On the actions of neurotrophic factors on the chromaffin cells of the adrenal medulla

Abstract

Chromaffin cells producing adrenaline or noradrenaline are the predominant celltype in the adrenal medulla, but ganglion cells and Schwann cells are also present. All cells of the adrenal medulla are derived from the neural crest, which is a transient neuroectoderm-derived embryonic structure. During the migration of the pluripotent neural crest cells they differentiate into their mature phenotype. The differentiation is regulated by signal molecules, such as neurotrophic factors and steroid hormones. The pluripotency of the neural crest cells is to some extent maintained in the mature chromaffin cells. In this thesis chromaffin cells were used to study the effects of nerve growth factor (NGF) and other neurotrophic factors on the transformation of adult chromaffin cells into neuronal-like phenotypes, as well as the expression and regulation of neurotrofic factors and their receptors. Furthermore, striatal neuronal responses to intraparenchymal infusion of NGF was studied. In situ hybridization, immunohistochemistry, neurite outgrowth assay, TGFß and NGF assays, and HPLC were used to study the chromaffin cells in situ and the effects of neurotrophic factors on intraocular transplanted and primary cultured adult chromaffin cells. Dose-dependent reinnervation of host irides in response to different concentrations of NGF was shown. Nerve fiber formation was saturated when transplants were treated with NGF (100 [my]g/ml), and the half-maximal effect was achieved when host eyes were injected with 30 [my]g/ml NGF. As a result of the first study a baseline of neurite formation in response to NGF had been documented, and therefore other neurotrophic factors were tested using the same experimental model. The results revealed that ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), neurotrophin 4 (NT-4), glial cell line-derived neurotrophic factor (GDNF), at concentrations of 100200 [my]g/ml, stimulated nerve fiber formation from chromaffin grafts. Neurotrophin 3 (NT-3) was the only neurotrophic factor tested that had no effect on neurite outgrowth. In the next series of experiment the relationship between NGF and transforming growth factor-ß (TGFß) was studied on the chromaffin cells. No synergistic effect was found on neurite formation after co-administration of TGFß and NGF in the chromaffin grafts. Instead grafts treated with NGF and antibodies against TGFß resulted in enlarged area of reinnervation. TGFß and NGF had thus antagonistic effects on neurite formation from the chromaffin cells. Using in situ hybridization CNTF receptor alpha (CNTFR[alpha]) mRNA was detected in P1, and adult adrenal medullary cells. Increased level of CNTFR[alpha] mRNA was seen after NGF treatment. Furthermore, GDNF family receptor alpha I (GFR[alpha]1) mRNA was present in the ganglion cells of the adrenal medulla, when adult adrenal medullary tissue was explanted, many chromaffin cells became GFR[alpha]1 mRNA-positive. Factors within cell culture medium were important for the expression of GFR[alpha]1 in the cultured chromaffin cells. GDNF induced neurite formation from intraocular chromaffin grafts, and this neurite-promoting capacity of GDNF, was confirmed in primary cultures of adult chromaffin cells. Intrastriatal transplants of chromaffin tissue as a source of catecholamines, have been proposed as an alternative treatment strategy in Parkinsion's disease. In clinical trials intrastriatal chromaffin transplant have been stimulated by co-administration of NGF. A transient reduction of some typical parkinsonian symptoms was reported. In the last article of this thesis NGF was intraparenchymally infused into the normal rat striatum, to evaluate the role of NGF on the striatal neurons. NGF diffused from the infusion site to the overlying cortex and to the hippocampus. The results revealed that increased levels of mRNA corresponding to trkA, p75, muscarin receptor 2, ChAT and increased levels of acetylcholine esterase in the NGF infused striatum compared to contralateral side were found. Thus, NGF stimulates the cholinergic system, which is believed to be excitatory in the striatum. Surprisingly, a decreased spontaneous firing rate was found in the NGF infused animals, although the cholinergic markers were upregulated. This downregulation of spontaneous activity in the striatum could explain some of the beneficial effects, seen in patients suffering from Parkinson's disease, when NGF was co-administrated to the intra striatal chromaffin graft