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2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces rapid actin polymerization in HL-60 cells differentiated into macrophage-like cells.

ABSTRACT: Delta9-Tetrahydrocannabinol, a major psychoactive constituent of marijuana, interacts with specific receptors, i.e. the cannabinoid receptors, thereby eliciting a variety of pharmacological responses. To date, two types of cannabinoid receptors have been identified: the CB1 receptor, which is abundantly expressed in the nervous system, and the CB2 receptor, which is predominantly expressed in the immune system. Previously, we investigated in detail the structure-activity relationship of various cannabinoid receptor ligands and found that 2-AG (2-arachidonoylglycerol) is the most efficacious agonist. We have proposed that 2-AG is the true natural ligand for both the CB1 and CB2 receptors. Despite the potential physiological importance of 2-AG, not much information is available concerning its biological activities towards mammalian tissues and cells. In the present study, we examined the effect of 2-AG on morphology as well as the actin filament system in differentiated HL-60 cells, which express the CB2 receptor. We found that 2-AG induces rapid morphological changes such as the extension of pseudopods. We also found that it provokes a rapid actin polymerization in these cells. Actin polymerization induced by 2-AG was abolished when cells were treated with SR144528, a CB2 receptor antagonist, and pertussis toxin, suggesting that the response was mediated by the CB2 receptor and G(i/o). A phosphoinositide 3-kinase, Rho family small G-proteins and a tyrosine kinase were also suggested to be involved. Reorganization of the actin filament system is known to be indispensable for a variety of cellular events; it is possible that 2-AG plays physiologically essential roles in various inflammatory cells and immune-competent cells by inducing a rapid actin rearrangement.

SUBMITTER: Gokoh M

PROVIDER: S-EPMC1134878 | biostudies-other | 2005 Mar

REPOSITORIES: biostudies-other

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Project description:Synthetic lipopeptides activate superoxide-anion (O2-) formation in human neutrophils in a pertussis-toxin (PTX)-sensitive manner, suggesting the involvement of G-proteins of the Gi family in the signal-transduction pathway. We compared G-protein activation by lipopeptides and the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in dibutyryl-cyclic-AMP-differentiated HL-60 cells. The lipopeptide (2S)-2-palmitoylamino-6-palmitoyloxymethyl-7-palmitoyloxy heptanoyl-SK4 (Pam3AhhSK4) and fMLP activated high-affinity GTPase, i.e. the enzymic activity of G-protein alpha-subunits, in HL-60 membranes in a time- and protein-dependent manner, but they had no effect on Mg(2+)-ATPase and Na+/K(+)-ATPase. Pam3AhhSK4 and fMLP increased Vmax. of GTP hydrolysis. Pam3AhhSK4 activated GTP hydrolysis with half-maximal and maximal effects at about 2 microM and 10 microM respectively. Other lipopeptides activated GTP hydrolysis as well. Lipopeptides were less effective than fMLP to activate GTPase. In membranes from PTX-treated cells, the stimulatory effects of lipopeptides and fMLP on GTPase were abolished. In N-ethylmaleimide-treated membranes, the relative stimulatory effect of Pam3AhhSK4 on GTP hydrolysis was enhanced, whereas that of fMLP was diminished. fMLP and Pam3AhhSK4 activated GTPase in an over-additive manner in N-ethylmaleimide-treated membranes. Unlike fMLP, Pam3AhhSK4 did not enhance incorporation of GTP azidoanilide into, and cholera-toxin-catalysed ADP-ribosylation of Gi-protein alpha-subunits in, HL-60 membranes and did not induce rises in cytosolic Ca2+ concentration. Pam3AhhSK4 and fMLP stimulated phosphatidic acid formation in a PTX-sensitive manner. Pam3AhhSK4 itself did not activate O2- formation, but potentiated the stimulatory effects of fMLP. Our data suggest that (i) lipopeptides activate the GTPase of Gi-proteins, (ii) lipopeptides and fMLP activate Gi-proteins differently, (iii) lipopeptides stimulate phospholipase D via Gi-proteins, and (iv) phosphatidic acid formation is not sufficient for activation of O2- formation.

Project description:HL-60 cells differentiated with DMSO increased their rates of uptake of ascorbate when they were activated with PMA. The rates observed after this activation were essentially the same as those with dehydroascorbic acid as the original transport substrate. The effect of activation was sensitive to the antioxidant enzymes superoxide dismutase and catalase. When ascorbate was oxidized in situ by chemical or enzymic oxidation, the rates of uptake were similar to those after activation of the cells by phorbol ester; however, in the latter case the extracellular vitamin remained largely in the reduced form and there was very little loss by degradation, whereas after immediate oxidation no more reduced ascorbate could be found outside the cells after a few minutes and a significant part of the total vitamin was lost. The generation of superoxide by xanthine/xanthine oxidase stimulated the uptake of ascorbate much less than the activation by phorbol ester; H(2)O(2) was even less effective. Stimulation of the uptake by phorbol ester was also insensitive to GSH, in contrast with stimulation by the chemical oxidation of ascorbate. Stimulation of ascorbate uptake by phorbol ester was sensitive to the respiratory-burst inhibitor diphenyliodonium as well as the protein kinase C inhibitor staurosporine, indicating the respiratory burst as the cause of stimulation. Activation of the cells by the phorbol ester also stimulated the uptake of dehydroascorbate as the original substrate, in a manner insensitive to antioxidants or inhibitors of the respiratory burst. In all cases the intracellular vitamin was completely in the reduced form. Kinetic characterization by the calculation of maximal velocities and apparent K(m) values and assaying for the dependence of uptake rates on the ionic milieu and for inhibition by glucose analogues and inhibitors of glucose transport revealed that after treatment with phorbol ester the uptake of total vitamin C in differentiated HL-60 cells was largely due to the low-affinity high-capacity glucose transporter. In contrast, in non-stimulated cells reduced ascorbate was taken up by the Na(+)-dependent high-affinity low-capacity ascorbate transporter. This change was probably due to the oxidation of ascorbate and, simultaneously, the recruitment of additional transporter molecules to the cell surface.

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2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces rapid actin polymerization in HL-60 cells differentiated into macrophage-like cells.

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