Oral H, Dorn G W, Mann D L
Cardiology Section, Department of Medicine, Veterans Administration Medical Center, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 1997 Feb 21;272(8):4836-42. doi: 10.1074/jbc.272.8.4836.
To determine whether activation of the neutral sphingomyelinase pathway was responsible for the immediate (<30 min) negative inotropic effects of tumor necrosis factor-alpha (TNF-alpha), we examined sphingosine levels in diluent and TNF-alpha-stimulated cardiac myocytes. TNF-alpha stimulation of adult feline cardiac myocytes provoked a rapid (<15 min) increase in the hydrolysis of [14C]sphingomyelin in cell-free extracts, as well as an increase in ceramide mass, consistent with cytokine-induced activation of the neutral sphingomyelinase pathway. High performance liquid chromatographic analysis of lipid extracts from TNF-alpha-stimulated cardiac myocytes showed that TNF-alpha stimulation produced a rapid (<30 min) increase in free sphingosine levels. Moreover, exogenous D-sphingosine mimicked the effects of TNF-alpha on intracellular calcium homeostasis, as well as the negative inotropic effects of TNF-alpha in isolated contracting myocytes; time course studies showed that exogenous D-sphingosine produced abnormalities in cell shortening that were maximal at 5 min. Finally, blocking sphingosine production using an inhibitor of ceramidase, n-oleoylethanolamine, completely abrogated the negative inotropic effects of TNF-alpha in isolated contracting cardiac myocytes. Additional studies employing biologically active ceramide analogs and sphingosine 1-phosphate suggested that neither the immediate precursor of sphingosine nor the immediate metabolite of sphingosine, respectively, were likely to be responsible for the immediate negative inotropic effects of TNF-alpha. Thus, these studies suggest that sphingosine mediates the immediate negative inotropic effects of TNF-alpha in isolated cardiac myocytes.
为了确定中性鞘磷脂酶途径的激活是否是肿瘤坏死因子-α（TNF-α）即刻（<30分钟）负性肌力作用的原因，我们检测了稀释液和TNF-α刺激的心肌细胞中的鞘氨醇水平。TNF-α刺激成年猫心肌细胞可使无细胞提取物中[14C]鞘磷脂的水解迅速（<15分钟）增加，同时神经酰胺量增加，这与细胞因子诱导的中性鞘磷脂酶途径激活一致。对TNF-α刺激的心肌细胞脂质提取物的高效液相色谱分析表明，TNF-α刺激可使游离鞘氨醇水平迅速（<30分钟）升高。此外，外源性D-鞘氨醇模拟了TNF-α对细胞内钙稳态的影响，以及TNF-α对分离的收缩性心肌细胞的负性肌力作用；时间进程研究表明，外源性D-鞘氨醇在5分钟时对细胞缩短产生的异常最为明显。最后，使用神经酰胺酶抑制剂n-油酰乙醇胺阻断鞘氨醇的产生，完全消除了TNF-α对分离的收缩性心肌细胞的负性肌力作用。使用生物活性神经酰胺类似物和1-磷酸鞘氨醇的其他研究表明，鞘氨醇的直接前体和鞘氨醇的直接代谢产物都不太可能是TNF-α即刻负性肌力作用的原因。因此，这些研究表明鞘氨醇介导了TNF-α对分离的心肌细胞的即刻负性肌力作用。
