Abstract
Acid ceramidase (AC) has been reported to be a critical sphingolipid metabolizing enzyme in the regulation of cellular activity under physiological and pathological conditions. However, it remains unknown whether AC is functioning in coronary arterial myocytes (CAMs) to regulate vascular function. In the present study, we first characterized the expression and activity of AC in CAMs. By real time RT‐PCR, Western blot analysis and immunhistochemistry, AC was found highly expressed in the primary cultures of mouse CAMs and in the middle layer of mouse coronary arterial wall. Confocal microscopy showed that AC, in particular, its alpha subunit was mainly colocalized with lysosome marker, Lamp‐1, indicating a main location of this enzyme in lysosomes. Treatment of CAMs with AC inducer, genistein significantly increased AC expression in CAMs, while inhibition of AC gene expression by N‐oleoylethanolamine (NOE) markedly reduced the level of AC in CAMs. Using LC/MS analysis, we demonstrated that basal ceramide level in CAMs was 707.49 ± 103.94 pmol/1 million cells and that genistein mainly reduced the concentration of C‐14 ceramide (−35%) and C1–6 ceramide (−18%) and NOE increased both ceramide species by 2.4 and 3.4 folds, respectively. Given recent reports on the implication of a typical alarmin, high‐mobility‐group box 1 (HMGB1) in tissue injury and inflammation, we tested whether AC and related ceramide signaling is involved in the action of HMGB1. It was found that HMGB1 decreased AC mRNA and protein levels in a concentration‐dependent manner. Correspondingly, cellular total, C‐14‐, and C‐16 ceramide concentrations in HMGB1‐treated CAMs increased by 65%, 51% and 77%, respectively. This HMGB1‐indcued increase in ceramide accumulation in CAMs was blocked by AC inducer, genistein, but further enhanced by AC inhibitor, NOE. These results suggest that AC is enriched in CAMs and its activation or inhibition alters cellular ceramide levels and that AC may be a signaling enzyme mediating the action of HMGB1 in the regulation of CAM function in response to atherogenic or inflammatory stimuli.Support or Funding InformationSupported by NIH grants HL057244, HL075316 and HL122937
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