Acid sphingomyelinase and inhibition by phosphate ion: role of inhibition by phosphatidyl‐myo‐inositol 3,4,5‐triphosphate in oligodendrocyte cell signaling

Abstract

There is ample evidence that both acid (ASMase) and neutral (NSMase) sphingomyelinases play a role in cell death so inhibitors of either enzyme could have significant value as protectors against neurodegeneration. We used a fluorogenic sphingomyelinase substrate, 6-hexadecanoylamino-4-methylumbelliferyl-phosphorylcholine, and a [ 14 C]choline-labeled sphingomyelin substrate to screen large numbers of phosphocompounds for inhibition of ASMase in extracts of human oligodendroglioma cells (HOG) and neonatal rat oligodendrocytes. Non-competitive inhibition was observed with inorganic phosphate and AMP, which was a more potent inhibitor of ASMase than cyclic AMP, ADP or ATP. However, other nucleotide phosphates, sugar phosphates, nucleotide sugars and glycerol phosphate did not inhibit ASMase. Our key finding was that phosphatidyl-myo-inositol 3,4,5-triphosphate [PtdIns (3,4,5)P 3 ] was a much more potent inhibitor of ASMase than lysophosphatidic acid or phosphatidyl-myo-inositol 4,5-diphosphate [PtdIns(4,5)P 2 ]. When PtdIns(3,4,5)P 3 was added to cultured cells we observed 50% inhibition of ASMase but no inhibition of other lysosomal hydrolases. After transfection of HOG cells with the tumor supressor phosphatase and tensin homolog protein (PTEN), which hydrolyses PtdIns(3,4,5)P 3 to PtdIns(4,5)P 2 , we observed a two-fold increase in ASMase activity. Furthermore, the phosphatidylinositol-3-kinase inhibitor wortmannin (which reduces PtdIns(3,4,5)P 3 levels) also resulted in activation of ASMase. We propose that the small amount of ASMase activity associated with detergent-resistant cell membranes (Rafts) is regulated by PtdIns(3,4,5)P 3 and is most likely involved in receptor clustering and capping. Keywords: phosphate, phosphatidylinositol-3-kinase, phosphatidyl-myo-inositol 3,4,5-triphosphate, PTEN, sphingomyelinase. Abbreviations used: ASMase, acid sphingomyelinase; DISC, deathinducing signaling complex; HMU-P-Chol, 6-hexadecanoylamino-4-methylumbelliferyl-phosphorylcholine; HOG, human oligodendroglioma cells; Ins(1,4,5)P 3 , myo-inositol 1,4,5-triphosphate; lyso-PA, lysophosphatidic acid; NGF, nerve growth factor; NSMase, neutral sphingomyelinase; NT-3, neurotropin-3; NTR, neurotropin receptor; OPC, oligodendrocyte primary cultures; PI3 kinase, phosphatidylinositol-3-kinase; PtdIns(4,5)P 2 , phosphatidyl-myo-inositol 4,5-diphosphate; PtdIns(3,4,5)P 3 , phosphatidyl-myo-inositol 3,4,5-triphosphate; PTEN, phosphatase and tensin homolog protein; Raft, Triton X-100-insoluble, sphingolipid-rich membrane fraction from sucrose density gradient; TNF, tumor necrosis factor.

Ceramide‐1‐phosphate promotes cell survival through activation of the phosphatidylinositol 3‐kinase/protein kinase B pathway

“…The effects of C1P on inhibition of A-SMase and activation of PI3-K are consistent with previous observations by Testai et al [31] who have recently demonstrated that inhibition of PI3-K leads to A-SMase activation in oligodendrocytes.…”

Secretory sphingomyelinase in health and disease

“…Inorganic phosphate, other nucleotides, dolichol phosphate and phosphoinositides are potent noncompetitive inhibitors of ASM Watanabe et al, 1983). Interestingly, while phosphatidylinositol 3,5-bisphosphate (Kölzer et al, 2003) and phosphatidylinositol 3,4,5-trisphosphate (Testai et al, 2004) both inhibit ASM in the low mm range, the enzyme is potently activated by other phosphate-containing lipids such as endocytosed bismonoacylgycerophosphate (Linke et al, 2001) or by plasma membrane-derived phosphatidylglycerol and phosphatidic acid (Oninla et al, 2014). Cholesterol, another endogenous lipid, potently inhibits ASM (Reagan et al, 2000).…”

Functional Inhibitors of Acid Sphingomyelinase (FIASMAs): A Novel Pharmacological Group of Drugs with Broad Clinical Applications

“…Therefore, only few examples of inhibitors directly interacting with ASM are currently known. These substances include physiological inhibitors of ASM such as phosphatidyl-myo-inositol-3,4,5-triphosphate [79], L-α-phosphatidyl-D-myoinositol-3,5-biphosphate [80], compounds isolated from plants, such as α-mangostin [81,82] and non-natural direct inhibitors of ASM, such as SMA-7 [66], AD2765 [83] and synthetic phosphoinositide analogues [84]. Several biphosphonates are potent and selective inhibitors of ASM [85], among them zoledronic acid, which is clinically used in the treatment of osteoporosis.…”