Glioma Hybrid NG108-15 Research Articles

Enhanced calcium signalling events in neuroblastoma × glioma hybrid NG108-15 cells after treatment with dibutyryl cyclic AMP

The effects of dibutyryl cyclic AMP (dbcAMP) treatment on Ca2+ channel activities, Ca2+ accumulation by intracellular Ca2+ pools, and sizes of IP3- and GTP-releasable pools in neuroblastoma × glioma hybrid NG108-15 cells were studied. High extracellular K+ induced a greater rise in intracellular calcium concentration ([Ca2+]i) in dbcAMP-treated cells than in control cells. In dbcAMP-treated cells, the initial phase of the high K+-induced [Ca2+]i rise displayed a much higher sensitivity to ω-conotoxin than it did in control cells, whereas the plateau phase of the [Ca2+]i rise was sensitive only to nifedipine. These results indicate that predominantly L-type Ca2+ channels exist in control cells, and that N-type channels develop only after dbcAMP treatment. In dbcAMP-treated cells, mitochondria showed an increased Ca2+ uptake capacity (5.3 nmol Ca2+/mg protein) compared with that in control cells (4.2 nmol Ca2+/mg protein). However, dbcAMP treatment did not cause significant change in the affinity for Ca2+. Dibutyryl cAMP treatment enhanced the Ca2+ accumulation activity by nonmitochondrial pools (from 0.84 to 0.97 nmol Ca2+/mg protein) and increased the affinity for Ca2+ (EC50 for Ca2+ decreased from 0.146 μM to 0.063 μM). Our data also indicate that the pool that is sensitive to both IP3 and GTP was enlarged. The affinities for IP3 and GTP in causing Ca2+ release remained the same before or after dbcAMP treatment.

Agonist regulation of cellular Gs alpha-subunit levels in neuroblastoma x glioma hybrid NG108-15 cells transfected to express different levels of the human beta 2 adrenoceptor.

Neuroblastoma x glioma hybrid NG108-15 cells endogenously express at least three receptors which activate adenylate cyclase via the intermediacy of the stimulatory G-protein, Gs. Sustained exposure of the cells to agonists at the IP prostanoid receptor results in a substantial decrease in cellular levels of the alpha-subunit of Gs (Gs alpha) [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093; Adie, Mullaney, McKenzie and Milligan (1992) Biochem J. 285, 529-536]. By contrast, equivalent treatments of the cells with agonists at either the A2 adenosine receptor or the secretin receptor have no measurable effect on cellular amounts of Gs alpha. To examine whether this is a feature specific to the IP prostanoid receptor or is related to the level of expression of the individual receptors, NG108-15 cells were transfected with a construct containing a human beta 2-adrenoceptor cDNA under the control of the beta-actin promoter. Two clones of these cells were examined in detail, beta N22, which expressed some 4000 fmol/mg of membrane protein, and clone beta N17, which expressed approx. 300 fmol/mg of membrane protein of the receptor. Exposure of beta N22 cells to the beta-adrenergic agonist isoprenaline resulted maximally in some 55% decrease in membrane-associated levels of Gs alpha, without effect on membrane levels of Gi2 alpha, Gi3 alpha, G(o) alpha or Gq alpha/G11 alpha. Dose-response curves to isoprenaline in beta N22 cells indicated that half-maximal down-regulation of Gs alpha was produced by approx. 1 nM agonist. Equivalent exposure of beta N17 cells to isoprenaline did not significantly modify levels of any of the G-protein alpha subunits, including Gs alpha. In beta N22 cells the IP prostanoid receptor was expressed at similar levels to those in wild-type NG108-15 cells, and treatment with iloprost resulted in a similar down-regulation of cellular Gs alpha levels. Iloprost was also effective in causing down-regulation of Gs alpha levels in clone beta N17. Concurrent addition of both isoprenaline and iloprost to clone beta N22 resulted in less than additive down-regulation of Gs alpha. These results demonstrate that the phenomenon of agonist-induced specific G-protein down-regulation is determined by the levels of expression of the receptor.

Calcium signaling induced by bradykinin is synergistically enhanced by high K+ in NG108-15 cells

We report a novel phenomenon in which the cytosolic Ca2+ concentration ([Ca2+]i) rise induced in neuroblastoma x glioma hybrid NG108-15 cells by bradykinin is synergistically enhanced by elevated extracellular K+ concentrations. Presence of extracellular Ca2+ during high-K+ treatment, but not after high-K+ treatment, was required for the synergism. In addition, when thapsigargin was added concurrently with high K+, bradykinin still induced a significantly higher [Ca2+]i rise than in cells treated with thapsigargin only. Both bradykinin-induced inositol 1,4,5-trisphosphate (IP3) generation and the size of the internal Ca2+ pool were increased by high-K+ treatment. Our data suggest that changes in membrane potential itself induced by high K+ probably do not cause the synergistic effect. The synergistic effect is apparently due to the stimulatory effects of high K+ on [Ca2+]i, which in turn modulates IP3 generation and increases the size of intracellular Ca2+ pools. If bradykinin is added following high K+, the synergism can be accounted for by increases both in IP3 production and in the size of the internal Ca2+ pools. If bradykinin is added simultaneously with high K+, enhanced Ca2+ release triggered by enhanced IP3 production is the major cause of the synergistic effects.

Presence of [formula omitted] (NMDA) receptors in neuroblastoma × glioma hybrid NG108-15 cells-analysis using [ 45Ca 2+]influx and [ 3H]MK-801 binding as functional measures

In the present study, we have attempted to clarify whether neuroblastoma glioma hybrid NG 108-15 cells (NG cells) possess the NMDA receptor complex using [ 45Ca 2+]influx and [ 3H]MK-801 binding as functional measures. Glutamate and NMDA dose-dependently increased [ 45Ca 2+]influx and these increases were further enhanced by glycine. Scatchard analysis revealed the presence of a high-affinity binding site for [ 3H]MK-801 with a K D of 18.8 nM and a B max of 0.328 pmol/mg protein. This [ 3H]MK-801 binding was also increased by NMDA in a dose-dependent manner and this increase was further enhanced by glycine. Both ketamine and MK-801 inhibited glutamate- and NMDA-induced [ 45Ca 2+]influx as well as the increase of [ 3H]MK-801 binding in a dose-dependent manner. Similarly, Mg 2+ and Zn 2+ dose-dependently reduced both glutamate-induced [ 45Ca 2+]influx and [ 3H]MK-801 binding. Spermine, one of the polyamines, showed a biphasic stimulatory effects on glutamate-induced [ 45Ca 2+]influx and [ 3H]MK-801 binding. These results indicate that NG cells possess a pharmacologically distinct NMDA receptor complex and suggest that these cells may be useful for the analyses on pharmacological and biochemical characteristics of the NMDA receptor complex.

Ethanol inhibits neural cell-cell adhesion.

Gestational exposure to ethanol causes defects in neuronal migration, fasciculation, and synaptogenesis, developmental events that depend on the patterned expression and function of cell adhesion molecules (CAMs). Recombinant human osteogenic protein-1 (hOP-1) increases cell-cell adhesion and promotes cell clustering in proliferating neuroblastoma x glioma hybrid NG108-15 cells by strongly inducing N-CAM and L1. Here we show that concentrations of ethanol achieved during social drinking inhibit hOP-1-induced cell clustering without affecting cell proliferation, the induction and cell surface expression of N-CAM and L1, or the alternative splicing and sialylation of N-CAM. This inhibition was reproduced by other alcohols in proportion to their chain length, but not by teratogenic anticonvulsants or phenylalanine. Ethanol inhibition of hOP-1 morphogenesis was inversely proportional to the concentration of hOP-1 and, hence, to the levels of N-CAM and L1. Low concentrations of ethanol (IC50 5-10 mM) inhibited cell-cell adhesion in hOP-1-treated cells, and this action too was reproduced more potently by propanol and butanol. Ethanol may perturb brain and skeletal development by inhibiting CAM-mediated cell-cell interactions.

Contribution of a H+ pump in determining the resting potential of neuroblastoma cells.

The aim of this work was to examine the effects of changes in external K+ concentration (Ko) around its physiological value, of various K+ channels blockers, including internal Cs+, of vacuolar H(+)-ATPase inhibitors and of the protonophore CCCP on the resting potential and the voltage-dependent K+ current of differentiated neuroblastoma x glioma hybrid NG108-15 cells using the whole-cell patch-clamp technique. The results are as follows: (i) under standard conditions (Ko = 5 mM) the membrane potential was -60 +/- 1 mV. It was unchanged when Ko was decreased to 1 mM and was depolarized by 4 +/- 1 mV when Ko was increased to 10 mM. (ii) Internal Cs+ depolarized the membrane by 21 +/- 3 mV. (iii) The internal application of the vacuolar H(+)-ATPase inhibitors N-ethylmaleimide (NEM), NO3- and bafilomycin A1 (BFA) depolarized the membrane by 15 +/- 2, 18 +/- 2 and 16 +/- 2 mV, respectively. (iv) When NEM or BFA were added to the internal medium containing Cs+, the membrane was depolarized by 45 +/- 1 and 42 +/- 2 mV, respectively. (v) The external application of CCCP induced a transient depolarization followed by a prolonged hyperpolarization. This hyperpolarization was absent in BFA-treated cells. The voltage-dependent K+ current was increased at negative voltages and decreased at positive voltages by NEM, BFA and CCCP. Taken together, these results suggest that under physiological conditions, the resting potential of NG108-15 neuroblastoma cells is maintained at negative values by both voltage-dependent K+ channels and an electrogenic vacuolar type H(+)-ATPase.

Regulation of neural cell adhesion molecule and L1 by the transforming growth factor-beta superfamily. Selective effects of the bone morphogenetic proteins.

The transforming growth factor-beta (TGF-beta) superfamily plays a role in embryogenesis and regeneration. We have reported that osteogenic protein-1 (OP-1) promotes cell aggregation and induces the expression of the neural cell adhesion molecules N-CAM and L1 in proliferating neuroblastoma x glioma hybrid NG108-15 cells (Perides, G., Safran, R. M., Rueger, D. C., and Charness, M. E. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10326-10330; Perides, G., Hu, G., Rueger, D. C., and Charness, M. E. (1993) J. Biol. Chem. 268, 25197-25205). Here we show that the structurally homologous bone morphogenetic proteins (BMP) BMP-2 and BMP-4 are 10-50-fold more potent in these actions than the subfamily comprising BMP-5, BMP-6, and OP-1 (BMP-7). In contrast, members of the TGF-beta subfamily, activin-A, inhibin-A, and 29 additional growth factors and cytokines did not induce N-CAM. The addition of serum to cells growing in serum-free medium caused a concentration-dependent increase in N-CAM and L1 expression; however, serum did not potentiate the induction of N-CAM and L1 by 40 ng/ml OP-1. These findings suggest the presence in NG108-15 cells of a BMP-2/BMP-4 receptor that discriminates subtle differences in structure among homologous members of the TGF-beta superfamily. An endogenous ligand for this receptor may be present in serum.

Effects of Ca Channel Agonists on 45Ca Uptake Differ Depending on the State of NG108-15 Cells

We investigated the effects of various Ca channel agonists (Ca agonist) derived from 1,4-dihydropyridine on KCl-stimulated 45Ca uptake by differentiated and undifferentiated neuroblastoma x glioma hybrid NG108-15 cells with and without dibutyryl cAMP. Ca agonists Bay K 8644, YC-170 and CGP 28392 enhanced KCl-stimulated 45Ca uptake in differentiated NG108-15 cells, but only slightly in undifferentiated NG108-15 cells. The rank order of the enhancing effects was roughly Bay K 8644 > YC-170 > > CGP 28392. These results suggest that there is some difference between the mechanism by which these Ca agonists affect KCl-stimulated 45Ca uptake in differentiated and undifferentiated NG108-15 cells, although the nature of that difference is not clear.

Distribution and relative levels of expression of the phosphoinositidase-C-linked G-proteins G qα and G 11α: Absence of G 11α in human platelets and haemopoietically derived cell lines

Membranes of a variety of clonal cell lines, including neuroblastoma× glioma hybrid NG108-15, glioma C6, Rat 1 and CHO fibroblasts and the pituitary-derived cell lines αT3 and GH3 were immunoblotted with an antiserum (CQ) raised against a synthetic peptide corresponding to the C-terminal decapeptide of the α subunits of the phosphoinositidase-C-linked G-proteins G q and G 11. In SDS-PAGE conditions able to resolve these two polypeptides, direct evidence was obtained for co-expression of these two G-proteins in all of the above cell lines. The ratio of these two G-proteins varied substantially ( α 11 α q = 0.25–2.5 ) between the cell lines. In human platelets and in a range of haemopoietically derived human cell lines including U937 (monoblasts), Raji (Burkitts lymphoma) and Jurkat (mature T cell) expression of G 11α was not detected. This was not due to the inability of the antiserum to identify human G 11α as other human cell lines co-expressed both G-proteins. A third, unidentified CQ reactive polypeptide of similar mobility was resolved and present in all cell lines examined.

Osteogenic protein-1 regulates L1 and neural cell adhesion molecule gene expression in neural cells.

Osteogenic protein-1 (OP-1) is a member of the TGF-beta superfamily that is expressed in the nervous system. We recently showed that human recombinant osteogenic protein-1 (hOP-1) strongly promotes the aggregation of dividing neuroblastoma x glioma hybrid NG108-15 cells, in part by inducing the major isoforms of the neural cell adhesion molecule (N-CAM) (Perides, G., Safran, R. M., Rueger, D. C., and Charness, M. E. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10326-10330). Here we show that hOP-1 induces L1 expression approximately 6-fold in NG108-15 cells without changing the levels of N-cadherin, neurofilament 200, Thy-1, tau, and G alpha s. OP-1 induction of L1 and N-CAM was unassociated with changes in cell proliferation and was not reproduced by cellular differentiation. The increased adhesiveness of hOP-1-treated NG108-15 cells could be inhibited in part by Fab fragments of an anti-L1 polyclonal antiserum. L1 and N-CAM expression first increased 12-18 h after hOP-1 treatment, reached a maximum after 2-3 days, persisted for up to 5 days, and returned to control levels 3 days after hOP-1 withdrawal. The increases in L1 and N-CAM protein levels were preceded or accompanied by large increases in the abundance of L1 and all detectable N-CAM mRNAs. Actinomycin D prevented the induction by hOP-1 of L1 and N-CAM mRNAs, suggesting that hOP-1 regulates immunoglobulin CAM gene transcription. OP-1 is the first described growth factor that regulates both N-CAM and L1 gene expression.

Extracellular ATP Stimulates Calcium Influx in Neuroblastoma Glioma Hybrid NG108–15 Cells

ATP-induced changes in the intracellular Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108-15 cells were studied. Using the fluorescent Ca2+ indicator fura-2, we have shown that the [Ca2+]i increased in response to ATP. ATP at 3 mM caused the greatest increased in [Ca2+]i, whereas at higher concentrations of ATP the response became smaller. Two nonhydrolyzable ATP analogues, adenosine 5'-thiotriphosphate and 5'-adenylyl-beta, gamma-imidodiphosphate, could not trigger significant [Ca2+]i change, but they could block the ATP effect. Other adenine nucleotides, including ADP, AMP, alpha beta-methylene-ATP, beta, gamma-methylene-ATP, and 2-methylthio-ATP, as well as UTP and adenosine, all had no effect on [Ca2+]i at 3 mM. In the absence of extracellular Ca2+, the effect of ATP was inhibited totally, but could be restored by the addition of Ca2+ to the cells. Upon removal of Mg2+, the maximum increase in [Ca2+]i induced by ATP was enhanced by about 42%. Ca(2+)-channel blockers partially inhibited the ATP-induced [Ca2+]i rise. The ATP-induced [Ca2+]i rise was not affected by thapsigargin pretreatment, though such pretreatment blocked bradykinin-induced [Ca2+]i rise completely. No heterologous desensitization of [Ca2+]i rise was observed between ATP and bradykinin. The magnitude of the [Ca2+]i rise induced by ATP increased between 1.5 and 3.1 times when external Na+ was replaced with Tris, N-methyl-D-glucamine, choline, or Li+. The addition of EGTA or verapamil to cells after their maximum response to ATP immediately lowered the [Ca2+]i to the basal level in Na(+)-containing or Na(+)-free Tris solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and reconstitution of the δ opioid receptor

The δ opioid receptor has been purified, in an active form, by succinylmorphine affinity chromatography. The receptor was purified partially from bovine frontal cortex and to apparent homogeneity from neuroblastoma × glioma hybrid NG108-15 cells as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. Antiserum to the purified bovine receptor inhibited ligand binding to membranes and immunoprecipitated a 58 kDa protein from NG108-15 cells. Reconstitution of the receptor with lipids enhanced binding by 9-fold. The 58 kDa band protein after electroelution and reconstitution with lipids also showed specific binding, indicating that the receptor could be renatured even after SDS-PAGE in an appropriate lipid environment.

Specific bindings of [3H](+)PN200-110 and [125I]omega-conotoxin to crude membranes from differentiated NG108-15 cells.

The characteristics of the specific bindings of [3H](+)PN200-110 (PN: L-type Ca channel antagonist) and [125I]omega-conotoxin G VI A (omega-CgTX: neuronal L- or N-type Ca channel antagonist) to crude membranes from undifferentiated neuroblastoma X glioma hybrid NG108-15 (NG108-15) cells and differentiated cells induced with dibutyryl cAMP (Bt2cAMP) were examined, because we have already observed that the magnitude and rate of KCl-stimulated 45Ca uptake by NG108-15 cells increased progressively during differentiation of the cells induced with Bt2-cAMP (unpublished results). The specific binding of [3H](+)PN to these crude membranes was saturable at various concentrations of 2.5-5.0 nM [3H](+)PN. Scatchard analysis showed that the specific binding of [3H](+)PN at equilibrium was significantly increased after differentiation of the NG108-15 cells with Bt2cAMP, but that the apparent Kd value for the specific binding of [3H](+)PN was not influenced by treatment with Bt2cAMP. The specific binding of [3H](+)PN to crude membranes from Bt2cAMP-treated NG108-15 cells was inhibited by a calcium agonist and antagonists, the order of their inhibitory potencies being (+)PN > nitrendipine > (-)PN > or = Bay K 8644 > > diltiazem = verapamil. Thus, PNs showed significant stereoselective inhibition of the specific binding of [3H](+)PN. On the other hand, [125I]omega-CgTX at concentrations of 0.075-0.6 nM showed scarcely any specific binding to these crude membranes, although at 0.6 nM it showed specific binding to crude membranes from rat brain in the same experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of 45Ca uptake stimulated by high KCl of differentiated and undifferentiated NG108-15 and PC12h cells.

The characteristics of KCl-stimulated 45Ca uptake by neuroblastoma X glioma hybrid NG108-15 cells induced to differentiate with dibutyryl cAMP (Bt2cAMP) and of PC12h pheochromocytoma cells induced to differentiate with nerve growth factor (NGF) were studied. The extent and rate of KCl-stimulated 45Ca uptake by differentiated NG108-15 cells induced with Bt2cAMP were significantly higher than those of the undifferentiated cells. However, differentiation of PC12h cells induced with NGF did not enhance their extent or rate of KCl-stimulated 45Ca uptake. The effects of Ca agonist and antagonists indicated that the characteristics of KCl-stimulated 45Ca uptake by Bt2cAMP-treated NG108-15 cells and NGF-treated PC12h cells mainly reflected those of peripheral L-type voltage-sensitive calcium channels activated by high KCl. These results suggest that differentiated neural cells did not all show an enhanced capacity for KCl-stimulated 45Ca uptake, although the characteristic patterns of differentiation (extension of neurite-like processes, etc.) and that of effect by Ca agonist or antagonists on NG108-15 cells and PC12h cells were similar.

Refilling the inositol 1,4,5-trisphosphate-sensitive Ca2+ store in neuroblastoma x glioma hybrid NG108-15 cells

Bradykinin-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) were recorded in single NG108-15 cells with indo-1-based dual-emission microfluorimetry (50% effective concentration, 16 nM). A 1-min exposure to 30 nM bradykinin completely depleted the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store; refilling the store required extracellular Ca2+ (half time, 2 min). Refilling the IP3-sensitive store was completely blocked by 1 microM La3+ and 10 microM nitrendipine, but not 10 microM verapamil, 10 microM flunarizine, 1 microM nitrendipine, or 0.1 microM La3+. Thapsigargin irreversibly depleted the Ca2+ store and prevented its refilling (half-maximal inhibitory concentration, 3 nM). Influx of Ca2+ across the plasma membrane did not increase after depletion of the IP3-sensitive store by exposure to bradykinin, although maintained presence of the agonist produced significant Ca2+ influx. Similarly, Mn2+ and Ba2+ influx, as measured by indo-1 quenching and spectral shifts, did not increase following depletion of IP3-sensitive store. In contrast to depletion of the IP3-sensitive Ca2+ store by bradykinin, thapsigargin (10 nM) treatment produced Ca2+ and Ba2+ influx. We conclude that after Ca2+ mobilization, the IP3-sensitive Ca2+ store in NG108-15 cells is refilled with cytoplasmic Ca2+ via a thapsigargin-sensitive Ca(2+)-Mg(2+)-ATPase. Cytoplasmic Ca2+ is replenished by a persistent leak of Ca2+ across the plasma membrane. This leak is not modulated by the status of the intracellular Ca2+ store. In NG108-15 cells, agonist and thapsigargin-evoked Ca2+ entry are mediated by activation of plasmalemmal Ca2+ channels independent of the status of the IP3-sensitive intracellular Ca2+ store.

Additive induction of Egr-1 (zif/268) mRNA expression in neuroblastoma x glioma hybrid NG108-15 cells via cholinergic muscarinic, alpha 2-adrenergic, and bradykinin receptors.

Administration of carbachol, noradrenaline, and bradykinin induced Egr-1 mRNA expression within 1 h in mouse neuroblastoma x rat glioma hybrid NG108-15 cells. With specific receptor antagonists, the Egr-1 inductions by carbachol and noradrenaline were shown to be mediated via cholinergic muscarinic and alpha 2-adrenergic receptors, respectively. At their saturation levels for Egr-1 induction, the two agonists had additive effects when added together, but no prolongation of the effect on Egr-1 induction was observed. Addition of carbachol or noradrenaline 6 h after primary stimulation with carbachol or noradrenaline did not result in secondary Egr-1 induction, probably because of receptor desensitization. On the other hand, bradykinin consistently had an additive effect on Egr-1 induction, irrespective of the time of its addition, suggesting that the signal pathways for Egr-1 induction by carbachol or noradrenaline and by bradykinin are different. Treatment of cells with pertussis toxin or cholera toxin strongly inhibited Egr-1 induction by carbachol or noradrenaline but only partially inhibited the induction by bradykinin. Thus, the signals transduced in NG108-15 cells by different neurotransmitter receptors appear to have different effects on Egr-1 induction, depending on the times of stimulation and the combinations of receptors stimulated.

Developmental transition by spinal cord plasma membranes of embryonic chick from permissive to restrictive substrates for the morphological differentiation of neuroblastoma × glioma hybrid NG108-15 cell

Recent studies of spinal cord development and plasticity, in chick, have demonstrated a loss of regenerative ability correlating to embryonic day (E) 13 of the 21-day developmental period. Here we describe membrane fractions from embryonic chick spinal cords as permissive or restrictive substrates for the neuron-like differentiation of neuroblastoma × glioma hybrid NG108-15 cells, in vitro. Plasma membranes were purified from the thoracic spinal cord of embryos at a series of developmental stages (E10–E18). Micro-well plates were coated with the fractions and NG108-15 cells cultured thereon. Cells adhered to the E10-coated wells and began to differentiate after 2 h, becoming highly differentiated, with neurites 2–3 times longer than the diameter of the cell body after 24 h in culture. In contrast, cells cultured in E18-coated wells remained as clusters of undifferentiated cells of rounded morphology, even after 48 h in culture. As well, the permissive and restrictive plasma membranes were assessed semiquantitatively as the number of adhering cells after 20 h of culture. Adhesion of cells to the substrate decreased as the embryonic age of the plasma membrane substrate increased. Examination of the plasma membrane fractions, using SDS-PAGE, revealed several proteins in the 40–60 kDa range that varied substantially between E12, E14 and E18. Results of this study provide in vitro confirmation of previous in vivo findings; namely, that early embryonic spinal cord is initially permissive for neuritic outgrowth becoming restrictive around E13. As well, this study provides the foundation for several possible avenues of research to identify the proteins involved in the transition from permissive to restrictive environments for spinal cord repair in the embryonic chick.

Chapter 35: Postsynaptic actions of acetylcholine: the coupling of muscarinic receptor subtypes to neuronal ion channels

In recent years, the application of molecular biological techniques has established that there are at least five subtypes of muscarinic receptor, each with a unique pattern of distribution. Radio-ligand binding displacement studies of antagonist pharmacology of the individual receptors expressed in appropriate host cells have shown that each receptor does have a discrete antagonist profile, but that there are no single antagonists with sufficient selectivity to be clearly diagnostic for any particular receptor type. This has hampered the elucidation of the roles of muscarinic receptor subtypes, particularly, in cells which may simultaneously express more than one subtype, as is probably the case for neurones. The use of cell lines transfected with a gene encoding a single muscarinic receptor subtype has extended the characterization of muscarinic receptor subtypes beyond the pharmacology of the recognition site and has generated an additional classification on the basis of the downstream cellular mechanisms to which each subtype preferentially couples. This approach can clearly provide indications of likely coupling mechanisms of subtypes in, for example, the CNS neurones. Initial biochemical experiments with cell lines, such as the neuroblastoma x glioma hybrid NG108-15 have established two major groupings of muscarinic receptor subtypes, with ml, m3, and m5 receptors coupling preferentially to stimulation of phospholipase C (PLC) through a Pertussis toxin-insensitive G-protein, with subsequent elevation of IP3, while m2 and m4 receptors couple through a Pertussis toxin-sensitive G-protein to preferentially inhibit adenylyl cyclase.This chapter describes the electrophysiological consequences of muscarinic receptor activation using chemically differentiated NG108-15 cells transfected with ml-m4 receptor genes. These cells are especially useful for electrophysiological studies, because they express a range of K + and Ca 2+ channels similar to those found in many neuronal cell types. However, NGl08-15 cells do natively express the m4 muscarinic receptor.

Enhanced acetylcholine secretion in neuroblastoma x glioma hybrid NG108-15 cells transfected with rat choline acetyltransferase cDNA

Neuroblastoma x glioma hybrid NG108-15 cells and mouse neuroblastoma N18TG-2 and NIE-115 celIs were transiently transfected with the sense cDNA coding for rat choline acetyltransferase (ChAT). All transfected cell lines showed a high level of ChAT activity. ACh secretion was monitored by recording miniature end-plate potentials (MEPPs) in striated muscle cells that had been co-cultured with transfected cells. The number of muscle cells with synaptic responses and the MEPP frequency were higher in co-culture with transfected NG108-15 cells than with control or mock cells. No tsynaptic response was detected in muscle cells co-cultured with transfected N18TG-2 or N1E-115 cells. The results show that ACh secretion into the synaptic cleft was enhanced due to ChAT overexpression in NG108-15 hybrid cells but not in neuroblastoma cells.

Activation of Ca 2+/calmodulin-dependent protein kinase II by stimulation with bradykinin in neuroblastoma x glioma hybrid NG108-15 cells

To elucidate the mechanisms of the initracellular signal transduction elicited with bradykinin in NG108-15 neuroblastoma x glioma hybrid cells, we examined the activation of Ca 2+/calmodulin-dependent protein kinase II (CaM kinase II) by bradykinin stimulation. When the extract of NG108-15 cells was immunoprecipitated with the affinity-purified antibody to brain CaM kinase II, a 50-kDa protein in the immunoprecipitate mainly became autophosphorylated in a Ca 2+/calmodulin-dependent manner. The result suggest that the 50-kDa protein is the subunit of CaM kinasee II in NG108-15 cells.The Ca 2+/calmodulin-independent activity (autonomous activity) of the enzyme increased twice within 10 s by stimulation with 1 μM bradykinin in the cells. The increase in the autonomous activity of the enzyme had two phases: the transient early-peak phase and the long late-plateau phase. The former was abolished by the pretreatment of the cells with 10 mM caffeine or 20 μM BAPTA-AM, and the latter was abolished by the removal of the extracellular Ca 2+ with 1 mM EGTA or by the pretreatment with 1 μM nifedipine. Stimulation of 32P-labeled NG108-15 cells with 1 μM bradykinin increased the autophosphorylation of CaM kinase II and this increase was abolished by pretreatment with caffeine or BAPTA-AM. Theseesults suggest that CaM kinase II is activated via the inositol phospholipid signaling pathway induced with bradykinin in NG108-15 cells.