Changes in Glycosaminoglycans During the Neuritogenesis in PC12 Pheochromocytoma Cells Induced by Nerve Growth Factor

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Abstract
Previously, we had suggested that heparan sulfate (HS) makes some contribution to a flat-shaped morphology of PC12D cells. Therefore, we carried out quantitative and qualitative analysis of glycosaminoglycans (GAG''s), the polysaccharide moiety of proteoglycans, during neuritogenesis in PC12 cells that is induced by nerve growth factor (NGF). (a) In PC12 cells, NGF induced a flat-shaped morphology with a few short processes after 3 days of culture, and then it elicited short and long neurites after 6 (in .apprx. 30% of cells) and 9 (in 60-70%) days of culture, respectively. (b) HS and chondroitin sulfate (CS) were detected in the cell layer at all times. Only CS was found in the medium at 3 and 6 days, whereas a low level of HS, in addition to CS, was detectable on day 9. (c) In the NGF-treated cultures, the amount of cell-associated HS per cell were two to three times as high as those in the respective nontreated cultures at all times, whereas the amount based on phospholipid was about twofold higher after after 3 days of culture. (d) The levels of HS labelled with [35S]sulfate during the last 48 h of the culture were 1.5- to twofold higher in the NGF-treated cultures than in the respective controls at any time. (e) The amount of cell-associated CS per cell (or per unit of phospholipid), but not of labelled CS per cell, was transiently enhanced at 3 days in culture with or without NGF. At all times, NGF treatment caused an increase in the levels of total and [35S]sulfate-labeled CS associated with the cells and released into the medium. (f) NGF enhanced the amount of N-sulfation of glucosamine residues of HS at all times, but it did not change the ratio of 4-sulfate units to 6-sulfate units in CS. (g) At 3 days in culture, the uptake of [35S]sulfate by PC12 cells was lower in the NGF-treated culture than in nontreated control. (h) In chase experiments, the percentage of unrecovered CS was about twofold higher in the NGF-treated culture than in the non-treated control. These results suggest that the enhanced synthetic activity and the accumulation of GAGs as well as the structural changes of HS induced by NGF occur preceding the neurtie elongation from PC12 cells. Also, it is suggested that the increase in content of HS is closely correlated with the morphological change from round to flat in PC12 cells.