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1.
Bioorg Med Chem Lett ; 10(22): 2557-61, 2000 Nov 20.
Article in English | MEDLINE | ID: mdl-11086729

ABSTRACT

From two related series of 2-(alkylthio)-pyrimidones, a novel series of 1-((amidolinked)-alkyl)-pyrimidones has been designed as nanomolar inhibitors of human lipoprotein-associated phospholipase A2. These compounds show greatly enhanced activity in isolated plasma. Selected derivatives such as compounds 51 and 52 are orally active with a good duration of action.


Subject(s)
Enzyme Inhibitors/pharmacology , Lipoproteins/metabolism , Phospholipases A/antagonists & inhibitors , Pyrimidinones/pharmacology , Administration, Oral , Animals , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Molecular Structure , Phospholipases A/metabolism , Phospholipases A2 , Pyrimidinones/administration & dosage , Pyrimidinones/chemistry , Rabbits
2.
Eur J Pharmacol ; 392(3): 189-95, 2000 Mar 31.
Article in English | MEDLINE | ID: mdl-10762673

ABSTRACT

Recombinant fractalkine possesses both chemoattractive and adhesive properties in vitro. Previous studies have demonstrated an upregulation of this molecule on the membranes of activated human endothelial cells and hypothesised that fractalkine plays a role in the recruitment and adherence of monocytes to the activated endothelium. Here we present data analysing both the adhesive and chemoattractive properties of this chemokine expressed by activated human umbilical vein endothelial cells. We demonstrate that both recombinant fractalkine and endogenously produced fractalkine function as adhesion molecules, tethering monocytes to the endothelium. However, our data demonstrate that although recombinant fractalkine has the potential to function as a potent monocyte chemoattractant, the endogenous fractalkine cleaved from activated human umbilical vein endothelial cells is not responsible for the observed chemotaxis in this model. Instead, we show that monocyte chemoattractant protein-1 (MCP-1), secreted from the activated human umbilical vein endothelial cells, is responsible for the chemotaxis of these monocytes.


Subject(s)
Chemokines, CX3C , Chemokines, CXC/physiology , Endothelium, Vascular/physiology , Membrane Proteins/physiology , Monocytes/cytology , Blotting, Western , Cell Adhesion/drug effects , Cell Line , Chemokine CX3CL1 , Chemokines, CXC/genetics , Chemokines, CXC/pharmacology , Chemotactic Factors/physiology , Chemotaxis/drug effects , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Humans , Membrane Proteins/genetics , Membrane Proteins/pharmacology , Monocytes/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/pharmacology , Time Factors , Tumor Necrosis Factor-alpha/pharmacology , Umbilical Veins/cytology , Umbilical Veins/metabolism
3.
Biochem Pharmacol ; 59(5): 591-6, 2000 Mar 01.
Article in English | MEDLINE | ID: mdl-10660125

ABSTRACT

Human CC chemokine receptor 1 (CCR1) has been proposed as a receptor for CKbeta8. To obtain conclusive evidence, binding-displacement studies of 125I-CKbeta8 (25-99) were performed on membranes of Chinese hamster ovary cells expressing human CCR1. The Ic50 for displacement of 125I-CKbeta8 (25-99) with CKbeta8 (25-99) was 0.22 nM. The longer forms of CKbeta8 (24-99 and 1-99) also displaced 125I-CKbeta8, with Ic50 values of 6.5 and 16 nM, respectively. Displacement profiles of 125I-CKbeta8 (25-99) on freshly prepared human monocytes indicated that CCR1 was the major receptor for CKbeta8. We conclude that CCR1 is a receptor for different-length CKbeta8 and that CKbeta8 (25-99) has a similar affinity for CCR1 as macrophage inflammatory protein-1alpha (MIP-1alpha). The longer variants of CKbeta8 are significantly less potent than CKbeta8 (25-99) and MIP-1a on CCR1 and monocytes (P < 0.05).


Subject(s)
Chemokines, CC/metabolism , Receptors, Chemokine/metabolism , Animals , Binding, Competitive , CHO Cells , Cricetinae , Humans , Iodine Radioisotopes , Monocytes/metabolism , Peptides/metabolism , Receptors, CCR1 , Transfection
4.
Biochem J ; 338 ( Pt 2): 479-87, 1999 Mar 01.
Article in English | MEDLINE | ID: mdl-10024526

ABSTRACT

A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine.


Subject(s)
Azetidines/pharmacology , Enzyme Inhibitors/pharmacology , Lipoproteins, LDL/metabolism , Phospholipases A/metabolism , Sulfoxides/pharmacology , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Aryldialkylphosphatase , Chemotaxis, Leukocyte/drug effects , Esterases/antagonists & inhibitors , Humans , Oxidation-Reduction , Phosphatidylcholine-Sterol O-Acyltransferase/antagonists & inhibitors , Phospholipases A/antagonists & inhibitors , Phospholipases A2 , Protein Binding
5.
J Immunol ; 161(11): 6273-9, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9834116

ABSTRACT

A new CC chemokine, designated CKbeta-8 or myeloid progenitor inhibitor factor-1, was recently identified in a large scale sequencing effort and was cloned from a human aortic endothelial library. CKbeta-8 cDNA encodes a signal sequence of 21 amino acids, followed by a 99-amino acid predicted mature form. CKbeta-8 was expressed and purified from a baculovirus insect cell expression system, which resulted in the identification of different N-terminal variants of the secreted chemokine. The three major forms (containing amino acids 1-99, 24-99, and 25-99 of the secreted chemokine) showed a large variation in potency. CKbeta-8 activated both monocytes and eosinophils to mobilize intracellular calcium; however, the shortest form of CKbeta-8 (25-99) was >2 orders of magnitude more potent than the longest form. Cross-desensitization experiments in both monocytes and eosinophils suggested that the CCR1 receptor was probably the predominant receptor that mediates this chemokine's physiologic response. However, incomplete desensitization was encountered in both cell systems, suggesting involvement of an additional receptor(s). Interestingly, the short form of CKbeta-8 was the most potent chemotactic chemokine that we have ever evaluated in the monocyte system (EC50 = 54 pM). However, in contrast to its action on monocytes, CKbeta-8 was a very poor chemotactic factor for eosinophils.


Subject(s)
Chemokines, CC/chemistry , Chemokines, CC/isolation & purification , Amino Acid Sequence , Calcium/metabolism , Chemokines, CC/genetics , Chemokines, CC/metabolism , Chemotactic Factors, Eosinophil/physiology , Chemotaxis, Leukocyte/drug effects , Eosinophils/immunology , Eosinophils/metabolism , Humans , Molecular Sequence Data , Monocyte Chemoattractant Proteins/physiology , Monocytes/immunology , Monocytes/metabolism , Protein Isoforms/chemistry , Protein Isoforms/immunology , Protein Isoforms/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology
6.
Biochem J ; 289 ( Pt 3): 919-26, 1993 Feb 01.
Article in English | MEDLINE | ID: mdl-8435086

ABSTRACT

In this study, the effects of a series of phorbol esters with different spectra of biological activities and different patterns of activation of the isoenzymes of protein kinase C (PKC) have been studied in human neutrophils. The aim was to gain more information on which isoenzymes of PKC are involved in neutrophil activation, specifically inhibition of fMet-Leu-Phe (fMLP)-stimulated bivalent cation influx and stimulation of O2-. release (either alone or potentiation of the response to fMLP). Prior addition of both phorbol 12-myristate 13-acetate (PMA) and sapintoxin A (SAPA) inhibited fMLP-stimulated Mn2+ influx. Higher concentrations of resiniferatoxin (RX) were also inhibitory, inhibition being more apparent at longer preincubation times. However, 12-deoxyphorbol 13-O-phenylacetate (DOPPA) showed only a slight inhibitory effect and required a prolonged preincubation. PMA, SAPA and RX, but not DOPPA, stimulated O2-. release by themselves. Lower concentrations of PMA, SAPA and RX, which were ineffective alone, considerably potentiated O2-. release stimulated by fMLP, whereas DOPPA had little or no effect. These results rule out a major role for PKC-delta (not activated by SAPA) and PKC-beta 1 (activated by DOPPA), but suggest the involvement of RX kinase in addition to PKC in the inhibition of fMLP-stimulated Mn2+ influx and potentiation of fMLP-stimulated O2-. release. However, when the cytosolic free Ca2+ concentration ([Ca2+]i) was elevated with the Ca2+ ionophore ionomycin, DOPPA was able to stimulate O2-. release, which probably reflects the known Ca2+ requirement for activation of PKC-beta 1 by DOPPA in vitro. The effects of the other phorbols were also enhanced when [Ca2+]i was elevated; all of the phorbols synergize, to variable extents, with Ca2+ to activate PKC in vitro. Enhancement of RX-stimulated O2- release by elevation of [Ca2+]i was unexpected, since RX kinase has been reported to be inhibited by high concentrations of Ca2+ in vitro. Finally, use of fura-2 and SK&F 96365 to manipulate the fMLP-stimulated rise in [Ca2+]i showed that when fMLP was able to evoke its normal rise in [Ca2+]i (to a peak of 700-900 nM), O2-. release was potentiated by PMA, SAPA and RX. However, when fMLP was only able to evoke a small increase in [Ca2+]i (to a peak of 400 nM), potentiation by PMA was unaffected but potentiation by SAPA and RX was considerably reduced. This observation agrees with published data demonstrating that activation of PKC in vitro by SAPA is more Ca(2+)-dependent than activation by PMA.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Calcium/metabolism , Isoenzymes/metabolism , Neutrophils/physiology , Phorbol Esters/pharmacology , Protein Kinase C/metabolism , Diterpenes/pharmacology , Dose-Response Relationship, Drug , Enzyme Activation , Free Radicals , Humans , Imidazoles/pharmacology , Ionomycin/pharmacology , Manganese/metabolism , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Oxygen/metabolism , Platelet Aggregation Inhibitors/pharmacology , Tetradecanoylphorbol Acetate/pharmacology
8.
Eicosanoids ; 4(2): 89-94, 1991.
Article in English | MEDLINE | ID: mdl-1910866

ABSTRACT

Lipoxin A4 (LXA4), a lipoxygenase-derived metabolite of arachidonic acid, stimulated a dose-dependent elevation in cytosolic free Ca2+ concentration, [Ca2+]i, in fura-2-loaded human neutrophils, with an EC50 of 0.4-0.5 microM. The time for [Ca2+]i to peak was also dose-dependent. In the presence of extracellular Ca2+ (CaDT-PA added), the rise in [Ca2+]i was due to a combination of Ca2+ release from internal stores and influx of extracellular Ca2+. In the absence of extracellular Ca2+, the rise in [Ca2+]i was due to release from internal stores, which then became depleted. No response to LXA4 was seen in the absence of divalent cation chelators (EGTA or DTPA); this is presumably because LXA4 forms an inactive complex with heavy metal cations. In the presence of extracellular Ca2+, LXA4 had no effect on the subsequent response of neutrophils to the chemotactic peptide fmetleu-phe (fmlp). In the absence of extracellular Ca2+, LXA4 dose-dependently reduced the subsequent response of neutrophils to fmlp; this is presumably because LXA4 discharges the store, and so reduces the amount of Ca2+ available for subsequent release by fmlp.


Subject(s)
Calcium/metabolism , Cytosol/metabolism , Hydroxyeicosatetraenoic Acids/pharmacology , Lipoxins , Neutrophils/metabolism , Dose-Response Relationship, Drug , Humans , N-Formylmethionine Leucyl-Phenylalanine/pharmacology
9.
Cell Signal ; 3(3): 243-9, 1991.
Article in English | MEDLINE | ID: mdl-1892734

ABSTRACT

Stimulation of suspensions of fura-2-loaded human neutrophils with ATP resulted in an elevation in cytosolic free calcium concentration ([Ca2+]i) from a basal value of 0.1 microM to a transient peak of 1 microM. The response is due to Ca2+ release from intracellular stores and influx of extracellular Ca2+. Release from intracellular stores is shown by the response in the absence of extracellular Ca2+. The greater and more maintained response in the presence of extracellular Ca2+ is indicative of stimulated Ca2+ entry and a stimulated influx pathway was confirmed by using Mn2+ as a surrogate for Ca2+. A variety of purinergic agonists were used to characterize the pharmacology of this [Ca2+]i response. Their rank order of potency was ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) greater than ADP much greater than 2-methylthioadenosine 5'-triphosphate (2Me-SATP), where ATP had an EC50 value of 3 microM and 2MeSATP had an EC50 value of 1000 microM. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), adenylyl (alpha,beta-methylene)- diphosphonate (AMPCPP) and adenosine were inactive at 1 mM. These results suggest that neutrophils have a novel type of purinergic P2 receptor that is neither P2x nor P2y.


Subject(s)
Calcium/metabolism , Neutrophils/metabolism , Receptors, Purinergic/metabolism , Adenosine Triphosphate/metabolism , Animals , Erythrocytes/enzymology , Fura-2 , Humans , N-Formylmethionine Leucyl-Phenylalanine/metabolism , Turkeys , Type C Phospholipases/metabolism
10.
Biochem J ; 271(2): 515-22, 1990 Oct 15.
Article in English | MEDLINE | ID: mdl-2173565

ABSTRACT

A novel inhibitor of receptor-mediated calcium entry (RMCE) is described. SK&F 96365 (1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H- imidazole hydrochloride) is structurally distinct from the known 'calcium antagonists' and shows selectivity in blocking RMCE compared with receptor-mediated internal Ca2+ release. Human platelets, neutrophils and endothelial cells were loaded with the fluorescent Ca2(+)-indicator dyes quin2 or fura-2, in order to measure Ca2+ or Mn2+ entry through RMCE as well as Ca2+ release from internal stores. The IC50 (concn. producing 50% inhibition) for inhibition of RMCE by SK&F 96365 in platelets stimulated with ADP or thrombin was 8.5 microM or 11.7 microM respectively; these concentrations of SK&F 96365 did not affect internal Ca2+ release. Similar effects of SK&F 96365 were observed in suspensions of neutrophils and in single endothelial cells. SK&F 96365 also inhibited agonist-stimulated Mn2+ entry in platelets and neutrophils. The effects of SK&F 96365 were independent of cell type and of agonist, as would be expected for a compound that modulates post-receptor events. Voltage-gated Ca2+ entry in fura-2-loaded GH3 (pituitary) cells and rabbit ear-artery smooth-muscle cells held under voltage-clamp was also inhibited by SK&F 96365; however, the ATP-gated Ca2(+)-permeable channel of rabbit ear-artery smooth-muscle cells was unaffected by SK&F 96365. Thus SK&F 96365 (unlike the 'organic Ca2+ antagonists') shows no selectivity between voltage-gated Ca2+ entry and RMCE, although the lack of effect on ATP-gated channels indicates that it discriminates between different types of RMCE. The effects of SK&F 96365 on functional responses of cells thought to be dependent on Ca2+ entry via RMCE were also studied. Under conditions where platelet aggregation is dependent on stimulated Ca2+ entry via RMCE, the response was blocked by SK&F 96365 with an IC50 of 15.9 microM, which is similar to the IC50 of 8-12 microM observed for inhibition of RMCE. Adhesion and chemotaxis of neutrophils were also inhibited by SK&F 96365. SK&F 96365 is a useful tool to distinguish RMCE from internal Ca2+ release, and to probe the role of RMCE in mediating functional responses of cells. However, SK&F 96365 is not as potent (IC50 around 10 microM) or selective (also inhibits voltage-gated Ca2+ entry) as would be desirable, so caution must be exercised when using this compound.


Subject(s)
Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Calcium/metabolism , Imidazoles/pharmacology , Adenosine Diphosphate/pharmacology , Adenosine Triphosphate/pharmacology , Aminoquinolines , Animals , Blood Platelets/drug effects , Blood Platelets/metabolism , Cell Adhesion/drug effects , Chemotaxis, Leukocyte/drug effects , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Fluorescent Dyes , Fura-2 , Humans , Ion Channel Gating/drug effects , Manganese/metabolism , Neutrophils/drug effects , Neutrophils/metabolism , Platelet Aggregation Inhibitors/pharmacology , Rabbits , Umbilical Veins
11.
Biochem J ; 269(2): 513-9, 1990 Jul 15.
Article in English | MEDLINE | ID: mdl-2117443

ABSTRACT

A description is given of the methodology, and problems encountered, for the use of a new fluorescent Ca2(+)-indicator dye, fluo-3, in neutrophils and platelets. The higher Kd and longer excitation wavelength of fluo-3 can have significant advantages over fura-2. Although neutrophils and platelets are used as examples, these observations will be applicable to other cell types. The Kd of fluo-3 for binding Ca2+ at 37 degrees C was measured and found to be 864 nM; the previously published value was 400 nM at 22 degrees C. The Kd of fluo-3, like that of fura-2, is therefore very temperature-dependent. Protocols for loading cells, and preventing leakage of fluo-3, are described; probenecid, known to inhibit fura-2 leakage from cells, was found to be essential to get good fluo-3 signals from platelets. Calibration of fluo-3 fluorescence signals to [Ca2+] and methods for obtaining maximum and minimum fluorescence signals are described; these methods differ from those used with fura-2. Agonist-stimulated responses of fluo-3-loaded neutrophils and platelets are shown, and the calculated cytosolic [Ca2+] is comparable with that previously obtained with fura-2. Responses of cells in the presence of plasma are also shown; such measurements, unobtainable with quin2, fura-2 or indo-1, are possible with fluo-3, owing to its longer excitation wavelengths. Co-loading of cells with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid and fluo-3 is included as an example of how cytosolic [Ca2+] can be buffered and manipulated. Many of these observations will be of value when using fluo-3 (or other Ca2(+)-indicator dyes) in most cell types.


Subject(s)
Aniline Compounds , Blood Platelets/analysis , Calcium/blood , Cytosol/analysis , Fluorescent Dyes , Neutrophils/analysis , Xanthenes , Benzofurans , Blood , Blood Platelets/ultrastructure , Buffers , Digitonin/pharmacology , Egtazic Acid/pharmacology , Fura-2 , Humans , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/ultrastructure , Probenecid/pharmacology , Spectrometry, Fluorescence , Thrombin/pharmacology
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