Substrates for Kinin-Releasing Enzymes in Rat Plasma

Indirect evidence has been provided for the presence of 3 kininogen fractions: The average amounts of kinin released by rat plasma kallikrein (1.5 μg/ml plasma, S. E. M. = 0.03) and by rat urine kallikrein (1.4 μg/ml plasma, S. E. M. = 0.03) in 7 plasma batches corresponding to a total of 90 rats, when added up, significantly exceeded the total kininogen (2.0 μg/ml plasma, S. E. M. = 0.04). Methods and materials were as described by BRISEID, DYRUD & ÖIE (1970). It is suggested that plasma kallikrein released kinin from 2 kininogen fractions, S1″ and S1″, and that urine kallikrein released kinin from 2 kininogen fractions, S1″ and S2. Repeated incubation with each of the kininogenase preparations used did not increase the yield of kinin. Soybean trypsin inhibitor did not reduce the amount of kinin released by urine kallikrein; the plasma kallikrein, however, was strongly inhibited. In control experiments leucine aminopeptidase transformed kallidin to bradykinin, but did not increase the kinin activity of the urine kallikrein incubates.     

Evidence for the involvement of a plasma kallikrein-kinin system in the immediate hypotension produced by endotoxin in anaesthetized rats.

1. In vitro incubation of normal rat plasma with endotoxin from E. coli (3-10 mg ml-1) in the incubation mixture) caused a dose-dependent increase in levels of free kinin and plasma kallikrein in the presence of o-phenanthroline, together with a mirror-image, dose-dependent decrease in the residual levels of the precursors, plasma prekallikrein and high-molecular-weight kininogen. Low-molecular-weight kininogen levels were not modified. 2. Intravenous injection of endotoxin (3-30 mg kg-1) into the femoral vein of anaesthetized rats resulted in dose-dependent hypotension. In blood collected up to 15 min after injection, the levels of prekallikrein and high-molecular-weight kininogen in plasma were decreased while levels of the active forms, plasma kallikrein and free kinin, showed a transient increase in the blood 1 min after administration of endotoxin. 3. A degradation product of bradykinin, des-Phe8-Arg9-bradykinin, as measured by a newly developed enzyme immunoassay, was detectable up to 5 min after administration of endotoxin. 4. Intravenous infusion of soybean trypsin inhibitor inhibited both the formation of bradykinin and des-Phe8-Arg9-bradykinin and the initial hypotension. 5. It can be concluded from our results that plasma prekallikrein is activated in the blood immediately after administration of endotoxin to rats and that bradykinin is a major cause of the immediate hypotension.     

A new direct radioimmunoassay of rat urinary kininogen

A protein-binding radioimmunoassay (RIA) of rat low molecular weight (LMW) kininogen with the following characteristics has been developed: sensitivity, 2.5 ng/tube; inter-assay coefficient of variation, 12.4% (N = 28); and intra-assay coefficient of variation, 9.4% (N = 11). The new assay correlated (r = 1) with the determination of kinin equivalence of kininogen after trypsinization. The cross-reactivity of rabbit anti-rat LMW kininogen antibody was 2.5% with bovine LMW kininogen, 5.8% with rat plasma high molecular weight (HMW) kininogen, and none with kinin. Although the antibody appears to partially recognize des-kinin-kininogen, the low degree of cross-reactivity and the extremely low levels of kinin-free-kininogen allow accurate determination of total LMW kininogen in rat urines. The LMW kininogen formed 20% kinins with salivary kallikrein when compared with trypsin, suggesting that the preparation consists of both K- and T-kininogens (K = kallikrein susceptible; T = trypsin susceptible). The newly developed protein-binding RIA recognizes LMW kininogen of rat urine which consists of both K- and T-kininogens.     

The myostimulating effect of tissue kallikrein on rat uterus

The mechanism of the myostimulating activity of rat tissue kallikrein on rat uterus was re-examined using uterus from kininogen-deficient rats and HOE 140 (D-Arg[Hyp³, This, D-Tic⁷, Oic⁸]bradykinin), a specific bradykinin receptor-B₂ antagonist. The uterus from kininogen-deficient rats was 50 times less sensitive to rat kallikrein than that from normal rats. HOE 140 (6 to 60 nM) inhibited the contracting effects of bradykinin and of rat kallikrein. Porcine kallikrein had no effect on rat uterus. Bradykinin and rat kallikrein induced a relaxation of rat duodenum. The duodenum from kininogen-deficient rats was 100 times less sensitive to rat kallikrein than the duodenum from normal rats. HOE 140 (0.6 to 3 nM) inhibited the relaxing effects of bradykinin and of kallikrein. Preincubation of rat kallikrein with aprotinin (Trasylol) abolished the effects of kallikrein on smooth muscles. HOE 140 inhibited the amidolytic activity of tissue kallikrein with a K_i value of 220 W. HOE 140, at micromolar concentrations, suppressed the kininogenase activity of tissue kallikrein. Plasma of deficient rats contained 0.7% of the normal levels of kininogens. After washing the blood vessels with saline, kininogens were present in uterine homogenates but not in duodenal homogenates from both rat strains. Uterine kininogens from deficient rats amounted to 19% of the kininogen content of the uterus of normal rats. The blood pressure of anaesthetized rats was lowered by rat tissue kallikrein but not by porcine kallikrein.We conclude that the myostimulating activity of kallikrein mostly depends on kinin formation from kininogens present in the tissue. These kininogens arise from blood contamination in the duodenum and also from a local store in the uterus. The presence of a significant amount of kininogens in the uterus from deficient rats might suggest a local synthesis of kininogens. HOE 140 inhibits the enzymatic activity of kallikrein at micromolar concentrations.     

Measurement of urinary kallikrein activity by kinin radioimmunoassay

A method for measuring urinary kallikrein activity by kinin radioimmunoassay (RIA) is described. Kinins were generated by incubating urine with partially purified dog kininogen in the presence of peptidase inhibitors. Antibodies against kallidin were induced in rabbits by injecting kallidin coupled to ovalbumin. One of the antisera generated was used at a final dilution of 1:18,000 to obtain a 40 per cent binding of approximately 3000 cpm (10 pg) of bradykinin (S-tyrosin)-[¹²⁵I]triacetate ([¹²⁵I]bradykinin). Synthetic kallidin (10–500 pg) was used to construct standard curves. When kinins generated by urinary kallikrein were also used, the two displacement curves for [¹²⁵I]bradykinin were similar. The RIA was sensitive to 10 pg kinins. The antiserum cross-reacted with bradykinin, methionyl- kallidin, and kininogen, but not with angiotensin, oxytocin or SQ 20,881. To increase the specificity of the RIA, the kininogen was removed by ethanol precipitation followed by QAE-Sephadex-A50 chromatography. Kallikrein activity in 81 human urine samples and 8 samples obtained from a dog undergoing stop-flow procedure was measured by RIA and bioassay. Correlations of r = 0.81 and 0.94 were found. This RIA is useful for measuring kallikrein activity in rat, dog and human urine.     

Rodent kinin-forming enzyme systems—I: Purification and characterization of plasma kininogen

Low molecular weight (LMW) kininogen was purified 70-fold with a 16% yield from fresh rat plasma by DEAE-Sephadex chromatography, ammonium sulfate precipitation, Sephadex G-200 gel filtration, SP-Sephadex chromatography, CM-cellulose chromatography, and Sephadex G-200 gel filtration. Ferguson plots of polyacrylamide gel electrophoretic patterns revealed four bands with relative molecular weights of 64,000, 123,500, 252,436 and 357,900 (ratio of 1:2:4:6). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis provided a single protein band with a molecular weight of 72,000, suggesting that the four kininogen bands had been caused by the aggregation of a single oligomeric protein. The purified LMW rat kininogen Fraction B (3.9 μg bradykinin/mg) was used to elicit an antiserum in the rabbit. Monospecificity of the antiserum was demonstrated by immunoelec-trophoresis (Laurell rocket and Grabar methods) and, thus, the homogeneity of the kininogen was also. The purified kininogen (both Fractions A and B) formed kinin with human urinary kallikrein, rat urinary kallikrein and hog pancreatic kallikrein. Murphy-Sturm lymphosarcoma acid protease also formed kinin when incubated with the kininogen at pH 3.0. The isoelectric point for both fractions was at pH 4.3. Amino acid analyses showed the two kininogen fractions to be rich in acidic amino acids and to have a total carbohydrate content of 8.5% consisting of galactose (1.2 to 1.5%), mannose (1.9 to 2.1%), N-acetylglucosamine (4.3 to 5.1%), N-acetylgalactosamine (0.3%), and sialic acid (0.68%).     

Separation of Kininogens by Gel Filtration of Plasminogen–free Human Plasma

Abstract High molecular weight kininogen (S1) is useful as a sensitive substrate in assays of the kininogenase activity of plasma kallikrein, but considerable difficulties are encountered in the isolation of this kininogen due to loss by “spontaneous” activation. In the present work a satisfactory yield (40–60 %) of a crude preparation of S1 was obtained through gel chromatography on Sephadex G–200 of plasminogen–free 60°-heated human citrated plasma. Low molecular weight kininogen (S2) was also obtained by the same procedure. The two kininogen preparations did not contain significant amounts of inhibitors of human plasma kallikrein, hog pancreas kallikrein or human plasmin, as judged by the stability of the arginyl–esterase activities of these enzymes in contact with the kininogens. Both S1 and S2 were stable for at least one year when stored at -20°. A preparation of human plasma kallikrein reacted specifically with S1, while both hog pancreas kallikrein and human plasmin also reacted with S2. The quantitative data are in accordance with the assumption that hog pancreas kallikrein and plasmin release bradykinin from S1 and kallidin from S2. The rate of release of kinin produced by hog pancreas kallikrein in S2 occurred twice as fast as in S1, while the opposite was registered for plasmin.     

The application of bradykinin radioimmunoassay to plasma kininogen determination

Bradykinin radioimmunoassay was applied to plasma kininogen determination by measuring the bradykinin content of the trypsin hydrolysate of heat-acid denatured plasma. The bradykinin antibody was produced in rabbits. The immunogen was prepared by coupling bradykinin to human serum albumin with ethyl-carbo-di-imide. In the radioimmunoassay [¹²⁵I]tyr⁸-bradykinin was used as tracer. The antibody was suitable for serial determinations of kininogen from 0·2 ml of plasma. The dose-response pattern of bradykinin standard solutions was the same as that of kininogen hydrolysates and the recovery in the hydrolysates was 95–110%. The mean plasma kininogen content of 26 healthy human individuals was 3·36 μg/ml (S.D. 0·49) in bradykinin equivalents which is in good agreement with previous results of kininogen bioassay.     

Rat uterine contraction by kallikrein and its dependence on uterine kininogen.

Smooth muscle responses to kallikrein (EC 3.4.21.8) are generally considered to result from kinin formation. In the present study, this premise was reexamined with respect to the isolated rat uterus. Rat submandibular gland kallikrein produced contractions of the rat uterus but the contractions disappeared after successive additions of the same dose of the enzyme to the preparation. Kallikrein-induced rat uterine contractions as well as bradykinin-induced contractions were enhanced by rat submandibular gland bradykinin potentiating factor. The incubation of kallikrein with rat uterine extract in the presence of a kininogen-depleted rat uterus produced kinin which elicited the uterine contraction. An extract from uterine horns previously depleted of kininogen was prepared. Incubation of this extract with kallikrein in a bath containing a kininogen-depleted rat uterus did not evoke uterine contraction. The incubation of four rat uterine horns with kallikrein in the presence of a uterine horn previously depleted of kininogen elicited contractions of the depleted uterus. These results suggest that the contraction produced by kallikrein involves kinin release from the uterus.     

Mechanism of kinin release during experimental acute pancreatitis in rats: evidence for pro- as well as anti-inflammatory roles of oedema formation

1 Kinin B(2) receptor antagonists or tissue kallikrein (t-KK) inhibitors prevent oedema formation and associated sequelae in caerulein-induced pancreatitis in the rat. We have now further investigated the mechanism of kinin generation in the pancreas. 2 Kinins were elevated in the pancreatic tissue already before oedema formation became manifest. Peak values (421+/-59 pmol g(-1) dry wt) were reached at 45 min and remained elevated for at least 2 h; a second increase was observed at 24 h. Pretreatment with the B(2) receptor antagonist icatibant abolished kinin formation, while post-treatment was ineffective. 3 Total kininogen levels were very low in the pancreas of controls, but increased 75-fold during acute pancreatitis. This increase was absent in rats that were pretreated with icatibant. 4 During pancreatitis, t-KK-like and plasma kallikrein (p-KK)-like activity in the pancreas, as well as trypsinogen activation peptide (TAP) increased significantly. Icatibant pretreatment further augmented t-KK about 100-fold, while p-KK was significantly attenuated; TAP levels remained unaffected. 5 Endogenous protease inhibitors (alpha(1)-antitrypsin, alpha(2)-macroglobulin) were low in normal tissues, but increased 45- and four-fold, respectively, during pancreatitis. This increase was abolished when oedema formation was prevented by icatibant. 6 In summary, oedema formation is initiated by t-KK; the ensuing plasma protein extravasation supplies further kininogen and active p-KK to the tissue. Concomitantly, endogenous protease inhibitors in the oedema fluid inhibit up to 99% of active t-KK. Our data thus suggest a complex interaction between kinin action and kinin generation involving positive and negative feedback actions of the inflammatory oedema.     

Binding and dissociation of Hageman factor, prekallikrein and high molecular weight kininogen in human plasma during contact activation

A kaolin pellet was incubated with human plasma at room temperature and immediately washed to remove all unbound proteins. Whereas in normal plasma, 154 mU(PPAN) of kallikrein was bound to the kaolin surface, in Hageman factor (HF)-deficient plasma or normal plasma preincubated with polybrene the surface-bound kallikrein was undetected, while a trace of prekallikrein was bound to the kaolin surface. Dissociation of kinin and kallikrein from the kaolin surface occurs during varying periods of incubation of the kaolin suspension alone. The dissociation of the surface-bound kallikrein revealed two phases in a 15 min incubation: the first phase of dissociation which rapidly progressed until the kinin liberation reached a plateau was followed by the second phase where the kallikrein was slowly dissociated and kinin was no longer liberated. Treatment of the kaolin suspension with trypsin, plasmin or plasma kallikrein enhanced the kinin liberation and dissociation of kallikrein from the kaolin surface. Kallikrein-kinin-free high molecular weight (HMW) kininogen-activated HF complex, kalli-krein-kinin-free HMW kininogen complex, kallikrein, kinin-free HMW kininogen and two activated HFs were found on Sephacryl S-300 and Sephadex G-100 gel filtrations of the supernatant obtained after a 60 min incubation of the kaolin suspension alone. The ternary complex of kallikrein, kinin-free HMW kininogen and activated HF suggests the presence of prekallikrein-HMW kininogen-HF complex on the kaolin surface.     

Activation of the plasma kallikrein/kinin system on endothelial cell membranes

For more than three decades, it has been known that the plasma kallikrein/kinin system becomes activated when exposed to artificial, negatively charged surfaces. The existence of an encompassing in vivo, negatively charged surface capable of activation of the plasma kallikrein/kinin system has, however, never been convincingly demonstrated. In this report, we describe current knowledge on how the proteins of the plasma kallikrein/kinin system assemble to become activated on cell membranes. On endothelial cells, the activation of the plasma kallikrein/kinin system is not initiated by factor XII autoactivation as seen on artificial surfaces. On endothelial cells, prekallikrein is activated by an antipain sensitive protease. Prekallikrein activation is dependent on the presence of high molecular weight kininogen and an optimal free Zn2+ concentration. Kallikrein generated on the surface of endothelial cell is capable of activating factor XII. Further, kallikrein formed on endothelial cell membranes is capable of cleaving its receptor and native substrate, high molecular weight kininogen, liberating bradykinin and the HK PK complex from the endothelial cell surface. Endothelial cell-associated kallikrein also is capable of kinetically favorable pro-urokinase and, subsequent, plasminogen activation.     

Inhibition of Rat Acute Inflammatory Paw Oedema by Dihemiphthalate of Glycyrrhetinic Acid Derivatives: Comparison with Glycyrrhetinic Acid

Abstract— The anti-inflammatory profile of dihemiphthalate compounds of glycyrrhetinic acid derivatives in acute rat paw oedema induced by various vasoactive agents was compared with the parent compound. Three dihemiphthalate compounds (the di-sodium salt of 18 β-olean-12-ene-3β,30-diol di-O-hemiphthalate, 18β-olean-9(11),12-dione-3β,30-diol di-O-hemiphthalate and olean-11,13(18)-diene-3β,30-diol di-O-hemiphthalate), significantly inhibited development of carrageenan-induced rat paw oedema during the first 3 h (ED50 70, 90, and 108 mg kg^?1 respectively, p.o.), while glycyrrhetinic acid (ED50, 200 mg kg^?1) showed a significant inhibition of paw oedema 3 h after carrageenan treatment. The dihemiphthalate compounds also suppressed mouse paw oedema induced by histamine, bradykinin, and PAF acether at doses of less than 100 mg kg^?1. However, these compounds failed to inhibit 5-HT-induced mouse paw oedema. Glycyrrhetinic acid had little effect on mouse paw inflammation induced by the above irritants. The three compounds at 10^?7-10^?4 m , inhibited histamine-induced contraction of guinea-pig isolated ileum. However, concentration-response curves to 5-HT and bradykinin were not affected by the same compounds. These results suggest that the dihemiphthalate compounds modulate vascular permeability caused by endogenous vasoactive agents as one of the anti-inflammatory mechanisms. This action is quite different from that of glycyrrhetinic acid.     

Kinin-forming enzyme in rat brain mitochondria fraction and biological activity of a kinin released from rat plasma kininogen by this enzyme

The kinin-forming enzyme of rat brain was studied by bioassaying kinin using a rat uterus. The enzyme released a kinin from the partially purified kininogen of rat plasma. The activity is exclusively distributed in the mitochondrial fraction and was detected in the pH range of 2.5-4.0 (optimally at pH 3.0). The enzyme was potently inhibited by pepstatin, but not by aprotinin. Released kinin was extracted by n-butanol and it was purified using Amberlite CG-50 absorption and CM-cellulose column chromatography. The elution profile of kinin from the CM-cellulose column did not coincide with that of bradykinin, Lys-bradykinin or Met-Lys-bradykinin. Isolated kinin was inactivated by treatment with chymotrypsin, but not with trypsin. In addition to the contractile activity on rat uterus, the kinin caused contraction of guinea pig ileum, with the response being potentiated by the presence of bradykinin-potentiator B. It also relaxed a rat duodenum, decreased rat blood pressure, and increased the vascular permeability in guinea pigs. Relative potencies of kinin on these pharmacological activities did not coincide with those of bradykinin. From these results, it is concluded that a kinin-forming enzyme is present in the rat brain. It is a cathepsin D-like enzyme, and furthermore, the enzyme releases a kinin-like peptide from the plasma kininogen fraction.     

Components of the kallikrein-kinin system in rat urine

Using a direct radioimmunoassay and a kininogenase assay, we determined that 68% of rat urinary kallikrein was enzymatically active while 32% was in an inactive form which was activated by trypsin. Inorganic cations, at concentrations found in rat urine, were inhibitory in an amidase assay but appeared to potentiate kininogenase activity of pure rat urinary kallikrein. In random urines, kinin concentration was 4.2 +/- 0.7 ng/ml. Trypsinization of the urines generated 52.9 +/- 25.8 ng kinin/ml, indicating that kininogen was present. The rate of kinin formation in vivo may depend on the availability of kininogen and the concentration of inorganic cations in urine, as well as on other well-recognized factors, such as the kallikrein activity of the urine.     

On the ability of prostaglandin E1, and arachidonic acid to modulate experimentally induced oedema in the rat paw.

1 Prostaglandins E1 and E2 but not prostaglandin F2alpha, arachidonic acid or linolenic acid, produced slight oedema when injected into the rat hindpaw. 2 Prostaglandin E1 potentiated hindpaw oedema produced by carrageenan, kaolin, bradykinin and trypsin but not that produced by 5-hydroxytryptamine (5-HT), histamine, dextran B or compound 48/80. Carrageenan- and bradykinin-induced paw oedemas were also potentiated by prostaglandin E2. Arachidonic acid potentiated responses to carrageenan and kaolin but not responses to bradykinin, trypsin, 5-HT, histamine, dextran B or compound 48/80. Linolenic acid did not potentiate hindpaw oedema induced by carrageenan. 3 Potentiation of carrageenan-induced oedema by prostaglandin E1 was not diminished by pretreatment with indomethacin, hydrocortisone or cyproheptadine. However, arachidonic acid potentiation of carrageenan oedema was reduced by pretreatment with non-steroidal anti-inflammatory drugs but not by anti-inflammatory steroids or by paracetamol. 4 The enhancement of the response to carrageenan and kaolin by prostaglandins E1, E2 and arachidonic acid is discussed in terms of kinin mediation.     

Untersuchungen zur Existenz zweier verschiedener kininbildender Systeme in menschlichem Plasma

Zusammenfassung Die Entstehung von Plasmakininen in menschlichem Plasma wurde unter verschiedenen Bedingungen studiert. Nach erschöpfender Kininentwicklung mit Pankreaskallikrein kann durch Glaskontakt noch weiteres Kinin freigelegt werden und umgekehrt. Demnach sind zwei verschiedene Kininogene (I und II) vorhanden, die jeweils nur von Pankreaskallikrein bzw. dem durch Glaskontakt aktivierten Ferment (Kininogenase II) utilisiert werden.Kininogenase II ist nicht identisch mit Serumkallikrein. Serumkallikrein (Kininogenase I) ist aber in menschlichem Plasma ebenfalls enthalten. Es wird während der Glasbehandlung von Plasma inaktiviert. In aktiver Form liegt es in säurebehandeltem Plasma vor. Es wirkt auf das auch von Pankreaskallikrein benutzte Substrat Kininogen I ein, möglicherweise auch außerdem auf Kininogen II, das sonst von Kininogenase II umgesetzt wird.Es wird gefolgert, daß im menschlichen Plasma zwei verschiedene kininbildende Systeme existieren, die — in unterschiedlicher Verteilung — auch in tierischen Plasmen vorkommen. Hundeplasma enthält Kininogen I und Kininogenase I, ihm fehlt das System II. Umgekehrt enthält Rattenplasma das System II, aber offenbar kein Kininogen I.

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Summary The formation of plasma kinins in human plasma has been studied under various conditions. After completion of kinin development with pancreatic kallikrein contact of the plasma with glass leads to new kinin liberation andvice versa. Apparently, there are two kininogens (I and II) which are utilized only by one of the two enzymes, that activated by glass contact, or pancreatic kallikrein.Kininogenase II is not identical with serum kallikrein. Serum kallikrein is also present in human plasma. It is inactivated after glass contact, and is obtained in an active state by treating plasma with acid. It utilizes the same kininogen I as does pancreatic kallikrein, in addition it may also act upon kininogen II.It is concluded that human plasma contains two different kinin-forming systems which are also present in animal plasmas, in varying proportions. Dog plasma contains kininogen I and plasma kallikrein (kininogenase I), the kinin forming system II is lacking. In contrast, rat plasma contains the system II but not kininogen I.

     

Mechanism of the pro-inflammatory activity of sympathomimetic amines in thermic oedema of the rat paw

1 Thermic oedema induced by heating rat paws at 46.5 degrees C was potentiated by local injection of adrenaline, noradrenaline or high doses of isoprenaline. The pro-inflammatory effect of sympathomimetic amines was antagonized by phenoxybenzamine or phentolamine but not by propranolol.2 The subcutaneous space of heated rat paws was perfused with Tyrode solution and the perfusate collected and assayed for bradykinin, bradykininogen, kinin-forming activity and kininase activity. When adrenaline (0.5 mug/ml) was included in the perfusion fluid, kininase activity of the perfusate was increased by 76% and free bradykinin reduced by 46%.3 Increased vascular permeability induced by injection of bradykinin or kallikrein was reduced by adrenaline or noradrenaline, but isoprenaline had no significant effect.4 Pretreatment with soya bean trypsin inhibitor (SBTI) or heparin did not antagonize the pro-inflammatory effect of adrenaline or thermic oedema per se.5 Potentiation of thermic oedema similar to that induced by sympathomimetic amines was obtained by injecting paws with vasopressin prior to heating, or by applying a ligature to stop blood flow to the paw for the first 15 min of heating.6 Thermistor probes inserted beneath the paw skin showed that sympathomimetic amines increased the internal temperature of heated paws. This was significant, as small changes in temperature had a marked effect on the development of thermic oedema.7 It is suggested that sympathomimetic amines potentiate thermic oedema of rat paws heated at 46.5 degrees C by reducing blood flow to the paw, thereby causing a greater rise in paw temperature and consequently greater injury.     

Receptors mediating the increase in vascular permeability to kinins: comparative studies in rat,guinea-pig and rabbit

Summary The effect of bradykinin (BK) and a variety of kinin analogues and modified kinin fragments were assessed in several models representing the vascular permeability aspect of the inflammatory response. The rank order of potency of various kinin analogues to increase paw volume and skin vascular permeability in rats was \gZ-cyclo-(Lys¹-Gly⁶)-BK = \gZ-cyclo-kallidin > BK > kallidin = methionyllysyl-BKdes-Arg⁹-BK. The same order was seen for skin responses in day 21, rheumatoid-arthritic rats. Mepyramine, 10 mg · kg^–1 almost completely inhibited rat skin vascular responses to histamine, had no effect on BK and produced a small but significant inhibition of the responses to both cyclic-kinins. A different rank order of potency for the kinins was produced in both the guinea-pig and rabbit skin; this being kallidin > methionyl-lysyl-BK > BK >> Z-cyclo-(Lys¹-Gly⁶)-BK = Z-cyclo-kallidin>des-Arg⁹-BK. Neither of the cyclic kinins antagonised BK-induced increases in skin vascular permeability in guinea-pig or rabbit. Two modified kinin fragments, D-Pro-Phe-Arg-paranitroaniline and D-Pro-Phe-Arg-heptylamide, which have previously been demonstrated to be putative B2 receptor antagonists on in vitro tissues, enhanced the effect of BK in rat skin and when injected alone produced dose-related increases in skin vascular permeability in normal and rheumatoid-arthritic rats, both having approximately half the potency of BK. Neither of these fragments possessed agonist or antagonist action against BK in guinea-pig or rabbit skin. The B₁. receptor antagonist, des-Arg⁹-Leu⁸-BK, had no effect against BK-induced responses in any of the models used. It is concluded that the kinin receptor mediating the increase in paw volume of rat and the increase in vascular permeability in rat, rabbit and guinea-pig is not of the B₁-type. The difference in effects of the cyclic kinins and the modified kinin fragments may reflect a difference in the receptor mediating paw oedema and skin permeability in the rat compared to that mediating the same response in rabbit and guinea-pig skin. This requires further study. Send offprint requests to E. T. Whalley at the above address     

Quantitative Kininogenbestimmung in menschlichem Plasma

Zusammenfassung Es wurde nach Verfahren gesucht, die Inaktivierung von Kininen und Kallikrein im Plasma zu verhindern. Zur Blockade von Kininasen ist o-Phenanthrolin am besten geeignet. Inaktivierung von Kallikrein kann durch eine Wärme- und Säurebehandlung des Plasmas ausgeschaltet werden. Mit diesem Verfahren kann das gesamte, von Pankreaskallikrein angreifbare Kininogen durch eine einzige Inkubation mit 1 K.E./ml umgesetzt werden.Trypsin entwickelt aus dem gleichen Plasma etwa viermal so viel Kinin wie Pankreaskallikrein. Diese Differenz ist nur zu einem Teil durch die verschiedene Natur der entstandenen Kinine erklärbar. Ein Teil des im Plasma vorhandenen Kininogens wird von Pankreaskallikrein nicht umgesetzt, von Trypsin aber angegriffen. Diese Befunde weisen auf die Existenz zweier Kininogene im menschlichen Plasma.

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Summary Methods have been looked for to block the inactivation of kinins and of kallikrein in human plasma. For blockade of kininases o-phenanthroline is the most efficient compound. Systems which inactivate kallikrein can be destroyed by treating plasma with heat and acid. Using these procedures it is possible to convert the total accessible kininogen by incubating plasma with 1 unit pancreatic kallikrein/ml.In the same plasma specimen trypsin develops four times as much kinin activity as does pancreatic kallikrein. The difference is only partly accounted for by the different nature of the kinins formed. A fraction of the total kininogen is only attacked by trypsin, not by pancreatic kallikrein. This finding indicates the presence of two different kininogens in human plasma.