Protein expression, biochemical pharmacology of signal transduction, and relation to intraocular pressure modulation by bradykinin B(2) receptors in ciliary muscle

PURPOSE: To examine the bradykinin (BK) B(2)-receptor system in human and monkey ciliary muscle (CM) using immunohistochemical techniques, and to pharmacologically characterize the associated biochemical signal transduction systems in human CM (h-CM) cells. BK-induced modulation of intraocular pressure (IOP) in pigmented Dutch-Belt rabbits and cynomolgus monkeys was also studied. METHODS: Previously published procedures were used throughout these studies. RESULTS: The human and monkey ciliary bodies expressed high levels of B(2)-receptor protein immunoreactivity. Various kinins differentially stimulated [Ca(2+)](i) mobilization in primary h-CM cells (BK EC(50)=2.4±0.2 nM > Hyp(3),β-(2-thienyl)-Ala(5),Tyr(Me)(8)-(®)-Arg(9))-BK (RMP-7) > Des-Arg(9)-BK EC(50)=4.2 µM [n=3–6]), and this was blocked by B(2)-selective antagonists, HOE-140 (IC(50)=1.4±0.1 nM) and WIN-63448 (IC(50)=174 nM). A phospholipase C inhibitor (U73122; 10–30 µM) and ethylene glycol tetraacetic acid (1–2 mM) abolished the BK-induced [Ca(2+)](i) mobilization. Total prostaglandin (primarily PGE(2)) secretion stimulated by BK and other kinins in h-CM cells was attenuated by the cyclooxygenase inhibitors bromfenac and flurbiprofen, and by the B(2)-antagonists. BK and RMP-7 (100 nM) induced a twofold increase in extracellular signal-regulated kinase-1/2 phosphorylation, and BK (0.1–1 µM; at 24 h) caused a 1.4–3.1-fold increase in promatrix metalloproteinases-1–3 release. Topical ocular BK (100 µg) failed to alter IOP in cynomolgus monkeys. However, intravitreal injection of 50 µg of BK, but not Des-Arg(9)-BK, lowered IOP in rabbit eyes (22.9±7.3% and 37.0±5.6% at 5 h and 8 h post-injection; n=7–10). CONCLUSIONS: These studies have provided evidence of a functional endogenously expressed B(2)-receptor system in the CM that appears to be involved in modulating IOP.