Various glucocorticoids differ in their ability to induce gene expression, apoptosis and to repress NF-kappaB-dependent transcription

1. The nonpeptide bradykinin (BK) B2 receptor antagonist, FR165649 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl ]-N-methylamino]benzyloxy]-2-methylquinoline), and agonist, FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridyl methoxy)quinoline) have been identified. These compounds have a common chemical structure, and the 2-pyridylmethoxy group is the only structural difference between them. 2. Both FR165649 and FR190997 displaced [3H]-BK binding to B2 receptors in guinea-pig ileum membranes, with an IC50 of 4.7 x 10(-10) M and 1.5 x 10(-9) M, respectively. They also displaced [3H]-BK binding to B2 receptors in human lung fibroblast IMR-90 cells, with an IC50 of 1.6 x 10(-9) M and 9.8 x 10(-10) M, respectively. 3. In guinea-pig isolated ileum-preparations, FR165649 had no agonistic effect on contraction and caused parallel rightward shifts of the concentration-response curves to BK on contraction. Analysis of the data produced a nominal pA2 value of 9.2+/-0.1 (n=5) and a slope of 1.4+/-0.1 (n=5). On the other hand, FR190997 induced concentration-dependent contraction of guinea-pig ilea with a pD2 of 7.9+/-0.2 and the contraction was inhibited by a specific peptide bradykinin B2 receptor antagonist, Hoe 140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]BK) in a non-competitive manner. 4. In IMR-90 cells, FR165649 had no agonistic effect on phosphatidyl inositol (PI) hydrolysis and caused parallel rightward shifts (approximately 200 fold shift at 10(-7) M) of the concentration-response curves to BK on PI hydrolysis. FR190997 induced concentration-dependent PI hydrolysis in IMR-90 cells with a pD2 of 8.4+/-0.1, and this effect was inhibited by Hoe 140. 5. These results indicate that FR165649 and FR190997 are, respectively, a potent bradykinin B2 receptor antagonist and agonist, and that the agonistic activity depends on the small part of the nonpeptide ligand. FR165649 and FR190997 may be useful tools for studying the relationship between ligands and receptors.     

Selective labelling of bradykinin receptor subtypes in WI38 human lung fibroblasts

1. Binding of the B1 bradykinin receptor radioligand, [3H]-des-Arg10-kallidin (-KD) and the B2 receptor radioligand [3H]-bradykinin (-BK) was investigated in membranes prepared from WI38 human foetal lung fibroblasts. 2. One-site analysis of the saturation data for [3H]-des-Arg10-KD gave an equilibrium dissociation constant (KD) value of 0.51 +/- 0.12 nM and a maximum receptor density (Bmax) of 260 +/- 49 fmol mg-1 of protein. [3H]-des-Arg10-KD binding was displaced by ligands in the order: des-Arg10-KD > KD > > des-Arg9[Leu8]-BK > des-Arg9-BK > Hoe 140 > > BK, implying that it was binding selectively to B1 receptors. 3. One-site analysis of the binding of [3H]-BK to W138 membranes indicated that it had a KD value of 0.25 +/- 0.06 nM and a Bmax of 753 +/- 98 fmol mg-1 of protein. The potencies for displacement of [3H]-BK binding were: Hoe 140 > > BK = KD > > > des-Arg10-KD = des-Arg9[Leu8]-BK = des-Arg9-BK, which was consistent with binding to B2 receptors. 4. This is the first characterization of [3H]-des-Arg10-KD binding to include both kinetic and equilibrium data, and demonstrates that [3H]-des-Arg10-KD has a high affinity for human B1 bradykinin receptors and is sufficiently selective to be used as a radioligand for B1 receptors in human cells or tissues expressing an excess of B2 BK receptors.     

Adaptive survival trials

1. An orally active, nonpeptide bradykinin (BK) B2 receptor antagonist, FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2-4-dichloro-3-[(2-methyl-8-quinolin yl) oxymethyl]phenyl]-N-methylaminocarbonyl-methyl] acrylamide) has been identified. 2. This compound displaced [3H]-BK binding to B2 receptors present in guinea-pig ileum membranes with an IC50 of 5.6 x 10(-10) M and in rat uterus with an IC50 of 1.5 x 10(-9) M. It did not inhibit different specific radio-ligand binding to other receptor sites. 3. In human lung fibroblast IMR-90 cells, FR173657 displaced [3H]-BK binding to B2 receptors with an IC50 of 2.9 x 10(-9) M and a Ki of 3.6 x 10(-10) M, but did not reduce [3H]-des]Arg10-kallidin binding to B1 receptors. 4. In guinea-pig isolated preparations, FR173657 antagonized BK-induced contractions with an IC50 of 7.9 x 10(-9) M, but did not antagonize acetylcholine or histamine-induced contractions even at a concentration of 10(-6) M. FR173657 caused parallel rightward shifts of the concentration-response curves to BK at concentrations of 10(-9) M and 3.2 x 10(-9) M, and a little depression of the maximal response in addition to the parallel rightward shift of the concentration-response curve at a concentration of 10(-8) M. Analysis of the data yield a pA2 of 9.2 +/- 0.2 (n = 5) and a slope of 1.5 +/- 0.2 (n = 5). 5. In vivo, the oral administration of FR173657 inhibited BK-induced bronchoconstriction dose-dependently in guinea-pigs with an ED50 of 0.075 mg kg-1, but did not inhibit histamine-induced bronchoconstriction even at 1 mg kg-1. FR173657 also inhibited carrageenin-induced paw oedema with an ED50 of 6.8 mg kg-1 2 h after the carrageenin injection in rats. 6. These results show that FR173657 is a potent, selective, and orally active bradykinin B2 receptor antagonist.     

Purification and characterization of a human bradykinin binding protein from inflammatory cells

Bradykinin (BK) is a potent mediator with a broad spectrum of pharmacological and inflammatory actions which are exerted through cell surface receptors. We report here the affinity chromatographic purification of a novel 14 kDa BK binding protein from human blood neutrophils and also peripheral blood mononuclear cells (PBMC), 80% of which are lymphocytes. Radioreceptor crosslinking experiments using bifunctional crosslinkers and radiolabelled BK identified a 14 kDa protein in these cell types both on the cell surface, in glycerol purified plasma membranes and in detergent solubilized cell extracts. Purification by BK affinity chromatography from a variety of BK responsive human cell types i.e. CCD-16Lu lung fibroblasts, HL60 promyelocytes, U937 myelomonocytes and Jurkat T lymphocytes also demonstrated a 14 kDa protein. Purified material obtained from three different BK affinity columns all demonstrated three major proteins at 190, 50 and 14 kDa when eluted with either excess BK or mild acid. Neutrophil fractions from detergent solubilized cell extracts contained an additional 150 kDa protein when eluted with mild acid. Neutrophil and PBMC crude plasma membrane BK affinity column purifications yielded only a single 14 kDa protein. Radioreceptor dot assays of the purified neutrophil eluates containing the 14 kDa protein revealed specific binding to [125I]-BK with a 160 fold excess signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific binding protein expressed on the surface of inflammatory cells.     

Specific RecA amino acid changes affect RecA-UmuD''C interaction

1 The characterization of the B1 kinin receptor, and some mediators involved in the inflammatory response elicited by intrathoracic (i.t.) administration of des-Arg9-bradykinin (BK) in the mouse model of pleurisy, was investigated. 2 An i.t. injection of des-Arg9-BK (10-100 nmol per site), a selective B1 agonist, caused a significant and dose-related increase in the vascular permeability observed after 5 min, which peaked at 1 h, associated with an increase in cell influx, mainly neutrophils, and, to a lesser extent, mononuclear cell influx, peaking at 4 h and lasting for up to 48 h. The increase in fluid leakage caused by des-Arg9-BK was completely resolved 4 h after peptide injection. I.t. injection of Lys-des-Arg9-BK (30 nmol per site) caused a similar inflammatory response. 3 Both the exudation and the neutrophil influx elicited by i.t. injection of des-Arg9-BK were significantly antagonized (P<0.01) by an i.t. injection of the selective B1 antagonists des-Arg9-[Leu8]-BK (60 and 100 nmol per site) or des-Arg9-NPC 17731 (5 nmol per site), administered in association with des-Arg9-BK (P<0.01), or 30 and 60 min before the cellular peak, respectively. In contrast, an i.t. injection of the B2 bradykinin selective receptor antagonist Hoe 140 (30 nmol per site), at a dose which consistently antagonized bradykinin (10 nmol per site)-induced pleurisy, had no significant effect on des-Arg9-BK-induced pleurisy. 4 An i.t. injection of the selective tachykinin receptor antagonists (NK1) FK 888 (1 nmol per site), (NK2) SR 48968 (20 nmol per site) or (NK3) SR 142801 (10 nmol per site), administered 5 min before pleurisy induction, significantly antagonized neutrophil migration caused by i.t. injection of des-Arg9-BK. In addition, FK 888 and SR 142801, but not SR 48968, also prevented the influx of mononuclear cells in response to i.t. injection of des-Arg9-BK (P<0.01). However, the NK3 receptor antagonist SR 142801 (10 nmol per site) also significantly inhibited des-Arg9-BK-induced plasma extravasation. An i.t. injection of the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP8-37 (1 nmol per site), administered 5 min before pleurisy induction, inhibited des-Arg9-BK-induced plasma extravasation (P<0.01), without significantly affecting the total and differential cell migration. 5 The nitric oxide synthase inhibitors L-NOARG and L-NAME (1 pmol per site), administered 30 min beforehand, almost completely prevented des-Arg9-BK (i.t.)-induced neutrophil cell migration (P<0.01), and, to a lesser extent, mononuclear cell migration (P<0.01). The D-enantiomer D-NAME had no effect on des-Arg9-BK-induced pleurisy. At the same dose range, L-NOARG and L-NAME inhibited the total cell migration (P<0.01). L-NAME, but not L-NOARG caused significant inhibition of des-Arg9-BK-induced fluid leakage. Indomethacin (1 mg kg(-1), i.p.), administered 1 h before des-Arg9-BK (30 nmol per site), inhibited the mononuclear cell migration (P<0.05), but, surprisingly, increased the neutrophil migration at 4 h without interfering with plasma extravasation. The administration of terfenadine (50 mg kg(-1), i.p.), 30 min before des-Arg9-BK (30 nmol per site), did not interfere significantly with the total cell migration or with the plasma extravasation in the mouse pleurisy caused by i.t. injection of des-Arg9-BK. 6 Pretreatment of animals with the lipopolysaccharide of E. coli (LPS; 10 microg per animal, i.v.) for 24 h did not result in any significant change of the inflammatory response induced by i.t. injection of des-Arg9-BK compared with the saline treated group. However, the identical treatment of mice with LPS resulted in a marked enhancement of des-Arg9-BK induced paw oedema (P<0.01). 7 In conclusion, we have demonstrated that the inflammatory response induced by i.t. injection of desArg9-BK, in a murine model of pleurisy, is mediated by stimulation of constitutive B1 receptors. (These responses are largely mediated by release of neuropeptides such as substanceP or CGRP and also by NO, but products derived from cyclo-oxygenase pathway and histamine seem not to be involved. Therefore, these results further support the notion that the B1 kinin receptor has an important role in modulating inflammatory responses, and it is suggested that selective B1 antagonists may provide therapeutic benefit in the treatment of inflammatory and allergic conditions.     

Fungal infection of human organs by resistant melanin-synthesizing species is one of the pathogenic factors and one of the real consequences of the accident at Chernobyl power plant

1. Bradykinin (BK) and Lys-BK are peptides which are released at high nanomolar concentrations into the tear-film of ocular allergic patients. We hypothesized that these peptides may activate specific receptors on the ocular surface, especially the corneal epithelium (CE) and thus the CE cells may represent a potential target tissue for these kinins. 2. The purpose of the present studies, therefore, was to determine the presence of and the pharmacological characteristics of bradykinin receptors on normal cultured primary and SV40 virus-transformed human corneal epithelial (CEPI) cells by use of the accumulation of [3H]-inositol phosphates ([3H]-IPs) as a bioassay. 3. Bradykinin (BK) induced a maximal 1.95 +/- 0.24 fold (n = 17) and 2.51 +/- 0.29 fold (n = 26) stimulation of [3H]-IPs accumulation in normal, primary (P-CEPI) and SV40-immortalized (CEPI-17-CL4) cells, respectively. This contrasted with a maximal 3.2-4.5 fold and 2.0-2.9 fold stimulation by histamine (100 microM) and platelet activating factor (100 nM) in both cell-types, respectively. 4. The molar potencies of BK and some of its analogues in the CEPI-17-CL4 cells were as follows: BK (EC50 = 3.26 +/- 0.61 nM, n = 18), Lys-BK (EC50 = 0.95 +/- 0.16 nM, n = 5), Met-Lys-BK (EC50 = 2.3 +/- 0.42 nM, n = 5), Ile-Ser-BK (EC50 = 5.19 +/- 1.23 nM, n = 6), Ala3-Lys-BK (EC50 = 12.7 +/- 2.08 nM, n = 3), Tyr8-BK (EC50 = 19.3 +/- 0.77 nM, n = 3), Tyr5-BK (EC50 = 467 +/- 53 nM, n = 4) and des-Arg9-BK (EC50 = 14.1 +/- 2.7 microM, n = 4). The potencies of BK-related peptides in normal, P-CEPI cells were similar to those found in transformed cells, thus: BK, EC50 = 2.02 +/- 0.69 nM (n = 7), Tyr8-BK, EC50 = 14.6 +/- 2.7 nM (n = 3), Tyr5 = BK, EC50 = 310 +/- 70 nM (n = 4) and des-Arg9-BK, EC50 = 12.3 +/- 3.8 microM (n = 3). 5. The bradykinin-induced responses were competitively antagonized by the B2-receptor selective BK antagonists, Hoe-140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]BK; Icatibant; molar antagonist potency = 2.9 nM; pA2 = 8.54 +/- 0.06, n = 4; and slope = 1.04 +/- 0.08) and D-Arg0[Hyp3,Thi5,8, DPhe7]-BK (KB = 371 nM; pKB = 6.43 +/- 0.08, n = 4) in CEPI-17-CL4 cells. The antagonist potency of Hoe-140 against BK in normal, P-CEPI cells was 8.4 +/- 1.8 nM (pKi = 8.11 +/- 0.12, n = 4), this being similar to the potency observed in the immortalized cells. 6. This rank order of potency of agonist BK-related peptides, coupled with the antagonism of the BK-induced [3H]-IPs by the specific B2-receptor antagonists, strongly suggests that a B2-receptor subtype is involved in mediating functional phosphoinositide (PI) responses in the CEPI-17-CL4 and P-CEPI cells. 7. In conclusion, these data indicate that the P-CEPI and CEPI-17-CL4 cells express BK receptors of the B2-subtype coupled to the PI turnover signal transduction pathway. The CEPI-17-CL4 cells represent a good in vitro model of the human corneal epithelium in which to study further the role of BK receptors in its physiology and pathology, such as in allergic/inflammatory conditions, potential wound healing and other functions of the cornea.     

Bradykinin antagonists in human systems: correlation between receptor binding, calcium signalling in isolated cells, and functional activity in isolated ileum

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.     

Mechanisms of kinin B1-receptor-induced hypotension in the anesthetized dog

Our study was performed to investigate the mechanism underlying the phypotensive effect of kinin B1-receptor activation with des-Arg9-bradykinin (des-Arg9-BK), in comparison with B2-receptor activation with bradykinin (BK), in anesthetized dogs. Bolus intravenous and intraarterial injections of both kinins were compared. BK (0.6 microgram/kg) produced a transient hypotension of the same magnitude, regardless of the route of administration (from 110 +/- 6 mm Hg to 66 +/- 6 mm Hg, or -41 +/- 5%). In contrast, intraarterial injection of des-Arg9-BK (0.6 microgram/kg) induced a weaker hypotension compared with its intravenous injection (-27 +/- 2% vs. -39 +/- 3%, p < 0.05). The hypotension induced by both kinins was accompanied by increases in heart rate, maximum left ventricular dP/dt, and aortic blood flow, suggesting a reduction in peripheral resistance. The positive inotropic and chronotropic effects of BK and des-Arg9-BK were found to be mediated by the sympathetic nervous system, because they were abolished by propranolol. The hypotension induced by intravenous and intraarterial injections of BK and intravenous injections of des-Arg9-BK was only slightly reduced after nitric oxide (NO) synthase inhibition with NG-nitro-L-arginine (L-NNA). In contrast, the hypotensive effect of intraarterial injection of des-Arg9-BK was reduced by half after treatment with L-NNA (p < 0.05). Neither bilateral vagotomy nor ganglionic blockade with pentolinium reduced the hypotension induced by both kinins. In conclusion, the hypotensive effect of des-Arg9-BK and BK results from a peripheral vasodilation. The contribution of NO in this vasodilation is substantial for des-Arg9-BK when administered intraarterial but limited for BK and intravenous des-Arg9-BK.     

Involvement of endogenous kinins in the pathogenesis of peptidoglycan-induced arthritis in the Lewis rat

OBJECTIVE: To investigate the pathophysiologic roles of endogenous bradykinin (BK) and des-Arg9-BK on local and systemic inflammatory responses in a rat model of acute arthritis induced by peptidoglycan-polysaccharide (PG-APS). METHODS: Female Lewis rats were injected intraperitoneally with PG-APS. Selective antagonists of B1 (Lys-[Leu8]-des-Arg9-BK) and B2 (Hoe 140) receptors were infused at 500 microg/kg and 5 mg/kg per day for 6 days, starting 3 days before induction of inflammation, with subcutaneous micro-osmotic pumps. The local inflammatory response was assessed by paw edema, joint swelling, and tissue content of BK and des-Arg9-BK. These peptides were measured by highly sensitive and specific chemiluminescent enzyme immunoassays. Systemic inflammatory reaction was evaluated by the hepatic concentration of the type 2 acute-phase protein T-kininogen. RESULTS: PG-APS induced significant paw edema and joint swelling 24-72 hours after intraperitoneal injection. The maximal responses to PG-APS observed at 72 hours were significantly reduced (31-38%) by the combination of both B1 and B2 receptor antagonists at 5 mg/kg per day. PG-APS induced a significant increase of BK (up to 5.3-fold) and des-Arg9-BK (up to 4.1-fold) 72 hours after challenge. Liver T-kininogen content was increased by 5.3-, 7.7-, and 5.8-fold at 24, 48, and 72 hours, respectively, after PG-APS injection. At 24 hours, Hoe 140 and Lys-[Leu8]-des-Arg9-BK increased liver T-kininogen content by 43% and 45%, respectively, but they had no effect at 72 hours. CONCLUSION: The results indicate that endogenous kinins are involved in local and systemic acute inflammatory responses, through both B1 and B2 kinin receptors, in the model of PG-APS-induced arthritis.     

B1 receptor involvement in the effect of bradykinin on venular endothelial cell proliferation and potentiation of FGF-2 effects

1. Bradykinin (BK) contributes to the inflammatory response inducing vasodilation of postcapillary venules and has been demonstrated to induce neovascular growth in subcutaneous rat sponges. 2. In this study the ability of BK to stimulate cell growth and migration in cultured endothelium from coronary postcapillary venules (CVEC) has been investigated. 3. [3H]-thymidine incorporation in subconfluent and synchronised CVEC was used to monitor DNA synthesis over 24 h. BK promoted a concentration-dependent increase of DNA synthesis with maximal activity at 100 nM. At this concentration BK also induced 18 fold accumulation of c-Fos protein immunoreactivity in the nucleus within 1 h from peptide exposure. 4. The total number of cells recovered after 48 h exposure to BK was increased in a concentration-dependent manner. Maximal effect was produced by 100 nM concentration of the peptide which produced 50% increase in cell number. The selective B1 receptor agonist Des-Arg9-BK mimicked the proliferative effect of BK, while the B2 receptor agonist kallidin was devoid of any activity. The proliferation induced by BK was abolished in a concentration-dependent manner by the addition of the B1 selective antagonist Des-Arg9-Leu8-BK, while the selective B2 receptor antagonist HOE140 did not modify BK-induced growth. 5. DNA synthesis and growth promoted by a threshold concentration of fibroblast growth factor-2 (FGF-2) (0.25 nM) were potentiated by increasing concentrations of BK and Des-Arg9-BK. 6. Endothelial cell migration assessed by the Boyden Chamber procedure was not promoted by BK or the selective B1 and B2 receptor agonists. 7. These data are the first demonstration that BK promotes growth of endothelial cells from postcapillary venules. The mitogenic activity of BK involves c-Fos expression and potentiates the growth promoting effect of FGF-2. Only the B1 receptor appears to be responsible for the proliferation induced by BK and suggests that this type of receptor might be implicated in favouring angiogenesis of coronary venules.     

A novel technique for long-term vascular access in the unrestrained rat

125I-labelled recombinant human interferon alpha 2 (rHuIFN-alpha 2) capable of high-affinity binding (Kd = 2.46 +/- 0.18 x 10(-10) M) with receptors expressed on mouse thymocytes was obtained. Prothymosin alpha (proTM-alpha) but not cholera toxin was found to compete with radiolabelled IFN-alpha 2 for binding to the same receptor (Ki = 3.68 +/- 0.21 x 10(-11) M). The synthetic peptide covering the sequence 130-137 of IFN-alpha 2 (authors' definition: alpha-peptoferon) was shown to have the capacity to displace the labelled IFN-alpha 2 from the IFN-alpha 2/receptor complex (Ki = 7.19 +/- 0.12 x 10(-11) M). It was shown that receptors of this type are localized in plasmatic membrane fraction. Using [125I]-alpha-peptoferon, specific and saturable binding was detected on human fibroblasts and the data fitted a single binding site. Scatchard analysis yielded a Kd of 9.63 +/- 0.17 x 10(-8) M. The binding was competitively inhibited by IFN-alpha 2 (the Ki value in competition assays was 1.37 +/- 0.12 x 10(-8) M), proTM-alpha(Ki = 2.2 +/- 0.2 x 10(-7) M) and cholera toxin B subunit (Ki = 5.5 +/- 0.2 x 10(-7)). The present study has demonstrated that the sequence 130-137 of HuIFN-alpha 2 is involved in the competition of HuIFN-alpha 2, proTM-alpha and cholera toxin B subunit for common receptors on human fibroblasts.     

Antagonists of bradykinin that stabilize a G-protein-uncoupled state of the B2 receptor act as inverse agonists in rat myometrial cells

Several B2 bradykinin (BK) receptor-specific antagonists including HOE140, NPC17731, and NPC567 exhibited negative intrinsic activity, which was observed as a decrease in basal phosphoinositide hydrolysis in primary cultures of rat myometrial cells, and this response was opposite to that elicited by the agonist BK. The order of potency of the antagonists in attenuating basal activity was essentially the same as that in competing both [3H]BK and [3H]NPC17731 for binding to B2 receptors on both intact rat myometrial cells and bovine myometrial membranes. We previously proposed a three-state model for the binding of agonists to G-protein-coupled B2 receptors in bovine myometrial membranes (Leeb-Lundberg, L. M. F. and Mathis, S. A. (1990) J. Biol. Chem. 265, 9621-9627). This model was based on the ability of BK to promote the sequential formation of three receptor binding states where formation of the third, equilibrium state was blocked by Gpp(NH)p (guanyl-5'-yl imidodiphosphate) identifying it as the G-protein-coupled state of the receptor. Here, we show that, in contrast to BK, these antagonists bound preferentially to a G-protein-uncoupled state of the receptor. These results indicate that B2 receptor antagonists that stabilize a G-protein-uncoupled state of the receptor act as inverse agonists. Furthermore, these results provide strong evidence that endogenous G-protein-coupled receptors exhibit spontaneous activity in their natural environment in the absence of agonist occupancy.     

Accelerated healing of gingival incisions by the helium-neon diode laser: a preliminary study

Platelet-activating factor (PAF) concordantly primes neutrophils (PMNs) for superoxide generation and elastase release. beta-Adrenergic stimulation of PMNs enhances cAMP-dependent protein kinase A (PKA) activity and has been shown to inhibit PAF-mediated NADPH-oxidase activity. PMN superoxide generation is thought to play a predominate microbicidal role, whereas elastase is known to mediate untoward PMN-endothelial interactions. We hypothesized that beta-adrenergic neutrophil stimulation has disparate effects on PAF-mediated PMN superoxide generation versus elastase release. Human PMNs were isolated using a standard Ficoll/Hypaque gradient. PMNs were then primed with PAF (200 nM) and activated with fMLP (1 microM). Subsets of PMNs were pretreated for 5 min with a beta agonist (10(-4) M isoprotereno) or an adenylate cyclase agonist (10(-5) M forskolin). Superoxide generation was determined by superoxide dismutase inhibitive cytochrome c reduction. Elastase activity was measured by the cleavage of n-methoxylsuccinyl-A-A-P-V-p-nitroanilide. Pretreatment with isoproterenol and forskolin yielded superoxide generation of 3.2 +/- 0.6 and 3.1 +/- 1.2 nmole/2.5 x 10(5) PMN/min compared to 9.0 +/- 0.6 nmole/2.5 x 10(5) PMN/min for PAF/fMLP alone, whereas isoproterenol and forskolin did not significantly affect PAF-mediated neutrophil elastase release, 22.4 +/- 5.3 and 24.0 +/- 3.6%, respectively, compared to 39.4 +/- 9.1% for PAF/fMLP alone. Disparate PMN signal transduction for superoxide generation versus elastase release may explain the SICU clinical paradox, in which patients are both susceptible to infection and vulnerable to PMN-mediated multiple organ failure.     

Studies of skin-window exudate human neutrophils: complex patterns of adherence to serum-coated surfaces in dependence on FMLP doses

Human neutrophils were isolated both from peripheral blood (PB) and from aseptic inflammatory exudates obtained by the Senn's skin-window (SW) technique. The respiratory burst (O2- release) and the adherence to serum-coated wells of culture microplates was investigated using a simultaneous assay. Unstimulated PB resting neutrophils did not produce a significant amount of O2- and were incapable of adhering to serum-coated plastic surfaces, while unstimulated SW neutrophils showed augmented adhesion to serum-coated culture wells. SW neutrophils were primed to enhanced FMLP-dependent O2- release in response to n-formyl-methionyl-leucylphenylalanine (FMLP). Adhesion of SW neutrophils was significantly decreased by addition of low doses (10(-10)-10(-8) M) of FMLP (from 17.1% to 8.4%, P < 0.01, N = 12), while fully activating doses (> 5 x 10(-8) M) of FMLP induced a marked increase of the cell adhesion, more pronounced in SW (39.2%) than in PB cells (27.2%). Low (5 x 10(-9) M) and high (5 x 10(-7) M) FMLP doses induced morphological changes (polarization) and actin polymerization in the neutrophils from both sources. Biphasic dose-response curves of SW neutrophil adherence were observed using FMLP, but not using concanavalin A or phorbol myristate acetate as stimulatory agents. Therefore, the adherence of SW cells appears to be regulated in a complex fashion, nonlinearly dependent on the chemotactic peptide doses and specifically regulated according to the receptors involved.     

Measurement of the kinetics of protein uptake by proximal tubular cells using an optical biosensor

BACKGROUND: The affinity and specificity of protein reabsorption by proximal tubular cells have been investigated using techniques for monitoring endocytosis, demonstrating a high capacity but low affinity process. It is not known whether uptake is through binding to a single binding site/receptor with differing affinities, or if there are several classes of binding sites receptors, each specific for differing proteins or groups, such as, high or low molecular weight proteins. METHODS: We have developed a novel technique for analyzing the kinetics of protein binding to tubular cells using a optical biosensor system. We have studied the binding of cultured LLCPK cells to albumin and RBP immobilized onto the sensor. By adding increasing concentrations of competing proteins [varying in molecular weight from 66,000 to 11,800 D and pI from 4.6 to 9.2 as represented by albumin, alpha1-microglobulin (alpha1M), retinol binding protein (RBP), cystatin C and beta2-microglobulin (beta2m)], specific and inhibitable cell binding was demonstrated. RESULTS: Equilibrium constants, KA, could be calculated from the reciprocal of the protein concentration causing 50% inhibition in binding rate. These were: albumin = 8.0 x 10(4) M(-1), alpha1M = 2.0 x 10(5) M(-1), RBP = 2.7 x 10(4) M(-1), cystatin C = 2.0 x 10(4) M(-1), beta2m = 4.2 x 10(3) M(-1). There were no significant differences between the measured KA's whether RBP or albumin were immobilized on the surface. CONCLUSIONS: All the proteins gave similar shaped inhibition profiles, suggesting that there is one binding site/receptor for all proteins studied, regardless of molecular weight or charge, but there are differing affinities for each protein.     

Phase angle convergence of multiple monophasic action potential recordings precedes spontaneous termination of ventricular fibrillation

Cell-surface expression of endothelial P-selectin increases adhesion and migration of leukocytes and thus may participate in the pathogenesis of reperfusion injury and atherosclerosis. Angiotensin II (Ang II) is also thought to be involved in such disease states. Nitric oxide (NO) downregulates P-selectin expression, and bradykinin (BK) is known to stimulate NO release from endothelial cells. The objective of this study was to determine the effects of 10-min stimulation of cultured human umbilical endothelial cells (HUVECs) with Ang II, BK, or both on P-selectin expression. Ang II (10(-9)-10(-5) M) stimulated P-selectin expression in a concentration-dependent manner, exhibiting a significant effect at 10(-7) M and reaching a plateau at 5 x 10(-5) M. Pretreatment of HUVECs with the AT1 antagonist losartan and the AT1/AT2 antagonist saralasin but not the AT2 antagonist PD123319 (all at 10(-5) M) markedly attenuated the effect of 10(-7) M Ang II. The effects of Ang II on P-selectin expression were not affected by the presence of the NO synthase inhibitor nitro-L-arginine (L-NA, 5 x 10(-4) M) but were abolished by pretreatment with superoxide dismutase (SOD). BK (10(-6) M) abolished the effects of 10(-7) M Ang II on P-selectin expression but did not affect P-selectin expression induced by desmopressin (0.01-10 microM). L-NA obliterated the blunting effect of BK on the Ang II-induced P-selectin membrane expression. BK alone slightly stimulated P-selectin expression, but in the presence of L-NA, BK markedly enhanced P-selectin expression. The effects of BK in the presence of NA were not altered by SOD, indicating that at difference with Ang II, it acts by a mechanism other than superoxide generation. Thus, Ang II acting on AT1 receptors stimulates superoxide generation, which, in turn, induces expression of P-selectin on the endothelial cell surface. BK inhibits the effects of Ang II, likely acting via NO. We conclude that the balance between Ang II, BK, and NO can regulate P-selectin expression on the endothelial cell membrane, an important component of the cascade leading to leukocyte adhesion to the vascular endothelium.     

Evaluation of a latex kit for the detection of rheumatoid factor: Rheumatest

A novel binding assay to kinin B1 receptors was developed, based on the design of a high-affinity agonist ligand, [125I]Tyr-Gly-Lys-Aca-Lys-des-Arg9-BK. Binding to rabbit aortic smooth muscle cells is highly temperature-dependent (optimal at 37 degrees C); apparent binding equilibrium is reached within 30 min, and competition by kinin analogs reveals the expected correlation with the B1 receptor pharmacology. The dissociation constant (Kd) of the labeled ligand is approx. 0.2 nM and this value does not change significantly as a function of cytokine pretreatment. However, the receptor abundance (Bmax) is significantly increased (1.5-fold) by pretreating the cells with interleukin-1 (IL-1), while oncostatin M (OSM) produces a marginal increase of the Bmax. This assay may be useful in documenting the regulation of B1 receptors in pathology.     

A single position in the third transmembrane domains of the human B1 and B2 bradykinin receptors is adjacent to and discriminates between the C-terminal residues of subtype-selective ligands

In order to identify agonist- and antagonist-binding epitopes in the human B1 and B2 bradykinin (BK) receptors, we exploited the ability of these receptors to discriminate between peptide ligands that differ only by the absence (B1) and presence (B2) of a C-terminal Arg. This was done by constructing chimeric proteins in which specific domains were exchanged between these receptors as recently described by us (Leeb, T., Mathis, S. A., and Leeb-Lundberg, L. M. F. (1997) J. Biol. Chem. 272, 311-317). The constructs were then expressed in HEK293 and A10 cells and assayed by radioligand binding and by agonist-stimulated inositol phospholipid hydrolysis and intracellular Ca2+ mobilization. Substitution of the third transmembrane domain (TM-III) of the B1 receptor in the B2 receptor (B2(B1III)) dramatically reduced the affinities of B2-selective peptide ligands including both the agonist BK and the antagonist NPC17731. High affinity binding of both ligands to B2(B1III) was fully regained when one residue, Lys111, in TM-III of this chimera was replaced with the corresponding wild-type (WT) B2 receptor residue, Ser (B2(B1IIIS111)). Replacement of Ser111 with Lys in the WT B2 receptor decreased the affinities of BK and NPC17731 and increased the affinity of the B1-selective des-Arg10 analog of NPC17731, NPC18565. The results show that the C-terminal residue of peptide agonists and antagonists when bound to the B2 receptor is adjacent to Ser111 in the receptor. A Lys at this position, as is the case in the WT B1 receptor, provides a positive charge that repels the C-terminal Arg in B2-selective peptides and attracts the negative charge of the C terminus of B1-selective peptides, which lack the C-terminal Arg. Therefore, the residues at this one single position are crucial in determining the peptide selectivity of B1 and B2 BK receptors.