Abstract
Serotonin, or 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter found in blood platelets, the gastrointestinal (GI) tract, and the central nervous system (CNS) of animals and humans. The signaling pathways of 5-hydroxytryptamine (5-HT)-induced contractions in cat esophageal smooth muscle cell (ESMC)s have been identified, but the downstream components of the 5-HT signaling pathway remain unclear. DA-9701 is the standardized extract of the Pharbitis nil Choisy seed (Pharbitidis Semen, Convolvulaceae) and the root of Corydalis yahusuo W.T. Wang (Corydalis Tuber, Papaveraceae). DA-9701 is known to have strong gastroprokinetic effects and a good safety profile. In this study, we investigated the 5-HT signaling pathway at the G-protein level, and we explored the mechanisms by which DA-9701 induces smooth muscle contraction. Freshly isolated smooth muscle cells were harvested from the feline esophagus, and cells were permeabilized to measure their length. 5-HT produced esophageal smooth muscle contractions in a dose-dependent manner. Furthermore, 5-HT produced a relatively long-acting contraction. 5-HT binds to the 5-HT2, 5-HT3 and 5-HT4 receptors to induce smooth muscle contraction in feline ESMCs. These receptors, which are located in esophageal smooth muscle, are coupled to Gαq, Gαo and Gαs. These G proteins activate PLC, which leads to Ca2+/calmodulin-dependent MLCK activation, resulting in MLC20 phosphorylation and cell contraction. Conversely, DA-9701 inhibits 5-HT-induced contraction by inhibiting MLC20 phosphorylation.
Highlights
Serotonin, or 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter found in blood platelets, the GI tract and the central nervous system (CNS) of animals and humans [1]
We observed that esophageal smooth muscle cell (ESMC) were separated from the esophageal smooth muscle tissue squares (Figure 1)
5-Hydroxytryptamine hydrochloride, methysergide maleate salt, ketanserin (+)-tartrate salt, ondansetron hydrochloride dihydrate, GR113808 (1-[2-[(methylsulfonyl)amino]ethyl]-4-piperidinyl]methyl-1-methyl1H-indole-3-carboxylate), α-methylserotonin, mosapride, U73122, ML-9, Y27632, and chelerythrine were from Sigma Chemical Co. (St Louis, MO, USA). 2-methyl-5-hydroxytryptamine HCl (Tocris, Bristol, UK), DA-9701 (Dong-A Pharmaceutical Co., Seoul, Korea ), G protein antibodies (Gi1, Gi2, Gi3, Gq, Gs, Go, and G β), PLC isozyme antibodies (β1, 3, γ1), phosphospecific Ser19-MLC20 antibody, and GAPDH antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and
Summary
5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter found in blood platelets, the GI tract and the central nervous system (CNS) of animals and humans [1]. Isolated ESMCs were permeabilized with saponin to allow G protein antibodies to enter into the cytoplasm These antisera, raised against synthetic peptides corresponding to the amino acid sequence of the carboxyl terminal of the G protein α subunit, have been used as effective probes for G protein structure and function [11]. Contraction of the lower esophageal sphincter by acetylcholine (ACh) is mediated by activation of M3 muscarinic receptors linked to Gq and to phosphatidylinositol-specific phospholipase. Acetylcholine-induced contraction of esophageal muscle is mediated by muscarinic M2 receptors linked to Gi3-type G proteins, which activate phosphatidylcholine-specific phospholipase C (PC-PLC). The signaling pathways initiated by 5-HT in feline esophageal muscle cells were identified. 5-HT-induced contraction, and MLC20 phosphorylation was used to determine the effect of DA-9701
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