A new kinin moiety in human plasma kininogens

Recently, we isolated a new kinin from human urine and tentatively identified it as [Ala3]-Lys-bradykinin. However, there were inconsistencies between the properties of the naturally occurring new kinin and synthetic [Ala3]-Lys-bradykinin. In the present work, we determined whether the new kinin was released from human plasma kininogen, and further investigated the structure of the new kinin. After incubation of plasma (n = 6) with human urinary kallikrein, kinins were separated by HPLC and measured by RIA. The new kinin and Lys-bradykinin were found representing 23 +/- 3 and 76 +/- 6%, respectively, of total kinins released (2.0 +/- 0.4 micrograms/ml). The new kinin was also released from both purified low- and high-molecular-weight kininogens, representing 40-42% of total kinins released. Amino acid sequencing and composition analysis indicated that the structure of the new kinin was [Hyp3]-Lys-bradykinin (Lys-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg) and not [Ala3]-Lys-bradykinin. We conclude that an important proportion of human kininogens contain hydroxyproline instead of proline in position three of the bradykinin moiety.     

Kininogen-derived peptides for investigating the putative vasoactive properties of human cathepsins K and L.

Macrophages at an inflammatory site release massive amounts of proteolytic enzymes, including lysosomal cysteine proteases, which colocalize with their circulating, tight-binding inhibitors (cystatins, kininogens), so modifying the protease/antiprotease equilibrium in favor of enhanced proteolysis. We have explored the ability of human cathepsins B, K and L to participate in the production of kinins, using kininogens and synthetic peptides that mimic the insertion sites of bradykinin on human kininogens. Although both cathepsins processed high-molecular weight kininogen under stoichiometric conditions, only cathepsin L generated significant amounts of immunoreactive kinins. Cathepsin L exhibited higher specificity constants (kcat/Km) than tissue kallikrein (hK1), and similar Michaelis constants towards kininogen-derived synthetic substrates. A 20-mer peptide, whose sequence encompassed kininogen residues Ile376 to Ile393, released bradykinin (BK; 80%) and Lys-bradykinin (20%) when incubated with cathepsin L. By contrast, cathepsin K did not release any kinin, but a truncated kinin metabolite BK(5-9) [FSPFR(385-389)]. Accordingly cathepsin K rapidly produced BK(5-9) from bradykinin and Lys-bradykinin, and BK(5-8) from des-Arg9-bradykinin, by cleaving the Gly384-Phe385 bond. Data suggest that extracellular cysteine proteases may participate in the regulation of kinin levels at inflammatory sites, and clearly support that cathepsin K may act as a potent kininase.     

Identification of [hydroxyproline3]-lysyl-bradykinin released from human kininogens by human urinary kallikrein

The types of kinins released from purified native, single chain human high and low molecular mass kininogens (HMMKs and LMMKs, respectively) by purified human urinary kallikrein were separated by reverse-phase HPLC and quantitated by the rat uterus bioassay. [Hyp3]-lysyl-bradykinin, a recently discovered kinin, represented up to 58% of the biological activity released from 4 individual HMMK preparations purified from 4 different healthy volunteers. In contrast, the majority of the biological activity released from LMMKs purified from pooled plasma was identified as Lys-bradykinin and [Hyp3]-lysyl-bradykinin represented only 6.4 +/- 3.8%. These findings indicate posttranslation hydroxylation of human kininogens and suggest a preference of HMMKs for this modification.     

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

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

Bradykinin receptors in isolated rat duodenum

Pharmacological properties of the bradykinin receptors in the isolated rat duodenum were investigated by examining the relaxant and contractile responses to bradykinin and [des-Arg9]-bradykinin, an agonist of B1 receptors. A specific desensitization and de novo formation for B1 receptors were observed. Changes in medium pH caused a decrease in the responses to bradykinin and [des-Arg9]-bradykinin of rat duodenum. Urea incubation in test tube inhibited the responses to bradykinin and [des-Arg9]-bradykinin of rat duodenum, while urea in bathing medium was ineffective. These findings strongly suggested that (a) ionic bonds are important in the interaction between bradykinin and its receptors, and (b) B2 receptors in rat duodenum are different from those in guinea pig ileum.     

Pharmacological and functional characterization of bradykinin receptors in rat cultured vascular smooth muscle cells

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant (K(D)) of 1.2 +/- 0.2 nM and a maximum receptor density (Bmax) of 47.3 +/- 4.4 fmol/mg protein. The specific binding of [3H]-bradykinin to VSMCs was inhibited by the B2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140) and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin, but not by a B1 receptor-selective agonist (des-Arg9-bradykinin) and antagonist ([Leu8, des-Arg9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca2+]i with half-maximal responses (pEC50) were 7.53 and 7.69, respectively. B2 receptor-selective antagonists (Hoe 140 and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca2+]i changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, verapamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.     

The role of kinin B1 in the plasma extravasation of carrageenin-induced pleurisy

The role of des-Arg9-bradykinin (des-Arg9-BK) and kinin B1 receptor in the plasma extravasation of rat carrageenin-induced pleurisy was investigated employing B1 receptor agonist and antagonists and kininogen-deficient rats. Expression of the B1 receptor mRNA in pleura was induced from 3 to 5 h after the injection of carrageenin into the pleural cavity of Sprague-Dawley rats. Exogenous injection of des-Arg9-BK into the pleural cavity provoked a significant increase in plasma extravasation in 5 h carrageenin-induced pleurisy, but not in 20 min kaolin-induced pleurisy. The level of immunoreactive des-Arg9-BK in the exudate of 5 h carrageenin-induced pleurisy was higher than that of bradykinin (BK). Administration of the B1 receptor antagonists, des-Arg9-[Leu8]-BK or des-Arg9-D-Arg-[Hyp3, Thi5, D-Tic7,Oic8]-BK significantly reduced the exudation rate. However, intrapleural administration of des-Arg9-BK to plasma kininogen-deficient. Brown Norway-Katholiek rats did not result in a further increase in the plasma extravasation. In conclusion, endogenously generated des-Arg9-BK could contribute to the plasma extravasation in carrageenin-induced pleurisy via mediation of the inducible B1 receptor.     

Purification and identification of [hydroxyprolyl3]bradykinin in ascitic fluid from a patient with gastric cancer

Kinins in the ascitic fluid from a patient with gastric cancer were purified by gel filtration and reversed-phase high-performance liquid chromatography (HPLC). Two fractions (fractions I and II) showed kinin activity. Fraction I did not correspond to either bradykinin or other known kinins, whereas fraction II corresponded to bradykinin. Fraction I contained 8 amino acid residues from bradykinin minus 1 proline plus 1 additional hydroxyproline. Sequence analysis of fraction I showed that the proline at the third amino acid residue of bradykinin was replaced by hydroxyproline. The retention time of fraction I on reversed-phase HPLC was exactly the same as that of synthetic [hydroxyprolyl3]bradykinin (Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg) and was distinguishable from des-Pro3-bradykinin. Thus, these results demonstrate for the first time the presence of [hydroxyprolyl3]bradykinin in vivo. This is also the first report of the presence of bradykinin in human tumor ascites.     

Activation of bradykinin B2 receptors increases calcium entry and intracellular mobilization in C9 liver cells.

In C9 rat liver cells bradykinin and kallidin increased (approximately 2-fold) the intracellular concentration of calcium, but the B1 agonist, des-Arg9-bradykinin did not. The effect of bradykinin was inhibited by the B2 antagonists, Hoe 140 and N-alpha-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin, but not by the B1 antagonist, des-Arg9-[Leu8]-bradykinin. The action of bradykinin was diminished, but not abolished, in medium without calcium. The peptide was able to increase intracellular calcium concentration in cells treated with thapsigargin. Bradykinin action was not observed in cells previously stimulated with this local mediator: however, under the same conditions, angiotensin II induced a clear increase in intracellular calcium concentration. Our data indicate that activation of bradykinin B2 receptors increase intracellular calcium concentrations by inducing both gating of the cation and intracellular mobilization in C9 liver cells. In addition, homologous desensitization was observed.     

Kinin B1 receptor-mediated motor responses in normal or inflamed rat urinary bladder in vivo

The rat urinary bladder is one of the few in vivo preparations in which kinin B1 receptor-mediated contractile responses have been described, but the nature (local or reflex) of these responses has not been characterized. We have investigated the motor effects of i.v. or topical (onto the bladder serosa) administration of the selective kinin B1 receptor agonist [des-Arg9]-bradykinin ([des-Arg9]-BK) in the normal or inflamed (cyclophosphamide-induced) urinary bladder in urethane-anaesthetized rats. In both normal and inflamed bladders [des-Arg9]-BK produced a tonic contraction of low amplitude (< 15 mmHg) with phasic contractions of high amplitude (> or = 15 mmHg) superimposed (micturition reflex contractions). In inflamed bladders, the response to [des-Arg9]-BK was more prominent than in controls. Similar observations were made after the topical administration of [des-Arg9]-BK. In order to evaluate any time-dependency in the expression of B1 receptor-mediated bladder responses, [des-Arg9]-BK was administered in separate groups of control animals at 30 and 240 min after the completion of surgical procedures required for set-up of the preparation: no bladder contraction was detected at 30 min whereas both local and reflex contractions could be elicited by [des-Arg9]-BK at 240 min after the set up. In ganglionectomized rats, the response to [des-Arg9]-BK or the selective tachykinin NK2 receptor agonist [betaAla8]NKA(4-10) was evaluated at 30 and 240 min after the set up in inflamed or in control animals. The response to [des-Arg9]-BK was greater after inflammation although a time-dependent increase was evident in both groups; in contrast, the response to [betaAla8]NKA(4-10) was similar in both groups and remained constant over the observation period. After induction of inflammation, the tonic contraction induced by [des-Arg9]-BK in ganglionectomized rats was dose-dependently reduced by the kinin B1 receptor antagonist [desArg10]Hoe 140. The contractile response (number of micturition reflex contractions) induced by [des-Arg9]-BK in normal rats with intact pelvic nerves at 240 min from the set up was not changed after the administration of the selective B2 receptor antagonist Hoe 140. These results indicate that stimulation of bladder kinin B1 receptors evokes a local, tonic-type contraction with reflex contractions superimposed in both normal and inflamed bladders, but in the latter situation the motor responses are magnified.     

Characterization of bradykinin receptors in peripheral organs.

Bradykinin (BK) and related kinins are potent stimulants of the rabbit jugular vein, the hamster urinary bladder, and the guinea pig trachea. The characterization of kinin receptors in these tissues was made with agonists and antagonists. Results obtained with agonists indicate that bradykinin and kallidin are much more active than des-Arg9-BK and suggest the presence of B2 receptors in the three organs. Some new agonists were also tested and the BK analogue, [Hyp3,Tyr(Me)8]BK, was found to be a potent and selective stimulant of the three preparations, with pD2 values of 8.56, 8.00, and 8.39, respectively, but inactive on the rabbit aorta (a B1-receptor system). Contractile effects of kinins in the rabbit jugular vein and hamster urinary bladder were reduced or eliminated by B2-receptor antagonists but at different concentration levels; e.g., acetyl-D-Arg[Hyp3,D-Phe7]BK showed pA2 values of 7.78 on the rabbit jugular vein but only 5.72 on hamster urinary bladder. This compound contracted the guinea-pig trachea and was found to be inactive as an antagonist on this preparation. Contractions of the hamster urinary bladder and the guinea-pig trachea in response to bradykinin were markedly reduced or eliminated by indomethacin and by BW 755C, while those of the rabbit jugular vein were not modified. The present findings indicate that the myotropic effect of kinins on the rabbit jugular vein depends on the activation of B2 receptors and suggest that B2 receptors are largely responsible also for the response of the hamster urinary bladder. B2 receptors and (or) a nonreceptor mechanism appear to be involved in the stimulant effects of the kinin agonists and some antagonists in the guinea-pig trachea.     

Polyphosphoinositide hydrolysis in endothelial cells and carotid artery segments. Bradykinin-2 receptor stimulation is calcium-independent

Bovine aortic and cerebral microvascular endothelial cells and cultured segments of canine common carotid artery possess functional receptors for the nonapeptide bradykinin which mediate a rapid increase in the formation of [3H]inositol 1-phosphate, [3H]inositol 1,4-bisphosphate, and [3H]inositol 1,4,5-trisphosphate from cell membranes containing isotopically labeled myo-inositol. Bradykinin stimulated the formation of [3H]inositol phosphates from cells in culture or tissues at threshold concentrations of 0.1 nM and 1 nM, and with a half-maximal effective concentration of 0.6-1.0 nM and 30 nM, respectively. In cultured cells, the formation of [3H]inositol trisphosphate and [3H]inositol bisphosphate preceded the formation of [3H]inositol monophosphate. Similarly, [3H]inositol phosphate formation was not inhibited by addition of calcium channel blockers, a calcium chelator, or an intracellular calcium antagonist. Calcium ionophore A23187 did not promote [3H]inositol phosphate accumulation. The receptor selectivity of the bradykinin response in cultured cells was most compatible with a type-2 mediated response. Kallidin stimulated with the same potency as bradykinin but was more potent than methionyl-lysyl-bradykinin or des-Arg9-bradykinin. The B1 receptor antagonists des-Arg9-[Leu8]-bradykinin and des-Arg10-[Leu9]-kallidin were without effect. The rapidity of the inositol phosphate response as well as the close correspondence between the bradykinin type-2 receptor mediated hydrolysis of polyphosphoinositides and changes in prostacyclin synthesis, vessel dilation, and permeability suggests that breakdown products of inositol lipids serve as second messengers mediating the effects of bradykinin on the vascular endothelium.     

Tissue kallikrein KLK1 is expressed de novo in endothelial cells and mediates relaxation of human umbilical veins.

Bradykinin released by the endothelium is thought to play an important local role in cardiovascular regulation. However, the molecular identity of endothelial proteases liberating bradykinin from its precursors remained unclear. Using RT-PCR and Southern blotting techniques we detected mRNA for tissue kallikrein (KLK1) in human umbilical vein endothelial cells and in bovine aortic endothelial cells. Protein expression was confirmed by precipitation of KLK1 from lysates of endothelial cells pre-labeled with [35S]-cysteine/methionine. Partial purification of tissue kallikrein from total endothelial cell extracts resulted in a protein triplet of about 50 kDa in Western blots using specific anti-KLK1 antibodies. The immunodetection of tissue kallikrein antigen in the fractions from ion exchange chromatography correlated with the presence of amidolytic tissue kallikrein activity. Stimulation of endothelial cells with angiotensin II (ANG-II), which recently has been shown to activate the vascular kinin system and to cause vasodilation, resulted in the release of bradykinin and kallidin. ANG-II-dependent relaxation of pre-constricted rings from human umbilical veins was abolished in the presence of a specific tissue kallikrein inhibitor. We conclude that endothelial cells de novo express significant amounts of tissue kallikrein, which likely serves in the local generation of vasoactive kinins.     

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.     

Tissue kallikrein and kinin infusion promotes neovascularization in limb ischemia

Adenovirus-mediated kallikrein delivery has been shown to promote blood vessel growth in the limb under both ischemic and normoperfused conditions. Here we investigated whether a continuous supply of kallikrein and kinin peptide can induce neovascularization in a rat model of hindlimb ischemia. Rats underwent femoral artery ligation and localized injection of tissue kallikrein, bradykinin or B1 receptor agonist, followed by infusion of proteins by osmotic minipump. Regional blood flow was monitored weekly by laser Doppler perfusion imaging. Three weeks after surgery, rats receiving kallikrein and kinins showed a significant increase in the perfusion ratio of ischemic vs. normoperfused limb compared to control rats. Similarly, a microsphere assay showed that kallikrein and kinins significantly increased regional blood flow without altering blood pressure. Moreover, kallikrein and kinins significantly augmented capillary and arteriole densities, as quantified by immunostaining with CD-31 and smooth muscle alpha-actin. Both tissue kallikrein and bradykinin increased hemoglobin content in Matrigel implants in mice, providing further evidence of the angiogenic properties. Kinins, when delivered subcutaneously via Matrigel in rats, also increased regional perfusion. This is the first demonstration that local application of tissue kallikrein protein or kinin peptide has therapeutic value in the treatment of ischemic disease by promoting neovascularization.     

Concentrations of glandular kallikrein in human nasal secretions increase during experimentally induced allergic rhinitis

We have previously demonstrated that a mixture of bradykinin and lysylbradykinin is generated in nasal secretions during the immediate allergic response to allergen. The present studies were performed to determine whether glandular kallikrein plays a role in kinin formation during the allergic reaction. Allergic individuals (n = 7) and nonallergic controls (n = 7) were challenged intranasally with appropriate allergen, and nasal lavages obtained before and after challenge were assayed for immunoreactive glandular kallikrein as well as for histamine, kinins, and N-alpha-tosyl-L-arginine methyl esterase (TAME-esterase) activity. The increase in postchallenge immunoreactive glandular kallikrein levels above baseline was significantly greater (p less than 0.01) for the allergic group (16.3 +/- 14 ng/ml; means +/- SD) than for the nonallergic controls (1.0 +/- 1.9 ng/ml). Increased levels of immunoreactive glandular kallikrein correlated with increases in kinins, histamine, and TAME-esterase activity and with the onset of clinical symptoms. Characterization of immunoreactive glandular kallikrein purified from postchallenge lavages by immunoaffinity chromatography confirmed the identity of this material as an authentic glandular kallikrein on the basis of its inhibition by protease inhibitors and by monospecific antibody to tissue kallikrein, its chromatographic behavior on gel filtration, and its ability to generate lysylbradykinin from highly purified human low m.w. kininogen. The specific activity of this purified material, in terms of kinin generation from kininogen, was very similar to that for authentic glandular kallikrein, suggesting that most if not all of the immunoreactive material purified from nasal lavages represented active enzyme. Inhibition studies by using pooled postchallenge lavages suggest that the majority of the kinin generating activity in these samples was due to glandular kallikrein. We conclude, therefore, that glandular kallikrein is secreted during the allergic response and can contribute to the formation of the lysylbradykinin produced during the allergic reaction.     

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.     

Role of kallikrein-kininogen system in insulin-stimulated glucose transport after muscle contractions.

Serum proteins [molecular weight (MW) > 10,000] are essential for increased insulin-stimulated glucose transport after in vitro muscle contractions. We investigated the role of the kallikrein-kininogen system, including bradykinin, which is derived from kallikrein (MW > 10,000)-catalyzed degradation of serum protein kininogen (MW > 10,000), on this contraction effect. In vitro electrical stimulation of rat epitrochlearis muscles was performed in 1) rat serum +/- kallikrein inhibitors; 2) human plasma (normal or kallikrein-deficient); 3) rat serum +/- bradykinin receptor-2 inhibitors; or 4) serum-free buffer +/- bradykinin. 3-O-methylglucose transport (3-MGT) was measured 3.5 h later. Serum +/- kallikrein inhibitors tended (P = 0.08) to diminish postcontraction insulin-stimulated 3-MGT. Contractions in normal plasma enhanced insulin-stimulated 3-MGT vs. controls, but contractions in kallikrein-deficient plasma did not. Supplementing rat serum with bradykinin receptor antagonist HOE-140 during contraction did not alter insulin-stimulated 3-MGT. Muscles stimulated to contract in serum-free buffer plus bradykinin did not have enhanced insulin-stimulated 3-MGT. Bradykinin was insufficient for postcontraction-enhanced insulin sensitivity. However, results with kallikrein inhibitors and kallikrein-deficient plasma suggest kallikrein plays a role in this improved insulin action.     

Extracellular aspartic protease SAP2 of Candida albicans yeast cleaves human kininogens and releases proinflammatory peptides, Met-Lys-bradykinin and des-Arg9-Met-Lys-bradykinin

Abstract Bradykinin-related peptides, universal mediators of inflammation collectively referred to as the kinins, are often produced in excessive amounts during microbial infections. We have recently shown that the yeast Candida albicans, the major fungal pathogen to humans, can exploit two mechanisms to enhance kinin levels at the sites of candidial infection, one depending on adsorption and activation of the endogenous kinin-generating system of the host on the fungal cell wall and the other relying on cleavage of kinin precursors, the kininogens, by pathogen-secreted proteases. This work aimed at assigning this kininogenase activity to the major secreted aspartic protease of C. albicans (SAP2). The purified SAP2 was shown to cleave human kininogens, preferably the low molecular mass form (LK) and optimally in an acidic environment (pH 3.5-4.0), and to produce two kinins, Met-Lys-bradykinin and its derivative, [Hydroxyproline3]-Met-Lys-bradykinin, both of which are capable of interacting with cellular bradykinin receptors of the B2 subtype. Additionally, albeit with a lower yield, des-Arg9-Met-Lys-bradykinin, an effective agonist of B1-subtype receptors, was released. The pathophysiological potential of these kinins and des-Arg-kinin was also proven by presenting their ability to stimulate human promonocytic cells U937 to release proinflammatory interleukin 1β (IL-1β) and IL-6.