Biomedical subjects

I Paegelow

Publications and source records attributed to I Paegelow.

At least 19 recordsLinked to original sources

PubMed2002

Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues.

A series of conformationally constrained cyclic analogues of the peptide hormone bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) was synthesized to check different turned structures proposed for the bioactive conformation of BK agonists and antagonists. Cycles differing in the size and direction of the lactam bridge were performed at the C- and N-terminal sequences of the molecule. Glutamic acid and lysine were introduced into the native BK sequence at different positions for cyclization through their side chains. Backbone cyclic analogues were synthesized by incorporation of N-carboxy alkylated and N-amino alkylated amino acids into the peptide chain. Although the coupling of Fmoc-glycine to the N-alkylated phenylalanine derivatives was effected with DIC/HOAt in SPPS, the dipeptide building units with more bulky amino acids were pre-built in solution. For backbone cyclization at the C-terminus an alternative building unit with an acylated reduced peptide bond was preformed in solution. Both types of building units were handled in the SPPS in the same manner as amino acids. The agonistic and antagonistic activities of the cyclic BK analogues were determined in rat uterus (RUT) and guinea-pig ileum (GPI) assays. Additionally, the potentiation of the BK-induced effects was examined. Among the series of cyclic BK agonists only compound 3 with backbone cyclization between positions 2 and 5 shows a significant agonistic activity on RUT. To study the influence of intramolecular ring closure we used an antagonistic analogue with weak activity, [D-Phe7]-BK. Side chain as well as backbone cyclization in the N-terminus of [D-Phe7]-BK resulted in analogues with moderate antagonistic activity on RUT. Also, compound 18 in which a lactam bridge between positions 6 and 9 was achieved via an acylated reduced peptide bond has moderate antagonistic activity on RUT. These results support the hypothesis of turn structures in both parts of the molecule as a requirement for BK antagonism. Certain active and inactive agonists and antagonists are able to potentiate the bradykinin-induced contraction of guinea-pig ileum.

Acylation↗

PubMed2002

Migratory responses of polymorphonuclear leukocytes to kinin peptides.

The present study examines the influence of kinins on the migratory capacity of human polymorphonuclear leukocytes under in vitro conditions using the Boyden chamber technique. By means of checkerboard analysis the migration of neutrophils induced by bradykinin could be characterized as true chemotaxis. The stimulation of human neutrophils with bradykinin, with the nonpeptide B(2) receptor agonist FR190997 as well as with des-Arg(9)-bradykinin and des-Arg(10)-kallidin results in a concentration-dependent migration. Pretreatment of the neutrophils with the B(2) receptor antagonist HOE-140 (icatibant) inhibited the bradykinin-induced migration but not that induced by B(1) receptor agonists, whereas the B(1 )receptor antagonist des-Arg(10)HOE-140 abolished the migration elicited by des-Arg(9)-bradykinin or des-Arg(10)-kallidin but not that evoked by bradykinin. Pretreatment of the neutrophils with the leukotriene B(4) (LTB(4)) antagonist ZK158252 inhibited the LTB(4)-induced chemotaxis as well as the chemotaxis produced by bradykinin and des-Arg(10)-kallidin. An involvement of interleukin-1beta and of the chemokine IL-8 in the bradykinin-induced migration in vitro could be excluded during the migration time of the neutrophils. In conclusion, the present study provides pharmacological evidence showing that B(1) and B(2) kinin receptors are involved in the migration of human neutrophils in vitro, that LTB(4) participates in the downstream pathway and that the B(1) kinin receptor seems to be expressed already under physiological conditions.

Cell Movement↗

PubMed2000

Structure activity relationships for bradykinin antagonists on the inhibition of cytokine release and the release of histamine.

Highly potent bradykinin antagonists were found to inhibit bradykinin-induced release of cytokines but to stimulate histamine release. Both actions show structural requirements completely different from those for bradykinin B1 and B2 receptors, indicating that the release of some cytokines from spleen mononuclear cells and of histamine from rat mast cells is not mediated by these receptors. Most potent bradykinin antagonists release histamine at lower concentrations than does bradykinin itself. Dimers of bradykinin antagonists are the most potent compounds for histamine release. In contrast to enhanced histamine release, potent inhibition of cytokine release enhances the applicability of these compounds as anti-inflammatory drugs. Many of the peptides designed for high B2-receptor antagonism were found to be compared by their concentrations far more potent for inhibition of cytokine release than for smooth muscle contraction. Thus, for some antagonists inhibition of cytokine release was detected at concentrations as low as 10(-15) M. The rational design of peptide and nonpeptide bradykinin antagonists for therapeutic use requires not only knowledge about the potency but also knowledge about the structure-activity relationships of such important side effects as cytokine and histamine release.

Amino Acid Substitution↗

PubMed2000

Degradation of bradykinin by peritoneal and alveolar macrophages of the guinea pig.

Peptidase inhibitors and identification of the peptide fragments were used for the characterization of the bradykinin metabolism by alveolar and peritoneal macrophages. Both cell types show differences in the rate of inactivation and in the quantity of the metabolites generated. BK(1-5), BK(1-8), and BK(1-7) are the predominant direct metabolites. Metalloendopeptidase 24.15, carboxypeptidase M, and an unidentified peptidase are responsible for their formation. Angiotensin-converting enzyme and neutral endopeptidase 24.11 do not play a crucial role in the degradation of bradykinin by macrophages. In the bronchoalveolar space, other cells than the macrophages are more important to the breakdown of this peptide.

Animals↗

PubMed2000

Kinins and kinin receptors: importance for the activation of leukocytes.

In this article, we analyzed the role of kinins and kinin receptors with respect to the activation of leukocytes. In these cells, the biological effects of kinin peptides are mediated by kinin receptor subtypes B1, B2, or both, depending on species and cell type. In contrast to the other leukocytes, neutrophils contain the complete system for the synthesis and release of bioactive kinins. Consequently, very high concentrations of these peptides can be reached in the close neighborhood of the kinin receptors, in particular at the site of inflammation. Kinins are responsible for many effects in leukocytes including the release of other inflammatory mediators, such as cytokines, prostaglandins, leukotrienes, and reactive oxygen species. Obviously, the potency of kinins to stimulate leukocytes is dependent on the differentiation and especially on the activation stage of these cells. An upregulation of kinin receptors on neutrophils and macrophages appears to be involved in increasing the sensitivity of these cells to kinins at the site of inflammation.

Animals↗

PubMed1999

Influence of interleukin-1 beta on bradykinin-induced responses in guinea pig peritoneal macrophages.

OBJECTIVE AND DESIGN: The endogenous nonapeptide bradykinin is implicated in the pathogenesis of a number of inflammatory diseases. Recently, it could be shown that bradykinin is a potent stimulus for the generation of arachidonic acid, prostaglandin E2 (PGE2) and superoxide radical via the bradykinin B2 receptor from macrophages depending on their stage of maturation or activation. The present study was designed to characterize the effect of the proinflammatory cytokine interleukin-1 beta (IL-1 beta) on responsiveness of macrophages to bradykinin. MATERIAL: Guinea pig peritoneal macrophages were collected 7 days after i.p. injection of paraffinum subliquidum. RESULTS: The bradykinin-induced increase in cytosolic free calcium concentration ([Ca2+]i) and activation of the respiratory burst were significantly enhanced by interleukin-1 beta (100 U/ml) added to cultures of macrophages 24 h before stimulation with bradykinin. However, the EC50 values of bradykinin-induced calcium signal of 23 +/- 8 nM and 29 +/- 17 nM and superoxide radical formation of 64 +/- 22 nM and 34 +/- 29 nM in control and interleukin-1 beta-treated cells, respectively, were not changed. In contrast to the influence of IL-1 beta on these functions, saturation and competition experiments measured by binding of [3H]bradykinin ([3H]BK) to intact macrophages demonstrate that cell exposure to interleukin-1 beta for 24 h caused no change in the density and affinity of the bradykinin B2 receptors. CONCLUSIONS: The most likely explanation for the cytokine effects is an influence on the level of the receptor-effector coupling downstream of the agonist binding.

Animals↗

PubMed1998

Bradykinin antagonists with dehydrophenylalanine analogues at position 5.

Continuing the studies on structural requirements of bradykinin antagonists, it has been found that analogues with dehydrophenylalanine (deltaPhe) or its ring-substituted analogues (deltaPhe(X)) at position 5 act as antagonists on guinea pig pulmonary artery, and on guinea pig ileum. Because both organs are considered to be bradykinin B2 receptor tissues, the analogues with deltaPhe or deltaPhe(X) at position 5, but without any replacement at position 7, seem to represent a new structural type of B2 receptor antagonist. All the analogues investigated act as partial antagonists; they inhibit the bradykinin-induced contraction at low concentrations and act as agonists at higher concentrations. Ring substitutions by methyl groups or iodine reduce both the agonistic and antagonistic activity. Only substitution by fluorine gives a high potency. Incorporation of deltaPhe into different representative antagonists with key modifications at position 7 does not enhance the antagonist activity of the basic structures, with one exception. Only the combination of deltaPhe at position 5 with DPhe at position 7 increases the antagonistic potency on guinea pig ileum by about one order of magnitude. Radioligand binding studies indicate the importance of position 5 for the discrimination of B2 receptor subtypes. The binding affinity to the low-affinity binding site (KL) was not significantly changed by replacement of Phe by deltaPhe. In contrast, ring-methylation of deltaPhe results in clearly reduced binding to KL. The affinity to the high-affinity binding site (KH) was almost unchanged by the replacement of Phe in position 5 by deltaPhe, whereas the analogue with 2-methyl-dehydrophenylalanine completely failed to detect the KH-site. The peptides were synthesized on the Wang-resin according to the Fmoc/Bu(t) strategy using Mtr protection for the side chain of Arg. The dehydrophenylalanine analogues were prepared by a strategy involving PyBop couplings of the dipeptide unit Fmoc-Gly-deltaPhe(X)-OH to resin-bound fragments.

Animals↗

PubMed1998

Differential sensitivity of macrophages to bradykinin.

In this report, we investigated the responsiveness of subpopulations of elicited peritoneal macrophages between each other compared to resident tissue macrophages of alveoli of guinea pig to the action of bradykinin. Bradykinin stimulated the secretion of superoxide radical, arachidonic acid and prostaglandin E2 (PGE2) via the bradykinin B2 receptor subtype in peritoneal macrophages, indicated by complete inhibitory effect of the bradykinin B2 receptor antagonist HOE 140. The extent of the secretion, however, varied substantially between macrophages of different size. The highest level of the secretion was observed in the fraction containing the intermediate-size macrophages, while progressively lower level of the secretion was observed with decreasing size. In contrast, large macrophages obviously lost their secretory ability. Additionally, the bradykinin-stimulated release of cyclooxygenase products exerted an inhibitory action on NADPH-oxidase activity depending on size and stage of maturation/activation of macrophages, as judged by an increase in superoxide radical generation by indomethacin (100 microM) preincubation of cells. Furthermore, the investigation of resident tissue macrophages of alveoli has shown that these cells also express the bradykinin B2 receptor subtype. However, the receptor activity measured by bradykinin-induced increase in intracellular free calcium [Ca2+]i was very low compared to elicited peritoneal macrophages. These findings indicate that the stage of differentiation/maturation and activation of macrophages may be important for the ability of bradykinin to stimulate these cells to inflammatory responses in vivo.

Animals↗

PubMed1996

Highly selective bradykinin agonists and antagonists with replacement of proline residues by N-methyl-D- and L-phenylalanine.

For further studies on the structural and conformational requirements of positions 2,3, and 7 in the bradykinin sequence, we replaced the proline residues by the more hydrophobic and conformationally restricted N-methyl-L- and D-phenylalanine (NMF). The biological activities of the new analogs were evaluated on rat uterus, guinea pig ileum, and guinea pig lung strip. Receptor binding of the analogs was studied in membranes from rat uterus and guinea pig ileum. Influence of bradykinin analogs on the release of cytokines from mouse spleen cell cultures was also measured. Bradykinin analogs were synthesized by the solid phase method, using Boc strategy on PAM or Merrifield resins. The best results in the formation of the N-methylamide bond were obtained with the coupling reagent PyBrop. In position 7 the substitution of D-Phe by D-NMF, retaining the configuration of the amino acid, converts bradykinin antagonists into agonists. The bradykinin analogs with D-NMF at position 7 gave the highest known tissue selectivity for rat uterus among agonists. [L-NMF(2)]bradykinin has moderate agonist activity on rat uterus but antagonist activity on guinea pig lung strip. It represents a new antagonist for B(2) receptors without any replacement at position 7. The same analog completely inhibits bradykinin-evoked cytokine expression by mononuclear cells.

Amino Acid Sequence↗

PubMed1996

A new type of bradykinin B2 receptor antagonists: bradykinin analogs with N-alkyl amino acids at position 2.

It is commonly assumed that bradykinin B2 receptor antagonists bind to a receptor site partially different from that for agonists. Thus, it is likely that there exists more than one key modification to convert bradykinin receptor agonists into antagonists. In this respect, [L-NMePhe2]-BK represents the basic structure of a new type of bradykinin B2 receptor antagonists without any replacement at position 7. This compound inhibits both in vitro bradykinin-induced contraction of the guinea pig lung strip and in vivo bradykinin-induced bronchoconstriction. Furthermore, this analog shows analgesic activity, blocks in a dose-dependent manner the bradykinin-induced Ca2+ release from macrophages and inhibits at a concentration of 10(-13) M the bradykinin-induced cytokine release from mononuclear cells. Combinations with structural modifications previously performed for other B2 receptor antagonists rather reduce than enhance the potency.

Amino Acid Sequence↗

PubMed1996

Induction of histamine release from rat mast cells by bradykinin analogues.

Independently of their agonistic or antagonistic activity on different isolated tissue preparations, the kinin analogues investigated induce histamine release on rat peritoneal mast cells. The effectivity of most compounds is 10 to 100 times higher than that of bradykinin. Beside the positively charged amino acids, the elongation at the N-terminus with hydrophobic amino acids and the replacement of amino acids in the bradykinin sequence (especially at position 7) with aromatic residues is important for a high histamine-releasing activity.

Amino Acid Sequence↗

PubMed1995

Bradykinin receptors in signal transduction pathways in peritoneal guinea pigs macrophages.

The presence of a bradykinin receptor on guinea pig peritoneal macrophages was evidenced by binding studies and by the effect of bradykinin on activation of the phospholipase C and the increase in intracellular calcium concentration ([Ca2+]i). Binding studies demonstrated a specific, saturable binding for [3H]bradykinin inhibited by the bradykinin B2 (HOe 140) but not bradykinin B1 (des-Arg9[Leu8]bradykinin) receptor antagonist. Scatchard analysis revealed a single class B2 bradykinin binding site with a binding affinity (kd) of 0.8 nM and a receptor concentration (Bmax) of 35 fmol/5 x 10(6) cells, representing approximately 4000 bradykinin receptors per cells. Kinetic studies confirmed the presence of this single binding site by the determination of similar binding affinity. Activation of peritoneal macrophages by bradykinin resulted in a time- and dose-dependent release of inositol phosphates determined by anion exchange chromatography and intracellular calcium analyzed using fura-2/AM. The increase in [Ca2+]i induced by bradykinin was blocked by the specific bradykinin B2 receptor antagonist HOE 140 but not the bradykinin B1 receptor antagonist des-Arg9[leu8]-BK. These studies provide novel information regarding the nature of kinin receptors on guinea pig peritoneal macrophages and their signal transduction pathways.

Animals↗

PubMed1995

Effects of bradykinin and bradykinin analogues on spleen cells of mice.

The present study was undertaken to examine the effects of bradykinin and selected bradykinin analogues on mononuclear cells derived from mouse spleen. Bradykinin as well as des-Arg9-bradykinin, a bradykinin B1 receptor agonist, were able to induce the release of so-called charge-changing lymphokines, which could be identified as interleukin-1, interleukin-6, interleukin-2 and as interleukin-2 receptor. The cytokine release evoked by bradykinin and all analogues showed a bell-shaped dose dependence in a range of 10(-8) M to 10(-6) M and could be inhibited by the specific bradykinin receptor antagonist, D-Arg0[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HOE140), and by bradykinin analogues with N-methyl-phenylalanine at position 2 in concentrations as low as 10(-12) M and 10(-13) M, respectively. Obviously the N-terminus of bradykinin seems to be responsible for the interaction with the mononuclear cells concerning all peptides investigated.

Amino Acid Sequence↗

PubMed1995

Release of cytokines from isolated lung strips by bradykinin.

Bradykinin as an inflammatory mediator was assayed for its ability to release cytokines from isolated lung tissue derived from guinea pigs, mice and in some cases from patients. Bradykinin elicited in concentrations, which were able to induce a contraction of isolated lung strips, a secretion of different cytokines from the tissue into organ baths as well as from lung tissue incubated in petri dishes (4h, 37 degrees C). Using enzyme immuno assays and the tanned erythrocyte electrophoretic mobility (TEEM)-test in combination with monoclonal antibodies the cytokines could be identified preferably as interleukin(IL)-1, IL-2, sIL-2R and IL-6. Tyrode solution as a control and carbachol in a concentration causing also a contraction were not able to release cytokines in a significant amount. The bradykinin B2 receptor antagonist icatebant (HOE 140) inhibited the bradykinin-induced IL-2 and IL-6 release. The results show that bradykinin can elicit the secretion of the cytokine cascade via a receptor-mediated process.

Animals↗

PubMed1995

New cyclic bradykinin antagonists containing disulfide and lactam bridges at the N-terminal sequence.

Continuing our studies of the bioactive conformation of bradykinin (BK) antagonists, we synthesized a first series of analogues with side-chain cyclization in the N-terminal sequence. Through this conformational constraint it should be possible to gain insight into their three-dimensional structure. The cycles were proposed on the basis of existing ideas and hypotheses about the receptor bound conformation of BK and its antagonists. The reported peptides contain D-Phe at position 7 or D-Tic-Oic (D-Tetrahydroisoquinoline-3 -carboxyl-octahydroindole-2-carboxylic acid) at positions 7 and 8, respectively, and a disulfide or lactam bridge between positions 0 and 6. Syntheses, including cyclization reactions, were carried out on PAM resin. The biological activity of the lead compound [DPhe7]-BK, the linear precursors and the cyclic peptides, as estimated on isolated rat uterus, guinea pig ileum and lung strips, are in the same range. The conformational properties of the new cyclic analogues were studied through energy minimization on a model compound. The results of the calculations support the existence of low-energy structures containing a beta-turn. Therefore, such a turn in the N-terminal segment of the molecule can be proposed as an important structural feature of the bioactive conformation of BK antagonists.

Amino Acid Sequence↗

PubMed1995

New photoaffinity labelled agonists of bradykinin.

Several photoaffinity labelled agonists of the peptide hormone bradykinin (BK) were synthesized by solid phase methods. Their biological activities and binding affinities were determined in both the isolated rat uterus (RUT) and guinea pig ileum (GPI). As photoreactive groups p-benzoyl-phenylalanine (Bpa) and the arylazides azidobenzoic acid (ABA) and azidosalicylic acid (ASA) were attached to the N-terminus of the BK agonists. In addition, Bpa was incorporated at different positions of the BK sequence. Three different types of BK agonists were used. Firstly, the photolabels ASA and ABA were attached to BK or to Lys-BK (kallidin). Secondly, tyrosine containing BK analogues, suitable for radioiodination, were labelled. This series is derived from the naturally occurring analogue phyllokinin [BK-Ile-Tyr(SO3H)] and from BK analogues with tyrosine at position 0 and 3. The third series includes several analogues with D-N-methyl-phenylalanine (D-NMe-Phe) at position 7, which selectively discriminate between the RUT and GPI bradykinin B2 receptors. Among the photoaffinity labelled BK agonists, the iodinatable Lys(ASA)-BK (50.8% on RUT, 73.0% on GPI), ASA-BK (26.3% on RUT), Bpa-BK-Ile-Tyr (13.6% on RUT, 14.0% on GPI) and the iodinated [D-Bpa-1, 3-I-Tyr0]-BK (15.5% on RUT, 19.0% on GPI) retained a relatively high biological activity compared with BK (100%). Thus, although BK agonists are known to allow only very restricted modifications without a strong reduction in biological activity, these compounds should be useful candidates for receptor labelling.

Affinity Labels↗

PubMed1995

Potent photoaffinity labelled and iodinated antagonists of bradykinin.

Continuing the studies on photoaffinity labelled analogues of the peptide hormone bradykinin (BK), several labelled antagonists were synthesized and characterized regarding their biological activities on rat uterus (RUT) and guinea pig ileum (GPI). The photoreactive amino acid p-benzoyl-phenylalanine (Bpa) was incorporated in potent, iodinated BK analogues at positions -2, -1, 0 and 7. The newly synthesized BK antagonists were derived from HOE 140 ([DArg0, Hyp3, Thi5, D-Tic7, Oic8]-BK) or [D-Phe7]-BK. Because the application of Bpa requires an additional group for the introduction of 125I, iodinated tyrosine was inserted at different positions as a model for radioiodination. Suitable positions for incorporation of tyrosine residues are -1, 0, 3 and 7, whereas the compound with 3-I-Tyr at position 4 had only a low biological activity. The antagonists obtained by modification of HOE 140 generally retained a high antagonistic potency. In this group [D-Bpa-2, 3-I-D-Tyr-1, D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-BK (pA2 values 8.06 on RUT and 8.15 on GPI) and [Bpa-1, D-Arg0, 3-I-Tyr3, Thi5, D-Tic7, Oic8]-BK (pA2 values 7.55 on RUT and 8.07 on GPI) belong to the most active compounds. The incorporation of D-Bpa at position 7 also resulted in potent analogues. The antagonists [3-I-Tyr-1, D-Arg0, D-Bpa7]-BK (pA2 on RUT 7.69) and [3-I-Tyr-1, D-Arg0, D-Bpa7, Oic8]-BK (pA2 on GPI 7.53) are an alternative to the N-terminal modified HOE 140 analogues. Compounds with D-Bpa7 act as pure competitive antagonists, whereas the HOE 140 derivatives show a mixed antagonism. The comparison of the results between photoaffinity labelled agonists and antagonists suggests that modifications in the series of BK antagonists were better tolerated.

Affinity Labels↗

PubMed1993

Pharmacological and molecular actions of the bradykinin B2 receptor antagonist, Hoe 140, in the rat uterus.

The type of antagonism exhibited by the potent bradykinin B2 receptor antagonist, Hoe 140, on the rat uterus was investigated using various approaches. In the isolated rat uterus the concentration-response curve of bradykinin was shifted to the right and the maximum effect was reduced after pretreatment with Hoe 140 for 5 min. Shorter times of antagonist pretreatment failed to decrease the maximum effect of bradykinin. In the [3H]BK binding assay Hoe 140 bound with pM affinity to a single site and did not discriminate multiple bradykinin receptors. Studying the bradykinin-induced G protein activation we could verify that Hoe 140 at subnanomolar concentrations selectively antagonized the low-affinity BK receptor in the rat myometrium. At nM concentrations Hoe 140 itself was able to stimulate G proteins. The results suggest that in the rat uterus, differently from guinea pig ileum, Hoe 140 possibly acts as a mixed competitive as well as functional antagonist.

Animals↗