phosphatidylethanol and Astrocytoma

In [3H]myristic acid-prelabeled Chinese hamster ovary cells stably expressing the rat NK1 tachykinin receptor, the selective NK1 agonist [Pro9]substance P ([Pro9]SP) time and concentration dependently stimulated the formation of [3H]phosphatidylethanol in the presence of ethanol. This [Pro9]SP-induced activation of phospholipase D (PLD) was blocked by NK1 receptor antagonists and poorly or not mimicked by NK2 and NK3 agonists, respectively. In confirmation of previous observations, [Pro9]SP also stimulated the hydrolysis of phosphoinositides, the release of arachidonic acid, and the formation of cyclic AMP (cAMP). All these [Pro9]SP-evoked responses could be mimicked by aluminum fluoride, but they remained unaffected in cells pretreated with pertussis toxin, suggesting that a Gi/Go protein is not involved in these different signaling pathways. The activation of PLD by [Pro9]SP was sensitive to external calcium and required an active protein kinase C because the inhibition of this kinase (Ro 31-8220) or its down-regulation (long-term treatment with a phorbol ester) abolished the response. In contrast, a cAMP-dependent process was not involved in the activation of PLD because the [Pro9]SP-evoked response was neither affected by Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate nor mimicked by cAMP-generating compounds (cholera toxin or forskolin) or by 8-bromo-cyclic AMP. A functional coupling of NK1 receptors to PLD was also demonstrated in the human astrocytoma cell line U 373 MG stimulated by SP or [Pro9]SP. These results suggest that PLD activation could be an additional signaling pathway involved in the mechanism of action of SP in target cells expressing NK1 receptors.

Topics: Animals; Astrocytoma; Calcium; CHO Cells; Cricetinae; Cyclic AMP; Enzyme Activation; Glycerophospholipids; GTP-Binding Proteins; Humans; Myristic Acid; Phosphatidic Acids; Phosphatidylcholines; Phospholipase D; Protein Kinase C; Rats; Receptors, Neurokinin-1; Substance P; Tachykinins; Tumor Cells, Cultured

Activation of the M3 muscarinic receptor in 1321N1 human astrocytoma cells leads to increased phospholipase D (PLD)-catalyzed hydrolysis of phosphatidylcholine, which is maximal within 1 min of exposure to agonist. Studies examining the kinetics of phosphatidylethanol formation indicate that there is no further PLD activation beyond this time. Thrombin, a mitogen for quiescent 1321N1 cells, also activates PLD only transiently. The PLD response does not recover for up to 1 hr and cells that have been exposed to carbachol or thrombin do not respond to subsequent challenge with the heterologous agonist. In contrast to the desensitization observed with agonists, phorbol-12-myristate-13-acetate induces a sustained stimulation of PLD. In addition, cells pretreated with carbachol or thrombin show a normal response to phorbol-12-myristate-13-acetate, suggesting that the enzymatic activity of PLD is not compromised. Guanosine-5'-O-(3-thio)triphosphate activation of PLD in cell-free homogenates is also unaffected by prior treatment of the cells with agonist. Agonist-stimulated PLD activation in 1321N1 cells is mediated by protein kinase C (PKC). Thrombin and carbachol cause comparable changes in redistribution of both PKC-alpha and PKC-epsilon. The increase in membrane-associated PKC-alpha is transient and is reinitiated by addition of the heterologous agonist, whereas PKC-epsilon remains membrane associated for at least 60 min and is not further increased by addition of the heterologous agonist. We suggest that desensitization of PLD activation results from the down-regulation of an as yet undefined mediator required for agonist receptor coupling to PLD and that PKC-epsilon might participate in this down-regulation.

Topics: Astrocytoma; Carbachol; Chemical Fractionation; Down-Regulation; Enzyme Activation; Ethanol; Glycerophospholipids; Humans; Hydrolysis; Immunoblotting; Muscarinic Agonists; Phosphatidic Acids; Phosphatidylcholines; Phospholipase D; Protein Kinases; Receptors, Thrombin; Tetradecanoylphorbol Acetate; Thrombin; Tumor Cells, Cultured