Inhibition of glutamine synthetase decreases proliferation of cultured rat intestinal epithelial cells.

The importance of glutamine synthetase (GS) for cell proliferation was examined in rat intestinal crypt cells (IEC-6) by inhibiting its activity with 10 mmol/L methionine sulfoximine (MS) at varying extracellular glutamine (Q) concentrations. In uninhibited cultures, cell number, protein, and DNA accumulation and synthesis showed a dependence on extracellular Q over a concentration range of 0.06 to 1.06 mmol/L, with apparent half-maximal responses of 0.46 mmol/L extracellular Q. In contrast, proliferation of GS-inhibited cultures required >/=1.06 mmol/L extracellular Q, with an apparent half-maximal response of 2 mmol/L. MS inhibited GS activity >97% in extracts of washed cells and appeared to be specific because its effects on proliferation were overcome by 4.06 mmol/L Q and were reversible. The increased dependence of IEC-6 cells on extracellular Q when GS was inhibited suggests that Q derived from GS (GS-Q) contributes importantly to cell proliferation at physiologic levels of extracellular Q (0.6 mmol/L). The unexpectedly high concentration of extracellular Q required to rescue maximal proliferation during GS-inhibition, relative to a reported Km for Q-transport into the cell, indicates that intracellular Q derived from the extracellular medium (exo-Q) is inefficiently utilized. In a previous study, we found that GS-protein and mRNA are concentrated in the proliferative crypt region of the small intestine in vivo, and predicted that GS activity is important for crypt cell proliferation. Here, we show that enzyme activity is important for cell proliferation at physiologic concentrations of Q in this cell culture model. Finally, we speculate that exo-Q and GS-Q are utilized differently in the cell.

Degradation of chondroitin sulfate proteoglycan enhances the neurite-promoting potential of spinal cord tissue.

The contribution of chondroitin sulfate proteoglycan (CSPG) in the suppression of axonal growth in rat spinal cord has been examined by means of an in vitro bioassay in which regenerating neurons are grown on tissue section substrata. Dissociated embryonic chick dorsal root ganglionic neurons were grown on normal and injured adult spinal cord tissue sections treated with chondroitinases. Neuritic growth on normal spinal cord tissue was meager. However, both the percentage of neurons with neurites and the average neurite length were substantially greater on sections treated with chondroitinase ABC. Enzymes that specifically degraded dermatan sulfate or hyaluronan were ineffective. Neuritic growth was significantly greater on injured (compared to normal) spinal cord and a further dramatic increase resulted from chondroitinase ABC treatment. Neurites grew equally within white and gray matter regions after chondroitinase treatment. Observed increases in neurite outgrowth on chondroitinase-treated tissues were largely inhibited in the presence of function-blocking laminin antibodies. These findings indicate that inhibitory CSPG is widely distributed and predominant in both normal and injured spinal cord tissues. Additionally, inhibitory CSPG is implicated in negating the potential stimulatory effects of laminin that might otherwise support spinal cord regeneration.