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Research Report

Pioglitazone potentiates development ofmorphine-dependence in mice: Possiblerole of NO/cGMP pathway

Shiva Javadia, Shahram Ejtemaeimehra, Hamid Reza Keyvanfara, Peiman Moghaddasa,Atefeh Aminiana, Alaleh Rajabzadeha, Ali R. Manic, Ahmad Reza Dehpoura,b,n

aDepartment of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, IranbExperimental Medicine Research Center, School of Medicine, Tehran University of Medical Sciences, Tehran, IrancDepartment of Physiology, School of Medicine, Tarbiat Modares University, Tehran, Iran

a r t i c l e i n f o

Article history:

Accepted 13 December 2012

Peroxizome proliferator-activated receptor gamma (PPARg) is highly expressed in the central

nervous system where it modulates numerous gene transcriptions. Nitric oxide synthase (NOS)

Available online 8 February 2013

Keywords:

PPARg

Morphine-dependence

Withdrawal symptom

Nitric oxide

Hippocampus

Mice

U87 cell line

nt matter & 2013 Publish.1016/j.brainres.2012.12.0

uthor at: Department of, Tehran, Iran. Fax: þ9821dehpour@sina.tums.ac.ir

a b s t r a c t

expression could be modified by simulation of PPARg which in turn activates nitric oxide (NO)/

soluble guanylyl–cyclase (sGC)/cyclic guanosine mono phosphate (cGMP) pathway. It is well

known that NO/cGMP pathway possesses pivotal role in the development of opioid dependence

and this study is aimed to investigate the effect of PPARg stimulation on opioid dependence in

mice as well as human glioblastoma cell line. Pioglitazone potentiated naloxone-induced

withdrawal syndrome in morphine dependent mice in vivo. While selective inhibition of PPARg,

neuronal NOS or GC could reverse the pioglitazone-induced potentiation of morphine with-

drawal signs; sildenafil, a phosphodiesterase-5 inhibitor amplified its effect. We also showed

that nitrite levels in the hippocampus were significantly elevated in pioglitazone-treated

morphine dependent mice. In the human glioblastoma (U87) cell line, rendered dependent to

morphine, cAMP levels did not show any alteration after chronic pioglitazone administration

while cGMP measurement revealed a significant rise. We were unable to show a significant

alteration in neuronal NOS mRNA expressions by pioglitazone in mice hippocampus or U87

cells. Our results suggest that pioglitazone has the ability to enhance morphine-dependence

and to augment morphine withdrawal signs. The possible pathway underlying this effect is

through activation of NO/GC/cGMP pathway.

& 2013 Published by Elsevier B.V.

ed by Elsevier B.V.35

Pharmacology, School of Medicine, Tehran University of Medical Sciences,6402569.(A.R. Dehpour).

1. Introduction

The chronic use of opioids is regularly associated with the

development of dependence. This social and clinical issue

consists of two distinctive psychological and physical parts.

Psychological dependence is operationally defined by obses-

sive, out of control drug use, whereas physical dependence is

expressed by withdrawal symptoms caused by cessation or

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 7 23

decrease in the use of the substance (Camı and Farre, 2003).

Since physical dependence is the major cause of obsessive

drug-taking behavior and short term relapse, clarification of

the diverse mechanisms concerned with physical depen-

dence is still essential for the progression of the treatment

strategies to inhibit opioid dependence. While all the

mechanisms by which morphine induced dependence have

yet to be verified, a growing body of evidence suggests the

involvement of the nitric oxide (NO)—cyclic guanosine mono

phosphate (cGMP) pathway.

Nitric oxide (NO) is a crucial intra- and inter-cellular

messenger that takes part in a number of physiological and

pathological conditions within the nervous system. Neuro-

nal, endothelial and inducible forms of this enzyme (respec-

tively, nNOS, eNOS and iNOS), have been identified in the

brain and spinal cord. A rising body of evidence suggests that

NO through the NO-NOS/GC/cGMP (nitric oxide/nitric oxide

synthase-guanyly cyclase-cyclic guanosine mono phosphate)

pathway has an important function in the development of

opioid physical dependence in addition to opioid withdrawal

syndrome (Bredt and Snyder, 1989; Buccafusco et al., 1995;

Cao et al., 2005; Gartaite, 1991; Machelska et al., 1997; Morris

et al., 1991).

Moreover, it has been shown that naloxone-induced with-

drawal syndrome as well as morphine-dependence is signifi-

cantly diminished by NOS inhibitors through the suppression

of the NO-cGMP system (Adams et al., 1993; Babey et al., 1994;

Cappendijk et al., 1993; Elliott et al., 1994; Kimes et al., 1993;

Kolesnikov et al., 1993; Thorat et al., 1994).

Accumulating evidence reveals that as a result of chronic

morphine administration NO synthesis is altered in various

CNS areas due to the modified expressions of diverse NOS

enzymes. It has been reported that all of these enzymes

participate in the development of opiate dependence and

naloxone-induced withdrawal signs (Cuellar et al., 2000; Leza

et al., 1995, 1996; Liang and Clark., 2004; Machelska et al.,

1997).

Several in vivo and in vitro experiments have assessed cyclic

adenosine mono phosphate (cAMP) and cGMP levels to recognize

the roles of these second messengers in morphine-dependence

Table 1 – The effect of repeated doses of pioglitazone on the ndependent mice (n¼8–10 for each group).

Treatment %Weight loss Numbe

Saline �1.7970.47 0.570

Vehicle �0.0170.15 0.470

Morphine 8.970.67��� 14.671

Pioglitazne2.5 �1.1470.88 0.670

Pioglitazone2.5þmorphine 6.2671.9 16.771

Pioglitazne5 �1.5471.33 0.770

Pioglitazone5þmorphine 12.8370.82 17.571

Pioglitazne10 �2.5973.49 171

Pioglitazone10þmorphine 11.6771.02 2279

Pioglitazne20 �3.8972.34 1.471

Pioglitazone20þmorphine 17.4671.72a 57.372

nnn Po0.001 compared to saline control group.a Po0.05 compared to morphine group.b Po0.01 compared to morphine group.c Po0.001 compared to morphine group.

(Fang et al., 1998, 2000; Gu et al., 2004; Gullis et al., 1975; Sharma

et al., 1975, 1977; Shijun et al., 2009). It has been a long time since

scientists discovered the importance of adenylyl cyclase (AC)/

cAMP-protein kinase (PKA) system and its up-regulation in

morphine-dependence (Sharma et al., 1975, 1977; Shijun et al.,

2009). In spite of these findings, it was recently proposed that up-

regulation of NO-NOS/GC/cGMP system following chronic use of

morphine has a key function in increasing the activity of

neurons and modifying the animal behaviors (Shijun et al., 2009).

Peroxisome proliferator-activated receptors (PPARs) are

parts of the nuclear hormone receptor family of ligand

activated transcription factors (Rosen and Spiegelman,

2001). To date, three mammalian PPAR subtypes have been

identified including PPARa, PPARb/d, and PPARg (Berger and

Moller, 2002). Several in vitro and in vivo studies have

revealed that pharmacological activation of PPARg by various

ligands of this receptor like tiazolidindiones (Lehmann et al.,

1995), alters the expression or activity of NOS enzymes

(Cernuda-Morollon et al., 2002; Colville-Nash et al., 1998;

Dobrian et al., 2004; Hattori et al., 1999; Li et al., 2000; Marx

and Walcher, 2007; Ricote and Glass, 2007; Smiley et al., 2004).

In addition, antinociceptive mechanism underlying 15-

deoxy-D12, 14 –PGJ2 (an endogenous PPARg ligand) involves

activation of the L-arginine/NO/cGMP pathway (Napimoga

et al., 2008; Pena-dos-Santos et al., 2009; Quinteiro et al.,

2012).

Despite growing evidence in establishing the possible

involvement of PPARg in CNS diseases (Heneka and

Landreth, 2007), there is no information regarding the parti-

cipation of these receptors in morphine-dependence.

Considering the abundance of PPARg in hippocampus

neurons (Inestrosa et al., 2005) along with the fact that

hippocampus plays an important role in drug addiction

(Koob and Volkow, 2010; Moron and Green, 2010), strongly

support the interest for studying the effect of pioglitazone as

a PPARg agonist on development of morphine-dependence.

In the present study we aim to examine: (i) pioglitazone

effect on development of morphine-dependence, using

morphine-dependent mice and human glioblastoma (U87)

cell line; ( ii) the involvement of PPARg receptors in the

aloxone-induced withdrawal manifestations in morphine-

r of jumping Grooming score Diarrhea score

.71 0.370.15 0.270.13

.70 0.370.15 0.470.16

0.3��� 1.970.41��� 1.870.29���

.8 070 0.170.1

0.4 270.42 1.370.15

.8 0.270.13 0.370.15

1.4 1.970.28 1.670.34

.1 0.470.16 0.570.22

.9 2.170.28 1.770.21

.4 0.670.22 0.870.25

.78c 3.470.22b 370.26c

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 724

probable effect of pioglitazone on morphine-dependence; (iii)

the participation of NO-NOS/GC/cGMP system in mediating

piogliatzone effects on morphine-dependence; (iv) the effects

of pioglitazone on mRNA expression of NOS isoforms in

hippocampus of morphine-dependent mice; and (v) the effect

of pioglitazone on cAMP and cGMP levels in morphine-

dependent U87 cell line.

2. Results

2.1. Effects of repeated doses of pioglitazone on naloxone-induced withdrawal manifestations in morphine-dependentmice

The results depicted in Table 1 show the effects of pioglita-

zone pretreatment (2.5–20 mg/kg, 45 min before daily mor-

phine injections) on the development of morphine induced

dependence. Our results show that 20 mg/kg, pioglitazone

potentiated significantly all the symptoms observed through

naloxone-induced withdrawal syndrome including weight

loss, grooming, jumps and diarrhea (Po0.05, Po0.01 and

Po0.001, respectively, Table 1).

Moreover, data from Table 1 show that treatment of

animals with various doses of pioglitazone or vehicle (DMSO

1%) alone did not result in any withdrawal signs in control

mice after receiving naloxone on 5th day (P40.05, Table 1).

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Fig. 1 – Animals received GW-9662 (GW) (2 mg/kg, i.p.) 30 min

(DMSO 1%), which were injected 45 min before i.p. administrati

injected on 5th day after the last dose of morphine. Values are m

MOR values; ffPo0.01 and fffPo0.001 compared to PIO20 þMOR

2.2. GW-9662 antagonized pioglitazone effect on thedevelopment of morphine withdrawal symptoms

To investigate the possibility of PPARg participation in the

effect of pioglitazone on naloxone-induced morphine with-

drawal signs, GW-9662, a PPARg antagonist, was co-

administered with pioglitazone. As shown in Fig. 1, adminis-

tration of 2 mg/kg GW-9662, 30 min before pioglitazone

(20 mg/kg) significantly antagonized its effect on the devel-

opment of withdrawal manifestations (Po0.01 and Po0.001,

Fig. 1).

2.3. NOS inhibition reversed pioglitazone action onnaloxone-induced withdrawal signs in morphine-dependentmice

It is evident from Fig. 2 that pretreatment with a non-selective

NOS inhibitor (L-NAME, 10 mg/kg, i.p.) could reverse the effect of

pioglitazone (20 mg/kg) on the development of morphine with-

drawal manifestations following naloxone challenge (Po0.001,

Fig. 2). It was also observed that L-NAME (10 mg/kg) was not

effective in morphine group (P40.05, Fig. 2). We also used two-

way ANOVA to investigate the interaction between the effect of

L-NAME and pioglitazone on morphine withdrawal sings and

showed that the interaction is statistically significant (Po0.05) in

most tested withdrawal signs (data not shown).

MOR MOR+PIO20 GW GW+PIO20+MOR

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before either pioglitazone (PIO) (20 mg/kg, i.p.) or its vehicle

on of morphine (MOR) thrice daily for 4 days. Naloxone was

eans7SEM. (n¼8–10); ##Po0.01 and ###Po0.001 compared to

values.

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Fig. 2 – L-NAME (10 mg/kg, i.p.) was injected 30 min before either pioglitazone (PIO) (20 mg/kg, i.p.) or its vehicle (DMSO 1%),

which were administered 45 min before i.p. injection of morphine (MOR) thrice daily for 4 days. Naloxone was injected into

mice on 5th day after the last dose of morphine. Values are means7SEM. (n¼8–10); ##Po0.01 and ###Po0.001 compared to

MOR values; &&&Po0.001 compared to PIO20þMOR values.

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 7 25

2.4. Pretreatment with a selective nNOS inhibitorprevented pioglitazone effect on naloxone-inducedwithdrawal signs in morphine-dependent mice

To investigate the possible involvement of nNOS in effect of

pioglitazone on morphine withdrawal signs, 7-NI was used

either alone or in combination with pioglitazone in morphine-

dependent mice.

Daily administration of 7-NI (20 mg/kg, i.p.) 30 min before

pioglitazone (20 mg/kg) significantly repealed its effect on the

naloxone-induced withdrawal symptoms (Po0.001, Fig. 3). It

should be mentioned that 7-NI (20 mg/kg) was not effective in

morphine group (P40.05, Fig. 3). We used two-way ANOVA to

investigate the interaction between the effect of 7-NI and

pioglitazone on morphine withdrawal sings and showed that

the interaction is statistically significant (Po0.05) in all tested

withdrawal signs.

2.5. Selective sGC inhibition attenuated the effectof pioglitazone on naloxone-induced withdrawal signsin morphine-dependent mice

The effect of pretreatment with a selective sGC inhibitor

(ODQ, 10 mg/kg, i.p.) on the effect of pioglitazone (20 mg/kg)

on the development of morphine-dependence is shown

in Fig. 4. It can be seen that ODQ significantly attenu-

ated withdrawal signs in morphine-dependent mice

which were treated with pioglitazone (Po0.001, Fig. 4) but it

did not change the withdrawal symptoms in vehicle-treated

morphine-dependent group (P40.05, Fig. 4). Two way

ANOVA was used to test the interaction between the effect

of ODQ and pioglitazone on morphine withdrawal sings

and showed that the interaction is statistically signifi-

cant (Po0.05) in most of tested withdrawal signs (data not

shown).

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Fig. 3 – Animals received 7-NI (20 mg/kg, i.p.) 30 min before either pioglitazone (PIO) (20 mg/kg, i.p.) or its vehicle (DMSO 1%),

which were injected 45 min before i.p. administration of morphine (MOR) thrice daily for 4 days. Naloxone was injected on0

5th day after the last dose of morphine. Values are means7SEM. (n¼8–10); #Po0.01, ##Po0.01 and ###Po0.001 compared to

MOR values; ^^^Po0.001 compared to PIO20þMOR values.

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 726

2.6. Phosphodiesterase-5 inhibition enhanced the effectof pioglitazone on naloxone-induced withdrawal signsin morphine-dependent mice

The results represented in Fig. 5 show the effects of

sildenafil (a selective phosphodiesterase – 5 inhibitor) pre-

treatment (3–30 mg/kg, 30 min before daily morphine injec-

tions) on naloxone-induced withdrawal signs. These data

show that 30 mg/kg, sildenafil potentiated significantly all

the symptoms observed through naloxone-induced withdra-

wal syndrome (P40.05, P40.01 and P40.001, Fig. 5). Sildenafil

alone did not result in any withdrawal signs after receiving

naloxone in intact mice (P40.05, Fig. 5). The sub-effective

dose of sildenafil, (10 mg/kg i.p.), was used to explore the

probable mechanism of pioglitazone on the morphine with-

drawal manifestations through sGC-cGMP signaling. The

effects of pioglitazone on the naloxone-induced with-

drawal symptoms were dramatically raised by sildenafil

(Po0.01, Po0.001, Fig. 6). While co-administration of sildena-

fil (10 mg/kg) with pioglitazone (10 mg/kg) led to an

enhanced effect, this dose of sildenafil did not alter the

withdrawal symptoms in the morphine treated group

(P40.05, Fig. 6).

2.7. Pioglitazone did not have a significant effect onmRNA expression of NOS isoforms in the hippocampusin morphine-dependent mice

We used real time RT-PCR to look at expression of NOS isoforms

in the hippocampus and our results indicated nNOS mRNA was

significantly up-regulated in the morphine-dependent compared

to independent animals (Po0.05, Fig. 7A). On the other hand,

neither eNOS nor iNOS mRNA expression were significantly

different in morphine-dependent mice compared to control

(P40.05, Fig. 7B and C).

The results in Fig. 7 illustrated no considerable differences in

the mRNA expression of NOS isoforms between pioglitazone

(20 mg/kg) and vehicle treated morphine-dependent mice

(P40.05, Fig. 7). Furthermore, pioglitazone (20 mg/kg) alone did

not make significant changes in the expression of NOS isoforms

in mice hippocampus (P40.05, Fig. 7).

2.8. Pioglitazone increased nitrite levels in thehippocampus of morphine-dependent mice

Nitrite levels were radically increased in morphine-dependent

mice compared to vehicle treated group (F (1, 10)¼83.12;

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Fig. 4 – ODQ (10 mg/kg, i.p.) was administered 30 min before either pioglitazone (PIO) (20 mg/kg, i.p.) or its vehicle (DMSO 1%),

which were injected 45 min before i.p. administration of morphine (MOR) thrice daily for 4 days. Naloxone was injected on

5th day after the last dose of morphine. Values are means7SEM. (n¼8–10); #Po0.05; ##Po0.01 and ###Po0.001 compared to

MOR values; $$$Po0.001 compared to PIO20þMOR values.

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 7 27

Po0.001, Fig. 8). Pretreatment with pioglitazone (20 mg/kg)

greatly elevated the increased nitrite levels in morphine-

dependent mice (F (1, 10)¼84.36; Po0.01, Fig. 8).

However, pioglitazone (20 mg/kg) did not alter nitrite

levels in the hippocampus of saline treated group (P40.05,

Fig. 8).

2.9. U87 cells expressed PPARc receptors

To explore whether U87 cells express PPARg receptors or not,

RT-PCR for PPARg mRNA was carried out.

Our results clearly illustrated the expression of PPARgreceptors in this human cell line (Fig. 9).

2.10. cGMP and cAMP productions could be modified byacute and chronic morphine treatment in U87 cells

Since in our pilot studies, the basal levels of cAMP and cGMP

in U87 cell line were undetectable due to their instability and

short life, we stimulated the cells with either forskolin (FSK)

or sodium nitroprusside (SNP) and then measured cAMP and

cGMP respectively. This approach enabled us to measure AC

and sGC activity within our experimental setting. Applica-

tions of FSK (1.0 mM) and SNP (10.0 mM) 10 min before harvest-

ing U87 cells resulted in a drastic rise in cAMP and cGMP

levels in cultures treated with 1 mM morphine (Po0.001,

Fig. 10). We also showed that 10 min incubation with mor-

phine (1 mM) could significantly decrease AC (Po0.001,

Fig. 10A) as well as GC activity (Po0.01, Fig. 10B) in U87 cells.

However, after 18 h exposure with morphine, radical

increases in AC (Po0.01, Fig. 10A) and GC (Po0.001, Fig. 10B)

activities were observed.

2.11. Effect of acute and chronic treatment withpiogltazone on cAMP and cGMP levels in U87 morphine-dependent cells

These experiments were conducted to study whether piogli-

tazone (0.1–10 mM) in U87 culture leads to any alteration in

chronic morphine-mediated signal transduction through

AC or GC.

Neither acute (45 min) nor chronic (18 h) incubation with

pioglitazone altered FSK-stimulated cAMP levels in chronic

morphine-treated cells (data not shown). Likewise, acute

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Fig. 5 – Sildenafil (1–30 mg/kg) or saline (10 ml/kg) was administered 30 min before i.p. injection of morphine (MOR) thrice

daily for 4 days. Naloxone was injected on 5th day after the last dose of morphine. Values are means7SEM. (n¼8–10);�Po0.05; ��Po0.01 and ���Po0.001 compared to saline values. DPo0.05; DDPo0.01 and DDDPo0.001 compared with MOR

values.

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 728

incubations with pioglitazone (45 min) did not change the

SNP-induced cGMP levels in chronic morphine-treated cells.

However chronic (18 h) exposure of U87 cells with pioglita-

zone (10 mM) significantly increased SNP-induced cGMP con-

centration in chronic morphine-incubated cells (F (4, 25)¼8.977,

Po0.001, Fig. 11).

Although, pioglitazone could affect GC activity in

morphine-dependent cells, it did not induce any momentous

alterations in AC or GC activity in intact U87 cells. In other

words, no differences were seen between control and piogli-

tazone treated cells in FSK and SNP-induced increase in

cAMP and cGMP levels without incubation with morphine

(data not shown).

2.12. PPARc receptor Is involved in the effect ofpioglitazone on morphine-dependent U87 cells

The PPARg receptor antagonist, GW-9662 (10 mM), completely

prevented PPAR g-mediated cGMP overshoot in morphine-

treated cells (F (3, 20)¼10.007, Po0.05, Fig. 12). This finding

indicates that PPARg receptor stimulation is necessary for the

development of increased morphine mediated GC activation,

which occurred by pioglitazone pretreatment.

2.13. Pioglitazone did not affect nNOS mRNA level in U87morphine-dependent cells

To investigate whether pioglitazone affects nNOS mRNA

expression in U87 morphine-dependent cells, real time RT-

PCR for nNOS mRNA was carried out. The outcomes specify

no meaningful alteration between experimental groups. Nor-

malized expression level of nNOS (nNOS/GAPDH) for

morphine-dependent cells was 1.1970.69 which was not

significantly different in pioglitazone treated morphine-

dependent cells (1.3970.79).

3. Discussion

In this study we employed in vivo and in vitro models to test

the hypothesis that PPARg receptors participate in develop-

ment of morphine-dependence. This is the first report

to demonstrate that chronic administration of pioglitazone

%W

EIG

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LO

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20

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80

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enaf

il10

+PIO

10+M

OR

Sild

enaf

il10+

MO

R

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10+M

OR

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Fig. 6 – Animals received sildenafil 30 min before either pioglitazone (PIO) (10 mg/kg, i.p.) or its vehicle (DMSO 1%), which

were injected 45 min before i.p. administration of morphine (MOR) thrice daily for 4 days. Naloxone was injected on 5th day

after the last dose of morphine. Values are means7SEM. (n¼8–10); OOPo0.01 and OOOPo0.001 compared to PIO20þMOR

values.

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 7 29

(a PPARg agonist) during morphine treatment protocol enhances

naloxone-induced opioid withdrawal syndrome in mice and

potentiates GC activity in morphine-dependent U87 cells. We

also explored the possible mechanisms through which pioglita-

zone potentiates morphine withdrawal syndrome.

First, the possible involvement of PPARg receptors in this

effect was assessed by administration of GW-9662, a selective

PPARg antagonist. While co-administration of pioglitazone

with morphine significantly intensified the expression of the

major withdrawal behaviors, including jumps, diarrhea,

grooming and weight loss, GW-9662 reversed these effects

indicating a substantial role of PPARg receptors. Considering

the dynamic role of PPARg receptors, we attempted to clarify

the possibility of NO/GC/cGMP pathway in the pioglitazone-

amplified naloxone-induced withdrawal signs. It is well

established that NO/GC/cGMP pathway contributes to neuro-

nal adaptations in response to repeated exposure to mor-

phine and that NOS particularly nNOS inhibition attenuates

morphine withdrawal symptoms in a variety of animal

paradigms including morphine-dependent rat and mouse

(Bredt and Snyder, 1989; Buccafusco et al., 1995; Cao et al.,

2005; Garthwaite, 1991; Kumar and Bhargava, 1997;

Machelska et al., 1997; Morris et al., 1991; Yang et al., 2000).

GC, one of the main mediators of this pathway, was reported

to be involved in the adaptation to chronic morphine admin-

istration (Hoskins and Ho, 1987; Pasternak et al., 1995;

Sullivan et al., 2000) and inhibition of GC activity resulted in

compromised opium tolerance and dependence (Zang and

Meng, 1999).

Present data strongly indicate that the effect of pioglita-

zone is dependent on the activity of this pathway, since this

event is severely reduced following pioglitazone co-

administration with L-NAME, a non-selective NOS inhibitor

or ODQ, a selective sGC inhibitor. Moreover, the possible

involvement of calcium-sensitive neuronal NOS enzyme

(nNOS) in mediating the effects of pioglitazone on

naloxone-induced opioid withdrawal syndrome was indir-

ectly examined using pretreatment with 7-NI which signifi-

cantly prevented the effect of pioglitazone on naloxone-

induced opioid withdrawal signs.

Rel

ativ

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vel o

f tra

nscr

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nal

diffe

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e (n

NO

S/ß-

actin

)

0

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20+M

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diff

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ce (i

NO

S/ß-

actin

)

0

1

2

3

4

MO

R

PIO

20

PIO

20+M

OR

Salin

e

Rel

ativ

e le

vel o

f tra

nscr

iptio

nal

diff

eren

ce (e

NO

S/ß-

actin

)

0

1

2

3

4

MO

R

PIO

20

PIO

20+M

OR

Salin

e

Fig. 7 – Animals received PIO (pioglitazone; 20 mg/kg) i.p. 45 min before morphine (MOR) daily treatment. nNOS (neuronal

nitric oxide synthase) is up-regulated in morphine-dependent mice (in comparison to saline-treated independent mice )

(Fig.7A). Values are means7SEM. (n¼5–8); �Po0.05 compared to saline values. The mRNA expression of iNOS (inducible

nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) did not show any considerable changes in Fig.7B and C.

Values are means7SEM. (n¼5–8); P40.05 compared to saline values.

Nitr

ite(μ

M)

0

20

40

60

80

100DMSOPIO20mg/kg

Saline MOR

##

+++

Fig. 8 – Four groups of animals were used in this study: MOR

(morphine)—dependent mice receiving either 20 mg/kg PIO

(pioglitazone) or its vehicle (DMSO 1%), i.p. 45 min before

morphine daily treatment and saline groups receiving

either 20 mg/kg PIO (pioglitazone) or its vehicle (DMSO 1%),

i.p. three times a day for 4 days. Values are mean7SEM.

(n¼6); ##Po0.01 compared to vehicle treated control (DMSO

1%) group.þþþPo0.01 compared to MOR (morphine) treated

control (DMSO 1%) group.

Fig. 9 – RNA was isolated from U87 cell. cDNA was amplified

using specific PPARc and GAPDH primers. PCR was

performed. PCR products for PPARc (150 bp, lanes 3 and 4)

were visible after agarose gel electrophoresis. GAPDH

(266 bp lane 2) was used as a housekeeping gene.

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 730

Due to the instability and short life of the cGMP and cAMP

levels in the hippocampus, we run cellular studies to inves-

tigate the role of these two cyclic nucleotides in the pioglita-

zone effects on the development of morphine-dependence.

Although previous studies are mainly focused on cAMP, the

role of cGMP in this respect still has received little attention.

The present study gave us the opportunity not only to

examine the role of cGMP in morphine-dependence but also

to evaluate the involvement of this nucleotide in molecular

signaling occurred by chronic treatment of pioglitazone in

morphine-dependent U87 cells.

ACUTE CHRONIC

0

20

40

60

80

100

120

cAM

P(μM

)

CO

NT

RO

L

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R

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R

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100

120

cGM

P(μM

)

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NT

RO

L

MO

R

***

ACUTE CHRONIC

ΔΔ

ΔΔΔ

MO

R

MO

R+

SNP

MO

R+

SNP

SNP

Fig. 10 – Application of FSK (forskolin; 1.0 lM) or SNP (sodium nitroprusside;10 lM)10 min before harvesting cells

significantly increased cAMP or cGMP levels in all control cultures and cultures treated with 1 lM MOR (morphine).

Morphine was incubated for 10 min in acute and for 18 h in chronic treatment. All data represent mean7SEM of six

experiments. ###Po0.001 and ���Po0.001 compared respectively to cAMP and cGMP control values; fffPo0.001; ffPo0.01

and DDPo0.01; DDDPo0.001 compared respectively to FSK and SNP values.

cGM

P(%

SNP

Stim

ulat

ed)

0

20

40

60

80

100

120

MO

R

PIO

0.1+

MO

R

* * *

PIO

1+M

OR

PIO

10+M

OR

VE

HIC

LE

+MO

R

Fig. 11 – U87 cells were treated by MOR (Morphine; 1 lM) and

PIO (pioglitazone; 0.1–10 lM) for 18 h. GC activity was

stimulated with SNP (sodium nitroprusside; 10 lM) for

10 min. Vehicle contained 1% DMSO (v/v) in saline. All data

represent mean7SEM of six experiments. ���Po0.001

compared to MOR values.

cGM

P(%

SNP

Stim

ulat

ed)

0

20

40

60

80

100

120

MO

R

PIO

10 +

MO

R

**

GW

+PIO

10+

MO

R

GW

#

Fig. 12 – U87 cells were incubated by MOR (Morphine; 1 lM),

PIO (pioglitazone; 10 lM) and GW-9662 (10 lM) for 18 h. GC

activity was stimulated with SNP (sodium nitroprusside;

10 lM) for 10 min. All data represent mean7SEM of six

experiments. ��Po0.01 compared to MOR values; #Po0.05

compared to PIOþMOR values.

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 7 31

It was shown that chronic exposure to morphine induced

dependence in cells, and the altered levels of cGMP in

addition to cAMP could confirm this event (Gullis et al.,

1975; Sharma et al., 1975, 1977). In agreement with previous

mentioned studies, our results suggest that both cyclic

nucleotides could have roles in morphine-dependence. How-

ever, assessing these nucleotide levels following chronic

pioglitazone treatment showed a profound increase in cGMP

levels while cAMP levels did not significantly change.

Alternatively, based upon the previous study, 15-deoxy-

D12, 14-PGJ2 (a natural ligand for PPARg) could affect the NO/

cGMP/PKG pathway (Napimoga et al., 2008). PPARg agonists

are able to lower blood pressure in spontaneously hyperten-

sive rats (SHR) which is accomplished by stimulation of NO

production (Grinsell et al., 2000; Igarashi et al., 1997; Marx and

Walcher, 2007; Verma et al., 1998). Despite the prevailing

scientific belief that AC/cAMP-PKA up-regulation has the

most important contribution to the mechanism of mor-

phine-dependence, we add further data supporting the

potential role of cGMP in this complex event.

Although we did not measure cGMP levels in the hippo-

campus of morphine treated animals, our behavioral obser-

vations using selective sGC inhibitor (ODQ), provided indirect

evidence for involvement of cGMP in potentiating effect of

pioglitazone on development of morphine-dependence in

mice. Alternatively pretreatment of mice with a selective

PDE-5 inhibitor (sildenafil) enhanced the effect of pioglita-

zone on the development of morphine-dependence. It is

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 732

known that sildenafil administration increases the cGMP

level in the rat dorsal hippocampus (Prickaerts et al., 2002;

Rutten et al., 2005) and was previously reported to potentiate

the antinociceptive effect of morphine in experimental mod-

els (Jain et al.,2003; Mixcoatl-Zecuatl et al., 2000). Therefore

our findings further reinforce the hypothesis that pioglita-

zone exerts its effect on the development of morphine-

dependence by increasing cGMP levels, likely as a conse-

quence of elevating the NO synthesis.

To confirm a role for involvement of NO in our in vivo

model, we measure nitrite levels in the hippocampus and

observed that this NO end product significantly increased

following PPARg activation in morphine treated mice. This

indicates that either NOS activity or its expression is

increased following pioglitazone treatment. We therefore

looked at NOS isoforms expression in the hippocampus and

found that nNOS mRNA expression increased dramatically

following chronic morphine treatment whereas no significant

increase occurred in eNOS or iNOS mRNA expressions. This

pattern of results is in agreement with previous studies

showing that nNOS expression is enhanced with opioid

agonists (Leza et al., 1995; Machelska et al., 1997; Wong

et al., 2000). Although some reports exist to show that

pioglitazone has the ability to increase the expression of

various NOS enzymes in a diverse laboratorial models

(Cernuda-Morollon et al., 2002; Dobrian et al., 2004; Marx

and Walcher, 2007; Smiley et al., 2004), we were unable to

demonstrate a significant increase in nNOS expression in

nether hippocampus nor U87 cells after chronic PPARg activa-

tion. This might indicate that nNOS gene is less likely to be

the main target for PPARg in our experimental models.

However, pioglitazone may increase nNOS enzymatic activity

through increased expression of other proteins that are

involved in modulation of NO/cGMP pathway (e.g. sGC,

calmodulin, CAPON, hsp90, caveolins, etc). We did not sys-

tematically investigate the genes that can be targeted by

PPARg during development of morphine dependence and this

can be studied in future experiments. Another limitation of

our study is that we only looked at mRNA expression of NOS

isoforms in the hippocampus. Although hippocampus is an

important brain region in morphine dependence, other brain

areas are also involved (Koob and Volkow, 2010; Moron and

Green, 2010).Thus, future studies can be carried out to

PPARγ ligand

PPARγ

PPARγ

Target genes?

nNOS/sGScGMP

OpioidRs

Opioids

Opioid dependenceWithdrawal

Fig. 13 – PPAR-c, Peroxizome proliferator-activated receptor

gamma; Opioid-Rs, Opioid receptor; nNOS, Neuronal nitric-

oxide synthase.

investigate the pattern of expression of NOS isoforms follow-

ing PPARg activation in other relevant brain regions.

In summary, the results from the present study which are

schematically presented in Fig. 13 indicate that pioglitazone

pretreatment increases morphine-dependence and withdra-

wal signs possibly through activation of PPARg and NO-nNOS/

sGC/cGMP pathway.

4. Experimental procedures

4.1. Animals

Male adult NMRI mice weighing 25–30 g, obtained from

Pasteur Institute of Iran, were used throughout the study.

Animals were housed in groups of four to five in a

temperature-controlled room (2471 1C) on a 12-h light/dark

cycle (Lights turned on at 7:00 h). Animals had free access to

food and water apart from short time of test. All behavioral

experiments were conducted between 8:00 and 16:00 h to

minimize diurnal variations. Each animal was used only once

and the number of animals in each group was 8–10. All the

animal studies were approved by the Ethics Committee of

Tehran University of Medical Sciences (TUMS) and experi-

ments were performed in compliance with the National

Institutes of Health Guide for Care and Use of Laboratory

Animals (Publication no. 85-23, revised 1985).

4.2. Drugs and chemicals

The following drugs were used in this study: morphine

sulfate (Sigma, UK); pioglitazone, a PPAR g agonist(Osveh

Company, Tehran, Iran); GW-9662, a PPAR g antagonist

(Sigma, St. Louis, MO, USA); N(G)-nitro-L-arginine methyl

ester (L-NAME), a non-specific NOS inhibitor; 7-nitroindazole

(7-NI), a selective nNOS inhibitor (Sigma, St. Louis, MO, USA);

1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selec-

tive inhibitor of solubale guanylyl–cyclase (Alexis Corpora-

tion, Lausen, Switzerland); sildenafil citrate, a selective

inhibitor of phosphodiesterase—5 (Poursina, Tehran, Ira-

n),and naloxone hydrochloride (Tolid-daru, Iran). Forskolin

(FSK), Sodium Nitroprusside (SNP), Dulbecco’s Modified Eagle

Medium (DMEM) and Fetal Bovine Serum (FBS) from Sigma

(Gibco, Gaithersburg, MD, USA). cAMP and cGMP enzyme

immunoassay (EIA) system kits from Enso Life Science (Alexis

Corporation, Lausen, Switzerland). The human brain glio-

blastoma/astrocytoma cell line U87 MG (ATCC; HTB-14) was

obtained from National Cell Bank of Iran (Tehran, Iran).

All drugs were freshly diluted in physiological saline

except for pioglitazone, GW-9662 and ODQ, which were

dissolved in 1% dimethyl sulfoxide (DMSO) (Fermentas Life

Sciences, Lithuania) and were diluted up to 10 times of the

main volume with normal saline. DMSO and saline were used

as controls.

All the drugs were administered intraperitoneally (i.p.) in a

volume of 10 ml/kg of body weight. The doses used in both

in vivo and in vitro studies were chosen based on previous

published studies and pilot experiments (Avidor-Reiss et al.,

1996, 1997; Bhargava et al., 1997; Dambisya and Lee, 1996;

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 7 33

Ghasemi et al., 2008; Hajhashemi et al., 2007; Hazra et al.,

2007; Jain et al., 2003; Maeda et al., 2008; Orasanu et al., 2008).

Griess reagent (Enzo lise sciences, ALX-400-004-LO50) was

used in this study in order to assay hippocampus nitrite

levels.

4.3. Induction of morphine-dependence and withdrawal

The animals were rendered dependent on morphine using

the method described previously (Sadeghi et al., 2009) with

some modification. Morphine sulfate was injected i.p. three

times daily at 08.00, 11.00 and 16.00 h at the doses of 50, 50

and 75 mg/kg, respectively. The higher dose at the third daily

injection was aimed to minimize any overnight withdrawal.

Morphine administration was carried out over 4 days for all

groups of mice. A dose of 100 mg/kg of morphine sulfate was

also injected on the test (5th) day (1 h before naloxone

injection). Naloxone (4 mg/kg, i.p.) was used in order to

induce withdrawal signs. An equal volume of saline was

intraperitoneally injected in to the control group. The ani-

mals were placed in a plexiglass cylinder (40 cm long, 25 cm

wide and 45 cm high) for habituation to the test environment

1 hour prior to the naloxone injection. Immediately, after the

injection of naloxone, the animals were placed separately in

those platforms and signs of withdrawal: jumps, diarrhea

and grooming were studied as previously described during a

60-min period (Broseta et al., 2002; Marshall and Grahame-

Smith, 1971; Rehni et al., 2008a,b; Rehni and Singh, 2011; Way

et al., 1969).

% body weight loss was determined according to the

weight before and 60 min after administration of the nalox-

one. It was reported as another important withdrawal sign

(Broseta et al., 2002; Hajhashemi et al., 2007).

4.4. Real time-PCR for evaluation the mRNA expressionsof NOS hippocampus enzymes

The expression of iNOS, nNOS and eNOS were evaluated in

the hippocampus in control, morphine-dependent and pio-

glitazone treated morphine-dependent mice. The hippo-

campi briefly were isolated and kept at -80 1C. Frozen

tissues were then homogenized and total RNA was extracted

using RNeasy mini kit (Qiagen, USA) following the manufac-

turer’s instructions. First strand cDNA was generated using

reverse transcriptase and oligo (dT) 15 and the expression of

iNOS, nNOS, eNOS and b-actin (as housekeeping gene) were

assessed using quantitative real time PCR. Oligonucleotide

primers used for PCR amplification were as follows: iNOS

sense, 50–GAC GAG ACG GAT AGG CAG AG-30; antisense, 50-

CTT CAA GCA CCT CCA GGA AC-30; nNOS sense, 5- TAT GTG

GCA GAA GCT CCA GA-30; antisense, 50- CGG CTG GAT TTA

GGA CTT TG-30; eNOS sense, 50- GGA AAT GTC AGG CCC GTA

CA-30; and antisense, 50- GGT CTG AGC AGG AGA CAC TGT

TG-30. The relative expression of each gene was normalized

against b-actin (50–TTC CTC CCT GGA GAA GAG-30, 50–TGC

CAC AGG ATT CCATAC-30).

Reaction conditions were 95 1C for 10 min, followed by

denaturing for 15 s at 95 1C and annealing and extension at

60 1C for 1 min for 36 cycles. The level of transcriptional

difference between treated groups and control group was

calculated relative to the level of b-actin expression.

4.5. Nitric oxide quantification in the mice hippocampus

The hippocampi were isolated and kept at �80 1C. Frozen

tissues were then homogenized. Weighed samples of tissues

(approximately 80 mg) were placed in 1.5 ml microtubes and

homogenized with the gradual addition of 4–6 times the

tissue weight of lysis buffer solution (Tris buffer pH¼8). Homo-

genates were incubated at room temperature (20710 1C) for

10 min. Homogenates were centrifuged at 13,400 RCF (Model 3-

30 K Microcentrifuge, Sigma) for 15 min. Following centrifuga-

tion, the supernatants were assayed for nitrite contents. Nitrite is

generated by the rapid oxidation of NO. To assay nitrite, we used

a modified version of a previously published Griess reaction

method (Kumral et al., 2004, 2006). Results are presented as the

average of the triplicate readings for each sample followed by the

standard error of mean.

4.6. Cell culture studies

The U87 cell line (Human glioblastoma–astrocytoma,

epithelial-like cell line) is highly qualified for in vitro study

of the pioglitazone effect on the opioid dependence (Leach

et al., 2002; Luth et al., 2001; Mahajan et al., 2002; Mueller

et al., 1998; Panigrahy et al., 2002; Shidona and Whittle, 2001;

Strakova et al., 2004).

This cell line (ATCC; HTB-14) was obtained from National

Cell Bank of Iran (Tehran, Iran).

The cells were grown in DMEM supplemented with 10%

fetal bovine serum, 100 unit/ml penicillin and streptomycin

at 37 1C in a humidified atmosphere of 5% CO2/95% air.

Thereafter, cells that reaching confluence followed by grow-

ing up in 75 cm2 flasks were counted and plated in 96 multi

well plates (Nunc, Roskilde, Denmark) at density of

2�105cells/well and allowed to adhere 48 h at 37 1C to be

prepared for studying pharmacological interventions. In

order to induce dependence in U87 cells, cells incubated with

morphine 1 mM for 18 h (Avidor-Reiss et al., 1997).

4.7. cAMP and cGMP assays

The two key second messengers, cAMP and cGMP which have

critical functions in the process of morphine-dependence and

withdrawal (Fang et al., 1999; Nestler et al., 1994; Shijun et al.,

2009) were assessed in this study.

Using FSK (1 mM) and SNP (10 mM), 10 min before evaluat-

ing the amounts of cAMP and cGMP in treated cells, we could

have the opportunity to evaluate the severity of morphine-

dependence in cells by a crystal clear observing of not only

the AC superactivity but also the GC rising activity which

were followed by inducing morphine-dependence in cells

(Avidor-Reiss et al., 1996, 1997; Chu et al., 2004; Durand

et al., 2010; Matsumoto et al., 2007; Nevo et al., 1998; Varga

et al., 2003).

At the end of drug treatment periods, cells were washed,

lysed in 0.1 M HCl (500 ml/well), and centrifuged at 600g.

Supernatants were collected and assayed for cAMP and cGMP

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 734

using Direct Cyclic AMP and Direct Cyclic GMP Enzyme

Immunoassay Kits, according to the manufacturer’s instruc-

tions (Alexis Corporation, Lausen, Switzerland).

In brief, samples or standards (0–20 pmol/ml) were loaded

in the wells pre-coated with an antibody mounted against

cAMP or cGMP. Afterwards, we added cAMP or cGMP con-

jugate and cAMP or cGMP antibody and let the reaction

proceed at room temperature for 2 h. Washing three times,

we added the chromogenic substrate p-Npp and allowed

color to be developed for 1 h. Optical density (OD) was read

at 405 nm for cAMP or cGMP directly after addition of stop

solutions. The intensity of the created yellow color was

inversely proportional to the concentration of cAMP or cGMP

in either standards or samples.

The results are presented as the mean7S.E.M of three

duplicate independent experiments (Seyedabadi et al., 2012).

4.8. Evaluation of mRNA expressions of PPARc and nNOSin U87 cells

To clarify whether U87 cells express PPARg receptors or not,

the expression of PPARg was evaluated in U87 intact cells by

using RT-PCR method. Furthermore, the expression of nNOS

was assessed in morphine-dependent and pioglitazone trea-

ted morphine-dependent U87 cells using real time RT-PCR.

Oligonucleotide primers used for PCR amplification were as

follows: PPARg sense, 50-AAA GAA GCC GAC ACT AAA CC-30;

antisense, 50-CTT CCA TTA CGG AGA GAT CC-30; nNOS sense,

50-CCT CCC GCC CTG CAC CAT CTT-30; antisense, 50-CTT GCC

CCA TTT CCA TTC CTC GTA-30 and GAPDH sense, 50-ACA GTC

CAT GCC ATC ACT GCC-30; antisense, 50-GCC TGC TTC ACC

ACC TTC TTG-30.

PCR products were separated by 1% agarose gel electro-

phoresis followed by staining with ethidium-bromide for

PPARg.

5. Experimental protocols

5.1. In vivo mouse study

5.1.1. Behavioral animal studies: naloxone-inducedwithdrawal manifestationsTo determine the pioglitazone effect on naloxone-induced

withdrawal manifestations in morphine-dependent mice, the

effects of four different doses of pioglitazone (2.5, 5, 10 and

20 mg/kg) were investigated. Mice were treated with DMSO

1% or pioglitazone (2.5–20 mg/kg) 45 min before morphine or

its vehicle.

Considering the involvement of PPARg receptors in piogli-

tazone effect on the morphine withdrawal syndrome, we

used GW-9662 (2 mg/kg). It was administered 30 min before

administration of either pioglitazone (20 mg/kg) or its vehicle

(DMSO 1%).

The possible involvement of NOS/GC/cGMP pathway on

the pioglitazone activity in naloxone-induced withdrawal

signs was studied using the co-administration of L-NAME

(10 mg/kg), 7-NI (20 mg/kg) and ODQ (10 mg/kg) with piogli-

tazone (20 mg/kg).

In another set of experiments, to confirm the importance

of cGMP in mediating pioglitazone effect on the development

of morphine dependence, we investigated the effects of the

combined administration of the sub-effective dose of piogli-

tazone (10 mg/kg) with the sub-effective dose of sildenafil

(10 mg/kg, i.p., a selective PDE-5 inhibitor).

All of the drugs were administered 30 min before either

pioglitazone or its vehicle.

5.1.2. In vivo real time PCR and Griess assay studiesFor the real time PCR studies of mRNA expression of NOS

isoforms and analyzing of hippocampus nitrite content, four

groups of animals were used: normal, morphine-dependent,

pioglitazone (20 mg/kg) treated and pioglitazone treated

morphine-dependent mice (n¼5–8 in each group).

5.2. In vitro cell culture study

5.2.1. Evaluation of the FSK and SNP -stimulated AC and GCactivities in U87 cellsTo investigate the effect of pioglitazone on morphine-

dependent cells; we used human brain glioblastoma/astro-

cytoma (U87) cell line. First, we studied the effects of acute

(45 min) and chronic (18 h) pioglitazone treatment (0.1–10 mM)

on the FSK -stimulated AC and SNP- stimulated GC activities

in U87 cells.

Then, to demonstrate the effect of pioglitazone on

morphine-mediated AC and GC increased activities, cells

were pre incubated with morphine (1 mM) for 18 h (Avidor-

Reiss et al.,1996).

Pioglitazone was incubated for 45 min or for 18 h before

10-min assay (started by addition of FSK and SNP).

Finally to clarify the involvement of PPARg receptor in

pioglitazone effect on morphine-dependent U87cells, we

used GW-9662 (10 mM) 30 min before pioglitazone.

All experiments were repeated twice.

5.2.2. Real time-PCR studiesIntact U87 cells were used to evaluate the mRNA expressions

of PPARg receptors. To study mRNA expression of nNOS in

U87 cells, morphine-dependent and pioglitazone treated

morphine-dependent cells were used (n¼3 in each group).

6. Data analysis

The results were expressed as the mean7S.E.M. The data

were analyzed using the Graphpad Prism data analysis

program (Graphpad Software San Diego, CA, USA).

Differences within experimental groups in withdrawal

signs were analyzed by one-way analysis of variance

(ANOVA), whereas each of between groups differences (the

interaction between pioglitazone and the corresponding

interventions) were analyzed by two-way ANOVA, both was

followed by Tukey’s post test. Po0.05 was considered statis-

tically significant in all experiments.

For analysis of the real time PCR results; we used the

2�DDCT (relative quantification) method. The principles of

calculations are discussed elsewhere (Schmittgen and Livak,

2008).

b r a i n r e s e a r c h 1 5 1 0 ( 2 0 1 3 ) 2 2 – 3 7 35

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