Structural

and

functional

evolution

of

the

receptor

group

Metabotropic

pyrimidine

and

purine

nucleotide

receptors

-LRB-

receptors

-RRB-

belong

to

the

superfamily

of

G

protein-coupled

receptors

-LRB-

GPCR

-RRB-

They

are

distinguishable

from

adenosine

receptors

-LRB-

-RRB-

as

they

bind

adenine

and/or

uracil

nucleotide

triphosphates

or

diphosphates

depending

on

the

subtype

Over

the

past

decade

receptors

have

been

cloned

from

a

variety

of

tissues

and

species

and

as

many

as

eight

functional

subtypes

have

been

characterized

Most

recently

several

members

of

the

receptor

group

which

includes

the

clopidogrel-sensitive

ADP

receptor

have

been

deorphanized

The

receptor

group

comprises

several

structurally

related

GPCR

which

however

display

heterogeneous

agonist

specificity

including

nucleotides

their

derivatives

and

lipids

Besides

the

established

function

of

in

platelet

activation

expression

in

macrophages

neuronal

and

glial

cells

as

well

as

recent

results

from

functional

studies

implicate

that

several

members

of

this

group

may

have

specific

functions

in

neurotransmission

inflammation

chemotaxis

and

response

to

tissue

injury

This

review

focuses

specifically

on

the

structure-function

relation

and

shortly

summarizes

some

aspects

of

the

physiological

relevance

of

receptor

members

Introduction

Most

cellular

effects

of

nucleotides

are

mediated

through

two

main

families

of

specific

cell

surface

receptors

the

ionotropic

and

metabotropic

nucleotide

receptors

The

metabotropic

receptors

belong

to

the

superfamily

of

G

protein-coupled

receptors

-LRB-

GPCR

-RRB-

The

GPCR

superfamily

comprises

at

least

five

structurally

distinct

families/subfamilies

-LRB-

GRAFS

classification

-RRB-

which

share

little

sequence

homology

among

each

other

-LSB-

-RSB-

The

rhodopsin-like

receptors

-LRB-

also

called

family

A

or

-RRB-

form

the

largest

family

in

vertebrates

Eight

different

mammalian

-LRB-

-RRB-

receptor

subtypes

all

belonging

to

the

rhodopsin-like

receptors

-LRB-

family

A

-RRB-

have

been

identified

and

proven

to

function

as

receptors

for

extracellular

nucleotides

so

far

-LSB-

-RSB-

Based

on

their

preferential

agonists

receptors

have

been

subclassified

into

adenine

nucleotide-activated

-LRB-

-RRB-

pyrimidine

nucleotide-activated

-LRB-

-RRB-

ATP/UTP-activated

-LRB-

-RRB-

and

UDP-sugar-activated

-LRB-

-RRB-

receptors

Most

structural

and

functional

studies

were

performed

with

the

classic

receptors

and

-LSB-

-RSB-

However

in

comparison

to

other

GPCR

large-scale

structure/function

data

are

rare

mainly

due

to

difficulties

in

finding

suitable

heterologous

expression

systems

-LSB-

-RSB-

Pharmacological

characterization

on

both

native

and

recombinant

receptors

is

further

hampered

by

the

release

of

endogenous

nucleotides

and

the

hydrolysis

or

conversion

of

exogenous

and

endogenous

nucleotides

by

ectoenzymes

-LRB-

for

review

see

-LSB-

-RSB-

-RRB-

ATP

and

AMP

are

metabolized

by

cell

surface

enzymes

the

ecto-NTPDase

-LRB-

nucleoside

triphosphate

diphosphohydrolase

ectoapyrase

metabolizes

ATP

to

AMP

-RRB-

and

the

ectonucleotidase

-LRB-

metabolizes

AMP

to

adenosine

-RRB-

In

addition

a

nucleoside

diphosphokinase

catalyzes

the

transfer

of

the

gamma

phosphate

of

nucleoside

triphosphates

to

nucleoside

diphosphates

For

example

in

the

presence

of

ATP

nucleoside

diphosphokinase

catalyzes

the

conversion

of

UDP

to

UTP

These

specific

problems

may

have

contributed

to

many

false

and

controversial

declarations

of

receptors

being

activated

by

nucleotides

-LSB-

-RSB-

Recent

success

in

expressing

GPCR

in

yeast

which

is

discussed

below

may

circumvent

these

problems

and

may

engage

structure-function-relation

studies

Although

in-depth

phylogenetic

analysis

of

GPCR

groups

indicates

clustering

of

receptors

when

compared

with

other

GPCR

of

family

A

-LSB-

-RSB-

the

adenine

nucleotide-activated

and

for

example

display

only

minor

structural

relation

at

the

protein

level

Phylogenetic

analysis

even

revealed

that

nucleotide

specificity

e.g.

for

ADP

evolved

independently

in

and

So

we

suggest

that

grouping

of

receptors

should

be

performed

on

phylogenetic

aspects

rather

than

on

ligand

specificity

Based

on

their

amino

acid

sequences

receptors

can

be

subdivided

into

at

least

two

groups

-LRB-

Fig.

-RRB-

One

comprises

and

the

second

group

contains

Additionally

several

other

structurally

related

GPCR

such

as

and

cluster

into

both

groups

-LRB-

Fig.

-RRB-

These

receptors

are

activated

by

structurally

different

ligands

such

as

leukotrienes

organic

acids

and

phospholipids

Probably

because

of

their

structural

relation

several

receptors

display

agonist

promiscuity

It

was

shown

that

not

only

ADP

but

also

leukotriene

-LRB-

-RRB-

activates

at

nanomolar

concentrations

-LSB-

-RSB-

Cysteinyl

leukotriene

receptors

-LRB-

and

-RRB-

have

been

described

to

be

activated

by

UDP

in

addition

to

activation

by

cysteinyl

leukotrienes

-LSB-

-RSB-

Further

there

is

evidence

that

antagonists

such

as

pranlukast

and

montelukast

inhibit

activation

of

and

by

-LRB-

-RRB-

and

UDP

respectively

-LSB-

-RSB-

Although

and

cysteinyl

leukotriene

receptors

are

clinically

used

targets

for

prevention

of

thrombocyte

aggregation

-LRB-

clopidogrel

-RRB-

and

treatment

of

asthma

-LRB-

montelukast

-RRB-

respectively

the

structural

basis

and

-LRB-

patho

-RRB-

physiological

relevance

of

dual/multiple

agonist

specificity

is

obscure

Close

structural

relations

between

and

other

non-nucleotide

receptors

as

well

as

the

mentioned

examples

of

ligand

promiscuity

indicate

that

nucleotides

are

probably

only

one

of

many

natural

agonists

on

the

genuine

receptors

Fig.

relation

of

human

receptors

To

evaluate

the

structural

relation

of

receptors

and

related

GPCR

amino

acid

sequences

of

human

orthologs

were

aligned

and

phylogenetic

relations

were

estimated

using

CLUSTAL

W

The

derived

tree

was

essentially

identical

to

former

analysis

-LSB-

-RSB-

The

receptors

cluster

into

a

group

-LRB-

framed

-RRB-

that

is

distinct

from

other

nucleotide

receptors

such

as

and

Structural

evolution

of

GPCR

Discovery

and

deorphanization

of

group

members

The

ADP

-LRB-

-RRB-

like

receptor

group

is

the

most

recently

identified

receptor

group

and

includes

and

-LRB-

see

Fig.

Table

-RRB-

Based

on

high

structural

similarity

the

lyso-phosphatidylserine

receptor

-LRB-

-RRB-

and

several

orphan

GPCR

-LRB-

-RRB-

are

considered

members

of

the

structural

receptor

group

-LRB-

-RRB-

Most

members

of

this

group

were

deorphanized

by

the

so-called

reverse

pharmacology

approach

The

orphan

receptors

predicted

from

sequence

data

are

now

often

used

to

ascertain

the

ligands

by

testing

them

on

tissue

extracts

and

subsequent

fractionation

or

on

huge

libraries

of

bioactive

compounds

whereas

traditionally

the

bioactive

ligand

was

used

to

identify

the

receptor

-LRB-

classic

approach

-RRB-

Table

of

the

receptor

familyReceptorG

protein

couplingNatural

agonistTissue

phosphoribosyl

pyrophosphatePlatelets

brain

-LRB-

glia

-RRB-

-LSB-

-RSB-

diadenosine

triphosphateSpleen

and

adult

brain

lower

expression

in

placenta

lung

liver

spinal

cord

thymus

spleen

small

intestine

uterus

stomach

testis

fetal

brain

adrenal

gland

lymph

node

bone

marrow

peripheral

blood

mononuclear

cells

leukocytes

-LSB-

-RSB-

UDP-galactose

UDP-glucuronic

acid

UDP-N-acetylglucosamineBrain

-LRB-

astrocytes

glia

-RRB-

placenta

adipose

tissue

stomach

intestine

spleen

lung

-LRB-

epithelium

-RRB-

heart

-LSB-

-RSB-

thymus

small

intestine

colon

prostate

uterus

testis

peripheral

blood

leukocytes

-LSB-

-RSB-

glia

cells

mast

cells

spleen

heart

kidney

liver

-LSB-

-RSB-

epididymis

-LRB-

unpublished

own

results

-RRB-

Based

on

phylogenetic

analyses

and

structural

similarities

the

seven

GPCR

clusters

in

a

group

which

is

distinct

from

other

receptors

-LRB-

see

Fig.

-RRB-

Although

they

share

structural

features

it

appears

from

current

studies

that

not

all

members

are

receptors

for

nucleotides

The

table

lists

the

currently

known

receptor

agonists

the

receptor

's

G

protein

specificity

and

expression

patternn.k

not

known

As

the

first

member

of

the

receptor

group

-LRB-

synonyms

VTR

-RRB-

was

identified

-LSB-

-RSB-

and

then

deorphanized

in

the

year

-LSB-

-RSB-

UDP-glucose

was

found

to

activate

by

screening

multiple

receptors

each

expressed

in

individual

yeast

strains

against

a

large

library

of

over

known

and

putative

natural

GPCR

agonists

In

-LRB-

initially

named

-RRB-

was

discovered

and

identified

as

the

platelet

clopidogrel-sensitive

ADP

receptor

by

a

number

of

groups

-LSB-

-RSB-

The

was

deorphanized

with

both

the

classic

and

the

reverse

pharmacology

approaches

Hollopeter

et

al.

used

subfractionated

transcripts

from

platelets

and

isolated

the

cRNA

-LRB-

complementary

RNA

encoding

e.g.

the

receptor

-RRB-

which

mediated

ADP-induced

increases

in

K

current

after

injection

into

Xenopus

oocytes

-LSB-

-RSB-

In

contrast

fractionated

rat

spinal

cord

extracts

were

assayed

for

mobilization

in

cells

transiently

transfected

with

and

chimeric

Gα

subunit

-LSB-

-RSB-

Almost

in

parallel

ADP

was

shown

to

be

an

agonist

for

-LRB-

former

-RRB-

a

very

close

structural

neighbor

of

-LSB-

-RSB-

is

the

most

potent

agonist

at

followed

by

ADP

adenosine

and

ATP

-LRB-

N

-RRB-

a

highly

potent

agonist

at

exhibited

no

and

only

minor

agonist

activity

at

and

respectively

-LSB-

-RSB-

Based

on

structural

relations

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

and

-LRB-

synonym

-RRB-

-LSB-

-RSB-

also

belong

to

the

group

-LRB-

see

Fig.

-RRB-

Except

for

which

was

recently

identified

as

a

receptor

for

lyso-phosphatidylserine

-LSB-

-RSB-

by

a

reverse

pharmacology

approach

all

the

other

receptors

are

still

orphan

GPCR

The

evolutionary

origins

Based

on

structural

similarities

receptors

cluster

into

a

group

distinct

from

other

receptors

-LRB-

see

Figs.

and

-RRB-

This

receptor

group

can

be

subdivided

into

two

subgroups

One

subgroup

encompasses

and

the

other

subgroup

and

-LRB-

Fig.

-RRB-

This

may

suggest

that

both

subgroups

evolved

from

gene

duplications

starting

from

two

related

members

The

grouping

is

further

supported

by

shared

genomic

localization

Chromosomal

clustering

in

the

human

genome

is

found

for

and

at

as

well

as

for

and

at

This

clustering

is

evolutionary

well

preserved

In

the

zebrafish

genome

and

cluster

at

chromosome

and

type

and

are

tandemly

arranged

at

chromosome

Gene

clustering

is

not

only

found

for

vertebrate

receptors

but

also

for

several

other

GPCR

genes

Multiple

copies

of

related

GPCR

genes

such

as

the

human

protease-activated

receptors

on

trace

amine-associated

receptors

-LRB-

TAAR

human

-RRB-

and

CC-chemokine

receptors

-LRB-

human

-RRB-

are

the

result

of

intrachromosomal

gene

duplications

The

most

impressive

example

is

found

in

odorant

receptors

where

in

humans

chromosome

contains

nearly

half

of

the

odorant

receptor

repertoire

including

a

single

cluster

of

more

than

odorant

receptors

-LSB-

-RSB-

Gene

amplification

can

be

viewed

as

a

dynamic

and

reversible

regulatory

mechanism

that

facilitates

adaptation

to

variable

environments

Clustered

genes

may

confer

selective

benefits

via

their

ability

to

be

co-regulated

and

co-amplified

Indeed

even

transcripts

encoding

a

fusion

protein

of

and

have

been

observed

-LRB-

own

unpublished

results

-RRB-

However

the

biological

relevance

of

receptor

clustering

in

respect

to

transcriptional

activity

at

their

respective

genomic

loci

co-regulation

and

transcript

diversity

need

to

be

determined

in

the

future

Fig.

origin

of

receptors

To

subclassify

and

evaluate

the

phylogenetic

relations

between

the

receptors

the

amino

acid

sequences

of

orthologs

from

one

species

of

each

vertebrate

class

-LRB-

if

available

-RRB-

were

aligned

using

CLUSTAL

W

-LRB-

identity

matrices

-RRB-

and

a

phylogenetic

tree

was

constructed

-LRB-

iterations

-RRB-

Bootstrap

values

were

considered

as

significant

to

support

a

new

branch

Since

and

are

all

found

in

sharks

-LRB-

Mustelus

manazo

Carcharodon

carcharias

-RRB-

and

chimeras

-LRB-

Callorhinchus

milii

-RRB-

one

can

assume

that

receptors

arose

more

than

million

years

-LRB-

Myr

-RRB-

ago

before

cartilaginous

and

bony

fish

split

Our

own

polymerase

chain

reaction

-LRB-

PCR

-RRB-

amplification

attempts

as

well

as

the

ongoing

lamprey

-LRB-

Petromyzon

marina

-RRB-

and

sea

urchin

-LRB-

Strongylocentrotus

purpuratus

-RRB-

genome

projects

revealed

no

sequences

yet

suggesting

their

origin

in

the

very

early

Gnathostomata

or

less

likely

a

loss

in

agnate

and

all

non-vertebrates

Gene

duplication

can

be

a

primary

source

of

the

genetic

material

from

which

genes

with

new

functions

evolve

One

copy

of

a

duplicated

gene

may

become

mutated

and

acquire

unique

functionality

without

risking

the

fitness

of

the

organism

ensured

by

the

homolog

On

the

other

hand

if

not

advantageous

continuous

accumulation

of

mutations

-LRB-

neutral

drift

-RRB-

will

eliminate

one

of

the

genes

a

process

named

pseudogenization

In

fish

genomes

there

is

only

one

respective

ortholog

with

relation

to

and

to

In

the

case

of

the

fish

ortholog

is

more

closely

related

to

implicating

that

evolved

later

in

tetrapod

evolution

probably

by

duplication

However

one

can

not

rule

out

that

the

gene

was

eliminated

in

the

common

ancestor

of

sharks

and

bony

fishes

The

evolutionary

mechanism

of

and

evolvement

remains

unsolved

since

their

structural

relation

to

the

fish

orthologs

is

similar

The

absence

of

in

the

African

craw

frog

-LRB-

Xenopus

-RRB-

genome

but

the

presence

of

ortholog

sequences

in

all

other

sequenced

tetrapod

genomes

does

not

necessarily

implicate

that

derived

from

in

early

tetrapod

evolution

Again

gene

loss

in

amphibians

must

be

considered

Orthologs

of

and

are

found

in

all

tetrapods

and

bony

and

cartilaginous

fishes

investigated

so

far

However

not

all

members

of

the

group

appear

to

be

present

in

all

vertebrate

classes

has

an

ortholog

in

zebrafish

but

is

not

yet

found

in

the

genomes

of

pufferfishes

and

the

lizard

-LRB-

Anolis

carolinensis

-RRB-

genome

Although

is

found

in

reptiles

birds

mammals

zebrafish

and

chimera

it

appears

to

be

absent

in

African

craw

frog

stickleback

and

pufferfishes

This

may

implicate

specific

functions

of

some

receptors

which

are

less

important

in

some

species

or

are

compensated

by

other

receptors

and

mechanisms

Signatures

of

gene

inactivation

-LRB-

pseudogenization

-RRB-

are

rarely

found

for

members

of

the

group

Following

gene

duplication

in

the

common

ancestor

of

evolutionary

basic

fishes

like

eels

and

carps

-LSB-

-RSB-

pseudogenization

of

one

subtype

was

identified

in

a

salmon

species

-LRB-

Keta

salmon

-RRB-

-LRB-

own

unpublished

results

-RRB-

The

receptor

is

an

evolutionary

old

receptor

being

present

in

cartilaginous

and

bony

fishes

amphibians

and

birds

but

not

in

mammals

-LRB-

Fig.

-RRB-

Sequences

with

residual

relation

to

the

receptor

are

found

in

the

platypus

genome

but

it

is

most

likely

a

pseudogene

Genomic

organization

of

receptors

Comparison

of

transcript

and

genomic

sequences

provides

information

on

intron/exon

structure

of

a

GPCR

gene

Since

the

gain

or

loss

of

spliceosomal

introns

are

unique

events

in

evolution

they

can

serve

as

markers

for

phylogenetic

analysis

Further

such

analyses

may

reveal

splice

variants

and

may

be

informative

about

the

promoter

structure

and

gene

regulation

Introns

are

the

basis

of

alternative

splicing

exon

skipping

and

RNA

editing

events

and

therefore

can

contribute

to

receptor

diversity

at

a

supragenomic

level

Most

coding

regions

of

human

receptors

do

not

contain

introns

-LRB-

Table

-RRB-

One

exception

is

where

the

genomic

sequence

encoding

the

receptor

's

N

terminus

is

interrupted

by

an

intron

in

mammalian

and

avian

orthologs

Another

intron

which

is

rarely

removed

-LRB-

cryptic

intron

-RRB-

has

been

identified

in

the

N

terminus-encoding

sequence

of

-LSB-

-RSB-

Table

organization

of

GPCR

n.a.

not

analyzed

because

of

lack

of

mRNA

informationMemberNumber

of

introns

in

the

non-coding

regionNumber

of

introns

in

the

coding

regionApprox

size

of

genomic

kbp

-LRB-

different

transcript

starts

-RRB-

kbp

Chickenn.a

bn.a

-LRB-

kbp

-RRB-

African

clawed

frog

-LRB-

N

terminus

-RRB-

kbp

Zebrafishn.a

bn.a

-LRB-

kbp

-RRB-

-LRB-

-RRB-

kbp

Pufferfishn.a

-LRB-

-RRB-

Human

-LRB-

rare

transcript

N

terminus

-RRB-

kp

-LRB-

different

transcript

starts

-RRB-

kbp

Chickenn.a

bn.a

-LRB-

kbp

-RRB-

African

clawed

frog

-LRB-

N

terminus

-RRB-

kbp

-LRB-

different

transcript

starts

-RRB-

kbp

Chickenn.a

bn.a

-LRB-

kbp

-RRB-

-LRB-

N

terminus

-RRB-

kbp

-LRB-

N

terminus

-RRB-

kbp

-LRB-

N

terminus

-RRB-

kbp

African

clawed

frogn.a

bn.a

-LRB-

kbp

-RRB-

Zebrafishn.a

bn.a

-LRB-

kbp

-RRB-

Pufferfishn.a

bn.a

-LRB-

kbp

-RRB-

kbp

kbp

kbp

Zebrafishn.a

bn.a

-LRB-

kbp

-RRB-

kbp

kbp

Chickenn.a

bn.a

-LRB-

kbp

-RRB-

African

clawed

frogn.a

bn.a

-LRB-

kbp

-RRB-

Zebrafishn.a

bn.a

-LRB-

kbp

-RRB-

cryptic

-LRB-

N

terminus

-RRB-

kbp

kbp

kbp

African

clawed

kbp

Zebrafish

Type

kbp

Type

kbp

Pufferfishn.a

bn.a

-LRB-

kbp

-RRB-

Except

for

human

receptor

members

contain

no

intron

in

the

coding

region

However

this

intronless

gene

structure

is

not

well

preserved

in

all

vertebrates

The

table

summarizes

the

number

of

introns

within

the

and

coding

region

of

selected

vertebrate

genes

and

estimates

the

size

of

the

geneaBased

on

the

longest

transcriptbComplete

open

reading

frame

within

the

genomic

sequence

but

intron

within

the

very

N

terminus

can

not

be

ruled

out

because

of

lack

of

mRNA

information

The

genomic

organization

of

the

individual

receptor

genes

is

not

well

conserved

during

vertebrate

evolution

-LRB-

see

Table

-RRB-

For

example

the

gene

gained

an

intron

in

bony

fish

evolution

disrupting

the

open

reading

frame

of

the

transmembrane

domain

-LRB-

-RRB-

encoding

part

Similarly

one

of

the

two

paralog

genes

acquired

an

intron

in

the

more

recent

bony

fish

evolution

-LSB-

-RSB-

The

two

long-standing

alternative

explanations

for

the

origin

of

introns

the

intron-early

theory

and

intron-late

theory

remain

a

matter

of

continuous

debate

not

only

for

GPCR

-LSB-

-RSB-

The

intron-early

theory

suggests

that

introns

are

extremely

ancient

characteristics

of

genes

and

that

early

genes

were

created

through

the

intron-mediated

shuffling

of

exons

However

numerous

gene

and

genome

comparison

studies

provided

evidence

that

at

least

some

introns

are

more

recently

acquired

-LRB-

intron-late

theory

-RRB-

The

receptor

group

is

therefore

a

nice

example

where

introns

were

acquired

in

the

coding

region

in

some

species

during

more

recent

evolution

In

contrast

to

the

coding

region

the

region

of

most

members

of

the

receptor

group

displays

a

distinct

intron/exon

organization

with

sometimes

multiple

transcription

starts

Also

the

gene

sizes

strongly

differ

between

the

individual

and

even

closely

related

receptors

Introns

in

the

untranslated

region

-LRB-

UTR

-RRB-

of

receptors

have

not

been

found

yet

Large

and

complex

organized

non-coding

regions

of

a

gene

may

provide

the

basis

for

multiple

promoter

regions

cis-acting

elements

and

a

variable

UTR

of

the

mRNA

Alternative

structures

of

the

UTR

can

contribute

to

expression

regulation

and

alternative

translation

start

points

For

example

UTRs

often

contain

small

open

reading

frames

-LRB-

ORF

-RRB-

which

can

be

translated

via

leaky

scanning

at

the

ribosome

Such

leaky

scanning

can

reduce

translation

of

the

downstream

main

ORF

as

shown

for

several

genes

including

GPCR

-LSB-

-RSB-

It

is

of

interest

to

note

that

both

genes

and

are

located

in

antisense

orientation

within

a

large

intron

of

the

CASK

gene

This

position

is

conserved

during

vertebrate

evolution

The

CASK

gene

encodes

a

calcium/calmodulin-dependent

serine

protein

kinase

that

is

a

member

of

the

membrane-associated

guanylate

kinase

-LRB-

MAGUK

-RRB-

protein

family

Since

and

transcripts

are

antisense

orientated

to

CASK

one

can

speculate

that

transcripts

may

regulate

expression

of

CASK

or

vice

versa

Such

hypotheses

will

be

addressed

in

receptor-deficient

mouse

models

-LRB-

see

below

-RRB-

in

the

future

Key

residues

defining

the

individual

member

Many

attempts

have

been

made

to

identify

structural

signatures

which

are

helpful

in

annotation

and

grouping

of

receptors

On

the

basis

of

available

sequence

data

for

validated

receptors

key

residues

were

extracted

to

define

and

receptor

groups

-LSB-

-RSB-

In

recent

studies

we

have

shown

for

several

GPCR

that

a

significant

number

of

orthologs

is

required

for

identification

of

functional

motifs

and

key

residues

-LSB-

-RSB-

By

mining

public

databases

and

by

amplifying

and

sequencing

orthologs

we

have

acquired

large

sets

of

sequences

of

-LRB-

orthologs

-RRB-

-LRB-

orthologs

-RRB-

-LRB-

orthologs

-RRB-

-LRB-

orthologs

-RRB-

-LRB-

orthologs

-RRB-

-LRB-

orthologs

-RRB-

and

-LRB-

orthologs

-RRB-

to

determine

structural

conservation

of

the

members

and

to

identify

amino

acid

sequence

motifs

and

key

residues

that

are

unique

for

the

receptor

group

and

the

individual

members

-LRB-

Fig.

-RRB-

These

data

sets

were

obtained

from

vertebrate

species

ranging

from

evolutionary

old

bony

and

cartilaginous

fishes

to

the

more

modern

mammals

representing

Myr

of

evolution

The

overall

identity

between

members

is

rather

low

ranging

from

amino

acid

identity

-LRB-

human

vs

human

-RRB-

to

-LRB-

human

and

human

-RRB-

Between

the

respective

fish

and

mammal

receptor

orthologs

-LRB-

-RRB-

shows

the

highest

identity

followed

by

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

Since

not

all

GPCR

have

orthologs

in

all

vertebrate

classes

we

analyzed

the

structural

conservation

within

this

receptor

group

by

comparing

Ka/Ks

values

-LRB-

ratio

of

the

number

of

nonsynonymous

substitutions

per

nonsynonymous

site

and

the

number

of

synonymous

or

silent

substitutions

per

synonymous

or

silent

site

-RRB-

of

the

receptor

ortholog

set

from

species

containing

all

group

members

-LRB-

Table

-RRB-

Although

all

receptors

display

a

purifying

selection

mode

of

evolution

-LRB-

Ka/Ks

indicates

high

conservation

and

elimination

of

deleterious

mutations

-RRB-

there

are

significant

differences

between

the

members

of

this

receptor

group

and

were

kept

most

conserved

during

evolution

in

birds

and

mammals

By

contrast

appears

to

be

less

constrained

as

already

indicated

by

a

relatively

low

conservation

at

the

amino

acid

level

-LRB-

see

above

-RRB-

Fig.

residues

in

receptors

To

identify

conserved

group

and

member-specific

positions

the

amino

acid

sequences

of

-LRB-

orthologs

-RRB-

-LRB-

orthologs

-RRB-

-LRB-

orthologs

-RRB-

-LRB-

orthologs

-RRB-

-LRB-

orthologs

-RRB-

-LRB-

orthologs

-RRB-

and

-LRB-

orthologs

-RRB-

were

aligned

using

CLUSTAL

W.

Residues

that

are

conserved

among

the

respective

orthologs

were

boxed

Only

a

few

positions

are

almost

fully

conserved

among

all

members

of

the

group

-LRB-

position

number

refers

to

the

relative

numbering

system

by

Ballesteros

and

Weinstein

-LSB-

-RSB-

-RRB-

and

the

two

Cys

residues

bridging

extracellular

loops

and

The

approximate

positions

of

the

seven

transmembrane

domains

-LRB-

TMD

-RRB-

are

given

above

the

sequencesTable

conservation

of

GPCRReceptorKa/KsPi

-LRB-

mean

SD

-RRB-

-LRB-

mean

SD

-RRB-

To

compare

the

sequence

conservation

of

receptors

ortholog

sequences

-LSB-

relative

positions

-LRB-

in

-RRB-

to

-LRB-

in

C-term

-RRB-

-RSB-

from

species

were

aligned

and

DNA

polymorphism

analyses

were

performed

using

DnaSP

-LRB-

version

-RRB-

The

Ka/Ks

ratio

is

calculated

from

the

number

of

nonsynonymous

substitutions

per

nonsynonymous

site

-LRB-

Ka

-RRB-

and

the

number

of

synonymous

substitutions

per

synonymous

site

-LRB-

Ks

-RRB-

for

any

pair

of

sequences

The

nucleotide

diversity

-LRB-

Pi

-RRB-

is

the

average

number

of

nucleotide

differences

per

site

between

two

sequences

The

orthologs

of

the

following

species

were

analyzed

because

their

genome

contained

all

members

of

the

group

Bos

taurus

Equus

caballus

Canis

familiaris

Pteropus

vampyrus

Ornithorhynchus

anatinus

Monodelphis

domestica

Callithrix

jacchus

Pan

troglodytes

Homo

sapiens

Macaca

mulatta

Microcebus

murinus

Pongo

pygmaeus

Tarsius

syrichta

Cavia

porcellus

Mus

musculus

Rattus

norvegicus

Tursiops

truncates

Gallus

gallus

As

shown

in

Fig.

members

of

the

group

share

only

eight

fully

conserved

residues

-LRB-

and

both

Cys

residues

bridging

extracellular

loops

and

-RRB-

when

sequences

of

more

than

receptors

of

this

group

are

compared

Although

the

ADP

receptors

and

share

more

than

fully

conserved

residues

of

these

conserved

residues

is

exclusively

found

in

and

but

rather

present

also

in

other

receptors

-LRB-

see

Fig.

-RRB-

Further

only

a

few

residues

are

member

specifically

conserved

-LRB-

and

and

-RRB-

and

have

not

been

found

in

other

receptors

so

far

These

facts

suggest

that

ligand

and

signaling

specificity

is

determined

by

a

combination

of

many

more

or

less

conserved

determinants

It

has

been

proposed

that

and

within

and

the

respectively

may

present

such

motifs

required

for

nucleotide

binding

-LSB-

-RSB-

However

such

residue

combination

is

also

present

in

some

and

orthologs

which

are

not

activated

by

ADP

This

does

not

rule

out

that

these

residues

are

involved

in

nucleotide

binding

of

e.g.

but

it

implicates

additional

positions

which

determine

ligand

specificity

In-depth

structure-function

analysis

e.g.

by

mutagenesis

studies

are

required

to

identify

key

positions

and

their

structural

properties

Physiological

relevance

of

receptors

Functional

characterization

of

receptors

in

heterologous

expression

systems

Signal

transduction

of

the

ADP

receptor

via

pertussis

toxin-sensitive

Gi

proteins

and

adenylyl

cyclase

inhibition

is

well

established

-LSB-

-RSB-

Similar

Gi

protein-coupling

specificity

was

found

for

-LSB-

-RSB-

-LSB-

-RSB-

and

lyso-PS

receptor

-LSB-

-RSB-

Because

adenylyl

cyclase

inhibition

assays

are

usually

less

sensitive

and

robust

several

other

experimental

setups

were

established

to

measure

function

of

GPCR

It

has

been

shown

that

and

can

be

activated

by

a

wide

variety

of

GPCR

-LSB-

-RSB-

The

ability

of

to

bypass

the

selectivity

of

receptor/G

protein

interaction

was

also

useful

to

measure

activation

of

-LSB-

-RSB-

It

has

been

demonstrated

that

replacement

of

the

four

or

five

C-terminal

amino

acids

of

Gαq

with

the

corresponding

Gαi

residues

-LRB-

referred

to

as

-LSB-

-RSB-

-RRB-

confers

the

ability

to

stimulate

the

PLC-β

pathway

onto

Gi-coupled

receptors

-LSB-

-RSB-

Successful

heterologous

expression

and

activation

by

applying

chimeric

has

been

demonstrated

for

and

lyso-PS

receptor

-LSB-

-RSB-

We

have

previously

shown

that

as

well

as

the

lyso-PS

receptor

display

increased

basal

activity

in

functional

assays

when

compared

with

other

GPCR

-LSB-

-RSB-

This

high

basal

activity

can

be

either

discussed

as

the

natural

ground

state

of

the

receptor

activity

equilibrium

or

as

the

effect

of

receptor

agonists

that

are

present

in

the

cell

culture

medium

or

that

are

released

from

cells

Analyzing

mutations

in

the

highly

conserved

DRY

motif

of

we

recently

showed

that

basal

activity

is

abolished

in

the

DHY

mutant

but

agonist-induced

activation

remains

intact

-LRB-

Fig.

-RRB-

We

conclude

that

the

basal

activity

of

the

ADP

receptor

is

rather

a

genuine

property

of

this

receptor

and

not

due

to

continuous

stimulation

by

agonists

High

basal

activity

appears

to

be

a

general

feature

of

all

members

of

the

receptor

group

In

our

initial

ortholog

screen

we

identified

two

receptors

carp

types

and

-LRB-

-RRB-

which

differ

in

only

ten

amino

acid

positions

Interestingly

carp

type

displays

higher

basal

activity

and

studies

are

ongoing

to

identify

residues

that

promote

constitutive

activity

Fig.

basal

activity

is

a

genuine

property

of

Basal

activity

of

the

wild-type

-LRB-

DRY

motif

-RRB-

and

a

mutant

-LRB-

DHR

motif

-RRB-

was

determined

in

transiently

transfected

cells

Gi

coupling

of

was

rerouted

to

IP

production

by

co-transfection

of

a

chimeric

G

protein

-LSB-

-RSB-

Because

many

standard

mammalian

expression

systems

endogenously

express

receptors

clear-cut

functional

studies

are

difficult

to

perform

One

exception

is

the

human

astrocytoma

cell

line

which

does

not

express

receptors

However

transient

expression

of

GPCR

in

cells

is

limited

by

low

transfection

efficiency

and

stable

transfection

of

these

cells

is

usually

required

Heterologous

expression

of

GPCR

in

yeast

was

initially

established

for

large-scale

purification

of

receptor

proteins

These

advances

in

the

expression

of

heterologous

GPCR

in

the

yeast

Saccharomyces

cerevisiae

have

led

to

the

development

of

sensitive

and

selective

assays

of

ligand-induced

GPCR

activation

-LRB-

reviewed

in

-LSB-

-RSB-

-RRB-

To

facilitate

a

more

systematic

genetic

analysis

of

GPCR

function

e.g.

by

saturating

random

mutagenesis

we

took

advantage

of

a

yeast

expression

system

in

which

parts

of

the

mammalian

GPCR

signaling

system

-LRB-

GPCR

and

chimeric

G

protein

-RRB-

are

linked

to

a

modified

yeast

pheromone

pathway

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

The

coupling

of

receptor

activity

to

the

genetically

engineered

yeast

pathway

allows

for

rapid

and

economical

screening

of

substance

libraries

and

randomly

modified

receptor

libraries

Functional

studies

in

yeast

may

have

an

advantage

especially

for

receptors

because

one

can

-LRB-

at

least

partially

-RRB-

circumvent

specific

problems

in

working

with

nucleotide

receptors

-LRB-

see

above

-RRB-

Only

two

NTPDases

-LRB-

and

-RRB-

have

been

found

within

the

entire

yeast

genome

which

are

mainly

expressed

in

the

Golgi

apparatus

-LSB-

-RSB-

Although

conversion

of

nucleotides

by

nucleotidases

and

ectoapyrases

occur

also

in

yeast

and

one

can

not

exclude

conversion

of

extracellular

nucleotides

by

yeast

enzymes

there

is

no

endogenous

plasma

membrane

receptor

in

yeast

which

can

mediate

and

therefore

interfere

with

transmembrane

signaling

of

heterologously

expressed

receptors

in

yeast

We

and

others

have

successfully

used

this

system

for

expression

and

functional

study

of

-LSB-

-RSB-

-LSB-

-RSB-

and

lyso-PS

receptor

-LRB-

unpublished

data

-RRB-

As

shown

in

Fig.

yeast

cells

grow

only

in

the

presence

of

the

agonist

diphosphate

-LRB-

-RRB-

Fig.

expression

and

in

vitro

evolution

of

GPCR

in

yeast

Genetically

modified

yeast

cells

are

transformed

with

a

mammalian

GPCR

Following

agonist

activation

and

constitutive

receptor

activity

the

receptor

couples

to

a

chimeric

G

protein

-LSB-

backbone

yeast

G

protein

in

which

the

C-terminal

five

amino

acids

were

replaced

by

the

respective

mammalian

G

protein

sequence

-LRB-

e.g.

from

the

Gi

protein

-RRB-

-RSB-

Activation

of

the

chimeric

G

protein

enables

yeast

cells

to

grow

on

histidine-free

medium

by

utilizing

parts

of

the

yeast

mating

pathway

-LSB-

-RSB-

Gpa

yeast

ortholog

of

the

mammalian

G

protein

alpha

subunit

yeast

ortholog

of

the

mammalian

G

protein

beta

subunit

yeast

ortholog

of

the

mammalian

G

protein

gamma

subunit

transcription

factor

that

is

activated

by

a

MAP

kinase

signaling

cascade

cell

cycle

regulator

that

directly

inhibits

the

yeast

cyclin-dependent

kinase

expression

and

random

mutagenesis

of

in

yeast

a

The

human

receptor

was

transformed

into

modified

yeast

-LRB-

see

Fig.

-RRB-

The

agonist

diphosphate

-LRB-

-RRB-

induces

a

robust

yeast

cell

growth

b

The

entire

coding

region

of

the

human

was

subjected

to

random

mutagenesis

and

transformed

yeast

cells

were

selected

for

growth

in

agonist-free

U

H

medium

Colonies

that

grow

under

this

condition

contain

a

constitutively

active

Exemplarily

mutations

of

a

triple

mutant

-LRB-

-RRB-

were

individualized

and

tested

separately

for

constitutive

activity

The

data

indicate

that

mainly

contributes

to

the

constitutive

activity

of

the

triple

mutant

Constitutively

active

GPCR

are

useful

tools

in

studying

the

action

of

inverse

agonists

and

activation

mechanisms

in

GPCR

A

constitutively

active

was

generated

by

replacing

the

endogenous

C

terminus

with

the

corresponding

part

of

the

human

receptor

-LSB-

-RSB-

Pharmacological

evaluation

of

several

antagonists

revealed

-LRB-

an

adenosine

derivative

-RRB-

as

a

potent

inverse

agonist

Traditional

mutagenesis

approaches

are

limited

to

screen

for

activating

mutations

because

the

number

of

mutant

proteins

that

can

be

investigated

is

usually

relatively

small

primarily

because

of

technical

reasons

and

the

time

and

costs

needed

to

generate

and

analyze

large

numbers

of

mutant

receptors

To

circumvent

these

limitations

yeast

has

emerged

as

a

highly

useful

host

for

the

in

vivo

reconstitution

of

mammalian

GPCR

Therefore

we

applied

a

random

mutagenesis

and

screening

approach

in

yeast

to

identify

key

residues

in

maintaining

ground

stage

of

the

human

PCR-based

random

mutagenesis

was

optimized

to

induce

approximately

four

mutations

in

kbp

Several

dozen

clones

were

selected

and

are

now

under

in-depth

investigation

For

example

we

identified

a

triple

mutant

-LRB-

-RRB-

and

individual

characterization

revealed

-LRB-

-RRB-

to

be

mainly

responsible

for

constitutive

activity

in

the

yeast

expression

system

-LRB-

Fig.

-RRB-

These

promising

results

in

applying

the

yeast

expression

system

await

further

efforts

to

identify

functionally

relevant

determinants

in

receptors

Establishing

in

vivo

function

of

receptors

using

mouse

models

Selective

receptor

ligand

and

receptor-deficient

animal

models

are

suitable

tools

to

evaluate

the

physiological

relevance

of

distinct

GPCR

Enormous

efforts

have

been

undertaken

in

the

development

of

selective

and

clinically

useful

ADP

and

other

nucleotide

receptor

ligands

But

except

for

dissection

of

the

physiological

relevance

of

all

other

GPCR

is

at

the

very

early

stage

Since

the

pharmacological

properties

of

ligands

have

been

reviewed

in

detail

elsewhere

-LSB-

-RSB-

we

only

shortly

summarize

available

data

on

receptor-deficient

animal

models

Based

on

the

clinical

success

of

irreversibly

bound

antagonists

such

as

clopidogrel

the

pivotal

role

of

ADP

in

arterial

thrombogenesis

is

well

established

Although

the

combined

action

of

and

is

necessary

for

the

full

platelet

aggregation

response

to

ADP

mice

deficient

for

already

display

reduced

platelet

adhesion/activation

thrombus

growth

and

stability

-LSB-

-RSB-

Except

for

the

altered

platelet

functionality

mice

appear

normal

under

standard

laboratory

conditions

In

consent

with

this

finding

we

occasionally

identified

a

frameshift

-LRB-

T

insertion

at

base

pair

position

-RRB-

in

the

coding

sequence

of

several

individuals

of

the

Asian

house

mouse

-LRB-

inbred

laboratory

strain

of

Mus

musculus

castaneus

from

of

the

Laboratoire

Génome

Populations

Interactions

Adaptation

at

the

Universite

Montpellier

France

-RRB-

during

ortholog

screening

which

showed

no

obvious

phenotype

in

standard

laboratory

captivity

The

platelet

function

was

however

not

tested

The

identified

inactivation

in

some

Asian

house

mice

may

reflect

either

natural

polymorphisms

present

in

the

wild

population

or

a

new

polymorphism

that

has

been

introduced

during

captivity

To

distinguish

between

these

possibilities

we

analyzed

the

position

in

from

wild

M.

m.

castaneus

trapped

in

Taiwan

-LSB-

-RSB-

All

contained

the

intact

allele

indicating

that

the

inactive

allele

is

very

rare

or

absent

in

this

wild

M.

m.

castaneus

population

and

favors

the

hypothesis

of

its

acquisition

in

captivity

-LRB-

unpublished

observations

-RRB-

The

fact

that

receptor

deficiency

is

per

se

compatible

with

viability

and

fertility

appears

to

be

true

also

for

other

receptor-deficient

species

and

mouse

models

For

example

mice

individually

lacking

and

are

viable

fertile

and

produced

viable

offspring

-LRB-

own

unpublished

observation

-RRB-

Further

there

are

several

vertebrate

classes

and

species

naturally

deficient

in

distinct

receptors

For

example

is

absent

in

fish

genomes

sequenced

so

far

is

not

present

in

pufferfish

genomes

and

is

absent

in

pufferfish

and

Xenopus

genomes

It

appears

that

many

functions

of

receptors

are

more

distinct

and

their

disclosure

requires

specific

challenging

conditions

Indeed

mice

revealed

an

unexpected

phenotype

when

specifically

challenged

CNS

injury

is

accompanied

by

release

of

nucleotides

serving

as

signals

for

microglial

activation

or

chemotaxis

Microglia

cells

express

several

purinoceptors

including

Microglia

in

mice

showed

significantly

diminished

directional

branch

extension

toward

sites

of

cortical

damage

in

the

living

mouse

These

results

imply

that

is

a

primary

site

at

which

nucleotides

act

to

induce

microglial

chemotaxis

at

early

stages

of

the

response

to

local

CNS

injury

-LSB-

-RSB-

Variants

of

receptor

genes

within

human

populations

Activating

and

inactivating

mutations

in

GPCR

have

been

made

responsible

for

more

than

different

human

diseases

-LSB-

-RSB-

As

expected

from

its

pivotal

role

in

platelet

activation

inactivating

mutations

in

can

cause

a

congenital

bleeding

disorder

Only

a

few

missense

-LRB-

-RRB-

and

frameshifting

-LRB-

frameshift

at

amino

acid

position

-RRB-

mutations

have

been

reported

-LSB-

-RSB-

Unexceptionally

the

missense

mutations

found

are

at

highly

conserved

positions

and

cause

an

impairment

of

receptor

function

There

are

no

other

human

diseases

identified

yet

which

are

associated

with

dysfunction

of

receptors

The

antithrombotic

effect

of

clopidogrel

is

considerably

variable

and

the

gene

was

screened

for

possible

sequence

variants

Five

nucleotide

variations

were

found

in

the

human

gene

two

of

them

silent

substitutions

in

the

coding

region

-LSB-

-RSB-

Several

studies

were

initiated

to

investigate

the

impact

of

polymorphisms

on

atherosclerosis

-LSB-

-RSB-

and

clopidogrel

efficiency

in

preventing

neurological

events

-LSB-

-RSB-

showing

no

or

some

association

to

one

haplotype

In

single

nucleotide

polymorphism

-LRB-

SNP

-RRB-

projects

such

as

HapMap

-LSB-

-RSB-

SeattleSNP

etc.

silent

variations

have

been

identified

at

amino

acid

position

and

of

For

example

the

C/T

variation

at

amino

acid

position

is

only

found

in

the

African

population

whereas

the

G/T

variation

at

position

is

present

only

in

European

and

Asian

populations

One

missense

mutation

-LRB-

receptor

C

terminus

-RRB-

has

been

detected

in

SNP

projects

This

variant

is

absent

in

European

and

Asian

populations

but

displays

an

allelic

frequency

of

about

in

the

African

population

The

functional

relevance

of

this

missense

mutation

has

not

been

studied

yet

but

a

Gly

at

position

is

naturally

found

in

many

other

orthologs

including

primate

almost

ruling

out

a

specific

input

in

receptor

function

One

silent

variant

-LRB-

C/T

at

-RRB-

and

one

substituting

variant

-LRB-

T/C

at

-RRB-

were

identified

in

the

coding

region

of

The

variant

is

less

frequent

in

African

populations

-LRB-

-RRB-

when

compared

to

European

-LRB-

-RRB-

and

Asian

-LRB-

-RRB-

populations

is

not

conserved

and

a

Thr

at

this

position

is

found

in

the

dolphin

-LRB-

Tursiops

truncates

-RRB-

Only

two

silent

substitutions

-LRB-

A/G

at

and

T/C

at

-RRB-

have

been

identified

in

so

far

The

gene

contains

two

more

frequent

silent

substitutions

-LRB-

T/C

at

A/C

-RRB-

and

one

missense

variant

-LRB-

A/G

at

frequency

about

-RRB-

which

is

located

within

the

DPXXY

motif

of

is

quite

conserved

among

vertebrate

orthologs

The

functional

relevance

of

has

not

been

studied

yet

but

it

occurs

naturally

in

the

zebrafish

ortholog

In

the

human

gene

three

silent

polymorphisms

-LRB-

G/A

at

G/A

at

C/T

at

-RRB-

and

one

nonsynonymous

polymorphism

-LRB-

C/T

at

-RRB-

are

present

The

position

is

not

conserved

and

is

found

in

several

rodents

Only

rare

silent

substitutions

in

the

coding

regions

have

been

found

in

-LRB-

C/T

at

-RRB-

and

in

-LRB-

A/G

at

-RRB-

-LSB-

-RSB-

SNP

stochastically

occur

in

individual

genomes

and

can

amount

to

a

reasonable

frequency

in

populations

by

drift

but

also

by

selection

There

are

several

approaches

and

methods

which

are

suitable

to

distinguish

between

drift

and

selection

Population

genetic

models

predict

that

selection

can

leave

footprints

in

closely

linked

genomic

regions

Several

methods

were

developed

to

detect

signatures

of

selective

sweeps

in

genomic

sequences

-LSB-

-RSB-

All

methods

require

data

on

allele

variation

-LRB-

mainly

SNP

-RRB-

within

populations

Large

genome-wide

analyses

have

scanned

the

human

genome

for

signatures

of

positive

selection

on

the

basis

of

nonsynonymous

and

synonymous

substitution

ratios

or

single

nucleotide

polymorphism

-LRB-

SNP

-RRB-

data

Several

loci

which

contain

GPCR

genes

have

been

identified

using

such

methods

but

the

receptor-containing

loci

showed

no

strong

signatures

of

recent

positive

selection

in

these

studies

-LSB-

-RSB-

Conclusion

receptors

which

are

grouped

mainly

by

phylogenetic

relations

have

been

an

inherent

part

of

the

vertebrate

GPCR

repertoire

since

more

than

Myr

Although

sharing

features

in

respect

to

structural

determinants

and

signal

transduction

the

activating

ligands

are

heterogeneous

such

as

nucleotides

nucleotide

derivatives

leukotrienes

and

phospholipids

We

are

at

the

very

beginning

of

understanding

the

physiological

importance

of

the

individual

members

The

nature

of

the

ligands

first

functional

data

and

expression

of

several

members

in

migrating

cells

point

at

functions

in

immunologic

response

and

tissue

damage

response

Doubtless

upcoming

receptor-deficient

mouse

models

and

selective

receptor

ligands

will

help

to

unveil

the

functions

of

receptor

members

Further

one

should

consider

combined

receptor-deficient

mouse

models

within

this

group

but

also

with

other

receptors

to

uncover

phenotypes

which

are

hidden

by

receptor

redundancy

e.g.

in

the

case

of

ADP

receptors

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

evolution
nucleotide receptor
orphan gpcr


Progressive improvement of impaired visual acuity during the first year after transsphenoidal surgery for non-functioning pituitary macroadenoma Objective Improvement of visual field defects continues even years after the initial surgical treatment . Because this process of continuing improvement has not been documented for visual acuity , we audited our data to explore the pattern of recovery of visual acuity until 1 year after transsphenoidal surgery for non-functioning pituitary macroadenoma . Introduction Non-functioning pituitary macroadenomas -LRB- NFMA -RRB- are the most prevalent macroadenomas -LSB- 1 , 2 -RSB- . Patients with NFMA mainly present with decreased visual acuity , visual field defects and hypopituitarism , caused by mass effects of the tumor -LSB- 3 -- 5 -RSB- . Transsphenoidal surgery is the treatment of choice , resulting in improvement of visual field defects in 75 -- 100 % of all patients -LSB- 3 -- 5 -RSB- . Accordingly , visual acuity improves in the majority of patients -LSB- 6 -- 10 -RSB- , although visual field defects and visual acuity may worsen in a limited number of patients after surgery -LSB- 6 , 7 , 9 , 10 -RSB- . The process of visual fields recovery starts directly after surgery and can already be documented on the second postoperative day -LSB- 11 -RSB- . This process of recovery is probably due to restoration of the velocity of conduction . However , improvement of visual field defects continues even years after the initial surgical treatment -LSB- 12 , 13 -RSB- . Although a reduced visual acuity is correlated with the extent of visual field defects -LSB- 13 , 14 -RSB- , postoperative changes in visual acuity do not parallel changes in visual fields in every case -LSB- 6 -RSB- . To our knowledge , the process of gradual visual improvement , has only been observed for visual field defects , but not for visual acuity -LSB- 12 -RSB- . This is , however , clinically relevant , since improvement of minor visual field defects can occur unnoticed , whereas improvement in visual acuity is almost invariably noticed , and can potentially overcome thresholds to previous impairments in daily life . We therefore audited our data to explore the pattern of recovery of visual acuity until 1 year after transsphenoidal surgery for non-functioning pituitary macroadenoma . Patients and methods Patient selection Forty-three patients , mean age 56 ± 14 years , were included in this retrospective analysis . In order to obtain a homogenous cohort for assessment , the inclusion criteria were the following : Compression of the optic chiasm on MRI by the pituitary tumor prior to surgeryTranssphenoidal surgery for non-functioning pituitary macroadenoma -LRB- diameter > 1 cm -RRB- for decompression of the optic chiasmAssessment of visual acuity at least once before surgery , and at least twice in the first year after surgeryThe availability of two postsurgical MR scans , within a time frame of maximal 3 months of the ophtalmological assessments We did not restrict the inclusion criteria to patients with visual field defects , but also included patients with clear compression of the optic chiasm without decreased visual function , for two reasons . First , we were interested in the pattern of change in visual function prior to surgery . Second , it is , at least theoretically , possible that in patients with compression of the optic chiasm , surgical treatment may decrease visual function . To overcome the potential effect of postoperative tumor-regrowth on visual outcome , patients were excluded from analysis if the 1 year post-operative scans revealed growth of residual tumor . For complete assessment , we reviewed the patient records of all departments involved in the treatment of NFMA -LRB- Endocrinology , Neurosurgery , Ophthalmology -RRB- . Endocrine -LRB- pituitary function -RRB- and ophthalmologic data -LRB- visual acuity and visual fields -RRB- were assessed before surgery , 3 and 12 months after surgery . An MRI was performed before surgery , 3 -- 6 months and 12 -- 15 months after surgery . Transsphenoidal surgery was performed by one of two neurosurgeons Corrected visual acuity was determined by the Snellen chart -LSB- 15 , 16 -RSB- and was scored for both eyes . Visual fields were assessed by Humphrey perimetry in all patients . Goldman perimetry was used as an additional tool to assess peripheral visual field defects . To increase the reliability of the visual field testing , assessment of the visual fields , and thereby also visual acuity , was preferably performed twice prior to surgical treatment . However , surgical treatment was not delayed due to double ophthalmologic testing in patients with severe impaired visual acuity . Definitions The diagnosis of non-functioning pituitary macroadenoma was based on two criteria : the presence of a pituitary macroadenoma -LRB- > 1 cm -RRB- on MRI , and the absence of overproduction of any of the pituitary hormones . In all cases the diagnosis was histologically confirmed . Tumor size was classified according to Hardy -LSB- 17 -RSB- . Tumor extension was classified as suprasellar -LRB- Hardy II0 , A , B , C -RRB- or parasellar/infrasellar -LRB- Hardy IID , E , Hardy III0-E and Hardy IV0-E -RRB- . In all patients , visual acuity was scored on a scale between 0 and 1.25 . Visual field defects were scored semi-quantitative and classified as mild , moderate or severe . Visual field defects were classified as mild , if there were peripheral defects in only one quadrant . Defects were classified as moderate if the upper quadrants were affected , whereas in combined upper and lower quadrant field defects , these were classified as severe . Growth hormone -LRB- GH -RRB- deficiency was defined as an insufficient rise in GH levels -LRB- absolute value < 3 μg/l -RRB- after stimulation during an insulin tolerance test -LRB- ITT -RRB- . When secondary amenorrhoea was present for more than 1 year premenopausal women were defined as LH/FSH deficient . Postmenopausal women were defined as LH/FSH deficient , when gonadotropin levels were below the normal post-menopausal range -LRB- LH < 10 U/l , FSH < 30 U/l -RRB- . In men , LH/FSH deficiency was defined , as a testosterone level below the reference range -LRB- 8.0 nmol/l -RRB- . TSH deficiency was defined as a total or free T4 level below the reference range . ACTH deficiency was defined as a basal cortisol level at 8.00 AM of < 0.12 μmol/l and/or an insufficient increase in cortisol levels -LRB- absolute value < 0.55 μmol/l -RRB- after an ITT . Statistical analysis The paired t-test was used for paired samples . SPSS software version 12.0 -LRB- SPSS Inc. , Chicago , IL , USA -RRB- was used . A P-value of < 0.05 was considered statistically significant . Results Preoperative patient characteristics -LRB- Table 1 -RRB- Forty-three patients , 51 % male , were included in this study . Radiological imaging by MRI revealed a macroadenoma in all patients , with suprasellar extension in 100 % and parasellar/infrasellar extension in 42 % of cases . Hypopituitarism was present in 79 % of all patients , and panhypopituitarism in 23 % . Table 1Patient characteristics before transsphenoidal surgeryMale/female22/21Age -LRB- years ± SD -RRB- 56 ± 14Visual acuity Right eye -LRB- mean ± SD -RRB- 0.65 ± 0.37 Left eye -LRB- mean ± SD -RRB- 0.60 ± 0.32 Visual field defectsSevere60 % Moderate17 % Mild14 % None9 % Pituitary functionGH deficiency76 % LH/FSH deficiency69 % ACTH deficiency43 % TSH deficiency36 % Panhypopituitarism23 % MRI characteristicsSuprasellar extension100 % Infrasellar/parasellar extension42 % Pre-operative assessment of visual function -LRB- Table 1 -RRB- Visual fields were normal in 4 patients . Transsphenoidal surgery in these patients was performed because compression of the optic chiasm was evident on the MR scan -LRB- n = 4 -RRB- . In these 4 patients visual acuity was at least 1.0 for both eyes . Of all patients with visual field defects 60 % were classified as severe , 17 % as moderate , and 14 % as mild . Mean visual acuity was 0.65 ± 0.37 for the right eye , 0.60 ± 0.32 for the left eye . Visual acuity was assessed twice before surgery in 34 patients -LRB- 79 % of total -RRB- . The median time between the first and the second pre-surgical assessment was 4 weeks -LRB- range 1 -- 45 -RRB- . No significant decrease in visual acuity was observed within this time period . The mean visual acuity for the two pre-surgical assessments was : 0.65 ± 0.37 vs. 0.66 ± 0.38 -LRB- right eye -RRB- , and 0.60 ± 0.32 vs. 0.62 ± 0.33 -LRB- left eye -RRB- . Moreover , no decrease in visual acuity was observed in patients -LRB- n = 13 -RRB- with a time interval of > 8 weeks between the first and the second pre-surgical assessment . In 4 of these patients there were no visual field defects . Surgical treatment All patients were treated by transsphenoidal surgery . Repeat surgery within 6 months after initial treatment was performed twice -LRB- large residual tumor mass -LRB- n = 1 -RRB- and persisting liquor leakage -LRB- n = 1 -RRB- -RRB- . Repeat surgery was performed by transcranial approach . Transsphenoidal surgery was followed by radiotherapy in 10 patients in order to prevent recurrence . Patients received 40 Gy -LRB- n = 5 -RRB- or 46 Gy -LRB- n = 5 -RRB- . Three months postoperative assessment of visual function After transsphenoidal operation in patients with preoperative visual field defects improvement was observed in 60 % and normalization of the visual fields in 30 % . In 1 patient there was a slight increase in visual field defects . Visual acuity improved significantly 3 months after transsphenoidal surgery -LRB- Fig. 1 -RRB- . The mean visual acuity increased from 0.65 ± 0.37 to 0.75 ± 0.36 -LRB- P < 0.01 -RRB- -LRB- right eye -RRB- , and from 0.60 ± 0.32 to 0.82 ± 0.30 -LRB- P < 0.01 -RRB- -LRB- left eye -RRB- . Fig. 1The pattern of improvement of visual acuity after transsphenoidal surgery for non-functioning pituitary macroadenomas -LRB- n = 43 -RRB- One-year postoperative assessment of visual function One year after initial surgical therapy visual field defects showed continuous improvement in 36 % of patients , compared to early postoperative results . In 80 % of them , this improvement was accompanied by continuous improvement of visual acuity . In 56 % of all patients -LRB- n = 24 -RRB- visual acuity shows continuing improvement until 1 year after surgery . In 8 of them , this improvement was not accompanied by further improvement of visual field defects . Visual acuity was improved 1 year after transsphenoidal surgery compared to the 3 months postoperative values -LRB- Fig. 1 -RRB- . The mean visual acuity increased from 0.75 ± 0.36 to 0.82 ± 0.34 -LRB- P < 0.05 -RRB- -LRB- right eye -RRB- , and from 0.82 ± 0.30 to 0.88 ± 0.27 -LRB- P < 0.05 -RRB- -LRB- left eye -RRB- . Discussion The main aims of surgery in non-functioning pituitary macroadenoma are restoration of visual acuity and visual field defects by decompression of the optic chiasm . Nonetheless , our data indicate that there is no necessity for immediate decompression , since postponement of surgery for several weeks did not result in deterioration of visual acuity . Moreover , in this series of 43 patients , we demonstrated a continuing improvement of visual acuity until 1 year after transsphenoidal surgery . There is a significant correlation between the severity of visual loss prior to surgery and persisting visual field defects -LSB- 9 , 13 , 14 -RSB- . In our patients in whom visual acuity was assessed twice before surgery , no decrease of visual acuity was observed with a median time interval between two measurements of 4 weeks . This indicates that postponing surgery for 1 month does not negatively influence visual outcome , which is in line with the slow growth pattern of non-functioning adenomas -LSB- 4 -RSB- . Nonetheless , the delay of surgery should not unnecessarily be prolonged because a significant , inverse , correlation between visual outcome and the prolonged duration of symptoms has been reported -LSB- 9 -RSB- . Moreover , especially in patients with rapid loss of visual function or decreased visual acuity due to apoplexy , urgent surgical intervention is indicated . The initial event in the pathogenesis of decreased visual function in pituitary macroadenomas is compression of the optic chiasm . Nerve compression leads to decreased conduction and to demyelination . In an experimental setting , the process of demyelination after nerve compression has been observed even after 2 days -LSB- 18 , 19 -RSB- . In case of continuous nerve compression remyelination can be observed after several weeks , although remyelinated fibers do not seem to reach normal thickness and organization structure , and complete demyelinated fibers co-exist -LSB- 18 , 19 -RSB- . Re-myelinated fibers restore conduction , at least partially , even if the causative nerve lesion is still existing -LSB- 20 , 21 -RSB- . The improvement of visual dysfunction after surgical treatment is supposed to consist of two , or probably even three , phases -LSB- 12 -RSB- . There is an early phase , comprising the first hours and days after surgery . In this early fast phase , the improvement is caused by decompression of the visual pathways , leading to a restoration of signal conduction . Visual recovery has been demonstrated in the first days after surgical treatment -LSB- 11 , 12 -RSB- . The second phase , i.e. , delayed recovery , is pathophysiologically caused by restoration of axonal transport and remyelination , and based on remyelination of the optic nerve vessels . This phase of delayed recovery may last for several years -LSB- 12 , 13 -RSB- . A precise boundary between the end of the fast phase of recovery and the start of the delayed recovery seems to be artificial , because these two phases reflect different pathophysiological mechanisms , which may co-exist for a certain time-period . The contribution of the first phase of recovery might be larger , given the fact that more than 50 % of eventual recovery takes place within the first 3 months after surgery -LSB- 13 -RSB- . It is already known that visual acuity improves in the first months after surgical treatment -LSB- 6 -- 10 -RSB- and that the improvement of visual field defects is a continuing process for at least 1 year -LSB- 12 , 13 -RSB- . Kerrison et al. -LSB- 12 -RSB- showed progressive improvement of visual fields even more than 2 years after surgical decompression of the optic chiasm . However , they did not demonstrate this same pattern of recovery for visual acuity . This might be due to the relative small number of patients during prolonged follow-up . In the present study we demonstrate that also improvement of visual acuity continued 1 year after surgical treatment . The clinical consequences of the delayed phase of recovery for both visual field defects and visual acuity are obvious . Follow-up of patients after surgical treatment for pituitary macroadenomas should include ophthalmologic assessment within several weeks after surgery , as well as subsequent assessments after one and 2 years , in order to estimate the final effect of surgery on visual function . Moreover , patients should be told that visual function can continue to improve , at least until 1 year after surgery . The relevance of these findings are obvious and is of importance to all patients , given the impact of a decreased visual acuity as an independent predictor for a decreased quality of life -LSB- 22 , 23 -RSB- . Moreover , these data are essential in order to evaluate potential effects of recurrent pituitary adenomas on visual function . Ten patients in our series received postoperative radiotherapy . However , it is unlikely that this treatment affected the results of our study . In a series reported by Gnanalingham et al. -LSB- 13 -RSB- , in which 34 % of all patients received postoperative radiotherapy , persistent improvement of visual field defects was documented even years after surgical therapy . In another series of 21 patients , 2 years after pituitary irradiation , there were no cases of radiation-induced visual field or visual acuity deterioration -LSB- 24 -RSB- . In conclusion , this study demonstrates that the improvement of visual acuity , after transsphenoidal treatment for non-functioning pituitary macroadenomas , consists of both an early and a delayed phase of recovery . After initial post-surgical recovery , a progressive delayed improvement of visual acuity at least until 1 year after transsphenoidal surgery , is likely to occur .

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