Ciliary Muscle (CM) Bradykinin B2-Receptors: Protein Expression, Pharmacology of Biochemical Signaling Processes and Relation to IOP Modulation

Abstract

Purpose: : Studies were conducted to examine the bradykinin (BK) B₂-receptor system in ciliary muscle (CM) using immunohistochemical techniques, and to pharmacologically characterize the associated biochemical signal transduction systems in human CM (h-CM) cells. Some of the latter elements were compared with pharmacological aspects of human cloned B₂-receptor expressed in Chinese hamster ovary cells (CHO-B2). BK-induced modulation of intraocular pressure (IOP) of Dutch-Belt rabbits and Cynomolgus monkeys was also studied.

Methods: : Previously published procedures were used throughout these studies.

Results: : Human and monkey CB (ciliary muscle [CM] and non-pigmented ciliary epithelium) expressed a high level of B₂-receptor protein immunoreactivity. BK and its analogs differentially stimulated mobilization of [Ca²⁺]_i in primary cultured h-CM cells: BK EC₅₀ = 2.4 ± 0.2 nM = Hyp³-BK > Lys-BK EC₅₀ = 3.2 nM = Hyp³,β-(2-thienyl)-Ala⁵,Tyr(Me)⁸-(®)-Arg⁹)-BK (RMP-7) > Met-Lys-BK EC₅₀ = 16.1 nM >> Des-Arg⁹-BK EC₅₀ = 4.2 µM (n = 3-6). BK-induced [Ca²⁺]_i mobilization was blocked by B₂-receptor-selective antagonists, HOE-140 (IC₅₀ = 1.4 ± 0.1nM) and WIN-63448 (IC₅₀ = 174 ± 18 nM) (n = 3-7). A similar rank order of potency of agonists and antagonists was observed for [Ca²⁺]_i mobilization in CHO-B2 cells. In h-CM cells, BK-induced [Ca²⁺]_i mobilization response was abolished when all extracellular Ca²⁺ was chelated with 1 - 2.5 mM EGTA, and similarly abolished after a phospholipase C inhibitor (U73122; 10-30 µM) was added to the cells. A similar rank order of potency of the peptides was observed for the synthesis and release of endogenous total PGs from h-CM cells as noted above for the [Ca²⁺]_i mobilization response. Total PGs release stimulated by BK in h-CM and CHO-B2 cells was attenuated in a concentration-dependent manner by the cyclooxygenase inhibitors, bromfenac and flurbiprofen, and also by the B₂-antagonists. BK (100 nM) induced a 2-fold increase in extracellular signal-regulated kinase-1/2 phosphorylation, and RMP-7 (1-10 µM; at 24 hrs) caused a 1.4-2.1-fold increase in pro-MMP-1 and pro-MMP-2 levels above baseline in h-CM cells. Topical ocular dosing of BK (50 µg) to Cynomolgus monkeys did not alter IOP. However, intravitreal injection of 50 µg of BK (B₂-agonist), but not Des-Arg⁹-BK or Sar-[D-Phe⁹]-Des-Arg⁹-BK (B₁-agonists), resulted in a maximum 37.0 ± 5.6% reduction in IOP in rabbit eyes 8 hrs post-injection, relative to vehicle-injected eyes (n = 10/group).

Conclusions: : These results support a role for endogenous kallikrein/kinin components associated with CM, in particular the B₂-receptor, in aqueous humor modulation and thus IOP regulation.

Keywords: ciliary muscle • receptors: pharmacology/physiology • intraocular pressure