Vitamin D3, lactose, and xylitol stimulate chromaffin cell proliferation in the rat adrenal medulla.

Chronic consumption by rats of diets rich in sugars or sugar alcohols leads to an increased incidence of pheochromocytomas. This relationship is hypothesized to be based on altered Ca2+ homeostasis due to increased intestinal Ca2+ absorption. Other agents associated with pheochromocytomas in rats in long-term toxicity studies have been shown to increase chromaffin cell proliferation, leading to the suggestion that the tumors occur secondarily to increased chromaffin cell turnover. We have demonstrated marked stimulation of chromaffin cell proliferation by vitamin D3, a potent stimulus to Ca2+ absorption not previously associated with adrenal medullary toxicity. This effect is detectable during the first week of dietary supplementation and persists throughout a 4-week time course. Lactose and xylitol, representative of sugars and sugar alcohols associated with pheochromocytomas, are also mitogenic but to a lesser extent, with their effects first detectable during Week 4 of dietary supplementation. Vitamin D3, its active metabolite calcitriol, lactose, and xylitol all fail to stimulate proliferation of rat chromaffin cells in vitro. The mitogenic effects of these agents may be mediated presynaptically in vivo. The data suggest that altered Ca2+ homeostasis may increase chromaffin cell proliferation and support the hypothesis that diets containing high concentrations of sugars and sugar alcohols cause pheochromocytomas in rats secondarily by this mechanism.

Mitogenic and antimitogenic effects of pituitary adenylate cyclase-activating polypeptide (PACAP) in adult rat chromaffin cell cultures.

The neurotransmitter, pituitary adenylate cyclase-activating polypeptide (PACAP), is present in the rat adrenal medulla and is a potent stimulus for catecholamine secretion. Previous studies have suggested that neurally derived signals stimulate proliferation of chromaffin cells in adult rats. To determine whether PACAP might be involved in mitogenic signalling, its effects on bromodeoxyuridine incorporation were studied in adrenal medullary cell cultures from adult female rats. Both PACAP 27 and PACAP 38 are able to stimulate proliferation of adult rat chromaffin cells in vitro, either alone or in conjunction with PMA, an activator of protein kinase C. BrdU-labelled nuclei are observed in both epinephrine and norepinephrine cells, and proliferation of both cell types is stimulated by the same concentrations of PACAP that elicit secretion of catecholamines. The mitogenic effects of PACAP are potentiated by indolidan, a phosphodiesterase inhibitor known to cause pheochromocytomas in rats, and are inhibited by H-89, an inhibitor of protein kinase A. Mitogenic concentrations of PACAP inhibit mitogenic effects of nerve growth factor. These findings support the hypothesis that neurally derived signals regulate chromaffin cell proliferation in adult rats. Indolidan and a variety of nongenotoxic agents that cause pheochromocytomas in rats may do so indirectly by increasing neurally mediated chromaffin cell turnover. The antagonism between PACAP and NGF suggests that neurotransmitters may supersede growth factors in regulating chromaffin cell proliferation during development by suppressing or co-opting portions of growth factor signaling pathways.

Multiple mitogenic signalling pathways in chromaffin cells: a model for cell cycle regulation in the nervous system.

Adult rat chromaffin cells proliferate in vivo in response to neurally derived signals. Their proliferation in vitro is stimulated either by peptide growth factors or by activators of adenylate cyclase or protein kinase C that mimic the effects of neurotransmitters in adrenal medullary nerve endings. Differing susceptibilities to inhibitors and potentiators suggest that growth factors, cyclic AMP-dependent protein kinases and protein kinase C act via partially distinct and partially overlapping signalling pathways. Depolarization inhibits the mitogenic response to NGF, through a mechanism that apparently involves activation of voltage-gated calcium channels, while sparing the response to phorbol esters that activate PKC. Activators of adenylate cyclase also inhibit the response to NGF. The findings suggest that during normal development, neurally derived signals supersede growth factors in regulating proliferation of chromaffin cells by selectively inhibiting or co-opting portions of growth factor signalling pathways. This model might be generally applicable to the development of the nervous system.

Polyoma-induced neoplasms of the mouse adrenal medulla. Characterization of the tumors and establishment of cell lines.

BACKGROUND: Pheochromocytomas that are usually noradrenergic arise commonly in the adult rat adrenal medulla. The widely studied PC12 cell line, that is representative of these rat adrenal tumors, is also noradrenergic. The reasons for the absence of epinephrine production by most rat pheochromocytoma cells are unknown, and there are currently no adrenergic adrenal medullary cell lines. Pheochromocytomas are rare in mice. EXPERIMENTAL DESIGN: Tumors induced by polyoma virus in the adrenal medullas of postnatal mice were studied immunocytochemically for catecholamine biosynthetic enzymes in order to determine how their profiles of catecholamine production compared with those of rat pheochromocytomas. Clonal cell lines were established from a representative tumor and were evaluated for responsiveness to agents known to affect the development and function of normal and neoplastic rat chromaffin cells. RESULTS: Although adrenal medullary cells from normal rodents produce epinephrine before birth, polyoma-induced mouse adrenal tumor cells are immature or poorly differentiated. They synthesize norepinephrine, but not epinephrine, which during normal development is produced later than norepinephrine. They also produce relatively large quantities of dihydroxyphenylalanine, suggesting an abnormality of catecholamine biosynthesis such that tyrosine hydroxylase is not rate-limiting. Secretory granules are sparse, as demonstrated by electron microscopy or by staining for chromogranin A, and catecholamine stores are low. Further, the tumor cells appear to be phenotypically unstable, as judged from heterogeneous staining for tyrosine hydroxylase even in early passage, twice-cloned cell lines. Tumor cell morphology and catecholamine profiles appear to be unaffected or minimally affected by nerve growth factor, forskolin or dexamethasone, which are known to affect normal or neoplastic rat chromaffin cells. However, tumors formed after subcutaneous injection of cell lines into mice show up to a 10-fold increase in catecholamine stores, suggesting that the cells are subject to some forms of regulation. The cloned cell lines do not produce detectable polyoma virus, but express all three viral T antigens, including a characteristic, truncated form of large T. CONCLUSIONS: The findings suggest that the process of neoplastic transformation and/or the presence of polyoma virus T antigens results in suppression of the adrenergic phenotype in mouse adrenal chromaffin cells. T antigens might therefore be useful as tools for studying mechanisms that regulate the differentiation and maturation of chromaffin cells in normal and neoplastic states. Furthermore, although polyoma virus cannot be readily used to produce adrenergic cell lines from the mouse adrenal medulla, the lines that are produced might substitute for PC12 cells in some types of studies that require a mouse model.

Nerve growth factor is a potent inducer of proliferation and neuronal differentiation for adult rat chromaffin cells in vitro.

Adult rat chromaffin cells in vitro show a large proliferative response to NGF, followed by neuronal differentiation. Infection of replicating chromaffin cells with a retrovirus carrying the Escherichia coli beta-galactosidase (beta-gal) gene demonstrates beta-gal expression in cells that continue to multiply, that differentiate into neurons, and that become static. The effects of NGF on proliferation and differentiation are abolished by the protein kinase inhibitors K252a and staurosporine, and by cholera toxin, an activator of adenylate cyclase. They are diminished, but not abolished, by high concentrations of dexamethasone. Both cholera toxin alone and phorbol myristate acetate (PMA), an activator of protein kinase C, elicit small and inconsistent mitogenic responses. The responses to PMA cannot be shown to be additive with the effects of NGF. NGF is a known mitogen and neuritogen for chromaffin cells from neonatal rats, but has not previously been believed to affect similarly chromaffin cells from adults. The present findings suggest that portions of NGF signaling pathways might continue to be involved in regulating proliferation of adult rat chromaffin cells in vivo, and might be constitutively activated in PC12 cells and other adrenal medullary tumors. They further suggest that rat chromaffin cells might be propagated in vitro to obtain large numbers of sympathetic neurons expressing normal or exogenous genes.

Immunocytochemical analysis of chromaffin cell proliferation in vitro.

The bromodeoxyuridine (BrdU) incorporation technique for immunocytochemical labeling of S-phase nuclei was optimized for the study of chromaffin cell proliferation. Sequential fixation in ethanol followed by paraformaldehyde, and the use of DNAse to render incorporated BrdU accessible to antibody, permitted permanent double staining for BrdU and tyrosine hydroxylase. The efficacy of the technique was demonstrated in microcultures of dissociated neonatal rat adrenal glands, in which chromaffin cells exhibited proliferative responses to nerve growth factor and fibroblast growth factor similar to those previously demonstrated by autoradiography. Growth factor responsiveness was observed in both serum-containing and serum-free medium.