The

E-NTPDase

family

of

ectonucleotidases

Structure

function

relationships

and

pathophysiological

significance

Ectonucleotidases

are

ectoenzymes

that

hydrolyze

extracellular

nucleotides

to

the

respective

nucleosides

Within

the

past

decade

ectonucleotidases

belonging

to

several

enzyme

families

have

been

discovered

cloned

and

characterized

In

this

article

we

specifically

address

the

cell

surface-located

members

of

the

ecto-nucleoside

triphosphate

diphosphohydrolase

-LRB-

-RRB-

family

-LRB-

and

-RRB-

The

molecular

identification

of

individual

NTPDase

subtypes

genetic

engineering

mutational

analyses

and

the

generation

of

subtype-specific

antibodies

have

resulted

in

considerable

insights

into

enzyme

structure

and

function

These

advances

also

allow

definition

of

physiological

and

patho-physiological

implications

of

NTPDases

in

a

considerable

variety

of

tissues

Biological

actions

of

NTPDases

are

a

consequence

-LRB-

at

least

in

part

-RRB-

of

the

regulated

phosphohydrolytic

activity

on

extracellular

nucleotides

and

consequent

effects

on

signaling

It

further

appears

that

the

spatial

and

temporal

expression

of

NTPDases

by

various

cell

types

within

the

vasculature

the

nervous

tissues

and

other

tissues

impacts

on

several

patho-physiological

processes

Examples

include

acute

effects

on

cellular

metabolism

adhesion

activation

and

migration

with

other

protracted

impacts

upon

developmental

responses

inclusive

of

cellular

proliferation

differentiation

and

apoptosis

as

seen

with

atherosclerosis

degenerative

neurological

diseases

and

immune

rejection

of

transplanted

organs

and

cells

Future

clinical

applications

are

expected

to

involve

the

development

of

new

therapeutic

strategies

for

transplantation

and

various

inflammatory

cardiovascular

gastrointestinal

and

neurological

diseases

Introduction

Extracellular

nucleotides

modulate

a

multiplicity

of

tissue

functions

including

development

blood

flow

secretion

inflammation

and

immune

reactions

Indeed

signaling

via

extracellular

nucleotides

has

been

recognized

for

over

a

decade

as

one

of

the

most

ubiquitous

intercellular

signaling

mechanisms

-LSB-

-RSB-

Essentially

every

cell

in

a

mammalian

organism

leaks

or

releases

these

mediators

and

carries

receptors

for

nucleotides

of

which

seven

ionotropic

-LRB-

-RRB-

and

at

least

eight

metabotropic

-LRB-

-RRB-

receptor

subtypes

have

been

identified

and

characterized

to

date

Whereas

receptors

respond

to

ATP

receptors

can

be

activated

by

ATP

ADP

UTP

UDP

ITP

and

nucleotide

sugars

albeit

agonist

specificity

varies

between

subtypes

and

the

multiple

animal

species

-LSB-

-RSB-

Depending

on

the

receptor

subtype

and

signaling

pathways

involved

these

receptors

trigger

and

mediate

short-term

-LRB-

acute

-RRB-

processes

that

affect

cellular

metabolism

adhesion

activation

or

migration

In

addition

purinergic

signaling

also

has

profound

impacts

upon

other

more

protracted

responses

including

cell

proliferation

differentiation

and

apoptosis

such

as

seen

in

atherosclerosis

degenerative

neurological

diseases

and

in

several

inflammatory

conditions

-LSB-

-RSB-

The

effects

of

extracellular

nucleotides

appear

to

overlap

at

least

in

part

with

those

of

vascular

growth

factors

cytokines

-LRB-

inflammatory

-RRB-

adhesion

molecules

and

nitric

oxide

-LRB-

NO

-RRB-

Nucleotide-mediated

activation

may

be

also

synergistic

with

polypeptide

growth

factors

-LRB-

PDGF

bFGF

-RRB-

and

insulin

the

signaling

being

mediated

via

phospholipase

C

and

D

diacylglycerol

protein

kinase

C

ERKs

phosphatidylinositol

-LRB-

-RRB-

MAP

kinases

-LRB-

MAPK

-RRB-

and

Rho

-LSB-

-RSB-

The

situation

concerning

extracellular

nucleotide-signaling

can

be

suitably

contrasted

with

the

unique

specificity

of

peptide

hormones

or

vasoactive

factors

for

often

single

defined

receptors

-LSB-

-RSB-

Within

purinergic/pyrimidinergic

signaling

events

specificity

is

dictated

by

three

essential

modulatory

components

-RRB-

The

derivation

or

source

of

the

extracellular

nucleotides

-LSB-

-RSB-

-RRB-

the

expression

of

specific

receptors

for

these

molecular

transmitters

-LRB-

and

for

the

nucleotide

and

nucleoside

derivatives

-RRB-

-LSB-

-RSB-

-LRB-

See

also

Molecular

Recognition

Section

of

National

Institutes

of

Health

also

and

-RRB-

and

-RRB-

select

ectonucleotidases

that

dictate

the

cellular

responses

by

the

stepwise

degradation

of

extracellular

nucleotides

to

nucleosides

-LSB-

-RSB-

Ensembles

of

ectonucleotidases

associated

receptors

and

signaling

molecules

Within

the

past

decade

ectonucleotidases

belonging

to

several

enzyme

families

have

been

discovered

cloned

and

functionally

characterized

by

pharmacological

means

Specifically

we

refer

here

to

members

of

the

ecto-nucleoside

triphosphate

diphosphohydrolase

-LRB-

ENTPDase

-RRB-

family

-LRB-

EC

-RRB-

as

ectoenzymes

that

hydrolyze

extracellular

nucleoside

tri-and

diphosphates

and

have

a

defined

pharmacological

profile

Most

notably

in

many

tissues

and

cells

NTPDases

comprise

dominant

parts

of

a

complex

cell

surfacelocated

nucleotide

hydrolyzing

and

interconverting

machinery

This

ensemble

includes

the

ecto-nucleotide

pyrophosphatase

phosphodiesterases

-LRB-

E-NPPs

-RRB-

NADglycohydrolases

alkaline

phosphatases

dinucleoside

polyphosphate

hydrolases

adenylate

kinase

nucleoside

diphosphate

kinase

and

potentially

ATP

synthases

-LSB-

-RSB-

that

may

interact

in

various

tissues

and

cellular

systems

The

ectonucleotidase

chain

or

cascade

as

initiated

by

NTPDases

can

be

terminated

by

nucleotidase

-LRB-

EC

-RRB-

with

hydrolysis

of

nucleoside

monophosphates

-LSB-

-RSB-

Together

nucleotidase

and

adenosine

deaminase

-LRB-

ADA

EC

-RRB-

another

ectoenzyme

that

is

involved

in

purine

salvage

pathways

and

converts

adenosine

to

inosine

closely

regulate

local

and

pericellular

extracellular

and

plasma

concentrations

of

adenosine

-LSB-

-RSB-

Several

of

these

ectonucleotidase

families

and

additional

functions

of

NTPDases

-LSB-

-RSB-

are

addressed

elsewhere

in

this

issue

in

detail

This

review

focuses

on

the

surface-located

mammalian

members

of

the

E-NTPDase

protein

family

It

starts

with

a

brief

introduction

of

molecular

structure

and

functional

properties

followed

by

an

analysis

of

the

physiological

and

pathophysiological

roles

at

various

sites

with

an

emphasis

on

vasculature

and

neural

tissues

Molecular

identities

unraveled

The

literature

on

the

molecular

and

functional

characterization

of

the

E-NTPDase

family

has

been

intensively

reviewed

-LSB-

-RSB-

and

will

not

be

repeated

here

in

detail

Our

intent

is

to

summarize

principal

properties

of

the

enzymes

that

will

be

of

use

for

the

reader

new

to

this

field

Eight

different

ENTPD

genes

-LRB-

Table

and

Fig.

-RRB-

encode

members

of

the

NTPDase

protein

family

Four

of

the

NTPDases

are

typical

cell

surface-located

enzymes

with

an

extracellularly

facing

catalytic

site

-LRB-

-RRB-

NTPDases

and

exhibit

intracellular

localization

and

undergo

secretion

after

heterologous

expression

NTPDases

and

are

entirely

intracellularly

located

facing

the

lumen

of

cytoplasmic

organelles

-LRB-

Fig.

-RRB-

The

molecular

identification

of

individual

NTPDase

subtypes

genetic

engineering

mutational

analyses

and

the

generation

of

subtypespecific

antibodies

have

not

only

led

to

considerable

insight

into

enzyme

structure

and

function

These

advances

have

also

defined

physiological

and

pathophysiological

functions

of

NTPDases

in

a

considerable

variety

of

tissues

Table

of

mammalian

members

of

the

E-NTPDase

family

and

chromosomal

localizationProtein

nameAdditional

namesGene

name

human

mouseChromosome

location

human

mouseAccession

number

human

ATPDase

ecto-apyrase

-LSB-

-RSB-

ecto-ATPase

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

ER-UDPase

PCPH

-LSB-

-RSB-

-LRB-

-RRB-

-LSB-

-RSB-

-LSB-

-RSB-

-LRB-

-RRB-

canalicular

ecto-ATPase

hATPDase

-LSB-

-RSB-

is

provided

for

the

human

genome

from

GenBank

-LRB-

-RRB-

and

mouse

genome

informatics

-LRB-

MGI

-RRB-

for

the

mouse

genome

-LRB-

-RRB-

Since

the

mouse

genome

represents

a

composite

assembly

that

continues

to

undergo

updates

and

changes

from

build

to

build

the

computed

map

locations

may

be

corrected

in

the

future.a

For

mouse

and

the

BLAST

analysis

displayed

in

Map

Viewer

indicates

a

different

map

location

-LRB-

in

brackets

-RRB-

when

compared

with

the

mapping

data

reported

on

MGI

records

using

cytoband

information

based

on

experimental

evidence.Fig

phylogenetic

tree

derived

for

selected

members

of

the

E-NTPDase

family

-LRB-

to

-RRB-

from

rat

-LRB-

r

-RRB-

human

-LRB-

h

-RRB-

and

mouse

-LRB-

m

-RRB-

following

alignment

of

amino

acid

sequences

The

length

of

the

lines

indicates

the

differences

between

amino

acid

sequences

The

graph

depicts

a

clear

separation

between

surface-located

-LRB-

top

-RRB-

and

intracellular

-LRB-

bottom

-RRB-

NTPDases

In

addition

the

major

substrate

preferences

of

individual

subtypes

and

the

predicted

membrane

topography

for

each

group

of

enzymes

is

given

-LRB-

one

or

two

transmembrane

domains

indicated

by

barrels

-RRB-

Modified

from

-LSB-

-RSB-

The

presence

of

ATP

and/or

ADP

hydrolyzing

activity

at

the

surface

of

many

cell

types

had

been

recognized

for

several

decades

-LSB-

-RSB-

However

the

molecular

identity

of

the

first

member

of

the

ENTPDase

family

-LRB-

-RRB-

was

not

unraveled

and

determined

until

the

The

prototypic

member

of

the

enzyme

family

had

first

been

cloned

and

sequenced

as

a

lymphocyte

cell

activation

-LRB-

-RRB-

antigen

of

undetermined

function

-LSB-

-RSB-

Final

success

came

from

three

independent

approaches

Handa

and

Guidotti

-LSB-

-RSB-

purified

and

cloned

a

soluble

ATP

diphosphohydrolase

-LRB-

apyrase

-RRB-

from

potato

tubers

and

noted

that

this

protein

was

related

not

only

to

similar

enzymes

of

some

protozoans

plants

and

yeast

but

also

to

human

They

also

recognized

conserved

sequence

domains

and

the

relation

to

members

of

the

superfamily

This

was

then

followed

by

the

functional

expression

of

human

and

the

demonstration

that

this

protein

was

in

fact

an

ecto-apyrase

-LSB-

-RSB-

In

parallel

ectonucleotidases

-LRB-

termed

ATP

diphosphohydrolases

-RRB-

from

porcine

pancreas

and

bovine

aorta

were

purified

The

partial

amino

acid

sequences

for

both

ATP

diphosphohydrolases

revealed

identity

with

the

cloned

cDNA

sequence

of

-LSB-

-RSB-

The

cDNA

was

isolated

from

human

endothelial

cells

and

functional

thromboregulatory

studies

confirmed

that

the

dominant

vascular

ectonucleotidase

-LRB-

ATP

diphosphohydrolase

-RRB-

activity

was

identical

to

the

previously

described

and

cloned

human

-LSB-

-RSB-

Several

internal

peptide

sequences

obtained

from

the

purified

human

placental

ATP

diphosphohydrolase

-LSB-

-RSB-

revealed

that

in

retrospect

this

protein

was

also

identical

to

It

was

originally

thought

that

there

existed

a

single

ectonucleotidase

of

the

NTPDase

type

with

potential

post-translational

modifications

-LSB-

-RSB-

However

a

close

molecular

relative

was

soon

cloned

that

revealed

functional

properties

of

an

ecto-ATPase

-LRB-

now

-RRB-

rather

than

of

an

ecto-ATP

diphosphohydrolase

-LSB-

-RSB-

Further

human

genomic

analysis

of

expressed

sequence

tags

-LRB-

ESTs

-RRB-

allowed

the

identifi-cation

of

additional

members

of

the

gene

family

-LSB-

-RSB-

These

genes

were

originally

named

-LRB-

ike

-RRB-

to

Then

followed

the

identification

cloning

and

functional

expression

of

all

members

of

the

ENTPDase

family

the

last

to

date

being

-LSB-

-RSB-

Potential

splice

variants

have

been

isolated

for

the

surface-located

and

-LSB-

for

references

see

-RSB-

It

should

be

further

noted

that

heterologous

expression

of

potential

splice

variants

does

not

necessarily

result

in

the

formation

of

a

functional

protein

-LSB-

-RSB-

The

initially

proposed

nomenclature

-LSB-

-RSB-

has

been

somewhat

confusing

as

it

did

not

meet

with

generally

accepted

norms

for

human

cell

differentiation

molecules

-LSB-

-RSB-

While

-LRB-

now

-RRB-

indeed

belongs

to

the

cluster

of

differentiation

antigens

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

do

not

Scientists

at

the

Second

International

Workshop

on

Ecto-ATPases

proposed

that

all

E-NTPDase

family

members

be

termed

as

NTPDase

proteins

and

classi-fied

in

order

of

discovery

and

characterization

-LSB-

-RSB-

The

nomenclature

should

fall

away

for

all

but

the

prototypic

member

that

already

has

a

long

history

of

use

in

the

Immunology

and

Oncology

fields

Further

revisions

are

however

inevitable

Catalytic

properties

The

individual

NTPDase

subtypes

differ

in

cellular

location

and

functional

properties

The

four

cell

surface-located

forms

-LRB-

-RRB-

can

be

differentiated

according

to

substrate

preference

divalent

cation

usage

and

product

formation

All

surfacelocated

NTPDases

require

or

ions

in

the

millimolar

range

for

maximal

activity

and

are

inactive

in

their

absence

-LSB-

-RSB-

They

all

hydrolyze

nucleoside

triphosphates

including

the

physiologically

active

ATP

and

UTP

Notably

the

hydrolysis

rates

for

nucleoside

diphosphates

vary

considerably

between

subtypes

-LRB-

Figs.

and

-RRB-

Whereas

hydrolyzes

ATP

and

ADP

about

equally

well

and

reveal

a

preference

for

ATP

over

ADP

as

substrate

stands

out

for

its

high

preference

for

nucleoside

triphosphates

and

therefore

has

previously

also

been

classified

as

an

ecto-ATPase

-LSB-

-RSB-

In

contrast

to

and

murine

and

are

preferentially

activated

by

over

-LSB-

-RSB-

Presumably

differences

in

sequence

but

also

in

secondary

tertiary

and

quaternary

structure

account

for

differences

between

subtypes

in

the

catalytic

properties

-LSB-

-RSB-

Fig.

surface-located

catabolism

of

extracellular

nucleotides

and

potential

activation

of

receptors

for

nucleotides

-LRB-

receptors

-RRB-

and

adenosine

-LRB-

receptors

-RRB-

The

figure

depicts

the

principal

catalytic

properties

of

members

of

the

E-NTPDase

family

and

of

nucleotidase

NTPDases

sequentially

convert

ATP

to

ADP

Pi

and

ADP

to

AMP

Pi

is

distinct

among

these

enzymes

as

it

dephosphorylates

ATP

directly

to

AMP

without

the

release

of

significant

amounts

of

ADP

Hydrolysis

of

the

nucleoside

monophosphate

to

the

nucleoside

is

catalyzed

by

nucleotidase

NTPDases

NPPs

and

alkaline

phosphatase

sometimes

co-exist

and

it

seems

likely

that

they

can

act

in

concert

to

metabolize

extracellular

nucleotides

ATP

can

activate

both

receptors

and

subtypes

receptors

whereas

UTP

activates

subtypes

of

receptors

only

After

degradation

ADP

or

UDP

may

activate

additional

subtypes

of

receptors

The

adenosine

formed

can

potentially

act

on

four

different

types

of

receptors

and

is

either

deaminated

to

inosine

or

directly

recycled

via

nucleoside

transporters

Bottom

Profiles

of

nucleotide

hydrolysis

and

substrate

formation

by

plasma

membrane-located

NTPDases

The

figure

compares

catalytic

properties

of

human

and

murine

and

following

expression

in

cells

The

principal

catalytic

properties

of

the

respective

human

and

murine

enzymes

are

similar

ATP

-LRB-

-RRB-

ADP

-LRB-

-RRB-

AMP

-LRB-

-RRB-

Modified

from

-LSB-

-RSB-

Membrane-bound

hydrolyzes

ATP

almost

directly

to

AMP

with

the

transient

production

of

minor

amounts

of

free

ADP

-LRB-

Fig.

-RRB-

This

functional

property

largely

circumvents

activation

of

for

nucleoside

diphosphates

Interestingly

significant

amounts

of

UDP

are

accumulated

when

UTP

is

hydrolyzed

by

-LSB-

-RSB-

In

contrast

ADP

is

released

upon

ATP

hydrolysis

by

then

accumulates

and

is

slowly

dephosphorylated

to

AMP

On

the

one

hand

this

results

in

the

removal

of

agonists

for

nucleoside

triphosphate-sensitive

-LRB-

Fig.

-RRB-

On

the

other

hand

it

generates

agonists

for

nucleoside

diphosphate-sensitive

receptors

such

as

platelet

and

receptors

-LSB-

-RSB-

The

actions

of

and

result

in

intermediate

patterns

of

product

formation

leading

to

a

transient

accumulation

of

diphosphonucleosides

with

the

simultaneous

presence

of

triphosphonucleosides

Principal

structural

features

The

hallmarks

of

all

NTPDases

are

the

five

highly

conserved

sequence

domains

known

as

apyrase

conserved

regions

abbreviated

and

termed

to

-LSB-

-RSB-

that

are

involved

in

the

catalytic

cycle

This

notion

is

supported

by

a

considerable

variety

of

deletion

and

mutation

experiments

-LSB-

for

reviews

see

-RSB-

NTPDases

share

two

common

sequence

motifs

with

members

of

the

kinase

superfamily

the

actin-HSP

b-and

g-phosphate

binding

motif

-LSB-

-LRB-

I/L/V

-RRB-

X

-LRB-

I/L/V

C

-RRB-

DXG

-LRB-

T/S/G

-RRB-

-LRB-

T/S/G

-RRB-

XX

-LRB-

R/K/C

-RRB-

-RSB-

-LSB-

-RSB-

with

the

DXG

sequence

strictly

conserved

These

motifs

are

identified

in

and

Furthermore

there

are

striking

similarities

in

secondary

structure

with

members

of

the

kinase

superfamily

-LSB-

-RSB-

These

proteins

are

soluble

have

ATP

phosphotransferase

or

hydrolase

activity

depend

on

divalent

metal

ion

and

tend

to

form

oligomeric

structures

In

spite

of

negligible

global

sequence

identity

they

share

the

principal

structure

of

two

major

domains

-LRB-

I

and

II

possibly

resulting

from

gene

duplication

-RRB-

of

similar

folds

on

either

side

of

a

large

cleft

They

reveal

similar

conserved

secondary

structure

topology

-LRB-

β1β2β3α1β4α2β5α3

-RRB-

repeated

in

each

domain

and

fold

into

a

pocket

for

substrate

binding

at

the

bottom

-LSB-

-RSB-

Presumably

NTPDases

share

not

only

secondary

structure

but

also

major

elements

of

tertiary

structure

with

members

of

the

kinase

superfamily

-LRB-

Fig.

-RRB-

Homology

modeling

of

the

sequence

reveals

high

degrees

of

structural

fold

similarity

with

a

bacterial

exopolyphosphatase

-LRB-

PDB

-RRB-

that

further

refine

structural

predictions

for

members

of

the

E-NTPDase

family

-LSB-

-RSB-

Fig.

membrane

topology

of

a

surface-located

NTPDase

with

two

transmembrane

domains

A

comparison

of

the

conserved

secondary

structure

reveals

duplicate

conservation

of

two

major

domains

related

to

subdomains

Ia

and

IIa

of

actin

and

other

members

of

the

kinase

superfamily

-LSB-

-RSB-

In

contrast

to

the

other

members

of

the

superfamily

surface-located

NTPDases

are

anchored

to

the

plasma

membrane

by

terminal

hydrophobic

domains

The

figure

takes

into

account

the

close

distance

of

the

N-and

C-terminus

of

actin

at

domain

I

and

the

binding

of

ATP

-LRB-

red

-RRB-

in

the

cleft

between

domains

I

and

II

-LSB-

-RSB-

These

two

domains

are

expected

to

undergo

conformational

changes

involving

movement

relative

to

each

other

NTPDases

readily

form

homo-oligomeric

assemblies

to

were

found

as

dimers

to

tetramers

-LSB-

-RSB-

In

contrast

to

the

receptors

that

share

a

similar

membrane

topography

hetero-oligomeric

complexes

between

NTPDases

have

not

been

reported

to

date

Oligomeric

forms

reveal

increased

catalytic

activity

-LSB-

-RSB-

and

the

state

of

oligomerization

can

affect

catalytic

properties

-LSB-

-RSB-

and

are

firmly

anchored

to

the

membrane

via

two

transmembrane

domains

that

in

the

instance

of

are

important

for

maintaining

catalytic

activity

and

substrate

specificity

-LSB-

-RSB-

The

two

transmembrane

domains

interact

both

within

and

between

monomers

They

may

also

undergo

coordinated

motions

during

the

process

of

nucleotide

binding

and

hydrolysis

-LSB-

-RSB-

This

could

in

turn

induce

conformational

changes

-LSB-

-RSB-

involving

movement

of

the

two

major

domains

-LRB-

I

and

II

-RRB-

relative

to

each

other

-LRB-

Fig.

-RRB-

Alterations

in

quarternary

structure

and

subunit

interactions

may

thus

affect

the

impact

or

interaction

of

ACRs

involved

in

substrate

binding

and

hydrolysis

Whether

posttranslational

modifications

such

as

protein

phosphorylation

contribute

to

this

dynamic

behavior

remains

to

be

investigated

Functional

modifications

Biologically

active

is

subject

to

differential

forms

of

surface

modification

under

conditions

of

oxidative

stress

that

inhibit

enzymatic

activity

as

influenced

by

unsaturated

fatty

acids

-LSB-

-RSB-

It

also

undergoes

limited

proteolysis

that

increases

enzyme

activity

and

differential

glycosylation

reactions

that

appear

to

be

required

for

membrane

expression

-LSB-

-RSB-

Since

the

surface-located

ATP-hydrolyzing

members

of

the

NTPDase

family

pass

through

the

endoplasmic

reticulum

and

Golgi

apparatus

the

associated

catalytic

activity

might

abrogate

ATP-dependent

luminal

functional

processes

becomes

catalytically

active

on

reaching

the

cell

surface

and

glycosylation

reactions

appear

crucial

in

this

respect

-LSB-

-RSB-

The

N-terminal

intracytoplasmic

domain

of

is

palmitoylated

Truncated

forms

of

lacking

the

N-terminal

intracytoplasmic

region

and

the

associated

residue

are

not

subject

to

palmitoylation

This

post-translational

modification

appears

to

be

constitutive

and

to

contribute

to

the

integral

membrane

association

of

this

ectonucleotidase

in

lipid

rafts

-LSB-

-RSB-

This

raises

the

possibility

that

may

be

recycled

to

and

from

cell

membranes

via

sequential

actions

of

putative

palmitoyltransferases

and

palmitoyl-protein

thioesterases

-LSB-

-RSB-

in

order

to

fine

tune

and

modulate

purinergic

signaling

responses

In

contrast

to

and

does

not

have

the

required

intra-cytoplasmic

Cys

to

undergo

this

post-translational

modification

The

potential

multimerization

of

-LSB-

-RSB-

may

be

facilitated

by

acylation

with

intermolecular

interactions

within

the

cholesterol

and

sphingolipid-rich

microdomains

of

the

plasma

membrane

-LSB-

-RSB-

Experiments

using

endothelial

cells

from

deficient

mice

suggest

that

caveolae

are

not

essential

for

the

enzymatic

activity

or

for

the

targeting

to

the

plasma

membrane

of

However

cholesterol

depletion

results

in

a

strong

inhibition

of

the

enzyme

-LSB-

-RSB-

The

targeting

of

palmitoylated

to

lipid

rafts

could

influence

defined

G-protein

coupled

receptors

within

this

plasmalemmal

microenvironment

and

thus

regulate

cellular

signal

transduction

pathways

Furthermore

the

caveolar

co-localization

of

nucleotidase

receptors

and

could

serve

to

modulate

signaling

via

both

ATP

and

adenosine

at

the

cell

surface

and

possibly

also

within

endosomal

compartments

-LSB-

-RSB-

Transcriptional

regulation

of

expression

Members

of

the

E-NTPDase

family

are

constitutively

expressed

in

many

tissues

To

date

there

is

only

scattered

evidence

on

promoters

and

the

factors

controlling

NTPDase

expression

-LSB-

-RSB-

The

transcription

of

is

constitutive

in

venous

arterial

and

certain

non-fenestrated

microvascular

endothelium

and

certain

immune

cells

e.g.

B

cells

dendritic

cells

and

defined

T-cell

subsets

-LSB-

-RSB-

The

modulated

expression

of

has

been

closely

associated

with

inflammatory

cytokines

oxidative

stress

and

hypoxia

in

vitro

and

in

vivo

-LSB-

-RSB-

Expression

of

is

increased

in

differentiating

melanomas

followed

by

a

gradual

decrease

with

tumor

progression

-LSB-

-RSB-

and

enhanced

activity

of

stimulated

endothelial

and

mesangial

cells

is

downregulated

by

glucocorticosteroids

-LSB-

-RSB-

Activity

of

ecto-ATP

diphosphohydrolase

in

human

endothelial

cells

in-vitro

is

increased

by

aspirin

-LSB-

-RSB-

and

glomerular

ecto-ATP

diphosphohydrolase

immunoreactivity

might

well

be

modulated

by

estradiol

-LSB-

-RSB-

Transcription

of

in

mouse

hepatoma

cells

is

inducible

by

-LRB-

TCDD

-RRB-

-LSB-

-RSB-

These

cells

contain

both

constitutive

and

TCDD-inducible

activity

The

core

promotor

reveals

constitutive

activity

that

is

independent

of

TCDD

-LSB-

-RSB-

TCDD

does

not

increase

expression

of

or

other

ectonucleotidases

and

apparently

fails

to

induce

in

a

variety

of

other

cell

lines

derived

from

varying

species

-LSB-

-RSB-

In

rat

Sertoli

cells

is

upregulated

by

follicle-stimulating

hormone

and

cAMP

-LSB-

-RSB-

and

it

is

selectively

downregulated

in

biliary

cirrhosis

-LSB-

-RSB-

Human

epidermoid

carcinoma

cells

increase

the

cascade

for

extracellular

nucleotide

hydrolysis

when

periodically

treated

with

extracellular

ATP

suggesting

that

the

substrate

itself

may

affect

the

expression

of

its

own

hydrolysis

chain

-LSB-

-RSB-

Inhibitors

A

considerable

number

of

compounds

alter

and

inhibit

extracellular

nucleotide

hydrolysis

by

NTPDases

These

include

non-hydrolysable

nucleotide

analogues

and

inhibitors

of

receptors

Ideally

NTPDase

inhibitors

should

not

be

receptor

agonists

or

antagonists

and

not

be

subject

to

dephosphorylation

by

the

ectoenzyme

-LSB-

-RSB-

The

only

commercially

available

compound

reported

to

effectively

inhibit

hydrolysis

of

ATP

in

a

variety

of

tissues

without

significantly

acting

on

purinoceptors

is

the

structural

analogue

of

ATP

ARL

-LRB-

FPL

-RRB-

-LRB-

N-diethyl-D-β

γ-dibromomethylene

ATP

-RRB-

-LSB-

-RSB-

Other

potential

inhibitors

include

triphosphate

-LRB-

-RRB-

-LSB-

-RSB-

and

acid

-LRB-

-RRB-

-LSB-

-RSB-

Periodate-oxidized

ATP

inhibits

ecto-ATPase

activity

in

human

astrocytoma

cells

-LSB-

-RSB-

and

Gadolinium

ions

have

been

found

to

effectively

inhibit

the

ecto-nucleoside

triphosphate

diphosphohydrolase

from

Torpedo

electric

organ

as

well

as

potato

apyrase

-LSB-

-RSB-

It

is

noteworthy

that

the

potency

of

inhibitors

can

vary

considerably

between

individual

members

of

the

E-NTPDase

family

-LSB-

-RSB-

This

necessitates

a

functional

evaluation

of

each

inhibitor

for

the

enzyme

investigated

in

a

given

tissue

or

cell

type

The

failure

to

develop

specific

inhibitors

remains

a

major

impediment

to

ongoing

discoveries

Principal

functional

contexts

Cell

surface-located

NTPDases

are

considered

to

be

of

major

importance

for

controlling

the

availability

of

extracellular

nucleotide

agonists

at

receptors

They

also

contribute

to

recycling

of

nucleosides

derived

from

extracellular

nucleoside

phosphates

and

metabolic

salvage

pathways

The

number

of

studies

that

define

a

functional

impact

of

individual

NTPDases

in

purinergic

signaling

in

situ

is

limited

and

has

been

dependent

to

date

on

global

genetic

modifications

of

mice

and

swine

to

delete

or

upregulate

the

NTPDase

or

gene

of

interest

-LSB-

-RSB-

Subtype-specific

inhibitors

siRNA

approaches

and

animals

in

which

the

encoding

gene

can

be

inactivated

or

selectively

induced

in

specific

tissues

will

be

of

major

importance

There

is

increasing

experimental

evidence

that

ectonucleotidases

compete

with

receptors

for

a

limited

pool

of

endogenously

released

nucleotide

-LSB-

-RSB-

and

by

hydrolyzing

released

nucleotide

terminate

or

modulate

the

function

of

receptors

-LSB-

-RSB-

Portal

fibroblasts

regulate

receptormediated

bile

duct

epithelial

proliferation

via

expression

of

-LSB-

-RSB-

-LRB-

see

liver

section

below

-RRB-

NTPDases

functionally

interact

with

-LSB-

-RSB-

and

may

also

co-localize

with

these

G-proteincoupled

receptors

-LRB-

GPCR

-RRB-

in

lipid

rafts

and

possibly

caveolae

-LSB-

-RSB-

The

modulatory

effects

of

NTPDases

are

complex

as

the

enzymes

differentially

regulate

agonist

availability

in

a

process

that

is

dependent

upon

receptor

subtype

by

either

degrading

ATP/UTP

or

by

generating

ADP/UDP

-LRB-

Fig.

-RRB-

Recent

experiments

suggest

that

plasma

membranebound

NTPDases

may

have

functions

distinct

from

their

catalytic

properties

alone

In

a

yeast

two-hybrid

system

using

techniques

developed

by

Zhong

for

yeast

apyrases

-LSB-

-RSB-

the

N-terminus

of

human

-LRB-

used

as

bait

protein

-RRB-

has

been

shown

to

interact

with

truncated

Ran

Binding

Protein

M

-LRB-

RanBPM

otherwise

known

as

-RRB-

in

the

human

library

screened

-LSB-

-RSB-

RanBPM

contains

conserved

SPRY

-LRB-

repeats

in

splA

and

RyR

-RRB-

domains

that

appear

to

be

crucial

for

the

interaction

with

and

is

preferentially

distributed

in

human

heart

tissues

-LSB-

-RSB-

RanBPM

is

known

to

interact

with

Sos

and

regulate

ERK/Ras

signaling

interacts

with

RanBPM

to

directly

modulate

Ras

activation

and

cellular

proliferation

in

liver

regeneration

following

partial

hepatectomy

-LSB-

-RSB-

The

N-termini

of

NTPDases

also

have

consensus

sequences

for

protein

phosphorylation

by

protein

kinase

C

-LSB-

-RSB-

that

could

have

additional

functional

impacts

Furthermore

the

C-terminal

sequence

of

contains

a

putative

PDZ

domain

-LRB-

K-DM-V

-RRB-

This

may

have

utility

in

determining

interactions

with

select

receptors

e.g.

the

purinergic

and

receptors

that

terminate

in

D-T-S-L

and

D-I-R-L

respectively

-LSB-

-RSB-

PDZ

domains

are

most

often

found

in

combination

with

other

protein

interaction

domains

-LRB-

for

instance

PTB

WW

-RRB-

participating

in

complexes

that

facilitate

signaling

or

determine

the

localization

of

receptors

-LSB-

-RSB-

Finally

the

general

membrane

topography

of

and

oligomeric

assembly

resemble

the

morphology

of

channel

forming

proteins

such

as

nucleotide

receptors

and

members

of

the

epithelial

channel/degenerin

gene

superfamily

-LSB-

-RSB-

This

raises

the

question

whether

in

addition

to

their

catalytic

activity

NTPDases

could

function

as

channels

Release

of

ATP

from

Xenopus

oocytes

induced

by

hyperpolarizing

pulses

requires

functional

ecto-ATPase

activity

-LSB-

-RSB-

To

what

extent

this

functional

property

is

shared

by

the

structurally

related

and

has

not

been

investigated

Vasculature

The

normal

vascular

endothelium

provides

a

barrier

that

separates

blood

cells

and

plasma

factors

from

highly

reactive

elements

of

the

deeper

layer

of

vessel

wall

The

vessel

wall

maintains

blood

fluidity

and

promotes

flow

by

inhibiting

coagulation

platelet

activation

and

promoting

fibrinolysis

-LSB-

-RSB-

These

properties

are

governed

by

important

thromboregulatory

mechanisms

key

biological

activities

of

the

vasculature

have

been

already

identified

and

shown

to

be

ecto-nucleotide

catalysts

that

generate

the

respective

nucleosides

by

phosphohydrolysis

-LSB-

-RSB-

The

dominant

ectonucleotidases

of

the

vasculature

have

now

more

fully

been

characterized

as

NTPDases

This

important

biological

property

expressed

by

the

endothelium

and

associated

cells

is

responsible

for

the

regulation

of

extracellular

and

plasma

levels

of

nucleotides

-LSB-

-RSB-

Over

the

past

decade

extracellular

nucleotides

have

been

recognized

as

important

mediators

of

a

variety

of

processes

including

vascular

inflammation

and

thrombosis

with

varying

impacts

in

different

systems

-LSB-

-RSB-

Adenosine

and

ATP

mediated

effects

or

mechanisms

can

be

implicated

in

the

local

control

of

vessel

tone

as

well

as

in

individual

vascular

cell

migration

proliferation

and

differentiation

As

an

example

ATP

may

be

released

from

sympathetic

nerves

-LRB-

see

later

sections

-RRB-

and

results

in

constriction

of

vascular

smooth

muscle

through

effects

mediated

by

receptors

In

contrast

ATP

released

from

endothelial

cells

during

changes

in

flow

-LRB-

shear

stress

-RRB-

or

following

exposure

to

hypoxic

conditions

activates

receptors

in

a

paracrine

manner

to

release

NO

resulting

in

vessel

relaxation

Any

nucleotide

released

will

be

ultimately

hydrolyzed

to

adenosine

and

will

result

in

vasodilatation

via

the

effects

of

smooth

muscle

receptors

receptors

also

appear

on

vascular

cells

and

are

associated

with

changes

in

cell

adhesion

and

permeability

-LSB-

-RSB-

These

cellular

processes

and

nucleotidetriggered

events

are

modulated

during

angiogenesis

-LRB-

Fig.

-RRB-

and

influence

the

development

of

atherosclerosis

and

restenosis

following

angioplasty

-LSB-

-RSB-

Fig.

with

expression

of

in

the

vasculature

of

syngeneic

islet

transplants

Mouse

islets

were

prepared

from

wild

type

and

mice

as

described

by

T.

Maki

et

al.

and

transplanted

under

the

renal

capsule

-LSB-

-RSB-

Islets

were

harvested

at

four

weeks

-LRB-

n

per

group

-RRB-

and

stained

for

immunoactivity

and

other

markers

of

EC

Substantially

diminished

levels

of

staining

vascular

elements

were

also

present

in

to

grafts

indicating

a

defect

in

new

vessel

growth

-LRB-

not

depicted

here

-RRB-

A

-RRB-

Wild

type

to

wild

type

showing

grafted

islet

vasculature

staining

for

with

adjacent

normal

renal

vascular

pattern

B

-RRB-

Wild

type

to

mouse

showing

intrinsic

vasculature

of

islet

has

persisted

within

the

graft

and

even

entered

the

renal

parenchyma

C

-RRB-

to

wild

type

grafts

showing

infiltrating

macrophages

and

positive

endothelium

migrating

from

recipient

-LRB-

confirmed

by

other

stains

not

shown

-RRB-

is

the

major

ectonucleotidase

in

the

vasculature

-LSB-

-RSB-

Other

NTPDases

associated

with

the

vasculature

are

the

cell-associated

and

the

soluble

monocyte

expressed

-LSB-

-RSB-

The

phosphohydrolytic

reaction

of

limits

the

platelet

activation

response

that

is

dependent

upon

the

paracrine

release

of

ADP

and

activation

of

specific

purinergic

receptors

-LSB-

-RSB-

In

contrast

a

preferential

nucleoside

triphosphatase

activates

platelets

by

converting

the

competitive

antagonist

-LRB-

ATP

-RRB-

of

platelet

ADP-receptors

to

the

specific

agonist

of

the

and

receptors

In

keeping

with

these

biochemical

properties

is

dominantly

expressed

by

endothelial

cells

and

the

associated

vascular

smooth

muscle

where

it

serves

as

a

thromboregulatory

factor

In

contrast

is

associated

with

the

adventitial

surfaces

of

the

muscularized

vessels

microvascular

pericytes

of

some

tissues

and

organs

as

the

heart

and

the

stromal

cells

and

would

potentially

serve

as

a

hemostatic

factor

-LSB-

-RSB-

Extracellular

nucleotide

stimulation

of

receptors

represents

components

of

platelet

endothelial

cell

and

leukocyte

activation

that

culminate

in

vascular

thrombosis

and

inflammation

in

vivo

-LSB-

-RSB-

In

these

inflammatory

settings

with

oxidant

endothelial

injury

biochemical

function

is

substantially

albeit

temporarily

decreased

because

of

post-translational

changes

reconstitution

of

vascular

NTPDase

activity

occurs

following

transcriptional

upregulation

of

in

endothelium

-LSB-

-RSB-

This

functional

change

may

relate

at

least

in

part

to

alterations

in

acylation

and

associated

membrane

lipid

association

with

consequent

disruption

of

multimer

structure

Interestingly

palmitate

supplementation

may

protect

against

loss

of

NTPDase

activity

following

cellular

activation

in

vitro

-LSB-

-RSB-

These

observations

may

provide

several

avenues

of

research

to

augment

NTPDase

activity

within

the

vasculature

at

sites

of

injury

-LSB-

-RSB-

Mechanisms

of

endothelial

cell

activation

by

nucleotides

ATP

and

UTP

increases

intracellular

calcium

levels

results

in

cytoskeletal

rearrangements

and

stimulates

phosphorylation

of

several

proteins

in

human

endothelial

cells

-LRB-

EC

-RRB-

that

are

also

associated

with

integrin

signaling

-LSB-

-RSB-

These

include

the

focal

adhesion

kinase

-LRB-

FAK

-RRB-

and

paxillin

proline-rich

tyrosine

kinase

-LRB-

-RRB-

-LRB-

also

named

related

adhesion

focal

tyrosine

kinase

RAFTK

-RRB-

and

MAP

kinase

Further

UTP

preferentially

increases

EC

migration

in

a

and

ERK-dependent

manner

Moreover

extracellular

nucleotide-mediated

EC

activation

involves

cytoskeletal

rearrangements

and

increases

in

cell

motility

comparable

to

that

seen

with

ligation

of

integrins

by

extracellular

matrix

proteins

-LSB-

-RSB-

These

phenotypic

changes

-LRB-

seen

in

both

nucleotide-and

matrix-mediated

activation

-RRB-

are

associated

with

tyrosine

phosphorylation

of

FAK

paxillin

and

Crk-associated

substrate

-LRB-

-RRB-

and

down-stream

activation

of

MAP

kinases

FAK

has

been

implicated

to

play

an

important

role

in

integrin-mediated

signal

transduction

pathways

-LSB-

-RSB-

suggesting

that

are

implicated

in

inside-out

integrin

signaling

in

EC

as

well

as

platelets

-LSB-

-RSB-

Therapeutic

considerations

To

test

how

extracellular

nucleotide-mediated

signaling

influences

pathophysiological

events

several

techniques

have

been

developed

and

validated

to

manipulate

expression

in

the

vasculature

and

to

study

conditions

of

inflammatory

stress

The

first

mutant

mouse

derived

and

studied

concerned

the

global

deletion

of

the

gene

encoding

the

dominant

ectonucleotidase

-LRB-

-RRB-

The

mutant

mice

exhibit

major

perturbations

of

receptormediated

signaling

in

the

vasculature

and

immune

systems

-LSB-

-RSB-

These

phenomena

manifest

as

hemostatic

defects

thromboregulatory

disturbances

heightened

acute

inflammatory

responses

with

a

failure

to

generate

cellular

immune

responses

that

are

all

associated

with

vascular

endothelium

monocyte

dendritic

cell

and

platelet

integrin

dysfunction

-LSB-

-RSB-

The

therapeutic

potential

of

to

regulate

receptor

function

in

the

vasculature

and

mitigate

against

thrombotic/inflammatory

stress

has

been

further

established

by

the

generation

of

transgenic

mice

and

swine

-LSB-

-RSB-

the

use

of

adenoviral

vectors

to

upregulate

in

cardiac

grafts

-LSB-

-RSB-

and

the

use

of

soluble

derivatives

of

and

apyrases

-LSB-

-RSB-

The

beneficial

effects

of

administered

NTPDases

have

been

determined

in

several

animal

models

of

vascular

inflammation

-LSB-

-RSB-

Exogenous

infusions

of

soluble

NTPDases

are

able

to

rescue

mice

from

systemic

toxicity

induced

by

ischemia

reperfusion

injury

and

after

stroke

induction

-LSB-

-RSB-

Angiogenesis

requires

the

dynamic

interaction

of

endothelial

cell

proliferation

and

differentiation

with

orchestrated

interactions

between

extracellular

matrix

and

surrounding

cells

-LRB-

such

as

vascular

smooth

muscle

and/or

pericytes

-RRB-

-LSB-

-RSB-

appears

crucial

in

the

co-ordination

of

angiogenic

responses

in

inflammation

organ

remodeling

and

transplantation

-LSB-

-RSB-

For

example

in

syngeneic

pancreatic

islet

transplantation

the

maintenance

and

revascularization

of

grafted

islets

appears

dependent

upon

expression

of

by

the

developing

vasculature

within

the

islet

-LRB-

Fig.

-RRB-

In

summary

multiple

experimental

studies

largely

reveal

beneficial

effects

of

over-expression

of

NTPDases

within

the

vasculature

or

by

their

pharmacological

administration

-LSB-

-RSB-

Clinical

studies

of

these

soluble

thromboregulatory

factors

are

in

planning

-LSB-

-RSB-

Immune

system

There

are

multiple

and

receptor

subtypes

expressed

by

monocytes

and

dendritic

cells

whereas

lymphocytes

express

only

receptors

-LSB-

-RSB-

was

first

described

as

a

B

lymphocyte

activation

marker

and

also

shown

to

be

expressed

on

activated

T

cells

-LSB-

-RSB-

and

dendritic

cells

-LSB-

-RSB-

The

enzymatic

function

on

dendritic

cells

is

involved

in

the

recruitment

activation

and

polarization

of

naive

T

cells

ATP

is

released

by

and

T

cells

upon

stimulation

with

Con

A

or

mAb

while

functions

as

an

additional

recognition

structure

on

haptenated

target

immunocytes

for

hapten-specific

cytotoxic

T

cells

-LSB-

-RSB-

In

mice

there

are

major

defects

in

dendritic

cell

function

antigen

presentation

and

T-cell

responses

to

haptens

-LRB-

type

IV

hypersensitivity

reactions

-RRB-

-LSB-

-RSB-

Immunocyte-associated

may

play

an

immunoregulatory

role

by

hydrolyzing

ATP

-LRB-

and

perhaps

ADP

-RRB-

released

by

T

cells

during

antigen

presentation

and

thereby

generating

adenosine

-LSB-

-RSB-

Ectoenzymes

including

ectonucleotidases

are

known

to

play

an

important

role

in

leukocyte

trafficking

-LRB-

for

an

excellent

review

on

this

topic

see

-LSB-

-RSB-

-RRB-

Recent

work

has

indicated

that

regulatory

ve

ve

T

cells

-LRB-

Treg

cells

-RRB-

play

important

roles

in

the

maintenance

of

immunological

reactivity

and

tolerance

-LSB-

-RSB-

The

selective

expression

of

by

Treg

and

the

question

whether

this

ectonucleotidase

and/or

extracellular

nucleotides

influence

-LRB-

s

-RRB-

the

function

of

these

interesting

cells

is

a

focus

of

current

work

Digestive

and

renal

systems

Released

nucleotides

are

polarized

and

do

not

re-enter

cells

They

have

to

be

transformed

into

the

corresponding

nucleosides

that

enter

cells

via

specific

transporters

to

rebuild

nucleoside

pools

If

this

did

not

occur

they

would

be

lost

from

the

metabolic

pool

The

same

may

pertain

to

dietary

ingestion

of

nucleotides

where

NTPDases

are

potential

participants

in

the

digestion

of

exogenous

nucleotides

and

intestinal

function

In

addition

extracellular

nucleotide

and

adenosine

receptors

are

highly

expressed

in

the

digestive

and

renal

systems

so

these

molecules

are

likely

to

have

homeostatic

functions

-LSB-

-RSB-

An

important

nucleotide-mediated

mechanism

that

seems

common

to

various

epithelia

as

well

as

to

hepatocytes

involves

the

autocrine

regulation

of

cell

volume

by

ATP

via

receptors

-LSB-

-RSB-

As

receptors

are

expressed

by

epithelia

in

a

polarized

manner

and

can

be

linked

to

several

digestive

and

homeostatic

functions

-LSB-

-RSB-

the

presence

of

NTPDases

in

the

immediate

environment

may

serve

as

regulatory

switches

Liver

In

the

liver

extracellular

nucleotides

are

potentially

involved

in

several

functional

contexts

-LSB-

-RSB-

There

is

evidence

that

extracellular

nucleotides

regulate

glycogenolysis

through

activation

of

glycogen

phosphorylase

and

inactivation

of

glycogen

synthase

by

inhibition

of

the

glucagon

effect

on

cAMP

and

by

the

activation

of

phospholipase

D

-LSB-

-RSB-

In

addition

nucleotides

may

be

involved

in

the

regulation

of

canalicular

contraction

and

bile

flow

-LSB-

-RSB-

Concentrations

of

canalicular

adenine

nucleotides

in

bile

samples

and

effluents

from

hepatic

cell

lines

are

estimated

to

be

around

to

µM

-LSB-

-RSB-

Hepatocytes

and

bile

duct

cells

have

been

shown

to

interact

and

communicate

via

local

ATP

release

in

vitro

-LSB-

-RSB-

Extracellular

ATP

acts

as

a

hepatic

mitogen

and

activates

JNK

signaling

and

hepatocyte

proliferation

both

in

vitro

and

in

vivo

-LSB-

-RSB-

Several

ectonucleotidases

are

expressed

in

liver

Of

the

nucleotide

pyrophosphatase/phosphodiesterases

-LRB-

-RRB-

is

expressed

on

the

basolateral

membrane

of

hepatocytes

while

the

closely

related

-LRB-

-RRB-

has

a

predominant

canalicular

in

distribution

-LSB-

-RSB-

is

highly

expressed

on

larger

vessels

and

more

weakly

on

sinusoids

as

well

as

in

Kupffer

cells

-LSB-

-RSB-

In

the

quiescent

liver

is

expressed

by

cells

of

the

subendothelium

of

veins

and

adventitial

cells

of

arteries

but

not

in

sinusoids

In

addition

is

expressed

by

portal

fibroblasts

near

basolateral

membranes

of

bile

duct

epithelia

-LSB-

-RSB-

Activated

but

not

quiescent

hepatic

stellate

cells

express

at

the

protein

level

-LSB-

-RSB-

Only

low

expression

of

could

be

demonstrated

at

the

mRNA

level

in

the

liver

-LSB-

-RSB-

expression

in

portal

fibroblasts

the

primary

fibroblastic

cell

type

of

the

portal

area

suggests

a

role

in

the

regulation

of

bile

ductular

signaling

and

secretion

-LSB-

-RSB-

Jhandier

et

al.

tested

the

hypothesis

that

portal

fibroblast

regulates

epithelial

cell

proliferation

Using

co-cultures

of

cholangiocytes

-LRB-

human

cholangiocarcinoma

cells

-RRB-

and

primary

portal

fibroblasts

from

rat

liver

increased

expression

decreased

cell

proliferation

and

knockdown

of

by

siRNA

increased

proliferation

receptor

blockade

also

attenuated

proliferation

-LSB-

-RSB-

These

experiments

defined

a

novel

cross-talk

signaling

pathway

between

bile

duct

epithelial

cells

and

underlying

portal

fibroblasts

regulated

by

Because

they

are

the

chief

fibrogenic

cells

of

the

liver

hepatic

stellate

cells

and

portal

fibroblasts

are

important

targets

of

liver

disease

therapy

Loss

of

expression

in

human

biliary

cirrhosis

as

well

as

in

models

of

bile

duct

ligation

in

rat

has

been

observed

expression

also

shifts

from

the

portal

area

to

bridging

fibrous

bands

in

cirrhosis

with

hepatitis

C

-LSB-

-RSB-

Functional

ATPases

were

previously

shown

to

be

associated

with

bile

canalicular

plasma

membranes

by

histochemical

techniques

-LSB-

-RSB-

the

corresponding

enzyme

was

subsequently

incorrectly

identified

as

-LSB-

-RSB-

More

recent

studies

revealed

that

the

canalicular

ecto-ATPase

corresponds

to

-LSB-

-RSB-

also

referred

to

as

hepatic

ATP

diphosphohydrolase

-LRB-

ATPDase

-RRB-

-LSB-

-RSB-

is

the

mammalian

orthologue

of

the

chicken

ecto-ATPDase

cloned

from

oviduct

and

liver

-LSB-

-RSB-

In

tandem

with

nucleotidase

has

the

potential

to

regulate

the

concentration

of

nucleotides

in

the

hepatic

canalicule

The

ultimate

generation

of

extracellular

adenosine

from

dephosphorylated

ATP

not

only

activates

adenosine

receptors

but

also

produces

the

key

molecule

for

purine

salvage

and

consequent

replenishment

of

ATP

stores

within

many

cell

types

-LSB-

-RSB-

Adenosine

transporters

are

of

major

importance

to

organs

and

cells

incapable

of

de

novo

nucleotide

synthesis

such

as

brain

muscle

intestinal

mucosa

and

bone

marrow

-LSB-

-RSB-

As

the

liver

appears

to

be

a

major

source

of

purines

for

these

tissues

curtailment

of

nucleotide

loss

into

the

bile

may

be

important

to

maintain

appropriate

nucleotide/nucleoside

concentrations

within

hepatocytes

-LSB-

-RSB-

Thus

dephosphorylation

of

nucleotides

by

ectonucleotidases

may

be

critical

for

appropriate

systemic

purine

homeostasis

-LSB-

-RSB-

The

presence

of

nucleotidase

and

nucleoside

transporters

in

the

canalicular

domain

of

hepatocytes

would

be

consistent

with

an

important

role

of

in

purine

salvage

The

exocrine

pancreas

The

exocrine

pancreas

secretes

digestive

enzymes

and

a

fluid

Acini

release

ATP

and

the

excurrent

ducts

express

several

types

of

receptors

-LSB-

-RSB-

Thus

ATP

may

function

as

a

paracrine

mediator

between

pancreatic

acini

and

ducts

Ectonucleotidase

activity

in

pancreatic

tissues

was

first

detected

in

the

rat

in

the

-LSB-

-RSB-

followed

by

analyses

in

the

pig

-LSB-

-RSB-

Cytochemical

and

biochemical

observations

have

corroborated

the

association

of

ATPase

activity

with

zymogen

granules

-LSB-

-RSB-

In

other

studies

of

small

intercalated/interlobular

ducts

immuno-fluorescence

can

be

localized

on

the

luminal

membranes

while

in

larger

ducts

it

is

localized

on

the

basolateral

membranes

-LSB-

-RSB-

Upon

stimulation

with

cholecystokinin

-LRB-

-RRB-

acinar

relocalized

in

clusters

towards

the

lumen

and

is

secreted

into

the

pancreatic

juice

as

an

active

form

associated

with

particulate

fractions

-LSB-

-RSB-

As

revealed

by

electron

microscopy

is

located

on

epithelial

cells

myoepithelial

cells

and

the

basolateral

membrane

of

acini

Interestingly

could

be

also

detected

at

the

basolateral

surface

of

endothelial

cells

-LSB-

-RSB-

Salivary

glands

There

are

only

few

studies

on

the

localization

of

NTPDases

in

salivary

glands

NTPDases

might

play

a

role

in

the

transport

of

electrolytes

by

modulating

the

extracellular

ATP

concentration

in

the

salivary

gland

ducts

reveals

to

be

mainly

vascular

in

expression

was

immunodetected

on

myoepithelial

cells

and

in

nerves

-LSB-

-RSB-

The

immunolocalization

of

NTPDases

and

in

salivary

glands

has

not

yet

been

determined

Kidney

The

kidney

reveals

a

complex

cellular

profile

of

expression

for

and

receptors

as

well

as

of

ectonucleotidases

Both

ATP

and

adenosine

have

been

invoked

in

the

regulation

of

tubuloglomerular

feedback

-LSB-

-RSB-

This

feedback

system

links

the

salt

concentrations

in

the

tubular

fluid

at

the

macula

densa

to

the

vascular

tone

of

the

afferent

arteriole

of

the

same

nephron

As

depicted

by

their

localization

NTPDases

may

participate

in

the

regulation

of

several

biological

functions

of

the

kidney

including

vascular

perfusion

In

mouse

rat

and

porcine

kidneys

can

be

detected

in

vascular

structures

including

blood

vessels

of

glomerular

and

peritubular

capillaries

-LSB-

-RSB-

is

detected

on

the

Bowman

's

capsules

of

mouse

and

rat

-LSB-

-RSB-

and

on

the

luminal

side

of

porcine

renal

tubules

-LSB-

-RSB-

More

recently

an

immunohistochemical

analysis

of

various

ectonucleotidases

of

the

rat

nephron

revealed

expression

of

both

and

in

the

thick

ascending

limb

the

distal

tubule

and

the

inner

medullary

collecting

ducts

In

addition

is

located

in

the

cortical

and

outer

medullary

collecting

ducts

-LSB-

-RSB-

The

nervous

system

All

cell

types

of

the

nervous

system

express

nucleotide

receptors

-LSB-

-RSB-

It

is

increasingly

apparent

that

NTPDases

are

distributed

in

the

nervous

system

as

ubiquitously

as

are

receptors

and

that

these

ectoenzymes

are

directly

involved

in

the

control

of

receptor

function

in

nervous

tissues

-LSB-

-RSB-

Signaling

via

nucleotides

is

widespread

both

in

the

peripheral

and

central

nervous

system

Major

nucleotide

receptormediated

functions

in

the

central

nervous

system

include

the

modulation

of

synaptic

signal

transmission

-LSB-

-RSB-

the

propagation

of

waves

between

glial

cells

-LSB-

-RSB-

or

the

control

and

activation

of

astrocytes

and

microglia

-LSB-

-RSB-

In

addition

ATP

can

contribute

to

synaptic

signal

transmission

-LSB-

-RSB-

In

the

sympathetic

nervous

system

ATP

acts

as

a

fast

neurotransmitter

together

with

catecholamines

-LSB-

-RSB-

it

is

an

important

mediator

of

central

and

peripheral

chemosensory

transduction

including

pain

-LSB-

-RSB-

and

it

is

involved

in

the

control

of

myelination

formation

of

peripheral

axons

-LSB-

-RSB-

Central

nervous

system

ATP

can

be

rapidly

hydrolyzed

to

adenosine

at

brain

synapses

that

in

turn

activates

pre-or

postsynaptic

receptors

thereby

modulating

neuronal

transmission

Adenine

nucleotides

undergo

conversion

to

adenosine

within

a

few

hundred

milliseconds

in

the

extracellular

-LRB-

synaptic

-RRB-

space

of

rat

brain

slices

-LSB-

-RSB-

Complex

synaptic

interactions

in

the

central

nervous

system

may

thus

be

modulated

both

by

the

activation

of

and

-LRB-

after

hydrolysis

of

the

nucleotide

-RRB-

receptors

that

may

be

located

at

identical

or

different

cellular

targets

-LSB-

-RSB-

Based

on

immunoblotting

and

in

situ

hybridization

and

are

expressed

in

the

mammalian

brain

-LSB-

-RSB-

and

have

been

purified

from

porcine

brain

-LSB-

-RSB-

But

the

exact

cellular

allocation

of

individual

subtypes

is

still

a

challenge

There

is

ample

evidence

from

early

enzyme

histochemical

investigations

that

surface-located

catalytic

activity

for

the

hydrolysis

of

nucleoside

tri-and

diphosphates

can

be

allocated

to

all

cell

types

of

the

nervous

system

-LSB-

for

reviews

see

-RSB-

This

catalytic

activity

can

be

localized

to

synapses

including

the

synaptic

cleft

at

the

surface

of

neurosecretory

nerve

terminals

in

the

pituitary

or

at

peripheral

nerve

terminals

These

data

imply

a

wide

distribution

of

cell

surface-located

ATP

hydrolyzing

activity

in

the

CNS

Neurons

Ecto-ATPase

activity

has

been

observed

in

synaptosomal

fractions

isolated

from

various

sources

implying

endogenous

ectonucleotidase

activity

of

nerve

cells

Biochemical

studies

on

isolated

synaptosomes

permit

the

determination

of

the

ratios

of

ATP

to

ADP

hydrolysis

rates

as

well

as

the

analysis

of

product

formation

Total

synaptosome

fractions

isolated

from

rat

brain

cortex

and

immunopurified

cholinergic

striatal

synaptosomes

revealed

ratios

of

and

respectively

-LSB-

-RSB-

ADP

was

found

to

transiently

accumulate

after

addition

of

ATP

and

was

subsequently

metabolized

to

AMP

and

adenosine

Similar

results

were

obtained

with

hippocampal

synaptosomes

-LSB-

-RSB-

This

strongly

argues

against

a

major

contribution

by

and

and

would

rather

be

compatible

with

a

neuronal

expression

of

-LRB-

comp

Fig.

-RRB-

A

recent

immunocytochemical

study

allocates

to

neurons

including

axon-like

structures

of

various

brain

regions

-LSB-

-RSB-

Astrocytes

oligodendrocytes

and

microglia

The

ratio

of

ATP

to

ADP

hydrolysis

is

clearly

different

in

cultured

astrocytes

Astrocytes

cultured

from

cortex

or

hippocampus

display

a

ratio

of

-LSB-

-RSB-

Furthermore

cultured

rat

cortical

astrocytes

accumulate

ADP

from

ATP

that

is

only

very

slowly

further

degraded

to

AMP

-LSB-

-RSB-

This

would

be

largely

compatible

with

as

the

major

ectonucleotidase

of

cultured

astrocytes

Immunocytological

investigations

of

adult

rat

and

mouse

brain

sections

assign

solely

to

the

astrocyte-like

stem

cells

in

the

subventricular

zone

of

the

lateral

ventricles

and

the

dentate

gyrus

of

the

hippocampus

and

to

astrocytes

in

few

distinct

additional

brain

regions

-LSB-

-RSB-

Thus

cultured

astrocytes

may

reveal

functional

properties

that

differ

from

the

in

situ

situation

as

they

tend

to

rapidly

alter

their

protein

expression

profile

-LSB-

-RSB-

Enzyme

histochemistry

assigns

ecto-ATPase

activity

to

both

central

and

peripheral

myelin

-LSB-

-RSB-

but

fully

supplementary

immunocytochemical

data

are

lacking

Enzyme

histochemical

staining

for

surface-located

nucleoside

diphosphate

activity

has

long

been

used

to

identify

microglia

in

tissue

sections

of

the

adult

and

developing

brain

-LSB-

-RSB-

The

major

microglial

ectonucleotidase

has

been

identified

as

-LSB-

-RSB-

Stem

cells

in

the

adult

mammalian

brain

In

the

adult

rodent

brain

neurogenesis

persists

in

two

restricted

regions

the

subventricular

zone

-LRB-

SVZ

-RRB-

of

the

lateral

ventricles

and

the

dentate

gyrus

of

the

hippocampus

These

regions

contain

stem

cells

that

give

rise

to

neurons

throughout

the

life

span

of

the

animal

Interestingly

these

cells

share

astrocytic

properties

-LSB-

-RSB-

They

generate

highly

proliferating

intermediate

cell

types

and

finally

mature

neurons

is

highly

and

selectively

expressed

by

the

stem

cells

-LRB-

type

B

cells

-RRB-

of

the

SVZ

-LSB-

-RSB-

-LRB-

Fig.

-RRB-

as

well

as

by

the

progenitor

cells

-LRB-

residual

radial

glia

-RRB-

of

the

dentate

gyrus

-LSB-

-RSB-

In

the

presence

of

epidermal

growth

factor

-LRB-

EGF

-RRB-

and

fibroblast

growth

-LRB-

-RRB-

SVZ-derived

stem

cells

can

be

cultured

as

free

floating

cellular

aggregates

-LRB-

neurospheres

-RRB-

Cultured

stem

cells

express

and

functional

receptors

Agonists

of

and

receptors

augment

cell

proliferation

whereas

inhibition

of

the

receptors

attenuates

cell

proliferation

in

spite

of

the

presence

of

mitogenic

growth

factors

-LSB-

-RSB-

These

data

suggest

that

and

nucleotides

together

with

other

signaling

pathways

contribute

to

the

control

of

neurogenesis

in

the

adult

mammalian

brain

Fig.

of

arrangement

of

neuronal

stem

cells

and

neuroblasts

at

the

lateral

lining

of

the

mouse

subventricular

zone

-LRB-

SVZ

-RRB-

-LRB-

triple

labeling

-RRB-

A

-RRB-

DAPI

staining

of

all

nuclei

Arrow

heads

mark

endymal

lining

B

-RRB-

Stem

cells

-LRB-

type

B

cells

-RRB-

immunopositive

for

form

tube-like

sheeths

around

clusters

of

migrating

immature

neurons

-LRB-

type

A

cells

-RRB-

that

immunostain

for

the

microtubule-associated

protein

doublecortin

-LRB-

DCX

-RRB-

-LRB-

C

-RRB-

The

spaces

covered

by

type

A

cells

remain

dark

in

-LRB-

B

-RRB-

and

are

indicated

with

stars

D

-RRB-

Merge

of

B

-RRB-

and

C

-RRB-

E

-RRB-

Merge

of

A

-RRB-

B

-RRB-

and

C

-RRB-

Bar

µm

-LRB-

by

courtesy

of

David

Langer

Frankfurt

am

Main

-RRB-

Apparently

individual

enzyme

isoforms

govern

cell

surface-located

nucleotide

hydrolysis

in

the

various

cell

types

of

the

central

nervous

system

This

does

not

exclude

however

the

possibility

that

individual

cell

types

express

more

than

one

isoform

with

one

of

the

enzymes

predominating

For

example

cells

express

mRNA

for

But

the

ATP/ADP

hydrolysis

ratio

the

pattern

of

product

formation

and

the

immunocytochemical

surface

staining

suggest

that

is

the

major

functional

isoform

-LSB-

-RSB-

Similarly

cultured

normal

and

immortalized

pituitary

and

hypothalamic

cells

express

-LSB-

-RSB-

The

future

planned

use

of

transgenic

mice

expressing

fluorescent

protein

under

the

promoter

of

the

respective

NTPDase

isoform

will

greatly

facilitate

the

identification

of

the

expression

pattern

of

individual

enzyme

isoforms

in

the

developing

and

adult

nervous

system

Peripheral

nervous

system

Noradrenaline

and

ATP

are

co-released

from

sympathetic

nerve

terminals

of

the

guinea

pig

heart

whereby

ATP

enhances

noradrenaline

release

by

a

mechanism

controlled

by

ectonucleotidases

possibly

-LSB-

-RSB-

Interestingly

stimulated

sympathetic

nerves

of

the

guinea

pig

vas

deferens

release

not

only

ATP

and

noradrenaline

but

also

enzyme

activity

that

degrades

ATP

to

adenosine

The

latter

exhibits

similarities

to

NTPDases

and

nucleotidase

but

their

molecular

identity

has

not

been

defined

-LSB-

-RSB-

associates

with

immature

and

non-myelinating

Schwann

cells

of

peripheral

nerves

whereas

immunoreactivity

is

absent

-LSB-

-RSB-

is

also

expressed

by

the

satellite

glial

cells

in

dorsal

root

ganglia

and

sympathetic

ganglia

and

by

the

enteric

glia

surrounding

cell

bodies

of

ganglionic

neurons

of

the

myenteric

and

submucous

plexus

-LSB-

-RSB-

Sensory

systems

The

most

comprehensive

investigation

of

expression

of

NTPDases

within

sensory

systems

concerns

the

inner

ear

Ectonucleotidase

activity

is

associated

with

the

tissues

lining

the

perilymphatic

compartment

of

the

cochlea

-LSB-

-RSB-

Immunohistochemical

analysis

of

the

murine

cochlea

has

assigned

to

the

cochlear

vasculature

and

primary

auditory

neurons

in

the

spiral

ganglion

whereas

is

associated

with

synaptic

regions

of

the

sensory

inner

and

outer

hair

cells

supporting

cells

of

the

organ

of

Corti

and

additional

tissue

elements

-LSB-

-RSB-

Interestingly

noise

exposure

induces

upregulation

of

and

in

the

rat

cochlea

-LSB-

-RSB-

Taste

buds

transduce

chemical

signals

in

the

mouth

into

neural

messages

Taste

cells

and

nerve

fibers

express

and

receptors

-LSB-

-RSB-

and

various

receptors

-LSB-

-RSB-

Genetic

elimination

of

and

receptors

revealed

that

ATP

is

a

key

neurotransmitter

in

this

system

-LSB-

-RSB-

is

expressed

at

the

mRNA

level

in

mouse

taste

papillae

-LSB-

-RSB-

Immunohistochemistry

and

enzyme

histochemical

staining

allocate

to

type

I

glial-like

cells

in

the

tongue

palate

and

larynx

Furthermore

immunostaining

is

associated

with

nearby

nerves

suggestive

of

Schwann

cells

implying

that

may

be

a

regulator

in

defined

taste

transmission

Pathological

implications

Cerebral

ischemia

The

interruption

of

blood

flow

accompanied

by

an

interrupted

supply

of

oxygen

and

glucose

initiates

a

sequence

of

events

resulting

in

structural

and

functional

damage

of

the

nervous

tissue

comparable

to

that

seen

at

other

sites

of

vascular

injury

-LSB-

-RSB-

Transient

global

cerebral

ischemia

of

the

rat

results

in

a

long-term

increase

in

extracellular

nucleotide

hydrolysis

pathways

-LSB-

-RSB-

Preconditioning

delays

the

postischemic

increase

in

ATP

diphosphohydrolase

activity

-LSB-

-RSB-

During

the

days

following

transient

forebrain

ischemia

mRNA

for

-LRB-

but

not

of

-RRB-

and

nucleotidase

becomes

upregulated

in

the

hippocampus

-LSB-

-RSB-

corresponding

to

the

upregulation

of

the

entire

ectonucleotidase

chain

for

the

hydrolysis

of

ATP

to

adenosine

The

data

suggest

that

the

increased

expression

of

ectonucleotidases

in

the

regions

of

damaged

nerve

cells

is

associated

with

activated

glia

mainly

microglia

-LSB-

-RSB-

The

upregulation

of

the

ectonucleotidase

chain

is

suggestive

of

an

ischemia-induced

increased

and

sustained

cellular

release

of

nucleotides

This

could

have

several

functional

implications

Since

microglial

cells

express

the

cytolytic

receptor

-LSB-

-RSB-

the

activity

of

these

cells

may

be

particularly

endangered

by

increased

levels

of

extracellular

ATP

Enhanced

activity

of

may

prevent

activated

microglia

from

overstimulation

by

ATP

released

from

the

injured

tissue

Alternatively

microglial

expression

of

might

contribute

to

preventing

receptor

desensitization

on

prolonged

exposure

to

elevated

ATP

levels

The

parallel

increase

in

activity

of

nucleotidase

would

facilitate

the

formation

of

the

final

hydrolysis

product

adenosine

that

exerts

neuro-modulatory

and

immunomodulatory

actions

and

contributes

to

the

protection

of

neurons

Alterations

following

plastic

changes

in

the

nervous

system

Additional

experiments

analyzing

synaptosome

fractions

suggest

that

changes

in

neural

plasticity

can

be

paralleled

by

changes

in

ecto-ATPase

activity

Enzyme

activity

is

reduced

following

avoidance

learning

-LSB-

-RSB-

and

status

epilepticus

-LSB-

-RSB-

It

is

altered

in

two

rat

models

of

temporal

lobe

epilepsy

-LSB-

-RSB-

and

on

pentylenetetrazol

kindling

-LSB-

-RSB-

Changes

in

synaptosomal

ectonucleotidase

activity

have

been

implicated

by

a

broad

variety

of

additional

treatments

including

acute

caffeine

treatment

-LSB-

-RSB-

Taken

together

these

experiments

suggest

that

expression

of

ectonucleotidases

can

be

altered

following

a

variety

of

physiological

or

pathological

stimuli

possibly

together

with

that

of

purine

receptors

Further

work

needs

to

define

the

enzyme

subtypes

involved

and

the

mechanisms

underlying

the

regulation

of

ectonucleotidase

expression

Conclusions

This

review

summarizes

components

of

extracellular

nucleotide-mediated

signaling

pathways

that

are

impacted

upon

largely

by

the

E-NTPDase

family

of

ectonucleotidases

Modulated

distinct

NTPDase

expression

appears

to

regulate

nucleotide-mediated

signaling

in

essentially

every

tissue

including

the

vasculature

and

of

immune

and

nervous

systems

For

example

extracellular

nucleotide-mediated

vascular

endothelial

and

accessory

cell

stimulation

might

have

important

consequences

for

platelet

activation

thrombogenesis

angiogenesis

vascular

remodeling

and

the

metabolic

milieu

of

the

vasculature

in

response

to

inflammatory

stress

and/or

immune

reactions

Nucleotides

are

also

of

significant

relevance

for

the

communication

of

nerve

cells

and

glial

cells

or

in

the

reciprocal

signaling

between

these

cells

These

purinergic

mechanisms

might

also

dictate

pathological

processes

of

the

nervous

system

or

following

vascular

injury

thromboregulatory

disturbances

and

defective

angiogenesis

with

associated

perturbations

in

tissue

remodeling

and

regeneration

There

is

a

wide

field

for

future

investigations

of

the

role

of

nucleotides

and

ectonucleotidases

in

other

tissues

Increasing

interest

in

this

field

may

open

up

new

avenues

for

investigation

and

the

development

of

new

treatment

modalities

for

a

large

variety

of

diseases

including

neurological

pathological

states

vascular

thrombotic

disorders

including

stoke

atherosclerosis

and

the

vascular

inflammation

seen

in

transplant-graft

failure

apyrase
brain
cd39
immunology
ischemia
kidney
liver
nervous tissue
ntpdase
platelet
vasculature