Role of nucleotide sugar pools in the inhibition of NCAM polysialylation by ammonia

Ammonia in animal cell cultures has been shown to specifically inhibit term inal sialylation of N- and O-linked oligosaccharides of glycoproteins. For example, we have previously shown that as little as 2.5 mM NH4Cl can decrea se neural cell adhesion molecule (NCAM) polysialylation in both small cell lung cancer (SCLC) and Chinese hamster ovary (CHO) cells. Besides its poten tial involvement in SCLC metastasis, polysialic acid (PolySia) is a sensiti ve marker for measuring changes in sialylation. The role of UDP-N-acetylglu cosamine (UDP-GlcNAc) in ammonia's inhibition of NCAM polysialylation was e xamined by adding glucosamine (GlcN) and uridine (Urd) to the cultures. Thi s bypassed feedback inhibition of GlcN-6-P synthase and increased UDP-GlcNA c content by 25-fold in SCLC cells. After 3 days, PolySia levels were reduc ed to 10% of control with little effect on NCAM protein content. The extens ive decrease in PolySia was confirmed in CHO cells. The effects of GlcN or Urd alone were less extensive, lending support to a specific role for UDP-G lcNAc in inhibition by ammonia. By comparison, 20 mM NH4Cl decreased PolySi a content by 45% and increased UDP-GlcNAc in SCLC cells by 2-fold. The disc repancy between the {GlcN+Urd} and NH4Cl effects on UDP-GlcNAc and PolySia suggests that accumulation of UDP-GlcNAc is only partially responsible for decreased polysialylation in response to NH4Cl. In an attempt to increase N CAM polysialylation, N-acetylmannosamine and cytidine were added to culture s in order to circumvent the feedback inhibition of CMP-sialic acid synthes is. However, this only slightly increased PolySia levels and failed to coun ter ammonia's inhibition of NCAM polysialylation.