INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 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 NO-related 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, t_1/2) 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 t_1/2 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-sGC-PKG 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).

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 t_1/2 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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. 2-(4-Carboxylphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO); SNP; 6-(2-hydroxy-1-methyl- 2-nitrosohydrazino)-N-methyl-1-hexanamine (NOC-9); 3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propana- mine (NOC-5); {FK409, (±)-E-ethyl-2-[(E)-hydroxyimino]-5-nitro- 3-hexeneamide} (NOR-3); 2,2'-(hydroxynitrosohydrazino)- bis-ethanamine (NOC-18); 3-(5'-hydroxymethyl-2'-furyl)-1- benzylindazole (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 recommendations of the manufacturer and immediately used. The NONOates half-life (t_1/2) 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'-²⁴⁷²GGA 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 semiquantitative 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.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 (t_1/2 = 3 min) and NOC-5 (t_1/2 = 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 (t_1/2 = 40 min), and SNP (t_1/2 = 2.5 min) during 2 h and with 1.0 mM of the slow NO generator NOC-18 (t_1/2 = 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 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 t_1/2 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).

View larger version (22K): [in this window] [in a new window]   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 µg 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.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effect of rapid versus slow NO donors on KCC3 mRNA expression in rat VSMCs. Semiquantitative RT-PCR analysis performed using 0.25 µg 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.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effect of rapid versus slow NO donors on KCC1 mRNA expression in rat VSMCs. Semiquantitative RT-PCR analysis performed using 0.50 µg total RNA from rat VSMCs under different treatments as noted. A: time-dependent 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.

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 NO radical, we tested both the requirement for NO and the potential effects of NO and its derived products (N₂O₄, NO, and NO) 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 NOC-5 actions, but also prevented the NOC-5-mediated increase in KCC3 mRNA expression in the time-frame tested (Fig. 4C). Thus the results presented here support the role of NO per se as the direct participant in the actions of NO donors, instead of the stable NO-derived by-products or NONOate metabolites.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of a NO scavenger on NONOate-induced KCC3 mRNA expression in rat VSMCs. Semiquantitative RT-PCR analysis performed using 0.25 µg 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.

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.

View larger version (21K): [in this window] [in a new window]   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 µM) and with YC-1 (25 µM) in the presence or absence of LY-83583 (50 µM) during 2 h. Total RNA from rat VSMCs were obtained, and 0.25 µg 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.

View larger version (18K): [in this window] [in a new window]   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 µM) during 2 h. RT-PCR was performed using 0.25 µg 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).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 t_1/2 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 t_1/2 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 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 t_1/2 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 NO, followed by N₂O₃/N₂O₄, and then a mixture of NO/NO, 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 NO in the induction of KCC mRNA. To further pinpoint the role of NO versus NO 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 NO (and its derivatives N₂O₄, NO, and NO) after reaction with NO (26), we can conclude that NO but not NO 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.

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 concentration-dependent 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-sGC-cGMP PKG-signaling pathway are involved in the regulation of KCC3 gene expression at the mRNA level in primary cultures of rat VSMCs.