Nerve Growth Factor Effect on Adenosine Transport in Cultured Chromaffin Cells

Chromaffin cells both recently isolated or in culture present a high-affinity adenosine transporter with a Km value of 1 microM. When cells were exposed to nerve growth factor (NGF; 10 ng/ml), the adenosine transporter affinity decreased to 3 microM. This value was maintained from 3 days after plating to the end of the culture period. A change in the transport capacity was observed, with a significant increase (approximately 200-260%) in NGF-cultured cells throughout the period studied.     

Purification of the Chick Eye Ciliary Neuronotrophic Factor

Dissociated 8-day chick embryo ciliary ganglionic neurons will not survive for even 24 h in culture without the addition of specific supplements. One such supplement is a protein termed the ciliary neuronotrophic factor (CNTF) which is present at very high concentrations within intraocular tissues that contain the same muscle cells innervated by ciliary ganglionic neurons in vivo. We describe here the purification of chick eye CNTF by a 2 1/2-day procedure involving the processing of intraocular tissue extract sequentially through DE52 ion-exchange chromatography, membrane ultrafiltration-concentration, sucrose density gradient ultracentrifugation, and preparative sodium dodecyl sulfate-polyacrylamide gradient electrophoresis. An aqueous extract of the tissue from 300 eyes will yield about 10-20 micrograms of biologically active, electrophoretically pure CNTF with a specific activity of 7.5 X 10(6) trophic units/mg protein. Purified CNTF has an Mr of 20,400 daltons and an isoelectric point of about 5, as determined by analytical gel electrophoresis. In addition to supporting the survival of ciliary ganglion neurons, purified CNTF also supports the 24-h survival of cultured neurons from certain chick and rodent sensory and sympathetic ganglia. CNTF differs from mouse submaxillary nerve growth factor (NGF) in molecular weight, isoelectric point, inability to be inactivated by antibodies to NGF, ability to support the in vitro survival of the ciliary ganglion neurons, and inability to support that of 8-day chick embryo dorsal root ganglionic neurons. Thus, CNTF represents the first purified neuronotrophic factor which addresses parasympathetic cholinergic neurons.     

Nerve Growth Factor and Dexamethasone Modulate Synthesis and Storage of Catecholamines in Cultured Rat Adrenal Medullary Cells: Dependence on Postnatal Age

Catecholamine content and in vitro activities of tyrosine hydroxylase (TH) and noradrenaline N-methyltransferase (NMT) were measured in cultures of isolated adrenal medullary cells from newborn and young postnatal rats to study the effects of the differentiation factors glucocorticoids and nerve growth factor (NGF). During the 4-day culture period the cellular catecholamine (CA) content and TH activity remained stable, whereas NMT activity dropped to about half of the initial level. In cells from 2- and 10-day-old rats 10 microM dexamethasone specifically prevented this loss in NMT activity. Furthermore, this glucocorticoid treatment increased, in a dose-dependent manner, the total CA content by 50-100% over control levels without changes in the adrenaline (A) proportion or TH activity. In contrast, NGF did not affect NMT activities at all. In cells from 10-day-old rats 100 ng/ml NFG elevated TH activity and total CA content to about 160% of controls and did not change the proportion of A. This increase in total CA content was linear with the NGF dose and required greater than 5 ng/ml NGF. In chromaffin cells from 2-day-old rats 100 ng/ml NGF affected neither TH activity nor the total content, whereas it significantly reduced the proportion of A by about 25%.     

Cholinergic Neuronotrophic Factors. Concurrent Activities on Certain Nerve Growth Factor-Responsive Neurons

Abstract: We had previously reported that in vitro survival of chick embryo ciliary ganglionic neurons can be assured by the addition to the culture medium of appropriate amounts of soluble macromolecular agents termed ciliary neuronotrophic factors. Particularly rich sources of one such factor are aqueous extracts from chick embryo intraocular tissues that include the smooth and striated musculature innervated by ciliary ganglionic neurons. We report here that this eye extract also contains agents that we term ganglionic neuronotrophic factors that support the survival of 11-day chick embryo sympathetic and neonatal mouse dorsal root ganglionic neurons, two traditional targets of nerve growth factor (NGF). Using a recently developed microassay procedure we found that these ganglionic activities are not inactivated by rabbit, rat, or guinea pig antisera raised against the 2.5S (beta) subunit of male mouse submaxillary NGF, rabbit antisera against 7S NGF, or quail antisera against cobra venom NGF. Both the ciliary and ganglionic activities can be quantitated simultaneously by using 24-h in vitro microassays, thus permitting a direct comparison of their respective properties. Both activities were found to (a) adsorb to DE52 cellulose and coelute at a similar salt concentration, (b) focus and be recovered from isoelectric polyacrylamide gels at exactly the same pH region, (c) be heat-and partially acid-labile, but base-stable, and (d) be inactivated by exposure to trypsin. These results suggest that the ciliary and ganglionic neuronotrophic activities are associated with the same protein.     

Ciliary Neuronotrophic Factor Stimulates Choline Acetyltransferase Activity in Cultured Chicken Retina Neurons

It has been demonstrated that cultured cholinergic retinal neurons from 8-day-old chicken embryos respond to a polypeptide factor present in retinal cell-conditioned medium (RCM) and in retinal extracts. Compared with control cultures, the activity of acetyl-CoA:choline O-acetyltransferase (EC 2.3.1.6; ChAT) is enhanced more than twofold in neuronal retinal cultures grown for 7 days in the presence of RCM. The present study demonstrates that both ciliary neuronotrophic factor (CNTF), which is characterized by its trophic activity on parasympathetic ciliary neurons, and RCM exhibit identical stimulatory effects on ChAT activity in retinal monolayer cultures. Similarly, RCM supports the in vitro survival of ciliary neurons to the same extent as CNTF. The active species in RCM has a molecular weight (20,900 +/- 1,000) identical to that of CNTF, as determined by preparative sodium dodecyl sulfate gel electrophoresis. The results indicate that cholinergic retinal neurons represent a central neuronal target for CNTF or a closely related protein.     

Mimicry and Inhibition of Nerve Growth Factor Effects: Interactions of Staurosporine, Forskolin, and K252a in PC12 Cells and Normal Rat Chromaffin Cells In Vitro

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)     

Effects of Reserpine and Tetrabenazine on Catecholamine and ATP Storage in Cultured Bovine Adrenal Medullary Chromaffin Cells

The in vivo storage relationship between catecholamines and ATP in chromaffin vesicles of cultured bovine adrenal medulla cells was investigated using drugs that block vesicular catecholamine uptake. Three-day treatments with reserpine and tetrabenazine causing 85-90% depletion of catecholamines resulted in 41-46% reductions in cellular ATP content. Subcellular fractionation of reserpine-treated cells indicated that the ATP is lost from the chromaffin vesicle pool. This was confirmed in experiments using metabolic inhibitors to differentiate the vesicular and extravesicular ATP pools. The vesicular ATP loss was not proportional to that of catecholamines, resulting in a reduction by 50% in the chromaffin vesicle mole ratio of catecholamines to ATP after 48 h of treatment. In metabolic labeling studies, it was found that reserpine treatment reduced the incorporation of [3H]adenosine into vesicular ATP selectively, but it reduced the incorporation of 32Pi into both the vesicular and extravesicular pools. The reduction of the [3H]adenosine incorporation was not due to diminished vesicular nucleotide uptake resulting from low catecholamine levels, because when the catecholamines were depleted by tetrabenazine pretreatment followed by removal of the drug before labeling, no reduction in [3H]adenosine incorporation was observed. When present during the labeling, tetrabenazine was found to be a reversible inhibitor of plasma membrane adenosine uptake. The observed loss of adenine nucleotides from catecholamine-depleted chromaffin vesicles in vivo provides evidence that interactions between ATP and catecholamines are important in the vesicular storage of high concentration of these compounds.     

Characterization of Histamine-Induced Catecholamine Secretion from Bovine Adrenal Medullary Chromaffin Cells

Histamine activation of H1 receptors stimulates 3H release from cultured bovine adrenal chromaffin cells preloaded with [3H]noradrenaline. The initial (1-min) release induced by a high concentration of histamine was unaffected by the removal of extracellular Ca2+, whereas the more sustained response (10 min) was largely inhibited. In contrast, release induced by nicotine was dependent on extracellular Ca2+ at all times. The protein kinase inhibitor staurosporine inhibited both the initial and sustained (10-min) phases of histamine-induced release (IC50 in the region of 200 nM) but was ineffective against a direct depolarizing stimulus (56 mM K+). In contrast, the calmodulin antagonist trifluoperazine was equally effective against both stimuli. These data indicate that although a staurosporine-sensitive event (perhaps involving protein kinase C) is essential for coupling histamine receptor activation to the release processes, it is not essential for exocytosis itself. A further distinction between histamine- and depolarization-induced release was demonstrated by the differential effect of the guanine nucleotide-binding protein inhibitor pertussis toxin. Pretreatment with pertussis toxin (0.1 microgram/ml for 16 h) enhanced depolarization-induced release by approximately 1.5-fold. This pertussis toxin pretreatment was, however, approximately twofold as effective in potentiating histamine-evoked release. Thus, the characteristics of the histaminergic response are distinct from those of a depolarizing stimulus, perhaps indicating the involvement of different mechanisms in the release process.     

Bovine Chromaffin Cells Have Insulin-Like Growth Factor-I (IGF-I) Receptors: IGF-I Enhances Catecholamine Secretion

The binding of 125I-insulin-like growth factor-I (125I-IGF-I) to bovine chromaffin cells was measured. Chromaffin cell cultures contained 111,000 +/- 40,000 IGF-I binding sites/cell. These sites bound IGF-I with a KD of 1.1 +/- 0.3 nM and had a much lower affinity for insulin. Cross-linking studies showed that 125I-IGF-I bound to a protein that had an Mr of approximately 125,000, similar to the Mr of the alpha subunit of the IGF-I receptor in other tissues. Cells cultured with IGF-I (10 nM) for 4 days exhibited an almost twofold increase in high K+-evoked catecholamine secretion. Insulin was much less potent than IGF-I in enhancing catecholamine secretion. These data indicate that binding of IGF-I to its receptors on chromaffin cells can modulate the function of these cells.     

Both Short- and Long-Term Effects of Nerve Growth Factor on Tyrosine Hydroxylase in Calf Adrenal Chromaffin Cells Are Blocked by S-Adenosylhomocysteine Hydrolase Inhibitors

We have previously shown that primary cultures of calf chromaffin cells respond to nerve growth factor (NGF) treatment with a selective induction of tyrosine hydroxylase (TH), which takes 48 h to be manifested. In the present study, we report that short exposure of calf chromaffin cells to NGF (5-60 min) results in TH activation, which involves a change in the Vmax of the enzyme with no change in the number of enzyme molecules, similar to an effect that has been previously reported in PC12 cells. This activation is markedly potentiated when the chromaffin cells are plated on a laminin substrate, such that after 5 min of NGF exposure, there is an approximately fourfold increase in the TH activity. Both short-term activation and long-term TH induction brought about by NGF treatment are blocked by 5'-deoxy-5'-methylthioadenosine and other drugs that act as S-adenosylhomocysteine (SAH) hydrolase inhibitors to block methylation by end-product inhibition. These drugs did not inhibit cyclic AMP-mediated TH activation or increases in the levels of TH. However, measurements of the degree of blockade of methylation in cells treated with these drugs, taken together with conceptual information regarding the nonregulatory nature of methylation in eukaryotic cells, were not consistent with inhibition of methylation as the crucial effect of the drugs to block the effects of NGF. Nonetheless, since SAH hydrolase inhibitors selectively inhibited NGF-mediated effects, and not comparable effects triggered by other stimuli, these compounds provide useful tools in future studies of the biochemical signalling mechanism of NGF.     

Regulation by Interleukin-1 of Nerve Growth Factor Secretion and Nerve Growth Factor mRNA Expression in Rat Primary Astroglial Cultures

Primary cultures of neonatal rat cortical astrocytes contain low cellular levels (about 2 pg/mg of protein) of nerve growth factor (NGF), but secrete significant amounts of NGF into the culture medium (about 540 pg of NGF/mg of cell protein/38-h incubation). Incubation of astrocytes with interleukin-1 (IL-1) increased the cellular content of NGF and the amount secreted by about threefold. In comparison, cerebellar astrocytes secreted significant amounts of NGF, and the secretion was also stimulated by IL-1. The stimulatory action of IL-1 on astrocytes prepared from cortex was dose- and time-dependent. Concentrations of IL-1 causing half-maximal and maximal stimulation of NGF secretion were 1 and 10 U/ml, respectively). Maximal NGF secretion induced by IL-1 (10 U/ml) was seen following 38 h of incubation. The basal secretion of NGF was reduced by about 50% under Ca2(+)-free conditions; however, the percent stimulation of NGF secretion by IL-1 was the same in the absence or presence of Ca2+. The stimulatory action of IL-1 was specific, because other glial growth factors and cytokines were almost ineffective in stimulating NGF secretion from cortical astroglial cells. IL-1 treatment also increased cellular NGF mRNA content twofold. The results indicate that IL-1 specifically triggers a cascade of events, independent of cell growth, which regulate NGF mRNA content and NGF secretion by astrocytes.     

Induction of Growth Factor Receptors on Cultured Human Melanocytes

Using a sensitive and selective culture system for human epidermal melanocytes, we have demonstrated that the morphologic changes induced by addition of phorbol 12-tetradecanoate 13-acetate (TPA) to proliferating newborn melanocytes are associated with induction of nerve growth factor (NGF) receptors, as measured by messenger RNA (mRNA) levels and protein accumulation and by cell surface immunofluorescent staining. Growth factor deprivation or addition of NGF similarly results in NGF receptor induction. NGF is believed to function in vivo and in vitro as a survival factor for many neural crest-derived cells and has been demonstrated to promote specific neural cell functions ranging from neurite outgrowth to enzyme induction, but to date no role for NGF has been identified with regard to normal human melanocytes. Our data demonstrate that, given appropriate stimulation, cultured human melanocytes may express the NGF receptor gene and therefore suggest that NGF may modulate human melanocyte behavior in vivo. This first demonstration of a growth factor receptor on human melanocytes provides an important opportunity to explore signal transduction relevant to their growth, differentiation, and malignant transformation.     

The Relationship Between Arachidonic Acid Release and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

Increased arachidonic acid release occurred during activation of catecholamine secretion from cultured bovine adrenal medullary chromaffin cells. The nicotinic agonist 1,1-dimethyl-4- phenylpiperazinium (DMPP) caused an increased release of preincubated [3H]arachidonic acid over a time course which corresponded to the stimulation of catecholamine secretion. Like catecholamine secretion, the DMPP-induced [3H]arachidonic acid release was calcium-dependent and was blocked by the nicotinic antagonist mecamylamine. Depolarization by elevated K+, which induced catecholamine secretion, also stimulated arachidonic acid release. Because arachidonic acid release from cells probably results from phospholipase A2 activity, our findings indicate that phospholipase A2 may be activated in chromaffin cells during secretion.     

Production of 1,2-Diacylglycerol in PC12 Cells by Nerve Growth Factor and Basic Fibroblast Growth Factor

The addition of nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) to PC12 cells prelabeled with [3H]inositol and preincubated for 15 min in the presence of 10 mM LiCl stimulated the production of inositol phosphates with maximal increases of 120-180% in inositol monophosphate (IP), 130-200% in inositol bisphosphate (IP2), and 45-50% in inositol trisphosphate (IP3) within 30 min. The majority of the overall increase (approximately 85%) was in IP; the remainder was recovered as IP2 and IP3 (approximately 10% as IP2 and 5% as IP3). Under similar conditions, carbachol (0.5 mM) stimulated about a 10-fold increase in IP, a sixfold increase in IP2, and a fourfold increase in IP3. The mass level of 1,2-diacylglycerol (DG) in PC12 cells was found to be dependent on the incubation conditions; in growth medium [Dulbecco's modified Eagle's medium (DME) plus serum], it was around 6.2 mol %, in DME without serum, 2.5 mol %, and after a 15-min incubation in Dulbecco's phosphate-buffered saline, 0.62 mol %. The addition of NGF and bFGF induced an increase in the mass level of DG of about twofold within 1-2 min, often rising to two- to threefold by 15 min, and then decreasing slightly by 30 min. This increase was dependent on the presence of extracellular Ca2+, and was inhibited by both phenylarsine oxide (25 microM) and 5'-deoxy-5'-methylthioadenosine (3 mM). Under similar conditions, 0.5 mM carbachol stimulated the production of DG to the same extent as 200 ng/ml NGF and 50 ng/ml bFGF. Because carbachol is much more effective in stimulating the production of inositol phosphates, the results suggest that both NGF and bFGF stimulate the production of DG primarily from phospholipids other than the phosphoinositides.     

Differential Responses of the Phosphorylation of Ribosomal Protein S6 to Nerve Growth Factor and Epidermal Growth Factor in PC 12 Cells

Previous studies from this laboratory have shown that the phosphorylation of the S6 protein of the ribosomes is catalyzed by at least two different and separable kinase activities in PC12 cells. One of these activities is increased by treatment of the cells with nerve growth factor, the other by treatment of the cells with epidermal growth factor. The present work shows that these two factors stimulate the phosphorylation of S6 with quite different kinetics, and that both the number of phosphates incorporated into S6 and the phosphopeptide pattern of S6 are different in cells treated with nerve growth factor than in cells treated with epidermal growth factor. The characteristics of the nerve growth factor-sensitive S6 kinase and of the epidermal growth factor-sensitive kinase were also clearly different. Substrate specificity and inhibitor studies indicated that neither was identical to cyclic AMP-dependent kinase, kinase C, or the calcium/calmodulin-dependent kinases. However, two major phosphopeptides produced by S6 phosphorylation in nerve growth factor-treated cells were also seen on phosphorylation of S6 by cyclic AMP-dependent kinase in vitro. In addition, when rat liver 40S ribosomal subunits were pretreated with cyclic AMP-dependent kinase in vitro, the action of the nerve growth factor-sensitive S6 kinase was increased about twofold.     

Differential Expression of Ciliary Neurotrophic Factor Receptor in Skeletal Muscle of Chick and Rat After Nerve Injury

Abstract: The activities of ciliary neurotrophic factor (CNTF) were initially thought to be restricted to cells in the nervous system. However, the recent identification of its receptor specificity-conferring α component (CNTFRα) in skeletal muscle has provided the clue to the unexpected actions of CNTF in the periphery. In the present study, we demonstrated that the mRNA expression of CNTFRα in chick skeletal muscle was decreased by ∼10-fold after nerve transection; this finding is in sharp contrast to the dramatic up-regulation observed in denervated rat muscle. As a first step toward investigating the differential regulation of CNTFRα in chick and rat, we examined the mRNA expression of CNTFRα in different types of muscle following nerve injury in young and adult animals. Our findings demonstrated that the differential expression of CNTFRα observed in denervated skeletal muscle of the chick and rat was not dependent on age or muscle type. The temporal profile of the changes in CNTFRα expression was, however, dependent on the age of the chick as well as the types of muscle. Furthermore, the low level of CNTFRα expression observed in denervated chick muscle recovered to almost control levels in regenerating skeletal muscle. Taken together, our findings provided the first extensive analysis on the mRNA expression of CNTFRα and the α subunit of the acetylcholine receptor in various skeletal muscles of the chick following nerve injury and regeneration.     

Differential Ras-Dependence of Gene Induction by Nerve Growth Factor and Second Messenger Analogs in PC12 Cells

Induction of neurite formation by nerve growth factor (NGF) in PC12 pheochromocytoma cells can be efficiently inhibited by expressing a dominant negative mutant form of the small guanine nucleotide binding Ha-Ras protein in these cells. The block in NGF-induced neuritogenesis caused by inhibition of endogenous Ras proteins was found to be partially relieved by simultaneous stimulation of cAMP- or Ca⁺⁺-dependent signaling pathways. Since expression of certain genes is believed to be involved in NGF-signaling leading to morphological differentiation, we decided to study the combined effects of NGF and second messenger analogs on gene expression in PC12 cell lines expressing different levels of the interfering Ras protein. We found NGF-second messenger combinations that induced normal c-fos, zif268 and nur77 early-response gene expression without neuritogenesis, and, conversely, cell lines in which certain combination treatments caused partial neuronal differentiation in the absence of substantial activation of these genes. Similarly, neurite outgrowth induced by combination treatments does not seem to require the activation of the late-response transin gene. Our results thus suggest a lack of strong correlation between NGF-stimulated early- and secondary-response gene induction and morphological differentiation.     

Differentiation Effects of Ciliary Neurotrophic Factor on Human Neuroblastoma Cells

Abstract: In the human neuroblastoma cell line LA-N-2, recombinant rat ciliary neurotrophic factor (CNTF) induced neurite growth and cholinergic differentiation that were both half-maximally saturated at <100 p M of the neurokine, but was not required for cell survival in serum-free conditions over a 13-day period. CNTF markedly stimulated choline acetyltransferase activity and acetylcholine synthesis, whereas high-affinity choline transport was only slightly enhanced and acetylcholinesterase activity was unchanged. Leukemia inhibitory factor had effects identical to CNTF on neurite growth and choline acetyltransferase activity, but interleukin 6 had no effect. Radioiodinated CNTF binding and affinity cross-linking studies were consistent with tripartite receptor activation as a mediator of the observed biological effects.