cyclic-gmp and Glioma

Histidyl-proline diketopiperazine [cyclo(His-Pro)] is a metabolite of thyrotropin releasing hormone (TRH). This review summarizes the literature concerning cyclo (His-Pro) and, in addition, some studies dealing with TRH and other peptide that are considered of interest. The enzymes concerned with the metabolism of TRH are discussed. Distribution studies of peptides by immunological methods show that, while TRH is concentrated in synaptosomes, cyclo (His-Pro) is not, suggesting that cyclo (His-Pro) is not a classical neurotransmitter. Rat brain contains approximately three times as much cyclo (His-Pro) as TRH, mainly localized in the pituitary and hypothalamus. While the TRH is found in a free form, the cyclo (His-Pro) is bound to a carrier of molecular weight approximately 70,000. While specific membrane receptors for TRH have been detected in pituitary cells, no such receptors for cyclo (His-Pro) have yet been found in brain or pituitary; however, there is a specific binding of cyclo (His-Pro) to adrenal cortex membranes. Both TRH and cyclo (His-Pro) have effects in the central nervous system or pituitary. These include effects on prolactin release, thermoregulation, CNS depression, stereotypic behavior and cyclic nucleotide levels. Possible mechanisms and interrelations of these effects are discussed.

Topics: Animals; Body Temperature Regulation; Brain; Cell Line; Cyclic AMP; Cyclic GMP; Dopamine; Ethanol; Glioma; Kinetics; Peptides, Cyclic; Piperazines; Pituitary Gland; Prolactin; Rats; Receptors, Cell Surface; Receptors, Thyrotropin-Releasing Hormone; Thyrotropin-Releasing Hormone; Tissue Distribution

Topics: Animals; Cell Membrane; Cells, Cultured; Clone Cells; Culture Media; Cyclic GMP; Glioma; Membrane Potentials; Mice; Neoplasms, Experimental; Neuroblastoma; Neurons; Rats; Receptors, Adrenergic, alpha; Receptors, Muscarinic

C-type natriuretic peptide (CNP) is the major natriuretic peptide of the central nervous system and acts via its selective guanylyl cyclase-B (GC-B) receptor to regulate cGMP production in neurons, astrocytes and endothelial cells. CNP is implicated in the regulation of neurogenesis, axonal bifurcation, as well as learning and memory. Several neurological disorders result in toxic concentrations of ammonia (hyperammonaemia), which can adversely affect astrocyte function. However, the relationship between CNP and hyperammonaemia is poorly understood. Here, we examine the molecular and pharmacological control of CNP in rat C6 glioma cells and rat GPNT brain endothelial cells, under conditions of hyperammonaemia. Concentration-dependent inhibition of C6 glioma cell proliferation by hyperammonaemia was unaffected by CNP co-treatment. Furthermore, hyperammonaemia pre-treatment (for 1 h and 24 h) caused a significant inhibition in subsequent CNP-stimulated cGMP accumulation in both C6 and GPNT cells, whereas nitric-oxide-dependent cGMP accumulation was not affected. CNP-stimulated cGMP efflux from C6 glioma cells was significantly reduced under conditions of hyperammonaemia, potentially via a mechanism involving changed in phosphodiesterase expression. Hyperammonaemia-stimulated ROS production was unaffected by CNP but enhanced by a nitric oxide donor in C6 cells. Extracellular vesicle production from C6 cells was enhanced by hyperammonaemia, and these vesicles caused impaired CNP-stimulated cGMP signalling in GPNT cells. Collectively, these data demonstrate functional interaction between CNP signalling and hyperammonaemia in C6 glioma and GPNT cells, but the exact mechanisms remain to be established.

Topics: Animals; Brain; Cyclic GMP; Endothelial Cells; Glioma; Hyperammonemia; Natriuretic Peptide, C-Type; Natriuretic Peptides; Phosphoric Diester Hydrolases; Rats; Signal Transduction

Although type I IFNs play critical roles in antiviral and antitumor activity, it remains to be elucidated how type I IFNs are produced in sterile conditions of the tumor microenvironment and directly affect tumor-infiltrating immune cells. Mouse de novo gliomas show increased expression of type I IFN messages, and in mice, CD11b(+) brain-infiltrating leukocytes (BIL) are the main source of type I IFNs that are induced partially in a STING (stimulator of IFN genes)-dependent manner. Consequently, glioma-bearing Sting(Gt) (/Gt) mice showed shorter survival and lower expression levels of Ifns compared with wild-type mice. Furthermore, BILs of Sting(Gt) (/Gt) mice showed increased CD11b(+) Gr-1(+) immature myeloid suppressor and CD25(+) Foxp3(+) regulatory T cells (Treg) and decreased IFNγ-producing CD8(+) T cells. CD4(+) and CD8(+) T cells that received direct type I IFN signals showed lesser degrees of regulatory activity and increased levels of antitumor activity, respectively. Finally, intratumoral administration of a STING agonist (cyclic diguanylate monophosphate; c-di-GMP) improved the survival of glioma-bearing mice associated with enhanced type I IFN signaling, Cxcl10 and Ccl5, and T-cell migration into the brain. In combination with subcutaneous OVA peptide vaccination, c-di-GMP increased OVA-specific cytotoxicity of BILs and prolonged their survival. These data demonstrate significant contributions of STING to antitumor immunity via enhancement of type I IFN signaling in the tumor microenvironment and suggest a potential use of STING agonists for the development of effective immunotherapy, such as the combination with antigen-specific vaccinations.

Topics: Animals; Cancer Vaccines; CD11b Antigen; CD11c Antigen; Cell Line, Tumor; Cyclic GMP; Disease Models, Animal; Gene Expression Profiling; Glioma; Interferon Type I; Membrane Proteins; Mice; Mice, Knockout; Signal Transduction; T-Lymphocyte Subsets; Tumor Microenvironment

Nitric oxide (NO) released from NO donors can be cytotoxic in tumor cells and can enhance the transport of drugs into brain tumors by altering blood-tumor barrier permeability. The NO donor JS-K [O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] releases NO upon enzymatic activation selectively in cells overexpressing glutathione-S-transferases (GSTs) such as gliomas. Thus, JS-K-dependent NO effects - especially on cell viability and vascular permeability - were investigated in U87 glioma cells in vitro and in an orthotopic U87 xenograft model in vivo by magnetic resonance imaging (MRI). In vitro experiments showed dose-dependent antiproliferative and cytotoxic effects in U87 cells. In addition, treatment of U87 cells with JS-K resulted in a dose-dependent activation of soluble guanylate cyclase and intracellular accumulation of cyclic guanosine monophosphate (cGMP) which was irreversibly inhibited by the selective inhibitor of soluble guanylate cyclase ODQ (1H-[1,2,4]oxadiazolo(4,3a)quinoxaline-1-one). Using dynamic contrast enhanced MRI (DCE-MRI) as a minimally invasive technique, we demonstrated for the first time a significant increase in the DCE-MRI read-out initial area under the concentration curve (iAUC60) indicating an acute increase in blood-tumor barrier permeability after i.v. treatment with JS-K. Repeated MR imaging of animals with intracranial U87 gliomas under treatment with JS-K (3.5 μmol/kg JS-K 3×/week) and of untreated controls on day 12 and 19 after tumor inoculation revealed no significant changes in tumor growth, edema formation or tumor perfusion. Immunohistochemical workup of the brains showed a significant antiproliferative effect of JS-K in the gliomas. Taken together, in vitro and in vivo data suggest that JS-K has antiproliferative effects in U87 gliomas and opens the blood-tumor barrier by activation of the NO/cGMP signaling pathway. This might be a novel approach to facilitate entry of therapeutic drugs into brain tumors. DCE-MRI is a non-invasive, repeatable imaging modality to monitor biological effects of NO donors and other experimental therapeutics in intracranial tumor models.

Topics: Animals; Azo Compounds; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclic GMP; Glioma; Humans; Immunohistochemistry; Magnetic Resonance Imaging; Nitric Oxide Donors; Piperazines; Rats; Rats, Nude; Xenograft Model Antitumor Assays

The mediators and cellular effectors of inflammation are important constituents of the local environment of tumors. In some occasions, oncogenic changes induce an inflammatory microenvironment that promotes the progression of tumors. In gliomas, the presence of microglia may represent tumor-related inflammation and microglia activation, and subsequent inflammatory responses may influence tumor growth and metastasis. Here, we found that C6 glioma--but not primary astrocyte-derived extracellular matrix (ECM) could activate microglia, including primary microglia and BV-2 cell line, and activated microglia-secreted interleukin (IL)-18, a potent inflammatory cytokine of the IL-1 family, to promote C6 migration. In addition, by coating purified ECM components, it was found that secretion of IL-18 by activated microglia was enhanced when microglia encountered with fibronectin and vitronectin. Furthermore, IL-18-induced C6 migration and microfilament disassembly were antagonized by iNOS inhibitor, guanylate cyclase inhibitor, and protein kinase G inhibitor. Taken together, these results indicate that IL-18 secreted by microglia, which was activated by C6 glioma-derived ECM, enhanced migration of C6 glioma through NO/cGMP pathway.

Topics: Actin Cytoskeleton; Animals; Animals, Newborn; Astrocytes; Cell Line, Tumor; Cell Movement; Cyclic GMP; Extracellular Matrix; Fibronectins; Gene Expression Regulation; Glioma; Interleukin-18; Microglia; Nitric Oxide; Rats; Vitronectin

In rat C6 glioma cells, the ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1), a modulator of purinergic receptor signaling, is down regulated after an increase in intracellular cAMP by addition of dibutyryl cAMP, a membrane-permeable cAMP-analog, or by activation of the β-adrenoceptor receptor with (-)-isoproterenol (Aerts et al., 2011, Eur. J. Pharmacol. 654, 1-9). In this communication we studied the effect of nitric oxide (NO)/cGMP, a pathway also affecting purinergic receptor signaling, on the level of NPP1 protein. Sodium nitroprusside (SNP), a NO donor, reduces NPP1 protein in a dose-dependent manner. A combination of SNP and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, demonstrated that NO-dependent down regulation of NPP1 was caused by NO-sensitive guanylyl cyclase. Treatment with Rp-pCPT-cGMPS, an inhibitor of protein kinase G (PKG), showed that PKG is not involved in the down regulation of NPP1. In addition, we have shown that the cAMP- and cGMP-dependent decrease in NPP1 expression is unrelated. These results indicate that NO/cGMP regulates the level of NPP1 protein by a pathway that differs from the cAMP-induced decrease in NPP1.

Topics: Animals; Cell Line, Tumor; Cyclic AMP; Cyclic GMP; Down-Regulation; Glioma; Guanylate Cyclase; Nitric Oxide; Phosphoric Diester Hydrolases; Pyrophosphatases; Rats; Signal Transduction; Solubility

eNOS expression is elevated in human glioblastomas and correlated with increased tumor growth and aggressive character. We investigated the potential role of nitric oxide (NO) activity in the perivascular niche (PVN) using a genetic engineered mouse model of PDGF-induced gliomas. eNOS expression is highly elevated in tumor vascular endothelium adjacent to perivascular glioma cells expressing Nestin, Notch, and the NO receptor, sGC. In addition, the NO/cGMP/PKG pathway drives Notch signaling in PDGF-induced gliomas in vitro, and induces the side population phenotype in primary glioma cell cultures. NO also increases neurosphere forming capacity of PDGF-driven glioma primary cultures, and enhances their tumorigenic capacity in vivo. Loss of NO activity in these tumors suppresses Notch signaling in vivo and prolongs survival of mice. This mechanism is conserved in human PDGFR amplified gliomas. The NO/cGMP/PKG pathway's promotion of stem cell-like character in the tumor PVN may identify therapeutic targets for this subset of gliomas.

Topics: Animals; Cell Line; Chickens; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Glioma; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Neoplastic Stem Cells; Nitric Oxide; Nitric Oxide Synthase Type III; Platelet-Derived Growth Factor; Receptors, Notch; Receptors, Platelet-Derived Growth Factor; Signal Transduction; Tumor Cells, Cultured

A nitrated guanine nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), is formed via nitric oxide (NO) and causes protein S-guanylation. However, intracellular 8-nitro-cGMP levels and mechanisms of formation of 8-nitro-cGMP and S-guanylation are yet to be identified. In this study, we precisely quantified NO-dependent formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. Treatment of cells with S-nitroso-N-acetylpenicillamine led to a rapid, transient increase in cGMP, after which 8-nitro-cGMP increased linearly up to a peak value comparable with that of cGMP at 24 h and declined thereafter. Markedly high levels (>40 microm) of 8-nitro-cGMP were also evident in C6 cells that had been stimulated to express inducible NO synthase with excessive NO production. The amount of 8-nitro-cGMP generated was comparable with or much higher than that of cGMP, whose production profile slightly preceded 8-nitro-cGMP formation in the activated inducible NO synthase-expressing cells. These unexpectedly large amounts of 8-nitro-cGMP suggest that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, may undergo guanine nitration. Also, 8-nitro-cGMP caused S-guanylation of KEAP1 in cells, which led to Nrf2 activation and subsequent induction of antioxidant enzymes, including heme oxygenase-1; thus, 8-nitro-cGMP protected cells against cytotoxic effects of hydrogen peroxide. Proteomic analysis for endogenously modified KEAP1 with matrix-assisted laser desorption/ionization time-of-flight-tandem mass spectrometry revealed that 8-nitro-cGMP S-guanylated the Cys(434) of KEAP1. The present report is therefore the first substantial corroboration of the biological significance of cellular 8-nitro-cGMP formation and potential roles of 8-nitro-cGMP in the Nrf2-dependent antioxidant response.

Topics: Animals; Antioxidants; Cell Line, Tumor; Chromatography, Liquid; Cyclic GMP; Glioma; Guanine; Mass Spectrometry; Nitric Oxide; Oxidative Stress; Protein Processing, Post-Translational; Rats; Reactive Oxygen Species; Tandem Mass Spectrometry

The blood-brain tumor barrier (BTB) significantly limits the delivery of chemotherapeutics to brain tumors. Nitric oxide (NO) is involved in the regulation of cerebral vascular permeability. We investigated the effects of NO donors, L-arginine and hydroxyurea, on BTB permeability in 9L gliosarcoma-bearing Fischer rats.. The rats implanted with 9L gliosarcoma were dosed orally with hydroxyurea and L-arginine. BTB permeability, defined by the unidirectional transport constant, Ki, for [14C]sucrose was measured. The expression of neural and endothelial NO synthase (NOS) in tumors and normal brain tissue was examined. Further, the levels of NO, L-citrulline, and cGMP in the tumor and normal brain tissue were measured.. Oral administration of l-arginine or hydroxyurea significantly increased BTB permeability when compared with the nontreated control. The selective effects were abolished by iberiotoxin, an antagonist of calcium-dependent potassium (KCa) channel that is a cGMP pathway effector. The expression of endothelial NOS, but not neural NOS, was higher in tumor vessels than in those of normal brain. Moreover, the levels of NO, L-citrulline, a byproduct of NO formation from L-arginine, and cGMP were enhanced in the tumor tissue by oral administration of L-arginine and/or hydroxyurea.. Oral administration of L-arginine or hydroxyurea selectively increased tumor permeability, which is likely mediated by alteration in cGMP levels. The findings suggest that use of oral NO donors may be a strategy to enhance the delivery of chemotherapeutics to malignant brain tumors.

Topics: Administration, Oral; Animals; Arginine; Blood-Brain Barrier; Brain Neoplasms; Capillary Permeability; Citrulline; Cyclic GMP; Drug Delivery Systems; Drug Evaluation, Preclinical; Drug Synergism; Female; Glioma; Hydroxyurea; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Rats; Rats, Inbred F344; Tumor Cells, Cultured

The blood-brain tumor barrier (BTB) significantly limits delivery of therapeutic concentrations of chemotherapy to brain tumors. A novel approach to selectively increase drug delivery is pharmacologic modulation of signaling molecules that regulate BTB permeability, such as those in cGMP signaling. Here we show that oral administration of sildenafil (Viagra) and vardenafil (Levitra), inhibitors of cGMP-specific PDE5, selectively increased tumor capillary permeability in 9L gliosarcoma-bearing rats with no significant increase in normal brain capillaries. Tumor-bearing rats treated with the chemotherapy agent, adriamycin, in combination with vardenafil survived significantly longer than rats treated with adriamycin alone. The selective increase in tumor capillary permeability appears to be mediated by a selective increase in tumor cGMP levels and increased vesicular transport through tumor capillaries, and could be attenuated by iberiotoxin, a selective inhibitor for calcium-dependent potassium (K(Ca)) channels, that are effectors in cGMP signaling. The effect by sildenafil could be further increased by simultaneously using another BTB "opener", bradykinin. Collectively, this data demonstrates that oral administration of PDE5 inhibitors selectively increases BTB permeability and enhances anti-tumor efficacy for a chemotherapeutic agent. These findings have significant implications for improving delivery of anti-tumor agents to brain tumors.

Topics: Animals; Antineoplastic Agents; Autoradiography; Blood Pressure; Brain Chemistry; Brain Neoplasms; Capillaries; Capillary Permeability; Cyclic GMP; Female; Glioma; Imidazoles; Microscopy, Electron, Transmission; Neovascularization, Pathologic; Phosphodiesterase 5 Inhibitors; Phosphodiesterase Inhibitors; Piperazines; Purines; Rats; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; Sildenafil Citrate; Sucrose; Sulfones; Survival Analysis; Tight Junctions; Triazines; Vardenafil Dihydrochloride

Angiogenesis is a prerequisite for tumour progression and is highly regulated by growth factors and cytokines a number of which also stimulate the production of nitric oxide. Asymmetric dimethylarginine is an endogenous inhibitor of nitric oxide synthesis. Asymmetric dimethylarginine is metabolised by dimethylarginine dimethylaminohydrolase. To study the effect of dimethylarginine dimethylaminohydrolase on tumour growth and vascular development, the rat C6 glioma cell line was manipulated to overexpress the rat gene for dimethylarginine dimethylaminohydrolase I. Enhanced expression of dimethylarginine dimethylaminohydrolase I increased nitric oxide synthesis (as indicated by a two-fold increase in the production of cGMP), expression and secretion of vascular endothelial cell growth factor, and induced angiogenesis in vitro. Tumours derived from these cells grew more rapidly in vivo than cells with normal dimethylarginine dimethylaminohydrolase I expression. Immunohistochemical and magnetic resonance imaging measurements were consistent with increased tumour vascular development. Furthermore, dimethylarginine dimethylaminohydrolase activity was detected in a series of human tumours. This data demonstrates that dimethylarginine dimethylaminohydrolase plays a pivotal role in tumour growth and the development of the tumour vasculature by regulating the concentration of nitric oxide and altering vascular endothelial cell growth factor production.

Topics: Amidohydrolases; Animals; Astrocytoma; Blotting, Northern; Blotting, Western; Brain Neoplasms; Cell Division; Cell Movement; Cells, Cultured; Cyclic GMP; DNA Primers; Endothelial Growth Factors; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Glioblastoma; Glioma; Humans; Lymphokines; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Neovascularization, Pathologic; Nitric Oxide; Rats; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Exisulind (sulindac sulfone) and two potent derivatives, CP248 and CP461, have been shown previously to cause growth inhibition and apoptosis in several types of human carcinoma cell lines. These and related compounds have not been previously studied with respect to glioma cell lines. In the present study, we found that these three compounds caused marked growth inhibition in four rat glioma and eight human glioma cell lines, with IC50 values of 150, 1, and 0.075 microm, respectively. When studied at these concentrations exisulind and CP461 had no significant effect on the cell cycle profile of glioma cells, but CP248 caused marked arrest in mitosis. Detailed studies of CP248 in the 9L rat gliosarcoma cell line indicated that treatment with 0.075 microM CP248 caused abnormalities in the spindle apparatus and activation of the spindle assembly check point. In interphase glioma cells, CP248 stabilized microtubules (MTs) at low concentrations (0.075 microM) and depolymerized MTs at higher concentrations (0.2-0.4 microM). In NIH 3T3 fibroblasts, 0.1 microM CP248 caused extensive MT depolymerization. CP248 also caused MT depolymerization when added to assembled MTs in vitro, which indicated that it can directly affect MTs, perhaps because it shares certain structural similarities with Colcemid. In glioma cells, the effects of CP248 on MTs were independent of the previously reported effects of this compound on activation of protein kinase G. Therefore, CP248 is a novel MT-active agent that may be useful in the treatment of glioblastoma, and possibly other types of cancer, because of its dual effects on protein kinase G and MTs.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; 3T3 Cells; Animals; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Cycle Proteins; Cell Division; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 2; Cyclic Nucleotide Phosphodiesterases, Type 5; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Glioma; Humans; Immunoenzyme Techniques; In Vitro Techniques; Interphase; Kinesins; Mice; Microtubules; Phosphoproteins; Phosphoric Diester Hydrolases; Rats; Spindle Apparatus; Sulindac; Thymidine

Methemoglobin (metHb) has been reported to be present in areas surrounding solid tumors. The effects of human metHb on the growth of one human hepatocellular carcinoma cell line and one human glioma cell line that simply replicate in Ham's nutrient mixture F12 (F12) were investigated. MetHb, depending on its concentration, stimulated or inhibited the in vitro growth of both cancer cell lines. The stimulatory or inhibitory effect was due to the release of hemin from metHb, which was recognized by its characteristic light absorption spectrum. The possibility of metHb or hemin acting initially through a 3', 5'-cyclic guanosine monophosphate- (cGMP-) or prostaglandin E2- (PGE2-) mediated pathway to enhance cell growth was excluded. Ferric iron derived from the catabolic degradation of hemin increased cell growth, whereas biliverdin (Bv) and its reduction product, bilirubin (Br), decreased cell growth. Hemoglobin oxidized to metHb in conditions found in tumors showing neovascularization and hemorrhage may contribute significantly to increased proliferation of cancerous cells.

Topics: Adult; Aged; Bilirubin; Biliverdine; Carcinoma, Hepatocellular; Cell Division; Cyclic GMP; Dinoprostone; Dose-Response Relationship, Drug; Female; Glioma; Globins; Hemin; Humans; Liver Neoplasms; Methemoglobin; Pituitary Neoplasms; Tumor Cells, Cultured

Previous studies have established that Ca2+-sensitive adenylyl cyclases, whether endogenously or heterologously expressed, are preferentially regulated by capacitative Ca2+ entry, compared with other means of elevating cytosolic Ca2+ (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155; Fagan, K. A., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444; Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998) J. Biol. Chem. 273, 9297-9305). These findings led to the suggestion that adenylyl cyclases and capacitative Ca2+ entry channels were localized in the same functional domain of the plasma membrane. In the present study, we have asked whether a heterologously expressed Ca2+-permeable channel could regulate the Ca2+-inhibitable adenylyl cyclase of C6-2B glioma cells. The cDNA coding for the rat olfactory cyclic nucleotide-gated channel was inserted into an adenovirus construct to achieve high levels of expression. Electrophysiological measurements confirmed the preservation of the properties of the expressed olfactory channel. Stimulation of the channel with cGMP analogs yielded a robust elevation in cytosolic Ca2+, which was associated with an inhibition of cAMP accumulation, comparable with that elicited by capacitative Ca2+ entry. These findings not only extend the means whereby Ca2+-sensitive adenylyl cyclases may be regulated, they also suggest that in tissues where they co-exist, cyclic nucleotide-gated channels and Ca2+-sensitive adenylyl cyclases may reciprocally modulate each other's activity.

Topics: Adenoviridae; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Calcium; Cyclic AMP; Cyclic GMP; Glioma; Membrane Potentials; Olfactory Pathways; Rats; Tumor Cells, Cultured

To investigate the mechanisms by which lipopolysaccharide (LPS) affects Ca2+ signaling systems, we studied the effects of LPS on the serotonin (5-HT)- or thrombin-induced intracellular Ca2+ ([Ca2+]i) increase in rat C6 glioma cells. Pretreatment of the cells with 1 microg/ml LPS for 24 hr significantly inhibited [Ca2+]i increase induced by 10 microM 5-HT- or 0.5 U/ml thrombin. Its inhibitory effects were both dose- and time-dependent. Treatment with 1 mM dibutyryl cGMP (dbcGMP) for 30 min also significantly inhibited the 5-HT- and thrombin-induced [Ca2+]i increase to approximately 60-70% of control. However, simultaneous pretreatment with LPS and dbcGMP did not show any synergistic inhibition. The simultaneous pretreatment with LPS and the potent cGMP-dependent protein kinase (PKG) inhibitors H-8 and KT5823 for 24 hr significantly antagonized the inhibitory effect of LPS. Pretreatment of the cells with 1 microg/ml LPS for 24 hr significantly enhanced cGMP accumulation, while dexamethasone and NMMA (NOS inhibitors) significantly attenuated the LPS-induced enhancement in cGMP accumulation. In addition, pretreatment of the cells with 100 nM dexamethasone for 24 hr significantly suppressed LPS-induced inducible nitric oxide synthase (iNOS; type II NOS, NOS-II) protein expression. These results indicate that LPS may inhibit both 5-HT- and thrombin-induced [Ca2+]i increase via iNOS expression and PKG activation pathway in rat C6 glioma cells.

Topics: Animals; Calcium; Cyclic GMP; Dexamethasone; Dibutyryl Cyclic GMP; Glioma; Intracellular Fluid; Isoquinolines; Lipopolysaccharides; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroprusside; Protein Kinase C; Rats; Serotonin; Signal Transduction; Thrombin; Tumor Cells, Cultured

1. The present study reexamines a previous notion on opioid stimulation of cyclic GMP (cGMP) formation and the retraction of the original findings. 2. The effect of opioid agonists on cGMP accumulation in two cell lines of neuronal origin was measured. The proportion of cGMP stimulation in NG108-15 neuroblastoma x glioma hybrid cells resembled the proportion of [Ca2+]in elevation by opioids in this culture. The failure of opioids to stimulate cGMP formation in SK-N-SH human neuroblastoma coincided with the lack of cGMP stimulation by other Ca2+ mobilizing agents in these cells. The nitric oxide donor nitroprusside elevated cGMP in both cell lines. 3. The implication of the opioid-Ca(2+)-NO-cGMP cellular pathway for opioid activity in vivo is discussed.

Topics: Animals; Bradykinin; Cell Line; Cyclic GMP; Etorphine; Glioma; Humans; Hybrid Cells; Neuroblastoma; Neurons; Nitroprusside; Potassium Chloride

Intracarotid infusion of bradykinin and its analogue, RMP-7, selectively increase the permeability of brain tumor capillaries though the nitrix oxide (NO) and cyclic GMP pathway. Maximum blood-tumor barrier (BTB) permeability induced by bradykinin is observed at 15 min after intracarotid infusion and this effect is decreased even if the infusion continues. The mechanism for this decreased effect with long term infusion has not been clearly defined. This study sought to determine the involvement of the NO-cyclic GMP pathway in this event. Regional permeability was investigated in 44 Wistar rats with implanted RG2 gliomas, using quantitative autoradiography to determine the unidirectional transfer constant (Ki) of radiolabeled 14C-dextran. Tumor bearing rats were treated by intracarotid infusion of bradykinin (10 micrograms kg-1 min-1) with or without pretreatment with bradykinin, the NO donor s-nitrosoglutathione (10 nmol kg-1 min-1), or the cyclic GMP analogue, 8Br-cyclic GMP (200 micrograms kg-1 min-1). At 30 min of bradykinin infusion, BTB permeability was significantly lower compared to 15 min of bradykinin infusion (3.79 +/- 0.99 vs. 16.20 +/- 3.43 microliters g-1 min-1, p < 0.001). Pretreatment with an NO donor significantly decreased BTB permeability in bradykinin infused rats (5.09 +/- 2.61 vs. 13.51 +/- 4.19 microliters g-1 min-1, p < 0.001), as did pretreatment with a cyclic GMP analogue (4.48 +/- 0.95 vs. 12.31 +/- 3.90 microliters g-1 min-1, p < 0.001). There was no increased permeability in nontumor brain areas. Increased tumor permeability by bradykinin appears to be regulated by NO and cyclic GMP which are second messengers involved in the bradykinin B2 receptor mediated cascade.

Topics: Animals; Bradykinin; Brain Neoplasms; Capillary Permeability; Cyclic GMP; Drug Resistance; Female; Glioma; Glutathione; Nitric Oxide; Nitroso Compounds; Rats; Rats, Wistar; S-Nitrosoglutathione

It is now widely accepted that cyclic nucleotide phosphodiesterases (PDEs) play fundamental roles in signal transduction pathways; they show a remarkable molecular complexity, different tissue distribution and complex regulatory mechanisms. Here we report PDE isoforms expression in two dibutyryl cyclic AMP differentiated murine cell lines: the hybrid neuroblastoma-glioma 108CC15 and the parental neuroblastoma N18TG2. They differ in the ability to establish functional synapses, a feature present only in the former. Ionic exchange chromatography elution profiles of N18TG2 and 108CC15 undifferentiated cell extracts show two main peaks of activity. The first one hydrolyzes cyclic GMP and is specifically inhibited by Zaprinast, thus representing a member of the PDE5 family. The second peak hydrolyzes cyclic AMP and is significantly inhibited by rolipram, as all the PDE4 family members. The induction of differentiation by dibutyryl cyclic AMP in both clonal lines results in an increase of PDE activities only after 3 hr of treatment, suggesting that protein neosynthesis is involved. Interestingly in both clones, besides the increase in cyclic AMP hydrolyzing specific activity (3.1-fold in 108CC15 and 2.5-fold in N18TG2), we also observed an increase in cyclic GMP hydrolyzing activity (1.7-fold in 108CC15 and 4.3-fold in N18TG2). While the induction of PDE4, previously reported also in other cellular systems, could be considered as a feedback response to the higher cyclic AMP levels, this is not true for the isoform that hydrolyzes cyclic GMP. These data suggest that the induction of PDE isoforms in neuroblastoma cells could be related to the activation of neuronal differentiative pathway.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Bucladesine; Cell Differentiation; Choline O-Acetyltransferase; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 4; Cyclic Nucleotide Phosphodiesterases, Type 5; Glioma; Hybrid Cells; L-Lactate Dehydrogenase; Mice; Neuroblastoma; Phosphoric Diester Hydrolases; Rats

The effect of endothelin-3 (ET-3) on C-type natriuretic peptide (CNP)-induced guanosine 3',5'-cyclic monophosphate (cGMP) was examined in C6 glioma cells, CNP-induced cGMP formation was both time- and dose-dependent, with an EC50 value of about 10 nM. While ET-3 and phorbol 12-myristate 13-acetate (PMA) had no effect on basal cGMP production, both compounds were potent inhibitors of CNP-induced cGMP formation, with IC50 values of approximately 10 and 2 nM, respectively. Although protein kinase C (PKC) inhibitors had no effect on basal cGMP formation, Ro 31-8220, a PKC inhibitor, reversed the ET-3 inhibition on CNP-induced cGMP formation by 63% and that of PMA almost completely. Our findings suggest that stimulation of cGMP formation by CNP in C6 glioma cells is negatively modulated by PKC activation, and that the inhibitory action of ET-3 on CNP-stimulated cGMP formation is mediated partly by PKC.

Topics: Animals; Atrial Natriuretic Factor; Cyclic GMP; Endothelin-3; Glioma; Natriuretic Peptide, C-Type; Proteins; Rats; Tumor Cells, Cultured

Capacitative Ca2+ entry, a main pathway of Ca2+ entry evoked by receptor activation, is widely confirmed in various types of cells. However, the mechanism of the activation of capacitative Ca2+ entry is unknown. We checked the several candidates for the mechanism of capacitative Ca2+ entry pathway in rat glioma C6 cells using thapsigargin (TG), a microsomal Ca(2+)-ATPase inhibitor. Pretreatment with pertussis toxin did not affect the peak and sustained elevation of [Ca2+]i evoked by TG. Sodium nitroprusside and 8-bromo cyclic GMP did not affect an elevation of [Ca2+]i induced by TG. Phorbol 12-myristate 13-acetate, an activator of protein kinase C (PKC), and staurosporine, an inhibitor of PKC, did not modify an increase in [Ca2+]i induced by TG. Okadaic acid, an inhibitor of phosphatase, did not affect an increase in [Ca2+]i evoked by TG. Pretreatment with colchicine and cytochalasin D, drugs disrupting cytoskeleton, had no effect on a rise of [Ca2+]i induced by TG. Genistein and erbastatin analog, inhibitors of tyrosine kinase, inhibited an elevation of [Ca2+]i evoked by TG in a dose-dependent manner. The present results suggest that tyrosine kinase regulates capacitative Ca2+ entry into rat glioma C6 cells.

Topics: Animals; Calcium; Cell Line; Cyclic GMP; Cytoskeleton; Enzyme Inhibitors; Glioma; GTP-Binding Proteins; Nitric Oxide; Pertussis Toxin; Phosphoprotein Phosphatases; Protein Kinase C; Protein-Tyrosine Kinases; Rats; Virulence Factors, Bordetella

We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca2+ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca2+-sensitive dye fura-2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration-dependent manner. This enhancement was abolished by pretreatment with 100 microM BAPTA tetraacetoxymethal ester or in the presence of W-7 in a concentration-dependent manner. N G-Monomethyl-L-arginine (NMMA), a competitive inhibitor or nitric oxide synthase (NOS), also inhibited the NaF-induced generation of cGMP. These results suggest that NaF-induced cGMP generation occurs via a calcium/calmodulin- and NOS-dependent pathway. (b) The basal intracellular Ca2+ concentration ([Ca2+]i) was transiently greater at 1 and 3 h after pretreatment with NaF. W-7 and W-13 antagonized the increase in [Ca2+]i, whereas NMMA had little effect. This suggests that the NaF-induced change in basal [Ca2+] was mediated by a calmodulin-dependent pathway but was independent of a NOS-sensitive pathway. (c) The serotonin (5-HT)-induced intracellular mobilization of Ca2+ was reduced by pretreating the cells with NaF. The reduction in Ca2+ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W-7, W-5, and H-8 had no effect. Results suggest that NaF differentially regulated the cGMP generation. basal [Ca2+]i, and 5-HT2A receptor function in C6 glioma cells.

Topics: Animals; Arginine; Calcium; Chelating Agents; Cyclic GMP; Egtazic Acid; Enzyme Inhibitors; Glioma; omega-N-Methylarginine; Rats; Serotonin; Signal Transduction; Sodium Fluoride; Sulfonamides; Time Factors; Tumor Cells, Cultured; Vasodilator Agents

A significant fraction of the beta-amyloid precursor protein is proteolytically processed to yield large secreted forms (sAPP). These proteins have pleiotropic effects which potentially involve control of gene expression. We have investigated the influence of sAPP on the class of transcription factors which bind kappa B enhancer sequences. Transcription dependent on a kappa B element was enhanced by sAPP in several cell lines, as measured by expression of a transfected chloramphenicol acetyltransferase reporter gene. Secreted APP also induced an increase in kappa B DNA-binding activity in hippocampal neurons treated with sAPP. Both effects were mimicked by an analog of cyclic GMP and inhibited by an antagonist of cyclic GMP-dependent protein kinase. Such activation of kappa B-dependent transcription was correlated in two ways with the ability of sAPP to protect neuronal cells against calcium-mediated damage: (1) tumor necrosis factor beta also protected against calcium-mediated insults and induced kappa B-dependent transcription; (2) antisense oligonucleotide-mediated reduction of an endogenous inhibitor of NF-kappa B activated kappa B-binding activity and attenuated calcium-mediated toxicity in both a neuronal cell line and in primary neurons. These findings suggest that a kappa B-binding transcription factor can act as a coordinator of neuroprotective gene expression in response to cytokines.

Topics: Amyloid beta-Protein Precursor; Base Sequence; Calcimycin; Calcium; Cell Line; Cell Survival; Chloramphenicol O-Acetyltransferase; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; DNA-Binding Proteins; Enhancer Elements, Genetic; Glioma; Humans; I-kappa B Proteins; Kidney; Kinetics; Neuroblastoma; Neurons; NF-kappa B; NF-KappaB Inhibitor alpha; Oligonucleotides, Antisense; Recombinant Fusion Proteins; Thionucleotides; Transcription, Genetic; Transfection; Tumor Cells, Cultured

To clarify the involvement of nitric oxide (NO) derived from nitrosothiols (RSNO) in 5-hydroxytryptamine (5-HT)-induced Ca(2+)-independent cGMP formation (CIGF) in NG108-15 cells, we investigated the effects of 5-HT on intracellular contents of RSNO as well as of NO metabolites. 5-HT stimulation resulted in an increase in the intracellular contents of nitrate and cGMP. RSNO was detected in NG108-15 cells and was decreased by 5-HT stimulation. Furthermore, the time course of nitrate increase was coincident with that of RSNO decrease. CarboxyPTIO inhibited 5-HT-induced CIGF, whereas oxyhemoglobin failed to inhibit it. The data suggest that NO is stored in a stable form as RSNO and that 5-HT stimulates NO generation from endogenous RSNO, which is followed by elevation of cGMP via activation of cytosolic guanylyl cyclase by NO in NG108-15 cells. We suggest the existence of a novel 5-HT signal transduction pathway involved in NO generation in NG108-15 cells.

Topics: Animals; Cyclic GMP; Glioma; Hybrid Cells; Kinetics; Neuroblastoma; Nitrates; Nitric Oxide; Nitrites; Nitroprusside; Nitroso Compounds; Oxyhemoglobins; Serotonin; Sulfhydryl Compounds

In the present study, we investigated the effects of chronic in vitro administration of amitriptyline, a tricyclic antidepressant, on cyclic GMP formation stimulated by 5-hydroxytryptamine (5-HT) in the neuroblastoma x glioma hybrid cell line, NG 108-15, 5-HT (0.01-100 microM)-stimulated cyclic GMP formation was concentration-dependent and was sensitive to ICS 205-930, a 5-HT3 receptor antagonist. Exposure of NG 108-15 cells to 5 microM amitriptyline for 3 days significantly reduced 5-HT-stimulated cyclic GMP formation. Acute treatment with amitriptyline had no effect on 5-HT-stimulated cyclic GMP formation. The reduction by chronic amitriptyline exposure of 10 microM 5-HT-stimulated cyclic GMP formation was concentration-dependent over the concentration range examined (0.5 to 10 microM). The IC50 of amitriptyline was 1.9 microM. In contrast, amitriptyline exposure, even at a concentration of 8 microM, failed to modify cyclic GMP formation stimulated by bradykinin, sodium nitroprusside, or atrial natriuretic peptide. Increases in intracellular Ca2+ concentration ([Ca2+]i) evoked by 10 microM 5-HT were attenuated in amitriptyline-exposed cells, while 100 nM bradykinin-induced [Ca2+]i increases were not affected. In addition, chronic exposure to 5 microM amitriptyline caused a decrease in affinity (Kd) of [3H]zacopride specific binding to 5-HT3 recognition sites. The Bmax for the labelled ligand remained unchanged. These results suggest that chronic amitriptyline exposure reduces 5-HT-stimulated cyclic GMP formation and [Ca2+]i increases, and this may reflect the functional changes of 5-HT3 receptors.

Topics: Amitriptyline; Antidepressive Agents, Tricyclic; Benzamides; Bradykinin; Brain Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cyclic GMP; Fluorescent Dyes; Fura-2; Glioma; Hybrid Cells; Indoles; Kinetics; Neuroblastoma; Receptors, Serotonin; Serotonin Antagonists; Tropisetron; Tumor Cells, Cultured

We studied the activity and the ultracytochemical localization of membrane-bound guanylate cyclase (GC) after stimulation with rat atrial natriuretic peptide (rANP), porcine brain natriuretic peptide (pBNP), rat brain natriuretic peptide (rBNP), or porcine C-type natriuretic peptide (CNP) in rat C6 glioma cells during proliferation or following exposure of confluent cells to dibutyryl cyclic AMP (db-cAMP) or retinoic acid (RA). Under our experimental conditions all peptides were activators of GC as demonstrated by the accumulation of cGMP within cells. During proliferation of C6 cells, the amounts of cGMP remained approximately constant. However, at subconfluency, confluency and postconfluency, the GC reaction product was located at different sites in C6 cells. At subconfluency, GC reaction product was on membranes of protoplasmic extensions, at postconfluency, GC reaction product was in association with membranes of cell bodies, and at confluency, both localizations of GC reaction product were detected. Incubation of confluent cells in culture medium containing db-cAMP or RA induced the appearance of long and slender protoplasmic extensions. Under these conditions, the GC reaction product was localized exclusively to these processes. These data suggest that GC is differentially located depending on the state of growth of glial cells, and that in differentiating glial cells GC is preferentially located in cell processes.

Topics: Animals; Atrial Natriuretic Factor; Bucladesine; Cell Division; Culture Media; Cyclic GMP; Glioma; Guanylate Cyclase; Guanylyl Imidodiphosphate; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Nerve Tissue Proteins; Proteins; Rats; Swine; Tretinoin; Tumor Cells, Cultured

The mechanism by which cyclic GMP synthesis is activated through a nucleotide receptor was studied in mouse neuroblastoma x rat glioma hybrid cells [108CC15 (NG 108-15)]. The transient increase in cyclic GMP level induced by ATP reached its maximum at 20 s and lasted for approximately 1 min. The maximal rise in cyclic GMP level achieved was highest for ATP and decreased in the following order: ATP = adenosine 5'(gamma-thio)triphosphate > UTP = 2-methylthio-ATP > ADP much greater than CTP, AMP, alpha,beta-methylene-ATP, 2'- and 3'-O-(4-benzoylbenzoyl)ATP. The EC50 of 1 +/- 0.2 microM for UTP was significantly lower than that for ATP (14 +/- 8 microM) and for all the other nucleotides tested. The rank order of potency is consistent with the pharmacology of a P2u receptor. At submaximal concentrations of the nucleotides ATP and UTP, the rise in cyclic GMP level was inhibited by suramin (IC50 = 40-60 microM) or the pyridoxal phosphate analogue pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (IC50 = 20-30 microM). Pretreatment of cells with the Ca2+ ionophore ionomycin or with 2,5-di(tert-butyl)-1,4-benzohydroquinone, an inhibitor of Ca(2+)-ATPase in the endoplasmic reticulum, a maneuver to deplete internal Ca2+ stores, suppressed the ATP- or UTP-induced stimulation of cyclic GMP synthesis. Similarly, loading of the cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid inhibited cyclic GMP formation by ATP. Preincubation with forskolin to raise the cyclic AMP level potentiated the ATP-induced rise in cyclic GMP level by 60%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Aminoquinolines; Animals; Bradykinin; Calcium; Colforsin; Cyclic GMP; Glioma; Hybrid Cells; Ionomycin; Mice; Neuroblastoma; Neurons; Nitric Oxide; Radioimmunoassay; Rats; Receptors, Purinergic P2; Signal Transduction; Suramin; Tritium; Tumor Cells, Cultured

In non-differentiated NG108-15 cells, both angiotensin II (Ang II) (100 nM) and CGP 42112 (100 nM) decreased the T-type calcium current amplitude by 24 +/- 2% and 21 +/- 3%, respectively. cGMP is not a mediator of the Ang II effect, since loading of cells with 50 microM cGMP did not prevent the inhibitory effects of Ang II. The effects of Ang II involves a non-identified GTPase activity since incubation with GDP beta S (3 mM) completely reversed the inhibitory effect of Ang II while GTP gamma S mimicked its effect. However, Ang II binding was not affected by GTP gamma S, and the effect of Ang II was not modified in pertussis toxin-treated cells. The inhibitory effect of Ang II on the T-type Ca2+ current involves a phosphotyrosine phosphatase activity since sodium orthovanadate prevented the effects of Ang II, although microcystin-LR, a selective Ser/Thr phosphatase 1 and 2A inhibitor, did not modify the effect of Ang II. These results provide the first evidence of a modulation of membrane conductance by Ang II through the AT2 receptor and demonstrate the involvement of a phosphotyrosine phosphatase and a G protein in the AT2 transduction mechanism in NG108-15 cells. Moreover, our data suggest that phosphotyrosine phosphatase activation is proximal to receptor occupation, since sodium orthovanadate inhibits both GTPase activity and T-type current blockage induced by Ang II or CGP 42112, while GTP gamma S inhibition of the T-type calcium current is not impaired.

Topics: Angiotensin II; Angiotensin Receptor Antagonists; Animals; Calcium Channels; Cyclic GMP; Glioma; GTP-Binding Proteins; Hybrid Cells; Kinetics; Membrane Potentials; Neuroblastoma; Oligopeptides; Patch-Clamp Techniques; Receptors, Angiotensin; Time Factors

C6 glioma cells possess endothelin ETA receptor and P2 purinoceptor coupled to two signaling pathways, i.e. phosphoinositide turnover and inhibition of adenylyl cyclase. In this study, the effects of raising cyclic AMP levels on the inositol phospholipid hydrolysis and adenylyl cyclase inhibition caused by endothelin-1 and ATP in C6 glioma cells were examined. Pretreatment with cAMP generating agents (forskolin, isoproterenol and cholera toxin) or dibutyryl cAMP for 10 min-3 h did not affect the inositol phosphate accumulation caused by endothelin and ATP. Long-term (8-24 h) pretreatment with isoproterenol, forskolin, cholera toxin or dibutyryl cAMP resulted in a 40-50% inhibition of endothelin- and ATP-stimulated inositol phosphate accumulation, whereas the EC50 values of endothelin and ATP were not affected. Consistent with the effects on endothelin and ATP, NaF-induced inositol phosphate formation was also inhibited by cAMP generating agents to a similar extent. Permeabilized cells from 24 h isoproterenol-or forskolin-pretreated C6 cells also showed a diminished Ca(2+)-sensitivity of phosphoinositide-specific phospholipase C and also attenuated the potentiation response caused by GTP gamma S. The inhibitory effects on adenylyl cyclase by endothelin, ATP and 2-methylthio-ATP were unaffected by 24 h pretreatment with isoproterenol or forskolin. Long-term treatment with dibutyryl cGMP did not affect the two signaling pathways caused by ATP and endothelin. It is concluded that the phosphoinositide turnover, but not the adenylyl cyclase inhibition caused by endothelin and ATP in C6 cells, was inhibited by protein kinase A-dependent pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Adenylyl Cyclase Inhibitors; Animals; Brain Neoplasms; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Endothelins; Enzyme Activation; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Phosphatidylinositols; Rats; Signal Transduction; Stimulation, Chemical; Tumor Cells, Cultured

Serotonin (5-HT)-2 receptor-mediated cGMP generation was investigated in comparison with calcium (Ca2+) mobilization in C6 glioma cells. 5-HT enhanced cGMP generation, and risperidone and ketanserin potently blocked the response. These results indicate that 5-HT-2 receptors are responsible for the cGMP generation. 5-HT-induced cGMP production was completely abolished by BAPTA, an intracellular Ca2+ chelating agent, or NG-mono-methyl-L-arginine(NMMA), a nitric oxide synthase (NOS) inhibitor, suggesting that 5-HT-induced cGMP generation was through nitric oxide (NO)-dependent pathway. 5-HT (10 microM)-elicited Ca2+ mobilization and cGMP generation were reduced to 40 and 15% after pretreatment with 10 microM 5-HT for 4 hours. NMMA did not modify 5-HT-induced desensitization of either Ca2+ mobilization or cGMP generation, suggesting that NO pathway is independent of the desensitization. The present study has demonstrated the nature of 5-HT-2 receptor-mediated cGMP generation in C6 glioma cells.

Topics: Animals; Arginine; Calcium; Chelating Agents; Cyclic GMP; Dopamine Antagonists; Edetic Acid; Egtazic Acid; Glioma; Haloperidol; Ketanserin; Nitric Oxide; omega-N-Methylarginine; Rats; Receptors, Serotonin; Risperidone; Serotonin Antagonists; Tropanes; Tumor Cells, Cultured

Lipopolysaccharide (LPS) or a combination of interleukin (IL)-1 beta and interferon (IFN)-gamma cause transcriptional induction of a calcium-independent nitric oxide synthase (NOS) in astrocytes and C6 glioma cells. LPS induction of NOS in C6 cells was evidenced by a small amount of nitrite accumulation as compared with cells exposed to IL-1 beta/IFN-gamma, but the maximal NOS activity achieved (as revealed by cGMP formation) was the same. The NOS activity induced by LPS in C6 cells was maximal at 4 to 8 hr and then rapidly decreased, while NOS activity induced by IL-1 beta/IFN-gamma slowly decreased after 4 hr. In addition, the effects of re-presenting IL-1 beta/IFN-gamma to both astrocytes and C6 cells after maximal induction of activity of the inducible form of NOS were studied. The re-addition of cytokines prolonged both NOS mRNA expression and also enzyme activity, suggesting effects at either the transcriptional (further induction) or translational level (mRNA stability). These results imply that the time course of NO production by induced astrocytes depends both upon the nature of the inducing stimulus and the frequency of the cells' exposure to it.

Topics: Amino Acid Oxidoreductases; Animals; Animals, Newborn; Astrocytes; Blotting, Northern; Cells, Cultured; Cyclic GMP; Enzyme Induction; Gene Expression; Glioma; Interferon-gamma; Interleukin-1; Kinetics; Lipopolysaccharides; Neuroglia; Nitric Oxide Synthase; Rats; RNA, Messenger; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

Receptor binding and cyclic GMP generation by three distinct natriuretic peptides (ANP, BNP, CNP) were studied in a cultured rat glioma cell line (C6). Binding studies revealed the presence of high-affinity binding sites for three natriuretic peptides with almost comparable affinities. In contrast, CNP and BNP were almost equipotent in stimulating intracellular cyclic GMP generation over the low concentration range, but CNP caused further elevation in the high concentration range, whereas ANP was minimally effective. Our data suggest that the glioma cells possess receptors more responsive to CNP than ANP and BNP despite no apparent correlation between receptor binding affinities and cyclic GMP responses.

Topics: Animals; Atrial Natriuretic Factor; Binding Sites; Cyclic GMP; Glioma; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Nerve Tissue Proteins; Rats; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; Tumor Cells, Cultured

HS-142-1 is a novel non-peptide antagonist for atrial natriuretic peptide (ANP) receptor. The effect of HS-142-1 on the cyclic GMP production elicited by natriuretic peptides in neuronal cell lines, PC12 and NG108-15 was examined. Natriuretic peptides such as ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) enhanced cyclic GMP production in a dose-dependent manner. HS-142-1 inhibited cyclic GMP accumulation elicited by natriuretic peptides in a dose-dependent fashion in both cells. The results suggest that HS-142-1 will be an important tool for identification and understanding of the mechanisms by which natriuretic peptides act in nervous systems.

Topics: Animals; Atrial Natriuretic Factor; Cell Line; Cyclic GMP; Dose-Response Relationship, Drug; Glioma; Hybrid Cells; Kinetics; Mice; Neuroblastoma; PC12 Cells; Polysaccharides; Rats

Characterization of the serotonin (5-HT)-induced cyclic GMP (cGMP) elevation was investigated in comparison with bradykinin- and ANP-induced elevations in NG108-15 cells. At 20 s, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM, 100 microM), a membrane-permeabilized Ca2+ chelator, or N-monomethyl-L-arginine (NMMA, 300 microM), an inhibitor of L-arginine-derived nitric oxide (NO) synthesis, inhibited 5-HT-induced elevation by approximately 40%, and completely inhibited bradykinin-induced response. Neither 5-HT- nor ANP-induced cGMP elevation at 10 min was affected by BAPTA-AM or NMMA. The cGMP elevated by 5-HT as well as by ANP was effluxed to the extracellular medium. These results and our previous report suggest that 5-HT stimulates two subtypes of 5-HT receptors in NG108-15: first, 5-HT3 subtype stimulating Ca(2+)-sensitive cytosolic guanylate cyclase through NO derived from L-arginine and second, a probably novel 5-HT receptor subtype involved in activation of membrane-bound guanylate cyclase.

Topics: Animals; Atrial Natriuretic Factor; Bradykinin; Cell Line; Cell Membrane; Chelating Agents; Cyclic GMP; Egtazic Acid; Glioma; Guanylate Cyclase; Hybrid Cells; Kinetics; Neuroblastoma; Receptors, Serotonin; Serotonin

Human glioblastoma cells secrete factors, such as prostaglandin E (PGE) and transforming growth factor beta type 2, which are capable of suppressing several immune functions. The present study investigated the effect of PGE2 and agents known to increase intracellular cyclic adenosine monophosphate (cAMP) levels on 1) the induction of lymphokine-activated killer (LAK) cell activity from the peripheral blood lymphocytes (PBL) of both normal and glioma patients and on 2) the cytolytic activities of tumor-infiltrating lymphocytes (TIL's) isolated from malignant gliomas after expansion in vitro with interleukin-2 (IL-2). Cytolytic activity was measured against autologous and allogeneic tumor cells and the natural killer-resistant Daudi cell line. The results demonstrate that PGE2 and agents known to increase intracellular cAMP levels can significantly suppress the IL-2-dependent generation of cytolytic activity from the PBL of normal and glioma patients and from glioblastoma-derived TIL's. The inhibitory effects of these agents could not be reduced by higher concentrations of IL-2 or by cyclic guanosine monophosphate. Although the suppressive effect of PGE2 was most significant during the early stages of LAK cell generation, an inhibitory effect was still evident when PGE2 was added directly to the cytotoxicity assay. Secretion of PGE2 by glioblastoma cells in vivo may regulate both the generation of an immune response and the effectiveness of adoptively transferred immune cells.

Topics: Bucladesine; Cell Division; Cell Survival; Cyclic AMP; Cyclic GMP; Dinoprostone; Glioma; Humans; Immunosuppressive Agents; Interleukin-2; Killer Cells, Lymphokine-Activated; Lymphocytes; Neurotransmitter Agents

Serotonin (5-HT) induced a transient rise of the cyclic GMP level in neuroblastoma X glioma hybrid cells, half-maximally at 1 microM 5-HT. 2-Methyl-5-HT displayed an about 5 times lower potency but equal efficacy. alpha-Methyl-5-HT and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) were completely ineffective at concentrations up to 30 microM. Antagonists specific for 5-HT3 receptors, ICS 205-930, GR 38032 F and MDL 72222, blocked the response to 5-HT at nanomolar concentrations but antagonists directed towards 5-HT1 and 5-HT2 receptors, ketanserin and methysergide, had no effect at concentrations up to 1 microM. Thus, 5-HT3 receptors are responsible for activating guanylate cyclase in the hybrid cells.

Topics: 8-Hydroxy-2-(di-n-propylamino)tetralin; Animals; Cell Line; Cyclic AMP; Cyclic GMP; Glioma; Mice; Neuroblastoma; Neurons; Receptors, Serotonin; Serotonin; Tetrahydronaphthalenes; Tumor Cells, Cultured

Serotonin (5-HT) evoked a rise of cytosolic Ca2+ activity in neuroblastoma X glioma hybrid cells, most probably due to the entry of extracellular Ca2+; cyclic GMP synthesis was also stimulated. The rise of both cytosolic Ca2+ activity and of cyclic GMP level was blocked by memantine (1-amino-3,5-dimethyladamantane). Memantine inhibited the rise of the cyclic GMP level non-competitively (Ki about 50 microM). Thus, memantine suppresses the effects of 5-HT in the neuronal cell line, most likely by blocking Ca2+-permeable ion channels. This interpretation is in line with the previously reported finding that memantine suppressed the 5-HT-induced depolarizing response in the same cell line.

Topics: Amantadine; Animals; Calcium; Cyclic GMP; Cytosol; Glioma; Memantine; Nervous System Neoplasms; Neuroblastoma; Neurons; Rats; Serotonin; Serotonin Antagonists; Tumor Cells, Cultured

The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 microM. The present study examined the effects of extracellular ATP in the concentration range of 0.1-1.0 mM on second-messenger-generating systems in cultured neural cells of the clones NG108-15 and N1E-115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin-2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCl. Extracellular ATP induced a concentration-dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin-2 signals. At the same concentration range (0.1-1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1-stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15-fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG108-15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half-maximal effects on Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Aequorin; Alprostadil; Aminoquinolines; Calcium; Cyclic AMP; Cyclic GMP; Fluorescent Dyes; Glioma; Hybrid Cells; Inositol; Inositol Phosphates; Neuroblastoma; Neurons; Phosphorylation; Potassium; Protein Kinases; Tumor Cells, Cultured

Various prostaglandins (PGs) (10 nM-30 microM) were added to NG108-15 cells in culture, and changes in the levels of intracellular cyclic GMP and Ca2+ were investigated. Exposure of the cells to PGF2 alpha, PGD2, and PGE2 (10 microM) transiently increased the cyclic GMP content 7.5-, 3.9-, and 3.1-fold, respectively. Furthermore, the increased levels of cyclic GMP correlated well with the rise in cytosolic free Ca2+ concentrations induced by the PGs. Other PGs (10 microM), including metabolites and synthetic analogs, which had no effect on intracellular Ca2+, failed to increase the cyclic GMP content in the cells. When extracellular Ca2+ was depleted from the culture medium, the PG-induced increase in cyclic GMP level was almost completely abolished. In addition, treatment of the cells with quin 2 tetraacetoxymethyl ester dose-dependently inhibited the PG-induced cyclic GMP formation. The increase in cyclic GMP content caused by treatment of the cells with a high K+ level (50 mM) was completely blocked by voltage-dependent Ca2+ entry blockers, such as verapamil (10 microM), nifedipine (1 microM), and diltiazem (100 microM); however, the PG (10 microM)-induced increase in cyclic GMP content was not affected by such Ca2+ entry blockers. These findings indicate that PG-induced cyclic GMP formation may require the rise in intracellular Ca2+ level and that the voltage-dependent Ca2+ channels may not be involved in the PG-induced rise in Ca2+ content.

Topics: Aminoquinolines; Animals; Calcium; Cyclic GMP; Diltiazem; Dinoprost; Dinoprostone; Fluorescent Dyes; Glioma; Hybrid Cells; Kinetics; Mice; Neuroblastoma; Nifedipine; Potassium; Prostaglandin D2; Prostaglandins; Prostaglandins D; Prostaglandins E; Prostaglandins F; Rats; Tumor Cells, Cultured; Verapamil

Atrial natriuretic factors (ANFs) were tested for their effects on cyclic GMP production in two neurally derived cell lines, the C6-2B rat glioma cells and the PC12 rat pheochromocytoma cells. These cell lines were selected because both are known to possess high amounts of the particulate form of guanylate cyclase, a proposed target of ANF in peripheral organs. Previous studies from our laboratory have shown that ANF selectively activates particulate, but not soluble, guanylate cyclase in homogenates of a variety of rat tissues and that one class of ANF receptor appears to be the same glycoprotein as particulate guanylate cyclase. In the present study we found that four analogs of ANF stimulate cyclic GMP accumulation in both C6-2B and PC12 cells with the rank order of potency being atriopeptin III = atriopeptin II greater than human atrial natriuretic polypeptide greater than atriopeptin I. Atriopeptin II (100 nM) for 20 min elevated cyclic GMP content in C6-2B cells fourfold and in PC12 cells 12-fold. Atriopeptin II (100 nM) for 20 min also stimulated the efflux of cyclic GMP from both C6-2B cells (47-fold) and PC12 cells (12-fold). Accumulation of cyclic GMP in both cells and media was enhanced by preincubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (250 microM). After 20 min of exposure to atriopeptin II, cyclic GMP amounts in the media were equal to or greater than the amounts in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Adrenal Gland Neoplasms; Animals; Atrial Natriuretic Factor; Cell Line; Cyclic GMP; Dose-Response Relationship, Drug; Glioma; Pheochromocytoma; Rats; Time Factors

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

The replication of HVJ (Sendai virus) in C6 rat glial cells was found to be inhibited by treatment of the cells with papaverine, an inhibitor of cAMP phosphodiesterase, but not with cAMP or dibutyryl cAMP. In addition, cyclic GMP which often manifests a reciprocal relationship to cAMP did not counteract the inhibition of HVJ yield by papaverine. Both viral genome replication and transcription were suppressed slightly by treatment of the cells with papaverine. In the cells cultured in the presence of papaverine, the synthesis of viral proteins and their phosphorylation occurred at normal rates. Membrane immunofluorescence and cell surface immunoprecipitation showed that the viral glycoproteins HN and F0 were expressed on the cell surface of the papaverine-treated cells. Moreover, all the viral structural proteins were associated with plasma membrane isolated from the treated cells. These results indicate that papaverine treatment suppresses some part of the process of virus budding at the plasma membrane.

Topics: Animals; Antigens, Surface; Antigens, Viral; Cell Line; Cell Membrane; Cyclic AMP; Cyclic GMP; Glioma; Papaverine; Parainfluenza Virus 1, Human; Phosphorylation; Rats; RNA, Viral; Viral Proteins; Virus Replication

Atriopeptin III and related atrial natriuretic peptide hormones strongly elevate the level of cyclic GMP in three neural tumor cell lines. At peptide concentrations of 1 microM clear-cut plateaus of the dose-response curves are not yet reached. Atriopeptin III increases the intracellular concentration of cyclic GMP to a maximum in the course of 30-40 min. The effect of atriopeptin III on the cellular cyclic GMP level is independent of the concentration of extracellular Ca2+ and is not affected by the Ca2+ ionophore A23187. These results suggest (1) that atrial natriuretic hormones may play an important role in the nervous system, and (2) that cultured neural cells may be useful tools in the elucidation of the mechanisms of action of these hormones.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Atrial Natriuretic Factor; Calcimycin; Calcium; Cell Line; Cyclic GMP; Glioma; Hybrid Cells; Kinetics; Neuroblastoma; Rats

Topics: Bucladesine; Cells, Cultured; Cyclic AMP; Cyclic GMP; Glioma; Nucleotides, Cyclic; Polyamines

It has been shown recently that astroglial cells of the mammalian CNS possess receptors for neurotransmitters. In order to analyze what sequences of cellular events occur upon activation of these glial receptors, we utilized a 5-HT receptor in a rat clonal cell of glial origin as a model system. When the C6BU-1 glioma cells were exposed to 5-HT, the cytosolic Ca2+ concentration ([Ca2+]i) was elevated and the cellular content of cGMP was increased in a dose-dependent manner. 5-HT receptor antagonists and a Ca2+ entry blocker suppressed the increases in both [Ca2+]i and cGMP. The magnitude of the cGMP increment depended on the environmental Ca2+ concentration and was totally blocked by Ca2+ depletion. Application of a Ca2+ ionophore increased [Ca2+]i and cGMP. There was a tendency for extremely high [Ca2+]i to suppress the cGMP increment. On the contrary, membrane-permeable cyclic nucleotide analogs failed to increase [Ca2+]i. These results suggest that the following sequence of events occurs in 5-HT-induced C6BU-1 cells: activation of 5-HT receptors, Ca2+ influx, a rise in [Ca2+]i, activation of guanylate cyclase, and, finally, activation of cyclic nucleotide phosphodiesterase.

Topics: Animals; Astrocytes; Brain Neoplasms; Calcium; Calcium Channel Blockers; Clone Cells; Cyclic GMP; Cytosol; Dose-Response Relationship, Drug; Ethers; Glioma; Ionomycin; Nucleotides, Cyclic; Rats; Serotonin; Serotonin Antagonists; Time Factors

The cellular cGMP content increased in response to a variety of receptor agonists, which activate [e.g., prostaglandin (PG) E1, E2, and F2 alpha] or inhibit (e.g., alpha-adrenergic, muscarinic, and opiate agonists) adenylate cyclase in neuroblastoma X glioma hybrid NG108-15 cells. The responses were additive when PGF2 alpha and enkephalin were mixed. The inhibitory guanine nucleotide regulatory protein (Ni) is involved in adenylate cyclase inhibition; this function of Ni is lost when it is ADP-ribosylated by islet-activating protein (IAP), pertussis toxin [H. Kurose, T. Katada, T. Amano, and M. Ui (1983) J. Biol. Chem. 258, 4870-4875]. The cGMP rise induced by stimulation of the receptors linked to adenylate cyclase inhibition was also diminished by IAP; the time course and dose response for the IAP-induced diminution were the same between adenylate cyclase inhibition and cGMP generation. Ni thus appears to mediate guanylate cyclase activation as well as adenylate cyclase inhibition initiated via the same receptors. Melittin also increased cGMP. No additivity was shown when enkephalin and melittin were combined, suggesting that phospholipase A2 might play a role in Ni-mediated guanylate cyclase activation. On the other hand, the PGF2 alpha-induced cGMP rise was associated with increased incorporation of 32Pi into phosphatidylinositol; was not affected by cholera toxin, IAP or forskolin; and showed no additivity when combined with A23187, which increased cGMP by itself. PGs would occupy receptors linked to phosphatidylinositol breakdown, thereby increasing the availability of intracellular Ca2+, which is responsible for guanylate cyclase activation. Thus, dual pathways are proposed for a receptor-mediated cGMP rise in NG108-15 cells.

Topics: Adenylate Cyclase Toxin; Animals; Bacterial Toxins; Calcimycin; Cyclic AMP; Cyclic GMP; Dinoprost; Enkephalins; Glioma; Hybrid Cells; Melitten; Mice; Neuroblastoma; Pertussis Toxin; Phospholipids; Prostaglandins F; Rats; Receptors, Cell Surface; Virulence Factors, Bordetella

The nonapeptide, bradykinin, elevated the level of cyclic GMP in two neural cell lines, neuroblastoma X glioma hybrid cells (clone 108CC15) and glioma cells (clone C6-4-2). In the hybrid cells the half-maximal stimulation occurred at 0.1 nM and the maximum was reached at 10 nM bradykinin. As soon as 30 s after the addition of bradykinin to the cultured cells, the intracellular concentration of cyclic GMP had increased maximally. The subsequent decline to the original level proceeded more slowly and lasted around 10 min. Hybrid cells incubated for 10 min in the presence of bradykinin and washed thereafter, did not respond at all to a subsequent 1 min challenge incubation with bradykinin. This nearly complete desensitization lasted for a period of 20 min. One hour after removal of bradykinin the original response to the peptide was restored. Modified and partial sequences of bradykinin were also investigated for their ability to induce the cyclic GMP response in the hybrid cells. Removal of amino acids from either terminus of bradykinin led to an almost complete loss of activity. The data are discussed with respect to our previous observation that bradykinin causes a slow hyperpolarization response in these cell lines and that on prolonged exposure to the peptide the membrane potential response of the cells is lost due to desensitization.

Topics: Animals; Bradykinin; Cell Line; Cyclic GMP; Glioma; Hybrid Cells; Kinetics; Mice; Neuroblastoma; Rats; Structure-Activity Relationship

The biochemical characteristics of the protein kinase (PK; adenosine triphosphate-protein phosphotransferase, EC 2.7.1.37) isozymes in subcellular preparations from normal human brain cortex and glioblastoma were investigated after chromatography on diethylaminoethyl cellulose, and the following results have been obtained. Two major isozyme forms, eluted by 50 and 200 mM phosphate buffer, are present in both cytosol and membrane-derived preparations from cerebral cortex. Furthermore, these isozyme forms have properties similar to those referred to as type I and type II cyclic adenosine 3':5'-monophosphate-dependent PK. In these chromatographic isozymes, cyclic adenosine 3';5'-monophosphate is more active in stimulating the basal PK enzyme than is cyclic guanosine 3':5'-monophosphate. In glioblastoma, the PK activity from cytosol and particulate preparations is resolved by diethylaminoethyl cellulose in four peaks. In cytosol, the major portion of the enzyme is eluted with a 300 mM buffer (about 50% of the total basal PK activity) and is cyclic nucleotide dependent. On the contrary, in glioblastoma particulate, the PK enzyme is mainly eluted at 50 and 100 mM buffer; neither of these isozymes is cyclic nucleotide dependent. As for cytosol, only the particulate isozyme eluted at 300 mM buffer is strongly activated by cyclic nucleotides. Finally, in both glioblastoma subcellular preparations, only a type II cyclic adenosine 3':5'-monophosphate-dependent PK is present.

Topics: Brain; Brain Neoplasms; Chromatography, DEAE-Cellulose; Cyclic AMP; Cyclic GMP; Glioma; Humans; Isoenzymes; Protein Kinases; Subcellular Fractions

Neuroblastoma growth inhibitory factor (NGIF) exists in the conditioned medium of normal rat glioblasts. When neuroblastoma cells (Neuro2a, NS-20Y, and N1E-115) were cultured in the presence of the factor, the cell growth rates and DNA synthesis were markedly inhibited and the morphological differentiation including neural process formation was induced. However, the factor neither altered the growth rate nor the morphology of non-neuronal cells such as glial cell lines (C6 and 354A) or fibroblast (3T3). The molecular weight of the factor was estimated to be 75,000 Mr by gel filtration with Bio-Gel P-200, and the isoelectric point was 5.8. The factor was devoid of esteropeptidase activity, and susceptible to protease and thermal treatment. The growth inhibitory action of the factor was unrelated to the intracellular contents of cyclic AMP and GMP. The ability of NGIF to suppress preferentially the neural growth suggests its regulatory role in normal brain development.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Choline O-Acetyltransferase; Cyclic AMP; Cyclic GMP; DNA Replication; Dopamine beta-Hydroxylase; Glioma; Nerve Tissue Proteins; Neuroblastoma; Neuroglia; Peptide Hydrolases; Rats

The hormonal control of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity has been studied by using as a model the isoproterenol stimulation of cyclic AMP phosphodiesterase activity in C6 glioma cells. A 2-fold increase in cyclic AMP phosphodiesterase specific activity was observed in homogenates of isoproterenol-treated cells relative to control. This increase reached a maximum 3 h after addition of isoproterenol, was selective for cyclic AMP hydrolysis, was reproduced by incubation with 8-Br cycl AMP but not with 8-Br cyclic GMP and was limited to the soluble enzyme activity. The presence of 0.1 mM EGTA did not alter the magnitude of the increase in phosphodiesterase activity. Moreover, the calmodulin content in the cell extracts was not changed after isoproterenol. DEAE-Sephacel chromatography of the 100000 X g supernatant resolved two peaks of phosphodiesterase activity. The first peak hydrolyzed both cyclic nucleotides and was activated by Ca2+ an purified calmodulin. The second peak was specific for cyclic AMP but it was Ca2+- and calmodulin-insensitive. Isoproterenol selectively increased the specific activity of the second peak. Kinetic analysis of the cyclic AMP hydrolysis by the induced enzyme revealed a non-linear Hofstee plot with apparent Km values of 2-5 microM. Cyclic GMP was not hydrolyzed by this enzyme in the absence or presence of calmodulin and failed to affect the kinetics of the hydrolysis of cyclic AMP. Gel filtration chromatography of the induced DEAE-Sephacel peak resolved a single peak of enzyme activity with an apparent molecular weight of 54000.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcium; Calmodulin; Cell Line; Chromatography, Ion Exchange; Cyclic AMP; Cyclic GMP; Glioma; Isoenzymes; Isoproterenol; Kinetics; Rats; Receptors, Adrenergic; Receptors, Adrenergic, beta; Subcellular Fractions

Cyclic-GMP-dependent protein kinase purified from bovine lung was radioiodinated by the Bolton-Hunter procedure yielding a specific radioactivity of 2200 Ci/mmol of enzyme, Using a specific precipitating rabbit antiserum to the cyclic-GMP-dependent protein kinase, a sensitive radioimmunoassay was developed which can detect 200 pg (1.33 fmol) of cyclic GMP-dependent protein kinase. Immunoreactivity like that of cyclic-GMP-dependent protein kinase was detectable in extracts of all rat tissues tested, in extracts of cultured rat brain and heart cells, and in extracts of rat glioma (C6) and neuroblastoma x glioma hybrid cells. In extracts of several tissues and cell lines the presence of cyclic-GMP-dependent protein kinase was also demonstrated by a photoaffinity-labeling procedure using 8-azidoinosine 3',5'-[32P]monophosphate. The results suggest that cyclic-GMP-dependent protein kinase is ubiquitously distributed although its level varies significantly from tissue to tissue and cell type to type. The results also support the hypothesis that cyclic-GMP-dependent protein kinase is involved in mediating some of the intracellular effects of those hormones, neurotransmitters and drugs which regulate the intracellular level of cyclic GMP.

Topics: Animals; Brain; Cattle; Cell Line; Chromatography, DEAE-Cellulose; Cyclic GMP; Cyclic IMP; Glioma; Hybrid Cells; Lung; Myocardium; Neuroblastoma; Protein Kinases; Rabbits; Radioimmunoassay; Rats; Tissue Distribution

Topics: 2-Chloroadenosine; Adenosine; Adenylyl Cyclases; Alprostadil; Cyclic AMP; Cyclic GMP; Cycloheximide; Enzyme Activation; Glioma; Hybrid Cells; Kinetics; Neoplasms, Experimental; Neuroblastoma; Prostaglandins E; Time Factors

Recent advances indicate that cyclic nucleotides and perhaps certain adrenergic metabolites might be directly involved in contact-inhibition mechanisms and tumoral cell growth. CSF levels of 3'-5' cAMP, 3'-5' cGMP, HVA, and 5-HIAA were investigated in patients with supratentorial malignant gliomas, brain stem gliomas and posterior fossa medulloblastoma. cAMP levels were slightly decreased only in medulloblastomas, whereas no significant difference was detectable in patients with supratentorial gliomas. cGMP values turned out to be significantly higher, with a very peculiar increase in the most anaplastic neoplasms. Postoperative follow-up showed a gradual decrease of cGMP values, with progressive restoring of a normal cAMP/cGMP ratio. The possible correlation of these findings with CSF levels of HVA and 5-HIAA is discussed.

Topics: Adult; Aged; Blood-Brain Barrier; Brain Neoplasms; Brain Stem; Cranial Fossa, Posterior; Cyclic AMP; Cyclic GMP; Female; Glioma; Homovanillic Acid; Humans; Hydroxyindoleacetic Acid; Male; Medulloblastoma; Middle Aged; Phenylacetates

Topics: Astrocytoma; Brain Neoplasms; Cyclic AMP; Cyclic GMP; Glioblastoma; Glioma; Humans; Meningeal Neoplasms; Meningioma

Topics: Animals; Cations, Divalent; Cell Line; Cell Membrane; Cyclic AMP; Cyclic GMP; Glioma; Kinetics; Magnesium; Membrane Proteins; Phosphorylation; Protein Kinases; Rats

(-)-Norepinephrine and other catecholamines inhibit basal and prostaglandin E1-stimulated adenylate cyclase activities by 35 to 60% in homogenates of NG108-15 neuroblastoma x gloma hybrid cells and markedly reduce adenosine 3'35:'-monophosphate levels of intact cells, but do not affect guanosine 3':5'-monophosphate levels. The specificity of the NG108-15 receptor for ligands is that of an alpha receptor, possibly a presynaptic alpha 2 receptor. The inhibition of adenylate cyclase by norepinephrine is reversed by alpha receptor antagonists such as dihydroergotamine or phentolamine, but not by the beta receptor antagonist propranolol. The effect of norepinephrine on adenylate cyclase activity initially is dependent on GTP; half-maximal inhibition of enzyme activity by norepinephrine is obtained with 0.2 micron GTP. The inhibition of adenylate cyclase activity by norepinephrine is reduced by 10 mM NaF and is abolished by 0.05 mM guanyl-5'-yl imidodiphosphate. Inhibitions of NG108-15 adenylate cyclase mediated by alpha receptors, opiate receptors, and muscarinic acetylcholine receptors are not additive; this suggests that the three species of receptors can be functionally coupled to the same adenylate cyclase molecules or molecules regulating the enzyme.

Topics: Adenylyl Cyclase Inhibitors; Adrenergic alpha-Antagonists; Animals; Cells, Cultured; Cyclic AMP; Cyclic GMP; Glioma; Hybrid Cells; Mice; Neoplasms, Experimental; Neuroblastoma; Norepinephrine; Rats; Receptors, Adrenergic; Receptors, Adrenergic, alpha; Receptors, Muscarinic; Receptors, Opioid

Recent evidence indicates that cyclic nucleotides are of importance for general and neurosurgical oncology, especially with respect to the contact-inhibition mechanisms and tumour cell growth. This preliminary report deals with the CSF levels of c-AMP and c-GMP in primary neoplasms in children and to problems related to the blood-brain barrier. Some cases of medulloblastoma were studied as well as a few cases of brain stem glioma and cystic astrocytoma. The importance of some rather unusual findings seems undebatable, i.e., the marked increase in c-GMP values usually observed in medulloblastoma and the decrease of c-AMP, that is fairly common in all malignant neoplasms. The main changes in the c-AMP/c-GMP ratio are also discussed.

Topics: Astrocytoma; Blood-Brain Barrier; Brain Neoplasms; Brain Stem; Cerebellar Neoplasms; Child; Child, Preschool; Cyclic AMP; Cyclic GMP; Glioma; Humans; Infant; Medulloblastoma

Since the effects of cyclic nucleotides are mediated via protein kinases activation, we have studied the properties and regulation of these enzymes in cytosol and particulate fraction of normal cerebral tissues and of some human brain tumors. We found that distribution and activity of cyclic nucleotide-dependent protein kinases are regulated differently among various brain tumors and in comparison to normal gray and white matter. Pathological tissues show an higher cGMP-dependent protein kinase and this biochemical pattern is particularly evident in tumors with more pronounced malignancy. These data further confirm the hypothesis of a correlation between the increase of cGMP function and cellular growth and malignancy.

Topics: Astrocytoma; Brain Neoplasms; Cyclic AMP; Cyclic GMP; Cytosol; Glioma; Humans; Kinetics; Neurilemmoma; Protein Kinases

Topics: Animals; Catecholamines; Cell Line; Cyclic AMP; Cyclic GMP; Glioma; L-Lactate Dehydrogenase; Neurotransmitter Agents; Psychotropic Drugs; Rats; Time Factors

Several reports have suggested that cylcic guanosine 3'-5' monophosphate (cGMP) and cyclic 3'-5' adenosine monophosphate (cAMP) are involved in the regulation of cellular proliferation. Following our previous reports on the cAMP system in human brain tumors, we decided to investigate the cGMP system in the same pathological tissues by studying the activity of guanylate cyclase and cGMP-phosphodiesterase (cGMP-PDE). We found that the activity of both enzymes is lower in neurinomas and glioblastomas than in meningiomas or in normal cerebral cortex. Furthermore, the subcellular distribution of guanylate cyclase in human cerebral cortex differs from that of neurinomas and glioblastomas. On the basis of such observations we have discussed the possibility that the regulatory mechanism of the enzymes related to the cyclic nucleotide metabolism is altered in brain tumors.

Topics: Brain Neoplasms; Cerebral Cortex; Cyclic GMP; Glioma; Guanosine; Guanylate Cyclase; Humans; Meningioma; Neurilemmoma; Phosphoric Diester Hydrolases

Topics: Animals; Cyclic AMP; Cyclic GMP; Dextrorphan; Dose-Response Relationship, Drug; Glioma; Hybrid Cells; Levorphanol; Mice; Morphine; Naloxone; Neuroblastoma; Neuroglia; Neurons; Rats; Receptors, Drug

A culture line of mouse glioblastoma cells changed morphologically to differentiated astrocyte-like cells when cultured in medium with dibutyryl cyclic adenosine monophosphate and theophylline. Morphological alteration occurred within only 5 hr when 3 mM dibutyryl cyclic adenosine monophosphate and 1 mM theophylline were used, and in 1 day when 1 mM theophylline were used. Cells showing this morphological change reverted completely to immature cells when they were transferred to medium without these two chemicals. Addition of 1 or 3 mM dibutyryl cyclic guanosine monophosphate with 1 mM theophylline to the medium also induced development of cytoplasmic processes from these cells and the cells became stellate, although the cytoplasmic processes were not as long or as numerous as those induced by dibutyryl cyclic adenosine monophosphate, and the altered cells could not be referred to as differentiated glia cells. Sodium butyrate induced morphological alterations similar to those induced by dibutyryl cyclic guanosine monophosphate, but fewer cells showed these alterations. Addition of cyclic adenosine monophosphate or cyclic guanosine monophosphate in the presence of theophylline or addition of theophylline alone did not induce morphological changes of the cells.

Topics: Animals; Bucladesine; Butyrates; Cell Differentiation; Cell Line; Cells, Cultured; Cyclic GMP; DNA, Neoplasm; Dose-Response Relationship, Drug; Glioma; Mice; Neoplasms, Experimental; Neuroglia; Theophylline; Thymidine; Time Factors

Topics: Animals; Cell Line; Cyclic AMP; Cyclic GMP; Glioma; Hybrid Cells; Mice; Morphine; Neuroblastoma; Neurons; Norepinephrine; Opium; Prostaglandins E; Protein Biosynthesis; Rats; Stimulation, Chemical; Time Factors

Topics: 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Adenine; Catecholamines; Cell Line; Cyclic AMP; Cyclic GMP; Dimethyl Sulfoxide; Epinephrine; Glioma; Isoproterenol; Neurons; Papaverine; Phenoxybenzamine; Prostaglandins E; Sotalol; Zeatin

Topics: Acetylcholine; Carbachol; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Drug Interactions; Glioma; Hybrid Cells; Neuroblastoma; Neuroglia; Neurons; Parasympathomimetics; Pilocarpine; Quaternary Ammonium Compounds; Sodium

Topics: Animals; Brain; Calcium; Calcium Radioisotopes; Cell Line; Chelating Agents; Chromatography, Ion Exchange; Clone Cells; Cyclic AMP; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Glioma; Kinetics; Neoplasm Proteins; Phosphoric Diester Hydrolases; Rats; Receptors, Drug; Subcellular Fractions; Swine; Tritium