Molecular cloning and expression of cDNAs coding for soluble guanylate cyclase from rat lung.

Complementary DNA clones corresponding to the 70- and 82-kDa subunits of soluble guanylate cyclase of rat lung have been isolated. Blot hybridization of total poly(A)+ RNA from rat tissues detected mRNA of about 3.4 kilobases for the 70-kDa subunit and about 5.5 kilobases for the 82-kDa subunit. Messenger RNA levels of both subunits were abundant in lung and cerebrum, moderate in cerebellum, heart, and kidney, and low in liver and muscle, consistent with previously described enzyme activities in these tissues. Southern blot analysis of high molecular weight genomic DNA from rat liver indicated that the genes for the 70- and 82-kDa subunits are different. The carboxyl-terminal region of the 70- and 82-kDa subunits showed a high degree of homology and also had a partial homology with the putative catalytic domain of particulate guanylate cyclase and adenylate cyclase, indicating that both the 70- and 82-kDa subunits have catalytic domains. The cDNAs were subcloned to an expression vector and transfected to L cells. The cells transfected with cDNA of the 70-kDa subunit or the 82-kDa subunit showed no guanylate cyclase activity, whereas the cells transfected with both the 70- and 82-kDa subunit cDNAs showed significant guanylate cyclase activity that was activated markedly by sodium nitroprusside. These data suggest that both subunits are required for both the basal catalytic and regulatory activity of soluble guanylate cyclase. Presumably both catalytic subunits must be present and interactive to permit synthesis of cyclic GMP and nitrovasodilator activation.     

Expression of soluble guanylyl cyclase. Catalytic activity requires two enzyme subunits

Purified soluble guanylyl cyclase consists of two subunits (70 and 73 kDa) whose primary structures were recently determined. The availability of cDNA clones coding for either subunit allowed to study the question of the functional roles of the two subunits in expression experiments. Enzyme subunits were expressed in COS-7 cells by transfection with expression vectors containing the coding region for the 70 of 73 kDa subunit of the enzyme. No significant elevation in the activity of soluble guanylyl cyclase was observed in cells transfected with cDNA coding for one of the subunits. In contrast, transfection of cells with cDNAs coding for both subunits resulted in a marked increase in activity of soluble guanylyl cyclase. Enzyme activity was stimulated about 50-fold by sodium nitroprusside. The results indicate that formation of cyclic GMP by soluble guanylyl cyclase requires both 70 and 73 kDa subunits.     

Radiation inactivation target-size analysis of soluble guanylate cyclase

The soluble form of guanylate cyclase, which is a heterodimer of two subunits with molecular weights of 82,000 and 70,000, was analyzed by radiation inactivation experiments to determine its functional size. Lyophilized crude extract from rat lung or the purified enzyme were irradiated with different doses from 60Co gamma-rays, and the residual activities were measured in the presence or absence of a potent activator, sodium nitroprusside. The target sizes for the basal activity and for the activity in the presence of sodium nitroprusside were calculated from the decay curve was 77 and 192 kDa, respectively, on the crude enzyme, or as 71 and 163 kDa, respectively, on the purified enzyme. The size for the activatable form of the enzyme was more than twice that of the basal activity and close to the size of the holoenzyme, implying that the enzyme activity must reside on one of the subunits and the activation by sodium nitroprusside requires interaction of both subunits.     

17β-Estradiol inhibits soluble guanylate cyclase activity through a protein tyrosine phosphatase in PC12 cells

Besides its involvement in reproductive functions, estrogen protects against the development of cardiovascular diseases. The guanylate cyclase/cGMP system is known to exert potent effects on the regulation of blood pressure and electrolyte balance. We examined whether 17β-estradiol can affect soluble guanylate cyclase in PC12 cells. The results indicate that 17β-estradiol decreases cGMP levels in PC12 cells. 17β-Estradiol decreases sodium nitroprusside (SNP)-stimulated, but not atrial natriuretic factor-stimulated cGMP formation in PC12 cells, indicating that 17β-estradiol decreases cGMP levels by inhibiting the activity of soluble guanylate cyclase. 17β-Estradiol also stimulates protein tyrosine phosphatase activities in PC12 cells and dephosphorylates at least three proteins. Addition of sodium vanadate, a protein tyrosine phosphatase inhibitor, blocks the inhibitory effects of 17β-estradiol on soluble guanylate cyclase activity in PC12 cells. Furthermore, transfection of SHP-1, a protein tyrosine phosphatase, into PC12 cells inhibits both basal and SNP-stimulated guanylate cyclase activity. Amino acid analysis also reveals that the 70-kDa subunit of soluble guanylate cyclase contains the SHP-1 substrate consensus sequence. These results suggest that 17β-estradiol inhibits soluble guanylate cyclase activity through SHP-1.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Cytochemical demonstration of guanylate cyclase activity in cardiac muscle. Preferential localization at sarcolemma and junctional sarcoplasmic reticulum

The localization of guanylate cyclase activity was cytochemically studied in heart tissue from guinea pig and pigeon. The method, based on a lead precipitation technique with GPPNHP as the substrate, was tested by quantitative biochemical analysis. The data obtained showed that in heart homogenates GPPNHP is an acceptable substrate for guanylate cyclase. The guanylate cyclase activity of glutaraldehyde prefixed heart tissue was also measured in the presence of 2 mM lead nitrate, in 30% of the untreated control hearts. The residual guanylate cyclase responded to the addition of sodium nitroprusside with a 7-fold increase in its activity. Furthermore, the guanylate cyclase requirement for Mn2+ ions was so changed by this activator that Mg2+ was as active as Mn2+. In heart muscle cells of guinea pigs and pigeons the plasma membrane of the sarcolemma and the junctional sarcoplasmic reticulum are the precipitation sites of the reaction product. In guinea pig hearts the T-tubule membranes were likewise covered with precipitates. Sodium nitroprusside stimulation of guanylate cyclase activity was indicated by increased precipitation and by shortening of the incubation time.     

Guanylate cyclase activators hemin and sodium nitroprusside stimulate cell growth in serum-free medium

Hemin and sodium nitroprusside, which strongly activate purified rat brain guanylate cyclase in vitro, were also found to stimulate glioma C6 and neuroblastoma M1 and N1E-115 cells to divide in serum-free medium. Hemin and sodium nitroprusside each stimulate C6 cell growth to a comparable extent. Sodium nitroprusside was less potent than hemin for inducing growth of neuroblastoma cells. Moreover, both agents when added together caused a synergic cell growth enhancement which is comparable to the synergism observed in their guanylate cyclase stimulation in vitro. These results suggest that activation of guanylate cyclase may play a role in the proliferative response to these compounds.     

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.     

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.     

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.     

Molecular cloning of a cDNA coding for 70 kilodalton subunit of soluble guanylate cyclase from rat lung

A complementary DNA clone corresponding to the 70 kDa subunit of soluble guanylate cyclase (EC 4.6.1.2) of rat lung has been isolated. The primary structure of the cDNA consisted of 3063 nucleotides including a 1857-nucleotide coding region for 619 amino acids, and the calculated molecular weight was 70476. Blot hybridization of total poly(A)+RNAs from rat tissues detected a mRNA of about 3.4 kilobases. The amount of mRNA was abundant in lung, cerebrum and cerebellum, moderate in heart and kidney, and low in liver and muscle. Southern blot analysis of high molecular weight genomic DNA from rat liver indicated the presence of one gene in the rat haploid genome. The amino acid sequence of the 70 kDa subunit has partial homology with particulate guanylate cyclase from sea-urchin sperm, and protein phosphatase inhibitor I.     

Reversible activation of soluble guanylate cyclase by oxidizing agents.

Soluble guanylate cyclase of human platelets was stimulated by thiol oxidizing compounds like diamide and the reactive disulfide 4, 4'-dithiodipyridine. Activation followed a bell-shaped curve, revealing somewhat different optimum concentrations for each compound, although in both cases, higher concentrations were inhibitory. Diamide at a concentration of 100 microM transiently activated the enzyme. In the presence of moderate concentrations of diamide and 4,4'-dithiodipyridine, causing a two- to fourfold activation by themselves, the stimulatory activity of NO-releasing compounds like sodium nitroprusside was potentiated. In contrast, higher concentrations of thiol oxidizing compounds inhibited the NO-stimulated activation of soluble guanylate cyclase. Activation of guanylate cyclase was accompanied by a reduction in reduced glutathione and a concomitant formation of protein-bound glutathione (protein-SSG). Both compounds showed an activating potency as long as reduced glutathione remained, leading to inhibition of the enzyme just when all reduced glutathione was oxidized. Activation was reversible while reduced glutathione recovered and protein-SSG disappeared. We propose that diamide or reactive disulfides and other thiol oxidizing compounds inducing thiol-disulfide exchange activate soluble guanylate cyclase. In this respect partial oxidation is associated with enzyme activation, whereas massive oxidation results in loss of enzymatic activity. Physiologically, partial disulfide formation may amplify the signal toward NO as the endogenous activator of soluble guanylate cyclase.