guanylyl-imidodiphosphate 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

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

Mutant isolates [designated desensitization resistant (DR)] from the Y1 mouse adrenocortical tumor cell line resist agonist-induced desensitization of adenylyl cyclase by preventing the uncoupling of receptors from their guanyl nucleotide-binding regulatory G proteins. In this study, we tested the hypothesis that an underlying G protein defect is associated with the DR phenotype. We found that the G protein reagent guanyl-5'-yl imidodiphosphate [Gpp(NH)p] shifted beta2-adrenergic receptors from a high affinity state to a low affinity state 4-fold more effectively in mutant DR cells than in parent Y1 cells. In the DR mutant, Gpp(NH)p was able to shift receptors to a low affinity state in the absence of NaCl, whereas the effect of Gpp(NH)p in parent Y1 cells was dependent upon the presence of NaCl. Moreover, these differences in sensitivity to Gpp(NH)p and NaCl were transferred to Gs alpha-deficient S49(CYC-) lymphoma cell membranes in G protein reconstitution assays. These observations suggested that the DR mutation was associated with altered activity of the stimulatory G protein, Gs. Cloning and sequence analysis demonstrated that Gs alpha transcripts in the DR mutant were normal, suggesting that another factor involved in guanyl nucleotide exchange is responsible for the altered G protein activity in DR mutant cells.

Topics: Adaptation, Physiological; Adrenal Cortex Neoplasms; Amino Acid Sequence; Animals; Base Sequence; DNA, Complementary; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Lymphoma; Mice; Molecular Sequence Data; Mutation; Phenotype; Receptors, Adrenergic, beta; Sodium Chloride; Tumor Cells, Cultured

The present study investigated whether high salt intake (8%) in Dahl salt-sensitive and salt-resistant rats with and without hypertension produces a heterologous desensitization of cardiac adenylyl cyclase as observed in various types of hypertension and human heart failure. In membranes from Dahl salt-sensitive rats on a high-salt diet (8%) basal, isoproterenol-, 5'-guanylylimidodiphosphate-, and forskolin-stimulated adenylyl cyclase was reduced compared with the low-salt (0.4%) group and Dahl salt-resistant rats on either 0.4% or 8% sodium chloride. The activity of the catalyst was depressed, and the expression of the immunodetectable inhibitory G proteins Gi alpha was increased in Dahl salt-sensitive rats on 8% sodium chloride, whereas the density of beta-adrenergic receptors and the activity of the stimulatory G protein Gs alpha reconstituted into Gs alpha-deficient S49 cyc- mouse lymphoma cell membranes were unchanged in any condition studied. We conclude that high salt intake in salt-sensitive hypertensive Dahl rats produces hypertension, cardiac hypertrophy, and heterologous desensitization of cardiac adenylyl cyclase. The latter alteration is due to an increase of Gi alpha proteins and a depressed catalyst activity of adenylyl cyclase. The results demonstrate that heterologous adenylyl cyclase desensitization can precede the development of contractile dysfunction in later stages and can occur independently of changes in beta-adrenergic receptors.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Amino Acid Sequence; Animals; Blood Pressure; Cardiomegaly; Cell Membrane; Colforsin; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Humans; Hypertension; Immune Sera; Kinetics; Lymphoma; Male; Mice; Molecular Sequence Data; Myocardium; NAD; Oligopeptides; Radioimmunoassay; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Adrenergic, beta; Recombinant Proteins; Sodium Chloride; Transducin; Transfection; Tumor Cells, Cultured; Virulence Factors, Bordetella

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

The present study describes the characterization of an opioid binding site on membranes prepared from the R1.1 cell line, a murine thymoma. Specific (-)[3H]bremazocine binding was saturable, stereoselective, and limited to a single high affinity binding site with a Kd value of 15.2 +/- 1.6 pM and a Bmax value of 54.8 +/- 6.0 fmol/mg of protein. The kappa-selective alkaloids and dynorphin peptides inhibited (-)[3H]bremazocine binding with Ki values of less than 1 nM, in contrast to mu- and delta-selective ligands. The high affinity of this site for alpha-neo-endorphin and U50,488 suggests that this kappa opioid binding site resembles the kappa 1b subtype. NaCl, as well as other mono- and divalent cations, inhibited (-)[3H]bremazocine binding. In the presence of NaCl, the nucleotides GTP, GDP, and the nonhydrolyzable analog guanylyl-5'-imidodiphosphate (Gpp(NH)p) also decreased (-)[3H]bremazocine binding, suggesting that this kappa opioid binding site is coupled to a G-protein. In summary, R1.1 cells possess a single high affinity kappa opioid receptor that shares many properties with brain kappa 1b opioid receptors.

Topics: Animals; Benzomorphans; Binding Sites; Guanine Nucleotides; Guanylyl Imidodiphosphate; Lymphoma; Mice; Receptors, Opioid, kappa; Sodium Chloride; Tumor Cells, Cultured

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

1. Functional vasoactive intestinal peptide (VIP)/helodermin receptors and beta 2-adrenoceptors coexist in membranes from a cultured cloned BL/VL3 cell line of murine T-cell lymphoma induced by a radiation leukemia virus (see preceding paper in this journal). 2. Short-term (5-30 min) exposures of BL/VL3 cells to VIP or isoproterenol induced both homologous and heterologous desensitization. The potency of VIP and isoproterenol to desensitize was similar to their potency to occupy receptors and activate adenylate cyclase. 3. Long-term (16-h) exposure of BL/VL3 cells to VIP induced homologous down regulation only, whereas isoproterenol induced both homologous and heterologous down regulation. The potency of VIP, peptide histidine isoleucinamide, helodermin, helospectin, and [D-Phe2]VIP on the one hand, and of isoproterenol on the other hand, to decrease homologous responses was comparable to their potency for receptor occupancy and adenylate cyclase activation.

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Drug Tolerance; Enzyme Activation; Guanylyl Imidodiphosphate; Intercellular Signaling Peptides and Proteins; Isoproterenol; Leukemia, Radiation-Induced; Lymphoma; Mice; Peptide PHI; Peptides; Receptors, Adrenergic, beta; Retroviridae; Sodium Fluoride; T-Lymphocytes; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

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

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

The effects of 4-beta phorbol 12-myristate 13-acetate (PMA) on hormone and forskolin-stimulated adenylate cyclase were evaluated in S49 lymphoma cells. Treatment of wild type (WT) S49 cells with PMA caused stimulation, inhibition or had no effect on epinephrine stimulation of cAMP accumulation. The effect observed was dependent on the length of PMA treatment, the concentration of PMA and the concentration of hormone (or forskolin) used to stimulate cAMP accumulation. Longer treatment times with PMA and higher PMA concentrations favored the inhibitory effects. Pretreating WT with 0.5 microM PMA for 18 min caused an increase in the EC50 and maximal levels for epinephrine stimulation of cAMP accumulation. Thus inhibition was seen at relatively low epinephrine concentrations and augmentation with high concentrations. The inhibitory effects of PMA on epinephrine-stimulated adenylate cyclase activity were observed only at low free Mg++ concentrations (0.75 mM). The effects of PMA on PGE1-stimulated cAMP accumulation were similar to those observed for epinephrine. In S49 WT cells 100 nM PMA augmented 5 microM forskolin-stimulated cAMP accumulation; however with 100 microM forskolin, PMA effects were minimal. PMA also attenuated Gi-mediated Gpp(NH)p inhibition of forskolin-stimulated adenylate cyclase in both WT and cyc- membranes, resembling the effects of pertussis toxin. The effects of various phorbol analogues on epinephrine-stimulated cAMP accumulation were as follows: 4 beta-phorbol 12,13-didecanoate had similar effects to PMA, 4 alpha-phorbol 12,13-didecanoate had no effects and 1-oleoyl, 2-acetylglycerol augmented epinephrine-stimulated cAMP accumulation at concentrations greater than or equal to 5 microM. Our results are consistent with a dual mechanism of PMA action on adenylate cyclase involving protein kinase C-mediated phosphorylation of Gi and of the beta-adrenergic receptor, the former leading to augmentation and the latter to inhibition of hormone-stimulated adenylate cyclase.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Alprostadil; Cells, Cultured; Colforsin; Cyclic AMP; Epinephrine; Guanylyl Imidodiphosphate; Lymphoma; Pertussis Toxin; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella

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

Brief (approximately 20-min) exposure of S49 lymphoma cells to beta-agonists such as isoproterenol leads to a homologous form of desensitization in which beta-agonist but not prostaglandin E1-sensitive or NaF-sensitive adenylate cyclase is reduced. The desensitized receptors (R) appear to be sequestered away from the effector system (guanine nucleotide regulatory protein (Ns) and adenylate cyclase (C)). Membrane perturbants such as polyethylene glycol are known to reorient membrane proteins and lipids. Thus, we fused agonist-desensitized S49 lymphoma cells to each other, using polyethylene glycol as fusogen, in an attempt to functionally reunite the R, N, and C components which might have become sequestered in microdomains of the plasma membrane during desensitization. Such treatment completely restored isoproterenol-stimulated adenylate cyclase to normal and re-established the ability of R and N to functionally couple as assessed by the ability to form a high affinity, guanine nucleotide-sensitive state of the receptor. These results support the concept that agonist-promoted sequestration plays a functionally significant role in the homologous desensitization of the beta-adrenergic receptor.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Isoproterenol; Lymphoma; Membrane Lipids; Membrane Proteins; Polyethylene Glycols; Receptors, Adrenergic, beta; Sodium Fluoride; Time Factors

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

Rat reticulocytes contain a cytosol activator protein (RCAP) that augments hormone-sensitive adenylate cyclase activity in the rat reticulocyte and other systems. In a previous publication, using a highly purified preparation of RCAP, we reported that the stimulatory guanine nucleotide-binding protein (Ns) was required for the actions of RCAP. We investigated this possibility by studying the actions of RCAP on cholera toxin-dependent ADP ribosylation of Ns. RCAP decreased cholera toxin-dependent ADP ribosylation of the 42,000-dalton subunit of Ns of reticulocyte [40.2 +/- 3.7 (+/-SEM) to 26.5 +/- 3.8 fmol/mg; n = 10; P less than 0.001], S49 wild-type (33.9 +/- 2.4 to 24.9 +/- 2.8 fmol/mg; n = 9; P less than 0.01), and UNC (25.3 +/- 3.5 vs. 17.6 +/- 3.1; n = 5; P less than 0.02) membranes. In contrast, pertussis toxin-dependent ADP-ribosylation of the inhibitory guanine nucleotide binding protein, Ni in reticulocyte, S49 wild-type lymphoma, and its UNC and cyc- variant membranes were all significantly augmented by RCAP. Moreover, when reticulocyte Ni was functionally ablated by exposure to pertussis toxin, RCAP no longer enhanced isoproterenol-responsive adenylate cyclase activity in reticulocyte membranes. These results suggest that RCAP stimulates adenylate cyclase activity by inhibiting Ni function, thus permitting enhanced Ns coupling to the adenylate cyclase enzyme (C).

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Bacterial Toxins; Blood Proteins; Cell Line; Cell Membrane; Cholera Toxin; Cytosol; Enzyme Activation; Fluorides; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Humans; Isoproterenol; Lymphoma; Pertussis Toxin; Rats; Reticulocytes; Virulence Factors, Bordetella

Forskolin clearly has effects on all three known components of adenylate cyclase, Ni, Ns, and the catalytic subunit (C). Forskolin can activate the catalytic activity of adenylate cyclase directly in the absence of the Ni or Ns subunit, and, therefore, forskolin is acting at a site that is on the catalytic subunit or a closely associated protein. A lack of forskolin stimulation of cyclic AMP in intact cells does not necessarily imply that it requires or acts via the Ns protein, since, as shown for the cyc- S49 cells, the enzyme may be in an inhibited state because of the presence of Ni. The presence of a site on adenylate cyclase that can regulate not only the absolute activity of the enzyme but also its sensitivity to hormones raises the possibility that there may be substances endogenous to the cell that can functionally interact at this site. It is too early to speculate as to the nature of these substances. However, they could be extracellular, originating from other cells, or they could be intracellular. The final determination will rely on the identification and physical disposition of the forskolin-binding site and also the identification of endogenous compounds with forskolin-like activities.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Blood Platelets; Cell Line; Colforsin; Diterpenes; Drug Interactions; Enzyme Activation; Ethylmaleimide; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hormones; Humans; Lymphoma; Manganese; Mice; Receptors, Cell Surface

The thermal inactivation of adenylate cyclase was investigated in human lymphocytes and in the N-protein deficient cyc- S49 mouse lymphoma cell line. The enzyme is rapidly inactivated at 37C with a t1/2 of 5.5 and 4.5 min respectively in human and cyc- membranes. Thermal inactivation is prevented by at least two mechanisms. The first mechanism involves ATP which stabilizes adenylate cyclase in a concentration dependent manner similar to the Km of ATP for cAMP formation. However, the inhibition of inactivation does not require Mg++ while the enzyme catalysis of ATP to cAMP does. The second mechanism involves substances which activate the enzyme. The human lymphocyte enzyme is equally stabilized by either NaF, GppNHp, or forskolin. In contrast, the cyc- enzyme is fully stabilized by forskolin but only partially stabilized by NaF. When human erythrocyte N-protein extract is added to cyc- membranes, NaF fully stabilizes the enzyme. These data suggest that an activated N-protein is instrumental in stabilizing adenylate cyclase and that there is some N-protein component in cyc- membranes through which NaF may be exerting its stabilizing action.

Topics: Adenosine Triphosphate; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cardiotonic Agents; Cell Line; Colforsin; Diterpenes; Enzyme Activation; Erythrocyte Membrane; Erythrocytes; Guanylyl Imidodiphosphate; Humans; Lymphocytes; Lymphoma; Mice; Neoplasms, Experimental; Sodium Fluoride; Temperature

cyc- S49 cell membranes contain an adenylyl cyclase activity which is stimulated by forskolin and inhibited by guanine nucleotides and NaF. These inhibitory effects are mediated by an inhibitory guanine nucleotide-binding regulatory component (Ni) affecting the adenylyl cyclase catalytic unit (Hildebrandt, J. D., Sekura, R. D., Codina, J., Iyengar, R., Manclark, C. R., and Birnbaumer, L. (1983) Nature (Lond.) 302, 706-709). Since cyc- S49 cells do not contain a stimulatory guanine nucleotide-binding regulatory component (Ns), these membranes were used to study the requirements and kinetics of activation of Ni in the absence of Ns. Activation of Ni by guanyl-5'-yl imidodiphosphate was time-dependent (i.e. hysteretic) and pseudo-irreversible. Although GTP and guanosine 5'-(beta-thio)diphosphate could prevent the inhibition caused by guanyl-5'-yl imidodiphosphate if added simultaneously with it, they could not reverse the inhibited state induced by previous exposure to guanyl-5'-yl imidodiphosphate. Activation of Ni had an absolute requirement for Mg2+. Unlike the activation of Ns, however, which requires millimolar concentrations of Mg2+ in the absence of hormonal stimulation, activation of Ni requires only micromolar concentrations of the divalent cation. These results support the contention that hormones which activate Ni or Ns do so by altering different parameters of a similar activation mechanism.

Topics: Adenylyl Cyclases; Animals; Antihypertensive Agents; Cell Line; Cell Membrane; Colforsin; Diterpenes; Guanine Nucleotides; Guanylyl Imidodiphosphate; Lymphoma; Magnesium; Manganese; Mice; Molecular Weight; Sodium Fluoride

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: Adenosine Triphosphate; Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Edetic Acid; Erythrocyte Membrane; Guanylyl Imidodiphosphate; Isoproterenol; Kinetics; Lymphoma; Magnesium; Manganese; Mice; Propranolol; Turkeys

Topics: Adenosine Triphosphate; Adenylyl Cyclase Inhibitors; Animals; Calcium; Cell Membrane; Enzyme Activation; Erythrocyte Membrane; Erythrocytes; Guanylyl Imidodiphosphate; Kinetics; Lymphoma; Mice; Neoplasms, Experimental; Turkeys

Topics: Adenylyl Cyclases; Adrenergic beta-Antagonists; Animals; Biological Transport; Cell Line; Cell Membrane; Cholera Toxin; Clone Cells; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Isoproterenol; Kinetics; Lymphoma; Magnesium; Mice; Receptors, Adrenergic; Receptors, Adrenergic, beta; Receptors, Cell Surface

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

Forskolin, a novel diterpene activator of adenylate cyclase in membranes and intact cells, activates the enzyme in membranes from mutant cyc-S49 murine lymphoma cells and the soluble enzyme from rat testes. Each of these enzymes consists only of the catalytic subunit and does not have a functional guanine nucleotide-binding protein. In both cases forskolin converts the manganese-dependent enzymes to a form which does not require manganese for activity. Forskolin can also stimulate a detergent-solubilized preparation of adenylate cyclase from rat cerebral cortex. Activation of adenylate cyclase by forskolin is therefore not dependent on a perturbation of membrane structure nor does it require a functional guanine nucleotide-binding subunit.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Colforsin; Diterpenes; Enzyme Activation; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Isoproterenol; Kinetics; Lymphoma; Manganese; Mice; Neoplasms, Experimental; Receptors, Cell Surface; Sodium Fluoride

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Cyclic AMP; Enzyme Activation; Guanylyl Imidodiphosphate; Hormones; Isoproterenol; Kinetics; Liver; Lymphoma; Mice; Rats; Receptors, Adrenergic; Receptors, Adrenergic, beta; Sodium Fluoride

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; Binding Sites; Cell Line; Cell Membrane; Cholera Toxin; Chymotrypsin; Cytidine Triphosphate; Cytosine Nucleotides; Enzyme Activation; Female; Fluorides; Guanylyl Imidodiphosphate; Kinetics; Liver; Lymphoma; Neoplasms, Experimental; Papain; Protein Binding; Rats

Topics: Adenylyl Cyclases; Cell Line; Cell Membrane; Clone Cells; Fluorides; Guanylyl Imidodiphosphate; Kinetics; Lymphoma; Membrane Proteins; Phenotype; Receptors, Adrenergic; Receptors, Adrenergic, beta

Adenylate cyclase can be resolved into at least two protein components, neither of which by itself catalyzes the formation of cyclic AMP with Mg-ATP as substrate. Mixture of the two reconstitutes Mg-ATP-dependent, fluoride- and Gpp(NH)p-stimulable activity. One, a heat-labile, N-ethylmaleimide-sensitive protein of molecular weight 190,000 can catalyze cyclic AMP formation with Mn-ATP as substrate, and is therefore proposed to be the catalytic moiety of the adenylate cyclase complex. The other protein (or proteins) is more resistant to heating or N-ethylmaleimide, and is proposed to confer upon the catalyst the ability to ultilize Mg-ATP as substrate. It is also required for the regulation of that activity by guanine nucleotides, hormones, and probably fluoride ion. The catalytic protein is found in a phenotypically adenylate cyclase-deficient (AC-) variant of S49 lymphoma cells. The thermostable regulatory protein can be resolved from the catalyst by heat treatment or N-ethylmaleimide treatment of plasma membranes of wild-type S49 cells, rat or rabbit liver, or avian erythrocytes, and is also found in a phenotypically adenylate cyclase-deficient hepatoma cell line. Mixture of AC- S49 membranes, which contain the beta-adrenergic receptor, with a crude detergent-solubilized preparation of the regulatory protein reconstitutes hormone-stimulable adenylate cyclase activity. Binding of the regulatory protein to the membranes is a time- and temperature-dependent process that requires an activating ligand of the adenylate cyclase system [fluoride, Gpp(NH)p].

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Cells, Cultured; Ethylmaleimide; Guanylyl Imidodiphosphate; Hot Temperature; Liver Neoplasms, Experimental; Lymphoma; Neoplasms, Experimental; Rats; Receptors, Adrenergic; Receptors, Adrenergic, beta; Receptors, Cyclic AMP

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