The Signaling Mechanism of Contraction Induced by ATP and UTP in Feline Esophageal Smooth Muscle Cells

P2 receptors are membrane-bound receptors for extracellular nucleotides such as ATP and UTP. P2 receptors have been classified as ligand-gated ion channels or P2X receptors and G protein-coupled P2Y receptors. Recently, purinergic signaling has begun to attract attention as a potential therapeutic target for a variety of diseases especially associated with gastroenterology. This study determined the ATP and UTP-induced receptor signaling mechanism in feline esophageal contraction. Contraction of dispersed feline esophageal smooth muscle cells was measured by scanning micrometry. Phosphorylation of MLC20 was determined by western blot analysis. ATP and UTP elicited maximum esophageal contraction at 30 s and 10 micrometer concentration. Contraction of dispersed cells treated with 10 mM ATP was inhibited by nifedipine. However, contraction induced by 0.1 mM ATP, 0.1 mM UTP and 10 micrometer UTP was decreased by U73122, chelerythrine, ML-9, PTX and GDPbetaS. Contraction induced by 0.1 mM ATP and UTP was inhibited by Galphai 3 or Galphaq antibodies and by PLCbeta 1 or PLCbeta 3 antibodies. Phosphorylated MLC20 was increased by ATP and UTP treatment. In conclusion, esophageal contraction induced by ATP and UTP was preferentially mediated by P2Y receptors coupled to Galphai 3 and G q proteins, which activate PLCbeta 1 and PLCbeta3 . Subsequently, increased intracellular Ca2+ and activated PKC triggered stimulation of MLC kinase and inhibition of MLC phosphatase. Finally, increased pMLC20 generated esophageal contraction.