Rapid agonist-induced beta-adrenergic receptor kinase translocation in C6 glioma cells.

Exposure of C6 glioma cells to 1 microM isoproterenol leads to fast desensitization of the beta-adrenergic receptor/adenylyl cyclase system and transient receptor sequestration. It also triggers a very rapid and transient translocation to the plasma membrane of beta-adrenergic receptor kinase (beta ARK), a specific cytoplasmic kinase that phosphorylates only the agonist-occupied form of several G protein-coupled receptors. beta ARK-mediated receptor phosphorylation appears to be a suitable mechanism for the rapid regulation of adrenergic receptor function in the nervous tissue.     

Differences in the beta-adrenergic responsiveness between high and low passage rat glioma C6 cells

Responsiveness to catecholamines was studied in two different strains of rat glioma C6 cells. The C6 cells of low passage possessed a high capacity to accumulate cyclic AMP in response to (-)-isoproterenol. Cholera toxin was also able to stimulate cyclic AMP accumulation in these cells. High passage C6 cells were unresponsive to (-)-isoproterenol or to cholera toxin except in the presence of a high concentration of phosphodiesterase inhibitor. The affinity of beta-adrenergic receptors on both strains for (-) [3H] dihydroalprenolol was similar; however, C6 low passage possessed several times the number of beta-adrenergic receptors found in C6 high passage. This difference correlated with the difference found in (-)-isoproterenol-stimulated adenylate cyclase between C6 low passage and high passage. The sodium fluoride-stimulated adenylate cyclase was similar in both strains. Cyclic AMP phosphodiesterase activity was 2-3 times higher in homogenates of C6 high passage than in low passage. In intact cells, the rate of breakdown of cyclic AMP was 5-times faster in C6 high passage than in low passage. Thus, differences in beta-adrenergic receptor number and phosphodiesterase activity explain in part the lack of responsiveness of C6 high passage. Our studies indicate that continuous subculturing of rat glioma C6 cells led to complex alterations in the beta-adrenergic receptor-adenylate cyclase system.     

Concanavalin A prevents phorbol-mediated redistribution of protein kinase C and beta-adrenergic receptors in rat glioma C6 cells

Exposure of rat glioma C6 cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused an activation of protein kinase C wherein the enzyme rapidly became membrane-bound (T 1/2 of 15 min). This translocation of protein kinase C from cytosol to membrane was followed by a sequestration of cell surface beta-adrenergic receptors and a loss of isoproterenol-stimulated adenylate cyclase activity. We had reported previously that prior exposure of rat glioma cells to concanavalin A prevents the TPA-mediated sequestration of receptors and desensitization of adenylate cyclase (Kassis et al., 1985). We now show that the concanavalin A treatment also prevents the translocation and activation of protein kinase C. These results are further evidence that in the TPA-treated cells, sequestration of beta-adrenergic receptors is mediated by membrane-bound protein kinase C.     

Serum catecholamines desensitize beta-adrenergic receptors of cultured C6 glioma cells

C6 glioma cells grown in medium containing fetal bovine serum have a decreased beta-adrenergic receptor number and beta-receptor-stimulated cyclic AMP accumulation as compared to cells grown in a serum-free, defined medium. The decreased number of receptors and decreased cAMP accumulation are attributable to a suppression of receptor binding and response by serum as opposed to increases produced by growth in the defined medium. Serum, when added to cells grown in the absence of serum, stimulated cellular cyclic AMP levels to 2-3 times basal levels. This direct stimulatory effect was blocked by incubation of the cells with the beta-adrenergic antagonist propranolol and was partially reversed by dialysis of the serum. In contrast, addition of serum to cells that have been grown with serum fails to stimulate cyclic AMP accumulation. The decrease in receptors following growth in serum can be mimicked by growing cells in serum-free medium in the presence of beta-adrenergic agonists such as isoproterenol or norepinephrine. Radioenzymatic assays indicate that fetal bovine serum contains approximately 0.3 nM norepinephrine and lower concentrations of epinephrine. It thus appears that growth of C6 cells in serum-containing media desensitizes the beta-adrenergic receptor/cyclic AMP system of these cells. This desensitized state appears to result primarily from the action of catecholamines present in serum. These data indicate that retained catecholamines are one component in serum that can modify expression of beta-adrenergic receptors and hormonal response of cultured glioma cells.     

Exfoliation of the beta-adrenergic receptor and the regulatory components of adenylate cyclase by cultured rat glioma C6 cells

Cultured rat glioma C6 cells exfoliate membrane vesicles which have been termed 'exosomes' into the culture medium. The exosomes contained both stimulatory and inhibitory GTP-binding components of adenylate cyclase (the stimulatory, Gs, and the inhibitory, Gi, regulatory components) and beta-adrenergic receptors but were devoid of adenylate cyclase activity. It was therefore apparent that the catalytic component of adenylate cyclase was either not exfoliated or was inactivated during the exfoliation process. The presence of Gs or Gi in the exosomes was detected by ADP ribosylation using [alpha-32P]NAD in the presence of cholera or pertussis toxins, respectively. The exosomal concentration of each of the two components was estimated to be about one fifth of that of the cell membrane when expressed on a per mg protein basis. Exosomal Gs was almost as active as the membrane-derived Gs in its ability to reconstitute NaF- and guanine nucleotide-stimulated adenylate cyclase activity in membranes of S49 cyc- cells, which lack a functional Gs. The ability of exosomal Gs to reconstitute isoproterenol-stimulated activity, however, was much lower than that of membrane Gs. The density of beta-adrenergic receptors in the exosomes was much less than that found in the membranes. Although the exosomal receptors bound the antagonist iodocyanopindolol with the same affinity as receptors from the cell membrane, the affinity for the agonist isoproterenol was 13- to 18-fold lower in the exosomes. In addition, this affinity was not modulated by GTP in the exosomes. Thus, exfoliated beta-adrenergic receptors seem to be impaired in their ability to couple to and activate Gs. This was directly tested by coupling the receptors to a foreign adenylate cyclase using membrane fusion. The fusates were then assayed for agonist-stimulated activity. While significant stimulation of the acceptor adenylate cyclase was obtained using C6 membrane receptors, the exosomal receptors were completely inactive. Thus during exfoliation, there appear to be changes in the components of the beta-adrenergic-sensitive adenylate cyclase that results in a nonfunctional system in the exosomes.     

Thrombin and PAR-1 acitvating peptide increase iNOS expression in cytokine-stimulated C6 glioma cells

Thrombin (THR) plays a key role in the brain under physiological and pathological conditions. Several of the biological activities of thrombin have been shown to be mainly driven through activation of protease-activated receptor-1 (PAR-1)-type thrombin receptor. Here we have studied the effect of THR and PAR-1-activating peptide (PAR1-AP), SFLLRN, on cytokine-induced expression of inducible nitric oxide (iNOS), a prominent marker of astroglial activation using the rat C6 glioma cells. In this cell line, THR (1-10 U/mL) and PAR1-AP (1-100 microM) induced a significant concentration-dependent increase both of IFN-gamma- (250 U/mL) or TNF-alpha- (500 U/mL) induced NO release. The observed increase of NO production was related to an enhancement of iNOS expression as measured in cell lysates prepared from different treatments by using SDS-PAGE followed by western blot analysis. The effect of THR, but not that of PAR1-AP, was significantly inhibited by hirulog(TM) (60 microg/mL), a specific and stochiometric THR inhibitor or by cathepsin-G (40 mU/mL), an inhibitor of PAR-1. In conclusion our data suggest a role for THR through activation of PAR-1 in the induction of astroglial iNOS, and further support the hypothesis that THR may function as an important pathophysiological modulator of the inflammatory response.     

Phorbol esters and beta-adrenergic agonists mediate desensitization of adenylate cyclase in rat glioma C6 cells by distinct mechanisms

Exposure of rat glioma C6 cells to either isoproterenol or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in desensitization of isoproterenol-stimulated adenylate cyclase activity. After either treatment, the affinity of beta-receptors for isoproterenol was reduced. Thus, desensitization by TPA or isoproterenol appeared to involve an "uncoupling" of the beta-receptor from the stimulatory regulatory component (Ns) of adenylate cyclase. The activity of Ns, assayed by reconstitution of S49 cyc- adenylate cyclase activity, was found to be unchanged after desensitization. The activity of beta-receptors was measured by inactivating Ns and the catalytic component of adenylate cyclase in C6 membranes and fusing them with membranes lacking beta-receptors. Receptors from isoproterenol-treated C6 cells were less active in "coupling" to the foreign adenylate cyclase than receptors from untreated cells, whereas receptors from TPA-treated cells were fully active. This unexpected latter result was explored further. Lysates from C6 cells were centrifuged on linear sucrose density gradients and the gradient fractions assayed for beta-receptor binding activity. Most of the receptors were recovered in a "heavy" plasma membrane peak but some receptors also appeared in a "light" membrane peak. After treatment of the cells with isoproterenol or TPA, the proportion of receptors in the light peak increased. Prior treatment of the cells with concanavalin A prevented the increase in light receptors caused by isoproterenol or TPA. In addition, the concanavalin A treatment prevented the desensitization of adenylate cyclase caused by TPA but not that caused by isoproterenol. Finally, desensitization of adenylate cyclase was reversed by polyethylene glycol-induced fusion of membranes from cells treated with TPA but not isoproterenol. We conclude that beta-agonists and phorbol esters desensitize adenylate cyclase by distinct mechanisms. Agonists cause a reduction in the functional activity of the beta-receptors followed by a segregation of the receptors into a light membrane fraction devoid of Ns. Phorbol esters do not alter the activity of the receptors but do cause their segregation.     

Intracellular zinc distribution and transport in C6 rat glioma cells

In mammalian cells, the intracellular availability of zinc influences numerous crucial processes. Its distribution has previously been visualized with several fluorescent probes, but it was unclear how these probes are compartmentalized within the cell. Here, we show that in C6 cells the zinc-specific probe Zinquin is evenly distributed. Thus, the significantly lower level of fluorescence in the nucleus and a punctuate vesicular staining are real differences in the concentrations of zinc. Chemical perturbation of the steady state by releasing intracellular protein-bound zinc with the sulfhydryl-reactive N-ethylmaleimide (NEM) resulted in a vanadate sensitive transport of zinc out of the nucleus and into zincosomes. If the zinc-release was performed with the histidine-reactive diethylpyrocarbonate, sequestration was reduced compared to treatment with NEM, indicating the importance of histidine within membrane zinc transporters. Another major factor regulating the zinc homeostasis is ion export. As determined by atomic absorption spectroscopy, up to 50% of the cellular zinc was exported by a mechanism sensitive to lanthanum ions. We conclude that different concentrations of labile zinc exist in different cellular compartments, which are maintained by export and intracellular transport of zinc.     

Ultrasound-mediated microbubble destruction facilitates gene transfection in rat C6 glioma cells

The goal of this study was to determine whether ultrasound (US) exposure combined with microbubble destruction could be used to enhance non-viral gene delivery in rat C6 glioma cells. Microbubbles were prepared and gently mixed with plasmid DNA. The mixture of the DNA and microbubbles was administered to cultured C6 cells under different US/microbubble conditions. Transfection efficiency and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, and Trypan blue staining. The results demonstrate that microbubble with US exposure could significantly enhance the reporter gene expression as compared with other groups. No statistical significant difference was observed in the glioma cell viability between different groups. Our in vitro findings suggest that US-mediated microbubble destruction has the potential to promote safe and efficient gene transfer into C6 cells. This non-invasive gene transfer method may be useful for safe clinical gene therapy of brain cancer without a viral vector system.     

beta-Adrenergic receptor desensitization stimulates glucose uptake in C6 rat glioma cells

When C₆ glioma cells were stimulated by β -adrenergic ligands, [³H]-deoxyglucose uptake by the cells decreased in the first 30 min, followed by its acceleration. The stimulation of deoxyglucose uptake was attributable to desensitization of β-adrenergic receptor-adenylate cyclase system. When the cells were treated with quinacrine or tetracaine, phospholipase inhibitors, the stimulation of deoxyglucose uptake by isoproterenol was diminished without changing the basal rate. On the other hand, when C₆ glioma cells were treated with melittin or phorbol ester, phospholipase A₂ activators, the deoxyglucose uptake increases even in the absence of isoproterenol. Since these compounds inhibit or enhance phospholipase A₂ as well as the desensitization of β -adrenergic receptors (Proc. Natl. Acad. Sci. USA 77, 1341–1345, 1980), these results suggest that turnovers of phospholipids in the vicinity of β -adrenergic receptors modify the glucose uptake of C₆ glioma cells.     

Zinc homeostasis in C6 glioma cells

Zinc homeostasis in mammalian cells is precisely regulated by cellular signal transduction mechanisms. The main result of this study is the finding that modulators of phospholipase C (PLC) activity affect cellular zinc export. Two different PLC inhibitors caused an increase of the total cellular zinc level whereas two different PLC activators caused a decrease. Furthermore, both the inhibition of cyclic nucleotide phosphodiesterases as well as the administration of 8-bromo-cAMP evoked a drop in the intracellular zinc level, indicating the involvement of cAMP in the control of cellular zinc export. It is concluded that the activity of PLC controls cellular zinc transport and that the effect of elevated zinc concentrations on PLC activity might be mediated by cAMP. However, modulation of other major signaling enzymes did not affect the cellular zinc homeostasis. These include activation and inhibition of guanylate cyclase, activation of protein kinase G, activation of protein kinase A, and activation or inhibition of protein kinase C. Furthermore there was no evidence for the existence of a zinc-sensing receptor in C6 glioma cells, which would stimulate PLC activity and evoke a mobilization of intracellular free-calcium levels.     

Induction of apoptosis by penta-acetyl geniposide in rat C6 glioma cells

Penta-acetyl geniposide, (Ac)(5)-GP, was produced by acetylation of a glycoside, isolated from an extract of Gardenia fructus. Previously, we have reported that C6 glioma cells could be inhibited in culturing as well as in bearing rats by treating with (Ac)(5)-GP. In this study, the effect and action of (Ac)(5)-GP on inducing cell death was examined in rat C6 glioma cells. Treatment of C6 glioma cells with (Ac)(5)-GP caused cell death, chromatin condensation, and internucleosomal DNA ladder. Also, cell cycle arrest at G(0)/G(1) phase revealed that (Ac)(5)-GP-induced cell death appears to be mediated by apoptosis. In addition, the results also showed that p53 and c-Myc increased due to treatment of (Ac)(5)-GP in a dose-response and time-dependent manner. Concomitant with the expression of p53 and c-Myc, decreased level of Bcl-2 and increased level of Bax protein were observed. These results suggest that cell death caused by (Ac)(5)-GP through apoptosis and cell cycle arrest at G(0)/G(1) may be associated with the induction of p53, c-Myc and may be mediated with apoptosis-related Bcl-2 family proteins.     

Glutamine transport in C6 glioma cells

Glutamine transport across the cell membranes of a variety of mammalian tissues is mediated by at least four transport systems: a sodium-independent system L, and sodium-dependent systems A, ASC and N, the latter occurring in different tissue-specific variants. In this study we assessed the contribution of these systems to the uptake of [(3)H]glutamine in C6 rat glioma cells. The sodium-dependent uptake, which accounted for more than 80% of the total uptake, was not inhibited by 2-methylaminoisobutyric acid (MeAIB), indicating that system A was inactive, possibly being depressed by glutamine present in the culture medium. About 80% of the sodium-dependent uptake was mediated by system ASC, which differed from system ASC common to other CNS- and non-CNS tissues by its pH-dependence and partial lithium tolerance. The residual 20% of sodium-dependent uptake appeared to be mediated by system N, which was identified as a component resistant to inhibition by MeAIB+threonine. The system N in C6 cells appeared to be neither fully compatible with the neuronal system Nb, nor with the N system described in astrocytes: it differed from the former in being strongly inhibited by histidine and showing fair tolerance for lithium, and from the latter in its pH-insensitivity and strong inhibition by glutamate. The sodium-independent glutamine uptake differed from the astrocytic or neuronal uptake in its relatively weak inhibition by system L substrates and a strong inhibition by system ASC substrates, indicating a possible contribution of a variant of the ASC system.     

Modulation of the beta-adrenergic response in cultured rat heart cells

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A₂-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A₂ inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A₂ in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclooxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A₂-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.Abbreviations NDGA nordihydroguaiaretic acid - HETE hydroeicosatetraenoic acid - HPETE hydroperoxyeicosatetraenoic acid     

Huperzine A protects C6 rat glioma cells against oxygen-glucose deprivation-induced injury

The protective effects of huperzine A against oxygen-glucose deprivation (OGD)-induced injury in C6 cells were investigated. OGD for 6h and reoxygenation for 6h enhanced phosphorylation and degradation of IkappaBalpha and nuclear translocation of nuclear factor-kappa B (NF-kappaB), triggered overexpression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nitric oxide (NO) in C6 cells. Along with inhibiting acetylcholinesterase activity, treatment with 1 microM huperzine A inhibited activation of NF-kappaB, attenuated iNOS, COX-2 and NO overexpression, and promoted survival in C6 cells subjected to OGD insult. The protective effects of huperzine A were partly mediated by "cholinergic anti-inflammatory pathway" through alpha7 nicotinic acetylcholine receptor.