guanosine-triphosphate and Lymphoma

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cell Line; Fluorides; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Lymphoma; Magnesium; Mice; Models, Biological; Molecular Weight; Receptors, Cell Surface

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Animals; Bacterial Toxins; Cell Line; Cholera Toxin; GTP Phosphohydrolases; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Hormones; Lymphoma; Mice; Neurotransmitter Agents; Receptors, Cell Surface; Stimulation, Chemical; Virulence Factors, Bordetella

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Animals; Bacterial Toxins; Cell Line; Chemical Phenomena; Chemistry; Cholera Toxin; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Lymphoma; Membrane Proteins; Mice; Rhodopsin; Rod Cell Outer Segment; Saccharomyces cerevisiae; Transducin; Virulence Factors, Bordetella

Topics: Adenylyl Cyclases; Animals; Binding Sites; Biological Transport; Calcium; Clone Cells; Enzyme Activation; Guanosine Triphosphate; Kinetics; Ligands; Lymphoma; Magnesium; Manganese; Models, Biological; Receptors, Cell Surface

Vav1 is normally and exclusively expressed in the hematopoietic system where it functions as a specific GDP/GTP nucleotide exchange factor (GEF), firmly regulated by tyrosine phosphorylation. Mutations and overexpression of Vav1 in hematopoietic malignancies, and in human cancers of various histologic origins, are well documented. To reveal whether overexpression of Vav1 in different tissues suffices for promoting the development of malignant lesions, we expressed Vav1 in transgenic mice by using the ubiquitous ROSA26 promoter (Rosa Vav1). We detected Vav1 expression in epithelial tissues of various organs including pancreas, liver, and lung. While carcinomas did not develop in these organs, surprisingly, we noticed the development of B-cell lymphomas. Rac1-GTP levels did not change in tissues from Rosa Vav1 mice expressing the transgenic Vav1, while ERK phosphorylation increased in the lymphomas, suggesting that signaling pathways are evoked. One of the growth factors analyzed by us as a suspect candidate to mediate paracrine stimulation in the lymphocytes was CSF-1, which was highly expressed in the epithelial compartment of Rosa Vav1 mice. The expression of its specific receptor, CSF-1R, was found to be highly expressed in the B-cell lymphomas. Taken together, our results suggest a potential cross-talk between epithelial cells expressing Vav1, that secrete CSF-1, and the lymphocytes that express CSF-1R, thus leading to the generation of B-cell lymphomas. Our findings provide a novel mechanism by which Vav1 contributes to tumor propagation.

Topics: Animals; Guanosine Triphosphate; Humans; Lymphoma; Lymphoma, B-Cell; Macrophage Colony-Stimulating Factor; Mice; Proto-Oncogene Proteins c-vav; Receptor Protein-Tyrosine Kinases

The 21-kD protein Ras of the low-molecular-weight GTP-binding (LMWG) family plays an important role in transduction of extracellular signals. Ras functions as a 'molecular switch' in transduction of signals from the membrane receptors of many growth factors, cytokines, and other second messengers to the cell nucleus. Numerous studies have shown that in multiple malignant tumors and hematopoietic malignancies, faulty signal transduction via the Ras pathway plays a key role in tumorigenesis. In this work, a non-radioactive assay was used to quantify Ras activity in hematologic malignancies. Ras activation was measured in six different cell lines and 24 patient samples, and sequence analysis of N- and K-ras was performed. The 24 patient samples comprised of seven acute myelogenous leukemia (AML) samples, five acute lymphocytic leukemia (ALL) samples, four myeloproliferative disease (MPD) samples, four lymphoma samples, four juvenile myelomonocytic leukemia (JMML) samples, and WBC from a healthy donor. The purpose of this study was to compare Ras activity determined by percentage of Ras-GTP with the mutational status of the Ras gene in the hematopoietic cells of the patients. Mutation analysis revealed ras mutations in two of the seven AML samples, one in codon 12 and one in codon 61; ras mutations were also found in two of the four JMML samples, and in one of the four lymphoma samples (codon 12). We found a mean Ras activation of 23.1% in cell lines with known constitutively activating ras mutations, which was significantly different from cell lines with ras wildtype sequence (Ras activation of 4.8%). Two of the five activating ras mutations in the patient samples correlated with increased Ras activation. In the other three samples, Ras was probably activated through "upstream" or "downstream" mechanisms.

Topics: DNA Mutational Analysis; Guanosine Diphosphate; Guanosine Triphosphate; Hematologic Neoplasms; Humans; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Juvenile; Lymphoma; Mutation; Myeloproliferative Disorders; Oncogenes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; ras Proteins; Signal Transduction; Tumor Cells, Cultured

The proliferation of human lymphoblastoma cell line (H9) was differently stimulated by Peptide Histidine Methionine (PHM) and Vasoactive Intestinal Peptide (VIP). PHM induced a cyclic AMP (cAMP) accumulation, abolished by Adenylate Cyclase (AC) inhibitors leading to a loss of proliferative effect. VIP mitogenic activity was Pertussis toxin (PTX) sensitive and AC inhibitors insensitive. Pharmacological experiments performed on H9 membranes with or without a GTP analogue indicated expression of both GTP-insensitive and -sensitive PHM/VIP high-affinity binding sites (HA). H9 cells expressed only the VPAC1 receptor. VIP(10-28), known as a VPAC1 antagonist, bond to all GTP-insensitive PHM sites and inhibited evenly the PHM and VIP mitogenic actions. These data strongly suggested different mechanisms initiated by VIP and PHM and highlighted the key role of GTP-insensitive binding sites in the control of cell proliferation.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Analysis of Variance; Blotting, Southern; Bromodeoxyuridine; Cell Line, Tumor; Cell Proliferation; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Imines; Iodine Isotopes; Lymphoma; Peptide Fragments; Peptide PHI; Pertussis Toxin; Protein Binding; Radioligand Assay; Receptors, Cell Surface; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Peptide, Type II; Receptors, Vasoactive Intestinal Polypeptide, Type I; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Vasoactive Intestinal Peptide

The regulatory G-proteins of adenylyl cyclase (AC), G(i) and G(s), are not only activated by GTP and the stable GTP analogs, guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) and guanosine 5'-[beta,gamma-imido]triphosphate (GppNHp), but also by hypoxanthine, xanthine, uracil and cytidine nucleotides. The latter nucleotides were previously used to analyze distinct active G-protein states. Surprisingly, recent studies have shown that inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate can also inhibit AC directly. Therefore, we systematically compared the interactions of nucleoside 5'-triphosphates (NTPs), nucleoside 5'-[gamma-thio]triphosphates (NTPgammaSs) and nucleoside 5'-[beta,gamma-imido]triphosphates (NppNHps) with G(i), G(s) and AC. NTPgammaSs exhibited up to 26,000-fold higher affinity for G-proteins than NTPs and NppNHps. NTPgammaSs were up to 150-fold more potent direct AC inhibitors than NTPs and NppNHps. G-proteins exhibited striking preference for guanine nucleotides compared to other purine and pyrimidine nucleotides, whereas base-selectivity of various ACs, particularly the purified catalytic subunits C1.C2, was rather poor. GTP, GTPgammaS and GppNHp exhibited much higher selectivity for G-proteins relative to AC than all other purine and pyrimidine nucleotides. We have energetically characterized the interactions of purine and pyrimidine nucleotides with AC in silico, constructing pharmacophore models that correlate well with experimental affinities and have elucidated specific amino acid residues with greatest influence on nucleotide binding. Collectively, both G-proteins and ACs bind purine and pyrimidine nucleotides, with G-proteins showing much higher base-selectivity than AC. Thus, direct inhibitory effects of nucleotides on AC should be understood and considered when probing distinct active G-protein states with non-guanine nucleotides.

Topics: Adenylyl Cyclases; Animals; Catalysis; Cell Line, Tumor; Colforsin; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Lymphoma; Models, Molecular; Protein Binding; Spodoptera

To study behavior of activated G(alpha)(s) in living cells, green fluorescent protein (GFP) was inserted within the internal amino acid sequence of G(alpha)(s) to generate a G(alpha)(s)-GFP fusion protein. The fusion protein maintained a bright green fluorescence and was identified by immunoblotting with antibodies against G(alpha)(s) or GFP. The cellular distribution of G(alpha)(s)-GFP was similar to that of endogenous G(alpha)(s). G(alpha)(s)-GFP was tightly coupled to the beta adrenergic receptor to activate the G(alpha)(s) effector, adenylyl cyclase. Activation of G(alpha)(s)-GFP by cholera toxin caused a gradual displacement of the fusion protein from the plasma membrane throughout the cytoplasm in living cells. Unlike the slow release of G(alpha)(s)-GFP from the membrane induced by cholera toxin, the beta-adrenergic agonist isoproterenol caused a rapid partial release of the fusion protein into the cytoplasm. At 1 min after treatment with isoproterenol, the extent of G(alpha)(s)-GFP release from plasma membrane sites was maximal; however, insertion of G(alpha)(s)-GFP at other membrane sites occurred during the same time period. Translocation of G(alpha)(s)-GFP fusion protein induced by isoproterenol suggested that the internalization of G(alpha)(s) might play a role in signal transduction by interacting with effector molecules and cytoskeletal elements at multiple cellular sites.

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Cells, Cultured; Cholera Toxin; Computer Systems; COS Cells; Enzyme Activation; Green Fluorescent Proteins; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Isoproterenol; Luminescent Proteins; Lymphoma; Recombinant Fusion Proteins; Spectrometry, Fluorescence; Transfection; Tumor Cells, Cultured

The synthesis and biological activity of selenophenfurin (5-beta-D-ribofuranosylselenophene-3-carboxamide, 1), the selenophene analogue of selenazofurin, are described. Glycosylation of ethyl selenophene-3-carboxylate (6) under stannic chloride-catalyzed conditions gave 2- and 5-glycosylated regioisomers, as a mixture of alpha- and beta-anomers, and the beta-2,5-diglycosylated derivative. Deprotected ethyl 5-beta-D-ribofuranosylselenophene-3-carboxylate (12 beta) was converted into selenophenfurin by ammonolysis. The structure of 12 beta was determined by 1H- and 13C-NMR, crystallographic, and computational studies. Selenophenfurin proved to be antiproliferative against a number of leukemia, lymphoma, and solid tumor cell lines at concentrations similar to those of selenazofurin but was more potent than the thiophene and thiazole analogues thiophenfurin and tiazofurin. Incubation of K562 cells with selenophenfurin resulted in inhibition of IMP dehydrogenase (IMPDH) (76%) and an increase in IMP pools (14.5-fold) with a concurrent decrease in GTP levels (58%). The results obtained confirm the hypothesis that the presence of heteroatoms such as S or Se in the heterocycle in position 2 with respect to the glycosidic bond is essential for both cytotoxicity and IMP dehydrogenase inhibitory activity in this type of C-nucleosides.

Topics: Animals; Antineoplastic Agents; Cell Division; Computer Simulation; Crystallography, X-Ray; Enzyme Inhibitors; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Inosine Monophosphate; Leukemia; Lymphoma; Magnetic Resonance Spectroscopy; Mice; Models, Molecular; Molecular Structure; Neoplasms; Organoselenium Compounds; Ribavirin; Ribonucleosides; Tumor Cells, Cultured

The extracellular accumulation of ATP after activation of T-lymphocytes, as well as the presence of ecto-protein kinases in these cells, led us to propose that T cell surface receptors could be regulated through the reversible phosphorylation of their extracellular domains (ectodomains). Here, in a model system, we used T cell transfectants which express T cell antigen receptor chains lacking intracellular and transmembrane protein domains and 32Pi metabolic labeling of cells to definitively demonstrate phosphorylation of ectodomains of T cell surface proteins. We show that alphabetaTCR ectodomains were phosphorylated intracellularly and constitutively on serine and threonine residues and were then expressed on the T cell surface in phosphorylated form. TCR ectodomains also could be phosphorylated at the cell surface when extracellular [gamma-32P]ATP or [gamma-32P]GTP were used as phosphate donors with the same cells. Consensus phosphorylation sites for serine and threonine protein kinases were found to be strongly evolutionary conserved in both alpha and beta TCR chains constant regions. These results are consistent with the hypothesis, where T cell surface proteins which are phosphorylated intracellularly on their ectodomains, could subsequently be expressed at the cell surface and then be reversibly modified by ectoprotein phosphatase(s) and by ectokinase(s). Such modifications may change T cells cognate interactions by, e.g. affecting TCR-multimolecular complex formation and antigen binding affinity. It is suggested that alphabetaTCR ectodomain phosphorylation could serve as a potential mechanism for regulation of alphabetaTCR-mediated T-lymphocytes response.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Antibodies, Monoclonal; Biological Evolution; Cell Line; Conserved Sequence; Guanosine Triphosphate; Humans; Lymphoma; Membrane Proteins; Phosphates; Phosphoprotein Phosphatases; Phosphorus Radioisotopes; Phosphorylation; Phosphoserine; Phosphothreonine; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes; Tumor Cells, Cultured; Type C Phospholipases

This study was undertaken to examine the effects of hydrogen peroxide on stimulatory guanine nucleotide-binding protein (Gs), and coupling in the beta-adrenergic receptor-Gs-adenylate cyclase system in rat heart, in vitro. Cardiac membranes were preincubated with various concentrations (0.1, 1, and 10 mM) of hydrogen peroxide at 30 degrees C for 5, 10, 30 and 60 min. Although the assay of beta-adrenergic receptors involving [3H]-dihydroalprenolol ([3H]-DHA) binding revealed that the maximal number of binding sites (Bmax) was not altered, the dissociation constant (Kd) for [3H]-DHA was increased in the presence of 1 mM and 10 mM hydrogen peroxide (control 0.68 +/- 0.16 nM, vs 1 mM H2O2 1.13 +/- 0.16, 10 mM H2O2 1.01 +/- 0.12). Conversely, no significant changes in Gs activities were observed in hydrogen peroxide-treated groups. Adenylate cyclase activity (stimulated by forskolin) was significantly reduced by 10 mM hydrogen peroxide after a 5 min preincubation period (control 277.1 +/- 19.2 pmol cAMP/mg protein/min, H2O2 230.3 +/- 14.9). The amounts of cyclic AMP produced by the stimulation of membranes with GTP, GTP+(l)-isoproterenol, guanylimidodiphosphate (Gpp(NH)p) or Gpp(NH)p+(l)-isoproterenol were significantly lower in 10 mM hydrogen peroxide-treated groups than those in controls (GTP: control 57.6 +/- 5.6 pmol cAMP/mg protein/min vs H2O2 46.4 +/- 6.9, GTP+(l)-isoproterenol: control 83.9 +/- 10.2 vs H2O2 67.7 +/- 10.3, Gpp(NH)p: control 77.5 +/- 8.8 vs H2O2 61.0 +/- 8.6, Gpp(NH)p+(l)-isoproterenol: control 105.0 +/- 13.1 vs H2O2 83.9 +/- 12.2, forskolin: control 223.2 +/- 13.8 vs H2O2 182.8 +/- 18.4).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Binding Sites; Colforsin; Cyclic AMP; Dihydroalprenolol; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Heart; Hydrogen Peroxide; In Vitro Techniques; Isoproterenol; Lymphoma; Male; Myocardium; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Tumor Cells, Cultured

Glycosylphosphatidylinositol (GPI) is biosynthesized by the sequential addition of carbohydrates to phosphatidylinositol (PI). In the first two reactions, GlcNAc is transferred from UDP-GlcNAc to PI and then deacetylated to form GlcNAc-PI and GlcN-PI, respectively. In this paper, stimulation of GlcNAc-PI deacetylation by GTP, is reported. Addition of this nucleotide triphosphate to incubations in which GPI precursors were synthesized from UDP-[6-3H]GlcNAc by microsomes prepared from the lymphoma cell line EL4 resulted in a shift in the relative amount of each intermediate formed such that [6-3H]GlcN-PI was the predominant product. GTP also increased the total synthesis of the first GPI intermediate, GlcNAc-PI, by inhibiting reactions that metabolize UDP-[6-3H]GlcNAc into non-GPI-related products. However, unlike the stimulation of GlcNAc-PI deacetylation, ATP was equally effective in increasing the formation of GlcNAc-PI. An additional product, tentatively identified as [6-3H]GlcN-PI(acyl-inositol), was also detected when GTP was present in the incubation. The synthesis of this GPI precursor, which is proposed to be the third intermediate in GPI biosynthesis in mammals, was increased by GTP because the level of GlcN-PI, the substrate for acylation, was elevated. To isolate the effects of GTP on the GlcNAc-PI deacetylation, this reaction was studied directly by using [6-3H]GlcNAc-PI as the substrate. The stimulation was found to be specific for the guanosine-containing nucleotide triphosphate and optimal with approximately 1 mM GTP. Both the reaction rate at early time points and the total amount of deacetylated product formed in 60 min were increased by GTP. The effect on the second reaction of the pathway does not appear to be coupled to the first reaction because GlcNAc-PI deacetylation was increased by GTP in microsomes from cells defective in the GlcNAc-PI synthesis. Finally, 0.5 mM GTP gamma S (guanosine 5'-O-(thiotriphosphate)) completely inhibited the stimulation of GlcNAc-PI deacetylation caused by 1 mM GTP, indicating that hydrolysis of the nucleotide triphosphate was required for this effect. Although the mechanism and role of the GTP stimulation of GlcNAc-PI deacetylation is not clear, this regulation could influence the biosynthesis of mature GPI precursors and the subsequent expression of GPI-anchored proteins.

Topics: Acetylglucosamine; Animals; Glycosylphosphatidylinositols; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kinetics; Lymphoma; Mice; Microsomes; Phosphatidylinositol Diacylglycerol-Lyase; Phosphatidylinositols; Phosphoric Diester Hydrolases; Ribonucleotides; Tumor Cells, Cultured; Uridine Diphosphate N-Acetylglucosamine

We reported [Ransnäs, Svoboda, Jasper & Insel (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7900-7903] that in intact S49 lymphoma cells the beta-adrenergic-receptor agonist isoprenaline dissociates the stimulatory guanine-nucleotide-binding protein, Gs, into its alpha s and beta gamma subunits, leading to redistribution of alpha s from plasma membranes to the cytoplasm. In the present studies we investigated the kinetics of Gs dissociation and membrane release in plasma membranes from S49 lymphoma cells. We analysed cholate extracts of membranes for alpha s levels by a competitive e.l.i.s.a. with a polyclonal antibody that selectively recognizes monomeric alpha s and we assayed supernatant fractions using both competitive e.l.i.s.a. and immunoblotting. The plasma membranes contained 19.3 +/- 1.4 pmol of alpha s/mg of membrane protein and lacked significant dissociation of Gs and activity of adenylate cyclase in the absence of guanine nucleotides. Mg2+ ions were obligatorily required for isoprenaline-induced dissociation of Gs in plasma membranes and for membrane release of alpha s. At a physiological concentration of free Mg2+ ions (100 microM), 100 microM-GTP induced a slow first-order (k = 0.038 +/- 0.004 min-1) dissociation of 17.8 +/- 1.2 pmol of Gs/mg of membrane protein. A substantial increase in the dissociation rate of Gs was achieved by addition of 1 microM-isoprenaline and 100 microM-GTP; 18.4 +/- 0.9 pmol of Gs/mg of membrane protein was dissociated, with a kappa of 1.49 +/- 0.22 min-1. The effect of isoprenaline on the dissociation rate and on membrane release of Gs was completely blocked by the beta-adrenergic receptor antagonist propranolol. The concentration-response relationship for isoprenaline-induced dissociation during the first 1 min after addition of hormone yielded a kappa act. of 16 +/- 5 nM, whereas the kappa act. for isoprenaline-induced membrane release was 10 nM. We conclude that release of alpha s from plasma membranes is likely to accompany Gs-subunit dissociation and constitutes a potentially important facet of Gs action.

Topics: Animals; Cell Line; Cell Membrane; Enzyme-Linked Immunosorbent Assay; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Isoproterenol; Kinetics; Lymphoma; Macromolecular Substances; Magnesium; Mice; Receptors, Adrenergic, beta; Tumor Cells, Cultured

In this study we have used several complementary techniques to isolate and characterize a lymphoma membrane-associated 41-kDa protein that shares a number of structural and functional similarities with the alpha i subunit of the guanosine 5'-triphosphate (GTP)-binding protein (e.g., Gi alpha-like protein). In addition, using permeabilized lymphoma cells, we have found that: 1) GTP or GTP-tau-S augments, and pertussis toxin inhibits, phospholipase C (PLC) activity and receptor capping; and 2) the addition of lymphoma 41-kDa Gi alpha-like protein stimulates PLC activity and receptor patching/capping, and reverses the inhibitory effect of pertussis toxin on both activity and receptor patching/capping. Additional cytochemical and biochemical data indicate that the lymphoma 41-kDa protein is closely associated with several cytoskeletal proteins (e.g., actin, myosin, and fodrin) all of which colocalize under receptor cap structures. Furthermore, both the 41-kDa-mediated phospholipase C activity and receptor patching/capping are inhibited by cytochalasin D (a microfilament disrupting drug) and W-7 drug (a calmodulin inhibitor). Together, these data provide strong evidence for a functional association between the lymphoma membrane cytoskeleton and the 41-kDa (Gi alpha-like) protein. Specifically, this association appears to be required for the activation of phospholipase C that results in inositol triphosphate production, subsequent internal Ca2+ release, and finally surface receptor patching and capping.

Topics: Animals; Antigens, Surface; Cell Line; Cytochalasin D; Cytoskeleton; GTP-Binding Proteins; Guanosine Triphosphate; Immunologic Capping; Lymphoma; Membrane Proteins; Mice; Molecular Weight; Peptide Mapping; Pertussis Toxin; Receptor Aggregation; Sulfonamides; Thy-1 Antigens; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

After ADP-ribosylation by cholera toxin which promotes dissociation of the subunits, the alpha-subunit of Gs (Gs alpha) remained strongly associated with plasma membranes of wild-type S49 cells, since its interaction with the membrane was insensitive to 1 M KCl. Its association with the membrane was partially disrupted by 6 M urea and totally abolished by treatment with alkali at pH greater than or equal to 11.5. In vitro translated Gs alpha could interact with plasma membranes from the cyc- mutant of S49 cells as revealed by its cosedimentation with the membrane fraction and incubation of reconstituted membranes with GTP gamma S did not alter anchorage of Gs alpha. The characteristics of the association of in vitro translated Gs alpha with cyc- membranes after GTP gamma S treatment, i.e. sensitivity to 1 M KCl, 6 M urea and alkali treatment, were very similar to those described for the ADP-ribosylated form in wild-type membranes. Restoration of the coupling between the adrenergic receptor and adenylate cyclase further confirmed the vectorial reconstitution of cyc- membranes by in vitro translated alpha-subunit of Gs.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Animals; Cell Membrane; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydrogen-Ion Concentration; Isoproterenol; Lymphoma; Mice; Molecular Weight; Mutation; NAD; Osmolar Concentration; Protein Biosynthesis; RNA, Messenger; Thionucleotides; Tumor Cells, Cultured

The role of a specific guanine nucleotide binding (G protein) protein in coupling murine B lymphocyte receptor immunoglobulin to inositol phospholipid hydrolysis was investigated. Using an in vitro system with isolated membranes, we have observed specific enhancement of GTP binding subsequent to ligand-induced receptor crosslinking. Induced increases were inhibited by pretreatment with pertussis toxin which catalyzed ADP-ribosylation of a 43 kDa substrate. Involvement of this G protein with receptor immunoglobulin-induced inositol phospholipid hydrolysis was evidenced by the ability of pertussis toxin to block this response. This report, then, indicates that the B lymphocyte antigen receptor belongs to a family of receptors which are linked to inositol phospholipid hydrolysis through a G protein.

Topics: Adenosine Diphosphate Ribose; Animals; B-Lymphocytes; Cell Membrane; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Immunoglobulin M; Immunoglobulin mu-Chains; Lymphoma; Mice; Pertussis Toxin; Phosphatidylinositols; Receptors, Immunologic; Thionucleotides; Tumor Cells, Cultured; Virulence Factors, Bordetella

GTP-binding proteins have been implicated as transducers of a variety of biological signaling processes. These proteins share considerable structural as well as functional homology. Due to these similarities, it was thought that a monoclonal antibody that inhibits the light activation of the rod outer segment GTP-binding protein, tranducin (Gt), might exert some functional effect upon the G proteins that regulate the adenylate cyclase system. Antibody 4A, raised against the alpha subunit of Gt, cross-reacted (by hybridization on nitrocellulose) with purified alpha subunits of other G proteins (Gi and Gs, regulatory guanyl nucleotide-binding proteins that mediate inhibition and stimulation of adenylate cyclase, respectively) as long as they were not denatured. This antibody, which interferes with rod outer segment cGMP phosphodiesterase activation by blocking interaction between rhodopsin and Gt, also interfered with actions of both the stimulatory and inhibitory G proteins of adenylate cyclase from rat cerebral cortex membranes. Effects of monoclonal antibody (mAb) 4A were dose-dependent and not reversed by washing. mAb 4A also blocked the Gi-mediated inhibition of adenylate cyclase in the cyc- variant of S49 lymphoma and in doing so raised the level of adenylate cyclase activity in both the cyc- variant and the S49 wild type. There was no effect of mAb 4A on adenylate cyclase activity of the resolved catalytic subunit. These results suggest that the well known sequence homologies among the G proteins involved in cellular signal transduction may extend to the sites that interact with other members of signal-transducing cascades (receptors and effector molecules). Therefore, antibody 4A may serve as a useful tool to probe the similarities and differences among the various systems.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Antibodies, Monoclonal; Antibody Specificity; Cell Membrane; Cerebral Cortex; Enzyme Activation; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; Immunoblotting; Light; Lymphoma; Mice; Photoreceptor Cells; Pineal Gland; Rats; Rhodopsin; Rod Cell Outer Segment; Thionucleotides; Transducin; Tumor Cells, Cultured

A new type of VIP receptor was characterized in human SUP-T1 lymphoblasts. The order of potency of unlabeled peptides, in the presence of [125I]helodermin, was: helodermin(1-35)-NH2 = helodermin(1-27)-NH2 greater than helospectin greater than VIP = PHI greater than [D-Ser2]VIP greater than [D-Asp3]VIP greater than [D-His1]VIP greater than or equal to [D-Ala4]VIP greater than or equal to secretin = GRF. This specificity was distinct from that of all VIP receptors described so far in that: (i) the affinity for helodermin (Kd = 3 nM) was higher than that of VIP (Kd = 15 nM) and PHI (Kd = 20 nM); and (ii) position 4 played an important role in ligand binding. The labeled sites were likely to be functional receptors as adenylate cyclase in crude lymphoblastic membranes (200-10,000 x g pellets) was stimulated by peptides, in the presence of GTP, with the following order of potency: helodermin(1-35)-NH2 greater than helodermin(1-27)-NH2 greater than helospectin = VIP = PHI.

Topics: Adenylyl Cyclases; Cell Membrane; Guanosine Triphosphate; Humans; Intercellular Signaling Peptides and Proteins; Kinetics; Lymphoma; Peptide Fragments; Peptide PHI; Peptides; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; T-Lymphocytes; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The effects of ethanol on the beta adrenergic receptor-coupled adenylate cyclase system were examined in vitro using membranes prepared from S49 lymphoma cells. Ethanol caused a dose-dependent increase in adenylate cyclase activity in membranes prepared from wild-type cells when the activity was measured in the presence of GTP. Activity measured in the presence of isoproterenol was also increased by ethanol, but the fold-stimulation by isoproterenol was lower in the presence of ethanol. Ethanol also shifted the dose-response curve for stimulation of the enzyme by isoproterenol to the right. This shift was due to a decrease in the affinity of the beta adrenergic receptor for isoproterenol. A decrease in the affinity of the receptor for the antagonists [125I]iodopindolol and propranolol was also observed, but the magnitude of this effect was less than that seen with the agonist isoproterenol. The density of binding sites for [125I]iodopindolol was not affected by ethanol. Dose-response curves for NaF and guanosine-5'-O-(3-thiotriphosphate), both of which stimulate adenylate cyclase activity through an effect on the stimulatory guanine nucleotide-binding protein (Gs), were shifted to the left by the addition of ethanol. In membranes prepared from the CYC- variant of S49 cells, which lacks the alpha subunit of Gs, guanosine-5'-O-(3-thiotriphosphate) inhibited forskolin-stimulated adenylate cyclase activity. The inhibition by guanosine-5'-O-(3-thiotriphosphate) was not affected by ethanol. In membranes prepared from both wild-type and CYC- S49 cells, ethanol inhibited forskolin-stimulated adenylate cyclase activity. Whereas the inhibition of this activity by GTP was greatly attenuated in membranes prepared from CYC- S49 cells which had been pretreated with pertussis toxin, the inhibition by ethanol was not affected by pretreatment with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Animals; Cell Line; Dose-Response Relationship, Drug; Ethanol; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Isoproterenol; Lymphoma; Molecular Weight; Propranolol; Receptors, Adrenergic, beta; Thionucleotides

Membrane-bound G proteins carry information from receptors on the outside of cells to effector proteins inside cells. The alpha subunits of these heterotrimeric proteins bind and hydrolyse GTP and control the specificity of interactions with receptor and effector elements. Signalling by G proteins involves a cycle in which the inactive alpha beta gamma-GDP complex dissociates to produce alpha*-GTP, which is capable of activating the effector enzyme or ion channel; the alpha*-GTP complex hydrolyses bound GTP and reassociates with beta gamma to form the inactive complex. We have characterized a mutation that interrupts this GTP-driven cycle in alpha s, the alpha-chain of Gs, the G protein that stimulates adenylyl cyclase. The mutation converts a glycine to an alanine residue in the presumed GDP-binding domain of alpha s. The location and biochemical consequences of this mutation suggest a common mechanism by which binding of GTP or ATP may induce changes in the conformation of a number of nucleoside triphosphate binding proteins.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Aluminum; Aluminum Compounds; Animals; Binding Sites; Cell Membrane; DNA; Fluorides; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Lymphoma; Magnesium; Mice; Mutation; Protein Conformation; Trypsin; Tumor Cells, Cultured

We have recently reported (Ransnäs, L.A., and Insel, P.A. (1988) J. Biol. Chem. 263, 9482-9485) development of antipeptide antibodies to the alpha s protein of the stimulatory guanine nucleotide binding regulatory protein, Gs, and use of one of these antibodies, GS-1, to quantitate Gs levels in S49 lymphoma cell membranes. Another of these antibodies, termed GS-2, appears to detect only dissociated alpha s, but not the heterotrimer alpha s beta gamma. Using a competitive enzyme-linked immunosorbent assay, we have found that the guanine nucleotides GTP and guanosine 5'-O-(thiotriphosphate) (GTP gamma S) (but not GDP) and the beta-adrenergic receptor agonist isoproterenol activate Gs in native S49 cell membrane by subunit dissociation. Evidence for this includes detection of dissociated alpha s in membrane extracts and release of alpha s from S49 cell membranes treated with GTP gamma S or isoproterenol. Moreover, the estimates of apparent stoichiometry for this dissociation indicate that each beta-adrenergic receptor is able to activate greater than or equal to 100 molecules of Gs in native membranes. Thus, receptor-mediated dissociation of Gs is likely to be the major site of amplification of signal transduction by agonists active at hormone receptors that link to Gs.

Topics: Animals; Cell Line; Cell Membrane; Enzyme-Linked Immunosorbent Assay; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Isoproterenol; Kinetics; Liver; Lymphoma; Macromolecular Substances; Rabbits; Thionucleotides

In S49 lymphoma cells, 12-O-tetradecanoyl phorbol-13-acetate (TPA) enhances adenylate cyclase activity and doubles cAMP accumulation in response to beta-adrenergic stimulation at 37 degrees C, putatively via the action of protein kinase C. At 27 degrees C, TPA has the opposite effect, inhibiting cAMP production in response to isoproterenol by approximately 25%. TPA also inhibits the response to prostaglandin E1 (PGE1), another stimulant of hormone-sensitive adenylate cyclase in these cells, by 30% at 37 degrees C and almost 50% at 27 degrees C. In contrast, TPA enhances responses to forskolin and cholera toxin at both 27 and 37 degrees C. In membranes from cells treated with TPA, PGE1-stimulated adenylate cyclase activity is inhibited by 50%, whereas the catalytic activity stimulated by NaF or forskolin is enhanced. TPA reduces the potency of both PGE1 and isoproterenol for cAMP generation by 50%. TPA causes a similar decrease in beta-adrenergic agonist affinity with no reduction in the density of either antagonist or agonist binding sites in wild type cells and in cells lacking the alpha-subunit of the stimulatory transducer protein (Gs) (cyc-) or lacking functional receptor Gs coupling (UNC). Therefore, TPA has at least three functionally distinct effects on hormone-sensitive adenylate cyclase in S49 cells: a 50% reduction in agonist affinity, attenuation of receptor-transducer coupling, and enhancement of GTP-dependent catalytic activity. We conclude that multiple and opposing effects of TPA on hormone-sensitive adenylate cyclase occur simultaneously within the same cell, affecting the responses to several agonists differently.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Alprostadil; Cell Line; Cholera Toxin; Colforsin; Cyclic AMP; GTP-Binding Proteins; Guanosine Triphosphate; Isoproterenol; Lymphoma; Phorbol Esters; Protein Kinase C; Receptors, Adrenergic, beta; Sodium Fluoride; Temperature; Tetradecanoylphorbol Acetate

12-O-Tetradecanoylphorbol-13-acetate (TPA) enhances the apparent maximal velocity of adenylate cyclase in S49 lymphoma cells, an effect that seems not to result from an increased rate of activation of the catalytic subunit by the stimulatory GTP-binding protein (Gs) (Bell, J. D., Buxton, I. L. O., and Brunton, L. L. (1985) J. Biol. Chem. 260, 2625-2628). In membranes from wild type S49 cells, this enhancing effect of TPA is largely GTP-dependent; TPA enhances forskolin-stimulated adenylate cyclase activity by 35% in the presence of guanine nucleotide but only slightly (approximately 10%) in its absence. TPA causes comparable results in membranes from the cyc- variant that lacks the GTP-binding subunit of Gs. Blockade of the activity of the inhibitory GTP-binding protein (Gi) by high concentrations of Mg2+ (100 mM) or Mn2+ (3 mM) abolishes the effect of TPA to enhance adenylate cyclase activity in wild type membranes. The potentiation by TPA of cAMP accumulation in intact cells is greater than and not additive with the similar effect of pertussis toxin (an agent known to abolish hormonal inhibition of adenylate cyclase). Kinetic experiments indicate that TPA decreases the rate of activation of Gi by guanine nucleotide. We conclude that the resultant withdrawal of tonic inhibition of adenylate cyclase is one mechanism by which phorbol esters enhance guanine nucleotide-dependent cAMP synthesis.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Cell Line; Chlorides; Colforsin; Cyclic AMP; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Isoproterenol; Kinetics; Lymphoma; Magnesium; Magnesium Chloride; Manganese; Manganese Compounds; Mathematics; Pertussis Toxin; Phorbol Esters; Somatostatin; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella

We have used two experimental approaches--receptor inactivation with an irreversible antagonist and changes in receptor expression during passage of cells through the cell cycle--to explore the relationship between beta-adrenergic receptor number and response in intact S49 lymphoma cells. beta-Receptors in asynchronous cultures of S49 cells were blocked to varying degrees with the irreversible antagonist bromoacetylalprenololmenthane (BAAM). Blockade by BAAM was noncompetitive and did not alter the affinity of receptors for the agonist isoproterenol. Intracellular accumulation of cAMP in response to 1 microM isoproterenol was proportional to receptor number both at times of initial and maximal accumulation. In contrast, when intracellular accumulation of cAMP in response to isoproterenol was measured in synchronized cultures of S49 cells (obtained by centrifugal elutriation), a notably different relationship was observed. Cells were least responsive, that is, receptors appeared "uncoupled," during S phase of the cell cycle. This attenuation of response was not due to alterations of receptor number, receptor affinity for agonist, or expression of the catalytic unit of adenylate cyclase. Use of the antibiotic mycophenolic acid, a selective inhibitor of the synthesis of GTP, elicited response patterns in asynchronous cells similar to those seen in synchronized cells. These results confirm that wild-type S49 cells do not possess spare receptors. In addition to the importance of total receptor number in determining maximal response to isoproterenol, receptors may show differential efficacy in promoting cAMP accumulation as cells traverse the cell cycle. Changes in cellular levels or utilization of GTP during the cell cycle may serve to regulate the coupling of receptors to the stimulation of adenylate cyclase.

Topics: Adenylyl Cyclases; Alprenolol; Cell Cycle; Cell Line; Colforsin; Cyclic AMP; GTP-Binding Proteins; Guanosine Triphosphate; Isoproterenol; Lymphoma; Receptors, Adrenergic, beta

Treatment of intact human platelets and S49 lymphoma cyc- cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, impairs GTP-dependent and hormone-induced inhibition of adenylate cyclase, an action mediated by the inhibitory coupling protein Ni. In contrast, receptor-independent activation of Ni with subsequent adenylate cyclase inhibition induced by the stable GTP analog, guanosine 5'-[gamma-thio]triphosphate, was affected in neither the potency nor onset of Ni activation by the stable GTP analog, in both membrane systems studied. The data indicate that modification of Ni following phorbol ester treatment does not impair its activation by stable GTP analogs.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclase Inhibitors; Blood Platelets; Epinephrine; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydrolysis; Lymphoma; Phorbols; Phosphorylation; Protein Kinase C; Tetradecanoylphorbol Acetate; Thionucleotides

Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to S49 lymphoma cells (wild type and a cyclic AMP-dependent protein kinase-lacking clone) has little effect alone but doubles accumulation of cyclic AMP in response to isoproterenol. The effect is immediate and has an apparent affinity and order of potency characteristic of the activation of protein kinase C by phorbol esters. Enhancement does not reflect an altered time course of the beta-adrenergic response, enhanced affinity of the cellular beta-receptor for agonist, or decreased degradation and export of cellular cyclic AMP. Reduction of the beta-adrenergic response by somatostatin does not remove the effect of TPA nor does TPA abolish the effect of somatostatin. Phorbol ester enhances cyclic AMP accumulation in response to cholera toxin in wild type and UNC clones but not in H21a or cyc-. TPA also enhances cAMP accumulation in response to forskolin in wild type cells. The effect of TPA is stable to rapid preparation of membranes. In adenylate cyclase assays on membranes from cells treated with TPA, the activation by guanosine 5'-(beta, gamma-imino)triphosphate is enhanced by 40% with no change in lag time; the effect of beta-agonist plus Gpp(NH)p is similarly enhanced; activation by Mn2+ is unchanged. We conclude that phorbol ester facilitates the productive interaction of the alpha subunit of the transducer protein Gs with the catalytic unit of adenylate cyclase, hypothetically via an action of protein kinase C.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cholera Toxin; Cyclic AMP; Drug Synergism; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Isoproterenol; Kinetics; Lymphoma; Manganese; Phorbols; Protein Kinase C; Protein Kinases; Somatostatin; Tetradecanoylphorbol Acetate; Time Factors

Topics: Adenosine Triphosphate; Adrenergic beta-Agonists; Albuterol; Animals; Binding, Competitive; Cell Line; Cell Membrane; Guanosine Triphosphate; Isoproterenol; Lymphoma; Mice; Pindolol; Receptors, Adrenergic, beta; Stereoisomerism; Structure-Activity Relationship

Rat reticulocytes contain a cytosol activator protein (RCAP) that augments catecholamine-sensitive adenylate cyclase activity in reticulocyte membranes. A highly purified preparation of RCAP, obtained by Sephacryl S-200 chromatography, was used to elucidate further its mechanism of action. The specific activity of the S-200 fraction to augment isoproterenol responsiveness was increased approximately 1,100-fold over the starting material, from 1.2 to 1,300 nmol cAMP formed per mg RCAP. The mol wt of RCAP is approximately 20,000. The effect of RCAP to enhance isoproterenol responsiveness was apparent within 20 sec, virtually abolishing the normal lag time of hormone-activated adenylate cyclase. In addition to its effects on catecholamine-responsive adenylate cyclase, RCAP significantly increased basal [21 +/- 3 (+/- SEM) to 41 +/- 4 pmol/mg protein X 30 min; P less than 0.02], guanyl-5'-yl-imidodiphosphate-associated (3173 +/- 213 to 4339 +/- 365 pmol/mg X 30 min; P less than 0.03), and fluoride-associated (5152 +/- 64 to 5807 +/- 58 pmol/mg X 30 min; P less than 0.05) adenylate cyclase activities. RCAP also altered the characteristics of agonist binding to the beta-adrenergic receptor of reticulocyte membranes, causing an increase in the apparent IC50 for isoproterenol from 0.7 +/- 0.2 to 7.9 +/- 1.6 microM (P less than 0.001). Similar to its effects on reticulocytes, RCAP enhanced isoproterenol- and prostaglandin E2-sensitive adenylate cyclase activity in the wild-type S49 lymphoma cell and shifted the binding isotherm for isoproterenol rightward. In cyc-, the mutant that lacks the stimulatory guanine nucleotide-binding protein (Ns) and in UNC, the mutant in which receptors are uncoupled from N, RCAP was ineffective. Moreover, RCAP decreased agonist affinity for the beta-adrenergic receptor in wild-type S49 cells, but not in cyc- or UNC cells. These observations suggest that RCAP requires a functional Ns unit for its effects on hormone-sensitive adenylate cyclase activity.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cytosol; Dinoprostone; Enzyme Activation; Erythrocyte Membrane; Fluorides; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Iodocyanopindolol; Isoproterenol; Lymphoma; Pindolol; Prostaglandins E; Proteins; Rats; Receptors, Adrenergic, beta; Reticulocytes

Four members of a family of GTP-binding proteins (G-proteins) which translate stimulation of extracellular receptors into regulation of intracellular enzymes were isolated from the bovine central nervous system. These proteins were examined for functional similarities and cross-reactivity with antibodies to the G-protein (transducin, Gt) from the photoreceptor system. Two proteins, Gs and Gi, can be distinguished by their respective abilities to stimulate or inhibit adenylate cyclase. The activated alpha subunits of Gt and a fourth member of the family, Go, did not affect this enzyme. Gt was shown to be unique in its ability to stimulate cGMP-dependent phosphodiesterase. While functionally diverse, the G-proteins were shown to have some common antigenic properties. Antibodies directed against the beta subunit of Gt recognize the beta 36 subunits of all preparations but not a putative second beta 35 subunit. Antibodies specific for the alpha subunit of Gt did not recognize other alpha subunits when immune blots from sodium dodecyl sulfate gels were examined. However, Go alpha, but not Gs alpha or Gi alpha, reacted strongly with the antibodies when the native subunit was spotted directly. This suggests that Go alpha and Gt alpha have homologous structural determinants. An antiserum that recognized Gt gamma did not recognize gamma subunits from other sources. These data support the proposed diversity of function and similarity of structure among the four G-proteins. The alpha and potentially gamma subunits appear to be responsible for the specificity of function.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenylyl Cyclases; Animals; Brain Chemistry; Cattle; Cell Line; Cell Membrane; Chromatography; Durapatite; Electrophoresis, Polyacrylamide Gel; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hydroxyapatites; Kinetics; Lymphoma; Mice; Molecular Weight; Photoreceptor Cells; Rod Cell Outer Segment; Thionucleotides

Ns and Ni have been purified without using NaF and Mg as stabilizing agents (Codina, J., Hildebrandt, J.D., Sekura, R.D., Birnbaumer, M., Bryan, J., Manclark, C.R. and Birnbaumer, L. [1984] J. Biol. Chem. 259, in press). Since the submission of that report, several modifications have been introduced to the purification procedure and additional fractions have been processed from which N proteins are obtained. This article describes the updated protocols and presents methodological details not included in the previous publication. The final products are Ns, the stimulatory N, Ni the inhibitory N, both of subunit structure alpha beta gamma, and a Mr = 40,000 protein of beta gamma composition. They are obtained from human erythrocytes.

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Guanine Nucleotides; Guanosine Triphosphate; Hormones; Humans; In Vitro Techniques; Kinetics; Ligands; Lymphoma; Magnesium; Membrane Proteins; Mice; Molecular Weight; Receptors, Cell Surface; Receptors, Neurotransmitter

The influence of sodium was studied on hormone and guanine nucleotide-induced stimulation and inhibition of adenylate cyclase and on beta-adrenoceptor binding in various membrane systems. Sodium exerted almost identical effects on stimulation and inhibition of adenylate cyclase by various stimulatory and inhibitory hormones in all of the systems studied. The potencies of the hormones and of GTP to increase or to decrease the enzyme activity were reduced by sodium ions, without changing the maximal degree of adenylate cyclase stimulation or inhibition. Stimulation and inhibition of adenylate cyclase by the stable GTP analog, GTP gamma S, was affected in an identical manner by sodium, causing a retardation in the onset without a change in final stimulation or inhibition by the analog. Similar to the well-known reduction in alpha 2-adrenoceptor affinity for agonists, sodium also reduced the apparent affinity of beta-adrenoceptors for the agonist, isoproterenol. It is concluded that sodium exerts identical effects on Ns and Ni, inhibiting the activation process of these two coupling components of the adenylate cyclase.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Adipose Tissue; Alprostadil; Animals; Blood Platelets; Cricetinae; Cyclic AMP; Epinephrine; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; In Vitro Techniques; Isoproterenol; Lymphoma; Prostaglandins E; Receptors, Adrenergic, beta; Receptors, Cell Surface; Sodium Chloride; Thionucleotides

The activation reaction of the inhibitory guanine nucleotide-binding regulatory site of the adenylate cyclase system was studied in membranes of rat adipocytes, S49 lymphoma wild-type cells and their cyc- variants, pretreated without and with the Bordetella pertussis toxin, islet-activating protein (IAP), by measuring the kinetics of adenylate cyclase inhibition by the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The IAP treatment, which caused a loss of GTP and hormone-induced adenylate cyclase inhibition, did not prevent enzyme inhibition by the stable GTP analogue. However, in either cell type studied, pretreated with IAP, the lag phase of GTP[S] inhibitory action was largely increased by about fivefold compared to control membranes. Similar to the controls, the lag phase of GTP[S] inhibition of adenylate cyclase in membranes of IAP-pretreated cells was shortened in the presence of an inhibitory hormone. Furthermore, the lag phase of inhibition by GTP[S] was decreased with increasing concentrations of Mg2+. The data indicate that the pertussis toxin does not principally prevent an interaction of the inhibitory guanine nucleotide regulatory site of the adenylate cyclase system with either the catalytic moiety or an inhibitory hormone receptor. The data, furthermore, suggest that the toxin inhibits the activation reaction (turn-on reaction) of the inhibitory coupling component. This inhibition, which may take place at a Mg2+-binding site, can account for the observed functional loss of GTP and hormone-induced adenylate cyclase inhibition after IAP treatment.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adipose Tissue; Animals; Bacterial Proteins; Binding Sites; Chemical Phenomena; Chemistry; GTP-Binding Proteins; Guanosine Triphosphate; Lymphoma; Magnesium; Mice; Pertussis Toxin; Rats; Receptors, Cell Surface; Somatostatin; Virulence Factors, Bordetella

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta subunits are functionally indistinguishable. GTP-dependent hormonal inhibition of adenylate cyclase and that caused by guanine nucleotide analogs seem to result from dissociation of the subunits of Gi. Such inhibition can be explained by reduction of the concentration of the free alpha subunit of Gs as a result of its interaction with the beta subunit of Gi in normal Gs-containing membranes. However, inhibition in S49 lymphoma cyc- cell membranes presumably cannot be explained by the Gi-Gs interaction, since the activity of the alpha subunit of Gs is not detectable in this variant. Several characteristics of Gi-mediated inhibition of adenylate cyclase have been studied in both S49 cyc- and wild type membranes. There are several similarities between inhibition of forskolin-stimulated adenylate cyclase by guanine nucleotides and somatostatin in cyc- and wild type membranes. 1) Somatostatin-induced inhibition of the enzyme is dependent on GTP; nonhydrolyzable GTP analogs are also effective inhibitors. 2) The effect of guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) is essentially irreversible, and somatostatin accelerates GTP gamma S-induced inhibition. 3) Inhibition of adenylate cyclase by somatostatin or Gpp(NH)p is attenuated by treatment of cells with islet-activating protein (IAP). 4) Both cyc- and wild type membranes contain the substrate for IAP-catalyzed ADP-ribosylation (the alpha subunit of Gi). 5) beta Subunit activity in detergent extracts of membranes is liberated by exposure of the membranes to GTP gamma S. The alpha subunit of Gi in such extracts has a reduced ability to be ADP-ribosylated by IAP, which implies that this subunit is in the GTP gamma S-bound form. The resolved subunits of Gi have been tested as regulators of cyc- and wild type adenylate cyclase under a variety of conditions. The alpha subunit of Gi inhibits forskolin-stimulated adenylate cyclase activity in cyc-, while the beta subunit stimulates; these actions are opposite to those seen with wild type membranes. The inhibitory effects of GTP plus somatostatin (or GTP gamma S) and the alpha subunit of Gi are not additive in cyc- membranes. In wild type, the inhibitory effects of the hormone and GTP gamma S are not additive with those of the beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Adenylyl Cyclases; Animals; Antihypertensive Agents; Cell Line; Cell Membrane; Colforsin; Diterpenes; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kinetics; Lymphoma; Mice; Molecular Weight; Mutation; Receptors, Cell Surface; Somatostatin; Thionucleotides

Pertussis toxin catalyzes the ADP-ribosylation of a single 41-kDa peptide of membranes prepared from rat hepatocytes, S49 mouse lymphoma wild-type and cyc-mutant cells. This 41-kDa peptide has been shown to be the alpha-subunit of the inhibitory, guanine nucleotide binding regulatory component of adenylate cyclase (Ni). Incubating membranes of rat fat cells with pertussis toxin and [32P]NAD+ radiolabels a 41- and a 40-kDa peptide. Possible homologies between these peptides were investigated by comparing the electrophoretic patterns of proteolytic fragments derived from each of them that are radiolabeled by [32P]NAD+ and pertussis toxin. The 40-kDa substrate for pertussis toxin-catalyzed ADP-ribosylation and the alpha-subunit of Ni in rat fat cells appear to be homologous, but non-identical peptides.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adipose Tissue; Animals; Bacterial Toxins; Cholera Toxin; Electrophoresis, Polyacrylamide Gel; Female; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Liver; Lymphoma; Membrane Proteins; Molecular Weight; NAD; Nucleoside Diphosphate Sugars; Peptide Hydrolases; Pertussis Toxin; Rats; Rats, Inbred Strains; Thionucleotides; Virulence Factors, Bordetella

The mechanism by which Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclases, regulate the activity of the catalytic component (C) of adenylyl cyclase was investigated using cyc-S49 cell membranes which contain a functional inhibitory regulatory protein (Ni) but not the active subunit of the stimulatory regulatory protein (Ns). To this end, purified Ns protein was preactivated (Ns) in solution with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and Mg2+, and then added to cyc- membranes under conditions where Ni was either unactivated or activated (Ni) by GTP gamma S and Mg2+. Activation of Ni in cyc- membranes resulted in a lowered expression of Ns activity under all conditions tested. Upon dilution of the reactants (Ns and cyc- membranes) the reconstituted activity declined in proportion to the dilution with an approximate t 1/2 of 30-45 min, being unaffected by activation of Ni. Postactivation of Ni after reconstitution of cyc- membranes with Ns resulted in a time-dependent decline in Ns activity to a level that was the same as that obtained when Ns was added to cyc- membranes with preactivated Ni. These data indicated that the effects of Ns on C are of a reversible type. The following indicated that Ns and Ni affect C activity in a noncompetitive manner: (a) the per cent reduction in Ns activity due to activation of Ni was constant and independent of the concentration of Ns, (b) double reciprocal plots of activities reconstituted in control and Ni-containing cyc- membranes versus Ns concentration were linear with an unaltered apparent Km for Ns, and (c) the onset of inhibition of C prereconstituted with Ns was much faster (approximate t 1/2 = 2-5 min) than expected if it were due to occupancy of a common site on C left vacant by Ns.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Lymphoma; Magnesium; Mice; Sodium Fluoride; Thionucleotides

N-Ethylmaleimide treatment of rat liver plasma membranes results in an adenylyl cyclase (EC 4.6.1.1) system that shows no measurable cyclizing activity but retains both an active glucagon receptor and a receptor-sensitive regulatory component N as assessed by reconstitution into cyclase-negative (cyc-) membranes from S49 murine lymphoma. Treatment of such N-ethylmaleimide-treated membranes, termed C- liver membranes, with guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] ) and Mg2+, followed by the removal of GTP[gamma S] by washing, yields an activated N which upon mixing with cyc- S49 membranes reconstitutes the cyc- S49 membrane adenylyl cyclase in the absence of added GTP[gamma S]. It was found that GTP[gamma S] activation of the N at saturating concentrations of GTP[gamma S] is slow at low Mg2+ concentration and accelerated by increasing Mg2+ concentrations. Addition of glucagon during the activation results in a lowering of the Mg2+ requirement for full activation from 25 mM to around 10 muM and in concomitant increases in both the rate and the extent of N activation. In contrast to its dramatic effect on Mg2+ requirement, glucagon has little (less than 2-fold) effect on the GTP[gamma S] requirement of N activation. These experiments indicate that the glucagon receptor facilitates activation of N by: (i) decreasing the apparent Km of N for Mg2+, and (ii) increasing the extent of activation that can be elicited by saturating concentrations of guanine nucleotide. It is postulated that the mechanism by which Mg2+ and receptors facilitate N activation involves dissociation of n alpha activated ADP-ribosylatable subunits (with guanine nucleotide bound to them) from n beta non-ADP-ribosylatable subunits (with receptor and Mg2+ bound to them).

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Ethylmaleimide; Glucagon; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kinetics; Liver; Lymphoma; Magnesium; Mice; Neoplasms, Experimental; Receptors, Cell Surface; Receptors, Glucagon; Thionucleotides

Several homogeneous cell systems contain distinct subpopulations of beta-adrenergic receptors, distinguished by their relative sensitivity to N-ethylmaleimide (NEM) in the presence of agonist but not antagonist (G. Vauquelin and M.E. Maguire (1980) Mol. Pharmacol. 18, 363-369). The sensitivity to agonist/NEM inactivation requires receptor interaction with the magnesium-guanine nucleotide coupling proteins (G/F). We have investigated the effects of agonist/NEM treatment on Mg2+ and GTP modulation of receptor affinity in two such systems, turkey erythrocytes and murine S49 lymphoma cells. In each systems, the agonist/NEM-sensitive beta-receptor subpopulation exhibits both Mg2+ and GTP modulation of beta-receptor affinity for agonist. Further, Mg2+ and GTP are not competitive with regard to alteration of receptor affinity; that is, GTP can block the effect of Mg2+, but not vice versa. In contrast, the agonist/NEM-resistant beta-receptor subpopulation shows distinct differences in Mg2+ and GTP effects when the turkey and S49 systems are compared. The agonist/NEM-resistant population in S49 shows no effect of Mg2+ or GTP on beta-receptor affinity for agonist whereas the resistant beta-receptors of turkey erythrocytes still exhibit modulation by both GTP and Mg2+. Moreover, in this receptor population the actions of GTP and Mg2+ are apparently competitive, with increasing Mg2+ concentrations able to overcome the decrease in affinity induced by GTP. Thus, beta-receptor interaction with the metal/nucleotide coupling proteins may differ significantly in the two systems examined. An additional result of these experiments is the demonstration for S49 beta-receptors that free, unchelated GTP or GDP rather than MgGTP or MgGDP modulates receptor affinity for agonist.

Topics: Animals; Blood Proteins; Cell Membrane; Edetic Acid; Erythrocyte Membrane; Erythrocytes; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Triphosphate; Isoproterenol; Kinetics; Lymphoma; Magnesium; Membrane Proteins; Mice; Neoplasms, Experimental; Receptors, Adrenergic; Receptors, Adrenergic, beta; Receptors, Cell Surface; Turkeys

Cyc- S49 mouse lymphoma cell membranes contain an adenylyl cyclase system which is deficient in the regulatory properties characteristic of a stimulatory guanine nucleotide-binding regulatory component (Ns), such as enhancement of activity in the presence of GTP, GTP analogues, and NaF. Detailed kinetic analysis of cycl- adenylyl cyclase showed, however, that it is not unresponsive to these agents, for their addition to assays elicited inhibitory effects. Inhibition of cyc- activity was seen in 11 different membrane preparations obtained from two different strains and was observable both in the absence and presence of the strongly stimulatory diterpene forskolin. The GTP analogues GTP gamma S and guanyl-5'-yl imidodiphosphate caused a maximum of 60% inhibition with IC50 values of 2 and 12 nM, respectively. GTP itself was less potent than its analogues, with an IC50 of 100 mM, and elicited less inhibition as well (a maximum of 25%). Cholera toxin treatment of cyc- S49 cell membranes, under conditions which appeared to maximally activate the adenylyl cyclase system of wild type S49 cell membranes, had no effect on inhibition of catalytic activity. Our results indicate the presence in cyc- S49 cell membranes of a guanine nucleotide-binding component which is inhibitory to adenylyl cyclase activity (Ni). This suggests (a) that these membranes cannot be considered as completely deficient of adenylyl cyclase guanine nucleotide-binding regulatory components, and (b) that these membranes offer a unique opportunity to study Ni-mediated effects of guanine nucleotides and fluoride in the functional absence of Ns-mediated effects of these agents.

Topics: Adenylyl Cyclase Inhibitors; Animals; Cell Line; Cell Membrane; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Lymphoma; Mice; Neoplasms, Experimental; Sodium Fluoride; Thionucleotides

Topics: Animals; Cell Membrane Permeability; Complement System Proteins; Cytarabine; Diphosphonates; Erythrocyte Membrane; Guanosine Monophosphate; Guanosine Triphosphate; Lymphoma; Mice; Osmolar Concentration; Rabbits; Sucrose

Topics: Adenylyl Cyclases; Animals; Cell Line; Enzyme Activation; Fluorides; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Isoproterenol; Kinetics; Lymphoma; Magnesium; Mice; Neoplasms, Experimental; Prostaglandins E; Receptors, Adrenergic; Receptors, Adrenergic, beta

Topics: Adenine; AMP Deaminase; Animals; Cell Division; Cell Line; Guanosine Triphosphate; Kinetics; Lymphoma; Mice; Neoplasms, Experimental; Nucleotide Deaminases; Purines; Ribonucleotide Reductases

Incubation of mouse T lymphoma (S-49) cells with the inosinate dehydrogenase inhibitor mycophenolic acid produced a depletion of both GTP and dGTP, and resulted in growth inhibition, partial reduction in RNA synthesis, and drastic inhibition of DNA synthesis. Similar results suggested to others that the depletion of dGTP is primarily responsible for toxicity. However, guanosine was as effective as deoxyguanosine at preventing mycophenolic acid toxicity although deoxyguanosine was more effective at elevating dGTP levels. Moreover, in hypoxanthine-guanine phosphoribosyltransferase-deficient mutants of S-49 (6MPR-3-3) deoxyguanosine was unable to prevent mycophenolic acid toxicity or to re-establish normal DNA synthesis, although it returned cellular dGTP but not GTP levels to normal. No other nucleotide levels changed in a way which could account for the toxicity. Incubation of cells with a combination of deoxyadenosine, deoxycytidine, and erythro-9-(2-hydroxy-3-nonyl)adenine produced a selective depletion of dGTP to levels similar to that produced by mycophenolic acid, but did not affect cell growth. Studies with cells synchronized by centrifugal elutriation show that the toxicity of mycophenolic acid is specific to the S-phase of the cell cycle. Addition of actinomycin D at a concentration that inhibited RNA synthesis increased the availability of GTP and re-established normal DNA synthesis in mycophenolic acid-treated S-49 cells. These results suggest that the depletion of GTP rather than that of dGTP produces toxic effects in S-49 cells and that GTP is required for DNA synthesis.

Topics: Animals; Cell Division; Cell Line; Deoxyguanine Nucleotides; DNA, Neoplasm; Guanosine Triphosphate; Hypoxanthine Phosphoribosyltransferase; Kinetics; Lymphoma; Mice; Mutation; Mycophenolic Acid; Neoplasms, Experimental

The rate of appearance and extent of reconstituted adenylyl cyclase activity in the presence of guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) upon mixing cholate extract of rat liver 10,000 X g particulate preparation and S49 cyc- cell membranes was dependent on Mg ion concentration. Increasing concentrations of Mg ion lead to progressively shorter lag times in the appearance of reconstituted activity. In contrast, at 20 mM MgCl2, the rate of appearance of reconstituted activity was insensitive to concentrations of GMP-P(NH)P, cyc- membrane protein, and untreated cholate extract protein, but became sensitive to cholate extract protein concentration after treatment with GMP-P(NH)P and Mg ion. Treatment of cholate extract with GMP-P(NH)P and Mg ion resulted in a faster appearance of reconstituted activity upon exposure to S49 cyc- membranes as compared to treatment in the presence of GMP-P(NH)P and EDTA. Activation of the regulatory component by GMP-P(NH)P occurred only in the presence of Mg ion. Mn2+, Ca2+, or Ba2+ did not appear to substitute for Mg. The effect of Mg ion allowing for the rapid appearance of reconstituted activity was reversible in the presence or absence of S49 cyc- membranes. However, while dilution of GMP-P(NH)P in the absence of S49 cyc- membranes resulted in relatively rapid reversal, treatment of cholate extract with 5 microM GMP-P(NH)P and 20 mM Mg ion followed by reconstitution at 5 or 0.1 microM GMP-P(NH)P showed similar reconstituted activities for up to 15 min. The rate of activation of the regulatory component was faster at 20 mM Mg ion as compared to that at 1 mM Mg ion. Similarly, the rate of activation of the regulatory component by NaF was also accelerated by increasing the concentration of Mg ion. These data indicate that the rate-limiting step in the activation of adenylyl cyclase is conversion of the regulatory component from its inactive to active form. In the presence of saturating concentration of guanine nucleotides and NaF, both the rate and extent of this activation is regulated by Mg ion.

Topics: Adenylyl Cyclases; Animals; Cations, Divalent; Cell Line; Cell Membrane; Enzyme Activation; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Liver; Lymphoma; Magnesium; Mice; Rats

Topics: Adenylyl Cyclases; Animals; Aprotinin; Carrier Proteins; Cell Line; Cell Membrane; Cholera Toxin; GTP-Binding Proteins; Guanosine Triphosphate; Liver Neoplasms, Experimental; Lymphoma; Mice; Molecular Weight; Peptide Fragments; Rats

GDP and GTP regulation of receptor-mediated stimulation of adenylyl cyclases in membranes of S49 murine lymphoma cells (S49), NS-20 murine neuroblastoma cells (NS-20), rabbit corpora lutea (CL), and turkey erythrocytes were studied under assay conditions which minimized conversion of added GTP to GDP and of added GDP to GTP. Hormonal stimulation in all systems required guanine nucleotide addition. In the presence of GTP, adenylyl cyclase activity in S49, NS-20, and CL was stimulated respectively by isoproterenol and prostaglandin E1 (PGE1), by PGE1 and the adenosine analog, phenylisopropyladenosine, and by PGE1 and isoproterenol, with the first of the listed stimulants eliciting higher activities than the second. Activity in turkey erythrocyte membranes was stimulated by isoproterenol. GDP was partially effective in promoting hormonal stimulation, being able to sustain stimulation by isoproterenol and PGE1 in S49 cell membranes and by PGE1 in CL membranes. In NS-20 membranes, both GDP and guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) were inhibitory on basal activity, yet promoted limited but significant stimulation by PGE1. In turkey erythrocytes, stimulation by isoproterenol could not be elicited with GDP or GDP beta S. Thus, although less effective than GTP in promoting hormonal stimulation of several adenylyl cyclase systems, GDP was clearly not inactive. Concentration effect curves for active hormone in the presence of GDP had higher apparent Ka values than in the presence of GTP. In spite of differences between the effects of GTP and GDP on hormonal stimulation of adenylyl cyclase activities, GTP and GDP affected equally well isoproterenol binding, regardless of whether or not its receptor could be shown to stimulate adenylyl cyclase in the presence of GDP. Determination of transphosphorylation of GDP to GTP showed that at saturating concentrations, the proportion of GDP converted to GTP is negligible and unaffected by hormonal stimulation. Concentrations giving 50% inhibition were determined for GTP- and GDP-mediated inhibition of guanyl-5'-yl imidodiphosphate stimulation in the absence and presence of stimulatory hormones. In all four systems studied, GTP and GDP interacted with about equal potency and hormonal stimulation was not accompanied by a selective decrease in affinity for GDP. One way to explain all of the results obtained is to view hormonally sensitive adenylyl cyclase systems as two-state enzymes whose activities are regulated by GT

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Corpus Luteum; Erythrocyte Membrane; Female; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Isoproterenol; Kinetics; Lymphoma; Mice; Prostaglandins E; Receptors, Cell Surface; Turkeys

Treatment of membranes from HeLa cells, rat adipocytes, and rat liver with organic mercurials results in complex effects on adenylate cyclase activity that are not mimicked by the reversible sulfhydryl reagent, tetrathionate. At low concentrations (0.1 mM or less 1 mercurials inactivate the enzyme; inactivation is reversed by the thiol-reducing agent, dithiothreitol. Treatment with higher concentrations of organic mercurials (1 mM and above) results in a time-dependent, irreversible change in the ability of guanine nucleotides and fluoride ion to stimulate adenylate cyclase activity. The irreversible changes are blocked by treatment of membranes with cholera toxin and NAD, suggesting that the GTP-regulatory component is the site of mercurial action. This is further suggested by the lack of irreversible effects of mercurials on adenylate cyclase activity in membranes from mouse lymphoma cells that lack this component. Irreversible effects of mercurials on the adipocyte cyclase system also include enhancement of basal activity and potentiation of the inhibitory effects of GTP on cyclase activity; the latter effects of GTP are mediated through a process independent from that mediating stimulation of activity by GTP. It is concluded that the GTP-regulatory proteins responsible for the modulation of adenylate cyclase activity by hormones and neurotransmitters contain the sites of action of organic mercurials. Their possible mode of action is discussed.

Topics: Adenylyl Cyclases; Adipose Tissue; Animals; Carrier Proteins; Cell Line; Cell Membrane; Chlormerodrin; Cholera Toxin; Dithiothreitol; GTP-Binding Proteins; Guanosine Triphosphate; HeLa Cells; Humans; Kinetics; Liver; Lymphoma; Mice; Organ Specificity; Rats; Tetrathionic Acid

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Cholera Toxin; Erythrocyte Membrane; Fluorides; Guanine Nucleotides; Guanosine Triphosphate; Humans; In Vitro Techniques; Isoproterenol; Kinetics; Lymphoma; Mice; Neoplasms, Experimental; Receptors, Adrenergic, beta; Turkeys

Topics: Adenylyl Cyclases; Cell Line; Cell Membrane; Enzyme Activation; Fluorides; Guanosine Triphosphate; Isoproterenol; Kinetics; Lymphoma; Magnesium; Receptors, Adrenergic; Receptors, Adrenergic, beta

Calcium dependent regulator is present in wild-type S49 lymphoma cells, in the variant that is deficient in adenylate cyclase activity (AC-), and in the uncoupled variant (UNC). The electrophoretic mobility and the ability to stimulate cyclic nucleotide phosphodiesterase of the calcium dependent regulator from each of these three clones are indistinguishable from those of the modulator protein isolated from bovine brain. Calcium dependent regulator does not appear to be involved in the defect responsible for the UNC or AC- variants.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenylyl Cyclases; Calcium; Calmodulin; Carrier Proteins; Cell Line; Clone Cells; Enzyme Activation; Fluorides; Guanosine Triphosphate; Isoproterenol; Lymphoma; Neoplasms, Experimental

A novel variant of the S49 mouse lymphoma has been selected from wild-type cells by growth in medium containing the beta-adrenergic agonist terbutaline and inhibitors of cyclic nucleotide phosphodiesterase. In contrast to the situation in the wild-type clone, synthesis of adenosine 3':5'-monophosphate (cyclic AMP) is not stimulated by beta-adrenergic agonists or by prostaglandin E1 either in intact variant cells or in membrane preparations of such clones. However, basal and NaF-stimulated activities of adenylate cyclase [ATP pyrophosphate-lyase (cyclizine), EC 4.6.1.1] are normal, enzyme activity is stimulated by guanyl-5'-yl imidodiphosphate [Gpp(NH)p], and intact cells accumulate cyclic AMP when exposed to cholera toxin. Furthermore, variant cell membranes possess ligand-binding activity consistent with the conclusion that a normal or an excessive number of beta-adrenergic receptors is present. Thus, interaction between the hormone-binding and the catalytic moieties of the adenylate cyclase system is lost. This variant phenotype, designated as uncoupled (UNC), has been stable for more than 100 generations without exposure to the drugs used for selection. Such cells should be useful for the elucidation of methanisms of transmission of information from hormone receptors to adenylate cyclase.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Animals; Bacterial Toxins; Binding, Competitive; Cell Line; Cyclic AMP; Fluorides; Guanosine Triphosphate; Isoproterenol; Karyotyping; Lymphoma; Mice; Mutation; Prostaglandins E; Receptors, Adrenergic; Receptors, Adrenergic, beta; Vibrio cholerae

Topics: Adenylyl Cyclases; Bacterial Toxins; Cell Line; Cell Membrane; Enzyme Activation; Guanosine Triphosphate; Isoproterenol; Kinetics; Ligands; Lymphoma; Mathematics; Mutation; Pindolol; Propranolol; Prostaglandins E; Receptors, Adrenergic; Receptors, Adrenergic, beta; Ribonucleotides; Vibrio cholerae

Topics: Adenylyl Cyclases; Binding Sites; Cell Line; Cell Membrane; Cholera Toxin; Cyclic AMP; Enzyme Activation; GTP Phosphohydrolases; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Isoproterenol; Lymphoma; NAD; Phosphoric Monoester Hydrolases; Propranolol

Mycophenolic acid and virazole are inhibitors of inosinate dehydrogenase and produce growth inhibition and loss of viability in cultured murine lymphoma L5178Y cells. Treatment with 1 muM mycophenolic acid produced the following changes in concentrations of acid-soluble nucleotides: (a) guanosine triphosphate decreased to less than 10% of control within 2 hr; (b) uridine triphosphate and cytidine triphosphate concentrations increased markedly; (c) adenosine triphosphate did not change; (d) deoxyguanosine triphosphate decreased; and (e) thymidine triphosphate increased. DNA synthesis was inhibited by 90% within 2 hr, whereas the incorporation of adenosine into RNA and of leucine into protein were much less affected. Virazole (100 muM) produces similar effects. These biochemical effects of mycophenolic acid, as well as its effects on cell growth, can be prevented by addition of guanylate to the medium. Mycophenolic acid treatment also appears to cause breakdown of high-molecular-weight DNA.

Topics: Adenosine Triphosphate; Cell Division; Cells, Cultured; Cytosine Nucleotides; DNA, Neoplasm; Guanine Nucleotides; Guanosine Triphosphate; IMP Dehydrogenase; Lymphoma; Mycophenolic Acid; Neoplasm Proteins; Neoplasms, Experimental; Ribavirin; Ribonucleosides; RNA, Neoplasm; Thymine Nucleotides; Uracil Nucleotides

Topics: Adenine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Cytosine Nucleotides; Guanosine Triphosphate; Hypoxanthines; Inosine; Lymphoma; Male; Mice; Mice, Inbred DBA; Neoplasms, Experimental; Purines; Sulfides; Uracil Nucleotides

In the presence of 10(-4) to 10(-5) molar adenosine, established cell lines of fibroblastic or lymphoid origin die of pyrimidine starvation. Less than lethal concentrations inhibit cell growth. Over a broad concentration range, the effects of adenosine are prevented by providing a suitable pyrimidine source. We suggest that the recently described immune deficiency disease associated with absence of adenosine deaminase may be the result of pyrimidine starvation induced by adenosine nucleotides in cells of the lymphoid system.

Topics: Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Adolescent; Aminohydrolases; Animals; Cell Line; Cytosine Nucleotides; Female; Fibroblasts; Guanine Nucleotides; Guanosine Triphosphate; Humans; Infectious Mononucleosis; Lymphocytes; Lymphoma; Mice; Pyrimidine Nucleotides; Uracil Nucleotides; Uridine