Expression of histamine H1 receptors on cultured histiocytic lymphoma cells

Histamine H1 receptors were identified in U937 human histiocytic lymphoma cells using a radioligand binding technique with [3H]mepyramine. Reversible high-affinity binding with this ligand was obtained, and specificity of binding for selected H1 agonists and antagonists was demonstrated. Competition binding experiments with mepyramine and histamine yielded results consistent with single-site binding for mepyramine and two-site binding for histamine. Dissociation constants for the high- and low-affinity histamine binding states were 6.8 X 10(-6) and 2.4 X 10(-4) M respectively. The high-affinity state for histamine binding was abolished when the membranes were coincubated with 100 microM guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S). Saturation binding studies yielded an average of 66 fmol/mg protein binding sites (6000 receptors per cell) with a [3H]mepyramine KD of 9.6 nM. When differentiation of these cells was induced by phorbol-myristate-acetate, receptor density increased by 73% to 114 fmol/mg protein. This increase in receptor density was inhibited by actinomycin D and cycloheximide. Exposure of native and differentiated U937 cells to 10(-5) and 10(-4) M histamine for 24 hr resulted in a dose-dependent down-regulation in receptor density. The data indicate that U937 cells may provide a model cell line for the study of histamine receptor gene expression.     

银杏内酯B对oxLDL刺激大鼠胸主动脉平滑肌细胞和U937细胞功能变化的作用

目的观察银杏内酯B对oxLDL或PAF引起的大鼠胸主动脉平滑肌细胞(VSMC)增殖及U937单核细胞趋化因子分泌的抑制作用。方法用³H-Tdr参入实验测定VSMC增殖。用ELISA和RT-PCR方法测定MCP-1和IL-8的蛋白和mRNA水平。Western blotting测定p65和IKB的蛋白量。放射受体配体结合实验观察oxLDL与PAF受体的结合。结果银杏内酯B呈剂量依赖性地抑制oxLDL诱导的VSMC增殖以及U937细胞MCP-1和IL-8分泌，并抑制oxLDL诱导的p65活化和IKB降解。并证实oxLDL可以抑制PAF与其在U937细胞上受体的结合。结论银杏内酯B在体外具有一定的抗oxLDL促进VSMC增殖及刺激单核细胞趋化因子分泌的作用。银杏内酯B抑制oxLDL的毒性作用可能部分通过抑制其与PAF受体的结合实现。     

A crystalline compound (BM-ANF1) from the Indian toad (Bufo melanostictus, Schneider) skin extract, induced antiproliferation and apoptosis in leukemic and hepatoma cell line involving cell cycle proteins.

In our earlier communication, it was reported that Indian toad (Bufo melanostictus) skin extract (TSE) possesses antiproliferative and apoptogenic activity in U937 and K562 cells [Giri et al., 2006. Antiproliferative, cytotoxic and apoptogenic activity of Indian toad (Bufo melanostictus, Schneider) skin extract in U937 and K562 cells. Toxicon 48 (4), 388-400]. In the present study, a compound (BM-ANF1) has been isolated from the TSE by alumina gel column chromatography, crystallized and evaluated for its antiproliferative and apoptogenic activity in U937, K562 and HepG2 cells. BM-ANF1 produced dose-dependent inhibition of U937, K562 and HepG2 cell growth. The antiproliferative activity was reflected by the MTT assay and demonstrated by the reduced expression of proliferative cell nuclear antigen (PCNA). Flow-cytometric analysis showed that BM-ANF1 arrested the cell cycle at G1 phase and enhanced annexin-V binding in U937 and K562 cells. Scanning electron microscopic and fluorescent microscopic analysis of U937 and K562 cells revealed the apoptogenic nature of the compound. Alkaline comet assay showed that BM-ANF1 produced DNA fragmentation. The dose-dependent expression of caspase 3 indicated that the apoptogenic properties of BM-ANF1 were mediated through the activation of downstream effector nucleases in the cancer cells. The increased expression of p53 and moderate expression of p21(Cip1)/p27(Kip1) due to BM-ANF1 treatment in HepG2 cells supported that the apoptogenic activity of BM-ANF1 was mediated through p53 tumor-suppressor gene expression followed by the expression of p21(Cip1) and p27(Kip1) and it was likely to be linked with cell cycle arrest at G1 phase in cancer cells. From the present study, it may be suggested that the crystalline compound, BM-ANF1, was antiproliferative and apoptogenic in human leukemic and hepatoma cells.     

Curcuma longa extract protects against gastric ulcers by blocking H2 histamine receptors

Curcuma longa has been commonly used as a traditional remedy for a variety of symptoms such as inflammation, gastritis and gastric ulcer. When C. longa extract was administered per os to pylori-ligated rat stomachs, it reduced gastric acid secretion and protected against the formation of gastric mucosal lesions. We therefore tested whether C. longa extract inhibits gastric ulcers by blocking the H(2) histamine receptor. Dimaprit, a H(2) histamine receptor agonist, induced intracellular cAMP production in U937 and HL-60 promyelocytes. Pretreatment with C. longa extract significantly blocked dimaprit-induced cAMP production in a concentration dependent manner, but had no effect on the elevation of cAMP levels triggered by isoproterenol-induced beta(2)-adrenoceptor activation in U937 cells. To identify the active component(s) of C. longa extract, we sequentially fractionated it by extraction with ethyl acetate, n-butanol and water. We found that the ethyl acetate extract showed the most potent H(2)R antagonistic effect against dimaprit-induced cAMP production. However, curcumin, a major component of C. longa extract, showed no H(2)R blocking effect. C. longa ethanol extract and ethylacetate extract also blocked the binding of [(3)H]-tiotidine to membrane receptors on HL-60 cells. These findings suggest that the extract from C. longa specifically inhibits gastric acid secretion by blocking H(2) histamine receptors in a competitive manner.     

Characterization of the leukotriene D4 receptor in dimethylsulphoxide-differentiated U937 cells: comparison with the leukotriene D4 receptor in human lung and guinea-pig lung

The leukotriene D₄ receptor has been fully characterized by radioligand binding in membrane preparations from dimethyl sulphoxide-differentiated U937 cells, a human monocyte leukemia cell line, and, in parallel experiments, compared with leukotriene D₄ receptor found in human lung and guinea-pig lung preparations. [³H]Leukotriene D₄ specific binding in differentiated U937 cell membranes is of high affinity (K_D = 0.35 nM), saturable (B_max = 287 fmol/mg protein), with differentiation resulting in a 3–5-fold increase in the number of detectable binding sites. [³H]Leukotriene D₄-specific binding in differentiated U937 cell membranes displays several features of G-protein-cupled receptors, being inhibited by GTP analogues and sodium ions, but increased by divalent cations. These characteristics are shared with [³H]leukotriene D₄-specific binding in human and guinea-pig lung preparations. However, differences between these leukotriene D₄ receptor types were observed. [³H]Leukotriene D₄ equilibrium binding to differentiated U937 cell membranes could be dissociated to non-specific binding levels by 1000-fold excess of competing ligand, whereas binding to guinea-pig lung membranes was only partially dissociated under these conditions. In addition, differences in potency were demonstrated in competition studies with leukotriene E₄ and leukotriene C₄, although leukotriene D₄ and the leukotriene D₄-receptor antagonists MK-571 and ICI 204,219 were equipotent in competing for [³H]leukotriene D₄-specific binding in all three membranes preparations. In conclusion, the leukotriene D₄ receptor in differentiated U937 cell membranes resembles that in human lung, validating the use of this cell line as a suitable source of receptor in the development of potent specific antagonists.     

Limitations of the single-cell gel electrophoresis assay to monitor apoptosis in U937 and HepG2 cells exposed to 7beta-hydroxycholesterol

The single-cell gel electrophoresis (comet) assay is a method which allows the detection of DNA strand breaks in individual cells. It has been suggested that the single cell gel electrophoresis assay, as an index of DNA fragmentation during cell death, may be applied to monitor apoptosis. The aim of the present study was to determine if the pattern of DNA fragmentation determined by the single cell gel electrophoresis assay can be used to discriminate between the mode of cell death in two cell lines (U937, a human monocytic blood cell line and HepG2, a human hepatocarcinoma cell line) which were treated with 30 microM 7beta-hydroxycholesterol (7betaOHC) over a 48 hr period. The single cell gel electrophoresis assay was compared with more established methods for the determination of apoptosis such as morphological examination, flow cytometry and DNA laddering. The percentage of maximally damaged nuclei as measured by the single cell gel electrophoresis assay was found to be similar at 48 hr in both U937 and HepG2 cells when treated with 7betaOHC. However, morphological examination, flow cytometry and DNA laddering techniques showed that 7betaOHC induced apoptosis in U937 cells but not in HepG2 cells. Thus, although the alkaline single cell gel electrophoresis assay detected DNA strand breaks occurring during cell death, these breaks were observed only when the process was fairly well advanced and a major part of the cells had lost membrane permeability. Therefore the present report demonstrates that the single cell gel electrophoresis assay, used in isolation, cannot accurately be used to distinguish between the mode of cell death induced by 7betaOHC in U937 cells (apoptosis), or HepG2 cells (cell lysis).     

Effects of IL-4 and IL-13 on adenosine receptor expression and responsiveness of the human mast cell line 1

BACKGROUND: Inhalation of adenosine-5'-monophosphate (AMP) causes bronchoconstriction in asthma but not in healthy subjects. Bronchoconstriction upon AMP inhalation is thought to occur by histamine release and subsequent binding to receptors on airway smooth muscle cells. METHODS: To explain enhanced sensitivity to AMP in asthma, mast cell expression of the adenosine A2A and A2B receptors and histamine release were measured after incubation of human mast cell line 1 (HMC-1) cells with AMP and the non-specific adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) for 1.5 and 6 h. To establish a Thelper-2 environment resembling the asthma phenotype, HMC-1 cells were additionally cultured with IL-4 and IL-13 alone or stimulated with the combination of both cytokines and AMP and NECA. To study effects of prolonged presence of the inflammatory environment, the cells were pre-incubated overnight (18 h) with IL-4 and IL-13 and additionally stimulated with AMP and NECA for 1.5 or 6 h. RESULTS: AMP and NECA hardly affected adenosine receptor expression but increased IL-8 secretion. Incubation with IL-4 and IL-13 for 6 h increased adenosine A2A receptor expression and histamine secretion, but decreased IL-8 secretion. The combination of IL-4, IL-13, and AMP/NECA for 6 h increased A2B receptor expression and IL-8 secretion. Overnight stimulation with IL-4, IL-13 and subsequent stimulation with AMP/NECA for 1.5 h decreased A2AR expression which was accompanied by increased histamine secretion. CONCLUSION: These results suggest a role for decreased A(2A)R expression in enhanced adenosine responsiveness as observed in asthma.     

Thrombin signalling in U937 human monocytic cells is coupled to inositol phosphate formation but not to thromboxane B2 synthesis nor to inhibition of adenylate cyclase: distinct differences in thrombin signalling between U937 cells and platelets.

The blood coagulation factor, human thrombin has been shown to have chemotactic and mitogenic effects on mononuclear phagocytic inflammatory cells. In the present study, we have used the U937 human monocytic cell line to explore the signal transduction mechanisms utilised by thrombin in these cells. In U937 cells differentiated into a macrophage-like phenotype, thrombin stimulated the formation of inositol trisphosphate (IP3) and the mobilisation of intracellular Ca2+ [( Ca2+]i) via a mechanism which was partially sensitive to pertussis toxin. Thrombin failed, however, to evoke thromboxane (Tx) B2 synthesis in the differentiated cells. In contrast, the chemotactic peptide N-formyl-L-methionylleucyl-L-phenylalanine (FMLP) stimulated TxB2 synthesis under conditions where it evoked increases in IP3 formation and [Ca2+]i mobilisation, via a pertussis toxin-sensitive mechanism, comparable in extent to those mediated by thrombin. Thrombin also failed to cause inhibitory guanine nucleotide binding protein (Gi)-mediated inhibition of adenylate cyclase activity in U937 cell membranes. These results indicate that U937 cells express receptors for thrombin which are in part coupled via a pertussis toxin-sensitive guanine nucleotide binding protein to phospholipase C activation, the formation of IP3 and the mobilisation of [Ca2+]i. However, the failure of thrombin to stimulate TxB2 synthesis or cause Gi-mediated inhibition of adenylate cyclase in U937 cells contrasts with its effects in human platelets and other thrombin-responsive cells. These results suggest that the thrombin receptor or receptor-effector coupling mechanism(s) in mononuclear cells is functionally distinct from the thrombin receptor or receptor-effector coupling mechanism(s) present in other thrombin-responsive cells.     

Chlorpyrifos induces apoptosis in human monocyte cell line U937

In order to investigate chlorpyrifos-induced cell death and its underlying mechanism in human immune cells, a human monocyte like cell line (U937) was treated with chlorpyrifos at 4.45-570microM for 0.5-24h at 37 degrees Celsius in a 5% CO(2) incubator. We first found that chlorpyrifos induced cell death of U937 in a dose- and time-dependent manner, as shown by LDH and MTT assays and PI uptake. Then, we investigated if chlorpyrifos-induced cell death consisted of apoptosis, as determined by analysis of Annexin-V staining and the intracellular level of active caspase-3 by flow cytometry, and DNA fragmentation analysis. We found that chlorpyrifos induced apoptosis in U937 in a time- and dose-dependent manner, as shown by Annexin-V staining. DNA fragmentation was detected when cells were treated with 71 to 284microM chlorpyrifos for 4 or 6h. Chlorpyrifos also induced an increase of intracellular active caspase-3 in U937 cells in a dose-dependent manner, and a caspase-3 inhibitor, Z-DEVD-FMK, significantly inhibited the chlorpyrifos-induced apoptosis. These findings indicate that chlorpyrifos can induce apoptosis in U937 cells, and this effect is partially mediated by activation of intracellular caspase-3.     

In vitro evaluation of the chemoprotective action mechanisms of leontopodic acid against aflatoxin B1 and deoxynivalenol-induced cell damage

Several in vitro studies showed that free radical scavengers possess chemopreventive properties against mycotoxin-induced cell damage which are at least partially associated with the induction of phase II detoxifying enzymes and antioxidant enzymes like glutathione S-transferase (GST) and glutathione peroxidase (GPx). The aim of this project was to study the chemopreventive effects of leontopodic acid (LA), a potent natural occurring free radical scavenger isolated from the aerial parts of Leontopodium alpinum. Different mycotoxins were evaluated in two different cell lines on the basis of their specific toxicity: aflatoxin B1 (AFB1) on HepG2 cells and deoxynivalenol (DON) on U937 cells. Cell viability and reactive oxygen species concentration were determined, and the effects of pre-treatment with LA on these parameters were investigated together with the GST and GPx activity as well as the concentration of reduced glutathione. The results show that LA protects U937 cells from DON-induced cell damage but not HepG2 cells from AFB1. Moreover LA is able to enhance GPx activity in U937, but not GST activity in HepG2. We hypothesize that the increase in detoxifying enzymes is probably the main mechanism of antioxidant mediated chemoprevention.     

Rapid desensitization and slow recovery of the cyclic AMP response mediated by histamine H(2) receptors in the U937 cell line

The present study focused on the desensitization process of the H(2) receptor in U937 cells and the recovery of the cyclic AMP (cAMP) response. Treatment of U937 leukemic cells with the H(2) histamine receptor agonists (+/-)-N(1)-[3-(3, 4-difluorophenyl)-3-(pyridin-2-yl)propyl]-N(2)-[3-(1H-imidazol-4-yl)p ropyl]guanidine (BU-E-75) and amthamine produced a rapid desensitization characterized by decreased cAMP production (T(1/2) = 20 min). Pretreatment with 10 microM BU-E-75 did not induce modifications in the responses to prostaglandin E(2), isoproterenol, or forskolin. H(2) receptor desensitization was not affected by protein kinase A and C inhibitors, but was reduced drastically by Zn(2+) and heparin, known to act as inhibitors of G protein-coupled receptor kinases. Recovery studies of the cAMP response showed that cAMP levels reached 50% of the initial values within 5 hr. Furthermore, desensitization produced an important decrease in the basal level of this cyclic nucleotide. The minimal value was observed 12 hr later, and corresponded to approximately 1.3% of the initial basal level (7.5 vs 0.1 pmol/10(6) cells). This result could be explained by an increase in phosphodiesterase activity following 10 microM BU-E-75 treatment. When cells were exposed for 2 hr to an H(2) agonist, binding assays showed no modification in the number of H(2) receptors; internalization began just after 8 hr. Although the initial desensitization seems to involve G protein-coupled receptor kinases, results indicate that additional mechanisms of regulation were triggered by the H(2) agonists.     

H1-histamine receptors on human astrocytoma cells

The H1-histamine receptor antagonist [3H]mepyramine bound with high affinity (Kd = 3-5 nM) to membranes derived from 1321N1 human astrocytoma cells. The H1-receptor antagonists triprolidine and diphenhydramine inhibited [3H]mepyramine binding with Kj values of 1-5 nM, whereas the Kj of the H2-histamine receptor antagonist cimetidine was greater than 100 microM. Histamine also inhibited [3H]mepyramine binding to 1321N1 cell membranes, and the histamine inhibition curve was shifted to the right and steepened in the presence of 1 microM guanosine 5'-O-(3-thiotriphosphate). Treatment of 1321N1 cells with pertussis toxin had no effect on the capacity of histamine to inhibit [3H]mepyramine binding either in the absence or presence of guanosine 5'-O-(3-thiotriphosphate). Therefore, agonist-occupied histamine receptors in these cells apparently interact with a guanine nucleotide regulatory protein that is not the inhibitory guanine nucleotide regulatory protein of adenylate cyclase. Although adenylate cyclase activity was not affected by histamine in a cell-free preparation, incubation of 1321N1 cells with histamine resulted in an attenuation of cyclic AMP accumulation. Analysis of cyclic AMP degradation in the presence of histamine indicated that the effects of histamine on cyclic AMP accumulation are mediated through activation of phosphodiesterase. This idea was supported by the fact that the phosphodiesterase inhibitor 1-isobutyl 3-methylxanthine blocked attenuation of cyclic AMP accumulation by histamine in a noncompetitive manner. Histamine also markedly increased phosphoinositide breakdown and 45Ca2+ efflux in 1321N1 cells. These histamine-induced effects apparently are mediated through H1-receptors, since triprolidine, but not cimetidine, potently inhibited histamine action. As for histamine interaction with its receptor, pertussis toxin had no effect on histamine-induced phosphoinositide breakdown, 45Ca2+ efflux, or attenuation of cyclic AMP accumulation. Taken together, these data indicate that 1321N1 human astrocytoma cells are a useful model system for the study of H1-histamine receptors and the biochemical responses mediated through these receptors.     

Calcium-dependence of histamine- and carbachol-induced inositol phosphate formation in human U373 MG astrocytoma cells: comparison with HeLa cells and brain slices.

1. Histamine (1 mM) induced an accumulation of inositol monophosphate ([3H]-IP1) in the U373 MG human astrocytoma cell line which increased with time in the presence of 30 mM Li+. After a 30 min incubation period with 1 mM histamine [3H]-IP1 was the major product detected (84 +/- 1% of total [3H]-IPx) and was present at a level 11 (+/- 1) fold of basal accumulation. 2. Concentration-response curves for histamine-induced [3H]-IP1 accumulation in U373 MG cells (EC50 5.4 +/- 0.5 microM) were shifted to the right in a parallel fashion by mepyramine (slope of a Schild plot 0.99 +/- 0.08), yielding a Kd for mepyramine of 3.5 +/- 0.3 nM, consistent with the involvement of histamine H1-receptors. 3. The temelastine-sensitive binding of [3H]-mepyramine to a membrane fraction from U373 MG cells was hyperbolic and had a mean Kd of 2.5 +/- 1.0 nM. The maximum amount of temelastine-sensitive binding was 86 +/- 19 pmol g-1 membrane protein. 4. Carbachol also induced [3H]-IP1 accumulation in U373 MG cells, 2.8 (+/- 0.1) fold of basal with 1 mM carbachol, with an EC50 of 48 +/- 8 microM. Pirenzepine shifted carbachol concentration-response curves to the right (slope of Schild plot 0.89 +/- 0.07) giving a Kd for pirenzepine of 0.10 +/- 0.01 microM, suggesting that phosphoinositide hydrolysis in U373 MG cells is mediated by the M3-, rather than the M1-, muscarinic receptor subtype. 5. [3H]-IP1 accumulation induced by both 1 mM histamine and by 1 mM carbachol increased when the Ca2+ concentration of the medium was increased from 'zero' (no added Ca2+) to 0.3 mM. Histamine-stimulated [3H]-IP1 accumulation was further increased, although not so markedly, as the Ca2+ was raised to 4 mM. The same pattern was apparent with histamine-induced accumulations of [3H]-IP2 and [3H]-IP3. In contrast, [3H]-IPx accumulation in response to carbachol increased between 0.3 and 1.3 mM, but thereafter remained unchanged ([3H]-IP1) or declined ([3H]-IP2 and [3H]-IP3). 6. In HeLa cells, [3H]-IP1 accumulations induced by 1 mM histamine and 1 mM carbachol showed the same pattern of Ca2+ dependence and were independent of extracellular Ca2+ above 0.3 mM (histamine) or 1.3 mM (carbachol). The response to carbachol appeared to be mediated by an M3-muscarinic receptor (apparent Kd for pirenzepine 0.09 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors

Montelukast and pranlukast are orally active leukotriene receptor antagonists selective for the CysLT1 receptor. Conversely, the hP2Y(1,2,4,6,11,12,13,14) receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937). Montelukast and pranlukast inhibited the effects of UTP with IC50 values of 7.7 and 4.3 microM, respectively, and inhibited the effects of UDP with IC50 values of 4.5 and 1.6 microM, respectively, in an insurmountable manner. Furthermore, ligand binding studies using [3H]LTD4 excluded the possibility of orthosteric nucleotide binding to the CysLT1 receptor. dU937 cells were shown to express P2Y2, P2Y4, P2Y6, P2Y11, P2Y13 and P2Y14 receptors. Therefore, these antagonists were studied functionally in a heterologous expression system for the human P2Y receptors. In 1321N1 astrocytoma cells stably expressing human P2Y(1,2,4,6) receptors, CysLT1 antagonists inhibited both the P2Y agonist-induced activation of phospholipase C and intracellular Ca2+ mobilization. IC50 values at P2Y1 and P2Y6 receptors were <1 microM. In control astrocytoma cells expressing an endogenous M3 muscarinic receptor, 10 microM montelukast had no effect on the carbachol-induced rise in intracellular Ca2+. These data demonstrated that CysLT1 receptor antagonists interact functionally with signaling pathways of P2Y receptors, and this should foster the study of possible implications for the clinical use of these compounds in asthma or in other inflammatory conditions.     

Involvement of protein kinase A in histamine-mediated inhibition of IL-2 mRNA expression in mouse splenocytes

The release of histamine from mast cells and basophils during allergic reactions can regulate functions of T cells and may influence the nature of the immune response to a given antigen. The effects of histamine on T lymphocytes are associated with its binding to H2-receptors linked with adenylate cyclase, elevation of cAMP levels and activation of cAMP-dependent protein kinase (PKA). In this report we explore the role of PKA in histamine-mediated effects on IL-2 mRNA expression and IL-2 protein secretion. Fresh isolated mouse splenocytes (C57Bl/6) were pretreated with histamine (10(-4) M) for 1 h in the presence or absence of Rp-cAMPS (50 microM), an inhibitor of PKA regulatory subunit. The cells were then washed thoroughly and activated with plate-bound anti-CD3 (5 microg/ml), or PHA (1:100) or PMA + ionomycin (10 ng/ml, 1 microg/ml) for 6 h. Pretreatment with histamine inhibited IL-2 mRNA expression and secretion in cells activated with anti-CD3 or PMA, but not in cells activated with PMA + ionomycin. Rp-cAMPS prevented histamine-mediated suppression and did not itself affect IL-2 production. These results provide evidence that histamine affected IL-2 production when the cells were activated via the T cell receptor (TCR)/CD3 complex, but did not interfere with signal transduction pathways downstream of PKC leading to production of IL-2. These effects of histamine on IL-2 secretion and mRNA expression were mediated via PKA.     

Stereoselectivity at the β2-adrenoceptor on macrophages is a major determinant of the anti-inflammatory effects of β2-agonists

Previous research has shown that beta-adrenoceptor (beta-AR) agonists have potent anti-inflammatory capabilities, e.g. represented by suppression of release of the proinflammatory cytokines. Aim of this research was to determine whether the effects of beta-agonists on LPS-induced TNFalpha and IL-10 release are influenced by their different stereochemistry. In addition, the role of the beta-AR subtypes was studied. The effect of two stereoisomers of the selective beta2-AR agonist TA2005 [(R,R)- and (S,S)-] on the LPS-induced TNFalpha and IL-10 release by U937 macrophages was compared. The (R,R)-stereoisomer was 277 times more potent in inhibiting the TNFalpha release than the (S,S)-form. The (R,R)-stereoisomer also appeared to be more potent in increasing the IL-10 release. In radioligand binding studies the affinity of (R,R)-TA2005 for the beta-adrenoceptor was 755 times higher than the (S,S)-TA2005 stereoisomer. In addition, the elevation of intracellular cAMP in U937 cells appeared to be stereoselective: (R,R)-TA2005 was more potent in elevating intracellular cAMP. The effect of both stereoisomers on the LPS-induced TNFalpha release could almost completely be antagonized by preincubation with the selective beta2-AR-antagonist ICI-118551. Further evidence that the effect of the beta-agonists is mediated via the beta2-adrenoceptor subtype exclusively was acquired by incubation of U937 cells with selective beta1- and beta3-agonists. None of these receptor subtype agonists showed significant suppressive effect on TNFalpha release. This study provides additional proof that the anti-inflammatory effects of beta2-agonists are mediated via the beta2-adrenoceptor and indicates that these effects are highly dependent on the stereoselectivity of the ligand.     

Histamine H4 receptor mediates eosinophil chemotaxis with cell shape change and adhesion molecule upregulation

1. During mast cell degranulation, histamine is released in large quantities. Human eosinophils were found to express histamine H(4) but not H(3) receptors. The possible effects of histamine on eosinophils and the receptor mediating these effects were investigated in our studies. 2. Histamine (0.01-30 microm) induced a rapid and transient cell shape change in human eosinophils, but had no effects on neutrophils. The maximal shape change was at 0.3 microm histamine with EC(50) at 19 nm. After 60 min incubation with 1 microm histamine, eosinophils were desensitized and were refractory to shape change response upon histamine restimulation. Histamine (0.01-1 microm) also enhanced the eosinophil shape change induced by other chemokines. 3. Histamine-induced eosinophil shape change was mediated by the H(4) receptor. This effect was completely inhibited by H(4) receptor-specific antagonist JNJ 7777120 (IC(50) 0.3 microm) and H(3)/H(4) receptor antagonist thioperamide (IC(50) 1.4 microm), but not by selective H(1), H(2) or H(3) receptor antagonists. H(4) receptor agonists imetit (EC(50) 25 nm) and clobenpropit (EC(50) 72 nm) could mimic histamine effect in inducing eosinophil shape change. 4. Histamine (0.01-100 microm) induced upregulation of adhesion molecules CD11b/CD18 (Mac-1) and CD54 (ICAM-1) on eosinophils. This effect was mediated by the H(4) receptor and could be blocked by H(4) receptor antagonists JNJ 7777120 and thioperamide. 5. Histamine (0.01-10 microm) induced eosinophil chemotaxis with an EC(50) of 83 nm. This effect was mediated by the H(4) receptor and could be blocked by H(4) receptor antagonists JNJ 7777120 (IC(50) 86 nm) and thioperamide (IC(50) 519 nm). Histamine (0.5 microm) also enhanced the eosinophil shape change induced by other chemokines. 6. In conclusion, we have demonstrated a new mechanism of eosinophil recruitment driven by mast cells via the release of histamine. Using specific histamine receptor ligands, we have provided a definitive proof that the H(4) receptor mediates eosinophil chemotaxis, cell shape change and upregulation of adhesion molecules. The effect of H(4) receptor antagonists in blocking eosinophil infiltration could be valuable for the treatment of allergic diseases. The histamine-induced shape change and upregulation of adhesion molecules on eosinophils can serve as biomarkers for clinical studies of H(4) receptor antagonists.     

Pharmacological characterization of the human histamine H2 receptor stably expressed in Chinese hamster ovary cells.

1. The gene for the human histamine H2 receptor was stably expressed in Chinese hamster ovary (CHO) cells and characterized by [125I]-iodoaminopotentidine binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. 2. After cotransfection of CHO cells with pCMVhumH2 and pUT626, a phleomycine-resistant clonal cell line (CHOhumH2) was isolated that expressed 565 +/- 35 fmol kg-1 protein binding sites with high affinity (0.21 +/- 0.02 nM) for the H2 antagonist, [125I]-iodoaminopotentidine. 3. Displacement studies with a variety of H2 antagonists indicated that the encoded protein was indistinguishable from the H2 receptor identified in human brain membranes and guinea-pig right atrium. The Ki-values observed in the various preparations correlated very well (r2 = 0.996-0.920). 4. Displacement studies with histamine showed that a limited fraction (32 +/- 6%) of the binding sites showed a high affinity for histamine (2 +/- 1.2 microM); the shallow displacement curves were reflected by a Hill-coefficient significantly different from unity (nH = 0.58 +/- 0.09). The addition of 100 microM Gpp(NH)p resulted in a steepening of the displacement curve (nH = 0.79 +/- 0.02) and a loss of high affinity sites for histamine. 5. Displacement studies with other agonists indicated that the recently developed specific H2 agonists, amthamine and amselamine, showed an approximately 4-5 fold higher affinity for the human H2 receptor than histamine. 6. Stimulation of CHOhumH2 cells with histamine resulted in a rapid rise of the intracellular cyclic AMP levels. After 10 min an approximately 10 fold increase in cyclic AMP could be measured.(ABSTRACT TRUNCATED AT 250 WORDS)