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

Abstract

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 mu M concentration. Contraction of dispersed cells treated with 10 mu M ATP was inhibited by nifedipine. However, contraction induced by 0.1 mu M ATP, 0.1 mu M UTP and 10 mu M UTP was decreased by U73122, chelerythrine, ML-9, PTX and GDP beta S. Contraction induced by 0.1 mu M ATP and UTP was inhibited by G alpha i(3) or G alpha q antibodies and by PLC beta(1) or PLC beta(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 G alpha i(3) and G alpha q proteins, which activate PLC beta(1) and PLC beta(3). Subsequently, increased intracellular Ca2+ and activated PKC triggered stimulation of MLC kinase and inhibition of MLC phosphatase. Finally, increased pMLC(20) generated esophageal contraction.