Ceramide 1-phosphate enhances calcium entry through voltage-operated calcium channels by a protein kinase C-dependent mechanism in GH4C1 rat pituitary cells.
Sphingomyelin derivatives modulate a multitude of cellular processes, including the regulation of [Ca2+]i (the intracellular free calcium concentration). Previous studies have shown that these metabolites often inhibit calcium entry through VOCCs (voltage-operated calcium channels). In the present study, we show that, in pituitary GH4C1 cells, C1P (C2-ceramide 1-phosphate) enhances calcium entry in a dose-dependent manner. The phospholipase C inhibitor U73122 attenuated the response. C1P invoked a small, but significant, increase in the formation of inositol phosphates. Pre-treatment of the cells with pertussis toxin was without an effect on the C1P-evoked increase in [Ca2+]i. The effect of C1P was critically dependent on extracellular calcium, since no increase in [Ca2+]i was observed when cells in a calcium-free buffer were stimulated with C1P. Furthermore, if the cells were retreated with 300 nM of the VOCC inhibitor nimodipine, the effect of C1P was almost totally abolished. In addition, ceramide C8-1-phosphate evoked an increase in [Ca2+]i, but the onset of the response was slow compared with that of C1P. In cells treated with 1 mM thapsigargin for 15 min, C1P still evoked an increase in [Ca2+]i. In patch-clamp experiments in the whole-cell mode, C1P enhanced calcium entry through the VOCCs compared with vehicle-treated cells. Dialysis of the cells with C1P did not enhance the calcium current. On-cell patch-clamp experiments showed an enhanced probability of the VOCCs being open (P(open)) in the presence of C1P. Inhibition of PKC (protein kinase C) with GF109203X and down-regulation of PKC with PMA attenuated the C1P-evoked increase in [Ca2+]i. Furthermore, down-regulation of PKC abolished the effect of C1P on P(open). This is the first report showing that a sphingomyelin derivative enhances calcium entry through VOCCs.
Törnquist
Kid
K
Department of Biology, Abo Akademi University, BioCity, Artillerigatan 6, 20520 Turku, Finland. kid.tornqvist@abo.fi
Blom
Tomas
T
Shariatmadari
Ramin
R
Pasternack
Michael
M
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005110:Extracellular Space / Q000737:chemistry / Q000378:metabolism; D018408:Patch-Clamp Techniques / Q000379:methods; D010902:Pituitary Gland / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats
D015220:Calcium Channels; D002518:Ceramides; C065576:ceramide 1-phosphate; D011493:Protein Kinase C; D002118:Calcium