DIFFERENTIAL SIGNAL-TRANSDUCTION PATHWAYS IN EAT LOWER ESOPHAGEAL SPHINCTER TONE AND RESPONSE TO ACH

Lower esophageal sphincter (LES) basal tone and contraction in response to maximally effective doses (E(max)) of acetylcholine (ACh) may be mediated by different intracellular transduction pathways. In the basal state resting tone, inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3] formation and levels of diacylglycerol (DAG) (C. Hillemeier, K. N. Bitar, and P. Biancani, unpublished data) are higher in LES circular muscle than in esophageal muscle, which does not maintain tone. In vitro resting tone and spontaneously elevated formation of Ins(1,4,5)P-3 in LES circular muscle strips decrease in a dose-dependent manner in response to the phospholipase C antagonist 1-[6-{[(17-beta)-3-methoxyestra-1,3, 5(10)-trien-17-yl] amino}hexyl]-1H-pyrrole-2, 5-dione (U-73122). Basal Ins(1,4,5)P-3 formation, however, is submaximal, since it can be increased by cholinergic stimulation. These data suggest that LES tone is associated with partial activation of phospholipase C. We therefore tested submaximal doses of Ins(1,4,5)P-3 and DAG in permeabilized LES muscle cells and found that they act synergistically; their interaction depends on calcium release and is mediated through a protein kinase C (PKC)-dependent pathway. In contrast, we have previously shown that contraction induced by E(max) of ACh is mediated through calmodulin-dependent mechanisms (14). To investigate these differences, we tested high and low doses of ACh. Contraction induced by high doses of ACh was inhibited by calmodulin but not by PKC antagonists, as previously reported, but low ACh doses were preferentially inhibited by PKC antagonists. Similarly, low Ins(1,4,5)P-3 concentrations activated a PKC-dependent pathway, whereas contraction induced by E(max) of Ins(1,4,5)P-3 was calmodulin dependent. These data suggest that lower calcium levels may be required for PKC activation than for calmodulin and are consistent with the finding that low calcium levels can support contraction induced by the PKC agonist DAG, but not by calmodulin, which requires micromolar calcium levels. We conclude that LES tone is associated with low-level phospholipase C activity, which results in the formation of inositol trisphosphate and the release of low Ca2+ concentrations from intracellular stores and potentiation of DAG to activate a PKC-dependent pathway. In contrast, maximal cholinergic stimulation activates a calmodulin-dependent pathway. The switch from a PKC-dependent to a calmodulin-dependent pathway may result from the different calcium requirements of these pathways.