Density-dependent regulation of cell surface gamma-glutamyl transpeptidase in cultured glial cells.
A decline in cell surface gamma-glutamyl transpeptidase specific activity was previously observed to be concomitant with C6 glial cell proliferation. To elucidate the underlying factor(s) mediating gamma-glutamyl transpeptidase down-regulation, the effects of C6 cell density and culture conditions on cell surface transpeptidase activity levels were investigated. After 24 h of culture, the transpeptidase specific activities were inversely related to the initial plating densities. The lower-density cultures showed an induction within 24 h of plating. As the cultures proliferated, the specific transpeptidase activities declined to a common low level at post-confluency. The gamma-glutamyl transpeptidase down-regulation was unrelated to cell growth rate and was most pronounced during logarithmic proliferation. Induction and down-regulation of gamma-glutamyl transpeptidase activity at low cell densities were not a result of trypsinization. Supplementation of low-density cultures with conditioned medium, use of matrix-coated wells, or periodic replacement of growth media to prevent conditioning had minor effects on the decline of cell surface activity. Kinetic analysis showed that the Michaelis constants and the reaction mechanism were unaltered by cell density, indicating that down-regulation was not due to allosteric factors or an alteration in enzyme character. A reduction in the maximal velocity of cell surface transpeptidation at higher cell densities suggested that gamma-glutamyl transpeptidase down-regulation is related to the concentration of enzyme at the cell surface. Immunocytochemical localization of gamma-glutamyl transpeptidase demonstrated that gamma-glutamyl transpeptidase antigen levels decrease as C6 cell density increases. These results led us to propose that cell-cell contact stimulates the disappearance of gamma-glutamyl transpeptidase from the surface of cultured C6 glial cells.
Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
K
K
Morgenstern
O
O
Hanson-Painton
B L
BL
Wang
L
L
De Bault
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000201:enzymology*; D001254:Astrocytoma; D002450:Cell Communication; D002452:Cell Count; D002455:Cell Division; D002462:Cell Membrane / Q000201:enzymology*; D003470:Culture Media; D015536:Down-Regulation; D007150:Immunohistochemistry; D007700:Kinetics; D051381:Rats; D014357:Trypsin; D014407:Tumor Cells, Cultured; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
D003470:Culture Media; D005723:gamma-Glutamyltransferase; D014357:Trypsin