Analogues and homologues of N-palmitoylethanolamide, a putative endogenous CB(2) cannabinoid, as potential ligands for the cannabinoid receptors

in Biochimica et Biophysica Acta (1999), 1440(2-3), 266-74

The presence of CB(2) receptors was reported in the rat basophilic cell line RBL-2H3 and N-palmitoylethanolamide was proposed as an endogenous, potent agonist of this receptor. We synthesized a series of ... [more ▼]

The presence of CB(2) receptors was reported in the rat basophilic cell line RBL-2H3 and N-palmitoylethanolamide was proposed as an endogenous, potent agonist of this receptor. We synthesized a series of 10 N-palmitoylethanolamide homologues and analogues, varying by the elongation of the fatty acid chain from caproyl to stearoyl and by the nature of the amide substituent, respectively, and evaluated the affinity of these compounds to cannabinoid receptors in the rat spleen, RBL-2H3 cells and CHO-CB(1) and CHO-CB(2) receptor-transfected cells. In rat spleen slices, CB(2) receptors were the predominant form of the cannabinoid receptors. No binding of [(3)H]SR141716A was observed. [(3)H]CP-55,940 binding was displaced by WIN 55,212-2 and anandamide. No displacement of [(3)H]CP-55,940 or [(3)H]WIN 55,212-2 by palmitoylethanolamide derivatives was observed in rat spleen slices. In RBL-2H3 cells, no binding of [(3)H]CP-55,940 or [(3)H]WIN 55,212-2 could be observed and conversely, no inhibitory activity of N-palmitoylethanolamide derivatives and analogues was measurable. These compounds do not recognize the human CB(1) and CB(2) receptors expressed in CHO cells. In conclusion, N-palmitoylethanolamide was, in our preparations, a weak ligand while its synthesized homologues or analogues were essentially inactive. Therefore, it seems unlikely that N-palmitoylethanolamide is an endogenous agonist of the CB(2) receptors but it may be a compound with potential therapeutic applications since it may act via other mechanisms than cannabinoid CB(1)-CB(2) receptor interactions. [less ▲]

Release of O2- by human umbilical cord blood-derived eosinophils: role of intra- and extracellular calcium

in Cell Calcium (1999), 25(5), 381-9

The aim of our study was to investigate the physiologic mechanisms involved in eosinophil activation as an essential prerequisite to disrupting the biochemical cascade that triggers inflammation, thereby ... [more ▼]

The aim of our study was to investigate the physiologic mechanisms involved in eosinophil activation as an essential prerequisite to disrupting the biochemical cascade that triggers inflammation, thereby attenuating the effect of this activation or, ideally, preventing it from occurring. We have, therefore, examined the nature of the fMLP- and PAF-induced [Ca2+]i rise and the relationship between the [Ca2+]i rise and O2- production in human umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5. These cells responded to fMLP or PAF (1 microM each) with an increase in [Ca2+]i (217.3 +/- 22.1 and 197.8 +/- 22.1 nM respectively) which was associated with production of O2- (40.2 +/- 8.2 and 35.2 +/- 7.6 pmol/min/10(6) cells respectively). The role of Ca2+ in the induced respiratory burst was studied by changing the availability of Ca2+ in the intra- and extracellular compartments. Removal or chelation of extracellular Ca2+ induced a reduction of both the fMLP and PAF-induced [Ca2+]i rise and O2- production. Chelation of intracellular Ca2+ induced a concentration-dependent inhibition of fMLP- and PAF-induced [Ca2+]i rise and caused a decrease in O2- production. SK&F 96365 had a stimulatory effect on PAF-induced [Ca2+]i rise and on fMLP-induced O2- production, this phenomenon was not observed with extracellular Ca2+ removal or chelation. Furthermore, Ni2+ exhibited an inhibition of both fMLP and PAF-induced [Ca2+]i rise and O2- production. Finally, both fMLP and PAF induced an increase in divalent cation influx that was further augmented by thapsigargin. Our results indicate that fMLP and PAF dependent O2- production in human eosinophils require intra- and extracellular Ca2+ and that Ca2+ influx is necessary for optimal activation. [less ▲]

Human umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5 respond to fMLP with [Ca2+]i variation and O2- production

in Journal of Immunological Methods (1997), 205(1), 1-9

In the presence of interleukin-3 and interleukin-5, eosinophil precursors from human umbilical cord blood mononuclear cells were regularly differentiated into mature eosinophil-like cells expressing ... [more ▼]

In the presence of interleukin-3 and interleukin-5, eosinophil precursors from human umbilical cord blood mononuclear cells were regularly differentiated into mature eosinophil-like cells expressing normal morphology and cyanide-resistant peroxidase. O2- production and [Ca2+]i rise were measured in these in vitro differentiated eosinophils after fMLP stimulation; with dihydrorhodamine-123 and fura-2, respectively. Umbilical cord blood-derived eosinophils responded to fMLP (0.01 nM to 3 microM) with a concentration-dependent production of O2- (EC50 = 63.1 +/- 17.2 nM; Emax = 71.0 +/- 6.2 pmol/min/10(6) cells). O2- production was correlated with an fMLP concentration-dependent increase in [Ca2+]i (EC50 = 32.5 +/- 14.9 nM; Emax = 200.0 +/- 23.9 nM). These results indicate that human umbilical cord blood-derived eosinophils demonstrate functional characteristics similar to adult human peripheral blood eosinophils after activation by fMLP. Therefore, the large numbers of eosinophils (2-3 x 10(6)/ml cord blood) which can be obtained by culture of human cord blood mononuclear cells may serve as a useful model for future studies which will provide insight into the pathogenesis of diseases associated with eosinophils. [less ▲]

A double-labelling fluorescent assay for concomitant measurements of [Ca2+]i and O2. production in human macrophages

in Biochimica et Biophysica Acta (1995), 1244(1), 79-84

To measure intracellular free Ca2+ concentration ([Ca2+]i) and superoxide (O2) production in human alveolar macrophages, we used the fluorescent Ca2+ indicator fura-2 and the O2-sensitive dye ... [more ▼]

To measure intracellular free Ca2+ concentration ([Ca2+]i) and superoxide (O2) production in human alveolar macrophages, we used the fluorescent Ca2+ indicator fura-2 and the O2-sensitive dye dihydrorhodamine-123, which becomes fluorescent in its oxidized form, rhodamine-123. We describe a new double-dye technique whereby the kinetics of both [Ca2+]i levels and O2. production can be monitored simultaneously. This technique was developed in the dimethylsulfoxide-differentiated monocytic-like U-937 cell line (not equal to U-937), validated by comparison with single dye measurements and applied to human alveolar macrophages. The chemotactic peptide N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine induced in both cell types a similar transient elevation in [Ca2+]i, followed within seconds by a sustained increase in O2 production, which was however 4-fold weaker in not equal to U-937 cells. These results indicate that O2 production is an early event following the stimulation of human alveolar macrophages. This new double-dye technique may be relevant to other O2 ion-producing cells and could help to define more precisely the kinetics of the events leading to this biological response. [less ▲]

Natural polyamines stimulate G-proteins

in Biochemical Journal (1992), 282 (Pt 2)

The natural polyamines spermine and spermidine, the biosynthetic precursor putrescine and their analogues cadaverine and tyramine stimulate the GTPase activity of purified GTP-binding proteins (Go/Gi ... [more ▼]

The natural polyamines spermine and spermidine, the biosynthetic precursor putrescine and their analogues cadaverine and tyramine stimulate the GTPase activity of purified GTP-binding proteins (Go/Gi) from calf brain reconstituted into phospholipid vesicles. The order of potency was spermine greater than spermidine greater than putrescine = cadaverine greater than tyramine. The physiological relevance of this observation was assessed, showing the same order of potency of polyamines in the stimulation of peritoneal and tracheal rat mast cells. The activation of rat mast cells by polyamines was inhibited by benzalkonium chloride or by a 2 h pretreatment of the cells with pertussis toxin. The increase in inositol phosphates evoked by polyamines was also inhibited by pertussis toxin. Therefore we propose that intracellular polyamines might control the basal level of second messengers and modulate extracellular signals transduced through G-protein-coupled receptors. [less ▲]

G-proteins as targets for non-immunological histamine releasers

in Agents and Actions (1991), 33(1-2), 81-3

The molecular mechanism of action of several non-immunological histamine releasers has been investigated using pertussis toxin which interfers, via ADP-ribosylation, with some G-proteins. Pertussis toxin ... [more ▼]

The molecular mechanism of action of several non-immunological histamine releasers has been investigated using pertussis toxin which interfers, via ADP-ribosylation, with some G-proteins. Pertussis toxin (100 ng/ml) inhibited histamine release induced by compound 48/80, substance P, mastoparan, peptide 401, bradykinin and spermine showing that a G-protein sensitive to pertussis toxin was involved in the non-immunological histamine release. All these compounds directly activate purified G-proteins. The sensitivity to pertussis toxin of this direct stimulatory effect was demonstrated for compound 48/80, mastoparan and substance P. Altogether these results suggest that a direct activation of G-protein might be the molecular mechanism of action of histamine secretagogues acting through a pertussis toxin sensitive G-protein and in this way mimic agonist-ligand receptor interaction. [less ▲]

Activation of Gi-like proteins, a receptor-independent effect of kinins in mast cells

in Molecular Pharmacology (1991), 38(6), 816-22

The peptide hormones bradykinin and kallidin (Lys-bradykinin), as well as their analogues [des-Arg9]-bradykinin, a selective B1 agonist, [des-Arg9,Leu8]-bradykinin, a selective B1 antagonist, and [Thi5,8 ... [more ▼]

The peptide hormones bradykinin and kallidin (Lys-bradykinin), as well as their analogues [des-Arg9]-bradykinin, a selective B1 agonist, [des-Arg9,Leu8]-bradykinin, a selective B1 antagonist, and [Thi5,8,D-Phe7]-bradykinin and D-Arg0-[Hyp3,D-Phe7]-bradykinin, two selective B2 antagonists, induced rapid histamine release from purified rat peritoneal mast cells. In contrast, the N-terminal fragment bradykinin-(1-5) was inactive. These peptides also activate the GTPase activity of GTP-binding proteins (G proteins) (Go/Gi) purified from calf brain, with an order of potency identical to that observed on mast cells, [Thi5,8,D-Phe7]-bradykinin much greater than kallidin greater than bradykinin greater than D-Arg0-[Hyp3,D-Phe7]-bradykinin greater than [des-Arg9]-bradykinin greater than [des-Arg9,Leu8]-bradykinin greater than bradykinin-(1-5). This correlation suggested that G proteins are the targets of kinins in mast cells. Accordingly, the concomitant increase in inositol trisphosphates and release of histamine elicited by kinins were inhibited by pertussis toxin pretreatment of mast cells. The inhibitory effect of benzalkonium chloride showed that the G proteins involved belong to the Gi type. GTPase activity was measured in the supernatant of homogenized mast cells but not in the membranous fraction. This activity was stimulated by kinins and by the venom peptide mastoparan. The potency of peptides was similar to that observed with purified bovine G proteins. Sodium dodecyl sulfate-gel electrophoresis of mast cell supernatant revealed pertussis toxin-induced ADP-ribosylation of two proteins, in the Mr 41,000 and 40,000 range, i.e., similar to purified alpha-subunits of Gi1 and Gi2 or Gi3 subtypes. The data support the proposal that bradykinin and analogues act like mastoparan, substance P, and compound 48/80, interacting first with sialic acid residues of the cell surface and then with Gi-like proteins, inducing phospholipase C activation and intracellular calcium mobilization. [less ▲]

Oxatomide and calcium: mechanisms involved in the secretion of mast cell mediators

in Annales de Dermatologie et de Vénéréologie (1990), 117 Suppl 1

Human cutaneous mast cells and their experimental model rat peritoneal mast cells, can be stimulated by an IgE-dependent process or by peptides through the direct activation of G proteins. Both activation ... [more ▼]

Human cutaneous mast cells and their experimental model rat peritoneal mast cells, can be stimulated by an IgE-dependent process or by peptides through the direct activation of G proteins. Both activation pathways lead to the increase of cytosolic Ca2+ level. This increase in dependent of the mobilisation of intracellular calcium stores of the endoplasmic reticulum involving the stimulation of IP3-sensitive calcium channels. Mast cells are characterized by the absence of calcium channels in the plasma membrane. Oxatomide has been synthetized as an analog of cinnarizine. However oxatomide is inactive on current calcium channels. In mast cells, oxatomide inhibits the increase of cytosolic calcium elicited during mast cell activation. Consequently mast cell exocytosis is inhibited altogether with the release of newly synthetized mediators. The authors propose several putative targets for oxatomide in mast cells. The therapeutic effect of oxatomide is also related to its property to antagonize the effects of anaphylactic mediators on their selective receptors. [less ▲]

Neuropeptides and inflammation: presumed mechanisms in neurogenic inflammation

in Pathologie et Biologie (1990), 38(1), 53-6

Neuropeptides, among which substance P, VIP (Vasoactive intestinal peptide), somatostatin, neurotensin, dynorphin and enkephalins, are able to modulate inflammatory processes. Increasing interest is now ... [more ▼]

Neuropeptides, among which substance P, VIP (Vasoactive intestinal peptide), somatostatin, neurotensin, dynorphin and enkephalins, are able to modulate inflammatory processes. Increasing interest is now devoted to these peptides in different inflammatory diseases, concerning skin, lung and joins. The effect of substance P can be dependent on its C-terminal moiety implicating by this way an interaction with specific neurokinin receptors or can be dependent on its N-terminal moiety which does not involve a specific membrane receptor. Such diversity of the action mechanisms of peptides should influence the evolution of the anti-inflammatory therapeutic. [less ▲]