The overall objective is to contribute to an understanding of the function of the cellular lipids by investigating whether regulated """"""""sphingolipid cycles"""""""" exist in mammalian cells. Such cycles would lead to the transient production of bioactive metabolites. Which would function in cell regulation as lipid mediators or second messengers. The hypothesis to be tested is that the cellular sphingolipids like the cellular glyserolipids exist, in part, as reservoirs for the transient production of bioactive metabolites which function in signal transduction processes. Sphingosine and lysosphingolipids inhibit protein kinase C in vitro. In cells, sphingosine inhibits proteins kinase C activity, [3H] PDBu binding, and protein kinase C dependent processes. A """"""""sphingomyelin cycle"""""""" was discovered in HL60 cells treated with 1,25-dihydroxyvitamin D3; D3 caused a transient 25% loss of sphingomyelin, the transient formation of ceramide and phosphocholine, and transient increase in a neutral Mg++-dependent sphingomyelinase (SMase) activity. Exogenously added sphingomyelinase and low levels of cell permeable ceramides potentiated HL60 cell differentiation at subthreshold levels of D3; at higher levels ,the cell permeable ceramides appeared to replace D3 and to cause differentiation.
The specific aims are: 1) to characterize, in detail, the enzymes in the metabolic pathways capable of generating bioactive sphingolipid metabolites with greatest emphasis being placed on the D3 regulatable SMase, 2) to characterize and elucidate the enzymes which attenuate the bioactive sphingosine/lysosphingolipids and ceramides and function to complete the """"""""sphingolipid cycles and their constituent enzymes are regulated; and to determine the range of pharmacologic activities of sphingolipid breakdown products nad whether they are of pathobiological significance. The studies will employ human HL60 cells, human and mouse keratinocytes, and other cells as appropriate for metabolic, biochemical and regulatory studies. The studies are of significance to the metabolic, biochemical and regulatory studies. The studies are of significance to the molecular basis of cell growth, differentiation and development. Alterations of sphingolipid metabolites are likely of importance to the sphingolipidosis, progressive dementias and other neurological disorders, cancer, psoriasis, immune disorders and inflammation.
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