Chen P F, Chin T Y, Chueh S H
Department of Biochemistry, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Kidney Int. 1998 Nov;54(5):1470-83. doi: 10.1046/j.1523-1755.1998.00162.x.
To elucidate the molecular mechanism underlying sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) mediated signaling, we compared their effects with those of adenosine triphosphate (ATP) and angiotensin II (Ang II) on the cytosolic free Ca2+ concentration ([Ca2+]i), inositol 1,4, 5-trisphosphate (IP3) generation and arachidonic acid release in rat glomerular mesangial cells.
The fluorescent Ca2+ indicator, Fura-2, was used to measure the [Ca2+]i changes in cultured rat glomerular mesangial cells either in suspension or attached to the coverslips.
SPC 5 microM, S1P 5 microM, ATP 100 microM and Ang II 90 nM all induced increases in the [Ca2+]i, and the effect showed marked homologous desensitization, while heterologous desensitization was less. After the initial exposure of the cells to SPC, the increase in [Ca2+]i induced by subsequent addition of ATP or Ang II was only reduced by about 14.3% and 4.8%, respectively. After the initial exposure to S1P, a greater reduction was seen (42. 1% and 47.7%, respectively). Both arachidonic acid release and IP3 generation were activated by all four agonists with an identical rank order of effectiveness of SPC >> S1P > ATP = Ang II; both were pertussis toxin-sensitive and cholera toxin-resistant. The arachidonic acid release induced by all four agonists showed identical susceptibility to removal of extracellular Ca2+, whereas IP3 generation displayed differential extracellular Ca2+ dependence. Only SPC-induced IP3 generation was highly sensitive to extracellular Ca2+ level, and this Ca2+ dependence was abolished after pretreatment of cells with arachidonyl trifluoromethyl ketone (AACOCF3), a phospholipase A2 inhibitor. Furthermore, the Mn2+ influx was markedly greater in SPC-stimulated cells than in either control or other agonist-stimulated cells, and was decreased by prior exposure of cells to AACOCF3. After phospholipase A2 was inhibited or in the absence of extracellular Ca2+, SPC displayed identical effectiveness as S1P on desensitizing the action of ATP or Ang II on the increase in [Ca2+]i. Conclusions. Our results indicate that all four agents primarily activate phospholipase C through their receptor occupancies, but that SPC alone also induces further significant Mn2+ influx and IP3 generation attributable to its primary stimulatory effect on arachidonic acid release. Thus, the heterologous desensitization to ATP or Ang II induced by SPC was less profound than that induced by S1P, since SPC induced a Ca2+ influx.
为阐明1-磷酸鞘氨醇（S1P）和鞘氨醇磷酰胆碱（SPC）介导的信号传导的分子机制，我们比较了它们与三磷酸腺苷（ATP）和血管紧张素II（Ang II）对大鼠肾小球系膜细胞胞质游离钙离子浓度（[Ca2+]i）、肌醇1,4,5-三磷酸（IP3）生成及花生四烯酸释放的影响。
采用荧光钙指示剂Fura-2测量悬浮或贴壁于盖玻片上的培养大鼠肾小球系膜细胞中[Ca2+]i的变化。
5μM的SPC、5μM的S1P、100μM的ATP和90nM的Ang II均可诱导[Ca2+]i升高，且该效应表现出明显的同源脱敏，而异源脱敏程度较小。细胞初次暴露于SPC后，随后添加ATP或Ang II诱导的[Ca2+]i升高仅分别降低约14.3%和4.8%。初次暴露于S1P后，降低幅度更大（分别为42.1%和47.7%）。花生四烯酸释放和IP3生成均被所有四种激动剂激活，其效力顺序相同：SPC >> S1P > ATP = Ang II；二者均对百日咳毒素敏感，对霍乱毒素耐药。所有四种激动剂诱导的花生四烯酸释放对去除细胞外钙的敏感性相同，而IP3生成表现出不同的细胞外钙依赖性。仅SPC诱导的IP3生成对细胞外钙水平高度敏感，在用磷脂酶A2抑制剂花生四烯酰三氟甲基酮（AACOCF3）预处理细胞后，这种钙依赖性被消除。此外，SPC刺激的细胞中Mn2+内流明显大于对照或其他激动剂刺激的细胞，且细胞预先暴露于AACOCF3后Mn2+内流减少。在磷脂酶A2被抑制或无细胞外钙的情况下，SPC在使ATP或Ang II对[Ca2+]i升高的作用脱敏方面与S1P表现出相同的效力。结论。我们的结果表明，所有四种药物主要通过占据其受体激活磷脂酶C，但只有SPC还可诱导进一步显著的Mn2+内流和IP3生成，这归因于其对花生四烯酸释放的主要刺激作用。因此，SPC对ATP或Ang II诱导的异源脱敏不如S1P诱导的深刻，因为SPC诱导了钙内流。
