BRADYKININ-STIMULATED CALCIUM MOBILIZATION IN CULTURED CANINE TRACHEAL SMOOTH-MUSCLE CELLS

Bradykinin (BDK)-induced increases in intracellular Ca2+ concentration ([Ca2+](i)) were monitored in cultured canine tracheal smooth muscle cells (TSMCs) using a fluorescent Ca2+ indicator, Fura-2. BDK and kall idin caused an initial transient peak followed by a sustained elevatio n of [Ca2+](i) in a concentration-dependent manner, with half-maximal stimulation (log EC(50)) obtained at -8.10 M and -8.04 M, respectively . The BDK-induced rise in [Ca2+](i) was not affected by the BDK B-1 re ceptor antagonist, des-Arg(9)[Leu(8)]-BDK (10 mu M). However, the BDK B-2 receptor antagonists des-Arg[Hyp(3), Thi(5,8), D-Phe(7)]-BDK and H oe 140 had high affinity in antagonizing BDK with pK(B) values of 7.5 +/- 0.3 and 8.7 +/- 0.3, respectively. The sustained phase of the rise in [Ca2+](i) was dependent on the presence of external Ca2+ as eviden ced by a decline to the resting level on addition of EGTA. In the abse nce of external Ca2+, only an initial transient peak was seen which th en declined to the resting level; a sustained elevation of [Ca2+](i) c ould then be evoked by addition of 1.8 mM Ca2+ in the continued presen ce of BDK. Ca2+ influx was required for the changes in [Ca2+](i), sinc e Ca2+-channel blockers, diltiazem, verapamil, and Ni2+, decreased bot h the initial and sustained elevation of [Ca2+](i) in response to BDK. In conclusion, these findings indicate that the initial increase in [ Ca2+](i) stimulated by BDK acting on BDK B-2 receptors is due to the r elease of Ca2+ from internal stores, followed by the influx of externa l Ca2+ into the cells. The influx of extracellular Ca2+ partially invo lves a diltiazem- and verapamil-sensitive Ca2+ channel.