Am J Physiol Heart Circ Physiol 284: H1686–H1692, 2003.
First published January 9, 2003; 10.1152/ajpheart.00710.2002.
NONOates regulate KCl cotransporter-1 and -3
mRNA expression in vascular smooth muscle cells
Mauricio Di Fulvio,1 Peter K. Lauf,2 Shalin Shah,3 and Norma C. Adragna1
Departments of 1Pharmacology and Toxicology, 2Physiology and Biophysics, and
3
Cardiology, School of Medicine, Wright State University, Dayton, Ohio 45435-0002
Submitted 13 August 2002; accepted in final form 30 December 2002
nitric oxide; soluble guanylyl cyclase
THE ELECTRONEUTRAL-COUPLED MOVEMENT of K and Cl ions
takes place at the membrane level via KCl cotransporters (KCC) (16, 17). KCC activity plays an important
role in cell volume regulation, epithelial transport, and
ion homeostasis. Different activators and inhibitors
regulate KCC activity through a putative kinase-phosphatase cascade (14, 16, 17). We (1–3, 9, 10) recently
found that the cGMP-dependent protein kinase (PKG)
pathway is involved in KCC regulation by nitric oxide
(NO) donors in erythrocytes and primary cultures of
rat vascular smooth muscle cells (VSMCs). However,
NO modulates genetic expression through cGMP-dependent and -independent mechanisms (6), and the NO
Address for reprint requests and other correspondence: N. C.
Adragna, Dept. of Pharmacology and Toxicology, Wright State
Univ., School of Medicine, Biological Sciences Bldg., Rm. 152-6,
3640 Colonel Glenn Highway, Dayton, OH 45435-0002 (E-mail:
[email protected]).
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donors used so far may have unspecific effects unrelated to NO release (6, 11).
The in vitro biological actions of NO donors are
numerous, complex, and frequently contradictory (6,
11). Most of the NO actions occur through direct activation of soluble guanylyl cyclase (sGC), cGMP generation, and the subsequent activation of PKG and ion
channels (4, 19, 20). However, many of the NO donors
effects are either sGC independent and/or related to
the compound-specific formation of metabolites or NOrelated by-products. Moreover, the pathways leading
to NO formation, the chemical reactivities, and kinetics of NO release differ among the individual classes of
NO donors: NONOates, sydnonimines, S-nitrosothiols,
organic nitrates, and sodium nitroprusside (SNP)
(6, 11).
NONOates belong to a family of NO donors that
spontaneously releases NO at physiological pH but
with different predictable first-order release rates (13).
The rate of dissociation of NO (referred as half-life, t1/2)
from NONOates as well as the properties of NO-derived by-products generated during NONOate decomposition are largely determinants of the biological effect. Moreover, the t1/2 of NONOates strongly correlates with vasorelaxant activity, the extent of mRNA
expression, and the degree of sGC activation in vitro (5,
7, 11, 21). These characteristics make NONOates optimal NO donors in the study of the mechanisms of
action of NO on gene expression in VSMCs in vitro.
Although the specific mechanism by which increases
in cGMP lead to vasorelaxation is still unknown (8),
the relevance of the NO signaling pathway in vascular
physiology and the relationship of NO with the vasorelaxant machinery is well established in vitro and in
vivo (22). Furthermore, a link between vasorelaxation
and KCC activity has been suggested because activation of KCC by commonly used nitrosovasodilators
decreases vascular smooth muscle tension (2). Moreover, in primary cultures of rat VSMCs, the NO-sGCPKG signaling pathway is involved in the acute upregulation of KCC1 mRNA expression (9), and a fast
PKG-dependent posttranscriptional upregulation of
KCC3 was also demonstrated in the same experimental model (10).
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Di Fulvio, Mauricio, Peter K. Lauf, Shalin Shah, and
Norma C. Adragna. NONOates regulate KCl cotransporter-1
and -3 mRNA expression in vascular smooth muscle cells. Am J
Physiol Heart Circ Physiol 284: H1686–H1692, 2003. First
published January 9, 2003; 10.1152/ajpheart.00710.2002.—
Nitric oxide (NO) donors regulate KCl cotransport (KCC)
activity and cotransporter-1 and -3 (KCC1 and KCC3)
mRNA expression in sheep erythrocytes and in primary
cultures of rat vascular smooth muscle cells (VSMCs),
respectively. In this study, we used NONOates as rapid
and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the
mRNA level. In addition, we used the expression of KCC3
mRNA to further investigate the mechanism of action of
these NO donors at the cellular level. Treatment of VSMCs
with rapid NO releasers, like NOC-5 and NOC-9, as well as
with the direct NO-independent soluble guanylyl cyclase
(sGC) stimulator YC-1, acutely increased KCC3 mRNA
expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3
gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression.
Inhibition of sGC with LY-83583 blocked the NONOateand YC-1-induced KCC3 mRNA expression. This study
shows that in primary cultures of rat VSMCs, the fast NO
releasers NOC-9 and NOC-5, but not the slow NO releaser
NOC-18, acutely upregulate KCC3 mRNA expression in a
NO/sGC-dependent manner.
NONOATES AND KCC GENE EXPRESSION IN VASCULAR SMOOTH MUSCLE
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Hence, we used primary cultures of freshly isolated
rat VSMCs expressing the main components of the
NO-signaling pathway, sGC and PKG (9), to determine
the role of several NONOates as pure NO donors with
different t1/2 on KCC3 mRNA expression. In addition,
and because NONOates are predictable and controllable NO releasers at physiological pH (13, 21), we used
the KCC3 mRNA expression to further investigate the
mechanism of action of this group of drugs at the
cellular level.
MATERIALS AND METHODS
Materials. 2-(4-Carboxylphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO); SNP; 6-(2-hydroxy-1-methyl2-nitrosohydrazino)-N-methyl-1-hexanamine (NOC-9); 3-[2hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propanamine (NOC-5); {FK409, (⫾)-E-ethyl-2-[(E)-hydroxyimino]-5-nitro3-hexeneamide} (NOR-3); 2,2⬘-(hydroxynitrosohydrazino)bis-ethanamine (NOC-18); 3-(5⬘-hydroxymethyl-2⬘-furyl)-1benzylindazole (YC-1); and 6-anilino-5,8-quinolinequinone
(LY-83583) were from Calbiochem (La Jolla, CA). DMEM,
TRIzol reagent for total RNA extraction, and all tissue culture grade or molecular biology reagents were purchased
from Invitrogen (Carlsbad, CA). Access RT-PCR kit and a
specific rat actin primer set were from Promega (Madison,
WI). Except when indicated, all the stock solutions of NONOates were freshly prepared in darkened vials following the
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recommendations of the manufacturer and immediately
used. The NONOates half-life (t1/2) values were obtained
from the source (Calbiochem) and from http://www.dojindo.
com/newsletter/review.html.
Primary culture of rat VSMCs. Primary cultures were
obtained according to protocols described previously (2, 15)
with modifications published in detail elsewhere (10).
VSMCs were plated in six-well culture plates and maintained in DMEM-10% fetal bovine serum antibiotics in a
controlled atmosphere of air-5% CO2 at 37°C until 90–95%
confluence (6–7 days). Only confluent cells at passages 0–3
were used for our experiments after 24 h of serum deprivation.
Total RNA extraction, RT-PCR, and semiquantitative KCC
mRNA expression in VSMCs. Total RNA from rat VSMCs in
primary culture was obtained by using the TRIzol reagent
following the instructions of the manufacturer. KCC1 and
KCC3 mRNA semiquantitation was performed by using specific RT-PCR reactions as described in detail elsewhere (9,
10) with modifications. The specific KCC3 primers used in
the present experiments were as follows: forward, 5⬘2472
GGA GGC AGA TAA TCC TTT CTC C2493-3⬘; reverse,
5⬘-3135CAC AGC AGT ATG CAT CCT CC3116-3⬘ (superscript
indicates the base pairs downstream of the start codon: A1TG
in the mouse KCC3 gene) (23). These primers (50 pmol each)
were used to obtain the first cDNA strand by reverse transcription, and the subsequent amplification of KCC3 mRNA
isoform present in VSMCs was done by PCR. The semiquan-
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Fig. 1. Action of rapid nitric oxide (NO) releasers on KCl cotransporter-3 (KCC3) mRNA expression in rat vascular
smooth muscle cells (VSMCs). Confluent VSMCs were deprived of serum for 24 h and subsequently were treated
with 0.5 and 1.0 mM NONOates (NOC-9 and NOC-5) during the indicated times. Relative KCC3 mRNA levels were
analyzed by semiquantitative RT-PCR using 0.25 ␮g total RNA at each point and the conditions described in
MATERIALS AND METHODS. A and B: representative RT-PCR products after treatment with the two different
concentrations of NOC-9 and NOC-5 separated in 1.8% agarose gel electrophoresis and stained with ethidium
bromide showing the bands of the expected sizes 663 bp (KCC3) and 285 bp (actin). C and D: densitometric analysis
normalized with respect to actin (optical density as a percentage of control, where control KCC-control actin ⫽
100%) representing the means ⫾ range (*P ⬍ 0.001) of 2 independent experiments for each NONOate and
concentration. Note actin mRNA levels were included as controls to minimize potential random changes in mRNA
expression.
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NONOATES AND KCC GENE EXPRESSION IN VASCULAR SMOOTH MUSCLE
RESULTS
Fast NO releasers upregulate KCC3 mRNA expression in VSMCs. The in vitro vasodilatory activity of
different NONOates correlates with the rate of NO
release, and the potency is linearly related to the quantity of NO release (21). Moreover, a direct correlation
among the rate of NO production, mRNA stability, and
sGC activation was recently demonstrated (5, 7). In
primary cultures of rat VSMCs, the spontaneous and
fast NO releaser SNP upregulates KCC1 mRNA expression in a sGC-dependent manner, and the PKG
agonist 8-bromo-cGMP acutely increases both KCC1
and KCC3 mRNA expression in the absence of active
transcription (9, 10). Thus the actions of two rapid NO
releasers, NOC-9 (t1/2 ⫽ 3 min) and NOC-5 (t1/2 ⫽ 93
min), on KCC3 mRNA expression were investigated in
the primary cultures of rat VSMCs. As shown in Fig. 1,
the two fast NO releasers were able to induce KCC3
mRNA expression in a concentration- and time-dependent fashion. Both NOC-9- and NOC-5-induced KCC3
mRNA expression followed a kinetics that resembles
the reported transcription-independent, PKG-dependent increase in KCC3 mRNA expression (10). Hence,
we treated VSMCs with 1.0 mM of several fast NO
releasers: NOC-9, NOC-5, NOR-3 (t1/2 ⫽ 40 min), and
SNP (t1/2 ⫽ 2.5 min) during 2 h and with 1.0 mM of the
slow NO generator NOC-18 (t1/2 ⫽ 3,400 min) during 0,
1, 2, 6, 12, and 24 h, and then KCC3 mRNA expression
was analyzed by semiquantitative RT-PCR. Figure 2, A
Fig. 2. Effect of rapid versus slow NO
donors on KCC3 mRNA expression in
rat VSMCs. Semiquantitative RT-PCR
analysis performed using 0.25 ␮g total
RNA from rat VSMCs under different
treatments as noted. A and B: actions
of 1.0 mM rapid NO releasers [NOC-9,
NOC-5, NOR-3, and sodium nitroprusside (SNP)] and time-dependent effect
of the slow NO generator NOC-18, respectively. Representative RT-PCR
products separated in 1.8% agarose gel
electrophoresis and stained with
ethidium bromide showing the bands
of the expected sizes 663 bp (KCC3)
and 285 bp (actin). C and D: densitometric analysis normalized with respect to actin (optical density as a percentage of control) representing the
means ⫾ range (*P ⬍ 0.001) of 2 independent experiments each. E: linear
regression analysis and correlation
(r2 ⫽ 0.902) between the theoretical
half-life of NO release (t1/2, min) from
NONOates [NOC-9 (t1/2 ⫽ 3), NOR-3
(t1/2 ⫽ 40), NOC-5 (t1/2 ⫽ 93), and
NOC-18 (t1/2 ⫽ 3,600)], and the levels
of KCC3 mRNA expression after 2 h of
stimulus.
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titative RT-PCR conditions were established in our laboratory to allow comparisons between the expression of 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. The relative expression levels of KCC3 mRNA
isoforms with respect to actin were determined by using 250
ng total RNA as a template, 0.2 mM dNTPs, 1.25 mM MgSO4,
5 U avian myeloblastosis virus reverse transcriptase-Thermus flavus DNA polymerase, and 28 cycles of PCR (GeneAmp
PCR System 2700). As a control, we analyzed the expression
of actin mRNA using specific rat primers (50 pmol), the same
condition as before, and 18 PCR cycles. These were optimal
conditions for the semiquantitative analysis of KCC3 mRNA,
and the analysis was limited to the products generated only
in the exponential phase of the amplification (10). As a
negative control for each set of primers, RT-PCR reactions
were performed in the absence of RT and/or RNA. After
RT-PCR, the content of each independent reaction tube was
analyzed by 1.8% agarose gel electrophoresis. The bands
(KCC3, 663 bp; and actin, 285 bp) were visualized with
fluorescent dye, and the stained gels were depicted as an
inverse image for clear results. Stained gels were scanned,
digitalized, and densitometrically analyzed with the National Institutes of Health Java-based ImageJ software
(Linux). KCC1 mRNA semiquantitation was performed as
previously described (9).
Statistical analysis. The analysis of multiple intergroup
differences in each experiment was conducted by one-way
analysis of variance followed by Student’s t-test. A P ⬍ 0.05
was considered statistically significant. Except when indicated, all values were obtained from two independent experiments in which each single value represents a pool of three
samples.
NONOATES AND KCC GENE EXPRESSION IN VASCULAR SMOOTH MUSCLE
and KCC1 mRNA levels after 2 h of stimulus was also
found.
NO per se is a mediator in KCC3 mRNA upregulation. NO released from NO donors generates several
compounds related or not to NO (NO by-products and
NO donor metabolites, respectively), which may be
responsible for the NO donor actions observed (6, 11).
Hence, to determine the role of NO in the induction of
KCC3 gene expression, we exposed VSMCs with 1.0
mM NOC-5 and NOC-9 in the presence or absence of
PTIO, a well-known NO scavenger (7). Because PTIO
reacts specifically with NO to produce the NO2䡠 radical,
we tested both the requirement for NO and the potential effects of NO2䡠 and its derived products (N2O4,
NO2⫺, and NO3⫺) in the regulation of KCC3 mRNA
expression. As shown in Fig. 4, A and B, coincubation
with PTIO not only effectively blocked the NOC-9- and
Fig. 3. Effect of rapid versus slow NO donors on KCC1 mRNA expression in rat VSMCs. Semiquantitative RT-PCR
analysis performed using 0.50 ␮g total RNA from rat VSMCs under different treatments as noted. A: timedependent actions of 1.0 mM NONOates (NOC-9, NOC-5, and NOC-18) on KCC1 mRNA expression levels.
Representative RT-PCR products separated in 1.8% agarose gel electrophoresis and stained with ethidium bromide
showing the bands of the expected sizes 409 bp (KCC1) and 285 bp (actin). B: densitometric analysis normalized
with respect to actin (optical density as a percentage of control) representing the means ⫾ range (*P ⬍ 0.001) of
2 independent experiments each. C: linear regression analysis and correlation (r2 ⫽ 0.931) between t1/2 (min) from
NOC-9 (t1/2 ⫽ 3), NOC-5 (t1/2 ⫽ 93), and NOC-18 (t1/2 ⫽ 3,600) and the extent of KCC1 mRNA expression after 2 h
of stimulus.
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and C, shows that only the rapid NO releasers (NOC-9,
NOC-5, NOR-3, and SNP) increased KCC3 mRNA expression, whereas NOC-18 had no effect under the
same experimental conditions (Fig. 2, B and D). Furthermore, the theoretical t1/2 of NO release from the
different NONOates strongly correlated (r2 ⫽ 0.902)
with the extent of KCC3 mRNA expression under our
experimental conditions (Fig. 2E).
Because the relevance of the NO-sGC-PKG signaling
pathway in the regulation of KCC1 gene expression in
VSMCs was recently demonstrated (9), we also tested the
actions of NONOates on KCC1 gene expression. As
shown in Fig. 3 and similar to the results for KCC3
mRNA expression (Figs. 1 and 2), only the fast NO
releasers, NOC-9 and NOC-5, but not NOC-18, increased
KCC1 mRNA expression. In addition, a strong correlation (r2 ⫽ 0.931) between the theoretical NONOate t1/2
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NONOATES AND KCC GENE EXPRESSION IN VASCULAR SMOOTH MUSCLE
NOC-5 actions, but also prevented the NOC-5-mediated increase in KCC3 mRNA expression in the timeframe tested (Fig. 4C). Thus the results presented here
support the role of NO per se as the direct participant
Fig. 5. Effect of YC-1 on KCC3 mRNA
expression levels in VSMCs. Cells were
treated with increasing concentrations
of YC-1 (5, 25, 50 ␮M) and with YC-1
(25 ␮M) in the presence or absence of
LY-83583 (50 ␮M) during 2 h. Total
RNA from rat VSMCs were obtained,
and 0.25 ␮g each was subjected to semiquantitative RT-PCR analysis. A: concentration-dependent effect of YC-1 on
KCC3 mRNA expression levels. B and
D: densitometric analysis normalized
with respect to actin (optical density in
arbitrary units as a percentage of control) from A and C, respectively, representing the mean and range for 2 independent experiments (*P ⬍ 0.001). C:
effect of LY-83583 on YC-1-induced
KCC3 mRNA expression: semiquantitative RT-PCR products were electrophoresed in 1.8% agarose gel and
stained with ethidium bromide to show
the bands of the expected sizes 663
(KCC3) and 285 bp (actin). See text for
definition of YC-1.
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Fig. 4. Effect of a NO scavenger on NONOate-induced KCC3 mRNA
expression in rat VSMCs. Semiquantitative RT-PCR analysis performed
using 0.25 ␮g total RNA from rat VSMCs under different treatments as
noted. A: VSMCs were exposed to 1.0 mM NOC-9 and NOC-5 in the
presence or absence of PTIO (1.0 mM) during 2 h. B: densitometric analysis
normalized with respect to actin (optical density as a percentage of control)
representing the mean and range for 2 independent experiments (*P ⬍
0.001). C: VSMCs incubated in the presence of 1.0 mM NOC-5 plus PTIO
(1.0 mM) during the indicated times (time 0 corresponds to mRNA
samples obtained immediately after addition of NOC-5 ⫹ PTIO). KCC3
and actin mRNA expression levels were tested by semiquantitative
RT-PCR using specific primers designed to amplify 663 bp (KCC3) or
285 bp (actin) products. See text for PTIO definition.
in the actions of NO donors, instead of the stable
NO-derived by-products or NONOate metabolites.
sGC is involved in the regulation of KCC3 gene expression in VSMCs. In previous studies, we have shown
the relevance of sGC/PKG in the regulation of KCC activity and KCC1 mRNA expression in red blood cells and
VSMCs (1–3, 9). However, the role of sGC on KCC3
mRNA expression remained to be shown. Thus we incubated VSMCs with YC-1, a direct stimulator of sGC (4,
18, 20). As shown in Fig. 5, A and B, YC-1 increased the
KCC3 mRNA expression levels in a concentration-dependent manner. As predicted, and in analogy to our previous findings (9), YC-1-induced KCC3 mRNA expression
in VSMCs was blocked when coincubated with LY-83583,
a well-characterized sGC inhibitor (4) (Fig. 5, C and D).
Thus our experiments suggest a direct role of sGC in the
regulation of KCC3 gene expression in VSMCs.
Many, but not all, of the biological actions of NO are
mediated by sGC (4, 20), and SNP increased KCC1
mRNA expression through a sGC-dependent mechanism (9). To further investigate whether NONOates
increase KCC3 mRNA expression in a sGC-dependent
manner, we used NOC-9 and NOC-5 in the presence or
absence of LY-83583 for 2 h. As shown in Fig. 6, A and
B, the NONOates-induced KCC3 mRNA expression in
VSMCs was effectively blocked by LY-83583, validating the role of sGC as a mediator of the NO donor
actions on KCC3 mRNA expression.
NONOATES AND KCC GENE EXPRESSION IN VASCULAR SMOOTH MUSCLE
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DISCUSSION
Unlike other NO donor families, NONOates spontaneously release NO in a predictable manner and independently of biological reactants making this class of
compounds ideal for NO research in vitro (5, 7, 11, 13,
21). However, much information is lacking concerning
their mechanism of action in vivo. The rate of NO
release from different NONOates directly correlates
with their vasorelaxant actions in vitro and probably in
vivo (6, 11, 21), as well as with the extent of sGC
activation (5) and mRNA stability (7). Interestingly,
both KCC1 and KCC3 mRNAs are maximally induced
in response to 8-bromo-cGMP after 60–120 min of
treatment, independently of de novo mRNA synthesis
(9, 10). In line with our previous reports (1–3, 10)
showing positive actions of NO donors on total KCC
activity and KCC1 gene expression in red blood cells
and VSMCs, the evidence presented here suggests that
the fast NO releasers like NOC-9 and NOC-5, but not
the slow generator of NO NOC-18, were able to upregulate both KCC1 and KCC3 mRNA expression (Figs. 1
and 2). Moreover and despite the fact that we did not
determine the different NO flux rates from each
NONOate under our specific experimental conditions,
we found a strong correlation between the theoretical
t1/2 of NO release from NONOates and KCC mRNA
expression levels (Figs. 2E and 3C), suggesting that the
NO flux rate is relevant for the regulation of both KCC
genes.
The fact that equimolar concentrations of NOC-18
had no effect on KCC1 and KCC3 gene expression
under the same experimental conditions is probably
related with the long t1/2 of NO release at physiological
pH (13, 24). However, longer incubation times of
VSMCs with 1.0 mM NOC-18 (6–12 h) resulted in
visible nuclear condensation and cell detachment, precluding the study of chronic actions of this NONOate
on KCC mRNA expression. Furthermore, lower concentrations of NOC-18 (0.01–0.1 mM) were also ineffective in the induction of KCC mRNA expression, and
AJP-Heart Circ Physiol • VOL
decayed NOC-18 solutions produced irreproducible results (data not shown). Thus whether or not the absence of a NOC-18 effect on KCC gene expression
correlates with the fact that under physiological conditions 1 mol of freshly prepared NOC-18 releases up to
2 moles of NO with a t1/2 of more than 3,000 min (25),
remains to be established.
Most of the biological actions of NONOates are mediated by NO (11), and the role of the NO-sGC-PKG
pathway in the regulation of KCC1 mRNA expression
was demonstrated (9). However, NONOate metabolites, as well as NO-related by-products may have
biological effects. Moreover and because NO can rapidly decompose to form the radical NO2䡠 , followed by
N2O3/N2O4, and then a mixture of NO3⫺/NO2⫺, the stimulatory actions of NOC-9 and NOC-5 observed on
KCC3 mRNA expression could be mediated by NO,
NONOate metabolites, and/or NO-related by-products.
However, only freshly added NONOates were able to
promote an increase in KCC mRNA expression (Figs. 1
and 2, A and B), whereas old NONOate solutions were
not effective or showed irreproducible results (NOC-18,
data not shown). These data point toward a direct
involvement of short-lived compound(s) likely NO,
and/or perhaps NO2䡠 in the induction of KCC mRNA. To
further pinpoint the role of NO versus NO2䡠 on KCC3
mRNA expression, we used PTIO, which scavenges
NO. As shown by our data, PTIO blocked NOC-5- and
NOC-9-stimulated KCC3 gene expression (Figs. 4, C
and D), and because PTIO produces NO2䡠 (and its derivatives N2O4, NO2⫺, and NO3⫺) after reaction with NO
(26), we can conclude that NO but not NO2䡠 or its
derivatives is directly involved in the induction of
KCC3 mRNA expression under our experimental conditions. In line with these findings, PTIO also prevented NOC-5-induced KCC3 mRNA expression during the time frame in which NOC-5 alone had positive
actions on KCC3 gene expression (Figs. 1, B and D and
3C), suggesting that neither NOC-5 metabolites nor
NO-derived by-products are responsible for the effect.
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Fig. 6. Effect of LY-83583 on NONOate-induced KCC3 mRNA expression
in rat VSMCs. VSMCs were treated
with 1.0 mM NONOates (NOC-9 and
NOC-5) in the presence or absence of
LY-83583 (50 ␮M) during 2 h. RT-PCR
was performed using 0.25 ␮g total
RNA from VSMCs. A: KCC3 and actin
mRNA expression levels were tested
by semiquantitative RT-PCR using
specific primers designed to amplify
663 bp (KCC3) or 285 bp (actin) products. B: densitometric analysis normalized with respect to actin (optical density as a percentage of each control)
representing the mean and range for 2
independent experiments (*P ⬍ 0.001).
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NONOATES AND KCC GENE EXPRESSION IN VASCULAR SMOOTH MUSCLE
We thank Jing Zhang (Graduate Student, Biomedical Sciences
Program) for making the primary cultures of rat VSMCs.
This work was supported by the National American Heart Association Grant 0050451N; Dayton Area Graduate Medical Education
Consortium, Wright State University Pruet Seed Grant; and Wright
State University Research Challenge Program Grant 99-623-10.
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sGC is one of the most important receptors for NO
(4), and a NO/sGC-dependent mechanism was implicated in KCC1 mRNA upregulation in VSMCs (9).
LY-83583, a known sGC inhibitor, effectively blocked
the positive actions of NOC-9 and NOC-5 (Fig. 6),
supporting the view of a NO-dependent sGC-mediated
effect of NONOates in KCC3 mRNA regulation. Additionally, a direct role of sGC was confirmed because
YC-1, a direct NO-independent stimulator of sGC (18),
increased KCC3 mRNA expression in a concentrationdependent manner (Fig. 5, A and B). Taken together
and because LY-83583 also inhibited the YC-1-stimulated KCC3 mRNA expression under our experimental
conditions (Fig. 5, C and D), these results support the
concept that in primary cultures of rat VSMCs NO per
se increases KCC3 mRNA expression via a sGC-dependent mechanism.
The increase in KCC mRNA expression correlated
with the half-time of NO release from several NONOates, although the same correlation at the KCC protein
level and/or cotransport activity remains to be shown
and is currently under investigation. Nevertheless, activation of sGC by NO produces cGMP (4, 20) and
stimulates PKG (12, 19), and the results presented
here in conjunction with our previous reports (1–3, 9,
10) suggest that NO per se and the classic NO-sGCcGMP PKG-signaling pathway are involved in the regulation of KCC3 gene expression at the mRNA level in
primary cultures of rat VSMCs.