From the Institute of Pharmacology, University of Vienna,
Währinger Str. 13a, A-1090 Vienna, Austria, the
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
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ABSTRACT |
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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 G 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 = G 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.
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
G 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), [
The source of soluble guanylyl cyclase was the cleared lysate of SF9
cells that had been co-infected with baculoviruses encoding the
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.
Comparison of IC1/IIC2 and VIIC1/IIC2 Heterodimers--
The
stimulatory effect of forskolin and G 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.
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.
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.
G 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
As can be seen from Table II, the
apparent potency of foscarnet depends on the nature of activating
ligand. In general, activation of GTP
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).
In both, myocardial and brain membranes, cAMP formation that was
stimulated by the addition of GTP 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
G 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
G 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.
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s; inhibitory = G
i;
-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
-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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s (12) and myristoylated rG
i-1 (13) were
purified from bacterial lysates, G protein
-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).
-32P]ATP (specific activity
10-100 cpm/pmol), 2.5 mM MgCl2, 1 mM MgSO4 (carry over from the preactivation of
rG
s or of rG
i-1), 0.01% Lubrol; unless
indicated otherwise in the figure legends, the concentrations of
[
-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, rG
s and
rG
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 GTP
S, and
0.01% Lubrol; free GTP
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
-subunits, are soluble in the absence of
detergent. However, Lubrol prevents adsorptive losses that occur at low
concentrations of G
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).
1
and
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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 G
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, G
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, G
s and the combination
thereof, respectively. The combination of forskolin and
G
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),
G
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,
rG 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 GTP
S-liganded rG
s (
), or
a combination of forskolin and rG
s (
). Panels
C-F, the amount of IIC2 (2 µg/assay) and IC1 (
; 20-30
ng/assay) or VIIC1 (
; 30 ng/assay) was kept constant and the
concentration of forskolin (C and D) and
preactivated rG
s (E and F) was
varied. In D and F, preactivated
rG
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.
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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 (
) and 250 µM Mg·foscarnet (
) or
0.3 (
) and 1.5 mM Mg·PPi (
). 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 (
) and VIIC1/IIC2 (
) was
plotted as a function of the concentration of foscarnet (C)
and PPi (D); error bars indicate
S.E.
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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
(
), 100 µM (
), or 1 mM (
)
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 (
), 150 µM (
),
or 500 µM (
) Mg·foscarnet. Data are means of
duplicate determinations in a representative experiment.
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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 1/
1-dimer was incubated in
the absence and presence of the indicated concentrations of
Mg·foscarnet (
) and Mg·PPi (
). 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
(
) 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.
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 G
s; we
have therefore also determined the inhibitory potency of foscarnet and
PPi on the activity stimulated by GTP
S-liganded
G
s, forskolin, and their combination. Preactivated G
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
rG
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 G
s. If
catalysis was further activated by the combination of forskolin and
G
s, foscarnet suppressed cAMP formation with an
intermediate potency (Table I); the IC50 values were lower than those observed in the presence of G
s but higher
than those seen with forskolin.
Inhibition by foscarnet and by pyrophosphate (PPi) of
cAMP formation catalyzed by IC1/IIC2- and VIIC1/IIC2-heterodimers
-dimers in the
presence of activated G
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 G
s (1), in membranes forskolin-stimulated catalysis reflects the sum of the direct action of
the compound and the basal level of G
s activation. (ii)
GTP
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 G
s was
preactivated by incubation with GTP
S and MgSO4, unbound
GTP
S was removed by gel filtration and GTP
S-liganded G
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).
S-liganded G
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 GTP
S and there
was a striking left-shift of the curve when compared with the activity
in the presence of GTP
S-liganded G
s. This was also
seen, albeit to a lesser extent, in cerebrocortical membranes. As
mentioned above, addition of GTP
S activates both, Gi and
Gs endogenous to the membranes. Hence, it appears likely that the presence of inhibitory subunits (G
i and/or
G
-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.
Inhibition by foscarnet of adenylyl cyclase activity in membranes
prepared from heart, brain cortex and platelets
View larger version (21K):
[in a new window]
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,
) and isoform the type V (ACV,
) as
well as the heterodimer (IM1C1/IIM2C2,
) 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.
S was more susceptible to
inhibition by foscarnet than catalysis activated by GTP
S-liganded G
s (Table II). This discrepancy can be rationalized if
the GTP
S-induced increase in inhibitory subunits (GTP
S-liganded
G
i and G
o and
-dimers) is assumed
to sensitize the catalyst to foscarnet. To test this conjecture, we
have mimicked this situation by adding GTP
S-liganded
G
i-1 and free
-dimers to Sf9 membranes
expressing the type I and type V isoforms in the presence of
preactivated G
s (Fig. 7).
Sole addition of G
i-1 caused a modest inhibition of the
G
s-activated type I enzyme which was substantially
augmented by the presence of free
-dimers (Fig. 7A).
Importantly, addition of G
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 rG
s,
rG
s + rG
i-1 and rG
s + rG
i-1 +
, respectively). In contrast, adenylyl
cyclase type V was only inhibited by G
i-1; G
had
no additional effect (Fig. 7B). Furthermore, the presence of
G
i-1 and G
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 rG
s, rG
s + rG
i-1 and rG
s + rG
i-1 +
, 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.
View larger version (22K):
[in a new window]
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 GTP S-liganded
rG
s in the absence (
) or presence of preactivated 2 µM rG
i-1 (
) or the combination of
preactivated rG
i-1 and 1 µM
-dimers
(
). 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.
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 G
s, adenylyl cyclase in HEK-A2A cells was
no longer susceptible to inhibition by foscarnet.
View larger version (26K):
[in a new window]
Fig. 8.
A2A-adenosine receptor and
forskolin-dependent cAMP accumulation in PC12 and
HEK-A2A cells. A, the adenine nucleotide
pool of PC12 ( ) 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 (
). 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
s relieved the inhibition of adenylyl cyclase by
foscarnet. The effect of G
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
G
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.
G
-Dimers (but not by Gi
-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 GTP
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
G
(38). However, previous reconstitution experiments also did not
detect an inhibitory action of
-dimers on adenylyl cyclase type V
(and VI) (29). The reconstitution assay may fail to restore the correct
interaction between
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.
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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;
GTPS, 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.
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