Participation of adenosine 5'-triphosphate in the activation of membrane-bound guanylate cyclase by the atrial natriuretic factor

The addition of ANF to Percoll-purified liver plasma membranes produced a slight activation of guanylate cyclase; the ANF-stimulated cyclase activity was further increased upon the addition of ATP to the enzyme assay mixture. The effect of ATP to potentiate the cyclase activation was concentration-dependent, required Mg2+ as a divalent cation, and was seen with membranes from various tissues and cells. ATP increased the maximal velocity of the cyclase without a change in the affinity for GTP or ANF. Phosphorylation by ATP might not be involved since ANF-stimulated guanylate cyclase was enhanced by non-phosphorylating ATP analogues as well. Thus, an allosteric ATP binding site is suggested to participate in ANF-induced regulation of membrane-bound guanylate cyclase.     

Atrial natriuretic factor and sodium nitroprusside increase cyclic GMP in cultured rat lung fibroblasts by activating different forms of guanylate cyclase.

We used cultured rat lung fibroblasts to evaluate the role of particulate and soluble guanylate cyclase in the atrial natriuretic factor (ANF)-induced stimulation of cyclic GMP. ANF receptors were identified by binding of 125I-ANF to confluent cells at 37 degrees C. Specific ANF binding was rapid and saturable with increasing concentrations of ANF. The equilibrium dissociation constant (KD) was 0.66 +/- 0.077 nM and the Bmax. was 216 +/- 33 fmol bound/10(6) cells, which corresponds to 130,000 +/- 20,000 sites/cell. The molecular characteristics of ANF binding sites were examined by affinity cross-linking of 125I-ANF to intact cells with disuccinimidyl suberate. ANF specifically labelled two sites with molecular sizes of 66 and 130 kDa, which we have identified in other cultured cells. ANF and sodium nitroprusside produced a time- and concentration-dependent increase in intracellular cyclic GMP. An increase in cyclic GMP by ANF was detected at 1 nM, and at 100 nM an approx. 100-fold increase in cyclic GMP was observed. Nitroprusside stimulated cyclic GMP at 10 nM and at 1 mM a 500-600-fold increase in cyclic GMP occurred. The simultaneous addition of 100 nM-ANF and 10 microM-nitroprusside to cells resulted in cyclic GMP levels that were additive. ANF increased the activity of particulate guanylate cyclase by about 10-fold, but had no effect on soluble guanylate cyclase. In contrast, nitroprusside did not alter the activity of particulate guanylate cyclase, but increased the activity of soluble guanylate cyclase by 17-fold. These results demonstrate that rat lung fibroblasts contain ANF receptors and suggest that the ANF-induced stimulation of cyclic GMP is mediated entirely by particulate guanylate cyclase.     

Atrial Natriuretic Factors Stimulate Accumulation and Efflux of Cyclic GMP in C6–2B Rat Glioma and PC12 Rat Pheochromocytoma Cell Cultures

Atrial natriuretic factors (ANFs) were tested for their effects on cyclic GMP production in two neurally derived cell lines, the C6-2B rat glioma cells and the PC12 rat pheochromocytoma cells. These cell lines were selected because both are known to possess high amounts of the particulate form of guanylate cyclase, a proposed target of ANF in peripheral organs. Previous studies from our laboratory have shown that ANF selectively activates particulate, but not soluble, guanylate cyclase in homogenates of a variety of rat tissues and that one class of ANF receptor appears to be the same glycoprotein as particulate guanylate cyclase. In the present study we found that four analogs of ANF stimulate cyclic GMP accumulation in both C6-2B and PC12 cells with the rank order of potency being atriopeptin III = atriopeptin II greater than human atrial natriuretic polypeptide greater than atriopeptin I. Atriopeptin II (100 nM) for 20 min elevated cyclic GMP content in C6-2B cells fourfold and in PC12 cells 12-fold. Atriopeptin II (100 nM) for 20 min also stimulated the efflux of cyclic GMP from both C6-2B cells (47-fold) and PC12 cells (12-fold). Accumulation of cyclic GMP in both cells and media was enhanced by preincubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (250 microM). After 20 min of exposure to atriopeptin II, cyclic GMP amounts in the media were equal to or greater than the amounts in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proof of guanylate cyclase activity in the coronary artery of cattle]

Guanylate cyclase activities were identified in a soluble fraction and a particular fraction obtained from the Arteria coronaria of cattle. The Km-value was 1.0 +/- 0.7 - 10(-4) M for the enzyme substrate complex of the guanylate cyclase of the soluble fraction and 9.2 +/- 1.5 - 10(-4) M for the particular fraction. For the enzyme activity of the soluble fraction Mn++ cannot be replaced by Ca++ or Mg++, whereas for the enzyme activity of the particulate fraction Mn++ can be replaced by Mg++ but not by Ca++. The guanylate cyclase of the particulate fraction can be activated by acetylcholine. This activation can be cancelled by atropine. Acetylcholine exerts no influence on the guanylate cyclase activity of the soluble fraction. ATP inhibits the enzyme activities of both fractions whereas cAMP shows no influence on the guanylate cyclase activity.     

Purification and characterization of two types of atrial natriuretic factor receptors from bovine adrenal cortex: guanylate cyclase-linked and cyclase-free receptors

Atrial natriuretic factor (ANF) receptors with and without guanylate cyclase activity were simultaneously purified to apparent homogeneity from bovine adrenal zona glomerulosa cell membrane fractions. The particulate guanylate cyclase which co-purified with the ANF receptor showed one of the highest specific activity reported. The receptors with or without the guanylate cyclase activity showed high affinities to ANF (99-126). The receptor without the cyclase showed a high affinity to truncated ANF analogs, ANF (103-123) and ANF (105-121), whereas the cyclase-linked receptor had a much lower affinity to these analogs. Both of the receptors migrated as a single band with a molecular weight of 135,000 daltons on SDS-gel electrophoresis under non-reducing conditions. The 135,000 daltons band of the receptor without the cyclase was shifted to a 62,000 daltons band under reducing conditions, but the band for the cyclase-linked receptor was not shifted. These results demonstrated the presence of two subtypes of ANF receptor in bovine adrenal cortex and indicate two different modes of intracellular action of ANF.     

The involvement of catalytic site thiol groups in the activation of soluble guanylate cyclase by sodium nitroprusside

Sodium nitroprusside, a potent activator of soluble guanylate cyclase, potentiated mixed disulfide formation between cystine, a potent inhibitor of the cyclase, and enzyme purified from rat lung. Incubation of soluble guanylate cyclase with nitroprusside and [35S]cystine resulted in a twofold increase in protein-bound radioactivity compared to incubations in the absence of nitroprusside. Purified enzyme preincubated with nitroprusside and then gel filtered (activated enzyme) was activated 10- to 20-fold compared to guanylate cyclase preincubated in the absence of nitroprusside and similarly processed (nonactivated enzyme). This activation was completely reversed by subsequent incubation at 37 degrees C (activation-reversed enzyme). Incorporation of [35S]cystine into guanylate cyclase was increased twofold with activated enzyme, while no difference was observed with activation-reversed enzyme, compared to nonactivated enzyme. Cystine decreased the activity of nonactivated and activation-reversed enzyme about 40% while it completely inhibited activated guanylate cyclase. Mg+2- or Mn+2-GTP inhibited the incorporation of [35S]cystine into nonactivated or activated guanylate cyclase. Also, diamide, a potent thiol oxidant that converts juxtaposed sulfhydryls to disulfides, completely blocked incorporation of [35S]cystine into nonactivated or activated guanylate cyclase. These data indicate that activation of soluble guanylate cyclase by nitroprusside results in an increased availability of protein sulfhydryl groups for mixed disulfide formation with cystine. Protection against mixed disulfide formation with diamide or substrate suggests that these groups exist as two or more juxtaposed sulfhydryl groups at the active site or a site on the enzyme that regulates catalytic activity. Differential inhibition by mixed disulfide formation of nonactivated and activated enzyme suggests a mechanism for amplification of the on-off signal for soluble guanylate cyclase within cells.     

Selective activation of particulate guanylate cyclase by a specific class of porphyrins

Guanylate cyclase was activated 3- to 10-fold by hemin in a dose-dependent manner in membranes prepared from homogenates of rat lung, C6 rat glioma cells, or B103 rat neuroblastoma cells. Maximum activation was observed with 50 to 100 microM hemin with higher concentrations being inhibitory. Activation was observed when Mg2+-GTP but not when Mn2+-GTP was used as the substrate. Increased enzyme activity reflected selective activation of the particulate form of guanylate cyclase; hemin inhibited the soluble form of guanylate cyclase 70 to 90% over a wide range of concentrations. Activation was not secondary to proteolysis since a variety of protease inhibitors failed to alter stimulation by hemin. Protophorphyrin IX had little effect on particulate guanylate cyclase activity and sodium borohydride almost completely abolished hemin-dependent activation. These data suggest a requirement for the ferric form of the porphyrin-metal chelate for activation. However, agents which interact with the iron nucleus of porphyrins, such as cyanide, had little effect on the ability of hemin to activate guanylate cyclase. The stimulatory effects of hemin were observed in the presence of detergents such as Lubrol-PX, and highly purified particulate enzyme could be activated to the same extent as enzyme in native membranes. These data suggest that the interaction of porphyrins with particulate guanylate cyclase is complex in nature and different from that with the soluble enzyme.     

Association of the atrial natriuretic factor receptor with guanylate cyclase in solubilized rat glomerular membranes

The elution profile of solubilized rat glomerular membranes from a gel filtration column showed two peaks of 125I-ANF (atrial natriuretic factor) binding (367 +/- 21, 156 +/- 12 KDa). Over 85% of the total binding for the extract was in the 367 KDa peak. Guanylate cyclase activity was correlated with 125I-ANF specific binding. ANF activation of guanylate cyclase was also observed. As observed previously with particulate membrane, Scatchard-analysis of ANF binding data with the solubilized extract was consistent with a two-site model. Both affinities (Kd's), 4 pM and 1 nM, are within the range of blood concentrations reported for ANF. These observations suggest that most rat glomerular ANF receptors are large molecular complexes coupled with guanylate cyclase in the 300-350 KDa size range.     

Vascular atrial natriuretic factor receptor subtypes are not independently regulated by atrial peptides

The regulation of the atrial natriuretic factor (ANF) receptor system in cultured rat vascular smooth muscle cells (RVSMC) was examined following long term pretreatment of these cells with rANF99-126 or with any one of a series of truncated and ring-deleted analogs. The latter analogs are reported to bind selectively the ANF-C or clearance receptor. Initial competition binding studies revealed that all analogs examined showed comparable apparent receptor binding affinities (Ki values did not differ by more than 10-fold). In contrast, the extent of interaction of the ANF analogs with the receptor pool coupled to particulate guanylate cyclase (the ANF-B receptor) was much more variable, with some ligands failing to stimulate cGMP production or particulate guanylate cyclase over the concentrations tested. Pretreatment of cells for 24 h with rANF99-126 or any of the truncated analogs that interact with the ANF-B receptor caused a dose- and time-dependent decrease in the number of ANF binding sites (99% of which are uncoupled in RVSMC) without any change in affinity. Examination of the binding activity following pretreatment of the cells with ANF suggested that the observed reduction in 125I-rANF99-126 binding capacity was not because of the retention of the peptide on its receptor. Furthermore, this down-regulation was associated with desensitization of particulate guanylate cyclase resulting in a decreased responsiveness of intracellular cGMP accumulation to ANF. In contrast, however, analogs selective for the ANF-C receptor pool failed to cause down-regulation or desensitization. These findings suggest that ANF-C receptors in RVSMC are not independently down-regulated by selective ligands but that nonselective analogs that down-regulate and desensitize the ANF-B receptor system can by some cooperative mechanism reduce the size of the predominant ANF-C receptor pool in these cells.     

YC-1-like potentiation of NO-dependent activation of soluble guanylate cyclase by derivatives of protoporphyrin IX

The influence of protoporphyrin IX derivatives—2,4-di(1-methoxyethyl)-deuteroporphyrin IX disodium salt (dimegin) and hematoporphyrin IX (HP)—on the activation of human platelet soluble guanylate cyclase by sodium nitroprusside was investigated. Dimegin and HP, like 1-benzyl-3-(hydroxymethyl-2-furyl)indazole (YC-1), produce synergistic effects on the activation of soluble guanylate cyclase by sodium nitroprusside. The synergistic activation of the enzyme by the combination of 10 μM sodium nitroprusside and 5 μM dimegin (or 5 μM HP) was 190 ± 19 and 134 ± 10%, respectively. The synergistic activation of guanylate cyclase by 3 μM YC-1 and 10 μM sodium nitroprusside was 255 ± 19%. Dimegin and HP had no effect on the activation of guanylate cyclase by YC-1; they did not change the synergistic effect of YC-1 (3 μM) and sodium nitroprusside (10 μM) on guanylate cyclase activity. The synergistic activation of NO-stimulated guanylate cyclase activity by dimegin and HP represents a new biochemical effect of these compounds that may have important pharmacotherapeutic and physiological significance. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 3, pp. 426–431.     

Under anaerobic conditions, soluble guanylate cyclase is specifically stimulated by glutathione

Various thiols exert non-specific effects on the activity of soluble guanylate cyclase under aerobic conditions. We studied the effects of thiols under anaerobic conditions (pO2 less than 6 Torr) on soluble guanylate cyclase, purified from bovine lung. Reduced glutathione stimulated the enzyme concentration-dependently with half-maximal enzyme stimulation at a concentration of about 0.5 mM. The extend of maximal enzyme stimulation (up to 80-fold) was comparable with the activation by NO-containing substances. The activation by glutathione was additive with the effect of sodium nitroprusside. Cysteine and various other thiols increased the enzyme activity 20-fold and 2- to 5-fold, respectively. The stimulatory effect of these thiols was not related to their reducing potency. Activation of soluble guanylate cyclase by glutathione was dose-dependently reduced in the presence of other thiols (cysteine greater than oxidized glutathione greater than S-methyl glutathione). Under aerobic conditions or with Mn-GTP as substrate, the effect of glutathione on soluble guanylate cyclase was suppressed. The results suggest a specific role for glutathione in the regulation of soluble guanylate cyclase activity and a modulation of this effect by redox reactions and other intracellular thiols.     

Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues

The effects on guanylate cyclase and cyclic GMP accumulation of a synthetic peptide containing the amino acid sequence and biological activity of atrial natriuretic factor (ANF) were studied. ANF activated particulate guanylate cyclase in a concentration- and time- dependent fashion in crude membranes obtained from homogenates of rat kidney. Activation of particulate guanylate cyclase by ANF was also observed in particulate fractions from homogenates of rat aorta, testes, intestine, lung, and liver, but not from heart or brain. Soluble guanylate cyclase obtained from these tissues was not activated by ANF. Trypsin treatment of ANF prevented the activation of guanylate cyclase, while heat treatment had no effect. Accumulation of cyclic GMP in kidney minces and aorta was stimulated by ANF activation of guanylate cyclase. These data suggest a role for particulate guanylate cyclase in the molecular mechanisms underlying the physiological effects of ANF such as vascular relaxation, natriuresis, and diuresis.     

Regulation of particulate guanylate cyclase by Escherichia coli heat-stable enterotoxin: receptor binding and enzyme kinetics

1. Escherichia coli heat-stable enterotoxin (ST) induces a secretory diarrhea by binding to receptors on brush borders of intestinal villus cells, activating particulate guanylate cyclase and increasing intracellular concentrations of guanosine 3',5'-cyclic monophosphate (cyclic GMP). 2. However, little is known concerning coupling of receptor-ligand interaction to enzyme activation. 3. This study compares the kinetics of toxin-receptor binding and enzyme activation to better understand this transmembrane signal cascade. 4. Toxin receptor binding was linear and saturable with 50% of maximum displacement of [125I]ST by unlabeled toxin observed at 1.1 x 10(-7) M. ST increased the maximum velocity (Vmax) of guanylate cyclase with magnesium or manganese as the cation substrate without altering the affinity of the enzyme for its substrate or its positive cooperativity. 5. The concentration of toxin yielding half-maximum stimulation of guanylate cyclase was 1.2 x 10(-6) M, 10-fold higher than the affinity of the ligand for its receptor. 6. These data are consistent with the suggestion that ST-receptor interaction is coupled to activation of particulate guanylate cyclase. 7. However, the discrepancy between the affinity of ST for its receptor and its efficacy in activating the enzyme suggests that this coupling is complex. 8. Possible mechanisms underlying this coupling are discussed.     

Biotin analogs activate guanylate cyclase

Summary The sulfur atom in the vitamin biotin has previously been suggested to be essential in biotin's mechanism of action. In a series of investigations on structure-function relationships with biotin analogs not containing the sulfur atom, the biotin analogs, azabiotin, bisnorazabiotin, carbobiotin and isoazabiotin enhanced guanylate cyclase, an enzyme that has recently been demonstrated to be activated by biotin. These analogs increased guanylate cyclase activity two-fold in liver, cerebellum, heart, kidney and colon at 1 M concentrations. The ED₅₀ for stimulation of guanulate cyclase activity occurred at 0.1 M for each of the biotin analogs. These data indicate that the sulfur atom is not essential in biotin's activation of guanylate cyclase since these analogs do not contain the sulfur atom. Studies on the ring structure of biotin revealed that even compounds with a single 5-membered ring (2-imidazolidone) could augment guanylate cyclase activity. The guanylate cyclase co-factor manganese was not essential for the enhancement of guanylate cyclase by these agents but a maximal activation of this enzyme by these analogs could not be obtained without manganese present.     

Benzodifuroxan as an NO-dependent activator of soluble guanylate cyclase and a novel highly effective inhibitor of platelet aggregation

The ability of benzodifuroxan (BDF) to activate human platelet guanylate cyclase was investigated. The maximal stimulatory effect (1160 +/- 86%) was observed at 0.01 mM concentration. Sodium nitroprusside produced the same stimulatory effect (1220 +/- 100%) but at a higher concentration (0.1 mM). 1-H-[1,2,4,]-Oxadiazolo[4, 3-alpha]quinoxalin-1-one (ODQ), an inhibitor of NO-dependent guanylate cyclase activation, attenuated the stimulatory effect of BDF (0.01 mM) by 75% and that of sodium nitroprusside (0.1 mM) by 80%. Increasing dithiothreitol concentration in the sample from 2. 10-6 to 2.10-4 M increased the stimulatory effect of BDF 2.7-fold. The possible involvement of sulfhydryl groups of low-molecular-weight thiols and guanylate cyclase in thiol-dependent activation of the enzyme is discussed. We have also found that BDF is a highly effective inhibitor of ADP-induced human platelet aggregation with IC50 of 6.10-8 M. The effect of sodium nitroprusside was much weaker (IC50, 5.10-5 M).     

Activation of guanyl cyclase during lipid peroxidation of biomembranes

The intensity of lipid peroxidation in the microsomal membranes of rat liver influences the activity of "soluble" guanylate cyclase preparations. The increased production of lipid peroxidation products after addition of Fe(II) results in a rise the guanylate cyclase activity; alpha-tocopherol causes a decrease of this activity. An addition of fatty acids hydroperoxides at concentrations above 10(-6) M activates both the membrane-bound and "soluble" guanylate cyclase. It was shown that the hydroperoxide degradation products--carbonyl derivatives responsible for the activation, at concentrations above 10(-9) M provide for activation of the enzyme. The blocking of the SH-groups in "soluble" enzyme preparations by N-ethylmaleimide completely prevents the enzyme activation by carbonyl.     

ANF stimulation of detergent-dispersed particulate guanylate cyclase from bovine adrenal cortex

Particulate guanylate cyclase from bovine adrenal cortex can be stimulated by ANF. A 2-fold stimulation of the enzyme was obtained with 100 nM ANF and a half-maximal stimulation, with a 5 nM dose. The stimulation by ANF persisted for at least 30 min. Various detergents, such as Triton X-100, Lubrol PX, cholate, CHAPS, digitonin and zwittergent, stimulated several-fold the activity of particulate guanylate cyclase. However, only Triton X-100 dispersed particulate guanylate cyclase without affecting its response to ANF. The dose-response curve of ANF stimulation of the particulate and the Triton X-100 dispersed enzyme was similar. The dispersion of a fully responsive guanylate cyclase to ANF will help us to uncover the type of interactions between guanylate cyclase and ANF. It will also be used as a first step for the purification of an ANF-sensitive particulate guanylate cyclase.     

Characterization of high affinity receptor sites for atrial natriuretic factor in anterior pituitary gland: evidence for the existence of two receptor forms

The present study shows that in rat anterior pituitary tissue atrial natriuretic factor (ANF) binds to two distinct receptor forms, with apparent molecular weights of about 166K and 58K. Binding assays carried out with (125I)-ANF revealed specific and high affinity non-interacting binding sites, with Ko values of 1-1.7 nM and a density of 10-15,000 sites/cell. rANF fragments (5-25), (5-27) and (5-28) exhibited apparent equipotency in displacing tracer binding, while fragment (13-28) and various other peptides were ineffective. ANF (5-25) was about 100-times less potent than ANF (8-33) in stimulating half-maximum pituitary cGMP production. These data indicate the presence of multiple binding sites for ANF in the pituitary gland and suggest that only part of these sites may be coupled to activation of guanylate cyclase.     

A sensitive method for the assay of guanylate cyclase activity.

A method for the assay of guanylate cyclase is described utilizing alpha-[32P]-GTP as substrate for the enzyme reaction. 100-150 microgram of enzyme protein is incubated in a 15.6 mM Tris-HCl buffer incubation mixture, pH 7.6. The reaction is stopped by the addition of EDTA. The [32P]-cyclic GMP formed is separated by a two-step column chromatography on Dowex 50W-X4 ion-exchange resin and neutral alumina. The recovery for cyclic GMP was about 70%. The blank values ranged from 0.001-0.003% of the added alpha-[32P]-GTP which had been purified by Dowex 50W-X4 column chromatography. This method was employed for the assay of guanylate cyclase activities in different tissues.     

Ascorbate activates soluble guanylate cyclase via H2O2-metabolism by catalase

Conditions necessary for the activation by ascorbic acid of soluble guanylate cyclase purified from bovine lung have been examined. Ascorbic acid (0.1-10 mM) did not directly activate the enzyme, nonetheless, pronounced activation by ascorbate (3-10 mM) was observed in incubation mixtures containing 1 microM bovine liver catalase. Superoxide dismutase (SOD) and mannitol did not affect the catalase-dependent activation of guanylate cyclase elicited by ascorbate, suggesting that superoxide anion and hydroxyl radical were not mediating the activation of the enzyme. However, SOD enhanced the relatively low level activation of the enzyme elicited by catalase in the absence of added ascorbate. Pronounced inhibition (both with and without added ascorbate) was observed of catalase-dependent activation of guanylate cyclase by either ethanol (100 mM) or a fungal catalase preparation. Neither ethanol nor fungal catalase inhibited activation of guanylate cyclase by S-nitrosyl-N-acetyl-penicillamine (SNAP), a source of the nitric oxide free radical. These observations indicate that autoxidation of ascorbic acid or thiols present with the guanylate cyclase preparation leads to generation of H2O2, and its metabolism by bovine liver catalase mediates the concomitant activation of guanylate cyclase. The mechanism of activation appears to be associated with the presence of Compound I of catalase and to be inhibited by superoxide anion.