Activation of guanylate cyclase from rat liver and other tissues by sodium azide.

Sodium azide, hydroxylamine, and phenylhydrazine at concentrations of 1 mM increased the activity of soluble guanylate cyclase from rat liver 2- to 20-fold. The increased accumulation of guanosine 3':5'-monophosphate in reaction mixtures with sodium azide was not due to altered levels of substrate, GTP, or altered hydrolysis of guanosine 3':5'-monophosphate by cyclic nucleotide phosphodiesterase. The activation of guanylate cyclase was dependent upon NaN3 concentration and temperature; preincubation prevented the time lag of activation observed during incubation. The concentration of NaN3 that resulted in half-maximal activation was 0.04 mM. Sodium azide increased the apparent Km for GTP from 35 to 113 muM. With NaN3 activation the enzyme was less dependent upon the concentration of free Mn2+. Activation of enzyme by NaN3 was irreversible with dilution or dialysis of reaction mixtures. The slopes of Arrhenius plots were altered with sodium azide-activated enzyme, while gel filtration of the enzyme on Sepharose 4B was unaltered by NaN3 treatment. Triton X-100 increased the activity of the enzyme, and in the presence of Triton X-100 the activation by NaN3 was not observed. Trypsin treatment decreased both basal guanylate cyclase activity and the responsiveness to NaN3. Phospholipase A, phospholipase C, and neuraminidase increased basal activity but had little effect on the responsiveness to NaN3. Both soluble and particulate guanylate cyclase from liver and kidney were stimulated with NaN3. The particulate enzyme from cerebral cortex and cerebellum was also activated with NaN3, whereas the soluble enzyme from these tissues was not. Little or no effect of NaN3 was observed with preparations from lung, heart, and several other tissues. The lack of an effect with NaN3 on soluble GUANYLATE Cyclase from heart was probably due to the presence of an inhibitor of NaN3 activation in heart preparations. The effect of NaN3 was decreased or absent when soluble guanylate cyclase from liver was purified or stored at -20degrees. The activation of guanylate cyclase by NaN3 is complex and may be the result of the nucleophilic agent acting on the enzyme directly or what may be more likely on some other factor in liver preparations.     

Activation of rat liver guanylate cyclase by proteolysis.

Guanylate cyclase activity (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.), measured in purified rat liver plasma membranes, was markedly increased by treatment with various purified proteases. The effect was maximal with trypsin, alpha-chymotrypsin, papain, and thermolysin (6- to 8-fold increase with 5 to 20 microgram of protease/ml) and lower with subtilisin and elastase (3- to 4-fold increase). The activation was due to an increase in the maximal velocity of the cyclizing reaction. No modification was observed either in the apparent affinity for the substrate MnGTP or in the cooperative behavior of the enzyme kinetics which displayed Hill coefficients of 1.6 for both basal and activated states. The Triton X-100-dispersed guanylate cyclase remained sensitive to papain, which suggests that the action of proteases was not restricted to an indirect action upon the membranous environment of the guanylate cyclase. In contrast, the cytosolic soluble guanylate cyclase, assayed in the presence or absence of sodium azide, was absolutely insensitive to papain. Thus, proteolysis represents a previously undescribed mechanism for activating membranous guanylate cyclase systems, which might be of importance in the physiological regulation of this enzyme.     

Fatty acid activation of guanylate cyclase from fibroblasts and liver.

Sodium arachidonate and sodium oleate increased particulate guanylate cyclase activity from homogenates of Balb 3T3 cells or rat liver. The fatty acids were about equipotent and were maximally effective at about 100 μm concentrations. Higher concentrations were less effective or inhibitory. Activation was similar in an air or nitrogen atmosphere and was unaltered by KCN, aspirin, or indomethacin. The dose-response curve was shifted to the right when arachidonate was preincubated prior to its addition to guanylate cyclase assays. Agents that facilitate fatty acid oxidation and the formation of malonyldialdehyde during preincubation such as glutathione, hemoglobin, Mn²⁺, Fe³⁺, or lipoxygenase shifted the dose-response curve further to the right. In contrast, agents that decreased or prevented arachidonate oxidation and malonyldialdehyde formation during preincubation such as butylated hydroxyanisole, propyl gallate, hydroquinone, and diphenylfuran prevented the shift in the dose-response curve or in some instances shifted the dose-response curve to the left. Activation of guanylate cyclase by arachidonate was reversed by the addition of lipoxygenase to incubations. These studies indicate that unsaturated fatty acids and not their oxidation products activate particulate enzyme from Balb 3T3 cells. The mechanism of fatty acid activation appears to be different from activation by nitro compounds. Fatty acids but not nitro compounds activated fibroblast preparations, and the effect of fatty acids in contrast to the activation by nitroprusside in liver preparations was not prevented with Lubrol PX.     

Butadiene diepoxide activation of guanylate cyclase.

A variety of nitroso chemical carcinogens increase the activity of guanylate cyclase (EC 4.6.1.2), the enzyme catalyzing the production of guanosine 3',5'-monophosphate. In the present report, the first non-nitroso chemical carcinogen, butadiene diepoxide, was shown to activate guanylate in a variety of tissues over the concentration range 1-100 mmol/l. At 20 mmol/l concentration, increases were 2- to 17-fold above control. These observations have potential importance since guanosine 3',5'-monophosphate may be involved in cell growth and malignant transformation.     

Characterization of ATP-stimulated guanylate cyclase activation in rat lung membranes

Many of the effects of ANP are mediated through the elevation of cellular cGMP levels by the activation of particulate guanylate cyclase. While the stimulation of this enzyme is receptor-mediated, the molecular mechanism of activation remains unknown. In this study we present evidence that ATP as well as its analogues adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) and adenylylimidophosphate (AMPPNP) activates guanylate cyclase from rat lung membranes and markedly potentiates the effect of ANP on the enzyme. The order of potency is ATP gamma S greater than ATP greater than AMPPNP. The enzyme activation by adenine nucleotide and ANP together is much more than the sum of the individual activations, suggesting that ATP may be the physiological component essential for the ANP-stimulated guanylate cyclase activation. The ATP gamma S-stimulated guanylate cyclase activity diminishes in the presence of various kinds of detergents, suggesting either that the conformation of an ATP binding site in guanylate cyclase is altered by detergents or that protein-protein interaction may be involved in the activation of guanylate cyclase by ATP. Guanylate cyclase from rat lung membranes is poorly activated by ANP and/or ATP gamma S after removing the cytosolic and weakly membrane-associated proteins or factors by centrifugation. Pre-incubation of the membranes with ATP gamma S retains enzyme activation after membrane washing. These results suggest either that ATP gamma S stabilizes the conformation of nucleotide binding site in guanylate cyclase from denaturation by membrane washing, or that the stimulatory effect of ATP on guanylate cyclase activity may be mediated by accessory proteins or non-protein cofactors which are lost during membrane washing, but remain bound to membranes by ATP gamma S pretreatment.     

Requirement for a macromolecular factor for sodium azide activation of guanulate cyclase.

Sodium azide, a highly nucleophilic agent and a potent metabolic inhibitor, markedly increased guanylate cyclase activity from supernatant fractions of rat liver homogenates. The effect of sodium azide was not observed with partially purified guanulate cyclase from liver or crude soluble guanylate cyclase from cerebral cortex. However, the effect of sodium azide could be restored by the readdition of a fraction isolated from rat liver homogenates. The macromolecular factor required for the sodium azide effect was separated from soluble guanylate cyclase of rat liver with DEAE-cellulose column chromatography, and some of its properties were examined. The factor was nondialyzable and heat labile.     

Phencyclidine stimulates guanylate cyclase activity.

The hallucinogenic agents, phencylidine (Angel's Dust), TCP¹ and their morpholine analogs enhanced the activity of guanylate cyclase {E.C.4.6.1.2}, the enzyme that catalyzes the production of guanosine 3′, 5′-monophosphate. This activation of guanylate cyclase by hencyclidine and TCP was observed over the concentration range of .00001 mM to 1 mM, while the morpholine analogs stimulated tha activity of guanylate cyclase in concentration of .0001 mM to 1 mM.     

Ultrastructural demonstration of guanylate cyclase in rat lung after activation by ANF.

We studied the cytochemical localization of particulate guanylate cyclase (GC) activity after stimulation with atrial natriuretic factor (ANF) in rat lung, at the electron microscope level. Samples incubated in the absence of ANF did not reveal any GC reaction product. These results indicate that ANF is a strong activator of the enzyme in this organ. In intrapulmonary bronchi, the ANF-activated GC reaction product was localized on mucus secreting goblet cells. GC was seen in bronchioles, alveoli and capillaries. All of the GC reaction product was associated with plasma membranes of Clara cells, of great alveolar cells and of endothelial cells in alveolar capillaries. Our data suggest that, by activation of particulate GC, ANF acts directly on cells where Na+ reabsorption occurs.     

Modulation of rat guanylate cyclase activity in vitro by chemical carcinogens.

The nucleotide cyclic GMP has been reported to be involved in cell proliferation and malignant transformation. Nitroso chemical carcinogens activate the enzyme guanylate cyclase (EC 4.6.1.2) which catalyzes the production of cyclic GMP. The present investigation demonstrates that compounds from other major classes of carcinogens including (1) alpha-halo ethers (chloromethyl methyl ether); (2) aromatic amines (benzidine and B-naphthylamine); (3) polycyclic hydrocarbons (1,2-benzanthracene and acridine); (4) azo dyes (p-dimethylaminoazobenzene), and (5) aflatoxins (B1, B2, G1, G2) produced a striking and significant inhibition of guanylate cyclase over a general concentration range of 0.5-13 mmol/1 in a variety of tissues. Some of the nitrosamides which increase guanylate cyclase activity, increase DNA synthesis whereas carcinogens which decrease guanylate cyclase activity inhibit DNA or RNA synthesis suggesting a relationship between cyclic GMP, DNA synthesis, and chemical carcinogenesis.     

Properties of guanylate cyclase in adult rat liver and several Morris hepatomas.

Guanylate cyclase (GTP pyrophyosphate-lyase (cyclizing), EC 4.6.1.2) activity was examined in preparations from normal rat liver and a series of Morris hepatomas. Homogenate gyanylate cyclase activites were 3.2, 1.6 and 1.2 nmol cyclic GMP formed per min/g tissue ihe non-substrate analogs of IMP were weak inhibitors of this enzyme, GMP and four of its analogs had Ki values ranging from 30 to 80 muM. The GMP analogs (8-azaGMP, 7-deaza-8-azaGMP, 2'-dGMP and beta-D-arabinosylGMP) and GMP were competitive inhibitors with respect to GTP.     

Activation of rat adipocyte plasma membrane adenylate cyclase by sodium azide

The adenylate cyclase of rat adipocyte plasma membrane is stimulated by sodium azide with a half maximal activation of 100–150% occuring at 50 mM NaN₃. Studies of the effects of azide and fluoride indicate different mechanisms of stimulation of the enzyme by these ions. Comparable stimulation of the activity is obtained by 100 mM NaN₃ or 10 mM NaF but unlike azide, higher concentrations of fluoride cause inhibition of the enzyme. Fluoride activated adenylate cyclase is further stimulated by azide. Epinephrine stimulation of the enzyme is absent in the presence of fluoride but the hormone enhances the activity in the presence of azide. Reversal of the inhibitory action of GTP on adenylate cyclase by epinephrine is demonstrated even in the presence of azide but not in the presence of fluoride.     

Receptor-mediated activation of spermatozoan guanylate cyclase

The sea urchin egg peptides speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Arg-Leu-NH2) bind to spermatozoa of the homologous species (Lytechinus pictus or Arbacia punctulata, respectively) and cause transient elevations of cyclic GMP concentrations (Hansbrough, J. R., and Garbers, D. L. (1981) J. Biol. Chem. 256, 1447-1452). The addition of these peptides to spermatozoan membrane preparations caused a rapid and dramatic (up to 25-fold) activation of guanylate cyclase. The peptide-induced activation of guanylate cyclase was transient, and the subsequent decline in enzyme activity coincided with conversion of a high Mr (phosphorylated) form of guanylate cyclase to a low Mr (dephosphorylated) form. When membranes were incubated at pH 8.0, the high Mr form was converted to the low Mr form without substantial changes in basal enzyme activity. However, the peptide-stimulated activity of the low Mr form of guanylate cyclase was much less than the peptide-stimulated activity of the high Mr form. Activation of the low Mr form by peptide was not transient and persisted for at least 10 min. In addition, the pH 8.0 treatment that caused the Mr conversion of guanylate cyclase also caused an increase in the peptide-binding capacity of the membranes. We propose a model in which activation of the membrane form of guanylate cyclase is receptor-mediated; the extent of enzyme activation is modulated by its phosphorylation state.     

Cation-dependent prolactin activation of guanylate cyclase

Prolactin enhanced guanylate cyclase [E.C.4.6.1.2] two- to threefold in ovary, testis, mammary gland, liver and kidney. Dose response relationships revealed that maximal activation of this enzyme was at a concentration of one nanomolar and that increasing prolactin's concentration to the millimolar range caused no further increase in activity. There was an absolute cation requirement for prolactin's enhancement of guanylate cyclase. Calcium or manganese allowed prolactin to increase guanylate cyclase activity. Greater enhancement of this enzyme's activity by prolactin was observed when manganese was the co-factor. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of prolactin.     

Receptor-mediated activation of detergent-solubilized guanylate cyclase

Here for the first time we report the successful detergent-solubilization of the speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) receptor and the subsequent activation of guanylate cyclase in response to receptor occupation. Sea urchin sperm membranes treated with a solution containing 0.5% LubrolR PX and 0.5% EmulphogeneR in the presence of MgCl2 and NaF released both the speract receptor and guanylate cyclase activity into solution. The solubilized apparent receptor was not sedimented at 400,000 x g x 15 min and was not retained by glass microfiber filters. In the presence of 125I-GGG(Y2)speract and dissuccinimidyl suberate, a major radioactive band at about Mr = 77,000 and minor bands at Mr = 35,000 and 150,000 were cross-linked. Speract but not resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-LeuNH2) competed in the cross-linking reaction. The amount of 125I-GGG(Y2)speract bound to solubilized receptor did not increase in a linear manner as a function of added protein but instead was concave upward. The addition of speract but not resact to the solubilized preparation resulted in the activation of the enzyme guanylate cyclase; the extent of stimulation was dependent on the amount of enzyme protein added and also was concave upward. Approximately 900 nM speract half-maximally activated guanylate cyclase. These data suggest that the speract receptor and guanylate cyclase are closely apposed, even in detergent, or that they are the same molecule.     

Differential stimulation of rat lung particulate guanylate cyclase activity by atrial natriuretic peptide and sodium nitroprusside

The effects of alpha-rat atrial natriuretic peptide (alpha-rANP) and sodium nitroprusside on the activity of rat lung particulate guanylate cyclase were examined. The particulate guanylate cyclase in partially purified rat lung membranes was stimulated by both alpha-rANP and nitroprusside. The effects of alpha-rANP and nitroprusside were, however, not additive. Diamide and N-ethylmaleimide almost completely abolished the nitroprusside-mediated stimulation, while they had only moderate effects on the alpha-rANP-mediated stimulation of the enzyme activity. ATP potentiated the enzyme stimulation by alpha-rANP, whereas it had no effect on the nitroprusside-mediated stimulation. These findings suggest that the stimulation of lung particulate guanylate cyclase activity by alpha-rANP and nitroprusside is mediated by different mechanisms.     

Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.