Neuronal differentiation of PC12 cells abolishes the expression of membrane androgen receptors

Sex steroids affect adrenal chromaffin cell function. In the present work, we have examined the expression and functional significance of membrane androgen receptor sites in normal rat adrenal chromaffin cells and in the PC12 rat pheochromocytoma cell line which can differentiate to either a neuronal or to an epithelial phenotype and expresses membrane estrogen receptor sites. Our data are as follows: (a) no cytosolic androgen receptors were found in both normal chromaffin and PC12 cells; (b) both types of chromaffin cells expressed high affinity membrane testosterone binding sites; (c) activation of these sites increased cytosolic Ca²⁺, decreased catecholamine secretion and induced apoptosis; (d) NGF-induced neuronal differentiation of PC12 cells resulted in the suppression of the number of membrane testosterone sites. In conclusion, our data provide evidence for the existence of specific membrane testosterone receptors on adrenal chromaffin cells via which androgens, (some of them originating in the cortex) modulate their function. Neuronal differentiation of chromaffin cells results in a significant attenuation of these effects, via suppression of the expression of membrane androgen receptors suggesting, that the latter are specific for epithelioid chromaffin cells.     

Localization of the Noradrenaline Transporter in Rat Adrenal Medulla and PC12 Cells

The noradrenaline transporter (NAT) is present in noradrenergic neurons and a few other specialized cells such as adrenal medullary chromaffin cells and the rat pheochromocytoma (PC12) cell line. We have raised antibodies to a 49-residue segment (NATM2) of the extracellular region (residues 184-232) of bovine NAT. Affinity-purified NATM2 antibodies specifically recognized an 80-kDa band in PC12 cell membranes by western blotting. Bands of a similar size were also detected in membranes from human neuroblastoma (SK-N-SH) cells expressing endogenous NAT and human embryonic kidney (HEK293) cells stably expressing bovine NAT. Immunocytochemistry of rat adrenal tissue showed that NAT staining was colocalized with tyrosine hydroxylase in medullary chromaffin cells. Most NAT immunoreactivity in rat adrenal chromaffin and PC12 cells was present in the cytoplasm and had a punctate appearance. Cell surface biotinylation experiments in PC12 cells confirmed that only a minor fraction of the NAT was present at the cell surface. Subcellular fractionation of PC12 cells showed that relatively little NAT colocalized with plasma membrane, synaptic-like microvesicles, recycling endosomes, or trans-Golgi vesicles. Most of the NAT was associated with [3H]noradrenaline-containing secretory granules. Following nerve growth factor treatment, NAT was localized to the growing tip of neurites. This distribution was similar to the secretory granule marker secretogranin I. We conclude that the majority of NAT is present intracellularly in secretory granules and suggest that NAT may undergo regulated trafficking in PC12 cells.     

Intracellular localization of tyrosine hydroxylase immunoreactivity in the rat adrenal chromaffin and pheochromocytoma cells

The localization of tyrosine hydroxylase (TH) immunoreactivity in rat adrenal chromaffin and pheochromocytoma (PC12) cells was investigated by immunoelectron microscopy using monoclonal and polyclonal antisera against TH purified from rat adrenal medulla. Strong TH immunoreactivity was found uniformly in the granules of the adrenaline cells; the immunoreactivity was visible mainly within the periphery, but not in the clear space of the granules of the noradrenaline cells. In the PC12 cells, strong TH immunoreactivity was also observed uniformly in the granules. In addition, TH immunoreactivity was seen in the cytoplasm, the ribosomes attached to the endoplasmic reticulum and the free ribosomes of both the rat adrenal chromaffin and PC12 cells. These results suggest that TH may be localized in the granules, cytoplasm and ribosomes of rat adrenal chromaffin and PC12 cells.     

Animal models of pheochromocytoma

Pheochromocytomas are neuroendocrine tumors of adrenal chromaffin cells. They are rare in all species except rats but occur with increased frequency in several human familial tumor syndromes. Concurrence of pheochromocytoma with other tumors sometimes parallels these human syndromes in rats, bovines, horses and dogs but a shared genetic basis for human and spontaneously occurring animal pheochromocytomas has thus far not been established. Pheochromocytomas are inducible in rats by a variety of non-genotoxic substances that may act indirectly by stimulating chromaffin cell proliferation. They are not known to be similarly inducible in other species but arise with increased frequency in transgenic and knockout mice that to varying degrees recapitulate human tumor syndromes. Preliminary evidence suggests that homologous somatic genetic changes might contribute to pheochromocytoma development in humans and some mouse models. The nerve growth factor-responsive PC12 cell line, established from a rat pheochromocytoma, has for almost 30 years served as a research tool for many aspects of neurobiology involving normal and neoplastic conditions. Recently developed pheochromocytoma cell lines from neurofibromatosis knockout mice supplement the PC12 line and have generated additional applications. Advantages of the mouse lines include expression of substantial levels of the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase and expression of high levels of the receptor tyrosine kinase, Ret, which is characteristic of sporadic and familial human pheochromocytomas but not of PC12 cells. Disadvantages include an apparently less stable phenotype. It is difficult to establish pheochromocytoma cell lines from any species, although the tumor cells persist in culture for many months. Understanding of factors that permit pheochromocytoma cells to proliferate might itself provide important insights for tumor biology.     

Specific adhesion between pheochromocytoma (PC12) cells and adrenal medullary endothelial cells in co-culture

Summary Chromaffin cells in the adrenal medulla are found in close proximity to capillary endothelial cells, thereby forming the classical endocrine complex. To examine the possible chemical basis of their interaction in more detail, we have grown bovine adrenal medullary endothelial (BAME) cells in monolayer cultures and added to them pheochromocytoma (PC12) cells, a chromaffin tumor cell line of rats. The PC12 cells were chosen because of the similarities they share with adrenal medullary chromaffin cells. PC12 cells rapidly attached to BAME cells cultures, their rate of adhesion being significantly enhanced over binding of PC12 cells to either uncoated plates or to monolayers of unrelated cell cultures. Consistent with this observation, we noted that the extracellular matrix (ECM) derived from the BAME cells did not enhance PC12 cell adhesion and did not promote neurite sprouting as previously described for ECM derived from corneal endothelial cells. The specific adhesion between PC12 and BAME cells could be abolished by cell surface extracts derived from these two cells but not by extracts derived from unrelated cell types. This activity was heat-labile, sensitive to trypsin and, to a lesser extent, to neuraminidase. We therefore conclude that PC12 cells may interact with BAME cells by specific proteinaceous adhesive factors associated with their plasma membranes. These interactions might represent the formative role of cell-cell contacts in the organization of the developing adrenal gland.Abbreviations BAME bovine adrenal medullary endothelial cells - DMEM Dulbecco's modified essential medium - ECM extracellular matrix - EMEM Eagle's modified essential medium - FCS fetal calf serum - PBS phosphate-buffered saline - PC12 rat pheochromocytoma cells     

The novel plasminogen receptor, plasminogen receptor(KT) (Plg-R(KT)), regulates catecholamine release

Neurotransmitter release by catecholaminergic cells is negatively regulated by prohormone cleavage products formed from plasmin-mediated proteolysis. Here, we investigated the expression and subcellular localization of Plg-R(KT), a novel plasminogen receptor, and its role in catecholaminergic cell plasminogen activation and regulation of catecholamine release. Prominent staining with anti-Plg-R(KT) mAb was observed in adrenal medullary chromaffin cells in murine and human tissue. In Western blotting, Plg-R(KT) was highly expressed in bovine adrenomedullary chromaffin cells, human pheochromocytoma tissue, PC12 pheochromocytoma cells, and murine hippocampus. Expression of Plg-R(KT) fused in-frame to GFP resulted in targeting of the GFP signal to the cell membrane. Phase partitioning, co-immunoprecipitation with urokinase-type plasminogen activator receptor (uPAR), and FACS analysis with antibody directed against the C terminus of Plg-R(KT) were consistent with Plg-R(KT) being an integral plasma membrane protein on the surface of catecholaminergic cells. Cells stably overexpressing Plg-R(KT) exhibited substantial enhancement of plasminogen activation, and antibody blockade of non-transfected PC12 cells suppressed plasminogen activation. In functional secretion assays, nicotine-evoked [(3)H]norepinephrine release from cells overexpressing Plg-R(KT) was markedly decreased (by 51 ± 2%, p < 0.001) when compared with control transfected cells, and antibody blockade increased [(3)H]norepinephrine release from non-transfected PC12 cells. In summary, Plg-R(KT) is present on the surface of catecholaminergic cells and functions to stimulate plasminogen activation and modulate catecholamine release. Plg-R(KT) thus represents a new mechanism and novel control point for regulating the interface between plasminogen activation and neurosecretory cell function.     

Quantitation of tyrosine hydroxylase, protein levels: spot immunolabeling with an affinity-purified antibody

Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and 125I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background 125I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.     

Corticotropin-releasing hormone induces Fas ligand production and apoptosis in PC12 cells via activation of p38 mitogen-activated protein kinase.

Recent experimental findings involve corticotropin-releasing hormone (CRH) in the cellular response to noxious stimuli and possibly apoptosis. The aim of the present work was to examine the effect of CRH on apoptosis and the Fas/Fas ligand system in an in vitro model, the PC12 rat pheochromocytoma cell line, which is widely used in the study of apoptosis and at the same time expresses the CRH/CRH receptor system. We have found the following. CRH induced Fas ligand production and apoptosis. These effects were mediated by the CRH type 1 receptor because its antagonist antalarmin blocked CRH-induced apoptosis and Fas ligand expression. CRH activated p38 mitogen-activated protein kinase, which was found to be essential for CRH-induced apoptosis and Fas ligand production. CRH also promoted a rapid and transient activation of ERK1/2, which, however, was not necessary for either CRH-induced apoptosis or Fas ligand production. Thus, CRH promotes PC12 apoptosis via the CRH type 1 receptor, which induces Fas ligand production via activation of p38.     

Insulin-like growth factors are mitogens for rat pheochromocytoma PC 12 cells

We have addressed the issue of a mitogenic effect of insulin-like growth factors IGF-I and IGF-II on the PC 12 line of rat pheochromocytoma cells. The proliferation of PC 12 cells cultured in serum-free medium is stimulated threefold by IGF-I and IGF-II with significantly higher potency than epidermal growth factor, whereas platelet-derived growth factor, nerve growth factor, growth hormone and bombesin are inactive. Two types of IGF receptor are present in PC 12 cells and the dose-response curves suggest that the mitogenic responses to IGF's are mediated by the IGF-I receptor. These results suggest that IGF-I and IGF-II act as mitogens on pluripotent chromaffin cells in the development of the peripheral nervous system and adrenal medulla as well as in promotion of in vivo growth of neural crest-derived tumors.     

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.     

The complement anaphylatoxin C5a induces apoptosis in adrenomedullary cells during experimental sepsis

Sepsis remains a poorly understood, enigmatic disease. One of the cascades crucially involved in its pathogenesis is the complement system. Especially the anaphylatoxin C5a has been shown to have numerous harmful effects during sepsis. We have investigated the impact of high levels of C5a on the adrenal medulla following cecal ligation and puncture (CLP)-induced sepsis in rats as well as the role of C5a on catecholamine production from pheochromocytoma-derived PC12 cells. There was significant apoptosis of adrenal medulla cells in rats 24 hrs after CLP, as assessed by the TUNEL technique. These effects could be reversed by dual-blockade of the C5a receptors, C5aR and C5L2. When rats were subjected to CLP, levels of C5a and norepinephrine were found to be antipodal as a function of time. PC12 cell production of norepinephrine and dopamine was significantly blunted following exposure to recombinant rat C5a in a time-dependent and dose-dependent manner. This impaired production could be related to C5a-induced initiation of apoptosis as defined by binding of Annexin V and Propidium Iodine to PC12 cells. Collectively, we describe a C5a-dependent induction of apoptotic events in cells of adrenal medulla in vivo and pheochromocytoma PC12 cells in vitro. These data suggest that experimental sepsis induces apoptosis of adrenomedullary cells, which are responsible for the bulk of endogenous catecholamines. Septic shock may be linked to these events. Since blockade of both C5a receptors virtually abolished adrenomedullary apoptosis in vivo, C5aR and C5L2 become promising targets with implications on future complement-blocking strategies in the clinical setting of sepsis.     

Orexins suppress catecholamine synthesis and secretion in cultured PC12 cells

New orexigenic peptides called orexin-A and -B have recently been described in neurons of the lateral hypothalamus and perifornical area. No orexins have been found in adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin-receptor 2 (OX2R) in the rat adrenal gland has been reported. To test the effects of orexins on peripheral organs, we investigated their effects on catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. Orexin-A and -B (100 nM) significantly reduced basal and PACAP-induced tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamines) mRNA levels. Orexin-A and -B (100 nM) also significantly inhibited the PACAP-induced increase in the cAMP level, suggesting that the suppressive effect on TH mRNA is mediated, at least in part, by the cAMP/protein kinase A pathway. Furthermore, orexin-A and -B (100 nM) significantly suppressed basal and PACAP-induced dopamine secretion from PC12 cells. Next, we examined whether orexin receptors (OX1R, OX2R) were present in the rat adrenal gland and PC12 cells. In the adrenal glands, OX2R was as strongly expressed as in the hypothalamus, but OX1R was not detected. On the other hand, neither OX1R nor OX2R was expressed in PC12 cells. However, binding assays showed equal binding of orexin-A and -B to PC12 cells, suggesting the existence in these cells of some receptors for orexins. These results indicate that orexins suppress catecholamine release and synthesis, and that the inhibitory effect is mediated by the cAMP/protein kinase A pathway.     

First continuous human pheochromocytoma cell line: KNA Biological,cytogenetic and molecular characterization of KNA cells

Pheochromocytomas are rare tumours, with an incidence of 1–2 per million which arise from chromaffin cells of the adrenal medulla. They occur sporadically or as part of dominantly inherited cancer syndromes like multiple endocrine neoplasia 2 (MEN2A and 2B) and others. Continuous cell lines, not available so far, are essential tools for studies in these tumours. A continuous cell line (KNA) was established from a sporadic pheochromocytoma of the right adrenal gland of a 73-year-old woman. The KNA cells grow as suspensions of spheroids and show the morphological and immunocytochemical characteristics of neuronal chromaffin cells, such as neuroendocrine granules, and positive reactions to chromogranin- and related peptide-, neuron specific enolase and vasoactive intestinal peptide antibodies. Neurite-like processes are formed after addition of nerve growth factor. Chromosomal analyses revealed a diploid (46,XX, n=50) to hypodiploid (43–45,XX, n=15) karyotype. In hypodiploid metaphases most frequently #19, #17, #21 and #22 were missing. Chromosome arms 1p and 4q showed apparently consistent interstitial deletions: 6q, 8q, 13q and 22q showed clonal interstitial deletions. The cell line shows a heterozygous sequence variant TGC (cysteine) to TGG (tryptophan) in codon 611 in exon 10 of the RET proto-oncogene. So far, PC-12, a rat adrenal pheochromocytoma, has been the only continuous pheochromocytoma cell line available. KNA represents the first report on a human continuous pheochromocytoma cell line, the first report of structural chromosome aberrations in pheochromocytomas and the first report of a RET mutation TGC to TGG in exon 10 of the RET proto-oncogene in a sporadic pheochromocytoma. © 1998 Chapman and Hall     

Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins

PC12 cells, a cell line derived from a rat pheochromocytoma, have both regulated and constitutive secretory pathways. Regulated secretion occurs via large dense core granules, which are related to chromaffin granules and are abundant in these cells. In addition, PC12 cells also contain small electron-lucent vesicles, whose numbers increase in response to nerve growth factor and which may be related to cholinergic synaptic vesicles. These could characterize a second regulated secretory pathway. We have investigated the trafficking of protein markers for both these organelles. We have purified and characterized the large dense core granules from these cells using sequential velocity and equilibrium gradients. We demonstrate the copurification of the major PC12 soluble regulated secretory protein (secretogranin II) with this organelle. As a marker for the synaptic vesicle-like organelles in this system, we have used the integral membrane glycoprotein p38 or synaptophysin. We show that the p38-enriched fraction of PC12 cells comigrates with rat brain synaptic vesicles on an equilibrium gradient. We also demonstrate that p38 purifies away from the dense core granules; less than 5% of this protein is found in our dense granule fraction. Finally we show that p38 does not pass through the dense granule fraction in pulse-chase experiments. These results rule out the possibility of p38 reaching the small clear vesicles via mature dense granules and imply that these cells may have two independently derived regulated pathways.     

Characterization of an endooligopeptidase A-like protein in PC12 cells: Activity modulation by cAMP but not by basic fibroblast growth factor

Endooligopeptidase A is a putative neuropeptide-metabolizing enzyme. It converts small enkephalin-containing peptides into the corresponding enkephalins and inactivates biopeptides such as bradykinin and neurotensin in vitro. We investigated the presence of endooligopeptidase A in PC12 cells. This cell line was derived from a rat pheochromocytoma tumor and resembles fetal chromaffin cell. Depending on the supplements added to the cell culture, this cell line can be differentiated into mature chromaffin cell or sympathetic neuron-like cell. Endooligopeptidase A activity was measured in soluble cellular extracts using a specific fluorogenic substrate QF-ERP7. The PC12 endooligopeptidase A-like activity shared similar but not identical biochemical properties with rabbit brain endooligopeptidase A. Similarly to rabbit brain endooligopeptidase A, the PC12 endooligopeptidase A-like activity was enhanced by DTT, totally inhibited by DTNB and 1-10 Phenanthroline, partially inhibited by cFP-AAF-pAb, and not affected by PMSF. Furthermore, the PC12 endooligopeptidase A-like activity displayed identical elution profile as rabbit brain endooligopeptidase A in gel filtration and anion-exchange chromatography. In addition, an antiserum raised against rabbit brain endooligopeptidase A cross-reacted with a 71 kDa component from PC12 cell extracts in Western blotting and was also able to partially neutralize the PC12 endooligopeptidase A-like activity. Treatment of PC12 cells with basic fibroblast growth factor (bFGF), a neurotrophic factor for this cell line, did not modify the specific activity of this enzyme. However, cAMP analogs decreased the specific activity of the enzyme. These results indicate the presence of an endooligopeptidase A-like activity in PC12 cells which is modulated by cAMP but not by bFGF.     

Searching for Depolarization-Induced Genes that Modulate Synaptic Plasticity and Neurotrophin-Induced Genes that Mediate Neuronal Differentiation

We identify and characterize two classes of immediate-early genes: (i) genes, induced by depolarization in neurons, that play a role in depolarization-induced neuronal plasticity and (ii) genes, induced in neuronal precursors by neurotrophins, that play a causal role in neurotrophin-directed neuronal differentiation. We use rat PC12 pheochromocytoma cells to identify (i) genes preferentially induced by [depolarization or forskolin] versus [Nerve Growth Factor (NGF) or Epidermal Growth Factor (EGF)] and (ii) genes preferentially induced by NGF versus EGF. We describe (i) a collection of genes preferentially induced by depolarization/forskolin in PC12 cells and by kainic acid in vivo, and (ii) a collection of genes preferentially induced by NGF. The synaptotagmin IV gene encodes a synaptic vesicle protein whose level is modulated by depolarization. NGF preferentially induces the urokinase-plasminogen activator receptor in PC12 cells. Antisense oligonucleotide and anti-UPAR antibody experiments demonstrate that NGF-induced UPAR expression is required for NGF-driven PC12 cell differentiation.     

Nestin expression in rat adrenal gland

The constituents of the intermediate filament network of adrenal gland cells have not been deeply investigated in vivo. Adrenocortical cells have been reported to express cytokeratins and vimentin, but the intermediate filament components of the adrenomedullary cells are still unknown. Nestin is an intermediate filament protein that is mainly expressed in the developing nervous and muscle systems. It has been reported to be unable to form filaments by itself and it co-assembles with vimentin. Using immunocytochemical and biochemical approaches, the present study demonstrates that nestin is expressed in situ either in the cortex or in the medulla of adult rat adrenal glands. Nestin-negative cells prevalently form the zona glomerulosa whereas the zona fasciculata and the zona reticularis are mainly nestin-immunoreactive. Nestin-positive cells always express vimentin-like immunoreactivity but several cells apparently expressing only vimentin are detectable too. Nestin is also expressed by adrenomedullary cells that also display a faint vimentin-like immunoreactivity. We hypothesise that the inconstant detection of nestin in adrenocortical cells depends on their different functional moments. Moreover, even though our data do not allow to confirm vimentin in adrenomedullary cells, in situ detection of nestin in the adrenal medulla indirectly supports in vivo expression of vimentin in chromaffin cells.