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Naj Sharif, Parvaneh Katoli, Linya Li, Shouxi Xu, Curtis Kelly, Richard Ornberg, Yu Wang, Laura Klekar, Daniel Scott, Shahid Husain; Ciliary Muscle (CM) Bradykinin B2-Receptors: Protein Expression, Pharmacology of Biochemical Signaling Processes and Relation to IOP Modulation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3671.
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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.
Previously published procedures were used throughout these studies.
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).
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.
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