Kinin-forming activity in rat brain

The present study shows that rat brain contains a kinin-forming activity which is distinguishable from plasma kallikrein. Kinin-forming activity was found in an acetone powder of frozen brain tissue (between 27 and 175.5 ng generated bradykinin/g fresh brain tissue/h). Analysis by high pressure liquid chromatography (HPLC) indicated that the kinin formed chromatographed like true bradykinin (BK). After subcellular fractionation using differential centrifugation of homogenized fresh brain tissue the kinin-forming activity was found mainly in a microsomal (P-3) fraction after preincubation with 2 μM melittin. Further fractionation of P-3 fraction using discontinuous sucrose gradient centrifugation identified activity in both the 1 M sucrose layer (5.8 ± 3.1 ng kinin/mg protein/h) and at the interface between the 0.8 and 0.3 sucrose layers (9.4 ± 4 ng kinin/mg protein/h). Melittin pretreatment did not change these values. The distribution pattern of the kallikrein-like activity was different from that of cathepsin d-like acid protease. The two kinin-forming activities were equally sensitive to treatment with various trypsin inhibitors but were clearly distinguishable from plasma kallikrein: brain activity was inhibited completely by Trasylol but not by soybean trypsin inhibitor (SBTI) or ovomucoid while plasma kallikrein was completely inhibited by SBTI and partially by ovomucoid and Trasilol. Our results clearly distinguish between plasma kallikrein, brain cathepsin d-like acid protease activity and an apparent brain kinin-forming activity, but do not by themselves establish a central biosynthetic pathway for kinin generation.     

Purification and characterization of plasma T-kininogen from Wistar and brown Norway rats

A rapid and convenient three-step purification scheme has been developed for the purification of T-kininogen (alpha 1-cysteine proteinase inhibitor) from rat plasma. The purification process includes chromatography on hydroxyapatite, immunoaffinity chromatography and gel filtration. This procedure is applied to plasma from the brown Norway rat which is known to be deficient in high and low molecular weight kininogens. The method furnished large amounts of T-kininogen from turpentine-treated Wistar rats as well as from untreated and turpentine-treated deficient brown Norway rats. The amino acid and hexose content of the three T-kininogens has been determined. While the composition of the molecules isolated from both injured rats was similar, the neutral sugar content of T-kininogen purified from untreated brown Norway rats was lower and its amino acid composition showed slight differences. The three molecules have identical behaviour and similar physicochemical and immunological properties when analysed by SDS electrophoresis, isoelectrofocusing and two-dimensional immunoelectrophoresis.     

T-kininogen--the major plasma kininogen in rat adjuvant arthritis

Total kininogen in plasma of Freund's adjuvant treated rats increased 20-fold 7 days following the injection. Analysis of the kininogens demonstrated that increases in T-kininogen was the major reason for the rise in kininogen. High molecular weight and low molecular weight kininogens showed little or no change. The increase in T-kininogen paralleled the inflammatory condition. Anti-inflammatory agents which reduced paw swelling also reduced plasma T-kininogen levels. Unidentified peaks on HPLC of kinin following plasma treatment by trypsin were shown to be oligopeptides containing T-kinin (Ile-serbradykinin). The relationship of T-kininogen to the inflammatory response is discussed.     

Kininogen substrates for trypsin and cathepsin D in human,rabbit and rat plasmas

Studies have compared “total”, HMW kininogen and leukokininogen levels in human, rabbit and rat plasmas using trypsin, glass powder and cathepsin D as kininogenases or activators of kininogenases. Rat plasma was found to have about 10 fold more leukokininogen than the other plasmas assayed. When trypsin was used to estimate total kininogen, rat plasma liberated maximal amounts of kinin only in the presence of high concentrations of trypsin (1 mg/ml incubation mixture). In addition, it was found that trypsin in these concentrations liberated from rat plasma both bradykinin and a previously unidentified kinin which we have termed “T-kinin”. The results overall indicate that in the case of rat and rabbit plasma, currently used methods for estimations of total kininogen may not be accurate. T-kinin may represent a leukokininogen or a hitherto undescribed kininogen.     

Amino liberation in Brown-Norway rats

When injected intravenously with dextran or O-hydroxyethyl derivatives of rutoside, rat Brown Norway BN/Mai Pfd f exhibit a fall in the blood pressure and an increase of vascular permeability with oedema formation. As the rat is genetically deprived of plasmatic prekallikrein an of kininogens, activation of the kinin system is not necessary neither for mast-cell degranulation nor for amine release.     

Attenuated kinin release from human neutrophil elastase-pretreated kininogens by tissue and plasma kallikreins

Components of kinin-forming systems operating at inflammatory sites are likely to interact with elastase that is released by recruited neutrophils and may, at least temporarily, constitute the major proteolytic activity present at these sites. The aim of this work was to determine the effect of kininogen degradation by human neutrophil elastase (HNE) on kinin generation by tissue and plasma kallikreins. We show that the digestion of both low molecular mass (LK) and high molecular mass (HK) forms of human kininogen by HNE renders them essentially unsusceptible to processing by human urinary kallikrein (tissue-type) and also significantly quenches the kinin release from HK by plasma kallikrein. Studies with synthetic model heptadecapeptide substrates, ISLMKRPPGFSPFRSSR and SLMKRPPGFSPFRSSRI, confirmed the inability of tissue kallikrein to process peptides at either termini of the internal kinin sequence, while plasma kallikrein was shown to release the kinin C-terminus relatively easily. The HNE-generated fragments of kininogens were separated by HPLC and the fractions containing internal kinin sequences were identified by a kinin-specific immunoenzymatic test after trypsin digestion. These fractions were analyzed by electrospray-ionization mass spectrometry. In this way, multiple peptides containing the kinin sequence flanked by only a few amino acid residues at each terminus were identified in elastase digests of both LK and HK. These results suggest that elastase may be involved in quenching the kinin-release cascade at the late stages of the inflammatory reaction.     

Direct radioimmunoassay for rat T-kininogen

Antibodies raised in rabbits against pure rat T-kininogen did not cross-react with Ile-Ser-Bradykinin, bradykinin, nor with kininogens from other mammalian species. They presented a 1 to 15% cross-reaction with pure rat HMW kininogen, depending on the quantity of HMW kininogen. A direct radioimmunoassay for rat T-kininogen in plasma was developed and it enabled 89 fmol of the protein to be detected. A good correspondence was obtained between the direct RIA and the T-kinin generating assay. By the direct assay, it was found that T-kininogen is increased about ten fold in rats subcutaneously injected with turpentine. These data were confirmed by HPLC analysis of the plasma kinins released by trypsin which demonstrated that only T-kinins are increased, bradykinin being unchanged. It was possible according to the results obtained by the direct RIA and HPLC analysis to estimate that in the normal rat, HMW and LMW kininogen represent about 35% and T-kininogen 65%. In the turpentine-treated rat, T-kininogen reached 95%. This RIA will allow the study of the regulation of T-kininogen in the rat and the synthesis of this protein in cells in culture.     

Two-dimensional electrophoresis protein profiling and identification in rat bronchoalveolar lavage fluid following allergen and endotoxin challenge

The protein content of bronchoalveolar lavage fluid (BALF) from actively sensitised Brown Norway (BN) rats challenged with allergen (ovalbumin, OA) and from na?ve Brown Norway rats challenged with endotoxin (lipopolysaccharide, LPS) was analyzed and compared to healthy controls treated with vehicle only. BALF proteins were analyzed by one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis and identified by peptide mass fingerprinting matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or nanoliquid chromatography-tandem MS (nanoLC-MS/MS) after in-gel trypsin digestion of selected 2-D gel spots. Our study shows that the BALF protein profile is significantly different in animals after allergen (OA) or endotoxin (LPS) challenge as compared to controls, concerning the content of proteins derived from plasma or produced locally in the lung. In both challenges the following proteins presented patterns which differed qualitatively compared to control: T-kininogen I and II, alpha-1-antitrypsin, calgranulin A, fetuin A and B, and haptoglobin. Other proteins were diminished in both challenges, such as Clara cell 10 kDa secretory protein (CC10) and pulmonary surfactant associated protein B (SP-B); c-reactive protein increased in the OA-challenge and decreased in the LPS-challenge, and pulmonary surfactant associated protein A (SP-A) was decreased in the OA-challenge and was not significantly changed in the LPS-challenge. The identified proteins could be important not only for the diagnosis but have also interesting implications for medical treatment of lung inflammatory conditions. Furthermore, even if based on a limited number of animals, our results are of interest for the identification of lung protein markers and a better understanding of the mechanisms involved in the pathogenesis of lung diseases.     

Limited proteolysis of T-kininogen (thiostatin). Release of comparable fragments by different endopeptidases

Limited proteolysis of T-kininogen by heterologous and homologous endopeptidases (bovine trypsin, human leukocyte elastase, rat submaxillary gland endopeptidase k, and rat mast cell chymase) produced similar fragmentation. Amino-terminal sequence analysis of whole T-kininogen lysates and purified proteolytic fragments identified four susceptible regions which contained all the preferential cleavage sites for these proteinases. Two of these susceptible regions were close to the junction between heavy chain cystatin-like domains, the third was in the kinin-containing region, and the fourth was close to the carboxyl terminus of the T-kininogen light chain. There was only one primary site for each proteinase in the kinin-containing region, which explains why catalytic amounts of these proteinases did not release immunoreactive kinin from this kininogen. However, preferential cleavage of T-kininogen close to the junction between cystatin-like domains released fragments which, provided they included cystatin-like domains 2 and/or 3, strongly inhibited papain and cathepsin L. The fragments were inhibitory even when parts of the amino-terminal ends of the domains were lacking. The highly conserved glycyl residue, thought to be involved in the inhibitory reactive site of cystatin-like inhibitors, was not required in purified domain 3 for inhibition of cathepsin L.     

Characterization of serine proteinases isolated from rat submaxillary gland: with special reference to the degradation of rat kininogens by these enzymes

From the homogenate of rat submaxillary gland, two kinds of serine proteinases, named tentatively proteinases A and B, were isolated and their chemical properties and activities toward rat kininogens were examined, in comparison with those of submaxillary kallikrein. Proteinase A with Mr of 28,200 rapidly cleaved high-molecular-weight (HMW) kininogen into a protein of 67 kDa, which retained thiol-proteinase inhibitory activity, but had lost the correcting activity of HMW kininogen on the prolonged clotting time of Fitzgerald trait plasma. It liberated bradykinin from HMW kininogen but did not liberate kinin from T-kininogen and did not degrade T-kininogen. On the other hand, proteinase B with Mr of 30,400 showed a very weak activity for the liberation of kinin from T-kininogen and the cleavage of T-kininogen at pH 8.0. However, the enzyme extensively degraded T-kininogen at pH 4.5. Proteinase B also degraded HMW kininogen at pH 4.5 and pH 8.0, but liberated bradykinin only at pH 8.0. Thiol-proteinase inhibitory activities of HMW kininogen and T-kininogen were inactivated after the incubation with proteinase B at pH 4.5 but not at pH 8.0, while the correcting activity of HMW kininogen on the Fitzgerald trait plasma was inactivated at pH 4.5 and 8.0. The NH2-terminal amino acid sequences of proteinases A and B were different from each other, and distinguishable with those of serine proteinases in rat submaxillary gland so far reported. These results provide evidence that in addition to the known kallikrein, there exist at least two kinds of serine proteinases in rat submaxillary gland, both of which liberate bradykinin from rat HMW kininogen at pH 8.0 and modulate the functional activities of HMW kininogen and T-kininogen, degrading these proteins at pH 8.0 or 4.5.     

Purification and characterization of rat low molecular weight kininogen

Low molecular weight (LMW) kininogen was isolated from pooled rat plasma by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Blue-Sepharose CL-6B, and Sephadex G-100. It was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoelectrophoresis. The molecular weight of rat LMW kininogen was determined to be 72,000 by SDS-PAGE. The LMW kininogen contained 83.5% protein, 4.0% hexose, 5.5% hexosamine, and 2.7% sialic acid. Kinin liberated from LMW kininogen by trypsin treatment was identified as an Ile-Ser-bradykinin(T-kinin) by analysis involving ion exchange column chromatography on CM-Sephadex C-25 and high performance liquid chromatography on a reverse-phase column (ODS-120T). LMW kininogen formed kinin with rat submaxillary gland kallikrein, but the kinin liberated was only 14% of the total kinin content, that is, that released by trypsin. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antiserum cross-reacted with high molecular weight (HMW) kininogen, but spur formation was observed between the LMW and HMW kininogens. The kininogen level in rat plasma was estimated to be 433 microgram/ml by a quantitative single radial immunodiffusion test.     

Release of T-kinin and bradykinin in carrageenin induced inflammation in the rat

Plasma and inflammatory fluid kininogen levels, and blood and inflammatory fluid free kinin levels were determined in rats 24 h after the injection of carrageenin into an air pouch. Plasma T-kininogen levels increased 7-fold. In the inflammatory fluid levels reached 8 μg/ml. Blood levels of free kinin showed a 5-fold increase. The kinins were identified on HPLC as T-kinin (Ile-Ser-bradykinin) and bradykinin, 63 and 37%, respectively. These results indicate for the first time that free T-kinin as well as bradykinin is released during an inflammatory response in rat and confirms our previous finding that T-kininogen may be a major acutephase protein in inflammation.

T-kinin T-kininogen Bradykinin Inflammation Acute-phase protein Carrageenin     

Clostridiopeptidase B inhibition by plasma marcroglobulins and microbial antiproteases.

Clostridiopeptidase B (EC 3.4.22.8) was not inhibited by stoichiometric amounts of lima bean trypsin inhibitor, ovomucoid trypsin inhibitor, Kuntiz bovine trypsin inhibotor, Kunitz soybean trypsin inhibitor or ovoinhibitor. Activity was diminished at relatively high concentrations of the three latter inhibitors. Human plasma alpha 2-macroglobulin inhibited both the amidase and protease activity of the enzyme. Rat and dog plasmas contained high molecular weight inhibitors, presumably macroglobulins as well. Inhibition by this component was greater in rat plasma than in dog plasma, which may be related to the observation that clostridiopeptidase B-induced generation of kinin activity is indirect in the former plasma, but direct in the later. Leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) and antipain ([S)-1-carboxy-2-phenylethyl] carbamoyl-L-arginyl-L-valyl-L-argininal) inhibited clostridiopeptidase B (Ki of 2 . 10(-8) and 3 . 10(-8) M, respectively). They were potent inhibitors of clostridiopeptidase B-induced kinin release in dog plasma.     

Proteomic analysis permits the identification of new biomarkers of arterial wall remodeling in hypertension

Hypertension represents one of the main risk factors for vascular diseases. Genetic susceptibility may influence the rate of its development and the associated vascular remodeling. To explore markers of hypertension-related morbidity, we have used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry to study changes in proteins released by the aorta of two rat strains with different susceptibilities to hypertension. Fischer and Brown Norway (BN) rats were divided into a control group and a group receiving low-dose N(Omega)-nitro-L-arginine methyl ester (L-NAME), a hypertensive drug, interfering with endothelial function. In spite of a significant elevation of blood pressure in both strains in response to L-NAME, BN rats exhibited a lower vascular remodeling in response to hypertension. Proteomic analysis of secreted aortic proteins by SELDI-TOF MS allowed detection of four mass-to-charge ratio (m/z) peaks whose corresponding proteins were identified as ubiquitin, smooth muscle (SM) 22alpha, thymosin beta4, and C-terminal fragment of filamin A, differentially secreted in Fischer rats in response to L-NAME. We have confirmed a strain-dependent difference in susceptibility to L-NAME-induced hypertension between BN and Fischer rats. The greater susceptibility of Fischer rats is associated with aortic wall hypertrophic remodeling, reflected by increased aortic secretion of four identified biomarkers. Similar variations in one of them, SM22alpha, also were observed in plasma, suggesting that this marker could be used to assess vascular damage induced by hypertension.     

Role of kinins in chronic heart failure and in the therapeutic effect of ACE inhibitors in kininogen-deficient rats

Using Brown Norway Katholiek (BNK) rats, which are deficient in kininogen (kinin precursor) due to a mutation in the kininogen gene, we examined the role of endogenous kinins in 1) normal cardiac function; 2) myocardial infarction (MI) caused by coronary artery ligation; 3) cardiac remodeling in the development of heart failure (HF) after MI; and 4) the cardioprotective effect of angiotensin-converting enzyme inhibitors (ACEI) on HF after MI. Two months after MI, rats were randomly treated with vehicle or the ACEI ramipril for 2 mo. Brown Norway rats (BN), which have normal kininogen, were used as controls. Left ventricular (LV) end-diastolic volume (EDV), end-systolic volume (ESV), end-diastolic pressure (EDP), and ejection fraction (EF) as well as myocardial infarct size (IS), interstitial collagen fraction (ICF), cardiomyocyte cross-sectional area (MCA), and oxygen diffusion distance (ODD) were measured. We found that 1) cardiac hemodynamics, function, and histology were the same in sham-ligated BN and BNK rats; 2) IS was similar in BN and BNK; 3) in rats with HF treated with vehicle, the decrease in LVEF and the increase in LVEDV, LVESV, LVEDP, ICF, MCA, and ODD did not differ between BNK and BN; and 4) ACEI increased EF, decreased LVEDV and LVESV, and improved cardiac remodeling in BN-HF rats, and these effects were partially blocked by the bradykinin B(2) receptor antagonist icatibant (HOE-140). In BNK-HF rats, ACEI failed to produce these beneficial cardiac effects. We concluded that in rats, lack of kinins does not influence regulation of normal cardiac function, myocardial infarct size, or development of HF; however, kinins appear to play an important role in the cardioprotective effect of ACEI, since 1) this effect was significantly diminished in kininogen-deficient rats and 2) it was blocked by a B(2) kinin receptor antagonist in BN rats.     

Ile-Ser-bradykinin (T-kinin) and Met-Ile-Ser-bradykinin (Met-T-kinin) are released from T-kininogen by an acid proteinase of granulomatous tissues in rats

An acid proteinase of granulomatous tissues in rats with carrageenin-induced inflammation released kinin from T-kininogen. The kinin isolated by n-butanol extraction was separated by reverse-phase high-performance liquid chromatography into T-kinin and a T-kinin derivative. From determination of its amino acid composition and its immunoreactivity toward anti-bradykinin antiserum, the T-kinin derivative was identified as Met-Ile-Ser-bradykinin (Met-T-kinin).     

Inactivation of kallikrein and kininases and stabilization of whole rat brain kinin levels following focused microwave irradiation

Focused microwave irradiation was employed to stabilize endogenous whole rat brain bradykinin levels prior to a simple extraction procedure. Skull microwave exposure (2450 MHz, 3.8 kW., 2.45 sec) resulted in inactivation to less than 5% of control of whole brain kallikrein and kininase activity. Using this adequate exposure duration whole rat brain kinin levels as measured by a sensitive radio-immunoassay were approximately 0.6 pmol/g (wet weight). Further purification of irradiated brain extracts using HPLC revealed that immunoreactive kinin eluted as a single peak that co-chromatographed with authentic bradykinin. Microwave fixation duration of 1.25 sec yielded greatly increased levels of immunoreactive kinin which following HPLC purification eluted in two peaks, corresponding to authentic bradykinin and T-kinin, respectively. The tissue injury resulting from incomplete microwave fixation resulted in the release of kinins. This excess immunoreactive kinin may be derived from cerebral blood, since the predominant form of kinin-generating protein in plasma is T-kininogen.     

Effect of macrophages and antibodies on in vivo growth of Moloney sarcoma in the rat.

Brown Norway and Lewis rats were challenged with a Brown Norway Moloney sarcoma tumor, MST-1, admixed with nonimmune peritoneal exudate macrophages syngeneic to the host; or admixed with nonimmune peritoneal exudate macrophages and hyperimmune anti-MST-1 antibodies. In vivo growth of MST-1 in BN and Lewis rats was inhibited by admixing Brown Norway or Lewis macrophages, respectively, with BN anti-MST-1 antibodies. The inhibiting BN antibodies were of the IgG2 class, lacking IgG2a antibodies. Brown Norway anti-MST-1 of IgG2 class without macrophages did not affect growth of MST-1. Brown Norway and Lewis anti-MST-1 antibodies of IgG2a class enhanced tumor growth, whether admixed with macrophages or not. Anti-MST-1 antibodies of IgM and IgG1 classes did not influence tumor growth. Peritoneal exudate macrophages removed from Lewis donors 8 to 10 days after inoculation of MST-1 inhibited completely growth of the challenge tumor; macrophages of Brown Norway origin were inhibitory only when harvested from hyperimmune donors, that is, 40 or more days after inoculation of MST-1. Macrophages from hyperimmune donors were specifically cytotoxic to MST-1 and did not inhibit an unrelated syngeneic BN tumor of chemical origin.     

Canine Plasma Kininogen: Evidence for the Presence of Two Kininogens and Purification of High Molecular Weight Kininogen and Characterization as Multi-Functional Molecule

The ratio of kininogen that is substrate of plasma kallikrein to kininogen, which is not substrate of plasma kallikrein in canine plasma, was about 1:3.6 by differential assay of kininogens. When the plasma was gel-filtered through a column of Sephacryl S-300 superfine, two fractions, which released kinin by trypsin, were obtained. These results indicate that two kininogens with different molecular weights are present in the plasma and they show different susceptibility to plasma kallikrein. One kininogen was purified by ion-exchange and zinc-chelating affinity chromatographies. Purified kininogen showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition and its molecular weight was 125 kDa. Released kinin from the kininogen by trypsin was bradykinin. The kininogen inhibited papain and ficin but did not inhibit bromelain at the concentration used. The kininogen bound to carboxymethylated-papain and this binding was dissociated by 3M NaSCN. Canine plasma shortened the abnormal clotting time of human high molecular weight kininogen-deficint plasma. The kininogen also shortened the abnormal clotting time of the plasma. From these results, the purified kininogen was high molecular weight kininogen and it was multi-functional protein.     

Measurement of T-kinin in rat plasma using a specific radioimmunoassay.

T-kinin (Ile-Ser-Bradykinin) has been isolated only from the plasma of the rat and it is unclear whether the peptide, or its biosynthetic precursor, T-kininogen, circulates in the human. An NH2-terminally directed antiserum to T-kinin was raised in rabbits using an immunogen prepared by coupling the free -SH group of T-kinin extended from its COOH-terminus by a cysteinyl residue to an -NH2 group on human serum albumin. A radioimmunoassay was developed using this antiserum and 125I-labelled [Tyr10]T-kinin as tracer that was sensitive (least-detectable concentration 3 fmol/tube) and relatively specific for T-kinin (cross-reactivity with bradykinin and kallidin less than 1%). Treatment of rat plasma with an excess of trypsin in the presence of a kininase inhibitor generated T-kinin immunoreactivity equivalent to 455 +/- 71 pmol/ml (mean +/- S.E.M.; n = 9) and this immunoreactivity was eluted from a reversed-phase HPLC column as a single peak with the same retention time as synthetic T-kinin. In contrast, treatment of plasma from healthy human subjects (n = 8) and from patients (n = 8) with inflammation due to acute or chronic gastrointestinal disease under the same conditions did not generate any detectable T-kinin immunoreactivity. It is concluded, therefore, that T-kininogen, the biosynthetic precursor of T-kinin in the rat, is either absent from the plasma of human subjects or is present in a concentration less than 30 fmol/ml. Similarly, T-kininogen is probably not an acute phase reactant in humans.