Identification of B2 bradykinin binding sites in the guinea pig nasal turbinate

Specific high affinity BK binding sites in the nasal turbinate of the guinea pig have been demonstrated. Specific [3H]BK binding (10-330 pM) was saturable, and nonlinear least squares analysis indicated the presence of a high affinity binding site with a Kd value of 60 (50-78) pM and a Bmax value of 13.1 = 2.0 fmol/mg protein. In inhibition experiments, D-Phe7-BK (a B2 antagonist) inhibited [3H]BK binding with a Ki value of 23 nM, while des-Arg9[Leu8]-BK (a B1 antagonist) had no effect up to a concentration of 10 microM. These studies indicate the presence of B2 BK receptors in the guinea pig nasal turbinate.     

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.     

Metabolism of bradykinin analogs by angiotensin I converting enzyme and carboxypeptidase N.

Bradykinin (BK) analogs such as Lys-Lys-BK, des-Arg9-BK and [Leu8]des-Arg9-BK were poor substrates for angiotensin I converting enzyme (ACE), and analogs containing D-Phe7 residues, or a pseudopeptide C-terminal bond, were completely resistant. However, many of these analogs were metabolized by carboxypeptidase N (CPN) including Lys-Lys-BK, [Tyr8(OMe)]BK and D-Phe7-containing analogs, with Km and Vmax values comparable to those for BK. The only analogs completely resistant to both ACE and CPN were the B2 agonist [Phe8 psi(CH2NH)Arg9]BK, the B2 agonist D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, and the B1 agonist [D-Phe8]des-Arg9-BK. These data indicate an important role for plasma CPN and vascular CPN-like activity in the metabolism of the widely used ACE-resistant/D-Phe7-containing antagonists of B2 kinin receptors.     

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.     

Bradykinin suppresses endothelin-induced contraction of coronary artery through its B2-receptor on the endothelium.

Bradykinin (BK), a powerful vasodilating peptide, has been known to be one of the factors regulating vascular contractility mainly through its action on the endothelium. The effects of BK on vascular contraction induced by endothelin-1 (ET-1), a potent vasoconstrictor peptide produced in endothelium, were investigated in vitro using the canine coronary ringed artery. ET-1 at concentrations of 10(-10) to 10(-7) M induced strong and persistent contraction dose-dependently. The ET-1-induced contraction was inhibited by BK at concentrations of 10(-7) and 10(-6) M only in the presence of endothelium. A B1-receptor agonist (des-Arg9-BK) and a B1-receptor antagonist (des-Arg9-[Leu8]-BK) did not affect these effects of BK, whereas a B2-receptor antagonist ([D-Arg0,Hyp3,Thi5,8,D-Phe7]-BK) inhibited the effect of BK on the ET-1-induced contraction. These results indicate that BK may be a potent counter-factor for the ET-1-induced coronary vasoconstriction through its B2-receptors on the endothelium.     

Characterization of bradykinin receptors in peripheral organs.

Bradykinin (BK) and related kinins are potent stimulants of the rabbit jugular vein, the hamster urinary bladder, and the guinea pig trachea. The characterization of kinin receptors in these tissues was made with agonists and antagonists. Results obtained with agonists indicate that bradykinin and kallidin are much more active than des-Arg9-BK and suggest the presence of B2 receptors in the three organs. Some new agonists were also tested and the BK analogue, [Hyp3,Tyr(Me)8]BK, was found to be a potent and selective stimulant of the three preparations, with pD2 values of 8.56, 8.00, and 8.39, respectively, but inactive on the rabbit aorta (a B1-receptor system). Contractile effects of kinins in the rabbit jugular vein and hamster urinary bladder were reduced or eliminated by B2-receptor antagonists but at different concentration levels; e.g., acetyl-D-Arg[Hyp3,D-Phe7]BK showed pA2 values of 7.78 on the rabbit jugular vein but only 5.72 on hamster urinary bladder. This compound contracted the guinea-pig trachea and was found to be inactive as an antagonist on this preparation. Contractions of the hamster urinary bladder and the guinea-pig trachea in response to bradykinin were markedly reduced or eliminated by indomethacin and by BW 755C, while those of the rabbit jugular vein were not modified. The present findings indicate that the myotropic effect of kinins on the rabbit jugular vein depends on the activation of B2 receptors and suggest that B2 receptors are largely responsible also for the response of the hamster urinary bladder. B2 receptors and (or) a nonreceptor mechanism appear to be involved in the stimulant effects of the kinin agonists and some antagonists in the guinea-pig trachea.     

Mechanism of the pressor response to intraperitoneal injection of bradykinin in guinea pigs.

Single intraperitoneal (IP) injection of bradykinin (BK) in anesthetized guinea pigs caused concentration-related pressor effects and slight, not significant tachycardia. Intravenous injections of BK in the same animal model evoked hypotension and a marked tachycardia. IP injection of des-Arg9-BK, a selective B1 receptor agonist, caused no changes of blood pressure or heart rate. The pressor response to IP BK was reduced by concomitant IP injection of lidocaine or of D-Arg[Hyp3,D-Phe7,Leu8]BK, a B2 receptor antagonist. It was also inhibited by acute animal pretreatment with sympatholytic drugs, by chronic animal exposure to capsaicin, or acute spinalization, but it was not affected by atropine, propranolol, indomethacin, [Leu8]des-Arg9-BK, a B1 receptor antagonist, or by acute cervical vagotomy. These results suggest that pressor responses to IP BK in anesthetized guinea pigs are reflex in nature, involving abdominal, capsaicin-sensitive, nonvagal visceral afferents, efferent components of the sympathetic nervous system and possibly supraspinal centers, and likely to be mediated by B2 receptors of kinins presumably located on abdominal visceral afferents.     

Buprenorphine: High-Affinity Binding to Dorsal Spinal Cord

The binding of the mixed opiate agonist-antagonist [3H]buprenorphine was compared with [3H]naloxone and [3H]dihydromorphine binding in membranes prepared from rat whole brain and dorsal spinal cord. Scatchard analysis of binding to whole brain yielded KD values close to 1.0 nM for all three 3H-ligands studied, although [3H]buprenorphine labelled five times as many binding sites. [3H]Naloxone and [3H]dihydromorphine bound to dorsal spinal cord with approximately the same affinity as to whole brain, although both 3H-ligands labelled fewer sites in the spinal cord. In contrast, Scatchard analysis of [3H]buprenorphine binding to spinal cord yielded curvilinear Scatchard plots, suggesting the presence of a very high-affinity (KD = 0.12 nM) binding site in addition to the high-affinity site (KD = 1.0 nM) present in the brain. Studies on the displacement of [3H]buprenorphine by opiates and D-Ala2,Met5-enkephalinamide supported the presence of two binding sites for this ligand in the spinal cord.     

Differential interaction of opiates to multiple "kappa" binding sites in the guinea-pig lumbo-sacral spinal cord

Binding characteristics of [3H]-etorphine and [3H]-ethylketocyclazocine are different in the lumbo-sacral spinal cord of guinea-pig. [3H]-etorphine binds to a single class of high affinity sites whereas [3H]-ethylketocyclazocine interacts with two components, a high affinity and a low affinity components. In the presence of 5 microM (D-Ala2, D-Leu5) enkephalin (DAL), the total high affinity sites can be resolved in two classes of sites, DAL sensitive sites (DALs sites) and DAL insensitive sites (DALI sites). In these conditions, [3H]-etorphine binding is completely abolished, and the binding capacity and properties of [3H]-etorphine correspond to the DALs sites. Pharmacological investigations indicated that DALI sites represent the kappa sites, whereas DALs sites closely correspond to benzomorphan sites described in rat brain and spinal cord. From these results, a new subclassification of "kappa" sites is proposed.     

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.     

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.     

Competitive antagonists of bradykinin

The first sequence-related competitive inhibitors of the classic kinin in vitro (rat uterus guinea pig ileum) and in vivo (rat blood pressure) assays have been developed. Replacement of the proline residue at position 7 of bradykinin (BK) with a D-phenylalanine residue is the key modification which converts BK agonists into antagonists. [D-Phe7]-BK exhibits moderate (pA2 = 5.0) inhibition of BK activity on the guinea pig ileum but possesses weak BK-like myotropic activity on the isolated rat uterus and 2-4% of BK depressor potency in the rat blood pressure assay. The additional replacement of the phenylalanine residues at positions 5 and 8 of [D-Phe7]-BK with the isosteric beta-(2-thienyl)-alanine residue produces a potent antagonist of BK activity on the uterus (pA2 = 6.4), ileum (pA2 = 6.3), and in the rat blood pressure assay. The antagonism of BK action on smooth muscle is specific for kinins (BK, kallidin, Met-Lys-BK), but neither inhibitor antagonizes the smooth muscle activity of angiotensin or substance P. Inhibition is competitive and fully reversible.     

Inhibition of human and rabbit arterial smooth muscle cell migration mediated by the kinin B1 receptor: role of receptor density and released mediators

Bradykinin (BK)-related peptides are suspected to negatively influence diverse functions in vascular smooth muscle cells (SMCs), notably via stimulation of the inducible B1 receptor (B1R), and have been shown to inhibit the migration of rat SMCs. The present study had several objectives: (i) to test whether B1R mediates the inhibition of migration of arterial SMCs from additional species (the human and the rabbit); (ii) whether B1R density influences this action and whether autocrine NO or prostanoid release modulate it; and (iii) the possible signaling interaction between the B1R and phosphatidylinositol-3 kinase (PI-3K) has been addressed. The peptidase resistant B1R agonist Sar-[D-Phe8]des-Arg9-BK (10 nmol/L - 1 micromol/L) was an inhibitor of migration in human or rabbit arterial SMCs in a wound closure assay, more effectively if the medium composition allowed a high B1R expression (20% fetal bovine serum (FBS) + interleukin-1beta (IL-1beta) in human SMCs, 10% FBS in rabbit cells). The effect of the B1R agonist on motility was abrogated by a B1R antagonist, B-9858, but not by the B2R antagonist Hoe 140; a peptidase-resistant B2R agonist, [Phe8Psi(CH2-NH)-Arg9]BK, had a marginal or no effect on migration. Sar-[D-Phe8]des-Arg9-BK (1 micromol/L) did not significantly influence SMC proliferation. The B1R-mediated inhibition of SMC migration was not affected by pharmacological inhibition of the nitric oxide synthases or cyclooxygenases-1 or -2, but was correlated to an inhibition of PI-3K in both types of SMCs. The inhibition of SMC migration mediated by the kinin B1R is likely independent from NO or prostanoid release, applicable to several species, and correlated to receptor density and the inhibition of PI-3K.     

A high affinity, highly selective ligand for the delta opioid receptor: [3H]-[D-Pen2, pCl-Phe4, d-Pen5]enkephalin

Binding characteristics of a new, conformationally constrained, halogenated enkephalin analogue, [3H]-[D-penicillamine2, pCl-Phe4, D-penicillamine5]enkephalin ([3H]pCl-DPDPE), were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C determined a dissociation constant (Kd) of 328 +/- 27.pM and a receptor density (Bmax) of 87.2 +/- 4.2 fmol/mg protein. Kinetic studies demonstrated biphasic association for [3H]pCl-DPDPE, with association rate constants of 5.05 x 10(8) +/- 2.5 x 10(8) and 0.147 +/- 10(8) +/- 0.014 x 10(8) M-1 min-1. Dissociation was monophasic with a dissociation rate constant of 2.96 x 10(-3) +/- 0.25 x 10(-3) min-1. The average Kd values determined by these kinetic studies were 8.4 +/- 2.7 pM and 201 +/- 4 pM. Competitive inhibition studies demonstrated that [3H]pCl-DPDPE has excellent selectively for the delta opioid receptor. [3H]pCl-DPDPE binding was inhibited by low concentrations of ligands selective for delta opioid receptor relative to the concentrations required by ligands selective for mu and kappa sites. These data show that [3H]pCl-DPDPE is a highly selective, high affinity ligand which should be useful in characterizing the delta opioid receptor.     

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.     

beta-Adrenergic receptors of brain cells. Membrane integrity implies apparent positive cooperativity and higher affinity

Beta-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [3H]dihydro-L-alprenolol ([3H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [3H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, beta-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of Bmax = 12.01 fmol/10(6) cells (7200 sites/cell), Kd = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with L-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k+ = 8.31 X 10(7) M-1 X min-1 and k- = 0.28 min-1 from the association results, and k- = 0.24 min-1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; Kd, as (k-/k+)1/n was found to be 101 pM. Analysis of the equilibrium binding of [3H]DHA to rat and mouse living brain cells resulted in values of Bmax = 13.04 fmol/10(6) cells (7800 sites/cell), Kd = 43.85 pM and n = 2.52, and Bmax = 8.08 fmol/10(6) cells (4800 sites/cell), Kd = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the beta-receptor in mammalian objects, too. The [3H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, Kd in the range 1-2 nM, and a Bmax of 10(3) - 10(4) sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the beta-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.     

Bradykinin induced a positive chronotropic effect via stimulation of T- and L-type calcium currents in heart cells

Using Fluo-3 calcium dye confocal microscopy and spontaneously contracting embryonic chick heart cells, bradykinin (10(-10) M) was found to induce positive chronotropic effects by increasing the frequency of the transient increase of cytosolic and nuclear free Ca2+. Pretreatment of the cells with either B1 or B2 receptor antagonists (R126 and R817, respectively) completely prevented bradykinin (BK) induced positive chronotropic effects on spontaneously contracting single heart cells. Using the whole-cell voltage clamp technique and ionic substitution to separate the different ionic current species, our results showed that BK (10(-6) M) had no effect on fast Na+ inward current and delayed outward potassium current. However, both L- and T-type Ca2+ currents were found to be increased by BK in a dose-dependent manner (10(-10)-10(-7) M). The effects of BK on T- and L-type Ca2+ currents were partially blocked by the B1 receptor antagonist [Leu8]des-Arg9-BK (R592) (10(-7) M) and completely reversed by the B2 receptor antagonist D-Arg[Hyp3,D-Phe7,Leu8]BK (R-588) (10(-7) M) or pretreatment with pertussis toxin (PTX). These results demonstrate that BK induced a positive chronotropic effect via stimulation of T- and L-type Ca2+ currents in heart cells mainly via stimulation of B2 receptor coupled to PTX-sensitive G-proteins. The increase of both types of Ca2+ current by BK in heart cells may explain the positive inotropic and chronotropic effects of this hormone.     

Bradykinin sensitization of colony-stimulating factor-1-responsive murine marrow progenitors to prostaglandin E. A property of the amino-terminal tetrapeptide fragment

The active sequence in bradykinin (BK) responsible for PGE-aided inhibition of CSF-1-stimulated clonal proliferation of murine mononuclear phagocyte progenitors was determined. In total marrow cultures, BK and (D-Phe7)-BK, a specific BK antagonist, inhibited colony formation by CSF-1 responsive precursors that require two signals, CSF and LPS, for clonal proliferation. (Lys1)-BK, an inactive BK analogue with Lys substituted for the amino-terminal Arg, was inactive. Arg-Pro-Pro-Gly, the amino-terminal tetrapeptide fragment of BK, was fully capable, on a molar basis, of replacing either BK or (D-Phe7)-BK as an inhibitor. Bk, (D-Phe7)-BK, and Arg-Pro-Pro-Gly were not inhibitory for colony formation in cultures containing indomethacin or in cultures depleted of adherent marrow cells. However, in these cultures addition of 10(-9) M PGE2 fully restored inhibition of two-signal-dependent colony formation. PGE2-dependent inhibition by the three peptides was equivalent on a molar basis indicating that Arg-Pro-Pro-Gly contains the sequence responsible for this inhibitory effect of BK and is sufficient to exert PGE-dependent inhibition of two-signal-dependent colony formation. The two-signal-dependent progenitors appear to be in transition to CSF competence suggesting that BK and PGE produced in an hematopoietic environment may act together to limit the production of new macrophages by inhibiting progenitors in transition to CSF competence.     

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 Receptors for Substance P in Human Astrocytoma Cells: Radioligand Binding and Inositol Phosphate Formation

UC11 cells, derived from a human astrocytoma, have a high density of functional substance P receptors. Radioligand binding studies were conducted with the highly selective neurokinin-1 receptor ligand [3H][Sar9,Met(O2)11]-substance P. Kinetic binding experiments conducted at 4 degrees C yielded an association rate constant k1 of 1.86 x 10(7) M-1 min-1, a dissociation rate constant k-1 of 0.00478 min-1, and a calculated kinetic KD of 257 pM. Saturation binding experiments yielded average values of KD = 447 +/- 103 pM, Bmax = 862 +/- 93 fmol/mg of protein. This Bmax corresponds to more than 150,000 binding sites/cell. Competition binding experiments with unlabeled [Sar9,Met(O2)11]-substance P yielded average values of KD = 491 +/- 48 pM and Bmax = 912 +/- 67 fmol/mg of protein. In [3H]inositol-labeled cells, substance P induced a robust inositol phosphate formation. Inositol trisphosphate levels increased as much as 20-fold within approximately 15 s of addition of substance P. This inositol trisphosphate formation was transient and had returned to baseline within the first 60-120 s. Inositol monophosphate formation, however, was linear for at least 2 h. Structure activity data on binding and inositol monophosphate formation confirmed the presence of a neurokinin-1 receptor subtype in these cells. Thus, the UC11 cell should be a useful model cell for delineating the physiological role of substance P receptors in astrocytes.