Craven P A, DeRubertis F R
Biochim Biophys Acta. 1984 Aug 17;804(4):450-8. doi: 10.1016/0167-4889(84)90073-9.
Previous studies have shown that hypertonic mannitol or NaCl increases the release of [3H]arachidonate and immunoreactive prostaglandin E in inner medullary slices incubated in Ca2+-free media containing EGTA. By contrast, the stimulation of these parameters by ionophore A23187 and by arginine-vasopressin are abolished in Ca2+-free media plus EGTA. In the present study, the effects of Ca2+ deprivation and the intracellular Ca2+ antagonist TMB-8 [8-N,N-diethylamino)octyl-3,4,5 -trimethoxybenzoate-HCl) were further examined to assess the Ca2+ dependence of the actions of different stimuli of prostaglandin E synthesis in rat renal inner medulla. Ca2+-free media without EGTA abolished increases in [3H]arachidonate and immunoreactive prostaglandin E release induced by ionophore A23187, but not those induced by arginine-vasopressin, suggesting that different pools of Ca2+ subserve expression of the actions of these two stimuli. At low concentrations, TMB-8 (10-25 microM) inhibited increases in [3H]arachidonate and immunoreactive prostaglandin E release induced by arginine-vasopressin, but did not influence effects of Ca2+ plus ionophore A23187 or hypertonicity on these parameters. At higher concentrations (100-500 microM), TMB-8 suppressed effects of ionophore A23187, hyperosmolar NaCl and mannitol on immunoreactive prostaglandin E and [3H]arachidonate release from slices. The effects of a sub-optimal inhibitory concentration of TMB-8 on ionophore A23187 actions were overcome by increasing Ca2+ in the media from 1.5 to 5 mM. Ca2+ deprivation, or concentrations of EGTA or TMB-8, that were effective in suppressing increases in immunoreactive prostaglandin E induced by ionophore A23187, arginine-vasopressin or hypertonicity, did not modify increases in immunoreactive prostaglandin E induced by exogenous arachidonate. Moreover, in microsomal fractions of inner medulla, TMB-8 suppressed Ca2+-dependent increases in phospholipase A2 and C activities, an effect which was competitive with Ca2+. Thus, Ca2+ deprivation and TMB-8 act at a step in the immunoreactive prostaglandin E synthetic pathway proximal to cyclooxygenase activity, and probably at the level of Ca2+-dependent acyl hydrolase activity. The results with TMB-8 indicate that an intracellular pool of Ca2+ is involved in expression of the actions of hypertonicity to increase [3H]arachidonate release and immunoreactive prostaglandin E in inner medulla.
先前的研究表明，在含有乙二醇双四乙酸（EGTA）的无钙培养基中孵育的内髓切片中，高渗甘露醇或氯化钠会增加[3H]花生四烯酸的释放以及免疫反应性前列腺素E的释放。相比之下，在无钙培养基加EGTA中，离子载体A23187和精氨酸加压素对这些参数的刺激作用被消除。在本研究中，进一步研究了钙缺乏和细胞内钙拮抗剂TMB - 8 [8 - N，N - 二乙氨基）辛基 - 3,4,5 - 三甲氧基苯甲酸 - HCl]的作用，以评估大鼠肾内髓中前列腺素E合成的不同刺激作用对钙的依赖性。不含EGTA的无钙培养基消除了离子载体A23187诱导的[3H]花生四烯酸和免疫反应性前列腺素E释放的增加，但未消除精氨酸加压素诱导的增加，这表明不同的钙库参与了这两种刺激作用的表达。在低浓度时，TMB - 8（10 - 25 microM）抑制精氨酸加压素诱导的[3H]花生四烯酸和免疫反应性前列腺素E释放的增加，但不影响钙加离子载体A23187或高渗对这些参数的影响。在较高浓度（100 - 500 microM）时，TMB - 8抑制离子载体A23187、高渗氯化钠和甘露醇对切片中免疫反应性前列腺素E和[3H]花生四烯酸释放的作用。通过将培养基中的钙从1.5 mM增加到5 mM，可以克服TMB - 8的次优抑制浓度对离子载体A23187作用的影响。能有效抑制离子载体A23187、精氨酸加压素或高渗诱导的免疫反应性前列腺素E增加的钙缺乏、EGTA或TMB - 8浓度，并未改变外源性花生四烯酸诱导的免疫反应性前列腺素E的增加。此外，在内髓的微粒体部分，TMB - 8抑制了磷脂酶A2和C活性的钙依赖性增加，这种作用与钙具有竞争性。因此，钙缺乏和TMB - 8作用于免疫反应性前列腺素E合成途径中靠近环氧化酶活性的步骤，可能作用于钙依赖性酰基水解酶活性水平。TMB - 8的结果表明，细胞内钙库参与了高渗作用在内髓中增加[3H]花生四烯酸释放和免疫反应性前列腺素E的表达。
