived frompled with not only Gs but also Gq, activated D1 receptor
causes the activation of PLC β family. Activated PLC β 4hydrolyzes phosphatidylinositol bisphosphate, which con-

Acknowledgements

stitute cell membrane, to diacylglycerol (DG) and inositol

numbers JP26860471, JP17K15884 and JP20K10567 and
Grant-in-Aid for Research from Fujita Health University.

This work was supported by JSPS KAKENHI Grant

trisphosphate (IP3), and subsequently DG is converted into
2-AG by DAGL α . On the other hand, IP3 activates IP3

Conﬂict of Interest

receptor of endoplasmic reticulum and thereby intracellular
calcium concentration rises. As the result, it seems that pro-

There are no conﬂict of interest relevant to this article.

duction of 2-AG of DA-treated C6 cells was signiﬁcantly
increased by synergistic eﬀect of change of mRNA expres-

References
1) Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Ka-sion state and condition of intracellular calcium.
Because there is much more amount of 2-AG than that ofminski NE, et al: Identiﬁcation of an endogenous 2-mono-

24)

AEA in the CNS , it is thought that main endocannabinoidaﬀecting the function of the CNS is 2-AG. We identiﬁed theglyceride, present in canine gut, that binds to cannabinoid

signiﬁcant increase of 2-AG production only when C6 cells
were exposed to highly-concentrated DA for a long term.

2) Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A,

receptors. Biochem Pharmacol 50: 83‒90, 1995.
et al: 2-Arachidonoylglycerol: A possible endogenous
9June 2021

Medical Mass Spectrometry Vol. 5 No. 1

cannabinoid receptor ligand in brain. Biochem Biophys16) Cavuoto P, McAinch AJ, Hatzinikolas G, Janovská A, GameRes Commun 215: 89‒97, 1995.

P, et al: The expression of receptors for endocannabinoids
in human and rodent skeletal muscle. Biochem Biophys

3) Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson

Res Commun 364: 105‒110, 2007.

L, et al: Isolation and structure of a brain constituent thatbinds to the cannabinoid receptor. Science 258: 1946‒1949,

17) Sütt S, Raud S, Areda T, Reimets A, Kõks S, et al: Catodour-induced anxiety̶A study of the involvement of

1992.
4) Lu HC, Mackie K: An introduction to the endogenous

the endocannabinoid system. Psychopharmacology 198:

cannabinoid system. Biol Psychiatry 79: 516‒525, 2016.

509‒520, 2008.

5) Morena M, Campolongo P: The endocannabinoid system:18) Higuchi S, Irie K, Nakano T, Sakamoto Y, Akitake Y, et al:

An emotional buﬀer in the modulation of memory function.

Reducing acyl migration during puriﬁcation of 2-arachi-

Neurobiol Learn Mem 112: 30‒43, 2014.

donoylglycerol from biological samples before gas chro-6) Bellocchio L, Cervino C, Pasquali R, Pagotto U: The en-

matography mass spectrometry analysis. Anal Sci 26:
1199‒1202, 2010.

docannabinoid system and energy metabolism. J Neuroendocrinol 20: 850‒857, 2008.

19) Bisogno T, Sepe N, Melck D, Maurelli S, De Petrocellis7) Wang L, Yang T, Qian W, Hou X: The role of endocanna-

L, et al: Biosynthesis, release and degradation of the novel

binoids in visceral hyposensitivity induced by rapid eye

endogenous cannabimimetic metabolite 2-arachidonoyl-

movement sleep deprivation in rats: Regional diﬀerences.

glycerol in mouse neuroblastoma cells. Biochem J 322:

Int J Mol Med 27: 119‒126, 2011.

671‒677, 1997.

8) Basu S, Dittel BN: Unraveling the complexities of canna-

20) Ashton JC, Friberg D, Darlington CL, Smith PF: Expression

binoid receptor 2 (CB2) immune regulation in health andof the cannabinoid CB2 receptor in the rat cerebellum: An

disease. Immunol Res 51: 26‒38, 2011.

immunohistochemical study. Neurosci Lett 396(2): 113‒
116, 2006.

9) Kwok CH, Devonshire IM, Imraish A, Greenspon CM,
Lockwood S, et al: Age-dependent plasticity in endocan-21) Nunez E, Benito C, Pazos MR, Barbachano A, Fajardo O,

nabinoid modulation of pain processing through postnatal

et al: Cannabinoid CB2 receptors are expressed by peri-development. Pain 158: 2222‒2232, 2017.

vascular microglial cells in the human brain: An immunohistochemical study. Synapse 53(4): 208‒213, 2004.

10) Fraga D, Zanoni CI, Rae GA, Parada CA, Souza GE: Endogenous cannabinoids induce fever through the activa-

22) Dinh TP, Carpenter D, Leslie FM, Freund TF, Katona I, ettion of CB1 receptors. Br J Pharmacol 157: 1494‒1501,

al: Brain monoglyceride lipase participating in endocan-

2009.

nabinoid inactivation. Proc Natl Acad Sci USA 99: 10819‒

11) Zlebnik NE, Cheer JF: Drug-induced alterations of endo-

10824, 2002.
23) Dinh TP, Kathuria S, Piomelli D: RNA interference sug-cannabinoid-mediated plasticity in brain reward regions. J
Neurosci 36: 10230‒10238, 2016.

gests a primary role for monoacylglycerol lipase in the
degradation of the endocannabinoid 2-arachidonoylglycerol.

12) Melis M, Perra S, Muntoni AL, Pillolla G, Lutz B, et al:

Mol Pharmacol 66: 1260‒1264, 2004.

Prefrontal cortex stimulation induces 2-arachidonoylglycerol-mediated suppression of excitation in dopamine24) Zoerner AA, Gutzki FM, Batkai S, May M, Rakers C, et al:neurons. J Neurosci 24: 10707‒10715, 2004.

Quantiﬁcation of endocannabinoids in biological systems13) Straiker A, Mackie K: Depolarization-induced suppression

by chromatography and mass spectrometry: A comprehen-of excitation in murine autaptic hippocampal neurons. J

sive review from an analytical and biological perspective.

Physiol 569: 501‒517, 2005.

Biochim Biophys Acta 1811: 706‒723, 2011.

14) Szabo B, Urbanski MJ, Bisogno T, Di Marzo V, Mendig-

25) Schultz W: Neuronal reward and decision signals: Fromuren A, et al: Depolarization-induced retrograde synaptic

theories to data. Physiol Rev 95: 853‒951, 2015.
26) Koob GF, Sanna PP, Bloom FE: Neuroscience of addiction.

inhibition in the mouse cerebellar cortex is mediated by

Neuron 21: 467‒476, 1998.

2-arachidonoylglycerol. J Physiol 577: 263‒280, 2006.
15) Perea G, Araque A: GLIA modulates synaptic transmission.

27) Lüscher C, Ungless MA: The mechanistic classiﬁcation

Brain Res Rev 63: 93‒102, 2010.

of addictive drugs. PLoS Med 3: 2005‒2010, 2006.

10