Search

The type 2 bradykinin receptor (B2R) is a G protein-coupled receptor (GPCR) in the cardiovascular system, and the dysfunction of B2R leads to inflammation, hereditary angioedema, and pain. Bradykinin and kallidin are both endogenous peptide agonists of B2R, acting as vasodilators to protect the cardiovascular system. Here we determine two cryo-electron microscopy (cryo-EM) structures of human B2R-G q in complex with bradykinin and kallidin at 3.0 Å and 2.9 Å resolution, respectively. The ligand-binding pocket accommodates S-shaped peptides, with aspartic acids and glutamates as an anion trap. The phenylalanines at the tail of the peptides induce significant conformational changes in the toggle switch W283 6.48 , the conserved PIF, DRY, and NPxxY motifs, for the B2R activation. This further induces the extensive interactions of the intracellular loops ICL2/3 and helix 8 with G q proteins. Our structures elucidate the molecular mechanisms for the ligand binding, receptor activation, and G q proteins coupling of B2R., (© 2022. The Author(s).)

Bradykinin and kallidin are endogenous kinin peptide hormones that belong to the kallikrein-kinin system and are essential to the regulation of blood pressure, inflammation, coagulation and pain control. Des-Arg 10 -kallidin, the carboxy-terminal des-Arg metabolite of kallidin, and bradykinin selectively activate two G protein-coupled receptors, type 1 and type 2 bradykinin receptors (B1R and B2R), respectively. The hyperactivation of bradykinin receptors, termed 'bradykinin storm', is associated with pulmonary edema in COVID-19 patients, suggesting that bradykinin receptors are important targets for COVID-19 intervention. Here we report two G protein-coupled complex structures of human B1R and B2R bound to des-Arg 10 -kallidin and bradykinin, respectively. Combined with functional analysis, our structures reveal the mechanism of ligand selectivity and specific activation of the bradykinin receptor. These findings also provide a framework for guiding drug design targeting bradykinin receptors for the treatment of inflammation, cardiovascular disorders and COVID-19., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The plasma contact system contributes to thrombosis in experimental models. Even though our standard blood coagulation tests are prolonged when plasma lacks contact factors, this enzyme system appears to have a minor (if any) role in hemostasis. In this review, we explore the clinical phenotype of C1 esterase inhibitor (C1-INH) deficiency. C1-INH is the key plasma inhibitor of the contact system enzymes, and its deficiency causes hereditary angioedema (HAE). This inflammatory disorder is characterized by recurrent aggressive attacks of tissue swelling that occur at unpredictable locations throughout the body. Bradykinin, which is considered to be a byproduct of the plasma contact system during in vitro coagulation, is the main disease mediator in HAE. Surprisingly, there is little evidence for thrombotic events in HAE patients, suggesting mechanistic uncoupling from the intrinsic pathway of coagulation. In addition, it is questionable whether a surface is responsible for contact system activation in HAE. In this review, we discuss the clinical phenotype, disease modifiers and diagnostic challenges of HAE. We subsequently describe the underlying biochemical mechanisms and contributing disease mediators. Furthermore, we review three types of HAE that are not caused by C1-INH inhibitor deficiency. Finally, we propose a central enzymatic axis that we hypothesize to be responsible for bradykinin production in health and disease., (© 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

The proteolytic processing of neuropeptides has an important regulatory function and the peptide fragments resulting from the enzymatic degradation often exert essential physiological roles. The proteolytic processing generates, not only biologically inactive fragments, but also bioactive fragments that modulate or even counteract the response of their parent peptides. Frequently, these peptide fragments interact with receptors that are not recognized by the parent peptides. This review discusses tachykinins, opioid peptides, angiotensins, bradykinins, and neuropeptide Y that are present in the central nervous system and their processing to bioactive degradation products. These well-known neuropeptide systems have been selected since they provide illustrative examples that proteolytic degradation of parent peptides can lead to bioactive metabolites with different biological activities as compared to their parent peptides. For example, substance P, dynorphin A, angiotensin I and II, bradykinin, and neuropeptide Y are all degraded to bioactive fragments with pharmacological profiles that differ considerably from those of the parent peptides. The review discusses a selection of the large number of drug-like molecules that act as agonists or antagonists at receptors of neuropeptides. It focuses in particular on the efforts to identify selective drug-like agonists and antagonists mimicking the effects of the endogenous peptide fragments formed. As exemplified in this review, many common neuropeptides are degraded to a variety of smaller fragments but many of the fragments generated have not yet been examined in detail with regard to their potential biological activities. Since these bioactive fragments contain a small number of amino acid residues, they provide an ideal starting point for the development of drug-like substances with ability to mimic the effects of the degradation products. Thus, these substances could provide a rich source of new pharmaceuticals. However, as discussed herein relatively few examples have so far been disclosed of successful attempts to create bioavailable, drug-like agonists or antagonists, starting from the structure of endogenous peptide fragments and applying procedures relying on stepwise manipulations and simplifications of the peptide structures., (© 2014 Wiley Periodicals, Inc.)

G protein-coupled receptor (GPCR) signaling is affected by formation of GPCR homo- or heterodimers, but GPCR regulation by other cell surface proteins is not well understood. We reported that the kinin B1 receptor (B1R) heterodimerizes with membrane carboxypeptidase M (CPM), facilitating receptor signaling via CPM-mediated conversion of bradykinin or kallidin to des-Arg kinin B1R agonists. Here, we found that a catalytically inactive CPM mutant that still binds substrate (CPM-E264Q) also facilitates efficient B1R signaling by B2 receptor agonists bradykinin or kallidin. This response required co-expression of B1R and CPM-E264Q in the same cell, was disrupted by antibody that dissociates CPM from B1R, and was not found with a CPM-E264Q-B1R fusion protein. An additional mutation that reduced the affinity of CPM for C-terminal Arg and increased the affinity for C-terminal Lys inhibited the B1R response to bradykinin (with C-terminal Arg) but generated a response to Lys(9)-bradykinin. CPM-E264Q-mediated activation of B1Rs by bradykinin resulted in increased intramolecular fluorescence resonance energy transfer (FRET) in a B1R FRET construct, similar to that generated directly by a B1R agonist. In cytokine-treated human lung microvascular endothelial cells, disruption of B1R-CPM heterodimers inhibited B1R-dependent NO production stimulated by bradykinin and blocked the increased endothelial permeability caused by treatment with bradykinin and pyrogallol (a superoxide generator). Thus, CPM and B1Rs on cell membranes form a critical complex that potentiates B1R signaling. Kinin peptide binding to CPM causes a conformational change in the B1R leading to intracellular signaling and reveals a new mode of GPCR activation by a cell surface peptidase.

The kallikrein-kinin system and the renin-angiotensin system interact at different levels and are linked by various molecules such as angiotensin-converting enzyme which degrades bradykinin into inactive peptides. Here we report that a cysteine-type carboxypeptidase, cathepsin X, is able to modulate the kallikrein-kinin system through carboxyterminal processing of the small peptide hormones bradykinin and kallidin. Both peptides are thereby converted from bradykinin B(2) receptor ligands to bradykinin B(1) receptor specific ligands. Cathepsin X, which has previously been recognized as an inflammatory marker may therefore act as a type I kininase. In addition, we have identified cathepsin X as an alternative possible link between the kallikrein-kinin system and the renin-angiotensin system in that it not only cleaves kinins C-terminally, but also converts angiotensin I to angiotensin II.

The kinin B(1) receptor plays an important role in mediating the inflammatory effects of the kallikrein-kinin pathway. The recent development of orally available non-peptidic antagonists and genetically modified mice deficient in B(1) receptor expression have demonstrated that the receptor plays a pivotal role in the cellular, particularly neutrophil, recruitment associated with an acute inflammatory response. These tools have also enabled elucidation of the pathways involved in mediating this effect and have highlighted a major role for chemokines, particularly CXCL5 and CCL2. Neutrophil recruitment is involved in the pathogenesis of renal disease and has very recently been implicated in the early stages of atherosclerosis. In this review we discuss the most recent evidence linking the B(1) receptor with the pathogenesis of these two inflammatory cardiovascular diseases and highlight the therapeutic potential of the kinin B(1) receptor in these disease states.

Insulin degrading enzyme (IDE) utilizes a large catalytic chamber to selectively bind and degrade peptide substrates such as insulin and amyloid beta (Abeta). Tight interactions with substrates occur at an exosite located approximately 30 A away from the catalytic center that anchors the N-terminus of substrates to facilitate binding and subsequent cleavages at the catalytic site. However, IDE also degrades peptide substrates that are too short to occupy both the catalytic site and the exosite simultaneously. Here, we use kinins as a model system to address the kinetics and regulation of human IDE with short peptides. IDE specifically degrades bradykinin and kallidin at the Pro/Phe site. A 1.9 A crystal structure of bradykinin-bound IDE reveals the binding of bradykinin to the exosite and not to the catalytic site. In agreement with observed high K(m) values, this suggests low affinity of bradykinin for IDE. This structure also provides the molecular basis on how the binding of short peptides at the exosite could regulate substrate recognition. We also found that human IDE is potently inhibited by physiologically relevant concentrations of S-nitrosylation and oxidation agents. Cysteine-directed modifications play a key role, since an IDE mutant devoid of all 13 cysteines is insensitive to the inhibition by S-nitrosoglutathione, hydrogen peroxide, or N-ethylmaleimide. Specifically, cysteine 819 of human IDE is located inside the catalytic chamber pointing toward an extended hydrophobic pocket and is critical for the inactivation. Thiol-directed modification of this residue likely causes local structural perturbation to reduce substrate binding and catalysis.

Unlabelled: An upregulation of the endogenous opioid, dynorphin A, in the spinal cord is seen in multiple experimental models of chronic pain. Recent findings implicate a direct excitatory action of dynorphin A at bradykinin receptors to promote hyperalgesia in nerve injured rats, and its upregulation may promote, rather than counteract, enhanced nociceptive input due to injury. Here we examined a model of inflammatory pain by unilateral injection of complete Freund's adjuvant (CFA) into the rat hind paw. Rats exhibited tactile hypersensitivity and thermal hyperalgesia in the inflamed paw by 6 hours after CFA injection, whereas a significant elevation of prodynorphin transcripts in the lumbar spinal cord was seen at day 3 but not at 6 hours. Thermal hyperalgesia at day 3, but not at 6 hours, after CFA injection was blocked by intrathecal administration of anti-dynorphin antiserum or by bradykinin receptor antagonists. The antihyperalgesic effect of the latter was not due to de novo production of bradykinin or upregulation of spinal bradykinin receptors. These data suggest that elevated spinal dynorphin on peripheral inflammation mediates chronic inflammatory hyperalgesia. The antihyperalgesic effect of bradykinin receptor antagonists requires the presence of upregulated spinal dynorphin but not of de novo production of bradykinin, supporting our hypothesis that pathological levels of dynorphin may activate spinal bradykinin receptors to mediate inflammatory hyperalgesia., Perspective: This study shows that chronic peripheral inflammation induces a significant upregulation of the endogenous opioid peptide dynorphin. Elevated levels of spinal dynorphin and activation of spinal bradykinin receptors are essential to maintain inflammatory hyperalgesia. The results suggest that blockade of spinal bradykinin receptors may have therapeutic potential in chronic inflammatory pain.

Background and Purpose: Kinins are implicated in many pathophysiological conditions, and recent evidence has suggested their involvement in colitis. This study assessed the role of the kinin B1 receptors in a mouse model of colitis., Experimental Approach: Colitis was induced in mice by 2,4,6-trinitrobenzene sulphonic acid (TNBS), and tissue damage and myeloperoxidase activity were assessed. B1 receptor induction was analysed by organ bath studies, binding assay and reverse transcription PCR., Key Results: TNBS-induced colitis was associated with tissue damage, neutrophil infiltration and time-dependent increase of colon B1 receptor-mediated contraction, with the maximal response observed at 72 h. The upregulation of the B1 receptor at this time point was also confirmed by means of binding studies. B1 receptor mRNA levels were elevated as early as 6 h after colitis induction and remained high for up to 48 h. TNBS-evoked tissue damage and neutrophil influx were reduced by the selective B1 receptor antagonist SSR240612, and in B1 receptor knockout mice. In vivo treatment with inhibitors of protein synthesis, nuclear factor-kappaB activation, inducible nitric oxide synthase (iNOS) or tumour necrosis factor alpha (TNFalpha) significantly reduced B1 receptor agonist-induced contraction. Similar results were observed in iNOS and TNF receptor 1-knockout mice., Conclusions and Implications: These results provide convincing evidence on the role of B1 receptors in the pathogenesis of colitis. Therefore, the blockade of kinin B1 receptors might represent a new therapeutic option for treating inflammatory bowel diseases.

Kinin B1 receptor (B1R) expression is induced by injury or inflammatory mediators, and its signaling produces both beneficial and deleterious effects. Kinins cleaved from kininogen are agonists of the B2R and must be processed by a carboxypeptidase to generate B1R agonists des-Arg(9)-bradykinin or des-Arg(10)-kallidin. Carboxypeptidase M (CPM) is a membrane protein potentially well suited for this function. Here we show that CPM expression is required to generate a B1R-dependent increase in [Ca(2+)](i) in cells stimulated with B2R agonists kallidin or bradykinin. CPM and the B1R interact on the cell membrane, as shown by co-immunoprecipitation, cross-linking, and fluorescence resonance energy transfer analysis. CPM and B1R are also co-localized in lipid raft/caveolin-enriched membrane fractions, as determined by gradient centrifugation. Treatment of cells co-expressing CPM and B1R with methyl-beta-cyclodextrin to disrupt lipid rafts reduced the B1R-dependent increase in [Ca(2+)](i) in response to B2R agonists, whereas cholesterol treatment enhanced the response. A monoclonal antibody to the C-terminal beta-sheet domain of CPM reduced the B1R response to B2R agonists without inhibiting CPM. Cells expressing a novel fusion protein containing CPM at the N terminus of the B1R also increased [Ca(2+)](i) when stimulated with B2R agonists, but the response was not reduced by methyl-beta-cyclodextrin or CPM antibody. A B1R- and CPM-dependent calcium signal in response to B2R agonist bradykinin was also found in endothelial cells that express both proteins. Thus, a close relationship of B1Rs and CPM on the membrane is required for efficiently generating B1R signals, which play important roles in inflammation.

In the kallikrein-kinin and renin-angiotensin systems the main receptors, B1 and B2 (kinin receptors) and AT1 and AT2 (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B1 agonists Des-Arg9-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg9-BK or Des-Arg10-KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT1 agonist (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) have the same two residues at the C-terminal region (i.e. Pro-Phe), we hypothesized that TM V and TM VI of the B1 receptor could play an essential role in agonist binding and activity, being these regions receptor sites for binding the C-terminal sequences of Des-Arg-kinins similarly to that observed to AT1 receptor. To investigate this hypothesis, we replaced Arg212 for Ala at the top of the TM V and the sequence 274-282 (CPYHFFAFL) in TM VI of the rat kinin B1 receptor by the B2 receptor homologous sequence, 289-297 (FPFQISTFL) and subsequently analyzed the consequences of these mutations by competition binding and functional assays. Despite correct expression, observed at the mRNA and protein level by RT-PCR and confocal microscopy, respectively, no agonist binding and function was verified for the mutated receptors. Therefore, our results suggest an important role for Arg212 in the TM V and a region of TM VI of rat B1 receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII.

Aminopeptidase A (APA) plays an important role in the regulation of blood pressure by mediating angiotensin II degradation in the renin-angiotensin system. The Ca2+-induced modulation of enzymatic activity is the most characteristic feature of APA among the M1 family of aminopeptidases. In this study, we used site-directed mutagenesis for any residues responsible for the Ca2+ modulation of human APA. Alignment of sequences of the M1 family members led to the identification of Asp-221 as a significant residue of APA among the family members. Replacement of Asp-221 with Asn or Gln resulted in a loss of Ca2+ responsiveness toward synthetic substrates. These enzymes were also unresponsive to Ca2+ when peptide hormones, such as angiotensin II, cholecystokinin-8, neurokinin B, and kallidin, were employed as substrates. These results suggest that the negative charge of Asp-221 is essential for Ca2+ modulation of the enzymatic activity of APA and causes preferential cleavage of acidic amino acid at the N-terminal end of substrate peptides.

The bradykinin B(1) receptor plays a critical role in chronic pain and inflammation, although efforts to demonstrate efficacy of receptor antagonists have been hampered by species-dependent potency differences, metabolic instability, and low oral exposure of current agents. The pharmacology, pharmacokinetics, and analgesic efficacy of the novel benzamide B(1) receptor antagonist 7-chloro-2-[3-(9-pyridin-4-yl-3,9-diazaspiro[5.5]undecanecarbonyl)phenyl]-2,3-dihydro-isoindol-1-one (ELN441958) is described. ELN441958 competitively inhibited the binding of the B(1) agonist ligand [(3)H]desArg(10)-kallidin ([(3)H]DAKD) to IMR-90 human fibroblast membranes with high affinity (K(i) = 0.26 +/- 0.02 nM). ELN441958 potently antagonized DAKD (but not bradykinin)-induced calcium mobilization in IMR-90 cells, indicating that it is highly selective for B(1) over B(2) receptors. Antagonism of agonist-induced calcium responses at B(1) receptors from different species indicated that ELN441958 is selective for primate over rodent B(1) receptors with a rank order potency (K(B), nanomolar) of human (0.12 +/- 0.02) approximately rhesus monkey (0.24 +/- 0.01) > rat (1.5 +/- 0.4) > mouse (14 +/- 4). ELN441958 had good permeability and metabolic stability in vitro consistent with high oral exposure and moderate plasma half-lives in rats and rhesus monkeys. Because ELN441958 is up to 120-fold more potent at primate than at rodent B(1) receptors, it was evaluated in a primate pain model. ELN441958 dose-dependently reduced carrageenan-induced thermal hyperalgesia in a rhesus monkey tail-withdrawal model, with an ED(50) approximately 3 mg/kg s.c. Naltrexone had no effect on the antihyperalgesia produced by ELN441958, indicating a lack of involvement of opioid receptors. ELN441958 is a novel small molecule bradykinin B(1) receptor antagonist exhibiting high oral bioavailability and potent systemic efficacy in rhesus monkey inflammatory pain.

The kallikrein-kinin system is subdivided into the plasma and tissue-kallikrein-kinin system, with bradykinin (BK) and kallidin (KAL) (Lys(0)-bradykinin) as functional peptides. This occurs in both humans and other mammals. Both peptides are released by plasma and tissue-kallikrein. BK, but not KAL, has been detected in rats until now. One can explain this observation by the structural differences found in the sequence of rat high- and low-molecular kininogen containing an Arg-residue instead of a Lys-residue in front of the N-terminus of the BK sequence. Nevertheless, we were able to measure a kallidin-like peptide (KLP), in rat plasma and urine, using a specific KAL antiserum. In order to confirm our data, we isolated low-molecular-weight kininogen from rat plasma and incubated it with purified rat glandular kallikrein. The generated peptide was retained on a high-pressure liquid chromatography column and displaced by an excess of angiotensin I. The KLP-containing fraction was identified with the KLP radioimmunoassay. A specific ion signal with a mass to charge ratio (m/z) of 1216.73 was detected with matrix-assisted laser desorption/ionization mass spectrometry. As proposed earlier, the structure of this peptide is Arg(1)-KAL, instead of Lys(1)-KAL. The structural similarity between the Lys- and the Arg-residue explains the high crossreactivity (80%) of KLP with the specific KAL antibody. The incubation of KLP with angiotensin-converting enzyme yields two molecules with masses of 913.4 and 729.3 containing the sequence H-Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-OH and H-Arg-Arg-Pro-Pro-Gly-Phe-OH. The enzymatic cleavage could be inhibited by captopril. The data suggest that in rats, as in other mammals, the tissue kallikrein-kinin system mediates its physiological effects via a kallidin-like peptide, which is Arg(1)-kallidin (Arg(0)-bradykinin).

Transforming growth factor (TGF)-beta and des-Arg(10)-kallidin stimulate the expression of connective tissue growth factor (CTGF), a matrix signaling molecule that is frequently overexpressed in fibrotic disorders. Because the early signal transduction events regulating CTGF expression are unclear, we investigated the role of Ca(2+) homeostasis in CTGF mRNA expression in TGF-beta1- and des-Arg(10)-kallidin-stimulated human lung myofibroblasts. Activation of the kinin B1 receptor with des-Arg(10)-kallidin stimulated a rise in cytosolic Ca(2+) that was extracellular Na(+)-dependent and extracellular Ca(2+)-dependent. The des-Arg(10)-kallidin-stimulated increase of cytosolic Ca(2+) was blocked by KB-R7943, a specific inhibitor of Ca(2+) entry mode operation of the plasma membrane Na(+)/Ca(2+) exchanger. TGF-beta1 similarly stimulated a KB-R7943-sensitive increase of cytosolic Ca(2+) with kinetics distinct from the des-Arg(10)-kallidin-stimulated Ca(2+) response. We also found that KB-R7943 or 2',4'-dichlorobenzamil, an amiloride analog that inhibits the Na(+)/Ca(2+) exchanger activity, blocked the TGF-beta1- and des-Arg(10)-kallidin-stimulated increases of CTGF mRNA. Pretreatment with KB-R7943 also reduced the basal and TGF-beta1-stimulated levels of alpha1(I) collagen and alpha smooth muscle actin mRNAs. These data suggest that, in addition to regulating ion homeostasis, Na(+)/Ca(2+) exchanger acts as a signal transducer regulating CTGF, alpha1(I) collagen, and alpha smooth muscle actin expression. Consistent with a more widespread role for Na(+)/Ca(2+) exchanger in fibrogenesis, we also observed that KB-R7943 likewise blocked TGF-beta1-stimulated levels of CTGF mRNA in human microvascular endothelial and human osteoblast-like cells. We conclude that Ca(2+) entry mode operation of the Na(+)/Ca(2+) exchanger is required for des-Arg(10)-kallidin- and TGF-beta1-stimulated fibrogenesis and participates in the maintenance of the myofibroblast phenotype.

To determine the muscular concentration of bradykinin and kallidin during static contraction, microdialysis probes were implanted bilaterally in the trapezius muscles of healthy women. Three hours after probe implantation, 200 microM of the angiotensin-converting enzyme (ACE) inhibitor enalaprilat were added to the perfusion solution in one of the sides for 30 min. Thirty minutes later, the subjects performed a sustained bilateral shoulder abduction at 10% of the maximal voluntary contraction until exhaustion. This protocol was repeated twice, with an interval of at least 17 days. High intersession repeatability was observed in the concentration of bradykinin but not of kallidin. Enalaprilat induced a significant increase in bradykinin levels in the dialysate, without affecting kallidin levels. The sustained contraction induced a significant increase in dialysate levels of both kinin peptides. The contraction also induced a significant increase in pain ratings, as measured by a visual analog scale. During contraction, positive correlations were found between pain ratings and levels of kinin peptides in dialysate, predominantly in the side previously perfused with enalaprilat. Subjects with the higher pain ratings also showed larger increases in kinin peptides in the side previously perfused with enalaprilat. The present results show that both plasma and tissue kinin-kallikrein are activated during muscle contraction, but that their metabolic pathways are differently regulated during rest and contraction, because they showed a different response to ACE inhibition. They also indicate that intramuscular kinin peptides levels, and ACE activity, may contribute to muscle pain.

Bradykinin and Lys-bradykinin are potent peptide mediators implicated in several physiopathological effects in mammals. They act through activation of G-protein-coupled constitutive B(2) or inducible kinin B(1) receptors linked to signaling pathways involving increased intracellular Ca(++) concentrations and/or release of mediators including arachidonic acid metabolites, NO and EDHF. In the cardiovascular system, the kallikrein-kinin system exerts a fine control of vascular smooth muscle tone and arterial blood pressure, and plays a significant cardioprotective effect. This has been lately confirmed in experimental studies employing transgenic mice overexpressing human tissue kallikrein and animals with knockout of kinin B(1) and B(2) receptor gene. Disturbances in this system are associated with arterial hypertension, myocardial ischaemia and other clinical complications. Inhibitors of kininase II (angiotensin-converting enzyme) have been prescribed successfully to patients with cardiovascular diseases, but there is still a great interest in developing drugs or pharmacological strategies that augment the activity of kininogen-kallikrein-kinin system in pathological conditions. Delivery of adenovirus vector containing the human tissue kallikrein gene (gene kallikrein therapy) has emerged as a great potential to satisfy these conditions. This review provides a summary of plasma and tissue kallikrein-kinin system, focusing on the pharmacological properties, kinin receptors and drugs reported to interfere with their actions. The modulatory effects of the kallikrein-kinin system on cardiovascular system, particularly in regulating smooth muscle tone and arterial blood pressure and in preventing myocardium ischaemia have also been explored in the review.

Phosphoglucose isomerase (PGI) with a novel lysyl aminopeptidase (LysAP) activity was recently isolated and partially characterized from the human pathogen, Vibrio vulnificus. This PGI is a heterodimer consisting of 60.8- and 23.4-kDa subunits, which together provide LysAP activity. The present study further characterizes the complex structure and functions of Vibrio PGI and draws parallels with rabbit and human PGI. A Proscan search of Vibrio PGI revealed 194 different structural motifs of which 124 and 127 were also found in rabbit and human PGI, respectively. Vibrio PGI contains motifs for the serine, histidine and aspartic acid active sites of the subtilase family of serine proteases which form a putative catalytic triad consisting of His534 and Ser159 on the 60.8-kDa subunit and Asp53 on the 23.4-kDa subunit. Together, they form one LysAP site for each heterodimer. Each active site motif is overlapped by motifs for EF-hand calcium binding domains. The LysAP activity was inhibited by the addition of > or =10 microM Ca2+, suggesting that the EF-hand calcium-binding domain may be a natural regulatory region for LysAP activity. In contrast, PGI's isomerase activity was enhanced at Ca2+ concentrations >100 microM. PGI-LysAP cleaved the amino-terminal lysyl residue from des-Arg10-kallidin producing des-Arg9-bradykinin; therefore, Vibrio PGI-LysAP may serve as a virulence factor to enhance Vibrio invasiveness. Together, these data provide a framework to account for PGI's LysAP activity and further demonstrate the structural complexity and functional importance of this molecule.

Compound A (N-[2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]ethyl]-2-[(2R)-1-(2-napthylsulfonyl)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetamide) is a member of a new class of aryl sulfonamide dihydroquinoxalinone bradykinin B1 receptor antagonists that should be useful pharmacological tools. Here we report on some of the pharmacological properties of compound A as well as the characterization of [35S]compound A as the first nonpeptide bradykinin B1 receptor radioligand. Compound A inhibited tritiated peptide ligand binding to the cloned human, rabbit, dog, and rat bradykinin B1 receptors expressed in CHO cells with Ki values of 0.016, 0.050, 0.56, and 29 nM, respectively. It was inactive at 10 microM in binding assays with the cloned human bradykinin B2 receptor. In functional antagonist assays with the cloned bradykinin B1 receptors, compound A inhibited agonist-induced signaling with activities consistent with the competition binding results, but had no antagonist activity at the bradykinin B2 receptor. Compound A was also found to be a potent antagonist in a rabbit aorta tissue bath preparation and to effectively block des-Arg9 bradykinin depressor responses in lipopolysaccharide-treated rabbit following intravenous administration. The binding of [35S]compound A was evaluated with the cloned bradykinin B1 receptors. In assays with human, rabbit, and dog receptors, [35S]compound A labeled a single site with Kd values of 0.012, 0.064, and 0.37 nM, respectively, and with binding site densities equivalent to those obtained using the conventional tritiated peptide ligands. Binding assays with the cloned rat bradykinin B1 receptor were not successful, presumably due to the low affinity of the ligand for this species receptor. There was no specific binding of the ligand detected in CHO cells expressing the human bradykinin B2 receptor. In assays with the cloned human bradykinin B1 receptor, the pharmacologies of the binding of [35S]compound A and [3H][Leu9]des-Arg10-kallidin were the same. The high signal-to-noise ratio obtained with [35S]compound A will allow this ligand to be a very useful tool for future investigations of the bradykinin B1 receptor.

We hypothesized that the inducible kinin B(1) receptor (B(1)R) is rapidly cleared from cells when its synthesis subsides. The agonist-independent degradation of the rabbit B(1)Rs and related B(2) receptors (B(2)Rs) was investigated. Endocytosis of the B(1)R-yellow fluorescent protein (YFP) conjugate was more intense than that of B(2)R-green fluorescent protein (GFP) based on fluorescence accumulation in HEK 293 cells treated with a lysosomal inhibitor. The cells expressing B(1)R-YFP contained more GFP/YFP-sized degradation product(s) than those expressing B(2)R-GFP (immunoblot, antibodies equally reacting with both fluorescent proteins). The binding site density of B(1)R-YFP decreased in the presence of protein synthesis or maturation inhibitors (anisomycin, brefeldin A), whereas that of B(2)R-GFP remained constant. Wild-type B(1)Rs were also cleared faster than B(2)Rs in rabbit smooth muscle cells treated with metabolic inhibitors. Contractility experiments based on brefeldin A-treated isolated rabbit blood vessels also functionally support that B(1)Rs are more rapidly eliminated than B(2)Rs (decreased maximal effect of agonist over 2 h). The highly regulated B(1)R is rapidly degraded, relative to the constitutive B(2)R.

The pharmacological properties of the kinin B1 receptor in binding the endogenous kinin peptides are known to differ across species. Molecular cloning has revealed that these pharmacological differences arise from the diversity within the BDKRB gene. In this report, the molecular diversity of the human BDKRB1 gene is expanded by the identification of eight single nucleotide polymorphisms (SNPs) in the coding sequence of the receptor, three of which change the amino acid sequence of the receptor. The molecular cloning and pharmacological characterization of two primate B1 receptors, rhesus and African Green monkey, reveals that they exhibit the same high degree of selectivity for des-Arg10 kallidin (Lys-bradykinin) relative to des-Arg9 bradykinin that is observed with the human kinin B1 receptor. Previous mutagenesis studies of the human B1 receptor have implicated extracellular domain (EC) IV in conferring this selectivity for des-Arg10 kallidin, by interacting with the N-terminal Lys residue of the peptide. The pharmacological analysis of chimeric B1 receptors, in which EC-IV of the human B1 receptor is replaced with the corresponding domain of either rat or dog, supports the proposal that EC-IV is an important determinant in conferring ligand selectivity.

In order to study the effects of the manipulation of various factors related to muscular activity on the concentration of kinins in muscular tissue, a microdialysis probe was implanted in the adductor muscle of the hindlimb in anaesthetized rats. After collection of baseline samples, the perfusion fluid was changed to a Ringer solution containing sodium lactate (10 or 20 mM), adenosine (50 or 100 microM) or a lower pH (7.0 or 6.6). Whereas perfusion with lactate did not have any significant effect on the concentration of kinins in the dialysate, the perfusion with a lower pH or with adenosine dose-dependently increased the kinin content in the samples. In a second microdialysis experiment, by using specific radioimmunoassays (RIA) for bradykinin and kallidin, we observed that about 70 % of the total kinins dialysed from rat muscle are a kallidin-like peptide. Also, the simultaneous perfusion with 100 microM caffeine totally abolished the increase in kinin levels induced by the perfusion at pH 6.6. In a third experiment, soleus muscles from rat were stimulated in vitro during 30 min in the presence or absence of 77 microM caffeine. Electrically stimulated contraction, but not the addition of 10 mU ml(-1) insulin, induced an increase in the concentration of the kallidin-like peptide in the buffer. This effect was totally prevented by the addition of the adenosine antagonist caffeine. These results show that a kallidin-like peptide is released from rat muscle, and that its production is enhanced by muscle activity. Furthermore, the increase in kinin peptides during muscle contraction may be mediated by an increase in adenosine levels.

Bradykinin B1 and B2 receptors, members of the G-protein coupled receptor superfamily, are involved in inflammation and pain. Chinese hamster ovary (CHO) cells stably expressing the human B2 bradykinin receptor (CHO-B2) were used to characterize the signal transduction pathways associated with this receptor and its regulation. The selective B2 antagonist [3H]NPC17731 but not the selective B1 antagonist [3,4-prolyl-3,4-(3)H(N)]-[des-Arg10,Leu9]kallidin ([3H]DALKD) bound to CHO-B2 cell membranes with a Kd of 0.77 nM and a Bmax of 1087 fmol/mg protein. [3H]NPC17731 binding was inhibited by bradykinin ligands in the order: NPC17731 > bradykinin > kallidin >> DALKD > [des-Arg10] kallidin (DAKD), consistent with the pharmacological profile of B2 bradykinin receptors. The B2 agonist bradykinin and the B1/B2 agonist kallidin, but not the B1 agonist DAKD, increased [35S]GTP gamma S binding to the CHO-B2 cell membranes. The B2 bradykinin receptors were co-immunoprecipitated with G alpha q/11. In response to bradykinin stimulation, coupling of the B2 receptors to G alpha q/11 was increased by 10-fold. Bradykinin and kallidin, but not DAKD, induced intracellular calcium release in CHO-B2 cells, which was blocked by NPC17731 but not by DALKD. These results demonstrate that B2 bradykinin receptors directly coupled to G alpha q/11 to regulate intracellular calcium release. CHO-B2 cell is a useful system that can be applied to study the effect of potential agents that may influence the B2 receptor function.

The bradykinin B1 receptor (BKB1R) gene is expressed in selected tissues such as lung and kidney. In these tissues it is expressed at a very low level until induced by inflammatory mediators. Our aim has been to understand the mechanism of this regulatory process. A human BKB1R minigene was constructed. It contained a 1.8 kb promoter, the entire exon I, 1.5 kb of intron I, the entire exon II and intron II, and the luciferase gene as a reporter. Transient transfection of the minigene into SV40-transformed IMR90 cells (IMRSV) resulted in a promoter activity which was activated by the mediators, lipopolysaccharide and (LPS) desArg(10)-kallidin. In contrast, these mediators did not induce the activity of the 1.8 kb promoter construct alone. Thus, motifs exclusive of the promoter such as 5'-UTR and/or intron regions are required for mediator-induced expression of this gene. Promoter activities of both the minigene and the 1.8 kb promoter construct were enhanced in a dose-dependent manner upon cotransfection with c-Jun. Furthermore, cotransfecting c-Jun with the minigene achieved the maximal promoter activity with no further increase in response to mediators. Conversely, the induction of the minigene promoter activity by mediators was abolished upon cotransfection with a dominant negative mutant of c-Jun. Other experiments suggest that multiple AP-1 sites are interactive with the c-Jun upregulation of this gene. Taken together, these results point to c-Jun as a key intermediary in the activation of the expression of this gene by mediators. However, participation of motifs outside of the promoter are necessary to obtain this inducible expression., (Copyright 2001 Wiley-Liss, Inc.)

Expression of the kinin B1 receptor is up-regulated in chronic inflammatory and fibrotic disorders; however, little is known about its role in fibrogenesis. We examined human embryonic lung fibroblasts that constitutively express the B1 receptor and report that engagement of the B1 receptor by des-Arg(10)-kallidin stabilized connective tissue growth factor (CTGF) mRNA, stimulated an increase in alpha1(I) collagen mRNA, and stimulated type I collagen production. These events were not observed in B2 receptor-activated fibroblasts. In addition, B1 receptor activation by des-Arg(10)-kallidin induced a rise in cytosolic Ca(2+) that is consistent with B1 receptor pharmacology. Our results show that the des-Arg(10)-kallidin-stimulated increase in alpha1(I) collagen mRNA was time- and dose-dependent, with a peak response observed at 20 h with 100 nM des-Arg(10)-kallidin. The increase in CTGF mRNA was also time- and dose-dependent, with a peak response observed at 4 h with 100 nM des-Arg(10)-kallidin. The increase in CTGF mRNA was blocked by the B1 receptor antagonist des-Arg(10),Leu(9)-kallidin. Inhibition of protein synthesis by cycloheximide did not block the des-Arg(10)-kallidin-induced increase in CTGF mRNA. These results suggest that engagement of the kinin B1 receptor contributes to fibrogenesis through increased expression of CTGF.

To investigate the molecular basis for the specificity of ligand recognition in human kinin B(1) (B(1)R) and B(2) (B(2)R) receptors, we constructed a series of chimeric receptors by progressively replacing, from the N to the C terminus, the human B(2)R domains by their B(1) counterparts. The chimeric construct possessing the C-terminal tail and the transmembrane domain VII (TM VII) of the B(2)R (construct 6) displayed 7- and 20- fold decreased affinities for the B(1) agonist [(3)H]desArg(10)-kallidin (desArg(10)-KD) and the B(1) antagonist [(3)H]desArg(10)-[Leu(9)]-KD respectively, as compared with the wild-type B(1)R. Moreover, the substitution of the B(1) TM VII by its B(2) homologue TM increased the affinity for the pseudopeptide antagonists, Hoe140 and NPC 567. High affinity for desArg(10)-KD binding was fully regained when the B(2) residue Thr(287) was replaced in construct 6 by the corresponding B(1) Leu(294) residue. When the B(2) residue Tyr(295) was exchanged with the corresponding B(1) Phe(302), high affinity binding for both agonist and antagonist was recovered. Moreover, the L294T and F302Y mutant B(1)R exhibited 69- and 6.5-fold increases, respectively, in their affinities for the B(2) receptor antagonist, Hoe140. Therefore we proposed that Leu(294) and Phe(302) residues, which may not be directly involved in the binding of B(1)R ligands and, hence, their Thr(287) and Tyr(295) B(2) counterparts, are localized in a receptor region, which plays a pivotal role in the binding selectivity of the peptide or pseudopeptide kinin ligands.

Angiotensin-converting enzyme (ACE) converts angiotensin I (Ang I) to angiotensin II (Ang II) and metabolizes bradykinin and kallidin peptides. Decreased Ang II levels and increased kinin peptide levels are implicated in the mediation of the therapeutic effects of ACE inhibition. However, alternative non-ACE pathways of Ang II formation have been proposed to predominate in human heart. We investigated the effects of ACE inhibition on cardiac tissue levels of angiotensin and kinin peptides. High-performance liquid chromatography-based radioimmunoassays were used to measure angiotensin peptides and hydroxylated and nonhydroxylated bradykinin and kallidin peptides in right atrial appendages of subjects who had been prepared for cardiopulmonary bypass. Peptide levels in subjects who received ACE inhibitor therapy were compared with those who did not receive ACE inhibitor therapy. ACE inhibition reduced Ang II levels, which was associated with an 80% reduction in the Ang II/Ang I ratio. ACE inhibition did not modify either bradykinin or kallidin peptide levels or the bradykinin-(1-7)/bradykinin-(1-9) ratio. The 80% reduction in the Ang II/Ang I ratio by ACE inhibition indicated a primary role for ACE in the conversion of Ang I to Ang II in atrial tissue. These data support a role for reduced Ang II levels but do not support a role for increased kinin peptide levels in mediating the direct cardiac effects of ACE inhibition.

This report describes the characterisation of cloned rat and human bradykinin B1 receptors in African green monkey kidney fibroblast (Cos-7) cells. A ligand binding assay with [3H]des-Arg10-kallidin was used to compare their pharmacology with respect to known bradykinin B1 and B2 receptor ligands. In addition, the pharmacology of T-kinin and its' derivative des-Arg11-T-kinin was investigated. The cloned rat receptor had a similar pharmacology to that of the recently described mouse receptor and differs from that described for the human receptor. The rat receptor had a higher affinity for des-Arg11-T-kinin than the human receptor. These differences in pharmacological properties may relate to the presence of T-kinin, bradykinin and their des-Arg derivatives as the major physiological peptides in rat and the predominance of kallidin and its derivatives in human. We confirm that the rat bradykinin B1 receptor gene is organised in a two exon structure and differs from the human gene which has a three exon structure and we further examine the inducible expression of this gene in a wide range of tissues using Northern blotting.

Biologically active kinin peptides are released from precursor kininogens by kallikreins. Kinins act on kinin receptors to mediate diverse biological functions including smooth muscle contraction, inflammation, pain and mitogenicity. All components of the kallikrein-kinin system exist in human male genital secretions suggesting that these molecules participate in physiological and pathophysiological genitourinary function. The objective of this study was to assess the consequences of kinin action on prostate cells. Primary cultures of prostate secretory epithelial (PE) and prostate fibromuscular stromal (PS) cells were established from human prostate tissue. Transcripts encoding both the human B1 and B2 bradykinin receptor subtypes were detected in human prostate transition-zone tissue and in cultured cells by RT-PCR. In receptor binding assays, the B1 subtype predominated on PE cell membranes and the B2 subtype predominated on PS cell membranes. In PS cells, but not in PE cells, BK induced significant inositol phosphate accumulation and [3H]-thymidine uptake. These responses were mediated through the B2 receptor subtype. The use of signal transduction inhibitors indicated that mitogenic activation by BK occurred through both protein kinase C (PKC) and protein tyrosine kinase dependent mechanisms. PMA (phorbol 12-myristate 13-acetate) produced maximal [3H]-thymidine uptake by PS cells, resulted in cell elongation and caused the alpha-actin fibres present in PS smooth muscle cells to became organized into parallel arrays along the length of the elongated cells. In summary, the prostate contains a functional kallikrein-kinin system, which could be significant in physiological and pathophysiological prostate function.

Background: Since kinins kallidin (KD) and bradykinin (BK) appear to have cardioprotective effects ranging from improved hemodynamics to antiproliferative effects, inhibition of kinin-degrading enzymes should potentiate such effects. Indeed, it is believed that this mechanism is partly responsible for the beneficial effects of angiotensin-converting enzyme (ACE) inhibitors. In the heart, enzymes other than ACE may contribute to local degradation of kinins. The purpose of this study was to investigate which enzymes are responsible for the degradation of KD and BK in human heart tissue., Methods and Results: Cardiac membranes were prepared from the left ventricles of normal (n=5) and failing (n=10) hearts. The patients had end-stage congestive heart failure as the result of coronary heart disease or idiopathic dilated cardiomyopathy. Heart tissue was incubated with KD or BK in the presence or absence of enzyme inhibitors. We found no difference in the enzymes responsible for kinin metabolism or their activities between normal and failing hearts. Thus KD was mostly converted into BK by the aminopeptidase M-like activity. When BK was used as substrate, it was converted into an inactive metabolite BK-(1-7) mostly (80% to 90%) by the neutral endopeptidase (NEP) activity, with ACE unexpectedly playing only a minor role. The low enzymatic activity of ACE in the cardiac membranes, compared with that of NEP, was not due to chronic ACE inhibitor therapy, because the cardiac ACE activities of patients, whether receiving ACE inhibitors or not, and of normal subjects were all equal., Conclusions: The present in vitro study shows that in human cardiac membranes, the most critical step in kinin metabolism, that is, inactivation of BK, appears to be mediated mostly by NEP. This observation suggests a role for NEP in the local control of BK concentration in heart tissue. Thus inhibition of cardiac NEP activity could be cardioprotective by elevating the local concentration of BK in the heart.

We investigated the role of bradykinin B receptors in inducing urinary bladder contraction and maintaining bladder compliance in anaesthetized rats following cyclophosphamide-induced bladder inflammation and the influence of dexamethasone treatment on these responses. In the group treated with cyclophosphamide the amplitude of the contraction induced by the selective bradykinin B1 receptor agonist des-Arg9-bradykinin was larger than that in controls and dexamethasone prevented the up-regulation of this response induced by inflammation. The specific binding of [3H]des-Arg10-kallidin to bladder membranes was only detected in cyclophosphamide-treated rats: this binding was prevented by dexamethasone pretreatment. The bladder contraction induced by des-Arg9-bradykinin in cyclophosphamide-treated rats was antagonized by the bradykinin B1 receptor antagonist des-Arg9-D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (des-Arg10-Hoe 140). Cyclophosphamide treatment increased the bladder weight and dexamethasone reversed this effect. Bladder compliance was decreased in the bladder inflammation group and this effect was partially reversed by dexamethasone pretreatment. Neither des-Arg10-Hoe 140 nor the combined administration of des-Arg10Hoe 140 and the selective bradykinin B2 receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (Hoe 140) affected bladder compliance, thus excluding a role of kinins in the maintenance of bladder tone during inflammation. These results indicate that: (1) dexamethasone pretreatment ameliorates cyclophosphamide-induced bladder inflammation: (2) dexamethasone pretreatment prevents cyclophosphamide-induced up-regulation of bradykinin B receptors; (3) kinins do not contribute to the increased vesical tone during inflammation.

Kallidin (KD) is an important vasoactive kinin whose physiological effects are strongly dependent on its degradation through local kininases. In the present study, we examined the spectrum of these enzymes and their contribution to KD degradation in isolated perfused rat hearts. By inhibiting angiotensin-converting enzyme (ACE), aminopeptidase M (APM) and neutral endopeptidase (NEP) with ramiprilat (0.25 microM), amastatin (40 microM) and phosphoramidon (1 microM), respectively, relative kininase activities were obtained. APM (44%) and ACE (35%) are the main KD degrading enzymes in rat heart; NEP (7%) plays a minor role. A participation of carboxypeptidase N (CPN) could not be found.

Kinin B1 receptors are induced by various inflammatory mediators. The aim of the present study was to investigate the effect of the CXC chemokine IL-8 on kinin B1 receptor expression in IMR-90 cells, by performing binding studies and Northern blot analysis of B1 receptor mRNA levels. We demonstrated here that the density of the kinin B1 receptors could be increased by the chemokine IL-8 in a concentration- and time-dependent manner. IL-8 also increased the kinin B1 receptor mRNA level in IMR-90 cells. IL-8-induced B1 receptor expression could be totally abolished by pretreatment with the metabolic inhibitors. Furthermore, expression was markedly reduced by antibodies to human IL-1alpha. In conclusion, IL-8 increased the expression of kinin B1 receptors in IMR-90 cells and this effect is likely to be secondary to the production of IL-1beta.

The past decade has seen renewed interest in the potential role of kinins in airway diseases. The correlation between kinin generation and symptoms of inflammation, together with the demonstration that administration of kinins to the airway mucosa can induce relevant symptoms, provides strong circumstantial support for a role of kinins in the pathogenesis of airway diseases, such as allergic and viral rhinitis and asthma. Definitive studies of the effects of blockade of kinin actions on symptomatic responses, however, are still needed. The effects of kinins in the airways, and the mechanisms by which they exert their actions clearly vary depending on the presence of inflammation in the airways. Although a growing body of evidence implicates activation of sensory nerves as an important component of kinin effects in inflamed airways, the components of inflammation that modify the response of these sensory nerves, the mechanisms by which neuronal responsiveness alters, and the degree of selectivity of neuronal activation to bradykinin are all topics that require further delineation.

Bradykinin (BK) receptors are involved in pain and inflammation. Two BK receptor subtypes, B1 and B2, have been defined based on their pharmacological properties. Both B1 and B2 receptors are G-protein coupled membrane receptors. B1 receptors are present in smooth muscle tissue, whereas B2 receptors are found in both smooth muscle tissue and neurons. [Des-Arg10,Leu9]kallidin (DALKD) is a selective B1 receptor antagonist, and NPC17731 is a selective B2 receptor antagonist. To develop binding assays for the two known BK receptor subtypes, [3H]DALKD and [3H]NPC17731 were used as selective ligands for B1 and B2 receptors respectively. Both ligands bound to the CCD-16 human lung fibroblast membranes reaching equilibrium at 25 degrees C within 30 min. Binding was stable for at least 60 min. The Kd of [3H]DALKD was 0.33 nM and Bmax was 52 fmol/mg membrane protein. The Kd of [3H]NPC17731 was 0.39 nM and Bmax was 700 fmol/mg membrane protein. Competition for [3H]DALKD binding with BK receptor agonists was in the order: [des-Arg10]KD (DAKD) > KD >> [des-Arg9]BK (DABK) > BK, and competition for [3H]DALKD binding with BK receptor antagonists was in the order: DALKD > [des-Arg10]Hoe 140 (DAHoe 140) > [des-Arg9,Leu8]BK (DALBK) > NPC17731 > Hoe 140 > DNMFBK, suggesting that [3H]DALKD bound selectively to B1 receptors. By contrast, competition for [3H]NPC17731 binding by BK agonists was in the order: BK > KD >> DAKD > DABK, and competition for [3H]NPC17731 binding by BK antagonists was in the order: NPC17731 = Hoe 140 >> DNMFBK > DAHoe 140 > DALBK > DALKD, indicating that [3H]NPC17731 labeled B2 receptors selectively. These results demonstrate that [3H]DALKD and [3H]NPC17731 can be used with CCD-16 human lung fibroblast membranes to provide a pair of binding assays for the simultaneous evaluation of B1 and B2 BK receptor subtypes.

The major isoform of Trypanosoma cruzi cysteinyl proteinase (cruzipain) has generated Lys-bradykinin (Lys-BK or kallidin), a proinflammatory peptide, by proteolysis of kininogen. The releasing of this peptide was demonstrated by mass spectrometry, radioimmunoassay, and ileum contractile responses. The kinin-releasing activity was immunoabsorbed selectively by monoclonal antibodies to the characteristic COOH-terminal domain of cruzipain. To determine the hydrolysis steps that account for the kininogenase activity of cruzipain, we synthesized a fluorogenic peptide (o-aminobenzoyl-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-S er-Pro-Phe-Arg389-Ser390-Ser-Arg-Ile-NH2) based on the sequence Leu373 to Ile393 of the human high molecular weight kininogen. The hydrolysis products from this peptide were isolated by high performance liquid chromatography, and Lys-BK was characterized as the major released kinin by mass spectrometry. Intramolecularly quenched fluorogenic peptides spanning the Met379-Lys380 and Arg389-Ser390 bradykinin-flanking sequences were then used to assess the substrate specificity requirements of the parasite-derived protease compared with two COOH-terminal truncated recombinant isoforms (cruzain and cruzipain 2). In contrast to the high catalytic efficiency of parasite-derived cruzipain, the recombinant proteinases cleaved the bradykinin-flanking sites at markedly different rates. In addition, we also demonstrated that cruzipain activates plasmatic prekallikrein, which would be a second and indirect way of the parasite protease to release bradykinin.

Specific direct bradykinin (BK) binding and competitive inhibition was detected in human neutrophil and peripheral blood mononuclear cell (PBMC) detergent solubilized extracts and purified plasma membranes using in vitro radioreceptor ligand binding. Scatchard analyses of [125I]-BK binding revealed an equilibrium dissociation constant (Kd) of 2.9 x 10(-11) M for neutrophils and 5.6 x 10(-11) M for PBMC using [des-arg9]-BK a B1 agonist; 2.6 x 10(-11) M for neutrophils, 6.2 x 10(-11) M for PBMC with BK a B2 agonist; 5.4 x 10(-11) M for PBMC using Lys-BK a B2 agonist. The number of binding sites (Bmax) was calculated to be 0.113 fM/microgram protein (720 receptors per cell) for neutrophils and 0.200 fM/microgram protein (1289 receptors per cell) for PBMC with the B1 agonist while with the B2 agonists the values were 0.128 fM/microgram protein (818 receptors per cell) for neutrophils and 0.157 fM/microgram protein (1005 receptors per cell) for PBMC with BK, and 0.293 fM/microgram protein (1870 receptors per cell) with Lys-BK for PBMC. In a competitive binding inhibition assay using neutrophil and PBMC glycerol purified plasma membranes, high affinity binding in the nanomolar range was detected to Lys-BK and BK but with [des-arg9]-BK a 10-100 fold lower order affinity was observed this being indicative of pharmacologically defined B2 characteristics.

1. We compared the binding properties of [3H]-desArg10-[Leu9]-kallidin, a radiolabelled kinin B1 receptor antagonist, to membranes from IMR-90 human embryonic fibroblasts and from 293 cells transiently or stably transfected with the human B1 receptor. 2. The dissociation constant (KD) of [3H]-desArg10-[Leu9]-kallidin and the affinity of several kinin receptor agonists and antagonists were similar between the native and cloned receptor, either transiently or stably expressed in 293 cells. In IMR-90 cells, the rank order of potency was that expected for a kinin B1 receptor. 3. The receptors transiently or stably expressed in 293 cells were fully functional with respect to their signalling properties. Phosphoinositide hydrolysis was increased in a concentration-dependent manner by the B1 receptor agonist, desArg10-kallidin. Functional coupling to the calcium pathway was also demonstrated for the native and stably expressed human B1 receptor. 4. In conclusion, the established stable and functional 293 cell clone may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of the kinin B1 receptor.

The characteristic features of kinin B1 and B2 receptors are analyzed. Emphasis is placed on the pharmacologic profiles of B1 and B2 functional sites by the use of naturally-occurring kinins, some selective agonists, as well as peptide and non-peptide antagonists. Species differences are indicated, particularly between human, rabbit and mouse B1 receptors and also between human, rabbit and guinea pig B2 receptors. Extensive use has been made of the non-peptide B2 receptor antagonist, WIN-64338 (which is active in the guinea pig and inactive in the other species) and FR-173657 which shows high affinity in human, rabbit, mouse, pig and guinea pig B2 receptors.

To delineate ligand binding and functional characteristics of the human B1 kinin receptor, a stable clone of Chinese hamster ovary cells expressing a single class of binding sites for [3H]des-Arg10-lysylbradykinin with a Kd of 0.3 nM and a Bmax of 38 fmol/mg protein ( approximately 40,000 receptors/cell) was isolated. Studies with peptide analogs showed that a lysine residue at position 1 (based on the lysylbradykinin sequence) of ligands was essential for high affinity binding to the human B1 receptor. In marked contrast to cloned Chinese hamster ovary cells expressing the human kinin B2 receptor, which internalized approximately 80% of the ligand within 5 min upon exposure to 2 nM [3H]bradykinin, exposure of cells expressing the B1 receptor to 1 nM [3H]des-Arg10-lysylbradykinin resulted in minimal ligand internalization. Stimulation of the B1 receptor led to inositol phosphate generation and transient increases in intracellular calcium, confirming coupling to phospholipase C, while immunoprecipitation of photoaffinity-labeled G-proteins from membranes indicated specific coupling of the receptor to Galphaq/11 and Galphai1,2. The B1, unlike the B2, receptor does not desensitize (as demonstrated by continuous phosphoinositide hydrolysis), enhancing the potential role of this receptor during inflammatory events.

The tissue kallikrein inhibitors reported in the present work were derived by selectively replacing residues in Nalpha-substituted arginine- or phenylalanine-pNA (where pNA is p-nitroanilide), and in peptide substrates for these enzymes. Phenylacetyl-Arg-pNA was found to be an efficient inhibitor of human tissue kallikrein (Ki 0.4 microM) and was neither a substrate nor an inhibitor of plasma kallikrein. The peptide inhibitors having phenylalanine as the P1 residue behaved as specific inhibitors for kallidin-releasing tissue kallikreins, while plasma kallikrein showed high affinity for inhibitors containing (p-nitro)phenylalanine at the same position. The Ki value of the most potent inhibitor developed, Abz-Phe-Arg-Arg-Pro-Arg-EDDnp [where Abz is o-aminobenzoyl and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine], was 0.08 microM for human tissue kallikrein. Progress curve analyses of the inhibition of human tissue kallikrein by benzoyl-Arg-pNA and phenylacetyl-Phe-Ser-Arg-EDDnp indicated a single-step mechanism for reversible formation of the enzyme-inhibitor complex.

A genomic clone encoding the mouse B1 receptor was isolated by homology to the human B1 receptor cDNA. The deduced amino acid sequence of the mouse B1 receptor is 72% identical to the human B1 receptor and 73% identical to the rabbit B1 receptor. Ligand binding studies of the mouse B1 receptor expressed in COS cells indicate that it has the pharmacological properties associated with the B1 receptor subtype. However the pharmacology of the mouse receptor is unique in that it possesses a 2-3-fold selectivity for the 'classical' B1 agonist des-Arg9BK over the agonist des-Arg10 kallidin. In contrast, the human and rabbit B1 receptors exhibit an approx. 2000- and 150-fold selectivity, respectively, for des-Arg10kallidin over des-Arg9BK. Thus relative to the human and rabbit B1 receptors the mouse B1 receptor has the opposite selectivity for kinin agonists. The DNA sequence of the region encoding bradykinin was determined for two different mouse kininogen cDNA clones, both encode the sequence Arg-BK. Antipeptide antibodies directed against a C-terminal peptide of the human B1 receptor were produced. Initial characterization of this antibody indicates that it detects specific bands by Western blot analyses that are present in membranes prepared from COS cells transfected with the human B1 receptor cDNA but not from mock transfected COS cells.

B1 bradykinin receptors were visualized by using the B1 bradykinin receptor agonist [3H]des-Arg10-kallidin in receptor autoradiography experiments. Cryosections were prepared from arterial vessels from a healthy control pig, a pig with pre-existing inflammation and an animal with experimental sepsis induced by an infusion of bacterial lipopolysaccharide (LPS). Only diffusely scattered silver grains with no preference for a distinct tissue structure were detected on emulsion-coated coverslips above the cryosections from the healthy control animal. This indicates that under normal circumstances no or only minute amounts of B1 bradykinin receptors are present in these tissues. In contrast, a 3-fold increase in specific B1 bradykinin receptor binding was observed on both the corresponding preparations of the sick piglet and of that with experimentally induced sepsis. A similar enhancement of specific [3H]des-Arg10-kallidin binding occurred in preparations devoid of endothelium. By comparison with the stained cryosection on the slide the silver grains showed a preferential distribution above smooth muscle cells. Taken together our data are consistent with the hypothesis that B1 bradykinin receptors are induced in the muscle layer of large vessels not only after experimentally-induced sepsis but also in pre-existing inflammatory disease.

Kinetic data for the hydrolysis by human tissue kallikrein of fluorogenic peptides with o-aminobenzoyl-Phe-Arg (Abz-FR) as the acyl group and different leaving groups demonstrate that interactions with the S'1, S'2 and S'3 subsites are important for cleavage efficiency. In addition, studies on the hydrolysis of fluorogenic peptides with the human kininogen sequence spanning the scissile Met-Lys bond [Abz-M-I-S-L-M-K-R-P-N-(2,4-dinitrophenyl)ethylenediamine] and analogues with different residues at positions P'1, P'2 and P'3 showed that (a) the presence of a proline residue at P'3 and the interactions with the tissue kallikrein-binding sites S2 to S'2 are determinants of Met-Lys bond cleavage and (b) residues P3, P4 and/or P5 arc important for cleavage efficiency. The substitution of phenylalanine for methionine or arginine in substrates with scissile Met-Lys or Arg-Xaa bonds demonstrated that lysyl-bradykinin-releasing tissue kallikreins also have a primary specificity for phenylalanine. The replacement of arginine by phenylalanine in (D)P-F-R-p-nitroanilide (pNA) produced an efficient and specific chromogenic substrate, (D)P-F-F-pNA, for the lysyl-bradykinin-releasing tissue kallikreins as it is resistant to plasma kallikrein and other arginine hydrolases.

Lysylbradykinin (LBK), a potent bioactive peptide with pleiotropic actions, is the major kinin generated in the extravascular space. To explore possible mechanisms of inactivation of this peptide in tissues, we evaluated its degradation by endopeptidase 24.11 (EP 24.11) and endopeptidase 24.15 (EP 24.15), two zinc metalloenzymes widely distributed in tissues. EP 24.11 cleaved LBK at the Gly5-Phe6 and Pro8-Phe9 bonds, whereas EP 24.15 cleaved the Phe6-Ser7 bond. Determination of kinetic constants for degradation of LBK by the two enzymes yielded kcat/Km ratios of 5.2 x 10(5) and 8.4 x 10(5) for EP 24.15 and EP 24.11, respectively, indicating that LBK is a good substrate for both enzymes. The findings demonstrate that both EP 24.11 and EP 24.15 efficiently degrade LBK and thus may contribute to the inactivation of this peptide in tissues.

Excessive release of kinin (BK) in the synovial fluid can produce oedema, pain and loss of functions due to activation of B1 and B2 kinin receptors. Activation of the kinin forming system could be mediated via injury, trauma, coagulation pathways (Hageman factor and thrombin) and immune complexes. The activated B1 and B2 receptors might cause release of other powerful non-cytokine and cytokine mediators of inflammation, e.g., PGE2, PGI2, LTs, histamine, PAF, IL-1 and TNF, derived mainly from polymorphonuclear leukocytes, macrophages, endothelial cells and synovial tissue. These mediators are capable of inducing bone and cartilage damage, hypertrophic synovitis, vessel proliferation, inflammatory cell migration and, possibly, angiogenesis in pannus formation. These pathological changes, however, are not yet defined in the human model of chronic inflammation. The role of kinins and their interacting inflammatory mediators would soon start to clarify the detailed questions they revealed in clinical and experimental models of chronic inflammatory diseases. Several B1 and B2 receptor antagonists are being synthesized in an attempt to study the molecular functions of kinins in inflammatory processes, such as rheumatoid arthritis, periodontitis, inflammatory diseases of the gut and osteomyelitis. Future development of specific potent and stable B1 and B2 receptor antagonists or combined B1 and B2 antagonists with y-IFN might serve as a pharmacological basis for more effective treatment of joint inflammatory and related diseases.

A cDNA clone encoding a human B1 bradykinin receptor was isolated from a human embryonic lung fibroblast cDNA library by expression cloning. The photoprotein aequorin was utilized as an indicator of the ability of the B1 receptor agonist [des-Arg10]kallidin to mediate Ca2+ mobilization in Xenopus laevis oocytes injected with RNA. A clone was isolated with a 1307-nucleotide insert which contains an open reading frame encoding a 353-amino acid protein with the characteristics of a G-protein-coupled receptor. The amino acid sequence of the B1 bradykinin receptor is 36% identical to the amino acid sequence of the B2 bradykinin receptor. The cloned B1 bradykinin receptor expressed in mammalian cells exhibits high affinity binding for 3H-labeled [des-Arg10]kallidin and low affinity for bradykinin. The B1 receptor antagonist [des-Arg10,Leu9]kallidin effectively displaces 3H-labeled [des-Arg10]kallidin from the cloned receptor, whereas the B2 receptor antagonist Hoe-140 (D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin, where Thi is L-[3-(2-thienyl)alanyl], Tic is D-(1,2,3,4-tetrahydroisoquinolin-3-yl-carbonyl), and Oic is L-[(3aS, 7aS)-octahydroindol-2-yl-carbonyl]) does not. Therefore, the expressed receptor has the pharmacological characteristics of the B1 receptor subtype. The availability of both the cloned human B1 and B2 bradykinin receptors should allow the elucidation of the relative contributions of these two receptor subtypes in acute and chronic inflammatory processes.

1. Tissue kallikrein (TK) cleaves low molecular weight kininogen (LK) at two sites to release kallidin: site I (between Arg389 and Ser390) is a typical cleavage point for a trypsin-like enzyme whereas site II (between Met379 and Lys380) is unusual and unique to TK. In order to learn more about the structural requirements and mechanism of cleavage at site II, we studied the hydrolysis by TK of several synthetic LK fragments varying in length between 4 and 22 residues and containing either site II only or both sites I and II. 2. Blocking site I cleavage in LK fragments by substituting DArg for LArg at position 389 or omitting site I from the sequence still allowed cleavage to proceed at site II. Replacement or deletion of selected amino acid residues in these fragments demonstrated that the presence of Arg381 was essential for site II cleavage to occur whereas Pro383, Phe385 and Ser386 could be replaced with Ala without affecting binding or cleavage by TK. Ki values towards TK were determined for all LK fragments in order to compare their binding affinities to the enzyme. Short peptides containing site II only exhibited high Ki values (> or = 100 microM) whereas longer fragments containing both sites I and II had Ki values of 2-7 microM. 3. In order to bring sites I and II into close proximity spatially and thus facilitating efficient cleavage in the enzyme-substrate complex, we prepared several cyclic analogs of the longer LK fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Respiratory epithelial cell surface neutral endopeptidase 24.11 (NEP-24.11) degrades proinflammatory peptides, and it has been suggested that glucocorticoids may reduce airway inflammation, in part, by upregulation of NEP-24.11. Despite the potential importance of the epithelium as a metabolic barrier, little is known regarding what other peptidases may be present on the epithelial cell surface. Using an immortalized bronchial epithelial cell line (BEAS-2B), we have shown that human epithelial cells express no detectable angiotensin-converting enzyme, carboxypeptidase N, or dipeptidyl(amino)peptidase IV, but express significant levels of aminopeptidase M (AmM), as well as NEP-24.11. The presence of these enzymes was demonstrated via their degradation of biologically active peptides and by flow cytometry. Exposure of cells to the glucocorticoid budesonide (10(-7) M) for up to 5 days did not markedly alter the expression of NEP-24.11 or AmM, as assessed by flow cytometry, nor did glucocorticoid treatment modify rates of peptide hydrolysis by NEP-24.11 or AmM. Thus, BEAS-2B cells have both AmM and NEP-24.11 on their surface, and expression of these enzymes is not altered by glucocorticoids.