Cell culture of mammalian endometrium and synthesis of blastokinin in vitro.

Endometrial cells obtained from mature female estrous rabbits can be grown in cell culture with the aid of insulin. These cells, after several days in culture, are capable of synthesizing blastokinin by induction with progesterone for 48 hours.

Aspects of bradykinin radioimmunoassay.

125I-derivatives of Tyr1-kallidin, Tyr5-bradykinin, and Tyr8-bradykinin were prepared. A technique for purification of the monoiodinated derivative is described. Bradykinin antisera were tested for their ability to bind the mono-iodinated analogues. Each antiserum had a characteristic preference for one of the three labeled peptides. The sensitivity of each antiserum was greatest when it was used with the label bound most avidly by that antiserum. The specificity of an antiserum was not changed by varying the labeled analogue. Some common enzyme inhibitors had significant effects on the antigen-antibody reactions. Lecithin interfered with the reaction between antiserum and Tyr1-kallidin. The data suggest that antisera for bradykinin radioimmunoassay be tested with several radioactive iodobradykinins to maximize their usefulness. In addition, enzyme inhibitors used to stabilize levels of kinins in biological fluids should be tested for their effects on the assay. Biologic samples rich in lipid may give spurious radioimmunoassay results unless they are freed of those phospholipids that can bind labeled and unlabeled peptides.

The biological activity of diuretic factors in Rhodnius prolixus.

The rapid post-feeding diuresis of Rhodnius prolixus is under neurohormonal control and involves the integrated activity of the crop, Malpighian tubules and hindgut. One of the factors which is involved in this rapid diuresis is serotonin, however a peptide(s) is also considered to be involved. In other insects, corticotropin releasing factor (CRF)-like and kinin-like, calcitonin-like peptides and CAP(2b) have been demonstrated to be diuretic factors/hormones. In the present study, serotonin and CRF-like peptides increased secretion rate and cAMP content of Rhodnius Malpighian tubules, while the kinin-like peptides tested did not increase secretion rate or cAMP content of the tubules. Extracts of the CNS were processed and several HPLC fractions revealed kinin-like immunoreactivity but these fractions did not increase secretion rate when tested on Malpighian tubules. However, these same fractions did possess activity when tested on the hindgut contraction assay. In addition, material eluting at higher acetonitrile concentrations from the HPLC increased secretion and cAMP content of Rhodnius Malpighian tubules. This material eluted at concentrations of acetonitrile consistent with the elution time of CRF-like peptide standards. Synergism was demonstrated using the pharmacological agent forskolin and serotonin, tested on the rate of secretion of Rhodnius Malpighian tubules, in agreement with data of Maddrell et al. As well, synergism could be demonstrated using mesothoracic ganglionic mass (MTGM) homogenates and serotonin at some concentrations of serotonin. However, combinations of CRF-like material and serotonin increased secretion additively, not synergistically. Kinin-like peptides, tested along with CRF-like material and serotonin, at low concentrations, did not increase secretion above that of those factors tested alone.

Isolation and structural elucidation of eight kinins from the retrocerebral complex of the American cockroach, Periplaneta americana.

By monitoring the contractile activity of the hindgut of the American cockroach in vitro eight myotropic neuropeptides were isolated from the retrocerebral complex of the American cockroach. Peptide sequence analysis and mass spectrometry yielded the following structures: Arg- Pro-Ser-Phe-Asn-Ser-Trp-Gly-NH2 (Pea-K-1), Asp-Ala-Ser-Phe-Ser-Ser-Trp-Gly-NH2 (Pea-K-2), Asp-Pro-Ser-Phe-Asn-Ser-Trp-Gly-NH2 (Pea-K-3), Gly-Ala-Gln-Phe-Ser-Ser-Trp-Gly-NH2 (Pea-K-4), Ser-Pro-Ala-Phe-Asn-Ser-Trp-Gly-NH2 (Pea-K-5), Asp-Pro-Ala-Phe-Ser-Ser-Trp-Gly-NH2 (Lem-K-7), Gly-Ala-Asp-Phe-Tyr-Ser-Trp-Gly-NH2 (Lem-K-8) and Ala-Phe-Ser-Ser-Trp-Gly-NH2 (Lom-K). The C-terminal sequence Phe-X-Ser-Trp-Gly-NH2 characterized the peptides as members of the insect kinin family. All structures were confirmed by comparison of retention times between synthetic and natural peptides. The threshold concentration for stimulatory effects of the synthetic peptides on the isolated hindgut was about 10(-9) M and there was no significant difference measured between the different kinin forms. These neuropeptides are the first members of the insect kinin-family isolated from the American cockroach. Their occurrence in the retrocerebral complex suggests a physiological role as neurohormone.

Inhibition of plasma kallikrein. Kininase and kinin-like activities of preparations from the medicinal leeches.

The medicinal leech salivary gland secretion deprived of hirudin antithrombin activity inhibits amidolytic (substrate S-2302) and kininogenase (substrate kininogen) activities of plasma kallikrein, the main component of the intrinsic mechanism of blood coagulation. It therefore possesses high anticoagulant properties. Kininase (substrate bradykinin) activity of leech saliva and extracts from the medicinal leeches, as well as kinin-like effects of extracts heated at 100 degrees C have been detected. The last one is correlated with the hyperalgetic property of the heated extract. An analgetic effect was observed with the unheated extract but not with leech saliva after intranasal administration to rats.

Measurements of urinary kinins by HPLC-radioimmunoassay.

In this paper we describe a method for the individual determination of urinary kinins. Extraction from the urine is performed on an Amberlite CG-50 column and kinins are eluted with formic acid. The samples are further purified and kinins are separated by reversed phase HPLC. Bradykinin and lysylbradykinin are quantified by a sensitive radioimmunoassay capable of detecting 0.1 fmol of either peptide. Procedural losses are monitored by measuring the recovery of [3H]bradykinin and [3H]lysylbradykinin. Simple methods for labeling of bradykinin and lysylbradykinin with tritium are also presented. Recoveries of [3H]bradykinin and [3H]lysylbradykinin from biological material ranged between 77 and 91%. The combination of HPLC with radioimmunoassay makes it possible to determine kinin concentrations of biological samples with a higher sensitivity and greater specificity than previous methods.

Two kinins isolated from an extract of the venom reservoirs of the solitary wasp Megascolia flavifrons.

From an extract of the venom reservoirs of the wasp Megascolia flavifrons two kinins have been isolated. The sequences of amino acids are: Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg (Thr6-bradykinin) and Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-Lys-Ala (Thr6-bradykinin-Lys-Ala). The bradykinin-like effects of the venom on a number of vertebrate smooth muscle preparations can be explained by the actions of these kinins.

Capillary zone electrophoresis separation of kinins using a novel laser fluorescence detector.

1. Bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg9-bradykinin and des-Arg1-bradykinin were separated by capillary zone electrophoresis in an apparatus constructed in our laboratory which utilizes a novel N2 pulsed laser-induced fluorescence detector. 2. Detection limits of 1.2 fmol for fluorescamine-derivatized bradykinin and 90 attomol for O-phthaldialdehyde-derivatized bradykinin were achieved. 3. This powerful analytical tool is described and its successful application to the measurements of bradykinin after enzymatic release from blood is documented.

Evidence for the presence of a kininogen-like species in a case of total deficiency of low and high molecular weight kininogens.

Low and high molecular weight kininogens (LK and HK), containing 409 and 626 amino acids with masses of approximately 65 and 120 kDa after glycosylation, respectively, are coded by a single gene mapped to the human chromosome 3 by alternative splicing of the transcribed mRNA. The NH2-termini Glu1-Thr383 region, identical in LK and HK, contains bradykinin (BK) moieties Arg363-Arg371. LK, HK and their kinin products Lys-BK and BK are involved in several biologic processes. They are evolutionarily conserved and only 7 patients, all apparently normal, have been reported to lack them. In one of these patients (Williams' trait), a codon mutation (Arg178-->stop) has been blamed for the absence of LK and HK. However, using Western blots with 2 monoclonal anti-HK antibodies, one that recognizes the region common to LK and HK and the other that recognizes only HK, 1 detected approximately 110-kDa bands in the plasma of this LK/HK-deficient patient vs approximately 120-kDa bands in normal human and ape plasmas. With polyclonal anti-Lys-BK antibody, which strongly detects BK cleaved at its COOH-terminus in purified HK, 1 detected approximately 110-kDa bands in the normal and the deficient plasmas. Western blots with a monoclonal anti-prekallikrein (PK) antibody showed that surface activation of PK and distribution of PK activation products, both dependent on HK, were similar in these plasmas. These findings suggest that a mutant gene yielded a kininogen-like species possibly involving aberrant mRNA splicing-structurally different from normal HK, but apparently with the capacity to carry out seemingly vital HK functions.

Characterization of a new kinin potentiating peptide obtained from human plasma proteins.

Partially purified kinin potentiating peptide (KPP) obtained from kininogen-depleted human plasma inhibited lung angiotensin converting enzyme in vitro and potentiated guinea-pig ileum contractions induced by bradykinin (BK), Lys-BK, Met-Lys-BK, Ile-Ser-BK, and Lys-Lys-BK. Contractions evoked by angiotensin II, histamine, acetylcholine, and barium chloride were not potentiated. KPP also potentiated kinin-induced contractions of rat uterus and of guinea-pig ileum pre-incubated with 1,10-phenanthroline. It is suggested that KPP potentiation is due, at least in part, to a direct effect on kinin receptor(s).

Ornitho-kininogen and ornitho-kinin: isolation, characterization and chemical structure.

Ornitho-kininogen was purified from chicken and duck blood plasmas by a two-stage method using chromatography on columns of S-alkylated papain-Cellulofine and DEAE-5PW. The isolated preparation from chicken plasma gave a single band on SDS-PAGE with or without 2-mercaptoethanol and on disc-PAGE. The molecular weight of ornitho-kininogen was estimated as 74,000 on SDS-PAGE using the Ferguson plot method. Ornitho-kininogen was found to have the similar properties to those of mammalian high molecular weight kininogen (HMWK), in terms of the amino acid composition, molecular weight, and susceptibility to plasma kallikrein. No kininogen corresponding to mammalian low molecular weight kininogen (LMWK) and rat T-kininogen could be detected in chicken plasma. In fact, ornitho-kininogen was degraded rapidly by bovine plasma kallikrein, liberating a kinin. This kinin was isolated from the digest by reversed-phase HPLC. The primary structure of the isolated kinin was determined as Arg-Pro-Pro-Gly-Phe-Thr-Pro-Leu-Arg. The sequence of this peptide, named ornitho-kinin, was similar to that of bradykinin except for the substitution of Thr-6 and Leu-8 for Ser-6 and Phe-8. The isolated ornitho-kinin induced a contraction of chicken smooth muscle and had a strong hypotensive effect in the chicken. However, it did not contract the isolated rat uterus. It is suggested that this specificity difference is due to the replacement of Phe-8 by Leu-8. The sequence of residues 1-30 of ornitho-kininogen exhibited 43% identity with that of bovine kininogen.

Kinins as mediators of human allergic reactions.

We have demonstrated that kinins are generated following nasal challenge with allergen of allergic (5.6 +/- 0.17 ng/m-), but not nonallergic (0.04 +/- .02 ng/ml), individuals (n = 8 in each case). The presence of kinin was highly correlated with that of histamine and TAME-esterase activity and with clinical symptoms (p less than 0.001). In a double blind, placebo-controlled study, topical administration of the drug Azatadine, which inhibits mast cell mediator release in vitro, reduced the clinical response to allergen challenge and reduced the concentrations of kinins, histamine, and TAME-esterase activity observed following allergen challenge. In addition to the immediate response to allergen, some individuals experience a recurrence of symptoms some 3-12 hours after challenge; in seven such individuals (13.5 +/- 3.2 ng kinin/ml in the immediate reaction), there was a second increase in nasal kinins (2.95 +/- 1.4 ng/ml) during this late reaction, again correlating with increases in histamine and TAME-esterase activity. HPLC analysis revealed that a mixture of bradykinin and lysylbradykinin is produced during both responses. Finally, 12 subjects with a history of nasal symptoms upon exposure to cold, dry air (CDA) were compared to five asymptomatic individuals in a nasal challenge system involving nasal breathing of CDA and warm, moist air (WMA). For the symptomatic group the levels of kinin in nasal lavages were significantly increased after CDA (2.9 +/- 0.8 ng/ml) compared to baseline (0.06 +/- 0.01 ng/ml) or WMA (0.3 +/- 0.07 ng/ml). Kinin generation again correlated with increases in histamine, PGD2 and TAME-esterase activity and with onset of symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of [hydroxyproline3]Lysyl-bradykinin formed by kallikrein from human plasma protein.

[Hydroxyproline3]Lysyl-bradykinin ([Hyp3]Lys-BK), a new kinin was isolated, besides Lysyl-bradykinin (Lys-BK), from the reaction mixture of human plasma protein Cohn's fraction IV-4 with hog pancreatic kallikrein. The liberated kinins were isolated by procedures including ethanol extraction, Sephadex G-15, CM cellulose and reverse-phase high performance liquid chromatography and quantitated by radioimmunoassay. On HPLC, two peaks of immunoreactive kinins emerged. Peak 1, an unknown kinin proceeded to peak 2 which had an identical retention time to that of Lys-BK. The amino acid sequence of the unknown peak 1 proved to be Lys-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg, or [Hydroxyproline3]Lys-BK, and peak 2 Lys-BK. The ratio of the amounts of two kinins thus formed were [Hyp3]Lys-BK 25 +/- 4% and Lys-BK 75 +/- 4%. The existence of [Hyp3]Lys-BK suggests a presence of a new kininogen containing [Hyp3]Lys-BK in human plasma protein.

A micro-kininogenase assay for studies of kallikrein in renal micropuncture/microperfusion.

We report the development of a micro-kininogenase assay suitable in studying the dynamics of kallikrein at intranephron segmental level. The detection limit is 119 fg or 2.6 attomoles of kallikrein. Activation of microquantities of kallikrein is possible with the use of Triton X-100. Because of its extremely high sensitivity and reproducibility the assay is likely to also prove useful in physiological studies in which only very small amounts of kallikrein containing samples can be obtained.

The kinin-forming acid protease system in murine fibroblasts L-929.

Components of an acid protease kinin-forming enzyme system were isolated and purified from the murine fibroblast L-929 cell line grown in stationary cell culture. The enzyme, was purified from the 10,000 g supernatant cell fraction by sequential passage through G-200 Sephadex, hydroxylapatite, DEAE-A50 Sephadex and affinity chromatography columns. The specific activity was determined on a rat plasma kininogen purified on DEAE and G-100 Sephadex columns as assayed for kinin-forming activity on the isolated perfused rat uterus. Both enzyme and substrate showed a single band pattern by disc gel electrophoresis technique. Optimum protease activity was obtained at pH 3.8, and kinin release was both time- and enzyme-dependent at 37 degrees. The molecular weight of the protease and kininogen, estimated on a G-200 Sephadex column, was 39,000 and 115,000 respectively. Two fibroblast kinins were isolated from a mixture of 12,000 g fraction of fibroblast homogenate and rat plasma incubated at 37 degrees, pH 4.0 for 92 hours. The two fibroblast kinins (I, II) were separated and purified on G-25 Sephadex, CM-C50 Sephadex, and Biogel P-4 columns. The purity of the kinins was ascertained by thin layer chromatography. The molecular weight of fibroblast kinin I was estimated to be 1450 with a 14-amino acid composition consisting of AspThrSerProGluGlyAlaVal-LeuTyrPheLysHisArg. Fibroblast kinin II had an estimated molecular weight of 1000 with a 12-amino acid composition that included AspSerProGluGlyAlaLeuTyrLysHisArg. The kinins were relatively rich in arginine and did not contain the bradykinin sequence.

Purification and characterization of an acid protease and vasopeptide kinins from Murphy-Sturm lymphosarcoma.

An acid protease from the rat Murphy-Sturm lymphosarcoma (MSLS) tumor was isolated, purified and characterized. The protease hydrolyzed bovine hemoglobin and formed vasopeptide kinins when incubated with purified rat plasma kininogen. The purification was carried out in seven steps involving homogenization of the tumor tissue, acid precipitation with glacial acetic acid, ammonium sulfate precipitation to 60% saturation, DEAE-Sephadex batch treatment, chromatography on a QAE-Sephadex column, gel filtration on Sephadex G-200 and finally chromatography on CM-32 cellulose. The purification scheme resulted in a 640-fold purification and a homogeneous preparation as confirmed by disc gel electrophoresis and Ouchterlony immunodifussion. Sephadex G-200 elution profiles indicated two different protease fractions containing protease activity which showed a single band with identical relative mobility on polyacrylamide gel electrophoresis in the presence and absence of SDS, and also showed immunological cross-reactivity against anti-acid protease antiserum raised in the rabbit against one of the fractions. The acid protease is presumed to be a single protease which has a tendency to aggregate. An apparent molecular weight of 39,500 was estimated by gel filtration on Sephadex G-200, and 41,000 by polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis showed that the acid protease was comprised of a major band with a molecular weight of 4,000 and two fainter bands with molecular weights of 27,000 and 12,000. The purified enzyme showed three major isozymic forms (alpha, beta, gamma) which had isoelectric points (pI) of 5.2, 5.5 and 5.8 respectively, and had near identical amino acid compositions. The carbohydrate moiety contained 2 mol of N-acetylglucosamine and 8 mol of mannose per mol enzyme. The pH optimum for the digestion of bovine hemoglobin was approximately 3.0. The protease activity was very stable above pH 3.4. The acid protease released kinin from purified rat plasma kininogen at an initial rapid rate which plateaued at 460 ng bradykinin equivalents per mg substrate after 2 hr incubation at 37 degrees. The vasopeptide was purified to electrophoretic homogeneity by gel filtration on Sephadex G-50 and chromatography on a column of CM-Sephadex. The amino acid composition of vasopeptide was Ser2, Gly1, Pro4, Ile1, Leu1, Phe2, Arg2.

Study on the in vitro assay method for evaluating the inhibitory effect of various substances on the production of plasma kallikrein.

The assay method based on the principle of kallikrein-kinin cascade was established for evaluating the inhibitory effects of various substances on the production of plasma kallikrein. In this in vitro assay, it was found that indomethacin, ketoprofen, ibuprofen and an extract obtained from inflamed rabbit skin inoculated with vaccinia virus (NSP) had the inhibitory effect on the production of plasma kallikrein. Kinins generated in the reaction mixture were measured by RIA. It was shown that the generation of kinins was also inhibited by these substances. From these results, it is hoped that this assay method may be useful for screening the substances which inhibited the production of kinin.