Pharmacological Characteristics of Extracellular Ca<sup>2+</sup> Influx Pathways Responsible for Platelet-Activating Factor-Induced Contractions in Rat Esophagus Smooth Muscle: Involvement of L-Type, Receptor-Operated, and Store-Operated

We examined the contribution of L-type voltage-dependent Ca²⁺ channels (VDCCs) and non-VDCCs to platelet-activating factor (PAF)-induced contractions of rat esophageal smooth muscle (ESM). We also attempted to obtain more detailed information about the non-VDCC molecules involved in the PAF effect. PAF (10^-6 M)-induced contractions were abolished in Ca²⁺-free solution containing ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid and were attenuated by diltiazem (10^-5 M), a VDCC inhibitor. PAF-induced contractions in the presence of diltiazem were inhibited by approx. 50% by LOE-908 (3 × 10^-5 M), an inhibitor of receptor-operated Ca²⁺ channels (ROCCs), and were strongly inhibited by LOE-908 plus SKF-96365 (3 × 10^-5 M), an inhibitor of both ROCCs and store-operated Ca²⁺ channels (SOCCs). The contribution of each Ca²⁺ channel was estimated to be approx. 25% for VDCCs, approx. 30% for ROCCs, and approx. 35% for SOCCs. Among the non-VDCC-related molecular candidates examined in rat ESM, Trpv4, Trpc6, and Trpc3 were abundant ROCC-related mRNAs, and Orai1 was the most abundant SOCC-related mRNAs. However, PAF-induced contractions in the presence of diltiazem were not significantly inhibited by combination treatment with putative inhibitors of transient receptor potential V4 (TRPV4) (GSK 2193874, 3 × 10^-7 M), TRPC6 (SAR7334, 10^-6 M), and TRPC3 (Pyr10, 3 × 10^-5 M). In contrast, PAF-induced contractions in the presence of both diltiazem and LOE-908 were completely inhibited by Synta66 (10^-5 M), an Orai1 inhibitor, which also inhibited PAF-induced contractions by approx. 30% in the absence of Ca²⁺ channel inhibitors. These findings indicate that PAF-induced rat ESM contractions depend on extracellular Ca²⁺ influx through VDCCs, ROCCs, and SOCCs, with Orai1 being the key SOCC molecule.