d-609 and 1-oleoyl-2-acetylglycerol

1. In visceral smooth muscles, both M(2) and M(3) muscarinic receptor subtypes are found, and produce two major metabolic effects: adenylyl cyclase inhibition and PLCbeta activation. Thus, we studied their relevance for muscarinic cationic current (mI(CAT)) generation, which underlies cholinergic excitation. Experiments were performed on single guinea-pig ileal cells using patch-clamp recording techniques under conditions of weakly buffered [Ca(2+)](i) (either using 50 microm EGTA or 50-100 microm fluo-3 for confocal fluorescence imaging) or with [Ca(2+)](i) 'clamped' at 100 nm using 10 mm BAPTA/CaCl(2) mixture. 2. Using a cAMP-elevating agent (1 microm isoproterenol) or a membrane-permeable cAMP analog (10 microm 8-Br-cAMP), we found no evidence for mI(CAT) modulation through a cAMP/PKA pathway. 3. With low [Ca(2+)](i) buffering, the PLC blocker U-73122 at 2.5 microm almost abolished mI(CAT), in some cases without any significant effect on [Ca(2+)](i). When [Ca(2+)](i) was buffered at 100 nm, U-73122 reduced both carbachol- and GTPgammaS-induced mI(CAT) maximal conductances (IC(50)=0.5-0.6 microm) and shifted their activation curves positively. 4. U-73343, a weak PLC blocker, had no effect on GTPgammaS-induced mI(CAT), but weakly inhibited carbachol-induced current, possibly by competitively inhibiting muscarinic receptors, since the inhibition could be prevented by increasing the carbachol concentration to 1 mm. Aristolochic acid and D-609, which inhibit PLA(2) and phosphatidylcholine-specific PLC, respectively, had no or very small effects on mI(CAT), suggesting that these enzymes were not involved. 5. InsP(3) (1 microm) in the pipette or OAG (20 microm) applied externally had no effect on mI(CAT) or its inhibition by U-73122. Ca(2+) store depletion (evoked by InsP(3), or by combined cyclopiazonic acid, ryanodine and caffeine treatment) did not induce any significant current, and had no effect on mI(CAT) in response to carbachol when [Ca(2+)](i) was strongly buffered to 100 nm. 6. It is concluded that phosphatidylinositol-specific PLC modulates mI(CAT) via Ca(2+) release, but also does so independently of InsP(3), DAG, Ca(2+) store depletion or a rise of [Ca(2+)](i). Our present results explain the previously established 'permissive' role of the M(3) receptor subtype in mI(CAT) generation, and provide a new insight into the molecular mechanisms underlying the shifts of the cationic conductance activation curve.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenylyl Cyclase Inhibitors; Animals; Aristolochic Acids; Bridged-Ring Compounds; Caffeine; Calcium; Carbachol; Diglycerides; Estrenes; Guanosine Triphosphate; Guinea Pigs; Ileum; Indoles; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Isoenzymes; Isoproterenol; Male; Membrane Potentials; Muscle, Smooth; Norbornanes; Patch-Clamp Techniques; Phospholipase C beta; Phospholipases A; Pyrrolidinones; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Ryanodine; Thiocarbamates; Thiones; Type C Phospholipases

Tricyclodecan-9-yl-xanthogenate (D609) is a selective tumor cytotoxic agent. However, the mechanisms of action of D609 against tumor cells have not been well established. Using U937 human monocytic leukemia cells, we examined the ability of D609 to inhibit sphingomyelin synthase (SMS), since inhibition of SMS may contribute to D609-induced tumor cell cytotoxicity via modulating the cellular levels of ceramide and diacylglycerol (DAG). The results showed that D609 is capable of inducing U937 cell death by apoptosis in a dose- and time-dependent manner. The induction of U937 cell apoptosis was associated with an inhibition of SMS activity and a significant increase in the intracellular level of ceramide and decrease in that of sphingomyelin (SM) and DAG, which resulted in an elevation of the ratio between ceramide and DAG favoring the induction of apoptosis. In addition, incubation of U937 cells with C(6)-ceramide and/or H7 (a selective PKC inhibitor) reduced U937 cell viability; whereas pretreatment of the cells with a PKC activator, PMA or 1-oleoyl-2-acetylglycerol (OAG), attenuated D609-induced U937 cell apoptosis. These results suggest that SMS is a potential target of D609 and inhibition of SMS may contribute to D609-induced tumor cell death via modulation of the cellular levels of ceramide and DAG.

Topics: Antineoplastic Agents; Apoptosis; Bridged-Ring Compounds; Ceramides; Cytotoxins; Diglycerides; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Humans; Leukemia, Monocytic, Acute; Norbornanes; Protein Kinase C; Reaction Time; Tetradecanoylphorbol Acetate; Thiocarbamates; Thiones; Transferases (Other Substituted Phosphate Groups); U937 Cells; Up-Regulation

Phospholipase activities are thought to be involved in the activation of macrophages by lipopolysaccharide (LPS). Because our previous studies showed that the synthetic lipopeptide JBT3002 might activate macrophages via signaling pathways similar to those used by LPS, we investigated whether phospholipase activities are required for activation of macrophages by JBT3002. Treatment of RAW264.7 murine macrophage-like cells with JBT3002 stimulated expression of both inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner. The JBT3002-induced production of nitric oxide and TNF-alpha was significantly inhibited by tricyclodecan-9-yl xanthogenate (D609), a selective inhibitor of phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). JBT3002-induced expression of steady-state mRNA for both iNOS and TNF-alpha was inhibited by D609. Cells treated with JBT3002 had greater production of diacylglycerol (DAG) in 2 min, which lasted for at least 30 min and could be blocked by D609. Activation of RAW264.7 cells was not affected by butanol, a PC-specific phospholipase D inhibitor, and treatment with JBT3002 did not affect phosphatidic acid formation. RAW264.7 cells treated with DAG analogue 1-oleoyl-2-acetyl-sn-glycerol, in the presence of interferon-gamma, produced TNF-alpha. These results suggested that activation of RAW264.7 cells by JBT3002 requires PC-PLC activity.

Topics: 1-Butanol; Animals; Bridged-Ring Compounds; Cell Line; Diglycerides; Enzyme Activation; Enzyme Induction; Enzyme Inhibitors; Estrenes; Gene Expression Regulation; Interferon-gamma; Lipopeptides; Lipoproteins; Macrophage Activation; Macrophages; Mice; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Norbornanes; Phosphatidic Acids; Phosphatidylinositol Diacylglycerol-Lyase; Phospholipase D; Pyrrolidinones; RNA, Messenger; Thiocarbamates; Thiones; Tumor Necrosis Factor-alpha; Type C Phospholipases