Human ciliary muscle cell responses to kinins:Activation of ERK1/2 and pro-matrix metalloproteinases secretion  

作　　者：Najam A Sharif Rajkumar Patil Linya Li Shahid Husain 

机构地区：[1]Pharmaceutical Research, Alcon Research, Ltd. (A Novartis Company) [2]Ophthalmology, Medical University of South Carolina

出　　处：《World Journal of Ophthalmology》2016年第3期20-27,共8页世界眼科杂志

摘　　要：AIM To study activation of extracellular signal-regulated kinase-1/2(ERK1/2) and pro-matrix metalloproteinases(pro-MMPs) secretion from isolated primary human ciliary muscle(h-CM) cells in response to bradykinin(BK) and other agonists. METHODS Serum-starved h-CM cells were challenged with vehicle, BK agonists or antagonists. Cell lysates were evaluated for phosphorylated ERK1/2 using homogeneous timeresolved fluorescence technology based on a sandwich immunoassay. Rabbit polyclonal anti-pro-MMP antibodies were used to measure pro-MMPs using immunoblot analysis.RESULTS A 10 min incubation time using 5 × 104 h-CM cells/well was optimum condition for studying stimulation of ERK1/2 phosphorylation. BK(100 nmol/L) caused a 1.86 ± 0.26 fold(n = 3) increase in ERK1/2 phosphorylation above baseline. BK analogs, Met-Lys-BK and RMP-7(100 nmol/L), also stimulated ERK1/2 phosphorylation by 1.57 ± 0.04 and 1.55 ± 0.09 fold, respectively. However, DesArg9-Bradykinin, a B1 receptor-selective agonist(0.1-1 μmol/L), was essentially inactive. HOE-140 or WIN-64338(B2-antagonists) appreciably blocked phosphorylation of ERK1/2 induced by various BK agonists. Pre-treatmentof cells with a prostaglandin(PG) synthase inhibitor(bromfenac; 1 μmol/L) failed to alter kinin-induced ERK1/2 activation. BK and a non-peptide BK agonist(FR-190997)(10 nmol/L-1 μmol/L) also enhanced pro-MMPs secretion(pro-MMP-1 > pro-MMP-3 > pro-MMP-2; 1.45-1.75-fold over baseline) from h-CM cells. CONCLUSION These collective data suggest that B2 kinin receptors initiate signaling in h-CM cells by a relatively rapid mechanism(within minutes) involving ERK1/2 activation which in turn regulates MMPs production(within hours). The latter process does not involve PGs.AIM To study activation of extracellular signal-regulated kinase-1/2 （ERK1/2） and pro-matrix metalloproteinases （pro-MMPs） secretion from isolated primary human ciliary muscle （h-CM） cells in response to bradykinin （BK） and other agonists. METHODSSerum-starved h-CM cells were challenged with vehicle, BK agonists or antagonists. Cell lysates were evaluated for phosphorylated ERK1/2 using homogeneous time-resolved fluorescence technology based on a sandwich immunoassay. Rabbit polyclonal anti-pro-MMP antibodies were used to measure pro-MMPs using immunoblot analysis.RESULTSA 10 min incubation time using 5 × 104 h-CM cells/well was optimum condition for studying stimulation of ERK1/2 phosphorylation. BK （100 nmol/L） caused a 1.86 ± 0.26 fold （ n = 3） increase in ERK1/2 phosphorylation above baseline. BK analogs, Met-Lys-BK and RMP-7 （100 nmol/L）, also stimulated ERK1/2 phosphorylation by 1.57 ± 0.04 and 1.55 ± 0.09 fold, respectively. However, Des-Arg9-Bradykinin, a B1 receptor-selective agonist （0.1-1 μmol/L）, was essentially inactive. HOE-140 or WIN-64338 （B2-antagonists） appreciably blocked phosphorylation of ERK1/2 induced by various BK agonists. Pre-treatmentof cells with a prostaglandin （PG） synthase inhibitor （bromfenac; 1 μmol/L） failed to alter kinin-induced ERK1/2 activation. BK and a non-peptide BK agonist （FR-190997） （10 nmol/L-1 μmol/L） also enhanced pro-MMPs secretion （pro-MMP-1 〉 pro-MMP-3 〉 pro-MMP-2; 1.45-1.75-fold over baseline） from h-CM cells. CONCLUSIONThese collective data suggest that B2 kinin receptors initiate signaling in h-CM cells by a relatively rapid mechanism （within minutes） involving ERK1/2 activation which in turn regulates MMPs production （within hours）. The latter process does not involve PGs.

关 键 词：Extracellular signal-regulated kinase-1/2 BRADYKININ Ciliary muscle Matrix metalloproteinases B2-receptor 

分 类 号：R77[医药卫生—眼科]