betadex and lysophosphatidylinositol

Secretory phospholipase A2 (sPLA2) activity is present in the CNS and the sPLA2-IIA isoform has been shown to induce exocytosis in cultured hippocampal neurons. However, little is known about possible contributions of various lysophospholipid species to exocytosis in neuroendocrine cells. This study was therefore carried out to examine the effects of several lysophospholipid species on exocytosis on rat pheochromocytoma-12 (PC12) cells. An increase in vesicle fusion, indicating exocytosis, was observed in PC12 cells after external infusion of lysophosphatidylinositol (LPI), but not lysophosphatidylcholine or lysophosphatidylserine by total internal reflection microscopy. Similarly, external infusion of LPI induced significant increases in capacitance, or number of spikes detected at amperometry, indicating exocytosis. Depletion of cholesterol by pre-incubation of cells with methyl beta cyclodextrin and depletion of Ca2+ by thapsigargin and incubation in zero external Ca2+ resulted in attenuation of LPI induced exocytosis, indicating that exocytosis was dependent on the integrity of lipid rafts and intracellular Ca2+. Moreover, LPI induced a rise in intracellular Ca2+ suggesting that this could be the trigger for exocytosis. It is postulated that LPI may be an active participant in sPLA2-mediated exocytosis in the CNS.

Topics: Animals; beta-Cyclodextrins; Calcium; Cholesterol; Cytoplasmic Vesicles; Electric Capacitance; Enzyme Inhibitors; Exocytosis; Hypolipidemic Agents; Intracellular Space; Lysophosphatidylcholines; Lysophospholipids; Membrane Microdomains; PC12 Cells; Rats; Thapsigargin

Ostreolysin, a 15kDa pore-forming protein from the oyster mushroom (Pleurotus ostreatus), binds specifically to cholesterol-enriched membrane domains existing in the liquid-ordered phase, and lyses cells and lipid vesicles made of cholesterol and sphingomyelin. We have monitored binding of sub-lytic concentrations of ostreolysin to membranes of Chinese Hamster Ovary cells and rat somatotrophs, using primary anti-ostreolysin and fluorescence-labeled secondary antibodies detected by confocal microscopy. Depletion of more than 40% membrane cholesterol content by methyl-beta-cyclodextrin dramatically decreased ostreolysin binding. Immunostaining showed that ostreolysin is not co-localized with raft-binding proteins, cholera toxin B-subunit or caveolin, suggesting that natural membranes display heterogeneity of cholesterol-enriched raft-like microdomains. Impaired ostreolysin binding was also observed after treating the cells with lysophosphatidylinositol. Effects of lysophosphatidylinositol on binding of ostreolysin to immobilized large sphingomyelin/cholesterol (1/1, mol/mol) unilamellar vesicles were studied by a surface plasmon resonance technique. Injection of ostreolysin during the lysophosphatidylinositol dissociation phase showed an inverse relationship between ostreolysin binding and the quantity of lysophosphatidylinositol in the membranes of lipid vesicles. It was concluded that lysophospholipids prevent binding of ostreolysin to cell and to artificial lipid membranes resembling lipid rafts, by partitioning into the lipid bilayer and altering the properties of cholesterol-rich microdomains.

Topics: Animals; beta-Cyclodextrins; Cell Membrane Permeability; CHO Cells; Cholesterol; Cricetinae; Cricetulus; Fungal Proteins; Hemolysin Proteins; Lysophospholipids; Male; Membrane Microdomains; Membranes, Artificial; Mice; Protein Binding; Rats