Effects of NP-1815-PX, a P2X4 Receptor Antagonist, on Contractions in Guinea Pig Tracheal and Bronchial Smooth Muscles

Abstract

Administration of a P2X4 receptor antagonist to asthma model mice improved asthma symptoms, suggesting that P2X4 receptor antagonists may be new therapeutics for asthma. However, the effects of these antagonists on tracheal/bronchial smooth muscle (TSM and BSM) have not been investigated. This study examined the effects of NP-1815-PX, a selective P2X4 receptor antagonist, on guinea pig TSM and BSM contractions. In epithelium-intact TSM, NP-1815-PX (10 5 M) strongly suppressed ATP-induced contractions. ATP-induced contractions were strongly suppressed by indomethacin (3 10 6 M) and ONO-8130 (a prostanoid EP 1 receptor antagonist, 10 7 M). ATP-induced contractions were partially suppressed by SQ 29,548 (a prostanoid TP receptor antagonist, 3 10 7 M), although the difference was not significant. In contrast, ATP-induced contractions were not affected by AL 8810 (a prostanoid FP receptor antagonist, 10 5 M) or L-798,106 (a prostanoid EP 3 receptor antagonist, 10 8 M). NP-1815-PX (10 5 -10 4 M) strongly suppressed U46619 (a TP receptor agonist)-and prostaglandin F 2α (PGF 2α )-induced epithelium-denuded TSM and BSM contractions, which were largely inhibited by SQ 29,548. Additionally, NP-1815-PX (10 5 -10 4 M) strongly suppressed the U46619-induced increase in intracellular Ca 2 concentrations in human TP receptor-expressing cells. However, NP-1815-PX (10 4 M) did not substantially inhibit the TSM/BSM contractions induced by carbachol, histamine, neurokinin A, or 50 mM KCl. These findings indicate that NP-1815-PX inhibits guinea pig TSM and BSM contractions mediated through the TP receptor, in addition to the P2X4 receptor, whose stimulation mainly induces EP 1 receptor-related mechanisms. Thus, these findings support the usefulness of NP-1815-PX as a therapeutic drug for asthma.

Pharmacological Studies on the Ca²⁺ Influx Pathways in Platelet-Activating Factor (PAF)-Induced Mouse Urinary Bladder Smooth Muscle Contraction

“…Measurement of Intracellular Ca 2+ Concentrations ([Ca 2+ ] i ) in 293T Cells Measurement of [Ca 2+ ] i in 293T cells was performed as previously described. [13][14][15] Briefly, seeded 293T cells in a 96-well plate were incubated with recording medium [(mM): 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 20; NaCl, 115; KCl, 5.4; MgCl 2 , 0.8; CaCl 2 , 1.8; glucose, 13.8; pH 7.4] containing Fura-2 AM (5 × 10 −6 M) for 30-60 min at 37 °C/5% CO 2 . The cells were then rinsed with Fura-2 AM-free/Ca 2+ -free medium, and their fluorescence intensities were measured using a microplate reader (Nivo, PerkinElmer, Inc., MA, U.S.A.) at 25 °C in Ca 2+ -free medium.…”