hoe-k86-4321 and icatibant

1. Experiments were designed to differentiate the mechanisms and subtype of kinin receptors mediating the changes in intracellular Ca2+ concentration ([Ca2+]i) induced by bradykinin (BK) in canine cultured tracheal epithelial cells (TECs). 2. BK and Lys-BK caused an initial transient peak of [Ca2+]i in a concentration-dependent manner, with half-maximal stimulation (pEC50) obtained at 7.70 and 7.23, respectively. 3. Kinin B2 antagonists Hoe 140 (10 nM) and [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK (1 microM) had high affinity in antagonizing BK-induced Ca2+ response with pKB values of 8.90 and 6.99, respectively. 4. Pretreatment of TECs with pertussis toxin (100 ng ml(-1)) or cholera toxin (10 microg ml(-1)) for 24 h did not affect the BK-induced IP accumulation and [Ca2+]i changes in TECs. 5. Removal of Ca2+ by the addition of EGTA or application of Ca2+-channel blockers, verapamil, diltiazem, and Ni2+, inhibited the BK-induced IP accumulation and Ca2+ mobilization, indicating that Ca2+ influx was required for the BK-induced responses. 6. Addition of thapsigargin (TG), which is known to deplete intracellular Ca2+ stores, transiently increased [Ca2+]i in Ca2+-free buffer and subsequently induced Ca2+ influx when Ca2+ was re-added to this buffer. Pretreatment of TECs with TG completely abolished BK-induced initial transient [Ca2+]i, but had slight effect on BK-induced Ca2+ influx. 7. Pretreatment of TECs with SKF96365 and U73122 inhibited the BK-induced Ca2+ influx and Ca2+ release, consistent with the inhibition of receptor-gated Ca2+-channels and phospholipase C in TECs, respectively. 8. These results demonstrate that BK directly stimulates kinin B2 receptors and subsequently phospholipase C-mediated IP accumulation and Ca2+ mobilization via a pertussis toxin-insensitive G protein in canine TECs. These results also suggest that BK-induced Ca2+ influx into the cells is not due to depletion of these Ca2+ stores, as prior depletion of these pools by TG has no effect on the BK-induced Ca2+ influx that is dependent on extracellular Ca2+ in TECs.

Topics: Animals; Bradykinin; Bradykinin Receptor Antagonists; Calcium; Calcium Channel Blockers; Cells, Cultured; Cholera Toxin; Dogs; Enzyme Inhibitors; Epithelial Cells; Estrenes; Female; Hydrolysis; Imidazoles; Male; Pertussis Toxin; Phosphatidylinositols; Pyrrolidinones; Receptor, Bradykinin B2; Thapsigargin; Trachea; Type C Phospholipases; Virulence Factors, Bordetella

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 kallidin caused an initial transient peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner, with half-maximal stimulation (log EC50) obtained at -8.10 M and -8.04 M, respectively. The BDK-induced rise in [Ca2+]i was not affected by the BDK B1 receptor antagonist, des-Arg9[Leu8]-BDK (10 microM). However, the BDK B2 receptor antagonists des-Arg[Hyp3, Thi5,8, D-Phe7]-BDK and Hoe 140 had high affinity in antagonizing BDK with pKB 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 evidenced by a decline to the resting level on addition of EGTA. In the absence of external Ca2+, only an initial transient peak was seen which then declined to the resting level; a sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of BDK. Ca2+ influx was required for the changes in [Ca2+]i, since Ca(2+)-channel blockers, diltiazem, verapamil, and Ni2+, decreased both 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 B2 receptors is due to the release of Ca2+ from internal stores, followed by the influx of external Ca2+ into the cells. The influx of extracellular Ca2+ partially involves a diltiazem- and verapamil-sensitive Ca2+ channel.

Topics: Animals; Bradykinin; Calcium; Carbachol; Cells, Cultured; Diltiazem; Dogs; Dose-Response Relationship, Drug; Egtazic Acid; Female; Fura-2; Kallidin; Male; Muscle, Smooth; Trachea; Verapamil

Arthritic hyperalgesia and pain result from an increased activity of nociceptive afferents that may be induced and maintained by inflammatory mediators like bradykinin (BK). The B1 receptor antagonist des-Arg9-Leu8-BK and the B2 receptor antagonists Thi5,8-D-Phe7-BK and Hoe 140 were used to study the involvement of BK receptors in the generation of ongoing afferent activity in the cat's knee joint that was inflamed by kaolin and carrageenin. After i.a. bolus administration of BK receptor antagonists (26-260 micrograms) close to the joint, the ongoing activity did not significantly vary in any group III or group IV unit. We conclude that activation of BK receptors by endogenous BK is probably not the mechanism that is responsible for the increased ongoing activity of articular afferents in the inflamed joint.

Topics: Animals; Arthritis, Experimental; Bradykinin; Bradykinin Receptor Antagonists; Cats; Hindlimb; Neural Conduction; Neurons, Afferent; Nociceptors; Potassium Chloride