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INTRODUCTION |
Kinin peptides are released from kininogen precursors by the
action of kallikreins in response to tissue injury (1). Kinins induce
smooth muscle contraction, vasodilation, increased vascular permeability, and pain (1). Kinins exert their effects through selective activation of two seven-transmembrane domain
(TMD)1 G protein-coupled
receptors (GPCRs), B1 and B2 (2-4). The
B2 receptor is constitutively expressed, mediating the
actions of intact kinins, bradykinin (BK) in rodents and Lys-BK or
kallidin in humans (2). In contrast, the B1 receptor is
expressed at very low levels in normal tissues in most animal species
but is induced under the influence of inflammation or exposure of
tissues to noxious stimuli, mediating the effects of the
carboxypeptidase metabolites of intact kinins, des-Arg9-BK
(DABK), and des-Arg10-kallidin (2). The cellular responses
of kinin receptors to agonists are transduced primarily via coupling to
either Gq protein, which in turn activates phospholipase C
to stimulate inositol phosphate production, or the Gi
protein, acting through phospholipase A2 to stimulate
arachidonic acid pathway (5, 6).
Over the past years, transgenic and gene-targeting technologies
associated with molecular biology tools have provided important knowledge concerning the role of kinin receptors in vivo.
Transgenic mice expressing the human B2 receptor under the
control of the Rous sarcoma virus 3'-long terminal repeat promoter were
hypotensive compared with control littermates (7). Administration of
the B2 receptor antagonist Hoe-140 blunted the blood
pressure-lowering effect of the transgene, whereas intra-arterial bolus
injection of BK produced more pronounced blood pressure reduction (7). In contrast, deletion of the B2 receptor in mice produced
an unaltered blood pressure phenotype (8) but led to salt-sensitive
hypertension and altered nociception (9, 10). Using specific
antagonists, the B1 receptor has been implicated in toxic
shock, inflammation, and nociception (11). Studies of mice lacking the
B1 receptor provided support to these observations.
B1 receptor knockout animals were healthy, fertile, and
normotensive and exhibited hypoalgesia and reduced inflammatory
response (12).
Although much has been learned about the physiological role of the
B1 receptor, most studies are about the lipopolysaccharide (LPS)-induced B1 receptors, because the B1
receptor is expressed at very low levels, if at all, in normal tissue.
In such experimental set-ups, the animals are under systemic
inflammation conditions, which preclude the direct study of the
function of the B1 receptor. Therefore, the precise
physiological and pathophysiological roles of the B1
receptor remain elusive. To extend our understanding of the
physiological function of the B1 receptor, we have created a constitutively active mutant of the rat B1 receptor and
generated transgenic mice that overexpress the wild-type B1
receptor and the mutant receptor under the control of human
cytomegalovirus immediately early gene enhancer/promoter. The
transgenic mice were characterized, and the findings are reported here.
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EXPERIMENTAL PROCEDURES |
Materials--
Human embryonic kidney 293A cells were obtained
from Quantum Biotechnologies. LPS (Salmonella
enteritidis, LD50: 7.20 mg/kg) was from Difco
Laboratories. myo-[3H]Inositol was from
PerkinElmer Life Sciences. LipofectAMINE, culture medium, restriction
enzymes, and fetal calf serum were bought from Invitrogen. Puromycin
was from Clontech Lab. Hoe-140 was obtained from
Hoechst. Sar-Tyr-
Ahx-Lys-des-Arg9-bradykinin and
Sar-Tyr-
Ahx-Lys-[D-
Nal7,Ile8]-des-Arg9-bradykinin
were gifts from Dr. D. Regoli (13). All other chemicals were purchased
from Sigma unless stated otherwise.
Site-directed Mutagenesis--
The mutations were created by
using the QuikChange site-directed mutagenesis kit (Stratagene), and
the previously cloned wild-type rat B1 cDNA in
pcDNA3 (Invitrogen) was used as a template (14). The following
oligonucleotides were used as forward primers: 5'-GGC CTC TTG GGG
GCC CTT TTA GTC TTG TC-3' for the preparation of clone N54A
(nucleotide changes underlined), 5'-GTC ATC AAG GCC GCC CTG TTT GTC AG-3' for clone N130A, 5'-GCT ATC AGT CAG
CAA CGC TAC AGG CTC-3' for clone D144Q, and
5'-GCT ATC AGT CAG ACC CGC TAC AGG CTC-3' for clone D144T.
The reverse primers were complementary to the forward primers
described. All of the mutations were confirmed by DNA sequencing. The
NruI/NotI fragments in the resultant plasmids were released and inserted at NruI/NotI sites in
pIRESpuro (Clontech Lab), leading to vectors for
stable transfection.
Transfection and Selection--
For transient transfection, 293A
cells were seeded into 12-well trays and left to adhere overnight.
pcDNA3-derived expression vectors were transfected with LipofectAMINE
as previously described (14). Transfection mixtures were left on cells
for 5 h, and then the cells were treated with a change of standard
growth medium for 48 h before functional studies.
For stable transfection, 293A cells were transfected with
pIRESpuro-derived expression vectors. After 16 h, the medium was changed with complete medium containing puromycin (2 µg/ml) to start
the selection of stably transfected cells. The medium was changed every
3 days, and after about 12 days, the colonies surviving selection were
lifted into 12-well plates, expanded with a maintenance concentration
of 2 µg/ml puromycin, and screened for ligand specific binding. All
of the stock cultures were kept under constant selection pressure of 2 µg/ml puromycin, whereas cells seeded in dishes/wells were maintained
without puromycin and used within 2-3 days.
Total Inositol Phosphate Measurement--
Monolayers of
transfected 293A cells grown in 12-well trays were labeled for 24 h with 2 µCi of [3H]inositol in 0.5 ml of inositol-free
Dulbecco's modified Eagles' medium supplemented with 0.05% bovine
serum albumin and penicillin/streptomycin. After equilibrated in
prewarmed Dulbecco's modified Eagles' medium containing 140 µg/ml
bacitracin, 100 µM Captopril, and 25 mM LiCl for 15-30 min, the cells were stimulated with various ligands at the
indicated concentrations for 20 min at 37 °C. The released total
inositol phosphates (IP) were isolated using Bio-Rad AG1-X8 anion
exchange columns (1-ml volume) and quantified as described (15,
16).
Radioligand Binding to Intact Cells--
Transfected 293A cell
monolayers in 12-well plates were washed twice with Dulbecco's
modified Eagles' medium and incubated at 4 °C with radioligand
[125I]Sar-Tyr-
Ahx-Lys-des-Arg9-bradykinin
(14) in the presence or absence of 5 µM of the unlabeled ligand in 0.3 ml of Dulbecco's phosphate-buffered saline supplemented with 140 µg/ml bacitracin, 1 mg/ml bovine serum albumin, 1 mM 1,10-phenanthroline, and 100 µM Captopril.
The incubation lasted at least 3 h under gentle agitation. The
cells were then rinsed twice with ice-cold phosphate-buffered saline
with 0.3% bovine serum albumin followed by solubilization in 0.5 ml of
0.1 M NaOH. The radioactivity of the sample was quantified
with a 1261 Mutigamma counter (Pharmacia Corp.). The cell number was
determined in parallel wells.
cGMP and cAMP Assays--
Stably transfected 293A cells grown in
6-well plates were preincubated for 15 min with Dulbecco's modified
Eagles' medium containing 1 mM 3-isobutyl-1-methylxanthine
and 100 µM Captopril and then stimulated with 1 µM DABK for 15 min. The stimulation was terminated by
exchanging the incubation medium for 0.5 ml of ice-cold 0.1 M HCl. The cGMP and cAMP productions were determined by
radioimmunoassys as described (17, 18).
Construction of Wild-type and N130A Mutant Rat B1
Receptor Transgenes--
The bovine growth hormone poly(A) sequence in
pcDNA3 was released with ApaI and PvuII and
inserted at ApaI/EcoRV sites in pBluescript KS II
(Stratagene). The ApaI/SmaI bovine growth hormone poly(A) fragment was then released and inserted at
ApaI/SmaI sites in the pcDNA3-derived
wild-type and N130A receptor expression vectors described above. The
human 4F2 enhancer was amplified by PCR from genomic DNA with
the primers 5'-TAC TCG AGT GCA GCG CGC CCC CG-3' and 5'-CTG GGC CCT TCA
CCT TCA GAG AGC-3' (19). After being cut with XbaI and
ApaI, the 4F2 enhancer fragment was inserted at
XbaI/ApaI sites, resulting in final transgene vectors. The vectors were cut with NruI and SmaI,
and the linear transgenes were separated from the unneeded fragments
with agarose gel and prepared for injection by Qiaquick gel extraction
columns (Qiagen).
Generation of B1 Receptor Transgenic
Mice--
Transgenic mice were created by the Transgenic Facility at
the Medical University of South Carolina and the Transgenic
Facility of University of Ohio at Cincinnati. Linear
transgene was injected into the pronuclei of one-cell mouse embryos,
which were then surgically implanted into pseudopregnant female mice.
Transgenic founder mice were identified by Southern blot analysis of
genomic DNA isolated from tail biopsies. Positive founders identified from each line were bred with normal mice, and then F1 littermates were
crossed between themselves. Tail DNA was digested with restriction enzyme KpnI, run on 0.7% agarose gels containing ethidium
bromide, and transferred to Immobilon-N membrane by capillary action
with 10× SSC overnight, and the blots were hybridized to a rat kinin B1 receptor cDNA probe as described previously (14,
20).
Expression of B1 Receptor Transgene--
Total RNA
was extracted from mouse tissues using the RNeasyTM columns
(Qiagen). Reverse transcription-PCR/Southern blot analysis was
performed using the transgene-specific primers and internal probe as
previously described (21). The upstream primer is 5'-ATG GCG TCC GAG
GTC TT-3'; the downstream primer is 5'-GAC AAA CAC CAG ATC GG-3'; and
the internal probe is 5'-TGG CAG CAA CGA CAG AG-3'.
Membrane Preparation and Radioligand Binding Assays--
The
mice were sacrificed by cervical dislocation. The kidney was removed,
and the wet weight was determined. The tissue was homogenized using a
Polytron in ~20 volumes of ice-cold 20 mM HEPES, pH 7.4. The membranes from the tissue were prepared, and binding assays were
performed as described previously (7, 14).
Blood Pressure Measurements by Tail Cuff--
Systolic blood
pressure was measured using a computer system RTBP2000 (Kent
Scientific) according to the manufacturer's instruction. Briefly, the
mice were placed into the prewarmed harness (38 °C). The tail was
placed in the occlusion cuff/piezoelectric pulse sensor and distention
caused by arterial blood pulses was detected by the sensor and read out
onto the computer system. Pressure in the cuff was increased until the
pulse was lost. Actual blood pressure was measured as the pressure at
which a pulse was detected during cuff depressurization. Ten readings
were taken for each animal.
Blood Pressure Measurements by Arterial Cannulation--
The
mice were initially anesthetized with 2,2,2-tribromoethanol in
tert-amyl alcohol (Avertin, 20 mg/ml, 0.4 ml/25 g of body weight) and placed on a heated table to maintain body temperature. The
right jugular vein and the left carotid artery were cannulated with
PE-l0 catheters (Clay Adams). After the animals were allowed to
recover, blood pressure (in the carotid artery) and heart rate were
recorded using a computer system MP100 (Biopac systems Inc). The mice
were given a bolus injection of BK or DABK from right jugular vein. BK
or DABK was serially diluted and administered at doses of 75, 150, and
300 ng in a volume of 50 µl of saline/mouse.
Paw Edema--
Inflammation of one hind paw of mice was induced
by intraplantar injection of 20 µl of 1% carrageenan (dissolved in
saline), 3 µg of capsaicin in 10 µl (dissolved in 5% ethanol, 5%
Tween 80 and 90% saline), or DABK (50 or 300 nmol in saline), whereas the contralateral paw received the same volume of vehicle. Thirty min
post injection of capsaicin and DABK or 3 h after carrageenan administration, the mice were sacrificed, both hind paws were cut off
at the ankle, and the difference between their weights, representing
paw edema, was calculated.
Response to Endotoxic Shock--
LPS was dissolved in sterile
0.9% NaCl. The mice were injected intraperitoneally with a single dose
of LPS (24 mg/kg body weight), and the percentage of survivors was
determined at 12-h intervals. Both of the control groups were injected
with 0.9% NaCl.
Statistical Analysis--
The group data are expressed as the
means ± S.E. The data were compared between experimental groups
by one-way analysis of variance. Differences between groups were
further evaluated by Fisher's protected least squares differences.
Differences were considered significant at a value of p < 0.05.
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RESULTS |
Generation and Characterization of Rat B1 Receptor
Mutants--
To generate the constitutively active mutants of
B1 receptors, site-directed mutagenesis was directed at the
Asn54, Asn130, and Asp144 residues
in the rat B1 receptor (Fig.
1). The amino acid replacements were
N54A, N130A, D144Q, and D144T. The receptor expression vectors were
transiently transfected into 293A cells for assessing constitutive activity by determining agonist-independent IP production. At optimal
transfection conditions, all of the mutants were expressed at a
comparable level but significantly lower than the wild-type receptor
(data not shown). As shown in Table I,
the wild-type B1 receptor displayed a marked
ligand-independent, spontaneous activity (104% above control levels of
the mock-transfected cells), and the N54A mutant showed an impaired
basal activity compared with the wild-type receptor. Substitution of
Ala for Asn130 resulted in significantly constitutive
activation of the rat B1 receptor (409% above control
levels). In contrast, the agonist-independent IP accumulations in 293A
cells expressing D144Q and D144T mutants were similar to that of mock
transfected cells, and the constitutive activity of the wild-type
receptor was abolished by the mutations. Interestingly, the maximal
extent of DABK stimulation of IP production was significantly reduced
for all mutants compared with the wild-type receptor.

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Fig. 1.
Top panel, seven-TMD topography of the
rat kinin B1 receptor. Bottom panel, amino acids
assigned to the different receptor domains. The numbering of the amino
acid residues starts with the first N-terminal methionine as 1, and the
number refers to the last residue in each line. The seven putative TMDs
are underlined. The amino acids mutated in the study are
indicated with an asterisk.
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Table I
IP accumulation in 293A cells expressing wild-type or mutant
receptors
Rmax indicates the percentage increase in the 1 µM DABK-induced IP accumulation above basal levels in the
absence of DABK. Basal IP indicates the percentage of increase in IP
concentrations over those of mock-transfected cells (control) in the
absence of DABK. The expression levels of the receptors were comparable
except for the wild type (WT) with a higher level of expression. The
results are the means ± S.E. of three independent experiments,
each performed in duplicate.
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To better characterize the N130A mutant receptor, stable expression of
the N130A mutant in 293A cells was established. For comparison, stable
expression of the wild-type rat B1 receptor in 293A cells
was also established. Such stably transfected 293A cells were analyzed
for receptor density and their affinity for radioligand
[125I]Sar-Tyr-
Ahx-Lys-des-Arg9-bradykinin
and used in functional assays. The N130A receptor was expressed at a
level of about 25% of the wild-type receptor in the stably transfected
293A cells (Table II). The maximum
number of N130A receptor-binding sites
(Bmax) using
[125I]Sar-Tyr-
Ahx-Lys-des-Arg9-bradykinin
is 1.4 × 105 sites/cell versus 5.3 × 105 sites/cell for the wild-type receptor. In contrast, the
mutation of Asn130 into Ala significantly increased the
affinity for
[125I]Sar-Tyr-
Ahx-Lys-des-Arg9-bradykinin.
The dissociation constant (Kd) for the binding of
[125I]Sar-Tyr-
Ahx-Lys-des-Arg9-bradykinin
by the wild-type and N130A receptors in intact stably transfected 293A
cells were 2.54 ± 0.40 and 1.63 ± 0.18 nM,
respectively.
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Table II
Characterization of the wild-type and N130A mutant receptors
293A cells stably expressing the wild-type (WT) and the N130A mutant
(N130A) rat kinin B1 receptor were established, and the
receptor binding sites (Bmax) and dissociation
constant (Kd) were determined by using radioligand
[125I]Sar-Tyr- Ahx-Lys-des-Arg9-bradykinin as
described under "Experimental Procedures." The basal (Basal) and 1 µM DABK-stimulated (Maximal) intracellular cAMP and cGMP
levels were also measured. The results are the means ± S.E. of
three experiments.
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To assess the mode of coupling between the B1 receptor and
adenylate cyclase and guanylate cyclase and the effect of the mutation of Asn130 into Ala on the DABK-induced cAMP and cGMP
production, the intracellular cAMP and cGMP levels were measured in the
stably transfected 293A cells. As shown in Table II, the basal cAMP and
cGMP levels in the 293A cells stably expressing the wild-type receptor
were similar to those in the nontransfected 293A cells, whereas in the
293A cells stably expressing the N130A receptor, the basal cAMP and cGMP levels were elevated. DABK challenge increased intracellular cAMP
and cGMP production in the 293A cells stably expressing the wild-type
and N130A receptors: 116-fold increase in cAMP levels and 5.2-fold
increase in cGMP levels for the wild-type receptor versus
14-fold increase in cAMP levels and 1.7-fold increase in cGMP levels
for the N130A mutant.
Using the stably transfected 293A cells, the dose-dependent
DABK stimulation of IP production for the wild-type and N130A receptor
was investigated. As shown in Fig. 2, the
N130A mutant could be further activated by over 5-fold by saturation
doses of DABK, whereas the wild-type receptor could be amplified to a
even higher degree (~16-fold).

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Fig. 2.
Dose-dependent DABK stimulation
of IP production by the wild-type and N130A mutant B1
receptors. The stimulation of IP production by various
concentrations of DABK was analyzed in the 293A cells stably expressing
the wild-type (WT) or N130A mutant (N130A)
receptors (expression levels = 5.3 × 105 and
1.4 × 105 sites/cell, respectively) as described
under "Experimental Procedures." The results are representative of
three independent experiments, each performed in duplicate.
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The properties of some kinins to modulate IP production by the
wild-type and N130A receptors were evaluated. As shown in Fig. 3, kinin B2 receptor agonist
BK and antagonist Hoe-140 have no effects.
Des-Arg9,[Leu8]-bradykinin and
Sar-Tyr-
Ahx-Lys-[D-
Nal7,Ile8]-des-Arg9-bradykinin
are human B1 receptor-specific antagonists.
Sar-Tyr-
Ahx-Lys-[D-
Nal7,Ile8]-des-Arg9-bradykinin
is still an antagonist for the wild-type rat B1 receptor, whereas des-Arg9,[Leu8]-braykinin becomes a
partial agonist (70-80% of DABK), which provides support for the
early observation that
des-Arg9,[Leu8]-bradykinin has partial
agonist activity in a contraction assay of smooth muscle of rat
duodenum and ileum (22, 23). In contrast, Sar-Tyr-
Ahx-Lys-[D-
Nal7,Ile8]-des-Arg9-bradykinin
becomes a partial agonist, and
des-Arg9,[Leu8]-BK becomes a potent agonist
for the N130A receptor.

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Fig. 3.
Effects of various kinins on IP production by
the wild-type and N130A mutant B1 receptors. The
effects of kinin peptides (100 nM) on IP production were
analyzed in the 293A cells stably expressing the wild-type
(WT) or N130A mutant (N130A) receptors
(expression levels = 5.3 × 105 and 1.4 × 105 sites/cell, respectively) as described under
"Experimental Procedures." IP production by various ligand
stimulations was expressed as the percentage of IP production by DABK
(100 nM) stimulation for the wild-type and N130A receptors,
respectively. The results are representative of three independent
experiments, each performed in duplicate. a,
Sar-Tyr- Ahx-Lys-des-Arg9-BK; b,
des-Arg9,[Leu8]-BK; c,
Sar-Tyr- Ahx-Lys-[D- Nal7,Ile8]-des-Arg9-BK.
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Generation of Transgenic Mice--
Using the wild-type and N130A
cDNAs, we have constructed two transgenes for development of
transgenic mice. The transgene consists of the cytomegalovirus
immediately early gene enhancer/promoter, the wild-type or N130A rat
B1 receptor cDNA, the human 4F2 enhancer, and the
bovine growth hormone poly(A) sequence (Fig.
4). Three transmitting founder lines,
including one wild-type line, WT2510, and two N130A lines, N130A58 and
N130A2592, were identified by Southern blot analysis of genomic DNA.
Heterozygous wild-type and N130A transgenic mice showed no gross
phenotypic abnormalities. All of the transgenic mice were fertile.
However, mating F1 generation heterozygotes of both N130A lines
produced smaller litters compared with the nontransgenic control and
WT2510 line, and a non-Mendelian ratio against N130A mice was observed
in the offspring. In the following studies heterozygous transgenic mice
were used.

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Fig. 4.
Rat kinin B1 receptor transgene
constructs. The open bar represents wild-type
(WT) or mutant N130A rat kinin B1 receptor
cDNA. pCMV denotes the human cytomegalovirus immediately
early gene enhancer/promoter, 4F2 represents the human 4F2
heavy-chain gene enhancer, and BGH polyA denotes the bovine
growth hormone gene polyadenylation sequence.
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Expression of B1 Receptor Transgene--
The
distribution of transgene mRNA expression in F1 and F2 generation
heterozygous mice was determined by reverse transcription-PCR/Southern blot analysis. As expected, both male and female transgenic mice showed
significant overexpression of the transgene mRNA in the aorta,
kidney, liver, heart, brain, and lung and in the prostate of males and
the uterus of females. Fig. 5 shows the
result from line N130A58. Using specific B1 receptor
radioligand
[125I]Sar-Tyr-
Ahx-Lys-des-Arg9-BK (14),
strong B1 receptor binding activity was detected in membranes prepared from the kidneys of all transgenic lines (data not
shown). In contrast, neither transgene mRNA expression nor B1 receptor (including endogenous) binding activity could
be detected in the corresponding tissues of the nontransgenic control
mice.

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Fig. 5.
Expression of N130A mutant mRNA in
transgenic mice. Reverse transcription-PCR Southern blot revealed
the expression of mutant N130A receptor mRNA in mouse tissues. 2 µg of total RNA was reversely transcribed, and one-twentieth of the
resultant cDNA was then subjected to PCR for 30 cycles, followed by
Southern blot analysis. + represents N130A58 mice, and represents nontransgenic mice. F indicates female, and
M indicates male.
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Blood Pressure--
All of the heterozygous transgenic mice were
normotensive. The systolic blood pressures of transgenic mice 10 weeks
old were 78.7 ± 8.5 mmHg (n = 11) for line
WT2510, 75.0 ± 7.6 mmHg (n = 14) for line
N130A58, and 76.2 ± 6.3 mmHg (n = 11) for line
N130A2592 versus 80.9 ± 3.9 mmHg (n = 11) for age-matched nontransgenic control littermates. Intravenous
injection of B1 receptor agonist DABK via the jugular vein
produced a transient increase of mean arterial blood pressure (MABP) in
anesthetized transgenic mice but not in nontransgenic control
littermates (Fig. 6A). The
duration of MABP increase lasted over 5 min. 75 ng of DABK led to an
increase of blood pressure by up to 15 mmHg. In contrast, intravenous
injection of B2 receptor agonist BK into transgenic mice
caused a remarkable primary MABP reduction, followed by a blood
pressure bounce-back going beyond basal level, whereas in nontransgenic
mice the blood pressure just returned to basal level, after a similar
primary MABP reduction (Fig. 6B). Single or subsequent
injections of higher doses of DABK did not result in a further increase
of blood pressure in transgenic mice (data not shown).

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Fig. 6.
Effects of DABK and BK on the MABP of
mice. Typical tracing of the blood pressure response to 75 ng of
DABK (A) or 300 ng of BK (B) in anesthetized mice
is shown. Kinins were injected intravenously at time 0. NT,
nontransgenic mice.
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Inflammation--
Intraplantar injection of carrageenan resulted
in a marked inflammation seen by paw swelling in normal and transgenic
mice (Fig. 7). But the paw edema induced
in transgenic mice was more severe. The percentage of the weight
increase of the carrageenan-injected paw over the contralateral
vehicle-injected paw was 38.4 ± 6.7% for line WT2510, 42.1 ± 5.0% for line N130A58, and 37.4 ± 6.5% for line N130A2592
versus only 21.7 ± 5.8% for nontransgenic control mice. However, there was no significant difference in paw weight increase between transgenic mice and nontransgenic mice after induction
by either capsaicin or DABK (data not shown). We then evaluated the
response of transgenic mice to the lethal effects of endotoxic shock.
To this end, the mice were injected with a high dose of LPS (24 mg/kg
of body weight). Fig. 8 shows the
percentage of survivors after LPS injection. Within the first 36 h
about 82% of transgenic mice from lines WT2510 and N130A58, but only 45% of nontransgenic mice died. After 3 days, the mortality rate of
WT2510 and N130A58 mice reached 87 and 99%, respectively, whereas nontransgenic mice reached only 76%. In contrast, mock injection of
vehicle did not cause death in either group.

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Fig. 7.
Carrageenan-induced paw edema in mice.
Transgenic and nontransgenic control mice received intraplantar
injection of the carrageenan suspension into one hind paw and received
the vehicle into the other paw. After 3 h, the animals were
sacrificed, both hind paws were cut off at the ankle, and the
difference between their weights was determined. NT,
nontransgenic mice. (For nontransgenic mice versus three
transgenic lines, p < 0.05.)
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Fig. 8.
Survival rate of mice after endotoxic
shock. Transgenic and their nontransgenic control mice were
subjected to LPS injection (24 mg/kg, intraperitoneally). The
percentage of surviving mice was determined at 12-h intervals. (For
line WT2510, n = 53; for line N130A58,
n = 80; for nontransgenic mice (NT),
n = 76.)
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DISCUSSION |
In the absence of agonist, GPCRs spontaneously isomerize between
the inactive and active conformations, with the equilibrium shifted
toward the predominantly inactive conformation (24, 25). Some receptor
mutations induce an agonist-independent shift in isomerization
equilibrium toward the active conformation and evoke second messenger
responses in the absence of agonist. Such constitutive activations of
GPCRs are generally believed to result from an increase in receptor
conformational flexibility caused by the loss of intramolecular
constraints (26). Our present study shows that the wild-type rat kinin
B1 receptor displayed a marked constitutive activity in the
transiently or stably transfected 293A cells. During the preparation of
this manuscript, Leeb-Lundberg et al. (27) reported that the
human counterpart B1 receptor also exhibited a high level
of constitutive activity in transiently transfected 293 cells and so
did the rabbit wild-type B1 receptor, suggesting that a
high constitutive activity might be a common characteristic of the
B1 receptor.
Using single amino acid replacements based on findings from other
GPCRs, we generated four mutants of rat B1 receptors.
Asn54 in TMD I is highly conserved in the superfamily of
seven-TMD GPCRs (28). It was thought to be part of a highly conserved transmembrane "polar pocket," involved in receptor activation (29).
Substitution of the homologous Asn residue with Ala induced a moderate
constitutive activation of the
1B-adrenergic receptor (29). However, in the case of the rat B1 receptor, similar
mutation did not result in enhancement of constitutive activation.
Asp144 is part of the highly conserved DRY motif (triplet
of amino acids: Asp-Arg-Tyr) located at the boundary of TMD III and the
second intracellular loop (Fig. 1). The DRY motif has played a pivotal role in the signal transduction pathway of GPCRs (30-32). Mutations of
the aspartate residue are reported to lead to constitutive activity for
some GPCRs (31-33). Similar mutation in the homologous position in the
rat B1 receptor failed to induce detectable constitutive activity. However, this result is not unprecedented. For some adrenergic and muscarinic receptors, mutations in the aspartate residue
of the DRY motif did not result in agonist-independent constitutive
activity (34, 35). Asn130 in the rat B1
receptor is 14 residues N-terminal to the DRY motif. Mutations in this
homologous position such as Cys128 in the
1B-adrenergic receptor (36), Cys116 in the
2-adrenergic receptor (37), and Asn111 in
the angiotensin AT1 receptor (38) led to constitutive
activity, suggesting that this particular amino acid position may
function as a switch that regulates transition between distinct
receptor conformations (37). Based on molecular modeling of the
AT1 receptor and their finding that mutation of
Asn111 to Ala led to constitutive activation of the
AT1 receptor, Bonnafous and co-workers (38) proposed that
Asp74, Asn111, and Trp253 were
involved in the AT1 receptor activation. They found that these residues were conserved in the kinin B2 receptor and
that mutation of Asn113 to Ala in the homologous position
resulted in a high constitutive activation of the human kinin
B2 receptor (39). Interestingly, these residues were also
conserved in the B1 receptor (i.e.
Asp93, Asn130, and Trp273 for the
rat B1 receptor). Here we show that mutation of the
homologous Asn130 to Ala in the rat B1 receptor
induced a marked constitutive activation. Leeb-Lundberg et
al. (27) reported that mutation of homologous Asn121
to Ala in the human B1 receptor also caused a further
increase in constitutive activity, indicating that this Asn is indeed
involved in constraining the B1 receptor in an inactive
state. Our findings suggest that the molecular events associated to
their activation processes are probably conserved between kinin
B1 and B2 receptors.
The basal IP production by the N130A receptor in the stably transfected
293A cells was about 3-fold higher than that by the wild-type receptor
in the stably transfected 293A cells. But if one considers that the
expression level of the N130A receptor is about 25% of that of the
wild-type B1 receptor and that increasing the expression of
receptors could increase signal to phospholipase C as demonstrated by
other researchers with other GPCRs (27, 31, 39), the N130A receptor
would have a up to 12-fold increase in basal activity compared with the
wild-type receptor.
Constitutively active mutants have been shown to be responsible for
several hereditary and acquired diseases and have been used to produce
transgenic mice serving as unique experimental models (40). Because the
wild-type rat B1 receptor has a high basal activity and the
N130A mutant is highly constitutively active, we expect that
overexpressing these receptors in mice would allow the emergence of any
pathophysiological consequences associated with B1
receptors. B1 receptor-mediated hypotensive responses have
been documented in rabbit, rat, pig, and dog (11).
Unexpectedly, we found that all three B1 receptor
transgenic lines were normotensive. One explanation for this finding is
that some strong unknown compensatory in vivo mechanisms
exist and that any such compensation might more likely dilute the
manifestation of altered blood pressure phenotype. Alternatively, the
B1 receptor may not be as important in the normal
modulation of hemodynamics, which is compatible with the observation
that B1-deficient mice are normotensive (12). It should be
noted that the B1 receptor-mediated hypotensive effect is
generally only observed following an inflammatory stimulus, such as
endotoxin treatment (11).
Surprisingly, intravenous administration of DABK produced a MABP
increase in transgenic mice, and a subsequent injection of DABK was
almost as potent as the first injection, which can be explained by the
lack of desensitization of B1 receptors (41). Intravenous
injection of BK initially caused blood pressure reduction in transgenic
mice, and then the blood pressure bounced back and went beyond the
basal level, whereas in the control littermates BK only produced a
transient blood pressure reduction. This discrepancy could be due to
the conversion of some of the injected BK by kininase I into DABK
in vivo (42), which then acted on the constitutively expressed B1 receptor, causing a blood pressure increase in
transgenic mice. B1 receptor ligands have been reported to
cause vasoconstriction of a range of blood vessels from several species
(43). The DABK-mediated hypertensive effect in B1 receptor
transgenic mice probably resulted from B1 receptor-induced
peripheral resistance. However, the central role of B1
receptors in hypertension cannot be ruled out at this time. Alvarez
et al. (44) reported that brain B1 receptor
blockade lowers blood pressure in spontaneously hypertensive rats but
not in normotensive rats. Similarly, we demonstrated that
intracerebroventricular administration of B1 receptor
agonists increases blood pressure in both Wistar Kyoto rats and
spontaneously hypertensive rats, whereas B1 receptor
blockade with antisense oligonucleotides reduced blood pressure in
spontaneously hypertensive rats but not in Wistar Kyoto rats (45).
In B1 receptor transgenic mice, we found that
carrageenan-induced paw edema was significantly enhanced compared with
that in nontransgenic control littermates. It was reported that
carrageenan, a water-extractable polysaccharide obtained from various
seaweeds, could activate kinin release and induced the B1
receptor (46, 47). This enhancement in B1 receptor
transgenic mice is probably because released kinins could immediately
activate the constitutively expressed B1 receptors without
time delay for the B1 receptor induction as in
nontransgenic control. Overexpression of B1 receptors appears to have no significant effects on DABK or capsaicin-induced acute edema. However, this finding is in line with the observation that
the acute edema produced by kinins injection into rat paw showed
mediation by a form of B2 receptor, without a significant involvement of B1 receptors (48), even if the animal was
pretreated with LPS (49). Compared with nontransgenic control mice,
line WT2510 was prone to endotoxic shock, but N130A lines were more susceptible. The reason is not clear for the time being. But these findings clearly support the notion that B1 receptors play
an important role in modulating inflammatory responses.