Pharmacological characterization of bradykinin receptors coupled to phosphoinositide turnover in SV40-immortalized human trabecular meshwork cells.

This study sought to pharmacologically characterize bradykinin receptors on SV40-immortalized human trabecular meshwork (HTM3) cells. Phosphoinositide (PI) turnover studies were conducted using [3H]myo-inositol-labeled HTM3 cells and anion exchange chromatography to quantify [3H]inositol phosphates generated in response to bradykinin (BK) and various BK analogs. The blockade of these responses was studied using two potent and receptor-subtype selective antagonists. BK and T-kinin (Ile-Ser-BK:TK) induced a 4.2-4.4 fold stimulation of PI turnover above base levels at 1-10 microM. Several other peptides unrelated to BK, including angiotensin II, endothelin, cholecystokinin, bombesin and peptide YY tested at 1-10 microM were essentially inactive. The molar potencies (EC50) of BK, TK and close analogs were: BK = 4.5 +/- 0.5 nM (n = 6), Lys-BK = 6.5 +/- 0.7 nM (n = 3), TK = 38.8 +/- 6.6 nM (n = 8), Met-Lys-BK = 41.5 +/- 13.4 nM (n = 4), Des-Arg9-BK = 2093 +/- 626 nM (n = 4). All the latter BK-related peptides were full agonists. The actions of BK and TK were potently and competitively antagonized by Hoe-140 (molar potency = 0.6-1 nM; pA2 = 8.97-9.21. n = 3-4) and by D-Arg0[Hyp3,-Thi5.8,-DPhe7]-BK (molar potency = 251 nM; -log potency, pKb = 6.6), two selective B2-type BK antagonists. In conclusion, rank order of potency of BK agonists and the blockade of BK- and TK-induced PI turnover by the selective antagonists are consistent with the classification of the BK receptors on HTM3 cells as the B2-receptor subtype.