Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Ophthalmol. Aug 12, 2016; 6(3): 20-27
Published online Aug 12, 2016. doi: 10.5318/wjo.v6.i3.20
Human ciliary muscle cell responses to kinins: Activation of ERK1/2 and pro-matrix metalloproteinases secretion
Najam A Sharif, Rajkumar Patil, Linya Li, Shahid Husain
Najam A Sharif, Rajkumar Patil, Linya Li, Pharmaceutical Research, Alcon Research, Ltd. (A Novartis Company), Fort Worth, TX 76134, United States
Shahid Husain, Ophthalmology, Medical University of South Carolina, Charlston, SC 29425, United States
Author contributions: All authors contributed to this paper.
Institutional review board statement: Since this is a basic research in vitro study, there was no institutional board review needed.
Institutional animal care and use committee statement: Since these were in vitro studies, no animals were involved, and thus no IACUC approval was necessary.
Conflict-of-interest statement: None of the authors have any conflict of interest or financial interest to report.
Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author at Participants gave informed consent for data sharing by signing copyright assignment form.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Najam A Sharif, PhD, Pharmaceutical Research, Alcon Research, Ltd. (A Novartis Company), 6201 South Freeway, Fort Worth, TX 76134, United States.
Telephone: +1-817-5657686 Fax: +1-415-2689181
Received: May 26, 2016
Peer-review started: May 26, 2016
First decision: July 5, 2016
Revised: July 29, 2016
Accepted: August 7, 2016
Article in press: August 8, 2016
Published online: August 12, 2016

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.


Serum-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.


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, 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-treatment of 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.


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.

Keywords: Extracellular signal-regulated kinase-1/2, Bradykinin, Ciliary muscle, Matrix metalloproteinases, B2-receptor

Core tip: Bradykinin (BK), its peptide and non-peptide analogs and mimetic were potently and efficaciously able to stimulate extracellular signal-regulated kinase 1/2 phosphorylation in human ciliary muscle (h-CM) cells in vitro. Additionally, these agonists also induced the production and secretion of pro-matrix metalloproteinases in a prostaglandin-independent manner. Selective antagonists helped link these responses to be mediated via the B2-receptor sub-type in h-CM cells. These mechanistic studies show how BK can lower intraocular pressure in animals when delivered to the eye.