Yousufzai S Y, Gao G, Abdel-Latif A A
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100, USA.
Eur J Pharmacol. 2000 Oct 27;407(1-2):17-26. doi: 10.1016/s0014-2999(00)00713-5.
The purpose of this study was to investigate the potential role of mitogen-activated protein (MAP) kinase in contraction by monitoring MAP kinase phosphorylation (activation) and contraction during agonist stimulation of cat iris sphincter smooth muscle. Changes in tension in response to prostaglandin F(2alpha), latanoprost, a prostaglandin F(2alpha) analog used as an anti-glaucoma drug, and carbachol were recorded isometrically, and MAP kinase activation was monitored by Western blot using a phosphospecific p42/p44 MAP kinase antibody. We found that treatment of the muscle with 2'-Amino-3'-methoxyflavone (PD98059) (10 microM), a specific inhibitor of MAP kinase kinase (MEK), inhibited significantly prostaglandin F(2alpha)- and latanoprost-induced phosphorylation and contraction, but had little effect on those evoked by carbachol. Prostaglandin F(2alpha) increased MAP kinase phosphorylation in a concentration-dependent manner with EC(50) value of 1.1 x 10(-8) M and increased contraction with EC(50) of 0.92 x 10(-9) M. The MAP kinase inhibitors PD98059, Apigenin and 1,4-Diamino-2,3-dicyano-1, 4bis(2-aminophenylthio)butadiene (UO126) inhibited prostaglandin F(2alpha)-induced contraction in a concentration-dependent manner with IC(50) values of 2.4, 3.0 and 4.8 microM, respectively. PD98059 had no effect on prostaglandin F(2alpha)- or on carbachol-stimulated inositol-1,4,5-trisphosphate (IP(3)) production. In contrast, the MAP kinase inhibitor inhibited prostaglandin F(2alpha)-induced myosin-light chain (MLC) phosphorylation, but had no effect on that of carbachol. N-[2-(N-(4-Chloro-cinnamyl)-N-methylaminomethyl)phenyl]-N-[2- hydroxyethyl]-4-methoxybenzenesulfonamide (KN-93) (10 microM), a Ca(2+)-calmodulin-dependent protein kinase inhibitor, and Wortmannin (10 microM), an MLC kinase inhibitor, inhibited significantly (by 80%) prostaglandin F(2alpha)- and carbachol-induced contraction. It can be concluded that in this smooth muscle p42/p44 MAP kinases are involved in the mechanism of prostaglandin F(2alpha)-, but not in that of carbachol, induced contraction. In addition, these data clearly indicate that the stimulation of the iris sphincter with prostaglandin F(2alpha) and carbachol activate two distinct pathways, the MAP kinase pathway and the Ca(2+) mobilization pathway.
本研究的目的是通过监测丝裂原活化蛋白（MAP）激酶磷酸化（激活）以及猫虹膜括约肌平滑肌在激动剂刺激下的收缩情况，来探究MAP激酶在收缩过程中的潜在作用。以等长方式记录了前列腺素F(2α)、拉坦前列素（一种用作抗青光眼药物的前列腺素F(2α)类似物）和卡巴胆碱引起的张力变化，并使用磷酸特异性p42/p44 MAP激酶抗体通过蛋白质免疫印迹法监测MAP激酶的激活情况。我们发现，用MAP激酶激酶（MEK）的特异性抑制剂2'-氨基-3'-甲氧基黄酮（PD98059）（10微摩尔）处理肌肉，可显著抑制前列腺素F(2α)和拉坦前列素诱导的磷酸化和收缩，但对卡巴胆碱引起的磷酸化和收缩影响不大。前列腺素F(2α)以浓度依赖方式增加MAP激酶磷酸化，其半数有效浓度（EC(50)）值为1.1×10(-8) M，同时以0.92×10(-9) M的EC(50)增加收缩。MAP激酶抑制剂PD98059、芹菜素和1,4-二氨基-2,3-二氰基-1,4-双（2-氨基苯硫基）丁二烯（UO126）以浓度依赖方式抑制前列腺素F(2α)诱导的收缩，其半数抑制浓度（IC(50)）值分别为2.4、3.0和4.8微摩尔。PD98059对前列腺素F(2α)或卡巴胆碱刺激的肌醇-1,4,5-三磷酸（IP(3)）生成没有影响。相反，MAP激酶抑制剂抑制前列腺素F(2α)诱导的肌球蛋白轻链（MLC）磷酸化，但对卡巴胆碱诱导的MLC磷酸化没有影响。N-[2-(N-(4-氯肉桂基)-N-甲基氨基甲基)phenyl]-N-[2-羟乙基]-4-甲氧基苯磺酰胺（KN-93）（10微摩尔），一种钙调蛋白依赖性蛋白激酶抑制剂，以及渥曼青霉素（10微摩尔），一种MLC激酶抑制剂，可显著（80%）抑制前列腺素F(2α)和卡巴胆碱诱导的收缩。可以得出结论，在这种平滑肌中，p42/p44 MAP激酶参与前列腺素F(2α)诱导的收缩机制，但不参与卡巴胆碱诱导的收缩机制。此外，这些数据清楚地表明，用前列腺素F(2α)和卡巴胆碱刺激虹膜括约肌激活了两条不同的途径，即MAP激酶途径和钙动员途径。
