Identification of [hydroxyproline3]-bradykinin released from human plasma and plasma protein Cohn's fraction IV-4 by trypsin

Aside from bradykinin (BK), a novel kinin, [Hydroxyproline3]-bradykinin ( [Hyp3]-BK), was isolated from the reaction mixture of human plasma and plasma protein Cohn's fraction IV-4 with trypsin. The liberated kinins were isolated based on procedures which we previously described for the isolation of [Hyp3]-lysyl-bradykinin ( [Hyp3]-Lys-BK) formed by kallikrein. The ratio of the amounts of two kinins thus formed from human plasma protein Cohn's fraction IV-4 were [Hyp3]-BK 25 +/- 4% and BK 75 +/- 4%, similarly to that of [Hyp3]-Lys-BK and Lys-BK, formed by kallikrein, but it varied by persons. The isolation of [Hyp3]-BK and [Hyp3]-Lys-BK suggests that a novel kininogen containing hydroxyproline in the third position of the bradykinin sequence in human plasma protein, possibly undergone post-translational modifications.     

Solubilization and characterization of B2 bradykinin receptors from cultured human fibroblasts.

Active B2 bradykinin (BK) receptors were solubilized in high yields from intact monolayers or particulate fractions of cultured human foreskin fibroblasts using 4 mM of the non-denaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Other detergents showed only minor (digitonin) or no (Triton X-100, n-octyl glucopyranosid) efficacy at all. The stability of CHAPS-solubilized BK binding activity was temperature dependent being reduced to 30% of initial binding after 3 days of storage at 4 degrees C. CHAPS extracts, however, retained BK binding activity for at least several days when they were stored at -20 degrees C in the presence of 10% glycerol. The pharmacological characterization gave a rank order of potency for unlabeled BK, BK agonists, and antagonists to compete with [3H]BK for specific binding very similar to that observed in intact fibroblasts. Association and dissociation kinetics demonstrated that the binding of [3H]BK to the soluble CHAPS extracts was time dependent and reversible. Scatchard analysis of equilibrium binding data exhibited saturable binding of a single class of high affinity BK-binding sites with a Kd of 1.68 +/- 0.8 nM. Gel filtration revealed an apparent molecular weight of 250,000 for the solubilized BK receptor complex in CHAPS extracts. The ability to solubilize the B2 BK receptor in an active and stable form should allow for its future purification and for the characterization of its chemical properties.     

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.     

Characterization of bradykinin receptors in canine cultured corneal epithelial cells: Pharmacological and functional studies

The pharmacological properties of bradykinin (BK) receptors were characterized in canine cultured corneal epithelial cells (CECs) using [(3)H]-BK as a radioligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant of 0.34 +/- 0.07 nM and a maximum receptor density of 179 +/- 23 fmol/mg protein. Neither a B(1) receptor-selective agonist (des-Arg(9)-BK) nor antagonist ([Leu(8), des-Arg(9)]-BK) significantly inhibited [(3)H]-BK binding to CECs, thus excluding the presence of B(1) receptors in canine CECs. The specific binding of [(3)H]-BK to CECs was inhibited by B(2) receptor-selective agonists (BK and kallidin) and antagonists (Hoe 140 and [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK), with a best fit using a one-binding-site model. The order of potency for the inhibition of [(3)H]-BK binding was BK = Hoe 140 > kallidin > [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK. Stimulation of CECs by BK produced a concentration-dependent accumulation of inositol phosphates (IP) and an initial transient peak of intracellular Ca(2+). B(2) receptor-selective antagonist ([D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK) significantly antagonized the BK-induced responses with dissociation constants of 6.0-6.1. Pretreatment of CECs with pertussis toxin (PTX) or cholera toxin did not alter the BK-induced IP accumulation. Incubation of CECs in the absence of external Ca(2+) led to a significant attenuation of the IP accumulation induced by BK. These results demonstrate that BK directly stimulates phospholipase C-mediated signal transduction through BK B(2) receptors via a PTX-insensitive G protein in canine CECs. This effect may function as the transducing mechanism for BK-mediated cellular responses.     

Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor.

A human BK-2 bradykinin receptor was cloned from the lung fibroblast cell line CCD-16Lu. The cDNA clone encodes a 364 amino acid protein that has the characteristics of a seven transmembrane domain G-protein coupled receptor. The predicted amino acid sequence of the human BK-2 receptor is 81% identical to the smooth muscle rat BK-2 receptor (1). Transfection of the human BK-2 receptor cDNA into COS-7 cells results in the expression of high levels of specific BK binding sites. Saturation binding analysis indicates that the human BK-2 receptor expressed in COS-7 cells binds BK with a KD of 0.13 nM. Pharmacological characterization of the expressed BK receptor is consistent with the cDNA encoding a receptor of the BK-2 subtype. The BK-2 receptor antagonist Hoe 140 (2), D-Arg0[Hyp3, Thi5, D-Tic7, Oic8]BK has a high affinity (IC50 = 65 pM) for the cloned human receptor. The tissue distribution of the human BK-2 receptor was analyzed by competitive PCR with human tissue cDNA and is similar to that determined for the BK-2 receptor in the rat.     

Hyp3]-tuftsin ([Hyp3]-TU) synthesis and biological activity.

[Hyp3]-tuftsin (Thr-Lys-Hyp-Arg) has been synthesized by the liquid-phase method. In biological investigations performed on rats antinociceptive and diuretic effects have been determined. It has been suggested that the presence of hydroxyl substituent in pyrrolidine ring of proline slightly modifies antinociceptive TU effect and is responsible for the increased diuretic [Hyp3]-TU activity.     

Luminal receptors for bradykinin on the canine tracheal epithelium: functional subtyping

Luminal addition of bradykinin (BK) to the open-circuited canine tracheal epithelium produces a biphasic response in transmucosal potential difference (P.D.): a rapid, transient decrease (dip) followed by a subsequent, more sustained increase (rise), both phases being associated with an increase in conductance. We have attempted to characterise the receptor subtype mediating the bradykinin response. Lys-bradykinin (Lys-BK) elicited a similar response, and its EC50 as judged from concentration-response relations was similar to that of BK. Cross-tachyphylaxis between the two peptides confirmed a common receptor. Des-Arg9-BK (a B1-agonist) neither elicited a response nor inhibited responses to BK. The novel B2-antagonist [Thi6,9-D-Phe8]kallidin reversibly inhibited responses to both BK and Lys-BK. The rapid changes in P.D. (dips) were unaffected by Na+ removal, but were eliminated by replacing luminal Cl- with isethionate. Thus, BK, acting on B2-receptors, transiently increases anion permeability of the luminal membrane of the canine tracheal epithelium.     

Characterization of kinin binding sites: identity of B2 receptors in the epithelium and the smooth muscle of the guinea pig ileum.

Binding of [125I-Tyr8]bradykinin (BK) was measured in homogenates of epithelial and smooth muscle layers of the guinea pig ileum. Binding assays were performed at 4 degrees C for 40 min (smooth muscle) or 90 min (epithelium) in 25 mM PIPES buffer at pH 6.8 in the presence of 1 mM 1,10-phenanthroline, 140 micrograms/mL bacitracin, 1 mM captopril, 1 mM dithiothreitol, and 0.1% bovine serum albumin. Specific binding of [125I-Tyr8]BK (0.32 nM) to epithelial and smooth muscle cell membranes was linearly related to protein concentration between 0.05 and 0.5 mg/mL. Equilibrium experiments showed that specific binding of [125I-Tyr8]BK was saturable and Scatchard analysis indicated the presence of a high affinity site with a Kd value of 1.6 nM and a Bmax of 156 fmol/mg of protein in the epithelial cell membranes. In smooth muscle membranes, Kd was 1.8 nM and the maximum number of binding sites was 58 fmol/mg of protein. Unlabelled peptides, namely bradykinin, [Tyr8]BK, [Hyp3]BK, D-Arg[Hyp3]BK, [Hyp3,Tyr(Me8)]BK, and kallidin displaced [125I-Tyr8]BK binding while other peptides, angiotensin II and substance P, had no effect. A series of B2-receptor antagonists displaced [125I-Tyr8]BK from specific binding sites with IC50 values ranging from 16 to 152 nM on epithelial cell membranes; similar values were obtained from smooth muscle cell membranes. These findings suggest that the binding sites in both preparations are of the B2 type. B1-receptor agonists and antagonists were found to be inactive at concentrations up to 10(-4) M. Results obtained in the two preparations were compared and a positive highly significant correlation was demonstrated between the two sets of data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological and biochemical characterization of bradykinin B2 receptors in the mouse colon: influence of the TNBS-induced colitis

This study analyzed bradykinin (BK)-evoked contractile responses in the mouse colon under normal and inflammatory conditions. BK and the preferential B(2) receptor agonists Hyp(3)-BK, Lys-BK, Met-Lys-BK and Tyr(8)-BK produced a marked and concentration-related contraction of the normal mouse colon, whereas the selective B(1) receptor agonist des-Arg(9)-BK had no effect. BK-induced contraction was concentration-dependently antagonized (in a non-competitive manner) by both B(2) receptor antagonists Hoe 140 and FR173657, but not the B(1) receptor antagonist des-Arg(9)-[Leu(8)]-BK. Analysis of the possible mechanisms implicated in the contractile responses of BK in the mouse colon revealed the involvement of the neural release of acetylcholine, the activation of L- and N-type voltage-gated calcium channels, and the release of neuropeptides, prostanoids and leukotrienes. The contraction induced by BK was markedly increased in preparations obtained from TNBS-treated mice. The up-regulation of B(2) receptors following the induction of colitis was confirmed with binding studies using [(3)H]-BK, which revealed a marked increase in B(2) receptor densities, without alterations of affinity. We provide convincing evidence on the relevance of B(2) receptors in the mouse colon under normal conditions, as well as under an inflammatory profile of colitis. Selective B(2) receptor antagonists might well represent rational therapeutic options for treating inflammatory bowel diseases.     

Characterization of [3H]bradykinin binding sites in guinea-pig central nervous system: possible existence of B2 subtypes

Specific [3H]bradykinin(BK) binding was investigated in membranes from guinea-pig brain. In kinetic experiments, specific [3H]BK binding (100 pM) reached equilibrium within 15 min at 25 degrees C (k + 1 = 1.40 nM-1min-1) and the binding was reversed by the addition of 1 microM BK (k-1 = 0.069 min-1). The presence of a high affinity BK binding site was also revealed in the guinea-pig brain by equilibrium saturation studies with a Kd value of 75 pM and a Bmax value of 4.9 +/- 0.9 fmol/mg protein. In inhibition experiments, the B2 antagonists (D-Phe7-BK and Thi5,8,D-Phe7-BK) inhibited [3H]BK binding, but not the B1 antagonist (des-Arg9[Leu8]-BK). D-Arg[Hyp3, D-Phe7]BK (B4801) showed a pseudo Hill coefficient of less than one. The KH and KL values are 1.8 and 94 nM. The regional distribution study shows the highest density of BK binding sites in the pons + medulla oblongata and the spinal cord, a moderate density in the cerebral cortex and hippocampus, and a low density in other brain regions. These data support the presence of B2 BK receptors in the guinea-pig brain and spinal cord and suggest the existence of B2 subtypes in the brain. The presence of these receptors suggests that BK acts as a neurotransmitter or a neuromodulator in these tissues.     

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.     

B2-kinin receptor like binding in rat glomerular membranes

Incubation of a radiolabeled bradykinin analog, [125I]-Tyr8-BK with a crude membrane preparation obtained from isolated rat glomeruli revealed a time dependent binding. The binding was saturable, reversible and was a linear function of protein membrane concentration. The radiolabeled Tyr8-BK bound to a single class of binding sites with an equilibrium dissociation constant (KD) of 3.9 +/_ 0.7 nM and a density (Bmax) of 31 +/- 5 fmol/mg protein. The BK-receptor complex was not affected by angiotensin II or by arginine vasopressin and atrial natriuretic factor. BK binding was reversed by bradykinin (Ki = 0.3 10(-9) M), and by other kinin analogs in the following order of potency: Lys-BK, Met-Lys-BK, Thi5,8-D Phe7-BK. However, Des-Arg9-BK had no effect on binding of the radiolabelled BK. These results are consistent with the presence of a B2-kinin like receptor in rat glomeruli.     

Diversity of B2 bradykinin receptors with nanomolar affinity expressed in passaged IMR90 human lung fibroblasts.

IMR90 human fetal lung fibroblasts express bradykinin receptors activating the pathway for biosynthesis of PGE2. A receptor of the B2 subtype stimulates half-maximal PGE2 production at 4.8 nM bradykinin, and maximal output takes place at 25 nM bradykinin. Radioligand binding studies reveal a population of [3H]bradykinin binding sites whose affinity correlates with this B2 receptor's biologic activity, with a KD of 2.5 nM. As IMR90 cells reach 60% of their defined life span in culture, they spontaneously induce expression of a second site of lower affinity, with half-maximal binding of [3H]bradykinin at 44 nM. This second site displays a characteristic primary B2 receptor recognition profile, but differs from the 2.5 nM site on a secondary level in recognition among different B2 ligands. Bradykinin is the most potent ligand at both sites; they each preferentially recognize an N-terminal extended bradykinin peptide construct having selectivity for the rat myometrial B2 receptor, suggesting that both sites have structural features in common. However, they display diversity in their order of preference for Met-Lys-bradykinin versus Lys-Lys-bradykinin; at the 44 nM site this order is completely reversed from the order of potency exhibited at the 2.5 nM site. Expression of the second site changes the manner in which these fibroblasts control their PGE2 production; it affords a graded response of PGE2 production at bradykinin levels beyond those which would normally saturate the 2.5 nM site. The inducibility of the 44 nM site in cultured fibroblasts addresses in vivo conditions in an inflammatory environment where continuing generation of bradykinin-related peptides takes place and presents a possible mechanism for overriding constraints that would otherwise limit the progression of inflammation.     

Fluorescent and biotinylated probes for B2 bradykinin receptors: agonists and antagonists.

Peptide ligands carrying additional reporter groups are valuable research tools to facilitate biochemical and pharmacological studies of G protein-coupled receptors. B2 bradykinin receptors, widely distributed in mammalian tissues, regulate many physiological systems and are therapeutic targets. Acylation of the amino-terminus of bradykinin (BK) and a B2a-selective antagonist produced ligands derivatized with biotinamidocaproate or 7-Amino-4-methylcoumarin-3-acetate. These fluorescent and biotinylated peptides bound with high affinity to bovine and rodent B2 receptors. Analysis of second messenger production confirmed that fluorescent and biotinylated analogs of BK were B2 receptor agonists whereas derivatives of DArg0[Hyp3,DPhe7,Leu8]BK were BK receptor antagonists. The complimentary properties of these selective receptor probes will be useful in studying B2 receptor localization, expression and desensitization.     

Bradykinin binding to B2 kinin receptors and stimulation of phosphoinositide turnover and arachidonic acid release in primary cultures of cells from late pregnant rat myometrium.

Primary cultures of cells from late pregnant rat myometrium contain B2 kinin receptors through which bradykinin (BK) stimulates inositol phosphate (InsP) formation and arachidonic acid (20:4) release. Equilibrium binding at 4 degrees C revealed that [3H]BK identified a maximal number of cell surface B2 kinin receptor binding sites on rat myometrial cells of 308 +/- 78 fmol/10(6) cells with apparently a single equilibrium dissociation constant of 1.8 +/- 0.2 nM. At 37 degrees C, [3H]BK binding was associated with a time-dependent decrease in the reversibility of the binding. This decrease was due in part to formation of slowly dissociating cell surface receptor [3H]BK binding and in part to internalization of the receptor-bound [3H]BK. Exposure of labeled cells to BK resulted in dose-dependent increases in [3H]InsP3, [3H]InsP2 ([3H]Ins(1,4)P2), and [3H]InsP1 ([3H]Ins(1)P1) formation and [3H]20:4 release. Pretreatment with 100 ng/mL pertussis toxin did not perturb BK stimulation of [3H]InsP formation but partially (approximately 30%) inhibited BK stimulation of [3H]20:4 release. BK stimulation of [3H]20:4 release was directly proportional to the number of receptor sites occupied by BK. In contrast, stimulation of [3H]InsP formation required a threshold level of receptor occupancy, which decreased as a function of time of BK exposure. These results show that BK interacts with B2 kinin receptors on rat myometrial cells with apparently a single affinity through which BK stimulates [3H]InsP formation and [3H]20:4 release. BK stimulation of [3H]InsP formation requires a threshold BK concentration, which decreases with time, and we suggest that the decrease is due to a time-dependent formation of a BK receptor binding state from which BK slowly dissociates.     

B2 bradykinin receptor-like binding in rat renomedullary interstitial cells

A particulate fraction from cultured rat renomedullary interstitial cells (RRIC) was prepared for bradykinin (BK) binding studies. Incubation of three radiolabeled BK analogs, [125I-Tyr1]kallidin, [125I-Tyr5]-BK, and [125I-Tyr8]-BK, with the particulate fraction resulted in degradation of these peptides. Assay conditions which prevented hydrolysis of these radiolabeled kinins were determined. Under these conditions, direct binding studies were performed with [125I-Tyr1]kallidin (TlK) as the radioligand. BK binding affinity, apparent Kassoc. = 1.3 X 10(9) M-1, and specificity, determined with 51 BK analogs, were consistent with those expected of a B2 BK receptor.     

Studies on the angiotensin-converting enzyme and the kinin B2 receptor in the rabbit jugular vein: modulation of contractile response to bradykinin

The rabbit jugular vein (rbJV) was used as a bioassay system to validate some early and new hypothetical interactions between the angiotensin-converting enzyme (ACE) and the B2 receptor, which may be influenced by ACE inhibitors (ACE-I). These involve the potentiation of the contractile effect of bradykinin (BK) and BK analogues, which are inactivated by ACE (e.g., [Hyp3, Tyr(Me8)]-BK (R556)), the prevention of BK-induced B2 receptor desensitisation, and the restoration of receptor sensitivity in tissues desensitised with B2 receptor agonists. Enzymatic degradation studies performed in vitro and in vivo revealed that BK and R556 are readily degraded by rabbit ACE whereas [Phe8psi(CH2-NH)Arg9]-BK (R379) is totally resistant. BK, R556, and R379 contracted endothelium-denuded veins with similar potencies (pEC50 range 8.10-8.50). Tissues pretreated with ACE-I showed an increase in pEC50 values for BK and R556 but not for R379. ACE-I (captopril, enalaprilat) were unable to prevent B2 receptor desensitisation induced by BK (1 microM). ACE-I partially restored B2 receptor-mediated contraction in tissues initially exposed to BK but not to R379. These effects were antagonised by HOE 140 (0.1 microM) but were unaffected by AcLys[Dbeta-Nal7, Ile8]-desArg9BK (R715) (1 microM) or by Losartan (1 microM). In conclusion, the potentiation of BK and its analogues relates exclusively on prevention of their metabolism, B2 receptor desensitisation is not affected by ACE-I, and restoration of tissue responsiveness to BK by ACE-I may be attributed to changes in BK concentrations in the vicinity of the B2 receptor.     

Transduction of the bradykinin response in human fibroblasts: prolonged elevation of diacylglycerol level and its correlation with protein kinase C activation.

Stimulation of quiescent human fibroblasts with the peptide mitogen bradykinin (BK) led to a biphasic elevation in cellular 1,2-diacylglycerol (DAG), as estimated by either measurement of total DAG mass or [3H]arachidonate incorporation. A rapid initial transient that peaked 15 s after BK addition was followed by a decline to near basal levels then a second rise to a plateau phase during which DAG levels remained elevated for less than or equal to 45 min. The source of the initial DAG transient appeared to be primarily polyphosphoinositides as these phospholipids were rapidly hydrolyzed after BK addition. This transient correlates well temporally with previous observations of the kinetics of inositol trisphosphate accumulation and intracellular free [Ca2+] observed in the same cells. Cultures preincubated with [3H]myristic acid incorporated label predominantly into the phosphatidylcholine (PC) pool. Subsequent addition of BK under these conditions caused only a relatively slow accumulation of [3H]DAG to a plateau level, without an initial transient. Together with the observation that PC was found to decrease upon BK stimulation, these observations suggest that the late phase of DAG accumulation may involve breakdown of other phospholipids including PC. To investigate the consequences of DAG elevation we examined the phosphorylation of an acidic 80 kDa protein, whose phosphorylation is solely dependent on the activation of protein kinase C (PK-C). The 80 kDa fibroblast protein could be immunoprecipitated by an antibody to bovine brain "myristoylated and alanine-rich C-kinase substrate" (MARCKS) and phosphopeptide maps of brain and fibroblast MARCKS were similar. Stimulation of [32P]-prelabeled fibroblasts with serum, BK, vasopressin, or 12-O-tetradecanoyl phorbol acetate, but not epidermal growth factor or calcium ionophores, resulted in the rapid phosphorylation of MARCKS. With BK or serum this phosphorylation showed an initial transient peak at less than 1 min then rose again to a plateau level that was sustained for less than or equal to 45 min. Removal of BK resulted in a rapid decline in MARCKS phosphorylation. These studies show that the biphasic DAG signal in BK-stimulated human fibroblasts correlates well with the state of activation of PK-C. However, the persistent activation of PK-C does not appear to require continued high levels of Ca2+.     

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.     

Guanosine nucleotides regulate B2 kinin receptor affinity of agonists but not of antagonists: discussion of a model proposing receptor precoupling to G protein

The effect of nucleotides on binding of the B2 kinin (BK) receptor agonist [3H]BK and the antagonist [3H]NPC17731 to particulate fractions of human foreskin fibroblasts was studied. At 0 degrees C, particulate fractions exhibited a single class of binding sites with a Kd of 2.3 nM for [3H]BK and a Kd of 3.8 nM for the antagonist [3H]NPC17731. Incubation with radioligands at 37 degrees C for 5 min gave a reduction of agonist, as well as antagonist, binding that was between 0-40% depending on the preparation, even in the absence of guanosine nucleotides. As shown by Scatchard analysis, this reduction in specific binding was due to a shift in the affinity of at least a fraction of the receptors. The presence at 37 degrees C of the guanine nucleotides GTP, GDP and their poorly hydrolyzable analogs left [3H]NPC17731 binding unaffected, but reduced the receptor affinity for [3H]BK to a Kd of about 15 nM. The maximal number of receptors, however, was unchanged. This affinity change was strongly dependent on the presence of bivalent cations, in particular Mg2+. It was reversed by incubation at 0 degrees C. The rank order of the guanosine nucleotides for [3H]BK binding reduction was GTP[gammaS] = Gpp[NH]p > GTP = GDP > GDP[betaS]. GMP, ATP, ADP and AMP showed no influence on agonist binding. A model for the interaction of the B2 kinin receptor with G proteins is discussed.