Endogenous GM1 Ganglioside of the Plasma Membrane Promotes Neuritogenesis by Two Mechanisms

The influence of GM1 on the neuritogenic phase of neuronal differentiation has been highlighted in recent reports showing upregulation of this ganglioside in the plasma and nuclear membranes concomitant with axonogenesis. These changes are accompanied by alterations in Ca²⁺ flux which constitute an essential component of the signaling mechanism for axon outgrowth. This study examines 2 distinct mechanisms of induced neurite outgrowth involving plasma membrane GM1, as expressed in 3 neuroblastoma cell lines. Growth of Neuro-2a and NG108-15 cells in the presence of neuraminidase (N'ase), an enzyme that increases the cell surface content of GM1, caused prolific outgrowth of neurites which, in the case of Neuro-2a, could be blocked by the B subunit of cholera toxin (Ctx B) which binds specifically to GM1; however, the latter agent applied to NG108-15 cells proved neuritogenic and potentiated the effect of N'ase. With N18 cells, the combination was also neuritogenic as was Ctx B alone, whereas N'ase by itself had no effect. Neurite outgrowth correlated with influx of extracellular Ca²⁺, determined with fura-2. Treatment of NG108-15 and N18 cells with Ctx B alone caused modest but persistent elevation of intracellular Ca²⁺ while a more pronounced increase occurred with the combination Ctx B + N'ase. Treatment with N'ase alone also caused modest but prolonged elevation of intracellular Ca²⁺ in NG108-15 and Neuro-2a but not N18; in the case of Neuro-2a this effect was blocked by Ctx B. Neuro-2a and N18 thus possess 2 distinctly different mechanisms for neuritogenesis based on Ca²⁺ modulation by plasma membrane GM1, while NG108-15 cells show both capabilities. The neurites stimulated by N'ase + Ctx B treatment of N18 cells were shown to have axonal character, as previously demonstrated for NG108-15 cells stimulated in this manner and for Neuro-2a cells stimulated by N'ase alone.     

Neurite Differentiation Is Modulated in Neuroblastoma Cells Engineered for Altered Acetylcholinesterase Expression

Abstract: Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5-fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level ( p < 0.01). Furthermore, treatment of AChE-overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.     

Promotion of Neuritogenesis in Mouse Neuroblastoma Cells by Exogenous Gangliosides. Relationship Between the Effect and the Cell Association of Ganglioside GM1

Ganglioside GM1 promoted neuritogenesis of neuroblastoma cells, neuro-2a clone, in monolayer culture. GM1 bound to neuro-2a cells in three distinct forms, one removable by treatment with serum-containing solutions, one serum-resistant and labile to trypsin treatment, and one resistant to serum and trypsin treatments. The proportions among the three forms of cell-associated GM1 varied in relation to duration of exposure to ganglioside, ganglioside concentration in the medium, and number of cells in culture. The form removable by serum was predominant at the initial stages of association and at the highest ganglioside concentrations (over 10(-6)M); the trypsin-labile and -stable forms tended to increase with increasing cell number and decreasing ganglioside concentration. The neuritogenic effect of GM1 was higher when neuro-2a cells were incubated for 24 h in the presence of GM1 and fetal calf serum. Under this condition the percentage of neurite-bearing cells increased from 11% of control to 62% at the optimal ganglioside concentration of 10-4M. The effect was still present, although to a lower extent (from 11% to 28% of neurite-bearing cells), when cells were first exposed for only 2 h to GM1, then washed and incubated for 24 h in the presence of fetal calf serum. The trypsin-labile and -stable forms of cell-associated GM1 had a fundamental role in the effect, whereas the form removable by serum was not involved. The preparation of GM1 used was extremely pure (99%) and, in particular, had a peptide contamination, if any, less than 1:20,000-1:50,000.(ABSTRACT TRUNCATED AT 250 WORDS)     

GM1 Ganglioside in the Nuclear Membrane Modulates Nuclear Calcium Homeostasis During Neurite Outgrowth

Abstract: GM1 in the nuclear membrane, previously shown to be up-regulated during neurite outgrowth, has been found to influence nuclear Ca²⁺ flux during differentiation of Neuro-2a cells. Nuclei were isolated from cultured Neuro-2a cells before and after neuraminidase-induced neuritogenesis and incubated with ⁴⁵Ca²⁺ for varying periods to determine uptake/efflux of Ca²⁺. At 5, 10, and 15 min ⁴⁵Ca²⁺ levels in nuclei from differentiated cells were significantly lower than those in nuclei from untreated cells. The same result was obtained when the GM1 level was elevated artificially by preincubation of the nuclei in 10 µ M GM1. In experiments designed to measure efflux specifically, isolated nuclei preincubated in GM1 released ⁴⁵Ca²⁺ more rapidly than untreated nuclei. We conclude that one role of GM1 in the nuclear membrane is to alter Ca²⁺ regulatory mechanisms in the nucleus following onset of neuronal process outgrowth.     

Induction of Ganglioside Biosynthesis and Neurite Outgrowth of Primary Cultured Neurons by l-threo-1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol

Abstract: We reported previously that stereoisomers of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), the d - threo and l - threo forms, exerted inhibitory and stimulatory effects on glycosphingolipid (GSL) biosynthesis in B16 melanoma cells, respectively. In the present study, the primary cultured rat neocortical explants were treated with l - or d - threo -PDMP. These isomers exhibited opposite effects on neurite outgrowth: d -PDMP was inhibitory at concentrations ranging from 5 to 20 µ M , whereas l -PDMP was stimulatory over the same concentration range, and the maximal effect was observed at 10–15 µ M . Rat neocortical explants were doubly labeled with [¹⁴C]serine and [³H]galactose at 15 µ M l - or d -PDMP. l -PDMP increased the incorporations of both labels into sphinganine, sphingosine, ceramide, sphingomyelin, neutral GSLs, and gangliosides, whereas d -PDMP inhibited the glucosylation of ceramide resulting in a reduction of ganglioside biosynthesis and accumulation of precursors of glucosylceramide, ceramide, and sphingomyelin. To clarify the stimulatory effect of l -PDMP on GSL biosynthesis, serine palmitoyltransferase, sphingosine N -acyltransferase, glucosylceramide synthase, lactosylceramide synthase, GM3 synthase, and GD3 synthase were quantified in cell lysates of explants pretreated with this agent. Serine palmitoyltransferase was fully activated up to 150% of the control. Furthermore, marked increases in the activities of lactosylceramide synthase (200%), GM3 synthase (240%), and GD3 synthase (300%) were observed. These results suggest that the neurotrophic action of l -PDMP may be ascribable to its stimulatory effect on the biosynthesis of GSLs, especially that of gangliosides.     

Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady-state levels of phosphotyrosine-modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF-primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/ trk tyrosine kinase because the presence of NGF in cultures of NGF-primed cells was not necessary for genistein-stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF-primed PC12 cells. Video-enhanced differential interference contrast microscopy revealed that growth cones of genistein-treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.     

GM1 ganglioside modulates prostaglandin E1 stimulated adenylyl cyclase in neuro-2A cells

This study demonstrates modulation by GM1 ganglioside of prostaglandin E1 (PGE1)-induced cAMP formation in Neuro-2a neuroblastoma cells. Pretreatment of the cells with neuraminidase, an enzyme that increases cell surface GM1, resulted in significant elevation of PGE1-induced cAMP formation, as did preincubation of the cells with nmolar concentrations of GM1. Pretreatment with brain ganglioside mixture lacking GM1 had no effect. Cholera toxin B subunit, a specific GM1-binding ligand, inhibited adenylyl cyclase. When the concentration of exogenous GM1 in which the cells were preincubated was increased from nmolar to molar levels there was a dose-responsive fall off in cAMP elevation, attributed to progressive inhibition of adenylyl cyclase by increasing GM1. These results are interpreted as indicating modulation of this PGE1 receptor in Neuro-2a cells by plasma membrane-localized GM1 in a structure-specific manner.Abbreviations PGE1 prostaglandin E1 - Ctx B B subunit of cholera toxin - BBG bovine brain ganglioside mixture - DMEM Dulbecco's modified Eagle's medium - FBS fetal bovine serum - IBMX 3-isobutyl-1-methylxanthine - N'ase neuraminidase - D-PBS Dulbecco's phosphate-buffered saline     

Ganglioside GM1 Enhances Induction by Nerve Growth Factor of a Putative Dimer of TrkA

Abstract: GM1 enhances nerve growth factor (NGF)-stimulated neuritogenesis and prevents apoptotic death of PC12 cells; both may be due to enhancement of TrkA dimerization. In this study, we examined the effect of GM1 on NGF-induced TrkA dimerization in Trk-PC12 (6–24) cells. NGF increased tyrosine phosphorylation of the 140-kDa protein (TrkA monomer), and preincubation with GM1 potentiated this effect. Adding the protein cross-linker bis(sulfosuccinimidyl) suberate with NGF resulted in the appearance of two major bands (220 and 330 kDa) when probed with antibodies against TrkA or phosphotyrosine, and GM1 also enhanced this effect. We interpret the 330-kDa band as being a homodimer of TrkA. The identity of the 220-kDa band is still not certain but may consist of a posttranslationally modified form of TrkA. Our results suggest that GM1 is augmenting the effects of NGF on PC12 cells by enhancing the dimerization and activation of the TrkA receptor.     

Regulation of Transmembrane Signaling by Ganglioside GM1 :

Interaction of antibodies to ganglioside GM1 with Neuro2a cells was studied to investigate the role of GM1 in cell signaling. Binding of anti-GM1 to Neuro2a cells induced the formation of 3H-inositol phosphates (3H-IPs) and elevated the intracellular Ca2+ concentration [Ca2+]i. The rise in [Ca2+]i was due to the influx of Ca2+ from the extracellular medium and release from intracellular Ca2+ pools. The Ca2+ influx pathway did not allow the permeation of Na+ or K+. The influx was inhibited by amiloride, a specific blocker of T-type Ca2+ channels, whereas nifedipine and diltiazem, blockers of L-type Ca2+ channels, did not have any effect. Thus, anti-GM1 appears to activate a T-type Ca2+ channel in Neuro2a cells. The intracellular Ca2+ release was inhibited by pretreatment of cells with neomycin sulfate, phorbol dibutyrate, and pertussis toxin (PTx), which also inhibited the 3H-IP formation in Neuro2a cells. Addition of caffeine neither elevated the [Ca2+]i nor affected the anti-GM1-induced [Ca2+]i rise. The data reveal that the binding of anti-GM1 to Neuro2a cells activates phospholipase C via a PTx-sensitive G protein, which leads to formation of IPs and release of Ca2+ from inositol trisphosphate-sensitive pool of endoplasmic reticulum. Anti-GM1 also arrested the differentiation of Neuro2a cells in culture and significantly stimulated their proliferation. This stimulatory effect of anti-GM1 on cell proliferation was blocked by amiloride but not by PTx, suggesting that the influx of Ca2+ was essentially required for cell proliferation. Our data suggest a role for GM1 in the regulation of transmembrane signaling events and cell growth.     

Ganglioside GM1 Contributes to the State of Insulin Resistance in Senescent Human Arterial Endothelial Cells

Vascular endothelial cells (ECs) play central roles in physiologically important functions of blood vessels and contribute to the maintenance of vascular integrity. Therefore, it is considered that the impairment of EC functions leads to the development of vascular diseases. However, the molecular mechanisms of the EC dysfunctions that accompany senescence and aging have not yet been clarified. The carbohydrate antigens carried by glycoconjugates (e.g. glycoproteins, glycosphingolipids, and proteoglycans) mainly present on the cell surface serve not only as marker molecules but also as functional molecules. In this study, we have investigated the abundance and functional roles of glycosphingolipids in human ECs during senescence and aging. Among glycosphingolipids, ganglioside GM1 was highly expressed in abundance on the surface of replicatively and prematurely senescent ECs and also of ECs derived from an elderly subject. Insulin signaling, which regulates important functions of ECs, is impaired in senescent and aged ECs. Actually, by down-regulating GM1 on senescent ECs and overloading exogenous GM1 onto non-senescent ECs, we showed that an increased abundance of GM1 functionally contributes to the impairment of insulin signaling in ECs. Taken together, these findings provide the first evidence that GM1 increases in abundance on the cell surface of ECs under the conditions of cellular senescence and aging and causes insulin resistance in ECs. GM1 may be an attractive target for the detection, prevention, and therapy of insulin resistance and related vascular diseases, particularly in older people.     

Oligosaccharide Portion of GM1 Enhances Process Formation by S20Y Neuroblastoma Cells

The oligosaccharide portion of ganglioside GM1 was found to enhance neuritogenesis by S20Y murine neuroblastoma cells grown in vitro. The average length of the neurites produced by cells grown in the presence of the oligosaccharide portion of GM1 was comparable to that of cells grown in the presence of intact GM1. The processes of these cells were significantly longer (p less than 0.005, pooled t test) than those of cells grown in the presence of comparable concentrations of sialic acid, lactose, sialyllactose, GD1a, or the oligosaccharide moiety of GD1a. These results suggest that it is the oligosaccharide portion of GM1 that is responsible for the ability of GM1 to enhance process outgrowth by S20Y neuroblastoma cells.     

Identification of a GM1-Binding Protein on the Surface of Murine Neuroblastoma Cells

S20Y murine neuroblastoma cells appear to express a protein component(s) able to adhere specifically to the oligosaccharide portion of GM1 (oligo-GM1). To identify proteins with which the oligo-GM1 becomes closely associated, a radiolabeled (125I), photoactivatable derivative of oligo-GM1 was prepared. This was accomplished by reductive amination of the glucosyl moiety of oligo-GM1 to 1-deoxy-1-aminoglucitol, followed by reaction of the amine with sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). Crosslinking studies using the photoactivatable probe indicated that it came in close proximity to a protein with an apparent molecular mass of approximately 71 kDa. In competition experiments, as little as a 10-fold molar excess of oligo-GM1 resulted in a selective reduction in labeling of this protein; preincubation with a 200-fold molar excess of siayllactose was necessary to observe the same change in the labeling pattern, lending additional support to the hypothesis that the approximately 71-kDa protein specifically associates with oligo-GM1. Cell surface location of the oligo-GM1 binding protein was confirmed using subcellular fractionation and morphological analyses.     

Increased Intracellular Cyclic AMP Differentially Modulates Nerve Growth Factor Induction of Three Neuronal Recognition Molecules Involved in Neurite Outgrowth

The relative expression of the immunoglobulin superfamily members Thy-1 and L1 and the neural cell adhesion molecule (N-CAM) in PC12 cells grown in the presence of nerve growth factor (NGF), cholera toxin, or both has been quantified. Whereas NGF treatment induced increases in the cell surface expression of all three glycoproteins, treatment with cholera toxin resulted in the specific induction of L1. During the first few days of culture, cholera toxin acted synergistically with NGF to promote increases in neuritic outgrowth and the synthesis and cell surface accumulation of the 140- and 180-kilodalton subunits of N-CAM. In contrast, over the same period of culture, cholera toxin inhibited the NGF induction of Thy-1 and L1. Over longer periods of culture (3-5 days), cholera toxin inhibited the NGF induction of N-CAM and neurite outgrowth. A similar pattern of synergistic and inhibitory responses was observed when differentiation was induced by fibroblast growth factor (FGF) rather than NGF or when cholera toxin was replaced with forskolin. These data suggest that intracellular cyclic AMP can differentially modulate cell surface glycoprotein expression induced by either NGF or FGF. Of the three cell surface glycoproteins we have studied, temporal changes in N-CAM expression correlate best with the morphological differentiation status of PC12 cells.     

Inositol Metabolism During Neuroblastoma B50 Cell Differentiation: Effects of Differentiating Agents on Inositol Uptake

Inositol uptake was studied in the rat CNS neuroblastoma B50 cell line. Eadie-Hofstee analysis of the uptake pattern reveals two defined modes of inositol entry into the cell. The high-affinity uptake component requires the presence of extracellular sodium and is inhibited by phloridzin. Analysis of the uptake velocities of the high-affinity uptake component provided the following apparent kinetic parameters: Km = 13.7 microM and Vmax = 14.7 pmol/mg of protein/min (without correcting for residual diffusion) and Km = 12.9 microM and Vmax = 12.3 pmol/mg of protein/min (with correction). At physiological concentrations, the high-affinity transport process contributes approximately 70% to total uptake; the remainder is due to a low-affinity diffusion-like process. Uptake inhibition studies reveal that the uptake process is sensitive to ouabain, amiloride, and dichlorobenzamil inhibition but relatively insensitive to cytochalasin B or phloretin. When neuroblastoma B50 cells are induced to differentiate morphologically with high extracellular calcium or with dibutyryl cyclic AMP, a significant decrease in inositol uptake is observed. The dibutyryl cyclic AMP-mediated inhibition of uptake affects only the high-affinity uptake component and is noncompetitive in nature. The high extracellular calcium-mediated inhibition is less specific; it involves "disappearance" of the high-affinity process, some inhibition of the low-affinity process, and an increase of inositol efflux. The significance of these observations is discussed in the context of neuroblastoma B50 cell differentiation.     

The Production of Arachidonic Acid Can Account for Calcium Channel Activation in the Second Messenger Pathway Underlying Neurite Outgrowth Stimulated by NCAM, N-Cadherin, and L1

Abstract: We have used monolayers of control 3T3 fibroblasts and 3T3 fibroblasts expressing transfected cell adhesion molecules (CAMs)—NCAM, N-cadherin, and L1—as a culture substrate for cerebellar neurones. The transfected CAMs promote neurite outgrowth by activating a second messenger pathway that culminates in calcium influx into neurones through N-and l -type calcium channels. We show that the same neurite outgrowth response can be directly induced by arachidonic acid (10 μ M ) and that this response can be inhibited by N-and l -type calcium channel antagonists. In cells, arachidonic acid can be generated by phospholipase A₂ or by the sequential activities of a phospholipase C (to generate diacylglycerol) and diacylglycerol lipase. In the present study we show the neurite outgrowth stimulated by CAMs (but not by various other agents) can be abolished by an inhibitor of diacylglycerol lipase acting at a site upstream from calcium channel activation. The results suggest that arachidonic acid and/or one of its metabolites is the second messenger that activates calcium channels in the CAM signalling pathway leading to axonal growth, and this is supported by recent evidence that shows the same concentrations of arachidonic acid can increase voltage-dependent calcium currents in cardiac myocytes.     

Identification of Two Distinct Isoforms of the Guanine Nucleotide Binding Protein Go in Neuroblastoma × Glioma Hybrid Cells: Independent Regulation During Cyclic AMP-Induced Differentiation

Three distinct antipeptide antisera generated against synthetic peptides that represent parts of the primary sequence of the alpha-subunit of the (pertussis toxin-sensitive) guanine nucleotide binding protein G0 were used in two-dimensional immunoblots of membranes of neuroblastoma X glioma (NG108-15) cells. Each antiserum identified two distinct polypeptides of some 39 kDa. These had apparent isoelectric points of 5.5 and 5.8. Differentiation of NG108-15 cells in response separately to dibutyryl cyclic AMP (cAMP), 8-bromo cAMP, forskolin, and prostaglandin E1 produced elevated levels of G0 alpha, as has previously been noted in one-dimensional immunoblots. Two-dimensional analysis demonstrated that the cAMP-induced increases in levels of G0 alpha were only of the more acidic isoform. The two isoforms were both substrates for pertussis toxin-catalysed ADP-ribosylation and did not appear to represent differentially phosphorylated forms of the same polypeptide. Separation of the two forms of G0 alpha could be achieved in one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis when 4 M deionized urea was included in the resolving gel. The more slowly migrating band was the acidic form and corresponded exactly in mobility with the major form of G0 from both rat and mouse brain. There was no equivalent in brain of the more rapidly migrating form of G0 from the cells. In agreement with the data from two-dimensional gels, only the more slowly migrating form was expressed in considerably higher amounts following cAMP-induced differentiation of NG108-15 cells. Of these two forms of "G0," the acidic species is equivalent to G0 from brain, but the basic form is not identical with G0*, which has been purified from bovine brain.     

Stimulation of Inositol Phosphate Production by Neurotensin in Neuroblastoma N1E115 Cells: Implication of GTP-Binding Proteins and Relationship with the Cyclic GMP Response

The association of neurotensin to its receptor in differentiated neuroblastoma N1E115 cells led to a fast and transitory increase of the intracellular concentration in inositol triphosphate and inositol biphosphate, followed by a slower and more stable increase inositol monophosphate. The action of inositol 1,4,5-triphosphate on digitonin-permeabilized N1E115 cells resulted in a stimulation of cyclic GMP levels that mimicked that induced by neurotensin. Therefore, the cyclic GMP stimulation is probably a consequence of the initial inositol triphosphate formation triggered by neurotensin. Fluoroaluminate ions and pertussis toxin had the capacity to modulate positively and negatively, respectively, the formation of inositol triphosphate induced by neurotensin, indicating that GTP-binding proteins are involved in the regulation of inositol phosphate levels by neurotensin receptors.     

Imaging metabolism of phosphatidylinositol 4,5-bisphosphate in T-cell GM1-enriched domains containing Ras proteins

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and Ras proteins are involved in signalling pathways originating at the plasma membrane. The localisation and metabolism of PI(4,5)P(2) was studied in Jurkat T cells using fluorescence microscopic imaging with EGFP-tagged and antibody probes. Software was developed to objectively quantitate colocalisation and was used to show that plasma membrane PI(4,5)P(2) was enriched in lipid raft-containing patches of GM1 ganglioside, formed by crosslinking cholera toxin B-subunit (CT-B). The PI(4,5)P(2) metabolites phosphatidylinositol 3,4,5-trisphosphate and diacylglycerol appeared in plasma membrane CT-B-GM1 patches upon induction of signalling. Transferrin receptor and the CD45 tyrosine phosphatase did not colocalise with CT-B-GM1 patches, whereas the tyrosine kinase Lck, the scaffolding protein LAT, and endogenous Ras proteins did partially colocalise with CT-B-GM1 patches as did transfected EGFP-K-Ras(4B) and EGFP-H-Ras. The results demonstrate that T-cell PI(4,5)P(2) metabolism is occurring in GM1-enriched domains and that Ras proteins are present in these domains in vivo.     

Evidence that the Modulation of Membrane-Associated Protein Kinase C Activity by an Endogenous Inhibitor Plays a Role in N1E-115 Murine Neuroblastoma Cell Differentiation

Abstract: Murine neuroblastoma cells, N1E-115, were induced to differentiate into neuron-like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro "direct" assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane-associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane-associated PKC activity in cells and thereby modulate PKC-related neuronal functions.