ORIGINAL PAPER

Theophylline Potentiates Lipopolysaccharide-Induced NOProduction in Cultured Astrocytes

Mizue Ogawa • Katsura Takano • Kenji Kawabe •

Mitsuaki Moriyama • Hideshi Ihara •

Yoichi Nakamura

Received: 12 August 2013 / Revised: 15 October 2013 / Accepted: 6 November 2013 / Published online: 15 November 2013

� Springer Science+Business Media New York 2013

Abstract Elucidation of the functions of astrocytes is

important for understanding of the pathogenic mechanism

of various neurodegenerative diseases. Theophylline is a

common drug for bronchial asthma and occasionally

develops side-effects, such as acute encephalopathy;

although the pathogenic mechanism of the side-effects is

unknown. The lipopolysaccharide (LPS)-induced nitric

oxide (NO) production is generally used for an index of the

activation of astrocyte in vitro. In this study, in order to

elucidate the effect of theophylline on the astrocytic

functions, we examined the LPS-induced NO production

and the expression of iNOS in cultured rat cortex astro-

cytes. Theophylline alone could not induce the NO pro-

duction; however, NO production induced by LPS was

enhanced by theophylline in a dose-dependent manner; and

by isobutylmethylxanthine, a phosphodiesterase inhibitor.

The theophylline enhancement of LPS-induced NO pro-

duction was further increased by dibutyryl cyclic AMP, a

membrane-permeable cAMP analog; and by forskolin, an

adenylate cyclase activator. When the cells were preincu-

bated with Rp-8-Br-cAMP, an inhibitor of protein kinase

A, the theophylline enhancement of LPS-induced NO

production was decreased. The extent of iNOS protein

expression induced by LPS was also enhanced by the-

ophylline. It is likely that phosphodiesterase inhibition is a

major action mechanism for the theophylline enhancement

of LPS-induced NO production in astrocytes. Theophyl-

line-induced acute encephalopathy might be due to the

hyper-activation of astrocytes via cAMP signaling to pro-

duce excess amount of NO.

Keywords Astrocyte � Theophylline � NO

Abbreviations

DAN 2,3-Diaminonaphthalene

dbcAMP Dibutyryl cyclic adenosine monophosphate

DMEM Dulbecco’s modified Eagle medium

FCS Fetal calf serum

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

GFAP Glial fibrillary acidic protein

IBMX Isobutylmethylxanthine

iNOS Inducible NO synthase

LPS Lipopolysaccharide

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide

NF-jB Nuclear factor-kappa B

NO Nitric oxide

NO2- Nitrite

PDE Phosphodiesterase

PKA cAMP dependent protein kinase

PKG cGMP dependent protein kinase

TAS Theophylline-associated seizure

TNF Tumor necrosis factor

M. Ogawa � K. Takano (&) � K. Kawabe � M. Moriyama �Y. Nakamura

Laboratory of Integrative Physiology in Veterinary Sciences,

Graduate School of Life and Environmental Sciences, Osaka

Prefecture University, 1-58, Rinku-Ourai Kita, Izumisano,

Osaka 598-8531, Japan

e-mail: takano@vet.osakafu-u.ac.jp

Present Address:

M. Ogawa

Department of Veterinary Pathology, The University of Tokyo,

Tokyo, Japan

H. Ihara

Department of Biological Science, Graduate School of Science,

Osaka Prefecture University, Sakai, Japan

123

Neurochem Res (2014) 39:107–116

DOI 10.1007/s11064-013-1195-9

Introduction

Theophylline, one of methylxanthine derivatives, has been

used in the treatment of asthma and chronic obstructive

pulmonary disease because of its bronchodilator activity. At

least three mechanisms of action are known for methylxan-

thine derivatives; phosphodiesterase (PDE) inhibition,

adenosine receptor antagonism and Ca2? mobilization from

intracellular store [1]. Although the mechanism of bron-

chodilation is not completely established, it is likely due to

increase in intracellular cAMP, resulted by PDE inhibition,

while adenosine receptor inhibition has fewer roles for it [1].

In addition, it is reported that low-dose theophylline has anti-

inflammatory effect for bronchus [2, 3]. On the other hand,

theophylline is also known to pass through blood–brain

barrier easily and cause some side-effects to CNS such as

seizure so-called theophylline-associated seizure (TAS) or

acute encephalopathy. These side-effects happen more often

in the cases that have family history or anamneses of CNS

disorders such as epilepsy [4–6]. Although the usage of

theophylline is decreasing globally because of these serious

side-effects, it is still used in Japan for asthma treatment [7].

As one of the mechanisms of side-effects of theophyl-

line in CNS, it is reported that it reduces cerebral blood

flow [8]. Also, theophylline is known to reduce serum

concentrations of the pyridoxal-50-phosphate, a co-factor of

glutamate decarboxylase which synthesize c-aminobutyric

acid (known to have antiepileptic effect) [9]. Meanwhile,

benzodiazepines such as diazepam are known to be rela-

tively ineffective in controlling TAS [10].

It is well known that functional changes of astrocytes are

involved in mechanisms of various neurodegenerative

diseases such as Alzheimer’s disease or Parkinson’s dis-

ease [11]. Recent studies have shown that astrocytes have

important roles in not only neurodegenerative diseases but

also epilepsy or encephalopathy [12–15]. For example, it is

reported that proliferation of astrocytes has been observed

in hippocampi in patients of epilepsy [16], or glutamate

release from astrocytes may have a key role in the epileptic

seizure [17]. Furthermore, some studies reported that

activation of astrocytes play an important role in influenza-

associated encephalopathy [18–20].

Nitric oxide (NO) production by astrocytes via inducible

NO synthase (iNOS) is well known as an example of

functional changes in cell activation. It is known that iNOS

is induced in astrocytes by various stimulation, including

lipopolysaccharide (LPS) or proinflammatory cytokines

such as interferon (IFN)-c or tumor necrosis factor (TNF)-

a [21]. NO produced by astrocytes promotes glutamate

release from neurons and astrocytes [22] and induces cell

death of them [23]. Therefore, excessive production of NO

from astrocytes might cause various CNS diseases.

Taken together, functional changes of astrocytes by

theophylline might be involved in its side-effects. Thus, we

investigated the effect of theophylline on the cell functions

of astrocytes, such as LPS-induced iNOS expression and

NO production in cultured astrocytes as indices.

Materials and Methods

Materials

LPS (from Salmonella serovar Enteritidis or Escherichia

coli), polyethyleneimine, DNase I, trypsin, and anti-b-actin

antibody were obtained from Sigma Chemical (St. Louis, MO,

USA). Fetal calf serum (FCS) was from PAA Laboratories

(Pasching, Austria) and Nichirei Biosciences (Tokyo, Japan).

Dulbecco’s modified Eagle medium (DMEM) and horse

serum were from Gibco BRL (Grand Island, NY, USA). 2,3-

Diaminonaphthalene (DAN) was from Dojindo (Kumamoto,

Japan). Theophylline, isobutylmethylxanthine (IBMX), di-

butyryl cAMP (dbcAMP), forskolin, and 3-(4,5-dimethylthi-

azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were

from Wako Pure Chemical Industries (Osaka, Japan).

8-Bromoadenosine-30,50-cyclic monophosphorothioate, Rp-

isomer (Rp-8-Br-cAMPS) and 8-bromoguanosine-30,50-cyc-

lic monophosphorothioate, Rp-isomer (Rp-8-Br-cGMPS)

were from Biolog Life Science Institute (Bremen, Germany).

RNeasy Mini kit, Omniscript Reverse Transcription kit, and

Taq PCR Master Mix kit were from Qiagen (Hilden, Ger-

many). SYBR Green Realtime PCR Master Mix was from

Toyobo (Osaka, Japan). iNOS and glyceraldehydes-3-phos-

phate dehydrogenase (GAPDH) primers were obtained from

Operon Biotechnologies (Tokyo, Japan). Horseradish perox-

idase conjugated anti-mouse IgG antibody was from Bio-Rad

(Hercules, CA, USA). iNOS antibody was prepared as

described previously [24].

Preparation of Astrocytes Culture

The present study was carried out in compliance with the

Guide for Animal Experimentation at Osaka Prefecture

University. Cultures of rat astrocyte were prepared as

described previously [25]. Briefly, brain cortices from

20-day-old embryos of Wistar rats were treated with

0.25 % trypsin in Ca2?, Mg2?-free phosphate-buffered

saline containing 5.5 mM glucose for 15 min at 37 �C with

gentle shaking. An equal volume of horse serum supple-

mented with 0.1 mg/ml of DNase I was added to the

medium to inactivate the trypsin, and the tissues were

centrifuged at 3509g for 5 min. The tissue sediments were

triturated with DMEM containing 10 % FCS, 100 mg/l

streptomycin, and 5 9 104 unit/l penicillin. The cells

108 Neurochem Res (2014) 39:107–116

123

were plated on polyethyleneimine-coated plastic dishes

(100 mm diameter, Iwaki), and cultured at 37 �C in

humidified atmosphere of 5 % CO2 and 95 % air. After

1 week, astrocytes were replated to remove neurons. On

day 14, they were replated onto appropriate culture dishes

or plates for each experiment (Corning or Iwaki) at a

density of 4 9 105 cells/ml. In our culture, approximately

95 % of the cells showed immunoreactivity to GFAP, an

astrocyte marker.

Cell Viability

To evaluate cell viability, we measured total mitochondrial

activity with the so-called MTT assay as described previ-

ously [26].

Nitrite Assay

NO release from astrocytes was analyzed by assaying the

levels of nitrite (NO2-), a relatively stable metabolite of

NO. Amounts of NO2- accumulated in the medium were

determined by a fluorometric method using DAN as

described previously [25]. Astrocytes replated onto 96-well

plate were stimulated with 100 ng/ml LPS for 48 h. An

aliquot 100 ll of cell-free supernatants was assayed.

Reverse Transcription-Polymerase Chain Reaction

(RT-PCR)

Total RNA from astrocytes was isolated using an RNeasy

Mini Kit (Qiagen, Hilden, Germany). Complementary

DNA (cDNA) was prepared using Omniscript Reverse

Transcription Kit (Qiagen) according to the manufacturer’s

protocol. PCR experiments were done using Taq PCR

Master Mix Kit. Sequences of the primers are shown

below;

iNOS: forward 50-GGA GAG ATT TTT CAC GAC

ACC CTT C-30

reverse 50-GGT TCC TGT TGT TTC TAT TTC CTT

TGT TAC-30

GAPDH: forward 50-TGC TGA GTA TGT CGT GGA

GTC T-30

reverse 50-AAT GGG AGT TGC TGT TGA AGT C-30

All PCR products were resolved in agarose gels (1.8 %),

and analyzed. Changes in the levels of each PCR product

were calculated as percentages of control using values

normalized to the intensity of the corresponding GAPDH

PCR products.

Western Blotting

Cultured astrocytes were homogenized in 20 mM Tris–HCl

(pH 7.5) buffer containing 1 mM EDTA and protease

inhibitor cocktail (Sigma). Each homogenate was added at

a volume ratio of 4:1–50 mM Tris–HCl buffer (pH 6.8)

containing 50 % glycerol, 10 % sodium dodecyl sulfate,

0.05 % bromophenol blue and 25 % 2-mercaptoethanol,

followed by mixing and boiling at 100 �C for 5 min. Each

aliquot of 20 lg proteins was loaded on a 10 % poly-

acrylamide gel for electrophoresis at a constant voltage of

120 V for 2 h at room temperature and subsequent blotting

to a polyvinylidene fluoride membrane previously treated

with 100 % methanol. After blocking by 5 % skimmed

milk dissolved in 20 mM Tris–HCl buffer (pH 7.5) con-

taining 137 mM NaCl and 0.05 % Tween 20, the mem-

brane was reacted with antibodies against iNOS or b-actin

followed by a reaction with anti-mouse IgG antibody

conjugated with peroxidase. Proteins reactive with those

antibodies were detected with the aid of ECL detection

reagents (Millipore) through exposure to X-ray films, or

analyzed with lumino-image-analyzer (LAS-4000, Fuji-

film). Laser densitometric analysis was performed to

standardize the results of Western blotting. Protein con-

centrations were determined by the method of Bradford

using CBB color solution (Nacalai Tesque, Kyoto, Japan),

according to the manufacturer’s protocol, with bovine

serum albumin as the standard.

Gel Retardation Electrophoresis

Assays were carried out using one of the double stranded

oligonucleotides with a base length of 22-m containing

consensus core sequences for NF-jB as probe for detection

of the corresponding DNA binding activity [26]. Nucleo-

tide sequences of the probe were designed in a manufac-

turer’s kit; Gel Shift Assay Systems (Promega, USA) and

shown below with the respective consensus elements in

bold letters:

NF-jB: 50-AGT TGA GGG GAC TTT CCC AGG C-30

30-TCA ACT CCC CTG AAA GGG TCC G-50

These oligonucleotides were labeled with [c-32P]ATP.

The labeling reaction was carried out using T4 polynu-

cleotide kinase in 70 mM Tris–HCl buffer (pH 7.6) con-

taining 10 mM MgCl2 and 5 mM DTT at 37 �C for

10 min, stopped by the addition of EDTA and TE buffer,

and followed by purification with gel filtration chroma-

tography on a spin column.

Nuclear extracts were prepared according to the proce-

dures described by Takano et al. [26] and stored at -80 �C

until use. Protein concentrations were determined by the

method of Bradford (described above). An aliquot of

nuclear extracts was incubated at a fixed amount of 5 lg

protein in 10 ll 10 mM Tris–HCl buffer (pH 7.5) con-

taining 4 % glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5

mM DTT, 50 mM NaCl, 0.5 lg poly(dI-dC)�poly(dI-dC)

Neurochem Res (2014) 39:107–116 109

123

and 1 ll labeled probe for 30 min at room temperature.

Bound and free probes were separated by electrophoresis on

a 4 % polyacrylamide gel in TBE buffer (pH 8.3) at a con-

stant voltage of 350 V for 15 min at room temperature.

Gels were fixed, followed by exposure to an imaging plate

(Fujifilm) for different periods to obtain autoradiograms

most adequate for quantitative densitometry. Densitometric

analysis for quantification of these autoradiograms was car-

ried out with the aid of a fluoro-image analyzer FLA-7000

(Fujifilm).

Data Analysis

For statistical evaluation, Scheffe’s F test following one-

way ANOVA or Student’s t test was employed. Differences

between treatments were considered statistically significant

when p \ 0.05.

Results

Effects of Theophylline on NO Production

We evaluated the effects of theophylline on NO production

in LPS-stimulated astrocytes. NO2- accumulation was

significantly increased when astrocytes were incubated

with 100 ng/ml LPS for 48 h. Although NO production

was not observed when astrocytes were incubated with

theophylline alone, LPS (100 ng/ml)-induced NO produc-

tion was increased by addition of 500 lM theophylline

(Fig. 1a). The drugs at the concentration tested did not

affect the cell viability (data not shown).

Time course of LPS-induced NO production was

examined. Although theophylline was not significantly

affected LPS-induced NO production for 24 h, it remark-

ably enhanced after 36 or 48 h incubation (Fig. 1b).

Potentiation iNOS mRNA and iNOS Protein

Expression by Theophylline

To assess whether enhancement of NO production by

theophylline is through the increase of iNOS mRNA and

protein, we assayed by RT-PCR and Western blotting

procedures. The cells were stimulated by 100 ng/ml LPS

with or without 500 lM theophylline for 6 h. The LPS-

induced expression of iNOS mRNA was significantly

enhanced by theophylline (Fig. 2a). Similarly, the expres-

sion level of iNOS protein was examined, in stimulated

cells by 100 ng/ml LPS with or without 500 lM theoph-

ylline for 24 h. The expression levels of iNOS protein were

significantly enhanced more than twice by theophylline

(Fig. 2b).

NF-jB Transcription Activity

To analyze more detail of mechanisms of enhancement of

LPS-induced NO production by theophylline, we assessed

LPS-induced NF-jB activation by gel shift assay. Cultured

astrocytes were stimulated by 100 ng/ml LPS with or

without 500 lM theophylline for 1 h and the nuclear

extracts were prepared from each condition. Samples

incubated with the radiolabeled probe for NF-jB, followed

by gel retardation electrophoresis. The NF-jB-specific

DNA binding activity was not detected in nuclear extracts

obtained from control or theophylline-treated cells. LPS

markedly increased the NF-jB-specific DNA binding

activity, and theophylline potentiated the LPS-stimulated

increase in DNA binding activity (Fig. 3).

0

2

4

6

8

10

0 30 100 300 500

theophylline ( M)

NO

2(

M)

(B)(A)controltheophylline 500 MLPS 100 ng/mlLPS 100 ng/ml + theophylline 500 M

Time (hrs)

0

5

10

15

0 10 20 30 40 50

NO

2(

M)

LPS -LPS 100 ng/ml

μ

μ

μ

μ μ

Fig. 1 Effect of theophylline on LPS-induced NO production in

cultured astrocytes. a Dose-dependence: Astrocytes were stimulated

with 100 ng/ml LPS in the presence of various concentrations of

theophylline for 48 h. b Time course: The cells were stimulated

100 ng/ml LPS and 500 lM theophylline for indicated time. After the

stimulation, the concentration of nitrite in the medium was assayed.

Data are mean ± SEM of eight samples from independent experi-

ments. *p \ 0.05 significantly different from 100 ng/ml LPS by

ANOVA/Scheffe’s F test

110 Neurochem Res (2014) 39:107–116

123

Potentiation of NO Production and iNOS Expression

by IBMX

To determine whether the effect of theophylline is due to

PDE inhibition, we examined the effect of IBMX, a typical

non-specific PDE inhibitor for research use. When astro-

cytes were incubated with LPS (100 ng/ml) and various

concentrations of IBMX for 48 h, LPS-induced NO pro-

duction was enhanced by IBMX dose-dependently;

whereas IBMX alone did not induce NO production

(Fig. 4a). In addition, the extent of iNOS protein expres-

sion in astrocytes stimulated with LPS for 24 h was

enhanced about twice by the presence of 100 lM IBMX

(Fig. 4b).

Potentiation of NO Production and iNOS Expression

by dbcAMP

Next, we examined the effect of dbcAMP, a membrane-

permeable cAMP analog. dbcAMP potentiated LPS-

induced NO production in a dose-dependent manner

(Fig. 5a). Furthermore, the enhanced effect of 30 lM

dbcAMP on LPS-induced NO production was potentiated

by addition of theophylline (Fig. 5b). However, when

dbcAMP was 100 lM, no further potentiation was seen by

theophylline. dbcAMP alone or dbcAMP/theophylline

stimulation did not induce NO production (data not

shown).

LPS-induced iNOS protein expression was enhanced by

30 lM dbcAMP. This enhancement of iNOS protein

expression was increased further by 500 lM theophylline

(Fig. 5c). dbcAMP alone or dbcAMP/theophylline did not

induce iNOS protein.

iNOS

GAPDH

iNOS mRNA

–theophylline

iNO

S/G

AP

DH

rat

io(%

of L

PS

)

LPS 100 ng/ml

0

50

100

150

(A) (B)

iNO

S/

-act

in r

atio

(% o

f LP

S)

iNOS Protein

0

100

200

300

LPS 100 ng/ml

iNOS

-actin

theophylline+ – + – + – +

β

β

Fig. 2 Effect of theophylline on LPS-induced expressions of iNOS

mRNA and protein. a The cells were stimulated by 100 ng/ml LPS

with or without 500 lM theophylline for 6 h. After stimulation, iNOS

mRNA was analyzed by RT-PCR methods. The lower graph shows

the results from semi-quantitative analysis of the intensity of detected

bands; GAPDH as internal standard and the values in the presence of

100 ng/ml LPS as 100 %. b The cells were stimulated by 100 ng/ml

LPS with or without 500 lM theophylline for 24 h. After stimulation,

the cell lysates were subjected to western blotting with anti-iNOS and

anti-b-actin antibodies. The intensity of iNOS protein was quantified

and standardized with that of b-actin. The graph shows the values in

the presence of 100 ng/ml LPS as 100 %. Data are mean ± SEM of

five (a) or nine (b) samples from independent experiments. *p \ 0.05

significantly different from 100 ng/ml LPS by ANOVA/Scheffe’s

F test

LPS

– + – +

LPS 100 ng/ml

NF

-B

act

ivity

(%

of L

PS

)

0

50

100

150

200

NF- B

theophylline

κ

κ

Fig. 3 Effects of theophylline on LPS-induced NF-jB activation.

The cells were stimulated with 100 ng/ml LPS with or without

500 lM theophylline for 1 h. After the stimulation, nuclear extracts

were obtained from the cells and subjected to gel shift assay with

radiolabeled consensus oligonucleotide for NF-jB (see ‘‘Materials

and Methods’’). Quantification of the autoradiogram is shown in a

graph. The graph shows the values in the presence of 100 ng/ml LPS

as 100 %. Data are mean ± SEM of four samples from independent

experiments. *p \ 0.05 significantly different from 100 ng/ml LPS

by ANOVA/Scheffe’s F test

Neurochem Res (2014) 39:107–116 111

123

Potentiation of NO Production and iNOS Expression

by Forskolin

We examined the effect of forskolin, an activator of

adenylate cyclase, on NO production and iNOS expression.

Forskolin potentiated LPS-induced NO production in a

dose-dependent manner (Fig. 6a). Moreover, the enhanced

effect by 1.0 lM forskolin on LPS-induced NO production

was further potentiated by addition of theophylline, and

forskolin dose-dependently potentiated the effect of

250 lM theophylline (Fig. 6b). Forskolin alone or for-

skolin/theophylline stimulation did not induce NO pro-

duction (data not shown).

LPS-induced iNOS protein expression was enhanced by

1.0 lM forskolin. This enhancement of iNOS protein

expression was increased more by 500 lM theophylline

(A) (B)

LPS -LPS 100 ng/ml

IBMX ( M)

0

4

8

12

Cont DMSO 10 30 100

NO

2(

M)

iNOS

-actin

0

50

100

150

200

250

– IBMX – IBMX

iNO

S/

-act

in r

atio

(% o

f LP

S 1

00 n

g/m

l)

LPS 100 ng/ml

β

β

μ

μ

Fig. 4 Effects of IBMX on LPS-induced NO production. a The cells

were stimulated with 100 ng/ml LPS in the presence of various

concentrations of IBMX for 48 h. After the stimulation, the concen-

tration of nitrite in the medium was assayed. b The cells were

stimulated by 100 ng/ml LPS with or without 100 lM IBMX for

24 h. After stimulation, the cell lysates were subjected to western

blotting with anti-iNOS and anti-b-actin antibodies. The intensity of

iNOS protein was quantified and standardized with that of b-actin.

The graph shows the values in the presence of 100 ng/ml LPS as

100 %. Data are mean ± SEM of eight (a) or four (b) samples from

independent experiments. *p \ 0.05 significantly different from

100 ng/ml LPS by ANOVA/Scheffe’s F test

(A) (B) (C)

Fig. 5 Effects of dbcAMP on LPS-induced NO production. a The

cells were stimulated with 100 ng/ml LPS in the presence of various

concentrations of dbcAMP for 48 h. b The cells were stimulated with

100 ng/ml LPS in the presence of various concentrations of theoph-

ylline and/or dbcAMP for 48 h. After the stimulation, the concen-

tration of nitrite in the medium was assayed. c The cells were

stimulated by 100 ng/ml LPS with or without 500 lM theophylline

and/or 100 lM dbcAMP for 24 h. After stimulation, the cell lysates

were subjected to western blotting with anti-iNOS and anti-b-actin

antibodies. The intensity of iNOS protein was quantified and

standardized with that of b-actin. The graph shows the values in

the presence of 100 ng/ml LPS as 100 %. Data are mean ± SEM of

four samples from independent experiments. **p \ 0.01 significantly

different from 100 ng/ml LPS; #p \ 0.05 significantly different from

100 ng/ml LPS ? 30 lM dbcAMP by ANOVA/Scheffe’s F test

112 Neurochem Res (2014) 39:107–116

123

(A) (B) (C)

Fig. 6 Effects of forskolin on LPS-induced NO production. a The

cells were stimulated with 100 ng/ml LPS in the presence of various

concentrations of forskolin for 48 h. b The cells were stimulated with

100 ng/ml LPS in the presence of various concentrations of theoph-

ylline and/or forskolin for 48 h. After the stimulation, the concen-

tration of nitrite in the medium was assayed. c The cells were

stimulated by 100 ng/ml LPS with or without 500 lM theophylline

and/or 1 lM forskolin for 24 h. After stimulation, the cell lysates

were subjected to western blotting with anti-iNOS and anti-b-actin

antibodies. The intensity of iNOS protein was quantified and

standardized with that of b-actin. The graph shows the values in

the presence of 100 ng/ml LPS as 100 %. Data are mean ± SEM of

four (a, b) or three (c) samples from independent experiments.

*p \ 0.05 significantly different from 100 ng/ml LPS; #p \ 0.05

significantly different from 100 ng/ml LPS ? 0.1 lM forskolin;$p \ 0.05 significantly different from 100 ng/ml LPS ? 1.0 lM

forskolin by ANOVA/Scheffe’s F test

(A)

(B)

(C)

Fig. 7 Effects of PKG and PKA inhibitors on LPS-induced NO

production. The cells were preincubated for 20 min with 50 lM Rp-

8-Br-cGMPS (a) or Rp-8-Br-cAMPS (b) and stimulated by 100 ng/ml

LPS with or without 500 lM theophylline for 48 h. After the

stimulation, the concentration of nitrite in the medium was assayed.

c The cells were preincubated with Rp-8-Br-cAMPS and stimulated

by 100 ng/ml LPS with or without 500 lM theophylline for 24 h.

After stimulation, the cell lysates were subjected to western blotting

with anti-iNOS and anti-b-actin antibodies. The intensity of iNOS

protein was quantified and standardized with that of b-actin. The

graph shows the values in the presence of 100 ng/ml LPS as 100 %.

Data are mean ± SEM of eight (a, b) or three (c) samples from

independent experiments. *p \ 0.05, **p \ 0.01 significantly differ-

ent from 100 ng/ml LPS; #p \ 0.05, ##p \ 0.01 significantly different

from 100 ng/ml LPS ? 500 lM theophylline by ANOVA/Scheffe’s

F test (a, b) or Student’s t test (c)

Neurochem Res (2014) 39:107–116 113

123

(Fig. 6c). Forskolin alone or forskolin/theophylline did not

induce iNOS protein.

PKA Inhibitor Inhibited Enhancement of LPS-Induced

NO Production by Theophylline

To assess the involvement of protein kinases, we examined

the effect of inhibitors for cAMP-dependent protein kinase

(PKA) and cGMP-dependent protein kinase (PKG).

Astrocytes were incubated with a PKA inhibitor; Rp-8-Br-

cAMPS (50 lM), or a PKG inhibitor; Rp-8-Br-cGMPS

(50 lM) for 20 min, and then 100 ng/ml LPS and/or

500 lM theophylline were added to culture medium, and

further incubated for 48 h. Rp-8-Br-cGMPS had no effect

on LPS-induced NO production or enhancement by the-

ophylline (Fig. 7a). On the other hand, when cells were

incubated with Rp-8-Br-cAMPS, it inhibited the enhanced

effect of theophylline on LPS-induced NO production

(Fig. 7b). Rp-8-Br-cAMPS or Rp-8-Br-cGMPS alone or

these inhibitors/theophylline did not induce NO

production.

Moreover, we assessed the effect of PKA inhibitor on

iNOS protein expression by western blotting. Pre-treatment

of 50 lM Rp-8-Br-cAMPS cancelled the theophylline

enhancement of LPS-induced iNOS protein expression

(Fig. 7c). Rp-8-Br-cAMPS alone or its pre-treatment/

theophylline did not induce iNOS protein expression (data

not shown).

Discussion

Exposure to LPS has been believed to stimulate intracel-

lular signaling pathways mediated by NF-jB through

activation of toll-like receptor-4, which is expressed on

cellular surfaces in cells other than neurons in the CNS [21,

27]. Moreover, activation of toll-like receptor-4 by LPS

induces a variety of cytokines, such as TNF-a [28] and

IFN-c [29], in addition to proinflammatory proteins

including iNOS [30], through NF-jB signaling pathway

[31]. In our preliminary experiments, LPS stimulated NF-

jB activation with time, and the activation was peaked at

1 h and decreased for several hours thereafter. NF-jB

activation induced iNOS mRNA and protein expressions,

they could be detected at 3 and 6 h, respectively, and

continued to increase by 24 h after LPS stimulation.

Moreover, theophylline significantly potentiated LPS-

induced NO production at 36 h. Therefore we assessed NF-

jB activation at 1 h, iNOS mRNA at 6 h, iNOS protein at

24 h after LPS stimulation, and all of these LPS-induced

changes were augmented by theophylline.

Theophylline, one of methylxanthine derivatives, is a

bronchodilator and three mechanisms of action have been

known; PDE inhibition, adenosine receptor antagonism and

Ca2? mobilization from intracellular store. Eleven iso-

zymes of PDE have been known; among them, three iso-

zymes are specific for cAMP and the other three are

specific for cGMP, and the rest five isozymes are active for

both of cAMP and cGMP [32]. Theophylline is a nonspe-

cific inhibitor of PDE. It has been reported that the increase

of intracellular cAMP levels suppressed proinflammatory

cytokines- or LPS-stimulated iNOS expression in several

cells [33]. However, iNOS expression and NO production

in astrocytes have been reported to be both enhanced [34,

35] and suppressed [36, 37] by cAMP. In the present study,

theophylline might enhance LPS-induced iNOS expression

and NO production through intracellular cAMP levels and

PKA activation via PDE inhibition activity because a PKA

inhibitor blocked enhanced NO production by theophylline

but not a PKG inhibitor. It has been reported that intra-

cellular cAMP levels decrease via adenosine A1 and A3

receptors and increase via A2A and A2B receptors and that

astrocytes express all of these four adenosine receptor

subtypes [38]. Furthermore, adenosine receptors have been

reported to modulate intracellular Ca2? levels in astrocytes

[39]. Therefore, complicated actions might be associated

with the enhancement effect of theophylline on LPS-

induced NO production, especially in long-lasting cell

incubation for more than 24 h. Further investigation should

be needed on the mechanisms of theophylline action other

than PDE inhibition in astrocytes.

Recent studies have shown that astrocytes play impor-

tant roles in epilepsy or encephalopathy [12]. It is reported

that activation of astrocytes such as increase of iNOS and

GFAP expressions was detected in influenza-associated

encephalopathy [19] and that NO release in cerebrospinal

fluid from astrocytes may cause influenza-associated

encephalopathy [18, 20]. Influenza infection has been

thought to be a risk factor of TAS and theophylline-asso-

ciated encephalopathy [40]; conformed with our present

study that theophylline did not induce NO production itself

but enhanced LPS-induced NO production. Moreover, it

also reported that proinflammatory cytokines such as TNF-

a and IFN-c were induced in brain of epilepsy [41, 42].

Proinflammatory cytokines could induce activation of

astrocytes, glutamate release and iNOS expression [21].

NO production might mediate cytokine-induced enhance-

ment calcium-dependent glutamate release in activated

astrocytes [43]. In astrocytes, intracellular Ca2? levels and

NO production could modulate each other and mediate

glutamate release [44, 45]. Therefore, enhancement of

iNOS expression and NO production by theophylline might

bring on aggravation of convulsive seizure through aug-

mentation of glutamate release from astrocytes.

It was reported that theophylline showed an increasing

acute bronchodilator response above plasma concentrations of

114 Neurochem Res (2014) 39:107–116

123

10 mg/l (55 lM) and that the upper recommended plasma

concentration was 20 mg/l due to unacceptable side effects

above this level [1]. In Japan, it is regarded that theophylline

metabolism varies according to fever and other factors when

administering aminophylline, and that the target serum the-

ophylline concentration is 5–15 mg/l [7]. In the present study,

theophylline potentiated LPS-induced NO production signif-

icantly at 500 lM and had upward tendency at 300 lM.

Moreover, the increase of intracellular cAMP levels by

dbcAMP or forskolin augmented the enhancement of LPS-

induced iNOS expression and NO production by theophylline.

On the basis of these results, we believed that enhancement of

NO production could be involved in side effects of

theophylline.

In summary, theophylline enhanced LPS-induced NO

production through the enhancement of NF-jB activation,

and iNOS mRNA and protein expressions in cultured

astrocytes. PKA activation was also involved in the

enhanced effects of theophylline. Excess NO production

from astrocytes by theophylline might be a cause of side-

effects such as TAS.

Acknowledgments This work was supported in part by Grants in

Aid for Scientific Research to Y.N., 24621008 and to M.M.,

23580408 from the Ministry of Education, Science and Culture of

Japan.

References

1. Barnes PJ (2003) Theophylline: new perspectives for an old drug.

Am J Respir Crit Care Med 167:813–818

2. Sullivan P, Bekir S, Jaffar Z, Page C, Jeffery P, Costello J (1994)

Anti-inflammatory effects of low-dose oral theophylline in atopic

asthma. Lancet 343:1006–1008

3. Watanabe S, Yamakami J, Tsuchiya M, Terajima T, Kizu J, Hori

S (2008) Anti-inflammatory effect of theophylline in rats and its

involvement of the glucocorticoid–glucocorticoid receptor sys-

tem. J Pharmacol Sci 106:566–570

4. Korematsu S, Miyahara H, Nagakura T, Suenobu S, Izumi T

(2008) Theophylline-associated seizures and their clinical char-

acterizations. Pediatr Int 50:95–98

5. Kuwahara H, Noguchi Y, Inaba A, Mizusawa H (2008) Case of

an 81-year-old woman with theophylline-associated seizures

followed by partial seizures due to vitamin B6 deficiency. Rinsho

Shinkeigaku 48:125–129

6. El-Bitar MK, Boustany RM (2009) Common causes of uncom-

mon seizures. Pediatr Neurol 41:83–87

7. Morikawa A, Nishima S (2007) New Japanese pediatric guide-

lines for the treatment and management of bronchial asthma.

Pediatr Int 49:1023–1031

8. Usui H, Okubo O, Hashimoto K, Harada K (2002) Effects of theoph-

ylline on cerebral blood flow. J Jpn Pediatr Soc 106:854–859 Japanese

9. Tanaka I, Ito Y, Hiraga Y, Fujino M, Kobayashi K (1996) Serum

concentrations of the pyridoxal and pyridoxal-50-phosphate in

children during sustained–release theophylline therapy. Arerugi

45:1098–1105

10. Yoshikawa H (2007) First-line therapy for theophylline-associ-

ated seizures. Acta Neurol Scand 115:57–61

11. Rossi D, Volterra A (2009) Astrocytic dysfunction: insights on

the role in neurodegeneration. Brain Res Bull 80:224–232

12. Seifert G, Schilling K, Steinhauser C (2006) Astrocyte dysfunc-

tion in neurological disorders: a molecular perspective. Nat Rev

Neurosci 7:194–206

13. Sidoryk-Wegrzynowicz M, Wegrzynowicz M, Lee E, Bowman

AB, Aschner M (2011) Role of astrocytes in brain function and

disease. Toxicol Pathol 39:115–123

14. Verkhratsky A, Rodrıguez JJ, Parpura V (2013) Astroglia in

neurological diseases. Future Neurol 8:149–158

15. Eid T, Tu N, Lee TS, Lai JC (2013) Regulation of astrocyte

glutamine synthetase in epilepsy. Neurochem Int 63:670–681

16. Choi J, Koh S (2008) Role of brain inflammation in epilepto-

genesis. Yonsei Med J 49:1–18

17. Tian G-F, Azmi H, Takano T, Xu Q, Peng W, Lin J, Oberheim N,

Lou N, Wang X, Zielke HR, Kang J, Nedergaard M (2005) An

astrocytic basis of epilepsy. Nat Med 11:973–981

18. Kawashima H, Amaha M, Ioi H, Yamanaka G, Kashiwagi Y,

Sasamoto M, Takekuma K, Hoshika A, Watanabe Y (2005)

Nitrite/nitrate (NOx) and zinc concentrations in influenza-asso-

ciated encephalopathy in children with different sequela. Neu-

rochem Res 30:311–314

19. Watanabe C, Kawashima H, Takekuma K, Hoshika A, Watanabe

Y (2008) Increased nitric oxide production and GFAP expression

in the brains of influenza A/NWS virus infected mice. Neuro-

chem Res 33:1017–1023

20. Kakita H, Aoyama M, Hussein MH, Kato S, Suzuki S, Ito T,

Togari H, Asai K (2009) Diclofenac enhances proinflammatory

cytokine-induced nitric oxide production through NF-jB signal-

ing in cultured astrocytes. Toxicol Appl Pharmacol 238:56–63

21. Saha RN, Pahan K (2006) Signals for the induction of nitric oxide

synthase in astrocytes. Neurochem Int 49:154–163

22. Bal-Price A, Brown GC (2001) Inflammatory neurodegeneration

mediated by nitric oxide from activated glia-inhibiting neuronal

respiration, causing glutamate release and excitotoxicity. J Neu-

rosci 21:6480–6491

23. Boje KM (2004) Nitric oxide neurotoxicity in neurodegenerative

diseases. Front Biosci 9:763–776

24. Nakamura Y, Kitagawa T, Ihara H, Kozaki S, Moriyama M,

Kannan Y (2006) Potentiation by high potassium of lipopoly-

saccharide-induced nitric oxide production from cultured astro-

cytes. Neurochem Int 48:43–49

25. Murakami K, Nakamura Y, Yoneda Y (2003) Potentiation by

ATP of lipopolysaccharide-stimulated nitric oxide production in

cultured astrocytes. Neuroscience 117:37–42

26. Takano K, Sugita K, Moriyama M, Hashida K, Hibino S, Choshi

T, Murakami R, Yamada M, Suzuki H, Hori O, Nakamura Y

(2011) A dibenzoylmethane derivative protects against hydrogen

peroxide-induced cell death and inhibits lippolysaccharide-

induced nitric oxide production in cultured rat astrocytes. J Neu-

rosci Res 89:955–965

27. Okun E, Griffioen KJ, Lathia JD, Tang S-C, Mattson MP,

Arumugam TV (2009) Toll-like receptors in neurodegeneration.

Brain Res Rev 59:278–292

28. Martin L, Pingle SC, Hallam DM, Rybak LP, Ramkumar V

(2006) Activation of the adenosine A3 receptor in RAW 264.7

cells inhibits lipopolysaccharide-stimulated tumor necrosis fac-

tor-alpha release by reducing calcium-dependent activation of

nuclear factor-kappaB and extracellular signal-regulated kinase

1/2. J Pharmacol Exp Ther 316:71–78

29. Faure E, Thomas L, Xu H, Medvedev A, Equils O, Arditi M

(2001) Bacterial lipopolysaccharide and IFN-gamma induce Toll-

like receptor 2 and Toll-like receptor 4 expression in human

endothelial cells: role of NF-kappa B activation. J Immunol

166:2018–2024

Neurochem Res (2014) 39:107–116 115

123

30. Lee JY, Lowell CA, Lemay DG, Youn HS, Rhee SH, Sohn KH,

Jang B, Ye J, Chung JH, Hwang DH (2005) The regulation of the

expression of inducible nitric oxide synthase by Src-family tyrosine

kinases mediated through MyD88-independent signaling pathways

of Toll-like receptor 4. Biochem Pharmacol 70:1231–1240

31. Frantz S, Kobzik L, Kim YD, Fukazawa R, Medzhitov R, Lee RT,

Kelly RA (1999) Toll4 (TLR4) expression in cardiac myocytes in

normal and failing myocardium. J Clin Invest 104:271–280

32. Bollen E, Prickaerts J (2012) Phosphodiesterases in neurode-

generative disorders. IUBMB Life 64:965–970

33. Galea E, Feinstein DL (1999) Regulation of the expression of the

inflammatory nitric oxide synthase (NOS2) by cyclic AMP.

FASEB J 13:2125–2137

34. Lee J, Ryu H, Ferrante RJ, Morris SM Jr, Ratan RR (2003)

Translational control of inducible nitric oxide synthase expres-

sion by arginine can explain the arginine paradox. Proc Natl Acad

Sci USA 100:4843–4848

35. Hsiao H-Y, Mak O-T, Yang C-S, Liu Y-P, Fang K-M, Tzeng S-F

(2007) TNF-a/IFN-c-induced iNOS expression increased by

prostaglandin E in rat primary astrocytes via EP2-evoked cAMP/

PKA and intracellular calcium signaling. Glia 55:214–223

36. Pahan K, Namboodiri AMS, Sheikh FG, Smith BT, Singh I

(1997) Increasing cAMP attenuates induction of inducible nitric-

oxide synthase in rat primary astrocytes. J Biol Chem

272:7786–7791

37. Kozuka N, Kudo Y, Morita M (2007) Multiple inhibitory path-

ways for lipopolysaccharide- and pro-inflammatory cytokine-

induced nitric oxide production in cultured astrocytes. Neuro-

science 144:911–919

38. Dare E, Schulte G, Karovic O, Hammarberg C, Fredholm BB

(2007) Modulation of glial cell functions by adenosine receptors.

Physiol Behav 92:15–20

39. Porter JT, McCarthy KD (1995) Adenosine receptors modulate

[Ca2?]i in hippocampal astrocytes in situ. J Neurochem

65:1515–1523

40. Mizoguchi M, Yamanouchi H, Ichiyama T, Shiomi M (2007)

Acute encephalopathy associated with influenza and other viral

infections. Acta Neurol Scand Suppl 186:45–56

41. Vezzani A, Granata T (2005) Brain inflammation in epilepsy:

experimental and clinical evidence. Epilepsia 46:1724–1743

42. Vezzani A, Balosso S, Ravizza T (2008) The role of cytokines in

the pathophysiology of epilepsy. Brain Behav Immun 22:

797–803

43. Ida T, Hara M, Nakamura Y, Kozaki S, Tsunoda S, Ihara H

(2008) Cytokine-induced enhancement of calcium-dependent

glutamate release from astrocytes mediated by nitric oxide.

Neurosci Lett 432:232–236

44. Li N, Sul J-Y, Haydon PG (2003) A calcium-induced calcium

influx factor, nitric oxide, modulates the refilling of calcium

stores in astrocytes. J Neurosci 23:10302–10310

45. Santello M, Volterra A (2009) Synaptic modulation by astrocytes

via Ca2?-dependent glutamate release. Neuroscience 158:253–259

116 Neurochem Res (2014) 39:107–116

123