Interleukin-2 receptors and interleukin-2-mediated signaling in myelin: activation of diacylglycerol kinase and phosphatidylinositol 3-kinase.

Myelin was previously shown to possess neurotransmitter and cytokine receptors that trigger well-defined signaling mechanisms within the multilamellar structure. The present study reveals the presence of an interleukin-2 (IL-2) receptor in isolated mouse CNS myelin that responds to recombinant mouse IL-2 by activating diacylglycerol kinase (DAGK) and phosphoinositide 3-kinase (PI3K); additional evidence suggests participation by protein tyrosine kinase. Activation of myelin DAGK by IL-2 occurred in brain stem tissue mince and was blocked by chelerythrin chloride, indicating an essential role for myelin-localized protein kinase C. Two inhibitors of PI3K, wortmannin and LY294002, blocked endogenous PI3K as well as that enhanced by IL-2. Activation of PI3K by IL-2 was also blocked by tyrphostin A25, a selective inhibitor of PTK, suggesting activation of the latter by IL-2 is upstream to PI3K activation. This reaction resulted in tyrosine phosphorylation of a protein tentatively identified as the p85 subunit of PI3K. Developmental changes were noted in that receptor density and signaling activity were robust during the period of rapid myelination and declined rapidly thereafter.

Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase.

Despite its growing use as a radiological indicator of neuronal viability, the biological function of N-acetylaspartate (NAA) has remained elusive. This is due in part to its unusual metabolic compartmentalization wherein the synthetic enzyme occurs in neuronal mitochondria whereas the principal metabolizing enzyme, N-acetyl-L-aspartate amidohydrolase (aspartoacylase), is located primarily in white matter elements. This study demonstrates that within white matter, aspartoacylase is an integral component of the myelin sheath where it is ideally situated to produce acetyl groups for synthesis of myelin lipids. That it functions in this manner is suggested by the fact that myelin lipids of the rat optic system are well labeled following intraocular injection of [14C-acetyl]NAA. This is attributed to uptake of radiolabeled NAA by retinal ganglion cells followed by axonal transport and transaxonal transfer of NAA into myelin, a membrane previously shown to contain many lipid synthesizing enzymes. This study identifies a group of myelin lipids that are so labeled by neuronal [14C]NAA, and demonstrates a different labeling pattern from that produced by neuronal [14C]acetate. High performance liquid chromatographic analysis of the deproteinated soluble materials from the optic system following intraocular injection of [14C]NAA revealed only the latter substance and no radiolabeled acetate, suggesting little or no hydrolysis of NAA within mature neurons of the optic system. These results suggest a rationale for the unusual compartmentalization of NAA metabolism and point to NAA as a neuronal constituent that is essential for the formation and/or maintenance of myelin. The relevance of these findings to Canavan disease is discussed.

Mutant NG108-15 cells (NG-CR72) deficient in GM1 synthase respond aberrantly to axonogenic stimuli and are vulnerable to calcium-induced apoptosis: they are rescued with LIGA-20.

The neuroblastoma x glioma NG108-15 hybrid cell line, a widely used model for the study of neuronal differentiation, contains a variety of gangliosides including GM1 and its sialosylated derivative, GD1a. To investigate the role of these a-series gangliotetraose gangliosides in neuritogenesis, we have obtained a mutated subclone of NG108-15 that is deficient in that family of gangliosides. NG108-15 cells were grown in the presence of cholera toxin, which killed the large majority of cells, and from the cholera-resistant survivors we isolated a clone, NG-CR72, that lacks GM1 and GD1a in the plasma and nuclear membranes. GM2 concentration was significantly higher in the plasma membrane. Enzyme assay indicated deficiency of UDP-Gal:GM2 galactosyltransferase (GM1 synthase), which was confirmed by incorporation studies with [3H]sphingosine. These cells resembled wild-type NG108-15 in extending dendritic processes in response to dendritogenic agents (retinoic acid, dibutyryl cAMP) but responded aberrantly to axonogenic stimuli (KCl, ionomycin) by extending unstable neurites that showed the cytoskeletal staining characteristic of dendrites. Moreover, mutant cells treated with the Ca2+ elevating axonogenic agents underwent apoptosis over time, attributed to dysfunction of Ca2+ regulatory mechanisms normally mediated by GM1. Such agents caused dramatic and sustained elevation of intracellular Ca2+ in mutant cells, in contrast to modest and temporary elevation in wild-type cells. Exogenous GM1, inserted into the plasma membrane, had no discernable protective effect on NG-CR72 cells whereas LIGA-20, a membrane-permeant derivative of GM1 that entered both plasma and nuclear membranes, blocked apoptosis, permitted extension of stable neurites, and attenuated the abnormal elevation of intracellular Ca2+.

Cerebellar neurons lacking complex gangliosides degenerate in the presence of depolarizing levels of potassium.

Mice engineered to lack GM2/GD2 synthase (GalNAc-T), with resultant deficit of GM2, GD2, and all gangliotetraose gangliosides, were originally described as showing a relatively normal phenotype with only a slight reduction in nerve conduction. However, a subsequent study showed that similar animals suffer axonal degeneration, myelination defects, and impaired motor coordination. We have examined the behavior of cerebellar granule neurons from these neonatal knockouts in culture and have found evidence of impaired capacity for Ca2+ regulation. These cells showed relatively normal behavior when grown in the presence of physiological or moderately elevated K+ but gradually degenerated in the presence of high K+. This degeneration in depolarizing medium was accompanied by progressive elevation of intracellular calcium and onset of apoptosis, phenomena not observed with normal cells. No differences were detected in cells from normal vs. heterozygous mice. These findings suggest that neurons from GalNAc-T knockout mice are lacking a calcium regulatory mechanism that is modulated by one or more of the deleted gangliosides, and they support the hypothesis that maintenance of calcium homeostasis is one function of complex gangliosides during, and perhaps subsequent to, neuronal development.

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.

Axonogenesis in neuro-2a cells correlates with GM1 upregulation in the nuclear and plasma membranes.

GM1 ganglioside was previously shown to occur in the nuclear membrane, as well as the plasma membrane, of central nervous system (CNS) and peripheral nervous system (PNS) neurons undergoing morphological differentiation in culture. NG108-15 neuroblastoma cells showed the same phenomenon when induced to extend axon-like but not dendrite-like processes, although in both cases terminal differentiation was evidenced by failure of extended neurites to retract following washout of neuritogenic agent. The present study of Neuro-2a neuroblastoma cells subjected to similar treatments has revealed both similarities and differences compared to NG108-15 cells. Similar to the latter, Neuro-2a cells responded to neuraminidase and ionomycin with axon-like outgrowth together with upregulation of nuclear GM1, and to three other agents (retinoic acid, dibutyryl cyclic AMP, exogenous GM1) with dendrite-like outgrowth that was unaccompanied by nuclear GM1 increase. Although both cell types responded to low serum by extending neurites of mixed axonal-dendritic properties, Neuro-2a, in keeping with its greater tendency to extend some neurites of axonal character in low serum, showed elevated nuclear GM1 in a significant number of such differentiated cells. All three axonogenic agents induced parallel upregulation of GM1 in plasma-, nuclear-, and Golgi membranes, and these increases were stable to washout. Neurites generated in Neuro-2a cells by the three dendritogenic agents lacked stability, unlike those produced by the same agents in NG108-15 cells. This study also amplified the differences in response triggered by exogenous GM1 compared to that resulting from enzyme-mediated elevation of endogenous GM1.

Developmental appearance of nuclear GM1 in neurons of the central and peripheral nervous systems.

Previous studies demonstrated expression of GM1 ganglioside in the nuclear envelope of differentiating neuroblastoma cells and cultured cerebellar granule cells from neonatal rat brain. In the present study, relatively few of the latter cells were shown to possess a nucleus with appreciable GM1 during the first few days in culture, but increasing numbers of such cells possessed GM1-expressing nuclei as morphological differentiation progressed. This phenomenon reached a plateau by the 8th day in culture, approximately 90% of observed nuclei showing cytochemical evidence of GM1 at that time. Cerebral cortical neurons from embryonic rat brain in culture also gave clear evidence of GM1 in the nuclear membrane. Similar results were obtained with cultured neurons from the superior cervical ganglion from embryonic rats, demonstrating developmental appearance of GM1 in the nuclear envelope of PNS neurons. Cytochemical evidence for GM1 in purified nuclei from freshly isolated cortical neurons of neonatal rat brain indicated that expression of nuclear GM1 is not an artifact of cell culture. Study of NG108-15 neuroblastoma x glioma hybrid cells showed upregulation of nuclear GM1 to lag somewhat behind neurite outgrowth, suggesting nuclear GM1 to have a functional role subsequent to onset of morphological differentiation.

Upregulation of nuclear GM1 accompanies axon-like, but not dendrite-like, outgrowth in NG108-15 cells.

Recent work has demonstrated that induced neurite outgrowth in neuroblastoma cells and spontaneous differentiation of primary neurons in culture are accompanied by upregulation of GM1 ganglioside in the nuclear envelope. Previous reports have depicted morphological variations in the nature of stimulated neurites resulting from different neuritogenic agents, and a recent study by this laboratory demonstrated that such stimulants could be divided into two categories: those which induce axon-like neurites (group I) as opposed to those that stimulate dendrite-like outgrowths (group II). The former includes KCl, ionomycin, neuraminidase, and cholera toxin B subunit (all agents which elevate intracellular Ca2+), while the latter group is comprised of retinoic acid, dibutyryl cAMP, exogenous GM1, and low serum treatment. The present study was undertaken to determine whether differences in neuritic phenotype could be correlated with upregulation of nuclear GM1. The neuroblastoma x glioma NG108-15 cell line was employed because of its ability to respond robustly to a variety of neuritogenic stimuli. It was found that although both groups of stimulants are capable of inducing stable neurites (terminal differentiation) in this cell line, nuclear GM1 is elevated only in the presence of group I stimulants. Thus, a correlation is indicated between axonogenesis and upregulation of GM1 in the nuclear envelope. Additionally, these two events appear to coincide with elevation of intracellular Ca2+. Conversion of cells to the differentiated phenotype, with or without nuclear GM1 elevation, was found to depend in some cases on concentration of stimulant and duration of treatment.

The role of GM1 ganglioside in regulating excitatory opioid effects.

Our studies with cultured cells have provided new insight into the particular role of GM1 in regulating excitatory opioid responses. GM1 is significantly elevated in chronic opioid-treated cells via Gs/adenylyl cyclase activation. Such GM1 elevation promotes coupling of opioid receptor with Gs, resulting in attenuation of inhibitory opioid effects and induction of a sustained excitatory response. Application of exogenous GM1, but not other gangliosides, induces excitatory opioid responses not only in neurons and neuroblastoma cells that bear intrinsic opioid receptors but also in nonneuronal cells that are transfected with delta-opioid receptor. The latter system provides evidence that allosteric binding of GM1 changes receptor conformation from a Gi-coupled to a Gs-coupled mode. This is supported by preliminary experiments with a mutated delta-opioid receptor.

The role of GM1 and other gangliosides in neuronal differentiation. Overview and new finding.

The pronounced increases in gangliosides belonging to the gangliotetraose family during the neurite outgrowth phase of neuronal differentiation have suggested a functional requirement for these substances related to process extension, arborization, and possibly synaptogenesis. Support for this hypothesis has come from a variety of experimental paradigms utilizing neuroblastoma cell lines, primary neuronal cultures, and observations on the developing nervous system. We have recently observed that differentiation of both primary neurons and neuroblastoma cells by Ca(2+)-elevating stimulants is characterized by upregulation of GM1 in the nuclear membrane. Immunostaining revealed these Ca(2+)-induced neurites to have axonal characteristics. Recent work has indicated that nuclear GM1 facilitates efflux of nuclear Ca2+, thereby contributing to the reduced level of nuclear Ca2+ that characterizes the differentiated neuron. Thus, while GM1 is generally recognized as a pluripotent molecule with several modulatory roles in the plasma membrane of developing and mature neurons, regulation of Ca2+ flux across the nuclear membrane is proposed as another critical function of this ganglioside in neuronal development, with special relevance to axonogenesis.

Induction of axon-like and dendrite-like processes in neuroblastoma cells.

Neuroblastoma cells are widely utilized models for the study of the neuritic outgrowth phase of neuronal differentiation, but relatively few such studies have attempted to identify the nature of the process outgrowths. This identification will be necessary in developing strategies for utilizing these models to distinguish the underlying mechanisms involved in axonogenesis vs dendritogenesis. In an effort to identify procedures for inducing specific types of neurite outgrowth, and for distinguishing axon- from dendrite-like processes, we have subjected two neuroblastoma cell lines to a variety of stimuli previously shown to induce neurite outgrowth in these cells. These include neuraminidase, ionomycin, KCl+dibutyryl cAMP, cholera toxin B subunit, retinoic acid, dibutyryl cAMP (alone), GM1 ganglioside, and low serum. The first four of these (group 1) gave rise to neurites with axon-like characteristics, including immunostaining that was positive for phosphorylated high molecular weight neurofilament protein (NF-H) and synaptic vesicle protein-2 (SV2), but negative for microtubule-associated protein-2 (MAP2). The next three treatments (group 2) resulted in dendrite-like processes, as evidenced in immunostaining that was positive for MAP2 and negative for NF-H and SV2. Neurites produced by low serum had mixed properties. These cytoskeletal differences were supported by immunoblot analysis with antisera to the above cytoskeletal proteins. Striking morphological differences were also noted, group 2-induced neurites being significantly shorter with more branch points than those generated by group 1 stimulants. Time of exposure to stimulatory agent was crucial in determining expression of the neuritic phenotype. Correlation with previous studies suggests that axon-like neurites result from stimulants which elevate intracellular Ca2+, a dependence not previously reported to our knowledge. Dendrite-like process outgrowth, on the other hand, does not appear to depend on altered intracellular Ca2+.

Cytokines, signal transduction, and inflammatory demyelination: review and hypothesis.

The mechanism of focal demyelination in multiple sclerosis has been a long-standing enigma of this disorder. Cytokines, a diverse family of signalling molecules, are viewed as potential mediators of the process based on clinical observations and studies with animal models and tissue/cell culture systems. Myelin and oligodendrocyte (OL) destruction occur in cultured preparations subjected to cytokines such as tumor necrosis factor-alpha (TNF alpha) and lymphotoxin (LT). Many studies have shown these and other cytokines to be elevated at lesion sites and in the CSF of multiple sclerosis (MS) patients, with similar findings in animal models. Some variability in the nature of MS lesion formation has been reported, both OLs and myelin being primary targets. To account for myelin destruction in the presence of apparently functional OLs we hypothesize that cytokines such as TNF alpha and LT alpha contribute to myelin damage through triggering of specific reactions within the myelin sheath. We further propose that neutral sphingomyelinase (SMase) is one such enzyme, two forms of which have been detected in purified myelin. An additional event is accumulation of cholesterol ester, apparently a downstream consequence of cytokine-induced SMase. The resulting lipid changes are viewed as potentially destabilizing to myelin, which may render it more vulnerable to attack by invading and resident phagocytes.

Myelin contains neutral sphingomyelinase activity that is stimulated by tumor necrosis factor-alpha.

Purified myelin from mouse brain was found to contain two forms of neutral sphingomyelinase, one Mg2+ dependent and the other Mg2+ independent. The former had a pH optimum of 7.5 and Km of 0.35 mM, whereas the corresponding values for the latter were pH 8.0 and Km 3.03 mM. Specific activity of the Mg(2+)-dependent enzyme showed a rostral-caudal gradient, ranging from 75 nmol/mg protein/hr in myelin from cerebral hemispheres to 21 nmol/mg protein/hr in myelin from spinal cord. Relative specific activity was approximately 20% that of brain stem or cerebral hemisphere homogenate. Treatment of myelin with taurocholate or high salt concentration did not significantly reduce activity of the Mg(2+)-dependent enzyme. The activity of that enzyme did not change with time or in the presence or absence of protease inhibitors; by contrast, that of Mg(2+)-independent enzyme decreased sharply in the absence of protease inhibitors but rose in their presence. To test for the effect of tumor necrosis factor-alpha (TNF alpha) on myelin sphingomyelinase, mouse brain myelin was labeled in vivo by intracerebral injection of [3H]acetate into 18-20-day-old mice. After 40 hr, brain stems were removed, minced, and treated with TNF alpha in Krebs-Ringer solution, after which myelin was immediately isolated. Separation and counting of individual lipids revealed TNF alpha treatment to cause increased labeling of myelin ceramide and cholesterol ester with concomitant decrease in myelin sphingomyelin. Western blotting of myelin proteins using antibodies to the two TNF alpha receptors as probes revealed the presence of the p75 receptor. Implications of these findings in relation to possible mechanisms of autoimmune demyelination are discussed.

Opioid receptor and calcium channel regulation of adenylyl cyclase, modulated by GM1, in NG108-15 cells: competitive interactions.

GM1 ganglioside was previously shown to function as a specific regulator of excitatory opioid activity in dorsal root ganglion neurons and F11 hybrid cells, as seen in its facilitation of opioid-induced activation of adenylyl cyclase and its ability to dramatically reduce the threshold opioid concentration required to prolong the action potential duration. The elevated levels of GM1 resulting from chronic opioid exposure of F11 cells were postulated to cause the ensuing opioid excitatory supersensitivity. We now show that GM1 promotes opioid (DADLE)-induced activation of adenylyl cyclase in NG108-15 cells which possess the delta-type of receptor. In keeping with previous studies of other systems, this can be envisioned as conformational interaction of GM1 with the receptor that results in uncoupling of the receptor from Gi and facilitated coupling to Gs. This would also account for the observation that DADLE-induced attenuation of forskolin-stimulated adenylyl cyclase was reversed by GM1, provided the cells were not pretreated with pertussis toxin. When the cells were so pretreated, GM1 evoked an unexpected attenuation of forskolin-stimulated adenylyl cyclase attributed to GM1-promoted influx of calcium which was postulated to inhibit a calcium-sensitive form of adenylyl cyclase. This is concordant with several studies showing GM1 to be a potent modulator of calcium flux. Pertussis toxin in these experiments exerted dual effects, one being to promote interaction of the delta-opioid receptor with Gs through inactivation of Gi, and the other to enhance the GM1-promoted influx of calcium by inactivation of Go; the latter is postulated to function as constitutive inhibitor of the relevant calcium channel. NG108-15 cells thus provide an interesting example of competitive interaction between two GM1-regulated systems involving enhancement of both opioid receptor excitatory activity and calcium influx.

Interaction of the delta-opioid receptor with GM1 ganglioside: conversion from inhibitory to excitatory mode.

Previous studies have shown GM1 ganglioside to play a crucial role in regulating excitatory opioid receptor function, which may underlie some aspects of opioid dependence, tolerance, and supersensitivity. To study the mechanism of this receptor modulation we have employed CHO cells containing a single, transfected opioid receptor of the delta-type. When forskolin was employed to elevate cAMP the reduction affected by 10 microM DADLE was counteracted by preincubation of the cells with GM1. No effect was observed with GD1a, GD1b, GT1b GM3, or the GM1 derivative, GM1-OH. In pertussis toxin-treated cells 10 nM DADLE increased basal levels of cAMP after preincubation with as little as 10 nM GM1. The results suggest conformational alteration of the opioid receptor from a form coupled primarily to G(i)/G(o) to one also capable of interacting with G(s).

Trophic effect of cholera toxin B subunit in cultured cerebellar granule neurons: modulation of intracellular calcium by GM1 ganglioside.

Survival of cerebellar granule cells (CGC) in culture was significantly improved in the presence of cholera toxin B subunit (Ctx B), a ligand which binds to GM1 with specificity and high affinity. This trophic effect was linked to elevation of intracellular calcium ([Ca2+]i), and was additive to that of high K+. Survival was optimized when Ctx B was present for several days during the early culture period. 45Ca2+ and cell survival studies indicated the mechanism to involve enhanced influx of Ca2+ through L-type voltage-sensitive channels, since the trophic effect was blocked by antagonists specific for that channel type. Inhibitors of N-methyl-D-aspartate receptor/channels were without effect. During the early stage of culture Ctx B, together with 25 mM K+, caused [Ca2+]i to rise to 0.2-0.7 microM in a higher proportion of cells than 25 mM K+ alone. A significant change in the nature of GM1 modulation of Ca2+ flux occurred after 7 days in culture, at which time Ctx B ceased to elevate and instead reduced [Ca2+]i below the level attained with 25 mM K+. GM1 thus appears to serve as intrinsic inhibitor of one or more L-type Ca2+ channels during the first 7 days in vitro, and then as intrinsic activator of (possibly other) L-type channels after that period. This is the first demonstration of a modulatory role for GM1 ganglioside affecting Ca2+ homeostasis in cultured neurons of the CNS.

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 mu 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.

Characterization of guanylyl cyclase in purified myelin.

This study was undertaken to characterize the enzymatic properties of the particulate guanylyl cyclase previously shown to be present at a high level of activity in purified rat brain myelin. Significant activation was achieved by both Lubrol-PX and Triton X-100, the latter being somewhat more effective. A pH optimum of 7.8 was observed, compared to 7.4 for microsomes. Employing 1.2 mM GTP with 1% Triton X-100, linearity of response was observed up to 60 min and approximately 1.2 mg of myelin protein. Kinetic analysis revealed Km values of 0.258mM and 0.486mM for myelin and microsomes, respectively, similar values being obtained by Lineweaver-Burke analysis or Direct Linear Plot. Vmax values were 20 and 266 pmol/mg protein/min for myelin and microsomes, respectively. Washing of the myelin with 0.5 M NaCl or 0.1% Na taurocholate did not remove a significant amount of guanylyl cyclase activity, indicating the enzyme to be intrinsic to the myelin sheath.

Chronic opioid treatment of neuroblastoma x dorsal root ganglion neuron hybrid F11 cells results in elevated GM1 ganglioside and cyclic adenosine monophosphate levels and onset of naloxone-evoked decreases in membrane K+ currents.

Prolongation of the action potential duration of dorsal root ganglion (DRG) neurons by low (nM) concentrations of opioids occurs through activation of excitatory opioid receptors that are positively coupled via Gs regulatory protein to adenylate cyclase. Previous results suggested GM1 ganglioside to have an essential role in regulating this excitatory response, but not the inhibitory (APD-shortening) response to higher (microM) opioid concentrations. Furthermore, it was proposed that synthesis of GM1 is upregulated by prolonged activation of excitatory opioid receptor functions. To explore this possibility we have utilized cultures of hybrid F11 cells to carry out closely correlated electrophysiological and biochemical analyses of the effects of chronic opioid treatment on a homogeneous population of clonal cells which express many functions characteristic of DRG neurons. We show that chronic opioid exposure of F11 cells does, in fact, result in elevated levels of GM1 as well as cyclic adenosine monophosphate (AMP), concomitant with the onset of opioid excitatory supersensitivity as manifested by naloxone-evoked decreases in voltage-dependent membrane K+ currents. Such elevation of GM1 would be expected to enhance the efficacy of excitatory opioid receptor activation of the Gs/adenylate cyclase/cyclic AMP system, thereby providing a positive feedback mechanism that may account for the remarkable supersensitivity of chronic opioid-treated neurons to the excitatory effects of opioid agonists as well as antagonists. These in vitro findings may provide novel insights into the mechanisms underlying naloxone-precipitated withdrawal syndromes and opioid-induced hyperalgesia after chronic opiate addiction in vivo.

GM1 ganglioside in the nuclear membrane modulates nuclear calcium homeostasis during neurite outgrowth.

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 microM 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.