Properties of growth-related acetylcholinesterase in a cell line of fibroblastic origin.

Abstract

We have previously reported the presence and regulation of an acetylcholinehydrolyzing enzyme in high density suspension cultures of WRL-10A fibroblasts where its activity increases 100-fold when growth is arrested. Substrate specificity, substrate inhibition, and product identification studies indicate that this enzyme is acetylcholinesterase (ACHE, EC 3.1.1.7). Treatment of whole cells with 5 mM diazotized sulfanilic acid revealed that most of the AChE is located on the external surface of the cell membrane. It was also found that the enzyme is released in the medium at a rate of 0.5 U/h/mg cell protein and that within a 24-h period the de novo synthesized and liberated AChE is equivalent to 90% of the activity associated with the cells. No similar synthesis of AChE was found in six other fibroblastic cell lines examined. These and related findings indicating that acetylcholine is also present in high density populations of WRL-10A cells suggest that this unique phenotype may be used profitably in exploring further the relationship between components of the cholinergic system and non-neuronal cell growth.Growth-inhibited dense attached cultures of normal euploid mammalian cells and some established cell lines are characterized by a marked depression of total protein synthesis (24, 42, 45), while the synthesis of specialized proteins such as collagen (15,23,26,34), "SI00-protein" (33), and certain enzymes (6, 16, 35, 37) is increased.We have previously reported that densitydependent regulation of growth comparable to that of attached cell populations occurs also in suspension cultures of WRL-10A cells (10, I1, 44), a subline of L-929 mouse fibroblasts (9). In the search for additional criteria for evaluating increased synthesis of specialized proteins in this system, acetylcholinesterase (ACHE) was considered because, besides there being a description of such an increase in neuroblastoma cells by Blume et al. (5), there is suggestive evidence that the activity of this enzyme is inversely related to the rate of cell division in attached cultures of cells of mesenchymal origin as well (14), and that small but quantifiable amounts of AChE are present in L-929 cells (1,28,49). In a previous paper we demonstrated that density-dependent metabolic regulation in suspension cultures of WRL-10A cells does indeed extend to the activity of ACHE, which was found to be approximately 100-fold greater in high density growth-inhibited populations than in low density exponentially growing cultures (8).

Quantitation of megakaryocytopoiesis in liquid culture by enzymatic determination of acetylcholinesterase

“…Since megakaryocytes will proliferate in liquid culture, we believed that a similar assay method based on the unique marker enzyme AchE was feasible (Jackson, 1973;Long and Williams, 1981;Long et al, 1982;Nagawara et al, 1982). AchE has been measured in cultured cells by other investigators in the past (Bartos and Glinos, 1976;Wilson et al, 1972). Moreover, we believed it was possible to semiautomate this assay, since the growth of cells in microwell cultures is well established, and new photometric methods commonly used for enzyme-linked immunoassays permit rapid measurements of OD in 96-well microtiter plates.…”

Modulation of the Distribution of Acetylcholinesterase Molecular Forms in a Murine Neuroblastoma × Sympathetic Ganglion Cell Hybrid Cell Line

“…Secretion of AChE has been demonstrated from brain into the cerebrospinal fluid (Chubb et al, 1976), and by nerve terminals at the neuromuscular junction (Skau and Brimijoin, 1978), as well as in organotypic cultures of rat superior cervical ganglia (Gisiger and Vigny, 1977), in primary cultures of avian muscle (Wilson et al, 1973;Rotundo and Fambrough, 1979) and in a cell line of fibroblastic origin (Bartos and Glinos, 1976). In contrast with T28 cells, which secrete the three globular forms, rat sympathetic ganglia secrete specifically the G4 form of AChE (Gisiger and Vigny, 1977).…”

Secretion of acetylcholinesterase and butyrylcholinesterase from the guinea‐pig isolated ileum

“…The release of AChE has also been demonstrated in the periphery, e.g. from the adrenal medulla (Chubb & Smith, 1975b), the phrenic nerve-diaphragm preparation (Skau & Brimijoin, 1978), sympathetic ganglia (Gisiger & Vigny, 1977), fibroblast cells (Bartos & Glinos, 1976) and platelets (Chuang et al, 1976).…”