New

insights

into

purinergic

receptor

signaling

in

neuronal

differentiation

neuroprotection

and

brain

disorders

Ionotropic

and

metabotropic

purinergic

receptors

are

expressed

in

the

central

nervous

system

and

participate

in

the

synaptic

process

particularly

associated

with

acetylcholine

GABA

and

glutamate

neurotransmission

As

a

result

of

activation

the

receptors

promote

the

elevation

of

free

intracellular

calcium

concentration

as

the

main

signaling

pathway

Purinergic

signaling

is

present

in

early

stages

of

embryogenesis

and

is

involved

in

processes

of

cell

proliferation

migration

and

differentiation

The

use

of

new

techniques

such

as

knockout

animals

in

vitro

models

of

neuronal

differentiation

antisense

oligonucleotides

to

induce

downregulation

of

purinergic

receptor

gene

expression

and

the

development

of

selective

inhibitors

for

purinergic

receptor

subtypes

contribute

to

the

comprehension

of

the

role

of

purinergic

signaling

during

neurogenesis

In

this

review

we

shall

discuss

the

participation

of

purinergic

receptors

in

developmental

processes

and

in

brain

physiology

including

neuron-glia

interactions

and

pathophysiology

Introduction

During

the

last

two

decades

evidence

for

the

participation

of

ATP

as

neurotransmitter

in

neuronal

signaling

was

collected

by

Drs.

Surprenant

-LSB-

-RSB-

and

Silinsky

-LSB-

-RSB-

Purine-sensitive

receptors

were

first

classified

as

G-coupled

receptors

which

are

activated

by

adenosine

and

receptors

responding

to

stimulation

of

ATP

-LSB-

-RSB-

Based

on

receptor

cloning

and

studying

of

receptor-induced

signal

transduction

receptors

were

divided

into

receptors

as

ATP-gated

ion

channels

and

G

protein-coupled

receptors

-LSB-

-RSB-

The

expression

of

purinergic

receptors

has

been

identified

during

development

and

differentiation

processes

-LSB-

-RSB-

Nucleotides

exert

a

synergic

effect

on

cell

proliferation

in

association

with

growth

factors

chemokines

or

cytokines

in

early

stages

of

development

-LSB-

-RSB-

by

parallel

activation

of

the

MAP

kinase

pathway

and/or

by

transactivation

of

growth

factor

receptors

-LSB-

-RSB-

The

complete

role

of

ATP

action

in

developmental

processes

still

needs

to

be

elucidated

It

is

known

that

ATP

activates

purinergic

receptors

resulting

in

many

cases

in

increases

of

intracellular

free

calcium

concentration

-LSB-

-RSB-

i.

Changes

in

-LSB-

-RSB-

i

are

involved

in

several

events

of

differentiation

and

the

embryogenesis

process

-LSB-

-RSB-

Spitzer

et

al.

-LSB-

-RSB-

showed

that

naturally

occurring

patterns

of

transients

encoded

neuronal

differentiation

Distinct

frequency

patterns

of

-LSB-

-RSB-

i

elevations

were

sufficient

to

promote

neuronal

differentiation

including

physiological

neurotransmitter

receptors

expression

-LSB-

-RSB-

ATP

and

UTP

are

the

main

purinergic

agonists

activating

or

receptors

These

nucleotides

can

be

rapidly

degraded

in

the

extracellular

space

by

ectoenzymes

to

ADP

or

UDP

subsequently

activating

distinct

receptors

or

be

finally

degraded

to

adenosine

which

is

known

to

induce

physiological

responses

via

activation

of

G

protein-coupled

receptors

-LSB-

-RSB-

-LRB-

Fig.

-RRB-

Fig.

signaling

pathway

involves

the

activation

of

adenosine

and

purinergic

receptors

and

purine

hydrolysis

by

ectonucleotidases

The

scheme

demonstrates

purinergic

receptor

activity

present

in

glia-glia

neuron-glia

and

neuron-neuron

interaction

during

neurogenesis

as

well

as

in

the

metabolism

of

the

adult

brain

In

this

review

article

we

shall

discuss

the

roles

of

purinergic

signaling

in

neurogenesis

such

as

cell

cycle

control

during

neural

progenitor

proliferation

and

differentiation

as

well

as

in

maintaining

physiology

of

neurons

and

glial

cells

and

the

involvement

of

purinergic

receptors

in

pathophysiology

In

addition

we

shall

outline

state-of-the-art

approaches

used

in

investigation

of

receptor

function

in

physiological

processes

such

as

the

use

of

antisense

oligonucleotides

generation

of

knockout

animals

and

identification

of

new

purinergic

receptor

subtype-selective

drugs

Study

of

purinergic

receptor

function

during

in

vitro

differentiation

During

the

development

of

the

mammalian

nervous

system

neural

stem

cells

and

their

derivative

progenitor

cells

generate

neurons

by

asymmetric

and

symmetric

divisions

-LSB-

-RSB-

receptors

were

shown

to

be

one

of

the

first

functionally

active

membrane

receptors

in

chick

embryo

cells

during

gastrulation

in

which

ATP

caused

rapid

accumulation

of

inositol

triphosphate

and

mobilization

in

a

similar

way

as

acetylcholine

-LRB-

Ach

-RRB-

did

via

activation

of

muscarinic

acetylcholine

receptors

whereas

other

endocrine-acting

substances

such

as

insulin

and

noradrenaline

-LRB-

-RRB-

induced

much

weaker

effects

in

terms

of

intracellular

calcium

signaling

-LSB-

-RSB-

The

induction

of

transient

fluctuation

in

-LSB-

-RSB-

i

also

denominated

as

calcium

wave

signaling

allows

for

a

coupling

of

spatial

and

temporal

information

Thus

calcium

waves

have

been

proposed

to

play

a

role

in

mapping

of

neuronal

networks

-LSB-

-RSB-

and

to

modulate

neurogenesis

during

embryonic

cortical

development

-LSB-

-RSB-

Neurotransmitters

are

prominent

candidates

for

transcellular

signals

that

could

influence

the

development

of

embryonic

neurons

as

they

surround

neural

cells

throughout

brain

development

-LSB-

-RSB-

In

addition

functional

ligand-gated

ionic

channel

receptors

have

been

identified

in

neural

progenitor

cells

prior

to

establishing

cortical

and

subcortical

synapses

-LSB-

-RSB-

In

this

context

the

extracellular

signaling

mechanisms

controlling

the

various

transition

steps

involved

in

adult

neurogenesis

are

still

poorly

understood

One

approach

used

to

identify

the

function

of

receptors

during

development

and

differentiation

is

the

use

of

in

vitro

models

for

neuronal

and

glial

differentiation

such

as

embryonic

and

adult

neural

progenitor

cells

-LRB-

NPC

-RRB-

also

known

as

neural

stem

cells

-LRB-

NSC

-RRB-

embryonic

stem

-LRB-

ES

-RRB-

and

embryonal

carcinoma

-LRB-

EC

-RRB-

cells

ES

cells

are

obtained

from

the

inner

mass

cell

of

the

blastocyst

The

differentiation

of

these

cells

closely

resembles

the

in

vivo

process

and

therefore

provides

stable

models

for

embryonic

growth

and

development

-LSB-

-RSB-

ATP

promotes

cell

proliferation

acting

through

and

receptors

in

murine

ES

cells

-LSB-

-RSB-

Tissue-nonspecific

alkaline

phosphatase

-LRB-

TNAP

-RRB-

was

also

detected

in

these

cells

and

used

as

a

marker

for

their

undifferentiated

stage

-LSB-

-RSB-

The

neuronal

differentiation

of

EC

cells

originated

from

irradiated

embryo

cells

-LSB-

-RSB-

also

resembles

early

neuronal

development

in

vivo

mouse

EC

cells

express

stem

cell-specific

marker

proteins

and

their

phenotypic

changes

in

specific

differentiation

stages

are

similar

to

those

of

stem

cells

-LSB-

-RSB-

Recently

our

laboratory

-LSB-

-RSB-

has

determined

gene

and

protein

expression

of

receptor

subtypes

throughout

in

vitro

neuronal

differentiation

of

cells

as

well

as

in

the

undifferentiated

cell

stage

suggesting

the

participation

of

purinergic

signaling

in

initiating

and

directing

differentiation

Differential

expression

and

activity

of

subtypes

and

subunits

were

reported

during

neuronal

maturation

of

cells

-LSB-

-RSB-

As

direct

evidence

for

participation

of

purinergic

receptors

in

neuronal

differentiation

the

presence

of

the

antagonists

disulfonic

acid

-LRB-

PPADS

-RRB-

reactive

blue

or

suramin

during

differentiation

of

neural

progenitor

cells

-LRB-

NPC

-RRB-

to

neurons

resulted

in

reduced

activity

of

cholinergic

and

glutamate

NMDA

receptors

in

differentiated

cells

pointing

at

a

participation

of

and

receptors

Other

in

vitro

neuronal

and

glial

differentiation

models

used

to

understand

the

purinergic

signaling

are

neural

stem

cells

or

progenitor

cells

which

are

isolated

from

the

subventricular

region

-LRB-

SVZ

-RRB-

located

in

the

lateral

ventricles

-LRB-

type

B

cells

-RRB-

or

in

the

subgranular

region

of

the

gyrus

dentatus

of

the

hippocampus

-LRB-

residual

radial

glia

-RRB-

or

even

from

the

subcortical

parenchyma

of

the

cerebral

cortex

of

embryonic

and

adult

brain

-LSB-

-RSB-

These

regions

in

the

adult

brain

act

as

neural

stem

cell

reservoirs

These

cells

are

already

advanced

in

their

differentiation

stage

when

compared

to

ES

or

EC

cells

Since

NSC

and

NPC

are

capable

of

differentiating

in

both

functional

neurons

and

glial

cells

they

possess

potential

therapeutic

applications

such

as

ES

cells

in

regeneration

therapy

following

neuronal

loss

These

NPC

differentiate

into

olfactory

cerebellar

and

retinal

neurons

-LSB-

-RSB-

in

the

presence

of

growth

factors

neurotransmitters

vasoactive

peptides

in

vivo

-LSB-

-RSB-

and

growth

factors

such

as

epidermal

growth

factor

-LRB-

EGF

-RRB-

fibroblast

growth

factor

-LRB-

-RRB-

and

leukemia

inhibitory

factor

-LRB-

LIF

-RRB-

in

vitro

When

exposed

to

a

high

concentration

of

in

suspension

proliferating

NPC

form

tridimensional

cell

aggregates

denominated

as

neurospheres

which

following

induction

to

differentiation

express

neuronal

marker

proteins

such

as

β-III-tubulin

microtubule-associated

-LRB-

-RRB-

and

synaptophysin

-LSB-

-RSB-

and

express

and

receptors

which

may

contribute

to

early

-LSB-

-RSB-

i

transients

as

prerequisites

for

further

differentiation

-LSB-

-RSB-

Shukla

et

al.

-LSB-

-RSB-

identified

functional

receptors

in

adult

mouse

hippocampal

progenitors

in

situ

and

the

nucleoside

triphosphate-hydrolyzing

ectoenzyme

-LRB-

NTPDase

-RRB-

in

type

B

cells

of

the

SVZ

-LSB-

-RSB-

and

in

hippocampal

progenitor

cells

In

adult

murine

NPC

of

SVZ

receptor

activity

mainly

contributes

to

-LSB-

-RSB-

i

transients

with

some

participation

of

receptors

The

presence

of

the

specific

receptor

antagonist

MRS

resulted

in

diminished

cell

proliferation

in

neurospheres

due

to

reductions

of

-LSB-

-RSB-

i

transients

Similar

results

were

obtained

with

NPC

from

SVZ

of

receptor

knockout

mice

-LSB-

-RSB-

receptor-deficient

mice

are

viable

however

they

have

deficits

in

platelet

aggregation

-LSB-

-RSB-

It

is

suggested

that

the

purine

signaling

underlies

autocrine

or

paracrine

mechanisms

and

and

receptors

are

important

for

NPC

differentiation

-LSB-

-RSB-

These

models

are

useful

tools

to

study

the

roles

of

receptor

signaling

in

early

stages

of

development

and

differentiation

The

importance

of

ATP

release

and

purinergic

signaling

has

not

only

been

demonstrated

in

developmental

progenitor

cell

expansion

and

neurogenesis

but

also

in

persistent

progenitor

cells

of

the

adult

brain

-LSB-

-RSB-

Expression

of

purinergic

receptors

during

development

of

the

central

nervous

system

Purinergic

signaling

pathways

are

also

involved

in

embryonic

neurogenesis

in

much

the

same

way

as

already

discussed

for

in

vitro

differentiation

models

ATP

mediates

elevation

of

-LSB-

-RSB-

i

and

proliferation

of

immortalized

human

stem

cells

from

embryonic

telencephalon

and

mouse

embryonic

neurospheres

-LSB-

-RSB-

waves

through

radial

glial

cells

in

slices

of

the

embryonic

rat

ventricular

zone

are

mediated

by

receptors

Disrupting

waves

between

embryonic

NPC

reduced

ventricular

zone

cell

proliferation

during

the

peak

of

embryonic

neurogenesis

-LSB-

-RSB-

ATP

directly

contributes

to

modulate

network-driven

giant

depolarizing

potentials

in

the

rat

hippocampus

during

early

stages

of

postnatal

development

-LSB-

-RSB-

In

the

developing

hippocampal

system

a

trophic

role

of

ATP

and

the

involvement

of

receptor

subtypes

in

shaping

interneuronal

connections

during

neuronal

differentiation

have

been

suggested

-LSB-

-RSB-

Alterations

of

the

regulation

of

embryonic

growth

by

purinergic

receptors

might

be

involved

in

the

onset

of

morphological

malformations

-LSB-

-RSB-

During

rat

postnatal

development

ectonucleotidase

activity

in

the

cerebral

cortex

steadily

increases

reaching

maximum

values

at

days

of

age

-LSB-

-RSB-

Several

and

receptors

were

shown

to

be

dynamically

expressed

in

the

pre

and

postnatal

central

and

peripheral

nervous

system

-LSB-

-RSB-

ATP

inhibited

motor

axon

outgrowth

during

early

embryonic

neurogenesis

most

likely

through

the

receptor

and

it

was

speculated

that

receptors

might

be

involved

in

programmed

cell

death

during

embryogenesis

-LSB-

-RSB-

From

all

of

the

studied

receptors

homomeric

receptors

were

the

first

expressed

in

the

rat

central

nervous

system

-LRB-

CNS

-RRB-

on

embryonic

day

-LRB-

-RRB-

-LSB-

-RSB-

On

heteromeric

receptors

were

formed

by

receptor

subunits

receptor

immunoreactivity

was

detected

in

cranial

motor

neurons

as

early

as

on

when

neurons

exited

the

cell

cycle

and

started

axon

outgrowth

as

well

as

postnatally

on

days

and

-LRB-

and

-RRB-

-LSB-

-RSB-

Moreover

expression

of

heteromeric

receptors

and

other

subunits

was

developmentally

regulated

in

nucleus

ambiguous

motoneurons

-LSB-

-RSB-

From

onwards

receptors

were

also

expressed

in

the

embryonic

brain

For

instance

in

primary

cultures

of

human

fetal

astrocytes

basal

levels

of

receptor

mRNA

transcription

and

protein

expression

were

detected

-LSB-

-RSB-

Sperlágh

et

al.

-LSB-

-RSB-

have

demonstrated

that

ATP

regulates

glutamate

release

via

activation

of

receptors

receptor-induced

excessive

glutamate

release

alters

homeostasis

subsequently

resulting

in

activation

of

the

apoptosis-related

caspase

cascade

-LSB-

-RSB-

receptor

expression

was

downregulated

in

Purkinje

cells

and

deep

cerebellar

nuclei

at

and

rat

embryonic

stages

with

the

exception

of

receptors

whose

immunoreactivity

in

granular

cells

was

increased

-LSB-

-RSB-

Evidence

for

participation

of

receptors

in

different

developmental

processes

such

as

neurite

outgrowth

-LRB-

involving

receptors

-RRB-

postnatal

neurogenesis

-LRB-

related

to

and

receptor

expression

-RRB-

and

cell

death

-LRB-

possibly

involving

receptors

-RRB-

was

collected

However

and

receptor

subunits

may

not

play

a

role

in

neuronal

development

-LSB-

-RSB-

Neocortical

neurons

from

rats

possess

a

quite

elaborated

purine-triggered

signaling

system

which

includes

both

and

receptor

activation

-LSB-

-RSB-

Weissman

et

al.

-LSB-

-RSB-

showed

that

-LSB-

-RSB-

i

waves

and

subsequent

ATP

release

with

consequent

receptor

activation

accompanied

radial

glial

cell-derived

neurogenesis

in

cultured

slices

of

the

developing

rat

forebrain

as

mentioned

above

Moreover

the

importance

of

calcium

signaling

for

differentiation

of

NPC

has

been

studied

-LSB-

-RSB-

and

direct

evidence

for

the

participation

of

receptor-activated

pathways

in

the

early

development

has

been

provided

by

Scemes

et

al.

-LSB-

-RSB-

receptors

-LRB-

particularly

the

subtype

-RRB-

were

widely

expressed

in

the

embryonic

rat

brain

as

early

as

on

-LSB-

-RSB-

There

was

a

marked

decrease

in

the

concentration

of

mRNA

coding

for

receptors

and

upregulation

of

mRNA

transcription

coding

for

receptors

in

freshly

isolated

astrocytes

of

developing

rat

hippocampus

-LSB-

-RSB-

Functional

interactions

between

neurons

and

glia

a

physiological

overview

An

increasing

amount

of

evidence

initiated

by

the

neuron-glia

unit

idea

proposed

by

Hyden

-LSB-

-RSB-

indicates

that

glial

cells

once

referred

to

as

a

simple

support

portion

in

the

CNS

are

now

considered

indispensable

functional

partners

of

neurons

-LSB-

-RSB-

both

in

physiological

and

pathological

conditions

However

many

questions

remain

unanswered

-LRB-

-RRB-

how

glia

detects

and

interacts

with

neural

function

-LRB-

-RRB-

does

neuron-glia

signaling

play

a

significant

role

in

synaptic

transmission

and

plasticity

and

-LRB-

-RRB-

how

glial

cells

can

communicate

with

other

glial

cells

Another

important

subject

related

to

the

interaction

between

glia

and

neurons

emerges

in

neurogenesis

There

is

now

a

general

agreement

that

the

adult

mammalian

nervous

system

possesses

many

characteristics

of

astrocytes

The

importance

of

glia

in

neuronal

development

was

confirmed

in

a

recent

study

showing

that

the

number

of

GFAP

-LRB-

glial

fibrillary

acidic

protein

-RRB-

containing

cells

was

reduced

following

transgenic

targeting

of

adult

mouse

subependymal

and

subgranular

zones

resulting

in

an

almost

complete

loss

of

neurogenesis

-LSB-

-RSB-

In

addition

to

assisting

migration

of

neurons

to

their

correct

position

and

managing

neurite

outgrowth

to

their

final

communication

targets

-LSB-

-RSB-

glial

cells

have

become

an

essential

key

for

understanding

neuronal

differentiation

by

promoting

initial

stem

cell

proliferation

and

instructing

undifferentiated

cells

to

adopt

a

neuronal

fate

-LSB-

-RSB-

In

the

mature

brain

the

proximity

of

astrocytes

to

neuronal

synapses

or

to

the

blood-brain

barrier

makes

these

cells

appropriate

to

control

water

diffusion

and

ion

concentration

in

extracellular

spaces

-LSB-

-RSB-

In

particular

astrocytes

regulate

homeostatic

environment

and

neurotransmitter

levels

by

functional

syncytium

in

which

gap

junctions

and

specific

membrane

carriers

play

an

important

role

-LSB-

-RSB-

In

addition

glial

cells

produce

and

release

a

vast

number

of

neurotrophins

including

fibroblast

growth

factor

nerve

growth

factor

and

transforming

growth

factor

which

directly

interfere

in

neuron

physiology

and

coordinate

developmental

processes

-LSB-

-RSB-

ATP

release

and

degradation

connecting

adenosinergic

and

purinergic

systems

As

already

mentioned

it

is

well

documented

that

glial

cells

may

directly

alter

neuronal

activity

by

releasing

neurotrophins

and

consequently

modulating

neurotransmitter

release

in

the

synapse

-LSB-

-RSB-

One

of

the

main

mechanisms

connecting

the

neuron-glia

system

is

believed

to

be

mediated

by

the

release

of

glutamate

from

glial

cells

-LSB-

-RSB-

In

this

context

growing

evidence

indicates

that

purinergic

receptor

ligands

are

widely

involved

in

the

cell-cell

signaling

mechanism

by

acting

as

neurotransmitters

or

neuromodulators

released

by

glial

cells

to

control

synaptic

transmission

in

the

CNS

as

part

of

multiple

functions

of

astrocytes

-LSB-

-RSB-

-LRB-

Fig.

-RRB-

ATP

is

an

ideal

molecule

for

cell

signaling

due

to

its

intrinsic

properties

such

as

its

small

size

diffusing

molecule

rate

instability

and

low

concentration

in

the

extracellular

environment

and

impossibility

to

cross

the

plasma

membrane

-LSB-

-RSB-

These

properties

imply

the

presence

of

particular

pathways

for

ATP

release

that

could

be

associated

with

cellular

excitation/response

and

cell-cell

signaling

-LSB-

-RSB-

First

ATP

may

be

stored

in

synaptic

vesicles

alone

or

with

other

neurotransmitters

and

then

released

as

a

classic

synaptic

mechanism

in

the

peripheral

or

central

nervous

system

-LSB-

-RSB-

Second

a

nonvesicular

mechanism

of

ATP

release

could

be

observed

through

gap

junction

hemichannels

ATP-binding

cassette

proteins

receptor

pores

in

glial

cells

and

via

chloride

channels

-LSB-

-RSB-

Third

ATP

could

be

released

due

to

cytolysis

or

cell

damage

While

this

is

not

a

physiological

mechanism

it

takes

place

following

biological

trauma

and

contributes

to

pathological

conditions

-LSB-

-RSB-

Subsequent

to

these

mechanisms

the

metabolism

of

the

released

ATP

is

regulated

by

a

vast

number

of

different

families

of

ectonucleotidases

in

the

synaptic

cleft

including

the

ectonucleoside

triphosphate

diphosphohydrolase

-LRB-

E-NTPDase

-RRB-

and

the

ectonucleotide

pyrophosphatase

phosphodiesterase

-LRB-

E-NPP

-RRB-

which

catalyze

the

degradation

of

ATP

to

ADP

or

AMP

The

degradation

to

adenosine

is

mediated

by

nucleotidase

-LRB-

NT

-RRB-

and

alkaline

phosphatase

-LSB-

-RSB-

-LRB-

Fig.

-RRB-

Consequently

the

reaction

products

resulting

from

the

ATP

hydrolysis

may

bind

to

receptors

in

the

case

of

ADP

or

to

receptors

in

the

case

of

adenosine

-LSB-

-RSB-

The

adenosinergic

receptor

ligand

adenosine

is

recognized

as

an

important

regulator

of

cellular

homeostasis

in

the

CNS

and

may

be

involved

in

the

prevention

or

induction

of

apoptosis

-LSB-

-RSB-

The

reduction

of

ectonucleotidase

activity

in

certain

pathological

conditions

provided

additional

evidence

for

the

accumulation

of

ATP

in

the

extracellular

environment

-LSB-

-RSB-

Therefore

the

complexity

of

the

communication

of

neural

and

nonneural

cells

expands

the

functional

significance

by

the

interaction

of

the

purinergic

receptors

in

association

with

a

variety

of

neurotransmitter

systems

ATP-mediated

neuron-glia

signaling

Novel

studies

in

the

purinergic

field

began

to

converge

with

glial

research

as

it

became

more

widely

accepted

that

ATP

is

released

through

synaptic

vesicles

and

thus

accessible

to

perisynaptic

glial

cells

allowing

them

to

detect

neuronal

activity

In

particular

glial

cells

are

responsive

to

ATP

as

all

types

of

glia

such

as

astrocytes

oligodendrocytes

microglia

and

Schwann

cells

express

purinergic

receptors

-LSB-

-RSB-

In

Schwann

cells

and

oligodendrocytes

ATP-mediated

signaling

predominantly

occurs

through

receptors

which

in

turn

trigger

intracellular

release

-LSB-

-RSB-

However

the

function

of

receptors

in

astrocytes

remains

unclear

although

currents

could

be

detected

in

astrocyte

cells

in

culture

and

receptors

are

widespread

in

these

cells

with

possible

contribution

to

pathological

conditions

-LSB-

-RSB-

Glial

cells

express

many

types

of

neurotransmitter

receptors

and

conventionally

are

considered

to

be

nonexcitable

-LSB-

-RSB-

However

a

surprising

observation

was

reported

by

Dani

et

al.

-LSB-

-RSB-

that

synaptic

transmission

may

propagate

to

glial

cells

as

calcium

waves

inducing

membrane

depolarization

and

regulating

neurotransmitter

release

These

properties

of

glial

cells

suggest

possible

rapid

communications

between

neurons

and

glia

during

synaptic

transmission

This

glial

communication

mechanism

allows

the

released

ATP

to

act

onto

adjacent

astrocytes

and

neurons

thus

supporting

the

propagation

of

waves

in

glial

syncytium

-LSB-

-RSB-

For

example

in

neuronal-glial

cocultures

prepared

from

hippocampus

ATP

secreted

by

astrocytes

was

shown

to

inhibit

glutamatergic

synapses

through

activation

of

receptors

-LSB-

-RSB-

The

glial

communication

mechanism

based

on

wave

propagation

could

be

inhibited

by

receptor

blockers

or

enzymes

that

rapidly

hydrolyze

extracellular

ATP

-LSB-

-RSB-

A

stimulation

applied

to

a

single

astrocyte

in

cocultures

of

rat

forebrain

astrocytes

and

associated

neurons

caused

an

elevation

of

-LSB-

-RSB-

i

and

induced

wave

propagation

in

dorsal

spinal

cord

through

receptor

activation

-LSB-

-RSB-

and

glutamate

release

-LSB-

-RSB-

This

new

finding

provided

a

parallel

mechanism

of

intercellular

communication

that

could

allow

astrocytes

to

detect

synaptic

function

propagate

the

information

through

neighboring

glial

cells

and

then

influence

synaptic

function

in

a

distant

part

of

the

nervous

system

Purinergic

receptor-calcium

signaling

in

glial

cells

plays

important

roles

during

CNS

development

and

receptors

were

expressed

in

microglia

at

rat

embryonic

stage

-LRB-

-RRB-

-LSB-

-RSB-

Moreover

changes

in

receptor

expression

in

microglial

cells

during

postnatal

development

of

the

rat

cerebellum

have

been

reported

receptor

immunoreactivity

was

also

upregulated

in

microglia

and

granular

cells

Both

and

receptors

contribute

to

the

modulation

of

oligodendrocyte

-LRB-

OP

-RRB-

development

since

they

have

been

shown

to

exert

similar

effects

on

OP

proliferation

and

differentiation

-LSB-

-RSB-

The

majority

of

the

studies

of

ATP

action

have

been

concerned

with

the

short-term

receptor

signaling

that

occurs

in

neurotransmission

and

in

secretion

-LSB-

-RSB-

Furthermore

there

is

increasing

evidence

that

purines

and

pyrimidines

can

have

trophic

roles

in

neuritogenesis

-LSB-

-RSB-

regeneration

-LSB-

-RSB-

and

proliferation

-LSB-

-RSB-

However

some

purines

by

themselves

have

limited

trophic

effects

in

a

few

types

of

cells

they

appear

to

be

much

more

effective

as

neuritogenic

agents

when

they

are

combined

with

other

trophic

factors

such

as

NGF

For

instance

inosine

and

AMP

alone

do

not

elicit

neurite

extensions

in

cells

-LSB-

-RSB-

Heine

et

al.

-LSB-

-RSB-

demonstrated

that

receptor

activation

induced

fiber

outgrowth

in

organotypic

cocultures

in

rat

hippocampus

Fiber

outgrowth

was

inhibited

in

the

presence

of

the

purinergic

antagonist

PPADS

suggesting

the

involvement

of

receptors

In

another

study

the

synergistic

interaction

between

bFGF

and

ATP

was

reported

on

DNA

synthesis

in

primary

cultures

of

rat

cortical

astrocytes

ATP

and

bFGF

induced

a

twofold

and

tenfold

incorporation

of

-LSB-

-RSB-

thymidine

into

astrocytes

respectively

but

when

ATP

and

bFGF

were

added

at

the

same

time

a

increase

in

-LSB-

-RSB-

thymidine

incorporation

was

observed

-LSB-

-RSB-

Neuroprotection

ATP

can

activate

receptors

in

astrocytes

to

release

glutamate

GABA

and

also

ATP

which

might

regulate

the

excitability

of

neurons

in

certain

pathological

conditions

-LSB-

-RSB-

It

has

been

suggested

that

astrocytes

can

sense

the

severity

of

damage

in

the

CNS

by

the

amount

of

ATP

released

from

damaged

cells

and

that

extracellular

ATP

concentration

and

the

corresponding

subtype

of

activated

astrocytic

receptor

modulate

the

tumor

necrosis

factor-α

-LRB-

TNF-α

-RRB-

mediated

inflammatory

response

-LSB-

-RSB-

After

mechanical

brain

injury

the

administration

of

PPADS

facilitated

the

recovery

of

pathologically

changed

electroencephalograms

-LSB-

-RSB-

These

results

suggest

that

interference

with

the

ATP-induced

excitatory

responses

could

provide

neuroprotection

and

possible

therapeutic

consequences

Evidence

for

a

neuroprotective

role

was

also

found

for

the

adenosine

receptor

in

hippocampus

This

cerebral

region

is

highly

sensitive

to

hypoxia

and

ischemia

The

study

of

the

action

of

hypoxia

on

synaptic

transmission

in

hippocampal

slices

has

suggested

that

substances

being

released

during

hypoxia

such

as

GABA

ACh

and

even

glutamate

may

also

play

neuroprotective

roles

However

the

actions

of

these

neurotransmitters

become

evident

only

when

activation

of

receptors

is

impaired

suggesting

a

critical

role

for

this

receptor

during

hypoxic

events

These

substances

can

operate

in

a

redundant

or

even

overprotective

manner

acting

as

a

substitute

for

some

adenosine

actions

when

the

nucleoside

is

not

operative

-LSB-

-RSB-

Neuroimmune

interactions

Microglia

the

immune

cells

of

the

CNS

can

be

activated

by

purines

and

pyrimidines

to

release

inflammatory

cytokines

such

as

and

TNF-α

However

hyperstimulation

of

the

immune

reaction

in

the

brain

may

accelerate

neuronal

damage

The

receptor

is

considered

to

have

a

potentially

pivotal

role

in

the

regulation

of

various

inflammatory

conditions

ATP

selectively

suppresses

the

synthesis

of

the

inflammatory

protein

microglial

response

factor

through

calcium

influx

via

receptors

in

microglia

-LSB-

-RSB-

which

also

leads

to

enhancement

of

interferon-γ

-LRB-

IFN-γ

-RRB-

induced

type

II

nitric

oxide

synthase

-LRB-

NOS

-RRB-

activity

-LSB-

-RSB-

receptor

activity

also

participated

in

ATP-induced

release

from

macrophages

and

microglia

that

had

been

primed

with

substances

such

as

bacterial

endotoxin

-LSB-

-RSB-

and

was

shown

to

stimulate

the

transcription

of

nuclear

factor

κB

TNF-α

-LSB-

-RSB-

the

stress-activated

protein

kinases

-LRB-

SAPK

-RRB-

JNK

pathway

-LSB-

-RSB-

and

the

production

of

which

is

also

involved

in

inflammation

induction

by

microglial

cells

rather

than

receptors

seem

to

have

a

major

role

in

the

production

by

microglial

cells

-LSB-

-RSB-

ATP

evoked

the

release

of

plasminogen

-LSB-

-RSB-

and

-LSB-

-RSB-

The

stimulation

of

microglia

by

either

ATP

or

BzATP

revealed

neurotoxic

properties

and

the

involvement

of

the

receptor

has

been

reported

in

excitotoxic/necrotic

and

apoptotic

degeneration

-LSB-

-RSB-

Neurological

disorders

Epilepsy

Several

anti-epileptic

agents

reduce

the

ability

of

astrocytes

to

transmit

waves

raising

the

possibility

that

blockade

of

ATP-induced

-LSB-

-RSB-

i

transients

in

astrocytes

by

purinergic

receptor

antagonists

could

offer

new

treatments

for

epileptic

disorders

Antiepileptic

effects

of

adenosine

are

mostly

due

to

the

well-known

inhibitory

actions

of

receptors

on

synaptic

transmission

in

the

hippocampus

However

as

recently

pointed

out

adenosine

actions

are

not

limited

to

presynaptic

actions

on

glutamate

release

-LSB-

-RSB-

The

intraventricular

injection

of

high

doses

of

ATP

in

rats

evoked

severe

chronic-tonic

convulsions

whereas

lower

doses

of

ATP

or

adenosine

elicited

a

kinetic

state

with

muscle

weakness

-LSB-

-RSB-

and

receptor

expression

in

the

hippocampus

of

seizure-prone

gerbils

was

significantly

reduced

compared

with

that

of

normal

gerbils

-LSB-

-RSB-

GABAA

receptors

mediated

modulation

of

expression

of

both

and

receptors

which

may

play

an

important

role

in

the

regulation

of

seizure

activity

in

the

gerbil

hippocampus

-LSB-

-RSB-

receptors

are

thought

to

play

a

definite

but

not

yet

well

defined

role

in

epilepsy

Treatment

with

the

GABAB

receptor

agonist

baclofen

and

antagonist

phaclofen

resulted

in

increased

and

decreased

receptor

expression

in

hippocampus

respectively

-LSB-

-RSB-

These

purinergic

receptor

responses

were

interpreted

as

compensatory

responses

to

the

modulation

of

GABAB

receptor

function

-LSB-

-RSB-

It

is

noteworthy

to

mention

that

this

positive

relationship

between

and

GABAA

receptors

was

also

reported

for

the

spinal

cord

-LSB-

-RSB-

and

dorsal

root

ganglia

-LRB-

DRG

-RRB-

-LSB-

-RSB-

In

these

populations

of

neurons

ATP-mediated

receptor

function

may

participate

in

neuronal

transmission

accompanied

by

GABA-mediated

actions

-LSB-

-RSB-

Pain

The

heteromeric

channel

comprised

of

and

subunits

was

expressed

almost

exclusively

in

a

subset

of

primary

afferents

implicated

in

nociception

-LSB-

-RSB-

It

has

been

observed

that

mechanical

allodynia

is

reduced

in

mice

with

deleted

receptor

genes

-LSB-

-RSB-

in

agreement

with

data

obtained

in

rats

that

have

been

treated

with

intrathecal

antisense

oligonucleotides

reducing

expression

of

receptors

-LSB-

-RSB-

or

with

the

selective

antagonist

for

and

receptors

-LSB-

-RSB-

receptor

knockout

mice

showed

additional

defects

in

afferent

pathways.The

receptor

is

also

implicated

in

pain

sensation

Activation

of

dorsal

horn

microglia

and

tactile

allodynia

developing

several

days

after

ligation

of

a

spinal

nerve

were

greatly

reduced

when

gene

expression

of

receptor

in

the

dorsal

horn

had

been

inhibited

by

the

presence

of

intrathecal

antisense

oligonucleotides

-LSB-

-RSB-

Accordingly

intraspinal

administration

of

microglia

following

induction

of

expression

and

activity

of

receptors

produced

tactile

allodynia

in

naive

rats

Intrathecal

administration

of

cultured

brain

microglia

produced

allodynia

but

only

when

the

cells

had

been

pretreated

with

ATP

-LSB-

-RSB-

The

inhibition

of

receptor

activity

in

microglia

might

be

a

new

therapeutic

strategy

for

pain

induced

by

nerve

injury

Alzheimer

's

disease

Alzheimer

's

disease

-LRB-

AD

-RRB-

is

caused

by

extracellular

deposition

of

amyloid

β-peptide

which

can

damage

neurons

leading

to

their

dysfunction

and

death

-LSB-

-RSB-

ATP

and

in

particular

aluminum-ATP

promoted

the

formation

of

thioflavin

T-reactive

fibrils

of

β-amyloid

and

an

unrelated

amyloidogenic

peptide

which

could

be

blocked

by

suramin

-LSB-

-RSB-

Microglial

cells

are

believed

to

contribute

to

the

progression

of

AD

and

are

known

to

release

proinflammatory

neurotoxic

substances

Extracellular

ATP

acting

through

the

receptor

can

alter

β-amyloid

peptide-induced

cytokine

secretion

from

human

macrophages

and

microglia

and

thus

may

be

an

important

modulator

of

neuroinflammation

in

AD

-LSB-

-RSB-

receptors

mediate

superoxide

production

in

primary

microglia

and

the

expression

of

this

receptor

subtype

was

specifically

upregulated

around

β-amyloid

plaques

in

a

transgenic

mouse

model

of

AD

-LSB-

-RSB-

In

contrast

to

the

control

human

brain

the

receptor

was

colocalized

with

a

number

of

characteristic

AD

structures

such

as

neurofibrillary

tangles

neuritic

plaques

and

neuropil

threads

in

the

hippocampus

and

cortex

-LSB-

-RSB-

In

general

control

brain

tissue

exhibited

a

greater

and

more

abundant

level

of

receptor

immunostaining

than

AD

tissue

did

probably

due

to

severe

neuronal

cell

degeneration

in

most

AD

brains

The

intense

receptor

staining

observed

over

pathological

AD

structures

might

imply

that

this

receptor

is

involved

either

directly

or

indirectly

in

signaling

events

mediating

neurodegeneration

of

pyramidal

cells

Alternatively

receptors

might

have

other

diverse

signaling

roles

possibly

involved

in

the

production

of

intracellular

tau

deposits

or

might

even

serve

to

stabilize

these

tangle

structures

in

some

way

-LSB-

-RSB-

Ischemia/hypoxia

Under

pathological

conditions

of

hypoxia

or

ischemia

extracellular

purine

nucleotides

leak

from

damaged

cells

and

thereby

may

reach

high

concentrations

in

the

extracellular

space

-LSB-

-RSB-

A

direct

participation

of

extracellular

ATP

and

receptors

in

ischemic

stress

has

been

reported

in

various

cellular

systems

-LSB-

-RSB-

For

example

and

receptor

expression

in

neurons

and

microglia

respectively

in

the

hippocampus

of

gerbils

was

upregulated

following

transient

global

ischemia

-LSB-

-RSB-

Increased

receptor

expression

in

astrocytes

microglia

and

neurons

appears

to

contribute

to

the

mechanisms

of

cell

death

caused

by

in

vivo

and

in

vitro

ischemia

-LSB-

-RSB-

Following

induction

of

ischemia

receptor

mRNA

transcription

and

protein

expression

were

elevated

in

cultured

cerebellar

granule

neurons

and

organotypic

hippocampal

cultures

-LSB-

-RSB-

Hence

the

receptor

is

apparently

an

important

element

in

the

mechanisms

of

cellular

damage

induced

by

hypoxia/ischemia

In

many

cell

types

the

activation

of

the

receptor

led

to

rapid

cytoskeletal

rearrangements

such

as

membrane

blebbing

and

cell

lysis

-LSB-

-RSB-

receptors

are

intensely

expressed

in

Purkinje

cells

in

deep

layers

of

the

cerebral

cortex

and

in

ischemia-sensitive

areas

of

the

hippocampus

-LSB-

-RSB-

In

conclusion

extensive

evidence

demonstrates

a

postischemic

time

and

region-dependent

upregulation

of

and

receptor

subtypes

in

neurons

and

glial

cells

and

suggests

a

direct

role

of

receptors

in

the

pathophysiology

of

cerebral

ischemia

in

vitro

and

in

vivo

Trauma

and

axotomy

receptors

are

suggested

to

be

involved

in

neuronal

reactions

after

axotomy

Colocalization

and

temporal

coactivation

of

purinergic

and

nitrergic

markers

support

this

idea

indicating

possible

interactions

between

these

two

systems

-LSB-

-RSB-

Following

peripheral

nerve

lesions

receptor

expression

in

DRG

neurons

was

changed

-LSB-

-RSB-

The

increased

expression

of

receptor

mRNA

in

intact

neurons

indicates

a

role

of

this

subtype

in

the

post-injury

pathomechanism

in

primary

sensory

neurons

-LSB-

-RSB-

After

spinal

cord

injury

large

regions

of

the

peritraumatic

zone

were

characterized

by

a

sustained

process

of

pathologically

high

ATP

release

-LSB-

-RSB-

Spinal

cord

neurons

express

receptors

and

exposure

to

ATP

led

to

high-frequency

spiking

irreversible

increases

in

-LSB-

-RSB-

i

and

cell

death

The

administration

of

receptor

antagonists

-LRB-

PPADS

oxATP

-RRB-

after

acute

impact

injury

significantly

improved

functional

recovery

and

diminished

cell

death

in

the

peritraumatic

zone

-LSB-

-RSB-

The

involvement

of

and

receptors

in

neuronal

reactions

after

hemicerebellectomy

was

also

described

-LSB-

-RSB-

Furthermore

neuronal

NOS

and

receptors

were

colocalized

and

showed

temporal

coactivation

after

cerebellar

lesions

indicating

a

close

relationship

between

these

two

systems

-LSB-

-RSB-

In

addition

in

this

mixed

model

of

differentiation

and

axotomy

the

colocalization

of

-LRB-

involved

in

resistance

to

degeneration

phenomena

which

may

be

lost

after

mutation

-RRB-

immunopositive

cells

and

receptors

was

demonstrated

in

neurons

and

post-lesional

induction

of

receptor

and

immunoreactivity

was

reported

as

well

-LSB-

-RSB-

In

vivo

treatment

of

receptor-expressing

sciatic

nerves

with

ATP-γS

increased

expression

levels

of

the

growth-associated

protein

-LRB-

-RRB-

as

a

marker

for

axonal

growth

in

wild-type

but

not

in

mice

-LSB-

-RSB-

Possible

therapeutic

manipulations

to

modulate

astrocytic

proliferation

and

to

diminish

glial

scar

formation

in

the

adult

brain

and

during

development

include

the

use

of

drugs

known

to

interfere

with

nucleotide

synthesis

Pekovic

et

al.

-LSB-

-RSB-

showed

that

treatment

with

the

purine

nucleoside

analogue

ribavirin

-LRB-

Virazole

1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide

-RRB-

downregulates

the

process

of

reactive

gliosis

after

sensory

motor

cortex

lesion

of

the

adult

brain

and

facilitates

re-establishing

synaptic

connections

with

the

denervated

cells

at

the

lesion

site

This

may

be

a

useful

approach

for

improving

neurological

recovery

from

brain

damage

The

antiproliferative

effect

of

ribavirin

is

due

to

the

inhibition

of

de

novo

nucleic

acid

synthesis

after

depletion

of

GTP

and

dGTP

pools

with

consequent

impairment

of

specific

transduction

pathways

ATP-induced

effects

on

cell

cycle

progression

There

is

evidence

showing

that

extracellular

ATP

enhances

the

expression

of

cell

cycle

regulating

proteins

-LSB-

-RSB-

Progression

of

the

cell

cycle

is

highly

controlled

Cyclins

are

synthesized

and

degraded

in

a

synchronous

way

due

to

changing

transcription

or

proteolysis

rates

thereby

directing

the

periods

of

the

cellular

cycle

Cyclins

interact

with

cyclin-dependent

kinases

-LRB-

cdks

-RRB-

resulting

in

activation

of

their

kinase

activity

phosphorylating

their

targets

and

themselves

and

regulating

the

specific

progression

of

the

cell

cycle

through

checkpoints

-LSB-

-RSB-

Proliferation

rates

in

mammalians

are

largely

determined

during

the

phase

of

the

cell

cycle

The

relevant

proteins

include

three

D-type

cyclins

-LRB-

and

-RRB-

that

in

different

combinations

bind

to

and

allosterically

regulate

one

of

two

cdk

subunits

and

as

well

as

the

E-type

cyclins

-LRB-

and

-RRB-

which

govern

the

activity

of

a

single

catalytic

subunit

-LSB-

-RSB-

Various

combinations

of

D-type

cyclins

are

expressed

in

different

cell

types

whereas

cyclin

complexes

are

ubiquitously

expressed

-LSB-

-RSB-

Two

families

of

cdk

inhibitors

regulate

the

activity

of

cyclins-cdks

complexes

the

family

-LRB-

and

-RRB-

which

blocks

the

activity

of

cyclin

complexes

and

the

Cip/Kip

family

-LRB-

and

-RRB-

which

preferentially

inhibits

cyclin

complexes

and

also

acts

as

a

scaffold

for

the

catalytically

active

cyclin

complexes

In

addition

to

cyclins

and

cdks

mitogen-activated

protein

kinase

-LRB-

MAPK

-RRB-

is

also

believed

to

have

a

role

in

induction

of

cell

proliferation

Therefore

cyclin

D-dependent

kinases

may

play

a

role

in

controlling

the

cell

cycle

of

embryonic

and

maybe

neural

progenitor

cells

In

addition

MAPK

is

also

believed

to

have

a

role

in

induction

of

cell

proliferation

Extracellular

ATP

induces

dependent

MAPK

activation

via

stimulation

of

receptors

in

neonatal

rat

astrocytes

-LSB-

-RSB-

On

the

other

hand

cell

proliferation

is

associated

with

activation

of

diverse

proteins

Positive

regulators

include

cyclins

and

their

partners

with

catalytic

activity

-LRB-

cdks

-RRB-

which

are

essential

for

progression

of

the

cells

through

each

phase

of

the

cell

cycle

and

various

cell

cycle

checkpoints

-LSB-

-RSB-

The

regulation

of

cyclin

expression

is

also

mediated

by

the

Ras/ERK

signaling

pathways

-LSB-

-RSB-

Raf/MEK/ERK

and

signaling

pathways

can

act

in

synergy

to

promote

the

phase

cell

cycle

progression

in

both

normal

and

cancer

cells

-LSB-

-RSB-

The

promoter

for

cyclin

contains

an

site

and

the

ectopic

expression

of

either

c-fos

or

c-jun

induces

cyclin

mRNA

expression

-LSB-

-RSB-

In

many

cell

types

phosphatidylinositol

-LRB-

PI

-RRB-

signaling

pathways

also

regulate

cyclin

expression

-LSB-

-RSB-

It

was

also

reported

that

the

control

of

the

cell

cycle

regulatory

proteins

was

dependent

on

and

MAPK

pathways

indicating

that

extracellular

ATP

alone

is

sufficient

to

induce

cell

cycle

progression

beyond

the

phase

of

the

cell

cycle

These

findings

also

suggest

that

once

receptors

are

activated

protein

kinase

C

-LRB-

PKC

-RRB-

transmit

signals

to

the

nucleus

through

one

or

more

of

the

MAPK

cascades

which

may

include

MEK

and

ERK

and

stimulate

transcription

factors

such

as

myc

max

fos

and

jun.

Moreover

MAPKs

are

upstream

regulators

of

and

expression

It

has

been

reported

that

MAPK

phosphorylation

is

essential

and

sufficient

for

the

increase

in

-LSB-

-RSB-

and

decrease

in

expression

-LSB-

-RSB-

However

Delmas

et

al.

-LSB-

-RSB-

provided

evidence

that

MAPK

activation

triggers

degradation

independently

from

E

in

NIH

cells

As

described

above

ATP

regulation

of

the

MAPK

and

cdk-cyclin

complex

has

not

been

elucidated

in

other

types

of

cells

-LSB-

-RSB-

It

is

documented

in

the

literature

that

purinergic

receptor

inhibitors

interfere

with

the

S

phase

of

the

cell

cycle

Neurospheres

treated

with

the

purinergic

receptor

antagonists

reactive

blue

or

suramin

are

mostly

in

S

phase

-LRB-

or

-RRB-

when

compared

to

untreated

control

neurospheres

with

of

the

cells

being

in

S

phase

Moreover

neurosphere

cultures

treated

with

suramin

or

reactive

blue

showed

an

increase

in

the

expression

of

the

tumor

suppressor

as

a

strong

regulator

of

cell

division

-LSB-

-RSB-

The

discussed

findings

led

to

the

suggestion

that

extracellular

ATP

plays

an

important

physiological

role

during

mammalian

embryonic

development

by

stimulating

proliferation

of

ES

cells

and

therefore

receptor

agonists

and

antagonists

might

provide

novel

and

powerful

tools

for

modulating

embryonic

cell

functions

In

conclusion

and

purinergic

receptors

can

promote

proliferation

of

ES

cells

as

well

as

of

progenitor

cell

types

by

a

mechanism

by

that

ATP

induces

increases

in

-LSB-

-RSB-

i

leading

to

activation

of

PKC

and

MAPK

followed

by

an

alteration

in

the

cdk-cyclin

complex

with

and

which

are

involved

in

stimulation

of

cell

proliferation

Pharmacological

approaches

Most

purinergic

receptors

do

not

have

specific

inhibitors

Therefore

receptor

agonists

and

antagonists

acting

on

most

of

the

purinergic

receptor

subtypes

are

widely

used

in

experimental

approaches

to

study

biological

functions

of

these

receptors

Such

approaches

are

feasible

since

these

compounds

mostly

have

higher

affinities

to

some

receptor

subtypes

than

to

other

ones

As

an

example

we

have

used

suramin

PPADS

and

reactive

blue

to

study

the

participation

of

and

receptors

in

neuronal

differentiation

of

EC

cells

-LSB-

-RSB-

One

possible

approach

towards

a

subtype-specific

inhibitor

would

be

based

on

results

from

receptor

structure

determination

Using

site-directed

mutagenesis

it

has

been

possible

to

understand

which

amino

acids

are

involved

in

ATP

binding

and

to

identify

allosteric

sites

in

purinergic

receptors

The

knowledge

obtained

on

location

and

structural

features

of

ligand

and

inhibitor

binding

sites

is

used

in

rational

based

drug

design

of

selective

purinergic

subtype

antagonists

Alternatively

combinatorial

libraries

formed

by

vast

amounts

of

possible

ligands

can

be

employed

for

discovery

of

subtype-specific

inhibitors

was

identified

as

a

specific

inhibitor

for

and

receptors

In

the

presence

of

both

thermal

hyperalgesia

and

mechanical

allodynia

were

attenuated

after

chronic

nerve

constriction

injury

in

which

homomeric

and

heteromeric

receptor

activities

were

involved

Although

active

in

chronic

pain

models

was

ineffective

in

reducing

nociception

in

animal

models

of

acute

postoperative

pain

and

visceral

pain

indicating

that

and

receptor

activation

may

not

be

a

major

mediator

of

acute

postoperative

or

visceral

pain

-LSB-

-RSB-

MRS

-LRB-

bisphosphate

-RRB-

was

discovered

as

a

specific

inhibitor

of

receptor

activity

-LSB-

-RSB-

This

compound

has

an

efficient

antithrombotic

action

in

which

receptors

are

involved

-LSB-

-RSB-

Based

on

structure

design

or

combinatorial

library

approaches

specific

agonists

or

antagonists

may

be

discovered

for

other

purinergic

receptor

subtypes

For

instance

the

SELEX

-LRB-

systematic

evolution

of

ligands

by

exponential

enrichment

-RRB-

technique

provides

a

particularly

promising

approach

for

the

discovery

of

such

compounds

This

technique

is

based

on

the

reiterative

presentation

of

a

partial

random

RNA

or

single-stranded

DNA

library

to

a

protein

preparation

containing

a

particular

purinergic

receptor

subtype

RNA

or

DNA

molecules

bound

to

a

target

site

on

the

receptor

are

displaced

from

the

receptor

and

eluted

by

addition

of

an

excess

concentration

of

an

unspecific

purinergic

receptor

antagonist

and

amplified

by

reverse

transcription

polymerase

chain

reaction

-LRB-

PCR

-RRB-

or

PCR

to

restore

the

library

used

for

the

next

in

vitro

selection

cycle

Using

this

approach

it

was

possible

to

identify

inhibitors

specific

for

isoforms

of

a

target

protein

-LSB-

-RSB-

Our

group

prepared

membrane

protein

fractions

of

cells

stably

transfected

with

rat

receptors

and

coupled

them

onto

an

immobilized

artificial

membrane

-LRB-

IAM

-RRB-

as

matrix

for

affinity

chromatography

The

equilibrium

binding

to

the

receptor

and

competition

between

ATP

and

the

purinergic

antagonists

suramin

and

O

-LRB-

-RRB-

adenosine

triphosphate

-LRB-

TNP-ATP

-RRB-

were

analyzed

by

a

chromatographic

assay

using

-LSB-

-RSB-

ATP

as

a

radioligand

Our

data

indicate

that

suramin

does

not

compete

with

ATP

for

the

ligand

binding

site

and

TNP-ATP

is

a

competitive

antagonist

as

already

shown

by

Trujillo

et

al.

-LSB-

-RSB-

Moreover

this

chromatographic

assay

can

be

used

in

in

vitro

selection

procedures

for

RNA

aptamers

binding

to

receptors

from

a

combinatorial

SELEX

RNA

library

-LSB-

-RSB-

The

development

of

a

subtype-specific

receptor

antagonist

by

using

the

SELEX

technique

or

another

combinatorial

library-based

approach

shall

serve

as

proof

of

principle

and

encourage

further

works

to

obtain

such

specific

antagonists

for

all

receptor

subtypes

as

tools

for

elucidating

their

biological

functions

and

for

possible

therapeutic

applications

Conclusion

receptor

function

is

involved

in

most

physiological

processes

and

participates

in

neurotransmission

in

the

CNS

Results

obtained

with

mouse

ES

and

EC

and

neural

progenitor

cells

suggest

an

important

role

of

purinergic

signaling

in

early

embryogenesis

especially

in

cell

proliferation

migration

and

differentiation

with

different

subtypes

of

receptors

participating

in

these

processes

Our

understanding

of

the

biological

functions

of

specific

receptor

subtypes

during

CNS

development

and

in

the

adult

brain

has

increased

due

to

the

availability

of

knockout

animals

and

specific

inhibition

of

gene

expression

or

activity

of

purinergic

receptor

subtypes

The

importance

of

receptor

signaling

in

neuroprotection

neuroimmunity

and

guiding

neuronal

differentiation

especially

in

glial

and

microglial

cells

has

been

related

to

purinergic

receptor

expression

Most

importantly

specific

agonists

and

antagonists

for

individual

receptor

subtypes

are

both

needed

for

studying

their

involvement

in

biological

processes

The

discovery

of

such

selective

compounds

will

elucidate

yet

unknown

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open

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for

therapeutic

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to

disease

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in

which

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receptor

activity

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involved

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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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 B 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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knockout animal
p19 embryonal carcinoma cells