Relation between structure and correcting activity of bovine high molecular weight kininogen upon the clotting time of Fitzgerald-trait plasma.

Bovine high molecular weight kininogen (bHMWK) partially corrects the activated plasma thromboplastin time (aPTT) of Fitzgerald trait plasma which is congenitally deficient in HMWK. The relationship between the structure and activity of HMWK was clarified by studying the effects of different fragments of bHMWK on the aPTT of Fitzgerald-trait plasma. The peptides studied were lys-bradykinin-free HMWK, bradykinin-fragment 1-2-free HMWK, heavy chain, fragment 1-2-light chain, and light chain. All fragments were tested in equimolar concentrations. Bradykinin-fragment 1-2-free HMWK, heavy chain, and light chain have little or no correcting activity upon Fitzgerald-trait plasma aPTr. Fragment 1-2 light chain has the same correcting activity as intact bHMWK, while that of lys-bradykinin-free HMWK appears to be higher. Both fragment 1-2 and fragment 2 inhibit the clotting time of normal human plasma. When compared on a molar basis, fragment 2 is a more active inhibitor than fragment 1-2. When the effects of bovine plasma kallikrein upon bHMWK and hHMWK were studied, it was found that it released kinins from both kininogens. However, while the correcting activity of bHMWK was completely destroyed after 60 min of incubation, that of hHMWK was fully retained. These data suggest that: (a) the active part of bHMWK is comprised of the fragment 1-2 light chain portion; (b) fragment 1-2 or fragment 2 is the binding site to negatively charged surfaces, while the light chain interacts with other components of the surface-mediated reactions; and (c) bovine plasma kallikrein releases kinins, but probably does not cause the release of fragment 1-2 from human HMWK.

Inhibition of cell adhesion by high molecular weight kininogen.

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.

Factor XI activation in a revised model of blood coagulation.

Coagulation factor XI is activated in vitro by factor XIIa in the presence of high molecular weight kininogen (HMWK) and a negatively charged surface. Factor XII deficiency is not associated with bleeding, which suggests that another mechanism for factor XI activation exists in vivo. A revised model of coagulation is proposed in which factor XI is activated by thrombin. In the absence of cofactors, thrombin is more effective (kcat/Km = 1.6 x 10(5)) than factor XIIa (1.7 x 10(4)) in activating factor XI. Dextran sulfate enhances activation of factor XI by thrombin 2000-fold; part of this effect is due to autoactivation of factor XI by activated factor XI.

Inactivation of factor XIa by plasma protease inhibitors: predominant role of alpha 1-protease inhibitor and protective effect of high molecular weight kininogen.

Factor XIa is a plasma protease that, by activating Factor IX, plays an important role in the early phase of the intrinsic pathway of blood coagulation. Four plasma protease inhibitors, alpha(1)-protease inhibitor, antithrombin III, C1-inhibitor, and alpha(2)-plasmin inhibitor, have been reported to inactivate human Factor XIa, but their quantitative contribution to the inactivation of Factor XIa in plasma has not been fully assessed. Using purified systems, we observed that the second-order rate constants for the reaction of Factor XIa with alpha(1)-protease inhibitor, antithrombin III, and CI-inhibitor were 4.08, 10, and 14.6 M(-1) min(-1) x 10(3), respectively. The pseudo-first-order rate constants, at plasma concentration of the inhibitors, were 1.86 x 10(-1), 4.68 x 10(-2), and 2.4 x 10(-2) min(-1), respectively. These kinetic data predict that alpha(1)-protease inhibitor should account for 68%, antithrombin III for 16%, and C1-inhibitor and the equipotent alpha(2)-plasmin inhibitor each for 8% of the total inhibitory activity of plasma against Factor XIa. The rate of inactivation of Factor XIa in various plasma samples specifically deficient in inhibitors was consistent with these predictions. Factor XI, the zymogen form of Factor XIa, circulates in plasma associated with the contact system cofactor, high molecular weight kininogen (HMW kininogen). Kinetic analysis indicated the existence of a reversible bimolecular Factor XIa-HMW kininogen complex with a dissociation constant (K(d)) = 0.17 muM. The light chain derived from HMW kininogen decreased the inactivation rate of Factor XIa by C1-inhibitor with a K(d) of 0.08 muM for a complex of Factor XIa and the light chain derived from HMW kininogen. The protective effect of HMW kininogen was confirmed by the finding that the inactivation rate of Factor XIa in kininogen-deficient plasma was increased over normal plasma. The present study confirms that alpha(1)-protease inhibitor is the major inhibitor of Factor XIa in plasma, and that the formation of a reversible complex between Factor XIa and HMW kininogen decreases the rate of inactivation of the enzyme by its inhibitors.

Potentiation of the function of Hageman factor fragments by high molecular weight kininogen.

Patients lacking high molecular weight (HMW) kininogen have profound abnormalities of the Hageman factor-dependent pathways of coagulation, kinin formation, and fibrinolysis. The ability of HMW kininogen to potentiate the Hageman factor fragments (HFf) activation of prekallikrein and Factor XI in plasma was studied. HFf only partially converted Factor XI to XIa and prekallikrein to kallikrein in plasma deficient in HMW kininogen (Williams trait), while enhanced activation of Factor XI and prekallikrein by HFf resulted after reconstitution with HMW kininogen. In a system using highly purified components, HMW kininogen increased the initial rate of prekallikrein activation whether the kallikrein formed was assayed by arginine esterase activity or kininforming ability. The potentiation of prekallikrein activation occurred over a 12-fold range of enzyme (HFf) concentration and was nonhyperbolic with respect to substrate (prekallikrein). HMW kininogen exerted its effect even in the absence of prekallikrein since the hydrolysis of acetylglycyl-lysine methyl ester by HFf was increased by HMW kininogen. These results suggest that one of the functions of HMW kininogen is to augment the catalytic action of HFf.

Mechanism of activation of coagulation factor XI by factor XIIa studied with monoclonal antibodies.

The interaction of Factor XIIa with Factor XI was investigated using two monoclonal antibodies, one (3Cl) directed against the heavy chain of Factor XIa and the other (5F4) against its light chain. 3C1 either as intact IgG or as Fab' fragment, enhanced the rate of Factor XIa generation in the fluid phase but inhibited it in the presence of kaolin and high molecular weight (HMW) kininogen. In contrast, the Fab' fragments of 5F4 inhibited only the fluid phase activation and had no effect on the surface-mediated activation. 3C1 was found to block the binding of Factor XI to HMW kininogen, whereas 5F4 did not. We conclude: a domain on the heavy chain region of Factor XI is essential for binding to HMW kininogen and for optimal surface-mediated activation by Factor XIIa; and binding of 3C1 to Factor XI changes its conformation rendering it a more favorable substrate for Factor XIIa in the fluid phase.

High molecular weight kininogen is an inhibitor of platelet calpain.

Recent studies from our laboratory indicate that a high concentration of platelet-derived calcium-activated cysteine protease (calpain) can cleave high molecular weight kininogen (HMWK). On immunodiffusion and immunoblot, antiserum directed to the heavy chain of HMWK showed immunochemical identity with alpha-cysteine protease inhibitor--a major plasma inhibitor of tissue calpains. Studies were then initiated to determine whether purified or plasma HMWK was also an inhibitor of platelet calpain. Purified alpha-cysteine protease inhibitor, alpha-2-macroglobulin, as well as purified heavy chain of HMWK or HMWK itself inhibited purified platelet calpain. Kinetic analysis revealed that HMWK inhibited platelet calpain noncompetitively (Ki approximately equal to 5 nM). Incubation of platelet calpain with HMWK, alpha-2-macroglobulin, purified heavy chain of HMWK, or purified alpha-cysteine protease inhibitor under similar conditions resulted in an IC50 of 36, 500, 700, and 1,700 nM, respectively. The contribution of these proteins in plasma towards the inhibition of platelet calpain was investigated next. Normal plasma contained a protein that conferred a five to sixfold greater IC50 of purified platelet calpain than plasma deficient in either HMWK or total kininogen. Reconstitution of total kininogen deficient plasma with purified HMWK to normal levels (0.67 microM) completely corrected the subnormal inhibitory activity. However, reconstitution of HMWK deficient plasma to normal levels of low molecular weight kininogen (2.4 microM) did not fully correct the subnormal calpain inhibitory capacity of this plasma. These studies indicate that HMWK is a potent inhibitor as well as a substrate of platelet calpain and that the plasma and cellular kininogens may function as regulators of cytosolic, calcium-activated cysteine proteases.

Activation and function of human Hageman factor. The role of high molecular weight kininogen and prekallikrein.

The activation and function of surface-bound Hageman factor in human plasma are dependent upon both high molecular weight (HMW) kininogen and prekallikrein. HMW kininogen does not affect the binding of Hageman factor to surfaces, but it enhances the function of surface-bound Hageman factor as assessed by its ability to activate prekallikrein and Factor XI. The initial conversion of prekallikrein to kallikrein by the surface-bound Hageman factor in the presence of HMW kininogen is followed by a rapid enzymatic activation of Hageman factor by kallikrein. The latter interaction is also facilitated by HMW kininogen. Kallikrein therefore functions as an activator of Hageman factor by a positive feedback mechanism and generates most of the activated Hageman factor during brief exposure of plasma to activating surfaces. HMW kininogen is a cofactor in the enzymatic activation of Hageman factor by kallikrein and it also augments the function of the activated Hageman factor generated. The stoichiometry of the Hagman factor interaction with HMW kininogen suggests that it enhances the activity of the active site of Hageman factor. Since HMW kininogen and prekallikrein circulate as a complex, HMW kininogen may also place the prekallikrein in an optimal position for its reciprocal interaction with Hageman factor to proceed. The surface appears to play a passive role upon which bound Hageman factor and the prekallikrein-HMW kininogen complex can interact.

T-kininogen can either induce or inhibit proliferation in Balb/c 3T3 fibroblasts, depending on the route of administration.

T-kininogen (T-KG) is a precursor of T-kinin, the most abundant kinin in rat serum, and also acts as a strong and specific cysteine proteinase inhibitor. Its expression is strongly induced during aging in rats, and expression of T-KG in Balb/c 3T3 fibroblasts results in inhibition of cell proliferation. However, T-KG is a serum protein produced primarily in the liver, and thus, most cells are only exposed to the protein from the outside. To test the effect of T-KG on fibroblasts exposed to exogenous T-KG, we purified the protein from the serum of K-kininogen-deficient Katholiek rats. In contrast to the results obtained by transfection, exposure of Balb/c 3T3 fibroblasts to exogenously added T-KG leads to a dose-dependent increase in [3H]-thymidine incorporation. This response does not require kinin receptors, but it is clearly mediated by activation of the ERK pathway. As a control, we repeated the transfection experiments, using a different promoter. The results are consistent with our published data showing that, under these circumstances, T-KG inhibits cell proliferation. We conclude that T-KG exerts opposite effects on fibroblast proliferation, depending exclusively on the way that it is administered to the cells (transfection versus exogenous addition).

A review on bradykinin-related peptides isolated from amphibian skin secretion.

Amphibian skin secretion has great potential for drug discovery and contributes hundreds of bioactive peptides including bradykinin-related peptides (BRPs). More than 50 BRPs have been reported in the last two decades arising from the skin secretion of amphibian species. They belong to the families Ascaphidae (1 species), Bombinatoridae (3 species), Hylidae (9 speices) and Ranidae (25 species). This paper presents the diversity of structural characteristics of BRPs with N-terminal, C-terminal extension and amino acid substitution. The further comparison of cDNA-encoded prepropeptides between the different species and families demonstrated that there are various forms of kininogen precursors to release BRPs and they constitute important evidence in amphibian evolution. The pharmacological activities of isolated BRPs exhibited unclear structure-function relationships, and therefore the scope for drug discovery and development is limited. However, their diversity shows new insights into biotechnological applications and, as a result, comprehensive and systematic studies of the physiological and pharmacological activities of BRPs from amphibian skin secretion are needed in the future.

Mechanisms of the flare reaction in human skin.

The diffuse area of arteriolar vasodilation surrounding a region of recently injured human skin (axon reflex flare) is dependent upon the integrity of nerve fibers with cell bodies located in dorsal root ganglia. Evidence is presented to indicate that a vasodilator peptide similar to a kinin, neurotensin, or substance P, is implicated in the chain of biochemical events responsible for the transient shift in vascular tonus observable as the flare reaction.

Role of endothelial kinins in control of coronary nitric oxide production.

The purpose of the present study was to determine whether interventions that promote kinin production or decrease kinin inactivation affect nitric oxide production in isolated canine coronary microvessels. Accordingly, bradykinin (10[-8] to 10[-5] mol/L), ramiprilat (10[-10] to 10[-8] mol/L), A23187 (10[-8] to 10[-6] mol/L), kallikrein (1 to 20 U/mL), and kininogen (0.5 to 10 microg/mL) were used to stimulate endothelium-dependent nitric oxide production. Receptor antagonists, serine protease inhibitors, and a kinin antibody were used to inactivate local kallikrein-kinin activity. Nitrite, the metabolite of nitric oxide in aqueous solution, was measured using the Griess reaction. All the agonists significantly increased nitrite release. For instance, the highest dose of bradykinin, ramiprilat, A23187, kallikrein, and kininogen markedly increased nitrite production, from 60+/-10 to 156+/-12, 153+/-11, 161+/-15, 176+/-15, and 168+/-16 pmol/mg (all P<.05), respectively. The increased nitrite production caused by these agents was not only blocked by N omega-nitro-L-arginine methyl ester (L-NAME) and HOE 140 (which blocks B2 kinin receptor) but by the kinin antibody also. For instance, nitrite production elicited by bradykinin, ramiprilat, A23187, and kininogen was reduced to 95+/-8, 87+/-8, 94+/-11, and 85+/-11 pmol/mg (all P<.05), respectively, by the kinin antibody. Carbachol-induced nitrite production (from 66+/-8 to 144+/-13) was blocked by L-NAME but not by HOE 140 or the kinin antibody. These results suggest that either increasing kininogen to promote endogenous kinin formation or inhibiting angiotensin-converting enzyme to decrease kinin breakdown, increases nitric oxide production in isolated coronary microvessels. These data indicate that a microvessel kallikrein-kinin system has an important role in the control of nitric oxide production in coronary microvessels.

High sensitivity to salt in kininogen-deficient brown Norway Katholiek rats.

Brown Norway Katholiek rats, which have very low levels of plasma kininogens, excreted a much smaller amount of kinin in the urine than normal rats of the same strain. The systolic blood pressure of 7-week-old kininogen-deficient rats fed low (0.3%) NaCl diets (131 +/- 4 mm Hg, n = 12) was not different from that in normal rats. Two percent NaCl diets given from 7 weeks of age for 4 weeks caused rapid increases in blood pressure (167 +/- 4 mm Hg, n = 12, 9 weeks old) in deficient rats, although the same diets induced no blood pressure increase in normal rats. Urinary excretion of active kallikrein and prokallikrein remained constant in both rat groups throughout NaCl loading. During this period, the deficient rats secreted less urine (9 weeks old, P < .05) and less urinary sodium (11 weeks old, P < .05). Serum levels of sodium in deficient rats were higher (P < .05) than in normal rats at 9 weeks of age. Intracellular concentrations of sodium in the erythrocytes of deficient rats were higher (P < .05) than in normal rats throughout NaCl loading. Subcutaneous infusion of bovine low molecular weight kininogen with an osmotic pump in NaCl-loaded deficient rats induced a reduction (P < .01) in blood pressure and increases (P < .05) in urine volume and urinary sodium and kinin levels. By contrast, subcutaneous infusion of the bradykinin antagonist Hoe 140 or of aprotinin in NaCl-loaded normal rats induced a hypertensive response. This antagonist treatment reduced urine volume and urinary sodium. These results indicate that the lack of kinin generation observed in the kininogen-deficient rats was related through sodium retention to the hypertensive response to NaCl loading.

A peptide released by pepsin from kininogen domain 1 is a potent blocker of ANP-mediated diuresis-natriuresis in the rat.

A 20-amino acid peptide, KYEIKEGDCPVQSGKTWQDC (PU-D1), released by pepsin hydrolysis of LMW kininogen domain 1 was tested for its ability to antagonize the diuretic and natriuretic effect of ANP(103-125) in anesthetized rats. A single dose of 10.8 or 21.6 pmol (25 or 50 ng) PU-D1 given intravenously or into the duodenal lumen suppressed the diuresis-natriuresis induced by 209 pmol (500 ng) ANP by 43% to 59% and 69% to 96%, respectively. None of the doses tested (2.16 to 432 pmol, 5 ng to 1 microg) modified systemic blood pressure. Strikingly, a single IV dose of 10.8 pmol PU-D1 blocked the action of ANP for more than 3 hours. ANP blockade by PU-D1 was annulled completely by the bradykinin (BK) B2 receptor inhibitor Hoe 140. On a molar basis, PU-D1 is more effective than BK and kinins of 15, 16, and 18 amino acids for blocking the ANP-mediated diuresis-natriuresis. As with BK and other kinins, the inhibitory effect of Pu-D1 on ANP is obtained only within a small range of picomol doses. A single dose of 2.16 or 4.32 pmol PU-D1 or 47 pmol (50 ng) BK is ineffective against ANP if injected alone. However, when both substances are administered concomitantly at these subthreshold doses, they totally suppress ANP-induced diuresis-natriuresis. These results raise the question of whether PU-D1, released from kininogen domain 1, either alone or associated with BK, may interact with ANP in the regulation of urinary water and electrolyte excretion in physiological and pathological conditions.

Human plasma kininogens are identical with alpha-cysteine proteinase inhibitors. Evidence from immunological, enzymological and sequence data.

Human high- and low-Mr kininogens were shown to be potent inhibitors of cysteine proteinases such as cathepsin L and papain (Ki = 17-48 pM). A strong immunological cross-reaction between the kininogens and low-Mr alpha-cysteine proteinase inhibitor from human plasma was found. Comparison of partial amino acid sequences from high- and low-Mr kininogen and low-Mr alpha-cysteine proteinase inhibitor demonstrated sequence identity for all segments analyzed. These findings suggest that the kininogens and the alpha-cysteine proteinase inhibitors from human plasma are identical proteins.

An immunochemical approach to investigating the mechanism of inhibition of cysteine proteinases by members of the cystatin superfamily.

Antibodies were raised against a synthetic dodecameric peptide KGAGQVVAGPWK (K12K), encompassing sequences thought to be important for the function of the cysteine proteinase inhibitors of the cystatin superfamily. These antibodies specifically recognized molecules of family 3, i.e., kininogens, in the serum of seven mammalian species tested in this study. The only notable exception was that of rat thiostatin (T kininogen) which is structurally related to the kininogen family. The antibodies also discriminated between family 2 (cystatins) and family 3 (kininogens) of the cystatin superfamily, since neither chicken cystatin nor human and rat cystatins C and S, which all belong to family 2 were recognized. The cystatin-like inhibitory domains resulting from fragmentation of human low molecular weight kininogen by bovine trypsin, were still recognized by antibodies, indicating that discrimination does not require two neighbouring inhibitory sites on the kininogen heavy chain. The antibodies blocked the capacity of kininogens to inhibit papain, suggesting that they recognize a conformational epitope at or near the kininogen inhibitory sites. The inhibitory properties of family 2 cystatins remained unchanged, confirming that members of this family do not interact with anti K12K antibodies. These antibodies are thus a new tool able to discriminate functionally and structurally between the members of the cystatin superfamily.

Mapping the binding site of tissue kallikrein: preparation and testing of all possible substrate analog inhibitors homologous with the sequence of kininogen between Ser386 and Gln392.

Programs aimed at converting peptide inhibitors of proteolytic enzymes into more traditional drug structures require an understanding of the role played by the individual amino acid residues in the inhibitor. To this end, all possible substrate analogues occurring within the sequence Ser386-Pro-Phe-Arg-Ser-Val-Gln392 from bovine kininogen were synthesized and tested as inhibitors of tissue kallikrein (EC 3.4.21.35, beta-PPK). Of the 21 sequences which can be formed from the heptapeptide, 11 have inhibitory constants which could be measured in the chromogenic assay employed in these studies. No dipeptide and only one tripeptide, Ac-Phe-Arg-Ser-NH2 (Ki = 718 microM), measurably inhibits the enzyme. All longer peptides inhibit beta-PPK. The heptapeptide Ac-Ser-Pro-Phe-Arg-Ser-Val-Gln-NH2 is the most effective inhibitor in this series (Ki = 101 microM). Each amino acid residue in the sequence appears to alter binding in a relatively independent manner. The N-terminal seryl residue (P4) and the prolyl residue (P3) slightly improve the Ki of the various inhibitors. The phenylalanyl residue at P2 appears to have a more pronounced effect on Ki. The arginyl residue at P1 and the seryl residue at P1' appear to be the most important residues in the inhibitory sequence. They contribute approximately one-third and one-fourth of the binding energy to the interaction between the substrate analogues and beta-PPK, respectively. The valyl residue at P2', and the C-terminal glutaminyl residue improve Ki of each of the peptides tested. Almost 80% of the binding energy of the substrate analogue inhibitors comes from the core sequence Phe-Arg-Ser which occurs between P2 and P1'. Molecular models developed from the Chen-Bode coordinates of the aprotinin-beta-PPK complex have been used to interpret the results of these studies.

Surface independent factor XI activation by thrombin in the presence of high molecular weight kininogen.

A deficiency of one of the proteins of the contact system of blood coagulation does not result in a bleeding disorder. For this reason activation of blood coagulation via this system is believed to be an in vitro artefact. However, patients deficient in factor XI do suffer from variable bleeding abnormalities. Recently, an alternative pathway for factor XI activation has been described. Factor XI was found to be activated by thrombin in the presence of dextran sulfate as a surface. However, high molecular weight kininogen (HK), to which factor XI is bound in plasma, and fibrinogen were shown to block this activation suggesting it to be an in vitro phenomenon. We investigated the thrombin-mediated factor XI activation using an amplified detection system consisting of factors IX, VIII and X, which was shown to be very sensitive for factor XIa activity. This assay is approximately 4 to 5 orders of magnitude more sensitive than the normal factor XIa activity assay using a chromogenic substrate. With this assay we found that factor XI activation by thrombin could take place in the absence of dextran sulfate. The initial activation rate was approximately 0.3 pM/min (using 25 nM factor XI and 10 nM thrombin). The presence of dextran sulfate enhanced this rate about 8500-fold. A very rapid and complete factor X activation was observed in the presence of dextran sulfate. Although only minute amounts of factor XIa were formed in the absence of dextran sulfate, significant activation of factor X was detected in the amplification assay within a few minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible influence of intrarenal generation of kinins on prostaglandin release from the rabbit perfused kidney.

The effects of bradykinin and kininogen on renal prostaglandin release were studied in rabbit isolated kidneys perfused with oxygenated Krebs solution. The concentration of prostaglandin-like material in kidney effluent was determined by bioassay after extraction of the samples with organic solvents. In 7 experiments the samples were assayed after separation of prostaglandins E and F by thin layer chromatography. Addition of bradykinin to the perfusing fluid increased the venous and urinary effluxes of prostaglandin E-like substance by sixfold and fivefold, respectively, but efflux of prostaglandin F-like material was unaffected. Addition of kininogen to the perfusing fluid augmented the venous and urinary release of prostaglandin E-like substances by fifteenfold and ninefold respectively and caused a twofold increase in the efflux of prostaglandin F-like material into the venous effluent. Aprotinin, a kallikrein inhibitor, reduced the prostaglandin releasing action of kininogen but not of bradykinin. In contrast, inhibition of prostaglandin synthesis by indomethacin suppressed the release of prostaglandin evoked by either bradykinin or kininogen. This study suggests that augmented release of prostaglandins in response to kininogen is a consequence of renal generation of kinins. Thus, changes in the intrarenal activity of the kallikreinkinin system may modulate renal prostaglandin release.

Formation of a factor increasing vascular permeability during electrical stimulation of the saphenous nerve in rats.

1 Increased vascular permeability following electric antidromic stimulation of the rat saphenous nerve was observed in the skin area supplied by the nerve, confirming previous results by other authors.2 The phenomenon was not affected by pretreatment of the rats with diphenhydramine, burimamide or their combination; atropine, methysergide, methysergide plus diphenhydramine, carboxypeptidase B, acetylsalicylic acid, indomethacin or methiazinic acid. It was partially reduced by previous injection of cellulose-sulphate, a kininogen-depleting agent.3 Perfusates from the subcutaneous tissue of the paw area supplied by the saphenous nerve contained permeability increasing activity as shown by intradermal tests in other rats. This activity was present in perfusates collected during nerve stimulation but not in those collected before stimulation. It was not destroyed by heating to 100 degrees C, or by alpha-chymotrypsin or trypsin.4 Bradykinin-like activity may appear later in the perfusates, depending on the intensity of the stimuli.5 It is concluded that following electrical antidromic stimulation of the saphenous nerve a permeability increasing factor is released, possibly from nerves. It is dialysable and can be distinguished from acetylcholine, histamine, 5-hydroxytryptamine, plasma kinins, substance P, prostaglandins and high molecular weight proteins. The increased vascular permeability induced by this factor leads to plasma exudation and activation of the kinin system.