Inhibition of Adenylyl and Guanylyl Cyclase Isoforms by the Antiviral Drug Foscarnet*

Oliver Kudlacek, Thomas Mitterauer, Christian Nanoff, Martin Hohenegger, Wei-Jen TangDagger , Michael Freissmuth§, and Christiane Kleuss

From the Institute of Pharmacology, University of Vienna, Währinger Str. 13a, A-1090 Vienna, Austria, the Dagger  Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, Illinois 60637, and the  Institute of Pharmacology, Freie Universität Berlin, Thielallee 67-73, 14195 Berlin, Germany

Received for publication, August 29, 2000, and in revised form, October 19, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The pyrophosphate (PPi) analog foscarnet inhibits viral DNA-polymerases and is used to treat cytomegalovirus and human immunodeficiency vius infections. Nucleotide cyclases and DNA-polymerases catalyze analogous reactions, i.e. a phosphodiester bond formation, and have similar topologies in their active sites. Inhibition by foscarnet of adenylyl cyclase isoforms was therefore tested with (i) purified catalytic domains C1 and C2 of types I and VII (IC1 and VIIC1) and of type II (IIC2) and (ii) membrane-bound holoenzymes (from mammalian tissues and types I, II, and V heterologously expressed in Sf9 cell membranes). Foscarnet was more potent than PPi in suppressing forskolin-stimulated catalysis by both, IC1/IIC2 and VIIC1/IIC2. Stimulation of VIIC1/IIC2 by Galpha s relieved the inhibition by foscarnet but not that by PPi. The IC50 of foscarnet on membrane-bound adenylyl cyclases also depended on their mode of regulation. These findings predict that receptor-dependent cAMP formation is sensitive to inhibition by foscarnet in some, but not all, cells. This was verified with two cell lines; foscarnet blocked cAMP accumulation after A2A-adenosine receptor stimulation in PC12 but not in HEK-A2A cells. Foscarnet also inhibited soluble and, to a lesser extent, particulate guanylyl cylase. Thus, foscarnet interferes with the generation of cyclic nucleotides, an effect which may give rise to clinical side effects. The extent of inhibition varies with the enzyme isoform and with the regulatory input.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The second messenger cAMP controls an array of cellular responses ranging from lipid and glucose metabolism, motility and contraction, proliferation and differentiation, to synaptic transmission and memory formation. The formation of cAMP is catalyzed by the enzyme adenylyl cyclase. In mammals, there are at least 9 membrane-bound isoforms; these differ in their susceptibility to regulation by G proteins (stimulatory = Galpha s; inhibitory = Galpha i; beta gamma -dimers = dual action), Ca2+, Ca2+-liganded calmodulin, protein kinases, and the plant diterpene forskolin (1). However, all isoforms share a similar channel- or transporter-like topology: two hydrophobic domains (of ~20 kDa each) contain 6 putative transmembrane spanning alpha -helices. These are linked by a cytosolic portion (of ~40 kDa) which contains the first catalytic domain (referred to as C1, of ~30 kDa). The carboxyl terminus (also of ~40 kDa) comprises the second catalytic domain (referred to as C2, of ~30 kDa) which is internally homologous to C1. Each domain is per se enzymatically inactive, but catalysis is restored, if the two domains are combined. In addition, there is a soluble isoform, the expression of which is restricted to sperms; this enzyme is not regulated by G proteins and is more closely related to the bacterial isoforms (2).

The substrate for the reaction catalyzed by adenylyl cyclase is Mg·ATP, the reaction product is cAMP and PPi (pyrophosphate). The formation of the intramolecular phosphodiester bond (5'-PO4 linked to the ribose 3'-OH) is analogous to the reaction catalyzed by DNA-polymerases (phosphodiester bond between incoming nucleotide and DNA strand). Thus, although adenylyl cyclases and DNA-polymerases have little, if any, sequence homology, the overall topology of the active site is similar in the two classes of enzymes (3) and catalysis is thought to involve two metal ions (Mg2+ as the physiological ligand which can be substituted for by Mn2+) that are bound in the active site of both, adenylyl cyclases and DNA-polymerases (4).

The PPi analog foscarnet was originally discovered as an inhibitor of herpesvirus DNA-polymerase (5) but later also found to inhibit the reverse transcriptase of HIV, the human immunodeficiency virus (for review, see Ref. 6). Viral DNA-polymerases are more sensitive to foscarnet than their mammalian counterparts; depending on the virus strain, IC50 values in the range of ~10 to ~250 µM have been observed. Foscarnet is currently used to treat infections with cytomegalovirus, other herpesviruses, and human immunodeficiency virus. The most common side effect of foscarnet is reversible nephrotoxicity. The phosphaturia associated with foscarnet treatment has been linked to a direct inhibition of Na+/Pi symport in the renal brush border (7). The mechanism underlying the many additional side effects (in particular the neurological abnormalities) is unknown. Previous experiments rule out an effect of foscarnet on cAMP accumulation induced by parathyroid hormone in proximal renal tubules (7). In contrast, the mechanism by which foscarnet blocks the action of antidiuretic hormone is consistent with inhibition of cAMP formation (8). Because adenylyl (and guanylyl) cyclases and DNA-polymerases catalyze related reactions, it is reasonable to assume that foscarnet can, in principle, inhibit adenylyl and guanylyl cyclases. In the present work, we show that this is the case; the inhibitory potency of foscarnet depends on the nature of the isoform and varies with the state of enzyme activation.


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ABSTRACT
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Materials-- Radioactively labeled compounds were from PerkinElmer Life Sciences (Boston, MA). Guanine nucleotides and adenosine deaminase were from Roche Molecular Biochemicals (Federal Republic of Germany). Adenosine, ATP, 2',3'-dideoxyadenosine, and 2',5'-dideoxyadenosine, forskolin, RO2017241 were from Sigma, CGS21680 was from Tocris Cookson Ltd. (Bristol, United Kingdom), diethylamine/nitric oxide was from RBI (Natick, MA). Foscarnet was purchased from Mayerhofer Pharmaceuticals (Linz, Austria). The materials required for protein purification (9), cell culture, and transfection (10), expression of adenylyl cyclase and guanylyl cyclase in Sf9 cells (11) have been described.

Protein Purification and Membrane Preparations-- The catalytic domains of adenylyl cyclase were expressed in Escherichia coli BL21 and purified from bacterial lysates using metal affinity chromatography, anion exchange chromatography on MonoQ, and gel filtration on Superose HR12 (9). Similarly, recombinant Galpha s (12) and myristoylated rGalpha i-1 (13) were purified from bacterial lysates, G protein beta gamma -dimers from porcine brain membranes (14). Membranes were prepared from the following sources: guinea pig cerebral cortex (15), calf myocardium (16), and human platelets (17). Sf9 cells were infected with recombinant baculoviruses encoding adenylyl and guanylyl cyclases, lysed, and fractionated into membranes and cytosol as described previously (11).

Cell Culture and cAMP Formation-- CHO-K1 cells were grown in Ham's F-12 nutrient mixture supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin G, and 100 µg/ml streptomycin; cells were transiently transfected by electroporation with the plasmid pSVL encoding the particulate guanylyl cyclase-A/atrial natriuretic factor receptor (18) and harvested 55 h later. The generation and propagation of stably transfected HEK293 cells that express the A2A-adenosine receptor (HEK-A2A) has been described (10). PC12 cells were propagated in Opti-MEM medium containing 10% horse serum, 5% fetal calf serum, L-glutamine, penicillin G, and streptomycin. The adenine nucleotide pool was metabolically labeled by incubating confluent monolayers for 16 h with [3H]adenine (1 µCi/well); if this preincubation period was varied between 12 and 24 h, there was no appreciable difference in the amount of [3H]cAMP formed in response to the receptor agonist or forskolin; hence, the adenine pool was assumed to be labeled to equilibrium. After the preincubation, fresh medium was added that contained adenosine deaminase (1 unit/ml), 100 µM RO201724, and the indicated foscarnet concentrations; after 30 min, cAMP formation was stimulated by the A2A-selective agonist CGS21680 or 25 µM forskolin for 15 min. Assays were performed in triplicate. The formation of [3H]cAMP was determined according to Salomon (19).

Enyzme Assays-- For measuring the generation of cAMP by the purified catalytic domains, IC1 or VIIC1 (each at 20-60 ng/assay) were combined with IIC2 (0.02-2 µg/assay) and incubated for 5 min at 20 °C in a final volume of 50 µl containing 50 mM Hepes/NaOH (pH 7.5), [alpha -32P]ATP (specific activity 10-100 cpm/pmol), 2.5 mM MgCl2, 1 mM MgSO4 (carry over from the preactivation of rGalpha s or of rGalpha i-1), 0.01% Lubrol; unless indicated otherwise in the figure legends, the concentrations of [alpha -32P]ATP-Mg and forskolin were 0.5 and 0.1 mM, respectively. The concentration of foscarnet and PPi was varied between 0.01 and 10 mM; prior to dilution, equimolar MgCl2 was added to the stock solution of foscarnet and PPi to keep the free Mg2+ concentration constant. Where applicable, rGalpha s and rGalpha i-1 (each at 20 µM) were preactivated at 30 °C for 30 and 120 min, respectively, in buffer containing 50 mM Hepes/NaOH (pH 7.5), 1 mM EDTA, 10 mM MgSO4, 100 µM GTPgamma S, and 0.01% Lubrol; free GTPgamma S and GDP (released from the proteins) was removed by gel filtration over Sephadex G-50 pre-equlibrated in 50 mM Hepes/NaOH (pH 7.5), 1 mM MgSO4, and 0.01% Lubrol (20). Both, the catalytic domains of adenylyl cyclase and the G protein alpha -subunits, are soluble in the absence of detergent. However, Lubrol prevents adsorptive losses that occur at low concentrations of Galpha subunits (20). The presence of Lubrol had no effect on the activity of C1/C2 heterodimers. The activity of membrane-bound adenylyl cyclase was assayed in a similar manner with the following modifications: the incubation time was 20 min, the assay mixture contained 10 mM MgCl2, 0.1 mM RO201724, 10 mM creatine phosphate, 1 mg/ml creatine kinase. The formation of [32P]cAMP was quantified according to Johnson and Salomon (21).

The source of soluble guanylyl cyclase was the cleared lysate of SF9 cells that had been co-infected with baculoviruses encoding the alpha 1 and beta 1 subunit (22). Guanylyl cylase assays were performed as described (23). Briefly, soluble guanylyl cyclase (2-4 µg of cytosolic protein) was incubated for 20 min at 37 °C in a final volume of 100 µl containing 50 mM triethanolamine-HCl (pH 7.4), 0.1 mM 3-isobutyl-1-methylxanthine, 0.5 mM GTP, 1 mM cGMP, 0.5 mM EDTA, 0.1 mg/ml bovine serum albumin, 5 mM creatine phosphate, 0.25 mg/ml creatine kinase, and where indicated, 100 µM diethylamine/nitric oxide. Activity of particulate guanylyl cyclase was measured in the absence and presence of 100 nM atrial natriuretic factor on membranes (30-40 µg/assay) from CHO-K1 cells transiently expressing GC-A (19).

If not otherwise indicated, experiments were done at least three times. Data were subjected to nonlinear least-squares curve-fitting using the appropriate equations (rectangular hyperbola, Hill equation) to obtain parameter estimates.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Comparison of IC1/IIC2 and VIIC1/IIC2 Heterodimers-- The stimulatory effect of forskolin and Galpha s differs in individual adenylyl cyclase isoforms (1, 24). To rule out that these differences may distort the results obtained in subsequent experiments, we have defined the conditions under which IC1 and VIIC1 were saturated with IIC2 and activators (Fig. 1). We observed a (slightly) higher potency of forskolin in stimulating catalysis by the dimer IC1/IIC2 than that by VIIC1/IIC2 (Fig. 1C; EC50 = 8.0 ± 0.9 and 25.8 ± 6.0 µM, respectively; n = 3). Conversely, the apparent affinity of preactivated Galpha s was modestly higher for VIIC1/IIC2 than for IC1/IIC2 (Fig. 1E; EC50 = 72 ± 28 and 245 ± 87 nM, respectively; n = 3). Accordingly, the apparent affinity of each C1 domain for IIC2 varied with the activating agent employed: for IC1 (Fig. 1A), the EC50 of IIC2 was 227 ± 109, 276 ± 24, and 153 ± 37 nM in the presence of forskolin, Galpha s, and the combination thereof, respectively. For VIIC1 (Fig. 1B) the EC50 of IIC2 was 510 ± 149, 39 ± 2, and 17 ± 7 nM in the presence of forskolin, Galpha s and the combination thereof, respectively. The combination of forskolin and Galpha s resulted in overadditive stimulation regardless of the C1-subtype (Fig. 1, D and F). In summary, these experiments indicated (modest) differences in the affinity of IC1 and VIIC1 for IIC2 which depended on the activator; thus, enzymatic activity was subsequently assessed under conditions where the C1 domain was limiting (~30 ng/assay), while IIC2 (~2 µg/assay), Galpha s (2 µM), and forskolin (100 µM) were saturating.



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Fig. 1.   Complementation of adenylyl cyclase activity of IC1 and VIIC1 by IIC2 in the presence of the activators forskolin, rGalpha s, and the combination thereof. A and B, increasing amounts of IIC2 were added to IC1 (A) or VIIC1 (B) (each at 30 ng/assay); catalysis was activated by 100 µM forskolin (), 2 µM GTPgamma S-liganded rGalpha s (open circle ), or a combination of forskolin and rGalpha s (black-down-triangle ). Panels C-F, the amount of IIC2 (2 µg/assay) and IC1 (; 20-30 ng/assay) or VIIC1 (open circle ; 30 ng/assay) was kept constant and the concentration of forskolin (C and D) and preactivated rGalpha s (E and F) was varied. In D and F, preactivated rGalpha s and forskolin were held constant at 0.3 and 30 µM, respectively. Data are means of duplicate determinations; each comparison of IC1 and VIIC1 was done in parallel. The experiment is representative for two additional experiments.

Foscarnet Inhibits cAMP Formation Catalyzed by IC1/IIC2 and VIIC1/IIC2 via Interaction with the PPi-binding Site-- Adenylyl cyclases are subject to product inhibition by both, cAMP and PPi. Product release is random and, at least in part, rate-limiting (25). In the forward reaction, i.e. the formation of cAMP + PPi from ATP, the product PPi is not a competitive inhibitor of adenylyl cyclase with respect to the substrate ATP. Depending on the assay conditions, the inhibition is noncompetitive or mixed competitive (25). If foscarnet acted as a PPi analog, the two compounds should inhibit the reaction in a similar way. This was the case. For both, IC1/IIC2 (Fig. 2A) and VIIC1/IIC2 (Fig. 2B), the Vmax of the forskolin stimulated activity was suppressed by raising the concentration of foscarnet and PPi. Similarly, the Km for ATP increased with higher concentrations of foscarnet and PPi; this is more readily seen from the replots shown in Fig. 2, C and D.



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Fig. 2.   Kinetic analysis of the inhibition by foscarnet and PPi. IC1 (30 ng/assay; A) or VIIC1 (60 ng/assay; B) were combined with IIC2 (2 µg/assay); catalysis was activated by 100 µM forskolin and the reaction was carried out in the absence () and presence of 50 (open circle ) and 250 µM Mg·foscarnet (black-down-triangle ) or 0.3 (down-triangle) and 1.5 mM Mg·PPi (black-square). Data are means of duplicate determinations in a representative experiment carried out in parallel. The Km for ATP calculated from three such experiments IC1/IIC2 (open circle ) and VIIC1/IIC2 () was plotted as a function of the concentration of foscarnet (C) and PPi (D); error bars indicate S.E.

The product inhibition imposed by (nonphysiological concentrations of) cAMP is mimicked by adenosine and some analogs (3'-phosphorylated and deoxyadenosine analogs, see Refs. 25 and 26), which are referred to as P-site inhibitors. When combined, PPi and P-site ligands inhibit adenylyl cyclase synergistically and this is thought to reflect the formation of a dead-end complex (25, 27). If foscarnet inhibited adenylyl cyclase via interaction with the binding site for PPi, the combination of foscarnet and a P-site ligand also should act in a synergistic manner; in contrast, the combination of PPi and foscarnet is expected to result in mutual antagonism. Dixon plots (where the reciprocal of enzymatic velocity is plotted as a function of one inhibitor at several fixed concentrations of the second inhibitor) allow to test if two inhibitors can occupy an enzyme simultaneously or whether their binding is mutually exclusive (28). If foscarnet was combined with fixed concentrations of 2',3'-dideoxyadenosine, the slope of the individual regression lines depended on the concentration of this second inhibitor for both, IC1/IIC2 (Fig. 3A) and VIIC1/IIC2 (Fig. 3B); this observation shows that the two compounds can be bound simultaneously (28). As expected, this was also seen for the combination of 2',3'-dideoxyadenosine and PPi (Fig. 3, C and D). In all cases, the lines intersected above the x axis indicating that 2',3'-dideoxyadenosine facilitated inhibition by foscarnet (or by PPi). A similar synergism was observed with adenosine and 2',5'-dideoxyadenosine (data not shown). In contrast with both, IC1/IIC2 (Fig. 4A) and VIIC1/IIC2 (Fig. 4B), Dixon plots for the combination of PPi and foscarnet yielded a family of parallel regression lines. This is the diagnostic feature indicative of mutually exclusive binding (28). Thus, the presence of a fixed concentration of foscarnet impeded the inhibitory action of PPi. Taken together, the data are consistent with the interpretation that foscarnet and PPi occupy the same site in the catalytic core of adenylyl cyclases.



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Fig. 3.   Inhibition of forskolin-activated adenylyl cyclase activity by the combination of foscarnet (A and B) or PPI (C and D) with 2',3'-dideoxyadenosine (ddA). IC1 (A and C, 20 ng/assay) or VIIC1 (B and D, 40 ng/assay) were combined with IIC2 (2 µg/assay); catalysis was activated by 100 µM forskolin. The reaction was carried out with the indicated concentrations of Mg·foscarnet (A and B) or of Mg·PPi (C and D) in the absence () and presence of 10 µM (open circle ), 100 µM (black-down-triangle ), or 1 mM (down-triangle) 2',3'-dideoxyadenosine (2',3'-ddA). Data are means of duplicate determinations in a representative experiment carried out in parallel.



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Fig. 4.   Mutually exclusive inhibition of adenylyl cyclase activity by foscarnet and PPI. IC1 (A, 50 ng/assay) or VIIC1 (B, 60 ng/assay) were combined with IIC2 (2 µg/assay); catalysis was activated by 100 µM forskolin. The reaction was carried out with the indicated concentrations of Mg·PPi in the absence () and presence of 50 µM (open circle ), 150 µM (black-down-triangle ), or 500 µM (down-triangle) Mg·foscarnet. Data are means of duplicate determinations in a representative experiment.

Inhibition of cGMP Formation by Foscarnet and PPi-- The other cyclic nucleotide second messenger in cells, cGMP, is generated by the isoforms of guanylyl cyclase. Although not investigated in detail, the catalytic mechanism of guanylyl cyclase is generally thought to resemble that of adenylyl cyclase. It is evident from Fig. 5A that both, PPi and foscarnet, inhibit basal soluble guanylyl cyclase; the difference in potency between PPi and foscarnet was very modest (IC50 = 0.35 ± 0.06 and 0.52 ± 0.09 mM for PPi and foscarnet, respectively). Addition of the NO-donor diethylamine/nitric oxide (0.1 mM) stimulated catalysis 8.2 ± 1.8-fold; the IC50 of PPi (0.40 ± 0.06 mM) was not affected, but the potency of foscarnet was somewhat lower (IC50 = 0.90 ± 0.17 mM) in the presence of the NO-donor (not shown). In contrast, cGMP formation catalyzed by atrial natriuretic peptide-stimulated particulate guanylyl cyclase was resistant to inhibition by PPi; the enzyme was only inhibited by foscarnet (Fig. 5B), albeit with lower potency (IC50 = 3.5 ± 0.3 mM) than the soluble isoform.



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Fig. 5.   Effect of foscarnet and of PPi on soluble (A) and particulate guanylyl cyclase (B). A, cytosol (2.5 µg/assay) of Sf9 cells that expressed the alpha 1/beta 1-dimer was incubated in the absence and presence of the indicated concentrations of Mg·foscarnet () and Mg·PPi (open circle ). B, enzyme activity was determined with membranes (30 µg/assay) of CHO-K1 cells that transiently expressed particulate guanylyl cyclase-A in the absence and presence of the indicated concentrations of Mg·foscarnet (open circle ) and Mg·PPi (). Catalysis was activated by 100 nM atrial natriuretic peptide; basal activity was 42 pmol/20 min (=70 pmol mg-1 min-1) Data are means of duplicate determinations in a single experiment which was reproduced twice.

Galpha s Affects the Potency of Foscarnet-- Foscarnet was ~5-6-fold more potent than PPi in inhibiting the basal as well as the forskolin-stimulated activity of IC1/IIC2 and VIIC1/IIC2 (Table I). However, the physiological activator of adenylyl cyclase is Galpha s; we have therefore also determined the inhibitory potency of foscarnet and PPi on the activity stimulated by GTPgamma S-liganded Galpha s, forskolin, and their combination. Preactivated Galpha s was employed at a saturating concentration to eliminate the difference in affinity of IC1/IIC2 and VIIC1/IIC2 (see Fig. 1). The inhibition by foscarnet was blunted in the presence of rGalpha s while the IC50 of PPi decreased. This effect was more pronounced for the heterodimer VIIC1/IIC2 than for IC1/IIC2 (Table I). We rule out that the difference between the IC50 can simply be accounted for by the higher activity that is achieved through stimulation by Galpha s. If catalysis was further activated by the combination of forskolin and Galpha s, foscarnet suppressed cAMP formation with an intermediate potency (Table I); the IC50 values were lower than those observed in the presence of Galpha s but higher than those seen with forskolin.


                              
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Table I
Inhibition by foscarnet and by pyrophosphate (PPi) of cAMP formation catalyzed by IC1/IIC2- and VIIC1/IIC2-heterodimers
IC1 or VIIC1 (each at 20-60 ng/assay) were combined with IIC2 (2 µg/assay); activity was determined in the absence (basal) and presence of the indicated concentrations of activators under assay conditions described in the legend to Fig. 1. Enzymatic activity is expressed as cAMP formation/mg of IC1 or of VIIC1; 6 logarithmically spaced concentrations of foscarnet (10 µM to 3 mM) or PPi (30 µM to 10 mM) were used. Data are mean ± S.D. from three experiments.

Inhibition of Membrane-bound Adenylyl Cyclase Isoforms-- Because the C1/C2-dimers employed are artificial, we have also analyzed the effect of foscarnet on adenylyl cyclase in membranes prepared from guinea pig cerebral cortex, calf ventricular myocardium, and human platelets. These preparations are likely to contain a mixture of several isoforms due to the cellular heterogeneity and due to the fact that many cells express more than one isoform. However, brain membranes are enriched in the type I isoform (1). Platelets contain an isoform that is activated by beta gamma -dimers in the presence of activated Galpha s (14). This is a characteristic feature of the type II and type IV isoforms (1, 29). The myocardium expresses predominantly the type V (and to lesser extent the type VI) isoform (1, 30). Adenylyl cyclase in these membrane preparations was assayed under four different conditions. (i) The enzyme was directly stimulated by forskolin. Since the stimulation is greatly augmented by the presence of Galpha s (1), in membranes forskolin-stimulated catalysis reflects the sum of the direct action of the compound and the basal level of Galpha s activation. (ii) GTPgamma S was added to the reaction; this results in activation of both Gi (and Go in brain membranes) and Gs; thus the activity reflects the combined effect of inhibition and stimulation. (iii) Purified Galpha s was preactivated by incubation with GTPgamma S and MgSO4, unbound GTPgamma S was removed by gel filtration and GTPgamma S-liganded Galpha s was added to the membranes. (iv) Basal activity was assessed without any exogenous activator. It has to be pointed out, though, that this basal activity does not only reflect the intrinsic rate of catalysis of the enzyme, but also the sum of stimulatory and inhibitory input occurring at low level. Trace amounts of GDP are present in the membrane (e.g. bound to monomeric and heterotrimeric G proteins); commercial ATP preparations are contaminated by low levels of GTP (15). In the assay, GTP is also formed by transphosphorylation of GDP (31).

As can be seen from Table II, the apparent potency of foscarnet depends on the nature of activating ligand. In general, activation of GTPgamma S-liganded Galpha s reduced the sensitivity of the enzyme to foscarnet. This effect was most pronounced in cardiac membranes. In addition, the IC50 values varied which presumably reflected the expression of different adenylyl cyclase isoforms. Basal activity, for instance, was most readily inhibited in brain membranes. In cardiac membranes, the enzyme was most susceptible to foscarnet in the presence of GTPgamma S and there was a striking left-shift of the curve when compared with the activity in the presence of GTPgamma S-liganded Galpha s. This was also seen, albeit to a lesser extent, in cerebrocortical membranes. As mentioned above, addition of GTPgamma S activates both, Gi and Gs endogenous to the membranes. Hence, it appears likely that the presence of inhibitory subunits (Galpha i and/or Gbeta gamma -dimers) renders the enzyme more sensitive to foscarnet. Regardless of the underlying mechanism, it is safe to conclude that the potency of foscarnet is affected by the activation state of the individual isoform.


                              
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Table II
Inhibition by foscarnet of adenylyl cyclase activity in membranes prepared from heart, brain cortex and platelets
Membranes (10-20 µg) were incubated in the absence of any exogenous activator (basal) and in the presence of the indicated activators; the carry-over of detergent and MgSO4 was corrected by adding appropriate amounts to the other reactions; 6 logarithmically spaced concentrations of foscarnet (10 µM to 3 mM) were used. The data represent mean ± S.D. of three separate experiments that were done in duplicate and conducted in parallel.

A comparison of Tables I and II shows that foscarnet inhibited the purified catalytic domains more potently than the membrane-bound holoenzymes. This discrepancy may result from the fact that the heterodimers represent nonphysiological forms of the enzyme. Alternatively, domains of the holoenzyme that are not part of the catalytic core may modify access of foscarnet to the PPi-binding sites. To differentiate between these two possibilities, we have used the adenylyl cyclase isoforms type I and type II. These were expressed in Sf9 cells either as intact holoenzymes or as half-molecules (12). In the latter case, the site of truncation is within the region preceding the second hydrophobic domain (positions 571 and 556 in type I and type II, respectively). Thus, IM1C1 comprises the entire amino-terminal half of type I (including the first transmembrane domain M1) and IIM2C2 the entire COOH-terminal half of type II (including the second transmembrane domain M2). Sf9 cells were simultaneously infected with baculoviruses encoding IM1C1 and IIM2C2. The control consisted of cells infected with viruses encoding either type I or type II holoenzyme. In addition, the type V isoform was also investigated because it is the predominant enzyme in the myocardium (1, 30). Membranes prepared from these cells were used to evaluate the effects of foscarnet and PPi on forskolin-stimulated catalysis (Fig. 6). Foscarnet inhibited holoenzymes type I and type II with comparable potency (Fig. 6A). Similarly, the variation in the IC50 values of PPi was trivial (Fig. 6B). Importantly, the combination of IM1C1 and IIM2C2 yielded an enzymatic activity that was inhibited over the same concentration range as the holoenzymes. Finally, the potency of foscarnet (IC50 = 0.4 ± 0.1 mM) and PPi (IC50 = 1.3 ± 0.2 mM) was lower than on the catalytic core IC1/IIC2 (see Table I).



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Fig. 6.   Inhibition by foscarnet of membrane-bound adenylyl cyclase isoforms expressed in Sf9 cells. Membranes (2-4 µg) prepared from baculovirus-infected Sf9 cells expressing the type I (ACI, ), the type II (ACII, open circle ) and isoform the type V (ACV, down-triangle) as well as the heterodimer (IM1C1/IIM2C2, black-down-triangle ) were incubated in the presence of 100 µM forskolin and the indicated concentrations of Mg·foscarnet (A) and Mg·PPi (B). Data are means of duplicate determinations in an experiment that is representative of two additional experiments. The data were normalized to account for the different levels of activities by setting the respective activity in the absence of foscarnet 100%; the specific activities were 4.6 ± 0.3, 2.5 ± 0.2, 13.5 ± 1.4, and 3.4 ± 0.2 nmol min-1 mg-1 for CI, CII, VC, and IC1M1/IIC2M, respectively.

In both, myocardial and brain membranes, cAMP formation that was stimulated by the addition of GTPgamma S was more susceptible to inhibition by foscarnet than catalysis activated by GTPgamma S-liganded Galpha s (Table II). This discrepancy can be rationalized if the GTPgamma S-induced increase in inhibitory subunits (GTPgamma S-liganded Galpha i and Galpha o and beta gamma -dimers) is assumed to sensitize the catalyst to foscarnet. To test this conjecture, we have mimicked this situation by adding GTPgamma S-liganded Galpha i-1 and free beta gamma -dimers to Sf9 membranes expressing the type I and type V isoforms in the presence of preactivated Galpha s (Fig. 7). Sole addition of Galpha i-1 caused a modest inhibition of the Galpha s-activated type I enzyme which was substantially augmented by the presence of free beta gamma -dimers (Fig. 7A). Importantly, addition of Gbeta gamma shifted the concentration-response curve of foscarnet to the left; this is most readily seen from the inset in Fig. 7A, where the data were normalized (IC50 = 0.9 ± 0.1, 1.0 ± 0.1, and 0.3 ± 0.1 mM in the presence of rGalpha s, rGalpha s + rGalpha i-1 and rGalpha s + rGalpha i-1 + beta gamma , respectively). In contrast, adenylyl cyclase type V was only inhibited by Galpha i-1; Gbeta gamma had no additional effect (Fig. 7B). Furthermore, the presence of Galpha i-1 and Gbeta gamma did not affect the potency of foscarnet; the inset of Fig. 7B shows that the three concentration-response curves were superimposable (IC50 = 1.2 ± 0.1, 1.3 ± 0.1, and 1.3 ± 0.2 mM in the presence of rGalpha s, rGalpha s + rGalpha i-1 and rGalpha s + rGalpha i-1 + beta gamma , respectively). Hence, the type V isoform did not adequately reproduce the properties of the catalyst(s) present in cardiac membranes. However, the results with the type I isoform clearly showed that an inhibitor was capable of rendering the enzyme more susceptible to foscarnet.



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Fig. 7.   Effect of foscarnet on membrane-bound adenylyl cyclase isoforms expressed in Sf9 cells. Sf9 cell membranes (each at 3 µg/assay) containing the type I (IC, panel A) and the type V (panel B, VC) isoform were incubated with 0.2 µM GTPgamma S-liganded rGalpha s in the absence () or presence of preactivated 2 µM rGalpha i-1 (open circle ) or the combination of preactivated rGalpha i-1 and 1 µM beta gamma -dimers (black-down-triangle ). Data are means of duplicate determinations in a representative experiment. Insets, to account for differences in activities, the data in panels A and B were normalized by setting the activity in the absence of 100% foscarnet.

Effect of Foscarnet in Intact Cells-- Taken together, the observations with the isolated catalytic domains and the membrane-bound enzymes suggested that the inhibitory potency of foscarnet depended on the mode of regulation of individual adenylyl cyclase isoforms. If this interpretation were correct, the susceptibility to foscarnet of receptor-dependent cAMP accumulation ought to vary in intact cells. This prediction was verified by testing foscarnet at concentrations encompassing the therapeutic range on two cell lines that express the same receptor, namely PC12 cells (which endogenously express the A2A-adenosine receptor) and HEK-A2A cells (HEK293 cells in which the receptor was introduced by stable transfection, Ref. 11). The A2A-selective agonist CGS 21680 stimulated cAMP accumulation in cells with a maximum effect that was comparable to the response elicited by forskolin in each cell line (Fig. 8A); the similar potency of CGS21680 (EC50 = 23 ± 9 and 24 ± 6 nM in HEK-A2A and PC12 cells, respectively) indicates that the A2A-receptor is efficiently coupled to Galpha s in both PC12 and HEK-A2A. However, the action of foscarnet was clearly distinct. In PC12 cells, forskolin- and A2A-agonist-dependent cAMP formation were suppressed by foscarnet over a similar concentration range (Fig. 8B). In contrast, in HEK-A2A cells, the A2A-agonist-dependent stimulation was resistant to foscarnet, while the response to forskolin was blunted (Fig. 8C). Thus, after receptor-dependent activation of Galpha s, adenylyl cyclase in HEK-A2A cells was no longer susceptible to inhibition by foscarnet.



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Fig. 8.   A2A-adenosine receptor and forskolin-dependent cAMP accumulation in PC12 and HEK-A2A cells. A, the adenine nucleotide pool of PC12 (open circle ) and HEK-A2A cells () was metabolically labeled by incubation with [3H]adenine and cAMP production was stimulated by the indicated concentrations of the A2A-selective agonist CGS21680 or by 25 µM forskolin: the response to forskolin in each cell line is shown on the left. Data are means of triplicate determinations in an experiment that was reproduced twice. B and C, PC12 (B) and HEK-A2A cells (C) were preincubated for 30 min in the absence or presence of the indicated concentrations of foscarnet; cAMP production was subsequently activated by 25 µM forskolin () or by 1 µM CGS21680 (open circle ). Data are mean ± S.D. of three separate experiments carried out in triplicate. Data were normalized by setting the cAMP levels in the absence of 100% foscarnet.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The observations demonstrate that foscarnet directly inhibits adenylyl cyclase isoforms; several lines of evidence indicate that foscarnet binds to the PPi-binding site. (i) Similar to PPi, foscarnet caused mixed-competitive inhibition of the forward reaction; (ii) foscarnet substituted for PPi in synergizing with P-site ligands; (iii) adenylyl cyclase could only be inhibited by either foscarnet or PPi indicating that they bound to the catalytic core in a mutually exclusive manner. The PPi-binding site is formed by a loop in the C1 domain (3, 4). We have observed that foscarnet (and PPi) prevented binding of the fluorescent ATP analog TNP-ATP (9) to VIIC1 but not to IIC2.2 Taken together, our findings are consistent with the notion that foscarnet and PPi bind to the same site. In addition, the data suggest that amino acid residues, which are not part of the catalytic core, impinge on the PPi-binding site to regulate catalysis. We observed that the soluble heterodimers formed by the C1 and C2 domains were more susceptible to inhibition by foscarnet and by PPi than were the holoenzymes (i.e. the molecules comprising the catalytic core, the additional cytoplasmic stretches and the transmembrane domain); this difference was seen regardless of whether intact holoenzymes or the artificial holoenzyme IM1C1/IIM2C2 were employed. Several lines of arguments suggest that the stretch that links the first catalytic C1 domain to the second transmembrane portion participates in the regulation of catalysis (32). This region, for instance, is required for activation of the type I isoform by calmodulin (33, 34) and is thought to contain the inhibitory Ca2+ site of the type V (and VI) isoforms (35). Foscarnet also inhibited soluble guanylyl cyclase and, to a lesser extent, particulate guanylyl cyclase-A. This is to be anticipated. Adenylyl cyclases and guanylyl cyclases catalyze the same reaction; accordingly, the substrate specificity can be switched by exchanging appropriate residues between adenylyl cyclase and soluble (36) or membrane-bound guanylyl cyclases (37).

Previous studies drew opposite conclusions, namely that foscarnet did (9) or did not (8) inhibit receptor-dependent cAMP production; our experiments resolve this controversy. We observed that Galpha s relieved the inhibition of adenylyl cyclase by foscarnet. The effect of Galpha s on the inhibitory potency of foscarnet, however, varied with individual isoforms; it was, for instance, more pronounced with the VIIC1/IIC2 than with the IC1/IIC2 heterodimer. In PC12 and HEK-A2A cells, the coupling efficiency of the signaling cascade A2A-adenosine receptor/Gs/adenylyl cyclase was similar as reflected by the virtually identical EC50 for the agonist. Nevertheless, foscarnet discriminated between the receptor (and hence Galpha s-)-dependent cAMP formation in PC12 and HEK-A2A cells. We thus conclude that the potency of foscarnet in intact cells depends on the cellular complement of adenylyl cyclase isoforms. In addition, an inhibitory input renders some isoforms more susceptible to the action of foscarnet. Gbeta gamma -Dimers (but not by Gialpha -1) enhanced the potency of foscarnet on adenylyl cyclase type I. In contrast, the experiments with the type V isoform failed to reproduce the sensitization that occurred in cardiac membranes upon activation of endogenous G proteins by GTPgamma S. The reason for this discrepancy is not clear. Suppression of cAMP accumulation was observed, if cells were co-transfected with plasmids encoding adenylyl cyclase type V and Gbeta gamma (38). However, previous reconstitution experiments also did not detect an inhibitory action of beta gamma -dimers on adenylyl cyclase type V (and VI) (29). The reconstitution assay may fail to restore the correct interaction between beta gamma and the type V enzyme and this may account for our inability to recapitulate the sensitization to foscarnet that was seen in cardiac membranes. The alternative explanation is the cellular heterogeneity of the myocardium. A large proportion of cardiac membranes is actually derived from the endothelium (39); hence, the presence of ubiquitously expressed isoforms such as type VII (30) may give rise to the distinct findings when cardiac membranes are compared with the type V isoform.

It has recently been appreciated that inhibition of adenylyl cyclase may account for side effects of antiviral and cytostatic adenosine analogs, which are converted to acyclic adenine nucleoside phosphonates (40). Contrary to these experimental drugs, foscarnet is widely used in man. Foscarnet permeates into cells, the volume of distribution is in the range of 0.5 liters/kg (7) which is indicative of distribution in the total body water. Hence, plasma concentrations (in the range of 0.25 to 0.5 mM) presumably reflect the intracellular levels. The experiments that were carried out in intact cells show that adenylyl cyclase inhibition can occur within the therapeutic concentration range. Thus, our findings indicate that some clinical side effects of foscarnet may be linked to inhibition of cAMP and/or cGMP accumulation. At the cellular level, the actual adenylyl cyclase activity reflects the integrated response to stimulatory and inhibitory input and is presumably subject to wide interindividual variation. The same consideration holds true for guanylyl cyclase isoforms. It is attractive to speculate that our observations can, in principle, explain the variable extent to which foscarnet elicits untoward reactions in individual patients. This is, in particular, relevant to the neurological manifestations of foscarnet toxicity. Since cAMP and cGMP levels in neurons are subject to diverse regulatory influences, the sensitivity to foscarnet may vary depending on the individual level of catalytic activity.


    ACKNOWLEDGEMENTS

We thank D. Koesling and D. L. Garbers for generously providing plasmids.


    FOOTNOTES

* This work was supported by Science Foundation of the Austrian National Bank Grant 8520 (to M. F.) and Deutsche Forschungsgemeinschaft Grant DFGKI773/4-1,2 (to C. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Institute of Pharmacology, University of Vienna, Währinger Str. 13a; A-1090 Vienna, Austria. Tel.: 43-1-4277-64171; Fax: 43-1-4277-9641; E-mail: michael.freissmuth@univie.ac.at.

Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M007910200

2 M. Freissmuth and T. Mitterauer, unpublished observation.


    ABBREVIATIONS

The abbreviations used are: RO201724, DL-4-(3-butoxy-4-methoxybenzyl)-2-imidazidinone; ANF, atrial natriuretic factor; CGS21680, N-ethylcarboxamido-2-[4-(2-carboxyethyl)phenylethyl]adenosine; GTPgamma S, guanosine 5'-(3-O-thio)triphosphate; HEK-A2A, HEK293 cells that express the A2A-adenosine receptor; TNP-ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Sunahara, R. K., Dessauer, C. W., and Gilman, A. G. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 461-480[CrossRef][Medline] [Order article via Infotrieve]
2. Buck, J., Sinclair, M. L., Schapal, L., Cann, M. J., and Levin, L. R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 79-84[Abstract/Free Full Text]
3. Tesmer, J. J. G., Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997) Science 278, 1907-1916[Abstract/Free Full Text]
4. Tesmer, J. J. G., Sunahara, R. K., Johnson, R. A., Gosselin, G., Gilman, A. G., and Sprang, S. R. (1999) Science 285, 756-760[Abstract/Free Full Text]
5. Helgstrand, E., Eriksson, B., Johansson, N. G., Lannero, A., Misony, A., Noren, J. O., Sjoberg, B., Stenberg, K., Stening, G., Stridh, S., and Oberg, B. (1978) Science 201, 819-821[Medline] [Order article via Infotrieve]
6. Wadstaff, A. J., and Bryson, H. M. (1994) Drugs 48, 199-226[Medline] [Order article via Infotrieve]
7. VanScoy, M., Loghman-Adham, M., Onsgard, M., Szczepanska-Konkel, M., Homma, S., Knox, F. G., and Dousa, T. P. (1988) Am. J. Physiol. 255, F984-F994[Abstract/Free Full Text]
8. Hoch, B. S., Shahmehdi, S. J., Louis, B. M., and Lipner, H. I. (1995) Antimicrob. Agents Chemother. 39, 2008-2012[Abstract]
9. Mitterauer, T., Hohenegger, M., Tang, W.-J., Nanoff, C., and Freissmuth, M. (1998) Biochemistry 37, 16183-16191[CrossRef][Medline] [Order article via Infotrieve]
10. Seidel, M. G., Klinger, M., Freissmuth, M., and Höller, C. (1999) J. Biol. Chem. 274, 25833-25841[Abstract/Free Full Text]
11. Weitmann, S., Wursig, N., Navarro, J. M., and Kleuss, C. (1999) Biochemistry 38, 3409-3413[CrossRef][Medline] [Order article via Infotrieve]
12. Graziano, M. P., Freissmuth, M., and Gilman, A. G. (1989) J. Biol. Chem. 264, 409-418[Abstract/Free Full Text]
13. Mumby, S. M., and Linder, M. E. (1994) Methods Enzymol. 237, 254-268[Medline] [Order article via Infotrieve]
14. Nanoff, C., Waldhoer, M., Roka, F., and Freissmuth, M. (1997) Neuropharmacology 36, 1211-1219[CrossRef][Medline] [Order article via Infotrieve]
15. Hausleithner, V., Freissmuth, M., and Schütz, W. (1985) Biochem. J. 232, 501-504[Medline] [Order article via Infotrieve]
16. Hohenegger, M., and Suko, J. (1993) Biochem. J. 296, 303-308[Medline] [Order article via Infotrieve]
17. Nanoff, C., Boehm, S., Hohenegger, M., Schütz, W., and Freissmuth, M. (1994) J. Biol. Chem. 269, 31999-32007[Abstract/Free Full Text]
18. Chinkers, M., Garbers, D. L., Chang, M. S., Lowe, D. G., Chin, H. M., Goeddel, D. V., and Schulz, S. (1989) Nature 338, 78-83[CrossRef][Medline] [Order article via Infotrieve]
19. Salomon, Y. (1991) Methods Enzymol. 195, 22-28[Medline] [Order article via Infotrieve]
20. Freissmuth, M., and Gilman, A. G. (1989) J. Biol. Chem. 264, 21907-21914[Abstract/Free Full Text]
21. Johnson, R. A., and Salomon, Y. (1991) Methods Enzymol. 195, 3-21[Medline] [Order article via Infotrieve]
22. Harteneck, C., Koesling, D., Söling, A., Schultz, G., and Böhme, E. (1990) FEBS Lett. 272, 221-223[CrossRef][Medline] [Order article via Infotrieve]
23. Domino, S. E., Tubb, D. J., and Garbers, D. L. (1991) Methods Enzymol. 195, 345-355[Medline] [Order article via Infotrieve]
24. Harry, A., Chen, Y., Magnusson, R., Iyengar, R., and Weng, R. (1997) J. Biol. Chem. 272, 19017-19021[Abstract/Free Full Text]
25. Dessauer, C. W., and Gilman, A. G. (1997) J. Biol. Chem. 272, 27787-27795[Abstract/Free Full Text]
26. Désaubry, L., Shoshani, I., and Johnson, R. A. (1996) J. Biol. Chem. 271, 2380-2382[Abstract/Free Full Text]
27. Florio, V. A., and Ross, E. M. (1983) Mol. Pharmacol. 23, 195-203
28. Segel, I. H. (1975) Enzyme Kinetics , John Wiley and Sons, New York
29. Taussig, R., Tang, W.-J., Hepler, J. R., and Gilman, A. G. (1994) J. Biol. Chem. 269, 6093-6100[Abstract/Free Full Text]
30. Ishikawa, Y., and Homcy, C. J. (1997) Circ. Res. 80, 297-304[Free Full Text]
31. Hohenegger, M., Mitterauer, T., Voss, T., Nanoff, C., and Freissmuth, M. (1996) Mol. Pharmacol. 49, 73-80[Abstract]
32. Hurley, J. H. (1999) J. Biol. Chem. 274, 7599-7602[Free Full Text]
33. Vorherr, T., Knopfel, L., Hofmann, F., Mollner, S., Pfeuffer, T., and Carafoli, E. (1993) Biochemistry 32, 6081-6088[Medline] [Order article via Infotrieve]
34. Wu, Z., Wong, S. T., and Storm, D. R. (1993) J. Biol. Chem. 268, 23766-23768[Abstract/Free Full Text]
35. Scholich, Barbier, A. J., Mullenix, J. B., and Patel, T. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2915-2920[Abstract/Free Full Text]
36. Sunahara, R. K., Beuve, A., Tesmer, J. J., Sprang, S. R., Garbers, D. L., and Gilman, A. G. (1998) J. Biol. Chem. 273, 16332-16338[Abstract/Free Full Text]
37. Tucker, C. L., Hurley, J. H., Miller, T. R., and Hurley, J. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5993-5997[Abstract/Free Full Text]
38. Bayewitch, M. L., Avidor-Reiss, T., Levy, R., Pfeuffer, T., Nevo, I., Simonds, W. F., and Vogel, Z. (1998) FASEB J. 12, 1019-1025[Abstract/Free Full Text]
39. Freissmuth, M., Hausleithner, V., Nees, S., Böck, M., and Schütz, W. (1986) Naunyn Schmiedeberg's Arch. Pharmacol. 334, 56-62[Medline] [Order article via Infotrieve]
40. Shoshani, I., Laux, W. H. G., Périgaud, C., Gosselin, G., and Johnson, R. A. (1999) J. Biol. Chem. 274, 34742-34744[Abstract/Free Full Text]


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