Cell contact-mediated regulation of tyrosine hydroxylase synthesis in cultured bovine adrenal chromaffin cells.

Abstract

The specific activity of tyrosine hydroxylase (TH) in bovine adrenal chromaffin cells can be controlled by changing cell density. Chromaffin cells initially plated at low density (2-3 x 104 cells/cm2), and subsequently replated at a 10-fold higher density showed a sixfold increase in specific TH activity within 48 h, resulting from-enhanced synthesis (increased number of TH molecules as demonstrated by immunotitration and blockade by cycloheximide) rather than activation. The density-mediated TH induction was blocked by inhibitors of both messenger RNA synthesis (o~-amanitin) and processing (9-#-arabinofuranosyladenine), indicating a transcriptional level of regulation. Medium conditioned by high density replated cells could not mimic the effect of high density plating itself, thus direct cell contact, rather than a diffusible factor, is responsible for the densitymediated TH induction. Since neither acetylcholinesterase nor lactate dehydrogenase specific activities were increased by high cell density, it can be concluded that the contact-mediated induction of TH is rather specific, and not the result of a general process of enzyme induction.Cell-cell interactions are known to be important for the formation of structures and functions of various integrated cellular systems during development. The role played by cellular interactions in developing neural systems is generally recognized to be of significance for the organotypic arrangement of neurons and glial cells, the path-finding of outgrowing neurites and the formation of correct connections between neurons (1-7). Moreover, direct cell contact has been demonstrated to be responsible for the mitogenic action of neurons on glial cells (8-10). With regard to the regulation of enzyme synthesis, cell contact has been shown to play a permissive role in hormone-mediated enzyme induction (11), but has not generally been considered as a direct regulatory stimulus on its own. We have reported that the specific activity of tyrosine hydroxylase (TH) ~ in cultured bovine adrenal chromaffin cells depends on cell density (12). For example, there was a 5-to 10-fold difference in TH specific activity between cells plated at low density (2 x 104 cells/cm 2) vs. high density (50 x 104 cells/cm 2) after 1 wk in culture. To study this effect in more detail, in the present study we developed a procedure in which cells originally plated at low density are harvested and replated either at the same low density or at a 10-fold higher density. This has allowed us to characterize the densitydependent increase in TH activity with regard to time course, enzyme activation vs. induction, level of regulation, role of I Abbreviations used in this paper." ACHE, acetylcholinesterase; LDH, lactate dehydrogenase; and TH, tyrosine hydroxylase. direct cell contact vs. possible humoral factors, and selectivity for TH. MATERIALS AND METHODSCell Culture: Adult bovine adrenal medullary cells were isolated and cultivated using a modification of the method of Naujoks et al. (12) which will be de...

Solubilization of a membrane factor that stimulates levels of substance P and choline acetyltransferase in sympathetic neurons.

“…Levels of ChoAcTase activity were stimulated by increasing cell density in PC12 (16,17), striatal (18), septal (19), and sympathetic neuronal (13,20) cultures, and TyrOHase was increased in high-density cultures of adrenal chromaffin cells (10,21). These changes apparently were mediated by cell-surface molecules rather than soluble diffusible factors because treatments with isolated plasma membranes or culture on layers of aldehyde-fixed cells he ChoAcTase-stimulating activity was eluted from a e hadex G-75 column in a single peak; NaDodSO4/polyc ylamide gel electrophoresis of the active peak revealed a Lnle predominant band at 29 kDa.…”

Sympathetic neuron density differentially regulates transmitter phenotypic expression in culture.

“…The temporal association of cellular aggregation and transmitter expression raised the possibility that these processes are causally related. This possibility has gained indirect support from the observations that growth of bovine adrenal chromaffin cells (2) or PC12 rat pheochromocytoma cells (3) in high-density cell culture selectively increased tyrosine hydroxylase specific activity.…”

Differential effects of neurotrophic factors on neurotransmitter development in the IMR-32 human neuroblastoma cell line

“…Thus, bFGF's effects on phenotypic properties appear to be variable and dependent on cell type. Cell density has been shown to influence the induction CAT and/or TH in several different cell types (Edgar and Thoenen, 1978;Lucas et al, 1979;Acheson and Thoenen, 1983;Hefti et al, 1985b, Adler andBlack, 1986;Kessler, 1986;Kirshner et al, 1986;C&per and Davies, 1988). Although the mechanisms of this effect are unknown, specific membrane proteins may mediate the density-dependent actions.…”