Effect of LIF-withdrawal on acetylcholine synthesis in the embryonic stem cell line CGR8 is not mediated by STAT3, PI3Ks or cAMP/PKA pathways

Acetylcholine (ACh) acts as a local cellular signaling molecule and is widely expressed in nature, including mammalian cells and embryonic stem cells. The murine embryonic stem cell line CGR8 synthesizes and releases substantial amounts of ACh. Particularly during early differentiation - a period associated with multiple alterations in geno-/phenotype functions - synthesis and release of ACh are increased by 10-fold. In murine stem cells second messengers of the STAT-3, PI3K and cAMP/PKA pathways are involved in maintaining self-renewal and pluripotency. The present experiments were designed to test whether blockers of these signaling pathways enhance ACh cell content in the presence of LIF, i.e. when CGR8 is pluripotent. NSC74859, an inhibitor of STAT-3, affected neither the proliferation rate nor ACh cell content, whereas the more sensitive STAT-3 inhibitor FLLL31 reduced the proliferation rate and increased ACh cell content by about 3-fold. The PI3K inhibitor LY294002 reduced the proliferation rate but did not modify the ACh cell content, whereas the PKA inhibitor H89 produced effects comparable to FLLL31. Interestingly, in control experiments a strong inverse correlation was found between cell density and ACh cell content, which could explain the 3-fold increase in the ACh cell content observed in the presence of FLLL31 and H89. Forskolin, a PKA activator, had no effect. In conclusion, it appears unlikely that the 10-fold increase in ACh cell content induced by LIF removal, i.e. during early differentiation, is mediated by second messengers of the STAT-3, PI3K and cAMP/PKA pathways. However, the PI3K pathway appears to be involved in control of the inverse relation between cell density and ACh cell content, because this correlation was significantly attenuated in the presence of LY294002.