Evidence that muscarinic cholinergic receptors selectively interact with either the cyclic AMP or the inositol phosphate second-messenger response systems.

The relative capacities of muscarinic cholinergic receptor (MR) and bradykinin (BK)-receptor activation to increase phosphoinositide hydrolysis and to increase cytosolic Ca2+ were compared in NG108-15 neuroblastoma x glioma and 1321N1 human astrocytoma cells. In 1321N1 cells, the muscarinic cholinergic agonist carbachol and BK each stimulated a concentration-dependent accumulation of inositol phosphates (K0.5 approximately 10 microM and approximately 10 nM respectively) and a rapid increase in cytosolic Ca2+ as determined by quin2 fluorescence. In NG108-15 cells, BK alone stimulated a pertussis-toxin-insensitive accumulation of inositol phosphates (K0.5 approximately 10 nM) under conditions in which pertussis toxin completely inhibited MR-mediated inhibition of adenylate cyclase. BK also stimulated a rapid increase in cytosolic Ca2+ in NG108-15 cells. In contrast, no MR-mediated increase in phosphoinositide hydrolysis or change in cytosolic Ca2+ concentration was observed in NG108-15 cells. These results support the idea that MR selectively interact with either the cyclic AMP or the inositol phosphate second-messenger systems.     

Cell interactions of enkephalin/polypeptide conjugates.

Enkephalin molecules were bound to poly(Lys) (poly-K) or poly(Ala-Lys-Ala-Leu) (poly-A) and their interactions with NG108-15 cells, platelets, erythrocytes and fibroblast cells were investigated. A fluorescent probe, rhodamine, also was bound to the conjugates for monitoring interactions with these cells. Observations by fluorescence microscopy revealed that NG108-15 cells, platelets, and fibroblast cells were labelled by the conjugates, whereas erythrocytes were not. Since polypeptides without enkephalin moieties were only weakly adsorbed on the cells, it was concluded that the enkephalin/polypeptide conjugates were bound specifically to receptors on the cell membrane. Interestingly, when the enkephalin/poly-K conjugate was bound to NG108-15 and fibroblast cells, fluorescent patches appeared on the membrane. Such patch formation was not clearly observed with an enkephalin/rhodamine or enkephalin/poly-A conjugate. In the case of fibroblast cells, the fluorescence converged to a large cluster, which was ultimately internalized. The results suggest that clustering of the receptors in cell membranes is influenced by the carrier polymer presumably due to cross-linking of the receptors and/or the effect of the cationic polypeptides.     

Overexpression of Adhesion Molecule L1 in NG108-15 Neuroblastoma × Glioma Hybrid Cells Enhances Dibutyryl Cyclic AMP-Induced Neurite Outgrowth and Functional Synapse Formation with Myotubes

Abstract: The role of adhesion molecule L1 in synapse formation was examined by transient transfection of L1 cDNA in neuroblastoma × glioma hybrid NG108-15 cells. L1 overexpression was found in ∼50% of the transfected NG108-15 cell population. Neurite outgrowth induced by 0.25 m M dibutyryl cyclic AMP (cAMP) was much greater in L1-transfected NG108-15 cells than that in nontransfected and mock-transfected cells. The proportion of cells with neurites and the number of neurites per cells were increased in L1-transfected cells after 2 days of dibutyryl cAMP treatment. The proportion of cells with branched neurites and the average length of neurites were higher at day 4. A significantly higher rate of synapse formation with myotubes was apparent in the late phase of coculture (days 4–7) in L1-transfected cells than in control cells. The miniature end-plate potential frequency in myotubes was the same for the three types of NG108-15 cells. These results show that overexpression of L1 in NG108-15 cells facilitates synaptic connections by enhancing branching and elongation of neurites induced with dibutyryl cAMP, rather than by increasing probability of acetylcholine release.     

The lipid-anchored ectodomain of influenza virus hemagglutinin (GPI-HA) is capable of inducing nonenlarging fusion pores

GPI-linked hemagglutinin (GPI-HA) of influenza virus was thought to induce hemifusion without pore formation. Cells expressing either HA or GPI-HA were bound to red blood cells, and their fusion was compared by patch-clamp capacitance measurements and fluorescence microscopy. It is now shown that under more optimal fusion conditions than have been used previously, GPI-HA is also able to induce fusion pore formation before lipid dye spread, although with fewer pores formed than those induced by HA. The GPI-HA pores did not enlarge substantially, as determined by the inability of a small aqueous dye to pass through them. The presence of 1,1'-dioctadecyl-3, 3,3',3'-tetramethylindocarbocyanine perchlorate or octadecylrhodamine B in red blood cells significantly increased the probability of pore formation by GPI-HA; the dyes affected pore formation to a much lesser degree for HA. This greater sensitivity of pore formation to lipid composition suggests that lipids are a more abundant component of a GPI-HA fusion pore than of an HA pore. The finding that GPI-HA can induce pores indicates that the ectodomain of HA is responsible for all steps up to the initial membrane merger and that the transmembrane domain, although not absolutely required, ensures reliable pore formation and is essential for pore growth. GPI-HA is the minimal unit identified to date that supports fusion to the point of pore formation.     

Cyclic AMP induces activity increase of kinase FA (a transmembrane signal of insulin) during NG108-15 hybrid cell differentiation

The ATP.Mg-dependent protein phosphatase activating factor (protein kinase FA) has been identified to exist in neuroblastoma x glioma hybrid 108-15 cells (NG108-15 cells). More importantly, when NG cells were induced to differentiate with N6, O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (dibutyryl cAMP), the cellular activity of kinase FA was found to increase dramatically. Time course study further revealed that induction of differentiation in NG cells by dibutyryl cAMP treatment increased the FA activity to over 3 times the levels found in undifferentiated cells and in a linear day-dependent manner, indicating that the FA activity level is correlated with the state of differentiation of NG108-15 cells. This is the first report providing initial evidence that protein kinase FA (a transmembrane signal of insulin) is involved in the induction of neuronal cell differentiation.     

Caveolin-1 expression and membrane cholesterol content modulate N-type calcium channel activity in NG108-15 cells

Caveolins are the main structural proteins of glycolipid/cholesterol-rich plasmalemmal invaginations, termed caveolae. In addition, caveolin-1 isoform takes part in membrane remodelling as it binds and transports newly synthesized cholesterol from endoplasmic reticulum to the plasma membrane. Caveolin-1 is expressed in many cell types, including hippocampal neurons, where an abundant SNAP25-caveolin-1 complex is detected after induction of persistent synaptic potentiation. To ascertain whether caveolin-1 influences neuronal voltage-gated Ca2+ channel basal activity, we stably expressed caveolin-1 into transfected neuroblastoma x glioma NG108-15 hybrid cells [cav1(+) clone] that lack endogenous caveolins but express N-type Ca2+ channels upon cAMP-induced neuronal differentiation. Whole-cell patch-clamp recordings of cav1(+) cells demonstrated that N-type current density was reduced in size by approximately 70% without any significant change in the time course of activation and inactivation and voltage dependence. Moreover, the cav1(+) clone exhibited a significantly increased proportion of membrane cholesterol compared to wild-type NG108-15 cells. To gain insight into the mechanism underlying caveolin-1 lowering of N-current density, and more precisely to test whether this was indirectly caused by caveolin-1-induced enhancement of membrane cholesterol, we compared single N-type channel activities in cav1(+) clone and wild-type NG108-15 cells enriched with cholesterol after exposure to a methyl-beta-cyclodextrin-cholesterol complex. A lower Ca2+ channel activity was recorded from cell-attached patches of both cell types, thus supporting the view that the increased proportion of membrane cholesterol is ultimately responsible for the effect. This is due to a reduction in the probability of channel opening caused by a significant decrease of channel mean open time and by an increase of the frequency of null sweeps.     

Bradykinin stimulates GTP hydrolysis in NG108-15 membranes by a high-affinity, pertussis toxin-insensitive GTPase

In membranes of neuroblastoma x glioma hybrid (NG108-15) cells, bradykinin (EC50 approximately equal to 5 nM) stimulates GTP hydrolysis by a high-affinity GTPase (Km approximately equal to 0.2 microM). The octapeptide, des-Arg9-bradykinin, was inactive. Stimulation of GTP hydrolysis by bradykinin and an opioid agonist was partially additive. Treatment of NG108-15 cells with pertussis toxin, which inactivates Ni, eliminated GTPase stimulation by the opioid agonist but not by bradykinin. The data suggest that bradykinin activates in NG108-15 membranes a guanine nucleotide-binding protein which is not sensitive to pertussis toxin and which may be involved in bradykinin-induced stimulation of phosphoinositide metabolism in these cells.     

Expression of Functional Bradykinin Receptors in Xenopus Oocytes

mRNA prepared from various tissues and cultured cells was injected into Xenopus laevis oocytes. Three to five days after injection, the response of the oocytes to the peptide bradykinin was monitored. The oocytes were voltage clamped and the membrane currents generated on application of agonist were recorded. mRNA from NG108-15, rat uterus, and human fibroblast cell line WI38 gave similar responses to bradykinin (1 microM), with an initial inward current (10-20 nA) followed by a prolonged period of membrane current oscillations. The same pattern of response was given by total RNA from rat dorsal root ganglia. No response to bradykinin (10 microM) was recorded from oocytes injected with rat brain mRNA, although these oocytes gave peak inward currents of about 75 nA in response to serotonin (10 microM). mRNA from both NG108-15 cells and rat uterus was fractionated on sucrose gradients. This resulted in an approximately five-fold increase in the size of the response compared to that given by unfractionated mRNA. The largest responses were given by mRNA fractions with a size of approximately 4.5 kb. Data were obtained consistent with the expression of both B1 and B2 receptors by WI38 human fibroblasts and with the expression of only the B2 type of receptor by NG108-15 cells.     

125I-beta-endorphin binding to neuroblastoma X glioma NG108-15 cells: distribution of delta opioid receptors.

The mouse neuroblastoma x rat glioma hybrid NG108-15 was previously shown to express delta opioid receptors. Because neuroblastoma cells display different phenotypes and cloned cell lines are heterogenous, we studied the characteristics and distribution of human 125I-beta-endorphin (125I-beta E) binding sites in cultures of NG108-15 cells with the use of micro-autoradiography and light microscopy. 125I-beta E labeled delta sites in NG108-15 in the presence of the non-opioid blocking peptide, beta-endorphin (6-31) (beta E (6-31)). Silver grains resulting from 125I-beta E binding to the opioid sites occurred in diffuse patches over several cells, with preferential location in dense cell patches. Pretreatment of NG108-15 with the delta agonist DADLE, previously shown to decrease beta E binding to delta sites on intact cells, also reduced silver grain density; however, some cells located in dense cell clusters were resistant to substantial agonist induced loss of labeling. These results suggest that delta opioid binding has a heterogenous cellular distribution in NG108.     

Heterogeneity of Nucleotide Receptors in NG108-15 Neuroblastoma and C6 Glioma Cells for Mediating Phosphoinositide Turnover

Abstract: We have compared the characteristics of receptors for nucleotide analogues and the involvement of phospholipase C (PLC) in the effector mechanism in NG108-15 neuroblastoma and C6 glioma cells. The relative potency of these analogues to stimulate inositol phosphate (IP) formation is UTP > UDP ? 2-methylthio-ATP (2-MeSATP), GTP > ATP, CTP > ADP > UMP in NG108-15 cells and ATP > UTP > ADP > GTP > UDP ? 2Me-SATP, CTP, UMP in C6 glioma cells. α,β-Methylene-ATP, β,γ-methylene-ATP, AMP, and adenosine had little or no effect in both types of cells. The EC₅₀ values were 3 and 106 µM for UTP in NG108-15 and C6 glioma cells, respectively. The EC₅₀ value for ATP in C6 glioma cells was 43 µM. 2-MeSATP was threefold more potent than ATP in NG108-15 cells but had little effect in C6 glioma cells at 1 mM. In NCB-20 cells, a similar rank order of potency to that found in NG108-15 cells, i.e., UTP ? GTP > ATP > CTP, was observed. In both NG108-15 and C6 glioma cells, preincubation with ATP or UTP caused a pronounced cross-desensitization of subsequent nucleotide-stimulated IP production. ATP and UTP displayed no additivity in terms of IP formation at maximally effective concentrations. In contrast, endothelin-1, bradykinin, and NaF interacted in an additive manner with either nucleotide in stimulating PI hydrolysis. Pretreatment with pertussis toxin did not affect ATP-, UTP-, and GTP-stimulated IP generation in these cells, indicating that nucleotide receptors coupled to PLC by a pertussis toxin-resistant G protein in both cell types. Short-term treatment of the cells with protein kinase C (PKC) activators [phorbol 12-myristate 13-acetate (PMA) and octylindolactam V] produced a dose-dependent inhibition of ATP- and UTP-induced IP formation with a greater extent and higher susceptibility in C6 glioma cells than in NG108-15 cells. Furthermore, a 24-h exposure of the cells to PMA resulted in an obvious attenuation of nucleotide-induced IP formation in C6 glioma cells but failed to change the response in NG108-15 cells. These results suggest that distinct nucleotide receptors that respond to ATP and UTP with different selectivity exist in NG108-15 and C6 glioma cells. These heterogeneous nucleotide receptors coupled to PLC undergo discriminative modulation by PKC. NG108-15 and NCB-20 neuroblastoma are two cell lines that showed the highest specificity to extracellular UTP rather than ATP among the nucleotide receptors so far studied in various cells, suggesting the presence of a pyrimidine receptor in these cells.     

Transmembrane potential measurements on plant cells using the voltage-sensitive dye ANNINE-6

The charging of the plasma membrane is a necessary condition for the generation of an electric-field-induced permeability increase of the plasmalemma, which is usually explained by the creation and the growth of aqueous pores. For cells suspended in physiological buffers, the time domain of membrane charging is in the submicrosecond range. Systematic measurements using Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) protoplasts stained with the fast voltage-sensitive fluorescence dye ANNINE-6 have been performed using a pulsed laser fluorescence microscopy setup with a time resolution of 5 ns. A clear saturation of the membrane voltage could be measured, caused by a strong membrane permeability increase, commonly explained by enhanced pore formation, which prevents further membrane charging by external electric field exposure. The field strength dependence of the protoplast’s transmembrane potential V _M shows strong asymmetric saturation characteristics due to the high resting potential of the plants plasmalemma. At the pole of the hyperpolarized hemisphere of the cell, saturation starts at an external field strength of 0.3 kV/cm, resulting in a measured transmembrane voltage shift of ?V _M?=??150 mV, while on the cathodic (depolarized) cell pole, the threshold for enhanced pore formation is reached at a field strength of approximately 1.0 kV/cm and ?V _M?=?450 mV, respectively. From this asymmetry of the measured maximum membrane voltage shifts, the resting potential of BY-2 protoplasts at the given experimental conditions can be determined to V _R?=??150 mV. Consequently, a strong membrane permeability increase occurs when the membrane voltage diverges |V _M|?=?300 mV from the resting potential of the protoplast. The largest membrane voltage change at a given external electric field occurs at the cell poles. The azimuthal dependence of the transmembrane potential, measured in angular intervals of 10° along the circumference of the cell, shows a flattening and a slight decrease at higher fields at the pole region due to enhanced pore formation. Additionally, at the hyperpolarized cell pole, a polarization reversal could be observed at an external field range around 1.0 kV/cm. This behavior might be attributed to a fast charge transfer through the membrane at the hyperpolarized pole, e.g., by voltage-gated channels.     

Induction of differentiation by radiation and hyperthermia in neuroblastoma—glioma hybrid cells

The effects of either radiation or hyperthermia on the differentiation potential of NG108-15, a neuroblastoma-glioma hybrid cell line, were studied. After radiation and hyperthermia, the outgrowth of neurites from NG108-15 cells was potentiated, and polarizing current and voltage pulses induced a distinct action potential and a diphasic (inward following outward) current, respectively. An increase in the specific activity of acetylcholinesterase was also observed. In addition, both treatments induced an elevation of the concentration of intracellular calcium in some cells. The increase in intracellular calcium concentration caused by applying the calcium ionophore, A23187, induced differentiation. It is suggested that both the radiation- and the hyperthermia-induced increases of electrical excitability and acetylcholinesterase activity may have originated from an increase in intracellular Ca²⁺ concentration.     

Cell permeabilization and inhibition of voltage-gated Ca(2+) and Na(+) channel currents by nanosecond pulsed electric field

Previous studies have found that nanosecond pulsed electric field (nsPEF) exposure causes long-term permeabilization of the cell plasma membrane. In this study, we utilized the whole-cell patch-clamp method to study the nsPEF effect on currents of voltage-gated (VG) Ca(2+) and Na(+) channels (I(Ca) and I(Na)) in cultured GH3 and NG108 cells. We found that a single 300 or 600 ns pulse at or above 1.5-2 kV/cm caused prolonged inhibition of I(Ca) and I(Na). Concurrently, nsPEF increased a non-inactivating "leak" current (I(leak)), presumably due to the formation of nanoelectropores or larger pores in the plasma membrane. The nsPEF effects were similar in cells that were exposed intact and subsequently brought into the whole-cell recording configuration, and in cells that were first brought into the whole-cell configuration and then exposed. Although both I(leak) and the inhibition of VG currents were enhanced at higher E-field levels, these two nsPEF effects showed relatively weak correlation with each other. In some cells, I(leak) increased 10-fold or more while VG currents remained unchanged. At longer time intervals after exposure (5-15 min), I(Ca) and I(Na) could remain inhibited although I(leak) had largely recovered. The causal relation of nsPEF inhibitory effects on VG currents and permeabilization of the plasma membrane is discussed.     

Puroindoline-a and alpha1-purothionin form ion channels in giant liposomes but exert different toxic actions on murine cells

Puroindoline-a (PIN-a) and alpha1-purothionin (alpha1-PTH), isolated from wheat endosperm of Triticum aestivum sp., have been suggested to play a role in plant defence mechanisms against phytopathogenic organisms. We investigated their ability to form pores when incorporated into giant liposomes using the patch-clamp technique. PIN-a formed cationic channels (approximately 15 pS) with the following selectivity K(+) > Na(+) > Cl(-). Also, alpha1-PTH formed channels of approximately 46 pS and 125 pS at +100 mV, the selectivity of which was Ca(2+) > Na(+) approximately K(+) > Cl(-) and Cl(-) > Na(+), respectively. In isolated mouse neuromuscular preparations, alpha1-PTH induced muscle membrane depolarization, leading to blockade of synaptic transmission and directly elicited muscle twitches. Also, alpha1-PTH caused swelling of differentiated neuroblastoma NG108-15 cells, membrane bleb formation, and disorganization of F-actin. In contrast, similar concentrations of PIN-a had no detectable effects. The cytotoxic actions of alpha1-PTH on mammalian cells may be explained by its ability to induce cationic-selective channels.     

Coupling of the Cloned μ-Opioid Receptor with the ω-Conotoxin-Sensitive Ca2+ Current in NG108-15 Cells

Abstract: Voltage-dependent Ca²⁺ currents were measured in NG108-15 neuroblastoma × glioma hybrid cells transformed to express the rat μ-opioid receptor by the whole-cell configuration of the patch-clamp technique with Ba²⁺ as charge carrier. A μ-opioid receptor-selective agonist, [ d -Ala², N -Me-Phe⁴,Gly⁵-ol]enkephalin caused significant inhibition of voltage-dependent Ca²⁺ currents in μ-receptor-transformed NG108-15 cells but not in nontransfected or vector-transformed control cells. On the other hand, a δ-opioid receptor-selective agonist, [ d -penicillamine², d -penicillamine⁵]enkephalin, induced inhibition of voltage-dependent Ca²⁺ currents in both control and μ-receptor-transformed cells, which is mediated by the δ-opioid receptor expressed endogenously in NG108-15 cells. The inhibition of voltage-dependent Ca²⁺ currents induced by [ d -Ala², N -Me-Phe⁴,Gly⁵-ol]enkephalin and [ d -penicillamine², d -penicillamine⁵]enkephalin was reduced by pretreatment of the cells with pertussis toxin or ω-conotoxin GVIA. These results indicate that the μ-opioid receptor expressed from cDNA functionally couples with ω-conotoxin-sensitive N-type Ca²⁺ channels through the action of pertussis toxin-sensitive G proteins in NG108-15 cells.