Endocrinology, Vol 136, 4894-4902, Copyright © 1995 by Endocrine Society

ARTICLES

Sphingosine derivatives inhibit depolarization-evoked calcium entry in rat GH4C1 cells

K Tornquist, M Pasternack and K Kaila
Department of Biosciences, University of Helsinki, Finland.

Several investigations have suggested that sphingosine (SP) derivatives are potent inhibitors of protein kinase C. In GH4C1 cells, protein kinase C is a potent modulator of voltage-operated calcium channels (VOCCs). The aim of the present study was to investigate whether SP derivatives could modify calcium entry via VOCCs. Using fura-2-loaded cells and 45Ca2+ flux studies, we show that several SPs potently and rapidly inhibit depolarization-evoked calcium entry in a dose-dependent manner. The effect was not due to an enhanced efflux of calcium from the cells, as the depolarization-evoked entry of Ba2+ was inhibited by the SPs. A similar inhibition was observed with 1,2-dioctanoylglycerol, an activator of sphingomyelinase in GH3 cells. Phorbol myristate acetate and 1-oleyl-2-acetylglycerol had only a modest inhibitory effect. Furthermore, whole cell patch-clamp experiments showed that sphingosinephosphorylcholine (SPC) potently attenuated calcium entry via VOCCs. In experiments using cells grown on coverslips, we found that the inhibitory effect of SPC on calcium entry was reversible. The addition of sphingomyelinase or hexanoyl ceramide, a cell-permeable ceramide, only modestly inhibited the depolarization-evoked entry of calcium, whereas arachidonic acid and phosphatidic acid had no effect. The SP metabolite sphingosine-1-phosphate had no effect on the entry of calcium. The results suggest that the effects of the SPs were probably not the result of a conversion to ceramide or of the production of other lipid second messengers. In cells with down-regulated protein kinase C, SPC, SP, and 1,2-dioctanoylglycerol inhibited depolarization- evoked calcium entry, suggesting that the inhibition was independent of an action mediated via protein kinase C. The SPs per se did not induce any changes in intracellular free calcium, and they did not inhibit the TRH-evoked release of sequestered calcium in the cells. However, TRH- evoked calcium entry was inhibited. The results suggest that SPs are potential regulators of calcium entry mediated by VOCCs in GH4C1 cells.

This article has been cited by other articles:

				S.-N. Wu, Y.-K. Lo, B. I.-T. Kuo, and H.-T. Chiang Ceramide Inhibits the Inwardly Rectifying Potassium Current in GH3 Lactotrophs Endocrinology, November 1, 2001; 142(11): 4785 - 4794. [Abstract] [Full Text] [PDF]

				T. Negishi, C. L. Chik, and A. K. Ho Ceramide Enhances Growth Hormone (GH)-Releasing Hormone-Stimulated Cyclic Adenosine 3',5'-Monophosphate Accumulation but Inhibits GH Release in Rat Anterior Pituitary Cells Endocrinology, December 1, 1999; 140(12): 5691 - 5697. [Abstract] [Full Text]

				H. M. Himmel, D. M. z. Heringdorf, B. Windorfer, C. J. v. Koppen, U. Ravens, and K. H. Jakobs Guanine Nucleotide-Sensitive Inhibition of L-Type Ca2+ Current by Lysosphingolipids in RINm5F Insulinoma Cells Mol. Pharmacol., May 1, 1998; 53(5): 862 - 869. [Abstract] [Full Text]

				A. Titievsky, I. Titievskaya, M. Pasternack, K. Kaila, and K. Tornquist Sphingosine Inhibits Voltage-operated Calcium Channels in GH4C1 Cells J. Biol. Chem., January 2, 1998; 273(1): 242 - 247. [Abstract] [Full Text] [PDF]

				K. Tornquist, P. Saarinen, M. Vainio, and M. Ahlstrom Sphingosine 1-Phosphate Mobilizes Sequestered Calcium, Activates Calcium Entry, and Stimulates Deoxyribonucleic Acid Synthesis in Thyroid FRTL-5 Cells Endocrinology, October 1, 1997; 138(10): 4049 - 4057. [Abstract] [Full Text] [PDF]