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J. Biol. Chem., Vol. 267, Issue 22, 15475-15484, 08, 1992
RA Sabbadini, R Betto, A Teresi, G Fachechi-Cassano and G Salviati
In this study, we report that sphingosine is a potent inhibitor of
sarcoplasmic reticulum (SR) calcium release. Evidence is presented
demonstrating a direct effect of sphingosine on the SR ryanodine receptor.
Calcium release from "skinned" rabbit skeletal muscle fibers and isolated
junctional SR derived from the terminal cisternae (TC) was measured in
response to caffeine, doxorubicin, 5'-adenylyl-beta,gamma- imidodiphosphate
or calcium. Sphingosine inhibited caffeine-induced release in a
dose-dependent manner with an IC50 of 0.1 microM for the single muscle
fibers and 0.5 microM for the isolated TC vesicles. Near complete blockage
of TC calcium release rate was observed with 3 microM sphingosine. Neither
sphingomyelin nor sphingosylphosphorylcholine had any effect at the 3
microM level, suggesting that the sphingosine effect was specific.
Doxorubicin-induced calcium release and spontaneous calcium release were
also blocked by sphingosine. Sphingosine was also capable of stimulating
calcium transport in the isolated TC vesicles without an effect on
Ca-ATPase activity. Ruthenium red was not capable of substantial additional
stimulation of calcium transport nor inhibition of calcium release beyond
the action of sphingosine. Sphingosine's blockage of calcium release was
not reversed by the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-
methylpiperazine dihydrochloride, suggesting that the action of sphingosine
on calcium release was not dependent on ryanodine receptor phosphorylation.
Sphingosine significantly increased (8-fold) the Kd for specific
[3H]ryanodine binding to TC membranes and decreased the Bmax with a dose
dependence similar to the inhibition of calcium release, but sphingosine
did not affect the pCa tension relationship of skinned skeletal muscle
fibers. These data are consistent with a direct effect of submicromolar
sphingosine on the ryanodine receptor. Substantially higher concentrations
of sphingosine (30-50 microM) or sphingosylphosphorylcholine (10-20 microM)
were capable of inducing calcium release by themselves. Preliminary data
indicate that the transverse tubule and not the SR contain substantial
sphingomyelinase activity consistent with a transverse tubule source of
sphingosine production. Considering that sphingosine is found in micromolar
concentrations in some cells, our data indicate that sphingosine generated
by the transverse tubule membranes may be a physiologically relevant
mechanism for modulating SR calcium release.
The effects of sphingosine on sarcoplasmic reticulum membrane calcium release
Department of Biology, San Diego State University, California 92182.
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