Ansari Habib R, Kaddour-Djebbar Ismail, Abdel-Latif Ata A
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA.
Exp Eye Res. 2004 Feb;78(2):285-96. doi: 10.1016/j.exer.2003.10.015.
A potential role for myosin light chain kinase (MLCK) in regulating intraocular pressure and outflow function has recently been reported in living monkey eye and rabbit eye. There is little information about the effects of the ocular hypotensive agents, prostaglandin F2alpha (PGF2alpha) and latanoprost on this signaling pathway in ocular tissues. The aim of this study was to determine the agonist activity of PGF2alpha, latanoprost and carbachol (CCh) on the MLCK pathway in isolated bovine iris sphincter and furthermore to investigate the existence of the FP receptor in this tissue. In the present studies on the MLCK pathway four signal transduction mechanism assays were employed, phosphoinositide (PI) turnover, p42/p44 MAP kinase phosphorylation and activation, MLC phosphorylation and contraction. In the studies on the existence of the FP receptor in the bovine iris sphincter, the pharmacology and expression of the FP receptor protein, using a polyclonal anti-FP-receptor antibody and Western blot analysis, were determined. The data obtained on the MLCK pathway showed that the three agonists stimulated the biochemical and pharmacological responses in a concentration and time-dependent manner and that the order of potency and efficacy is PGF2alpha>latanoprost>CCh. The EC50 values in the PI turnover, MAP kinase phosphorylation, MLC phosphorylation and contraction assays were for PGF2alpha: 9, 42, 200 and 140 nM, respectively, for latanoprost: 13, 59, 250 and 828 nM, respectively, and for CCh: 22, 200, 630 and 910 nM, respectively. Wortmannin, a selective inhibitor of MLCK, dose-dependently inhibited MLC phosphorylation and contraction induced by PGF2alpha, demonstrating a close relationship between activation of the MLCK pathway and contraction. The pharmacological studies showed that in the concentration range of 1 nM to 10 microM, the FP-receptor agonists caused concentration-response curves with the following order of potencies: 17-phenyl trinor PGF2alpha (bimatoprost acid)>PGF2alpha>cloprostenol>latanoprost>latanoprost acid>bimatoprost amide>>fluprostenol. Immunoblot analysis of the FP receptor demonstrated expression of the prostaglandin FP receptor protein in this smooth muscle. These results clearly indicate that the MLCK signaling pathway is involved in the FP-receptor function of the bovine iris sphincter and furthermore demonstrate that functional FP receptors exist and are expressed in this tissue.
最近有报道称，肌球蛋白轻链激酶（MLCK）在调节活猴眼和兔眼的眼压及房水流出功能中可能发挥作用。关于降眼压药物前列腺素F2α（PGF2α）和拉坦前列素对眼组织中该信号通路的影响，目前所知甚少。本研究的目的是确定PGF2α、拉坦前列素和卡巴胆碱（CCh）对离体牛虹膜括约肌中MLCK途径的激动剂活性，并进一步研究该组织中FP受体的存在情况。在目前关于MLCK途径的研究中，采用了四种信号转导机制检测方法，即磷酸肌醇（PI）周转、p42/p44丝裂原活化蛋白激酶磷酸化和激活、MLC磷酸化以及收缩。在关于牛虹膜括约肌中FP受体存在情况的研究中，使用多克隆抗FP受体抗体和蛋白质印迹分析，确定了FP受体蛋白的药理学特性和表达情况。在MLCK途径上获得的数据表明，这三种激动剂以浓度和时间依赖性方式刺激生化和药理反应，其效力和效能顺序为PGF2α>拉坦前列素>CCh。在PI周转、丝裂原活化蛋白激酶磷酸化、MLC磷酸化和收缩检测中的半数有效浓度（EC50）值，PGF2α分别为9、42、200和140 nM，拉坦前列素分别为13、59、250和828 nM，CCh分别为22、200、630和910 nM。渥曼青霉素是MLCK的选择性抑制剂，它能剂量依赖性地抑制PGF2α诱导的MLC磷酸化和收缩，表明MLCK途径的激活与收缩之间存在密切关系。药理学研究表明，在1 nM至10 μM的浓度范围内，FP受体激动剂产生的浓度-反应曲线效力顺序如下：17-苯基三去甲PGF2α（比马前列素酸）>PGF2α>氯前列醇>拉坦前列素>拉坦前列素酸>比马前列素酰胺>>氟前列醇。对FP受体的免疫印迹分析表明，在这种平滑肌中存在前列腺素FP受体蛋白的表达。这些结果清楚地表明，MLCK信号通路参与了牛虹膜括约肌的FP受体功能，并且进一步证明了功能性FP受体在该组织中存在并表达。
