Preferential suppression of delayed-type hypersensitivity by L-156,602, a C5a receptor antagonist.

In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hind-footpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.     

Preferential Suppression of Delayed-type Hypersensitivity by L-156,602, a C5a Receptor Antagonist

In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hind-footpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.     

L-364,718, a cholecystokinin-A receptor antagonist, suppresses feeding-induced sleep in rats

Feeding induces increased sleep in several species, including rats. The aim of the study was to determine if CCK plays a role in sleep responses to feeding. We induced excess eating in rats by 4 days of starvation and studied the sleep responses to refeeding in control and CCK-A receptor antagonist-treated animals. Sleep was recorded on 2 baseline days when food was provided ad libitum. After the starvation period, sleep was recorded on 2 refeeding days when the control rats (n = 8) were injected with vehicle and the experimental animals (n = 8) received intraperitoneal injections of L-364,718 (500 microg/kg, on both refeeding days). In the control group, refeeding caused increases in rapid eye movement sleep (REMS) and non-REMS (NREMS) and decreases in NREMS intensity as indicated by the slow-wave activity (SWA) of the electroencephalogram. CCK-A receptor antagonist treatment completely prevented the SWA responses and delayed the NREMS responses to refeeding; REMS responses were not simply abolished, but the amount of REMS was below baseline after the antagonist treatment. These results suggest that endogenous CCK, acting on CCK-A receptors, may play a key role in eliciting postprandial sleep.     

Effects of intracerebroventricular administration of D,L-2-amino-5-phosphonovaleric acid, an N-methyl-D-aspartate receptor antagonist, on luteininizing hormone release in ovariectomized lambs.

To determine whether endogenous glutamate and aspartate control LH secretion via N-methyl-D-aspartate (NMDA) receptors in the sheep, we evaluated the effects of the NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (AP5) on secretion of LH in ovariectomized lambs. Twelve lambs were ovariectomized and surgically implanted with lateral cerebroventricular cannulae. At the time of the experiment, (38 wk of age) they received intracerebrally 4 injections of either 50 (n = 4), 100 (n = 4), or 200 micrograms (n = 4) of AP5. Blood samples were collected every 10 min for 8 h with animals receiving AP5 at hours 4, 5, 6, and 7. Patterns of LH during the preinjection period were compared to those during the period encompassing AP5 injections. Mean concentrations of LH were lower during AP5 injections than during the preinjection periods, a response that was not influenced by dose (0.87 +/- 0.08 vs. 0.69 +/- .07 ng/ml; p < 0.01). LH pulse amplitude decreased during AP5 treatment relative to the preinjection periods, but this difference was not statistically significant (0.79 +/- 0.11 vs. 0.68 +/- 0.10 ng/ml; p = 0.09). There were no effects of AP5 on LH pulse frequency (1.00 +/- 0.10 vs. 0.83 +/- 0.15 pulses/h for injection and preinjection periods; p > 0.10). A second experiment was done to evaluate a higher dose of AP5. Four animals were chosen to receive 4 injections of 2 mg of AP5 in a design identical to that used in the first experiment.     

Solution structure of a unique C5a semi-synthetic antagonist: implications in receptor binding.

The tertiary structure of a unique C5a receptor antagonist was determined by two-dimensional NMR spectroscopy. The core domain of this 8-kDa antagonist exists as an antiparallel helical bundle, similar to recombinant human (rh)-C5a. However, unlike C5a, the antagonist's C terminus was found to be conformationally restricted along a groove between helices one and four in the core domain. This conformational restriction situates C-terminal D-Arg 75 in a wedge between core residues Arg 46 and His 15. Correlation of the antagonist's tertiary structure with point mutation analysis revealed the formation of a positively charged contiguous contact surface comprised of D-Arg 75, Arg 46, Lys 49, and His 15. The significance of this surface in generating antagonist properties implies a single binding site with the C5a receptor and provides a structural template for drug design.     

Site-specific disulfide capture of agonist and antagonist peptides on the C5a receptor

The manner by which peptidic ligands bind and activate their corresponding G-protein-coupled receptors is not well understood. One of the better characterized peptidic ligands is the chemotactic cytokine complement factor 5a (C5a), a 74-amino acid helical bundle. Previous studies showed 6-mer peptide analogs derived from the C terminus of the C5a ligand can bind to C5aR (Kd values approximately 0.1-1 microm) and either agonize or antagonize the receptor (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-3400). Here, we provide direct biochemical data using disulfide trapping to support a model that these peptides bind within a transmembrane helical triad formed by alpha-helices III, VI, and VII. We show that the three amino acids on the C terminus of the peptide analogs bind too weakly to exert a functional effect themselves. However, when a cysteine residue is placed on their N terminus they can be trapped by disulfide interchange to specific cysteines in helix III and VI and not to other cysteines, engineered into the C5aR. The trapped peptides function as agonists or partial antagonists, similar to the non-covalent parents from which they were derived. These data help to further refine the binding mode for C5a to the C5aR and suggest an approach and a binding site that may be applicable to studying other peptide binding receptors.     

Structural study of Ac-Phe-[Orn-Pro-dCha-Trp-Arg], a potent C5a receptor antagonist, by NMR

The cyclic hexapeptide Ac(0)-Phe(1)-[Orn(2)-Pro(3)-dCha(4)-Trp(5)-Arg(6)] (the square brackets denote cyclization) is a potent antagonist against C5a (the a-fragment of complement protein C5) binding to C5a receptor (C5aR) and an excellent candidate to become a therapeutic agent against diseases that involve unregulated activation of the complement system. We present the solution structure determination of this cyclic C5aR peptide antagonist (cC5aR-pa), using nuclear magnetic resonance (NMR) data and restrained molecular dynamics-based simulated annealing in torsion angle space with NMR-derived distance and torsion angle restraints. The calculated NMR ensemble of structures demonstrates the presence of a predominant conformation of a distorted type II' beta-turn in the segment Pro(3)-dCha(4)-Trp(5)-Arg(6). We critically examine the calculated structure with measured NMR parameters, such as nuclear Overhauser enhancement (NOE) connectivity patterns and intensities characteristic of specific structures, (3)J(H(N)-H(alpha)) scalar coupling constants, temperature coefficients for NH groups, and differences between observed chemical shifts and their random coil values. The raw NMR data are consistent with the presence of the type II' beta-turn, but also indicate the presence of conformational inter-conversion. The calculated three-dimensional coordinates for cC5aR-pa will form the basis for further computational studies and for the development of pharmacophore models.     

The platelet-activating factor receptor antagonist L-659,989 inhibits phospholipase D activity.

The platelet-activating factor (PAF) receptor antagonist L-659,989 [(+/-)-trans-2-(3-methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran)] has been reported to be a specific inhibitor of the PAF receptor and as such, it is widely used for assessment of PAF receptor mediated biological effects. We report here that L-659,989 may not be as specific as previously reported because it is also a potent inhibitor of phospholipase D activity. At concentrations of 30 micrograms/ml, L-659,989 inhibited basal and agonist-stimulated phospholipase D activity by about 55% and 70-100% respectively, through a mechanism that may involve the generation of intracellular ceramides. Another PAF receptor antagonist, WEB-2086, did not affect phospholipase D activity at concentrations up to 50 micrograms/ml. Either of these inhibitors when present at 20 micrograms/ml are reported to fully block the effects of PAF. Furthermore, L-659,989 directly inhibited the activity of bacterial PLD in vitro. These results indicate that caution is required in the interpretation of results derived from the use of L-659,989.     

RP S19 C-terminal peptide trimer acts as a C5a receptor antagonist

We have demonstrated that ribosomal protein S19 (RP S19) polymer, when crosslinked between Lys122 and Gln137 by activated coagulation factor XIII, acts as a C5a receptor (C5aR) antagonist/agonist. Based on experimental data obtained using RP S19 analog peptide and recombinant protein monomer, we suggested that L₁₃₁DR, I₁₃₄AGQVAAAN and K₁₄₃KH moieties in the RP S19 C‐terminus act in, respectively, C5aR binding, penetration of the plasma membrane, and interaction with either an apoptosis-inducing molecule in neutrophils (delta lactoferrin) or a calcium channel-activating molecule (annexin A3) to induce the p38 MAPK pathway in macrophages. Recently, we observed RP S19 trimer in serum. To study the effects of this RP S19 trimer on C5aR, we prepared mutant RP S19 C‐terminal peptide (RP S19^122-145) dimer and trimer, and examined their chemotactic activities and signal transduction pathways in human C5aR-overexpressing squamous cell carcinoma HSC-1 (HSC-1^C5aR) cells using 24 trans-well chamber and western blotting assays, respectively. HSC-1^C5aR cells were attracted by RP S19^122-145 dimer and vice versa by RP S19^122-145 trimer. The RP S19^122-145 dimer-induced attraction was competitively blocked by pre-treatment with RP S19^122-145 trimer. Moreover, RP S19^122-145 trimer-induced p38 MAPK phosphorylation was stronger than RP S19^122-145 dimer-induced p38 MAPK phosphorylation. RP S19^122-145 trimer appeared to act as a C5aR antagonist. The agonistic and antagonistic effects of RP S19^122-145 dimers and trimers were reflected by monocytic, THP-1-derived macrophage-like cells. Unlike the C5aR agonist C5a, which acts at the inflammation phase of acute inflammation, RP S19 trimer might act as a C5aR antagonist at the resolution phase.     

The pharmacology of L-670,596, a potent and selective thromboxane/prostaglandin endoperoxide receptor antagonist

L-670,596 ((-)6,8-difluoro-9-rho-methylsulfonyl benzyl-1,2,3,4- tetrahydrocarbazol-1-yl-acetic acid) has been shown to be a potent receptor antagonist as evidenced by the inhibition of the binding of 125I-labeled PTA-OH to human platelets (IC50, 5.5 x 10(-9) M), inhibition of U-44069 induced aggregation of human platelet rich plasma (IC50, 1.1 x 10(-7) M), and competitive inhibition of contractions of the guinea pig tracheal chain induced by U-44069 (pA2,9.0). The compound was also active in vivo as shown by inhibition of arachidonic acid and U-44069 induced bronchoconstriction in the guinea pig (ED50 values, 0.04 and 0.03 mg/kg i.v., respectively), U44069 induced renal vasoconstriction in the pig (ED50, 0.02 mg/kg i.v.), and inhibition of ex vivo aggregation of rhesus monkey platelets to U-44069 (active 1-5 mg/kg p.o.). The selectivity of the compound was indicated by the failure to inhibit, first, ADP-induced human or primate platelet aggregation and, second, bronchoconstriction in the guinea pig in vivo and contraction of the guinea pig tracheal chain in vitro to a variety of agonists. It is concluded that L-670,596 is a potent, selective, orally active thromboxane A2/prostaglandin endoperoxide receptor antagonist.     

Mapping the ligand-binding site on the C5a receptor: arginine74 of C5a contacts aspartate282 of the C5a receptor.

The interaction between the anaphylatoxin C5a and its receptor involves two distinct sites. One site is formed by acidic residues at the receptor N-terminus and contributes to only ligand binding. The second site, responsible for activation, is less well defined. In this study, we demonstrate that the receptor residue D(282), near the extracellular face of transmembrane domain VII, is a component of the second ligand-binding site. Mutation of D(282) to A decreases the sensitivity of the receptor to activation by intact C5a but not by its less potent metabolite, C5adR(74), which lacks the C-terminal arginine(74). The mutation of the R(74) residue of C5a to A causes a 60-fold decrease in wild-type receptor sensitivity, but only a 2-fold decrease for the receptor mutated at D(282). In contrast, the mutation of R(74) to D makes C5a completely inactive on both wild-type and A(282) C5a receptors. The mutation of D(282) to R partly restores the response to C5a[D(74)], which is a more effective ligand than C5a at the mutant receptor. A peptide mimic of the C5a activation domain with a C-terminal R potently activates the wild type but is only a weak agonist at the mutant D(282)R-C5a receptor. Conversely, a peptide with D at the C-terminus is a more effective activator of D(282)R than wild-type C5a receptors. These data indicate that the R(74) side chain of C5a makes an interaction with receptor D(282) that is responsible for the higher potency of intact C5a versus that of C5adR(74).     

Inhalation of an endothelin receptor A antagonist attenuates pulmonary inflammation in experimental acute lung injury

We recently demonstrated that inhalation of the endothelin receptor A (ETA) antagonist LU 135252 improved arterial oxygenation and reduced pulmonary artery pressure in experimental acute lung injury (ALI). In this study we analyzed potential immune modulatory effects of inhaled LU 135252 in experimental ALI. ALI was induced by repeated lung lavage in intubated (100% O2) and anesthetized piglets. Animals were randomly assigned to inhale either nebulized LU 135252 (0.3 mg.kg-1, ALI + LU group, n = 8) or saline buffer (ALI control group, n = 16), both for 30 min. Surviving animals were sacrificed 6 h after induction of ALI, and lung tissue specimens were obtained from all animals for histology and immunhistochemistry. Induction of ALI significantly decreased arterial oxygenation in all animals. Inhalation of LU 135252 significantly reduced mortality and induced significant and sustained increase in Pao2 (316 +/- 47 mm Hg vs. control 53 +/- 3 mm Hg, p < 0.001). We measured a significant reduction in the number of pulmonary leukocyte L1 antigen-positive cells in ALI + LU animals (8% +/- 1% positive cells vs. control 12% +/- 2% positive cells, p < 0.05). The number of CD3-positive cells was not altered by treatment with LU 135252. Pulmonary tissue concentration of IL-6 was significantly suppressed by LU 135252 inhalation (4 +/- 1 pg.100 mg-1 wet weight vs. control 7 +/- 1 pg.100 mg-1 wet weight, p < 0.05). Concentrations of TNF-alpha, IL-1beta, and ET-1 in pulmonary tissue were not influenced by inhalation of LU 135252. In conclusion, we demonstrated that inhalation of LU 135252 not only improves mortality and gas exchange, but also blunts the local immune response in experimental ALI.     

Treatment of hypertension with ketanserin,a new selective 5-HT2 receptor antagonist.

The new selective 5-HT2 receptor blocking agent ketanserin was given in a dose of 10 mg intravenously to 12 patients with essential hypertension. It caused a distinct fall in supine systemic arterial, right atrial, pulmonary artery, and pulmonary capillary "wedge" pressures. Cardiac output, renal blood flow, and glomerular filtration rate showed no persistent changes. Thus 5-HT2 receptor blockade caused dilatation of both resistance and capacitance vessels and of the renal vascular bed. Heart rate and plasma concentrations of renin and noradrenaline rose after ketanserin. These data suggest that 5-HT may have a role in maintaining high blood pressure.     

Angiotensin II receptor antagonist, L-158,809, prevents intimal hyperplasia in dog grafted veins

We investigated the levels of the angiotensin II-forming enzymes, chymase and angiotensin converting enzyme (ACE), in dog grafted veins, and studied the effect of an angiotensin II type 1 receptor antagonist, L-158,809, on vascular proliferation in the grafted veins. The right external jugular vein was grafted to the ipsilaterial carotid artery. In the group treated with L-158,809, the drug (10 mg/kg per day, p.o.) were administered orally from 7 days before the operation to 28 days after it, while the others were administrated placebo. In the placebo-treated group, the chymase activity in the grafted veins was increased about 10-fold and the ACE activity was doubled. The areas of intima and media were significantly increased in the grafted veins in the placebo-treated group. L-158,809 significantly reduced the intimal area of the grafted veins. An angiotensin II receptor antagonist, L-158,809, prevented the vascular proliferation in the grafted veins, and the development of the proliferation may depend on activation of local angiotensin II formation.     

Chemically induced mouse models of intestinal inflammation

Animal models of intestinal inflammation are indispensable for our understanding of the pathogenesis of Crohn disease and ulcerative colitis, the two major forms of inflammatory bowel disease in humans. Here, we provide protocols for establishing murine 2,4,6-trinitro benzene sulfonic acid (TNBS)-, oxazolone- and both acute and chronic dextran sodium sulfate (DSS) colitis, the most widely used chemically induced models of intestinal inflammation. In the former two models, colitis is induced by intrarectal administration of the covalently reactive reagents TNBS/oxazolone, which are believed to induce a T-cell-mediated response against hapten-modified autologous proteins/luminal antigens. In the DSS model, mice are subjected several days to drinking water supplemented with DSS, which seems to be directly toxic to colonic epithelial cells of the basal crypts. The procedures for the hapten models of colitis and acute DSS colitis can be accomplished in about 2 weeks but the protocol for chronic DSS colitis takes about 2 months.     

Expression and function of C5a receptor in mouse microvascular endothelial cells

The complement-derived anaphylatoxin, C5a, is a potent phlogistic molecule that mediates its effects by binding to C5a receptor (C5aR; CD88). We now demonstrate specific binding of radiolabeled recombinant mouse C5a to mouse dermal microvascular endothelial cells (MDMEC) with a K(d50) of 3.6 nM and to approximately 15,000-20,000 receptors/cell. Recombinant mC5a competed effectively with binding of [(125)I]rmC5a to MDMEC. Enhanced binding of C5a occurred, as well as increased mRNA for C5aR, after in vitro exposure of MDMEC to LPS, IFN-gamma, or IL-6 in a time- and dose-dependent manner. By confocal microscopy, C5aR could be detected on surfaces of MDMEC using anti-C5aR Ab. In vitro expression of macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) by MDMEC was also measured. Exposure of MDMEC to C5a or IL-6 did not result in changes in MIP-2 or MCP-1 production, but initial exposure of MDMEC to IL-6, followed by exposure to C5a, resulted in significantly enhanced production of MIP-2 and MCP-1 (but not TNF-alpha and MIP-1alpha). Although LPS or IFN-gamma alone induced some release of MCP-1 and MIP-2, pre-exposure of these monolayers to LPS or IFN-gamma, followed by addition of C5a, resulted in synergistic production of MIP-2 and MCP-1. Following i.v. infusion of LPS into mice, up-regulation of C5aR occurred in the capillary endothelium of mouse lung, as determined by immunostaining. These results support the hypothesis that C5aR expression on MDMEC and on the microvascular endothelium of lung can be up-regulated, suggesting that C5a in the co-presence of additional agonists may mediate pro-inflammatory effects of endothelial cells.     

Changes in the novel orphan, C5a receptor (C5L2), during experimental sepsis and sepsis in humans

Sepsis is associated with extensive complement activation, compromising innate immune defenses, especially in neutrophils (PMN). Recently, a second C5a receptor (C5L2) was detected on PMN without evidence of intracellular signaling. The current study was designed to determine changes in C5L2 in blood PMN during sepsis. In vitro exposure of PMN to C5a, but not to fMLP, led to reduced content of C5L2. Following cecal ligation and puncture-induced sepsis in rats, PMN demonstrated a time-dependent decrease in C5L2. In vivo blockade of C5a during experimental sepsis resulted in preservation of C5L2. Similarly, PMN from patients with progressive sepsis showed significantly reduced C5L2 expression (n = 26), which was virtually abolished in patients who developed multiorgan failure (n = 10). In contrast, sepsis survivors exhibited retention of C5L2 (n = 12/13). The data suggest that C5L2 on PMN diminishes during sepsis due to systemic generation of C5a, which is associated with a poor prognosis.     

Effects of cefotaxime,clindamycin, mezlocillin,and piperacillin on mouse sarcoma L-1 tumor

Summary For the study described in the paper, the effects of 10 days' chemotherapy with cefotaxime, clindamycin, mezlocillin, and piperacillin on local tumor growth and on spontaneous or artificial metastatic spread into the lungs were studied. For the animal tumor model Balb/c mice and the mouse sarcoma L-1 tumor were used. Chemotherapy was administered before, immediately after, or some time after the injection of tumor cells. The antibiotic dosage given to mice was calculated on a body weight basis from the doses recommended for humans. Cefotaxime and clindamycin did not influence the animal tumor model, whereas mezlocillin and piperacillin showed positive or negative effects depending on the chemotherapy schedule. In vitro none of the four antibiotics caused cytotoxic activity in cell cultures of mouse sarcoma L-1, human lung cancer E-14, or human malignant melanoma MEW.Fellows of the Alexander von Humboldt Foundation