Peptide and Non-Peptide Bradykinin (BK) Agonists and Antagonists Help Define Functional BK Receptors in Human Trabecular Meshwork and Ciliary Body

Abstract

Purpose: 1) to examine bradykinin (BK) receptor system in human trabecular meshwork (h-TM), human ciliary muscle (h-CM) and ciliary process (CP) by immunohistochemistry; 2) to pharmacologically characterize the associated signaling mechanisms in isolated cells from these tissues using peptide and non-peptide BK agonists (Compounds 1 & 2) and antagonists; 3) to study the intraocular pressure (IOP)-lowering effects of Compound 1 (CMPD-1)

Methods: Published methods were utilized throughout

Results: Human and Cynomolgus monkey TM, CM and CP expressed a high level of B₂-receptor protein immunoreactivity. In isolated h-TM, h-CM and human non-pigmented epithelial (h-NPE) cells, BK and related analogs (e.g. Lys-BK; Hyp³-BK) exhibited a high potency (EC₅₀ = 2-8 nM, n = 3-6), while Des-Arg⁹-BK (B₁-receptor agonist) was much weaker (EC₅₀= 4.2 µM), at stimulating intracellular Ca²⁺ ([Ca²⁺]_i) mobilization. Two non-peptide B₂-receptor agonists (CMPD-1 & 2) had lower potencies (CMPD-1 EC₅₀s = 150-276 nM; CMPD-2 EC₅₀s = 25-74 nM; n = 3-29) than BK in these assays. While BK peptides and CMPD-2 were full agonists, CMPD-1 was a partial agonist (E_max = 38% in NPE; 64% in CM; 80% in TM cells). These effects of BK, CMPD-1 & 2 were blocked by HOE-140 (peptide B₂-antagonist; K_i = 1-8 nM; n = 4-6) and WIN-63448 (non-peptide B₂-antagonist; K_i = 157 - 450 nM, n = 4-5) in all these ocular cells. While h-CM and h-TM cells responded to BK, CMPD-1 & 2 by secreting prostaglandins (PGE₂, PGF_2α) (e.g. EC₅₀s = 4 - 61 nM in h-TM cells, n = 3-5; CMPD-1 E_max = 28%), NPE cells failed to release any PGs. The latter PG secretion effects of the BK agonists were also blocked by HOE-140 and WIN-63448, and were attenuated by cyclooxygenase inhibitors (bromfenac and flurbiprofen). BK, CMPD-1 & 2 also increased secretion of Pro-MMP-3 by 1.3-1.6 fold above basal levels in h-CM cells 24 hr post incubation. CMPD-1 lowered IOP in conscious ocular hypertensive Cynomolgus monkeys (e.g. 37.7 ± 5.4% with 30 µg, 24 hrs post topical ocular dosing).

Conclusions: These data support the existence of functionally active B₂-receptors in h-TM, h-CM and h-NPE cells that mediate [Ca²⁺]_i mobilization, PG secretion (not in NPE cells) and pro-MMP-3 release (h-CM). These data help explain the potent, efficacious, and prolonged IOP-lowering effects of CMPD-1.

Keywords: 735 trabecular meshwork • 675 receptors: pharmacology/physiology • 457 ciliary processes