Published online Aug 12, 2016. doi: 10.5318/wjo.v6.i3.20
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.
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.