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Time of flight secondary ion mass spectrometry (ToF-SIMS) is a powerful technique for the nanoanalysis of biological samples, but improvements in sensitivity are needed in order to detect large biomolecules, such as peptides, on the individual cell level at physiological concentrations. Two promising options to improve the sensitivity of SIMS to large peptides are the use of cluster primary ions to increase desorption of intact molecules or the use of matrix-assisted laser desorption/ionization (MALDI) matrices to increase the ionization probability. In this paper, the authors have combined these two approaches in order to improve understanding of the interaction between ionization and fragmentation processes. The peptides bradykinin and melittin were prepared as neat monolayers on silicon, in a Dextran-40 matrix and in two common MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and a-cyano-4-hydroxy cinnamic acid (HCCA). ToF-SIMS spectra of these samples were collected using a range of small Bi cluster primary ions and large Ar cluster primary ions. The trends observed in the molecular ion yield and the [M+H]+/C4H8N+ ratio with primary ion cluster size were sample system dependent. The molecular ion yield of the bradykinin was maximized by using 30 keV Bi3+ primary ions in a DHB matrix but in the HCCA matrix, the maximum molecular ion yield was obtained by using 30 keV Bi7+ primary ions. In contrast, the molecular ion yield for melittin in both matrices was greatest using 20 keV Ar2000+ primary ions. Improvements in the molecular ion yield were only loosely correlated with a decrease in small fragment ions. The data indicate a complex interplay between desorption processes and ion formation processes which mean that the optimal analytical conditions depend on both the target analyte and the matrix. [ABSTRACT FROM AUTHOR]

(1) Background: Co-morbidities such as hypertension and cardiovascular disease are major risk factors for severe COVID-19. The renin–angiotensin system (RAS) is critically involved in their pathophysiology and is counter-balanced by both angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV-2, and the kallikrein–kinin system (KKS). Considerable research interest with respect to COVID-19 treatment is currently being directed towards the components of these systems. In earlier studies, we noticed significantly reduced carboxypeptidase N (CPN, KKS member) activity and excessive angiotensin-converting enzyme (ACE, RAS member) activity in the sera of both hospitalized COVID-19 patients and a subgroup of convalescent patients. The data had been obtained using labeled bradykinin (BK) as a reporter peptide, which is a target of both CPN and ACE. The data were supplemented with mass-spectrometry-based serum proteomic analysis. Here, we hypothesize that the degree of BK serum degradation could be indicative of Long COVID. (2) Review and Discussion: The recent literature is briefly reviewed. The fact that the levels of the BK serum degradation products did not reach normal concentrations in almost half of the patients during convalescences could have been partially due to a dysregulated RAS. (3) Conclusions: Standard tests for routine patient care in Long COVID come often back normal. We suggest that the measurement of selected members of the RAS such as ACE and angiotensin II or the use of our BK degradation assay could identify Long COVID candidates. Clinical studies are required to test this hypothesis. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is one of the most lethal malignancies worldwide, so the attempts to find novel therapeutic approaches are necessary. The aim of our study was to analyze how chemical modifications influence physical, chemical, and biological properties of the two peptides, namely, bradykinin (BK) and neurotensin (NT). For this purpose, we used fourteen modified peptides, and their anti-cancers features were analyzed on the HCT116 CRC cell line. Our results confirmed that the spherical mode of a CRC cell line culture better reflects the natural tumour microenvironment. We observed that the size of the colonospheres was markedly reduced following treatment with some BK and NT analogues. The proportion of CD133+ cancer stem cells (CSCs) in colonospheres decreased following incubation with the aforementioned peptides. In our research, we found two groups of these peptides. The first group influenced all the analyzed cellular features, while the second seemed to include the most promising peptides that lowered the count of CD133+ CSCs with parallel substantial reduction in CRC cells viability. These analogues need further analysis to uncover their overall anti-cancer potential. [ABSTRACT FROM AUTHOR]

Hereditary angioedema (HAE) is a condition characterized by episodes of cutaneous and submucosal edema. Angioedema of the extremities and abdominal attacks are the most common manifestations of the disease. It can also affect the upper airways with the potential of becoming life-threatening. The two most common causes of HAE are a deficiency of C1 inhibitor (classified as type 1 HAE) or a dysfunction of C1 inhibitor (type 2 HAE). A malfunction or deficiency of C1 inhibitor leads to an overactivated plasma kallikrein (an inflammatory vasoactive peptide), that increases bradykinin, mediating the angioedema episodes in patients with HAE. To minimize the difficulties of this pathology and to improve patients' quality of life, prevention of this condition is essential. Berotralstat is a unique option for oral administration for routine prophylaxis. This drug acts by binding to kallikrein and reducing its plasma activity, lowering bradykinin levels. Open-label studies have demonstrated the effectiveness of a single daily dose of berotralstat 150 mg in preventing HAE attacks. This review aims to examine studies performed to elucidate the efficacy, safety, and tolerability of berotralstat. [ABSTRACT FROM AUTHOR]

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-Gq 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 W2836.48, 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 Gq proteins. Our structures elucidate the molecular mechanisms for the ligand binding, receptor activation, and Gq proteins coupling of B2R. Type 2 bradykinin receptor (B2R) is essential in vasodilation and cardioprotection. Here the authors present two cryo-EM structures of human B2R-Gq in complex with bradykinin and kallidin to elucidate the mechanisms for ligand binding, receptor activation, and Gq proteins coupling. [ABSTRACT FROM AUTHOR]

Human kallikrein-kinin system (KKS) is a proteolytic cascade with two serine-protease zymogen couples (Factor XII and prekallikrein (PK) and their activated forms, FXIIa, PKa, respectively), releasing bradykinin by cleavage of native high-molecular-weight kininogen (nHK) into cleaved HK. For KKS investigation in human plasma, this cascade is usually triggered on ice eventually by mixing with purified proteins. It has been established that purified FXIIa, PK, and nHK required a fixed order and timing for mixing protein on ice to ensure reproducibility of testing, we investigated the activation kinetics of both enzymes. The activation process of this in vitro minimal reconstitution of KKS was studied by progress curve analysis, in condition of high enzyme/substrate ratio and by using on natural rather than peptide substrates. FXIIa and PKa were found five-times less active on ice than at 37°C: kcat = 0.133 +- 0.034 and 0.0119 +- 0.0027 s-1, KM = 672 +- 150 and 115 +- 24 nM, respectively. The progress curve analysis of our in vitro KKS reconstitutions differed from a Michaelis--Menten mathematical simulation by a faster initial rate and a slower late rate. These two features were also observed ex vivo by using dextran sulfate-activated plasma and could reinforce the hypothesis of a maximal local effect (bradykinin release) and a minimal systemic consequence (PK preservation) in KKS activation process. Analyzing the complete curve of cold KKS activation would provide valuable information for ex vivo investigation of KKS in samples from patients presenting with hereditary angioedema and other inflammatory conditions. [ABSTRACT FROM AUTHOR]

Background and Purpose: Bradykinin (BK‐(1–9)) is an endogenous nonapeptide involved in multiple physiological and pathological processes. Peptide fragments of bradykinin are believed to be biologically inactive. We have now tested the two major peptide fragments of bradykinin in human and animals. Experimental Approach BK peptides were quantified by MS in male rats. NO release was quantified from human, mouse and rat cells loaded with DAF‐FM. Rat aortic rings were used to measure vascular reactivity. Changes in BP and HR were measured in conscious male rats. To evaluate pro‐inflammatory effects both vascular permeability and nociception were measured in adult mice. Key Results: BK‐(1–7) and BK‐(1–5) are produced in vivo from BK‐(1–9). Both peptides induced NO production in all cell types tested. However, unlike BK‐(1–9), NO production elicited by BK‐(1–7) or BK‐(1–5) was not inhibited by B1 or B2 receptor antagonists. BK‐(1–7) and BK‐(1–5) induced concentration‐dependent vasorelaxation of aortic rings, without involvement of B1 or B2 receptors. Intravenous or intra‐arterial administration of BK‐(1–7) or BK‐(1–5) induced similar hypotensive response in vivo. Nociceptive responses of BK‐(1–7) and BK‐(1–5) were reduced compared to BK‐(1–9), and no increase in vascular permeability was observed for BK‐(1–9) fragments. Conclusions and Implications: BK‐(1–7) and BK‐(1–5) are endogenous peptides present in plasma. BK‐related peptide fragments show biological activity, not mediated by B1 or B2 receptors. These BK fragments could constitute new, active components of the kallikrein–kinin system. [ABSTRACT FROM AUTHOR]

Background: The kallikrein-kinin system is assumed to have a multifunctional role in health and disease, but its in vivo role in humans currently remains unclear owing to the divergence of plasma kinin level data published ranging from the low picomolar to high nanomolar range, even in healthy volunteers. Moreover, existing data are often restricted on reporting levels of single kinins, thus neglecting the distinct effects of active kinins on bradykinin (BK) receptors considering diverse metabolic pathways. A well-characterized and comprehensively evaluated healthy cohort is imperative for a better understanding of the biological variability of kinin profiles to enable reliable differentiation concerning disease-specific kinin profiles.Methods: To study biological levels and variability of kinin profiles comprehensively, 28 healthy adult volunteers were enrolled. Nasal lavage fluid and plasma were sampled in customized protease inhibitor prespiked tubes using standardized protocols, proven to limit inter-day and interindividual variability significantly. Nine kinins were quantitatively assessed using validated LC-MS/MS platforms: kallidin (KD), Hyp4-KD, KD1-9, BK, Hyp3-BK, BK1-8, BK1-7, BK1-5, and BK2-9. Kinin concentrations in nasal epithelial lining fluid were estimated by correlation using urea.Results: Circulating plasma kinin levels were confirmed in the very low picomolar range with levels below 4.2 pM for BK and even lower levels for the other kinins. Endogenous kinin levels in nasal epithelial lining fluids were substantially higher, including median levels of 80.0 pM for KD and 139.1 pM for BK. Hydroxylated BK levels were higher than mean BK concentrations (Hyp3-BK/BK = 1.6), but hydroxylated KD levels were substantially lower than KD (Hyp4-KD/KD = 0.37). No gender-specific differences on endogenous kinin levels were found.Conclusions: This well-characterized healthy cohort enables investigation of the potential of kinins as biomarkers and would provide a valid control group to study alterations of kinin profiles in diseases, such as angioedema, sepsis, stroke, Alzheimer's disease, and COVID-19. [ABSTRACT FROM AUTHOR]

Background and Purpose: A peptide bradykinin (BK) B(2) receptor agonist partially resistant to degradation, B-9972, down-regulates this receptor subtype. We have used another recently described non-peptide agonist, compound 47a, as a tool to study further the effects of metabolically more stable and thus persistent, agonists of the BK B(2) receptor on signalling, desensitization and down-regulation of this receptor.Experimental Approach and Key Results: Compound 47a was a partial agonist at the B(2) receptor in the human umbilical vein, where it shared with B-9972 a very slow relaxation on washout, and in HEK 293 cell lines expressing tagged forms [myc, green fluorescent protein (GFP)] of the rabbit B(2) receptor. Compound 47a desensitized the umbilical vein to BK. In the cellular systems, the inactivation-resistant agonists induced [Ca(2+)](i) transients as brief as those of BK but affected other functions with a longer duration than BK [12 h; receptor endocytosis, endosomal beta-arrestin(1/2) translocation, protein kinase C-dependent extracellular signal-regulated kinases (ERK)1/2 phosphorylation and c-Fos expression]. The B(2) receptor-GFP was degraded in cells exposed to B-9972 or compound 47a for 12 h. The non-peptide B(2) receptor antagonist LF 16-0687 prevented all effects of compound 47a, which were also absent in cells lacking recombinant B(2) receptors.Conclusion and Implications: Inactivation-resistant agonists revealed a long-lasting assembly of the agonist-B(2) receptor-beta-arrestin complexes in endosomal structures and induce 'biased signalling' (in terms of activation of ERK and c-Fos) as a function of time. Further, B-9972 and compound 47a, unlike BK, efficiently down-regulated BK B(2) receptors. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), continues to be a world‐wide pandemic with overwhelming socioeconomic impact. Since inflammation is one of the major causes of COVID‐19 complications, the associated molecular mechanisms have been the focus of many studies to better understand this disease and develop improved treatments for patients contracting SARS‐CoV‐2. Among these, strong emphasis has been placed on pro‐inflammatory cytokines, associating severity of COVID‐19 with so‐called "cytokine storm." More recently, peptide bradykinin, its dysregulated signaling or "bradykinin storm," has emerged as a primary mechanism to explain COVID‐19‐related complications. Unfortunately, this important development may not fully capture the main molecular players that underlie the disease severity. To this end, in this focused review, several lines of evidence are provided to suggest that in addition to bradykinin, two closely related vasoactive peptides, substance P and neurotensin, are also likely to drive microvascular permeability and inflammation, and be responsible for development of COVID‐19 pathology. Furthermore, based on published experimental observations, it is postulated that in addition to ACE and neprilysin, peptidase neurolysin (Nln) is also likely to contribute to accumulation of bradykinin, substance P and neurotensin, and progression of the disease. In conclusion, it is proposed that "vasoactive peptide storm" may underlie severity of COVID‐19 and that simultaneous inhibition of all three peptidergic systems could be therapeutically more advantageous rather than modulation of any single mechanism alone. [ABSTRACT FROM AUTHOR]

In order to evaluate the utility of copper surfaces as spray initiators for mass spectrometric ionization sources, it is necessary to establish how efficient and reproducible the methodology is. To do this, a superhydrophobic and bare copper surface were used to ionize the singly and doubly charged peptides, bradykinin and leucine-enkephalin spiked with substance P as an internal standard, respectively. Both treated and bare copper surfaces were cut to 90, 60, and 30° angles. Calibration curves were constructed for both peptides on a superhydrophobic surface using covalently bonded trichloro(1H, 1H, 2H, 2H-perflourooctyl)silane-treated copper, and bare copper surface. The highest sensitivity for all peptides was observed using a 30 or 60° tip with the silane-treated copper. Limits of detection and quantitation for the 60° silane-treated surface for bradykinin were 6.01 × 10−3 μM and 2.00 × 10−2 μM respectively, with a R2 of 0.9959. Limits of detection and quantitation for the 30° silane-treated surface for leucine-enkephalin were 3.28 × 10−2 μM and 1.09 × 10−1 μM respectively, with a R2 of 0.9993. Image 1 • Copper surfaces were used as spray initiators similar to paper spray ionization. • Surfaces are reusable, have fixed geometry, and can be functionalized, and require minimal solvent to initiate a spray event. • Metalspray ionization was used to test sensitivity between bare copper and modified copper surfaces. • Copper was functionalized with trichloro(1 H , 1 H , 2 H , 2 H -perflourooctyl)silane to create a superhydrophobic surface. • Superhydrophobic surface displayed greater sensitivity and lower limits of quantitation compared to bare copper surface. [ABSTRACT FROM AUTHOR]

Bradykinin-related peptides (BRPs) are significant components of the defensive skin secretions of many anuran amphibians, and these secretions represent the source of the most diverse spectrum of such peptides so far encountered in nature. Of the many families of bioactive peptides that have been identified from this source, the BRPs uniquely appear to represent homologues of counterparts that have specific distributions and receptor targets within discrete vertebrate taxa, ranging from fishes through mammals. Their broad spectra of actions, including pain and inflammation induction and smooth muscle effects, make these peptides ideal weapons in predator deterrence. Here, we describe a novel 12-mer BRP (RVALPPGFTPLR-RVAL-(L1, T6, L8)-bradykinin) from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis). The C-terminal 9 residues of this BRP (-LPPGFTPLR) exhibit three amino acid substitutions (L/R at Position 1, T/S at Position 6 and L/F at Position 8) when compared to canonical mammalian bradykinin (BK), but are identical to the kinin sequence present within the cloned kininogen-2 from the Chinese soft-shelled turtle (Pelodiscus sinensis) and differ from that encoded by kininogen-2 of the Tibetan ground tit (Pseudopodoces humilis) at just a single site (F/L at Position 8). These data would imply that the novel BRP is an amphibian defensive agent against predation by sympatric turtles and also that the primary structure of the avian BK, ornithokinin (RPPGFTPLR), is not invariant within this taxon. Synthetic RVAL-(L1, T6, L8)-bradykinin was found to be an antagonist of BK-induced rat tail artery smooth muscle relaxation acting via the B2-receptor. [ABSTRACT FROM AUTHOR]

The renin angiotensin system (RAS) is a network of enzymes and peptides that coalesce primarily on the angiotensin II type 1 receptor (AT1R) to induce cell proliferation, angiogenesis, fibrosis, and blood pressure control. Angiotensin-converting enzyme (ACE), the key peptidase of the RAS, is promiscuous in that it cleaves other substrates such as substance P and bradykinin. Accumulating evidence implicates ACE in the pathophysiology of carcinogenesis. While the role of ACE and its peptide network in modulating angiogenesis via the AT1R is well documented, its involvement in shaping other aspects of the tumor microenvironment remains largely unknown. Here, we review the role of ACE in modulating the immune compartment of the tumor microenvironment, which encompasses the immunosuppressive, cancer-promoting myeloidderived suppressor cells, alternatively activated tumor-associated macrophages, and T regulatory cells. We also discuss the potential roles of peptides that accumulate in the setting of chronic ACE inhibitor use, such as bradykinin, substance P, and N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), and how they may undercut the gains of anti-angiogenesis from ACE inhibition. These emerging mechanisms may harmonize the often-conflicting results on the role of ACE inhibitors and ACE polymorphisms in various cancers and call for further investigations into the potential benefit of ACE inhibitors in some neoplasms. [ABSTRACT FROM AUTHOR]

The reaction of some physiologically active peptides with bacterial strain B-9 has been investigated. Bradykinin, β-endorphin, and [Leu]enkephalin were quickly degraded, with half-lives of <5 min. Somatostatin, substance P, and angiotensin I were degraded relatively smoothly, with half-lives of 10 min to 1 h, whereas oxytocin and insulin were slowly degraded, with half-lives of 1 and 4 days, respectively. Vasopressin was barely degraded, with a half-life of >7 days. Linearized vasopressin, prepared by the reductive cleavage of the disulfide bond followed by alkylation with iodoacetamide, was degraded significantly faster than intact vasopressin, with a half-life of 2.5 h. A loop formed by disulfide bond formation was regarded as one of the degradation-resistant factors. Hydrolysis of the peptides in this study took place through cleavage of various peptide bonds, and the strain B-9 may bear similarities to the neutral endopeptidase in terms of its broad selectivity. [ABSTRACT FROM AUTHOR]

Background: Kinins, with bradykinin and des-Arg9-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg9-bradykinin as well as Lys-des-Arg9-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-Daspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices. Principal Findings: Bradykinin at 10 μM and 1 μM concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 μM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3- kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 μM Lys-des-Arg9-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spike recovery. The inhibition of population spikes recovery was reversed by PD98059, showing that MEK/MAPK was involved in the induction of apoptosis mediated by the B1 receptor. Conclusions: Bradykinin exerted protection against NMDA-induced excitotoxicity which is reversed in the presence of a kinin-B1 receptor agonist. As bradykinin is converted to the kinin-B1 receptor metabolite des-Arg9-bradykinin by carboxypeptidases, present in different areas including in brain, our results provide a mechanism for the neuroprotective effect in vitro despite of the deleterious effect observed in vivo. [ABSTRACT FROM AUTHOR]

Bradykinin-related peptides (BRPs) constitute one of the most studied groups of bioactive peptides in amphibian skin secretions. The present study describes the successful isolation of a novel BRP (hylaranakinin TE) from the skin secretion of the Indian bronzed frog Hylarana temporalis. The deduced open reading frame consisted of 115 amino acid residues with a putative signal peptide of 22 amino acid residues, followed by a spacer region and mature peptide regions that encode for two BRPs: a canonical bradykinin R-9-R with a C-terminal extension of FVPASSL and Thr6-BK. The Thr6-BK reported in the present study had an unusual FP-insertion in the N-terminal part and ended in FAPEII, which is very different from the IAPAIV sequence reported in other ranid frogs. Unlike the mammalian bradykinin and its precursor, amphibian BRPs and their precursors are extremely variable, as evident from the present study. This forms the first report of BRPs from Hylarana temporalis, endemic to India and Sri Lanka. [ABSTRACT FROM AUTHOR]

Thimet oligopeptidase (EC 3.4.24.15) is a zinc(II) endopeptidase implicated in the processing of numerous physiological peptides. Although its role in selecting and processing peptides is not fully understood, it is believed that flexible loop regions lining the substrate-binding site allow the enzyme to conform to substrates of varying structure. This study describes mutant forms of thimet oligopeptidase in which Gly or Tyr residues in the 599–611 loop region were replaced, individually and in combination, to elucidate the mechanism of substrate selection by this enzyme. Decreases in kcat observed on mutation of Tyr605 and Tyr612 demonstrate that these residues contribute to the efficient cleavage of most substrates. Modeling studies showing that a hinge-bend movement brings both Tyr612 and Tyr605 within hydrogen bond distance of the cleaved peptide bond supports this role. Thus, molecular modeling studies support a key role in transition state stabilization of this enzyme by Tyr605. Interestingly, kinetic parameters show that a bradykinin derivative is processed distinctly from the other substrates tested, suggesting that an alternative catalytic mechanism may be employed for this particular substrate. The data demonstrate that neither Tyr605 nor Tyr612 is necessary for the hydrolysis of this substrate. Relative to other substrates, the bradykinin derivative is also unaffected by Gly mutations in the loop. This distinction suggests that the role of Gly residues in the loop is to properly orientate these Tyr residues in order to accommodate varying substrate structures. This also opens up the possibility that certain substrates may be cleaved by an open form of the enzyme. [ABSTRACT FROM AUTHOR]

Background and Purpose: Inhibition of bradykinin metabolizing enzymes (BMEs) can cause acute angioedema, as demonstrated in a recent clinical trial in patients administered the antihypertensive, omapatrilat. However, the relative contribution of specific BMEs to this effect is unclear and confounded by the lack of a predictive pre-clinical model of angioedema.Experimental Approach: Rats were instrumented to record blood pressure and heart rate; inhibitors were infused for 35 min and bradykinin was infused during the last 5 min to elicit hypotension, as a functional marker of circulating bradykinin and relative angioedema risk.Key Results: In the presence of omapatrilat bradykinin produced dose-dependent hypotension, an effect abolished by B(2) blockade. In the presence of lisinopril (ACE inhibitor), but not candoxatril (NEP inhibitor) or apstatin (APP inhibitor), bradykinin also elicited hypotension. Lisinopril-mediated hypotension was unchanged with concomitant blockade of NEP or NEP/DPPIV (candoxatril+A-899301). However, hypotension was enhanced upon concomitant blockade of APP and further intensified in the presence of NEP inhibition to values not different from omapatrilat alone.Conclusions and Implications: We demonstrated that bradykinin is degraded in vivo with an enzyme rank-efficacy of ACE>APP>>NEP or DPPIV. These results suggest the effects of omapatrilat are mediated by inhibition of three BMEs, ACE/APP/NEP. However, dual inhibition of ACE/NEP or ACE/NEP/DPPIV elicits no increased risk of angioedema compared to ACE inhibition alone. Thus, novel BME inhibitors must display no activity against APP to avoid angioedema risk due to high prevalence of ACE inhibitor therapy in patients with diabetes and cardiovascular disease. [ABSTRACT FROM AUTHOR]

Background and purpose:In this work, a neuroactive peptide from the venom of the neotropical wasp Polybia occidentalis was isolated and its anti-nociceptive effects were characterized in well-established pain induction models.Experimental approach:Wasp venom was analysed by reverse-phase HPLC and fractions screened for anti-nociceptive activity. The structure of the most active fraction was identified by electron-spray mass spectrometry (ESI-MS/MS) and it was further assessed in two tests of anti-nociceptive activity in rats: the hot plate and tail flick tests.Key Results:The most active fraction contained a peptide whose structure was Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH, which corresponds to that of Thr6-BK, a bradykinin analogue. This peptide was given by i.c.v. injection to rats. In the tail flick test, Thr6-BK induced anti-nociceptive effects, approximately twice as potent as either morphine or bradykinin also given i.c.v. The anti-nociceptive activity of Thr6-BK peaked at 30 min after injection and persisted for 2 h, longer than bradykinin. The primary mode of action of Thr6-BK involved the activation of B2 bradykinin receptors, as anti-nociceptive effects of Thr6-BK were antagonized by a selective B2 receptor antagonist.Conclusions and implications:Our data indicate that Thr6-BK acts through B2 bradykinin receptors in the mammalian CNS, evoking antinociceptive behaviour. This activity is remarkably different from that of bradykinin, despite the structural similarities between both peptides. In addition, due to the increased metabolic stability of Thr6-BK, relative to that of bradykinin, this peptide could provide a novel tool in the investigation of kinin pathways involved with pain.British Journal of Pharmacology (2007) 151, 860–869; doi:10.1038/sj.bjp.0707275; published online 29 May 2007 [ABSTRACT FROM AUTHOR]

The potential cardioprotective effect of ACE inhibitors has been attributed to the inhibition of bradykinin degradation. Recent data in rats documented a kallidin-like peptide, which mimics the cardioprotective effect of ischaemic preconditioning. This study investigates in isolated Langendorff rat heart the effect of the ACE inhibitor captopril, the role of bradykinin, kallidin-like peptide, and nitric oxide (NO).The bradykinin level in the effluent of the control group was 14.6 pg ml−1 and was not affected by captopril in the presence or absence of kinin B2-receptor antagonist, HOE140.The kallidin-like peptide levels were approximately six-fold higher (89.8 pg ml−1) and increased significantly by treatment with captopril (144 pg ml−1), and simultaneous treatment with captopril and HOE140 (197 pg ml−1).Following 30 min ischaemia in the control group, the creatine kinase activity increased from 0.4 to 53.4 U l−1. In the captopril group and in the captopril+L-NAME group, the creatine kinase activity was significantly lower (18.5 and 22.8 U l−1). This beneficial effect of captopril was completely abolished by the kinin B2-receptor antagonist, HOE140, as well as by the kallidin antiserum.Perfusion of the hearts with kallidin before the 30 min ischaemia, but not with bradykinin, yielded an approximately 50% reduction in creatine kinase activity after reperfusion.Pretreatment with L-NAME alone and simultaneously with captopril, and with kallidin, respectively, suggests a kinin-independent action of NO before the 30 min ischaemia on coronary flow and a kinin-dependent action after ischaemia.These data show that captopril increases kallidin-like peptide in the effluent. Kallidin-like peptide via kinin B2 receptor seems to be the physiological mediator of cardioprotective actions of captopril against ischaemic reperfusion injury. HOE140 as well as the kallidin antiserum abolished the cardioprotective effects of captopril.British Journal of Pharmacology (2006) 148, 825–832. doi:10.1038/sj.bjp.0706799; published online 12 June 2006 [ABSTRACT FROM AUTHOR]

Kinins (peptides related to bradykinin, BK) are formed from circulating substrates, the kininogens, by the action of two proteases, the kallikreins. The only clinical application of a BK receptor ligand, the B2 receptor antagonist icatibant, is the treatment of the rare hereditary angioedema (HAE) caused by the deficiency of C1-esterase inhibitor (C1-INH). Less common forms of HAE (genetic variants of factor XII, plasminogen, kininogen) are presumably mediated by increased BK formation. Acquired forms of BK-mediated angioedema, such as that associated with angiotensin-I converting enzyme (ACE) inhibition, are also known. Antibody-based analytical techniques are briefly reviewed, and support that kinins are extremely short-lived, prominently cleared by ACE. Despite evidence of continuous activation of the kallikrein–kinin system in HAE, patients are not symptomatic most of the time and their blood or plasma obtained during remission does not generate excessive immunoreactive BK (iBK), suggesting effective homeostatic mechanisms. HAE-C1-INH and HAE-FXII plasmas are both hyperresponsive to fibrinolysis activation. On another hand, we suggested a role for the alternate tissue kallikrein–kinin system in patients with a plasminogen mutation. The role of the BK B1 receptor is still uncertain in angioedema states. iBK profiles under in vitro stimulation provide fresh insight into the physiopathology of angioedema. [ABSTRACT FROM AUTHOR]