Stimulation of Phosphoinositide Hydrolysis by Serotonin in C6 Glioma Cells

5-Hydroxytryptamine (serotonin or 5-HT) stimulated the incorporation of 32Pi into phosphatidylinositol (PI) but not into polyphosphoinositides in C6 glioma cells with an EC50 of 1.2 X 10(-7) M. The phosphoinositide response was blocked by the 5-HT2 antagonists ketanserin and spiperone but inhibited only partly by methysergide and mianserin. Atropine, prazosin, and yohimbine did not block the response, whereas fluphenazine and haloperidol did so partially but also inhibited basal incorporation by approximately 30%. The 5-HT1A agonist 8-hydroxy-2(di-n-propylamino)tetralin did not cause stimulation. Incubation with 5-HT (1 microM) for 1 h increased the incorporation of [2-3H]myoinositol into all phosphoinositides but not into inositol phosphates (IPs). Li+ alone at 10 mM increased labeling in inositol bisphosphate (IP2) and trisphosphate (IP3), whereas labeling in IP and phosphoinositides remained unaltered. Addition of 5-HT had no effect on this increase. Mn2+ at 1 mM enhanced labeling in PI, PI-4-phosphate, lyso-PI, glycerophosphoinositol, and IP, but the presence of 5-HT again did not cause further stimulation. 5-HT also stimulated the release of IPs in cells prelabeled with [2-3H]myo-inositol, incubated with LiCl (10 mM) and inositol (10 mM), and then exposed to 5-HT (1 microM). Radioactivity in IP2 and IP3 was very low, was stimulated approximately 50% as early as 30 s, and remained elevated for at least 20 min. Radioactivity in IP was at least 10 times as high as in IP3 but was increased only from 3 min on with a peak at 20 min, when the elevation was approximately 40 times that in IP3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of Formation of Inositol Phosphates in Primary Cultures of Bovine Adrenal Chromaffin Cells by Angiotensin II, Histamine, Bradykinin, and Carbachol

Histamine, bradykinin, and angiotensin II stimulate release of catecholamines from adrenal medulla. Here we show, using bovine adrenal chromaffin cells in culture, that these agonists as well as carbachol (with hexamethonium) stimulate production of inositol phosphates. The histamine response was mepyramine sensitive, implicating an H1 receptor, whereas bradykinin had a lower EC50 than Met-Lys-bradykinin, and [Des-Arg9]-bradykinin was relatively inactive, implicating a BK-2 receptor. Total inositol phosphates formed in the presence of lithium were measured, with histamine giving the largest response. The relative contribution of chromaffin cells and nonchromaffin cells in the responses was assessed. In each case chromaffin cells were found to be responding to the agonists; in the case of histamine the response was solely on chromaffin cells. When the inositol phosphates accumulating over 2 or 5 min, with no lithium present, were separated on Dowex anion-exchange columns, bradykinin gave the greatest stimulation in the inositol trisphosphate fraction, whereas histamine gave a larger inositol monophosphate accumulation. On resolution of the isomers of stimulated inositol trisphosphate after 2 min of stimulation, the principal isomer present was inositol 1,3,4-trisphosphate in each case. Two hypotheses for the differential responses to histamine and bradykinin are discussed.     

Lithium Effects on Inositol Phospholipids and Inositol Phosphates: Evaluation of an In Vivo Model for Assessing Polyphosphoinositide Turnover in Brain

Administration of lithium chloride to rats injected intracerebrally with [3H]inositol led to time- and dose-dependent increases in levels of labeled inositol monophosphates in brain. Quantitative analysis of the inositol phosphates by ion chromatography revealed 37- and 20-fold increases in the mass of myo-inositol 1-phosphate and 4-phosphate, respectively, at 4 h intraperitoneal after injections of 6 mEq/kg of lithium chloride. Albeit to a much lesser extent, lithium administration also resulted in an increase in the level of myo-inositol, 1,4-bisphosphate in brain. The lithium-induced increase in content of labeled inositol monophosphates was marked by a concomitant decrease in content of labeled inositol, and after injections of high doses of lithium, e.g., 10 mEq/kg, this was followed by a general decrease in labeling of the inositol phospholipids. In general, animals injected with [3H]inositol but not lithium did not reveal obvious differences in labeling of inositol monophosphates on stimulation by mecamylamine or pilocarpine. However, when animals were injected with [3H]inositol and then lithium, there were large increases in the levels of labeled inositol monophosphates on administration of these compounds. Administration of atropine to the lithium-treated mice led to a partial reduction in the amount of labeled inositol monophosphates accumulated due to the administration of lithium alone. Furthermore, atropine was able to block the pilocarpine-induced increase in level of labeled inositol monophosphates. These results demonstrate the suitable use of the radiotracer technique together with lithium administration for assessing the effects of drugs and receptor agonists on the signaling system involving polyphosphoinositide turnover in brain.     

Effects of Phorbol Esters and Forskolin on Basal and Histamine-induced Accumulation of Inositol Phosphates in Cultured Bovine Adrenal Chromaffin Cells

The effect of phorbol esters and forskolin pretreatment on basal and histamine-induced accumulation of inositol phosphates and catecholamine release was examined in cultures of bovine adrenal chromaffin cells. Histamine caused a dose-dependent, Ca2+-dependent accumulation of total inositol phosphates with an EC50 at approximately 1 microM and an eight- to 10-fold increase at 100 microM within 30 min of incubation. Histamine (10 microM) also caused the release of cellular catecholamines amounting to some 2.8% of cellular stores released over a 20-min period. Both the inositol phosphate and catecholamine responses were completely blocked by the H1-antagonist mepyramine and were insensitive to the H2-antagonist cimetidine. Examination of the time course of accumulation of the individual inositol phosphates stimulated by histamine revealed an early and sustained rise in inositol 1,4-bisphosphate content but not inositol 1,4,5-trisphosphate content at 1 min and the overall largest accumulation of inositol monophosphate after 30 min of stimulation. Pretreatment with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent, time-dependent inhibition of histamine-induced inositol phosphate formation and catecholamine secretion. In this inhibitory action, PMA exhibited high potency (IC50 of approximately 0.5 nM), an effect not shared by the inactive phorbol ester 4-alpha-phorbol 12,13-didecanoate. Pretreatment with forskolin, on the other hand, only marginally inhibited the histamine-induced inositol phospholipid metabolism and catecholamine secretion. These data suggest that protein kinase C activation in chromaffin cells may mediate a negative feedback control on inositol phospholipid metabolism.     

Histamine-Stimulated Inositol Phospholipid Metabolism in Bovine Adrenal Medullary Cells: A Kinetic Analysis

Abstract: Histamine stimulation of bovine adrenal medullary cells rapidly activated phospholipase C. [³H]Inositol 1,4,5-trisphosphate [[³H]Ins(1,4,5)P₃] levels were transiently increased (200% of basal values between 1 and 5 s) before declining to a new steady-state level of ～140% of basal values. [³H]Inositol 1,4-bisphosphate [[³H]Ins(1,4)P₂] content increased to a maximal and maintained level of 250% of basal values after 1 s, whereas levels of [³H]inositol 1,3,4-trisphosphate [[³H]-Ins(1,3,4)P₃], [³H]inositol 1,3-bisphosphate, and [³H]-inositol 4-monophosphate ([³H]Ins4P) increased more slowly. The rapid responses were not reduced by the removal of extracellular Ca²⁺, but they were no longer sustained over time. The turnover rates of selected inositol phosphate isomers have been estimated in the intact cell. [³H]Ins(1,4,5)P₃ was rapidly metabolized (t_1/2 of 11 s), whereas the other isomers were metabolized more slowly, with t_1/2 values of 113, 133, 104, and 66 s for [³H]Ins(1,3,4)P₃, [³H]Ins(1,4)P₂, an unresolved mixture of [³H]inositol 1- and 3-monophosphate ([³H]Ins1/3P), and [³H]Ins4P, respectively. The calculated turnover rate of [³H]Ins(1,4,5)P₃ was sufficient to account for the turnover of the combination of both [³H]Ins(1,4)P₂ and [³H]Ins(1,3,4)P₃ but not that of [³H]Ins1/3P or [³H]Ins4P. These observations demonstrate that histamine stimulation of these cells results in a complex Ca²⁺-dependent and -independent response that may involve the hydrolysis of inositol phospholipids in addition to phosphatidylinositol 4,5-bisphosphate.     

Neuronotrophic Factors Released by C6 Glioma Cells

Glial cells have been shown previously to release factors that promote survival of central and peripheral neurons [neuronotrophic factors (NTFs)]. We have investigated the release of NTFs by C6 cells, a rat glioma cell line, under different modes of conditioning. Media conditioned in the presence or absence of serum [C6 cell conditioned media (C6CMs)] were analyzed using biological, biochemical, and immunological assays. We report that (a) nuclear and cytoskeletal proteins were not present in C6CMs, indicating that C6CM proteins result from release by C6 cells rather than from cell death; (b) C6CM contained 1-3 micrograms protein/ml, corresponding to a secretion rate of about 0.5 pg protein per cell and day; (c) C6CM contained the neurite-promoting factor laminin and low amounts of nerve growth factor; (d) the presence of fetal calf serum in the culture medium was essential for synthesis and release of NTFs; and (e) our C6CM contained at least three NTFs differing by their temporal secretory patterns and three NTFs differing by biochemical properties, indicating that C6 cells produce and secrete six different NTFs. Within these, nerve growth factor seems to be the only established NTF.     

Differential Effects of Lithium on Muscarinic Receptor Stimulation of Inositol Phosphates in Rat Cerebral Cortex Slices

The accumulation of labelled inositol mono-, bis-, and trisphosphate in rat cerebral cortex slices was examined following preincubation with [3H]inositol. The muscarinic receptor agonist carbachol produced a rapid and sustained increased accumulation of each labelled inositol phosphate both in the presence and absence of 5 mM lithium. Lithium potentiated carbachol-stimulated accumulation of inositol monophosphate (EC50 0.5 mM) and inositol bisphosphate (EC50 4 mM) in a concentration-dependent manner. However, exposure to lithium in the presence of the muscarinic agonist produced a concentration- and time-dependent inhibition of inositol trisphosphate accumulation that was not related to receptor desensitisation. Although the present data do suggest that polyphosphoinositides are substrates for agonist-stimulated phospholipase C in brain, these results may not be entirely consistent with the production of inositol mono- and bisphosphate through inositol trisphosphate dephosphorylation. Furthermore, these data suggest site(s) additional to inositol monophosphatase that are affected by lithium.     

Differential Stimulation of Inositol Phospholipid Turnover in Brain by Analogs of Oxotremorine

Structural analogs of oxotremorine have been employed to examine the relationship between the binding of agonists to muscarinic receptors in guinea pig cerebral cortex and the enhancement of inositol lipid turnover. Large differences were observed in the ability of the analogs to stimulate inositol phospholipid turnover, as measured both by the increase in labeling of phosphatidate and phosphatidylinositol from 32Pi in a nerve-ending fraction, and by the stimulated release of labeled inositol phosphates from slices of cerebral cortex, a direct measure of inositol lipid breakdown. The quaternary N+ analogs, oxotremorine-M and its N-methylacetamide derivative, were five to thirteen times as effective as oxotremorine. In contrast, methyl substitution of the pyrrolidone ring of oxotremorine resulted in a complete loss of agonist activity. Receptor occupancy data obtained from the displacement of labeled quinuclidinyl benzilate bound to receptors in a nerve-ending fraction indicated that the more efficacious agonists interacted with at least two affinity forms of the muscarinic receptor, whereas the less effective agonists bound to a single affinity form. Dose-response curves obtained in the presence of oxotremorine-M for inositol lipid turnover in both the nerve-ending fraction and slice preparation correlated with the occupancy of a single low-affinity form of the muscarinic receptor. The results suggest that the differential abilities of analogs of oxotremorine to enhance inositol lipid turnover in brain are closely related to the extent of agonist-induced conformational change in the muscarinic receptor.     

Effect of Desipramine on a Glycoprotein Sialyltransferase Activity in C6 Cultured Glioma Cells

The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin-dependent kinases, gave the same concentration-dependent inhibition profile of sialyltransferase as desipramine, whereas H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide-dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin-dependent system.     

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophosphate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mM Ca2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+-dependent [3H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3-10 microM) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catecholamine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and myo-inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.     

A Novel Inhibitory Role for Glucocorticoids in the Secretion of Angiotensinogen by C6 Glioma Cells

Abstract: Astrocytes have been identified as the primary source of brain angiotensinogen (Ao), but the regulation of the secretion of this protein from astrocytes is poorly defined. In this study, the rat C6 glioma cell line was used as an astrocyte model to investigate the regulation of Ao secretion. C6 cultures secreted Ao at a rate of 4.05 ± 1.52 (mean ± SD) ng of Ao/10⁶ cells/24 h as determined by a direct radioimmunoassay. This rate was not significantly altered by the hormones thyroxine, estradiol, angiotensin II, growth hormone, and prostaglandins or by increased levels of intracellular cyclic AMP. Treatment with the synthetic glucocorticoid dexamethasone (DEX; 10–⁶M) reduced the rate of Ao secretion to 1.82 ± 0.28 ng of Ao/10⁸ cells/24 h. By comparison, the basal secretion rate for rat H4 hepatoma cells was 142.4 ± 10.0 ng of Ao/10⁶ cells/24 h, and this increased fourfold (572.4 ± 173.1 ng/10⁶ cells/ 24 h) in the presence of 10–⁶M DEX. Both these inhibitory (C6) and stimulatory (H4) actions of DEX were dose related. The inhibition observed in C6 cells was mimicked by RU28362, a pure glucocorticoid agonist, and reversed by the antagonist RU486, demonstrating that DEX was functioning as a true glucocorticoid. The action of DEX was also antagonized by the cyclic AMP analogue N⁶,2′-O- dibutyryladenosine 3′:5′-cyclic monophosphate (dBcAMP) (control, DEX, and DEX + dBcAMP, 3.58 ± 0.73, 1.69 ± 0.82, and 4.93 ± 1.88 ng of Ao/10⁶ cells/24 h, respectively, and by the β-adrenergic agonist isoprenaline, which stimulates cyclic AMP production. It was concluded that glucocorticoids inhibit Ao secretion, possibly by interacting with a cyclic AMP-responsive pathway. The inhibition of Ao production by DEX is a novel observation supporting the view that regulation of Ao is tissue specific.     

Stimulus-Induced Accumulation of Inositol Tetrakis-, Pentakis-, and Hexakisphosphates in N1E-115 Neuroblastoma Cells

When [3H]inositol-prelabelled N1E-115 cells were stimulated with carbamylcholine (CCh) (100 microM), high K+ (60 mM), and prostaglandin E1 (PGE1) (10 microM), a transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached its maximum level at 15 s and had declined to the basal level at 2 min. CCh, high K+, and PGE1 also caused accumulations of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], [3H]inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and [3H]inositol hexakisphosphate (InsP6). Muscarine and CCh induced accumulations of [3H]Ins(1,4,5)P3, [3H]-Ins(1,3,4,6)P4, [3H]InsP5, and [3H]InsP6 with a similar potency and exerted these maximal effects at 100 microM, whereas nicotine failed to do so at 1 mM. With a slower time course, CCh, high K+, and PGE1 caused accumulations of [3H]-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In an N1E-115 cell homogenate, [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3 were converted to [3H]InsP5 through [3H]-Ins(1,3,4,6)P4. The above results indicate that Ins(1,3,4,6)P4, InsP5, and InsP6 are rapidly formed by several kinds of stimulants in N1E-115 cells.     

Direct and Continuous Electrochemical Measurement of Noradrenaline-Induced Nitric Oxide Production in C6 Glioma Cells

1. Nitric oxide (NO) production in C6 glioma cells was directly monitored in real time by electrochemical detection with a NO-specific biosensor.2. We present here the first direct evidence that noradrenaline elicits long-lasting NO production in C6 cells pretreated with lipopolysaccharide and interferon-, an effect blocked by N ^G-monomethyl-L-arginine, a NO synthase inhibitor.3. This direct electrochemical measurement of glia-derived NO should facilitate our understanding of the kinetics of glial signaling in glia-glia and glia-neuron networks in the brain.     

Stimulation by ATP of Inositol Trisphosphate Accumulation and Calcium Mobilization in Cultured Adrenal Chromaffin Cells

Effects of ATP on accumulation of inositol phosphates and Ca2+ mobilization were investigated in cultured bovine adrenal chromaffin cells. When the cells were stimulated with 30 microM ATP, a rapid and transient rise in intracellular Ca2+ concentration was observed. At the same time, ATP rapidly increased accumulation of inositol phosphates. The concentration-response curve for the ATP-induced Ca2+ mobilization was similar to that for inositol trisphosphate (IP3) accumulation. ATP exerted its maximal effects at 30 microM for either IP3 accumulation or Ca2+ mobilization. The order of the efficacy of the agonists for IP3 accumulation and Ca2+ mobilization at 100 microM was ATP greater than ADP greater than AMP approximately adenosine, AMP (100 microM) and adenosine (300 microM) failed to induce IP3 accumulation and Ca2+ mobilization. Although 100 microM GTP and 100 microM UTP also induced IP3 accumulation and Ca2+ mobilization, their efficacy was less than that of ATP. CTP (100 microM) induced a slight IP3 accumulation, but it did not induce Ca2+ mobilization. Nifedipine (10 microM), a Ca2+ channel antagonist, and theophylline (100 microM), a P1-purinergic receptor antagonist, failed to inhibit the ATP-induced IP3 accumulation and Ca2+ mobilization. The above two cellular responses induced by ATP were also observed in the Ca2+-depleted medium. ATP induced a rapid and transient accumulation of 1,4,5-IP3 (5s), followed by a slower accumulation of 1,3,4-IP3. These results suggest that ATP induces the formation of 1,4,5-IP3 through the P2-purinergic receptor and consequently promotes Ca2+ mobilization from intracellular storage sites in cultured adrenal chromaffin cells.     

Calmodulin Antagonist W-7 Inhibits Lysosomal Sphingomyelinase Activity in C6 Glioma Cells

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) is known to be a potent calmodulin antagonist and inhibitor of calmodulin-dependent protein kinases. W-7 and 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7) are inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. In C6 glioma cells, W-7 and not H-7 inhibited dose-dependently acid sphingomyelinase, a result indicating the modulation of this lysosomal enzyme by a calmodulin-dependent system. Other lysosomal enzymes, such as beta-glucosidase, alpha-galactosidase, and arylsulfatase A, were unaffected by W-7 and H-7, a finding indicating a selective effect of W-7 on sphingomyelinase.     

Stimulation of Inositol Incorporation into Lipids of PC 12 Cells by Nerve Growth Factor and Bradykinin

The effects of bradykinin (BK) and lithium on the phosphatidylinositol cycle were examined in PC12 cells cultured for 20 h in the presence [PC12(+)] or in the absence [PC12(-)] of nerve growth factor (NGF). BK (1 microM) induced a small stimulation of the incorporation of myo-[2-3H]inositol into the lipids of PC12(-) cells and a three- to fourfold stimulation of such incorporation into the lipids of PC12 (+) cells. About 15 h of incubation with NGF and greater than 10 min of incubation with BK were needed for maximal stimulation of inositol incorporation by BK. In the presence of 25 mM LiCl, BK stimulated the inositol monophosphate levels nine-fold in PC12 (-) and 30-fold in PC12 (+) cells. After incubation for 20 h with NGF, an increased binding of [3H]BK to the PC12 (+) cells was observed at 4 degrees C. Exposure of the cells for 30 min to 25 mM LiCl enhanced the effect of BK on the inositol incorporation into total inositol lipids, especially in PC12(+) cells. In these cells, LiCl in the presence of BK also increased several-fold the intracellular levels of inositol bisphosphate and inositol trisphosphate.     

Products of Cultured Neuroglial Cells. III. Release of an 85,000 Molecular Weight Glycoprotein by C6 Glioma Cells In Vitro

Abstract: With [³H]fucose as a marker, C6 glioma cells in culture released an 85,000 molecular weight molecule into the medium as the major extracellular glycoprotein. The quantity and extracellularkytoplasmic ratio of this glycoprotein suggest that its cellular processing is different from that of five other released glycoproteins of molecular weights 55,000, 115,000, 130,000, 150,000, and 170,000. Nearly 40% of newly synthesized glycoproteins in the cells was released into the culture medium. Major glycoproteins retained by the cells migrated electrophoretically to molecular weight positions of 82,000, 110,000, 120,000, 140,000, and 160,000, and approximately one-third of these retained glycoproteins were labile to trypsinization. Both synthesis and release of these macromolecules were inhibited more than 95% with cycloheximide treatment, demonstrating that nearly all fucosylation was linked to protein synthesis. Since 40% of all glycoproteins was released under conditions of more than 99% cellular viability, it is likely that these extracellular glycoproteins are physiological products of membrane turnover and secretion, but not of cell lysis. The results provide a basis for the further study of glial differentiation and of shed glioma antigens.     

Late-Phase Accumulation of Inositol Phosphates Stimulated by Prostaglandins D2 and F2α in Neuroblastoma × Glioma Hybrid NG108-15 Cells

The accumulation of inositol phosphates (IPs) in response to prostaglandins (PGs) was studied in NG108-15 cells preincubated with myo-[3H]inositol. As a positive control, bradykinin caused accumulation of IPs transiently at an early phase (within 1 min) and continuously during a late phase (15-60 min) of incubation in the cells. PGD2 and PGF2 alpha did not significantly cause the accumulation of IPs at an early phase but significantly stimulated inositol bisphosphate (IP2) and inositol monophosphate (IP) formation at late phase of incubation. The maximum stimulation was obtained at greater than 10(-7) M concentrations of these PGs, the levels being three-and twofold for IP2 and IP1, respectively. 9 alpha, 11 beta-PGF2 has a slight effect but PGE2 and the metabolites of PGD2 and PGF2 alpha have no effect up to 10(-6)M. The effects of PGD2 and PGF2 alpha were not additive, but the effect of each PG was additive to that of bradykinin at a late phase of incubation. Inositol 1-monophosphate was mainly identified in the stimulation by 10(-5) M PGD2 and 10(-5) M PGF2 alpha, whereas both inositol 1-monophosphate and inositol 4-monophosphate were produced in the stimulation by 10(5) M bradykinin. Depletion of extracellular Ca2+ diminished the stimulatory effect of PGD2 and PGF2 alpha and late-phase effect of bradykinin, but simple Ca2+ influx into the cells by high K+, ionomycin, or A23187 failed to cause such late-phase effects. These results suggest that PGD2 and PGF2 alpha specifically stimulate hydrolysis of inositol phospholipids.     

Fra-1对大鼠C6胶质瘤细胞侵袭转移能力的影响

目的：观察Fra-1对C6胶质瘤侵袭转移能力的影响。方法：以大鼠C6胶质瘤细胞为研究对象,Fra-1siRNA通过脂质体转染C6,realtimeRT-PCR和westernblot法检测C6细胞中Fra-1的表达、Elisa法检测细胞培养上清中MMP一9的含量,Transwell小室观察C6细胞的转移侵袭能力。结果：干扰Fra-1后能降低C6细胞中Fra-1的表达,显著降低C6细胞中MMP-9的含量,降低C6细胞的转移侵袭能力。结论：干扰Fra-1能抑制C6细胞的转移侵袭。