Metabolism of bradykinin analogs by angiotensin I converting enzyme and carboxypeptidase N.

Bradykinin (BK) analogs such as Lys-Lys-BK, des-Arg9-BK and [Leu8]des-Arg9-BK were poor substrates for angiotensin I converting enzyme (ACE), and analogs containing D-Phe7 residues, or a pseudopeptide C-terminal bond, were completely resistant. However, many of these analogs were metabolized by carboxypeptidase N (CPN) including Lys-Lys-BK, [Tyr8(OMe)]BK and D-Phe7-containing analogs, with Km and Vmax values comparable to those for BK. The only analogs completely resistant to both ACE and CPN were the B2 agonist [Phe8 psi(CH2NH)Arg9]BK, the B2 agonist D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, and the B1 agonist [D-Phe8]des-Arg9-BK. These data indicate an important role for plasma CPN and vascular CPN-like activity in the metabolism of the widely used ACE-resistant/D-Phe7-containing antagonists of B2 kinin receptors.     

Differential processing of substance P and neurokinin A by plasma dipeptidyl(amino)peptidase IV, aminopeptidase M and angiotensin converting enzyme

In addition to plasma metabolism of substance P (SP) by angiotensin converting enzyme (ACE; EC 3.4.15.1) (<1.0 nmol/min/ml), the majority of SP hydrolysis by rat and human plasma was due to dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) (3.15–5.91 nmol/min/ml), which sequentially converted SP to SP(3–11) and SP(5–11). In turn, the SP(5–11) metabolite was rapidly hydrolyzed by rat and human plasma aminopeptidase M (AmM; EC 3.4.11.2) (24.2–25.5 nmol/min/ml). The K_m values of SP for DAP IV and of SP(5–11) for AmM ranged from 32.7 to 123 μM. In contrast, neurokinin A (NKA) was resistant to both ACE and DAP IV but was subject to N-terminal hydrolysis by AmM (3.76–10.8 nmol/min/ml; K_m=90.7 μM. These data demonstrate differential processing of SP and NKA by specific peptidases in rat and human plasma.     

Angiotensin-(1-7) potentiates responses to bradykinin but does not change responses to angiotensin I

Angiotensin-(1-7) (Ang-(1-7)), a bioactive peptide in the renin-angiotensin system, has counterregulatory actions to angiotensin II (Ang II). However, the mechanism by which Ang-(1-7) enhances vasodepressor responses to bradykinin (BK) is not well understood. In the present study, the effects of Ang-(1-7) on responses to BK, BK analogs, angiotensin I (Ang I), and Ang II were investigated in the anesthetized rat. The infusion of Ang-(1-7) (55 pmol/min i.v.) enhanced decreases in systemic arterial pressure in response to i.v. injections of BK and the BK analogs [Hyp3, Tyr(Me)8]-bradykinin (HT-BK) and [Phe8psi (CH2-NH) Arg9]-bradykinin (PA-BK) without altering pressor responses to Ang I or II, or depressor responses to acetylcholine and sodium nitroprusside. The angiotensin-converting enzyme (ACE) inhibitor enalaprilat enhanced responses to BK and the BK analog HT-BK without altering responses to PA-BK and inhibited responses to Ang I. The potentiating effects of Ang-(1-7) and enalaprilat on responses to BK were not attenuated by the Ang-(1-7) receptor antagonist A-779. Ang-(1-7)- and ACE inhibitor-potentiated responses to BK were attenuated by the BK B2 receptor antagonist Hoe 140. The cyclooxygenase inhibitor sodium meclofenamate had no significant effect on responses to BK or Ang-(1-7)-potentiated BK responses. These results suggest that Ang-(1-7) potentiates responses to BK by a selective B2 receptor mechanism that is independent of an effect on Ang-(1-7) receptors, ACE, or cyclooxygenase product formation. These data suggest that ACE inhibitor-potentiated responses to BK are not mediated by an A-779-sensitive mechanism and are consistent with the hypothesis that enalaprilat-induced BK potentiation is due to decreased BK inactivation.     

Different inhibition of enalaprilat and imidaprilat on bradykinin metabolizing enzymes

Effects of angiotensin-converting enzyme (ACE) inhibitors, enalaprilat and imidaprilat, on bradykinin (BK) metabolizing enzymes, aminopeptidase P (APP), neutral endopeptidase (NEP) and carboxypeptidase N (CPN), were examined. APP activity in the mouse lung was inhibited by enalaprilat in a concentration-dependent manner while imidaprilat did not influence the enzyme activity. The inhibitory effects of these ACE inhibitors on the NEP activity in the mouse lung and the CPN activity in the mouse serum were negligible. These data suggested that the influence of enalaprilat on the APP activity and subsequent BK metabolism are different from those of imidaprilat.     

ACE activity during the hypotension produced by standardized aqueous extract of Cecropia glaziovii Sneth: A comparative study to captopril effects in rats

To evaluate the effect of the standardized aqueous extract (AE) of Cecropia glaziovii Sneth on the plasma angiotensin I converting enzyme (ACE-EC 3.4.15.1) activity, rats were treated with a single dose of AE (1 g/kg, p.o.) or repeatedly (0.5 g/kg/bid, p.o.) for 60 days. Captopril (50 mg/kg, p.o.) was used as positive control on the same animals. The effects on the blood pressure were recorded directly from the femoral artery (single dose), or indirectly by the tail cuff method (repeated doses) in conscious rats. The plasma ACE activity was determined spectrofluorimetrically using Hypuril-Hystidine-Leucine as substrate. The arterial blood pressure, heart rate and plasma ACE activity were not significantly modified within 24 h after a single dose administration of AE. Comparatively, blood pressure in captopril treated rats was reduced by 7-16% and heart rate was increased by 10-20% from 30 min to 24 h after drug administration. ACE activity after captopril presented a dual response: an immediate inhibition peaking at 30 min and a slow reversal to 32% up-regulation after 24 h. To correlate the drug effects upon repeated administration of either compound, normotensive rats were separated in three groups: animals with high ACE (48.8+/-2.6 nmol/min/ml), intermediate ACE (39.4+/-1.4 nmol/min/ml) and low ACE (23.5+/-0.6 nmol/min/ml) activity, significantly different among them. Repeated treatment with AE reduced the mean systolic blood pressure (121.7+/-0.5 mm Hg) by 20 mm Hg after 14 days. The hypotension was reversed upon washout 60 days afterwards. Likely, repeated captopril administration decreased blood pressure by 20 mm Hg throughout treatment in all groups. After 30 days treatment with AE (0.5 g/kg/bid, p.o.) the plasma ACE activity was unchanged in any experimental group. After captopril (50 mg/kg/bid, p.o.) administration the plasma ACE activity was inhibited by 50% within 1 h treatment but it was up-regulated by 120% after 12 h in all groups. It is concluded that the hypotension produced by prolonged treatment with AE of C. glaziovii is unrelated to ACE inhibition.     

Pathways of bradykinin degradation in blood and plasma of normotensive and hypertensive rats

Kinins are vasoactive peptide hormones that can confer protection against the development of hypertension. Because their efficacy is greatly influenced by the rate of enzymatic degradation, the activities of various kininases in plasma and blood of spontaneously hypertensive rats (SHR) were compared with those in normotensive Wistar-Kyoto rats (WKY) to identify pathogenic alterations. Either plasma or whole blood was incubated with bradykinin (10 microM). Bradykinin and kinin metabolites were measured by high-performance liquid chromatography. Kininase activities were determined by cumulative inhibition of angiotensin I-converting enzyme (ACE), carboxypeptidase N (CPN), and aminopeptidase P (APP), using selective inhibitors. Plasma of WKY rats degraded bradykinin at a rate of 13.3 +/- 0.94 micromol x min(-1) x l(-1). The enzymes ACE, APP, and CPN represented 92% of this kininase activity, with relative contributions of 52, 25, and 16%, respectively. Inclusion of blood cells at physiological concentrations did not extend the activities of these plasma kininases further. No differences of kinin degradation were found between WKY and SHR. The identical conditions of kinin degradation in WKY and SHR suggest no pathogenic role of kininases in the SHR model of genetic hypertension.     

N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes: structure-activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes

Peptide agonists and antagonists of both bradykinin (BK) B(1) and B(2) receptors (B(1)R, B(2)R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent ligands by extending both agonists and antagonist peptides at both receptor subtypes with 5(6)-carboxyfluorescein (CF) and the ε-aminocaproyl (ε-ACA) optional spacer. Alternatively, kinin receptor ligands were extended with another carboxylic acid cargo (chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (AF350), ferrocenoyl, cetirizine) or with fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended peptides and the use of fluorophore-conjugated ligands in imaging of cell receptors and of angiotensin converting enzyme (ACE) in intact cells. Antagonists (B(1)R: B-10376: CF-ε-ACA-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; B(2)R: B-10380: CF-ε-ACA-D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B(2)R agonists CF-ε-ACA-BK, AF350-ε-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B(1)R agonist B-10378 (CF-ε-ACA-Lys-des-Arg(9)-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ε-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK. Metal- or drug-containing cargoes further show the prospect of ligands that confer special signaling to kinin receptors.     

Hypotensive effects of hemopressin and bradykinin in rabbits, rats and mice. A comparative study

Hemopressin is a novel vasoactive nonapeptide derived from hemoglobin's alpha-chain as recently reported by Rioli et al. [Rioli V, Gozzo FC, Heimann AS, Linardi A, Krieger JE, Shida CS, et al. Novel natural peptide substrates for endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme. J Biol Chem 2003;278(10):8547-55]. In anesthetized male Wistar rats, this peptide exhibited hypotensive actions similar to those of bradykinin (BK) when administered intravenously (i.v.), and was found to be metabolized both in vitro and in vivo by several peptidases, including the angiotensin-converting enzyme (ACE). In this study, these findings were expanded upon by examining: (i) the degradation kinetics following incubation with ACE purified from rabbit lung and (ii) the blood pressure lowering effects of HP and BK injected i.v. or intra-arterially (i.a.) in male rabbits, rats, and mice. Our findings demonstrate that, in vitro, HP and BK are both degraded by ACE, but at different velocity rates. Furthermore, both HP and BK induced transient hypotension in all animals tested, although the responses to HP relative to the administration sites were significantly lower (by 10-100-fold) on an equimolar basis compared to those of BK. In rabbits, the decrease of blood pressure induced by HP (10-100 nmol/kg) did not differ whether it was administered i.v. or i.a., suggesting an absence of pulmonary/cardiac inactivation in contrast to BK (0.1-1 nmol/kg). The in vivo effect of HP was significantly potentiated in rabbits immunostimulated with bacterial lipopolysaccharide (LPS), but was unaffected by both the B2 receptor antagonist HOE 140 (0.1 micromol/kg) and captopril (100 microg/kg), contrary to BK. Therefore, HP acts as a weak hypotensive mediator, which does not activate kinin B2 receptors, but uses a functional site and/or signaling paths appearing to be up-regulated by LPS.     

Potentiation of the effects of bradykinin on its receptor in the isolated guinea pig ileum

Angiotensin I-converting enzyme (ACE/kininase II) inhibitors potentiated guinea pig ileum's isotonic contractions to bradykinin (BK) and its analogues, shifting the BK dose-response curve to the left. ACE inhibitors added at the peak of the contraction immediately enhanced it further (343 +/- 40%), although the ileum inactivated BK slowly (t(1/2) = 12-16 min). Chymotrypsin and cathepsin G also augmented the activity of BK up to three- or four-fold, but in a manner slower than that of ACE inhibitors. The BK B(2) receptor blocker HOE 140 inhibited all effects. Histamine and angiotensin II were not potentiated. ACE inhibitors potentiate BK independent of blocking its inactivation by inducing crosstalk between ACE and the BK B(2) receptor; proteases activate the receptor by different mechanism.     

Studies on the angiotensin-converting enzyme and the kinin B2 receptor in the rabbit jugular vein: modulation of contractile response to bradykinin

The rabbit jugular vein (rbJV) was used as a bioassay system to validate some early and new hypothetical interactions between the angiotensin-converting enzyme (ACE) and the B2 receptor, which may be influenced by ACE inhibitors (ACE-I). These involve the potentiation of the contractile effect of bradykinin (BK) and BK analogues, which are inactivated by ACE (e.g., [Hyp3, Tyr(Me8)]-BK (R556)), the prevention of BK-induced B2 receptor desensitisation, and the restoration of receptor sensitivity in tissues desensitised with B2 receptor agonists. Enzymatic degradation studies performed in vitro and in vivo revealed that BK and R556 are readily degraded by rabbit ACE whereas [Phe8psi(CH2-NH)Arg9]-BK (R379) is totally resistant. BK, R556, and R379 contracted endothelium-denuded veins with similar potencies (pEC50 range 8.10-8.50). Tissues pretreated with ACE-I showed an increase in pEC50 values for BK and R556 but not for R379. ACE-I (captopril, enalaprilat) were unable to prevent B2 receptor desensitisation induced by BK (1 microM). ACE-I partially restored B2 receptor-mediated contraction in tissues initially exposed to BK but not to R379. These effects were antagonised by HOE 140 (0.1 microM) but were unaffected by AcLys[Dbeta-Nal7, Ile8]-desArg9BK (R715) (1 microM) or by Losartan (1 microM). In conclusion, the potentiation of BK and its analogues relates exclusively on prevention of their metabolism, B2 receptor desensitisation is not affected by ACE-I, and restoration of tissue responsiveness to BK by ACE-I may be attributed to changes in BK concentrations in the vicinity of the B2 receptor.     

Characterization of bradykinin receptors in canine cultured corneal epithelial cells: Pharmacological and functional studies

The pharmacological properties of bradykinin (BK) receptors were characterized in canine cultured corneal epithelial cells (CECs) using [(3)H]-BK as a radioligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant of 0.34 +/- 0.07 nM and a maximum receptor density of 179 +/- 23 fmol/mg protein. Neither a B(1) receptor-selective agonist (des-Arg(9)-BK) nor antagonist ([Leu(8), des-Arg(9)]-BK) significantly inhibited [(3)H]-BK binding to CECs, thus excluding the presence of B(1) receptors in canine CECs. The specific binding of [(3)H]-BK to CECs was inhibited by B(2) receptor-selective agonists (BK and kallidin) and antagonists (Hoe 140 and [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK), with a best fit using a one-binding-site model. The order of potency for the inhibition of [(3)H]-BK binding was BK = Hoe 140 > kallidin > [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK. Stimulation of CECs by BK produced a concentration-dependent accumulation of inositol phosphates (IP) and an initial transient peak of intracellular Ca(2+). B(2) receptor-selective antagonist ([D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK) significantly antagonized the BK-induced responses with dissociation constants of 6.0-6.1. Pretreatment of CECs with pertussis toxin (PTX) or cholera toxin did not alter the BK-induced IP accumulation. Incubation of CECs in the absence of external Ca(2+) led to a significant attenuation of the IP accumulation induced by BK. These results demonstrate that BK directly stimulates phospholipase C-mediated signal transduction through BK B(2) receptors via a PTX-insensitive G protein in canine CECs. This effect may function as the transducing mechanism for BK-mediated cellular responses.     

Identification of [hydroxyproline3]-bradykinin released from human plasma and plasma protein Cohn's fraction IV-4 by trypsin

Aside from bradykinin (BK), a novel kinin, [Hydroxyproline3]-bradykinin ( [Hyp3]-BK), was isolated from the reaction mixture of human plasma and plasma protein Cohn's fraction IV-4 with trypsin. The liberated kinins were isolated based on procedures which we previously described for the isolation of [Hyp3]-lysyl-bradykinin ( [Hyp3]-Lys-BK) formed by kallikrein. The ratio of the amounts of two kinins thus formed from human plasma protein Cohn's fraction IV-4 were [Hyp3]-BK 25 +/- 4% and BK 75 +/- 4%, similarly to that of [Hyp3]-Lys-BK and Lys-BK, formed by kallikrein, but it varied by persons. The isolation of [Hyp3]-BK and [Hyp3]-Lys-BK suggests that a novel kininogen containing hydroxyproline in the third position of the bradykinin sequence in human plasma protein, possibly undergone post-translational modifications.     

Inactivation of bradykinin by angiotensin-converting enzyme and by carboxypeptidase N in human plasma

Because bradykinin (BK) appears to have cardioprotective effects ranging from improved hemodynamics to antiproliferative effects, inhibition of BK-degrading enzymes should potentiate such actions. The purpose of this study was to find out which enzymes are responsible for the degradation of BK in human plasma. Human plasma from healthy donors (n = 10) was incubated with BK in the presence or absence of specific enzyme inhibitors. At high (micromolar) concentrations, BK was mostly (>90%) degraded by carboxypeptidase N (CPN)-like activity. In contrast, at low (nanomolar) substrate concentrations, at which the velocity of the catalytic reaction is equivalent to that under physiological conditions, BK was mostly (>90%) converted into an inactive metabolite, BK-(1-7), by angiotensin-converting enzyme (ACE). BK-(1-7) was further converted by ACE into BK-(1-5), with accumulation of this active peptide. A minor fraction (<10%) of the BK was converted into another active metabolite, BK-(1-8), by CPN-like activity. The present study shows that the most critical step in plasma kinin metabolism, i.e., inactivation of BK, is mediated by ACE. Thus inhibition of plasma ACE activity would be cardioprotective by elevating the concentration of BK in the circulation.     

Fluorescent and biotinylated probes for B2 bradykinin receptors: agonists and antagonists.

Peptide ligands carrying additional reporter groups are valuable research tools to facilitate biochemical and pharmacological studies of G protein-coupled receptors. B2 bradykinin receptors, widely distributed in mammalian tissues, regulate many physiological systems and are therapeutic targets. Acylation of the amino-terminus of bradykinin (BK) and a B2a-selective antagonist produced ligands derivatized with biotinamidocaproate or 7-Amino-4-methylcoumarin-3-acetate. These fluorescent and biotinylated peptides bound with high affinity to bovine and rodent B2 receptors. Analysis of second messenger production confirmed that fluorescent and biotinylated analogs of BK were B2 receptor agonists whereas derivatives of DArg0[Hyp3,DPhe7,Leu8]BK were BK receptor antagonists. The complimentary properties of these selective receptor probes will be useful in studying B2 receptor localization, expression and desensitization.     

Characterization of kinin binding sites: identity of B2 receptors in the epithelium and the smooth muscle of the guinea pig ileum.

Binding of [125I-Tyr8]bradykinin (BK) was measured in homogenates of epithelial and smooth muscle layers of the guinea pig ileum. Binding assays were performed at 4 degrees C for 40 min (smooth muscle) or 90 min (epithelium) in 25 mM PIPES buffer at pH 6.8 in the presence of 1 mM 1,10-phenanthroline, 140 micrograms/mL bacitracin, 1 mM captopril, 1 mM dithiothreitol, and 0.1% bovine serum albumin. Specific binding of [125I-Tyr8]BK (0.32 nM) to epithelial and smooth muscle cell membranes was linearly related to protein concentration between 0.05 and 0.5 mg/mL. Equilibrium experiments showed that specific binding of [125I-Tyr8]BK was saturable and Scatchard analysis indicated the presence of a high affinity site with a Kd value of 1.6 nM and a Bmax of 156 fmol/mg of protein in the epithelial cell membranes. In smooth muscle membranes, Kd was 1.8 nM and the maximum number of binding sites was 58 fmol/mg of protein. Unlabelled peptides, namely bradykinin, [Tyr8]BK, [Hyp3]BK, D-Arg[Hyp3]BK, [Hyp3,Tyr(Me8)]BK, and kallidin displaced [125I-Tyr8]BK binding while other peptides, angiotensin II and substance P, had no effect. A series of B2-receptor antagonists displaced [125I-Tyr8]BK from specific binding sites with IC50 values ranging from 16 to 152 nM on epithelial cell membranes; similar values were obtained from smooth muscle cell membranes. These findings suggest that the binding sites in both preparations are of the B2 type. B1-receptor agonists and antagonists were found to be inactive at concentrations up to 10(-4) M. Results obtained in the two preparations were compared and a positive highly significant correlation was demonstrated between the two sets of data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptide Y is a vasoconstrictor in human nasal mucosa.

Neuropeptide Y (NPY) is a neurotransmitter in sympathetic nerve fibers in human nasal mucosa. Like norepinephrine, NPY acts as a vasoconstrictor. An established method of nasal provocation was used to determine the effects of topically applied NPY on nasal resistance to airflow measured by anterior rhinomanometry, the protein content of nasal secretions, and the protein content of bradykinin-induced secretions. NPY (2.3 nmol) reduced the resistance to inspiratory airflow by 57 +/- 18% (P < 0.001) in 10 normal subjects and by 50 +/- 17% (P < 0.05) in 12 subjects with perennial rhinitis. In nasal provocations, NPY in doses of 0.1-10 nmol had no effect on vascular (albumin), glandular (lysozyme, glycoconjugate), or total proteins present in lavaged nasal secretions. Because the vasoconstrictor properties of NPY may only be apparent in the presence of increased vascular permeability and albumin exudation, bradykinin (BK) nasal provocation was performed. BK (500 nmol) significantly increase total protein (10- to 20-fold), albumin (10- to 30-fold), and glycoconjugate (2- to 5-fold) in lavage fluid. NPY (2.3 nmol) reduced BK-induced total protein by 59 +/- 15% (P < 0.05) and albumin by 63 +/- 17% (P < 0.02) but had no significant effect on glandular secretion. Therefore exogenous administration of NPY to the human nasal mucosa reduced nasal airflow resistance and albumin exudation without affecting submucosal gland secretion. NPY agonists may be useful for the treatment of mucosal diseases characterized by vasodilation, vascular permeability, and plasma exudation.     

Purification, structural characterization, and myotropic activity of a peptide related to des-Arg(9)-bradykinin from an elasmobranch fish, the little skate, Leucoraja erinacea

A bradykinin (BK)-related peptide was isolated from heat-denaturated plasma from an elasmobranch fish, the little skate, Leucoraja erinacea after incubation with porcine pancreatic kallikrein. The primary structure of the peptide (H-Gly-Ile-Thr-Ser-Trp-Leu-Pro-Phe-OH; skate BK) shows limited structural similarity to the mammalian B1 receptor agonist, des-Arg(9)-BK. The myotropic activities of synthetic skate BK, and the analog skate [Arg(9)]BK, were examined in isolated skate vascular and intestinal smooth muscle preparations. Skate BK produced a concentration-dependent constriction of the mesenteric artery (EC(50)=4.37x10(-8)M; maximum response=103.4+/-10.23% of the response to 60mM KCl) but the response to skate [Arg(9)]BK was appreciably weaker (response to 10(-6)M=73.0+/-23.4% of the response to 60mM KCl). Neither the first branchial gill arch nor the ventral aorta responded to either purified peptide. Skate BK also produced a concentration-dependent constriction of intestinal smooth muscle preparations (EC(50)=2.74x10(-7)M; maximum response 31.0+/-12.2% of the response to 10(-5)M acetylcholine). Skate [Arg(9)]BK was without effect on the intestinal preparation. The data provide evidence for the existence of the kallikrein-kinin system in a phylogenetically ancient vertebrate group and the greater potency of skate BK compared with the analog skate [Arg(9)]BK suggests that the receptor mediating vascular responses resembles the mammalian B1 receptor more closely than the B2 receptor.     

Replacement of the transmembrane anchor in angiotensin I-converting enzyme (ACE) with a glycosylphosphatidylinositol tail affects activation of the B2 bradykinin receptor by ACE inhibitors

To investigate further the relationship of angiotensin I-converting enzyme (ACE) inhibitors to activation of the B(2) bradykinin (BK) receptor, we transfected Chinese hamster ovary cells to stably express the human receptor and either wild-type ACE (WT-ACE), an ACE construct with most of the cytosolic portion deleted (Cyt-del-ACE), or ACE with a glycosylphosphatidylinositol (GPI) anchor replacing the transmembrane and cytosolic domains (GPI-ACE). BK or its ACE-resistant analogue were the agonists. All activities (arachidonic acid release and calcium mobilization) were blocked by the B(2) antagonist HOE 140. B(2) was desensitized by repeated administration of BK but resensitized to agonist by ACE inhibitors in the cells expressing both B(2) and either WT-ACE or Cyt-del-ACE. In GPI-ACE expressing cells, the B(2) receptor was still activated by the agonists, but ACE inhibitors did not resensitize. Pretreatment with filipin returned the sensitivity to inhibitors. In immunocytochemistry, GPI-ACE showed patchy, uneven distribution on the plasma membrane that was restored by filipin. Thus, ACE inhibitors were inactive as long as GPI-ACE was sequestered in cholesterol-rich membrane domains. WT-ACE and B(2) receptor in Chinese hamster ovary cells co-immunoprecipitated with antibody to receptor, suggesting an interaction on the cell membrane. ACE inhibitors augment BK effects on receptors indirectly only when enzyme and receptor molecules are sterically close, possibly forming a heterodimer.     

Solution conformations of bradykinin antagonists modified with Calpha-Calpha cyclized nonaromatic residues

The conformations of four BK antagonists, [D-Arg 0, Hyp3, Thi5, D-Phe7, Acc8]BK (1), Aaa[D-Arg 0, Hyp3, Thi5, D-Phe7, Acc8]BK (2), [D-Arg 0, Hyp3, Thi5, 8, Apc7]BK (3), and Aaa[D-Arg(0), Hyp(3), Thi(5, 8), Apc7]BK (4) were studied by using 2D NMR spectroscopy and MD simulations with time-averaged (TAV) restraints. According to the results of the NMR measurements, the BK antagonists contain 7-30% of minor conformation resulting from cis/trans isomerization of the peptide bonds preceding either Pro or Hyp residues. The major conformation of each peptide possesses all peptide bonds in trans configuration. Peptides modified with the Apc residue at position 7 (peptides 3 and 4) possess a higher percentage of minor isomer.Peptide 1 exhibits the strongest vasodepressor potency among the analogs studied and as a single one forms the betaII-turn in the 2-5 fragment, which is believed to be crucial for antagonistic activity. This peptide is also the most compact. The radius of gyration (Rg) amounts to 6.9 A and is by ca 1.5 A lower than that of the remaining analogs. With peptide 4, the ST-turn of type I within the Ser6-Thi8 fragment was found.     

Control of myocardial oxygen consumption in transgenic mice overexpressing vascular eNOS

Our objective was to investigate the potential role of selective endothelial nitric oxide (NO) synthase (eNOS) overexpression in coronary blood vessels in the control of myocardial oxygen consumption (MVO2). Transgenic (Tg) eNOS-overexpressing mice (eNOS Tg) (n=22) and wild-type (WT) mice (n=24) were studied. Western blot analysis indicated greater than sixfold increase of eNOS in cardiac tissue. Echocardiography in awake mice indicated no difference in cardiac function between WT and eNOS Tg; however, systolic pressure in eNOS Tg mice decreased significantly (126 +/- 2.3 to 109 +/- 2.3 mmHg; P <0.05), whereas heart rate (HR) was not different. Total peripheral resistance (TPR) was also decreased (9.8 +/- 0.8 to 7.6 +/- 0.4 4 mmHg.ml(-1).min; P <0.05) in eNOS Tg. Furthermore, female eNOS Tg mice showed even lower TPR (7.2 +/- 0.4 mmHg.ml(-1).min) compared with male eNOS mice (8.6 +/- 0.5, mmHg.ml.min(-1); P <0.05). Left ventricular slices were isolated from WT and eNOS Tg mice. With the use of a Clark-type oxygen electrode in an airtight bath, MVO2 was determined as the percent decrease during increasing doses (10(-10) to 10(-4) mol/l) of bradykinin (BK), carbachol (CCh), forskolin (10(-12) to 10(-6) mol/l), or S-nitroso-N-acetyl penicillamine (SNAP; 10(-7) to 10(-4) mol/l). Baseline MVO2 was not different between WT (181 +/- 13 nmol.g(-1).min(-1)) and eNOS Tg (188 +/- 14 nmol.g(-1).min(-1)). BK decreased MVO2 (10(-4) mol/l) in WT by 17% +/- 1.1 and 33% +/- 2.7 in eNOS Tg (P < 0.05). CCh also decreased MVO2, 10(-4) mol/l, in WT by 20% +/- 1.7 and 31% +/- 2.0 in eNOS Tg (P <0.05). Forskolin (10(-6) mol/l) or SNAP (10(-4) mol/l) also decreased MVO2 in WT by 24% +/- 2.8 and 36% +/- 1.8 versus eNOS 31% +/- 1.8 and 37% +/- 3.5, respectively. N-nitro-L-arginine methyl ester (10(-3) mol/l) inhibited the MVO2 reduction to BK, CCh, and forskolin by a similar degree (P <0.05), but not to SNAP. Thus selective overexpression of eNOS in cardiac blood vessels in mice enhances the control of MVO2 by eNOS-derived NO.