Kinins in humans

The kinin peptide system in humans is complex. Whereas plasma kallikrein generates bradykinin peptides, glandular kallikrein generates kallidin peptides. Moreover, a proportion of kinin peptides is hydroxylated on proline(3) of the bradykinin sequence. We established HPLC-based radioimmunoassays for nonhydroxylated and hydroxylated bradykinin and kallidin peptides and their metabolites in blood and urine. Both nonhydroxylated and hydroxylated bradykinin and kallidin peptides were identified in human blood and urine, although the levels in blood were often below the assay detection limit. Whereas kallidin peptides were more abundant than bradykinin peptides in urine, bradykinin peptides were more abundant in blood. Bradykinin and kallidin peptide levels were higher in venous than arterial blood. Angiotensin-converting enzyme inhibition increased blood levels of bradykinin, but not kallidin, peptides. Reactive hyperemia had no effect on antecubital venous levels of bradykinin or kallidin peptide levels. These studies demonstrate differential regulation of the bradykinin and kallidin peptide systems, and indicate that blood levels of bradykinin peptides are more responsive to angiotensin-converting enzyme inhibition than blood levels of kallidin peptides.     

Role of PGI2 and effects of ACE inhibition on the bradykinin potentiation by angiotensin-(1-7) in resistance vessels of SHR

The present study determined the participation of PGI2 in the angiotensin-(1-7) [Ang-(1-7)]/bradykinin (BK) interaction, in the presence and absence of Angiotensin Converting Enzyme (ACE) inhibition, trying to correlate it with tissue levels of both peptides. The isolated mesenteric arteriolar bed of Spontaneously Hypertensive Rats (SHR) was perfused with Krebs or Krebs plus enalaprilat (10 nM), and drugs were injected alone or in association. BK (10 ng)-induced relaxation was potentiated by Ang-(1-7) (2.2 microg) in the presence or absence of enalaprilat. Ang-(1-7) receptor blockade [A-779 (4.8 microg)] did not interfere with the BK effect in preparations perfused with normal Krebs, but reversed the increased BK relaxation observed after ACE inhibition. PGI2 release by mesenteric vessels was not altered by BK or Ang-(1-7) alone, but was increased when both peptides were injected in association, in the absence or in the presence of enalaprilat. ACE inhibition caused a 2-fold increase in the BK tissue levels, and a significant decrease in the Ang-(1-7) values. We conclude that endogenous Ang-(1-7) has an important contribution to the effect of ACE inhibitors participating in the enhancement of BK response. The mechanism of Ang-(1-7) potentiating effect probably involves an increased production of PGI2. Our results suggest that a different enzymatic pathway (non-related to ACE) is involved in the local Ang-(1-7) metabolism.     

Assay of kinins by their effects on canine femoral blood flow.

In pentobarbital anesthetized dogs, close arterial injections of bradykinin and kallidin elicit a dose related increase in femoral blood flow. Treatment with the kininase inhibitor BPP9alpha augments the femoral blood flow responses to bradykinin and kallidin by five and threefold respectively. The sensitivity of the preparation permits the detection of 0.5-1 ng of either bradykinin or kallidin in untreated dogs and as little as 0.1 ng of kinin peptides in animals receiving BPP9alpha. This sensitivity and the steepness of the dose response curves make this procedure suitable for the assay of kinins.     

Kallikrein activates bradykinin B2 receptors in absence of kininogen

Kallikreins cleave plasma kininogens to release the bioactive peptides bradykinin (BK) or kallidin (Lys-BK). These peptides then activate widely disseminated B2 receptors with consequences that may be either noxious or beneficial. We used cultured cells to show that kallikrein can bypass kinin release to activate BK B2 receptors directly. To exclude intermediate kinin release or kininogen uptake from the cultured medium, we cultured and maintained cells in medium entirely free of animal proteins. We compared the responses of stably transfected Chinese hamster ovary (CHO) cells that express human B2 receptors (CHO B2) and cells that coexpress angiotensin I-converting enzyme (ACE) as well (CHO AB). We found that BK (1 nM or more) and tissue kallikrein (1-10 nM) both significantly increased release of arachidonic acid beyond unstimulated baseline level. An enzyme-linked immunoassay for kinin established that kallikrein did not release a kinin from CHO cells. We confirmed the absence of kininogen mRNA with RT-PCR to rule out kininogen synthesis by CHO cells. We next tested an ACE inhibitor for enhanced BK receptor activation in the absence of kinin release and synthesized an ACE-resistant BK analog as a control for these experiments. Enalaprilat (1 microM) potentiated kallikrein (100 nM) in CHO AB cells but was ineffective in CHO B2 cells that do not bear ACE. We concluded that kallikrein activated B2 receptors without releasing a kinin. Furthermore, inhibition of ACE enhanced the receptor activation by kallikrein, an action that may contribute to the manifold therapeutic effects of ACE inhibitors.     

Potentiation of bradykinin effects and inhibition of kininase activity in isolated smooth muscle.

Prolongation of bradykinin half-life following kininase inhibition has been proposed as the reason for the potentiation of kinin effects. We have reassessed this assumption by using three different isolated smooth muscle preparations and simultaneously studying the inhibition of kininase activity and the potentiation of bradykinin effects by enalaprilat and BPP9a. Rat duodenum displayed higher total kininase activity, metabolizing half of the added bradykinin in 6.5 min, while this time for rat uterus was greater than 60 min. Guinea-pig ileum showed the intermediate value of 14.6 min. Enalaprilat and BPP9a slowed the metabolism of bradykinin by 50-100% in rat duodenum and by 50-180% in guinea-pig ileum, showing that a significant fraction of total kininase activity appears to be due to kininase II. In rat duodenum, an almost complete blockade of kininase activity was achieved when bacitracin and mergetpa were used together with enalaprilat. Enalaprilat and BPP9a potentiated bradykinin effects in guinea-pig ileum and rat uterus. In contrast, bradykinin-induced relaxations and contractions in rat duodenum were not potentiated by enalaprilat, BPP9a, or by the enzyme inhibitor mixture (enalaprilat--bacitracin--mergetpa). The results suggest that inhibition of bradykinin enzymatic metabolism by kininases does not necessarily lead to the potentiation of bradykinin effects.     

Role of intracellular antioxidant enzymes after in vivo myocardial ischemia and reperfusion

It has been recently claimed that the human B1 receptors for kinins bind angiotensin-converting enzyme (ACE) inhibitors via a potential zinc-binding domain and are pharmacologically stimulated by these drugs. We verified whether ACE inhibitors stimulate B1 receptors in vitro. The isolated rabbit aorta or mouse stomach responded by negligible contractions to the application of captopril, enalaprilat, or zofenoprilat. The human isolated umbilical vein also failed to respond to enalaprilat. All of these preparations were responsive to the B1 receptor agonists des-Arg9-bradykinin (BK) or Lys-des-Arg9-BK. Furthermore, enalaprilat applied continuously had no significant interaction with the effects of Lys-des-Arg9-BK on the rabbit aorta. Enalaprilat failed to stimulate [3H]arachidonate release, translocate the receptors (confocal microscopy), or stimulate ERK1/2 phosphorylation (immunoblot) in HEK-293 cells stably expressing the rabbit B1 receptor conjugated to yellow fluorescent protein. The phospho-ERK1/2 content of arterial smooth muscle cells of human or rabbit origin was increased by treatment with Lys-des-Arg9-BK but not with enalaprilat. ACE inhibitors do not act as bona fide agonists of the kinin B1 receptors.     

Anti-idiotypic antibodies bearing the internal image of a bradykinin epitope. Production, characterization, and interaction with the kinin receptor.

mAb against bradykinin, the prototypic member of the kinin family of vasodilator peptides, were generated by somatic cell fusion. The antibodies were isotyped as IgG1, kappa-type, and their target epitopes mapped with bradykinin, lysyl-bradykinin (kallidin), kinin receptor antagonists, and fragments thereof, revealing three distinct sets of mAb, i.e., mAb against bradykinin (MBK)1, MBK2, and MBK3. Comparison of the immunologic binding affinities and the known pharmacologic binding specificities of bradykinin derivatives disclosed a striking similarity in the binding profiles of mAb MBK3 and the B2 type of the kinin receptor. Anti-idiotypic antibodies against MBK1, MBK2, and MBK3 were raised in rabbit and sheep. Inhibition and competition experiments on the level of the Ag (ligand), the idiotype, and the anti-idiotype demonstrated the mutual specificity of the network system components. Anti-idiotypic antibodies against MBK3 recognized a particular idiotope that was conformation-dependent and associated with the Ag binding site of the antibody. Binding of anti-idiotypic antibodies to the B2 receptor expressed by human foreskin fibroblasts and guinea pig ileum demonstrated that the anti-idiotypes cross-react with the corresponding receptor across species. Specific stimulation of the inositol phosphate pathway in human fibroblasts and of the PG pathway in mouse fibroblasts, respectively, and inhibition of the latter effect by the B2 kinin receptor antagonist NPC 567 indicated that the anti-idiotypes bear the internal image of a bradykinin epitope. Furthermore, antibodies of the third generation (anti-anti-idiotypic antibodies) recognized the authentic Ag, i.e., bradykinin. Hence, the anti-idiotypic approach provides powerful tools to probe for the hitherto poorly characterized B2 kinin receptor.     

Disruption of the kinin B1 receptor gene affects potentiating effect of captopril on BK-induced contraction in mice stomach fundus

A transgenic mouse model, deficient in kinin B₁ receptor (B₁^−/−) was used to evaluate the role of B₂ receptor in the smooth muscle stomach fundus. The results showed that the potency of bradykinin (BK) to induce contraction in the gastric tissue was maintained whereas the efficacy was markedly reduced. The angiotensin converting enzyme (ACE) inhibitor captopril potentiated BK-induced effect in wild type (WT) but not in B₁^−/− fundus. However, ACE activity detected by the convertion of Ang I to Ang II was inhibited by captopril in both types of gastric tissues. Taking into account the hypothesis that captopril and ACE bind to the B₂ receptor, we suggest that this complex was not formed in the stomach deficient in B₁ receptor. Therefore, our finding strongly support the hypothesis that in smooth muscles that constitutively express the kinin B₁ and B₂ receptors, an interaction between captopril and ACE, B₁ and B₂ receptors should occur forming a complex protein interaction for the potentiating effect of ACE on kinin receptors.     

Effects of bradykinin on DNA synthesis in resting NIL8 hamster cells and human fibroblasts

Bradykinin stimulates [3H]thymidine incorporation and DNA synthesis in resting, serum-deprived NIL8 hamster cells. The ED50 for this stimulation is 4.52 +/- 2.91 nM. Other kinin peptides including lys-bradykinin (kallidin) and met-lys-bradykinin also stimulate [3H]thymidine incorporation in the NIL8 cells, whereas desarg9-bradykinin is without effect, suggesting action of the kinin peptides through type B2 receptors. Bradykinin also stimulates DNA synthesis in IMR-90 human fibroblasts; however, this effect is observed only in the presence of indomethacin, which blocks prostaglandin synthesis. These results suggest that prostaglandins act as negative modulators of the growth-stimulatory effects of bradykinin in the fibroblasts. This conclusion is supported by the observation that exogenously added PGE1, PGE2, PGA1, PGA2, PGB1, and PGB2 strongly inhibit [3H]thymidine incorporation in the human fibroblasts. The direct effect of bradykinin observed in the NIL8 cells may be attributable to the relative resistance of these cells to growth inhibition by prostaglandins.     

The renin-angiotensin and the kallikrein-kinin systems

The renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) each encompasses a large number of molecules, with several participating in both systems. The RAS generates a family of bioactive angiotensin peptides with varying biological activities. These include angiotensin-(1-8) (Ang II), angiotensin-(2-8) (Ang III), angiotensin-(3-8) (Ang IV), and angiotensin-(1-7) [Ang-(1-7)]. Ang II and Ang III act on type 1 (AT(1)) and type 2 (AT(2)) angiotensin receptors, whereas, Ang IV and Ang-(1-7) act on their own receptors. The KKS also generates a family of bioactive peptides with varying biological activities. These include hydroxylated and non-hydroxylated bradykinin and kallidin peptides and their carboxypeptidase metabolites des-Arg(9)-bradykinin and des-Arg(10)-kallidin. Whereas bradykinin and kallidin act mainly via the type 2 bradykinin (B(2)) receptor, des-Arg(9)-bradykinin and des-Arg(10)-kallidin act mainly via the type 1 bradykinin (B(1)) receptor. The AT(1) receptor forms heterodimers with the AT(2) and B(2) receptors and there is cross talk between the AT(1) and epidermal growth factor receptors. The B(2) receptor also interacts with angiotensin converting enzyme and nitric oxide synthase. Both angiotensin and kinin peptides are metabolised by many different peptidases that are important determinants of the activities of the RAS and KKS, and several of which participate in both systems.     

Bradykinin receptor gene variant and human physical performance.

Accumulating evidence suggests that athletic performance is strongly influenced by genetic variation. One such locus of influence is the gene for angiotensin-I converting enzyme (ACE), which exhibits a common variant [ACE insertion (I)/deletion (D)]. ACE can drive formation of vasoconstrictor ANG II but preferentially degrades vasodilator bradykinin. The ACE I allele is associated with higher kinin activity. A common gene variant in the kinin beta(2) receptor (B(2)R) exists: the -9 as opposed to +9 allele is associated with higher receptor mRNA expression. We tested whether this variant was associated with the efficiency of muscular contraction [delta efficiency (DE)] in 115 healthy men and women, or with running distance among 81 Olympic standard track athletes. We further sought evidence of biological interaction with ACE I/D genotype. DE was highly significantly associated with B(2)R genotype (23.84 +/- 2.41 vs. 24.25 +/- 2.81 vs. 26.05 +/- 2.26% for those of +9/+9 vs. +9/-9 vs. -9/-9 genotype; n = 25, 61, and 29, respectively; P = 0.0008 for ANOVA adjusted for sex). There was evidence for interaction with ACE I/D genotype, with individuals who were ACE II, with B(2)R -9/-9 having the highest DE at baseline. The ACE I/B(2)R -9 "high kinin receptor activity" haplotype was significantly associated with endurance (predominantly aerobic) event among elite athletes (P = 0.003). These data suggest that common genetic variation in the B(2)R is associated with efficiency of skeletal muscle contraction and with distance event of elite track athletes and that at least part of the associations of ACE and fitness phenotypes is through elevation of kinin activity.     

Activation of the kallikrein-kinin system by cardiopulmonary bypass in humans

We used cardiopulmonary bypass (CPB) as a model of activation of the contact system and investigated the involvement of the plasma and tissue kallikrein-kinin systems (KKS) in this process. Circulating levels of bradykinin and kallidin and their metabolites, plasma and tissue kallikrein, low and high molecular weight kininogen, and kallistatin were measured before, during, and 1, 4, and 10 h after CPB in subjects undergoing cardiac surgery. Bradykinin peptide levels increased 10- to 20-fold during the first 10 min, returned toward basal levels by 70 min of CPB, and remained 1.2- to 2.5-fold elevated after CPB. Kallidin peptide levels showed little change during CPB, but they were elevated 1.7- to 5.2-fold after CPB. There were reductions of 80 and 60% in plasma and tissue kallikrein levels, respectively, during the first minute of CPB. Kininogen and kallistatin levels were unchanged. Angiotensin-converting enzyme inhibition did not amplify the increase in bradykinin levels during CPB. Aprotinin administration prevented activation of the KKS. The changes in circulating kinin and kallikrein levels indicate activation of both the plasma and tissue KKS during activation of the contact system by CPB.     

Effects of peptidase inhibitors on bradykinin-induced bronchoconstriction in the rat

Objectives of this study were to determine if aerosolized bradykinin causes bronchoconstriction in anesthetized, mechanically ventilated rats, and if pretreatment with enalaprilat, an inhibitor of angiotensin-converting enzyme (ACE), or phosphoramidon, an inhibitor of endopeptidase 24.11 (EP 24.11), alters the response. We found that aerosolized bradykinin elicited a reproducible bronchoconstrictor response that was significantly amplified by pretreatment with aerosolized enalaprilat or phosphoramidon. Neither inhibitor alone affected airway tone or caused nonspecific airway hyperreactivity. These findings indicate that both ACE and EP 24.11 contribute to bradykinin degradation in rat airways.     

Bradykinin facilitates noradrenaline spillover during contraction in the canine gracilis muscle.

Noradrenaline spillover from skeletal muscle vascular areas increases during exercise but the underlying mechanisms are not well understood. Muscle contraction itself causes changes in many factors that could affect noradrenaline spillover. For instance, it has been reported that bradykinin is synthesized in skeletal muscle areas during contraction. Because the B2 bradykinin receptor facilitates noradrenaline spillover, it may be involved in the increase associated with contraction. In this experiment, we studied the effect of bradykinin on noradrenaline spillover in the in situ canine gracilis muscle, using the specific B2 antagonist HOE 140. The drug did not modify noradrenaline spillover at rest, but did cause a significant decrease during muscle contraction, from 558 to 181 pg min(-1). As reported previously in the literature, fractional extraction of noradrenaline decreased during muscle contraction. This effect was independent of HOE 140 treatment. In light of our results, it seems that bradykinin formation during muscle contraction may play an important part in the observed increase in noradrenaline spillover but does not affect fractional extraction.     

The temporal relationship between the neural and vascular actions of kallidin within the nose

The time course of effect of the B(2)-receptor agonist kallidin (K) on induced changes of nasal airflow, rhinorrhoea, nasal pain, sneezing and nasal microvascular leakage has been examined and compared with its B(2) metabolite agonist bradykinin (B) and the B(1)-agonist [des-arg(9)]-bradykinin (D). When administered as a single dose K and B induced an immediate sensation of pain, rhinorrhoea, elevations in lavage albumin and protein levels and a sustained increase in nasal airways resistance (NAR) for 5-40 min post-challenge. [des-arg(9)]-Bradykinin and vehicle placebo (V) were without effect on any of these indices. These studies identify the action of K and B within the nose and differentiate the neural and vascular effects of these kinins in addition to suggesting the potential that nasal blockage and nasal microvascular leakage represent alterations in differing vascular compartments. These findings have implications for the understanding and therapeutic manipulation of rhinitis.     

Kallidin applied to the human nasal mucosa produces algesic response not blocked by capsaicin desensitization.

Various kinins (dissolved in 50 microliters) were applied to the nasal mucosa of healthy human volunteers to test the algesic and proinflammatory effects of these peptides in an intact human tissue. [des-Arg9]-bradykinin (0.5 mumol) was found to be inactive, while bradykinin (0.05-0.5 mumol) and especially kallidin (0.005-0.5 mumol) induced: (a) a mild painful sensation described as burning and pricking (latency 30 s, duration 3-5 min), (b) perception of pulsatility and obstruction in the nasal cavity (onset 1 min, duration 6-8 min). Substance P (0.5 mumol) and neurokinin A (0.5 mumol) produced slight obstruction and weak pulsatile sensation but not pain. Capsaicin (0.05 nmol) produced pain and secretion of fluid, but not pulsatile sensation. The effects of kallidin were not affected by repeated (to induce desensitization) applications of capsaicin (0.5 mumol). Likewise, ipratropium bromide (80 mg in 100 microliters) did not affect responses to kallidin. In an intact human tissue, kallidin produces various effects, including an algesic response, that are apparently independent from activation of B1 receptors and from desensitization of capsaicin-sensitive primary afferents.     

Nonpeptide antagonists for kinin receptors

Kinins are a family of small peptides acting as mediators of inflammation and pain in the peripheral and central nervous system. The two main 'kinins' in mammals are the nonapeptide bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and the decapeptide kallidin (KD, [Lys0]-BK, Lys1-Arg2-Pro3-Pro4-Gly5-Phe6-Ser7-Pro8-Phe9- Arg10). Their biological actions are mediated by two distinct receptors, termed B1 and B2. Kinin B and B2 receptor antagonists may be useful drugs endowed with analgesic and anti-inflammatory properties, with potential use in asthma, allergic rhinitis and other diseases. The first nonpeptide kinin B2 receptor antagonist, WIN 64338, was reported in 1993. Despite its low selectivity, the compound provided a reference for pharmacological and modeling studies. Several quinoline and imidazo[1,2-a]pyridine derivatives have been shown by Fujisawa to possess high affinity and selectivity for kinin B2 receptors. Among them, FR 173657 displayed excellent in vitro and in vivo antagonistic activity, while FR 190997 emerged as the first nonpeptide agonist for B2 receptor. Two structurally related Fournier compounds were recently published. Other kinin B2 receptor ligands were obtained by rational design, through library screening or from natural sources. The only example of a nonpeptide kinin B1 receptor ligand has been reported in a patent by Sanofi.     

The release of bradykinin in bovine mastitis.

The kinin peptides are released during inflammation and are amongst the most potent known mediators of vasodilatation, pain and oedema. Despite early reports of the presence of kinins in milk, no previous study has investigated the role of the kinin system in bovine mastitis. The present study indicated that mastitis was accompanied by raised levels of bradykinin (BK) in milk and the increased levels of BK correlated with the severity of mastitis. Raised BK levels in mastitic milk were not dependent on the presence of inflammatory cells, nor were they secondary to changes in blood levels of BK. In milk from sub-clinically inflamed quarters, BK was raised in those milks where Staphylococcus aureus (S. aureus) was isolated but not in those milks where no pathogen was isolated. Increasing S. aureus artificially, also caused an increase in the milk BK. Increases in milk BK were not restricted only to the mastitic quarters of the udder. In udders in which mastitis was detected in one or more quarters, BK increases were also detected in the apparently uninvolved quarters.     

Tissue distribution and kininogen gene expression after acute-phase inflammation

A kinin-directed monoclonal antibody to kininogens has been developed by the fusion of murine myeloma cells with mouse splenocytes immunized with bradykinin-conjugated hemocyanin. The hybrid cells were screened by an enzyme-linked immunosorbent assay (ELISA) and a radioimmunoassay (RIA) for the secretion of antibodies to bradykinin. Ascitic fluids were produced and purified by a bradykinin-agarose affinity column. The monoclonal antibody (IgG1) bound to bradykinin, Lys-bradykinin, Met-Lys-bradykinin, and kininogens in ELISA. Further, this target-directed monoclonal antibody recognized purified low and high molecular weight bovine, human, or rat kininogens and T-kininogen in Western blotting. After turpentine-induced acute inflammation, rat kininogen levels increased dramatically in liver and serum as well as in the perfused pituitary, heart, lung, kidney, thymus, and other tissues, as identified by the kinin-directed kininogen antibody in Western blot analyses. The results were confirmed by measuring kinin equivalents of kininogens with a kinin RIA. During an induced inflammatory response, rat kininogens were localized immunohistochemically with the kinin-directed monoclonal antibody in parenchymal cells of liver, in acinar cells and some granular convoluted tubules of submandibular gland, and in the collecting tubules of kidney. Northern and cytoplasmic dot blot analyses using a kinin oligonucleotide probe showed that kininogen mRNA levels in liver but not in other tissues increase after turpentine-induced inflammation. The results indicated that rat kininogens are distributed in various tissues in addition to liver and only liver kininogen is induced by acute inflammation. The target-directed kininogen monoclonal antibody is a useful reagent for studying the structure, localization, and function of kininogens or any protein molecule containing the kinin moiety.     

Bradykinin release from contracting skeletal muscle of the cat

Results of previous studies from our laboratory suggest that bradykinin has a role in the exercise pressor reflex elicited by static muscle contraction. The purpose of this study was to quantify the release of bradykinin from contracting skeletal muscle. In 18 cats, blood samples were withdrawn directly from the venous effluent of the triceps surae muscles immediately before and after 30 s of static contraction producing peak muscle tensions of 33, 50, and 100% of maximum electrically stimulated contraction. Contractions producing muscle tensions of 50 and 100% of maximum increased muscle venous bradykinin levels by 27 +/- 9 and 19 +/- 10 pg/ml, respectively. Conversely, 33% maximum contraction did not alter muscle venous bradykinin concentrations. However, when captopril was administered to slow the degradation of bradykinin, muscle venous bradykinin increased from 68 +/- 15 pg/ml at rest to 106 +/- 18 after contractions of 33% of maximum. When muscle ischemia was induced by 2 min of arterial occlusion before and during 30 s of 33% of maximum contraction, muscle venous bradykinin increased by 15 +/- 5 pg/ml. In addition, contraction-induced changes in muscle venous pH and lactate strongly correlated with bradykinin concentrations (r = 0.80 and 0.83, respectively). These data demonstrate that static contraction of relatively high intensity evokes the release of bradykinin from skeletal muscle and that ischemia, decreased pH, and increased lactate are strongly correlated with this release.