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Published as doi: 10.1096/fj.06-7175com.
 (The FASEB Journal. 2007;21:1689-1698.)
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Mice deficient for both kinin receptors are normotensive and protected from endotoxin-induced hypotension

Cécile Cayla*, Mihail Todiras*, Radu Iliescu*, Vera V. Saul*, Volkmar Gross*, Bernhard Pilz{dagger}, Guixuan Chai*, Vanessa F. Merino*,{dagger}, João B. Pesquero{ddagger}, Ovidiu C. Baltatu* and Michael Bader*,1

* Max-Delbrück-Center for Molecular Medicine (MDC) and

{dagger} Helios-Klinikum Berlin, Franz-Volhard-Clinic, Berlin-Buch, Germany; and

{ddagger} Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil

1Correspondence: Max-Delbrück-Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, D-13092 Berlin-Buch, Germany. E-mail: mbader{at}mdc-berlin.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Kinins play a central role in the modulation of cardiovascular function and in the pathophysiology of inflammation. These peptides mediate their effects by binding to two specific G-protein coupled receptors named B1 and B2. To evaluate the full functional relevance of the kallikrein-kinin system, we generated mice lacking both kinin receptors (B1B2–/–). Because of the close chromosomal position of both kinin receptor genes, B1B2–/– mice could not be obtained by simple breeding of the single knockout lines. Therefore, we inactivated the B1 receptor gene by homologous recombination in embryonic stem cells derived from B2-deficient animals. The B1B2–/– mice exhibited undetectable levels of mRNAs for both receptors and a lack of response to bradykinin (B2 agonist) and des-Arg9-bradykinin (B1 agonist), as attested by contractility studies with isolated smooth muscle tissues. B1B2–/– mice are healthy and fertile, and no sign of cardiac abnormality was detected. They are normotensive but exhibit a lower heart rate than controls. Furthermore, kinin receptor deficiency affects the pathogenesis of endotoxin-induced hypotension. While blood pressure decreased markedly in wild-type mice and B2–/– and moderately in B1–/– mice after bacterial lipopolysaccharide (LPS) injection, blood pressure remained unchanged in B1B2–/– mice. These results clearly demonstrate a pivotal role of kinins and their receptors in hypotension induced by endotoxemia in mice.—Cayla, C., Todiras, M., Iliescu, R., Saul, V. V., Gross, V., Pilz, B., Chai, G., Merino, V. F., Pesquero, J. B., Baltatu, O. C., Bader, M. Mice deficient for both kinin receptors are normotensive and protected from endotoxin-induced hypotension.


Key Words: bradykinin • blood pressure • endotoxemia


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
KININS ARE A FAMILY OF PEPTIDES that participate in the function of the cardiovascular system and play a central role in the pathophysiology of inflammation (1) . They are released in the blood and tissues from kininogens, large protein precursors, by the action of two serine proteases, plasma and tissue kallikrein. Kinins have numerous biological effects, including vasodilation, increase of vascular permeability, and leukocyte recruitment. These effects are mediated in mammals through the interaction with two pharmacologically distinct G-protein coupled receptors, named B1 and B2 (2 ,3) . The B2 receptor has a high affinity for bradykinin (BK) and Lys-bradykinin (Lys-BK), whereas metabolites lacking the C-terminal arginine residue (des-Arg9-BK (DABK) and Lys-des-Arg9-BK) are more potent ligands at the B1 receptor. The two receptors differ further in their expression pattern: the B2 receptor is broadly and constitutively expressed and can become desensitized, whereas the B1 receptor is weakly expressed in most tissues under basal conditions but strongly upregulated following inflammation. Both receptors play important roles during inflammation, tissue trauma, and injury; the constitutively expressed B2 receptor is thought to be preferentially involved in the early phase and the inducible B1 receptor in later phases of inflammation.

Parallel to antagonist studies, genetically deficient animals have been extremely helpful to study the role of endogenously produced kinins (4) . A rat strain deficient in the kinin precursor kininogen (Brown Norway Katholiek rats, BN-Ka) was first described (5) , and more recently, mice lacking one of the receptors (B1–/– and B2–/–) or tissue kallikrein (TK–/–) have been created (6 ,7) . These models, however, do not permit us to study the complete relevance of the kinin system because either only one receptor is deleted or kinins can still be generated, by plasma kallikrein in TK–/– mice and by hydrolysis of T-kininogen in BN-Ka rats (8) . Therefore, even if the role of the kinin system in blood pressure regulation and cardiac function has been extensively studied in these models, the full importance of kinins in basal and inflammatory conditions remains to be elucidated.

Even though some studies reported high blood pressure in B2–/– mice (9 10 11) , and a recent study reported a slight increase in blood pressure of mice treated with antagonists for both receptors (12) , in general, chronic blockade of kinin receptors and studies in knockout mice offered little support for an essential role of kinins in the maintenance of normal blood pressure (13 , 14) . The involvement of the kallikrein-kinin system in cardiac hypertrophy is also controversial as the absence of the B2 receptor has been reported to induce cardiac hypertrophy (10 , 11) or not (15 , 16) , depending on the age, the sex, and the strain of mice. Compensatory effects of B1 receptors in the B2–/– mice have been reported (9 , 17) and might bias the effect of B2 receptor absence. Also, TK–/– mice were initially reported to exhibit cardiac abnormalities (7) , but these abnormalities were not detected on a pure C57BL/6 background (15) .

In this context, generation of mice lacking the two kinin receptors was essential to study the consequences of the complete absence of kinin function in vivo. The close proximity of the genes encoding the two receptors on chromosome 12 (18) prevented until now the generation of the double knockout mice by breeding the single knockout animals already existing (6 , 19) . In this study, we generated B1 and B2 double knockout mice (B1B2–/–) by inactivating the B1-receptor gene in embryonic stem (ES) cells isolated from B2-deficient animals. The effect of the resulting kinin-receptor deficiency was investigated first with regard to blood pressure and cardiac morphology. Next, because both kinin receptors are involved in cardiovascular dysfunctions induced by endotoxemia, we evaluated the hypotensive response to endotoxins (bacterial lipopolysaccharides, LPS) in the double knockout model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of the B1 and B2 kinin-receptor-deficient mice
ES cells were isolated from B2–/– mice on a 129Sv genetic background. B2–/– blastocysts were flushed out from the uteri of 3.5-day postcoitum pregnant B2–/– female, seeded onto inactivated embryonic fibroblasts, and left unattended for 3 or 4 days in Dulbecco’s modified Eagle’s medium (DMEM), 15% FCS, 1,000 U/ml of LIF (ESGRO, Life Technologies, Gaithersburg, MD, USA) with supplements (20) . The inner cell mass outgrowths with an ES cell-like appearance were gently trypsinized. After 4–7 days of culture, ES colonies were again trypsinized and transferred into fresh feeder plates. After one and half months, a growing ES cell line was obtained and expanded.

The cells were transfected with a targeting vector generated by inserting the hygromycin resistance gene into the vector used to generate the B1 knockout mice (6) . The vector contains a 1.0-kb genomic fragment 5' of the B1-coding region and a 7.0-kb fragment 3' of the B1-coding region. The construct was linearized and transfected into B2–/– ES cells by electroporation (single pulse of 0.5 mF, 240 V). Gancyclovir- and hygromycin- double-resistant clones were selected after 10 days of treatment with hygromycin (75 µg/ml, Sigma, St. Louis, MO, USA) and gancyclovir (2 µM). Positive clones were microinjected into C57BL/6 blastocysts, which gave rise to a germ-line chimera with offspring heterozygous for the targeted mutation. Transmission of the mutant allele was determined by Southern blot analysis of tail DNA. Heterozygotes were mated to establish the B1B2–/– mice. Mice backcrossed 3 times to the C57BL/6 strain were used for the initial characterization of the model and animals on a pure C57BL/6 genetic background (8 times backcrossed) were employed for physiological measurements (blood pressure, endotoxemia). Care and handling of all mice were carried out according to the guidelines for the humane use of laboratory animals by the Max-Delbrück-Center for Molecular Medicine and approved by a local ethical committee.

Receptor expression
Expression of kinin receptor mRNAs was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and ribonuclease protection assay (RPA). Total RNAs were isolated from organs with the TRIzol reagent (Life Technologies) according to the manufacturer’s protocol. RT-PCR was performed with primers for the B1 receptor mRNA (sense in exon 1: 5'-CAGAAACCTCCTAAGACAGC-3', antisense in exon 2: 5'-ACACCAGATCAGAAGCTGCC-3') and primers for the B2 receptor mRNA (sense in exon 2: 5'-GAAGCTACTCGGGTTTCTGTC-3', antisense in exon 3: 5'-ACCTCTCCAAACACCCAGTC-3'). B1 and B2 mRNA expression was also determined by RPA using the RPA II kit (Ambion, Austin, TX) with total RNA (50 µg) extracted from animals with or without LPS treatment (6 h, 5 mg/kg). The antisense probes used for RPA were a 190-bp probe complementary to 150 bp of the B1-receptor mRNA and a 460-bp probe complementary to 370 bp of the B2-receptor mRNA.

Receptor function
The functionality of B1 and B2 receptors was assayed by testing the contractile response of smooth muscle strips (stomach fundus, ileum) to BK and DABK (Bachem, King of Prussia, PA, USA). Briefly, strips were extracted from mice and placed at 37°C in oxygenated (95% O2-5% CO2) modified Krebs buffer (in mM: 119 NaCl, 4.7 KCl, 1.2 MgSO4: 7 H2O, 2.5 CaCl2: 2 H2O, 1.2 KH2PO4, 25.0 NaHCO3, and 5.5 glucose). After a 60–90 min equilibration period at a resting tension of 1 g, experiments were initiated by checking the viability of the preparation by exposure to carbachol (Sigma) at 10–5 M. Kinin agonists were then applied, BK (10–6 M) or DABK (10–6 M), and the contractile responses were recorded with an isometric transducer (model TRI202P, Letica) connected to a computerized system for data acquisition and analysis (PowerLab, ADInstruments, Colorado Springs, CO, USA).

Blood pressure and heart rate measurements
Two methods were used to determine arterial blood pressures in conscious animals. One set of experiments was performed with mice equipped with telemetric blood pressure transmitters (TA11PA-C20, Data Sciences International, Warwick, RI, USA). Under isoflurane anesthesia, the catheter of the telemetric device was implanted in the carotid artery (21) . The mice were allowed at least 9 days of recovery. Thereafter, baseline values were continuously recorded for 7 days, and the last 3 days of this period were used for statistical analyses. After recording of the baseline values, the mice were given a high-salt diet, first 4% NaCl in the chow alone complemented after a week by 0.9% NaCl in the drinking water for 4 wk. After this treatment, mice were given even a higher NaCl diet for another 2 wk (8% NaCl chow). Measurements of blood pressure and heart rate were performed at weekly intervals. In another set of experiments, animals were anesthetized (ketamine 100 mg/kg+xylazine 10 mg/kg), and modified cannulas (PE-10) were inserted into the femoral artery and exteriorized at the animal’s neck. After recovery, blood pressure and heart rate were recorded with a transducer (MLT 1050 model) connected to a computer system for data acquisition and analysis (PowerLab, ADInstruments). After baseline recording, LPS (Escherichia coli, serotype 011:B4, Sigma) was injected intravenously (i.v.) (5 mg/kg), and arterial pressure was continuously recorded.

We also assessed the changes in mean arterial pressure in response to the vasodilators acetylcholine (Ach) and sodium nitroprusside (SNP) in freely moving animals. The catheters (4 mm length, 0.1 mm inner diameter, and 0.25 mm outer diameter coupled to 3-cm-long PE-10 tubes) were placed in the descendent part of the aortic arc through the left carotid artery. The substances were given in 1 µl per 10 g body wt at the following doses: 25, 50, 100, and 200 ng/kg for Ach and 5 and 10 µg/kg for SNP.

Echocardiography
Transthoracic echocardiography was performed using a 12-MHz transducer (Acuson Sequoia, Erlangen, Germany) in anesthetized mice (ketamine/xylazine). End diastolic and systolic left ventricular diameters, as well as septum thickness and posterior wall thickness were measured from M-mode images using leading-edge conventions. All parameters were measured over at least 3 consecutive cardiac cycles. From these parameters, fractional shortening was calculated as [(LV diameterdiastole–LV diametersystole)/LV diameterdiastole)] x 100. Left ventricular mass was calculated as [(septum thicknessdiastole+LV diameterdiastole+LV posterior wall thicknessdiastole)3)–(LV diameter diastole)3] x 1.05, where 1.05 is the specific gravity of myocardium (22) .

Heart weight
After sacrifice, the hearts were excised, washed in saline, blotted dry, and weighed. The left and right ventricles were separated and weighed.

Survival after LPS injection
B1B2–/– and wild-type male mice were injected with LPS (intraperitoneally (i.p.), 10 mg /kg) and survival was monitored every 12 h for 4 days.

Nitrite and nitrate measurement
Plasma samples were analyzed for nitrite (NO2–) and nitrate (NO3–) using chemiluminescence, as described previously (23) . Briefly, samples and standards containing NO2– and NO3– were first reduced to NO, which was then quantified after reaction with ozone using a NO analyzer (NOA 280, Sievers). To determine total NO2– and NO3– concentrations, collectively termed "NO," samples were added to 0.1 M vanadium (III) chloride in 1 M hydrochloric acid refluxing at 90°C under nitrogen.

Quantitative real-time PCR
Levels of inducible NOS (iNOS), eNOS, and COX2 mRNA were determined by real-time quantitative PCR using SYBR Green reagent (Qiagen). The following primers were used: iNOS (RV: 5'-CAT TGG AAG TGA AGC GTT TCG-3' and FW: 5'-CAG CTG GGC TGT ACA AAC CTT-3'), eNOS (RV: 5'-CCT TCC GCTACCAGCCAGA-3' and FW: 5'-CAG AGA TCT TCA CTG CAT TGG CTA-3'), COX2 (FW: 5'-ATC CTG CCA GCT CCA CCG-3', RV: 5'-TGG TCA AAT CCT GTG CTC ATA CAT-3'). A standard curve (serial dilution of cDNA) was run and conditions were chosen to obtain a linear curve (correlation coefficients>0.98 and slopes between –3.3 and 3.7). Quantitative PCR was performed with 20 ng of cDNA on a Bio-Rad detection instrument, starting with 15 min at 95°C followed by 40 cycles of 95°C for 15 s and 58/60°C for 1 min. After the PCR reaction, a melting curve analysis was run, and the sizes of all PCR products were confirmed on agarose gels. The mRNA expression for all probes was normalized to ß-actin mRNA expression. The comparative CT method of Livak and Schmittgen (24) was applied to compare gene expression levels between samples. The amplification threshold cycle values (CT) were obtained and data analyzed using the equation 2–{Delta}{Delta}CT, where {Delta}{Delta}CT = (CT target gene–CT actin)knockout – (CT target gene–CT actin)wild-type.

Statistics
Results are expressed as mean ± SEM. Tests of significance (PRISM, GraphPad, San Diego, CA) were conducted by using either Student’s t tests or two-way ANOVA for multiple comparisons followed by Bonferroni’s post test analysis. Survival data were analyzed by a log-rank test to compare Kaplan-Meier survival curves. Criterion for significant differences between groups was a probability value <0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of kinin-B1B2 receptor knockout mice
We generated the B1B2–/– mice by targeting the B1 receptor gene in ES cells isolated from the B2–/– mice and replacing exon 2 by the hygromycin-resistance gene (Fig. 1 A). Breeding of mice heterozygous for the genetic alteration led to wild-type, heterozygous, and homozygous mutant offspring at the expected Mendelian ratio (50 +/+, 118 +/–, 46 –/–). B1B2–/– mice weighed the same as wild-type and gained weight at the same rate. Mature B1B2–/– mice produced litters at normal frequency and size.


Figure 1
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  		Figure 1. Generation of kinin receptor-deficient mice, B1B2–/–. A) Targeting strategy. Schematic representation of the B1 and B2 gene locus, the targeting vector and the predicted map of the disrupted genes. The targeting construct to disrupt the B1 gene contains the hygromycin resistance (Hygr) and the thymidine kinase (TK) genes. Restriction enzyme sites (B, BamHI; E, EcoRI), the coding region of each gene, the position of the probes used for Southern blot and the lengths of the EcoRI restriction fragments (E-E) are indicated. B) Southern blot analysis of genomic DNA from mice of different genotypes digested with EcoRI. C) Detection of B1 and B2 mRNA by RT-PCR in indicated tissues of wild-type (+/+) but not in B1B2–/– mice. D) RPA with 50 µg total RNA detecting B2 mRNA in untreated mouse kidney and B1 mRNA in stomach and ileum from LPS-treated wild-type (+/+) but not B1B2–/– mice. P, probes used for RPA.

Receptor expression and function
The disruption of the two genes was confirmed by Southern blot (Fig. 1B ) and the absence of the receptors was shown at the mRNA level by RT-PCR and RPA in tissues that normally express the receptors (Fig. 1C, D ). Even after LPS induction, which markedly increased B1-mRNA levels in wild-type mice, there was no evidence for B1-receptor expression in B1B2–/– animals (Fig. 1D ).

Previous studies have indicated that the stimulation of the two receptors elicits a contractile response of smooth muscles of the gastrointestinal and urogenital tracts and contraction or relaxation of vascular smooth muscles (1) . Different preparations were examined regarding their response to the preferential B1 and B2 agonists, DABK and BK, respectively. In the stomach fundus preparations from wild-type mice, both agonists induced contractile effects (Fig. 2 A). These effects were absent in stomachs isolated from knockout mice, confirming the absence of the two kinin receptors. Testing the response to the muscarinic agonist carbachol assessed the viability of the preparation. In ileum strips (Fig. 2B ), the response to BK is already present just after isolation, while DABK-induced contraction increases as a function of the in vitro incubation time (25) . In ileum from B1B2–/– mice, the responses to both agonists were abolished. Furthermore, no other preparations from B1B2–/– mice (urinary bladder, uterus, aorta, and portal vein) responded to BK or DABK.


Figure 2
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  		Figure 2. Kinin-induced smooth muscle contraction. Isometric contractile responses of isolated strips from stomach fundus (A) and ileum (B) after 3 h of in vitro incubation from wild-type and B1B2–/– mice to BK and DABK (10–6 M each). Only the contractile response to carbachol (Carb., 10–5 M) is preserved in B1B2–/– mice.

Blood pressure
No difference was observed in mean, systolic and diastolic arterial pressures in B1B2–/– compared to wild-type mice (Table 1 ). Mean arterial pressure (MAP) measured by femoral artery catheterization being 103.8 ± 1.5 vs. 107.5 ± 2.4 mmHg for B1B2–/– and wild-type mice, respectively, and 111.7 ± 1.2 vs. 112.8 ± 1.6 mmHg for B1B2–/– and wild-type mice, respectively, when measured by telemetry. Heart rate of B1B2–/– mice measured by telemetry was slightly but significantly lower compared to controls (B1B2–/–: 596.1±10.7 vs. wild-type mice: 626.5±5.0, P=0.037). This bradycardia was also detected by catheterization of the femoral artery (Table 1) , but the values were not significantly different.


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  		Table 1. Blood pressure of B1B2–/– mice

Mice with telemetric blood pressure transmitters were put on a high salt diet for 5 wk (first 4% NaCl chow alone, complemented after a week by 0.9% NaCl in the drinking water) and then for 2 wk on an even higher salt diet (8% NaCl). These diets did not increase the blood pressure of either wild-type mice or B1B2–/– mice (Table 1) .

Cardiac morphology
Cardiac morphology as assessed by echocardiography appeared normal in 3-mo-old B1B2–/– animals and was therefore studied further in older male and female mice (15–18 mo). Interventricular septum, posterior wall thickness, left ventricular diameter, and mass were not different between groups (Table 2 ). Additionally, left and right ventricular weights, measured after sacrifice, were comparable in the two groups (Table 2) . The overall function of the left ventricle determined by echocardiography was not different between B1B2–/– mice and controls, evidenced by similar fractional shortening values.


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  		Table 2. Cardiac structural features of B1B2–/– mice

Endotoxic shock
To study the importance of kinin receptors in the pathophysiology of endotoxic shock, LPS (5 mg/kg, i.v.) was administered to conscious mice instrumented with arterial catheters for blood pressure monitoring. In wild-type mice, two phases characterized the blood pressure drop induced by i.v. injection of LPS: a first phase lasting 20–30 min with a rapid fall of blood pressure (29.5±9.8 mmHg drop 10 min after LPS injection, P<0.01) followed by recovery, and a second phase, with a more progressive and sustained fall in blood pressure (Fig. 3 A). Three hours after LPS injection, blood pressure fell by 21.3 ± 1.9 mmHg, P < 0.01. In contrast, the hypotensive response to LPS injection in B1B2–/– mice was completely abolished; at no time point after LPS injection was blood pressure statistically significantly different from baseline. The hypotensive response to LPS was also assessed in the single kinin receptor knockout lines on the same genetic background (C57BL/6). In B2–/– mice, the response to LPS tended to be rather increased compared to controls, although the difference did not reach statistical significance. B1–/– mice presented an attenuated response to LPS but contrarily to B1B2–/– mice, the protection was not complete.


Figure 3
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  		Figure 3. Role of kinin receptors in endotoxemia. A) Blood pressure after intravenous injection of 5 mg/kg LPS in 3- to 4-mo-old male wild-type (+/+), B1–/–, B2–/–, and B1B2–/– mice (n=6/group). Changes in mean arterial pressure ({Delta}MAP vs. baseline) were monitored for 3 h following LPS injection in conscious freely moving mice by intra-arterial catheter. Data are mean ± SEM and were analyzed by two-way ANOVA repeated measures followed by Bonferroni’s post hoc test.*P < 0.05; **P < 0.01; ***P < 0.001 vs. wild-type, ###P < 0.001 B1B2–/– vs. B1–/–. B) Kaplan-Meier survival curves for 3- to 4-mo-old male wild-type (+/+) and B1B2–/– (n=15) mice after intraperitoneal injection of 10 mg/kg LPS. Results are shown as a percentage of surviving mice determined at 12-h intervals for 4 days.

Next, we evaluated the response of B1B2–/– mice to a lethal dose of LPS. Intraperitoneal injection of LPS (10 mg/kg) in control mice induced 87.5% mortality after 36 h. Kinin receptor deficiency did not improve survival, as LPS injection resulted in 86.6% mortality at 36 h in B1B2–/– mice (Fig. 3B ).

To understand the potential mechanisms of the lack of a blood pressure drop in B1B2–/–, we measured the plasma levels of NO in wild-type and B1B2–/– mice. As shown in Fig. 4 A, 3 h after LPS injection, NO plasma levels of B1B2–/– are as high as in control mice. Because NO levels are comparable, we tested the aptitude of the mice in response to NO. The hypotensive response to the endothelium-independent NO donor, SNP was assessed (Fig. 4B ), as well as the response to the endothelium-dependent vasodilator Ach. The hypotensive responses to SNP and Ach were similar in both B1B2–/– and wild-type mice (Fig. 4) , suggesting that the deficient response to LPS is not due to an altered response to NO. Tissue mRNA levels of inducible NO synthase (iNOS), endothelial NO synthase (eNOS), and of the inducible cyclooxygenase (COX2) were quantified by real-time PCR in the lung of animals subjected to LPS. Whereas eNOS and COX2 mRNA levels are unchanged, iNOS mRNA levels are lower in the B1B2–/– mice in comparison to controls, suggesting local changes of NO production (Fig. 4C ).


Figure 4
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  		Figure 4. NO system and endothelial function in B1B2–/– mice. A) NOx measurements in plasma of LPS-treated (5 mg/kg for 3 h) wild-type (+/+) and B1B2–/– (n=5) mice. Data are mean ± SEM and were analyzed by Student’s t test (B) Effect of SNP and Ach on mean arterial blood pressure (MAP) in wild-type (+/+) and B1B2–/– mice (n=4). Changes in mean arterial pressure ({Delta}MAP) are calculated between the maximal hypotensive responses to each dose of SNP or Ach vs. the baseline value. Data are expressed as mean ± SEM and were analyzed by two-way ANOVA repeated measures. C) The mRNA levels for iNOS, eNOS, and COX2 in lungs of LPS (5 mg/kg for 3 h) treated mice were quantified by real-time PCR. The data are expressed as a fold increase above control (wild-type-treated LPS) normalized to ß-actin. Data are expressed as mean ± SEM (n=7) and were analyzed by Student’s t test. **P < 0.05 B1B2–/– vs. wild-type


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study shows that mice lacking both kinin receptors, B1 and B2, have normal blood pressure and cardiac morphology and are protected against endotoxin-induced hypotension.

B1B2–/– mice represent the first mouse model not able to respond to kinins by lacking both kinin receptors. Even in the TK–/– mice, other enzymes like plasma kallikrein will produce kinins especially under inflammatory conditions (1) . In addition, the existence of two genes encoding kininogens in mice (26) will delay the generation of kininogen-free mice. The effective elimination of B1 and B2 receptors in our model was demonstrated both in terms of mRNA expression and classical contractile responses to BK and DABK. The B1B2–/– mice are healthy, have a normal gross morphology, are fertile, and produce usual litter sizes. Interestingly, a role of kinins in reproduction has been discussed (27 ,28) ; however, like B2–/–, B1–/–, and TK–/– mice, the B1B2–/– model shows that kinins are dispensable for development and reproduction, at least of mice.

B1B2–/– mice are normotensive (Table 1) , as assessed by two different methods of blood pressure measurement. Furthermore, no differences in day/night rhythm of cardiovascular parameters were found (data not shown). However, B1B2–/– mice have a lower heart rate, which is coherent with the suppression of the known central or peripheral effects of BK. Indeed, BK induces tachycardia (29) and enhances norepinephrine release via a presynaptic B2 receptor dependent mechanism (30 , 31) . The subtle bradycardia is in accordance with the study of Trabold et al. in B2–/– mice (15) but contrasts with reports showing either normal (14 , 21 , 32) or increased heart rate values in B2–/– mice (9 , 10) . The reduced heart rate in B1B2 –/– mice could also be the consequence of an increase in vascular resistance due to the dilatory actions of BK in resistance vessels. Our results indicate either that the kinin system does not play a major role in the control of blood pressure under basal conditions or that counter-regulatory systems have been set up. In this respect, we measured the activity of the renin-angiotensin system in the plasma of B1B2–/– mice but did not detect any difference to control animals (data not shown).

Double knockout B1B2–/– mice display normal echocardiographic parameters and ventricular weights at all ages assessed (Table 2) . This is in agreement with two studies on B2–/– mice (15) but contrast with other studies showing that B2–/– male mice display cardiac hypertrophy at 3 or 12 mo of age in 129J or C57BL/6 background, respectively (10 ,11) . These discrepancies are not entirely understood, and it would be interesting to assess the cardiovascular parameters of B1B2–/– mice on the 129J genetic background, as this background seems to be more capable of unraveling kinins’ cardiac functions (11) .

Kinins are known to increase renal blood flow and excretion of water and sodium from the kidney due to activation of the B2 receptor (33) . In B1B2–/– mice, the gross kidney morphology, water intake, urine excretion, and sodium excretion, as well as urinary osmolarity were assessed but did not differ from wild-type (data not shown). These parameters are also unchanged in B2–/– mice (34) . Further studies must be performed to assess renal function in more details (e.g., effect of high salt load during pregnancy). In rats, studies on the BN-Ka strain indicated that the renal kallikrein-kinin system may play a role in the sodium and water excretion mainly when excess amounts of sodium are given to the animals (35) . B1B2–/– mice, however, did not present a significant change in blood pressure after an increase in dietary salt intake.

A major finding of this study is that mice deficient in both B1 and B2 kinin receptors are substantially protected from hypotension induced by LPS, a cardinal feature of endotoxic shock. There is strong evidence that kinins are involved in the cardiovascular dysfunctions induced by endotoxemia. The activation of the kallikrein-kinin system has been documented by the consumption of plasma prekallikrein and high MW kininogen (36) and by the detection of kinin generation in endotoxemic animals (37) . The early cardiovascular changes accompanying endotoxic shock include a fall in blood pressure. Several studies have demonstrated a partial restoration of blood pressure following treatment with kinin antagonists and kininogen deficient BN-Ka rats also showed an attenuated hypotensive response to LPS (6 , 38 39 40) . Most of the acute inflammatory effects of kinins are thought to be mediated through the B2 subtype. The selective B2 antagonist, HOE140, has been shown to reduce LPS-induced hypotension (38 , 39) , however only partially. This contrasts with the sustained blood pressure fall we observed in the B2–/– mice (Fig. 3) . The reasons for these differences are unknown but may be due to an increased B1 receptor function in B2–/– mice as already shown in other studies (17) . B1 receptor expression is induced by endotoxins in several cell types, including vascular smooth muscle and endothelial cells (25) and it mediates vasodilation in the majority of vascular arterial beds (41) . Pharmacological blockade or knockout of the B1 receptor similarly to B2 blockade produce only a partial attenuation of LPS-induced hypotension (6 , 40) . In agreement with our present study, Whalley et al. showed that CP0127, a combined B1 and B2 antagonist blocks the development of hypotension in rats (42) . The B1B2–/– mice provide complementary evidence for the importance of the combined inhibition of both kinin receptors not based on pharmacological agents, which can have secondary targets. In endotoxemia, a proposed mechanism of plasma kallikrein-kinin system activation is that bacterial wall fractions activate prekallikrein through factor XII (43) and generate kinins from the precursor kininogens. This liberation of kinins results in vasodilation driven by the activation of both B1 and B2 receptors, thereby, potentiating the pathophysiological alterations associated with endotoxemia.

In wild-type mice, the profound hypotension induced by LPS may be elicited by NO and has been linked to changes in both inducible and constitutive NO pathways (44) . During endotoxemia, iNOS expression is upregulated and produces large amounts of NO. Animal models lacking this enzyme are protected from LPS-induced hypotension (45) showing that the hyperproduction of NO essentially contributes to the hypotension in endotoxic shock. Both, B1 and B2 receptors, can lead to NO generation in the vasculature (46 ,47) . The protection of the B1B2–/– mice against LPS-induced hypotension might therefore be related to an impairment of NO production or function; however, 3 h after LPS administration B1B2–/– plasma nitrate/nitrite concentrations, as a correlate for NO generation, were not found significantly different than the ones of wild-type mice (Fig. 4) . Moreover, the response of B1B2–/– to SNP and Ach were similar to wild-type mice, indicating that the protection of mice toward LPS-induced hypotension is not due to an impaired response to NO (Fig. 4B ). However, the iNOS mRNA level of the highly vascularized lung tissue was reduced in B1B2–/– by 63% compared to wild-type mice. In contrast, mRNA levels of eNOS and of the other major inflammatory enzyme, COX2, were unchanged. The reasons for the protection of B1B2–/– mice are therefore complex and will require further studies. It possibly involves an impairment of local production of NO due to a reduced iNOS expression, as well as other vasodilating mediators, such as endothelium-derived hyperpolarizing factor (17 , 48) , which might also account for some effects of LPS. Despite mRNA levels of eNOS in B1B2–/– being unchanged compared to wild-type, changes in enzymatic activity could be assumed. Interestingly, there is evidence showing that the B2 receptor increases eNOS activity through a complex set of molecular events in endothelial cells (47 , 49 , 50) . The functional status of eNOS following LPS administration is, however, not fully elucidated, some studies demonstrated a decrease in eNOS activity (51) and others an increase (52) . Additionally, because of the lack of receptors, it is possible that kinins produced during septicemia in the B1B2–/– mice will be more prone to degradation, raising the concentrations of a metabolic fragment of BK (BK1–5) that has been shown to protect against endotoxin-induced hypotension (53) .

Although the lack of the two receptors stabilized blood pressure in endotoxemia, this was not translated into improved survival. This raises the question of the mode of death in the B1B2–/–mice. Interestingly, the same observation was made for iNOS–/– mice (45) , and it indicates that the severe early hypotension (first 5 h) caused by LPS is not a major contributor to the mortality in endotoxic shock, at least in mice. Accordingly, treatment with 546C88, a non selective NOS inhibitor, despite improving mean arterial pressure, was associated with an increased mortality (54) . In fact, several clinical trials targeting single systems to treat septic shock, including inhibition of kinins (55) , NO, TNF-{alpha}, and prostaglandins, have yielded very little, if any, positive outcome. This indicates that tackling a single system does not seem to be successful in a complex and multifactorial inflammatory disease such as sepsis. Treatments of ongoing septic shock may require concurrent interruption of more than one pathway. In this context, the B2 receptor antagonist, NPC 17761, and a leukocyte recruitment inhibitor exerted synergistic effects on survival in animal models of endotoxemia (56) . Further studies with combined blockade of the two kinin receptors only or in association with other compounds might be very useful in the treatment of septic shock.

In conclusion, the double deficiency of the two kinin receptors, B1 and B2, has no effect on development, fertility, and long-term regulation of blood pressure in mice but protects them from the dramatic blood pressure fall induced by bacterial endotoxins. These results show the central role of the kallikrein-kinin system in endotoxic shock and may contribute to the understanding and treatment of this serious condition.


   ACKNOWLEDGMENTS
 
C.C. was supported by grants from the Institut National de la Santé de la Recherché Médicale/Deutsche Forschungsgemeinschaft and from Max-Delbrück-Center. The project was supported by Bundesministerium für Bildung und Forschung (01GR0104), VolkwagenStiftung, Deutscher Akademischer Austauschdienst (PROBRAL), and a joint grant of the ministries of science and technology of Germany and Brazil. We thank Dr. Ahluwalia for critical reading of the manuscript and Dr. Hobbs for his kind help to measure nitrate/nitrite.

Received for publication June 30, 2006. Accepted for publication December 25, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bhoola, K. D., Figueroa, C. D., Worthy, K. (1992) Bioregulation of kinins: Kallikreins, kininogens, and kininases. Pharmacol. Rev. 44,1-80[Medline]
  2. Regoli, D., Barabé, J. (1980) Pharmacology of bradykinin and related kinins. Pharmacol. Rev. 32,1-46[Medline]
  3. Leeb-Lundberg, L. M., Marceau, F., Muller-Esterl, W., Pettibone, D. J., Zuraw, B. L. (2005) International Union of Pharmacology. XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences. Pharmacol. Rev. 57,27-77[Abstract/Free Full Text]
  4. Pesquero, J. B., Bader, M. (2006) Genetically altered animal models in the kallikrein-kinin system. Biol. Chem. 387,119-126[CrossRef][Medline]
  5. Damas, J., Adam, A. (1979) The kallikrein-kininogens-kinins system in the Brown-Norway rat. Biomedicine 31,249[Medline]
  6. Pesquero, J. B., Araujo, R. C., Heppenstall, P. A., Stucky, C. L., Silva, J. A., Jr, Walther, T., Oliveira, S. M., Pesquero, J. L., Paiva, A. C., Calixto, J. B., et al (2000) Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors. Proc. Natl. Acad. Sci. U. S. A. 97,8140-8145[Abstract/Free Full Text]
  7. Meneton, P., Bloch-Faure, M., Hagege, A. A., Ruetten, H., Huang, W., Bergaya, S., Ceiler, D., Gehring, D., Martins, I., Salmon, G., et al (2001) Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice. Proc. Natl. Acad. Sci. U. S. A. 98,2634-2639[Abstract/Free Full Text]
  8. Hayashi, I., Oh-ishi, S., Kato, H., Enjyoji, K., Iwanaga, S., Nakano, T. (1985) Identification of T-kininogen in high and low molecular weight kininogens deficient rat (brown Norway Katholiek strain). Thromb. Res. 39,313-321[CrossRef][Medline]
  9. Madeddu, P., Varoni, M. V., Palomba, D., Emanueli, C., Demontis, M. P., Glorioso, N., Dessi Fulgheri, P., Sarzani, R., Anania, V. (1997) Cardiovascular phenotype of a mouse strain with disruption of bradykinin B2-receptor gene. Circulation 96,3570-3578[Abstract/Free Full Text]
  10. Emanueli, C., Maestri, R., Corradi, D., Marchioni, R., Minasi, A., Tozzi, M. G., Salis, M. B., Straino, S., Capogrossi, M. C., Olivetti, G., et al (1999) Dilated and failing cardiomyopathy in bradykinin B(2) receptor knockout mice. Circulation 100,2359-2365[Abstract/Free Full Text]
  11. Maestri, R., Milia, A. F., Salis, M. B., Graiani, G., Lagrasta, C., Monica, M., Corradi, D., Emanueli, C., Madeddu, P. (2003) Cardiac hypertrophy and microvascular deficit in kinin B2 receptor knockout mice. Hypertension 41,1151-1155[Abstract/Free Full Text]
  12. Duka, A., Duka, I., Gao, G., Shenouda, S., Gavras, I., Gavras, H. (2006) Role of bradykinin B1 and B2 receptors in normal blood pressure regulation. Am. J. Physiol. Endocrinol. Metab. 291,E268-E274[Abstract/Free Full Text]
  13. Bao, G., Gohlke, P., Unger, T. (1992) Chronic kinin receptor blockade attenuates the antihypertensive effect of ramipril. Hypertension 20,74-79[Abstract/Free Full Text]
  14. Yang, X. P., Liu, Y. H., Scicli, G. M., Webb, C. R., Carretero, O. A. (1997) Role of kinins in the cardioprotective effect of preconditioning: study of myocardial ischemia/reperfusion injury in B2 kinin receptor knockout mice and kininogen-deficient rats. Hypertension 30,735-740[Abstract/Free Full Text]
  15. Trabold, F., Pons, S., Hagege, A. A., Bloch-Faure, M., Alhenc-Gelas, F., Giudicelli, J. F., Richer-Giudicelli, C., Meneton, P. (2002) Cardiovascular phenotypes of kinin B2 receptor- and tissue kallikrein-deficient mice. Hypertension 40,90-95[Abstract/Free Full Text]
  16. Yang, X. P., Liu, Y. H., Mehta, D., Cavasin, M. A., Shesely, E., Xu, J., Liu, F., Carretero, O. A. (2001) Diminished cardioprotective response to inhibition of angiotensin-converting enzyme and angiotensin II type 1 receptor in B(2) kinin receptor gene knockout mice. Circ. Res. 88,1072-1079[Abstract/Free Full Text]
  17. Duka, I., Kintsurashvili, E., Gavras, I., Johns, C., Bresnahan, M., Gavras, H. (2001) Vasoactive potential of the b(1) bradykinin receptor in normotension and hypertension. Circ. Res. 88,275-281[Abstract/Free Full Text]
  18. Cayla, C., Merino, V. F., Cabrini, D. A., Silva, J. A., Jr, Pesquero, J. B., Bader, M. (2002) Structure of the mammalian kinin receptor gene locus. Int. Immunopharmacol. 2,1721-1727[CrossRef][Medline]
  19. Borkowski, J. A., Ransom, R. W., Seabrook, G. R., Trumbauer, M., Chen, H., Hill, R. G., Strader, C. D., Hess, J. F. (1995) Targeted disruption of a B2 bradykinin receptor gene in mice eliminates bradykinin action in smooth muscle and neurons. J. Biol. Chem. 270,13706-13710[Abstract/Free Full Text]
  20. Robertson, E. J. (1997) Derivation and maintenance of embryonic stem cell cultures. Methods. Mol. Biol. 75,173-184[Medline]
  21. Milia, A. F., Gross, V., Plehm, R., Silva, J. A., Jr, Bader, M., Luft, F. C. (2001) Normal blood pressure and renal function in mice lacking the bradykinin B(2) receptor. Hypertension 37,1473-1479[Abstract/Free Full Text]
  22. Tanaka, N., Dalton, N., Mao, L., Rockman, H. A., Peterson, K. L., Gottshall, K. R., Hunter, J. J., Chien, K. R., Ross, J., Jr (1996) Transthoracic echocardiography in models of cardiac disease in the mouse. Circulation 94,1109-1117[Abstract/Free Full Text]
  23. Ignarro, L. J., Fukuto, J. M., Griscavage, J. M., Rogers, N. E., Byrns, R. E. (1993) Oxidation of nitric oxide in aqueous solution to nitrite but not nitrate: comparison with enzymatically formed nitric oxide from L-arginine. Proc. Natl. Acad. Sci. U. S. A. 90,8103-8107[Abstract/Free Full Text]
  24. Livak, K. J., Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25,402-408[CrossRef][Medline]
  25. Marceau, F., Hess, J. F., Bachvarov, D. R. (1998) The B1 receptors for kinins. Pharmacol. Rev. 50,357-386[Abstract/Free Full Text]
  26. Cardoso, C. C., Garrett, T., Cayla, C., Meneton, P., Pesquero, J. B., Bader, M. (2004) Structure and expression of two kininogen genes in mice. Biol.Chem. 385,295-301[CrossRef][Medline]
  27. Monsees, T. K., Blocher, S., Heidorn, F., Winkler, A., Siems, W. E., Muller-Esterl, W., Hayatpour, J., Miska, W., Schill, W. B. (2002) Expression and location of the bradykinin B2 receptor in rat testis. Biol. Reprod. 67,1832-1839[Abstract/Free Full Text]
  28. Figueroa, C. D., Chacon, C., Corthorn, J., Ehrenfeld, P., Muller-Esterl, W., Valdes, G. (2001) Temporospatial changes of kinin b2 receptors during the estrous cycle and pregnancy in the rat uterus. Biol. Reprod. 64,1590-1599[Abstract/Free Full Text]
  29. Qadri, F., Baurle, L., Hauser, W., Rascher, W., Dominiak, P. (1999) Centrally bradykinin B2-receptor-induced hypertensive and positive chronotropic effects are mediated via activation of the sympathetic nervous system. J. Hypertens. 17,1265-1271[CrossRef][Medline]
  30. Chulak, C., Couture, R., Foucart, S. (1995) Modulatory effect of bradykinin on the release of noradrenaline from rat isolated atria. Br. J. Pharmacol. 115,330-334[Medline]
  31. Minshall, R. D., Yelamanchi, V. P., Djokovic, A., Miletich, D. J., Erdös, E. G., Rabito, S. F., Vogel, S. M. (1994) Importance of sympathetic innervation in the positive inotropic effects of bradykinin and ramiprilat. Circ. Res. 74,441-447[Abstract/Free Full Text]
  32. Rhaleb, N. E., Peng, H., Alfie, M. E., Shesely, E. G., Carretero, O. A. (1999) Effect of ACE inhibitor on DOCA-salt- and aortic coarctation-induced hypertension in mice: do kinin B2 receptors play a role?. Hypertension 33,329-334[Abstract/Free Full Text]
  33. Lortie, M., Regoli, D., Rhaleb, N. E., Plante, G. E. (1992) The role of B1- and B2-kinin receptors in the renal tubular and hemodynamic response to bradykinin. Am. J. Physiol. 262,R72-R76[Medline]
  34. Schanstra, J. P., Duchene, J., Praddaude, F., Bruneval, P., Tack, I., Chevalier, J., Girolami, J. P., Bascands, J. L. (2003) Decreased renal NO excretion and reduced glomerular tuft area in mice lacking the bradykinin B2 receptor. Am. J. Physiol. Heart. Circ. Physiol. 284,H1904-H1908[Abstract/Free Full Text]
  35. Majima, M., Yoshida, O., Muto, T., Mizogami, S., Kuribayashi, Y., Katori, M., Oh-ishi, S. (1993) High sensitivity to salt in kininogen-deficient Brown Norway Katholiek rats. Hypertension 22,705-714[Abstract/Free Full Text]
  36. Nies, A. S., Forsyth, R. P., Williams, H. E., Melmon, K. L. (1968) Contribution of kinins to endotoxin shock in unanesthetized Rhesus monkeys. Circ. Res. 22,155-164[Abstract/Free Full Text]
  37. Katori, M., Majima, M., Odoi Adome, R., Sunahara, N., Uchida, Y. (1989) Evidence for the involvement of a plasma kallikrein-kinin system in the immediate hypotension produced by endotoxin in anaesthetized rats. Br. J. Pharmacol. 98,1383-1391[Medline]
  38. Ueno, A., Ishida, H., Oh-ishi, S. (1995) Comparative study of endotoxin-induced hypotension in kininogen-deficient rats with that in normal rats. Br. J. Pharmacol. 114,1250-1256[Medline]
  39. Feletou, M., Jamonneau, I., Germain, M., Thurieau, C., Fauchere, J. L., Villa, P., Ghezzi, P., Canet, E. (1996) Bradykinin B2 receptor involvement in rabbit and murine models of septic shock. J. Cardiovasc. Pharmacol. 27,500-507[CrossRef][Medline]
  40. McLean, P. G., Perretti, M., Ahluwalia, A. (1999) Inducible expression of the kinin B1 receptor in the endotoxemic heart: mechanisms of des-Arg9bradykinin-induced coronary vasodilation. Br. J. Pharmacol. 128,275-282[CrossRef][Medline]
  41. McLean, P. G., Perretti, M., Ahluwalia, A. (2000) Kinin B(1) receptors and the cardiovascular system: regulation of expression and function. Cardiovasc. Res. 48,194-210[Abstract/Free Full Text]
  42. Whalley, E. T., Solomon, J. A., Modafferi, D. M., Bonham, K. A., Cheronis, J. C. (1992) CP-0127, a novel potent bradykinin antagonist, increases survival in rat and rabbit models of endotoxin shock. Agents Actions Suppl 38,413-420[Medline]
  43. Kalter, E. S., van Dijk, W. C., Timmerman, A., Verhoef, J., Bouma, B. N. (1983) Activation of purified human plasma prekallikrein triggered by cell wall fractions of Escherichia coli and Staphylococcus aureus. J. Infect. Dis. 148,682-691[Medline]
  44. Vincent, J. L., Zhang, H., Szabo, C., Preiser, J. C. (2000) Effects of nitric oxide in septic shock. Am. J. Respir. Crit. Care. Med. 161,1781-1785[Abstract/Free Full Text]
  45. MacMicking, J. D., Nathan, C., Hom, G., Chartrain, N., Fletcher, D. S., Trumbauer, M., Stevens, K., Xie, Q. W., Sokol, K., Hutchinson, N., et al (1995) Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81,641-650[CrossRef][Medline]
  46. Ignjatovic, T., Stanisavljevic, S., Brovkovych, V., Skidgel, R. A., Erdos, E. G. (2004) Kinin B1 receptors stimulate nitric oxide production in endothelial cells: signaling pathways activated by angiotensin I-converting enzyme inhibitors and peptide ligands. Mol. Pharmacol. 66,1310-1316[Abstract/Free Full Text]
  47. Venema, R. C. (2002) Post-translational mechanisms of endothelial nitric oxide synthase regulation by bradykinin. Int. Immunopharmacol. 2,1755-1762[CrossRef][Medline]
  48. D’Orleans-Juste, P., de Nucci, G., Vane, J. R. (1989) Kinins act on B1 or B2 receptors to release conjointly endothelium-derived relaxing factor and prostacyclin from bovine aortic endothelial cells. Br. J. Pharmacol. 96,920-926[Medline]
  49. Bae, S. W., Kim, H. S., Cha, Y. N., Park, Y. S., Jo, S. A., Jo, I. (2003) Rapid increase in endothelial nitric oxide production by bradykinin is mediated by protein kinase A signaling pathway. Biochem.Biophys.Res.Commun. 306,981-987[CrossRef][Medline]
  50. Fleming, I., Dambacher, T., Busse, R. (1992) Endothelium-derived kinins account for the immediate response of endothelial cells to bacterial lipopolysaccharide. J. Cardiovasc. Pharmacol. 20(Suppl. 12),S135-S138[Medline]
  51. Lu, J. L., Schmiege, L. M., III, Kuo, L., Liao, J. C. (1996) Downregulation of endothelial constitutive nitric oxide synthase expression by lipopolysaccharide. Biochem. Biophys. Res. Commun. 225,1-5[CrossRef][Medline]
  52. Salvemini, D., Korbut, R., Anggard, E., Vane, J. (1990) Immediate release of a nitric oxide-like factor from bovine aortic endothelial cells by Escherichia coli lipopolysaccharide. Proc. Natl. Acad. Sci. U. S. A. 87,2593-2597[Abstract/Free Full Text]
  53. Morinelli, T. A., Webb, J. G., Jaffa, A. A., Privitera, P. J., Margolius, H. S. (2001) A metabolic fragment of bradykinin, Arg-Pro-Pro-Gly-Phe, protects against the deleterious effects of lipopolysaccharide in rats. J. Pharmacol. Exp. Ther. 296,71-76[Abstract/Free Full Text]
  54. Lopez, A., Lorente, J. A., Steingrub, J., Bakker, J., McLuckie, A., Willatts, S., Brockway, M., Anzueto, A., Holzapfel, L., Breen, D., et al (2004) Multiple-center, randomized, placebo-controlled, double-blind study of the nitric oxide synthase inhibitor 546C88: effect on survival in patients with septic shock. Crit. Care. Med. 32,21-30[CrossRef][Medline]
  55. Fein, A. M., Bernard, G. R., Criner, G. J., Fletcher, E. C., Good, J. T., Jr, Knaus, W. A., Levy, H., Matuschak, G. M., Shanies, H. M., Taylor, R. W., et al (1997) Treatment of severe systemic inflammatory response syndrome and sepsis with a novel bradykinin antagonist, deltibant (CP-0127). Results of a randomized, double-blind, placebo-controlled trial. CP-0127 SIRS and Sepsis Study Group. JAMA 277,482-487[Abstract/Free Full Text]
  56. Otterbein, L., Lowe, V. C., Kyle, D. J., Noronha-Blob, L. (1993) Additive effects of a bradykinin antagonist, NPC 17761, and a leumedin, NPC 15669, on survival in animal models of sepsis. Agents Actions 39 Spec No,C125-C127[CrossRef][Medline]



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