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.