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.

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.

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 ~65 and 120120 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 Glu(1)-Thr(383) region, identical in LK and HK, contains bradykinin (BK) moieties Arg(363)-Arg(371). LK, HK and their kinin products Lys-BK and BK are involved in several biological 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 (Arg(178) r 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, I detected ~110-kDa bands in the plasma of this LK/HK-deficient patient vs ~120-kDa bands in normal human and ape plasmas. With polyclonal anti-Lys-BK antibody, which strongly detects BK eleaved at its COOH-terminus in purified HK, I detected ~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.

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.

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.

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-phthaldiadehyde-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

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.

Kallikrein-kinin system in the angiogenesis.

Using the bioassay to investigate the presence of mediators in the angiogenesis exudate to explain its property to cause vascular permeability and fall in the blood pressure we found the presence of histamine, bradykinin, prostaglandin E2 and angiotensin in the exudate. However, the role of these mediators in the angiogenesis process needs to be investigated.

Ile-Ser-bradykinin is an aberrant permeability factor in various human malignant effusions.

In this study we provide evidence for the presence of the aberrant peptide, Ile-Ser-bradykinin, in various human malignant exudates. The peptide was detected by deproteinisation of the effusion, application to reversed-phase HPLC, collection of the fractions containing Ile-Ser-bradykinin (retention time 6.90 min), degradation with carboxypeptidase B, and rechromatography of the resulting des-Arg-Ile-Ser-bradykinin (des-Arg-ISB) (retention time 13.5 min). In addition, all positive samples were confirmed by amino acid analysis and most of them (7/8) by amino-acid sequencing. In malignant effusions from 8 patients out of a group of 113 patients, Ile-Ser-bradykinin was found in concentrations between 12 and 520 mumol. In 44 malignant effusions, Ile-Ser-bradykinin was suspected, but could not be confirmed by the required additional methods (amino-acid analysis, sequencing) because of its low concentration. Sixty eight benign effusions were negative for Ile-Ser-bradykinin.

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.

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.

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.

Purification and identification of [hydroxyprolyl3]bradykinin in ascitic fluid from a patient with gastric cancer.

Kinins in the ascitic fluid from a patient with gastric cancer were purified by gel filtration and reversed-phase high-performance liquid chromatography (HPLC). Two fractions (fractions I and II) showed kinin activity. Fraction I did not correspond to either bradykinin or other known kinins, whereas fraction II corresponded to bradykinin. Fraction I contained 8 amino acid residues from bradykinin minus 1 proline plus 1 additional hydroxyproline. Sequence analysis of fraction I showed that the proline at the third amino acid residue of bradykinin was replaced by hydroxyproline. The retention time of fraction I on reversed-phase HPLC was exactly the same as that of synthetic [hydroxyprolyl3]bradykinin (Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg) and was distinguishable from des-Pro3-bradykinin. Thus, these results demonstrate for the first time the presence of [hydroxyprolyl3]bradykinin in vivo. This is also the first report of the presence of bradykinin in human tumor ascites.

Mechanism of kinin release from human low-molecular-mass-kininogen by the synergistic action of human plasma kallikrein and leukocyte elastase.

We have investigated the kinin release from human L-kininogen, a poor substrate for plasma kallikrein, by the synergistic action of human PMN elastase and plasma kallikrein. Although PMN elastase alone failed to generate kinin activity from L-kininogen, combination of PMN elastase with plasma kallikrein was found to be effective for the generation of kinin activity from L-kininogen. Two kinds of kinin, bradykinin and Met-Lys-bradykinin, were found to be released from L-kininogen by the synergistic action of PMN elastase and plasma kallikrein. Pretreatment of L-kininogen with PMN elastase facilitated the kinin release by plasma kallikrein, whereas pretreatment of L-kininogen with plasma kallikrein did not allow kinin release by the action of PMN elastase. These results suggested that PMN elastase would act firstly on L-kininogen to form a kinin containing fragment, from which kinin is released by the action of plasma kallikrein. The kinin-containing fragment was isolated by gel filtration and high-performance liquid chromatography of the elastase digest of L-kininogen. The amino-acid analysis and N-terminal amino-acid sequence analysis revealed that the kinin-containing fragment consisted of 26 amino-acid residues and is formed by cleavage of an Ile-Ser and a Ser-His bond of L-kininogen.

Effect of dexamethasone on kininogen production by a rat hepatoma cell line.

T Kininogen and High Molecular Weight Kininogen were characterized in the cell culture medium of Fao cells, a highly differentiated cell line derived from the Reuber H35 rat hepatoma. Immunoreactive T Kininogen and High Molecular Weight Kininogen identified by direct and specific RIAs were indistinguishable from standard kininogens. Immunoreactive T Kininogen was further identified by HPLC analysis of T kinin released after trypsin hydrolysis of the cell culture medium. The basal release rate of T kininogen was ten-fold higher than that of High Molecular Weight Kininogen. T Kininogen was not stored within the cells contrary to High Molecular Weight Kininogen. The production of the two kininogens in the cell medium was stimulated by dexamethasone up to five times in a dose-dependent manner. The specific antiglucocorticoid compound RU 38486 did not alter the basal rate of kininogen release by Fao cells, but abolished the stimulation by dexamethasone, indicating that dexamethasone exerts a true glucocorticoid type effect.