Heme deficiency of soluble guanylate cyclase in rat platelets]

Analysis of soluble guanylate cyclase of rat platelets (105,000 g supernatant) revealed no activating effect of sodium nitroprusside on the enzyme activity. Dithiothreitol (2 x 10(-4) H) added to the sample stimulated the basal activity of guanylate cyclase in the presence of Mg2+ but did not induce the enzyme activation by sodium nitroprusside. Hemoglobin added to the enzyme did not influence its basal activity or the activating effect of sodium nitroprusside. DEAE-Cellulose chromatography of the 105,000 g supernatant revealed two protein peaks, I and II, of which only peak II possessed a guanylate cyclase activity. Fraction I added to a partly purified enzyme did not change the enzyme activity, nor did it enhance the sodium nitroprusside-induced activation of guanylate cyclase. Spectral analysis of the 105,000 g supernatant revealed that the presence of a maximum at 415-425 nm (Soret band) depended on the degree of plasma hemolysis. In the absence of hemolysis the Soret band was unobserved either in the 105,000 g supernatant or in fractions I and II. It is suggested that rat platelet guanylate cyclase is present in these cells in a heme-deficient state.     

An endogenous regulator of the guanylate cyclase activity of human platelets

Chromatography of soluble human platelet guanylate cyclase (105,000 g supernatant) on DEAE-cellulose in a linear gradient of NaCl (0-0.5 M) in 50 mM Tris-HCl buffer pH 7.6 gave two protein peaks, I and II, of which only peak II possessed the guanylate cyclase activity (0.18-0.22 M NaCl). The protein fraction I was found to possess an inhibiting activity; its addition to the partially purified enzyme decreased the guanylate cyclase activity by 60-70% in the presence of Mg2+ with no effect on the enzyme activity in the presence of Mn2+. The isolated enzyme lost (by approximately 80%) its ability to be activated by sodium nitroprusside; the latter was reconstituted after addition of the inhibiting fraction. The data obtained testify to the heme origin of the endogenous inhibitor of human platelet guanylate cyclase.     

Effect of carnosine on the activation of human platelet soluble guanylate cyclase by sodium nitroprusside and protoporphyrin IX

Effect of carnosine on the activation of soluble guanylate cyclase by sodium nitroprusside and protoporphyrin IX was studied using human platelet 105000 g supernatants and partially purified heme-deficient guanylate cyclase preparations. In experiments with 105000 g supernatants, carnosine (1 mM) inhibited the enzyme activation by nitroprusside by about 70%. With the partially purified heme-deficient guanylate cyclase, the enzyme activation by nitroprusside was lowered by 86%, and the remaining insignificant stimulatory effect remained unchanged upon carnosine addition. The stimulatory effect of protoporphyrin IX on the partially purified heme-deficient enzyme preparation did not differ from that observed with the 105000 g supernatant; carnosine addition had no effect on activation of guanylate cyclase by protoporphyrin IX. It was concluded that the inhibitory effect of carnosine on the ability of the enzyme to be activated by nitroprusside is due to the interaction of carnosine with guanylate cyclase, and that it is heme directed.     

Soluble guanylate cyclase in molecular mechanisms underlying the therapeutic action of drugs

The influence of ambroxol (a mucolytic agent) on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside (SNP) and Sin-1) were investigated. Ambroxol in the range of concentrations from 0.1 to 10 ??M had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the SNP-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC₅₀ values of 3.9 and 2.1 ??M, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin (an antimalarial agent) on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1?100 ??M) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the SNP-induced activation of human platelet guanylate cyclase with the IC₅₀ value of 5.6 ??M. Artemisinin (10 ??M) also inhibited (by 71 ± 4.0%) the activation of the enzyme by a thiol-dependent NO-donor, the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 ??M), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the signaling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin.     

Conditions of activation of guanylate cyclase from human platelets

Preincubation (50 min, 0 degree C) with nitroprusside increases 12-fold the activity of human platelet guanylate cyclase. The stimulating effect of nitroprusside is enhanced two-fold by dithiothreitol (2 mM) and by 60% by hemoglobin (20 micrograms/ml). Storage of guanylate cyclase preparations (105000 g supernatant) for 2-3 days at 4 degrees C causes a progressive increase of the enzyme activity and diminishes the stimulating effect of nitroprusside. After storage of guanylate cyclase preparations for 3 days, hemoglobin (20 micrograms/ml) augments the stimulating effect of nitroprusside by 130%. It is concluded that the degree of activation of guanylate cyclase by nitroprusside reflects the functional state of the enzyme.     

Activation of guanylate cyclase and the regulatory role of cyclic 3',5'-guanosine monophosphate

The effect of dithiothreitol on the activity of soluble guanylate cyclase and on enzyme activation by sodium nitroprusside and free stable radical was studied. A higher degree of oxidation of guanylate cyclase from rat platelets in comparison with that of the enzyme from human platelets was found, which influences both the value of the enzyme activity and its regulation. It was shown that dithiothreitol enhanced the stimulating effect of nitroprusside but inhibited the activation of guanylate cyclase by free radical, which was suggestive of a difference in the mechanisms of the activating effect of these agents. A scheme of the biological role of cyclic 3',5'-guanosine monophosphate was proposed. On the basis of this scheme, different pathological states caused by disturbances in the functions of guanylate cyclase were identified and investigated.     

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.     

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.     

Potentiation of platelet aggregation by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) has binding sites on a variety of tissues, including human platelets. We have used a new, quenched-flow approach coupled to single-particle counting to investigate the effects of ANP (rat, 1-28) on the initial events (within the first several seconds) following human platelet activation. While ANP alone (1 pM-100 nM) had no effect, ANP significantly potentiated thrombin (0.4 units/ml)-, epinephrine (15 microM)- and ADP (2 or 10 microM)-induced aggregation. Maximum stimulation occurred between 10 to 100 pM. ANP had no influence on the thrombin or ADP-induced increase in platelet volume associated with the "shape change." Since ANP receptors are coupled to a particulate guanylate cyclase and some ANP-induced effects may be mediated through cyclic GMP, we studied how another activator of platelet guanylate cyclase, sodium nitroprusside, affected platelet activation and cyclic nucleotide levels. Sodium nitroprusside (1 microM) inhibited ADP, but not thrombin or epinephrine-induced aggregation. Both sodium nitroprusside (1 microM) and ANP (10 nM) increased cyclic GMP levels by 80% and 37%, respectively, within 60 sec in washed platelets. ANP had no effect on platelet cyclic AMP, while sodium nitroprusside induced a 77% increase. These data suggest that the platelet ANP receptor may be coupled to guanylate cyclase and the rise in cyclic GMP may potentiate platelet function.     

Potentiation of NO-dependent activation of soluble guanylate cyclase by polyamines

The influence of polyamines (putrescine, spermidine, and spermine) on the activity of human platelet soluble guanylate cyclase and the stimulation of the enzyme by sodium nitroprusside (SNP), YC-1 and their combination was investigated. All these polyamines stimulated the guanylate cyclase activity and potentiated its activation by sodium nitroprusside. The stimulatory effects of sodium nitroprusside and putrescine (or spermine) were addidive; spermidine produced a synergistic activation and increased the additive effect. All the polyamines inhibited the enzyme activation by YC-1 and decreased the synergistic activation of SNP-stimulated guanylate cyclase activity by YC-1 with nearly the same potency. The ability of the investigated polyamines to potentiate and to increase synergistically (similar to to YC-1, but less effective) NO-dependent activation of soluble guanylate cyclase represents a new biochemical effect of these compounds; this effect should be taken into consideration, especially due to the endogenous nature of polyamines. The data obtained suggest, that specific biological functions of polyamines in the processes of growth and differentiation of cells may be also related to the ability of compounds to activate soluble guanylate cyclase and to increase intracellular cGMP level.     

The role of carnosine in the function of soluble of guanylate cyclase]

The effect of carnosine on activation of human platelet soluble guanylate cyclase has been studied in 105,000 g supernatants and partially purified haem-deficient enzyme preparations. In the 105,000 g supernatant carnosine (1 mM) inhibited (by about 70%) the enzyme activation caused by sodium nitroprusside. In partially purified haem-deficient guanylate cyclase preparations the inhibition of enzyme activation by sodium nitroprusside was 86%; further addition of carnosine had no effect on the enzyme activity. The strength of the activating effect of protoporphyrin IX on partially purified haem-deficient guanylate cyclase did not differ from that for the 105,000 g supernatant; this stimulating effect did not change after carnosine addition. A conclusion is drawn that the inhibiting effect of carnosine on the ability of guanylate cyclase to be activated by sodium nitroprusside is due to the dipeptide interaction with the guanylate cyclase haem.     

Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine.

Sodium nitroprusside, nitroglycerin, sodium azide and hydroxylamine increased guanylate cyclase activity in particulate and/or soluble preparations from various tissues. While sodium nitroprusside increased guanylate cyclase activity in most of the preparations examined, the effects of sodium azide, hydroxylamine and nitroglycerin were tissue specific. Nitroglycerin and hydroxylamine were also less potent. Neither the protein activator factor nor catalase which is required for sodium azide effects altered the stimulatory effect of sodium nitroprusside. In the presence of sodium azide, sodium nitroprusside or hydroxylamine, magnesium ion was as effective as manganese ion as a sole cation cofactor for guanylate cyclase. With soluble guanylate cyclase from rat liver and bovine tracheal smooth muscle the concentrations of sodium nitroprusside that gave half-maximal stimulation with Mn2+ were 0.1 mM and 0.01 mM, respectively. Effective concentrations were slightly less with Mg2+ as a sole cation cofactor. The ability of these agents to increase cyclic GMP levels in intact tissues is probably due to their effects on guanylate cyclase activity. While the precise mechanism of guanylate cyclase activation by these agents is not known, activation may be due to the formation of nitric oxide or another reactive material since nitric oxide also increased guanylate cyclase activity.     

Separation of soluble adenylate and guanylate cyclases from the mature rat testis.

The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000. Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg(2+) as well as with Mn(2+). Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase. These experiments show that adenylate and guanylate cyclase activities are physically separable.     

The separation of the heme and apoheme forms of soluble guanylate cyclase

Two forms of soluble quanylate cyclase from mammalian tissues can be separated on DEAE Sephacel or Blue Sepharose CL-6B. The two forms, referred to as peak I or peak II, migrate identically during electrophoresis on polyacrylamide gels in the presence or absence of Na-dodecyl-SO₄. Peak I is markedly stimulated by sodium nitroprusside and is the heme-containing form of guanylate cyclase. Peak II is only weakly stimulated by nitroprusside and contains no heme absorbance. In fresh tissue extracts, peak I is the predominant form, but it can be converted to peak II by treatments (pH 5.0, storage at 4°C) that result in the loss of the heme absorbance from the enzyme. Peak II is not formed from peak I by proteolysis.     

Activation of guanylate cyclase and inhibition of platelet aggregation by endothelium-derived relaxing factor released from cultured cells

The aggregation of gel-filtered rabbit platelets by 50 microM ADP was inhibited by a labile factor produced by suspensions of cultured bovine pulmonary artery endothelial cells. Inhibition of aggregation occurred when indomethacin-treated endothelial cells (6.10(5) per ml) and rabbit platelets (3.2.10(8) per ml) were incubated together. This anti-aggregatory activity was characterized as similar to endothelium-derived relaxing factor (EDRF) in that it was unstable at neutral pH and by its inhibition by hemoglobin. The activity was unaffected by treatment of the platelets and endothelial cells with the cyclooxygenase inhibitor, indomethacin, and by the lipoxygenase inhibitor, BW755c. In association with the anti-aggregatory activity, the levels of cyclic GMP were elevated 4-fold. The effect of the EDRF-like product on the levels of cyclic nucleotides was mimicked by treatment of platelets with sodium nitroprusside, an activator of soluble guanylate cyclase; sodium nitroprusside had no measurable effect on the levels of cyclic nucleotides of endothelial cells. We conclude that a factor with the properties of EDRF inhibits platelet aggregation, and that this is associated with an activation of guanylate cyclase as in smooth muscle. Thus, EDRF may exert an inhibitory effect on platelets in a manner analogous to its actions on vascular smooth muscle.     

Activation of a cytosolic ADP-ribosyltransferase by nitric oxide-generating agents

Sodium nitroprusside is a vasodilator and an inhibitor of platelet activation. It is thought that these effects are mediated by the spontaneous release of nitric oxide and stimulation of cytosolic guanylate cyclase. We have found that sodium nitroprusside (5-200 microM) greatly increased a cytosolic ADP-ribosyltransferase that ADP-ribosylates a soluble 39-kDa protein. This activity causes the mono-ADP-ribosylation of the 39-kDa protein, since digestion with snake venom phosphodiesterase releases 5'-AMP. This enzyme is present in platelets, brain, heart, intestine, liver, and lung. The effect of sodium nitroprusside is not related to stimulation of soluble guanylate cyclase and the production of cyclic GMP because cyclic GMP, dibutyryl cyclic GMP, and 8-bromo-cyclic GMP are ineffective. 3-Morpholinosydnonimine (commonly known as SIN-1) (20-1000 micrograms/ml), another compound that acts through the spontaneous formation of nitric oxide as does sodium nitroprusside, also stimulates ADP-ribosylation of the 39-kDa protein. Hemoglobin, which binds nitric oxide, inhibits sodium nitroprusside's activation of the cytosolic ADP-ribosyltransferase. These studies demonstrate a novel action of nitric oxide related to the activation of an endogenous ADP-ribosyltransferase. The physiological role of this ADP-ribosylation needs further exploration.     

Calcium-blocking effect of nitro compounds in human platelets: correlation with changes in the cyclic guanosine monophosphate level

The effects of nitrates on Ca2+ increase and cyclic nucleotide content in human platelets were studied. Nitroglycerin, isosorbide dinitrate and sodium nitroprusside were found to inhibit the intracellular Ca2+ increase induced by the platelet activating factor, ADP and a stable thromboxane A2 analog--U46619. The inhibiting effect of sodium nitroprusside manifested itself at lower concentrations than those of nitroglycerin and isosorbide dinitrate. Nitroglycerin suppressed the Mn2+ entry into the cells and caused a 2-fold increase of the cGMP content which correlates with the calcium blocking activity. Methylene blue, a guanylate cyclase and glutathione reductase inhibitor, decreased the calcium blocking effect of nitroglycerin and its influence on the cyclic nucleotide content but failed to suppress the inhibitory effect of sodium nitroprusside. The data obtained suggest that the effects of nitrates on platelets are mediated by their influence on guanylate cyclase which leads to a cyclic nucleotide content increase and to a calcium blocking effect.     

Separation of soluble adenylate and guanylate cyclases from the mature rat testis

The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000.Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg²⁺ as well as with Mn²⁺. Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase.These experiments show that adenylate and guanylate cyclase activities are physically separable.     

Inhibition of soluble guanylate cyclase activity by citrate

Soluble guanylate cyclase activity from guinea pig heart is inhibited by increasing concentrations of sodium citrate. The Ki value was found to be 2.83 +/- 0.05 mM in the presence of 3 mM Mn2+ and 0.6 mM GTP. Citrate acts by lowering Vmax and increasing the apparent values of Km for GTP and K0.5 for Mn2+ and Mg2+. The soluble guanylate cyclase, activated by sodium nitroprusside, was also inhibited by citrate. This inhibitory action of citrate was not restricted to soluble guanylate cyclase activity of the heart and has been demonstrated also in the supernatant of lung, liver, diencephalon and in the homogenate of blood platelets. Since citrate is known to be an important intermediate of metabolism, its intracellular concentration may be also of relevance for guanylate cyclase activity.     

Activation of guanylate cyclase in cerebral cortex of rat by hydroxylamine.

Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral cortex of rat. Activation was most remarkable in crude mitochondrial fraction. When the crude mitochondrial fraction was subjected to osmotic shock and fractionated, guanylate cyclase activity recovered in the subfractions as assayed with hydroxylamine was only one-third of the starting material. Recombination of the soluble and the particulate fractions, however, restored guanylate cyclase activity to the same level as that of the starting material. When varying quantities of the particulate and soluble fractions were combined, enzyme activity was proportional to the quantity of the soluble fraction. Heating of the soluble or particulate fraction at 55 degrees for 5 min inactivated guanylate cyclase. The heated particulate fraction markedly activated guanylate cyclase activity in the native soluble fraction, while the heated soluble fraction did not stimulate enzyme activity in the particulate. The particulate fraction preincubated with hydroxylamine at 37 degrees for 5 min followed by washing activated guanylate cyclase activity in the soluble fraction in the absence of hydroxylamine. Further fractionation of the crude mitochondrial fraction revealed that the factor(s) needed for the activation by hydroxylamine is associated with the mitochondria. The mitochondrial fraction of cerebral cortex activated guanylate cyclase in supernatant of brain, liver, or kidney in the presence of hydroxylamine. The mitochondrial fraction prepared from liver or kidney, in turn, activated soluble guanylate cyclase in brain. Activation of guanylate cyclase by hydroxylamine was compared with that of sodium azide. Azide activated guanylate cyclase in the synaptosomal soluble fraction, while hydroxylamine inhibited it. The particulate fraction preincubated with azide followed by washing did not stimulate guanylate cyclase activity in the absence of azide. The activation of guanylate cyclase by hydroxylamine is not due to a change in the concentration of the substrate GTP, Addition of hydroxylamine did not alter the apparent Km value of guanylate cyclase for GTP. Guanylate cyclase became less dependent on manganese in the presence of hydroxylamine. Thus the activation of guanylate cyclase by hydroxylamine is due to the change in the Vmax of the reaction.