Alterations of calcium homoeostasis in cultured rat astrocytes evoked by bioactive sphingolipids.
OBJECTIVE
In the brain, alterations in sphingolipid metabolism contribute to several neurological disorders; however, their effect on astrocytes is largely unknown. Here, we identified bioactive sphingolipids that affect intracellular free calcium concentration ([Ca(2+)]i), mobility of peptidergic secretory vesicles, signalling pathways involved in alterations of calcium homoeostasis and explored the relationship between the stimulus-evoked increase in [Ca(2+)]i and attenuation of vesicle mobility.


METHODS
Confocal time-lapse images were acquired to explore [Ca(2+)]i signals, the mobility of fluorescently tagged peptidergic vesicles and the structural integrity of the microtubules and actin filaments before and after the addition of exogenous sphingolipids to astrocytes.


RESULTS
Fingolimod (FTY720), a recently introduced therapeutic for multiple sclerosis, and sphingosine, a releasable constituent of membrane sphingolipids, evoked long-lasting increases in [Ca(2+)]i in the presence and absence of extracellular Ca(2+); the evoked responses were diminished in the absence of extracellular Ca(2+). Activation of phospholipase C and inositol-1,4,5-triphosphate receptors was necessary and sufficient to evoke increases in [Ca(2+)]i as revealed by the pharmacologic inhibitors; Ca(2+) flux from the extracellular space intensified these responses several fold. The lipid-evoked increases in [Ca(2+)]i coincided with the attenuated vesicle mobility. High and positive correlation between increase in [Ca(2+)]i and decrease in peptidergic vesicle mobility was confirmed independently in astrocytes exposed to evoked, transient Ca(2+) signalling triggered by purinergic and glutamatergic stimulation.


CONCLUSIONS
Exogenously added cell-permeable sphingosine-like lipids exert complex, Ca(2+)-dependent effects on astrocytes and likely alter their homeostatic function in vivo.
Celica Biomedical Center d.o.o., Ljubljana, Slovenia; Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
M
M
Stenovec
S
S
Trkov
M
M
Kreft
R
R
Zorec
D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000187:drug effects; D000068876:Fingolimod Hydrochloride; D006706:Homeostasis / Q000187:drug effects / Q000502:physiology; D018613:Microscopy, Confocal; D011409:Propylene Glycols / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D013107:Sphingolipids / Q000494:pharmacology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*
D011409:Propylene Glycols; D013107:Sphingolipids; D000068876:Fingolimod Hydrochloride; D013110:Sphingosine; D002118:Calcium
FTY720; astrocytes; calcium; phospholipase C; sphingosine; vesicle traffic