Alkozi Hanan Awad, Perez de Lara María J, Pintor Jesús
Department of Biochemistry and Molecular Biology IV, Faculty of Optics and Optometry, Universidad Complutense de Madrid, Madrid, Spain.
Department of Biochemistry and Molecular Biology IV, Faculty of Optics and Optometry, Universidad Complutense de Madrid, Madrid, Spain.
Exp Eye Res. 2017 Sep;162:1-8. doi: 10.1016/j.exer.2017.06.018. Epub 2017 Jun 24.
Melatonin is a substance synthesized in the pineal gland as well as in other organs. This substance is involved in many ocular functions, giving its synthesis in numerous eye structures. Melatonin is synthesized from serotonin through two enzymes, the first limiting step into the synthesis of melatonin being aralkylamine N-acetyltransferase (AANAT). In this current study, AANAT phosphorylation after the activation of TRPV4 was studied using human non-pigmented epithelial ciliary body cells. Firstly, it was necessary to determine the adequate time and dose of the TRPV4 agonist GSK1016790A to reach the maximal phosphorylation of AANAT. An increase of 72% was observed after 5 min incubation with 10 nM GSK (**p < 0.05, n = 6) with a concomitant rise in N-acetyl serotonin and melatonin synthesis. The involvement of a TRPV4 channel in melatonin synthesis was verified by antagonist and siRNA studies as a previous step to studying intracellular signalling. Studies performed on the second messengers involved in GSK induced AANAT phosphorylation were carried out by inhibiting several pathways. In conclusion, the activation of calmodulin and calmodulin-dependent protein kinase II was confirmed, as shown by the cascade seen in AANAT phosphorylation (***p < 0.001, n = 4). This mechanism was also established by measuring N-acetyl serotonin and melatonin levels. In conclusion, the activation of a TRPV4 present in human ciliary body epithelial cells produced an increase in AANAT phosphorylation and a further melatonin increase by a mechanism in which Ca-calmodulin and the calmodulin-dependent protein kinase II are involved.
褪黑素是一种在松果体以及其他器官中合成的物质。这种物质参与多种眼部功能，在众多眼结构中均有合成。褪黑素由血清素通过两种酶合成，其合成的第一步限速酶是芳烷基胺N - 乙酰基转移酶（AANAT）。在本研究中，使用人非色素上皮睫状体细胞研究了TRPV4激活后AANAT的磷酸化情况。首先，有必要确定TRPV4激动剂GSK1016790A达到AANAT最大磷酸化的合适时间和剂量。与10 nM GSK孵育5分钟后，观察到AANAT磷酸化增加了72%（**p < 0.05，n = 6），同时N - 乙酰血清素和褪黑素的合成也随之增加。在研究细胞内信号传导的前一步，通过拮抗剂和小干扰RNA（siRNA）研究验证了TRPV4通道在褪黑素合成中的作用。通过抑制几种途径对参与GSK诱导的AANAT磷酸化的第二信使进行了研究。总之，如AANAT磷酸化过程中所见级联反应所示，钙调蛋白和钙调蛋白依赖性蛋白激酶II的激活得到了证实（***p < 0.001，n = 4）。通过测量N - 乙酰血清素和褪黑素水平也确立了这一机制。总之，人睫状体上皮细胞中存在的TRPV4的激活通过一种涉及钙 - 钙调蛋白和钙调蛋白依赖性蛋白激酶II的机制导致AANAT磷酸化增加以及褪黑素进一步增加。
