Bradykininase Activity Research Articles

Rat aortic smooth muscle cells in culture express kallikrein, kininogen, and bradykininase activity.

We have studied rat vascular smooth muscle (VSM) cells in culture for the presence of key elements of the glandular kallikrein-kinin system. Direct radioimmunoassay (RIA) using antiserum against rat urinary kallikrein detected a glandular kallikrein-like enzyme (GKLE) in VSM cells and in media. VSM homogenates and culture media had kininogenase activity, generating kinins from dog kininogen. About half of the GKLE was enzymatically inactive which could be activated with trypsin. Kininogenase activity was inhibited completely by aprotinin but only 20% by soybean trypsin inhibitor (SBTI). Trypsin liberated kinins from homogenates and media, demonstrating that VSM cells contain kininogen. Homogenates and media rapidly degrade bradykinin. GKLE, kininogen, and bradykininase activity were all present in VSM cells grown in defined media that contain no serum, thus eliminating any contamination or artefacts from fetal calf serum in standard culture media. Blood vessels of the rat have been reported to contain GKLE. Our observations indicate that GKLE is synthesized by VSM cells, not deposited from plasma. Furthermore, VSM cells synthesize kininogen and bradykininase(s), the other key elements of the glandular kallikrein-kinin system. Thus it is possible that the system functions as an autocoid mechanism that regulates local vascular tone.

Kallikrein and kininase activities in whole saliva from patients with periodontal disease (author's transl)

The kinins, a group of biologically active polypeptides, are generally considered as important chemical mediators in the inflammatory process. However, the role of these mediators in periodontal disease still remains unknown.To make these points clear, the present study was performed to measure kallikrein and bradykininase activities in whole saliva from patients with periodontal desease, and to correlate these data with various clinical conditions such as GI-score, OHI-score, Pocket depth and Bone loss score.The kallikrein activity in the samples was measured by colorimetric method of Moriya et al. and fluorometric method of Matsuda et al., Both methods were compared to find out the better way for the mesurement of kallikrein activity.The bradykininase activity in the samples was assayed by the biological method of Takeya et al., The enzyme activity was expressed as the percentage of inactivation of bradykinin.The following results were obtained:1. Kallikrein was measured in the range from 10 to 40μg/ml by colorimetric method, however, much lower amount of kallikrein, 0.5 to 12μg/ml, could be detected by fluorometric method.2. Statistically significant positive correlations were found between kallikrein activity and all the clinical conditions examined (p<0.001).3. Statistically significant negative correlations were obtaind between bradykininase activity and all the clinical conditions examined (-p<0.001), and also between kallikrein activity and bradykininase activity (-p<0.001).4. The kallikrein activities in the saliva increased following the progress of periodontal disease, but the bradykininase activities remarkably decreased.

スケーリングとブラッシングによる歯周ポケット浸出液中の Bradykininase の消長

Kinins are generally considered as important chemical mediators in inflammatory process. However, the role of these mediators in periodontal disease still remains unknown. Recently, Omori and Mizuno measured bradykininase activity in gingival fluid of the patients with periodontal disease and compared it with various clinical findings. They showed that this enzyme had a marked correlation with PMA-Index score (P<0.01), plaque score (P<0.001), calculus score (P<0.01) and pocket, depth (P<0.001).The present study was performed to know the effect of scaling and brushing on both the variation of bradykininase activity in pocket exudate of the patients with periodontal disease and their clinical findings.Twenty seven patients with periodontal disease (10 males and 17 females, aged 16 to 46) were examined in this study. The subjects were separated in two groups, the first group having only a scaling and the second group receiving a scaling and a brushing. The pocket exudate was collected from periodontal pocket by filter paper strips just before and a week, two weeks and three weeks after the treatment. After the strips were weighted, they were dipped in the inhibitor (Trasylol) and then homogenized by Polytron homogenizer. The mixture was centrifuged at 2, 500g for 20min. at O°C and the supernate was used as assay. The bradykininase activity was determined by measuring the amount of pocket exudate (mg) which inactivated 50% of added 1μg/ml bradykinin (biological method using guinea pig ileum)The results of study showed that PMA index, plaque index, pocket depth and exudate quantity decreased more significantly in scaling and brushing group than in only scaling group.In both groups, the bradykininase activity in pocket exudate had a tendency to increase with improvement of periodontal condition, paticularly in scaling and brushing group.

口腔清掃停止による全唾液, 歯垢および歯肉溝浸出液中の Kinins および Kininase の推移について

The present study was performed to elucidate the variation of kinin contents and kininase activities in the saliva, plaque and gingival fluid of the healthy people without oral hygiene.Nine male dental students, aged 20-24, were the experimental subjects. All of the participants had a tooth scaling and polishing, and then they were instructed not to brush their teeth for the following 10 days. At the 11th day, they were allowed to have a tooth brushing (Rolling method) 3 times a days for 5 days. The collection of samples (saliva, plaque and gingival fluid) and clinical examinations were performed daily for the entire experimental period (16 days). Kinin contents and bradykininase activities in the samples were assayed by a biological method of Takeya et al. The bradykininase activity was determined by measuring the percent of inactivation of added bradykinin (1μg/ml).The following results were obtained;1. The plaque index, gingival index and amount of gingival fluid were increased throughtout the period without oral hygiene (10 days). These clinical scores showed a rapid decline when the oral hygiene procedure was resumed.2. Kinin contents in the samples were almost zero for 5 or 6 days after stopping of the oral hygiene, and then it increased gradually for the following 4 or 5 days. The oral hygiene procedure was resumed at 10 days, and after that kinin contents decreased rapidly until these were not measured in the samples.3. Bradykininase activities in the samples decreased gradually after the oral hygiene was stopped, and these enzyme activities disappeared at 4-5 days after stopping of the oral hygiene. But, after the resume of oral hygiene, the enzyme activities rapidly increased to the level of the first days of the experiment.

キニンシステムにおける血液ｐＨの影響について

Effects of blood pH on the kinin system were studied fundamentally and clinically. Bradykinin was measured by bioassay of Miyanaga's method, and bradykininase was measured by bioassay of Streeten's method. Fundamental study showed that bradykinin recovery was 2.5 times higher in rabbits of acidosis than in rabbits of normal condition. However, there was not a significant difference in bradykinin recovery between rabbits of alkalosis and normal rabbits. Bradykininase in rabbits of acidosis was dropping, but in rabbits of al kalosis was slightly elevating. These facts suggest that bradykinin is very sensitive even to a little fluctuation of pH in the blood, but bradykininase might be hardly influenced even by pH of 7.0 in the blood. On the other hand, from the clinical point of view, the patients with Behcet disease were found to be acidosis, and bradykinin activity was slightly high, but bradykininase activity was low.In patients with diabets mellitus, there was not a significant differences between activities of bradykinin or bradykininase, and pH. In patients with myocardial infarction with strong acidosis, values of both bradykinin and bradykininase were high.

Subunits of human plasma kininase II generated by plasma kallikrein

Partially purified human plasma kininase II was incubated with proteolytic enzymes such as plasma kallikrein, prekallikrein activator, plasmin, thrombin and trypsin. Digestion of plasma kininase II by plasma kallikrein generated 180,000 (I) and 95,000 (II) mol. wt subunits while plasmin, thrombin and trypsin inactivated plasma kininase II. These subunits have two activities just as plasma kininase has: they hydrolyze bradykinin, and they convert angiotensin I into angiotensin II. K m values of the two subunits for bradykinin were 0.17 × 10 −6 M, indicating that this value was identical with plasma kininase II. V max values of the two subunits for bradykinin were higher than that of plasma kininase II. The bradykininase activities of the two subunits were significantly inhibited by inhibitors such as HgCl 2, ethylenediamine tetraacetic acid. p-chloromercuriphenyl sulfonic acid, SQ 20881(BPF9a), soy bean trypsin inhibitor and Trasylol. The behaviour of the two subunits towards these above inhibitors was also identical with plasma kininase II. A 180,000 mol. wt kininase was partially purified from human plasma. The kininase had bradykinin-inactivating and angiotensin I-converting activities. Its K m value and the behaviour towards the inhibitors described above were the same as the subunit I.

Peptide inhibitors of converting enzyme

Human plasma and atypical lung converting enzyme, and porcine plasma converting enzyme are substantially inhibited by other components of the renin-angiotensin system, and by angiotensin II and its analogues. Des-Asp 1 angiotensin II (angiotensin III) 0.1 mM and tridecapeptide renin substrate 0.1 mM are both effective inhibitors of human lung, plasma and porcine plasma converting enzymes. Des-Asp 1-Arg 2 angiotensin II also was an effective inhibitor of plasma enzymes. Bradykininase activity (kininase II) of the converting enzymes was also inhibited by angiotensin I, angiotensin III, tetradecapeptide renin substrate and tridecapeptide renin substrate. The substantial kininase and converting enzyme inhibitory effects of components of the renin-angiotensin system, suggest a potential close physiologic relationship between the kallikrein-kinin system and the renin-angiotensin system.

Bradykininase activity of aloe extract

Bradykininase activity of aloe extract

Hormone interactions in the isolated rabbit heart. Synthesis and coronary vasomotor effects of prostaglandins, angiotensin, and bradykinin.

In the present study, the synthesis and degradation of several potent vasoactive substances influencing coronary resistance were characterized in the isolated perfused rabbit heart. Prostaglandin synthetase activity, angiotensin converting enzyme activity, and bradykininase activity (without angiotensinase) were observed. A prostaglandin E2-like substance appeared to be the ednogenous mediator of the coronary vasodilation produced by bradykinin and angiotensin II (AII). (1) The concentration of this prostaglandinlike substance in the coronary venous effluent was directly proportional to the concentration of the coronary vasocilator stimulus (bradykinin or AII). (2) The prostaglandinlike substance released and the coronary dilation produced by the agonists correlated temporally and quantitatively. (3). Abolition of cardiac biosynthesis of the prostaglandinlike substance by indomethacin also abolished the decrease in coronary resistance produced by the agonists. AII, the most potent naturally occurring vasoconstrictor substance, produced a paradoxical coronary vasodilation because it stimulated cardiac prostaglandin biosynthesis, but the direct coronary vasoconstrictor action of AII could be readily unmasked by indomethacin, which blocks prostaglandin synthesis. The nonapeptide SQ-20881 blocked cardiac biosynthesis of AII (from angiotensin I) and enhanced the coronary vascular effects of bradykinin by interfering with bradykininase activity. Similarly, the AII-receptor antagonist, 1-Sar-8-Ile-AII, blocked the coronary vascular effect of AII.

Angiotensin I [Phe8-His9] hydrolase and bradykininase from human lung

Atypical angiotensin I “converting enzyme” (angiotensin I [Phe8-His9] hydrolase or APHH) was purified from human lung tissue. Two enzyme preparations from different lungs were found to fragment and inactive bradykinin. Fragmentation was demonstrated by electrophoretic techniques and biological inactivation was demonstrated by bioassay. Bradykininase activity was inhibited by low concentrations of 2,3-dimercaptol-propanol (BAL) and ethylenediaminetetraacetic acid (EDTA), but not by phenylmethylsulfonyl fluoride (PMSF) or p-chloromercuriphenyl sulfonic acid (CMPSA). Conversion of angiotensin I was inhibited by BAL, PMSF, and CMPSA but not by EDTA. APHH from a third lung preparation was free of significant bradykininase activity as determined by bioassay. It is concluded that these two enzymatic activities are probably associated with separate enzymes.

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Studies of plasma bradykininases using radiolabelled substrates.

SummaryThe activities of human plasma bradykininases (kininase I and II) have been investigated because of their importance as reactants in the kallikrein‐kinin system. The use of [14C] bradykinin as substrate allows radiometric analysis of the enzyme‐substrate reaction mixture. This is possible because separation of bradykinin from its hydrolysis products is achieved by chromatography over CM Sephadex C‐25. Use of this technique has allowed localization of bradykininase activities in plasma protein chromatograms and the separation and partial purification of kininase I and kininase II. Kininase I can be identified because it hydrofyses hippuryl‐L‐lysine, whereas kininase II converts angiotensin I to angiotensin II. The measurement of hydrolysis is simple and quantitative and has been used to demonstrate normal Michaelis‐Menten kinetics for both enzymes. The Michaelis‐Menten constants for the enzymes have been found to be 4·1 × 10‐7 M (kininase I) and 0·94 × 10‐7 M (kininase II).

Cobalt-induced alterations in plasma proteins, proteases and kinin system of the rat

Rats receiving an injection of cobalt (Co) showed marked changes in plasma kininogen (Brady kininogen, BKG) levels, and alterations in arginine-esterase and bradykininase activity. A significant increase in BKG concentrations was seen at 4 and 24 hr after Co treatment, with maximum titers occurring 4 hr after treatment. Plasma kallikrein activity was significantly increased between 1 and 24 hr after Co treatment. Co initiated a decrease in plasma kininase activity, with minimum activity occurring 8 hr after treatment. Plasma protease showed peak activity approximately 8 hr after Co administration. However, Co added in vitro failed to enhance the proteolytic activity of rat plasma from normal as well as cobalt-treated rats. Plasma protein concentrations were significantly increased above control levels at 18 and 24 hr after cobalt treatment. These observations would seem to suggest that Co. which is known to initiate a state of histotoxic (tissue) hypoxia, might be triggering a mechanism(s) responsible for the changes seen in the plasma.

Kinin contents and bradykininase activities in whole saliva and ginival fluid from patients with periodontal disease (author's transl)

Kinin contents and bradykininase activities in whole saliva and gingival fluid from patients with periodontal disease were measured, and compared with various clinical findings such as PMA-index by Schour and Massler, OHI-score by Greene and Vermillion, pocket depth (mm) by a pocket marker and bone loss score (%) by Schei et al.Whole saliva was collected by a vinyle tube after washing the oral cavity with water. For the measurement of kinin content or bradykininase activity, 1, 10-phenanthroline (kininase inhibitor) or Trasylol (kinin inhibitor) was added in the sample. The sample was centrifuged at 2, 500g for 20min. at 0°C and the supernate was used for the assay.Gingival fluid was collected by filter paper strips. After the strips were weighed, the above described inhibitor was added, then the sample strips were homogenized by a Polytron homogenizer, and the mixture was centrifuged as in the case of saliva.Kinin contents and bradykininase activities were assayed by a biological method using guinea pig ileum. The bradykininase activity was expressed by the amount of gingival fluid (mg) or whole saliva (ml) to produce 50% inactivation of added bradykinin (1μg).The following results were obtained:1. A statistically significant correlation was obtained only between kinin content in whole saliva or gingival fluid and pocket depth (p<0.01).2. Statistically significant correlations were obtained between bradykininase activities in whole saliva and the following clinical findings; calculus score (p<0.05), pocket depth (p<0.001) and bone loss score (p<0.05), except plaque score and PMA-index.3. Statistically significant correlations were obtained between bradykininase activites in gingival fluid and following clinical findings; PMA-index (p<0.01), plaque score (p<0.001), calculus score (p<0.01), pocket depth (p<0.01) and fluid quantity (p<0.001), except bone loss score.4. A statistically significant correlation was obtained between kinin content and bradykininase activity in gingival fluid (p<0.01), but there was no statistically significant correlation between kinin content and bradykininase activity in whole saliva.5. In the group of patients that were examined on the basis of clinical findings and X-ray photos, the kinin contents in whole saliva and gingival fluid were increased and the bradykininase activities were decreased following aggravation of the disease process.It is concluded from these results that the kinin content and bradykininase activity in whole saliva and gingival fluid is closely related to the process of periodontal disease, and that kinin content and bradykininase activity in gingival fluid is more closely related to each clinical finding.

Bradykininase activity in gingival fluid in the periodontal disease (author's transl)

The subjects in various periodontal conditions were examined based on the clinical symptoms and roentgenograms, and divided into five groups: clinically normal, gingivitis, slight, moderate and advanced periodontitis. The gingival fluid was collected from gingival sulcus by filter paper strips. The amount of gingival fluid and its bradykininase activity were assayed according to Takeya's method. The bradykininase activity was expressed as the amount of gingival fluid (mg) to produce 50% inactivation of added bradykinin (1μg/ml).The following results were obtained;1) The quantity of gingival fluid in clinically normal subjects averaged 1.74±0.38mg (mean±S. D.). Subjects with gingivitis, slight periodontitis, moderate periodontitis and advanced periodontitis showed the values of 4.68±3.13mg, 7.56±2.81mg, 10.68±7.67mg, and 19.18±12.89mg, respectively. The quantity of gingival fluid was increased with the progression of periodontal disease.2) The bradykininase activity in gingival fluid in clinically normal subjects expressed as the amount of gingival fluid to produce 50% inactivation of added bradykinin (1μg/ml) averaged 0.31±0.14mg (mean±S. D.). Subjects with gingivitis, slight periodontitis, moderate periodontitis and advanced periodontitis showed the values of 1.05±0.90mg, 1.47±0.59mg, 2.61±2.59mg and 3.61±2.87mg, respectively. The bradykininase activity was decrease with the progression of periodontal disease.3) The bradykininase activities in subjects with advanced periodontitis were significantly lower than those of clinically normal (P<0.05) and gingivitis subjects (P<0.05). No statistically significant difference was found between the normal and disease groups.4) Statistically significant correlations were obtained between the bradykininase activity expressed as the amount of gingival fluid to produce 50% inactivation of added bradykinin (1μg/ml) in diseases, and oral examination; gingival score (P<0.01), plaque score (P<0.05), calculus score (P<0.05), sulcus depth (P<0.001), and quantity of gingival fluid (P<0.001), except bone loss score.5) THe degrees of gingival score, oral hygiene score, sulcus depth, bone loss score, and quantity of gingival fluid were increased according to the disease process.It is concluded from these results that the measurement of the kininase activity which is thought to be an important factor for occurence and progression of periodontal disease may be a valuable mean.

The bradykininase activities of extracts of dog lung.

1. Homogenates of dog lungs freed from blood inactivate bradykinin. The bradykininase activity is present in soluble and particulate subcellular fractions.2. The fraction with the highest relative specific activity is that sedimenting between 8,700 g and 78,000 g. This fraction has been studied in more detail.3. There is evidence for two bradykinin inactivating enzymes in this fraction with apparent K(m) values of 7.5 muM and 120 muM.4. The bradykininase activity is not dependent on the concentration of chloride ion.5. The bradykininase activity is inhibited by EDTA, 2:3-dimercaptopropanol, nickel ions and some of the peptides from the venom of Bothrops jararaca and of Agkistrodon halys blomhoffii but not by 2-mercaptoethanol or N-ethylamaleimide.6. Angiotensin II in either 15 or 170 mM chloride ion is not an inhibitor of bradykininase activity but angiotensin I at either chloride concentration is an inhibitor. It is proposed that chloride is not necessary for binding of angiotensin I to converting enzyme but is necessary to ensure the correct orientation of substrate for hydrolysis to proceed.

The inactivation of bradykinin in the pulmonary circulation of isolated lungs.

1. The isolated lungs of guinea-pigs, rats and dogs, perfused with Krebs bicarbonate solution via the pulmonary artery, inactivated 95, 98 and 99% respectively of the bradykinin infused through them.2. This inactivation process was not inhibited by 2-mercaptoethanol (1 mM) or EDTA (500 muM).3. The inactivation was inhibited by 2:3-dimercaptopropanol (100 muM), N-ethylmaleimide (100 muM), and several of the bradykinin potentiating peptides from Bothrops jararaca, in both natural and synthetic forms.4. The effect of these compounds, 2-mercaptoethanol, EDTA, 2:3-dimercaptopropanol, N-ethylmaleimide and Bothrops peptides on the pulmonary bradykininase activity is the same as their effect on pulmonary angiotensin I converting enzyme.

Peptide inhibitors of pulmonary angiotensin I converting enzyme

A number of peptides have been found to inhibit a particulate angiotensin I converting enzyme preparation from rabbit lung; bradykinin-potentiating peptide C, bradykinin, and methionyl-lysyl-bradykinin inhibit competitively. The latter two peptides are hydrolyzed by the converting enzyme preparation; bradykinin-potentiating peptide C is not. Thus, it is possible that although bradykinin and methionyl-lysyl-bradykinin do compete with angiotensin I for the active site of the converting enzyme, they may not be hydrolyzed while bound to this enzyme. The converting enzyme activity is Cl − dependent; bradykininase is independent of the presence of Cl −; therefore, there does seem to be some distinction between converting enzyme activity and bradykininase activity.

Bradykininase Activity in Human Blood Plasma in Healthy Subjects and Asthmatic Patients

Bradykininase Activity in Human Blood Plasma in Healthy Subjects and Asthmatic Patients