Cooperation of G β and G αq protein in contractile response of cat lower esophageal sphincter (LES)

Abstract

We previously shown that LES contraction depends on M3 receptors linked to PTX insensitive Gq protein and activation of PLC. This results in production of IP3, which mediates calcium release, and contraction through a CaM dependent pathway. In the esophagus ACh activates M2 receptors linked to PTX sensitive Gi3 protein, resulting in activation of PLD, presumably, production of DAG. We investigated the role of PLC isozymes which can be activated by Gq or Gβ protein on ACh-induced contraction in LES and esophagus. Immunoblot analysis showed the presence of 3 types of PLC isozymes, PLC-β1, PLC-β3, and PLC-γ1, but not PLC-β2, PLC-β4, PLC-γ2, PLC-δ1, and PLC-δ2 from both LES and esophageal muscle. ACh produced contraction in a dose dependent manner in LES and esophageal muscle cells obtained by enzymatic digestion with collagenase. PLC-β1 or PLC-β3 antibody incubation reduced contraction in response to ACh in LES but not in esophageal permeabilized cells, but PLC-γ1 antibody incubation did not have an inhibitory effect. The inhibition by PLC-β1 or PLC-β3 antibody on Ach-induced contraction was antibody concentration dependent. The combination with PLC-β1 and PLC-β3 antibody completely abolished the contraction, suggesting that PLC-β1 and PLC-β3 have a synergism to inhibit the contraction in LES. PLC-β1, -β3 or -γ1 antibody did not reduce the contraction of LES cells in response to DAG (10-6 M), suggesting that this isozyme of PLC may not activate PKC. When Gq/11 antibody was incubated, the inhibitory effect of the incubation of PLC β3, but not of PLC β1 was additive (Fig. 6). In contrast, when Gβ antibody was incubated, the inhibitory effect of the incubation of PLC β1, but not of PLC β3 was additive. This data suggest that Gq/11 or Gβ may activate cooperatively different PLC isozyme, PLC β1 or PLC β3 respectively.