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INTRODUCTION |
K-Cl cotransport (KCC),1
the coupled movement of potassium and chloride, is involved in cell
volume maintenance, and its activity is highly regulated (1). We
recently found that KCC activity is present in VSMCs and that it is
activated by NO donors, an effect prevented by inhibitors of the cGMP
pathway, protein phosphatases and tyrosine kinases (1, 2). The cGMP/PKG
signaling pathway modulates the activity of ion transport where PKG
plays an important role (3, 4).
PKGs are classified into two main types: type 1 and type 2. Two type 1 isoforms, 1
and 1
, are the result of the alternative splicing of
a unique gene (5). These 75-kDa isoforms differ at the N terminus, but
they share the same C terminus, wherein the cGMP-binding sites and the
catalytic domain reside (6). In VSMCs, almost all of the PKG
immunoreactivity belongs to PKG-1, and only PKG-1
was
detected by Northern blot (7). Although PKG regulation of gene
expression remains largely unknown, a recent report indicates that PKG
may be a weak activator of cAMP-dependent transcription because of its cytoplasmic restriction. These data suggest that regulation of gene expression by PKG involves phosphorylation of
uncharacterized cytoplasmic protein(s) (8).
Both repetitive passage and chronic exposure of VSMCs to NO
donors are associated with reduced to undetectable expression levels of
PKG-1 (7). This enzyme determines the phenotype of VSMCs (9, 10). Thus,
PKG-expressing cells possess a contractile-like morphology, whereas
PKG-deficient cells become dedifferentiated and "synthetic."
Transfection of PKG-deficient VSMCs with cDNAs encoding the
full-length PKG-1 or its C-terminal catalytic domain restores their
original contractile morphology (11). On the other hand, in
PKG-deficient mice, the loss of PKG abolishes
NO/cGMP-dependent relaxation of VSM, resulting in severe
vascular dysfunction. However, PKG-deficient VSM responds normally to
cAMP, indicating that cAMP and cGMP signal via independent
pathways with PKG being the specific mediator of the NO/cGMP effects
(12).
Four isoforms of KCC have been characterized: KCC1 to KCC4, all members
of the superfamily of cation-chloride cotransporters. Northern blot
analysis was used to determine the tissue distribution of the KCC
isoform transcripts in rat. In contrast to the ubiquitous KCC1, the
KCC2 transcript has been found only in the brain (13). The transcript
for KCC3 is abundant in the kidney and heart but low in the brain,
whereas the KCC4 transcript has a more restricted expression pattern
than KCC1 and KCC3, confined primarily to the skeletal muscle, heart,
lung, and brain (14-20).
Based on our recent findings of an NO-dependent pathway of
KCC regulation in low potassium sheep red blood cells and in VSMCs (2,
21), our present studies are directed to the intracellular signaling
pathways utilized by NO in VSMCs and, in particular, to the role of PKG
in the regulation of KCC3 mRNA expression.
Here we report the mRNA expression pattern and relative abundance
of the KCC isoforms in low passaged VSMCs and the regulation of KCC3 at
the mRNA level by PKG. KCC1 and KCC3 mRNAs were found to be
expressed in normal, PKG+, and PKG
VSMCs. Furthermore, in all cell
types, KCC3 mRNA was expressed at lower levels than KCC1 mRNA,
and PKG appeared to be involved in the regulation of the KCC3 mRNA
isoform. KCC1 mRNA regulation by the NO/cGMP signaling pathway is
the subject of a separate study.
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EXPERIMENTAL PROCEDURES |
Materials--
The Renaissance NEL-100
immunochemiluminescence kit was from PerkinElmer Life Sciences.
Primary rabbit polyclonal anti-human PKG-1-specific antibody, secondary
horseradish peroxidase-conjugated mouse anti-rabbit IgG
antibody, KT5823, and actinomycin D were from Calbiochem. Dulbecco's
modified Eagle's medium, tissue culture grade, and molecular
biology reagents were purchased from Life Technologies, Inc. Total RNA
extraction, Access RT-PCR kit, rat actin PCR primers, and 100-bp DNA
ladder were from Promega Corp.
Primary Culture of Rat VSMCs--
Primary cultures were obtained
according to the protocols described previously (10) with some
modifications. Briefly, aortas from Harlan Sprague-Dawley rats
(150-200 g) sacrificed by asphyxiation in CO2-saturated
chambers were obtained from the Wright State University Animal
Facilities. Aortas were excised and placed in a wash medium of
Dulbecco's modified Eagle's medium with 20 mM HEPES, 5 µM amphotericin B, and 50 µg/ml gentamicin. The aortas were cleaned and placed in digestion medium (130 units/ml collagenase type IV and 5 µg/ml DNase I) for 10 min at 37 °C. The
tunica adventitia was removed, and the medial layers were minced and
further digested for 1-2 h in digestion medium containing 200 units/ml
collagenase until a single cell suspension was obtained. Cells were
washed twice in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum and antibiotics, 70 µM
streptomycin, 100 units/ml penicillin, 50 µg/ml gentamicin, and 2.5 µM amphotericin B and plated in 6-well culture plates.
Cells were maintained in Dulbecco's modified Eagle's medium plus 10%
fetal bovine serum as before in a controlled atmosphere of
air-CO2 (5%) at 37 °C until confluence (6-7 days).
Confluent VSMCs at passages 0-2 were used after 24 h of serum deprivation.
Culture of PKG+ and PKG
Rat VSMCs--
Normal
VSMCs expressing no endogenous PKG were transfected with the active
cGMP-independent catalytic domain of PKG-1 or with the empty vector, as
reported earlier (11). Transfected VSMCs were cultured in the same
culture medium and conditions as above but in the presence of 10%
CO2 and 1 mM geneticin until they reached confluence. The culture medium was changed every 48 h during 5-6 days. For the experiments reported here, at least three independent clones of cells expressing the PKG catalytic domain (PKG+ cells) or
vector only (PKG
cells) were examined. Agents to be tested were
incorporated on day 6-7 of culture after 24 h of serum
deprivation in triplicate dishes and for the time indicated. Cell
viability, as assessed by the trypan-blue exclusion test, ranged from
85 to 95%.
Total RNA Extraction, RT-PCR, and KCC Expression in
VSMCs--
Total RNA from normal, PKG
, and PKG+ VSMCs was obtained
by standard procedures (22). Specific sets of primers for all KCC mRNA isoforms were synthesized according to the sequences
previously published and used by others (14, 15, 17). These primers were used to obtain the first cDNA strand by RT, and the subsequent amplification of each KCC mRNA isoform present in VSMCs was done by
PCR. Briefly, RT: 45 min at 48 °C; denaturation, 2 min at 94 °C;
and PCR: denaturation, 1 min at 94 °C; annealing, 1 min at 55 °C;
and extension, 1 min at 68 °C, during 40 cycles. The
semiquantitative RT-PCR conditions were established in our laboratory
to allow comparisons between the expression of KCC1, KCC3, and actin
transcripts. Under these conditions, the efficiency of the RT-PCR
reaction for each gene did not plateau, and the numbers of cycles used in these experiments were kept to a minimum (Fig. 2B,
inset). The relative expression levels of KCC1 and KCC3 mRNA
isoforms were determined by using 1.0 µg of total RNA as
template and 30 cycles of PCR with the same thermal conditions as
before. As control, we analyzed the expression of actin mRNA using
specific rat primers, the same amount of total RNA as before, and 25 PCR cycles. These were optimal conditions for the semiquantitative
analysis of VSMC KCC mRNAs, and the analysis was limited to the
products generated only in the exponential phase of the amplification
(Fig. 2B, inset). The general semiquantitative RT-PCR
protocol was as follows: RT-PCR reactions were performed by preparing a
master RT-PCR mixture containing 3.0 µg of total RNA from VSMCs and
aliquoted in three separate tubes containing the specific set of
primers for KCC1, KCC3, and actin, respectively. As a negative control
for each set of primers, RT-PCR reactions were performed in the absence of reverse transcriptase and/or RNA. After RT-PCR, the content of each
independent reaction tube was analyzed by 2% agarose gel electrophoresis. The bands (KCC1, 233 bp; KCC3, 663 bp; and actin, 285 bp) were visualized with ethidium bromide. All of the ethidium bromide-stained gels were depicted as an inverse image for clear results. The identities of rat KCC1 and KCC3 were confirmed by restriction enzyme digestion according to the sequences published and
the expected DNA fragments obtained (data not shown).
Preparation of Total Cellular Protein Extract--
VSMCs were
rinsed twice with cold PBS and resuspended in cold lysis buffer
containing 10 mM HEPES (pH 7.4), 1 mM
MnCl2, 10 mM MgCl2, 0.1 mM EGTA, 0.5% Triton X-100, and protein phosphatase and
protease inhibitors (40 mM
-glycerol phosphate, 1 mM Na3VO4, 0.1 mM PMSF,
10 mg/liter leupeptin, and 5 µg/ml aprotinin A). Total
cellular protein extracts were obtained by successive passages through
a syringe with needle and centrifuged at 13,000 × g
(23). The protein content of the supernatant was determined by the BCA method (Pierce).
Expression of PKG-1 by Western Blot--
Expression of PKG-1 (75 kDa) was assayed in protein extracts from normal VSMCs by Western blot
by using a specific PKG-1 antibody directed against the last 15 amino
acids at the C terminus, according to published procedures (24).
Briefly, protein extracts (50 µg) from normal VSMCs were diluted with
1 M Tris-HCl buffer (pH 6.8), plus loading buffer
and subjected to 10% PAGE (Mini-Protean II, electrophoresis cell,
Bio-Rad) with 0.4% SDS, in parallel with prestained molecular weight
markers (Amersham Pharmacia Biotech). Proteins were transferred to a
nitrocellulose membrane at 4 °C during 70 min at 100 V in a
Trans-Blot cell (Mini Trans-Blot, electrophoretic transfer cell,
Bio-Rad). PKG-1 antibody was used at 1:10,000 dilution. Mouse
anti-rabbit IgG (1:5,000) was used as horseradish peroxidase-conjugated
secondary reagent. The horseradish peroxidase-reaction was
developed by immunochemiluminescence.
Statistical Analysis--
The analysis of multiple intergroup
differences in each experiment was conducted by one-way analysis of
variance (ANOVA) followed by Student-Newman-Keuls test. A
p < 0.05 was used as the criterion of statistical
significance. Except where indicated, all values were obtained from
three independent experiments in which at least triplicate samples were assayed.
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RESULTS |
KCC mRNA Isoform Expression in VSMCs--
Expression of the
different KCC mRNA isoforms in VSMCs is unknown. Thus, we performed
simple RT-PCR reactions with KCC isoform-specific primers to obtain
some information about the expression of KCC mRNAs in these cells,
as well as in PKG
and PKG+ VSMCs. Under normal conditions, KCC1 and
KCC3 mRNA isoforms of the expected sizes were detected in the three
VSMC types (Fig. 1A). To
corroborate the ability of our KCC-specific primers to amplify each KCC
isoform, we conducted simultaneously RT-PCR using total RNA extracted
from rat kidney and brain, because it has been reported that these tissues express mainly KCC1 and KCC3 and all of the mRNA isoforms, respectively (Fig. 1B). We also detected a faint band of the
size expected for the brain-specific KCC2 mRNA isoform in VSMCs
under our experimental conditions. Furthermore, semiquantitative RT-PCR revealed that KCC1 mRNA expression was more abundant than KCC3 mRNA, both relative to actin mRNA expression (Fig.
2). In this study, only results for KCC3
mRNA regulation have been presented. Parallel studies involving
KCC1 mRNA regulation will be reported separately.

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Fig. 1.
KCC mRNAs expression in rat VSMCs
(normal, PKG , and PKG+), kidney, and brain. A representative 2%
agarose gel electrophoresis stained with ethidium bromide is shown.
Reverse transcription and PCR were performed using total RNA
from the following sources. A, Normal, endogenous
PKG-depleted vector only (PKG )-, and PKG-catalytic domain
(PKG+)-transfected VSMCs. Cells were cultured as described
under "Experimental Procedures." B, whole rat kidney
(left) and brain (right). Each KCC (1-4)
mRNA expression was tested using specific primers designed to
amplify 233-bp (KCC1), 399-bp (KCC2), 663-bp
(KCC3), or 556-bp (KCC4) products.
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Fig. 2.
KCC1 and KCC3 mRNA expression levels in
normal VSMCs. Semiquantitative RT-PCR analysis was performed using
1.0 µg of total RNA from VSMCs and the conditions described under
"Experimental Procedures." A, representative RT-PCR
products separated in 2% agarose gel electrophoresis and stained with
ethidium bromide showing the bands of the expected sizes, 233 (KCC1), 663 (KCC3), and 285 bp
(Actin). B, bars (optical density in
arbitrary units) represent the mean ± S.E. from three independent
experiments, each done in triplicate. *p < 0.01 versus KCC1 mRNA. All of the results were normalized
with respect to actin. Inset, exponential amplification of
1.0 µg of total RNA as a function of PCR cycles. The kinetics
described for the three genes of interest are shown (KCC1,
KCC3, and Actin).
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KCC3 mRNA Expression in Normal VSMCs as a Function of Cellular
Passage--
To define the relationship between PKG and KCC3 mRNA
expression in VSMCs, we performed semiquantitative RT-PCR in normal
VSMCs as a function of cell passage, a well documented way to deplete gradually endogenous PKG-1 expression (7). Fig.
3A shows the semiquantitative
RT-PCR results as well as the densitometric analysis normalized with
respect to actin (Fig. 3B). The expression levels of PKG-1
protein in normal VSMCs were simultaneously examined by Western blot.
Fig. 3C shows a representative Western blot in which the
anti-PKG antibody detected a single major band with an apparent
molecular mass of 75 kDa corresponding to the native endogenous PKG.
These experiments demonstrate no correlation between endogenous PKG and basal KCC3 mRNA expression levels. Furthermore, no significant differences were found between the relative expression levels of KCC3 mRNA in normal, PKG+, and PKG
VSMCs (Fig.
4).

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Fig. 3.
KCC3 mRNA expression levels in normal
VSMCs as a function of cell passage. Semiquantitative RT-PCR
analysis was performed using 1.0 µg of total RNA from normal VSMCs at
different cell passage, as described under "Experimental
Procedures." A, representative RT-PCR products separated
in 2% agarose gel electrophoresis and stained with ethidium bromide
showing the bands of the expected sizes: 663 (KCC3) and 285 bp (Actin). B, densitometric analysis (optical
density in arbitrary units) representing the mean ± S.E. from
three independent experiments, each done in triplicate. All of the
results were normalized with respect to actin. C,
representative Western blot from a pool of two samples showing the
disappearance of PKG-1 protein levels in VSMCs as a function of cell
passage.
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Fig. 4.
KCC3 mRNA expression in PKG , normal,
and PKG+ VSMCs. PKG , PKG+, and normal VSMCs were cultured as
described under "Experimental Procedures." Total RNA was extracted,
and KCC3 and actin mRNA expression levels were tested by RT-PCR
using specific primers. A, representative RT-PCR products
separated in 2% agarose gel electrophoresis and stained with ethidium
bromide showing the bands of the expected sizes: 663 (KCC3)
and 285 bp (Actin). B, densitometric analysis
(optical density in arbitrary units as a % of Normal
cells) representing the mean ± S.E. from three
independent experiments, each done in triplicate. All of the results
were normalized with respect to actin.
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PKG Involvement in the Acute Regulation of KCC3 mRNA
Expression--
To establish a possible relationship between PKG
activity and KCC3 mRNA expression, we incubated normal VSMCs at
passage 0-2 with 8-Br-cGMP, a known stimulator of PKG activity. As
shown in Fig. 5, KCC3 mRNA expression
was acutely stimulated at the lowest cGMP analog concentration (0.1 mM) in normal VSMCs following the indicated exposure time
to the drug (Fig. 5, A and B). At higher 8-Br-cGMP concentrations, the effect reached a plateau (Fig. 5, C and D). These findings suggest for the first
time that PKG activity participates in acute regulation of KCC3
mRNA expression in normal VSMCs.

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Fig. 5.
Effect of 8-Br-cGMP on KCC3
mRNA expression levels in normal VSMCs. Cells were grown as
described under "Experimental Procedures." VSMCs were treated with
0.1 mM 8-Br-cGMP for the indicated periods of time or in
the presence of variable amounts of the cGMP analog (0.1-1.0
mM) for 1 h. Total RNA from normal VSMCs treated with
or without the cGMP analog was obtained and 1.0 µg each was subjected
to semiquantitative RT-PCR analysis. A and C,
semiquantitative RT-PCR products were electrophoresed in 2% agarose
gel and stained with ethidium bromide to show the bands of the expected
sizes: 663 (KCC3) and 285 bp (Actin).
B and D, densitometric analysis (optical density
in arbitrary units as a % of Control) representing the
mean ± S.E. from three independent experiments, each done in
triplicate; *, p < 0.001 versus control.
All of the results were normalized with respect to the actin
signal.
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The opposite situation was found in the presence of KT5823, a known
inhibitor of PKG actions. As shown in Fig.
6, A and B, KT5823
was able to inhibit the 8-Br-cGMP-induced effect on KCC3 mRNA
expression in normal VSMCs. Furthermore, KT5823 per se had no effect on KCC3 mRNA expression under these experimental
conditions (Fig. 6C).

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Fig. 6.
Effect of KT5823 on 8-Br-cGMP-induced KCC3
mRNA expression in normal VSMCs. Cells were grown as described
under "Experimental Procedures," and the confluent cells were
deprived of serum for 24 h. VSMCs were treated with 0.1-1.0
mM 8-Br-cGMP for 1 h with or without 1-5
µM KT5823. A, shown is a representative
RT-PCR from normal VSMCs in which the RT-PCR products were
electrophoresed in 2% agarose gel and stained with ethidium bromide to
show the bands of the expected sizes: 663 (KCC3) and 285 bp
(Actin). B, densitometric analysis (arbitrary
optical density units as % of Control) representing the
mean ± S.E. from two independent experiments, each done in
triplicate (*, p < 0.001 versus control;
**, p < 0.001 versus 0.1 mM
8-Br-cGMP). All of the results were normalized with respect to the
actin signal. C, shown is a representative RT-PCR from
normal VSMCs treated with 1-5 µM KT5823 alone as a
control.
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Post-transcriptional Regulation of KCC3 mRNA
Expression--
Because the onset of the effect of 8-Br-cGMP was fast,
we studied whether the changes observed in KCC3 mRNA expression
were due to transcriptional regulation. To this end, we incubated
normal VSMCs at passage 0-2 with 0.1 mM 8-Br-cGMP in the
presence or absence of 10 µg/ml actinomycin D, an inhibitor of
RNA polymerase II. As shown in Fig. 7,
A and B, no major changes induced by
actinomycin D in the presence of the cGMP analog were observed in our
experimental conditions. This finding indicates that the
transcriptional machinery is not needed for the 8-Br-cGMP-induced
effect on KCC3 mRNA expression.

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Fig. 7.
Effect of actinomycin D on
8-Br-cGMP-induced KCC3 mRNA expression in normal VSMCs. Cells
were grown as described under "Experimental Procedures." VSMCs were
treated with 8-Br-cGMP (0.1 mM) in the presence or absence
of actinomycin D (ActD, 10 µg/ml) or
actinomycin D alone for 1 h. Total RNA from normal VSMCs was
obtained, and 1.0 µg each was subjected to semiquantitative RT-PCR
analysis. A, semiquantitative RT-PCR products were
electrophoresed in 2% agarose gel and stained with ethidium bromide to
show the bands of the expected sizes: 663 (KCC3) and 285 bp
(Actin). B, densitometric analysis (optical
density in arbitrary units as a % of Control) representing
the mean ± S.E. from two independent experiments, each done in
triplicate; *, p < 0.001 versus control.
All of the results were normalized with respect to the actin
signal.
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DISCUSSION |
Analysis of rat normal, PKG
, and PKG+ VSMC mRNAs by RT-PCR
using specific sets of primers showed a specific expression pattern of
KCC isoforms. KCC1 and KCC3 mRNAs were found to be abundantly expressed, with higher KCC1 mRNA levels relative to KCC3 mRNA in VSMCs. Similar results have also been shown by others in human umbilical vascular endothelial cells in which KCC1 mRNA was found at highest abundance with respect to KCC3 mRNA (15). KCC1 mRNA has been found to be expressed in all tissues tested so far (20). Northern blot analysis of KCC3 mRNA expression revealed a more tissue-restricted expression pattern than KCC1 mRNA (15). However, the KCC1 mRNA levels relative to KCC3 mRNA in the different
tissues were not determined, and the reason for the selective VSM
distribution of these two different KCC mRNAs remains unknown. We
were unable to detect KCC4 mRNA, which suggests undetectable levels
or absence of KCC4 mRNA in VSMCs under our experimental conditions.
On the other hand, a faint band of the expected size for KCC2 mRNA
was detected by RT-PCR in our cells. This implies that using Northern blot as the major criteria for tissue-specific distribution of transcripts clearly needs further reconsideration.
The absence of correlation between KCC3 mRNA expression levels and
PKG protein expression as a function of cell passage and the finding of
a similar KCC3 mRNA expression level in normal, PKG
, and PKG+
VSMCs suggest no major involvement of this protein kinase in the
maintenance of basal KCC3 mRNA expression. Recently, it has been
shown that basal expression levels of the mitogen-activated protein
kinase remain without changes in normal VSMCs and in endogenous PKG-depleted VSMCs. However, the NO/cGMP signaling pathway increases mitogen-activated protein kinase activity only in normal VSMCs (24).
Besides, the absence of differences between KCC3 mRNA expression
levels in normal low and high passaged PKG-depleted VSMCs and in PKG
and PKG+ VSMCs suggests a lack of PKG-dependent phosphorylation of putative proteins involved in KCC3 mRNA regulation.
Little information is available on how the cGMP-activated
PKG-dependent signal transduction pathways may influence
gene expression. Recently, NO-releasing agents and membrane-permeable
analogs of cGMP at concentrations able to increase protein
phosphorylation in VSMCs have been shown to activate transcription from
activator protein-1 responsive promoters or by other mechanisms
in rodent fibroblast and epithelial cell lines (25-27). NO can also
regulate RNA polymerase II-independent gene expression by altering
mRNA stability (28-30).
The positive effect of 8-Br-cGMP on KCC3 mRNA expression suggests a
direct participation of PKG as a modulator of KCC3 mRNA expression.
The inhibitory actions of KT5823 on the 8-Br-cGMP-stimulated KCC3
mRNA expression levels in normal VSMCs occurred at concentrations that selectively block PKG-driven phosphorylations (24). Although the
mechanisms of action of these drugs are still obscure, our findings
suggest that the PKG pathway is important in KCC3 mRNA regulation.
Besides, the fact that KT5823 has little or no effect on
cAMP-dependent protein kinase (24) and that there is
no effect of 8-Br-cGMP in VSMCs lacking endogenous PKG but with
normal cAMP-dependent protein kinase (data not shown)
suggest that cGMP is not cross-talking with the cAMP-activated protein
kinase signaling pathways, as has been proposed for other effects of
the nucleotide (24, 31, 32). However, cAMP and other signaling pathways
should not be simply ignored in KCC regulation because both may target
the same factors before converging in the same final effect (8).
Further confirmation is needed to elucidate the molecular mechanisms
involved in the regulation of the KCC3 gene expression in VSMCs.
Post-transcriptional modifications may also contribute to the induction
of KCC3 mRNA expression in VSMCs. The inhibition of the
transcriptional machinery during the exposure time of the cGMP analog
by actinomycin D showed no effect on KCC3 mRNA expression. These
data suggest that the cGMP/PKG signaling pathway leads to an increase
in KCC3 mRNA expression by a post-transcriptional mechanism(s) in
VSMCs. Several lines of evidence support the notion that NO is able to
promote stabilization of different mRNAs against target
endonucleolytic degradation in the absence of active transcription (28,
30). Additionally, it has been recently demonstrated that the fate of
mRNAs are mostly controlled by interactions between specific
mRNA-binding proteins and certain mRNA 5'- or 3'-untranslated regions (33). Thus, it will be of interest to identify the
pertinent 5'- and/or 3'-untranslated control regions of the KCC3
mRNA and the PKG-regulated protein(s) that interact with them.
The fact that KT5823 inhibited the 8-Br-cGMP effect on KCC3 mRNA
expression, that these effects cannot be reproduced in VSMCs lacking
endogenous PKG (data not shown), and that there was a lack of effect of
actinomycin D on the 8-Br-cGMP-induced KCC3 mRNA expression are
consistent with the hypothesis that KCC3 gene expression is regulated
by PKG at the post-transcriptional level in VSMCs. Together these
data show for the first time a possible and direct role of PKG in the
acute regulation of KCC3 mRNA expression in VSMCs.