Phospholipase C digestion of rat liver plasma membrane stimulates adenylate cyclase

Brief treatment of rat liver plasma membranes with phospholipase C of Clostridium welchii increased both the ratio of saturated to unsaturated fatty acids and the ratio of cholesterol to phospholipids. Using 5-doxylstearic acid spin probes two breaks at 29 and 19.6 °C could be observed in the order parameter, S_A, vs temperature curve for untreated membranes. Upon phospholipase C digestion the lower phase transition temperature was shifted to 23 °C, while the higher phase transition temperature could not be detected up to 40 °C. The order parameter, S_A, was consistently higher at all temperatures in the phospholipase C-treated membranes. As phospholipase C is known to attack the outer lamella, these results can be interpreted as indicating an increase in ordering (i.e., decrease in fluidity) of the outer membrane lamella. On the other hand, an increase in basal activity of adenylate cyclase of the treated membranes was observed with an apparent reduction of the activation energies both below and above the break (at 20 °C) in the Arrhenius plot of enzyme activity. Phospholipase C treatment did not affect the temperature of the break in Arrhenius kinetics of the enzyme. The results are discussed in terms of the role of the ordering state of membrane lipids in adenylate cyclase activity.     

Adenylate cyclase activity is decreased in chick embryo fibroblasts transformed by wild type and temperature sensitive Schmidt-Ruppin Rous sarcoma virus

Chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV-SR) have decreased adenylate cyclase activity. In cells infected by a temperature-sensitive mutant of this virus (RSV-SR-T5), enzyme activity is near normal when the cells are grown at the non-permissive temperature (41°C) but decreases at the permissive temperature (36°). Adenylate cyclase activity decreases slowly over a 24 hr period to one half normal levels when CEF-RSV-SR-T5 are shifted from 41° to 36°C. The low enzyme activity in CEF-RSV-SR is not due to an alteration in the K_m ATP or a change in the kinetics of Mg⁺⁺ activation, and is not observed when the enzyme is assayed in the presence of NaF. We conclude that transformation by RSV-SR reduces adenylate cyclase activity by a different mechanism than the Bryan high-titer strain of RSV.     

The effect of temperature on the activity of the adenylate cyclase system of liver plasma membranes

The rat liver adenylate cyclase system shows a discontinuity in the Arrhenius plots at 20°C in the nonstimulated activity (basal) with activation energies of 16 and 28 Kcal/mole. The discontinuity disappears when the enzyme is stimulated either by glucagon, sodium fluoride, 5′ guanylyl-imidodiphosphate or glucagon plus 5′ guanylyl-imidodiphosphate and the energy of activation was the same with all the compounds tested. If the activator was initially in contact with the membranes at 0°C the energy of activation was similar to that observed below the break (26 Kcal/mole) but it changed to that above the break if the compound contacted the membranes at temperatures above the break (22–24°C). We discuss the possibility of two different conformations of the enzyme; both conformations can be “frozen” by any of the compounds tested, “isolating” the enzyme from any subsequent physical change of the membrane due to temperature.     

The Escherichia coli adenylate cyclase complex. Regulation by enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.

The activity of adenylate cyclase of Escherichia coli measured in toluene-treated cells under standard conditions is subject to control by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Sugars such as glucose, which are transported by the PTS, will inhibit adenylate cyclase provided the PTS is functional. An analysis was made of the properties of E. coli strains carrying mutations in PTS proteins. Leaky mutants in the PTS protein HPr are similar to wild-type strains with respect to cAMp regulation; adenylate cyclase activity in toluene-treated cells and intracellular cAMP levels are in the normal range. Furthermore, adenylate cyclase in toluene-treated cells of leaky HPr mutants is inhibited by glucose. In contrast, mutations in the PTS protein Enzyme I result in abnormalities in cAMP regulation. Enzyme I mutants generally have low intracellular cAMP levels. Leaky Enzyme I mutants show an unusual phosphoenolpyruvate-dependent activation of adenylate cyclase that is not seen in Enzyme I⁺ revertants or in Enzyme I deletions. A leaky Enzyme I mutant exhibits changes in the temperature-activity profile for adenylate cyclase, indicating that adenylate cyclase activity is controlled by Enzyme I. Temperature-shift studies suggest a functional complex between adenylate cyclase and a regulator protein at 30 °C that can be reversibly dissociated at 40 °C. These studies further support the model for adenylate cyclase activation that involves phosphoenolpyruvate-dependent phosphorylation of a PTS protein complexed to adenylate cyclase.     

Effect of proteases and protease inhibitors on adenylate cyclase activity in rat cerebral cortical membranes

Mild proteolysis of membrane preparations from rat cerebral cortex with low concentrations of endopeptidases such as trypsin or chymotrypsin caused a 50–400% increase in the basal adenylate cyclase activity. Maximal activation of adenylate cyclase was obtained by including the protease in the adenylate cyclase assay, although an activated preparation could be obtained by pretreatment of the membranes with proteolytic enzymes. The proteolytically activated enzyme showed an increased V, with very little change in the K_m for the substrate, ATP. The proteolytically activated enzyme retained responsiveness to activation by sodium fluoride and 5′-guanylylimidodiphosphate (GppNHp), but was no longer activated by gangliosides or calcium-dependent activator protein. Activation by alcohols and detergent was lost or reduced in magnitude. The activity of adenylate cyclase after protease treatment showed a very marked temperature dependence, with maximal activity expressed in the 30–40 °C range and no activation due to the prior protease treatment expressed at either 10 or 50 °C. Basal adenylate cyclase activity was usually slightly inhibited in the presence of various protease inhibitors. Activation by fluoride, gangliosides, or GppNHp was little affected by protease inhibitors although one inhibitor, N-α-tosyl-l-lysine chloromethyl ketone, caused an inhibition of the ganglioside and GppNHp responses, slightly inhibited the fluoride response, and blocked the norepinephrine response normally seen in the presence of gangliosides or GppNHp. This inhibitor caused a loss of β-adrenergic binding sites for dihydroalprenolol in rat cortical membranes which paralleled the loss of the responsiveness of adenylate cyclase to a GppNHp-norepinephrine combination.     

Adenylate cyclase in cilia from Paramecium: Localization and partial characterization

A particulate adenylate cyclase was identified in the excitable ciliary membrane from Paramecium tetraurelia. MnATP was preferentially used as substrate, the K_m was 67 μM, V_max was 1 nmol cAMP.min^?1.mg^?1, a marked temperature optimum of 37°C was observed. Adenylate cyclase was not inhibited by 100 μM EGTA or 100 μM La³⁺, whereas under these conditions guanylate cyclase activity was abolished. Fractionation of ciliary membrane vesicles by a Percoll density gradient yielded two vesicle populations with adenylate cyclase activity. In contrast, calmodulin/Ca-dependent guanylate cyclase was associated with vesicles of high buoyant density only.     

Stimulation and modulation of adenylate cyclase by Sendai virus.

Stimulation of adenylate cyclase activity occurs in membranes prepared from toad erythrocytes preincubated briefly (at 37° or 4°) with ultraviolet light-inactivated Sendai virus. Stimulation occurs with as few as five virions per cell, and it is blocked by pretreating the virus with the membrane glycolipid, ganglioside GM₁. Virus treatment also alters modulation of adenylate cyclase by hormones, nucleotides and sodium fluoride. Interactions of viral envelope antigens with plasma membrane components may thus elicit functional changes possibly important in the pathogenesis of viral infections.     

Changes in the form of Arrhenius plots of the activity of glucagon-stimulated adenylate cyclase and other hamster liver plasma-membrane enzymes occurring on hibernation.

1. Arrhenius plots of the glucagon-stimulated adenylate cyclase, 5'-nucleotidase, (Na+ + K+)-stimulated adenosine triphosphatase and Mg2+-dependent adenosine triphosphatase activities of control hamster liver plasma membranes exhibited two break points at around 25 and 13 degrees C, whereas Arrhenius plots of their activities in hibernating hamster liver plasma membranes exhibited two break points at around 25 and 4 degrees C. 2. A single break occurring between 25 and 26 degrees C was observed in Arrhenius plots of the activities of fluoride-stimulated adenylate cyclase, basal adenylate cyclase and cyclic AMP phosphodiesterase of liver plasma membranes from both control and hibernating animals. 3. Arrhenius plots of phosphodiesterase I activity showed a single break at 13 degrees C for membranes from control animals, and a single break at around 4 degrees C for liver plasma membranes from hibernating animals. 4. The temperature at which break points occurred in Arrhenius plots of glucagon- and fluoride-stimulated adenylate cyclase activity were decreased by about 7--8 degrees C by addition of 40 mm-benzyl alcohol to the assays. 5. Discontinuities in the Arrhenius plots of 4-anilinonaphthalene-1-sulphonic acid fluorescence occurred at around 24 and 13 degrees C for liver plasma membranes from control animals, and at around 25 and 4 degrees C for membranes from hibernating animals. 6. We suggest that in hamster liver plasma membranes from control animals a lipid phase separation occurs at around 25 degrees C in the inner half of the bilayer and at around 13 degrees C in the outer half of the bilayer. On hibernation a change in bilayer asymmetry occurs, which is expressed by a decrease in the temperature at which the lipid phase separation occurs in the outer half of the bilayer to around 4 degrees C. The assumption made is that enzymes expressing both lipid phase separations penetrate both halves of the bilayer, whereas those experiencing a single break penetrate one half of the bilayer only.     

Differential effects of temperature on cAMP-induced excitation, adaptation, and deadaptation of adenylate and guanylate cyclase in Dictyostelium discoideum

Extracellular cAMP induces excitation of adenylate and guanylate cyclase in Dictyostelium discoideum. Continuous stimulation with cAMP leads to adaptation, while cells deadapt upon removal of the cAMP stimulus. Excitation of guanylate cyclase by cAMP has a lag time of approximately 1 s; excitation of adenylate cyclase is much slower with a lag time of 30 s. Excitation of both enzyme activities is less than twofold slower at 0 degrees C than at 20 degrees C. Adaptation of guanylate cyclase is very fast (t1/2 = 2.4 s at 20 degrees C), and virtually absent at 0 degrees C. Adaptation of adenylate cyclase is much slower (t1/2 = 110 s at 20 degrees C) but not very temperature sensitive (t1/2 = 290 s at 0 degrees C). At 20 degrees C, deadaptation of adenylate cyclase is about twofold slower than deadaptation of guanylate cyclase (t1/2 = 190 and 95 s, respectively). Deadaptation of adenylate cyclase is absent at 0 degrees C, while that of guanylate cyclase proceeds slowly (t1/2 = 975 s). The results show that excitation, adaptation, and deadaptation of guanylate cyclase have different kinetics and temperature sensitivities than those of adenylate cyclase, and therefore are probably independent processes.     

Anti-microtubular agents as inhibitors of desensitization to catecholamine stimulation of adenylate cyclase in Ehrlich ascites tumor cells

Adenylate cyclase activity of the homogenate of Ehrlich ascites tumor cells pretreated with catecholamine at 37°C was not stimulated by the addition of the same catecholamine, whereas that of the cells without the pretreatment was stimulated. Such a desensitization was induced hardly at all when the pretreatment was performed at low temperature. The desensitization of adenylate cyclase activity to catecholamine stimulation was prevented by pretreatment. The effect of cholchine was dependent on the period of the treatment and concentration of colchicine. Vinblastine had a similar effect, whereas cytochalasin B was without effect. Thus, involvement of microtubules was suggested in the desensitization of the membrane-associated enzyme to external sitmulation.     

Effect of temperature and ionic environment on the specific binding of 3H(—)sulpiride to membranes from different rat brain regions

Sulpiride is an antipsychotic drug endowed with the properties of a dopamine antagonist. The failure of sulpiride to inhibit neostriatal dopamine stimulated adenylate cyclase activity indicated that this drug is a selective D₂ receptor antagonist. In this study we used a novel synthesized ²H(—)sulpiride with very high specific activity (72 Ci/mol) and characterized the temperature sensitivity of the binding sites labeled by this compound. Kinetic analysis of ³H(—)sulpiride binding in rat striatum showed unstable behavior when incubation was performed at 37 or 30°C. However when experiments were carried out at 15 or 10°C, binding reached a stable steady-state within 10 min. Scatchard analysis of binding isotherms obtained at 10°C showed a 5-fold increase in the maximum number of binding sites and a decrease in K_d values to one-third those obtained at 37°C. Pharmacological characterization of the binding sites labeled by ³H(—)sulpiride at 10°C showed a greater affinity for antagonists but not for agonists than 37°C. Under both experimental condition, ³H(—)sulpiride binding sites were Na⁺ and GTP-sensitive. The temperature sensitive binding phenomenon appeared to be area specific. ³H(—)sulpiride binding sites in tissues other than from striatum were influenced less or not at all by changes in incubation temperature.     

Regulation of adenylate cyclase in two rat liver cell lines, 3C4 and 62.

Adenylate cyclase (EC 4.6.1.1) activities were examined in membrane preparations from two rat liver cell lines (62 and 3C4) which were grown in monolayer cultures. The cells were epithelial-like in growth character. Adenylate cyclase from the line 62 was stimulated by epinephrine, Gpp(NH)p, and prostaglandins A1,A2,E1,E2, and F2alpha, but not by glucagon. Arrhenius plots of adenylate cylase activity from line 62 gave straight lines, except when epinephrine was present in the assay; epinephrine-stimulated activity gave a distinct break at 20 degrees C. Adenylate cyclase activity in line 3C4 was stimulated by glucagon ten times greater than by epinephrine. It was responsive to Gpp(NH)p and all the prostaglandins. Arrhenius plots of adenylate cyclase activity of line 3C4 always gave straight line curves. Prostaglandins flattened the straight line curves (allowed temperature independence) of adenylate cyclase activity in membranes from both cell lines.     

Protein kinase C and a viral K-RAS protein cooperatively enhance the response of adenylate cyclase to stimulators

The protein kinase C stimulator TPA (12-O-tetradecanoyl phorbol-13-acetate) enhanced the responsiveness of adenylate cyclase to IPR (isoproterenol) and PGE1 (prostaglandin E1) in quiescent tsKSV-NRK cells at the nonpermissive 41 degrees C. Reactivating the thermolabile mitogenic/oncogenic K-ras protein in tsKSV-NRK cells by dropping the temperature to 36 degrees C also enhanced the responsiveness of adenylate cyclase to IPR and PGE1. The enhancement was transient and peaked at 6 hours after the temperature shift. This enhanced responsiveness was specifically due to the reactivated viral K-ras protein rather than the temperature shift because the same temperature shift did not affect adenylate cyclase responsiveness in uninfected NRK cells, nor was it a result of the mitogenic stimulus since reacting the mitogenic pp60v-src protein in tsASV-NRK cells did not affect adenylate cyclase responsiveness. The increased responsiveness of adenylate cyclase at 6 hours after the temperature shift was not a result of elevated membrane-associated PKC activity. However, the reactivated viral K-ras protein strongly increased the stimulability of membrane-associated PKC by TPA and it further increased TPA's ability to enhance the responsiveness of adenylate cyclase to IPR and PGE1. Thus, a viral K-ras protein and membrane-associated protein kinase C can cooperate to increase the responsiveness of adenylate cyclase to agonists.     

Vasopressin-sensitive kidney adenylate cyclase: Modulation of the hormonal response

Vasopressin-sensitive pig kidney adenylate cyclase is sensitive to several effectors, such as Mg²⁺, other divalent cations, and guanyl nucleotides. The purpose of the present study was to compare the main characteristics of adenylate cyclase activation by vasopressin, Mg²⁺, and GMPPNP, respectively. Mg^2+·ions were shown to exert at least three different effects on adenylate cyclase. The substrate of the adenylate cyclase reaction is the Mg-ATP complex. Mg²⁺ interacts with an enzyme regulatory site. Finally, Mg²⁺ can modulate the hormonal response, with Mg²⁺ions affecting the coupling function–that is, the quantitative relationship between receptor occupancy and adenylate cyclase activation. At all the magnesium concentrations tested, from 0.25 mM to 16 mM, adenylate cyclase activation was not a direct function of receptor occupancy. At low Mg²⁺ concentrations, adenylate cyclase activation dose-response curve to the hormone tended to be superimposable to the hormone dose-binding curve. These results suggest a role of magnesium at the coupling step between the hormone-receptor complex and adenylate cyclase response. Cobalt, but not calcium, ions could exert the same effects as Mg²⁺ ions on this coupling step. GMPPNP induced considerable adenylate cyclase activation (15 to 35 times the basal value). Activation by GMPPNP was highly time and temperature dependent. At 30° C, a 20 to 60 min preincubation period in the presence of GMPPNP was needed to obtain maximal activation. The higher the dose of GMPPNP in the medium, the longer it took to reach equilibrium. At 15° C, activation was still increasing with time after 3 hr preincubation in the presence of the nucleotide. GMPPNP was active in a 10^?8 M to 10^?5 M concentration range. Unlike the results obtained with lysine vasopressin, the kinetic characteristics of dose-dependent adenylate cyclase activation curves by GMPPNP were unaffected by varying Mg²⁺ concentrations except for the increase in velocity when raising Mg²⁺ concentration. It was not clear whether or not the activation processes by the hormone and by GMPPNP had common mechanisms.     

Osteosarcoma cytosol factor promotes parathyroid hormone stimulation of adenylate cyclase independent of GTP

The adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1)-stimulating factor from rat osteosarcoma cytosol was purified 600-fold by ion-exchange chromatography. The factor has an apparent M_r of 20 000, is cold-labile, but retains activity at ?20°C in 10% glycerol.The factor enhanced parathyroid hormone stimulation of adenylate cyclase and restored hormone responsiveness to membranes washed with 0.5 M NaCl. These ‘GTP-like’ effects were not inhibited by 100 μM GDP-β-S, which completely abolished the GTP enhancement of both basal and hormone-stimulated adenylate cyclase.Adenylate cyclase activity in the presence of the stimulating factor was linear with time, and showed hyperbolic dependence on factor concentration. The factor also linearized (in double reciprocal plots) the downward-concave Mg²⁺-dependence of adenylate cyclase, increasing the apparent affinity of the enzyme for Mg²⁺.The presence of the factor in two clonal osteosarcoma cell lines correlated with parathyroid hormone-stimulatable adenylate cyclase. Factor stimulation was absent while GTP stimulation was retained in the hormone-nonresponsive clone. Factor and hormone sensitivity were restored by in vivo passage. This factor thus may represent a guanyl nucleotide-independent path for cellular regulation of hormone response.     

Effect of hyperthemia in combination with vitamin E and cyclic amp on neuroblastoma cells in culture

The effect of heat in combination with DL-alpha-tocopheryl (vitamin E) succinate and adenosine 3′, 5′-cyclic monophosphate (cAMP) stimulating agents on mouse neuroblastoma cells (NBP₂) in culture on the criterion of growth inhibition (due to cell death and inhibition of cell division) was studied. Heat (41°?40°) alone inhibited growth; however, the extent of growth inhibition was dependent upon the temperature and the time of heat treatment. Heat (41°?40°) in combination with vitamin E succinate (5 μg/ml) produced an additive effect on the criterion of growth inhibition. Vitamin C (100 μg/ml) failed to modify the effect of heat. Prostaglandin A₂, a stimulator of adenylate cyclase, and 4 - (3-butoxy-4-methoxybenzyl)-2-imidazolindinone (R020-1724), an inhibitor of cyclic nucleotide phosphodiesterase, are known to induce irreversible differentiation in mouse neuroblastoma cells in culture. These agents, in combination with heat (40°) produced a synergistic effect on the criterion of growth inhibition. These data suggest that the addition of vitamin E and cAMP stimulating agents may increase the effectiveness of hyperthermia protocol.     

Activation and stabilization of cardiac adenylate cyclase by GTP analog and fluoride.

The rate of cyclic AMP formation by rabbit heart membrane particles decreased at assay temperatures greater than 30 °C. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (assayed at 24 °C) decreased exponentially with time of preincubation at 30 or 37 °C, providing evidence for the instability of this enzyme. The half-life, t_1/2, of the enzyme at 37 °C was 9.9 min in the absence and 4.4 min in the presence of MgCl₂. The activity was most labile in the presence of 50 m m Mg²⁺ and 1 m m ATP, having t_1/2 = 1.3min. Prior incubation of membranes with the GTP analog, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], 0.1 m m, for 30 min at 37 °C produced maximal activation of adenylate cyclase; the rate of activation was temperature dependent and was increased in the presence of isoproterenol. The Gpp(NH)p-activated enzyme had increased thermal stability, t_1/2 = 170 min, and was also markedly more stable in the presence of Mg-ATP, t_1/2 = 72min, than nonactivated enzyme. Preactivation with F? (30 min at 24 °C) also stabilized the activity; t_1/2 > 70 min in the absence or presence of Mg-ATP. The Mg²⁺ concentration required for maximal activity was reduced from approximately 60 m m for nonactivated enzyme to 10 m m for the Gpp(NH)p- and F?activated enzyme.     

Supra-molecular organization of adenylate cyclase in a neuroblastoma × glioma hybrid

Studies on the thermal inactivation of adenylate cyclase from neuroblastoma x glioma hybrid cells have been carried out. Inactivation curves show marked deviation from first-order kinetics, and as a first approximation can be adequately described as a sum of two negative exponentials. Half-lives of the rapidly decaying component have been estimated to be 5, 3.4,1.2 and 0.5 min at 37, 40, 44 and 48°C, respectively. The corresponding values for the slow-decaying component are found to be 90, 30, 11 and 5 min. Plausible inactivation pathways responsible for multi-exponential decay curves are discussed. Kinetic curves describing fractional loss of stimulatory response of adenylate cyclase to prostaglandin E₁ are shifted downwards with reference to basal activity. In contrast, an upward shift is observed for the inhibitory response of the enzyme to etorphine. A quantitative analysis of the inactivation curves for prostaglandin and etorphine-responsiveness has led to definitive predictions regarding the heat-sensitivity of the ‘hypothetical’ temperature-labile component responsible for the observed shifts.     

Elevated membrane cholesterol concentrations inhibit glucagon-stimulated adenylate cyclase.

A method was devised which increases the cholesterol concentration of rat liver plasma membranes by exchange from cholesterol-rich liposomes at low temperature (4 degrees C). When the cholesterol concentration of liver plasma membranes is increased, there is an increase in lipid order as detected by a decrease in mobility of an incorporated fatty acid spin probe. This is accompanied by an inhibition of adenylate cyclase activity. The various ligand-stimulated adenylate cyclase activities exhibit different sensitivities to inhibition by cholesterol, with inhibition of glucagon-stimulated greater than fluoride-stimulated greater than basal activity. The bilayer-fluidizing agent benzyl alcohol is able to reverse the inhibitory effect of cholesterol on adenylate cyclase activity in full. The thermostability of fluoride-stimulated cyclase is increased in the cholesterol-rich membranes. Elevated cholesterol concentrations abolish the lipid-phase separation occurring at 28 degrees C in native membranes as detected by an incorporated fatty acid spin probe. This causes Arrhenius plots of glucagon-stimulated adenylate cyclase activity to become linear, rather than exhibiting a break at 28 degrees C. It is suggested that the cholesterol contents of both halves of the bilayer are increased by the method used and that inhibition of adenylate cyclase ensues, owing to the increase in lipid order and promotion of protein-protein and specific cholesterol-phospholipid interactions.