MPP

induced

cytotoxicity

in

neuroblastoma

cells

Antagonism

and

reversal

by

guanosine

Guanosine

exerts

neuroprotective

effects

in

the

central

nervous

system

Apoptosis

a

morphological

form

of

programmed

cell

death

is

implicated

in

the

pathophysiology

of

Parkinson

's

disease

-LRB-

PD

-RRB-

MPP

a

dopaminergic

neurotoxin

produces

in

vivo

and

in

vitro

cellular

changes

characteristic

of

PD

such

as

cytotoxicity

resulting

in

apoptosis

Undifferentiated

human

neuroblastoma

cells

had

been

used

as

an

in

vitro

model

of

Parkinson

's

disease

We

investigated

if

extracellular

guanosine

affected

MPP

induced

cytotoxicity

and

examined

the

molecular

mechanisms

mediating

its

effects

Exposure

of

neuroblastoma

cells

to

MPP

-LRB-

μM

mM

for

h

-RRB-

induced

DNA

fragmentation

in

a

time-dependent

manner

-LRB-

p

-RRB-

Administration

of

guanosine

-LRB-

μM

-RRB-

before

concomitantly

with

or

importantly

after

the

addition

of

MPP

abolished

MPP

induced

DNA

fragmentation

Addition

of

MPP

-LRB-

μM

-RRB-

to

cells

increased

activity

over

h

-LRB-

p

-RRB-

and

this

was

abolished

by

pre

or

co-treatment

with

guanosine

Exposure

of

cells

to

pertussis

toxin

prior

to

MPP

eliminated

the

anti-apoptotic

effect

of

guanosine

indicating

that

this

effect

is

dependent

on

a

Gi

protein-coupled

receptor

most

likely

the

putative

guanosine

receptor

The

protection

by

guanosine

was

also

abolished

by

the

selective

inhibitor

of

the

enzyme

Akt/PKB

-LRB-

-RRB-

confirming

that

this

pathway

plays

a

decisive

role

in

this

effect

of

guanosine

Neither

MPP

nor

guanosine

had

any

significant

effect

on

α-synuclein

expression

Thus

guanosine

antagonizes

and

reverses

MPP

induced

cytotoxicity

of

neuroblastoma

cells

via

activation

of

the

cell

survival

pathway

Akt/PKB

Our

results

suggest

that

guanosine

may

be

an

effective

pharmacological

intervention

in

PD

Introduction

Parkinson

's

disease

-LRB-

PD

-RRB-

is

a

progressive

neurodegenerative

disorder

caused

primarily

by

selective

degeneration

of

dopaminergic

neurons

in

the

substantia

nigra

pars

compacta

-LSB-

-RSB-

Apoptosis

a

morphological

form

of

programmed

cell

death

-LSB-

-RSB-

has

been

implicated

in

the

pathophysiology

of

PD

-LSB-

-RSB-

The

biochemical

pathways

that

mediate

apoptosis

may

be

initiated

by

extrinsic

-LRB-

e.g.

activation

of

the

death

receptors

-RRB-

or

intrinsic

factors

-LRB-

e.g.

mitochondrial

insult

-RRB-

-LSB-

-RSB-

and

result

in

the

activation

of

a

set

of

cysteine-dependent

aspartyl

proteases

-LRB-

caspases

-RRB-

-LSB-

-RSB-

Both

apoptotic

pathways

stimulate

the

effector

enzyme

-LSB-

-RSB-

which

in

turn

activates

a

specific

nuclear

DNAse

that

degrades

DNA

into

base

pair

oligonucleosomal

fragments

a

hallmark

of

apoptosis

-LSB-

-RSB-

Apoptosis-related

alterations

have

been

reported

in

the

dopaminergic

brain

regions

in

post-mortem

tissues

of

PD

patients

including

increased

activity

in

the

substantia

nigra

-LSB-

-RSB-

and

elevated

immunoreactivity

of

the

pro-apoptotic

protein

Bax

-LSB-

-RSB-

indicating

that

apoptosis

may

play

a

role

in

the

pathophysiology

of

PD

The

neurotoxin

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

-LRB-

MPTP

-RRB-

has

been

used

extensively

to

study

PD

-LSB-

-RSB-

MPTP

selectively

targets

and

damages

the

dopaminergic

neurons

causing

parkinsonism

in

humans

and

other

primates

-LSB-

-RSB-

MPTP

readily

crosses

the

blood-brain

barrier

and

is

converted

into

the

active

metabolite

pyridinium

-LRB-

MPP

-RRB-

-LSB-

-RSB-

MPP

is

taken

up

by

the

dopaminergic

neurons

and

accumulates

in

the

mitochondria

where

it

inhibits

complex

I

of

the

electron

transport

chain

and

ultimately

causes

neuronal

cell

death

-LSB-

-RSB-

The

mechanisms

of

MPP

induced

cytotoxicity

most

likely

involve

oxidative

stress

-LSB-

-RSB-

Several

groups

reported

that

treatment

of

mice

with

MPTP

induces

a

Parkinson-like

syndrome

-LSB-

-RSB-

accompanied

by

apoptosis

of

the

dopaminergic

neurons

in

the

substantia

nigra

-LSB-

-RSB-

and

up-regulation

of

α-synuclein

a

pre-synaptic

protein

which

plays

an

important

role

in

PD

pathogenesis

-LSB-

-RSB-

In

vitro

studies

using

human

neuroblastoma

cells

have

produced

comparable

results

-LSB-

-RSB-

Exposure

of

these

cells

to

MPP

led

to

fragmentation

of

nuclear

DNA

-LSB-

-RSB-

activation

of

-LSB-

-RSB-

and

to

up-regulation

of

α-synuclein

-LSB-

-RSB-

In

cultured

cells

dopamine-dependent

cytotoxicity

was

enhanced

by

the

accumulation

of

α-synuclein

-LSB-

-RSB-

Therefore

targeting

the

molecular

pathways

activated

during

apoptosis

may

lead

to

novel

treatment

strategies

for

PD

-LSB-

-RSB-

Guanosine

and

other

non-adenine-based

purines

exert

neuroprotective

effects

in

the

central

nervous

system

-LSB-

-RSB-

In

vitro

guanosine

also

protects

rat

astrocytes

from

staurosporine-induced

apoptosis

-LSB-

-RSB-

and

cells

from

β-amyloid-induced

apoptosis

-LSB-

-RSB-

In

both

cases

the

anti-apoptotic

effect

of

guanosine

was

mediated

by

stimulation

of

the

-LRB-

-RRB-

Akt/protein

kinase

B

-LRB-

PKB

-RRB-

and

mitogen-activated

protein

kinase

-LRB-

MAPK

-RRB-

cell

survival

pathways

-LSB-

-RSB-

These

pathways

were

activated

via

the

pertussis

toxin-sensitive

Gi

protein-coupled

putative

guanosine

receptor

that

we

have

recently

characterized

pharmacologically

-LSB-

-RSB-

In

PD

symptoms

appear

only

after

the

majority

of

the

dopaminergic

neurons

in

the

substantia

nigra

pars

compacta

have

degenerated

so

putative

neuroprotective

drugs

for

PD

should

be

tested

in

models

in

which

the

neurodegenerative

process

is

already

underway

-LSB-

-RSB-

Therefore

we

examined

the

effects

of

guanosine

on

MPP

induced

activation

and

DNA

fragmentation

in

human

neuroblastoma

cells

not

only

when

it

was

added

prior

to

or

at

the

same

time

as

MPP

but

also

when

it

was

added

to

the

cultures

up

to

h

after

MPP

treatment

when

the

pro-apoptotic

pathways

were

already

activated

We

also

studied

the

molecular

mechanisms

that

mediate

the

neuroprotective

effects

of

guanosine

Materials

and

methods

Tissue

culture

and

treatment

of

human

neuroblastoma

cells

Human

neuroblastoma

cells

-LRB-

ATCC

Manassas

VA

USA

-RRB-

were

cultured

using

minimum

essential

medium

Hamilton

's

medium

supplemented

with

fetal

bovine

serum

-LRB-

FBS

-RRB-

U/ml

of

penicillin

and

U/ml

of

streptomycin

at

a

pH

of

-LRB-

all

from

Invitrogen

Burlington

ON

Canada

-RRB-

Culture

medium

was

changed

every

days

and

cells

were

maintained

in

a

humidified

atmosphere

at

°C

and

subcultured

at

a

ratio

of

every

days

Culture

medium

was

changed

to

FBS

for

h

before

the

start

of

each

experiments

All

experiments

were

performed

using

confluent

cultures

Apoptosis

was

induced

by

adding

MPP

-LRB-

Sigma-Aldrich

-RRB-

to

neuroblastoma

cells

using

a

fresh

stock

solution

of

MPP

-LRB-

mM

in

reverse

osmosis

purified

-RRB-

In

all

experiments

guanosine

-LRB-

Sigma-Aldrich

-RRB-

was

dissolved

in

M

NaOH

and

added

to

the

culture

medium

at

a

final

concentration

of

M

NaOH

Some

cells

-LRB-

control

-RRB-

were

exposed

to

M

NaOH

In

experiments

in

which

the

protective

effect

of

guanosine

was

tested

guanosine

-LRB-

μM

-RRB-

was

added

to

neuroblastoma

cells

either

h

prior

to

the

administration

of

MPP

-LRB-

pre-treatment

-RRB-

or

at

the

same

time

as

MPP

-LRB-

co-treatment

-RRB-

or

or

h

after

the

addition

of

MPP

-LRB-

post-treatment

at

h

or

post-treatment

at

h

respectively

Fig.

-RRB-

Guanosine

and

MPP

remained

in

the

culture

medium

for

the

duration

of

the

experiment

-LRB-

or

h

-RRB-

In

the

first

post-treatment

experimental

group

at

h

cells

were

exposed

to

MPP

for

h

followed

by

MPP

plus

guanosine

for

an

additional

h

-LRB-

Fig.

-RRB-

In

the

second

post-treatment

experimental

group

at

h

cells

were

treated

with

MPP

for

h

followed

by

MPP

plus

guanosine

for

an

additional

h

-LRB-

Fig.

-RRB-

Fig.

design

for

guanosine

post-treatment

of

neuroblastoma

cells

added

at

or

h

after

MPP

neuroblastoma

cells

were

exposed

to

either

vehicle

or

guanosine

-LRB-

μM

-RRB-

or

MPP

-LRB-

μM

-RRB-

for

various

times

For

post-treatment

experiments

with

guanosine

at

h

cells

were

exposed

to

MPP

for

h

followed

by

MPP

plus

guanosine

for

an

additional

h

-LRB-

total

MPP

exposure

h

-RRB-

For

post-treatment

experiments

with

guanosine

at

h

cells

were

exposed

to

MPP

for

h

followed

by

MPP

plus

guanosine

for

a

further

h

-LRB-

total

MPP

exposure

h

-RRB-

Once

added

guanosine

and

MPP

remained

in

the

culture

medium

for

the

duration

of

the

experiment

DNA

fragmentation

or

activity

was

determined

at

or

h

as

described

in

the

Materials

and

methods

section

In

experiments

in

which

enzyme

inhibitors

were

tested

cells

were

pre-treated

with

various

agents

for

min

prior

to

the

addition

of

guanosine

These

treatments

included

the

potent

and

selective

inhibitor

of

-LSB-

-LRB-

-RRB-

-LRB-

-RRB-

-RSB-

-LRB-

μM

Sigma

-RRB-

or

the

selective

inhibitor

of

MAP

kinase

kinase

-LRB-

MEK

-RRB-

-LSB-

-LRB-

-RRB-

-RSB-

-LRB-

μM

Sigma

-RRB-

In

experiments

using

the

ADP-ribosylating

factor

of

the

inhibitory

guanosine

nucleotide

binding

protein

-LRB-

Gi

-RRB-

pertussis

toxin

-LSB-

PTX

ng/ml

in

glycerol

M

NaCl

and

M

Tris-glycine

-LRB-

pH

-RRB-

Sigma

-RSB-

was

added

overnight

-LRB-

h

-RRB-

and

were

dissolved

in

and

added

to

the

culture

medium

at

a

final

concentration

of

dimethyl

sulfoxide

-LRB-

DMSO

-RRB-

Evaluation

of

cell

death

and

DNA

fragmentation

In

initial

experiments

cell

death

was

determined

using

acridine

orange-ethidium

bromide

-LRB-

AO-EB

Sigma-Aldrich

-RRB-

staining

Cells

were

seeded

onto

plates

-LRB-

diameter

mm

cells/well

-RRB-

and

exposed

to

MPP

After

or

h

cells

were

washed

three

times

with

phosphate-buffered

saline

-LRB-

PBS

-RRB-

and

AO-EB

solution

-LRB-

μM/ml

-RRB-

was

added

to

the

wells

Cells

were

examined

using

a

fluorescence

microscope

-LRB-

Nikon

Tokyo

Japan

-RRB-

The

number

of

fragmented

nuclei

and/or

condensed

chromatin

was

determined

by

counting

cells

The

percentage

of

apoptotic

cells

is

defined

as

-LRB-

total

number

of

cells

with

apoptotic

nuclei/total

number

of

cells

-RRB-

In

subsequent

experiments

DNA

fragmentation

was

evaluated

by

the

oligonucleosomal

ELISA

-LRB-

Cell

Death

Detection

ELISAPLUS

Roche

Diagnostics

Laval

QC

Canada

-RRB-

and

carried

out

according

to

the

manufacturer

's

instructions

This

assay

measures

the

amount

of

oligonucleosomal

fragments

and

is

a

marker

of

apoptosis

-LSB-

-RSB-

Cells

were

exposed

to

MPP

alone

or

in

combination

with

guanosine

or

pre-treated

with

the

various

inhibitors

as

described

above

Following

various

treatments

cells

were

isolated

for

analysis

of

DNA

fragmentation

as

described

by

Pettifer

et

al.

-LSB-

-RSB-

For

the

oligonucleosomal

ELISA

viable

cells/treatment

were

lysed

and

centrifuged

-LRB-

g

for

min

-RRB-

to

isolate

fragmented

oligonucleosomal

DNA

The

cytosolic

fractions

of

cell

lysates

were

transferred

into

streptavidin-coated

microplate

wells

and

a

mixture

of

biotin-linked

anti-histone

antibody

and

peroxidase-linked

anti-DNA

antibody

was

added

and

incubated

for

h

at

room

temperature

Plates

were

washed

with

incubation

buffer

to

remove

the

unfixed

anti-DNA

antibody

and

the

peroxidase

activity

was

determined

spectrophotometrically

with

azino-bis

-LSB-

acid

-RSB-

-LRB-

ABTS

-RRB-

as

the

substrate

-LRB-

absorbance

of

nm

-RRB-

The

amount

of

DNA

fragmentation

is

expressed

as

a

percentage

of

the

positive

control

provided

with

the

kit

Determination

of

activity

activity

was

determined

using

the

colourimetric

assay

-LRB-

Sigma-Aldrich

-RRB-

Cells

were

exposed

to

different

treatments

as

described

above

then

cells

were

isolated

re-suspended

in

cells

of

lysis

buffer

-LRB-

mM

-LRB-

-RRB-

acid

-LRB-

HEPES

-RRB-

pH

mM

-LSB-

-LRB-

-RRB-

dimethylammonio

-RSB-

propanesulfonic

acid

-LRB-

CHAPS

-RRB-

mM

dithiothreitol

-LRB-

DTT

-RRB-

-RRB-

and

incubated

on

ice

for

min

Cell

lysates

were

centrifuged

-LRB-

g

for

min

at

°C

-RRB-

and

the

supernatants

-LRB-

μl/well

-RRB-

were

transferred

to

plates

A

selective

inhibitor

-LRB-

N-acetyl-Asp-Glu-Val-Asp-CHO

μl/well

at

μM

-RRB-

was

added

to

supernatants

to

control

for

the

nonspecific

hydrolysis

of

the

substrate

Purified

human

recombinant

protein

provided

with

the

assay

was

used

-LRB-

μl/well

at

μg/ml

-RRB-

as

a

positive

control

for

activity

Cell

lysates

were

incubated

in

an

assay

buffer

-LSB-

mM

HEPES

pH

CHAPS

mM

DTT

mM

ethylenediaminetetraacetic

acid

-LRB-

EDTA

-RRB-

-RSB-

containing

N-acetyl-Asp-Glu-Val-Asp

p-nitroaniline

-LRB-

μl/well

at

mM

-RRB-

overnight

at

°C

and

the

formation

of

p-nitroaniline

was

measured

at

nm

using

a

microtiter

plate

reader

The

activity

of

was

calculated

from

the

absorbance

values

using

a

calibration

curve

and

it

is

expressed

as

μg

of

substrate

cleaved/min

per

ml

As

the

number

of

cells

in

ml

of

cell

lysate

is

adjusted

to

cells

this

activity

is

generated

by

cells

Measurement

of

α-synuclein

protein

concentration

by

ELISA

The

concentration

of

α-synuclein

protein

in

neuroblastoma

cells

was

determined

by

an

ELISA

-LRB-

BioSource

San

Diego

CA

USA

-RRB-

Cells

were

exposed

to

various

treatments

as

described

above

and

subsequently

lysed

using

a

cell

extraction

buffer

-LRB-

mM

Tris

pH

mM

NaCl

mM

EDTA

mM

NaF

mM

mM

Triton

glycerol

sodium

dodecyl

sulfate

deoxycholate

BioSource

San

Diego

CA

USA

-RRB-

containing

protease

inhibitors

-LSB-

mM

phenylmethanesulfonyl

fluoride

mM

-LRB-

-RRB-

benzenesulfonyl

fluoride

μM

bestatin

μM

μM

leupeptin

μM

aprotinin

Sigma

-RSB-

Cells

were

incubated

on

ice

in

cell

extraction

buffer

for

min

and

centrifuged

-LRB-

g

for

min

at

°C

-RRB-

Aliquots

of

supernatants

-LRB-

μl

-RRB-

were

removed

for

the

determination

of

the

total

protein

concentration

using

the

bicinchoninic

acid

-LRB-

BCA

-RRB-

protein

assay

-LRB-

Pierce

Rockford

IL

USA

-RRB-

We

measured

α-synuclein

protein

concentration

using

an

ELISA

kit

according

to

the

manufacturer

's

instructions

Briefly

cell

lysates

-LRB-

diluted

-RRB-

were

added

to

α-synuclein

antibody-coated

wells

in

duplicate

allowed

to

bind

and

then

were

treated

with

the

anti-α-synuclein

antibody

This

was

followed

by

the

addition

of

an

anti-rabbit

IgG-horse

radish

peroxidase-linked

antibody

and

by

the

addition

of

a

stabilized

chromogen

-LRB-

tetramethylbenzidine

-RRB-

The

reaction

was

terminated

by

adding

M

HCl

-LRB-

stop

solution

-RRB-

provided

with

the

kit

and

the

absorbance

of

each

well

was

read

at

nm

Standard

curves

were

prepared

using

purified

recombinant

α-synuclein

protein

-LRB-

ng/ml

-RRB-

provided

with

the

kit

The

concentration

of

the

α-synuclein

protein

in

each

sample

was

determined

from

a

nonlinear

regression

and

it

is

expressed

as

a

ratio

of

α-synuclein

protein

-LRB-

in

ng/ml

-RRB-

to

total

protein

concentration

-LRB-

in

ng/ml

-RRB-

Statistical

analysis

Data

obtained

in

experiments

testing

the

concentration

dependence

of

the

protective

effect

of

guanosine

on

MPP

induced

DNA

fragmentation

-LRB-

Fig.

-RRB-

and

the

effect

of

PTX

and

inhibitors

of

the

and

the

MAPK

pathways

on

DNA

fragmentation

-LRB-

Fig.

and

Table

-RRB-

were

analyzed

by

a

one-way

analysis

of

variance

-LRB-

ANOVA

-RRB-

followed

by

a

Student-Newman-Keul

post-hoc

test

Two-way

ANOVA

followed

by

a

Bonferroni

post-hoc

test

was

used

to

analyze

the

data

obtained

from

experiments

evaluating

the

effects

of

guanosine

administered

at

different

time

points

on

MPP

induced

DNA

fragmentation

-LRB-

Fig.

-RRB-

and

on

MPP

induced

activation

-LRB-

Fig.

-RRB-

A

p

value

of

or

less

was

considered

statistically

significant

Results

represent

the

mean

SEM

of

a

minimum

of

three

independent

experiments

Table

of

selected

inhibitors

of

cell

survival

pathways

on

the

anti-apoptotic

effect

of

guanosine

in

cellsTreatmentDNA

fragmentation

-LRB-

of

positive

control

-RRB-

No

MPP

-LRB-

μM

-RRB-

a

Guanosine

bGuanosine

n.s.Guanosine

cells

were

exposed

to

MPP

-LRB-

μM

-RRB-

for

h

to

induce

DNA

fragmentation

Some

cells

were

treated

with

guanosine

-LRB-

μM

-RRB-

either

for

h

prior

to

MPP

-LRB-

pre-treatment

-RRB-

or

at

the

same

time

as

MPP

-LRB-

co-treatment

-RRB-

or

h

after

MPP

-LRB-

post-treatment

see

Fig.

-RRB-

Some

cultures

were

exposed

to

-LRB-

inhibitor

of

-RRB-

or

to

-LRB-

inhibitor

of

MAP

kinase

kinase

MEK

-RRB-

for

min

prior

to

the

addition

of

guanosine

DNA

fragmentation

was

determined

at

h

-LRB-

n

-RRB-

and

is

expressed

as

a

percentage

of

the

positive

control

abolished

the

protective

effect

of

guanosine

in

cells

pre-treated

co-treated

or

post-treated

with

this

nucleoside

also

abolished

the

protective

effect

of

guanosine

in

cells

pre-treated

with

this

nucleoside

but

had

no

significant

effect

in

cells

co-treated

or

post-treated

with

guanosineap

compared

to

controlbp

compared

to

same

treatment

without

inhibitorcp

compared

to

same

treatment

without

inhibitorn.s

not

significant

Results

MPP

induces

DNA

fragmentation

in

neuroblastoma

cells

neuroblastoma

cells

were

exposed

to

different

concentrations

of

MPP

-LRB-

μM

-RRB-

for

various

times

-LRB-

h

-RRB-

to

optimize

the

experimental

conditions

and

to

confirm

that

MPP

induced

DNA

fragmentation

was

concentration

and

time

dependent

-LRB-

Fig.

-RRB-

DNA

fragmentation

in

untreated

control

cells

was

around

and

remained

unchanged

for

up

to

h

-LRB-

Fig.

-RRB-

Treatment

of

cells

with

μM

MPP

increased

DNA

fragmentation

significantly

after

h

-LRB-

p

-RRB-

and

h

-LRB-

p

Fig.

-RRB-

compared

to

untreated

controls

Since

exposure

of

cells

to

μM

MPP

for

h

induced

robust

DNA

fragmentation

these

conditions

were

used

in

the

following

studies

Fig.

induces

DNA

fragmentation

in

cells

MPP

-LRB-

or

μM

-RRB-

was

added

to

cells

and

DNA

fragmentation

determined

after

or

h

by

oligonucleosomal

ELISA

as

described

in

the

Materials

and

methods

section

Data

are

presented

as

the

percentage

of

DNA

fragmentation

relative

to

a

positive

control

-LRB-

n

-RRB-

MPP

-LRB-

μM

-RRB-

increased

DNA

fragmentation

significantly

at

h

-LRB-

p

-RRB-

and

at

h

-LRB-

p

-RRB-

compared

to

that

of

untreated

cells

at

zero

time

Pre-treatment

of

neuroblastoma

cells

with

guanosine

attenuates

MPP

induced

DNA

fragmentation

cells

were

exposed

to

various

concentrations

of

guanosine

-LRB-

μM

-RRB-

for

h

followed

by

the

addition

of

μM

MPP

for

a

further

h

and

DNA

fragmentation

was

determined

at

h

by

oligonucleosomal

ELISA

As

we

observed

in

the

previous

experiment

the

addition

of

MPP

-LRB-

μM

-RRB-

led

to

significantly

increased

DNA

fragmentation

compared

to

controls

-LRB-

p

Fig.

-RRB-

Pre-treatment

of

cells

for

h

with

various

concentrations

of

guanosine

-LRB-

μM

-RRB-

significantly

reduced

MPP

induced

DNA

fragmentation

-LRB-

p

-RRB-

for

all

four

concentrations

of

guanosine

compared

to

cells

treated

with

MPP

alone

-LRB-

Fig.

-RRB-

Based

on

these

results

we

used

μM

guanosine

in

all

subsequent

experiments

Fig.

of

cells

with

guanosine

decreases

MPP

induced

DNA

fragmentation

cells

were

exposed

to

MPP

-LRB-

μM

-RRB-

for

h

to

induce

DNA

fragmentation

Some

cells

were

pre-treated

with

guanosine

-LRB-

μM

-RRB-

for

h

prior

to

MPP

Guanosine

and

MPP

remained

in

the

cultures

for

the

duration

of

the

experiment

DNA

fragmentation

was

determined

after

h

by

oligonucleosomal

ELISA

as

described

in

the

Materials

and

methods

section

and

is

expressed

as

the

percentage

of

the

positive

control

-LRB-

n

-RRB-

MPP

increased

DNA

fragmentation

significantly

at

h

compared

to

that

of

untreated

controls

-LRB-

p

-RRB-

Pre-treatment

with

guanosine

-LRB-

μM

-RRB-

reduced

significantly

the

MPP

induced

DNA

fragmentation

compared

to

that

in

cells

treated

with

MPP

for

the

same

time

-LRB-

p

at

all

four

concentrations

-RRB-

Post-treatment

with

guanosine

protects

neuroblastoma

cells

against

MPP

induced

DNA

fragmentation

We

next

examined

whether

guanosine

exerted

any

effect

on

MPP

induced

DNA

fragmentation

when

it

was

added

to

neuroblastoma

cells

after

apoptosis

was

already

in

progress

Cells

were

pre-treated

with

μM

guanosine

prior

to

MPP

or

co-treated

with

guanosine

plus

MPP

or

as

described

in

Fig.

guanosine

was

added

as

post-treatment

DNA

fragmentation

was

determined

in

cells

exposed

to

various

treatments

after

and

h

of

MPP

administration

-LRB-

Fig.

-RRB-

The

addition

of

MPP

led

to

a

significant

increase

in

DNA

fragmentation

compared

to

controls

at

h

-LRB-

p

-RRB-

and

h

-LRB-

p

-RRB-

-LRB-

Fig.

-RRB-

Guanosine

alone

had

no

significant

effect

on

DNA

fragmentation

-LRB-

Fig.

-RRB-

Pre-treatment

of

cells

with

guanosine

significantly

reduced

DNA

fragmentation

after

and

h

compared

to

MPP

treatment

alone

at

the

respective

time

points

-LRB-

p

at

both

time

points

Fig.

-RRB-

Similarly

co-treatment

of

cells

with

guanosine

plus

MPP

significantly

reduced

DNA

fragmentation

after

and

h

compared

to

cells

treated

with

MPP

alone

at

these

times

-LRB-

p

at

both

time

points

Fig.

-RRB-

Addition

of

guanosine

h

after

MPP

-LRB-

post-treatment

at

h

-RRB-

or

h

after

MPP

-LRB-

post-treatment

at

h

-RRB-

reduced

DNA

fragmentation

significantly

compared

to

cells

treated

with

MPP

alone

for

or

h

respectively

-LRB-

p

for

both

treatments

Fig.

-RRB-

These

results

demonstrate

that

pre-treatment

or

co-treatment

of

cells

with

guanosine

and

MPP

attenuates

MPP

induced

DNA

fragmentation

and

guanosine

added

or

h

after

the

start

of

the

DNA

fragmentation

-LRB-

post-treatment

-RRB-

can

actually

reverse

this

process

Fig.

antagonizes

and

reverses

MPP

induced

DNA

fragmentation

in

cells

cells

were

exposed

to

MPP

-LRB-

μM

-RRB-

for

h

to

induce

DNA

fragmentation

Some

cells

were

treated

with

guanosine

-LRB-

μM

-RRB-

either

for

h

prior

to

MPP

-LRB-

pre-treatment

-RRB-

or

at

the

same

time

as

MPP

-LRB-

co-treatment

-RRB-

or

or

h

after

MPP

-LRB-

post-treatment

see

Fig.

-RRB-

DNA

fragmentation

was

determined

after

or

h

of

MPP

addition

by

oligonucleosomal

ELISA

as

described

in

the

Materials

and

methods

section

and

it

is

expressed

as

a

percentage

of

the

positive

control

-LRB-

n

-RRB-

MPP

induced

a

significant

increase

in

DNA

fragmentation

at

-LRB-

p

-RRB-

and

at

h

-LRB-

p

-RRB-

compared

to

that

of

untreated

control

cells

at

the

same

time

Pre-treatment

or

co-treatment

of

cells

with

guanosine

significantly

reduced

MPP

induced

DNA

fragmentation

at

and

h

compared

to

that

in

cells

treated

with

MPP

alone

at

the

same

time

-LRB-

p

at

both

time

points

for

both

treatments

-RRB-

Post-treatment

with

guanosine

at

or

h

also

reduced

MPP

induced

DNA

fragmentation

at

and

h

compared

to

that

in

cells

treated

with

MPP

alone

at

the

same

time

-LRB-

p

at

both

time

points

-RRB-

Guanosine

inhibits

MPP

induced

activity

Exposure

of

cells

to

MPP

leads

to

apoptosis

via

activation

-LSB-

-RSB-

We

therefore

examined

if

under

our

experimental

conditions

treatment

of

neuroblastoma

cells

with

MPP

activated

and

whether

the

addition

of

guanosine

to

MPP

treated

cells

at

various

times

-LRB-

pre-treatment

co-treatment

or

post-treatment

-RRB-

had

any

effect

on

the

activity

of

this

enzyme

In

untreated

control

cells

activity

was

low

and

remained

unchanged

for

up

to

h

-LRB-

Fig.

-RRB-

Addition

of

guanosine

to

cells

had

no

significant

effect

on

activity

-LRB-

Fig.

-RRB-

In

MPP

treated

cells

activity

increased

significantly

from

to

h

-LRB-

p

at

all

time

points

Fig.

-RRB-

Pre-treatment

of

cells

with

guanosine

prior

to

MPP

addition

significantly

reduced

activity

after

or

h

compared

to

that

in

cells

treated

with

MPP

alone

for

the

same

time

-LRB-

p

at

both

time

points

Fig.

-RRB-

Similar

results

were

also

obtained

when

cells

were

co-treated

with

guanosine

and

MPP

for

and

h

-LRB-

p

at

both

time

points

Fig.

-RRB-

Post-treatment

with

guanosine

at

h

-LRB-

see

Fig.

-RRB-

also

reduced

activity

significantly

at

h

compared

to

that

in

cells

treated

with

MPP

alone

at

this

time

point

-LRB-

p

Fig.

-RRB-

When

guanosine

was

added

to

cells

h

after

the

addition

of

MPP

-LRB-

post-treatment

at

h

-RRB-

activity

determined

after

h

was

unaffected

compared

to

MPP

treatment

alone

at

this

time

point

-LRB-

Fig.

-RRB-

These

results

thus

demonstrate

that

pre-treatment

co-treatment

or

post-treatment

at

h

of

neuroblastoma

cells

with

guanosine

prevents

MPP

induced

activation

of

Administration

of

guanosine

to

cells

h

after

MPP

however

has

no

effect

on

activity

Fig.

inhibits

MPP

induced

activation

in

cells

cells

were

exposed

to

MPP

-LRB-

μM

-RRB-

for

h

to

induce

to

induce

activation

Some

cells

were

treated

with

guanosine

-LRB-

μM

-RRB-

either

for

h

prior

to

MPP

-LRB-

pre-treatment

-RRB-

or

at

the

same

time

as

MPP

-LRB-

co-treatment

-RRB-

or

or

h

after

MPP

-LRB-

post-treatment

see

Fig.

-RRB-

activity

was

determined

as

described

in

the

Materials

and

methods

section

and

is

expressed

as

the

amount

of

N-acetyl-Asp-Glu-Val-Asp

p-nitroaniline

cleaved

by

cells

in

μg/min

per

ml

-LRB-

n

-RRB-

MPP

significantly

increased

activity

from

to

h

-LRB-

p

at

all

three

time

points

-RRB-

compared

to

that

of

control

cells

at

the

same

time

Pre-treatment

or

co-treatment

of

cells

with

guanosine

significantly

reduced

MPP

induced

activity

at

and

h

compared

to

that

in

cells

treated

with

MPP

alone

at

the

same

time

-LRB-

p

at

both

time

points

for

both

treatments

-RRB-

Post-treatment

with

guanosine

at

h

reduced

MPP

induced

activity

at

h

compared

to

that

in

cells

treated

with

MPP

alone

at

the

same

time

-LRB-

p

-RRB-

Guanosine

added

to

cultures

h

after

MPP

had

no

effect

on

activity

Guanosine

has

no

effect

on

α-synuclein

protein

expression

in

MPP

treated

neuroblastoma

cells

Several

authors

reported

that

addition

of

MPP

to

cells

increased

α-synuclein

protein

expression

-LSB-

-RSB-

We

therefore

determined

α-synuclein

protein

concentration

in

neuroblastoma

cells

under

our

experimental

conditions

-LRB-

μM

MPP

for

h

-RRB-

and

evaluated

the

effects

of

guanosine

on

the

expression

of

this

protein

in

MPP

treated

cells

The

ratio

of

α-synuclein

protein/total

protein

in

untreated

control

cells

after

h

was

very

low

and

exposure

of

neuroblastoma

cells

to

MPP

for

h

had

no

significant

effect

on

the

expression

of

this

protein

-LRB-

data

not

shown

-RRB-

Treatment

of

cells

with

guanosine

prior

to

the

addition

of

MPP

or

at

the

same

time

as

MPP

or

h

after

MPP

had

no

significant

effect

on

of

α-synuclein

protein

expression

-LRB-

data

not

shown

-RRB-

These

results

indicate

that

under

the

present

experimental

conditions

neither

MPP

nor

a

combination

of

guanosine

plus

MPP

has

any

effect

on

α-synuclein

protein

expression

Pertussis

toxin

abolishes

the

anti-apoptotic

effect

of

guanosine

We

reported

recently

that

specific

binding

sites

exist

for

guanosine

in

rat

brain

membranes

-LSB-

-RSB-

in

cultured

rat

astrocytes

-LSB-

-RSB-

and

in

neuroblastoma

cells

-LRB-

Traversa

and

Caciagli

personal

communications

-RRB-

We

have

further

shown

that

guanosine

binding

in

these

cells

is

sensitive

to

treatment

with

PTX

-LSB-

-RSB-

indicating

that

the

putative

guanosine

receptor

is

coupled

to

Gi

protein

We

therefore

tested

whether

the

anti-apoptotic

effect

of

guanosine

in

the

MPP

treated

cells

is

mediated

via

this

putative

guanosine

receptor

Cells

were

exposed

to

PTX

-LRB-

ng/ml

for

h

-RRB-

prior

to

the

various

treatments

with

guanosine

and

MPP

-LRB-

pre-treatment

co-treatment

or

post-treatment

at

h

-RRB-

and

DNA

fragmentation

was

determined

at

h

As

in

previous

experiments

MPP

induced

a

significant

increase

in

DNA

fragmentation

and

the

addition

of

guanosine

alone

had

no

significant

effect

on

this

process

-LRB-

Fig.

-RRB-

Exposure

of

neuroblastoma

cells

to

PTX

alone

increased

DNA

fragmentation

slightly

under

basal

conditions

but

this

was

not

statistically

significant

Pre-treatment

co-treatment

or

post-treatment

at

h

of

cells

with

guanosine

and

MPP

significantly

reduced

DNA

fragmentation

compared

to

cells

treated

with

MPP

only

-LRB-

p

for

all

three

treatments

Fig.

-RRB-

Addition

of

PTX

to

neuroblastoma

cells

prior

to

any

of

the

three

guanosine

plus

MPP

treatments

reversed

the

anti-apoptotic

effect

of

guanosine

-LRB-

Fig.

-RRB-

These

results

demonstrate

that

regardless

of

the

time

of

its

addition

to

MPP

treated

cells

the

protective

effect

of

guanosine

is

abolished

by

PTX

These

findings

are

consistent

with

the

interpretation

that

these

effects

are

mediated

by

a

Gi

protein-coupled

receptor

most

likely

the

putative

guanosine

receptor

Fig.

toxin

abolishes

the

anti-apoptotic

effect

of

guanosine

in

cells

cells

were

exposed

to

MPP

-LRB-

μM

-RRB-

for

h

to

induce

DNA

fragmentation

Some

cells

were

treated

with

guanosine

-LRB-

μM

-RRB-

either

for

h

prior

to

MPP

-LRB-

pre-treatment

-RRB-

or

at

the

same

time

as

MPP

-LRB-

co-treatment

-RRB-

or

h

after

MPP

-LRB-

post-treatment

see

Fig.

-RRB-

Pertussis

toxin

-LRB-

PTX

ng/ml

for

h

-RRB-

was

added

to

some

cultures

prior

to

pre-treatment

or

co-treatment

or

post-treatment

with

guanosine

at

h.

DNA

fragmentation

was

determined

at

h

and

is

expressed

as

a

percentage

of

the

positive

control

-LRB-

n

-RRB-

MPP

increased

DNA

fragmentation

significantly

at

h

-LRB-

p

-RRB-

compared

to

that

of

untreated

control

cells

at

the

same

time

Pre-treatment

or

co-treatment

or

post-treatment

of

cells

with

guanosine

at

h

reduced

significantly

MPP

induced

DNA

fragmentation

compared

to

that

in

cells

treated

with

MPP

alone

at

the

same

time

-LRB-

p

-RRB-

PTX

reversed

the

protective

effect

of

guanosine

DNA

fragmentation

in

cells

exposed

to

PTX

plus

pre-treatment

or

co-treatment

or

post-treatment

with

guanosine

was

significantly

higher

compared

to

that

of

cells

without

PTX

-LRB-

p

for

all

three

treatments

-RRB-

These

values

were

comparable

to

that

obtained

in

cells

treated

with

MPP

alone

Guanosine

protects

against

MPP

induced

apoptosis

by

activating

the

cell

survival

pathways

We

have

shown

earlier

that

guanosine

activates

the

Akt/PKB

and

MAPK

pathways

in

neuroblastoma

cells

-LSB-

-RSB-

We

therefore

evaluated

if

the

protective

effects

of

guanosine

against

MPP

induced

DNA

fragmentation

were

also

mediated

via

these

signaling

pathways

Cells

were

exposed

to

either

or

to

selective

inhibitors

of

or

MEK

respectively

for

min

prior

to

various

treatments

with

guanosine

and

MPP

-LRB-

pre-treatment

co-treatment

or

post-treatment

at

h

-RRB-

Exposure

of

neuroblastoma

cells

to

alone

had

no

significant

effect

on

DNA

fragmentation

under

basal

conditions

Although

in

cells

exposed

to

alone

DNA

fragmentation

increased

slightly

this

was

not

statistically

significant

-LRB-

Table

-RRB-

As

in

previous

experiments

the

addition

of

MPP

induced

significant

DNA

fragmentation

and

treatment

of

cells

with

guanosine

under

various

conditions

significantly

reduced

DNA

fragmentation

compared

to

cells

treated

with

guanosine

-LRB-

Table

-RRB-

Exposure

of

cells

to

prior

to

pre-treatment

or

co-treatment

or

post-treatment

at

h

with

guanosine

and

MPP

reversed

the

protective

effect

of

guanosine

DNA

fragmentation

in

cells

exposed

to

plus

guanosine

plus

MPP

was

significantly

reduced

compared

to

cells

treated

with

guanosine

plus

MPP

without

the

inhibitor

-LRB-

p

for

pre-treatment

and

co-treatment

p

for

post-treatment

at

h

Table

-RRB-

Addition

of

to

cells

before

pre-treatment

with

guanosine

and

MPP

also

abolished

the

protective

effect

of

guanosine

-LRB-

p

Table

-RRB-

But

this

inhibitor

had

no

significant

effect

on

MPP

induced

DNA

fragmentation

in

cells

co-treated

or

post-treated

at

with

guanosine

and

MPP

-LRB-

Table

-RRB-

These

results

demonstrate

that

the

protective

effects

of

guanosine

are

abolished

by

in

MPP

treated

cells

regardless

of

the

time

of

its

administration

and

are

consistent

with

a

role

of

the

Akt/PKB

cell

survival

pathway

in

guanosine-mediated

reduction

of

DNA

fragmentation

In

contrast

the

effect

of

is

limited

to

pre-treatment

of

cells

with

guanosine

prior

to

MPP

thus

indicating

that

the

MAPK

pathway

contributes

to

the

protective

effects

of

guanosine

only

when

it

is

added

before

MPP

Discussion

We

have

shown

earlier

that

pre-treatment

with

guanosine

protects

different

cell

types

against

apoptosis

-LSB-

-RSB-

Here

we

report

that

guanosine

not

only

protects

human

neuroblastoma

cells

against

MPP

induced

apoptosis

but

more

importantly

it

can

reverse

MPP

induced

cytotoxicity

after

the

pro-apoptotic

pathways

have

already

been

activated

Thus

these

findings

are

novel

and

demonstrate

for

the

first

time

that

guanosine

can

in

fact

rescue

neuroblastoma

cells

from

apoptosis

when

it

is

added

to

cells

under

experimental

conditions

that

are

clinically

relevant

to

the

treatment

of

PD

Undifferentiated

dopaminergic

human

neuroblastoma

cells

have

been

used

widely

to

study

the

molecular

pathways

that

mediate

MPP

induced

apoptosis

-LSB-

-RSB-

MPTP

after

conversion

to

MPP

activates

the

same

intracellular

pathways

in

vivo

and

in

vitro

as

in

idiopathic

PD

-LSB-

-RSB-

Thus

MPP

is

taken

up

by

dopaminergic

neurons

where

it

inhibits

complex

I

of

the

mitochondrial

electron

transport

chain

This

leads

to

enhanced

production

of

reactive

oxygen

species

-LRB-

ROS

-RRB-

and

decreased

synthesis

of

ATP

-LSB-

-RSB-

Prolonged

exposure

to

MPP

results

in

the

up-regulation

of

the

pro-apoptotic

protein

Bax

and

following

its

translocation

to

the

mitochondria

it

promotes

the

release

of

mitochondrial

cytochrome

c

This

may

be

initiated

by

the

DNA

damage

caused

by

MPP

Cytochrome

c

together

with

is

incorporated

into

the

complex

the

apoptosome

which

activates

and

this

enzyme

in

turn

activates

-LSB-

-RSB-

At

the

same

time

the

anti-apoptotic

protein

is

down-regulated

-LSB-

-RSB-

Taken

together

these

data

demonstrate

that

exposure

of

dopaminergic

cells

to

MPP

leads

to

cytotoxicity

and

cell

death

by

apoptosis

-LSB-

-RSB-

Our

findings

are

consistent

with

previous

reports

that

MPP

induces

DNA

fragmentation

and

activation

of

in

cells

-LSB-

-RSB-

Interestingly

we

detected

no

significant

change

in

α-synuclein

protein

expression

in

MPP

treated

cells

-LRB-

data

not

shown

-RRB-

This

may

be

the

result

of

the

relatively

low

MPP

concentration

-LRB-

μM

-RRB-

and

short

duration

of

our

experiments

-LRB-

h

-RRB-

Others

reported

that

high

concentrations

of

MPP

-LRB-

mM

for

h

-LSB-

-RSB-

-RRB-

or

prolonged

exposure

of

neuroblastoma

cells

to

lower

concentrations

of

MPP

-LRB-

μM

for

days

-LSB-

-RSB-

or

mM

for

days

-LSB-

-RSB-

-RRB-

actually

increased

α-synuclein

protein

concentration

Mutations

of

this

protein

have

been

associated

with

a

familial

form

of

PD

-LSB-

-RSB-

and

the

wild-type

protein

is

a

major

constituent

of

Lewy

bodies

-LSB-

-RSB-

In

MPTP-treated

mice

α-synuclein

expression

is

up-regulated

-LSB-

-RSB-

and

in

cells

accumulation

of

this

protein

contributes

to

dopamine-dependent

apoptosis

-LSB-

-RSB-

Under

our

experimental

conditions

that

were

optimized

to

detect

DNA

fragmentation

we

observed

no

change

in

α-synuclein

protein

expression

The

molecular

mechanisms

by

which

guanosine

exerts

its

protective

effects

are

complex

Results

from

the

present

study

show

that

the

effects

of

both

pre-treatment

and

co-treatment

with

guanosine

are

pertussis

toxin

sensitive

and

these

may

depend

on

the

putative

plasma

membrane

localized

guanosine

receptor

-LSB-

-RSB-

that

is

coupled

to

Gi

proteins

There

may

be

other

possible

explanations

which

account

for

the

effects

of

pertussis

toxin

on

MPP

induced

DNA

fragmentation

Since

pertussis

toxin

enters

cells

and

ADP-ribosylates

the

α-subunit

of

Gi

proteins

-LSB-

-RSB-

it

may

also

attenuate

signaling

by

other

Gi

protein-coupled

receptors

resulting

in

the

elevation

of

intracellular

cAMP

-LSB-

-RSB-

and

this

in

turn

may

activate

cell

survival

pathways

such

as

the

MAPK

-LSB-

-RSB-

Guanosine

may

also

enter

neuroblastoma

cells

via

the

equilibrative

or

the

concentrative

nucleoside

transporters

-LSB-

-RSB-

cause

an

alteration

in

intracellular

concentrations

of

guanine

nucleotides

-LSB-

-RSB-

and

this

in

turn

may

affect

G

protein

activity

However

as

we

have

shown

before

guanosine

binding

to

its

cognate

receptor

leads

to

rapid

activation

of

the

cell

survival

pathways

Akt/PKB

and

MAPK

-LSB-

-RSB-

and

these

play

a

critical

role

in

protecting

neurons

against

cell

death

-LSB-

-RSB-

Although

these

signaling

pathways

are

most

often

triggered

by

growth

factor

binding

to

their

cognate

receptors

G

protein-coupled

receptors

have

also

been

shown

to

activate

them

-LSB-

-RSB-

Thus

binding

of

guanosine

to

its

Gi

protein-coupled

putative

receptor

may

activate

the

class

IB

-LRB-

-RRB-

via

the

dissociated

β,γ-subunits

of

Gi

proteins

which

promote

the

synthesis

of

phosphatidylinositol

-LRB-

-RRB-

trisphosphate

at

the

plasma

membrane

and

recruit

the

protein

kinase

Akt/PKB

-LSB-

-RSB-

Activation

of

Akt/PKB

by

phosphorylation

at

Thr

and

Ser

regulates

cell

survival

and

apoptosis

by

both

transcription-dependent

and

transcription-independent

pathways

-LSB-

-RSB-

Thus

Akt/PKB

may

suppress

apoptosis

by

phosphorylating

and

inactivating

the

pro-apoptotic

protein

Bad

-LSB-

-RSB-

inhibiting

the

release

of

mitochondrial

cytochrome

c

-LSB-

-RSB-

and

inhibiting

the

activation

of

and

-LSB-

-RSB-

Expression

of

cell

survival

genes

may

also

be

triggered

by

the

Akt/PKB

pathway

via

activation

of

the

transcription

factors

CREB

and

NF-κB

and

inhibition

of

Forkhead

transcription

factors

of

the

FOXO

family

-LSB-

-RSB-

Similarly

activation

of

the

MAPK

pathway

specifically

the

extracellular

signal-regulated

kinases

and

-LRB-

-RRB-

also

prevent

apoptosis

and

promote

cell

survival

by

both

transcription-dependent

and

transcription-independent

pathways

-LSB-

-RSB-

The

specific

downstream

targets

of

the

pathway

include

inhibition

of

by

phosphorylation

-LSB-

-RSB-

suppression

of

the

pro-apoptotic

protein

Bad

by

phosphorylation

and

activation

of

the

transcription

factor

CREB

-LSB-

-RSB-

Guanosine

binding

to

its

putative

receptor

may

also

stimulate

this

pathway

via

the

dissociated

β,γ-subunits

of

Gi

proteins

-LSB-

-RSB-

Reports

suggest

that

the

Akt/PKB

and

MAPK

pathways

do

not

function

independently

but

there

is

significant

cross-talk

between

them

-LSB-

-RSB-

It

has

been

suggested

that

whereas

the

Akt/PKB

pathway

is

the

main

signaling

pathway

for

maintaining

trophic

support

by

pro-survival

factors

mediates

protection

against

damage-induced

cell

death

-LSB-

-RSB-

Our

results

are

thus

consistent

with

the

interpretation

that

protection

of

cells

from

MPP

induced

DNA

fragmentation

by

guanosine

pre-treatment

is

dependent

on

the

activation

of

both

these

pathways

Following

co-treatment

with

guanosine

and

MPP

however

it

is

the

Akt/PKB

pathway

which

plays

the

essential

role

In

the

present

study

we

also

examined

the

effect

of

guanosine

on

MPP

induced

DNA

fragmentation

by

exposing

cells

to

guanosine

after

activity

was

elevated

and

DNA

fragmentation

was

already

underway

-LRB-

post-treatment

-RRB-

This

strategy

is

novel

and

is

based

on

a

recent

proposal

by

Meissner

et

al.

-LSB-

-RSB-

who

advocate

that

potential

neuroprotective

agents

for

the

treatment

of

PD

should

be

tested

under

conditions

that

are

relevant

to

the

clinical

situation

Because

clinical

symptoms

of

PD

are

apparent

only

after

the

majority

of

nigrostriatal

neurons

have

died

patients

do

not

present

for

treatment

until

after

the

process

of

cell

death

is

well

established

-LSB-

-RSB-

Most

in

vitro

studies

have

been

conducted

by

adding

potential

neuroprotective

agents

to

cells

prior

to

MPP

triggered

apoptosis

-LSB-

-RSB-

or

at

the

same

time

as

MPP

-LSB-

-RSB-

making

these

results

irrelevant

to

the

treatment

of

PD

where

the

process

of

neurodegeneration

has

already

begun

Addition

of

guanosine

to

cells

after

MPP

induced

DNA

fragmentation

is

already

in

progress

may

involve

additional

protective

mechanisms

As

DNA

fragmentation

is

a

late

event

in

apoptosis

-LSB-

-RSB-

some

cells

with

damaged

DNA

may

actually

undergo

DNA

repair

and

survive

-LSB-

-RSB-

The

preferred

survival

strategy

of

a

cell

is

to

attempt

to

restore

any

DNA

damage

by

using

a

complex

set

of

DNA

repair

pathways

-LSB-

-RSB-

Only

when

the

damage

to

the

DNA

becomes

excessive

and

the

repair

capacity

of

the

cell

is

overwhelmed

will

it

undergo

apoptosis

Therefore

adding

guanosine

or

h

after

DNA

fragmentation

has

been

initiated

may

activate

some

of

the

DNA

repair

mechanisms

antagonizing

and

reversing

the

MPP

induced

DNA

fragmentation

The

protective

effects

of

guanosine

in

these

cells

thus

may

include

activation

of

both

cell

survival

and

DNA

repair

pathways

In

contrast

post-treatment

with

guanosine

had

no

significant

effect

on

MPP

induced

activation

of

As

activation

of

this

enzyme

requires

a

cleavage

of

the

interdomain

region

of

the

inactive

dimer

of

once

this

process

is

initiated

it

can

not

be

reversed

-LSB-

-RSB-

So

addition

of

guanosine

to

cells

after

proteolytic

cleavage

of

this

enzyme

will

not

alter

activity

Recently

administration

of

MPTP

to

mice

has

been

reported

to

promote

caspase-independent

cell

death

of

dopaminergic

neurons

-LSB-

-RSB-

This

apoptosis-like

programmed

cell

death

-LSB-

-RSB-

is

mediated

by

the

release

of

the

apoptosis-inducing

factor

-LRB-

AIF

-RRB-

from

the

mitochondria

and

its

subsequent

translocation

to

the

nucleus

where

it

induces

DNA

fragmentation

-LSB-

-RSB-

If

this

process

plays

a

significant

role

in

MPP

induced

cell

death

of

neuroblastoma

cells

DNA

fragmentation

will

still

be

reversed

by

the

protective

action

of

guanosine

but

activation

will

not

be

affected

Several

authors

suggested

recently

that

targeting

programmed

cell

death

in

neurodegenerative

diseases

may

lead

to

successful

therapy

-LSB-

-RSB-

Drugs

that

will

be

effective

in

PD

should

protect

the

dopaminergic

cells

from

apoptotic

insult

by

either

interrupting

the

signaling

pathways

that

mediate

cell

death

or

activating

the

cell

survival

pathways

Our

present

results

show

that

the

non-adenine-based

purine

nucleoside

guanosine

when

added

to

MPP

treated

neuroblastoma

cells

promotes

their

survival

by

inhibiting

activation

and

DNA

fragmentation

via

activation

of

the

Akt/PKB

pathway

Furthermore

guanosine

exerts

these

effects

not

only

when

it

is

pre

or

co-administered

with

MPP

but

even

when

it

is

added

to

cells

after

is

activated

and

DNA

fragmentation

is

already

in

progress

Thus

these

findings

reveal

a

unique

neuroprotective

effect

of

guanosine

with

a

potential

for

effective

pharmacological

intervention

in

PD

O O O O B O O O O O O O O B B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O B O O O O O O O B O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O B O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O B I O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O

apoptosis
cell survival
cytotoxicity
dna fragmentation
guanosine

caspase-3
1-methyl–4-phenyl-pyridinium (mpp+)
parkinson’s disease
sh-sy5y human neuroblastoma cells


Transmural variations in gene expression of stretch-modulated proteins in the rat left ventricle The properties of left ventricular cardiac myocytes vary transmurally . This may be related to the gradients of stress and strain experienced in vivo across the ventricular wall . We tested the hypothesis that within the rat left ventricle there are transmural differences in the expression of genes for proteins that are involved in mechanosensitive pathways and in associated physiological responses . Real time reverse transcription polymerase chain reaction was used to measure messenger RNA -LRB- mRNA -RRB- levels of selected targets in sub-epicardial -LRB- EPI -RRB- and sub-endocardial -LRB- ENDO -RRB- myocardium . Carbon fibres were attached to single myocytes to stretch them and to record contractility . We observed that the slow positive inotropic response to stretch was not different between EPI and ENDO myocytes and consistent with this , that the mRNA expression of two proteins implicated in the slow response , non-specific cationic mechanosensitive channels -LRB- TRPC-1 -RRB- and Na/H exchanger , were not different . However , mRNA levels of other targets , e.g. the mechanosensitive K + channel TREK-1 , Brain Natriuretic Peptide and Endothelin-1 receptor B , were significantly greater in ENDO than EPI . No targets had significantly greater mRNA levels in EPI than ENDO . On the basis of these findings , we suggest that the response of the ventricle to stretch will depend upon both the regional differences in stimuli and the relative expression of the mechanosensitive targets and that generally , stretch sensitivity is predicted to be greater in ENDO . Introduction It is known that mechanical stimulation of the myocardium -LRB- stress and strain -RRB- can lead to both acute and chronic changes in the electrical and mechanical activity of the heart and contribute to the development of cardiac hypertrophy -LSB- 7 , 11 -RSB- . It is acknowledged that the ventricular myocardium is not homogenous and that there are regional differences in the basic properties of ventricular myocytes , e.g. -LSB- 1 , 10 -RSB- . In the whole heart , transmural gradients in ventricular wall stress and strain exist -LSB- 6 -RSB- , which may influence the expression of genes for proteins that are sensitive to mechanical stimuli . This may contribute to regional variations in physiological characteristics and thereby to transmural differences in the response to stimuli such as stretch . Therefore , regional variations in wall stress and strain , in conjunction with regional differences in gene expression , may regulate the response of the heart . To date , relatively little is known about these possibilities , although it has been shown that the messenger RNA -LRB- mRNA -RRB- expression and current density of the mechanosensitive twin-pore K + channel TREK-1 is greater in the rat sub-endocardium -LRB- ENDO -RRB- than the sub-epicardium -LRB- EPI -RRB- -LSB- 12 , 8 , 6 -RSB- . The purpose of this study was to test the hypothesis that the mRNA expression levels of several proteins , which are thought to modulate the mechanical and electrical response to stretch , vary across the rat left ventricular wall . Additionally , we wished to test whether the slow contractile response to axial stretch -LSB- 2 -RSB- , which is thought to be modulated by some of the chosen targets , differs between myocytes from different regions of the left ventricular free wall , thus potentially linking gene expression to function . Materials and methods All animal experimentation was carried out in accordance with the Animals -LRB- Scientific Procedures -RRB- Act 1986 and the local Institutional ethical review panel . Isolation of RNA Ten Sprague -- Dawley rats -LRB- 194 -- 240 g -RRB- were killed and the hearts removed . The left ventricular free wall was excised and snap frozen in liquid nitrogen between the large flat ends of a pair of tongs . The thickness of the left ventricular wall was measured with pre-cooled callipers , and 25-μm sections , equivalent to one third of the wall thickness , were collected on a cryostat from the EPI and ENDO surfaces , leaving a distinct mid-myocardial region . All samples were stored at − 80 °C until RNA extraction . Total RNA extraction was performed following the Qiagen mini-kit protocol for striated muscle , with the exception that an additional volume of RLT was added to the Proteinase K solution before binding of the RNA onto the mini-columns . Integrity and concentration of the isolated RNA were assessed by electrophoresis through 0.6 % formaldehyde/1 % agarose gels with ethidium bromide and the concentration of RNA adjusted to 1 g/l . Complementary DNA -LRB- cDNA -RRB- was prepared from 4 μg of total RNA with random priming using Superscript III first-strand synthesis system -LRB- Invitrogen , Life Technologies , Rockville , MD -RRB- and diluted 1:10 in TE -LRB- 10 mM Tris-HCl -LSB- pH 7.5 -RSB- , 1 mM ethylenediamine tetraacetic acid -RRB- before use in real-time polymerase chain reaction -LRB- PCR -RRB- . Real-time RT-PCR Real-time reverse transcription PCR -LRB- RT-PCR -RRB- was performed using TaqMan low-density arrays -LRB- Micro Fluidic Cards , Applied Biosystems , Foster City , CA -RRB- . Each card consisted of 384 wells , preloaded with pre-designed fluorogenic TaqMan probes and primers , configured to allow detection of 48 transcripts for 7 experimental and 1 calibrator samples . Our calibrator sample consisted of a mixture of cDNA from EPI and ENDO regions . Each of the eight sample lanes in a card was loaded with 100 μl of a 1:1 mixture of cDNA -LRB- equivalent to 77 ng input RNA -RRB- and TaqMan Universial PCR master mix -LRB- Applied Biosystems -RRB- . PCR was done according to the recommended protocol -LRB- 50 °C for 2 min and 94.5 °C for 10 min , followed by 40 cycles at 97 °C for 30 s and 59.7 °C for 1 min -RRB- on an ABI RISM 7900HT Sequence detection system -LRB- Applied Biosystems -RRB- . Data were collected with instrument spectral compensations by the Applied Biosystems SDS 2.2 software and analysed using the threshold cycle relative quantification method . Relative transcript expression was normalised to the housekeeper gene GAPDH . Mechanosensitive targets mRNA targets chosen for this investigation were : TREK-1 -LRB- a two-pore-domain K + - selective channel -RRB- and TRPC-1 -LRB- transient receptor potential canonical , a cationic non-selective mechanosensitive channel -LSB- MSC -RSB- -RRB- ; the Na/H exchanger , thought to be central to the slow increase in force that occurs upon axial stretch ; endothelin-1 and its receptor types A and B and the angiotensin II receptor A , known to regulate the hypertrophic response to mechanical stimulation and possibly the slow inotropic response ; caveolin-3 a critical component of caveolae , which are potential mechanosensors ; caveolin-1 , a major component of caveolae in non-cardiac muscle and recently identified in cardiac myocytes ; brain natruiretic peptide -LRB- BNP -RRB- and the α and β sub-types of myosin heavy chain -LRB- MHC -RRB- , the expression of both BNP and β-MHC are known to be increased by stretch -LSB- 11 -RSB- . Isolation of left ventricular myocytes The isolation of single rat left ventricular myocytes and the measurement of the rapid and slow responses to axial stretch by attached carbon fibres was performed as previously described -LSB- 2 -RSB- . In brief , excised hearts were perfused on a Langendorff apparatus with a collagenase/protease containing physiological solution . EPI and ENDO strips , each one third of the wall thickness , were manually cut from the left ventricular free wall with scissors leaving a distinct and intact mid-myocardial region . Further incubation with enzyme solution resulted in the isolation of separate populations of EPI and ENDO myocytes . Myocytes were attached to carbon fibres to record auxotonic force development in response to external stimulation and to axially stretch myocytes . Force was calculated from fibre motion , and stretch was calculated from the increase in sarcomere length . The rapid response to stretch was measured within 20 s of an increase in sacromere length , whereas the slow response was calculated as the further increase in force 5 min after the stretch . These experiments were carried out at a stimulation frequency of 1 Hz at a temperature of 22 -- 24 °C -LRB- to improve the experimental success rate -RRB- using a bicarbonate-based buffer containing -LRB- in mM -RRB- : NaCl 118.5 ; NaHCO3 14.5 ; KCl 4.2 ; KH2PO4 1.2 ; MgSO4 1.2 ; glucose 11.1 ; CaCl2 1 , equilibrated with 95 % O2/5 % CO2 -LRB- pH 7.4 -RRB- . Statistics Data were analysed using paired t tests to compare relative mRNA expression from the EPI vs ENDO region of each heart and unpaired t tests to compare the magnitude of the rapid and slow inotropic responses . Statistical significance was assumed when P < 0.05 . Results There was some consistency in the pattern of regional expression of mechanosensitive genes -LRB- Table 1 -RRB- . Although the expression levels of some targets were unchanged , others were significantly greater in ENDO but never so in EPI . This observation was not due to a systematic error of the technique as levels of mRNA of targets known to be greater in EPI , e.g. the potassium channel component , Kv4 .2 , were significantly greater in EPI than ENDO -LRB- results not presented -RRB- . Indeed , if α-MHC is excluded -LRB- being chosen as a counterpoint to β-MHC -RRB- , 9 of the 11 targets had a larger mean mRNA expression in ENDO than EPI ; this trend is statistically significant -LRB- P < 0.05 , sum of ranks test -RRB- . Table 1Comparison of mRNA expression of mechanosensitive proteins from the EPI and ENDO region of rat left ventricleAssay ID referenceCommon nameEPIENDOPaired t testMean ± SEMMean ± SEMSlow response and/or hypertrophic Agtr1a-Rn00578456_m1Angiotensin II receptor 1A1 .64 ± 0.161.30 ± 0.22 NS Edn1-Rn00561129_m1Endothelin-11.17 ± 0.141.58 ± 0.24 NS Ednra-Rn00561137_m1Endothelin-1 receptor type A1 .19 ± 0.141.38 ± 0.12 NS Ednrb-Rn00569139_m1Endothelin-1 receptor type B1 .27 ± 0.171.93 ± 0.220.001 ENDO > EPI Nppb-Rn00580641_m1Brain natriuretic peptide1 .42 ± 0.152.77 ± 0.420.002 ENDO > EPI Slc9a1-Rn00561924_m1Sodium -- hydrogen exchanger1 .33 ± 0.091.51 ± 0.17 NSMyofilaments Myh6-Rn00568304_m1α-myosin heavy chain1 .56 ± 0.121.37 ± 0.17 NS Myh7-Rn00568328_m1β-myosin heavy chain1 .20 ± 0.232.19 ± 0.360.006 ENDO > EPIMechanosensitive channels Kcnk2-Rn00597042_m1Kcnk2 -LRB- TREK-1 -RRB- 0.84 ± 0.041.04 ± 0.040.001 ENDO > EPI Trpc1-Rn00585625_m1Transient receptor potential canonical-10.72 ± 0.040.72 ± 0.05 NSMechanotransducers -LRB- caveolae -RRB- Cav-Rn00755834_m1Caveolin-10.90 ± 0.030.96 ± 0.030.015 ENDO > EPI Cav3-Rn0055343_m1Caveolin-30.81 ± 0.030.85 ± 0.05 NSData shown are mean ± SEM , expression is relative to GAPDH . The probability of paired t tests is shown in the table . P > 0.05 -LRB- n = 10 pairs -RRB- . NS Not significantly different When single myocytes were stretched from their resting sarcomere length -LRB- ENDO 1.83 ± 0.09 μm ; EPI 1.82 ± 0.07 μm -RRB- by an equivalent amount -LRB- ENDO 7.8 ± 0.9 % ; EPI 7.5 ± 0.5 % , n = 11 ENDO , 18 EPI myocytes -RRB- as illustrated in Fig. 1a , we observed that the magnitude of neither the rapid nor slow inotropic response was dependent upon regional origin -LRB- Fig. 1b -RRB- . Fig. 1The inotropic response of left ventricular myocytes to axial stretch . a Representative trace showing the typical biphasic inotropic response of a myocyte to a stretch of 8 % from a resting sacromere length -LRB- SL -RRB- of 1.85 μm . The trace shows changes in active force , resting force has been subtracted with a sample and hold device . The rapid increase in force is seen immediately upon an increase in SL , the slow response develops over the following minutes . b Mean data showing the rapid and slow response to stretch of ≅ 8 % from a resting SL of ≅ 1.82 μm in sub-epicardial -LRB- EPI -RRB- and sub-endocardial -LRB- ENDO -RRB- left ventricular myocytes . The responses from EPI and ENDO cells were not significantly different from each other . P > 0.05 , n = 18 EPI , n = 11 ENDO Discussion This investigation of transcripts for proteins sensitive to stretch , or involved in mechanosensitive pathways , has allowed us to detect a number of small but nevertheless significant differences in mRNA levels between ENDO and EPI samples . Rapid and slow inotropic responses to axial stretch Following equivalent amounts of axial stretch , the rapid inotropic response was not significantly different in EPI and ENDO rat myocytes . This observation is consistent with findings in untrained intact myocytes -LSB- 10 -RSB- . However , a greater stretch-induced increase in myofilament Ca2 + sensitivity -LRB- δpCa50 -RRB- has been reported in untrained skinned ENDO myocytes than EPI myocytes when larger stretches -LRB- from sarcomere lengths of 1.9 to 2.3 μm -RRB- are applied -LSB- 3 -RSB- . We observed that the slow response did not vary with myocyte regional origin . This is an important and novel observation because the multicellular cardiac preparations typically used to study the slow response -LRB- trabeculae and papillary muscles -RRB- are ENDO in nature . Additionally , previous whole heart studies recorded the aggregate response from the whole left ventricle while our own previous single cell experiments did not distinguish between myocyte origin . Consistent with the functional data , we observed that the mRNA expression levels for proteins implicated in the development of the slow inotropic response in rat -LRB- non-specific cationic MSCs , possibly TRPC-1 -RRB- and the Na/H exchanger , see -LSB- 2 -RSB- , are not regionally different . It should be noted however , that although TRPC-1 has been proposed as a non-specific cationic MSC -LSB- 9 -RSB- , such a role has not yet been established in the myocardium . MSCs and mechanotransducers Although levels of TRPC-1 mRNA were not regionally different , we found that TREK-1 mRNA expression was greater in ENDO than EPI . This latter finding agrees with the observations of Tan et al. -LSB- 12 -RSB- and Kelly et al. -LSB- 6 -RSB- , although the regional difference in our study was smaller than previously reported . We would therefore hypothesise that an equivalent mechanical stimulus that activated TRPC-1 and TREK-1 would produce a larger electrical response in ENDO tissue -LRB- see -LSB- 6 -RSB- -RRB- . Whether increased TREK-1 in ENDO would stabilise ENDO membrane potential during the diastolic period and be potentially anti-arrhythmic or reduce the transmural gradient in action potential duration and be potentially pro-arrhythmic -LSB- 1 -RSB- remains to be resolved . Although we found no evidence for a regionally different expression of caveolin-3 , an essential component of cardiac caveolae , the role of caveolae may also be influenced by regional expression of the receptors and other signalling molecules that they aggregate . The observation that mRNA for caveolin-1 is greater in ENDO is intriguing as this isoform has only recently been identified in cardiac myocytes , and its role in myocyte caveolae is unknown . Indicators of cardiac hypertrophy The ATII/ET -1 signalling pathways are known to play a role in the hypertrophic response to mechanical stimulation and , in some species , the slow inotropic response , see -LSB- 4 -RSB- for review . We only observed significantly greater levels of mRNA for the ET-1B receptor whereas it is the ET-1 A receptor that Cingolani et al. report to be important for the slow response . We also saw increased mRNA levels of BNP and of β-MHC in ENDO . Previous studies have variously reported increased -LSB- 5 -RSB- or similar -LSB- 3 -RSB- levels of β-MHC protein in normal rat ENDO vs EPI myocardium . Both BNP and β-MHC are indicators of hypertrophy provoked by mechanical stimuli . It is therefore interesting that wall stress and , in some studies , wall strain , is thought to be greater in ENDO -LSB- 6 -RSB- . Assumptions and limitations In this study , we have made the common , implicit assumption that there is a link between levels of mRNA expression and protein activity . This assumption can only be validated by measuring in situ protein activity , a huge task given the diverse nature of our targets . However , in measuring the slow inotropic response to stretch and the mRNA expression of the targets thought to underlie this effect , we have attempted to address this problem . We chose the rat for consistency with our previous work on regional differences and on the slow response to stretch -LSB- 10 , 2 -RSB- and because of the ready availability of mRNA sequences for this species . Given that regional variations exist in other species -LSB- 1 -RSB- , it seems reasonable to expect that our observations on mechanosensitive targets are relevant to other mammalian species , although investigation of other species is required to confirm this . Conclusions The level of mRNA expression of some proteins that are modulated by mechanical stimuli vary across the rat left ventricular wall . In vivo , the response to mechanical stimulation is likely to be complex . Targets -LRB- e.g. in the present study caveolin-3 and TRPC-1 -RRB- whose mRNA expression are not regionally different might still be differentially regulated by transmural variation in the mechanical stimuli , whereas other permutations of increased/decreased mRNA expression and protein activity with increased/decreased stimuli are possible . Based upon our present observations and those from the literature reporting increased stretch sensitivity of various responses in ENDO , e.g. -LSB- 5 , 10 , 3 , 6 -RSB- , it seems that in general , ENDO tissue is likely to be more influenced by , and be responsive to , mechanical stimulation than EPI tissue .

O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

stretch
cardiac myocytes
gene expression