27-Hydroxycholesterol inhibits neutral sphingomyelinase in cultured human endothelial cells

To study the effect of 27-hydroxycholesterol (27OHC) on the catabolism of sphingomyelin, we cultured endothelial cells (ECs) from human umbilical veins with 27OHC, then measured activities of acid sphingomyelinase (ASMase) and neutral sphingomyelinase (NSMase) and sphingomyelin consumption by using [14C]sphingomyelin, and determined NSMase mRNA expressions by RT-PCR method. The results indicated that [14C]sphingomyelin accumulated in cells treated with 27OHC, and that the activities of both NSMase and ASMase were inhibited in ECs cultured with 27OHC. To further study the effect of 27OHC on NSMase, we used desipramine, an inhibitor of ASMase, to exclude the possible interference of ASMase's residual activity at neutral condition. Also, we observed the significant inhibition of NSMase activity by using glutathione, an inhibitor of NSMase, but found no further impact when 27OHC was added later. To determine whether the inhibition of NSMase activity was directly due to the effect of 27OHC, we exposed cell homogenate to 27OHC, and found no inhibitive effect of 27OHC on the activity of NSMase. All of our data confirmed that 27OHC had only an indirect inhibitive effect on NSMase. Our finding that no change of the NSMase mRNA expression by 27OHC indicated that the inhibitive effect of 27OHC on NSMase activity occurred at a post-transcriptional level. We suggest that an altered membrane fluidity caused by 27OHC could be involved in the inhibited activity of NSMase.