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Duncan J. Campbell - One of the best experts on this subject based on the ideXlab platform.
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Genetic models provide unique insight into angiotensin and Bradykinin Peptides in the extravascular compartment of the heart in vivo
Clinical and experimental pharmacology & physiology, 2008Co-Authors: Duncan J. Campbell, Hong D. Xiao, Sebastien Fuchs, Kenneth E. BernsteinAbstract:There is continuing uncertainty about the tissue compartments where angiotensin and Bradykinin Peptide formation occurs. Mice with angiotensin converting enzyme (ACE) expression targeted to the cardiomyocyte membrane provide a unique experimental model to detect ACE substrates in the extravascular compartment of the heart in vivo. Angiotensin I and II, Bradykinin-(1–7), and Bradykinin-(1–9) were measured in blood and cardiac ventricles of wild type (WT) mice; mice with a nonfunctional somatic ACE gene promoter (KO); mice homozygous (8/8) and heterozygous (1/8) for cardiomyocyte-targeted ACE expression and a nonfunctional somatic ACE gene promoter; and mice heterozygous for cardiomyocyte-targeted ACE expression and heterozygous for the WT ACE allele (WT/8). Cardiac angiotensin II levels of 8/8, 1/8, WT/8, and WT mice were higher than KO levels. Cardiac angiotensin II levels in 8/8 and 1/8 mice were also higher than WT levels, but the levels in WT/8 mice were similar to WT levels. Cardiac Bradykinin-(1–9) levels of WT, but not 8/8 mice, were lower than in KO mice, whereas Bradykinin-(1–7) levels in 8/8 mice were lower than in KO mice. We conclude that angiotensin I and Bradykinin-(1–7) are present in the cardiac extravascular compartment of mice lacking vascular ACE, and extravascular ACE produces angiotensin II and metabolizes Bradykinin-(1–7) in this compartment. These data suggest the vascular compartment is the main site of angiotensin I and Bradykinin-(1–9) formation and metabolism, and vascular ACE may limit angiotensin I entry to the extravascular compartment of WT mice. Keywords: Heart, angiotensin, Bradykinin, angiotensin converting enzyme, genetic model Introduction Angiotensin and Bradykinin Peptides play important roles in cardiac physiology and disease,1–6 and the therapeutic effects of angiotensin converting enzyme (ACE) inhibitors and angiotensin type 1 receptor blockers (ARBs) are mediated in part by their modification of the levels and actions of these Peptides in the heart.1,2,4,5,7,8 Myocardial angiotensin and Bradykinin Peptide levels are higher than can be accounted for by the blood content of tissue,9,10 and are consistent with Peptide formation within the myocardium. There is, however, uncertainty about the location of angiotensin and Bradykinin Peptides in the myocardium and the contribution of the vascular and extravascular compartments to their formation and metabolism in this tissue (Figure 1).6,11 Nephrectomy models established that kidney-derived renin is the main mechanism of formation of cardiac angiotensin Peptides,9,12 and studies of angiotensin production by the heart showed most cardiac angiotensin II (Ang II) is produced at tissue sites by conversion of in situ-produced, rather than blood-derived, angiotensin I (Ang I).13 However, these studies did not identify the specific tissue compartments where Ang I is formed and converted to Ang II. Figure 1 Diagrammatic representation of the potential pathways of formation and metabolism of angiotensin and Bradykinin Peptides in the vascular and extravascular compartments of the heart. Solid arrows indicate the formation of angiotensin I (Ang I) and angiotensin ... Study of Peptides in the extravascular compartment of the heart presents special challenges because of the difficulties of access and sampling of this compartment in vivo. We recently reported the production of mice with cardiomyocyte-targeted ACE expression, in which the endogenous ACE gene was placed under the control of the α-myosin heavy chain promoter.14 These mice offer the possibility to use cardiomyocyte-targeted ACE as a reporter for the presence of ACE substrates in the extravascular compartment of the heart in vivo. ACE converts Ang I to Ang II, Bradykinin-(1–9) [BK-(1–9)] to Bradykinin-(1–7) [BK-(1–7)], and BK-(1–7) to Bradykinin-(1–5) [BK-(1–5)]. Ang II formation by cardiomyocyte-targeted ACE depends on Ang I having access to the extravascular compartment, due either to Ang I formation in the extravascular compartment or entry from the vascular compartment. Similarly, alteration in BK-(1–7) and BK-(1–9) levels by cardiomyocyte-targeted ACE indicates that these Peptides have access to the extravascular compartment of the heart. We present here the pooled data from 3 separate studies that comprised 5 genetic models of ACE gene expression in mouse heart. These were wild type (WT) mice, mice with a nonfunctional somatic ACE gene promoter (KO), mice homozygous (8/8) and heterozygous (1/8) for cardiomyocyte-targeted ACE expression and a nonfunctional somatic ACE gene promoter, and mice heterozygous for cardiomyocyte-targeted ACE expression and heterozygous for the wild type ACE allele (WT/8). Angiotensin Peptide data from these mice were previously reported.14–16 Bradykinin Peptide data from WT, KO, and some 8/8 mice were also reported,14,15 but Bradykinin Peptide data from 1/8, WT/8, and some of the 8/8 mice were not previously reported. Previous reports of these studies focused on the phenotype of the genetic models and the Peptide data from 8/8, 1/8 and WT/8 mice were compared with WT levels. However, given that 8/8 and 1/8 mice with cardiomyocyte-targeted ACE expression had a nonfunctional somatic ACE gene promoter, the present study examined the compartmentalization of angiotensin and Bradykinin Peptides in the heart by comparing these genetic models with KO mice. Peptide data from the 8/8, 1/8 and WT/8 mice14,16 were previously published separately from Peptide data from the KO mice,15 and this comparison was not reported.
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Angiotensin-Converting Enzyme C-Terminal Catalytic Domain Is the Main Site of Angiotensin I Cleavage In Vivo
Hypertension (Dallas Tex. : 1979), 2007Co-Authors: Sebastien Fuchs, Duncan J. Campbell, Hong D. Xiao, Pierre Corvol, Christine Hubert, Annie Michaud, Jonathan Adams, Mario R. Capecchi, Kenneth E. BernsteinAbstract:Angiotensin-converting enzyme (ACE) plays a central role in the production of the vasoconstrictor angiotensin II. ACE is a single polyPeptide, but it contains 2 homologous and independent catalytic domains, each of which binds zinc. To understand the in vivo role of these 2 domains, we used gene targeting to create mice with point mutations in the ACE C-domain zinc-binding motif. Such mice, termed ACE13/13, produce a full-length ACE protein with tissue expression identical to wild-type mice. Analysis of ACE13/13 mice showed that they produce ACE having only N-domain catalytic activity, as determined by the hydrolysis of domain specific substrates and by chloride sensitivity. ACE13/13 mice have blood pressure and blood angiotensin II levels similar to wild-type mice. However, plasma renin concentration is increased 2.6-fold and blood angiotensin I levels are increased 7.5-fold. Bradykinin Peptide levels are not different from wild-type levels. ACE13/13 mice have a reduced increase of blood pressure after intravenous infusion of angiotensin I. ACE13/13 mice have a normal renal structure, but they are not able to concentrate urine after dehydration as effectively as wild-type mice. This study shows that the C-domain of ACE is the predominant site of angiotensin I cleavage in vivo. Although mice lacking C-domain activity have normal physiology under laboratory conditions, they respond less well to the stress of dehydration.
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Angiotensinogen and angiotensin-converting enzyme gene copy number and angiotensin and Bradykinin Peptide levels in mice.
Journal of hypertension, 2005Co-Authors: Theodora Alexiou, Wee-ming Boon, Derek A. Denton, Robert Di Nicolantonio, Lesley L. Walker, Michael J. Mckinley, Duncan J. CampbellAbstract:ObjectiveTo test the hypothesis that changes in gene expression that may accompany angiotensinogen (AGT) and angiotensin-converting enzyme (ACE) gene polymorphism cause alteration in angiotensin and Bradykinin Peptide levels.DesignMice with one or two genes for AGT and ACE allow assessment of the ef
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Effect of Reduced Angiotensin-Converting Enzyme Gene Expression and Angiotensin-Converting Enzyme Inhibition on Angiotensin and Bradykinin Peptide Levels in Mice
Hypertension (Dallas Tex. : 1979), 2004Co-Authors: Duncan J. Campbell, Theodora Alexiou, Michael J. Mckinley, Hong D. Xiao, Sebastien Fuchs, Pierre Corvol, Kenneth E. BernsteinAbstract:There is uncertainty about the contribution of angiotensin-converting enzyme (ACE) to angiotensin II formation, with recent studies suggesting that non-ACE enzymes may be the predominant pathway of angiotensin II formation in kidney, heart, and lung. To investigate the role of ACE in angiotensin II formation, we measured angiotensin I and II levels in blood, kidney, and heart of 2 mouse genetic models (ACE.1 and ACE.4) of reduced somatic ACE gene expression and in blood, kidney, heart, lung, adrenal, and brain of mice administered the ACE inhibitor lisinopril. We also measured the levels of Bradykinin (1-9) and its ACE metabolite Bradykinin (1-7). Reduced ACE gene expression and ACE inhibition had similar effects on angiotensin and Bradykinin Peptide levels. Angiotensin II levels were reduced by 70% to 97% in blood, 92% to 99% in kidney, 93% to 99% in heart, 97% in lung, and 85% in adrenal and brain. The marked reductions in angiotensin II/angiotensin I ratio indicated that ACE was responsible for at least 90% of angiotensin I conversion to angiotensin II in blood, kidney, heart, lung, and brain, and at least 77% in adrenal. Blood Bradykinin (1-9) levels were increased 6.4-fold to 8.4-fold. Heart Bradykinin (1-9) levels were increased in ACE.4 mice and the Bradykinin (1-7)/Bradykinin (1-9) ratio was reduced in kidney and heart of ACE.4 mice and heart of lisinopril-treated mice. These studies demonstrate that ACE is the predominant pathway of angiotensin II formation in blood and tissues of mice and plays a major role in Bradykinin (1-9) metabolism in blood and, to a lesser extent, in kidney and heart.
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the kallikrein kinin system in humans
Clinical and Experimental Pharmacology and Physiology, 2001Co-Authors: Duncan J. CampbellAbstract:1. Kinin Peptides are implicated in many physiological and pathological processes, including the regulation of blood pressure and sodium homeostasis, inflammation and the cardioprotective effects of preconditioning. In humans, the plasma and tissue kallikrein-kinin systems (KKS) generate Bradykinin and kallidin Peptides, respectively. 2. We established methodology for the measurement of Bradykinin and kallidin Peptides and their metabolites in order to study the function of the plasma and tissue KKS in humans. 3. Bradykinin Peptides were more abundant than kallidin Peptides in blood and cardiac atrial tissue, whereas kallidin Peptides were predominant in urine. The levels of kinin Peptides in tissue were higher than in blood, confirming the primary tissue localization of the KKS. 4. Angiotensin-converting enzyme inhibition increased blood levels of Bradykinin and kallidin Peptides. 5. Blood levels of kallidin Peptides were suppressed in patients with severe cardiac failure, indicating that the activity of the tissue KKS is suppressed in this condition. 6. Bradykinin Peptide levels were increased in the urine of patients with interstitial cystitis, suggesting a role for these Peptides in the pathogenesis and/or symptomatology of this condition. 7. Cardiopulmonary bypass, a model of activation of the contact system, activated both the plasma and tissue KKS. 8. Measurement of individual Bradykinin and kallidin Peptides and their metabolites gives important information about the operation of the plasma and tissue KKS and their role in physiology and disease states.
Athena Kladis - One of the best experts on this subject based on the ideXlab platform.
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activation of the kallikrein kinin system by cardiopulmonary bypass in humans
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2001Co-Authors: Duncan J. Campbell, Barry Dixon, Athena Kladis, Michael Kemme, John D SantamariaAbstract:We used cardiopulmonary bypass (CPB) as a model of activation of the contact system and investigated the involvement of the plasma and tissue kallikrein-kinin systems (KKS) in this process. Circulating levels of Bradykinin and kallidin and their metabolites, plasma and tissue kallikrein, low and high molecular weight kininogen, and kallistatin were measured before, during, and 1, 4, and 10 h after CPB in subjects undergoing cardiac surgery. Bradykinin Peptide levels increased 10- to 20-fold during the first 10 min, returned toward basal levels by 70 min of CPB, and remained 1.2- to 2.5-fold elevated after CPB. Kallidin Peptide levels showed little change during CPB, but they were elevated 1.7- to 5.2-fold after CPB. There were reductions of 80 and 60% in plasma and tissue kallikrein levels, respectively, during the first minute of CPB. Kininogen and kallistatin levels were unchanged. Angiotensin-converting enzyme inhibition did not amplify the increase in Bradykinin levels during CPB. Aprotinin administration prevented activation of the KKS. The changes in circulating kinin and kallikrein levels indicate activation of both the plasma and tissue KKS during activation of the contact system by CPB.
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Interaction between neutral endopeptidase and angiotensin converting enzyme inhibition in rats with myocardial infarction: effects on cardiac hypertrophy and angiotensin and Bradykinin Peptide levels.
The Journal of pharmacology and experimental therapeutics, 1999Co-Authors: Ann-maree Duncan, Athena Kladis, Gail M. James, Frank Anastasopoulos, Todd A. Briscoe, Duncan J. CampbellAbstract:Combined inhibition of neutral endopeptidase 24.11 (NEP) and angiotensin converting enzyme (ACE) is a candidate therapy for hypertension and cardiac failure. Given that NEP and ACE metabolize angiotensin (Ang) and Bradykinin (BK) Peptides, we investigated the effects of NEP inhibition and combined NEP and ACE inhibition on Ang and BK levels in rats with myocardial infarction. We administered the NEP inhibitor ecadotril (0, 0.1, 1, 10, and 100 mg/kg/day), either alone or together with the ACE inhibitor perindopril (0.2 mg/kg/day) by 12-hourly gavage from day 2 to 28 after infarction. Ecadotril increased urine cyclic GMP and BK-(1–9) excretion. Perindopril potentiated the effect of ecadotril on urine cyclic GMP excretion. Neither perindopril nor ecadotril reduced cardiac hypertrophy when administered separately, whereas the combination of perindopril and 10 or 100 mg/kg/day ecadotril reduced heart weight/body weight ratio by 10%. Administration of ecadotril to perindopril-treated rats decreased plasma Ang-(1–7) levels, increased cardiac BK-(1–9) levels, and increased Ang II levels in plasma, kidney, aorta, and lung. These data demonstrate interactions between the effects of NEP and ACE inhibition on remodeling of the infarcted heart and on Ang and BK Peptide levels. Whereas increased cardiac BK-(1–9) levels may contribute to the reduction of cardiac hypertrophy, the reduction in plasma Ang-(1–7) levels and increase in Ang II levels in plasma and tissues may compromise the therapeutic effects of combined NEP/ACE inhibition.
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Increased levels of Bradykinin and its metabolites in tissues of young spontaneously hypertensive rats.
Journal of hypertension, 1995Co-Authors: Duncan J. Campbell, Athena Kladis, Ann-maree Duncan, Stephan B. HarrapAbstract:Objective : To determine whether tissue kinin levels in spontaneously hypertensive rats (SHR) differ from those in normotensive rats. Design and methods : The tissue levels of Bradykinin-(1-9) and its metabolites Bradykinin-(1-7) and Bradykinin-(1-8) were measured in kidney, and Bradykinin-(1-9) and Bradykinin-(1-7) were measured in adrenal, lung, heart, aorta, brown adipose tissue and brain of male SHR and the normotensive genetically homogeneous Donryu rat strain, at age 6, 10 and 20 weeks. Results : In comparison with Donryu rats, Bradykinin-(1-7), Bradykinin-(1-8) and Bradykinin-(1-9) levels were increased in kidney, and Bradykinin-(1-7) and Bradykinin-(1-9) levels were increased in adrenal, lung and heart of SHR aged 6 weeks. Bradykinin-(1-7) levels remained elevated in adrenal and lung of SHR aged 10 weeks. The Bradykinin-(1-7) : Bradykinin-(1-9) ratio for kidneys of SHR was reduced at all ages and the Bradykinin-(1-8) : Bradykinin-(1-9) ratio was reduced at age 10 and 20 weeks. Bradykinin Peptide levels in aorta, brown adipose tissue and brain were similar for SHR and Donryu rats. Conclusion : The increased levels of Bradykinin-(1-9) and its metabolites in kidney, adrenal, lung and heart tissues of young SHR suggest increased kallikrein activity in these tissues. Moreover, the reduced Bradykinin-(1-7) : Bradykinin-(1-9) and Bradykinin-(1-8) : Bradykinin-(1-9) ratios in kidneys of SHR indicate reduced endopeptidase- and carboxypeptidase-mediated metabolism of Bradykinin-(1-9), which may have contributed to the increased Bradykinin-(1-9) levels in this tissue. Together with the previously reported hypotensive effect of Bradykinin-(1-9) antagonism in young SHR, and the cosegregation of the SHR kallikrein gene with blood pressure, the increased Bradykinin-(1-9) levels in tissues of young SHR are consistent with a role for this Peptide in the pathogenesis of hypertension in those rats.
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Bradykinin Peptides in kidney, blood, and other tissues of the rat.
Hypertension (Dallas Tex. : 1979), 1993Co-Authors: Duncan J. Campbell, Athena Kladis, Ann-maree DuncanAbstract:The Bradykinin Peptide system is a tissue-based system with potent cardiovascular and renal effects. To investigate the regulation of this system, we developed a highly sensitive amino terminal-directed radioimmunoassay that, with high performance liquid chromatography, enables the measurement of Bradykinin-(1-7), Bradykinin-(1-8), and Bradykinin-(1-9). Together with a carboxy terminal-directed radioimmunoassay, we characterized Bradykinin Peptides in rat kidney and blood. The predominant Bradykinin Peptides in kidney were Bradykinin-(1-9) (approximately 100 fmol/g wet weight of tissue) and Bradykinin-(1-7) (approximately 70 fmol/g), with low levels of Bradykinin-(1-8) (approximately 8 fmol/g) and Bradykinin-(4-9) (approximately 12 fmol/g) detectable; Bradykinin-(2-9) and Bradykinin-(3-9) were below the limits of detection. In blood, the levels of Bradykinin-(1-9) were very low (approximately 2 fmol/ml), and other Bradykinin Peptides were below the limits of detection. Ile,Ser-Bradykinin and Met,Ile,Ser-Bradykinin were below the limits of detection in both kidney and blood, indicating that T-kininogen makes no detectable contribution to renal or circulating Bradykinin Peptides. Administration of the angiotensin converting enzyme inhibitor perindopril was associated with an approximate twofold increase in renal levels of Bradykinin-(1-8) and Bradykinin-(1-9) and a decrease in the Bradykinin-(1-7)/Bradykinin-(1-9) ratio. The amino terminal-directed radioimmunoassay was also applied to heart, aorta, brown adipose tissue, adrenal lung, and brain. For these tissues, Bradykinin-(1-7) and Bradykinin-(1-9) were of similar abundance (16-340 fmol/g), with lower levels of Bradykinin-(1-8). These studies demonstrate that tissue levels of Bradykinin Peptides are much higher than circulating levels, consistent with their formation at a local tissue site. Of Peptides derived from K-kininogen, Bradykinin-(1-9) is the predominant bioactive Peptide in all tissues, and a major pathway of Bradykinin-(1-9) metabolism involves the formation of Bradykinin-(1-7). In kidney, angiotensin converting enzyme plays an important role in Bradykinin-(1-9) metabolism, and increased Bradykinin-(1-9) and Bradykinin-(1-8) levels may mediate in part the renal effects of converting enzyme inhibition.
David G. Sawutz - One of the best experts on this subject based on the ideXlab platform.
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Synthesis, characterization, and conformational analysis of the D/L-Tic7 stereoisomers of the Bradykinin receptor antagonist D-Arg0[Hyp3,Thi5,D-Tic7,Oic8]Bradykinin.
Biochemistry, 1994Co-Authors: David G. Sawutz, Wayne T. Houck, Joseph M. Salvino, Peter R. Seoane, Brent Douty, Mark A. Bobko, Muriel S. Doleman, Roland E. Dolle, Henry R. WolfeAbstract:D-Arg0[Hyp3,Thi5,D-Tic7,Oic8]Bradykinin (HOE-140) is a potent (Ki = 0.11 nM) inhibitor of [3H]Bradykinin binding to Bradykinin B2 receptors found on human IMR-90 fetal lung fibroblasts. During the synthesis of this compound, we isolated and unambiguously identified the L-Tic7 stereoisomer (WIN 65365), which exhibits a 2000-fold lower binding affinity (Ki = 130 nM) than HOE-140 to the Bradykinin receptor. A similar decrease in potency is observed for WIN 65365 inhibition of Bradykinin-stimulated 45Ca2+ efflux from IMR-90 cells. Both HOE-140 and WIN 65365 appear to be competitive antagonists at the IMR-90 Bradykinin receptor. This is the first documentation of Bradykinin binding and functional antagonist activity by a Bradykinin Peptide analogue with an L amino acid replacing Pro7. In an attempt to rationalize the differences in binding affinities of HOE-140 and WIN 65365, a conformational analysis of the Peptides was undertaken using annealed molecular dynamics (AMD). Conformational analysis of HOE-140 reveals a strong preference for the formation of a type II' beta-turn in the carboxy-terminal region. Analogous modeling of WIN 65365 reveals that its conformation is strikingly different from HOE-140 in that the four carboxy-terminal residues of WIN 65365 do not form a beta-turn. These differences in low-energy conformations between the two Peptides may lead to a better understanding of the molecular interaction of antagonists with the Bradykinin receptor.
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Characterization of Bradykinin B2 receptors on human IMR-90 lung fibroblasts: Stimulation of 45Ca2+ efflux by D-Phe7 substituted Bradykinin analogues
European journal of pharmacology, 1992Co-Authors: David G. Sawutz, David M. Fauncc, Wayne T. Houck, Dean HaycockAbstract:[3H]Bradykinin binds to intact human IMR-90 fetal lung fibroblasts in a time and dose-dependent manner. Binding equilibrium was attained by 120 minutes at 4 degrees C. [3H]Bradykinin binding was saturable; Scatchard analysis of saturation binding data demonstrated a single binding site having a KD = 1.8 +/- 0.2 nM and a receptor concentration of 17.4 +/- 4.0 fmol/10(5) cells. The calculated value for KD(k-1/k1) from the association (k1 = 4.71 x 10(6) mol-1 min-1) and dissociation (k-1 = 1.13 x 10(-2) min-1) rate constants was 2.4 nM. The rank order of potency observed for Bradykinin Peptide agonists, Bradykinin > Lys-Bradykinin > Met,Lys-Bradykinin > Ile,Ser-Bradykinin >> des-Arg9-Bradykinin, is consistent with that of a Bradykinin B2 receptor. Bradykinin stimulated efflux of 45Ca2+ from IMR-90 cells dose dependently with an EC50 = 331 +/- 50 pM. 45Ca2+ efflux was also demonstrated with Lys-Bradykinin and Met-Lys-Bradykinin but not by des-Arg10-kallidin (100 nM) or NKA (1 microM). Hoe-140 inhibited Bradykinin-induced 45Ca2+ efflux (IC50 = 3 +/- 2 nM). D-Phe7-substituted Bradykinin analogues stimulated 45Ca2+ efflux dose dependently and this stimulation of 45Ca2+ efflux was inhibited by Hoe-140. These results suggest that D-Phe7 substituted Bradykinin analogues are agonists at the Bradykinin B2 receptor in IMR-90 cells.
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Synthesis and molecular characterization of a biotinylated analog of [Lys]Bradykinin.
Peptides, 1991Co-Authors: David G. Sawutz, John Yanni, Marian Kelley, Henry R. WolfeAbstract:Abstract We report the synthesis and molecular characterization of a biotinylated analog of kallidin, [Lys]Bradykinin. Bradykinin was prepared by solid phase Peptide synthesis. Before cleavage from the resin, a biotin moiety was coupled to the epsilon amino group of a lysine in the zeroth position of the Bradykinin Peptide. An ω-amino caproic acid spacer was incorporated between the biotin group and the N-terminal lysine. The biotinylated Peptide was deprotected, cleaved from the resin and purified by RP-HPLC. The identity of this analog was confirmed by amino acid analysis and FAB-mass spectrometry. Biotinyl [Lys]Bradykinin (BLBK, mol.wt. = 1528) inhibited [ 3 H]-Bradykinin binding to guinea pig ileum homogenates dose dependently, with an IC 50 of 28.9±6 nM. The IC 50 for [Lys]Bradykinin was approximately 10-fold lower, 3.2±0.6 nM. BLBK induced contractility in an isolated guinea pig smooth muscle preparation with an EC 50 of 129±14 nM; the corresponding value for [Lys]Bradykinin was 29±8 nM. These data are consistent with the difference in binding potency observed for BLBK compared to [Lys]Bradykinin. In an ELISA assay using BLBK and affinity-purified rabbit anti-Bradykinin antibody, BLBK bound to anti-Bradykinin antibody with an EC 50 =1.21±0.54 nM. Rank order potencies for several Bradykinin Peptide analogs suggest that the epitope on Bradykinin recognized by the antibody is likely to be at the carboxy terminus of the Peptide.
John D Santamaria - One of the best experts on this subject based on the ideXlab platform.
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activation of the kallikrein kinin system by cardiopulmonary bypass in humans
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2001Co-Authors: Duncan J. Campbell, Barry Dixon, Athena Kladis, Michael Kemme, John D SantamariaAbstract:We used cardiopulmonary bypass (CPB) as a model of activation of the contact system and investigated the involvement of the plasma and tissue kallikrein-kinin systems (KKS) in this process. Circulating levels of Bradykinin and kallidin and their metabolites, plasma and tissue kallikrein, low and high molecular weight kininogen, and kallistatin were measured before, during, and 1, 4, and 10 h after CPB in subjects undergoing cardiac surgery. Bradykinin Peptide levels increased 10- to 20-fold during the first 10 min, returned toward basal levels by 70 min of CPB, and remained 1.2- to 2.5-fold elevated after CPB. Kallidin Peptide levels showed little change during CPB, but they were elevated 1.7- to 5.2-fold after CPB. There were reductions of 80 and 60% in plasma and tissue kallikrein levels, respectively, during the first minute of CPB. Kininogen and kallistatin levels were unchanged. Angiotensin-converting enzyme inhibition did not amplify the increase in Bradykinin levels during CPB. Aprotinin administration prevented activation of the KKS. The changes in circulating kinin and kallikrein levels indicate activation of both the plasma and tissue KKS during activation of the contact system by CPB.
Kenneth E. Bernstein - One of the best experts on this subject based on the ideXlab platform.
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Genetic models provide unique insight into angiotensin and Bradykinin Peptides in the extravascular compartment of the heart in vivo
Clinical and experimental pharmacology & physiology, 2008Co-Authors: Duncan J. Campbell, Hong D. Xiao, Sebastien Fuchs, Kenneth E. BernsteinAbstract:There is continuing uncertainty about the tissue compartments where angiotensin and Bradykinin Peptide formation occurs. Mice with angiotensin converting enzyme (ACE) expression targeted to the cardiomyocyte membrane provide a unique experimental model to detect ACE substrates in the extravascular compartment of the heart in vivo. Angiotensin I and II, Bradykinin-(1–7), and Bradykinin-(1–9) were measured in blood and cardiac ventricles of wild type (WT) mice; mice with a nonfunctional somatic ACE gene promoter (KO); mice homozygous (8/8) and heterozygous (1/8) for cardiomyocyte-targeted ACE expression and a nonfunctional somatic ACE gene promoter; and mice heterozygous for cardiomyocyte-targeted ACE expression and heterozygous for the WT ACE allele (WT/8). Cardiac angiotensin II levels of 8/8, 1/8, WT/8, and WT mice were higher than KO levels. Cardiac angiotensin II levels in 8/8 and 1/8 mice were also higher than WT levels, but the levels in WT/8 mice were similar to WT levels. Cardiac Bradykinin-(1–9) levels of WT, but not 8/8 mice, were lower than in KO mice, whereas Bradykinin-(1–7) levels in 8/8 mice were lower than in KO mice. We conclude that angiotensin I and Bradykinin-(1–7) are present in the cardiac extravascular compartment of mice lacking vascular ACE, and extravascular ACE produces angiotensin II and metabolizes Bradykinin-(1–7) in this compartment. These data suggest the vascular compartment is the main site of angiotensin I and Bradykinin-(1–9) formation and metabolism, and vascular ACE may limit angiotensin I entry to the extravascular compartment of WT mice. Keywords: Heart, angiotensin, Bradykinin, angiotensin converting enzyme, genetic model Introduction Angiotensin and Bradykinin Peptides play important roles in cardiac physiology and disease,1–6 and the therapeutic effects of angiotensin converting enzyme (ACE) inhibitors and angiotensin type 1 receptor blockers (ARBs) are mediated in part by their modification of the levels and actions of these Peptides in the heart.1,2,4,5,7,8 Myocardial angiotensin and Bradykinin Peptide levels are higher than can be accounted for by the blood content of tissue,9,10 and are consistent with Peptide formation within the myocardium. There is, however, uncertainty about the location of angiotensin and Bradykinin Peptides in the myocardium and the contribution of the vascular and extravascular compartments to their formation and metabolism in this tissue (Figure 1).6,11 Nephrectomy models established that kidney-derived renin is the main mechanism of formation of cardiac angiotensin Peptides,9,12 and studies of angiotensin production by the heart showed most cardiac angiotensin II (Ang II) is produced at tissue sites by conversion of in situ-produced, rather than blood-derived, angiotensin I (Ang I).13 However, these studies did not identify the specific tissue compartments where Ang I is formed and converted to Ang II. Figure 1 Diagrammatic representation of the potential pathways of formation and metabolism of angiotensin and Bradykinin Peptides in the vascular and extravascular compartments of the heart. Solid arrows indicate the formation of angiotensin I (Ang I) and angiotensin ... Study of Peptides in the extravascular compartment of the heart presents special challenges because of the difficulties of access and sampling of this compartment in vivo. We recently reported the production of mice with cardiomyocyte-targeted ACE expression, in which the endogenous ACE gene was placed under the control of the α-myosin heavy chain promoter.14 These mice offer the possibility to use cardiomyocyte-targeted ACE as a reporter for the presence of ACE substrates in the extravascular compartment of the heart in vivo. ACE converts Ang I to Ang II, Bradykinin-(1–9) [BK-(1–9)] to Bradykinin-(1–7) [BK-(1–7)], and BK-(1–7) to Bradykinin-(1–5) [BK-(1–5)]. Ang II formation by cardiomyocyte-targeted ACE depends on Ang I having access to the extravascular compartment, due either to Ang I formation in the extravascular compartment or entry from the vascular compartment. Similarly, alteration in BK-(1–7) and BK-(1–9) levels by cardiomyocyte-targeted ACE indicates that these Peptides have access to the extravascular compartment of the heart. We present here the pooled data from 3 separate studies that comprised 5 genetic models of ACE gene expression in mouse heart. These were wild type (WT) mice, mice with a nonfunctional somatic ACE gene promoter (KO), mice homozygous (8/8) and heterozygous (1/8) for cardiomyocyte-targeted ACE expression and a nonfunctional somatic ACE gene promoter, and mice heterozygous for cardiomyocyte-targeted ACE expression and heterozygous for the wild type ACE allele (WT/8). Angiotensin Peptide data from these mice were previously reported.14–16 Bradykinin Peptide data from WT, KO, and some 8/8 mice were also reported,14,15 but Bradykinin Peptide data from 1/8, WT/8, and some of the 8/8 mice were not previously reported. Previous reports of these studies focused on the phenotype of the genetic models and the Peptide data from 8/8, 1/8 and WT/8 mice were compared with WT levels. However, given that 8/8 and 1/8 mice with cardiomyocyte-targeted ACE expression had a nonfunctional somatic ACE gene promoter, the present study examined the compartmentalization of angiotensin and Bradykinin Peptides in the heart by comparing these genetic models with KO mice. Peptide data from the 8/8, 1/8 and WT/8 mice14,16 were previously published separately from Peptide data from the KO mice,15 and this comparison was not reported.
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Angiotensin-Converting Enzyme C-Terminal Catalytic Domain Is the Main Site of Angiotensin I Cleavage In Vivo
Hypertension (Dallas Tex. : 1979), 2007Co-Authors: Sebastien Fuchs, Duncan J. Campbell, Hong D. Xiao, Pierre Corvol, Christine Hubert, Annie Michaud, Jonathan Adams, Mario R. Capecchi, Kenneth E. BernsteinAbstract:Angiotensin-converting enzyme (ACE) plays a central role in the production of the vasoconstrictor angiotensin II. ACE is a single polyPeptide, but it contains 2 homologous and independent catalytic domains, each of which binds zinc. To understand the in vivo role of these 2 domains, we used gene targeting to create mice with point mutations in the ACE C-domain zinc-binding motif. Such mice, termed ACE13/13, produce a full-length ACE protein with tissue expression identical to wild-type mice. Analysis of ACE13/13 mice showed that they produce ACE having only N-domain catalytic activity, as determined by the hydrolysis of domain specific substrates and by chloride sensitivity. ACE13/13 mice have blood pressure and blood angiotensin II levels similar to wild-type mice. However, plasma renin concentration is increased 2.6-fold and blood angiotensin I levels are increased 7.5-fold. Bradykinin Peptide levels are not different from wild-type levels. ACE13/13 mice have a reduced increase of blood pressure after intravenous infusion of angiotensin I. ACE13/13 mice have a normal renal structure, but they are not able to concentrate urine after dehydration as effectively as wild-type mice. This study shows that the C-domain of ACE is the predominant site of angiotensin I cleavage in vivo. Although mice lacking C-domain activity have normal physiology under laboratory conditions, they respond less well to the stress of dehydration.
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Effect of Reduced Angiotensin-Converting Enzyme Gene Expression and Angiotensin-Converting Enzyme Inhibition on Angiotensin and Bradykinin Peptide Levels in Mice
Hypertension (Dallas Tex. : 1979), 2004Co-Authors: Duncan J. Campbell, Theodora Alexiou, Michael J. Mckinley, Hong D. Xiao, Sebastien Fuchs, Pierre Corvol, Kenneth E. BernsteinAbstract:There is uncertainty about the contribution of angiotensin-converting enzyme (ACE) to angiotensin II formation, with recent studies suggesting that non-ACE enzymes may be the predominant pathway of angiotensin II formation in kidney, heart, and lung. To investigate the role of ACE in angiotensin II formation, we measured angiotensin I and II levels in blood, kidney, and heart of 2 mouse genetic models (ACE.1 and ACE.4) of reduced somatic ACE gene expression and in blood, kidney, heart, lung, adrenal, and brain of mice administered the ACE inhibitor lisinopril. We also measured the levels of Bradykinin (1-9) and its ACE metabolite Bradykinin (1-7). Reduced ACE gene expression and ACE inhibition had similar effects on angiotensin and Bradykinin Peptide levels. Angiotensin II levels were reduced by 70% to 97% in blood, 92% to 99% in kidney, 93% to 99% in heart, 97% in lung, and 85% in adrenal and brain. The marked reductions in angiotensin II/angiotensin I ratio indicated that ACE was responsible for at least 90% of angiotensin I conversion to angiotensin II in blood, kidney, heart, lung, and brain, and at least 77% in adrenal. Blood Bradykinin (1-9) levels were increased 6.4-fold to 8.4-fold. Heart Bradykinin (1-9) levels were increased in ACE.4 mice and the Bradykinin (1-7)/Bradykinin (1-9) ratio was reduced in kidney and heart of ACE.4 mice and heart of lisinopril-treated mice. These studies demonstrate that ACE is the predominant pathway of angiotensin II formation in blood and tissues of mice and plays a major role in Bradykinin (1-9) metabolism in blood and, to a lesser extent, in kidney and heart.