guanosine-diphosphate and Glioma

Studies with brain slices have provided evidence that synthesis of kynurenic acid (KYNA) from kynurenine (KYN), which occurs in astrocytes, is modulated by changes in the ionic composition of the medium and the presence of depolarizing agents or the excitatory amino acid glutamate (Glu). The present study analyzed the effects of changes in incubation medium on KYNA synthesis in cultured C6 glioma cells. The synthesis was not affected by omission of Na(+) and raising K(+) concentration to 50 mM, conditions that in brain slices stimulate or inhibit KYNA formation, respectively. KYNA synthesis in C6 cells was inhibited by the absence of Ca(2+), which contrasts with its Ca(2+) independence in brain slices. Also, lack of Mg(2+) and addition of a chloride channel blocker, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonate (SITS), did not affect the synthesis. KYNA synthesis in C6 cells was dose dependently inhibited by Glu. The inhibitory effect of Glu was not affected by GDPbetaS, an antagonist of metabotropic Glu receptors, the receptor class prevailing in C6 cells, suggesting that Glu acted intracellularly. NH(4)Cl and veratridine decreased KYNA production, mirroring the effects noted in brain slices. KYNA synthesis was strongly reduced in the presence of leucine (Leu), and the uptake of [(14)C]Leu was inhibited by the KYNA precursor KYN, which points to Leu as a potential endogenous modulator of KYNA formation in CNS cells.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Buffers; Calcium; Glioma; Glutamic Acid; Guanosine Diphosphate; Kynurenic Acid; Kynurenine; Leucine; Magnesium; Potassium; Rats; Receptors, Metabotropic Glutamate; Sodium; Thionucleotides; Tumor Cells, Cultured; Veratridine

The present study reports on G-protein activation by recombinant 5-HT receptors and by native 5-HT1A and 5-HT1B receptors in guinea-pig and rat brain using agonist-stimulated [35S]GTPgammaS binding responses mediated by a new 5-HT ligand, a dimer of sumatriptan. Dimerization of sumatriptan increased the binding affinity for h 5-HT1B (pKi: 9.22 vs. 7.79 for sumatriptan), h 5-HT1D (9.07 vs. 8.08) and also h 5-HT1A receptors (7.80 vs. 6.40), while the binding affinity for h 5-ht1E (6.67 vs. 6.19) and h 5-ht1F (7.37 vs. 7.78) receptors was not affected. Sumatriptan dimer (10 microM) stimulated [35S]GTPgammaS binding mainly in the superficial gray layer of the superior colliculi, hippocampus and substantia nigra of guinea-pig and rat coronal brain sections. This fits with the labelling by the 5-HT1B/1D receptor antagonist [3H] GR 125743. The observed [35S]GTPgammaS binding responses in the substantia nigra are likely to be mediated by stimulation of the 5-HT1B receptor subtype, since they were antagonized by the 5-HT1B inverse agonist SB 224289 (10 microM), and not by the 5-HT2A/1D antagonist ketanserin (10 microM). Quantitative assessment of the [35S]GTPgammaS binding responses in the substantia nigra of rat showed highly efficacious responses for both sumatriptan dimer and its monomer. In contrast, less efficacious agonist responses (51+/-10% and 35+/-13%, respectively) were measured in the guinea-pig substantia nigra. This may suggest that the G-protein coupling efficacy of 5-HT1B receptors is different between the substantia nigra of both species. In addition, the sumatriptan dimer also activated guinea-pig and rat hippocampal 5-HT1A receptors with high efficacy in contrast to sumatriptan. Therefore, dimerization of sumatriptan can be considered as a new approach to transform a partial 5-HT1A agonist into a more efficacious agonist. In conclusion, the sumatriptan dimer stimulates G-protein activation via 5-HT1B receptors besides 5-HT1A receptors in guinea-pig and rat brain. The magnitude of the 5-HT1B receptor responses is superior for sumatriptan and its dimer in rat compared to guinea-pig substantia nigra.

Topics: Animals; Autoradiography; Binding, Competitive; Brain Chemistry; Dimerization; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guinea Pigs; HeLa Cells; Humans; Male; Membrane Proteins; Oxadiazoles; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT1B; Receptors, Serotonin; Receptors, Serotonin, 5-HT1; Serotonin Receptor Agonists; Sulfur Radioisotopes; Sumatriptan; Tryptamines

Chronic treatment of neuroblastoma x glioma NG108-15 hybrid cells with the opioid agonist D-Ala,2 D-Leu5-enkephalin (DADLE) induces a homologous desensitization of the delta opioid receptors present in these cells. Since the Kd value of the delta opioid receptor's high-affinity state reflects the potency of the agonist, we examined the effect of receptor desensitization in NG108-15 cells on the percentage of receptor in the high-affinity state. When NG108-15 hybrid cells were treated with 10 or 100 nM DADLE for 4 hr at 24 degrees C, loss of DADLE's ability to inhibit adenylate cyclase was observed. However, when competition binding experiments were carried out with P2P3 membranes isolated from the delta opioid-desensitized hybrid cells, it was determined that 41.7 +/- 3.4% of the total binding sites remained in the high-affinity state, with no apparent alteration in the Kd value of either high- or low-affinity states. Similarly, when NG108-15 cells were treated with 100 ng/ml of pertussis toxin for 3 hr at 37 degrees C, 39.9 +/- 3.6% of the binding sites remained in the high-affinity state. This reduction in the percentage of receptor in high-affinity state was agonist specific, for chronic treatment of hybrid cells with levorphanol, a partial agonist, or the antagonist naloxone did not alter the percentage of opioid receptors in the high-affinity state. Furthermore, the delta opioid receptors remaining in the high-affinity state after chronic DADLE treatment were still sensitive to both Na+ and guanyldylimidodiphosphate, indicating that opioid ligand binding remained coupled to the G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylate Cyclase Toxin; Binding, Competitive; Enkephalin, Leucine-2-Alanine; Glioma; GTP-Binding Proteins; Guanosine Diphosphate; Guanylyl Imidodiphosphate; Humans; Hybrid Cells; Magnesium; Neuroblastoma; Pertussis Toxin; Receptors, Opioid; Receptors, Opioid, delta; Sodium; Virulence Factors, Bordetella

Regulation of [125I]beta h-endorphin binding by guanine nucleotides was investigated in membrane preparations from two opioid receptor-containing cell lines: NG108-15, which contains only delta opioid receptors, and SK-N-SH, which contains predominantly mu opioid receptors. In contrast to the binding of the delta-selective agonist [3H][D-penicillamine2,D-penicillamine5]enkephalin to NG108-15 cell membranes, and of the mu-selective agonist [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin to SK-N-SH cell membranes, [125I]beta h-endorphin binding to NG108-15 and SK-N-SH cell membranes was not altered by guanosine triphosphate (GTP) or guanylyl-5'-imidodiphosphate (Gpp(NH)p) in the absence of cations. However, in the presence of NaCl, [125I]beta h-endorphin binding to both cell lines was inhibited by GTP and Gpp(NH)p in a concentration-dependent manner. In SK-N-SH cell membranes, the ability of sodium to promote regulation of [125I]beta h-endorphin binding by GTP was mimicked by the monovalent cations lithium and potassium, but not by the divalent cations magnesium, calcium, or manganese. In NG108-15 cell membranes, only sodium was effective in promoting inhibition of [125I]beta h-endorphin binding by GTP. The effect of GTP or Gpp(NH)p in the presence of sodium was also observed with guanosine diphosphate, but not guanosine monophosphate or any of the non-guanine nucleotides tested. These results indicate that the presence of monovalent cations is required for regulation of [125I]beta h-endorphin binding by guanine nucleotides, and that the specificity of this cation requirement differs between the mu and delta receptor-containing cell lines.

Topics: beta-Endorphin; Cations; Cell Membrane; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Glioma; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Hybrid Cells; Neuroblastoma; Oligopeptides; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sodium Chloride; Tumor Cells, Cultured

Incubation of membranes of neuroblastoma x glioma hybrid, NG108-15 cells with GDP beta S followed by immunoblotting of resolved membrane and supernatant fractions with specific anti-peptide antisera showed essentially all of the alpha subunit of Go to be associated with the membrane. Similar experiments with poorly hydrolyzed analogues of GTP caused release of a significant fraction (some 50% within 60 minutes) of Go alpha into the supernatant. This was not mimicked by analogues of ATP. Antisera directed against peptides corresponding to the extreme N and C-termini of GO alpha demonstrated that the released polypeptide was not proteolytically clipped. These experiments show that the alpha subunit of GO need not be invariably bound to the plasma membrane and that guanine nucleotide activation can release the alpha subunit of GO from its site of membrane attachment.

Topics: Animals; Cell Line; Cell Membrane; Glioma; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Macromolecular Substances; Neuroblastoma; Thionucleotides

The cAMP content of intact cells as well as adenylate cyclase of the membrane-rich particulate fractions was studied with C6 glioma cells that had been exposed to the culture medium supplemented with islet-activating protein (IAP), one of the pertussis toxins. Both the increase in the cellular cAMP content in response to a beta-adrenergic agonist and the stimulation of membrane adenylate cyclase by the beta-agonist and/or GTP were markedly enhanced by the IAP treatment of C6 cells, but no change was induced in affinities of the agonist (or an antagonist) or GTP for their respective sites of action (or binding). The concentration of IAP required for the half-maximal enhancement was as low as 1 pg/ml, when the time of cell exposure to the toxin was prolonged to 18 h. No enhancement was observed for the basal cAMP content or basal enzyme activity, nor was activation of adenylate cyclase by Gpp(NH)p (or NaF) affected by IAP treatment. The Vmax value of a specific and low Km GTPase was significantly smaller in the membranes of IAP-treated cells than in those of control cells. Cholera toxin treatment of cells activated adenylate cyclase without exerting any influence on these IAP actions. Thus, IAP would appear to enhance beta-receptor-coupled stimulation of adenylate cyclase, in a manner distinct from cholera toxin, by rendering more GTP available to the GTP sites on the regulatory subunit of the receptor-enzyme system.

Topics: Adenine Nucleotides; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Bacterial Proteins; Calcium; Cell Line; Cell Membrane; Cyclic AMP; Glioma; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hypoglycemic Agents; Isoproterenol; Pertussis Toxin; Rats; Virulence Factors, Bordetella

The guanine nucleotides GDP, GTP, and guanosine-5'-(beta, gamma-imido)triphosphate inhibit binding of opiates and opioid peptides to receptors solubilized from membranes of neuroblastoma X glioma NG108-15 hybrid cells. The inhibition reflects decreased affinity of receptors for opioid ligands. Whereas in membranes, only opioid agonist binding is sensitive to guanine nucleotide inhibition, both agonist and antagonist binding is reduced in the case of soluble receptors. Furthermore, soluble receptors are more sensitive to the effects of guanine nucleotides than are membrane-bound receptors. These observations are consistent with the suggestion that solubilized receptors may be complexes of an opiate binding protein and a guanine nucleotide-sensitive regulatory component.

Topics: Animals; Cell Line; Cell Membrane; Diprenorphine; Etorphine; Glioma; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Mice; Morphinans; Neuroblastoma; Rats; Receptors, Opioid