Activation Of Erk Signaling Pathway By Bradykinin B2 Receptors In Isolated Human Primary Ciliary Muscle Cells

Abstract

Purpose: : Bradykinin (BK) stimulation of B₂ kinin receptors has been shown to initiate signaling in trabecular meshwork cells and increase conventional outflow facility. The ciliary body, which regulates aqueous outflow via uveo-scleral pathway, also expresses both B₁ and B₂ kinin receptors and is a potential target for kinin action. Here we investigated whether BK and related kinin peptides can initiate signaling pathways involving ERK1 and ERK2, upstream of matrix metalloproteinase (MMP) secretion in isolated primary human ciliary muscle (h-CM) cells. The results support the possibility that B₂ kinin receptors in ciliary body may contribute to the regulation of aqueous outflow.

Methods: : Primary h-CM cells were cultured in 96-well plates and incubated overnight, at 37°C in CO₂ atmosphere. Cells were then starved off serum overnight before being challenged with BK agonists or antagonists at room temperature. Treated cell lysates were then evaluated for phosphorylated ERK1/2 using HTRF technology based on a sandwich immunoassay using an anti-phospho-ERK1/2 antibody labeled with d2 and an anti-ERK1/2 antibody labeled with Eu3⁺-Cryptate. The fluorescence signals were recorded at 620 nm for the donor and 665 nm for the acceptor.

Results: : BK treatment of h-CM cells caused an increase in ERK1/2 phosphorylation. The stimulation of ERK1/2 phosphorylation was 1.86 ± 0.26 fold (n = 3) in the presence of 100 nM BK compared to basal ERK1/2 phosphorylation. The optimal time of stimulation of ERK1/2 phosphorylation was 10 min using 50K cells/well. In addition, BK analogs Met-Lys-BK, and RMP-7 (100 nM) also increased ERK1/2 phosphorylation in h-CM cells by 1.57 ± 0.04 and 1.55 ± 0.09 fold, respectively. However, Des-Arg⁹-Bradykinin, a B₁ receptor selective agonist (0.1-1µM), did not increase ERK1/2 phosphorylation in h-CM cells. Phosphorylation of ERK1/2 was significantly blocked when h-CM cells were pre-treated with 100 nM of a peptide B₂ receptor antagonist, HOE-140, or a non-peptide B₂ receptor antagonist, WIN-64338, before being exposed to BK agonists. HOE-140 and WIN-64338 alone had no effect on phosphorylation of ERK1/2 in h-CM cells.

Conclusions: : BK caused a concentration- and time-dependent increase in ERK1/2 phosphorylation, which was attenuated by two selective B₂ receptor antagonists (HOE140; WIN-64338) in h-CM cells. In addition, Des-Arg9 Bradykinin, a B₁ receptor selective agonist did not show any effect on ERK1/2 phosphorylation in h-CM cells. These observations suggests that B₂ kinin receptors initiate signaling in h-CM cells by activating ERK1/2 which in turn may regulate MMP release and ultimately regulate the uveo-scleral pathway resulting in modulation of intraocular pressure.

Keywords: ciliary muscle • receptors: pharmacology/physiology • phosphorylation