1-oleoyl-2-acetylglycerol and Glioma

Astrocytes express transient receptor potential channels (TRPCs), which have been implicated in Ca 2+ influx triggered by intracellular Ca 2+ stores depletion, a phenomenon known as capacitative Ca 2+ entry. We studied the properties of capacitative Ca 2+ entry in astrocytes by means of single-cell Ca 2+ imaging with the aim of understanding the involvement of TRPCs in this function. We found that, in astrocytes, capacitative Ca 2+ entry is not attributable to TRPC opening because the TRPC-permeable ions Sr2+ and Ba2+ do not enter astrocytes during capacitative Ca 2+ entry. Instead, natively expressed oleyl-acetyl-glycerol (OAG) (a structural analog of DAG) -sensitive TRPCs, when activated, initiate oscillations of cytosolic Ca 2+ concentration ([Ca 2+]i) pharmacologically and molecularly consistent with TRPC3 activation. OAG-induced [Ca 2+]i oscillations are not affected by inhibition of inositol trisphosphate (InsP3) production or blockade of the InsP3 receptor, therefore representing a novel form of [Ca 2+]i signaling. Instead, high [Ca 2+]i inhibited oscillations, by closing the OAG-sensitive channel. Also, treatment of astrocytes with antisense against TRPC3 caused a consistent decrease of the cells responding to OAG. Exogenous OAG but not endogenous DAG seems to activate TRPC3. In conclusion, in glial cells, natively expressed TRPC3s mediates a novel form of Ca 2+ signaling, distinct from capacitative Ca 2+ entry, which suggests a specific signaling function for this channel in glial cells.

Topics: Adenosine Triphosphate; Animals; Astrocytes; Biological Clocks; Calcium; Calcium Channels; Calcium Signaling; Cells, Cultured; Diglycerides; Enzyme Inhibitors; Fluorescent Dyes; Glioma; Immunohistochemistry; Intracellular Fluid; Ion Channels; Neuroglia; Oligonucleotides, Antisense; Protein Isoforms; Rats; Rats, Wistar; Strontium; Thapsigargin; TRPC Cation Channels

The cellular factors regulating the generation of beta-amyloid from the amyloid precursor protein (APP) are unknown. Activation of protein kinase C (PKC) by phorbol ester treatment inhibited the generation of the 4-kDa beta-amyloid peptide in transfected COS cells, a human glioma cell line, and human cortical astrocytes. An analogue of diacylglycerol, the endogenous cellular activator of PKC, also inhibited the generation of beta-amyloid. Activation of PKC increased the level of secreted APP in transfected COS cells but did not significantly affect the level of secreted APP in primary human astrocytes or in the glioma cell line. Cell-associated APP and the secreted APP derivative, but not beta-amyloid, were phosphorylated on serine residues. Activation of PKC did not increase the level of APP phosphorylation, suggesting that PKC modulates the proteolytic cleavage of APP indirectly by phosphorylation of other substrates. These results indicate that PKC activation inhibits beta-amyloid production by altering APP processing and suggest that beta-amyloid production can be regulated by the phospholipase C-diacylglycerol signal transduction pathway.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Astrocytes; Carcinogens; Cell Line; Cells, Cultured; Cerebral Cortex; Diglycerides; Enzyme Activation; Female; Fetus; Glioma; Humans; Phorbol Esters; Protease Inhibitors; Protein Kinase C; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

12-O-Tetradecanoylphorbol-13-acetate (TPA), a tumor promoter and potent activator of protein kinase C, stimulates [3H]choline incorporation into phosphatidylcholine (PtdCho) in NG108-15 cells (Liscovitch, M., Freese, A., Blusztajn, J. K. and Wurtman, R. J. (1986) J. Neurochem. 47, 1936-1941). In the present study we demonstrate that two cell-permeant diacylglycerols, sn-1-oleoyl-2-acetylglycerol and sn-1,2-dioctanoylglycerol, also stimulate [3H]choline incorporation into PtdCho. However, the effect of diacylglycerol is additional to that produced by a maximally effective concentration of TPA (0.5 microM), suggesting that the two agents may not act via the same mechanism. In addition, the protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (at 200 microM) inhibits the action of TPA by 59% while not affecting that of diacylglycerol. Finally, preincubation of the cells with TPA (0.1 microM) for 24 h reduces protein kinase C activity in the cells and completely abolishes the effect of additional TPA on choline incorporation. In contrast, diacylglycerol-induced stimulation of PtdCho biosynthesis was not inhibited in the cells that were desensitized to TPA. These results suggest that the effect of the two cell-permeant diacylglycerols on PtdCho biosynthesis either is not mediated by protein kinase C activation, or, is mediated by a TPA-insensitive isoenzyme of protein kinase C.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Choline; Chromatography, High Pressure Liquid; Diglycerides; Glioma; Glycerides; Hybrid Cells; Isoquinolines; Neuroblastoma; Phosphatidylcholines; Piperazines; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The effect of 1-oleoyl-2-acetyl-glycerol (OAG) and the phorbol diester 12-O-tetradecanoyl-phorbol-acetate (TPA) on the intracellular Ca2+ concentration ([Ca2+]i) in NG108-15 cells was studied using a Ca2+ indicator, quin 2. OAG and TPA induced an increase in [Ca2+]i from 100 +/- 19 to 187 +/- 24 nM and 192 +/- 15 nM, respectively, within 15 min. The increase in [Ca2+]i induced by activators of protein kinase C was dependent on the extracellular Ca2+ concentration [Ca2+]o) and was inhibited by the Ca2+ blockers, verapamil and nifedipine. These results indicate that the OAG- and TPA-induced [Ca2+]i increase is mediated by the influx of extracellular Ca2+ through voltage-sensitive Ca2+ channels.

Topics: Calcium; Diglycerides; Glioma; Glycerides; Humans; Hybrid Cells; Ion Channels; Kinetics; Nervous System Neoplasms; Neuroblastoma; Tetradecanoylphorbol Acetate

The addition of bradykinin to NG108-15 cells results in a transient hyperpolarization followed by prolonged cell depolarization. Injection of inositol 1,4,5-trisphosphate or Ca2+ into the cytoplasm of NG108-15 cells also elicits cell hyperpolarization followed by depolarization. Tetraethylammonium ions inhibit the hyperpolarizing response of cells to bradykinin or inositol 1,4,5-trisphosphate. Thus, the hyperpolarizing phase of the cell response may be due to inositol 1,4,5-trisphosphate-dependent release of stored Ca2+ into the cytoplasm, which activates Ca2+-dependent K+ channels. The depolarizing phase of the cell response to bradykinin is due largely to inhibition of M channels, thereby decreasing the rate of K+ efflux from cells and, to a lesser extent, to activation of Ca2+-dependent ion channels and Ca2+ channels. In contrast, injection of inositol 1,4,5-trisphosphate or Ca2+ into the cytosol did not alter M channel activity. Incubation of NG108-15 cells with pertussis toxin inhibits bradykinin-dependent cell hyperpolarization and depolarization. Bradykinin stimulates low Km GTPase activity and inhibits adenylate cyclase in NG108-15 membrane preparations but not in membranes prepared from cells treated with pertussis toxin. Reconstitution of NG108-15 membranes from cells treated with pertussis toxin with nanomolar concentrations of a mixture of highly purified No and Ni [guanine nucleotide-binding proteins that have no known function (No) or inhibit adenylate cyclase (Ni)] restores bradykinin-dependent activation of GTPase and inhibition of adenylate cyclase. These results show that [bradykinin . receptor] complexes interact with No or Ni and suggest that No and/or Ni mediate the transduction of signals from bradykinin receptors to phospholipase C and adenylate cyclase.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Bradykinin; Calcium; Cell Line; Diglycerides; Glioma; GTP-Binding Proteins; Humans; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Membrane Potentials; Neuroblastoma; Pertussis Toxin; Sugar Phosphates; Tetradecanoylphorbol Acetate; Tetraethylammonium; Tetraethylammonium Compounds; Virulence Factors, Bordetella