Cooperative regulation of neurotensin content in PC12 pheochromocytoma cell cultures: effects of nerve growth factor, dexamethasone, and activators of adenylate cyclase.

Nerve growth factor, dexamethasone, and forskolin or cholera toxin (CT) act cooperatively to increase the content of neurotensin (NT) in PC12 pheochromocytoma cells. Relatively small increases in NT content occur in the presence of NGF alone or dexamethasone alone, but not of forskolin or CT alone. Increases of 10- to 100-fold occur in the presence of NGF plus dexamethasone or NGF plus forskolin, and up to 600-fold increases occur in the presence of all three agents. These increases are extremely stable and persist for at least 2 weeks after removal of dexamethasone or forskolin. The complex regulation of NT stores in PC12 cells might reflect mechanisms that regulate NT content of normal chromaffin cells and/or neurons during development or in adult life. A small amount of stored NT is released in response to stimulation with 52 mM K+. This release is blocked in the presence of 2 mM Co2+, suggesting that it occurs via Ca2+-mediated exocytosis.

Nerve growth factor may function as a survival factor for human neuroblastoma cells in culture.

Cells from two human neuroblastomas were found to exhibit a marked dependency on nerve growth factor (NGF) for survival in primary cultures. Cells from both tumors also responded to NGF by forming processes, but survival was not necessarily associated with process outgrowth. Lines of replicating cells could not be obtained from either tumor. NGF-dependent survival has not previously been reported as a characteristic of NGF-responsive human neuroblastoma cells in primary cultures or in cultures of established cell lines. Our findings suggest that tumors which require NGF for survival might constitute a biologically distinctive subset of neuroblastomas, or that NGF might function as a survival factor for some human neuroblastomas only in suboptimal or deleterious environments.

Production of "ectopic" vasoactive intestinal peptide-like and neurotensin-like immunoreactivity in human pheochromocytoma cell cultures.

Neoplastic chromaffin cells from human pheochromocytomas can exhibit extensive spontaneous and nerve growth factor (NGF)-induced outgrowth of neurite-like processes in vitro, despite the absence of such processes in vivo. To determine whether acquisition of neuron-like features by human pheochromocytoma cells in culture is accompanied by functional alterations, process outgrowth, vasoactive intestinal peptide-like immunoreactivity ( VIPLI ), neurotensin-like immunoreactivity (NTLI), and catecholamine content were studied in freshly dissociated cells and in 21-day-old cultures from six human pheochromocytomas. All of the cultures produced VIPLI and exhibited spontaneous process outgrowth. NGF stimulated process outgrowth and enhanced production of VIPLI . Dexamethasone inhibited process outgrowth and tended to decrease production of VIPLI . NTLI was detected in cells from only one of the tumors, and its production appeared to be regulated comparably to that of VIPLI . Catecholamine content decreased markedly in all of the cultures and was not regulated in parallel with either VIPLI or NTLI. The findings suggest that human pheochromocytoma cultures may help to elucidate cellular and molecular mechanisms regulating ectopic and normal VIP production.

Kinetics of regulation of neurotensin content in PC12 cells.

The mechanism by which nerve growth factor (NGF) and dexamethasone synergistically increase the content of neurotensin (NT) in PC12 cells can be partially separated into NGF and dexamethasone-mediated components by study of cultures pre-treated with either agent. The detectable early response of NGF-treated cells to dexamethasone is at least 24 hours faster than that of dexamethasone-treated cells to NGF, and is also greater in both absolute and relative magnitude. Both in NGF-treated cultures exposed to dexamethasone and in dexamethasone-treated cultures exposed to NGF, however, the rates of increase in NT content are initially low and then accelerate, suggesting periods of enzyme induction preceding NT accumulation. This suggestion is further supported by the absence of increases in the presence of actinomycin D, but an apparently paradoxical increase in NT content is observed in the presence of camptothecin. The findings are consistent with a complex interaction between NGF and dexamethasone, which might involve induction of multiple enzymes.

Enkephalin-like immunoreactivity in human adrenal medullary cultures.

Normal adult human chromaffin cells in culture contain abundant immunoreactive enkephalin and exhibit spontaneous and nerve growth factor-induced outgrowth of processes. In cultures of adrenals from two age-matched white males, approximately 70 per cent of chromaffin cells from one case and 50 per cent from the other contained immunocytochemically demonstrable leuenkephalin-like material. There was no apparent correlation between enkephalin-like immunoreactivity and capacity for process outgrowth. Cells with and without processes stained for enkephalin, and the percentages of stained cells with processes appeared to change in proportion of overall process outgrowth. Numerous cells with and without processes also failed to stain for enkephalin. Marked variations in sizes of stained cells and in distribution and intensity of staining were observed in all of the cultures. These findings suggest that adult chromaffin cells are heterogeneous at a number of levels.

Ultrastructural effects of nerve growth factor on PC 12 pheochromocytoma cells in spinner culture.

PC12 pheochromocytoma cells treated with nerve growth factor (NGF) for two weeks in spinner cultures quickly begin to form processes after plating on an appropriate substrate, while cells freshly exposed to NGF in monolayer culture initiate neurite outgrowth only after a lag period of several days. The present ultrastructural studies indicate that PC 12 cells treated with NGF in spinner cultures do not form neurites, but do form short extensions comparable to those which have been reported within the first two days of exposure to NGF in monolayer cultures. These extensions contain organelles believed to be required for locomotion and for transport of cytoskeletal and membrane components and neurotransmitters. They also form bulbous distensions in which numerous chromaffin-type granules accumulate. These findings suggest that NGF may affect cells in spinner cultures by promoting development or activation of axonal transport mechanisms, and that the existence of these mechanisms may contribute to the neurite outgrowth which the cells exhibit when plated. NGF-treated PC 12 cells in spinner cultures do not accumulate the agranular synaptic-like vesicles, which are typically found in comparably treated monolayer cultures and which have been hypothesized to be sites of acetylcholine storage. These and other data demonstrate that attachment to a substrate can selectively modulate the responses of PC 12 cells to NGF.

Content and release of neurotensin in PC12 pheochromocytoma cell cultures: modulation by dexamethasone and nerve growth factor.

The production of neurotensin-like immunoreactivity was investigated in cultured PC12 rat pheochromocytoma cells with and without the addition of nerve growth factor (NGF) and dexamethasone. Both monolayer cultures and spinner cultures contained measurable amounts of neurotensin. In the monolayer cultures supplemented with NGF and dexamethasone, cell and medium concentrations of neurotensin increased approx. 100-fold. In the spinner cultures the effect of the additives was less potent and the increase in neurotensin content, while still considerable, was approximately an order of magnitude less. Chromatographic analysis of medium and extracts of the cells from monolayer cultures indicated that the majority of the immunoreactivity coeluted in the same position as synthetic neurotensin, with recoveries of 68 and 85%, respectively. The effect of NGF and dexamethasone on neurotensin production differs significantly from their effect on catecholamine production in PC12 cells, and this suggests the possibility of independent regulation.