The structurally similar compounds staurosporine and K252a are potent inhibitors of protein kinases. K252a has previously been reported to inhibit most or all of the effects of nerve growth factor (NGF) on PC12 pheochromocytoma cells, and staurosporine has been reported both to inhibit and to mimic NGF-induced neurite outgrowth from a PC12 cell subclone in a dose-dependent manner. We have studied the interactions of these agents with each other, with NGF, and with forskolin, an activator of adenylate cyclase, on the parent PC12 cell line and on normal neonatal and adult rat chromaffin cells. Staurosporine alone or in conjunction with forskolin induces outgrowth of short neurites from PC12 cells but does not substitute for NGF in promoting cell survival. It does not abolish NGF-induced neurite outgrowth but does reverse the effects of NGF on catecholamine synthesis. K252a abolishes NGF-induced neurite outgrowth but only partially decreases outgrowth induced by NGF plus forskolin. It does not inhibit neurite outgrowth produced by staurosporine or staurosporine plus forskolin. These findings with PC12 cells suggest that staurosporine might act downstream from K252a and NGF on components of one or more signal transduction pathways by which NGF selectively affects the expression of certain traits. Both neonatal and adult rat chromaffin cells show dramatic flattening and extension of filopodia in response to staurosporine, an observation suggesting that some of the same pathways might remain active in cells that do not exhibit a typical NGF response. Only a small amount of neurite outgrowth is observed, however, and only in neonatal cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Epinephrine and norepinephrine-containing chromaffin cells proliferate in the adrenal glands of normal adult rats throughout life. Moreover, their rate of proliferation is markedly increased by short-term administration of reserpine, one of many agents which in long-term experiments are associated with the development of adrenal medullary tumors. Current data suggest that chromaffin cell proliferation in the adult rat adrenal is mediated by the interaction of neurogenic and hormonal signals. Reserpine is known to directly deplete catecholamine stores, and to reflexively increase the activity of the splanchnic nerve endings innervating the adrenal medulla to stimulate both secretion and synthesis of catecholamines and other secretory granule constituents. Its effect on chromaffin cell proliferation suggests that the same signals may regulate chromaffin cell number to meet physiological needs. The reserpine model might shed light on signal transduction mechanisms which normally promote or prevent proliferation of chromaffin cells and of other neuroendocrine cells during development or in adult life, and on ways in which such mechanisms are altered in the course of the development and progression of tumors. It also suggests the possibility that chromaffin cells might be propagated in vitro for use in basic biological studies or in transplants for the treatment of Parkinson's disease.

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.

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