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J Biol Chem, Vol. 274, Issue 27, 18887-18892, July 2, 1999
, and
From the Departments of Biochemistry and Molecular Biology and
Pharmacological and Physiological Science, Saint
Louis University School of Medicine, St. Louis, Missouri 63104
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Nitric oxide (NO) donors inhibit hormone- and
forskolin-stimulated adenylyl cyclase activity in purified plasma
membrane preparations from N18TG2 neuroblastoma cells. Northern blot
analyses indicate that the predominant isoform of adenylyl cyclase in
N18TG2 cells is the type VI. Our experiments eliminate all the known
regulatory proteins for this isoform as possible targets of NO. NO
decreases the Vmax of the enzyme without
altering the Km for ATP. Occupancy of the
substrate-binding site protects the enzyme from the inhibitory effects
of NO, suggesting that the conformation of the enzyme determines its
sensitivity. The inhibition is reversed by reducing agents, implicating
a Cys residue(s) as the target for nitric oxide and an
S-nitrosylation as the underlying modification. These
findings implicate NO as a novel cellular regulator of the type VI
isoform of adenylyl cyclase.
Nitric oxide (NO)1 has
been attributed roles in a variety of cellular activities throughout
the body. For example, NO has been implicated as a regulator of
vasodilation, synaptic plasticity, and immune defense. In each case,
however, NO has potentially deleterious effects should its production
be disturbed, as occurs in excitotoxicity and ischemia (reviewed in
Refs. 1-3). To understand the means by which NO achieves its
paradoxical effects, and eventually to control its actions, attempts
have focused on characterizing the enzymes responsible for NO
production (NOS), identifying target molecules that are altered by NO,
and specifying the underlying mechanism(s) by which NO alters those targets.
One target of NO is the soluble guanylyl cyclase. It is the subsequent
increase in cGMP levels that is believed to mediate NO-induced
vasodilation. Given that an activation of soluble guanylyl cyclase
occurs almost universally in response to NO, changes in cGMP have
received primary consideration as the mechanism by which NO acts.
However, the multiplicity of NO actions are unlikely to be explained by
a common mechanism and a number of laboratories have suggested
alternative targets of NO. Generally, cellular components have been
implicated as targets of NO based largely on the ability of NO or
NO-releasing compounds to alter their activities in vitro.
Thus, whether they are altered in intact cells in response to NO, or
what the possible physiological consequences may be, remain
speculative. Our approach to discerning how NO functions has been to
examine its effects on intact cells. We previously demonstrated that NO
inhibits the production of cAMP pulses in Dictyostelium
discoideum and does so independently of any changes in guanylyl
cyclase activity (4, 5). The sum of the data indicated that NO
specifically alters either a regulatory domain of the adenylyl cyclase
itself or a distinct regulatory moiety. We have also observed that the
addition of NO gas or NO donor compounds to cultures of N18TG2
neuroblastoma cells inhibits Gs-coupled and
forskolin-stimulated cAMP accumulation (6). NO-mediated inhibition of
forskolin-stimulated cAMP production is unaltered by pretreating cells
with pertussis toxin, implying that the inhibition did not involve
Gi (6). Although experiments using intact cells were able
to eliminate a number of potential targets that NO could modulate that
would inhibit cAMP production, a number of possible targets still
remained. In particular, NO has been proposed to inhibit GAPDH and in
so doing limit the cellular levels of ATP (7). Similarly, NO-stimulated
poly-(ADP-ribosylation) has been proposed to limit ATP production as a
result of decreased availability of NAD (8). If such events were to
occur in N18TG2 cells in response to NO, the cell's ability to produce
cAMP could be compromised. NO has also been shown to alter the activity
of ion channels (9). Because a number of adenylyl cyclase isoforms are
regulated by Ca2+ (10), this could potentially influence
the activity of the activity in N18TG2 cells. For example, the type V
and VI isoforms are inhibited by Ca2+ by what appears to be
the binding of the cation directly to the enzyme (11). Thus, to further
address the mechanism by which NO regulates cAMP production in N18TG2
cells, and to identify the target of its actions, we have studied the
effects of NO on adenylyl cyclase activity in purified plasma membrane
preparations. In the present report, we identify the predominant
isoform of adenylyl cyclase present in N18TG2 cells and characterize
its inhibition by NO in purified plasma membranes. Our findings
demonstrate a novel, isoform-specific regulation of adenylyl cyclase
activity by NO.
SNP Pretreatment--
Sucrose gradient purified plasma membranes
(12) were resuspended in buffer A (50 mM NaHepes, pH 8, 3 mM MgCl2, 1 mM EDTA) at 0.5 mg/ml.
Protein determinations were done according to Bradford (13). Unless
indicated otherwise, membranes were preincubated with 3 mM
SNP for 30 min on ice. Other reagents were present as described in the
individual experiments. Where indicated, membranes were washed free of
any additions by diluting them 10-fold in ice-cold buffer A,
centrifuging at 700 × g for 10 min, and resuspending them in ice-cold buffer A at 0.5 mg/ml.
Adenylyl Cyclase Activity--
Adenylyl cyclase activity was
assayed by incubating membranes at 30 °C for 20 min and monitoring
the conversion of [ Northern Blot Analyses--
Nuclei were removed and total
cytoplasmic RNA was isolated according to Maniatis et al.
(16). RNA (30 µg) was fractionated on 1.25% formaldehyde-agarose
gels, and transferred to Duralon-UV membranes. Probes were labeled with
[32P]dCTP using the Prime-A-Gene kit. Northerns were
analyzed for adenylyl cyclase isoforms and GAPDH. The latter represents
a constitutively expressed gene that was monitored to verify loading
conditions. Loading was also monitored by ethidium bromide staining of
the rRNA bands. Hybridizations were done in BLOTTO (17) overnight at
37 °C. Membranes were washed in 2 × SSC, 0.5% SDS and then in
1 × SSC, 0.1% SSC at 55 °C and exposed to x-ray film
overnight (GAPDH) or for 1 week (adenylyl cyclase isoforms). 1 × SSC is 0.15 M NaCl, 0.015 M trisodium citrate,
pH 7.
Materials--
Sodium nitroprusside (SNP) and
S-nitroso-N-acetyl-D,L-penicillamine
(SNAP) were purchased from Alexis Biochemicals. PGE1 and secretin were purchased from Cayman Biochemicals and Bachem,
respectively. Duralon was purchased from Statagene and Prime-a-Gene was
from Promega. All other reagents were purchased from Sigma.
NO Inhibits Adenylyl Cyclase Activity in Purified Plasma Membrane
Preparations--
Plasma membranes were preincubated with increasing
concentrations of SNP, after which they were assayed for
forskolin-stimulated adenylyl cyclase activity. Forskolin stimulated
activity was also measured in the presence of GTP and will be discussed
in a later section. As seen in Fig. 1,
preincubation of membranes for 30 min with SNP attenuated the response
of adenylyl cyclase to forskolin and did so in a
concentration-dependent manner. Some inhibition was
observed when membranes were preincubated with 0.1 mM SNP while maximum inhibition occurred when SNP was present at approximately 3 mM. The maximum inhibition varied between 50 and 75%
with different membrane preparations, a range of inhibition similar to
that seen with intact cells (6). Basal enzyme activity is indicated for membranes that were not pretreated and for membranes pretreated with 10 mM SNP. Although in some experiments we did observe an inhibition of basal enzyme activity, in most cases this inhibition was
minimal (see figure legends for values in different experiments).
To confirm that the inhibition of forskolin-stimulated adenylyl cyclase
activity represented the action of NO and not another product of SNP,
we preincubated membranes with a chemically distinct NO donor, SNAP.
SNAP also inhibited adenylyl cyclase stimulation by forskolin in a
concentration-dependent manner. In general, both NO donors
were equally effective in their ability to do so (data not shown).
Other experiments indicated that the effects of NO donors were
unaltered when membranes were preincubated in the presence of Trolox
(0.1 mM) or superoxide dismutase (375 units/ml) plus/minus
catalase (100 units/ml) (data not shown). It would appear that NO,
rather than an oxidative product, is responsible for the inhibition of
adenylyl cyclase activity.
The finding that the effects of NO in intact cells (6) can be
faithfully reproduced in purified plasma membrane preparations limits
the possible targets of NO to components in those preparations. Experiments using intact cells had indicated that changes in cGMP or
PDE did not mediate the inhibition of cAMP accumulation in response to
hormone or forskolin (6). Although we do not expect soluble guanylyl
cyclase to be present in our membrane preparations, we confirmed the
lack of involvement of cGMP by adding cGMP to the adenylyl cyclase
reaction mixture. No changes in enzyme activity were observed (data not
shown). Activation of PDE cannot account for the decrease in adenylyl
cyclase activity in response to NO in purified membrane preparations
because assays are performed in the presence a PDE inhibitor specific
for the low Km cAMP-PDE isoform that is present in
N18TG2 cells (18), and an internal [3H]cAMP] standard is
used to normalize for any loss of newly generated [32P]cAMP.
NO Inhibits Adenylyl Cyclase Activity without Altering the
Regulation of Gi--
To identify the target of NO's
actions on adenylyl cyclase activity in plasma membranes, we performed
a series of studies examining the effects of NO on the regulation of
the enzyme by hormone-receptor complexes, GTP, and divalent cations. In
the experiment shown in Fig. 2, the
ability of SNP to attenuate hormone- and forskolin-stimulated adenylyl
cyclase activity was monitored at increasing concentrations of
effectors. SNP did not alter the EC50 for secretin,
PGE1, or forskolin. Thus, the apparent binding of hormone
to its receptor, or of forskolin to the adenylyl cyclase, was seemingly
unaltered by NO. In contrast, none of these effectors were able to
elicit maximum enzyme activity.
Forskolin stimulates adenylyl cyclase by directly binding to the
enzyme, and thus by-passes the hormone receptor-Gs protein signal transduction pathway. Although forskolin stimulation does not
require GTP, it has been shown that Gs activation can lead to additional stimulation of the enzyme (19). The addition of GTP to
the assay increased adenylyl cyclase activity above that seen with
forskolin alone (Fig. 1). We generally observed a 15-20% increase in
enzyme activity. This was true whether or not membranes had been
pretreated with SNP. It would appear from this experiment that there is
a population of Gs proteins in these membrane preparations that can be activated by the addition of GTP to enhance adenylyl cyclase activity and that SNP does not alter this ability.
Divalent cations have been shown to regulate the activation of
Gs by effecting the GDP/GTP exchange in the absence of a
hormone receptor stimulation (20, 21). Consistent with previously published data (20), Fig. 3 shows that
the stimulation of adenylyl cyclase activity was linear with increasing
concentrations of MgCl2, in the range of 3 to 75 mM. Also shown is that this linear enzyme stimulation was
not altered in membranes that had been pretreated with SNP, although
enzyme activity was significantly reduced. These data would suggest
that the divalent cation regulatory site(s) was not modified by NO. In
total, the above studies indicate that the ability of Gs to
regulate adenylyl cyclase is not altered by treatment with SNP.
N18TG2 Cells Express the Type VI Isoform of Adenylyl
Cyclase--
NO could attenuate adenylyl cyclase activity by targeting
the enzyme directly or by altering the action of a distinct regulatory protein. To identify other possible regulatory proteins of the enzyme
present in N18TG2 cells and to assess their role in this phenomenon, we
identified the isoform present in N18TG2 cells. The likelihood that
this would be a type V or VI isoform was already suggested by its
biochemical properties. The lack of a CaM regulation of cAMP production
in N18TG2 cells (6) was confirmed when plasma membranes were incubated
with calmidazolium and no change in adenylyl cyclase activity, or in
the ability of NO to inhibit that activity, was seen. This would
indicate that the enzyme is not of the type I, III, or VIII isoform
family (10, 23, 24). The enzyme in N18TG2 cells is regulated by hormone
receptors that stimulate via Gs and inhibit via
Gi (12, 14, 15, 22), a characteristic of the type V, VI
isoform family (10, 23, 24). This contrasts with the behavior of the
types II, IV, and VII isoforms, for which stimulation of Gi
enhances cAMP production in response to Gs stimulators (10,
23, 24). Finally, the adenylyl cyclase in N18TG2 cells is stimulated by
forskolin, which distinguishes it from the type IX isoform (24). To
confirm the biochemical data and to discern which member of the type V
and VI adenylyl cyclase family is predominant in N18TG2 cells, we
probed Northern blots of N18TG2 RNA with the cDNAs for the type V
and VI isoforms. As seen in Fig. 4, a
positive signal was observed when the blot was probed with the type VI cDNA (lane 4) but not when the type V cDNA was used
(lane 3). As expected, no mRNAs corresponding to the
type I and II isoforms were identified (lanes 1 and
2). If an isoform other than the type VI is expressed in
these cells, it is present at a level that is undetectable
biochemically or by Northern blot analyses and thus would not influence
the interpretation of our data concerning the regulation by NO.
Kinetic Analyses of the Effects of NO--
We first preincubated
membranes with SNP and determined forskolin-stimulated adenylyl cyclase
activity at increasing concentrations of substrate. The results are
depicted in Fig. 5. The kinetic analysis
of the rate of substrate utilization indicated that the Km values did not differ between control and
SNP-treated enzymes. The values were 0.48 ± 0.12 and 0.49 ± 0.091 mM for control and SNP-treated membranes,
respectively. This would suggest that the apparent binding of substrate
was not affected by the presence of NO. However, the
Vmax value for SNP-treated membranes (622 ± 44 pmol/min/mg) was reduced relative to that seen in control membranes (932 ± 87 pmol/min/mg), indicative of a reduced rate of
the enzyme conversion of substrate to product.
Although SNP inhibited adenylyl cyclase stimulation when membranes were
preincubated with the NO donor, no inhibition was observed when SNP was
added at the beginning of the assay. Fig. 6 shows that the extent to which SNP
inhibits enzyme activity was dependent upon the length of the
preincubation period. In that experiment, membranes were preincubated
with SNP for the indicated times and then assayed for forskolin
stimulated activity. Some degree of inhibition could be seen after a
10-min preincubation period while maximum inhibition occurred by 45 min. One conclusion of these observations is that SNP does not inhibit
the adenylyl cyclase by altering a reagent in the assay. This was
confirmed in other experiments in which SNP-treated membranes were
washed free of the NO-donor prior to the assay. The degree of
inhibition of forskolin-stimulated adenylyl cyclase activity was
unaltered compared with unwashed membranes (also see Figs.
7-9).
The fact that SNP was ineffective when added at the time of the assay
also raised the possibility that a component of the reaction mixture
served to modulate the effects of NO. Types III, V, and VI adenylyl
cyclases possess the consensus sequence for protein kinase A, and
incubation of those isoforms with protein kinase A alters their
activities. Added protein kinase C has also been shown to phosphorylate
recombinant adenylyl cyclases, altering their activities (16, 23, 24).
To test the hypothesis that a phosphorylation mechanism might alter the
response to NO, AMP-PNP, which cannot serve as a kinase substrate,
replaced ATP as the substrate for adenylyl cyclase. Whether AMP-PNP or
ATP served as substrate, SNP-mediated inhibition was observed (data not
shown), indicating that a phosphorylation event does not modulate the ability of NO to inhibit adenylyl cyclase.
We next addressed the possibility that occupancy of the
substrate-binding site offered protection of the enzyme against the effects of NO. To do so, we determined if the presence of ATP during
the preincubation period could limit the ability of SNP to inhibit the
enzyme. In the experiment shown in Fig. 7, membranes were preincubated
with SNP in the absence or presence of the indicated compounds.
Membranes were then washed and assayed for forskolin-stimulated activity. In the absence of any additions, SNP pretreatment resulted in
a 70% attenuation of enzyme activity. Adenylyl cyclase activity was
protected against the effects of SNP when Mg2+/ATP was
present during the preincubation period. The effect was specific for
ATP because neither Mg2+ alone, nor Mg2+/GTP
were effective. In contrast, protection was also afforded to the enzyme
when Mg2+/AMP-PNP was present at the time of SNP exposure,
indicating that the protective effects of ATP were not related to a
phosphorylation event. It would appear that the conformation of the
enzyme achieved upon occupancy of the substrate-binding site of the
enzyme renders it less susceptible to the effects of NO.
Given these results, we examined if the conformation of the enzyme, as
elicited by either forskolin or hormonal signaling, could also afford
the enzyme some protection against the effects of NO. To do so,
membranes were preincubated with SNP in the absence or presence of
forskolin or PGE1. When membranes were preincubated with
PGE1, Mg2+/GTP was also added to assure maximum
activation of Gs. After the 30-min preincubation period,
membranes were washed and assayed for their corresponding forskolin or
PGE1 stimulated activity. As shown in Fig.
8, preincubation of control membranes
with either PGE1 or forskolin did not alter their
respective effects on adenylyl cyclase activity when added to the
enzyme assay. Similarly, the SNP-mediated inhibition of forskolin and
PGE1 stimulated activity was unaltered when these effectors
were present during the preincubation period in the absence of added
ATP.
The Effects of NO on Adenylyl Cyclase Are Reversed by Reducing
Agents--
We previously demonstrated, in intact N18TG2 cells, that
NO-mediated inhibition of cAMP accumulation in response to hormone or
forskolin is readily reversible. Cells recover full ability to respond
to these stimulators when incubated for 20 min in the absence of NO
(6). As shown in Fig. 9, when SNP-treated
membranes were washed free of SNP and then incubated for 20 min in
buffer, forskolin-stimulated adenylyl cyclase activity remained
inhibited. However, when washed membranes were incubated with DTT,
forskolin stimulated activity was recovered in a
dose-dependent manner. Similar effects were observed when
Cys was added instead of DTT. In contrast, cystine did not allow for
recovery of forskolin-stimulated activity but was itself inhibitory to
the enzyme. We have verified that neither 10 mM Cys nor DTT
alter adenylyl cyclase activity when added directly to the assay. Thus,
the NO-mediated inhibition of adenylyl cyclase activity in plasma
membranes is reversible if an appropriate reducing agent is present. We
also observed that the ability of DTT to reverse the effects of NO are
rapid. When SNP-treated membranes were incubated for increasing times with 10 mM DTT prior to the determination of
forskolin-stimulated adenylyl cyclase activity, maximum recovery was
observed at 4 min, the shortest time point examined (data not
shown).
Nine isoforms of the mammalian enzyme have been cloned, each with
distinct regulatory properties, as revealed in reconstitution studies
of the recombinant enzyme expressed in Sf9 cell membranes, or in
co-transfection experiments (reviewed in Refs. 10, 23, and 24). With
the understanding that each enzyme is uniquely regulated by a variety
of factors, the view of a universal mechanism of adenylyl cyclase
regulation has been replaced by the recognition that
cell/tissue-specific changes in adenylyl cyclase activity require an
understanding of the profiles of isoforms present in a particular cell.
In the present study, we have identified the predominant isoform
present in N18TG2 cells as being a type VI enzyme and have described
its novel regulation by NO.
We had previously demonstrated that preincubation of intact N18TG2
cells with NO or NO donors attenuates their production of cAMP upon
stimulation with PGE1, secretin, or forskolin (6). The
effects of NO can be faithfully reproduced in purified plasma membrane
preparations, limiting its possible targets to components in those
preparations. Experiments (Ref. 6, and this work) eliminated guanylyl
cyclase, PDE, Gs, Gi/o, CaM, and protein
kinases as NO targets. In so doing, we have eliminated the known
regulators of the type VI isoform as targets of NO. That we are able to
demonstrate the NO-mediate attenuation of adenylyl cyclase activity in
plasma membranes also eliminates events such as changes in ATP levels or ion fluxes as possible mediators of the effects of NO on the enzyme.
We conclude that NO functions either via an as yet unidentified novel
regulator of the adenylyl cyclase in N18TG2 cells or that the enzyme
itself is the target of NO.
It is of interest to note that another isoform of adenylyl cyclase may
also be regulated by NO. Duhe et al. (25) reported that the
Ca2+/CaM regulation of recombinant type I adenylyl cyclase
in membranes from Sf9 cells is inhibited by NO although basal
enzyme activity is unaffected. The authors proposed that NO may
function by oxidizing a Cys residue(s) at the CaM-binding
site of the adenylyl cyclase. Vorherr et al. (26) had
identified a region of the type I adenylyl cyclase that possesses CaM
binding activity, a region that contains two Cys residues.
Currently it is unknown if either, or both, of these residues is
modified by NO, or if NO functions to inhibit CaM activation by
modifying another region of the protein. Since experiments were
restricted to analyses of Sf9 cell membranes, it also remains to
be determined if NO regulates type I adenylyl cyclase in either
membranes or in intact cells that normally express the enzyme and, if
so, if this results in a change in its activation by Ca2+.
This contrasts with the effects that we have observed in which NO
inhibits the forskolin-stimulated cAMP accumulation, in both intact
N18TG2 cells and in purified plasma membranes, and does so in a manner
that is independent of any CaM regulation of the enzyme.
Recent studies of truncated soluble constructs of adenylyl cyclase have
indicated that the enzyme exists in multiple conformational states,
elicited by the binding of effectors such as G The finding that occupancy of the substrate-binding site of the enzyme
protects it from inactivation by NO would indicate that optimal
attenuation of cAMP production will occur when cell exposure to NO
precedes stimulation of the enzyme by a Gs modulator. When
adenylyl cyclase stimulation precedes, or is simultaneous with that of
NO, the inhibitory effects of NO will be diminished. Our data indicate
that the effects of NO will be modulated also by the ability of cells
to reverse the inhibition of adenylyl cyclase. With intact cells, the
inhibitory effects of NO on cAMP accumulation are readily reversed when
cells are incubated in the absence of NO donors (6). In plasma membrane
preparations, the removal of NO donor compounds did not alleviate the
inhibition of adenylyl cyclase activity, suggesting that a stable
modification had occurred. The NO-mediated inhibition, however, was
reversed when membranes were washed free of NO donors and incubated in the presence of a reducing agent, either DTT or Cys, a more
physiological agent. Such observations strongly implicate
S-nitrosylation as the underlying mechanism of adenylyl
cyclase inhibition (3). They also indicate that the ability of NO to
regulate a cell's production of cAMP will depend upon the cell's
redox potential. Over recent years, a large body of evidence has
amassed to indicate that the cell's redox potential will significantly
alter a variety of signal transduction pathways involved in the control
of cell growth and death (29). These data extend such observations to now include signal transduction via cAMP.
Given the readily reversible nature of the effects of NO, it is likely
that the physiological consequences of NO exposure, with respect to
cAMP production, will depend upon the levels of NO and its stability.
If the source of NO results from the activity of a constitutive NOS,
such levels will be low and transient. The resulting changes in a
cell's responses to Gs modulators would also be transient
and thus more fitting of a homeostatic response. More chronic exposure,
or higher levels of NO as produced by inducible NOS, would result in a
longer lasting refractory period to Gs stimulators. In
general, high levels of NO are believed to be toxic, contributing to
the pathologies resulting from e.g. ischemic injury, heart
failure, and a number of neurodegenerative diseases (reviewed in Refs.
1-3).
In light of their varied and complex modes of regulation, adenylyl
cyclases have been proposed to serve as "co-incidence detectors" (30), where the enzymes are acted upon by varied hormones and then
integrate these influences into a change in cAMP. The response to
different signals could be synergistic, e.g. type II
adenylyl cyclase responds to Gs stimulators in a manner
that could be amplified by the simultaneous presence of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP to [32P]cAMP
at 30 °C (12, 14, 15). The standard reaction mixture contained 50 mM NaHepes, pH 8, 1 mM EDTA, 10 mM
MgCl2, 0.5 mM ATP containing 106
cpm of [-32P]ATP, 0.1 mM cAMP containing
10,000 cpm of [3H]cAMP, 0.1 mM Ro20-1724, 1.5 mM K+ phosphoenolpyruvate, 10 µg/ml pyruvate
kinase, 0.1 mg/ml bovine serum albumin, and 25 µg of membrane protein
in a final volume of 100 µl. Triplicate samples are assayed. Enzyme
activity is proportional to the concentration of membranes and the time
of incubation (12).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of increasing concentrations of SNP on
adenylyl cyclase stimulation. Membranes were preincubated with the
indicated concentrations of SNP for 30 min. Membranes were then assayed
for forskolin-stimulated adenylyl cyclase activity in the absence ( 
)
or presence (
) of 10
4 M GTP. Forskolin was
present at 105 M. Basal activities are
indicated (+). The data are presented relative to the highest activity
seen, i.e. when membranes were not pretreated with SNP and
adenylyl cyclase activity was assayed in the presence of GTP. The data
are the average of four experiments.
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Fig. 2.
Effect of SNP on the dose-response curve to
hormone and forskolin. Membranes were preincubated with or without
3 mM SNP for 30 min after which time they were assayed for
secretin (A), PGE1 (B), or
forskolin-stimulated adenylyl cyclase (C) activity at the
indicated concentration of stimulators. When hormone stimulated
activity was evaluated, the enzyme assay contained 10 4
M GTP. For control and SNP-treated membranes, the
EC50 values for secretin were 9.0 and 9.1 × 107 M, for PGE1 were 3.7 and
2.9 × 107 M, and for forskolin were 1.2 and 1.7 × 105 M. Unpaired t
test analyses indicated that, in each case, the EC50 values
for control and SNP-treated membranes were not significantly different
(p < 0.05). The respective Vmax
values, 96 and 26, 70 and 34, and 228 and 78 pmol/min/mg for control
and SNP-treated membranes, were significantly different. The data are
representative of three experiments.
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Fig. 3.
Effect of SNP on the divalent cation
regulation of adenylyl cyclase. Enzyme activity was determined at
500 µM ATP, and total Mg2+ was varied from 3 to 75 mM. The increase in activity with increasing
[Mg2+] was linear in this range with a slope of 0.66 ± 0.6 (mean ± S.E.) and r = 0.99 for control
( ) and a slope of 0.48 ± 0.12 and r = 0.92 for
SNP-treated (
). The dotted lines represent the 95%
confidence intervals on the lines. The slopes were not significantly
different at p = 0.05. The experiment is representative
of two experiments.
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Fig. 4.
Northern analyses demonstrate the type VI
isoform. Total RNA was isolated and probed with the rat cDNAs
of the type I, II, V, and VI isoforms as indicated (A). A
band of approximately 6 kilobases, as estimated by the position of the
large and small ribosomal subunits, is indicated by the
arrows. Total RNA was also probed with the rat GAPDH
cDNA (B). The data are representative of three
experiments.
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Fig. 5.
Effects of SNP on the
Vmax of the enzyme. Membranes were
preincubated for 30 min in the absence ( ) or presence () of 3 mM SNP. Membranes were then assayed for adenylyl cyclase
activity using 10 mM Mg2+ and increasing
concentrations of ATP ranging from 5 to 2000 µM. The data
were analyzed by nonlinear regression analysis of a rectangular
hyperbola. The inset shows the double-reciprocal plot of the
data points surrounding the Km. The experiment shown
is representative of four experiments.
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Fig. 6.
Time-dependent inhibition of
adenylyl cyclase by SNP. Membranes were preincubated with 3 mM SNP for the indicated time periods after which they were
assayed for adenylyl cyclase activity. The data are presented relative
to the activity achieved when membranes were not preincubated with SNP,
which was assigned the value of 100%. This value was unchanged when
membranes were assayed in the presence of 1 mM SNP, the
concentration that would be present due to carryover when membranes
were preincubated with 3 mM SNP. The data are the average
of five experiments.
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Fig. 7.
Protection of adenylyl cyclase activity by
occupancy of the substrate-binding site. Membranes were
preincubated with or without 3 mM SNP in the absence or
presence of the indicated compounds. After the 30-min preincubation,
membranes were washed and resuspended in buffer as described under
"Experimental Procedures." Membranes were assayed for
forskolin-stimulated (10 5 M) adenylyl cyclase
activity. 100% was assigned to the value seen when membranes were
preincubated in the absence of any additions (132 pmol/min/mg). Basal
activities were 11.5 and 12 pmol/min/mg in control and SNP-treated
membranes, respectively. The data are representative of five
experiments. ANOVA and Tukey's test indicated that the values for
control, SNP plus AMP-PNP, and SNP plus ATP-pretreated membranes were
not significantly different, but that they were different from the
values for SNP and SNP plus GTP-pretreated membranes, and that the
values for the latter two were not significantly different from each
other.
View larger version (19K):
[in a new window]
Fig. 8.
Failure of forskolin or PGE1 to
protect adenylyl cyclase from SNP. Membranes were preincubated
with or without SNP. Where indicated, either 10 5
M forskolin or 105 M
PGE1 was also present. When membranes were preincubated
with PGE1, GTP (104 M) was also
present. The absence or presence of GTP did not alter the results.
After 30 min membranes were washed and assayed for forskolin or
PGE1-stimulated adenylyl cyclase with 104
M GTP present in the assay. Basal activity was 19 and 21 pmol/min/mg in control and SNP-treated membranes. The data are
representative of three experiments.
View larger version (25K):
[in a new window]
Fig. 9.
Ability of reducing agents to reverse the
effects of SNP. Membranes were preincubated with 3 mM
SNP for 30 min. Membranes were then washed free of SNP and incubated
for 20 min in the absence or presence of the indicated concentrations
of DTT or Cys. Forskolin-stimulated (10 5 M)
adenylyl cyclase activity was measured. The results are presented
relative to the forskolin-stimulated adenylyl cyclase activity seen in
membranes that had not been preincubated with SNP or reducing agent.
The data are representative of three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits, forskolin,
or by occupancy of the ATP-binding site (27, 28). Our kinetic analyses
indicated that the inhibition by NO reflects an altered
Vmax, suggesting that the conformation of the
enzyme is altered in a manner that precludes its optimal catalytic
activity. The inhibitory effects of NO were not altered when membranes
were preincubated with SNP plus either forskolin or GTP. Thus, the conformations of the enzyme that are achieved as a result of forskolin or Gs binding do not alter the ability of NO to inhibit
adenylyl cyclase. Occupancy of the ATP-binding site, however, did
afford the enzyme significant protection from the inhibition by NO.
This would suggest that, under those latter conditions, the amino
acid(s) targeted by NO is no longer available, either because it is
sterically shielded upon ATP binding or because the enzyme conformation
induced upon ATP binding now renders it unavailable. Several
observations would suggest the latter to be the more reasonable
interpretation. If the catalytic site contained the residues targeted
by NO, then we would expect that basal cyclase activity would also be
inhibited by NO. This was not generally the case. Given the homologies
between the putative catalytic sites of the various isoforms, we would also expect other isoforms to be similarly regulated by NO. We note
that the basal activity of the type I recombinant enzyme is not altered
by NO (25), suggesting that, in their basal states, the type I and VI
isoforms share a conformation that precludes susceptibility to NO. In
contrast to the type VI isoform, it would appear that the type I
isoform of adenylyl cyclase, in which residues that modulate CaM
binding are susceptible to NO, exists in a conformation such that NO is
not able to inhibit forskolin stimulation.
generated by Gi stimulators. Alternatively, the response to
one signal may be attenuated by the presence of a second signal,
e.g. the type V and VI isoforms are stimulated by
Gs but inhibited by Ca2+ (10). Our data
indicate that the type VI isoform of adenylyl cyclase predominant in
N18TG2 cells is a coincidence detector for NO and that both the redox
potential of the cell and the order of input signals will influence the
ability of NO to regulate cAMP production.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. T. Misko (Monsanto) for stimulating conversations throughout the course of these experiments, Drs. R. Iyengar and R. Premont for the adenylyl cyclase cDNA clones, Dr. W. Sly for the cDNA clone for GAPDH, and John McAlpin for typing the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by the National Institutes of Health (to A. H.) and the American Heart Association (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: Dept. of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8155; Fax: 314-577-8156; E-mail: KleinC{at}slu.edu.
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ABBREVIATIONS |
|---|
The abbreviations used are:
NO, nitric oxide;
CaM, calmodulin;
Gs, guanine nucleotide-binding protein
that stimulates adenylyl cyclase;
Gi, guanine
nucleotide-binding protein that inhibits adenylyl cyclase;
PDE, phosphodiesterase;
NOS, nitric oxide synthetase;
SNAP, S-nitroso-N-acetyl-D,L-penicillamine;
SNP, sodium nitroprusside;
PGE1, prostaglandin
E1;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
DTT, dithiothreitol;
AMP-PNP, adenosine
5'-(,-imino)triphosphate.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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