Activation of bradykinin B2 receptors increases calcium entry and intracellular mobilization in C9 liver cells.

In C9 rat liver cells bradykinin and kallidin increased (approximately 2-fold) the intracellular concentration of calcium, but the B1 agonist, des-Arg9-bradykinin did not. The effect of bradykinin was inhibited by the B2 antagonists, Hoe 140 and N-alpha-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin, but not by the B1 antagonist, des-Arg9-[Leu8]-bradykinin. The action of bradykinin was diminished, but not abolished, in medium without calcium. The peptide was able to increase intracellular calcium concentration in cells treated with thapsigargin. Bradykinin action was not observed in cells previously stimulated with this local mediator: however, under the same conditions, angiotensin II induced a clear increase in intracellular calcium concentration. Our data indicate that activation of bradykinin B2 receptors increase intracellular calcium concentrations by inducing both gating of the cation and intracellular mobilization in C9 liver cells. In addition, homologous desensitization was observed.     

Pharmacological and functional characterization of bradykinin receptors in rat cultured vascular smooth muscle cells

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant (K(D)) of 1.2 +/- 0.2 nM and a maximum receptor density (Bmax) of 47.3 +/- 4.4 fmol/mg protein. The specific binding of [3H]-bradykinin to VSMCs was inhibited by the B2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140) and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin, but not by a B1 receptor-selective agonist (des-Arg9-bradykinin) and antagonist ([Leu8, des-Arg9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca2+]i with half-maximal responses (pEC50) were 7.53 and 7.69, respectively. B2 receptor-selective antagonists (Hoe 140 and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca2+]i changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, verapamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.     

Polyphosphoinositide hydrolysis in endothelial cells and carotid artery segments. Bradykinin-2 receptor stimulation is calcium-independent

Bovine aortic and cerebral microvascular endothelial cells and cultured segments of canine common carotid artery possess functional receptors for the nonapeptide bradykinin which mediate a rapid increase in the formation of [3H]inositol 1-phosphate, [3H]inositol 1,4-bisphosphate, and [3H]inositol 1,4,5-trisphosphate from cell membranes containing isotopically labeled myo-inositol. Bradykinin stimulated the formation of [3H]inositol phosphates from cells in culture or tissues at threshold concentrations of 0.1 nM and 1 nM, and with a half-maximal effective concentration of 0.6-1.0 nM and 30 nM, respectively. In cultured cells, the formation of [3H]inositol trisphosphate and [3H]inositol bisphosphate preceded the formation of [3H]inositol monophosphate. Similarly, [3H]inositol phosphate formation was not inhibited by addition of calcium channel blockers, a calcium chelator, or an intracellular calcium antagonist. Calcium ionophore A23187 did not promote [3H]inositol phosphate accumulation. The receptor selectivity of the bradykinin response in cultured cells was most compatible with a type-2 mediated response. Kallidin stimulated with the same potency as bradykinin but was more potent than methionyl-lysyl-bradykinin or des-Arg9-bradykinin. The B1 receptor antagonists des-Arg9-[Leu8]-bradykinin and des-Arg10-[Leu9]-kallidin were without effect. The rapidity of the inositol phosphate response as well as the close correspondence between the bradykinin type-2 receptor mediated hydrolysis of polyphosphoinositides and changes in prostacyclin synthesis, vessel dilation, and permeability suggests that breakdown products of inositol lipids serve as second messengers mediating the effects of bradykinin on the vascular endothelium.     

Effects of bradykinin on DNA synthesis in resting NIL8 hamster cells and human fibroblasts

Bradykinin stimulates [3H]thymidine incorporation and DNA synthesis in resting, serum-deprived NIL8 hamster cells. The ED50 for this stimulation is 4.52 +/- 2.91 nM. Other kinin peptides including lys-bradykinin (kallidin) and met-lys-bradykinin also stimulate [3H]thymidine incorporation in the NIL8 cells, whereas desarg9-bradykinin is without effect, suggesting action of the kinin peptides through type B2 receptors. Bradykinin also stimulates DNA synthesis in IMR-90 human fibroblasts; however, this effect is observed only in the presence of indomethacin, which blocks prostaglandin synthesis. These results suggest that prostaglandins act as negative modulators of the growth-stimulatory effects of bradykinin in the fibroblasts. This conclusion is supported by the observation that exogenously added PGE1, PGE2, PGA1, PGA2, PGB1, and PGB2 strongly inhibit [3H]thymidine incorporation in the human fibroblasts. The direct effect of bradykinin observed in the NIL8 cells may be attributable to the relative resistance of these cells to growth inhibition by prostaglandins.     

The renin-angiotensin and the kallikrein-kinin systems

The renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) each encompasses a large number of molecules, with several participating in both systems. The RAS generates a family of bioactive angiotensin peptides with varying biological activities. These include angiotensin-(1-8) (Ang II), angiotensin-(2-8) (Ang III), angiotensin-(3-8) (Ang IV), and angiotensin-(1-7) [Ang-(1-7)]. Ang II and Ang III act on type 1 (AT(1)) and type 2 (AT(2)) angiotensin receptors, whereas, Ang IV and Ang-(1-7) act on their own receptors. The KKS also generates a family of bioactive peptides with varying biological activities. These include hydroxylated and non-hydroxylated bradykinin and kallidin peptides and their carboxypeptidase metabolites des-Arg(9)-bradykinin and des-Arg(10)-kallidin. Whereas bradykinin and kallidin act mainly via the type 2 bradykinin (B(2)) receptor, des-Arg(9)-bradykinin and des-Arg(10)-kallidin act mainly via the type 1 bradykinin (B(1)) receptor. The AT(1) receptor forms heterodimers with the AT(2) and B(2) receptors and there is cross talk between the AT(1) and epidermal growth factor receptors. The B(2) receptor also interacts with angiotensin converting enzyme and nitric oxide synthase. Both angiotensin and kinin peptides are metabolised by many different peptidases that are important determinants of the activities of the RAS and KKS, and several of which participate in both systems.     

Synthesis and molecular characterization of a biotinylated analog of [Lys]bradykinin.

We report the synthesis and molecular characterization of a biotinylated analog of kallidin, [Lys]bradykinin. Bradykinin was prepared by solid phase peptide synthesis. Before cleavage from the resin, a biotin moiety was coupled to the epsilon amino group of a lysine in the zeroth position of the bradykinin peptide. An omega-amino caproic acid spacer was incorporated between the biotin group and the N-terminal lysine. The biotinylated peptide was deprotected, cleaved from the resin and purified by RP-HPLC. The identity of this analog was confirmed by amino acid analysis and FAB-mass spectrometry. Biotinyl [Lys]bradykinin (BLBK, mol, wt. = 1528) inhibited [3H]-bradykinin binding to guinea pig ileum homogenates dose dependently, with an IC50 of 28.9 +/- 6 nM. The IC50 for [Lys]bradykinin was approximately 10-fold lower, 3.2 +/- 0.6 nM. BLBK induced contractility in an isolated guinea pig smooth muscle preparation with an EC50 of 129 +/- 14 nM; the corresponding value for [Lys]bradykinin was 29 +/- 8 nM. These data are consistent with the difference in binding potency observed for BLBK compared to [Lys]bradykinin. In an ELISA assay using BLBK and affinity-purified rabbit anti-bradykinin antibody, BLBK bound to anti-bradykinin antibody with an EC50 = 1.21 +/- 0.54 nM. Rank order potencies for several bradykinin peptide analogs suggest that the epitope on bradykinin recognized by the antibody is likely to be at the carboxy terminus of the peptide.     

Bradykinin receptors in intestinal smooth muscles and their post-receptor events related to calcium

The effects of trifluoperazine and verapamil on bradykinin- and des-Arg(9)-bradykinin induced responses of isolated rat duodenum and guinea-pig ileum were investigated to elucidate post-bradykinin receptor events. Verapamil and trifluoperazine inhibited bradykinin induced relaxations and contractions and des-Arg(9)- bradykinin induced contractions in rat duodenum. Bradykinin induced contractions of ileum were also inhibited by trifluoperazine and. verapamil. Since non-competitive affinity constants of trifluoperazine and verapamil for the relaxant responses to bradykinin in duodenum and for the contractile responses to bradykinin in ileum are different, post-bradykinin receptor events related to calcium may be different in ileum and duodenum. In addition, affinity constants of bradykinin in guinea-pig ileum and rat duodenum are also disparate suggesting the presence of different types of bradykinin B(2) receptors in these two organs.     

Bradykinins and their cDNA from piebald odorous frog,Odorrana schmackeri,skin

Bradykinin, (des-Arg(9))-bradykinin and bradykinyl-VAPAS, were identified in the skin secretion of the piebald odorous frog, Odorrana schmackeri. Using 3'- and 5'-RACE reactions, bradykinin precursor cDNA was cloned and found to contain an open-reading frame of 311 amino acid residues. The preprobradykinin was found to consist of a putative signal peptide of approximately 20 amino acid residues, followed by seven tandem repeat coding domains of 43-44 amino acids. Bradykinin and its C-terminal extended molecular form were encoded on this single precursor and could be generated by differential post-translational processing.     

Kallidin applied to the human nasal mucosa produces algesic response not blocked by capsaicin desensitization.

Various kinins (dissolved in 50 microliters) were applied to the nasal mucosa of healthy human volunteers to test the algesic and proinflammatory effects of these peptides in an intact human tissue. [des-Arg9]-bradykinin (0.5 mumol) was found to be inactive, while bradykinin (0.05-0.5 mumol) and especially kallidin (0.005-0.5 mumol) induced: (a) a mild painful sensation described as burning and pricking (latency 30 s, duration 3-5 min), (b) perception of pulsatility and obstruction in the nasal cavity (onset 1 min, duration 6-8 min). Substance P (0.5 mumol) and neurokinin A (0.5 mumol) produced slight obstruction and weak pulsatile sensation but not pain. Capsaicin (0.05 nmol) produced pain and secretion of fluid, but not pulsatile sensation. The effects of kallidin were not affected by repeated (to induce desensitization) applications of capsaicin (0.5 mumol). Likewise, ipratropium bromide (80 mg in 100 microliters) did not affect responses to kallidin. In an intact human tissue, kallidin produces various effects, including an algesic response, that are apparently independent from activation of B1 receptors and from desensitization of capsaicin-sensitive primary afferents.     

Identification of the critical residues of bradykinin receptor B1 for interaction with the kinins guided by site-directed mutagenesis and molecular modeling

We report the critical residues for the interaction of the kinins with human bradykinin receptor 1 (B1) using site-directed mutagenesis in conjunction with molecular modeling of the binding modes of the kinins in the homology model of the B1 receptor. Mutation of Lys118 in transmembrane (TM) helix 3, Ala270 in TM6, and Leu294 in TM7 causes a significant decrease in the affinity for the peptide agonists des-Arg10kallidin (KD) and des-Arg9BK but not the peptide antagonist des-Arg10Leu9KD. In contrast, mutations in TM2, TM3, TM6, and TM7 cause a significant decrease in the affinity for both the peptide agonists and the antagonist. These data indicate that the B1 bradykinin binding pocket for agonists and antagonists is similar, but the manners in which they interact with the receptor do not completely overlap. Therefore, there is a potential to influence the receptor's ligand selectivity.     

Expression of bradykinin B2 receptor in the mouse ovary

The amounts of [1-5]-bradykinin in ovary extracts were determined using gonadotropin-treated immature female mice. The bradykinin levels in the ovary were high at 2, 6, and 48 hr after injection of human chorionic gonadotropin (hCG) into pregnant mare's serum gonadotropin (PMSG)-treated mice. Northern blot analysis of total RNAs isolated from the PMSG/hCG-treated mouse ovaries indicated that the B(2) receptor mRNA was constitutively expressed. Bradykinin B(2) receptor protein was detected by Western blot analysis of the ovary extracts. In situ hybridization analysis revealed that the B(2) receptor mRNA is expressed in the granulosa cells of all growing follicles of ovaries from both gonadotropin-treated immature and mature female mice. The effect of bradykinin on the expression of the B(2) receptor gene was examined by RT-PCR analysis with the ovary previously cultured in the presence of bradykinin. Bradykinin treatment of immature female, gonadotropin-treated immature female, and mature female mouse ovaries brought about no apparent changes in the B(2) receptor mRNA level. The present data indicate that the level of B(2) receptor expression in the ovary is fairly constant, and that the biological effect elicited by bradykinin in this organ may be dependent upon concentrations of the ligand produced by operation of the kinin-kallikrein system.     

B(1) and B(2) bradykinin receptors on adventitial fibroblasts of cerebral arteries are coupled to recombinant eNOS

Our previous ex vivo and in vivo studies reported that expression of the recombinant endothelial nitric oxide (NO) synthase (eNOS) gene in adventitial fibroblasts recovers NO production in arteries without endothelium in response to bradykinin. The present study was designed to characterize subtypes of bradykinin receptors on adventitial fibroblasts coupled to the activation of recombinant eNOS. Endothelium-denuded segments of canine basilar arteries were transduced with beta-galactosidase (beta-Gal) gene or eNOS gene ex vivo, using a replication-defective adenoviral vector (10(10) plaque-forming units/ml) for 30 min at 37 degrees C. Twenty-four hours later, isometric force recording or cGMP measurement was carried out. B(1) bradykinin receptor agonist (des-Arg(9)-bradykinin, 10(-10)-10(-8) mol/l) did not significantly affect vascular tone in control or beta-Gal gene-transduced canine basilar arteries without endothelium. In contrast, this agonist caused concentration-dependent relaxations in recombinant eNOS gene-transduced arteries without endothelium. Relaxations to B(1) receptor agonist in the eNOS arteries were abolished by B(1) receptor antagonist (des-Arg(9)-[Leu(8)]bradykinin, 6 x 10(-9) mol/l) but not by B(2) receptor antagonist (Hoe-140, 5 x 10(-8) mol/l). Bradykinin did not significantly alter vascular tone in control or beta-gal arteries without endothelium, whereas this peptide (10(-11)-10(-8) mol/l) induced concentration-dependent relaxations, as well as an increase in cGMP formation in endothelium-denuded eNOS-transduced arteries. Stimulatory effects of bradykinin were prevented in the presence of a B(2) receptor antagonist but not in the presence of a B(1) receptor antagonist. B(1) and B(2) receptor antagonists had no effect on relaxations to substance P, confirming the selectivity of the compounds. Our results suggest that B(1) and B(2) bradykinin receptors are coupled to activation of recombinant eNOS expressed in adventitial fibroblasts.     

Bradykinin receptors in isolated rat duodenum

Pharmacological properties of the bradykinin receptors in the isolated rat duodenum were investigated by examining the relaxant and contractile responses to bradykinin and [des-Arg9]-bradykinin, an agonist of B1 receptors. A specific desensitization and de novo formation for B1 receptors were observed. Changes in medium pH caused a decrease in the responses to bradykinin and [des-Arg9]-bradykinin of rat duodenum. Urea incubation in test tube inhibited the responses to bradykinin and [des-Arg9]-bradykinin of rat duodenum, while urea in bathing medium was ineffective. These findings strongly suggested that (a) ionic bonds are important in the interaction between bradykinin and its receptors, and (b) B2 receptors in rat duodenum are different from those in guinea pig ileum.     

Kininase I-type carboxypeptidases enhance nitric oxide production in endothelial cells by generating bradykinin B1 receptor agonists

Kininase I-type carboxypeptidases convert native kinin agonists for B(2) receptors into B(1) receptor agonists by specifically removing the COOH-terminal Arg residue. The membrane localization of carboxypeptidase M (CPM) and carboxypeptidase D (CPD) make them ideally situated to regulate kinin activity. Nitric oxide (NO) release from human lung microvascular endothelial cells (HLMVEC) was measured directly in real time with a porphyrinic microsensor. Bradykinin (1-100 nM) elicited a transient (5 min) peak of generation of NO that was blocked by the B(2) antagonist HOE 140, whereas B(1) agonist des-Arg(10)-kallidin caused a small linear increase in NO over 20 min. Treatment of HLMVEC with 5 ng/ml interleukin-1beta and 200 U/ml interferon-gamma for 16 h upregulated B(1) receptors as shown by an approximately fourfold increase in prolonged (>20 min) output of NO in response to des-Arg(10)-kallidin, which was blocked by the B(1) antagonist des-Arg(10)-Leu(9)-kallidin. B(2) receptor agonists bradykinin or kallidin also generated prolonged NO production in treated HLMVEC, which was significantly reduced by either a B(1) antagonist or carboxypeptidase inhibitor, and completely abolished with a combination of B(1) and B(2) receptor antagonists. Furthermore, CPM and CPD activities were increased about twofold in membrane fractions of HLMVEC treated with interleukin-1beta and interferon-gamma compared with control cells. Immunostaining localized CPD primarily in a perinuclear/Golgi region, whereas CPM was on the cell membrane. These data show that cellular kininase I-type carboxypeptidases can enhance kinin signaling and NO production by converting B(2) agonists to B(1) agonists, especially in inflammatory conditions.     

Identification of a new kinin in human urine

The types of kinins excreted in fresh urine of dogs, rats, and humans were compared. Urinary kinins were separated by reverse-phase (C18) high performance liquid chromatography and quantitated by radioimmunoassay using an antibody directed against the COOH-terminal region of the peptide. Kinins were found in the following proportions: 53 +/- 3% bradykinin, 23 +/- 4% Lys-bradykinin, and 13 +/- 7% des-Arg1-bradykinin in dog urine; 67 +/- 6% bradykinin, 6 +/- 3% Lys-bradykinin, and 10 +/- 3% des-Arg1-bradykinin in rat urine; and 12 +/- 4% bradykinin, 30 +/- 3% Lys-bradykinin, 2 +/- 1% des-Arg1-bradykinin, and 41 +/- 3% unknown kinin in human urine. The unknown kinin was purified from a pool of human urine. Amino acid sequencing revealed a structure similar to Lys-bradykinin except that proline in position 4 was replaced by alanine ([Ala3]Lys-bradykinin). Synthetic and endogenous [Ala3]Lys-bradykinins had similar high performance liquid chromotography elution volumes and both had vasodilator activity and contracted the rat uterus. Human urinary kallikrein incubated with semipurified human low molecular weight kininogen released 76% of the total kinins as Lys-bradykinin, 7% as bradykinin, and 17% as [Ala3]Lys-bradykinin. In contrast, rat urinary kallikrein released 86% bradykinin, 18% Lys-bradykinin, and negligible amounts of [Ala3]Lys-bradykinin. The study revealed the presence of a new kinin, [Ala3]Lys-bradykinin, in human urine and it also proves that the types of kinins generated intrarenally are species-dependent.     

Bradykinin receptors: functional similarities in guinea pig gut muscle and mucosa

It is well established that bradykinin can stimulate mucosal electrolyte transport. However, the receptor type which mediates this effect has not been fully characterized. Recent studies have suggested that bradykinin and related kinins may act at two types of receptors designated as B1 and B2. We have determined the effect of bradykinin on electrolyte secretion across guinea pig ileal mucosa and longitudinal muscle in vitro in the presence and absence of D-Phe7-bradykinin (B2 antagonist) and des-Arg9-(Leu8)-bradykinin (B1 antagonist). The B2 antagonist (less than 100 microM) did not affect resting muscle tension or basal electrolyte transport but at 6-30 microM it caused a parallel rightward shift in the concentration-response curves to bradykinin in the mucosa (Ki = 4 microM) and muscle (Ki = 6 microM). Changes in electrolyte transport and muscle contractility evoked by bethanechol and substance P were not affected by the B2 antagonist (30 microM) in either the muscle or the mucosa. Moreover, changes in electrolyte transport and muscle contractility produced by bradykinin were not altered by the B1 antagonist (30 microM). Finally, the B1 agonist des-Arg9-bradykinin (10 nM-1 microM) was not active in either preparation. These data suggest that under normal conditions, ileal secretion and smooth muscle contractility in the guinea pig are regulated by B2-type bradykinin receptors.     

ADP-ribosylation of bradykinin and effects on its biological activities

We investigated the ADP-ribosylation of bradykinin by hen liver nuclear ADP-ribosyltransferase. Two Arg residues of the peptide were modified by this enzyme. Arg1 was preferentially modified as compared to Arg9; the Vmax/Km for Arg1 was 3 times higher than that for Arg9. These results were given support by data observed in experiments with des-Arg1 and des-Arg9 bradykinin; the Vmax/Km for des-Arg9 bradykinin was 3 times that for des-Arg1 bradykinin. ADP-ribosylation suppressed the biological activity of bradykinin, as related to both binding and contractile activities. The extent of ADP-ribosylation-induced suppression of both activities was higher in the case of the modification of Arg1 than that of Arg9. In view of the observation of ADP-ribosyltransferase activity in skeletal, cardiac, and smooth muscles (Soman, G. et al. (1984) Biochem. Biophys. Res. Commun. 120, 973-980; Shimoyama, M. et al. (1987) in The 8th International Symposium on ADP-Ribosylation, Texas, abstract p. 13), bradykinin functioning in the contraction of smooth muscle may be modified in this way in vivo.     

Receptors for bradykinin and kallidin.

In order to establish if bradykinin (BK) and Lys-bradykinin (Lys-BK), alias kallidin, act on the same or on different receptors, experiments were performed on strips of cat terminal ileum and of rabbit aorta. The first preparation contains receptor B2 and the second has the newly identified receptor B1. The criterion used for establishing the identity of receptors for BK and Lys-BK in the cat ileum (receptor B2) was desensitization, while for the rabbit aorta (receptor B1) we measured the apparent affinity (pA2 value) of a competitive and specific inhibitor of BK, [Leu-OMe3,des-Arg9]-BK. Since cat ileum desensitized with BK or Lys-BK shows a significant decrease or a complete disappearance of the response to the other agent, while maintaining full sensitivity for histamine, and since the pA2 values of [Leu-OMe8,des-Arg9]-BK against BK and Lys-BK are identical in the rabbit aorta, we conclude that the two kinins act on the same types of receptors.     

Increase in blood bradykinin concentration after eccentric weight-training exercise in men.

The purpose of this study was to verify the possible appearance in the blood of bradykinin (BK) and des-Arg(9)-bradykinin (des-Arg(9)-BK) after eccentric exercise in 13 male subjects. Eccentric exercise (5 x 10 leg presses at 120% maximal voluntary concentric contraction) resulted in muscle damage and inflammation, as suggested by the significant increase in serum creatine kinase activity (from 204 +/- 41 to 322 +/- 63 U/l 12 h postexercise) and by severe lasting pain, which also peaked at 12 h postexercise. Blood BK and des-Arg(9)-BK concentrations were measured by competitive enzyme immunoassays using highly specific polyclonal rabbit IgG. Des-Arg(9)-BK concentration was not modified (preexercise: 44 +/- 14 pmol/l; pooled postexercise: 47 +/- 4 pmol/l). In contrast, BK concentration significantly increased immediately after the exercise session (68 +/- 9 vs. 42 +/- 3 pmol/l preexercise) and returned to basal values at 12, 24, and 48 h (pooled value: 40 +/- 4 pmol/l). This observation suggests that the inflammatory process due to eccentric exercise-induced muscle damage could be mediated in part by BK.     

Characterization of bradykinin receptors in a human osteoblastic cell line.

Bradykinin receptor subtypes linked to prostaglandin release have been assessed in a human osteosarcoma cell line with osteoblastic phenotype (MG-63). Bradykinin (BK; 1 micromol/l) caused a burst of prostaglandin E(2) release that was maximal at 10 min. When the effect on the burst of PGE(2) and PGI(2) release by a variety of kinins and kinin analogues was assessed, the following rank order of response was found: Lys-BK>BK> or =Met-Lys-BK>Ile-Ser-BK>[Tyr(8)]-BK> or =[Hyp(3)]-BK>des-Arg(9)-BK=des-Arg(10)-Lys-BK=des-Arg(1)-BK, [Thi(5,8),D-Phe(7)]-BK=Sar-[D-Phe(8)]-des-Arg(9)-BK=Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK. The rapid effect of BK on PGE(2) and PGI(2) release was unaffected by des-Arg(9)-[Leu(8)]-BK, des-Arg(10)-[Leu(9)]-Lys-BK and des-Arg(10)-[Hoe 140], but strongly inhibited by Hoe 140 in a concentration-dependent manner. When the incubation time was extended to 48 h, it was found that des-Arg(9)-BK and des-Arg(10)-Lys-BK caused a delayed enhancement of the formation of PGE(2). When PGE(2) formation was assessed in 24-h experiments, the following rank order of response was obtained: Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK>BK=Lys-BK>des-Arg(10)-Lys-BK>Sar[D-Phe(8)]-des-Arg(9)-BK>des-Arg(9)-BK. The stimulatory effect of BK at 24 h was unaffected by des-Arg(9)-[Leu(8)]-BK, des-Arg(10)-[Leu(9)]-Lys-BK and des-Arg(10)-[Hoe 140] but inhibited by Hoe 140. The stimulatory effect of des-Arg(10)-Lys-BK in 24-h experiments was inhibited by des-Arg(9)-[Leu(8)]-BK, des-Arg(10)-[Leu(9)]-Lys-BK and des-Arg(10)-[Hoe 140]. Similarly, the stimulatory effects of Sar[D-Phe(8)]-des-Arg(9)-BK and Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK was inhibited by des-Arg(10)-[Hoe 140].The following rank order of response was seen for inhibition of [3H]-BK binding to MG-63 cells: Lys-BK=BK=Hoe 140>des-Arg(10)-Hoe 140=des-Arg(10)-Lys-BK=des-Arg(9)-BK=Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK. Using [3H]-des-Arg(10)-Lys-BK, the following rank order of response for inhibition of binding was seen: des-Arg(10)-Lys-BK=Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK>des-Arg(10)-Hoe 140>des-Arg(9)-BK=Lys-BK=BK=Hoe 140. MG-63 cells expressed mRNAs for BK B1 and B2 receptors, as assessed by RT-PCR.These data indicate that the human osteoblastic osteosarcoma cell line MG-63 is equipped with functional BK receptors of both B1 and B2 receptor subtypes. The B2 receptors are linked to a burst of prostanoid release, whereas the B1 receptors mediate a delayed prostaglandin response, indicating that the two receptor subtypes are linked to different signal transducing mechanisms or that the molecular mechanisms involved in prostaglandin release are different.