Abstract. The localization of two type 1 angiotensin. II receptor subtype. mRNA,. ATIA and ATIB, was determined by reverse transcription-PCR on microdissected.
Expression of Type 1 Angiotensin Angiotensin TI-Induced Calcium Rat Nephron
II Receptor Mobilization
Subtypes along the
and
NADINE BOUBY,* ANNETTE HUSCITHAREL,t JEANNINE MARCHETTI, LISE BANKIR,* PIERRE CORVOL,t and CATHERINE LLORENSCORTESt Institut
Abstract.
The
subtype
mRNA,
National
localization
of two
ATIA
transcription-PCR
de
and
on
la Sante
type
ATIB,
la Recherche
M#{233}dicale U p36,
II receptor
in glomerulus
(12.2
determined
by reverse
are expressed,
and
glomeruli
and
nephron
segments. The coupling sensitivity of these two receptor subtypes was evaluated by measuring variations in intracellular calcium ([Ca2]) elicited by angiotensin II (Ang II) in structures expressing either ATIA or ATIB mRNA, using Fura-2 fluorescence.
The
in glomerulus,
highest
and
thick
mRNA
was
ascending
84%). II was
In
is approximately
The increase in [Ca211 elichighest in proximal segments
equivalent
to 300
to 400
and thick ascending limb (L [Ca2]1 is approximately lent to 200 nmoLfL). In glomerulus and collecting response was lower ( < 100 nmol/L). The median concentrations
found
limb.
AT1B mRNA were similarly expressed, segments ATIA mRNA expression was
(approximately l0 mollL Ang
[Ca2]1
(L
of AT1
tubule,
glomerulus, ATIA and whereas in all nephron dominant ited by
expression
proximal
for Ang
II were
*90
1 angiotensin
was
microdissected
et de
of the same
order
nmol/L)
AT1
receptor
AT2
antagonist
external mol/L
Ang
of the
[Ca2J1
II was
subtype is the in
In contrast,
have been identified, receptors. Most known be
mediated
function
by
of AT2
named type physiological
AT1
receptors
receptors
has
Interestingly, in adult rats, involved in pressure-natriuresis AT2 the
receptor
cDNA
(AT1-R). not been
renal
has revealed
seven-transmembrane In mouse and rat,
two
1 (AT1) effects
domain AT1-R
receptor subtypes
termed
(35%) are
acids
out
sequent
physiological elucidated. seem to be of AT1 and
family. have been
are,
which
March
et de Ia Recherche Paris, France. 1046-6673/0801
17. 1997. Accepted to Dr.
Catherine
Institut
M#{233}dicaleU 36, College
de France,
1 - l658$03.00/0
Journal of the American Society of Nephrology Copyright © 1997 by the American Society of Nephrology
the of
by l0 that a part of the
results
is the
AT1B the
demon-
predominant
receptor
AT1
subtypes
activation
Soc
National
de
3 Rue d’Ulm,
la Sante
75005
(2-6).
of
Nephrol
The
one
may
do
calcium
8: 1658-1667,
are
residues of the
homology
3 ‘-untranslated of these
by only
differences
are
(3).
in the
Of particular
inter-
serine
that
other
may
receptors
amino
distributed
residues,
in phosphorylation on the
recep-
22
prevalent
involving
a difference
coupling
and
particularly
domain
and,
‘-
sequences
differences
desensitization,
is limited
5
differing acid
but
hand,
suggest
acid
amino
intracellular
on
There in the
identical,
cysteine
and
hand,
be
important
to their
sub-
a difference for
effector
the
proteins
(2).
Comparison tors
June 5, 1997.
Llorens-Cortes,
95%
functional
of the pharmacology
in Chinese
either AT1-R
Received
by
induced
these
for
mRNA
involving
to
isolated
AT1B
molecule
the
(7-10). involve
family,
which
in intracellular regard that
(2,5,6,11,12).
to
of and
cells,
Ca2
revealed
pharmacologically
AT1B-R of
of AT1A-R cyclase
agonist/antagonist defined
transduction
G-proteins
pathways of
and protein results
have
the C, resulting
kinase
been
or AT1B-R according
recep-
B
expressing
phospholipase
mobilization
adenylate
and
the
activate
Divergent
A
specifically
has
activation
subsequently
to the coupling inhibits
that
ATIA-R the
of
ovary
receptor,
similar
mainly
tion
hamster
recombinant
potencies
Correspondence
not
absence
the mobilization
and
(J Am
the amino
than
carboxy-terminal
The
belong
and the two
of 359.
throughout
est
receptors
ATIA
between
and type 2 (AT2) of Ang II seem to
completely
these
reabwith antag-
II receptor
of Ang
AT2 receptors ( 1). The cloning that
but the
suggesting
from
ATIA
(3)
system.
more
types
and shortened,
The Ang by the
1997)
tors
distinct
of the response
efficiency
second-messenger
regions.
resistance, GFR, and proximal tubular Ang II effects are mediated through interaction receptors. Using nonpeptide and pseudopeptide pharmacologically
In
AT1B nmol/
of magnitude
of vascular
two
phase
pmol/L).
originated
and their
tion
onists,
and (10.3
expressed in the nephron segments, (2) gbonly structure with a relatively high AT1B
content,
differ
ATIA limb
(1 pmoWL)
(1
reduced
and
specific
losartan
increase
both
ascending
exclusively expressed. were totally abolished
123319
the peak
Angiotensin II (Ang II) exerts an essential contribution in controlling several physiological functions in different organs. In the kidney, this hormone is mainly involved in the regulasorption.
in which thick
is almost responses
PD
France.
Ca2 pool. In conclusion, in the rat kidney: (1) AT1A
receptor merulus not
Paris,
nmollL),
antagonist
Ca2,
intracellular strate that
p367,
in cortical
L), in which ATIA Il-induced calcium
mRNA
equivaduct, the effective
and
C activa-
obtained to a G
to the
tissues
with protein and
Renal
recombinant (1
cells However,
1,13,14).
signaling
pathways
coupling lipase
where there for
of ATIA-R for
ATIA
no study
ing
either
has been
pharmacological
Ca2 on cells
and
actions
of Ang
In the adult The distribution has
been
and
much
than
formed.
The
different
subtype
of
coupling
sensitivity
proteins
by
studied
renal
of ATIA
the
and
(I 6). (17-20)
distribution
zones
was
(2 1 ,22). subtype
the nephron
of the two
in
but
tified
by its attachment
receptor
the
inner
at levels
very
Until
variations
in
now,
no perthe
and (2) to study
the
subtypes
fresh
medium
intracellular
institutional guidelines Male Sprague Dawley 130 to 180 g were
Alimentation water
used.
con-
in agreement
were
fed a normal
Epinay-sur-Orge,
with
standard
France;
scribed
previously
diet
Animals
of Nephron anesthetized
barbital
(6 mg/bOO
nephron
microdissection
taming
0.3%
catheter
our
wt).
The
in the
previously
[Serva, aorta
(23).
kidney
Heidelberg,
just
below
kidney
The
the cortico-medullary
left
(Usine
axis.
the was
Small
prepared
left
renal
then
(con-
through artery,
removed
pyramids
for
medium”
Germany])
verse
conditions
nase
solution
tion
was
isolated
were
transcription
applied
(RT)-PCR,
according
incu-
at 35#{176}C for 60 mm; not
were
for calcium
performed.
Glomeruli
and
by microdissection
in basal
mediuma
4#{176}C under
stereomicroscopic
with for re-
incubated
in collage-
measurements, nephron without
observation.
incuba-
segments
were
collagenase
Pieces
Composition
of basal
medium
Na,HPO4, 0.44 KH,P04, lactate, 10 acetate, 1 pyruvate,
0.33
(in mmolIL): I MgCl2. 2 glutamine,
137 NaCI.
at
of the following
1 CaCI,. and
0.3
20
5 KC1, Hepes.
aspartate.
0.8 5
and
duct
the
inner
MgSO4.
glucose,
mRNA structures
to eliminate
were
cellular
transferred
debris.
of each
by Llorens-Cortes
France),
into
Approximately
tubule
segment
were
water. Ten microwas extracted and
RNA
ci al. (25).
cDNA
were
synthe-
with 200 U of Moloney (Gibco BRL, Life Tech-
carried
was
and
volume
stopped
sample
was
out using
ofeach
40
France),
in a final
of 20 l.
The
by heating used
with
for
for PCR
2.5 U of Taq
primer,
and
90 s, respectively,
RT reaction 10
amplification.
polymerase
20 mmollL
lasted
90
mm at 70#{176}C.Half of The
reaction and
0.1
(pH
8.3),
65
(Boehringer)
Tris-HC1
buffer
in a DNA
thermal
cycler
(Perkin
Elmer
Possible contamination by genomic DNA was additional RNA samples to PCR amplification
the complete
observing
after,
ATIA
and
digestion
AT1B
were
of an ATIA separated
by
cDNA gel
amplicon.
There-
electrophoresis
(1.5%
software.
where
there
assay
and
band
(not
[Ca2] 5
Total
AT1-R
mRNA
was
calculated
as the
sum
of
ATIA-R and ATIB-R mRNA. Amplifications were run for 35 cycles and on cDNA from 2.5-mm tubule or 2.5 glomeruli, because this was still in the exponential phase was
dissection,
ferred
a linear
the value
relationship
obtained
between
from
the length
densitometry
used
for the
of the amplification
shown).
Measurements a
ATIB
p.1 of total RNA sample, virus reverse transcriptase
Meylan,
(DTT)
Image
duct,
to measurements:
pyramids
(OMCD),
agarose gel in 1 x Tris borate ethylenediamine tetra-acetic acid buffer) and visualized under ultraviolet light. The image was captured, processed on computer, and analyzed with National Institutes of Health
sliced
cut and
bated in 0. 1% collagenase solution in basal mediuma bubbled filtered air at 30#{176}C for 15 mm. For inner medulla collecting special
a
as de-
and
were
the outer
collecting
without an RT step. To distinguish ATIA from ATIB, 20 pA of the PCR products were digested with EcoRI (2000 U/pA) for 90 mm at 37GC The efficiency of the digestion by EcoRI was checked by
of pento-
was
from
the iden-
KC1, 2.1 mmollL MgCI2, 0.5 mmol/L dNTP, 2 mmol/L dithiothreitol, 0.01% gelatin, and 10 U of RNase inhibitor in a final volume of 50 p1. The samples were covered with mineral oil. PCR amplification was conducted for 35 cycles at 94. 54, and 72#{176}C for 60,
and given
injection
of 5 ml of basal
froni and
mmol/L
Segments
by infusion
colbagenase
placed
scribed
g body
and
medulla
or 25 to 50 mm
480, Norwalk, CT). assessed by subjecting
by intraperitoneal
limb
(CTAL);
of diethylpyrocarbonate-treated tRNA was added. Total
at 37#{176}C and
60,
were
and
rinsed
Eragny,
tmol/L
animals. weighing
A03)
taken
medulla,
ascending
cortex
outer
(DTL)
inner
the RNA PLUS method (Bioprobe, Montreuil sous Bois, France) derived from the method Chomczynski and Sacchi (24). The RNA pellets were dissolved in an appropriate volume of diethylpyrocarbonate-treated water to obtain RNA of 2-mm tubule/pA or of 2 Glom/Ml, and the samples were stored at -80#{176}Cuntil RT-PCR. RT-PCR. ATIA-R and ATIB-R mRNA were quantified as dewith
threitol
ad libitum.
Microdissection
along
They
thick
the
the
early
U of RNase inhibitor (Boehringer 0.5 smol/L reverse primer in an RT-mix containing 50 mmollL Tris-HC1 buffer (pH 8.3), 75 mmollL KC1, 3 mmolIL MgCI,, 2.5 mmol/L dNTP, and 10 mmolIL dithio-
calcium
for the care and use of laboratory rats (Iffa Credo, L’Arbresle, France)
Rationnelle,
in 40 l of yeast
purified
was conducted
from
limb
and
Microdissected
and
the RT reaction
were
descending
medulla
0fATIA Isolation.
RNA
Mannheim,
by Ang II in fresh nephron segments of either ATIA-R or AT1B-R mRNA.
procedures
convoluted
(IMCD).
nologies,
to the
and Methods
animal
(CCD),
sized from 2.5 munne leukemia
Animals All
cortex
proximal
proximal tubule taken from the (CPST) and from the outer stripe of the
to OSPST:
and
Localization
mm
Materials
(Glom):
1659
of the
thin
of the outer
(MTAL) the
pooled grams
of
mRNA was (1) to localize
undertaken
along
measuring
medulla;
equally
receptor
work
mRNA
outer
present
of AT1
induced high levels
stripe
medulla
(OSPST);
Mobilization
similar
distribution and regulation of could mediate different biolog-
the
are
in other
present
inner
from
express-
to the
pathways
As
part
of the cortex
medulla
medulla
within each renal zone (21). AT1A-R subtype expressed in the cortex and
stripes
localization
([Ca2]1) taming
tissue
In contrast
previously
subtypes
bower
tubular
fresh
1).
rays
outer
and Ca24
glomerulus
straight
50 to 100 glomeruli
We
inner both
been
II.
described.
medulla,
(10,1
kidney, type 1 receptors are predominant of AT1 -R mRNA along the nephron
the two subtype mRNA mRNA is the predominant outer
from
not
isolated:
(PCT);
medullary
The
phospho-
has
were
tubule
the dihydropyri-
channel
signaling
AT1B receptors, the different their mRNA suggest that they
and
but
activate
receptors.
B
profiles
A2 (15)
subtypes
done
or
A
receptors.
ATIB
established
Both
voltage-dependent
yet,
ical
and
phospholipase
is clearly
ATIB-R.
dine-sensitive
the
to both
D activation
reported
segments
these receptors are expressed is no clear evidence for different
ATIA and AT8
of [Ca2 was measured each
individually
Glom
] as described
or isolated
previously
tubule
onto a thin glass
(0.2
microscope
(26).
to 0.3
mm)
coverslip
After was
microtrans-
in I l
of
1660
Journal
of the American
Society
of Nephrology
Glom
PCT
CPST
OSPST
DTL
MTAL
CTAL
CCD
OMCD
MCD
ATIB AT1A 1. Representative
Figure into
two
fragments
taken from and CTAL,
the medullary thick ascending
cortex,
outer
basal
medium
Then,
blot showing Glom,
the
the ATIA and ATIB receptor glomerulus; PCT, proximal
by EcoRI).
medulla,
and
inner
medulla,
2 mmol/L
was
jellied
each
CaCl,
and 1% agarose
by cooling
and
microscope
sample
was
was
the
placed
continuously
(type IX).
for 2 mm on ice.
slide
with 5 mol/L Fura-2 1 h. For fluorescence
on the
superfused
stage
of an inverted
at a rate
of 0.8
ml/min
which could be In most experi-
ments,
for
II,
Fura-2-loaded
Gloni
wavelengths source,
antagonists, and 380
and
Munich,
Germany).
objective
(Nikon)
and
passed
through
nm (every (PTI The
tion
of Grynkiewicz
a 75-W
the
dissociation
L,,111,, R,,,,,,
and R,i,a
were
light
K,
respectively.
by external
calibration
several
findings
were
each
SD in a few instances). test
for unpaired
data
for
comparison
among
effecti
Statistical the
ye concentration
(IC)
were
model
(least-squares
mercially
nm and
delimited by an using the equa-
nephron
and
L,m,x,
concentrations were
by fitting criterion,
the data
Gauss
et al.
software
SEM (or assessed using
samples
±
and Scheffe’s The
ATJB-R
algorithm)
mRNA
three
a typical
pattern
of
the
expression
OSPST,
CTAL, of
AT1-R
MTAL, mRNA.
and Moderate
OMCD
in
(72
3).
to 94%
AT1B
P
was
0.005).
