Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage.

Abstract

Focal brain ischemia is the most common event leading to stroke in humans. To understand the molecular mechanisms associated with brain ischemia, we applied the technique of mRNA differential display and isolated a gene that encodes a recently discovered peptide, adrenomedullin (AM), which is a member of the calcitonin gene-related peptide (CGRP) family. Using the rat focal stroke model of middle cerebral artery occlusion (MCAO), we determined that AM mRNA expression was significantly increased in the ischemic cortex up to 17.4-fold at 3 h post-MCAO (P < 0.05) and 21.7-fold at 6 h post-MCAO (P < 0.05) and remained elevated for up to 15 days (9.6-fold increase; P < 0.05). Immunohistochemical studies localized AM to ischemic neuronal processes, and radioligand (125I-labeled CGRP) displacement revealed high-affinity (IC50 = 80.3 nmol) binding of AM to CGRP receptors in brain cortex. The cerebrovascular function of AM was studied using synthetic AM microinjected onto rat pial vessels using a cranial window or applied to canine basilar arteries in vitro. AM, applied abluminally, produced dose-dependent relaxation of preconstricted pial vessels (P < 0.05). Intracerebroventricular (but not systemic) AM administration at a high dose (8 nmol), prior to and after MCAO, increased the degree of focal ischemic injury (P < 0.05). The ischemia-induced expression of both AM mRNA and peptide in ischemic cortical neurons, the demonstration of the direct vasodilating effects of the peptide on cerebral vessels, and the ability of AM to exacerbate ischemic brain damage suggests that AM plays a significant role in focal ischemic brain injury.

Full text

Proc.
Natl.
Acad.
Sci.
USA
Vol.
92,
pp.
11480-11484,
December
1995
Neurobiology
Discovery
of
adrenomedullin
in
rat
ischemic
cortex
and
evidence
for
its
role
in
exacerbating
focal
brain
ischemic
damage
XINKANG
WANG,
TIAN-LI
YUE,
FRANK
C.
BARONE,
RAYMOND
F.
WHITE,
ROBERT
K.
CLARK,
ROBERT
N.
WILLET-FE,
ANTONY
C.
SULPIZIO,
NAMBI
V.
AIYAR,
ROBERT
R.
RUFFOLO,
JR.,
AND
GIoRA
Z.
FEUERSTEIN
Department
of
Cardiovascular
Pharmacology,
SmithKline
Beecham
Pharmaceuticals,
King
of
Prussia,
PA
19406
Communicated
by
Stephen
J.
Benkovic,
The
Pennsylvania
State
University,
University
Park
PA,
August
7,
1995
(received
for
review
March
3,
1995)
ABSTRACT
Focal
brain
ischemia
is
the
most
common
event
leading
to
stroke
in
humans.
To
understand
the
molec-
ular
mechanisms
associated
with
brain
ischemia,
we
applied
the
technique
of
mRNA
differential
display
and
isolated
a
gene
that
encodes
a
recently
discovered
peptide,
adrenomedullin
(AM),
which
is
a
member
of
the
calcitonin
gene-related
peptide
(CGRP)
family.
Using
the
rat
focal
stroke
model
of
middle
cerebral
artery
occlusion
(MCAO),
we
determined
that
AM
mRNA
expression
was
significantly
increased
in
the
ischemic
cortex
up
to
17.4-fold
at
3
h
post-MCAO
(P
<
0.05)
and
21.7-fold
at
6
h
post-MCAO
(P
<
0.05)
and
remained
elevated
for
up
to
15
days
(9.6-fold
increase;
P
<
0.05).
Immuno-
histochemical
studies
localized
AM
to
ischemic
neuronal
processes,
and
radioligand
(12II-labeled
CGRP)
displacement
revealed
high-affinity
(IC50
=
80.3
nmol)
binding
of
AM
to
CGRP
receptors
in
brain
cortex.
The
cerebrovascular
function
of
AM
was
studied
using
synthetic
AM
microinjected
onto
rat
pial
vessels
using
a
cranial
window
or
applied
to
canine
basilar
arteries
in
vitro.
AM,
applied
abluminally,
produced
dose-
dependent
relaxation
of
preconstricted
pial
vessels
(P
<
0.05).
Intracerebroventricular
(but
not
systemic)
AM
administra-
tion
at
a
high
dose
(8
nmol),
prior
to
and
after
MCAO,
increased
the
degree
of
focal
ischemic
injury
(P
<
0.05).
The
ischemia-induced
expression
of
both
AM
mRNA
and
peptide
in
ischemic
cortical
neurons,
the
demonstration
of
the
direct
vasodilating
effects
of
the
peptide
on
cerebral
vessels,
and
the
ability
of
AM
to
exacerbate
ischemic
brain
damage
suggests
that
AM
plays
a
significant
role
in
focal
ischemic
brain
injury.
Adrenomedullin
(AM)
is
a
recently
discovered
peptide
that
was
initially
identified
from
human
pheochromocytoma
(1).
Biologically
active
AM
consists
of
52
amino
acids
in
humans
and
50
amino
acids
in
rats
(1,
2),
and
both
AMs
exhibit
potent
vasodilator
activity
in
vitro
and
in
vivo
(3-6).
AM
bears
homology
to
a
family
of
peptides
that
includes
calcitonin
gene-related
peptide
(CGRP)
(7-10)
and
amylin
(11,
12).
CGRP
is
a
widely
distributed
neuropeptide
best
known
for
its
potent
vasodilator
actions
(13-15)
and
its
effect
on
insulin
functions
(16).
Amylin
is
the
major
protein
found
in
islet
amyloid
(11,
16)
in
humans
with
non-insulin-dependent
dia-
betes
mellitus.
CGRP
and
amylin
have
been
found
to
have
a
wide
range
of
biological
activities,
including
energy
metabo-
lism,
central
nervous
system
and
cardiovascular
functions,
and
calcium
metabolism
(for
review
see
refs.
16
and
17).
In
contrast,
little
is
known
of
the
biological
function
of
AM
beyond
vasodilation.
In
contrast
to
CGRP,
AM
mRNA
and
peptide
have
not
been
detected
in
normal
brain
(1, 2).
Very
recently,
AM
has
been
associated
with
congestive
heart
failure,
since
elevated
levels
of
AM
were
found
in
cardiac
tissue
of
the
failing
hearts
(18).
In
the
present
report,
we
used
a
recently
developed
mRNA
differential
display
technique
(19)
to
identify
genes
expressed
The
publication
costs
of
this article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.
in
response
to
brain
ischemia
induced
by
permanent
occlusion
of
the
middle
cerebral
artery
(MCAO)
in
the
rat.
We
herein
report
the
upregulation
of
AM
mRNA
expression
and
peptide
production
after
focal
cerebral
ischemia.
Furthermore,
we
have
demonstrated
that
AM
is
a
potent
cerebral
vasodilator
and
that
intracerebroventricular
(i.c.v.),
but
not
i.v.,
adminis-
tration
of
AM
exacerbated
focal
ischemic
injury.
Taken
to-
gether,
our
data
suggest
a
significant
role
for
AM
in
the
evolution
of
stroke.
MATERIALS
AND
METHODS
Focal
Brain
Ischemia.
Focal
cerebral
ischemia
was
carried
out
in
spontaneously
hypertensive
rats
(Taconic
Farms)
by
permanent
MCAO
as
described
in
detail
previously
(20,
21).
mRNA
Differential
Display.
Total
cellular
RNA
was
iso-
lated
from
ipsilateral
(ischemic)
cortex
and
contralateral
(nonischemic)
cortex
samples
as
described
(22,
23).
RNA
samples
from
2
h
and
12
h
after
MCAO
were
used
for
differential
display
essentially
as
described
(19,
24)
using
an
RNAmap
kit
(GenHunter).
The
differentially
expressed
bands
of
interest
were
isolated,
reamplified,
and
subcloned
into
a
pCRII
vector
(Invitrogen).
Northern
Blot
Analysis.
Northern
hybridization
was
carried
out
as
described
earlier
(23,
25).
cDNA
Library
Construction,
Screening,
and
DNA
Sequence
Analysis.
A
rat
cerebral
ischemia
cDNA
library
in
A
ZAP
II
vector
(Stratagene)
was
constructed
according
to
manufactur-
er's
specifications
using
poly(A)
RNA
isolated
from
ischemic
cortex
2
and
12
h
after
MCAO.
This
library
was
screened
using
the
PMCAO-9
cDNA
probe
isolated
from
differential
display
as
described
in
detail
previously
(25).
Four
individual
positive
clones
were
isolated,
and
the
corresponding
phagemids
were
produced
by
in
vivo
excision.
The
complete
cDNA
sequence*
of
AM
was
determined
from
both
strands.
DNA
sequence
analysis
and
computer
data
base
searches
were
performed
using
the
Genetics
Computer
Group
program.
Primer
Extension
Analysis.
Primer
extension
was
carried
out
as
described
(24)
in
the
presence
of
a
32P-labeled
primer
complementary
to
bases
46-64
(5'-GATGAGAAGCCGAG-
AAACC-3'),
and
5
,tg
of
poly(A)
RNA
isolated
from
rat
ischemic
cortex
at
12
h
after
MCAO.
Immunohistochemical
Study.
Six-micron-thick
frozen
sec-
tions
were
incubated
with
rabbit
anti-AM-(1-52)
(human)
IgG
(Peninsula
Laboratories)
and
then
with
fluorescein-conjugated
goat
anti-rabbit
IgG
antiserum
(Organon
Teknika-Cappel).
Antisera
were
diluted
in
phosphate-buffered
saline
(PBS)
containing
3%
(wt/vol)
bovine
serum
albumin.
Parallel
sec-
tions
were
incubated
with
either
preabsorbed
antiserum
[i.e.,
preincubating
anti-AM
antiserum
with
human
AM-(1-52)
Abbreviations:
AM,
adrenomedullin;
CGRP,
calcitonin
gene-related
peptide;
MCAO,
middle
cerebral
artery
occlusion;
ET-1,
endothelin
1;
i.c.v.,
intracerebroventricular.
*The
sequence
reported
in
this
paper
has
been
deposited
in
the
GenBank
data
base
(accession
no.
U15419).
11480

Proc.
Natl.
Acad.
Sci.
USA
92
(1995)
11481
(Peninsula
Laboratories)
at
10
,g/ml]
or
secondary
antiserum
only.
Double
labeling
was
performed
on
sections
by
first
treating
for
AM
immunoreactivity
as
above
and
then
applying
monoclonal
antibodies
for
glial
fibrillary
acidic
protein
(Boehringer
Mannheim)
or
neurofilaments
(ICN)
followed
by
rhodamine-conjugated
goat
anti-mouse
IgG
secondary
anti-
serum.
Sections
were
analyzed
using
OPTIMAS
image
analysis
(BioScan,
Edmonds,
WA).
In
Vitro
Canine
Cerebral
Vessels.
Four
adult
mongrel
dogs
were
euthanized
via
an
intravenous
overdose
of
pentobarbital.
The
basilar
artery
with
intact
endothelium
was
removed,
and
segments
were
prepared
for
isometric
tension
recording
(for
details,
see
ref.
26).
An
optimum
resting
tension
of
750
mg
was
applied
to
each
segment. During
the
equilibration
period
(-2
h),
the
segments
included
in
this
study
generated
substantial
spontaneous
tone.
The
concentration-related
effects
of
AM
on
spontaneous tone
were
then
determined.
In
control
experiments,
pretreatment
of
the
tissues
with
the
appropriate
volume
of
the
AM
vehicle
(saline)
had
no
significant
effect
on
the
spontaneous
tone.
The
relaxation
observed
with
AM
was
expressed
as
the
mean
±
SEM
of
the
percentage
of
spontaneous
tone.
Rat
Pial
Arteries.
The
effects
of
AM
on
pial
arteries
in
the
rat
were
investigated
in
situ
with
video
microscopic
techniques
in
a
cranial
window
preparation
with
the
dura
intact
(27).
Three
male
Sprague-Dawley
rats
were
prepared
and
main-
tained
as
described
(28).
Since
in
preliminary
experiments
AM
produced
inconsistent
relaxation,
endothelin
1
(ET-1;
0.2
pmol)
in
saline
(0.5
,ul)
was
microinjected
to
elicit
local
vasospasm;
this
was
followed
by
a
second
dose
of
ET-1
(0.2
pmol)
in
the
control
group
or
ET-1
(0.2
pmol)
plus
AM
(50
pmol)
in
the
AM
group.
All
responses
were
recorded
on
video
tape
and,.analyzed
using
National
Institutes
of
Health
image
analysis
software.
AM
Administration
in
MCAO.
Focal
ischemia
was
produced
in
spontaneously
hypertensive
rats
as
described
earlier.
AM
(i.v.
at
1
,ug/kg
per
min;
n
=
6)
or
vehicle
(PBS
with
1%
bovine
serum
albumin;
n
=
6)
was
administrated
continuously
for
1
h
pre-MCAO
and
4
h
post-MCAO.
i.c.v.
AM
at
doses
of
0
(n
=
13),
0.2
(n
=
9),
2
(n
=
10),
or
8
(n
=
13)
nmol
in
5
,ul
was
administered
into
the
ipsilateral
cerebral
ventricle
at
1
h
pre-MCAO
and
6
h
post-MCAO.
After
24
h
of
MCAO,
forebrain
sections
were
removed,
stained,
and
analyzed
as
described
(20,
21)
for
percent
hemispheric
swelling
and
percent
hemispheric
infarct
(normalized
to
the
contralateral
control
hemisphere)
and
the
actual
infarct
volume
in
cubic
millimeters.
RESULTS
AND
DISCUSSION
Identification
of
Upregulated
PMCAO-9
Gene
Expression
in
Rat
Ischemic
Cortex
Using
mRNA
Differential
Display.
Fig.
1A
illustrates
a
representative
autoradiograph
showing
the
ischemia-induced
expression
of
a
band
designated
as
PM-
CAO-9
at
2
and
12
h
post-MCAO
using
mRNA
differential
display.
The
upregulation
of
this
gene
expressed
in
ischemic
cortex
was
confirmed
using
Northern
blot
analysis
(Fig.
1B).
Thereafter,
PMCAO-9
was
subcloned
into
a
pCRII
vector
and
subjected
to
DNA
sequencing
analysis.
Computer
data
base
searches
of
the
PMCAO-9
sequence
(Fig.
2)
demonstrated
that
the
sequence
represented
an
unreported
cDNA.
Isolation
and
Analysis
of
AM
cDNA.
A
rat
brain
ischemia
cDNA
library
was
constructed
and
screened
using
the
PM-
CAO-9
DNA
as
a
probe.
Four
individual
positive
clones
were
isolated,
which
differed
in
length
at
the
5'
ends
(Fig.
2).
Upon
completion
of
the
full-length
cDNA
sequencing,
we
found
that
the
clone
matched
a
rat
precursor
AM
cDNA
in
the
GenBank
data
base
(accession
no.
D15069).
However,
several
sequence
differences
were
noted:
18
bases
have
been
extended
at
the
5'
end
of
our
clone
compared
to
the
sequence
reported
previ-
ously
(2).
In
addition,
a
single
base
(T)
deletion
after
base
1085
and
a
base
replacement
of
C
for
G
at
base
982
were
observed
A
1
2
3
4
_
PMCAO-9
B
PMCAO-9
1
2 3
4
kb
-
1.5
j~~~~A
WkL_
A-
j
rpL32
-
0.6
FIG.
1.
Identification
of
altered
gene
expression
in
rat
ischemic
cortex
after
MCAO
by
mRNA
different
display.
The
differential
display
PCR
was
carried
out
using
a
5'
arbitrary
primer
(5'-
GACCGCTTGT-3')
and
a
3'
T12NA
primer
and
resolved
by
electro-
phoresis
in
the
following
order:
lane
1,
2
h
ischemic;
lane
2,
2
h
nonischemic;
lane
3,
12
h
ischemic;
lane
4,
12
h
nonischemic
(A).
The
candidate
gene
indicated
with
an
arrowhead
(PMCAO-9)
was
con-
firmed
by
Northern
analysis
(B).
Two
micrograms
of
poly(A)
RNA
per
lane
was
used
with
the
same
loading
order
as
shown
in
A.
The
ribosomal
protein
L32
(rpL32)
probe
was
used
as
a
loading
control
(23).
in
all
of
our
four
clones
compared
to
the
previously
reported
sequence
(2).
Also,
a
three-base
deletion
(CCT)
after
base
1227
was
observed
in
three
of
our
four
clones,
whereas
the
other
clone
has
five
bases
(CCTGT)
deleted
compared
to
the
published
rat
AM
sequence
(2).
The
position
of
this
deleted
sequence
is
tandemly
followed
by
23
GT
repeats
in
all
four
clones.
This
variation
is
likely
to
be
caused
by
in
vivo
recom-
bination
events
as
pointed
out
by
a
recent
study
(29).
Further-
more,
primer
extension
experiments
revealed
that
the
AM
mRNA
contains
a
single
transcription
initiation
site
located
about
47
bases
upstream
of
our
longest
cDNA
clone
(data
not
shown).
Temporal
Expression
of
AM
mRNA
in
Rat
Ischemic
Cortex
After
MCAO.
Fig.
3A
shows
a
representative
Northern
blot
for
the
AM
mRNA
expression
in
the
ipsilateral
(ischemic)
and
contralateral
(nonischemic)
cortical
samples
at
different
time
points
after
MCAO.
Quantitative
Northern
blot
data
(n
=
4),
after
normalizing
to
an
rpL32
probe,
are
illustrated
graphically
in
Fig.
3B.
Very
low
levels
of
AM
mRNA
were
detected
in
normal
(data
not
shown)
or
sham-operated
cortical
samples.
The
AM
mRNA
expression
was
induced
significantly
in
the
ischemic
cortex
3
h
after
MCAO
(17.4-fold
increase
compared
to
sham;
P
<
0.05),
reached
its
peak
expression
at
6
h
(21.7-fold
increase;
P
<
0.05),
and
remained
elevated
for
at
least
15
days
(9.6-fold
increase;
P
<
0.05)
after
MCAO
(Fig.
3).
The
temporal
expression
profile
of
AM
mRNA
is
distinctly
differ-
ent
from
that
of
the
immediate
early
genes,
such
as
c-fos
and
zif268
(30),
which
exhibit
a
more
acute
response
profile
(significant
increase
at
1
h
and
peak
at
3
h),
or
the
delayed
response
genes,
including
inflammatory
cytokines,
such
as
tumor
necrosis
factor
a
and
interleukin
1(3
(31,
32),
which
are
significantly
elevated
at
6
h
and
peak
at
12
h
after
MCAO,
in
the
same
focal
ischemia
model.
The
significantly
prolonged
increase
in
AM
mRNA
after
MCAO
(Fig.
3)
suggests
both
an
early
and
late
involvement
in
ischemic
injury.
As
is
the
case
for
c-fos
and
other
acute
response
genes,
AM
mRNA
contains
two
AUUUA
motifs
in
the
3'-untranslated
region,
which
are
believed
to
be
associated
with
rapid
degradation
of
mRNA
(33,
34),
suggesting
that
prolonged
elevation
of
AM
mRNA
expression
after
MCAO
may
represent
a
de
novo
transcriptional
event.
Neurobiology:
Wang
et
al.

11482
Neurobiology:
Wang
et
al.
Proc.
Natl.
Acad.
Sci.
USA
92
(1995)
1
GACACTAGGC
AGAACAACTC
CAGCCTTTAC
CGCTCCTGGT
TTCTCGGCTT
CTCATCGCAG
TCAGTCTTGG
ACTTTGCGGG
TTTTGCCGCT
91
GTCAGAAGGA
CGTCTCGGAC
TTTCTGCTTC
AAGTGCTTGA
CAACTCACCC
TTTCAGCAGG
GTATCGGAGC
ATCGCTACAG
A
172
ATG
AAG
CTG
GTT
TCC
ATC
GCC
CTG
ATG
TTA
TTG
GGT
TCG
CTC
GCC
GTT
CTC
GGC
GCG
GAC ACC
GCA
CGG
CTC
GAC
M
K
L
V
S
I
A
L
M
L
L
G
S
L
A
V
L
G
A
D
TA
R
T.
D
25
247
ACT
TCC
TCG
CAG
TTC
CGA AAG
AAG
TGG
AAT
AAG
TGG
GCG CTA
AGT
CGT
GGG
AAG
AGG
GAA
CTA
CAA
GCG
TCC
AGC
T
S
S
0
F
R
K
K
W
N
K
W A
L
S
R
G
K R
E L
Q
A
S
S
50
322
AGC
TAC
CCT
ACG
GGG
CTC
GTT
GAT
GAG
AAG
ACA
GTC
CCG
ACC
CAG ACT
CTT
GGG
CTC
CAG GAC
AAG
CAG
AGC
ACG
S
Y
P
T
G
L
V
D
E
K
T
V
P
T
Q
T
L
G
L
Q
D
K
Q
S
T
75
397
TCT
AGC
ACC
CCA CAA
GCC
AGC
ACT
CAG
AGC
ACA
GCC
CAC
ATT
CGA
GTC
AAA
CGC
TAC
CGC
CAG
AGC
ATG
AAC
CAG
S
S
T
P
Q
A
S
T
Q
S
T A
H
I
R
V
K R
Y R
0
S
M
N
0
100
472
GGG
TCC CGC
AGC
ACT
GGA
TGC
CGC
TTT
GGG
ACC
TGC
ACA
ATM
CAG
AAA
CTG
GCT
CAC
CAG
ATC
TAC
CAG
TTT
ACA
G
S
R
S
T
G
C
R
F
G
T
C
T
M
0
K
L
A
H
0
I
Y
0
F
T
125
547
GAC
AAA
GAC AAG
GAC GGC
ATG GCC
CCC
AGA
AAC
AAG
ATC
AGC
CCT
CAA
GGC
TAT
GGC
CGC
CGG
CGC
CGG
CGT
TCC
D
K
D
K
D
G
M
A
P
R
N
K
I
S
P
0
a
Y
G
R
R
R
R
R
S
150
622
CTG
CCA
GAG
GTC
CTC
CGA
GCC
CGG
ACT
GTG
GAG
TCC
TCC
CAG
GAG
CAG
ACA
CAC
TCA
GCT
CCA
GCC
TCC
CCG
GCG
L
P
E
V
L
R
A
R
T
V
E
S
S
Q
E
Q
T
H
S
A
P
A
S
P
A
175
697
CAC
CAA
GAC
ATC
TCC
AGA
GTC
TCT
AGG
TTA
TAG
GTGCGGGTGG
CAGCATTGAA
CAGTCGGGCG
AGTATCCCAT
TGGCGCCTGC
H
Q
D
I
S
R
V
S
R
L
*
186
780
GGAATCAGAG
AGCTTCGCAC
CCTGAGCGGA
CTGAGACAAT
CTTGCAGAGA
TCTGCCTGGC
TGCCCCTAGG
GGAGGCAGAG
GAACCCAAGA
870
TCAAGCCAGG
CTCACGTCAG
AAACCGAGAA
TTACAGGCTG
ATACTCTCTC
CGGGCAGGGG
TCTGAGCCAC
TGCCTTGCCC
GCTCATAAAC
960
TGGTTTTCTC
ACGGGGCATA
CGCCTCATTA
CTTACTTGAA
CTTTCCAAAA
CCTAGCGAGG
AAAAGTGCAA
TGCTTGTTAT
ACAGCCAAAG
1050
GTAACTATCA
T&A&AGTT
TGTTGATGTC
AAGAGGTTTT
TIMMTGTAA
CTTCAAATAT
ATAGAAATAT
T7TTGTACGT
TATATATTGT
1140
ATTAAGGGCA
ITTTTAAAGCG
ATTATATTGT
CACCTTCCCC
TATTTTAAGA
AGTGAATGTC
TCAGCAAGGT
GTAAGGTTGT
TTGGTTCCCC
1230
GTGTGTGTG
GTTTGT
GTGTGTGTGT
GTGTGTGTGT
GITIGTAAGG
TGGAGAGCGC
CTGATTACCG
CCTGTGGATG
AAGAAAAAAC
1320
ATTGTGTCTT
CTATAATCT&_.nCATAAA
ATATGTGATC
TGGGAAAAAG
CAAACCAAIA
AACTGTCTCA
ATGCTG
(A)
n
FIG.
2.
Nucleotide
sequence
of
rat
AM
cDNA.
The
numbers
to
the
left
refer
to
the
nucleotide
positions
and
those
to
the
right
refer
to
the
amino
acid
positions.
The
vertical
arrows
indicate
the
5'
end
of
the
individual
cDNA
clones,
and
the
horizontal
arrows
are the
primers
for
the
differential
display,
where
the
unmatched
bases
are
marked
with
a
dot.
The
position
of
the
two
AUUUA
motifs
in
the
3'-untranslated
region
are
bold-faced
and
underlined,
and
the
position
of
the
polyadenylylation
signal,
AATAAA,
is
bold-faced.
The
TG
repeats
are
underlined.
The
amino
acid
sequences
predicted
to
be
the
proteolytic
cleavage
products
are
underlined.
The
AM
peptide
(amino
acids
93-143)
has
been
shown
to
have
vasodilator
properties
and
was
used
for
functional
analysis
in
Fig.
5
and
Table
1.
Immunostaining
of
AM
in
the
Ischemic
Cortex
After
12
h,
24
h,
and
5
days
after
MCAO.
AM
immunoreactivity
MCAO.
Immunohistochemical
analysis
was
carried
out
using
revealed
the
presence
of
short
fiber
processes
(highly
fluores-
brain
tissues
after
sham
surgery
and
from
animals
(n
=
3)
6
h,
cent
in
a
granular
or
vacuolar
pattern
within
the
ischemic
A
lpsilateral:+
-
+
-
+
-
+ +
-
+
-
+
-
+
-
+
-
Contralateral:
-
+
-
+
-
+
-
+
-
+
-
+
-
+
-
+
-
+
-
+
Time:
S
S
lh
lh
3h
3h
6h
6h
12h
12h
24h 24h
2d
2d
Sd
5d
10d
10d
lSd
15d
kb
AM
.
-1.5
rpL32
*
B
S
20-
*
Ischemic
*
*
0
~~~~~~~Nonischemic
Z
10
*
*
E
Sham
1
h
3h
6h
1
2h
24h
2d
5d
1Od
15d
Time
post-MCAO
FIG.
3.
Time
course
study
of
AM
mRNA
induction
in
rat
ischemic
cortex
after
MCAO.
(A)
Representive
Northern
blot
for
AM
and
rpL32
probes
to
the
samples
isolated
at
various
time
points
and
conditions
from
rats
subjected
to
MCAO.
Total
cellular
RNA
(40
,ug
per
lane)
was
used
for
this
analysis.
Ipsilateral
and
contralateral
cortex
samples
(denoted
by
+)
from
individual
rats
after
sham
surgery
(S;
12
h
sacrifice)
or
1,
3,
6,
12,
and
24
h,
and
2,
5,
10,
and
15
days
(d)
of
MCAO
are
depicted.
(B)
Quantitative
Northern
blot
data
for
AM
mRNA
expression
after
focal
brain
ischemic
injury.
The
data
were
analyzed
using
a
Phosphorlmager
and
are
presented
as
the
mean
values
±
SE
of
four
animals
for
each
time
point.
The
data
were
analyzed
using
one-way
ANOVA
followed
by
Bonferroni-adjusted
post
hoc
t
test.
*,
P
<
0.05
vs
sham
samples.

Proc.
Natl.
Acad.
Sci.
USA
92
(1995)
11483
cortex).
These
fibers
were
rare
and
only
weakly
fluorescent
6
and
12
h
after
MCAO
but
were
intensely
immunoreactive
and
abundant
in
tissues
examined
24
h
and
5
days
after
MCAO
(Fig.
4A
and
C).
No
immunoreactivity
was
observed
in
sham-operated
rats
or
outside
of
the
ischemic
zone
after
MCAO.
When
AM
antiserum
was
preincubated
with
AM
prior
to
incubation
of
tissue
sections,
immunofluorescence
was
completely
eliminated
(Fig.
4,
E
compared
with
C).
Also,
no
immunoreactivity
was
observed
in
sections
incubated
with
the
second
antibody
only
(data
not
shown).
When
double
labeled
with
intermediate
filament
markers
for
neurons
(neurofila-
ments)
or
astroglia
(glial
fibrillary
acidic
protein),
the
immuno-
fluorescent
data
indicated
colocalization
of
AM
with
neuro-
filaments
(Fig.
4
B,
D,
and
F),
which
is
indicative
of
nerve
fiber
processes.
In
contrast,
no
colocalization
was
seen
between
AM
and
glial
fibrillary
acidic
protein
(data
not
shown).
AM
Effects
on
Rat
Pial
Arteries
in
Situ.
The
effects
of
rat
AM
on
rat
pial
microvessels
(43
±
7
,um)
were
determined
using
videomicroscopic
techniques
(Fig.
5A).
The
local
appli-
cation
of
ET-1
(0.2
pmol)
via
subarachnoid
microinjection
rapidly
elicited
a
prolonged
submaximal
constriction
of
adjacent
pial
arteries
(Fig.
5
A
and
B).
There
was
little
or
no
change
in
the
pial
artery
diameter
when
the
microinjection
of
ET-1
was
re-
peated
within
10
min
of
the
first
microinjection.
In
contrast,
when
AM
(50
pmol)
was
added
to
the
second
ET-1
microinjection,
a
significant
vasodilation
of
the
adjacent
pial
artery
was
observed.
AM
(up
to
10
,gmol)
did
not
interfere
with
ETA
receptor
binding
(data
not
shown).
The
percent
of
the
control
diameter
was
44%
±
8%
after
ET-1
alone
compared
to
98%
±
18%
after
ET-1
plus
AM
(Fig.
SB).
There
were
no
changes
in
systemic
arterial
pressure
after
ET-1
or
AM
microinjection.
In
addition,
AM
had
no
effect
on
cortical
perfusion
when
administered
systemically
at
the
same
doses
(data
not
shown).
AM
Effects
on
Cerebral
Vessels
in
Vitro.
The
effects
of
rat
AM
on
segments
of
the
canine
basilar
artery
were
determined
in
vitro.
The
canine
basilar
arteries
used
in
this
study
slowly
generated
spontaneous
tone,
which
reached
a
steady-state
A
CONTROL
ET-1
ET-1
+
AM
SOPm
B
120
100]
CD
-
E
80-
.U
L
a
5
60-
o
40-
20-
0-
I
(n=3)
ET-1
(0.2
pmoles)
ET-1
(0.2
pmoles)
AM
(50
pmoles)
C
CANINE
BASILAR
ARTERY
c
0
x
to
Ox
FIG.
4.
Immunohistochemical
detection
of
AM
expression
in
ischemic
cortex
after
MCAO
in
the
rats.
Matched
computer-captured
images
of
ischemic
rat
cerebral
cortex
immunolabeled
with
antiserum
against
AM
(A,
C,
and
E)
and
neurofilaments
(B,
D,
and
F).
A
and
B
are
from
the
same
matched
field,
as
are
C-F.
Ischemic
cortex
at
24
h
or
5
d
after
MCAO
demonstrated
intense
AM
immunoreactivity
in
a
granular
or
vacuolar
pattern
(A
and
C,
24
h
after
MCAO).
This
immunoflourescence
was
restricted
to
the
ischemic
cortex.
When
the
anti-AM
antiserum
was
preincubated
with
AM,
the
immunoreactivity
was
eliminated
(E
compared
with
C).
Combining
computer
images
of
brain
tissue
immunofluorescence
for
AM
(green
in
A
and
C)
and
neurofilaments
(red
in
F)
allow
demonstration
of
double-labeling
in
neuronal
processes
(yellow
in
double
labeled
B
and
D).
Arrows
indicate
location
of
neuronal
processes.
100-
90-/
80-
70
60
50
40
/
30
20
10-
/
n=4
3-
,
-
10
100
10
00
3
10
100
1000
AM
(nM)
FIG.
5.
The
cerebrovascular
effects
of
AM
were
assessed
in
rat
pial
arteries
in
situ
(A
and
B)
and
canine
basilar
artery
segments
in
vitro
(C).
Microinjection
of
ET-1
(0.2
pmol)
produced
significant
vasoconstric-
tion
of
rat
pial
arteries
(A
Middle
and
B).
AM
(50
pmol)
administration
reversed
ET-1-induced
vasoconstriction
(A
Right
and
B).
(C)
AM
also
produced
a
concentration-dependent
relaxation
of
spontaneous
tone
in
the
canine
basilar
artery.
*P
<
0.02,
compared
to
ET-1
treatment
alone
as
determined
by
Student's
t
test.
Neurobiology:
Wang
et
al.

Proc.
Natl.
Acad.
Sci.
USA
92
(1995)
Table
1.
Effects
of
AM
in
focal
ischemic
injury
i.v.,
ng/kg
per
min
i.c.v.,
nmol
Measure
0
1
0
0.2
2
8
Swelling,
%
1
±
1
2
±
2
2
±
1
0
±
1
0
±
1
5
±
1
Infarct,
%
18
±
2
17
±
2
16
±
1
13
±
1
13
±
1
20
±
2*
Infarct
volume,
mm3
110
±
13
105
±
14
92
±
8
67
±
8
72
±
9
120
±
9*
*P
<
0.05
compared
to
vehicle,
analyzed
by
a
one-way
ANOVA
followed
by
Fisher's
protected
least-significant
difference
post
hoc
t
test.
maximum
(841
±
115
mg)
prior
to
the
addition
of
AM
to
the
tissue
bath.
AM
produced
a
prolonged,
concentration-
dependent
relaxation
of
spontaneous
tone.
The
concentration
required
to
produce
half-maximal
relaxation
(EC5o)
was
56
nmol,
and
relaxation
was
complete
at
300
nmol
(Fig.
SC).
AM
produced
similar
concentration-dependent
vasodilation
of
the
canine
basilar
artery
segments
when
precontracted
with
0.1
nmol
ET-1
(data
not
shown).
Furthermore,
additional
studies
indicate
that
the
vasodilation
induced
by
AM
can
be
blocked
by
CGRP
antagonists
(CGRP8-37),
indicating
AM
action
at
CGRP
or
CGRP-like
receptors.
AM
Effects
on
MCAO
Injury.
Continuous
i.v.
infusion
of
AM
beginning
1
h
before
and
4
h
after
MCAO
did
not
produce
any
significant
changes
in
ischemic
injury
produced
by
focal
ischemia
(Table
1).
Low
doses
(0.2
and
2
nmol)
of
i.c.v.
AM
administration
at
1
h
prior
to
and
6
h
after
MCAO
tended
to
decrease
hemispheric
swelling,
hemispheric
infarct,
and
infarct
volume
(P
>
0.05;
not
significant),
whereas
the
high
i.c.v.
dose
(8
nmol)
of
AM
tended
to
increase
percent
hemispheric
swelling
(P
>
0.05;
not
significant)
and
significantly
increased
(P
<
0.05;
Table
1)
percent
hemispheric
infarct
(25.0%)
and
infarct
volume
(30.4%).
Furthermore,
we
have
established
that
AM
may
act
on
CGRP
or
CGRP-like
receptors
by
performing
radioligand
(125I-labeled
CGRP)
displacement
studies
(rat
cor-
tical
membranes)
with
AM;
these
binding
studies
(n
=
3)
revealed
an
IC50
=
80.3
nmol
for
AM
displacement
of
125I-labeled
CGRP.
Although
the
expression
of
AM
has
not
been
observed
in
the
normal
brain
(1,
2),
the
effects
of
AM
on
rat
pial
arteries
in
situ
and
on
canine
basilar
arteries
in
vitro
demonstrated
the
similar
vasodilation
function
of
AM
in
the
brain
as
in
the
non-central
nervous
system
vasculature
(1,
3-6).
i.v.
administration
of
CGRP,
a
related
vasodilator,
can
produce
a
significant
im-
provement
in
ischemic
blood
flow
to
cerebral
ischemic
tissue
and
reduce
brain
injury
after
focal
stroke
(35).
However,
dilation
of
the
microcirculation
at
the
site
of
injury
may
also
facilitate
inflammatory
cell
infiltration
that
occurs
in
the
ischemic
cortex
shortly
after
MCAO
(20,
36,
37).
Although
the
role
of
AM
in
ischemic
brain
injury
requires
further
explora-
tion,
unlike the
intraluminal
vasodilatory
and
protective
ef-
fects
of
CGRP
in
focal
ischemia
(35),
AM
vasodilatory
effects
are
abluminal.
AM
administration,
i.c.v.,
but
not
i.v.,
exacer-
bates
focal
ischemic
injury
at
a
relatively
high
dose
range
(8
nmol;
Table
1),
suggesting
that
ischemic
tissue
AM
may
contribute
to
permeability
and
disruption
of
the
blood
brain
barrier
and
thereby
to
increase
ischemic
brain
damage.
In
conclusion,
the
upregulation
of
AM
in
the
ischemic
cortex
suggests
its
involvement
in
the
acute
response
to
ischemia.
The
potential
action
of
AM
to
dilate
cerebral
vessels
and
to
increase
permeability
and
infarct
size
call
for
a
pathogenic
role
of
locally
expressed
AM
in
focal
stroke,
although
that
requires
further
exploration.
We
thank
G.
Sathe,
J.
Mao,
and
R.
Morris
for
oligonucleotide
synthesis
and
DNA
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11484
Neurobiology:
Wang
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