Sphinganine effects on chemoattractant-induced diacylglycerol generation, calcium fluxes, superoxide production, and on cell viability in the human neutrophil. Delivery of sphinganine with bovine serum albumin minimizes cytotoxicity without affecting inhibition of the respiratory burst.
Long chain bases (sphinganine and sphingosine) are potent inhibitors of protein kinase C in an in vitro mixed micelle-reconstituted system (Hannun, Y. A., Loomis, C. R., Merrill, A. H. J., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609) and block activation of the superoxide-generating respiratory burst in human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). In the present studies, we have investigated the effects of sphinganine on cellular levels of the second messengers related to phosphoinositide turnover: diacylglycerol and calcium. We find that sphinganine added from a stock solution containing equimolar or greater bovine serum albumin had no effect on either formyl-methionyl-leucyl-phenylalanine-stimulated calcium fluxes or diacylglycerol generation, at levels which completely blocked activation of superoxide generation. In addition, there was no effect of sphinganine on cell viability in this concentration range. These data indicate an inhibitory effect subsequent to the generation of second messengers and are consistent with protein kinase C as the locus of action. When sphinganine was added from a stock in dimethyl sulfoxide, significant cytotoxic effects (assayed by trypan blue exclusion, release of cellular lactate dehydrogenase, and leakage of Quin2) were seen at concentrations nearer those which inhibited the respiratory burst. Cytotoxicity was inversely proportional to cell concentration and was probably due to detergent micelle formation which occurs in the absence of albumin. These studies emphasize the importance of the method of delivery and the consideration of cytotoxic effects, but indicate that long-chain bases possess potent inhibitory properties which make them useful probes of signal transduction mechanisms.
Lambeth
J D
JD
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322.
Burnham
D N
DN
Tyagi
S R
SR
D002118:Calcium / Q000097:blood*; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism; D004075:Diglycerides / Q000097:blood*; D005989:Glycerides / Q000097:blood*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010101:Oxygen Consumption; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000097:blood / Q000494:pharmacology; D013481:Superoxides / Q000097:blood*
D004075:Diglycerides; D005989:Glycerides; D013481:Superoxides; D012710:Serum Albumin, Bovine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D013110:Sphingosine; C005682:safingol; D002118:Calcium