thapsigargin and lysophosphatidylserine

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

Previous reports have demonstrated that lysophosphatidylcholine (LPC) increases the intracellular concentration of calcium ([Ca++]i) in the heart; however, the mechanisms responsible for this increase are not clear. We examined the effect of exogenous LPC on [Ca++]i in freshly isolated cardiomyocytes from adult rats. Our results showed that LPC elevated the [Ca++]i in a dose-dependent (2.5-10 microM) manner. The LPC (10 microM)-induced increase in [Ca++]i was augmented upon increasing the concentration of extracellular Ca++ and was abolished by the removal of Ca++ from the medium. Preincubation of cardiomyocytes with sarcolemmal L-type Ca++ channel blocker, verapamil, did not affect the LPC-evoked increase in [Ca++]i significantly. On the other hand, ouabain, a Na(+)-K+ ATPase inhibitor, and low concentrations of extracellular Na+ enhanced the LPC response. The LPC-induced increase in [Ca++]i was attenuated significantly by the inhibitors of Na(+)-Ca++ exchanger such as Ni++ and amiloride. Depletion of the sarcoplasmic reticulum (SR) Ca++ stores by low micromolar concentrations of ryanodine (a SR Ca(++)-release channel activator) or by thapsigargin (a SR Ca(++)-pump ATPase inhibitor) depressed the LPC-mediated increase in [Ca++]i. Combined blockade of Na(+)-Ca++ exchanger and inhibition of SR Ca(++)-pump or ryanodine receptor had an additive effect on the LPC response. These observations suggest that the increase in [Ca++]i induced by LPC depends on both Ca(++)-influx from the extracellular space and Ca(++)-release from the SR stores. Furthermore, Na(+)-Ca++ exchange plays a critical role in the LPC-mediated entry of Ca++ into cardiomyocytes.

Topics: Amiloride; Animals; Calcium; Lysophospholipids; Male; Myocardium; Rats; Rats, Sprague-Dawley; Ryanodine; Sarcoplasmic Reticulum; Sodium-Calcium Exchanger; Thapsigargin