inositol-1-4-5-trisphosphate and Glioma

Topics: Acetylcholine; Animals; Bradykinin; Calcium; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Mice; Neuroblastoma; Rats; Tumor Cells, Cultured

Human serotonin 5A (5-HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca2+ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3-sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.

Topics: Adenylyl Cyclase Inhibitors; ADP-ribosyl Cyclase; Animals; Calcium; Cyclic AMP; Gene Expression; Glioma; GTP-Binding Proteins; Heparin; Humans; Inositol 1,4,5-Trisphosphate; Patch-Clamp Techniques; Potassium Channel Blockers; Rats; Receptors, Serotonin; Second Messenger Systems; Serotonin; Signal Transduction; Tumor Cells, Cultured

Capacitative Ca2+ entry exists in rat glioma C6 cells; however, how the information of depletion of Ca2+ in intracellular stores transmits to the plasma membrane is unknown. In the present study, we examined whether Ca2+ influx factor (CIF) causes capacitative Ca2+ entry in C6 cells. CIF was extracted from non-treated (Non-CIF), bombesin-treated (BBS-CIF) and thapsigargin-treated (TG-CIF) C6 cells by a reverse-phase silica cartridge. The addition of BBS-CIF and TG-CIF gradually increased cytoplasmic Ca2+ concentration ([Ca2+]i) but Non-CIF did not increase [Ca2+]i. Neither BBS-CIF nor TG-CIF elevated [Ca2+]i in the absence of extracellular Ca2+. Gd3+ inhibited the increase in [Ca2+]i induced by BBS-CIF and TG-CIF. Genistein abolished an elevation of [Ca2+]i induced by BBS-CIF and TG-CIF. BBS-CIF and TG-CIF did not increase inositol 1,4,5-trisphosphate accumulation. The results suggest that capacitative Ca2+ entry is caused by CIF in rat glioma C6 cells.

Topics: Animals; Calcium; Cyclic AMP; Genistein; Glioma; Inositol 1,4,5-Trisphosphate; Rats; Tumor Cells, Cultured

Metabotropic glutamate receptors (mGluRs) couple to heterotrimeric G-proteins and regulate cell excitability and synaptic transmission in the CNS. Considerable effort has been focused on understanding the cellular and biochemical mechanisms that underlie regulation of signaling by G-proteins and their linked receptors, including the mGluRs. Recent findings demonstrate that regulators of G-protein signaling (RGS) proteins act as effector antagonists and GTPase-activating proteins for Galpha subunits to inhibit cellular responses by G-protein-coupled receptors. RGS4 blocks Gq activation of phospholipase Cbeta and is expressed broadly in rat brain. The group I mGluRs (mGluRs 1 and 5) couple to Gq pathways to regulate several effectors in the CNS. We examined the capacity of RGS4 to regulate group I mGluR responses. In Xenopus oocytes, purified RGS4 virtually abolishes the mGluR1a- and mGluR5a-mediated but not the inositol trisphospate-mediated activation of a calcium-dependent chloride current. Additionally, RGS4 markedly attenuates the mGluR5-mediated inhibition of potassium currents in hippocampal CA1 neurons. This inhibition is dose-dependent and occurs at concentrations that are virtually identical to those required for inhibition of phospholipase C activity in NG108-15 membranes and reconstituted systems using purified proteins. These findings demonstrate that RGS4 can modulate mGluR responses in neurons, and they highlight a previously unknown mechanism for regulation of G-protein-coupled receptor signaling in the CNS.

Topics: Age Factors; Animals; Calcium; Calcium Channels; Calcium-Calmodulin-Dependent Protein Kinases; Cell Membrane; Chloride Channels; G Protein-Coupled Inwardly-Rectifying Potassium Channels; Glioma; GTP-Binding Proteins; Hippocampus; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Isoenzymes; Mice; Neurons; Oocytes; Patch-Clamp Techniques; Phospholipase C beta; Potassium Channels; Potassium Channels, Inwardly Rectifying; Proteins; Rats; Receptors, Metabotropic Glutamate; Receptors, Muscarinic; RGS Proteins; RNA, Messenger; Signal Transduction; Synapses; Tritium; Type C Phospholipases; Xenopus

An evanescent wave of ultraviolet light was successfully used to release biologically active molecules from caged compounds in living cells. The evanescent wave was generated by the total internal reflection in a limited region near the plasma membrane attached to the illuminated interface. At first, the photolysis efficiency of the evanescent wave of ultraviolet laser light was studied using caged glutamic acid in vitro. Then, caged Ca2+ introduced in the living cultured cell was similarly photolyzed by the evanescent wave and the resulting elevations of the concentration of intracellular Ca2+ in the proximity of the plasma membrane and in the cytosol were observed with a simultaneously introduced fluorescent calcium indicator. Inositol trisphosphate can also be photoreleased near the plasma membrane, which enables study of the temporal and spatial pathways of signal transduction. The method developed here provides a useful tool for studying signal transduction near the plasma membrane in a living cell.

Topics: Aniline Compounds; Animals; Calcium; Cell Membrane; Fluorescent Dyes; Glioma; Glutamic Acid; Inositol 1,4,5-Trisphosphate; Mice; Neuroblastoma; Photolysis; Rats; Second Messenger Systems; Tumor Cells, Cultured; Xanthenes

Lysophosphatidic acid (LPA) functions as an extracellular lipid mediator stimulating phospholipase C and affecting the structure of the cytoskeleton in several cell types. In rat glioma C6 cells, LPA mobilizes calcium from intracellular calcium stores and reverts morphological changes induced by elevated cytosolic cAMP-concentrations. Here we show that LPA-stimulation of C6 cells loaded with the calcium-sensitive fluorescent dye indo-1 results in calcium release from a subset of intracellular calcium stores that are not sensitive to the tumor promoter thapsigargin and do not overlap with calcium stores depleted during purinergic receptor stimulation with ATP. Furthermore, depletion of LPA-sensitive calcium stores does not induce capacitative calcium entry from the extracellular space into the cytosol to the same extent as ATP. These results indicate that inositol phosphate signaling induced by LPA or ATP may differ in kinetics or in spatial organisation within the cell. This may represent a possible explanation for the previous observation that only LPA, but not other calcium-mobilizing agonists, reverts cAMP-induced changes in the cytoskeletal organization in C6 cells.

Topics: Adenosine Triphosphate; Animals; Calcium; Electric Conductivity; Electrophysiology; Glioma; Inositol 1,4,5-Trisphosphate; Lysophospholipids; Rats; Signal Transduction; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

We examined the changes in phospholipid metabolisms in sodium butyrate-treated C6 glioma cells. Treatment of 2.5 mM sodium butyrate for 24 h induced an increase in the activity of glutamine synthetase, suggesting that these cells were under differentiation. Similar treatment was associated with (i) increased arachidonic acid incorporation into phosphatidylcholine, and (ii) decreased arachidonic acid incorporation into phosphatidylinositol and (iii) phosphatidylethanolamine. These effects were subsequently investigated by examining the acylation process, de novo biosynthesis, and the agonist-stimulated phosphoinositides hydrolysis in these cells. Our results indicated that sodium butyrate stimulated the acylation of arachidonic acid into lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylinositol. The glycerol incorporation into these lipids was not affected, but the inositol incorporation into total chloroform extracts and Pl and phosphatidylinositol 4-phosphate was decreased in the sodium butyrate-treated cells. Moreover, the accumulation of the rapid histamine-stimulated phosphoinositide metabolites, i.e., inositol monophosphate, inositol diphosphate, and inositol triphosphate (IP3) was decreased in these cells. To elucidate whether the decreased inositol phosphates were due to a decrease in the phosphoinositides hydrolysis, we measured the transient IP3 production directly by a receptor-binding assay. Our results indicated that histamine-stimulated transient IP3 formations were decreased. Taken together, these results indicated that multiple changes by multiple mechanisms of phospholipid metabolisms were found in sodium butyrate-treated C6 glioma cells. The decreased IP3 formation and its subsequent action, i.e., Ca2+ mobilization, may play an early but pivotal role by which sodium butyrate induces C6 glioma cell differentiation.

Topics: Acylation; Animals; Arachidonic Acid; Butyrates; Butyric Acid; Calcium; Chloroform; Glioma; Glutamate-Ammonia Ligase; Glycerol; Histamine; Hydrolysis; Inositol; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipids; Proteins; Rats; Thymidine; Tritium; Tumor Cells, Cultured

The effects of tetrandrine (TET), a Ca2+ antagonist of Chinese herbal origin, and hernandezine (HER), a structural analogue of TET, on Ca2+ mobilization were studied in rat glioma C6 cells. TET and HER alone did not affect the resting cytoplasmic Ca2+ concentration ([Ca2+]i). TET and HER inhibited the peak and sustained elevation of [Ca2+]i induced by bombesin and thapsigargin (TG), a microsomal Ca2+ ATPase inhibitor, in a dose-dependent manner. The doses of TET or HER needed to abolish the sustained and peak increase in [Ca2+]i induced by bombesin and TG were 30 microM and 300 microM, respectively. TET and HER did not increase inositol 1,4,5-trisphosphate (IP3) accumulation by themselves but inhibited IP3 accumulation elevated by bombesin. In permeabilized C6 cells, the addition of IP3 and TG released Ca2+ from intracellular stores. Pretreatment with TET or HER abolished Ca2+ release from intracellular stores induced by bombesin and TG. In the absence of extracellular Ca2+, the addition of 3 mM Ca2+ to extracellular medium slightly increased [Ca2+]i, which indicated Ca2+ entry due to leakage of Ca2+ at the plasma membrane but not Ca2+ influx through Ca2+ channels. TET and HER did not affect this leakage entry of Ca2+. The present results suggest that TET and HER inhibit Ca2+ release from intracellular stores as well as Ca2+ entry from extracellular medium evoked by bombesin and TG. In addition, TET and HER inhibit IP3 accumulation induced by bombesin in rat glioma C6 cells.

Topics: Alkaloids; Animals; Antineoplastic Agents, Phytogenic; Benzylisoquinolines; Bombesin; Calcium; Calcium Channel Blockers; Cell Division; Cell Membrane; Cell Membrane Permeability; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Enzyme Inhibitors; Glioma; Inositol 1,4,5-Trisphosphate; Rats; Thapsigargin; Tumor Cells, Cultured

In glioma C6 cells, extracellular ATP generates inositol 1,4,5-trisphosphate (InsP3), indicating the presence of purinergic receptors coupled to phosphoinositide turnover. To identify the effect of ATP (acting via InsP3) and thapsigargin (acting without InsP3 production as a specific inhibitor of the endoplasmic reticulum Ca(2+)-ATPase) on intracellular Ca2+ pools we used video imaging of Fura-2 loaded into single, intact glioma C6 cells. It has been shown that ATP and thapsigargin initiate Ca2+ response consistent with the capacitative model of Ca2+ influx. When the cells were stimulated by increasing concentrations of ATP (1, 10, 50 and 100 microM) the graded, quantal Ca2+ response was observed. In the absence of extracellular Ca2+ thapsigargin and ionomycin-releasable Ca2+ pools are overlapping, demonstrating that Ca2+ stores are located mainly in the endoplasmic reticulum. After maximal Ca2+ mobilization by ATP, thapsigargin causes further increase in cytosolic Ca2+ concentration, whereas emptying of thapsigargin-sensitive intracellular stores prevents any further Ca2+ release by ATP. Thus, the thapsigargin-sensitive intracellular pool of Ca2+ in glioma C6 cells seems to be larger than that sensitive to InsP3. Two hypothesis to explain this result are proposed. One postulates a presence of two different Ca2+ pools, sensitive and insensitive to InsP3 and both discharged by thapsigargin, and the other, the same intracellular pool of Ca2+ completely emptying by thapsigargin and only partially by InsP3. These results may contribute to understanding the mechanism of Ca2+ signalling mediated by ATP, the most potent intracellular Ca2+ mobilizing agonist in all types of glial cells.

Topics: Adenosine Triphosphate; Calcium; Drug Evaluation, Preclinical; Endoplasmic Reticulum; Enzyme Inhibitors; Glioma; Humans; Inositol 1,4,5-Trisphosphate; Signal Transduction; Thapsigargin; Tumor Cells, Cultured

The therapeutical efficacy of alpha-trinositol (D-myo-inositol-1,2,6-trisphosphate), an isomer of the intracellular messenger IP3, was analyzed on cytotoxic swelling and damage of glial cells in vitro from lactacidosis or glutamate. C6 glioma cells suspended in a physiological medium were either exposed to pH 5.0 by administration of lactic acid, or to 1 mM glutamate. Cell swelling and viability were quantified by flow cytometry. Lactacidosis of pH 5.0 led to an increase in cell volume to 139.7 +/- 1.3% within 20 min whereas alpha-trinositol was reducing the swelling response by approximately 25% (P < 0.01). In addition, at pH 5.0 the fraction of viable cells was lowered from 94.3 +/- 0.2% (control) to only 53.8 +/- 3.1% after 60 min. Alpha-trinositol was found to protect also cell viability; at 60 min of lactacidosis 70.2 +/- 1.6% of the cells still were viable (P < 0.01). The addition of glutamate (1 mM) to the cell suspension led to a steady increase in cell size, reaching 110% of control at 120 min, irrespectively of whether alpha-trinositol was added or not.

Topics: Acidosis, Lactic; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Size; Cell Survival; Glioma; Glutamic Acid; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Neuroglia; Tumor Cells, Cultured

delta-Opioids mobilize Ca2+ from intracellular stores in undifferentiated NG108-15 cells, but the mechanism involved remains unclear. Therefore, we examined the effect of [D-Pen 2,5] enkephalin on inositol 1,4,5-trisphosphate formation in these cells. [D-Pen 2,5] enkephalin caused a dose-dependent (EC50= 3.1 nM) increase in inositol 1,4,5-trisphosphate formation (measured using a specific radioreceptor mass assay), which peaked (25.7+/-1.2 pmol/mg of protein with 1 microM, n=9) at 30 s and returned to basal levels (10.6+/-0.9 pmol/mg of protein, n=9) within 4-5 min. This response was fully naloxone (1 microM) reversible and pertussis toxin (100ng/ml for 24 h) sensitive. Preincubation with Ni2+ (2.5 mM) or nifedipine (1 microM) had no effect on the [D-Pen 2,5] enkephalin (1 microM)-induced inositol 1,4,5-triphosphate response, and K+ (80mM) was unable to stimulate inositol 1,4,5-trisphosphate formation, indicating Ca2+ influx-induced activation of phospholipase C is not involved. Preincubation with the protein kinase C inhibitor Ro 31-8220 (1 microM) enhanced, whereas acute expo sure to phorbol 12,13-dibutyrate (1 microM) abolished, the [D-Pen 2,5] enkephalin (0.1 microM)-induced inositol 1,4,5-triphosphate response, suggesting protein kinase C exerts an autoinhibitory feedback action. [D-Pen 2,5] Enkephalin also dose-dependently (EC50 =2.8 nM) increased the intracellular [Ca2+], which was maximal (24 nM increase with 1 microM, n=5) at 30 s. This close temporal and dose-response relationship strongly suggests that delta-opioid receptor-mediated increases in intracellular [Ca2+] results from inositol 1,4,5-trisphosphate-induced Ca2+ release from intracellular stores, in undifferentiated NG108-15 cells.

Topics: Analgesics; Animals; Calcium; Calcium Channel Blockers; Dose-Response Relationship, Drug; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Glioma; Inositol 1,4,5-Trisphosphate; Neuroblastoma; Nickel; Nifedipine; Pertussis Toxin; Receptors, Opioid, delta; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

Confocal fluorescence microscopy was used to study the bradykinin-induced calcium signals in the neuroblastoma x glioma cell line NG 108-15. We found that bradykinin induced a rise in free calcium, not only in the cytoplasm but also in the nucleus. The nuclear and cytosolic calcium concentrations were not significantly different and rose to about 1.2 microM. The signal was mediated by the B2-receptor subtype as confirmed using the specific antagonist Hoe 140. Both the onset and the intensity of the calcium signals were concentration-dependent. The rise of nuclear calcium level was independent of extracellular calcium and suppressed by thapsigargin which is known to deplete inositol 1,4,5-trisphosphate-sensitive calcium stores. Bradykinin-induced calcium increase desensitizes rapidly. This desensitization was shown not to involve activation of protein kinase C.

Topics: Animals; Bradykinin; Calcium; Cell Nucleus; Fura-2; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Microscopy, Confocal; Neuroblastoma; Protein Kinase C; Rats; Signal Transduction; Tumor Cells, Cultured

Leucine-enkephalin (Leu-EK) dose-dependently elicited an increase in cytosolic Ca2+ concentration ([Ca2+]i) with an EC50 of 1.2 microM via the phosphoinositide cascade in NG108-15 cells. Chronic treatment of cells with [D-Ala2,D-Leu5]enkephalin caused time-dependent homologous desensitization. In the presence of extracellular Ca2+, ATP as well as bradykinin stimulated significantly higher increases in inositol 1,4,5-trisphosphate (IP3) generation than did Leu-EK; however, the magnitude of intracellular Ca2+ pools increased after ATP stimulation, whereas bradykinin depleted intracellular pools. Hence, cells lost their [Ca2+]i response to Leu-EK if bradykinin was first added to induce a [Ca2+]i increase, whereas the response was unchanged if Leu-EK was added after addition of ATP. When Leu-EK was added simultaneously with bradykinin or ATP, an additive response was observed in IP3 generation; however, the rise in [Ca2+]i reached the same level as that induced by bradykinin or ATP alone. In the absence of extracellular Ca2+ in which the replenishment of intracellular pools was not possible, ATP displayed an inhibitory effect similar to that of bradykinin on the Leu-EK-induced [Ca2+]i increase. Prior treatment of cells with Leu-EK slightly heterologously desensitized the action of bradykinin, but had no effect on the ATP response. Our results suggest that a shared intracellular Ca2+ pool is sensitive to the opioid, bradykinin and P2-purinoceptor agonists; however, a defined pool of phosphatidylinositol 4,5-bisphosphate or a specific phospholipase C is responsible for each receptor.

Topics: Adenosine Triphosphate; Bradykinin; Calcium; Cyclic AMP; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; Inositol 1,4,5-Trisphosphate; Neuroblastoma; Receptors, Opioid, delta; Tumor Cells, Cultured

The C6 glioma cell line possesses 5-HT2A receptors that have been shown to increase intracellular calcium levels. We have studied the electrophysiological response of these cells to 5-HT using the whole-cell recording method. Under voltage-clamp, 5-hydroxytryptamine (5-HT) produced an outward current in these cells which was inhibited by extracellularly applied ketanserin and spiperone and by EGTA (10 mM) in the recording electrode. The 5-HT induced response could be mimicked by intracellular photolytic release of inositol (1,4,5) trisphosphate (IP3) from caged molecules. The reversal potentials for the IP3- and 5-HT-induced responses were closely matched. The data indicates that the outward current is likely to be mediated by 5-HT2A receptors stimulating IP3 production which increases intracellular calcium leading to the opening of calcium-activated potassium channels.

Topics: Animals; Brain Neoplasms; Calcium; Electrophysiology; Glioma; Inositol 1,4,5-Trisphosphate; Ion Channels; Membrane Potentials; Potassium Channels; Rats; Receptors, Serotonin; Serotonin; Tumor Cells, Cultured

We found in cultured glioma (C6BU-1) cells that excitatory amino acids (EAAs) such as glutamate, N-methyl-D-aspartate (NMDA), aspartate, and metabotropic glutamate receptor agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylate caused an increase in the inositol 1,4,5-trisphosphate formation and the intracellular Ca2+ concentration ([Ca2+]i) in the absence of extracellular Mg2+ and Ca2+. Pertussis toxin treatment abolished this glutamate-induced [Ca2+]i increase. Various antagonists against NMDA receptor-ion channel complex, such as Mg2+, D-2-amino-5-phosphonovalerate (D-APV), HA-966, and MK-801, also inhibited the increase in [Ca2+]i induced by glutamate. These results indicate that these metabotropic EAA receptors coupled to pertussis toxin-susceptible GTP-binding protein and phospholipase C system in C6BU-1 glioma cells have the pharmacological properties of NMDA receptor-ion channel complexes. We also found that in the presence of Mg2+ these metabotropic receptors resemble the NMDA receptor-ion channel complex interacted with 5-hydroxytryptamine2 (5-HT2) receptor signaling. EAAs inhibited 5-HT2 receptor-mediated intracellular Ca2+ mobilization and inositol 1,4,5-trisphosphate formation in a concentration-dependent manner. The inhibitory effect of glutamate was reversed by various NMDA receptor antagonists (D-APV, MK-801, phencyclidine, and HA-966), but L-APV failed to block the inhibitory effect of glutamate. The same result was observed in the absence of extracellular Ca2+. In addition, this inhibitory effect on 5-HT2 receptor-mediated signal transduction was abolished by treatment of C6BU-1 cells with pertussis toxin, whereas 5-HT2 receptor-mediated [Ca2+]i increase was not abolished by pertussis toxin treatment. We can, therefore, conclude that the inhibitory effect of glutamate is not a result of the influx of Ca2+ through the ion channel and that it operates via metabotropic glutamate receptors, having NMDA receptor-ion channel complex-like properties and being coupled with pertussis toxin-sensitive GTP-binding protein and phospholipase C.

Topics: 2-Amino-5-phosphonovalerate; Animals; Aspartic Acid; Calcium; Cell Line; Cycloleucine; Dizocilpine Maleate; Glioma; Glutamic Acid; Inositol 1,4,5-Trisphosphate; Ion Channels; Kainic Acid; Kinetics; Magnesium Chloride; N-Methylaspartate; Neurotoxins; Pyrrolidinones; Rats; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Receptors, Serotonin; Signal Transduction; Tumor Cells, Cultured

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 x 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 omega-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 microM to 0.063 microM). 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.

Topics: Bucladesine; Calcium; Calcium Channels; Electrophysiology; Glioma; Guanosine Triphosphate; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Mitochondria; Neuroblastoma; Signal Transduction; Tumor Cells, Cultured

Cell culture models were used to study the effects of long-term ethanol exposure on neuronal cells. Effects on phospholipase C and phospholipase D mediated signal transduction were investigated by assaying receptor-binding, G protein function, activities of lipases, formation of second messengers and c-fos mRNA. The signal transduction cascades displayed abnormal activities from 2 to 7 days of exposure which differed from the acute effects. Phosphatidylethanol formed by phospholipase D is an abnormal lipid that may harmfully affect nerve cell function.

Topics: Acclimatization; Animals; Brain; Cell Line; Diglycerides; Ethanol; Genes, fos; Glioma; GTP-Binding Proteins; Humans; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Lipase; Models, Neurological; Neuroblastoma; Neurons; Phospholipase D; Proto-Oncogene Proteins c-fos; RNA, Messenger; Second Messenger Systems; Signal Transduction; Tumor Cells, Cultured; Type C Phospholipases

Opioids elicit an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108-15 cells, which, depending upon growth conditions, results from either Ca2+ influx in differentiated cells or Ca2+ release from internal stores in undifferentiated cells (Jin et al., 1992). In this report we describe fura-2-based digital imaging studies that demonstrate that opioid-evoked Ca2+ release in these cells results from the activation of phospholipase C (PLC) and subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive store. D-Ala2-D-Leu5-enkephalin (DA-DLE) evoked concentration-dependent increases in [Ca2+]i (EC50 approximately equal to 4 nM). The response was blocked by naloxone (1 microM). In single cells, sequential application of selective opioid agonists (10 nM) evoked responses of the rank order DADLE = D-Pen2, D-Pen5-enkephalin (DPDPE) > trans-(+/-) 3,4-dichloro-N-methyl-N-(2-[1- pyrrolidinyl]cyclohexyl) benzeneacetamide (U50488) > D-ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO), consistent with activation of a delta-opioid receptor. Forty percent (n = 198) of the cells responded to 100 nM DADLE with a net [Ca2+]i increase of 483 +/- 40 nM. Bradykinin (100 nM) elicited a response in 91% of the cells with a mean net amplitude of 707 +/- 36 nM. The DADLE-evoked responses were not blocked by removal of extracellular Ca2+; instead, they were abolished by treatment with 10 nM thapsigargin, an agent that depletes and prevents refilling of IP3-sensitive Ca2+ stores. A 1 microM concentration of U73122, an aminosteroid inhibitor of PLC, completely blocked the DADLE-evoked [Ca2+]i increase, while an inactive analog, U73433, was without effect. To explore the possible role of G-proteins in mediating opioid-induced [Ca2+]i increases in NG108-15 cells, we pretreated cells with pertussis or cholera toxin; pertussis toxin blocked the opioid-induced response while cholera toxin was without effect, consistent with a Gi- or Go-mediated effect. Activation of the opioid inhibitory pathway previously described for these cells appears to stimulate the phosphoinositide (PI) cascade as well. Including the PI cascade among the multiple second messenger systems modulated by opioids may be key to understanding the biochemical events that underlie acute and chronic opioid action.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Calcium; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Fluorescent Dyes; Fura-2; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Kinetics; Mice; Microscopy, Fluorescence; Naloxone; Narcotics; Neuroblastoma; Pyrrolidines; Rats; Tumor Cells, Cultured

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.

Topics: Bradykinin; Calcium; Drug Synergism; Egtazic Acid; Extracellular Space; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Ionomycin; Neuroblastoma; Osmolar Concentration; Potassium; Signal Transduction; Tumor Cells, Cultured

The effect of heat shock on agonist-stimulated intracellular Ca2+ mobilization and the expression of heat shock protein 72 (hsp72) in neuroblastoma x glioma hybrid cells (NG 108-15 cells) were examined. Hsp72 was expressed at 6 h after heat shock (42.5 degrees C, 2 h), reached a maximum at 12 h, and decreased thereafter. Bradykinin-induced [Ca2+]i rise was attenuated to 28% of control by heat shock at 2 h after heat shock, and reversion to the control level was seen 12 h later. When the cells were treated with quercetin or antisense oligodeoxyribonucleotide against hsp72 cDNA, the synthesis of hsp72 was not induced by heat shock, whereas bradykinin-induced [Ca2+]i rise was abolished and the [Ca2+]i rise was not restored. Recovery from this stressed condition was evident when cells were stimulated by the Ca(2+)-ATPase inhibitor thapsigargin, even in the presence of either quercetin or antisense oligodeoxyribonucleotide. Inositol 1,4,5-trisphosphate (IP3) production was not altered by heat shock at 12 h after heat shock, whereas IP3 receptor binding activity was reduced to 45.3%. In the presence of quercetin or antisense oligodeoxyribonucleotide, IP3 receptor binding activity decreased and reached 27.2% of the control 12 h after heat shock. Our working thesis is that heat shock transiently suppresses the IP3-mediated intracellular Ca2+ signal transduction system and that hsp72 is involved in the recovery of bradykinin-induced [Ca2+]i rise.

Topics: Animals; Biological Transport; Bradykinin; Calcium; Calcium Channels; Glioma; Heat-Shock Proteins; Hot Temperature; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Intracellular Membranes; Mice; Neuroblastoma; Oligonucleotide Probes; Quercetin; Rats; Receptors, Cytoplasmic and Nuclear; Shock; Tumor Cells, Cultured

1. A glial cell line derived from C6 rat glioma cells has been shown previously to respond to extracellular pulses of bradykinin or intracellular injection of inositol 1,4,5-trisphosphate (Ins-P3) with a slow hyperpolarizing response due to activation of a K+ current (G. Reiser et al., Brain Res. 506, 205-214; 1990). 2. We determined the ensuing single-channel activity, which is most likely caused by Ca2+ released from internal stores after bradykinin stimulation. Bradykinin-activated channels were selectively permeable to K+, but not to Na+ or to Cl-, and exhibited conductances of mainly 40 and 50 pS. In glioma cells the same type of channel was activated by intracellular injection of Ins-P3 and by extracellular bradykinin pulses.

Topics: Animals; Bradykinin; Calcium; Cell Line; Glioma; Inositol 1,4,5-Trisphosphate; Ion Channel Gating; Membrane Potentials; Microinjections; Potassium Channels; Rats; Tumor Cells, Cultured

Addition of endothelins (ETs) to neuroblastoma-glioma hybrid cells (NG108-15) induced increases in cytosolic free Ca2+ ([Ca2+]i) levels of labeled inositol monophosphates and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. The increases in [Ca2+]i elicited by the three ETs (ET-1, ET-2, and ET-3) were transient and did not show a sustained phase. Chelating extracellular Ca2+ in the medium by adding excess EGTA decreased the ET-mediated Ca2+ response by 40-50%. This result indicates that a substantial portion of the increase in [Ca2+]i was due to influx from an extracellular source. However, the increase in [Ca2+]i was not affected by verapamil or nifedipine (10(-5) M). A rank order potency of ET-1 > ET-2 > ET-3 is shown for the stimulated increase in [Ca2+]i, as well as labeled inositol phosphates, in these cells. ATP (10(-4) M) and bradykinin (10(-7) M) also induced the increases in [Ca2+]i and Ins(1,4,5)P3 in NG108-15 cells, albeit to a different extent. When compared at 10(-7) M, bradykinin elicited a five- to sixfold higher increase in the level of Ins(1,4,5)P3, but less than a twofold higher increase in [Ca2+]i than those induced by ET-1. Additive increases in both Ins(1,4,5)P3 and [Ca2+]i were observed when ET-1, ATP, and bradykinin were added to the cells in different combinations, suggesting that each receptor agonist is responsible for the hydrolysis of a pool of polyphosphoinositide within the membrane. ET-1 exhibited homologous desensitization of the Ca2+ response, but partial heterologous desensitization to the Ca2+ response elicited by ATP. On the contrary, ET-1 did not desensitize the response elicited by bradykinin, although bradykinin exhibited complete heterologous desensitization to the response elicited by ET-1. Taken together, these results illustrate that, in NG108-15 cells, a considerable amount of receptor cross talk occurs between ET and other receptors that transmit signals through the polyphosphoinositide pathway.

Topics: Adenosine Triphosphate; Animals; Bradykinin; Calcium; Calcium Channels; Egtazic Acid; Endothelins; Fura-2; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Kinetics; Neuroblastoma; Phosphatidylinositol Phosphates; Phosphatidylinositols; Radioligand Assay; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Time Factors

In an NG 108-15 neuroblastoma x glioma hybrid cell suspension, extracellular ATP (via P2-purinergic receptors) and bradykinin stimulated Ins(1,4,5)P3 formation, which was accompanied by an increase in the cytosolic Ca2+ concentration ([Ca2+]i). Leucine enkephalin (EK) also slightly increased [Ca2+]i in the absence, but not in the presence, of apyrase, which hydrolyses extracellular ATP and ADP to AMP. When the cells were stimulated by P2-agonists or bradykinin prior to the application of EK, EK induces a remarkable rise in [Ca2+]i. This P2-agonist- or bradykinin-assisted EK action was also observed in single cells on a coverslip. A decrease in the extracellular Ca2+ concentration only slightly lowered the EK-induced rise in [Ca2+]i, but treatment of the cells with thapsigargin, an agent which depletes Ca2+ in the Ins(1,4,5)P3-sensitive pool, almost completely abolished EK action. The observed permissive stimulation by EK of Ins(1,4,5)P3 formation induced by a P2-agonist or bradykinin may be a primary event for the EK-induced [Ca2+]i rise. These actions of EK were antagonized by naloxone and completely reversed by prior treatment of the cells with pertussis toxin, whereas the toxin hardly affected the actions of P2-agonists and bradykinin themselves. Thus EK can induce phospholipase C activation and subsequent Ca2+ mobilization, provided that the cells have been previously or are simultaneously stimulated by endogenous adenine nucleotides or by externally applied P2-agonists or bradykinin. In this cross-talk mechanism between opioid receptors and these Ca(2+)-mobilizing agonist receptors, pertussis toxin-sensitive G-proteins play a permissive role.

Topics: Adenosine Triphosphate; Bradykinin; Calcium; Enkephalin, Leucine; Enkephalins; Enzyme Activation; Glioma; GTP-Binding Proteins; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Naloxone; Neuroblastoma; Pertussis Toxin; Receptors, Bradykinin; Receptors, Neurotransmitter; Receptors, Opioid; Receptors, Purinergic; Terpenes; Thapsigargin; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

Phosphatidylethanol is formed by phospholipase D in animal cells exposed to ethanol. Previous reports have demonstrated that the degradation of phosphatidylethanol is slow, indicating that this lipid may be present in the cells after ethanol itself has disappeared. Accumulation of an abnormal alcohol metabolite may influence cellular functions. In the present study, cultivation of NG108-15 neuroblastoma x glioma hybrid cells in the presence of ethanol resulted in an accumulation of phosphatidylethanol and a simultaneous increase in basal inositol 1,4,5-trisphosphate levels. The direct effects of phosphatidylethanol on the phosphoinositide signal transduction system were examined through incorporation of exogenous phosphatidylethanol into membranes of ethanol-naive cells. An incorporation amounting to 2.8% of cellular phospholipids was achieved after a 5-h incubation with 30 microM phosphatidylethanol. Phosphatidylethanol was found to cause a time- and dose-dependent increase in the basal levels of inositol 1,4,5-trisphosphate. The effects on inositol 1,4,5-trisphosphate levels of exogenously added phosphatidylethanol and ethanol exposure for 2 days were not additive. No effect on bradykinin-stimulated inositol 1,4,5-trisphosphate production could be detected. However, the increase in basal inositol 1,4,5-trisphosphate levels indicates that phosphatidylethanol affects inositol 1,4,5-trisphosphate turnover and emphasizes the importance of considering phosphatidylethanol as a possible mediator of ethanol-induced effects on cellular processes.

Topics: Animals; Bradykinin; Dose-Response Relationship, Drug; Ethanol; Glioma; Glycerophospholipids; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Kinetics; Neuroblastoma; Phosphatidic Acids; Signal Transduction; Tetradecanoylphorbol Acetate; Time Factors

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.

Topics: Animals; Bradykinin; Calcium; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Flunarizine; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Mice; Neuroblastoma; Nitrendipine; Terpenes; Thapsigargin; Tumor Cells, Cultured; Verapamil

Under whole-cell recording, bradykinin (BK) produced an initial outward membrane current followed by an inward current in voltage-clamped NG108-15 cells. The initial outward current was associated with a rise in intracellular Ca2+ and was accompanied by the opening of Ca(2+)-dependent K(+)-channels recorded with a cell-attached patch electrode. This current was inhibited by intracellular Mg2+. The inward current was associated with inhibition of the voltage-dependent K(+)-current IK(M). These effects accord with those previously observed in microelectrode-impaled cells, with the difference that BK produced much more pronounced and long-lasting desensitization in the patch-clamped cells.

Topics: Acetylcholine; Animals; Apamin; Bradykinin; Calcium; Calcium Channels; Charybdotoxin; Fluorescent Dyes; Glioma; Hybrid Cells; Indoles; Inositol 1,4,5-Trisphosphate; Ionomycin; Membrane Potentials; Mice; Neuroblastoma; Neurotoxins; Norepinephrine; Potassium Channels; Rats; Scorpion Venoms; Virulence Factors, Bordetella

Continuous superfusion of rat glioma cells with medium containing bradykinin (from 0.2 nM) induced a transient hyperpolarization followed by regular hyperpolarizing oscillations of the membrane potential. Similar repetitive hyperpolarizing oscillations were caused by extracellularly applied bradykinin or muscarine or by intracellularly injected GTP-gamma-S. The frequency of the oscillations was 1 per minute at bradykinin concentrations ranging from 0.2 nM to 2 microM, but the amplitude and duration increased with rising peptide concentration. The muscarine-induced oscillations were blocked by atropine. In the presence of extracellular Ca2+, the substances thapsigargin, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), and ionomycin reversibly suppressed the bradykinin-induced oscillations. Thapsigargin and tBuBHA, which are known to block the Ca2+ ATPase of endoplasmic reticulum, caused a transient rise in cytosolic Ca2+ activity, monitored with Fura-2, in suspensions of rat glioma cells or of mouse neuroblastoma-rat glioma hybrid cells. After a transient Ca2+ rise caused by thapsigargin, tBuBHQ, or ionomycin, the Ca2+ response to bradykinin which is known to be due to release of Ca2+ from internal stores was suppressed. This indicates that thapsigargin and tBuBHQ deplete internal Ca2+ stores as already seen previously for ionomycin. Thus, the inhibition of the membrane potential oscillations by thapsigargin, tBuBHQ, and ionomycin indicates that the oscillations are associated with activation of InsP3-sensitive Ca2+ stores. In some cells composite oscillation patterns which consisted of two independent oscillations with different amplitudes that overlapped additively were seen. We discuss that this pattern and the concentration dependency of the oscillations could be due to "quantal" Ca2+ release from stores with different inositol 1,4,5-triphosphate sensitivities. Subsidence of the oscillations after omission of extracellular Ca2+ seems to be due to a lack of replenishment of the intracellular stores with Ca2+, which comes from the extracellular compartment.

Topics: Animals; Benzoquinones; Bradykinin; Calcium; Cytosol; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Mice; Muscarine; Neuroblastoma; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured

The homotetrameric complex of inositol 1,4,5-triphosphate (InsP3) receptors displays a Ca2+ release activity in response to InsP3 molecules. Structure-function relationships of the mouse cerebellar InsP3 receptor have been studied by analyses of a series of internal deletion or C-terminal truncation mutant proteins expressed in NG108-15 cells. Within the large cytoplasmic portion of the InsP3 receptor, approximately 650 N-terminal amino acids are highly conserved between mouse and Drosophila, and this region has the critical sequences for InsP3 binding that probably form the three-dimensionally restricted binding site. The N-terminal region of each InsP3 receptor subunit also binds one InsP3 molecule. Cross-linking experiments have revealed that InsP3 receptors are intermolecularly associated at the transmembrane domains and/or the successive C termini. The interaction between the receptor subunit and InsP3 may cause a conformational change in the tetrameric complex, resulting in the opening of Ca2+ channels.

Topics: Amino Acid Sequence; Animals; Calcium Channels; Cell Line; Cell Membrane; Cerebellum; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Macromolecular Substances; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Neuroblastoma; Protein Conformation; Rats; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Restriction Mapping; Sequence Homology, Nucleic Acid; Structure-Activity Relationship; Transfection

Topics: Acetylcholine; Animals; Glioma; Inositol 1,4,5-Trisphosphate; Ion Channels; Membrane Potentials; Neuroblastoma; Neurons; Pheochromocytoma; Phosphatidylinositols; Second Messenger Systems

In a neuronal cell line (108CC15, NG108-15) the levels of inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4), as measured by receptor binding assays, rise transiently after stimulation with bradykinin (EC50 approx. 150 nM). Maximal InsP3 level of 354 pmol/mg protein (15-fold basal level) is obtained at 10-15 s after addition of bradykinin, the InsP4 level rises maximally to 78 pmol/mg protein (14-fold basal level) at 20-30 s. In a rat glioma cell line, bradykinin (2 microM) causes a fast 6-fold increase in InsP3 and InsP4 levels. In the neuronal cells the bradykinin-dependent rise of the inositolphosphate levels is diminished with reduced extracellular Ca2+ concentration. However, depletion of internal Ca2+ stores does not affect the bradykinin-induced rise in InsP3 and InsP4 levels. Homologous desensitization to bradykinin occurs in the signal transduction pathway already at the production of inositolphosphates, since after a 2 min stimulation with bradykinin the rise in cellular masses of InsP3 and InsP4, inducible by a following second bradykinin stimulus, is substantially reduced.

Topics: Animals; Bradykinin; Calcium; Cell Line; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Mice; Neuroblastoma

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.

Topics: Animals; Biological Transport; Bradykinin; Calcium; Calcium Channel Blockers; Cell Line; Cell Membrane; Fluorescent Dyes; Fura-2; Glioma; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Neuroblastoma; Nimodipine; omega-Conotoxins; PC12 Cells; Peptides, Cyclic; Terpenes; Thapsigargin

Topics: Animals; Autoradiography; Cell Line; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Neuroblastoma; Phosphatidylinositols; Phosphorus Radioisotopes; Radioligand Assay; Tritium; Type C Phospholipases

External application of bradykinin (BK) to mouse neuroblastoma X mouse fibroblast hybrid NL308 cells and mouse neuroblastoma X rat glioma hybrid NG108-15 cells produced a transient outward (hyperpolarizing) current. In NG108-15 cells, BK also induced an inward (depolarizing) current associated with a decrease in input membrane conductance, which results from the inhibition of a voltage-sensitive potassium current, the M-current. However, in NL308 cells, either no depolarization was elicited by BK or, even if the BK-induced depolarization was evoked, it was associated with an increased conductance. To explain the above difference, the intracellular second messenger system of NL308 cells was examined in detail. BK induced the rapid accumulation (three- to fivefold higher than the control level) of inositol 1,4,5-trisphosphate (InsP3) in NL308 cells. The cytosolic Ca2+ concentration was also elevated to 540 nM from 180 nM at a basal level. This seems to be enough to activate a voltage-independent and Ca2(+)-sensitive K+ current, resulting in the hyperpolarization. Intracellular injection of InsP3 replicated the hyperpolarization. NL308 cells possess protein kinase C (C-kinase), with specific activities of C-kinase in cytosolic and membrane fractions being 233 and 24 pmol/min/mg protein, respectively. The activity associated with particulates became higher after phorbol dibutyrate (PDBu) treatment. But NL308 cells did not show the characteristic inward relaxation by step hyperpolarizations and the outward rectification in the current-voltage relationship, indicating that the M current is deficient in NL308 cells. Therefore, application of PDBu failed to mimic the inward current. The results suggest the role of InsP3 and C-kinase in controlling two K+ currents.

Topics: Animals; Bradykinin; Fibroblasts; Glioma; Hybridomas; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Mice; Neuroblastoma; Potassium; Second Messenger Systems

The intracellular nonmitochondrial calcium pools of saponin-permeabilized NG108-15 cells were characterized using inositol 1,4,5-trisphosphate (IP3) and GTP. IP3 or GTP alone induced release of 47 and 68%, respectively, of the calcium that was releasable by A23187. GTP induced release of a further 24% of the calcium after IP3 treatment, whereas IP3 induced release of a further 11% of the calcium after GTP treatment. Guanosine 5'-O-(3-thio)triphosphate had little effect on IP3-induced calcium release but completely inhibited GTP-induced calcium release. In contrast, heparin inhibited the action of IP3 but not that of GTP. The results imply the existence of at least three nonmitochondrial pools: (a) 31% is releasable by IP3 and GTP, (b) 11% is releasable by IP3 alone, and (c) 24% is releasable by GTP alone. GTP enhanced calcium uptake in the presence of oxalate with an EC50 of 0.6 microM and stimulated calcium release in the absence of oxalate with an EC50 of 0.32 microM. The similar EC50 values for these dual effects of GTP on calcium movement suggest that GTP exerts its dual action by the same mechanism.

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Calcimycin; Calcium; Calcium-Transporting ATPases; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Cell Membrane Permeability; Glioma; Guanosine Triphosphate; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Kinetics; Mice; Neuroblastoma; Oligomycins; Rats

In the neuroblastoma X glioma hybrid cell line NG108-15, bradykinin (BK) receptor stimulation induced a rapid and concentration-dependent rise in cytosolic free Ca2+ levels, as measured with the Ca2(+)-sensitive fluorescent dye fura-2. The Ca2+ transient was present in the absence of extracellular Ca2+ and was associated with a concentration-dependent production of inositol phosphates, particularly inositol trisphosphate (InsP3). Pretreatment of intact NG108-15 cells with forskolin or dibutyryl-cAMP plus isobutylmethylxanthine reduced BK-stimulated InsP3 production and the increase in cytosolic free Ca2+. Membranes prepared from forskolin- and [3H]inositol-pretreated NG108-15 cells also showed a diminished production of InsP3 elicited by guanosine 5'-[gamma-thio]triphosphate, NaF, or BK plus GTP. On the other hand, the Ca2+ sensitivity of membrane-associated phosphoinositide-specific phospholipase C (PI-PLC) was unaffected by forskolin pretreatment of intact NG108-15 cells. Collectively, these results suggest that A-kinase may inhibit receptor-mediated and postreceptor stimulation of PI-PLC in neuron-like cells, perhaps by impairing the coupling between a guanine nucleotide-binding protein and PI-PLC.

Topics: Adenylyl Cyclases; Bradykinin; Bucladesine; Calcium; Colforsin; Glioma; GTP-Binding Proteins; Humans; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Neuroblastoma; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases; Tumor Cells, Cultured

The effects of endothelin-1 (ET) on the signal transduction in rat and human glioma cell line cells have been investigated. ET was found to initiate the increase of intracellular calcium ([Ca2+]i) levels in both C6 and A-172 cells, which was concurrent with the formation of inositol 1,4,5-trisphosphate (IP3(1,4,5)). In the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in the incubation media, the duration of the intracellular calcium response was reduced, indicating that the ET-induced increase of intracellular calcium in glioma cells may be mediated by a dual mechanism, intracellular calcium mobilization and influx of extracellular calcium. These results suggest that ET may also act as a neuropeptide in the central nervous system.

Topics: Animals; Calcium; Cell Line; Endothelins; Glioma; Humans; Inositol 1,4,5-Trisphosphate; Neurons; Peptides; Rats; Signal Transduction

We examined the signal transduction mechanism responsible for the IFN-gamma-induced HLA class II molecule expressions on glioblastoma cell lines, T98G and A172. A series of experiments demonstrated that the activation of protein kinase C (PKC) is involved in the DR and DP molecule expressions on T98G cells. In addition to the activation of PKC, calcium influx appeared to be involved in the DR and DP molecule expressions on T98G. Northern blot analyses with actinomycin D or cycloheximide revealed that these second messengers induce the transcription of DRA and B and DPA and B genes without de novo protein synthesis. Furthermore, we examined the region of the DPB gene that is responsible for IFN-gamma-induced gene transcription by gene transfer of a series of 5' and 3' deletion mutants in which the upstream region of the DPB was linked to a reporter gene, chloramphenicol acetyltransferase. By using these deletion mutants, it appeared that the region between -152 and -126 bp contains a critical IFN-gamma-responsive element. Taken together, these results suggest that IFN-gamma activates PKC and stimulates calcium influx, resulting in the induction of transcription of DRA and B and DPA and B genes without de novo protein synthesis. In DPB gene, we speculate that preexiting protein(s) phosphorylated by PKC in the presence of Ca2+ might directly bind or indirectly interact with the region between -152 and -126 bp of the upstream sequence, leading to the induction of the transcription (possibly in concert with other nuclear protein(s) bound to the promoter sequences).

Topics: Biological Transport; Blotting, Northern; Calcimycin; Calcium; Cell Compartmentation; Enzyme Activation; Gene Expression Regulation, Neoplastic; Glioma; HLA-D Antigens; HLA-DP Antigens; HLA-DR Antigens; Humans; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Promoter Regions, Genetic; Protein Kinase C; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured

Extracellular application of bradykinin and injection of inositol-1,4,5-trisphosphate (Ins-P3) induced a hyperpolarization in polyploid rat glioma cells. Ins-1,4,5-P3 and Ins-2,4,5-P3 were effective but not Ins-4,5-P2, Ins-1,3,4,5-P4 and Ins-1,3,4,5,6-P5. The reversal potential of the hyperpolarizing response induced by bradykinin or by Ins-P3 increased to a comparable degree with increasing the extracellular K+ concentration. Certain blockers of K+ channels, for example charybdotoxin (5-50 nM), Ba2+ (5-20 mM), 4-aminopyridine (5-10 mM) and quinidine (0.1-0.5 mM) reversibly suppressed the membrane potential response to bradykinin or to Ins-P3; however, apamin (1 microM) and D-tubocurarine (0.5 mM) had no effect. Intracellular injection of EGTA made the glioma cells unresponsive to bradykinin. Superfusion of the cells with Ca2(+)-free medium gradually and reversibly abolished the response to bradykinin, but only slightly reduced the effect of Ins-P3. The Ca2+ channel blockers Co2+ (1-5 mM), Mn2+ (2-6 mM) and nifedipine (1-20 microM), but not desmethoxyverapamil (100 microM) inhibited the hyperpolarizing effect of bradykinin. The hyperpolarization induced by Ins-P3, however, was not influenced by Mn2+ (1-5 mM) or by Co2+ (7 mM). Injection of Ca2+ into the glioma cells induced a hyperpolarization susceptible to Ba2+ and quinidine. Treatment of glioma cells with an activator or with inhibitors of protein kinase C or with pertussis toxin did not affect the response to bradykinin. Incubation of the cells with the Ca2+ ionophore A23187 (0.1-1 microM) made the cells unresponsive to bradykinin and, somewhat less, to Ins-P3. At these concentrations the Ca2+ ionophore primarily depletes intracellular Ca2+ stores. In summary, bradykinin, via B2-receptors (blocked by [Thi5,8, D-Phe7]-bradykinin) activates a K+ conductance in glioma cells following a rise of cytosolic Ca2+ activity most likely due to Ins-P3-mediated release of Ca2+ from internal stores. Entry of extracellular Ca2+ appears also to be involved in this process.

Topics: Animals; Bradykinin; Calcium; Calcium Channel Blockers; Glioma; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Potassium; Rats; Tumor Cells, Cultured

The mechanism underlying the bradykinin (BK)-induced increase of acetylcholine (ACh) release was studied in neuroblastoma x glioma hybrid NG108-15 cells and their synapses formed onto mouse muscle cells. External application of BK or iontophoretic injection of extrinsic inositol 1,4,5-trisphosphate (InsP3) into the cytoplasm of NG108-15 cells produced membrane hyperpolarization in the hybrid cells and an increase in the frequency of miniature end-plate potentials (MEPPs) in paired myotubes. Ba2+ blocked the hyperpolarization in response to BK, but facilitation of MEPPs was still observed. InsP3-dependent facilitation of MEPPs was also observed in cells where the InsP3 injections produced no detectable hyperpolarization or even depolarization. Real-time quantitative monitoring of intracellular free Ca2+ concentration [( Ca2+]i) with fura-2 in single NG108-15 cells showed that BK application or InsP3 injection induced an elevation of [Ca2+]i which coincided in time with membrane hyperpolarization recorded from the same cell. The [Ca2+]i rise produced by InsP3 injection started from the single site of injection and that produced by BK began from a deep compartment of the cytoplasm of the NG108-15 cells. The BK- and InsP3-evoked facilitation of MEPPs and the [Ca2+]i rise were relatively independent of extracellular Ca2+. These findings suggest that the BK-induced ACh release results not from membrane potential changes but from a transient InsP3-dependent elevation of [Ca2+]i.

Topics: Acetylcholine; Animals; Barium; Bradykinin; Calcium; Cell Line; Evoked Potentials; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Mice; Neuroblastoma; Phorbol 12,13-Dibutyrate; Rats; Synapses

C6 rat glioma cells persistently infected with subacute sclerosing panencephalitis virus (C6/SSPE) were treated with measles antiserum and purified anti-measles IgG. This stimulated phosphoinositide breakdown and an increase in inositol phosphates. In uninfected C6 cells, however, only fetal calf serum (FCS), but not measles antiserum could induce inositol polyphosphate production.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Viral; Antigens, Viral; Glioma; Hemagglutinins, Viral; Immune Sera; Immunoglobulin G; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Measles virus; Membrane Proteins; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Rats; Signal Transduction; SSPE Virus; Tumor Cells, Cultured; Viral Proteins

The net content of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was measured in bradykinin (BK)-stimulated NIH3T3 fibroblasts and neuroblastoma-glioma hybrid cells (NG108-15). BK-mediated production of Ins(1,4,5)P3 was not affected by replacing the medium with Ca2+-free medium, but addition of EGTA (1mM) to Ca2+-free medium markedly prevented production of Ins(1,4,5)P3. Although pertussis toxin (PT) treatment caused ADP-ribosylation in both NIH3T3 cells and NG108-15 cells, the BK-induced Ins(1,4,5)P3 formation was considerably reduced in the former cells but not in the latter cells, suggesting that PT-sensitive and PT-insensitive GTP-binding proteins are involved in phosphoinositide phospholipase C (PI-PLC) activation in fibroblasts and neuroblastoma cells, respectively. In NG108-15 cells down-regulated in protein kinase C (PKC) by long-term exposure to phorbol 12-myristate 13-acetate (PMA), BK-stimulated Ins(1,4,5)P3 accumulation was significantly enhanced compared to control cells.

Topics: Adenosine Diphosphate Ribose; Bradykinin; Calcium; Enzyme Activation; Fibroblasts; Glioma; GTP-Binding Proteins; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Neuroblastoma; Pertussis Toxin; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases; Protein Kinase C; Sugar Phosphates; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Virulence Factors, Bordetella

Bradykinin (BK) activation of phosphatidylinositide breakdown in NG108-15 neuroblastoma x glioma hybrid cells in the generation of an outward K+ current through the release of Ca2+ by the intermediary messenger inositol 1,4,5-trisphosphate (InsP3). Channels mediating this outward current were identified using cell-attached patch electrodes. Intracellular iontophoretic injection of InsP3 or Ca2+, or extracellular application of BK, evoked bursts of K+ channel activity coincident with cell hyperpolarization measured with an intracellular recording micropipette. The most frequent channels had a mean single-channel conductance of about 40 pS in symmetrical K+ solutions; additional openings of lower conductance (18 pS) channels were also detected. Bath application of phorbol dibutyrate (PDBu, 1 microM) increased the number and opening probability of the InsP3-induced channels.

Topics: Animals; Bradykinin; Calcium; Electric Conductivity; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Mice; Neuroblastoma; Phorbol 12,13-Dibutyrate; Potassium Channels; Rats; Sugar Phosphates; Tumor Cells, Cultured

Application of bradykinin to voltage-clamped N1E-115 mouse neuroblastoma cells evoked sequential outward and inward membrane currents, accompanied by an increase and decrease of membrane conductance, respectively. Methacholine produced an inward current with a decreased conductance. The outward current response to bradykinin was imitated by intracellular inositol 1,4,5-trisphosphate (IP3). Bath application of phorbol dibutyrate induced an inward current and potentiated the response to IP3. We conclude that the response of these cells to bradykinin is identical to that of NG108-15 hybrid cells, and therefore may be attributed to the dual effects of inositol trisphosphate and diacylglycerol formed by hydrolysis of phosphatidylinositide.

Topics: Animals; Bradykinin; Cell Line; Electric Conductivity; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ion Channels; Membrane Potentials; Mice; Neuroblastoma; Phorbol Esters; Potassium

The effect of bradykinin on membrane potential, level of cyclic nucleotides and of cytosolic Ca2+-activity was determined in neural cell lines. Bradykinin induced a transient hyperpolarization followed by a depolarization in mouse neuroblastoma x rat glioma hybrid cells and in polyploid rat glioma cells. The reversal potential of the hyperpolarizing response depended on the extracellular K+ concentration. The K+ channel blockers, Ba2+, quinidine, and 4-aminopyridine, inhibited the response to bradykinin. This suggests that the hyperpolarization of ca. 1 min duration, which was accompanied by a decreased input resistance, is due to activation of K+ channels. Upon addition of bradykinin to the cells the cytosolic Ca2+-activity increased transiently. Ca2+ was involved in the induction of the hyperpolarization by bradykinin, since both removal of extracellular Ca2+ and injection of EGTA into the cells suppressed the membrane potential response. Bradykinin induced the formation of inositol-1,4,5-trisphosphate (IP3), an agent known to release Ca2+ from intracellular stores, and stimulated the uptake of 45Ca2+ into the cells. Therefore the increased level of intracellular Ca2+ activating the K+ conductance could be due to two components: release from intracellular pools and uptake. IP3 seems to be involved in the membrane potential response, because intracellular injection of either IP3 or Ca2+ into the glioma cells elicited a hyperpolarizing response which resembled that after application of bradykinin and was also susceptible to the K+ channel blocking agents listed above. However, the formation of cyclic GMP by bradykinin apparently plays no role in the membrane potential effect of bradykinin.

Topics: Animals; Bradykinin; Calcium; Cyclic GMP; Glioma; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ion Channels; Membrane Potentials; Neuroblastoma; Neurons; Potassium; Sugar Phosphates; Tumor Cells, Cultured

In neuroblastoma x glioma hybrid NG108-15 cells, bradykinin (BK) receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates and mobilization of intracellular calcium. Treatment of the cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) suppressed the spike phase of increases in intracellular calcium concentration. In radioligand binding studies, TPA treatment did not interfere with [3H]BK specific binding to intact cells or to cell membranes. The ability of guanyl-5'-yl-imidodiphosphate to promote the conversion of the high affinity sites of the BK receptors into a low affinity sites was unaffected by TPA. TPA treatment showed the dose-dependent, noncompetitive inhibition of BK-stimulated formation of inositol trisphosphate. In the membrane preparations from TPA-treated cells, guanosine 5'-(3-O-thio)triphosphate-stimulated inositol trisphosphate formation was inhibited by 50%. These data indicate that TPA exerts its inhibitory action on BK responses at the sites of guanine nucleotide-binding protein or phospholipase C or both.

Topics: Bradykinin; Calcium; Cell Line; Diglycerides; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Neuroblastoma; Protein Kinase C; Receptors, Bradykinin; Receptors, Neurotransmitter; Sugar Phosphates; Tetradecanoylphorbol Acetate; Thionucleotides; Type C Phospholipases

The addition of bradykinin to NG108-15 cells results in a transient hyperpolarization followed by prolonged cell depolarization. Injection of inositol 1,4,5-trisphosphate or Ca2+ into the cytoplasm of NG108-15 cells also elicits cell hyperpolarization followed by depolarization. Tetraethylammonium ions inhibit the hyperpolarizing response of cells to bradykinin or inositol 1,4,5-trisphosphate. Thus, the hyperpolarizing phase of the cell response may be due to inositol 1,4,5-trisphosphate-dependent release of stored Ca2+ into the cytoplasm, which activates Ca2+-dependent K+ channels. The depolarizing phase of the cell response to bradykinin is due largely to inhibition of M channels, thereby decreasing the rate of K+ efflux from cells and, to a lesser extent, to activation of Ca2+-dependent ion channels and Ca2+ channels. In contrast, injection of inositol 1,4,5-trisphosphate or Ca2+ into the cytosol did not alter M channel activity. Incubation of NG108-15 cells with pertussis toxin inhibits bradykinin-dependent cell hyperpolarization and depolarization. Bradykinin stimulates low Km GTPase activity and inhibits adenylate cyclase in NG108-15 membrane preparations but not in membranes prepared from cells treated with pertussis toxin. Reconstitution of NG108-15 membranes from cells treated with pertussis toxin with nanomolar concentrations of a mixture of highly purified No and Ni [guanine nucleotide-binding proteins that have no known function (No) or inhibit adenylate cyclase (Ni)] restores bradykinin-dependent activation of GTPase and inhibition of adenylate cyclase. These results show that [bradykinin . receptor] complexes interact with No or Ni and suggest that No and/or Ni mediate the transduction of signals from bradykinin receptors to phospholipase C and adenylate cyclase.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Bradykinin; Calcium; Cell Line; Diglycerides; Glioma; GTP-Binding Proteins; Humans; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Membrane Potentials; Neuroblastoma; Pertussis Toxin; Sugar Phosphates; Tetradecanoylphorbol Acetate; Tetraethylammonium; Tetraethylammonium Compounds; Virulence Factors, Bordetella

Subcellular fractions of neuroblastoma x glioma (NG108-15) hybrid cells were used to study the mechanism of inositol 1,4,5-trisphosphate-induced calcium release. A microsomal fraction, enriched in endoplasmic reticulum and plasma membranes and almost devoid of mitochondria, was the most active in inositol trisphosphate- or GTP-dependent release of calcium. Neither GTP nor inositol 1,4,5-trisphosphate affected the calcium efflux mediated by the other reagent, suggesting that inositol trisphosphate and GTP act on different calcium-sequestrating vesicles. The stimulation of calcium release by GTP was relatively slow (t1/2 = 90 s), dependent on polyethyleneglycol, and greater at 2 X 10(-5) M calcium (5 nmol X min-1 X mg-1) than at 10(-6) M calcium (0.8 nmol X min-1 X mg-1). The inositol trisphosphate-induced calcium efflux was not mimicked by inositol monophosphate; it was fast (t1/2 less than 10 s) and unaffected by 3% polyethyleneglycol. The amount of calcium released by inositol trisphosphate was greatest at 10(-6) M external calcium (1 nmol X min-1 X mg-1) and it was undetectable at 2 X 10(-5) M calcium. A feedback inhibition of the inositol trisphosphate-induced calcium release by cytoplasmic calcium provides a safety mechanism preventing deleterious effects of abnormally high calcium levels.

Topics: Animals; Calcium; Cell Line; Cell Membrane; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Mice; Microsomes; Neuroblastoma; Rats; Sugar Phosphates

Studies were undertaken to further elucidate the mechanism(s) by which bradykinin-dependent phosphoinositide metabolism takes place in neuroblastoma X glioma hybrid NG108-15 cells [(1984) J. Biol. Chem. 259, 10201-10207] using [3H]inositol-labelled cells. Bradykinin produced net increases in the level of [3H]inositol phosphates, especially of [3H]inositol trisphosphate which is formed transiently and most rapidly. The results indicate that bradykinin activates a phosphodiesterase to break down phosphatidylinositol 4,5-bisphosphate, generating two recently recognized intracellular messengers, 1,2-diacylglycerol and inositol trisphosphate.

Topics: Animals; Bradykinin; Cell Line; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Neuroblastoma; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphoric Diester Hydrolases; Receptors, Bradykinin; Receptors, Neurotransmitter; Sugar Phosphates