Chakraborti S, Michael J R, Sanyal T
Department of Biochemistry and Biophysics, University of Kalyani, India.
Eur J Biochem. 1992 Jun 15;206(3):965-72. doi: 10.1111/j.1432-1033.1992.tb17007.x.
We sought to investigate the mechanisms by which the calcium ionophore A23187 triggers arachidonic acid release in bovine pulmonary endothelial cells and to test the hypothesis that protein kinase C is involved in this process. Our results indicate that the mechanism by which A23187 increases phospholipase A2 activity and arachidonic acid release in bovine pulmonary arterial endothelial cells depends upon the concentration studied. At concentrations of 1 microM and 2.5 microM, A23187 increases phospholipase A2 activity and arachidonic acid release without stimulating protein kinase C. At concentrations of 5-12.5 microM, A23187 increases arachidonic acid release and phospholipase A2 activity in conjunction with a dose-dependent activation of membrane-bound protein kinase C. To test the hypothesis that these doses of A23187 increase phospholipase A2 activity by stimulating protein kinase C, we studied the effect of prior treatment with the protein kinase C inhibitor sphingosine. Sphingosine inhibits the increase in phospholipase A2 activity and arachidonic acid release caused by A23187 over the range 5-12.5 microM. To investigate further the potential role of protein kinase C, we studied the effects of the inactive phorbol ester 4 alpha-phorbol 12 beta-myristate 13 alpha-acetate (4 alpha-PMA) and an active phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (4 beta PMA). Neither 4 alpha-PMA nor 4 beta-PMA affected basal arachidonic acid release. 4 alpha-PMA also did not augment the effects of A23187. In contrast, 4 beta-PMA significantly augments the increase in phospholipase A2 activity and arachidonic acid release caused by lower doses of A23187. Under these conditions, sphingosine completely inhibits the stimulatory effects of 4 beta-PMA on protein kinase C translocation, phospholipase A2 and arachidonic acid release. Thus, at low doses (1 microM and 2.5 microM) A23187 increases phospholipase A2 activity and arachidonic acid release by a mechanism that does not involve protein kinase C. At these A23187 doses, activating membrane-bound protein kinase C with 4 beta-PMA causes a synergistic increase in phospholipase A2 activity and arachidonic acid release. At higher doses (5-12.5 microM), A23187 acts in large part by stimulating protein kinase C translocation. Overall, our results indicate that activating membrane-bound protein kinase C by itself is an insufficient stimulus to increase phospholipase A2 activity and arachidonic acid release in pulmonary endothelial cells, but activating protein kinase C can substantially augment the increase in phospholipase A2 activity and arachidonic acid caused by a small increase in intracellular calcium.
我们试图研究钙离子载体A23187在牛肺内皮细胞中触发花生四烯酸释放的机制，并检验蛋白激酶C参与此过程的假说。我们的结果表明，A23187增加牛肺动脉内皮细胞中磷脂酶A2活性和花生四烯酸释放的机制取决于所研究的浓度。在1微摩尔/升和2.5微摩尔/升的浓度下，A23187增加磷脂酶A2活性和花生四烯酸释放，而不刺激蛋白激酶C。在5 - 12.5微摩尔/升的浓度下，A23187增加花生四烯酸释放和磷脂酶A2活性，同时伴随着膜结合蛋白激酶C的剂量依赖性激活。为了检验这些剂量的A23187通过刺激蛋白激酶C来增加磷脂酶A2活性的假说，我们研究了用蛋白激酶C抑制剂鞘氨醇预先处理的效果。鞘氨醇抑制A23187在5 - 12.5微摩尔/升范围内引起的磷脂酶A2活性增加和花生四烯酸释放。为了进一步研究蛋白激酶C的潜在作用，我们研究了无活性佛波酯4α-佛波醇12β-肉豆蔻酸13α-乙酸酯（4α-PMA）和活性佛波酯4β-佛波醇12β-肉豆蔻酸13α-乙酸酯（4β-PMA）的作用。4α-PMA和4β-PMA均不影响基础花生四烯酸释放。4α-PMA也不增强A23187的作用。相反，4β-PMA显著增强较低剂量A23187引起的磷脂酶A2活性增加和花生四烯酸释放。在这些条件下，鞘氨醇完全抑制4β-PMA对蛋白激酶C易位、磷脂酶A2和花生四烯酸释放的刺激作用。因此，在低剂量（1微摩尔/升和2.5微摩尔/升）时，A23187通过一种不涉及蛋白激酶C的机制增加磷脂酶A2活性和花生四烯酸释放。在这些A23187剂量下，用4β-PMA激活膜结合蛋白激酶C会导致磷脂酶A2活性和花生四烯酸释放协同增加。在较高剂量（5 - 12.5微摩尔/升）时，A23187主要通过刺激蛋白激酶C易位起作用。总体而言，我们的结果表明，单独激活膜结合蛋白激酶C不足以刺激肺内皮细胞中磷脂酶A2活性增加和花生四烯酸释放，但激活蛋白激酶C可显著增强细胞内钙少量增加所引起的磷脂酶A2活性和花生四烯酸增加。
