McGhee J G, Shoback D M
Endocrine Research Unit, San Francisco Veterans Administration Medical Center, California 94121.
Endocrinology. 1990 Feb;126(2):899-907. doi: 10.1210/endo-126-2-899.
The observation that increases in extracellular Ca2+ or the addition of divalent cations, such as Ba2+, Mg2+, Mn2+, or Sr2+, stimulate the accumulation of inositol trisphosphate (InsP3) and its breakdown products in parathyroid cells strongly supports the idea that polyphosphoinositides are hydrolyzed under these conditions. Since phosphatidic acid is produced as a result of polyphosphoinositide hydrolysis, and it has been proposed that phosphatidic acid may be a second messenger for Ca2+ mobilization, we examined the effects of this compound on parathyroid cells. We assessed PTH release, intracellular free Ca2+ ([Ca2+]i), and inositol polyphosphate accumulation in response to phosphatidic acid. Natural phosphatidic acid reduced PTH release at 1.0 mM extracellular Ca2+ by 18 +/- 6%, 48 +/- 5%, 59 +/- 10%, and 79 +/- 6% at concentrations of 1, 10, 50, and 100 micrograms/ml, respectively (n = 5-11). The effect was not dependent on the presence of extracellular Ca2+, since phosphatidic acid (100 micrograms/ml) inhibited PTH secretion by 39 +/- 3% in medium with no added Ca2+ and 1.0 mM EGTA (n = 3). This agent rapidly and transiently increased [Ca2+]i in a dose-dependent manner, as determined by fura-2 fluorescence. At 1.0 mM extracellular Ca2+, [Ca2+]i rose from 309 +/- 8 to a peak of 356 +/- 26, 454 +/- 22, and 587 +/- 57 nM with the addition of 1, 10, and 100 micrograms/ml phosphatidic acid, respectively (n = 2-14). In the absence of extracellular Ca2+ (i.e. medium with 1 or 2 mM EGTA and no added Ca2+), phosphatidic acid produced a quantitatively smaller peak increment of 38 +/- 4% in [Ca2+]i, indicating that this compound could mobilize Ca2+ from intracellular stores (n = 3). At 1.0 mM extracellular Ca2+, phosphatidic acid (200 micrograms/ml) stimulated the accumulation of Inositol trisphosphate (InsP3), Inositol bisphosphate (InsP2), and Inositol monophosphate (InsP1) by 46 +/- 9%, 37 +/- 9%, and 59 +/- 11% after 60 sec, respectively (n = 5-7). Phosphatidic acid had no significant effect on forskolin-stimulated cAMP accumulation. We further determined whether the specific fatty acid composition of phosphatidic acid might influence its effects in parathyroid cells by testing several synthetic compounds. Dipalmitoyl phosphatidic acid (greater than or equal to 50 micrograms/ml) inhibited PTH release in a dose-dependent manner without significantly changing [Ca2+]i. Dioleoyl phosphatidic acid had modest biphasic effects on secretion, with 20 +/- 5% inhibition observed at lower doses (10 micrograms/ml) and a 27 +/- 8% stimulation of secretion at 100 micrograms/ml (n = 6).(ABSTRACT TRUNCATED AT 400 WORDS)
细胞外Ca2+增加或添加二价阳离子（如Ba2+、Mg2+、Mn2+或Sr2+）会刺激甲状旁腺细胞中肌醇三磷酸（InsP3）及其分解产物的积累，这一观察结果有力地支持了多磷酸肌醇在这些条件下会被水解的观点。由于多磷酸肌醇水解会产生磷脂酸，并且有人提出磷脂酸可能是Ca2+动员的第二信使，因此我们研究了该化合物对甲状旁腺细胞的影响。我们评估了磷脂酸对甲状旁腺激素（PTH）释放、细胞内游离Ca2+（[Ca2+]i）和肌醇多磷酸积累的影响。天然磷脂酸在细胞外Ca2+浓度为1.0 mM时，分别在浓度为1、10、50和100μg/ml时使PTH释放减少18±6%、48±5%、59±10%和79±6%（n = 5 - 11）。该作用不依赖于细胞外Ca2+的存在，因为在无添加Ca2+且含有1.0 mM乙二醇双四乙酸（EGTA）的培养基中，磷脂酸（100μg/ml）使PTH分泌减少39±3%（n = 3）。通过fura - 2荧光测定发现，该试剂以剂量依赖方式迅速且短暂地增加[Ca2+]i。在细胞外Ca2+浓度为1.0 mM时，添加1、10和100μg/ml磷脂酸后，[Ca2+]i分别从309±8上升至峰值356±26、454±22和587±57 nM（n = 2 - 14）。在无细胞外Ca2+（即含有1或2 mM EGTA且无添加Ca2+的培养基）时，磷脂酸使[Ca2+]i峰值增加量在数量上较小，为38±4%，表明该化合物可从细胞内储存库中动员Ca2+（n = 3）。在细胞外Ca2+浓度为1.0 mM时，磷脂酸（200μg/ml）在60秒后使肌醇三磷酸（InsP3）、肌醇二磷酸（InsP2）和肌醇单磷酸（InsP1）的积累分别增加46±9%、37±9%和59±11%（n = 5 - 7）。磷脂酸对福斯可林刺激的环磷酸腺苷（cAMP）积累无显著影响。我们通过测试几种合成化合物进一步确定磷脂酸的特定脂肪酸组成是否可能影响其在甲状旁腺细胞中的作用。二棕榈酰磷脂酸（≥50μg/ml）以剂量依赖方式抑制PTH释放，而不显著改变[Ca2+]i。二油酰磷脂酸对分泌有适度的双相作用，在较低剂量（10μg/ml）时观察到20±5% 的抑制作用，在100μg/ml时分泌增加27±8%（n = 6）。（摘要截取自400字）
