From the Departments of Pediatrics and
§ Medicine and the ¶ Cancer Center, University of
California, San Diego, La Jolla, California 92093-0652 and the
Alliance Pharmaceutical Company, La
Jolla, California 92037
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ABSTRACT |
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Nitric oxide (NO) regulates the
expression of multiple genes but in most cases its precise mechanism of
action is unclear. We used baby hamster kidney (BHK) cells, which have
very low soluble guanylate cyclase and cGMP-dependent
protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle
cells, which express these two enzymes, to study NO regulation of the
human fos promoter. The NO-releasing agent Deta-NONOate
(ethanamine-2,2'-(hydroxynitrosohydrazone)bis-) had no effect on a
chloramphenicol acetyltransferase (CAT) reporter gene under control of
the fos promoter in BHK cells transfected with an empty
vector or in cells transfected with a G-kinase I Nitric oxide (NO)1 is a
pluripotential molecule involved in regulating blood pressure,
neurotransmission, and immune function (1). One of its major
intracellular targets is the heme group of soluble guanylate cyclase
with NO markedly stimulating enzymatic activity and thereby increasing
the intracellular cGMP concentration (2, 3). Other NO targets are
thiol-containing proteins, iron sulfur proteins and non-heme iron; in
addition, NO can react with oxygen to produce peroxynitrite and
hydroxyl radical, both of which can have physiological effects (4).
NO regulates the expression of multiple genes including
c-fos, junB, heme oxygenase, smooth muscle
In this study we used baby hamster kidney (BHK) cells, which have very
low soluble guanylate cyclase and G-kinase activity, to determine
whether NO regulates the fos promoter via a cGMP/G-kinase signal transduction pathway. We found that maximal stimulation of the
fos promoter by NO in BHK cells required expression of both
guanylate cyclase and G-kinase. In cells transfected with guanylate
cyclase alone, NO caused a small activation of the fos promoter, apparently by cGMP activation of cAMP-dependent
protein kinase (protein kinase A), either directly by cross-activation or indirectly by inhibition of phosphodiesterase activity (12). In
cells containing endogenous guanylate cyclase and G-kinase, NO
stimulation of the fos promoter was inhibited by the
guanylate cyclase inhibitor ODQ. This, to our knowledge, is the first
demonstration that NO regulates gene expression via cGMP activation of
G-kinase.
Cell Culture and Transient Transfection Experiments--
BHK
cells were cultured and transfected as described previously (13). All
cells received 50 ng of pFos-CAT, which contains the chloramphenicol
acetyltransferase (CAT) gene under control of the human fos
promoter, and 50 ng of the luciferase expression vector pRSV luciferase
to serve as an internal control for transfection efficiency (5, 13). As
indicated, cells also received 300 ng of a human G-kinase I
CS-54 cells are rat pulmonary arterial smooth muscle cells that
maintain differentiated properties through multiple subcultures (16).
They were obtained from A. Rothman, University of California, San
Diego, and were transfected with pFOS-CAT and pRSV luciferase but not
with guanylate cyclase or G-kinase expression vectors. Where indicated,
ODQ (Calbiochem, La Jolla, CA) and/or Deta-NONOate were added 16 h
prior to cell harvest.
Measurement of Guanylate Cyclase, G-kinase, and Protein Kinase A
Activity--
Guanylate cyclase activity was measured as described
previously in the absence and presence of the NO donor
S-nitrosylpenicillamine (SNAP) using
[ Reporter Gene Assays--
CAT activity and luciferase activity
were measured as described previously using
[14C]chloramphenicol/butyryl-coenzyme A and
luciferin/ATP, respectively (5, 13). The data are expressed as relative
CAT activity, i.e. CAT activity/luciferase activity.
Measurement of cGMP and Nitrite/Nitrate Concentrations--
The
intracellular cGMP concentration was measured as described previously
by radioimmunoassay (5). After culturing cells for 24 h in low
serum medium, nitrite and nitrate concentrations in the medium were
measured according to the Griess reaction (20).
Data Analysis--
Comparison of data groups was done by the
two-tailed Student t test, with a p value of <0.05
considered significant.
Guanylate Cyclase and G-kinase Activity in Transfected BHK
Cells--
We found very low soluble guanylate cyclase activity in
extracts of BHK cells transfected with empty vector with about a 6-fold increase in enzyme activity on treating the extracts with 50 µM SNAP (Table I;
difference between minus and plus SNAP was significant at
p < 0.05). When the cells were transfected with
guanylate cyclase expression vectors, we found a large increase in
enzyme activity, which again was stimulated >6-fold by SNAP (Table I);
these latter experiments demonstrate that the transfected enzyme
contained a functional heme moiety. G-kinase activity was also very low in extracts of BHK cells transfected with empty vector and enzyme activity increased substantially when the cells were transfected with a
G-kinase expression vector (Table I). The guanylate cyclase and
G-kinase activities found in the transfected cells are comparable with
amounts of these enzymes expressed endogenously in several cell types,
e.g. smooth muscle cells, neuronal cells, and platelets (3,
21).
Intracellular cGMP in Guanylate Cyclase- and G-kinase-transfected
BHK Cells--
In BHK cells transfected with empty vector,
intracellular cGMP was low but measurable, consistent with the cells'
low guanylate cyclase activity, and Deta-NONOate caused a small
increase in cGMP, compatible with the low endogenous guanylate cyclase
being stimulated by NO (Table II). In
cells transfected with guanylate cyclase, intracellular cGMP increased
about 8-fold and Deta-NONOate increased cGMP ~250-fold (Table II;
throughout the text, -fold increase is relative to cells transfected
with empty vector receiving no drug treatment).
In cells transfected with both guanylate cyclase and G-kinase
expression vectors, intracellular cGMP increased ~60-fold and Deta-NONOate increased cGMP >600-fold (Table II). The higher cGMP in
cells co-transfected with guanylate cyclase and G-kinase compared with
cells transfected with guanylate cyclase alone is probably because the
cGMP assay measures total intracellular cGMP, including that which is
protein-bound: the intracellular G-kinase concentration in cells
transfected with this enzyme is 3-6 pmol/mg of protein corresponding
to a cGMP binding capacity of 12-24 pmol/mg of protein (13). In
addition, as shown below, BHK cells produce relatively high amounts of
NO. Thus, high cGMP in the guanylate cyclase- and
G-kinase-transfected cells in the absence of Deta-NONOate probably
represents the combination of high endogenous NO stimulating the
transfected guanylate cyclase with the cGMP produced binding to the
transfected G-kinase. These results suggest that in cells co-transfected with guanylate cyclase and G-kinase, G-kinase is likely
to be activated, even in the absence of exogenous NO.
Transactivation of the fos Promoter by NO in BHK Cells--
We
showed previously that NO-releasing agents activate synthetic
TRE-containing promoters in REF52 fibroblasts and rat thyroid cells,
and we and others (5, 6, 22-26) have shown that NO increases
c-fos mRNA in many cell types. We have also shown that cGMP regulates the fos promoter in BHK cells via G-kinase
activation and nuclear translocation (13, 27).
To test whether NO's effect on the fos promoter in BHK
cells was mediated by cGMP activation of G-kinase, we transfected cells with pFos-CAT in the absence or presence of guanylate cyclase and
G-kinase expression vectors. We found that the NO-generating agent
Deta-NONOate had no effect on CAT expression in cells transfected with
empty vector (Fig. 1). When the cells
were transfected singly with guanylate cyclase or G-kinase expression
vectors, CAT activity increased a minimal 1.3-fold in the absence of
drug treatment which was not statistically significant (Fig. 1).
Deta-NONOate increased CAT activity a small, but statistically
significant, 2.1-fold in the guanylate cyclase-transfected cells but
had no effect in the G-kinase-transfected cells (Fig. 1). The small
increase in CAT activity by Deta-NONOate in guanylate
cyclase-expressing cells points to a cGMP-mediated mechanism, and as
described above, we found that under these conditions Deta-NONOate
increased the intracellular cGMP concentration by ~250-fold (Table
II). This large increase in cGMP could activate the cells low
endogenous G-kinase or, as shown later, cross-activate protein kinase
A. Deta-NONOate increased CAT activity more than 8-Br-cGMP in the guanylate cyclase-transfected cells (Fig. 1); as discussed later, this
difference could be because endogenously produced cGMP is more
available than exogenously provided 8-Br-cGMP. As we showed previously (13), 8-Br-cGMP stimulated CAT expression about
4-fold in cells transfected with the G-kinase expression vector and was without effect in cells transfected with empty vector (Fig. 1), indicating that at the concentration used, 8-Br-cGMP did not
cross-activate protein kinase A (Fig. 1).
When the cells were co-transfected with guanylate cyclase and G-kinase
expression vectors, CAT expression increased 5.5-fold in the absence of
drug treatment (Fig. 1). This substantial increase in CAT activity can
be attributed to the large increase in intracellular cGMP under these
conditions (Table II) activating the transfected G-kinase (discussed
above). Deta-NONOate further increased CAT activity to about 7-fold in
cells co-transfected with guanylate cyclase and G-kinase expression
vectors (Fig. 1) and, as shown, further increased intracellular cGMP
(Table II). The increase in CAT activity by Deta-NONOate in the
guanylate cyclase- and G-kinase-transfected cells was similar to that
observed when these cells were treated with 8-Br-cGMP (Fig. 1; the
further increase in CAT activity by Deta-NONOate and 8-Br-cGMP was not
statistically significant compared with the untreated cells). Deta, the
parent compound of Deta-NONOate, which does not release NO, had no
effect on CAT expression in guanylate cyclase- and/or
G-kinase-expressing cells.
To examine whether high endogenous NO production in the guanylate
cyclase- and G-kinase-expressing cells could be the cause of increased
CAT expression in the absence of Deta-NONOate or 8-Br-cGMP, we treated
cells with the competitive NO synthase inhibitor NMEA. We found that
NMEA reduced CAT expression by >40% in cells transfected with
guanylate cyclase and G-kinase expression vectors (Fig. 1). To
eliminate possible effects of altered culture conditions, all cells
were grown in medium containing 400 µM arginine; this relatively high arginine concentration may have prevented more complete
inhibition of CAT expression by NMEA and other workers have also found
that NO synthase inhibitors are only partially effective at high
micromolar arginine concentrations (28). Corresponding to the 40%
reduction in CAT expression, NMEA reduced intracellular cGMP by ~50%
in the guanylate cyclase- and G-kinase-transfected cells (Table II).
When Deta-NONOate was added to the NMEA-treated cells, CAT expression
returned to the levels observed in the absence of NMEA, providing
evidence that NMEA was acting by inhibiting endogenous NO synthase
(Fig. 1). Consistent with recovery of CAT activity, Deta-NONOate almost
fully returned the intracellular cGMP concentration to the level
observed in the absence of NMEA (Table II).
NO Production by BHK Cells--
NO produced under physiological
conditions is converted stoichiometrically to nitrite and nitrate (29).
We found the sum of nitrite and nitrate in the medium of serum-starved
BHK cells to be >1 µM after 24 h of culture
corresponding to an NO production rate of 40 pmol/h/106
cells. This NO production rate is similar to what vascular endothelial cells and glioma cells, which have high NO synthase activity, produce
under nonstimulated conditions (30, 31).
Protein Kinase A Activation by Deta-NONOate in Guanylate
Cyclase-transfected BHK Cells--
At high intracellular
concentrations, cGMP can cross-activate protein kinase A and protein
kinase A activation can lead to transactivation of the fos
promoter (12, 13). The small increase in CAT expression on treating
guanylate cyclase-transfected cells with Deta-NONOate (Fig. 1) could,
therefore, be secondary to protein kinase A activation by high
intracellular cGMP concentrations (Table II). To address this question,
we measured the activity of the free catalytic subunit of protein
kinase A in Deta-NONOate-treated, guanylate cyclase-transfected cells
and found a 2.7-fold increase in enzyme activity (Table
III). This increase in free catalytic subunit activity by Deta-NONOate was about half that observed in cells
treated with 8-Br-cAMP (Table III; in control experiments we showed
that free catalytic subunit release by 8-Br-cAMP was not secondary to
drug carryover from the culture medium because adding 8-Br-cAMP
immediately prior to cell washing had no effect on free catalytic
subunit activity). In cells transfected with empty vector, Deta-NONOate
had no effect, while 8-Br-cAMP yielded the same increase in enzyme
activity as in cells transfected with guanylate cyclase expression
vectors (Table III). Thus, in cells containing soluble guanylate
cyclase, NO can increase the cGMP content sufficiently to
cross-activate protein kinase A.
Transactivation of the fos Promoter by NO in CS-54
Cells--
Having shown that NO activation of the fos
promoter in BHK cells required expression of both soluble guanylate
cyclase and G-kinase, we assessed NO's effect on the fos
promoter in cells expressing both of these enzymes endogenously. We
chose CS-54 cells, derived from the smooth muscle of rat pulmonary
arteries (16), because primary vascular smooth muscle cells contain
both soluble guanylate cyclase and G-kinase (11). We found that CS-54 cells contained 21 ± 3.4 and 335 ± 39 pmol/min/mg of
protein of guanylate cyclase and G-kinase activity, respectively
(mean ± S.D. of three independent experiments performed in
duplicate; guanylate cyclase activity was simulated 7-fold by
S-nitrosylpenicillamine). The activities of both of these
enzymes are considerably more than found in untransfected BHK cells,
and although less than in BHK cells transfected with guanylate cyclase
or G-kinase expression vectors (Table I), they are within the range
reported in primary smooth muscle cells and other mammalian cells (3,
21).
In CS-54 cells transfected with pFos-CAT, the guanylate cyclase
inhibitor ODQ reduced CAT activity by >60% compared with untreated cells (Fig. 2). This suggests that CAT
expression in untreated cells was determined in large part by
endogenously produced cGMP stimulating cellular G-kinase; ODQ was
clearly acting via guanylate cyclase inhibition because the
membrane-permeable cGMP analog 8-para-chlorophenylthio-cGMP reversed
ODQ's inhibitory effect (data not shown). When the cells were treated
with Deta-NONOate, CAT expression increased more than 2-fold compared
with untreated cells and more than 5-fold compared with cells treated
with ODQ (Fig. 2). Deta-NONOate added to ODQ-treated cells was without effect indicating that Deta-NONOate was acting via a
cGMP-dependent mechanism (Fig. 2).
We have shown that NO activation of the fos promoter is
dependent on both guanylate cyclase and G-kinase activity and have, therefore, defined a specific signal transduction pathway which mediates NO's effect on c-fos expression. We showed
previously that G-kinase I In the absence of drugs, fos promoter activity was not
significantly affected when BHK cells were transfected singly with either guanylate cyclase or G-kinase expression vectors. However, we
found a substantial increase in fos promoter activity when the cells were co-transfected with expression vectors for both enzymes,
even in the absence of drug treatment. These data suggest that the
transfected guanylate cyclase generated sufficient cGMP to activate the
transfected G-kinase, and indeed, we found a 60-fold increase in the
intracellular cGMP concentration compared with untransfected cells. For
guanylate cyclase to produce such high amounts of cGMP, it seemed
likely that the enzyme was stimulated by NO and we found high
constitutive NO production by BHK cells. Moreover, NMEA, a NO synthase
inhibitor, decreased CAT expression in the guanylate cyclase and
G-kinase transfected cells significantly and adding Deta-NONOate to the
NMEA-treated cells caused full recovery of CAT expression to maximal
levels. Although CAT activity was high in the guanylate cyclase- and
G-kinase-transfected cells in the absence of drugs, both Deta-NONOate
and 8-Br-cGMP further increased CAT activity, suggesting that the
amount of endogenously produced cGMP was not quite sufficient to
maximally stimulate G-kinase. Together, these data provide
evidence that NO activates the fos promoter through a
guanylate cyclase- and G-kinase-dependent mechanism.
In BHK cells transfected with guanylate cyclase, Deta-NONOate increased
the activity of the free catalytic subunit of protein kinase A,
although less effectively than 8-Br-cAMP. In vascular smooth muscle
cells, Cornwell et al. (32) also found that NO donors
activate protein kinase A, although they did not compare the effect of
the drugs to that of cAMP analogs. Protein kinase A activation by
8-Br-cAMP stimulates fos promoter activity about 6-fold in
BHK cells (13). Thus, protein kinase A activation by Deta-NONOate in
the guanylate cyclase-transfected cells likely caused the small
increase in fos promoter activity under these conditions;
the high amounts of cGMP produced by the cells could also have
activated the low endogenous G-kinase present in BHK cells.
The increase in CAT activity in cells transfected with guanylate
cyclase and G-kinase expression vectors was significantly more than the
increase in CAT activity in cells transfected with the G-kinase
expression vector only and treated with 8-Br-cGMP. This may be because
8-Br-cGMP, although more membrane-permeable than cGMP, still does not
diffuse readily across cell membranes and intracellular concentrations
achieved by the drug are relatively low.2,3
In addition, it may be that cGMP produced endogenously from guanylate cyclase is a more effective G-kinase activator than exogenous 8-Br-cGMP
because of the intracellular localization of the cyclase and kinase. We
found similar results in our previous work with REF52 and rat thyroid
cells where NO-releasing agents were more potent activators of
TRE-containing reporter genes than was 8-Br-cGMP (5). In the guanylate
cyclase- and G-kinase-transfected cells there is also likely some
degree of protein kinase A activation that we showed previously does
not occur in G-kinase-transfected cells treated with 8-Br-cGMP
(13).
NO plays an important role in regulating pulmonary vascular resistance
and growth of pulmonary arterial cells via activation of soluble
guanylate cyclase and G-kinase (33). In CS-54 cells, CAT expression
from the fos promoter was markedly reduced when the cells
were treated with the guanylate cyclase inhibitor ODQ, suggesting that
endogenously produced cGMP is sufficient to stimulate promoter activity
in these cells and that CAT activity observed in the presence of ODQ
may be the more correct "basal" level than that observed in
untreated cells. Although we did not define the type of G-kinase
expressed in CS-54 cells, vascular smooth muscle cells express
predominantly G-kinase I The findings in these studies are likely of physiological significance
for the following reasons. First, the amounts of guanylate cyclase and
G-kinase in the transfected BHK cells are comparable with the amounts
of these enzymes expressed endogenously in several cell types (3, 21).
Second, fos promoter activity was markedly stimulated by
endogenously produced NO in BHK cells expressing both guanylate cyclase
and G-kinase, and third, NO increased fos promoter activity
in CS-54 cells. Thus, we conclude that one mechanism whereby NO
regulates gene expression in vivo is via a guanylate cyclase/cGMP/G-kinase transduction pathway.
expression vector.
In BHK cells transfected with expression vectors for guanylate cyclase,
Deta-NONOate markedly increased the intracellular cGMP concentration
and caused a small (2-fold) increase in CAT activity; the increased CAT
activity appeared to be from cGMP activation of
cAMP-dependent protein kinase. In BHK cells co-transfected with guanylate cyclase and G-kinase expression vectors, CAT activity was increased 5-fold in the absence of Deta-NONOate and 7-fold in the
presence of Deta-NONOate. Stimulation of CAT activity in the absence of
Deta-NONOate appeared to be largely from endogenous NO since we found
that: (i) BHK cells produced high amounts of NO; (ii) CAT activity was
partially inhibited by a NO synthase inhibitor; and (iii) the
inhibition by the NO synthase inhibitor was reversed by exogenous NO.
In CS-54 cells, we found that NO increased fos promoter
activity and that the increase was prevented by a guanylate cyclase
inhibitor. In summary, we found that NO activates the fos
promoter by a guanylate cyclase- and G-kinase-dependent mechanism.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-actin, vascular endothelial growth factor, vascular cell adhesion
molecule-1 (VCAM-1), and mitogen-activated kinase phosphatase-1
(5-10). In regulation of VCAM-1, NO appears to act independently of
cGMP because it's effect is not mimicked by cGMP analogs, while in
regulation of c-fos and junB, NO and cGMP analogs
induce similar changes (5, 6, 10). In cases where NO and cGMP analogs
function similarly, it is likely that NO works through activation of
soluble guanylate cyclase, but this has not been shown definitively.
Moreover, it is not clear which of several cGMP target proteins,
e.g. cGMP-dependent protein kinases (G-kinase),
cGMP-gated ion channels, cGMP-activated phosphodiesterases, or
cGMP-inhibited phosphodiesterases, mediates the effects of NO on gene
expression (11).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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expression vector (14) and/or 100 ng each of expression vectors
encoding the
1 and
1 subunits of rat
soluble guanylate cyclase (15); variable amounts of the empty vector
pCB6 were used to keep the total amount of DNA constant. Where
indicated, NG-monoethyl-L-arginine
monoacetate (NMEA, Alexis Corp., San Diego, CA) was added 16 h
prior to cell harvest and 8-Br-cGMP (Biolog Life Sciences, Bremen,
Federal Republic of Germany), Deta-NONOate (ethanamine-2,2'-(hydroxynitrosohydrazone)bis-, Cayman Chemical, Ann
Arbor, MI), and Deta, the parent compound of Deta-NONOate that does not
release NO, were added 8 h prior to cell harvest.
-32P]GTP as substrate and separating substrate and
product by thin layer chromatography (17, 18). The activities of
G-kinase, protein kinase A holoenzyme, and the free catalytic subunit
of protein kinase A were measured as described previously following [
-32P]ATP phosphorylation of
Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) (13, 19). G-kinase activity was
measured in the presence of 0.01 mg/ml PKI (protein kinase inhibitor
from S. Taylor, University of California, San Diego) and 10 µM 8-Br-cGMP; protein kinase A holoenzyme activity was
measured in the presence of 1 µM cAMP. The activity of
the free catalytic subunit of protein kinase A was measured as
PKI-inhibitable phosphorylation in the absence of cAMP. All enzyme
assays were linear with time and protein concentration.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Guanylate cyclase and G-kinase activity in transfected BHK cells
Intracellular cGMP concentration in BHK cells
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Fig. 1.
Transactivation of the fos
promoter by NO in guanylate cyclase- and G-kinase-transfected BHK
cells. BHK cells were transfected as described under
"Experimental Procedures" with pFos-CAT and pRSV-luciferase and,
where indicated, with the parent vector pCB6 (empty vector) or with
expression vectors for guanylate cyclase (GC) and/or
G-kinase (GK). Cells were either left untreated
(Control, open bars) or were treated with 1 mM 8-Br-cGMP (wide diagonal-striped bars), 250 µM Deta-NONOate (Deta-NO, filled
bars), 20 mM NMEA (narrow diagonal-striped
bars), or the combination of 20 mM NMEA with 250 µM Deta-NONOate (filled bars with narrow diagonal
stripes). CAT activity was normalized to the luciferase activity
in each sample and the CAT/luciferase activity of untreated cells
transfected with empty vector was assigned a value of 1. The data are
the mean ± S.D. of at least three independent experiments
performed in duplicate; error bars are too small to be
observed in drug-treated cells transfected with empty vector.
Activity of A-kinase free catalytic subunit or holoenzyme in guanylate
cyclase-transfected BHK cells treated with 8-Br-cAMP or Deta-NONOate
View larger version (9K):
[in a new window]
Fig. 2.
Transactivation of the fos
Promoter by NO in CS-54 Cells. CS-54 cells were transfected
as described under "Experimental Procedures" with pFos-CAT and
pRSV-luciferase and were either left untreated (no additions) or were
treated with 10 µM ODQ (ODQ), 250 µM Deta-NONOate (Deta-NO), or the combination
of 10 µM ODQ plus 250 µM Deta-NONOate
(ODQ + Deta-NO). CAT activity was normalized to the
luciferase activity in each sample, and the CAT/luciferase activity of
untreated cells was assigned a value of 1. The data are the mean ± S.D. of at least three independent experiments performed in
duplicate.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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, the G-kinase isoform used in the present
studies, activates the fos promoter via translocation to the
nucleus and that this effect of G-kinase is mediated by several
sequence elements, including the serum response element, the AP-1
binding site, and the cAMP response element (13, 27).
, suggesting that the observed results are
applicable to cells expressing either G-kinase I
or I
(34-36).
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ACKNOWLEDGEMENTS |
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We thank S. Lohmann for the G-kinase cDNA, F. Murad and M. Nakane for the guanylate cyclase cDNAs, and S. Taylor for providing purified recombinant PKI.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant R01GM055586 (to R. B. P.) and American Heart Association Grant 9650582N (to G. R. B.).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. Tel.: 619-534-8805; Fax: 619-534-1421; E-mail: gboss{at}UCSD.edu.
2 G. R. Boss and R. B. Pilz, unpublished observations.
3 M. Bartsch, J. Kruppa, C. Schultz, and B. Jastorff, 11th Protein Kinase Seminar: Cyclic Nucleotide-dependent Signaling Mechanisms, June 4-7, 1998, Bergen, Norway.
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ABBREVIATIONS |
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The abbreviations used are: NO, nitric oxide; BHK, baby hamster kidney; G-kinase, cGMP-dependent protein kinase; protein kinase A, cAMP-dependent protein kinase; CAT, chloramphenicol acetyltransferase; SNAP, S-nitrosylpenicillamine; 8-Br-cGMP, 8-bromo-cGMP; PKI, protein kinase inhibitor; Deta-NONOate, ethanamine-2,2'-(hydroxynitrosohydrazone)bis-; Deta, non-NO-releasing parent compound of Deta-NONOate; NMEA, NG-monoethyl-L-arginine monoacetate; ODQ, [1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one]; TRE, phorbol ester response element.
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