The expression of functionally‐coupled B2‐bradykinin receptors in human corneal epithelial cells and their pharmacological characterization with agonists and antagonists

Bradykinin (BK) and Lys‐BK are peptides which are released at high nanomolar concentrations into the tear‐film of ocular allergic patients. We hypothesized that these peptides may activate specific receptors on the ocular surface, especially the corneal epithelium (CE) and thus the CE cells may represent a potential target tissue for these kinins. The purpose of the present studies, therefore, was to determine the presence of and the pharmacological characteristics of bradykinin receptors on normal cultured primary and SV40 virus‐transformed human corneal epithelial (CEPI) cells by use of the accumulation of [3H]‐inositol phosphates ([3H]‐IPs) as a bioassay. Bradykinin (BK) induced a maximal 1.95±0.24 fold (n=17) and 2.51±0.29 fold (n=26) stimulation of [3H]‐IPs accumulation in normal, primary (P‐CEPI) and SV40‐immortalized (CEPI‐17‐CL4) cells, respectively. This contrasted with a maximal 3.2–4.5 fold and 2.0–2.9 fold stimulation by histamine (100 μM) and platelet activating factor (100 nM) in both cell‐types, respectively. The molar potencies of BK and some of its analogues in the CEPI‐17‐CL4 cells were as follows: BK (EC50=3.26±0.61 nM, n=18), Lys‐BK (EC50=0.95±0.16 nM, n=5), Met‐Lys‐BK (EC50=2.3± 0.42 nM, n=5), Ile‐Ser‐BK (EC50=5.19±1.23 nM, n=6), Ala3‐Lys‐BK (EC50=12.7±2.08 nM, n=3), Tyr8‐BK (EC50=19.3±0.77 nM, n=3), Tyr5‐BK (EC50=467±53 nM, n=4) and des‐Arg9‐BK (EC50=14.1±2.7 μM, n=4). The potencies of BK‐related peptides in normal, P‐CEPI cells were similar to those found in transformed cells, thus: BK, EC50=2.02±0.69 nM (n=7), Tyr8‐BK, EC50=14.6±2.7 nM (n=3), Tyr5=BK, EC50=310±70 nM (n=4) and des‐Arg9‐BK, EC50=12.3± 3.8 μM (n=3). The bradykinin‐induced responses were competitively antagonized by the B2‐receptor selective BK antagonists, Hoe‐140 (D‐Arg‐[Hyp3,Thi5,D‐Tic7, Oic8]BK; Icatibant; molar antagonist potency=2.9 nM; pA2=8.54±0.06, n=4; and slope=1.04±0.08) and D‐Arg0[Hyp3,Thi5,8, DPhe7]‐BK (KB=371 nM; pKB=6.43±0.08, n=4) in CEPI‐17‐CL4 cells. The antagonist potency of Hoe‐140 against BK in normal, P‐CEPI cells was 8.4±1.8 nM (pKi=8.11±0.12, n=4), this being similar to the potency observed in the immortalized cells. This rank order of potency of agonist BK‐related peptides, coupled with the antagonism of the BK‐induced [3H]‐IPs by the specific B2‐receptor antagonists, strongly suggests that a B2‐receptor subtype is involved in mediating functional phosphoinositide (PI) responses in the CEPI‐17‐CL4 and P‐CEPI cells. In conclusion, these data ind