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Endothelial dysfunction has been linked to a decrease in nitric oxide (NO) bioavailability and attenuated endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation. The small (SKCa) and intermediate (IKCa) calcium-activated potassium channels play a key role in endothelium-dependent relaxation. Since the repressor element 1-silencing transcription factor (REST) negatively regulates IKCa expression, we hypothesized that augmented REST and decreased IKCa expression contributes to impaired endothelium-dependent vasodilation associated with hypertension. Acetylcholine (ACh) responses were slightly decreased in small mesenteric arteries from male stroke-prone spontaneously hypertensive rats (SHRSP) vs. arteries from Wistar Kyoto (WKY) rats. Incubation with L-NAME (100µM) and indomethacin (100µM) greatly impaired ACh responses in vessels from SHRSP. Iberiotoxin (0.1µM), selective inhibitor of large-conductance KCa (BKCa) channels, did not modify EDHF-mediated vasodilation in SHRSP or WKY. UCL-1684 (0.1µM), selective inhibitor of SKCa channels almost abolished EDHF-mediated vasodilation in WKY, and decreased relaxation in SHRSP. TRAM-34 (10µM) and charybdotoxin (0.1µM), both IKCa inhibitors, produced a small decrease of EDHF relaxation in WKY, but completely abrogated EDHF vasodilation in SHRSP. EDHF-mediated relaxant responses were completely abolished in both groups by simultaneous treatment with UCL-1684 and TRAM-34 or charybdotoxin. Relaxation to SKCa/IKCa channels agonist NS-309 was decreased in SHRSP arteries. Expression of SKCa was decreased, whereas IKCa was increased in SHRSP mesenteric arteries. REST expression was reduced in arteries from SHRSP. Vessels incubated with TRAM-34 (10µM) for 24 hours, displayed reduced REST expression, and no differences in IKCa. In conclusion, IKCa channels upregulation, via decreased REST, seems to compensate deficient activity of SKCa channels in the vasculature of spontaneously hypertensive rats.
The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing vasodilator factors, such as prostacyclin (PGI2), nitric oxide (NO), and a yet unidentified endothelium-derived hyperpolarizing factor (EDHF) (1). Because EDHF appears to decline with advancing age and to be targeted in diseases such as hypertension and diabetes, knowledge of the ionic mechanisms underlying EDHF actions would be expected to improve understanding of the nature of EDHF and how vascular tone is regulated during health or disease conditions (2).
Extensive studies have been performed in order to better understand EDHF actions. Accordingly, different hypotheses try to explain the mechanisms that mediate EDHF-induced vasodilatation. One of these hypothesis suggest that EDHF leads to endothelial hyperpolarization via activation of Ca2+-activated K+ channels (KCa). Hyperpolarization of the vascular smooth muscle cells can be elicited via myo-endothelial gap junctions to result in hyperpolarization of the smooth muscle cells (3–6), and reduced Ca2+ influx via voltage-operated Ca2+ channels (7–9).
Ca2+-activated K+ (KCa) channels are especially important in EDHF-mediated relaxation and hyperpolarization in resistance-sized arteries but the role of these channels during hypertension is far from clear (10–14). Changes in expression or activity of KCa channels may therefore be a fundamental event contributing to the progression of arterial dysfunction during hypertension. It was previously shown that small-mesenteric arteries from angiotensin II hypertensive rats maintain EDHF-like responses, despite reduced expression of intermediate KCa (IKCa) and small KCa (SKCa), (15). Whether these changes in KCa channel function and expression during hypertension occur at the level of resistance-sized arteries, and whether they are needed to regulate vascular tone and hence local blood flow, is not completely understood and requires further studies (14).
Repressor element 1-silencing transcriptional factor (REST, also known as NRSF) is a transcription factor that regulates IKCa channel gene expression (16–18). The IKCa channel gene sequence is encoded by the KCNN4 sequence and REST has been shown to regulate this sequence. Cheong and colleagues (2005) showed that this transcription factor modulates potassium channel expression in vascular smooth muscle cells (17). Therefore, alterations in REST regulation can have an important impact in the development of vascular disease by KCa channels alterations. Therefore, we hypothesized that increased REST expression during hypertension negatively modulates IKCa channel expression, contributing to reduced endothelium-dependent vasodilation in hypertension.
Five month-old male stroke-prone spontaneously hypertensive rats (SHRSP) were obtained from the breeding colony at the Medical College of Georgia. Age-matched male Wistar-Kyoto (WKY) rats were purchased from Harlan (Indianapolis IN). Rats were maintained on a 12-hour light dark cycle, housed two per cage and allowed access to normal chow and water ad libitum. Systolic blood pressure (SBP) was measured in non-anesthetized animals by tail cuff using a RTBP1001 blood pressure system (Kent Scientific Corporation, Connecticut, MA, USA). All procedures were performed in accordance with the Guiding Principles in the Care and Use of Animals, approved by the Medical College of Georgia Committee on the Use of Animals in Research and Education.
After euthanasia, the mesentery was rapidly excised and placed in an ice-cold physiological salt solution (PSS) containing: 130 mM NaCl, 14.9 mM NaHCO3, 5.5 mM dextrose, 4.7 mM KCl, 1.18 mM KH2PO4, 1.17 mM MgSO4.7H2O, 1.6 mM CaCl2.2H2O, and 0.026 mM EDTA. Second-order mesenteric arteries were carefully dissected and mounted as ring preparations on two stainless steel wires (200 mm in length with internal diameter 100 to 150 µm) in an isometric Mulvany-Halpern small-vessel myograph (Danish MyoTech, Aarhus, Denmark). One wire was attached to a force transducer and the other to a micrometer. Both dissection and mounting of the vessels were carried out in cold (4°C) PSS. The segments were adjusted to maintain a passive force of 3.5 mN. Vessels were equilibrated for 45 min in PSS at 37°C. Arterial integrity was assessed first by stimulation of vessels with KCl (120 mM) and, after washing and a new stabilization period, by contracting the segments with phenylephrine (10µM) followed by stimulation with acetylcholine (ACh; 10µM).
After being washed, the arterial rings were contracted with the thromboxane analog 9,11-dideoxy-9α,11α-methanoepoxyprostaglandin F2α (U-46619, 0.3µM). Cumulative concentration-response curves to ACh were performed under control conditions and in the combined presence of indomethacin (inhibitor of cyclooxygenase, 10 µM) and Nω-nitro-l-arginine methyl ester [L-NAME, inhibitor of NO synthase, 100µM]. To study the role of KCa channels in EDHF-mediated relaxations, ACh-induced vasorelaxation was determined in the presence of L-NAME and indomethacin. This procedure rules out any potential interference of NO and prostaglandins on EDHF-mediated responses (19). The effects of IKCa inhibitors (TRAM-34, 10µM and charybdotoxin, 0.1µM), a SKCa inhibitor (UCL-1684, 0.1µM) and a BKCa inhibitor (Iberiotoxin, 0.1µM) on ACh-induced relaxations were determined. These concentrations were chosen based on previous studies from our laboratory (14).
Mesenteric arteries from hypertensive and control rats were isolated, cleaned from fat, dissected and frozen in liquid nitrogen. Proteins (40 µg) extracted from the mesenteric bed were separated by electrophoresis on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Nonspecific binding sites were blocked with 5% skim milk in Tris-buffered saline solution with Tween (0.1%) for 1 hour at 24°C. Membranes were then incubated with antibodies overnight at 4°C. Antibodies were as follows: anti-potassium channel KCa2.3 (also known as SK3) and KCa3.1 (also known as IK1); (1:200, Sigma-Aldrich, MO, USA); REST/NRSF (1:500, Abcam) and β-actin (1:1000, Sigma-Aldrich, MO, USA). After incubation with secondary antibodies, signals were revealed with chemiluminescence, visualized by autoradiography, and quantified densitometrically. Results were normalized by β-actin expression and expressed as percentage of control.
Experimental values were calculated relative to the maximal changes from the contraction produced by U-46619 in each segment. Agonist concentration-response curves were fitted using a nonlinear interactive fitting program (Graph Pad Prism 3.0; GraphPad Software Inc., San Diego, CA). Agonist potencies and maximum response are expressed as negative logarithm of the molar concentration of agonist producing 50% of the maximum response (pD2) and maximum effect elicited by the agonist (Emax), respectively. Data are expressed as means ± SEM (n), where n is the number of experiments performed. Statistical analysis of the concentration-response curves was performed by using two-way analysis of variance (ANOVA) for comparison between the groups. Western blot data were analyzed by one-sample t test. Values of P<0.05 were considered a statistically significant difference.
L-Phenylephrine hydrochloride, ACh, l-NAME, iberiotoxin, indomethacin, sodium nitroprusside and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) were all purchased from Sigma-Aldrich. 9,11-Dideoxy-9α,11α-methanoepoxyprostaglandin F2α (U-46619) was purchased from Calbiochem (San Diego, CA) and 6,12,19,20,25,26-Hexahydro-5,27:13,18:21,24- trietheno-11,7-metheno-7H-dibenzo [b,n] [1,5,12,16] tetraazacyclotricosine-5,13-diium ditrifluoroacetate (UCL-1684) was from Tocris (Ellisville, MI). Indomethacin was dissolved in ethanol, and TRAM-34 and UCL-1684 were dissolved in DMSO. All other stock solutions were prepared by using PSS. Control solutions containing vehicle levels of ethanol and DMSO were also used through the experimental protocol.
At 24 weeks, SHRSP displayed higher systolic blood pressure (mmHg), in comparison with WKY rats (211±7.6, n=15 vs. 119±1.8, n=15; respectively). Body weight of SHRSP was significantly decreased (296 ±6.1g; n=15) when compared with WKY rats (345±5.2g; n=15),
Concentration-response curves to ACh were performed in vessels contracted with U-46619 (0.3µM) to evaluate endothelium-dependent relaxation. ACh-induced relaxation was slightly impaired in small mesenteric vessels from SHRSP vs. those from normotensive rats [Emax 87.7±4% vs. 102.2±1.4%, respectively, (p<0.05)], as shown at Figure 1A.
EDHF-mediated relaxation is particularly apparent when NO and prostaglandins production are blocked. Therefore, simultaneous inhibition of prostaglandins and NO production (by indomethacin; 10µM and L-NAME; 100µM, respectively) were performed in order to assess EDHF-mediated relaxation. At this condition, ACh-induced relaxation was greatly diminished in SHRSP, but not in WKY vessels [Emax 38.45±1.7% vs. 100.6±0.6%, respectively, (p<0.05), Figure 1B]. These results suggest impairment of EDHF production in vessels from hypertensive animals.
It has been shown that relaxations observed in the presence of NO-synthase and cyclooxygenase inhibitors are not necessarily attributed to EDHF-mediated responses, but may result of residual NO release (20, 21). To rule out this hypothesis, ACh-concentration response curve were performed in arteries from WKY and SHRSP, incubated with L-NAME (100µM) and indomethacin (10µM), in the presence or absence of Carboxy-PTIO (100µM), a NO scavenger. Addition of Carboxy-PTIO did not result in greater inhibition of ACh-induced relaxation in WKY [Emax 96.9±7.5% vs 89.1±4.1% (without Carboxy_PTIO)] or SHRSP [Emax 42.5±5.5% vs. 38.4±4.7% (without Carboxy_PTIO)]. These results show that residual NO does not seem to contribute to the differences observed in ACh responses in small-mesenteric arteries from WKY and SHRSP rats in the presence of L-NAME and indomethacin, reinforcing that relaxation is attributed to EDHF-mediated effects.
As previously discussed, KCa channels are especially important for EDHF-mediated relaxation and hyperpolarization in resistance arteries. Accordingly, the effects of KCa channel blockade on EDHF-mediated actions were always studied on arteries treated with both indomethacin (10µM) and L-NAME (100µM). The contribution of different subtypes of KCa channels, BKCa, SKCa and IKCa to ACh-induced relaxation was determined by using selective inhibitors. The effects of these inhibitors on pD2 and Emax values for ACh are summarized at Table 1.
Iberiotoxin (0.1µM), a BKCa channel inhibitor, did not significantly change EDHF-mediated relaxation either in arteries from SHRSP or WKY rats (Figure 2), suggesting a minor contribution of BKCa channel to EDHF-mediated relaxation.
UCL-1684 (0.1µM), a SKCa channel inhibitor, greatly impaired EDHF-induced relaxation in WKY small arteries [82.8% of inhibition of Emax, (p<0.05), Figure 3A], as well as reduced the relaxation in SHRSP arteries [61.9% inhibition of Emax, (p<0.05), Figure 3B]. These data suggest that the SKCa channels mediate EDHF-induced relaxation in arteries from both normotensive and hypertensive rats.
The blockade of IKCa channel with TRAM-34 (10µM) significantly inhibited EDHF-mediated relaxation in arteries from SHRSP [80.5% inhibition of Emax, (p<0.05), Figure 4B], but not in WKY (18.9% inhibition of Emax – Figure 4A). Inhibition of IKCa channel with charybdotoxin (0.1µM) produced similar results. Charybdotoxin significantly inhibited EDHF-mediated relaxation in arteries from SHRSP [78.9% inhibition of Emax, (p<0.05), Figure 4D], but not in WKY (5.3% inhibition of Emax – Figure 4C). These findings suggest that the IKCa channels greatly contribute to EDHF-mediated relaxation in small arteries from SHRSP, but play a minor role in arteries from WKY.
Concentration-response curves to NS309, a SKCa and IKCa channels opener, were also performed. The response to NS309 was decreased in arteries from SHRSP, when compared to those in control arteries [Emax 77.9±3.2 vs. 103.1±3.1%, respectively, (p<0.05), Figure 6]. NS309-induced relaxation was abolished with concomitant incubation of UCL-1684 (0.1µM) and charybdotoxin (0.1µM) – (data not shown).
Responses to NS1619 (a BKCa channel opener) were similar in vessels from SHRSP and WKY rats (Emax 96.1±2.1, n=5 and 92.8±1.4, n=5; respectively) and were fully blocked by incubation with iberiotoxin (0.1µM).
The functional studies performed in small-mesenteric arteries from normotensive and hypertensive animals indicated that EDHF-mediated relaxation has different profiles and is mediated by different subtypes of KCa channel. Analysis of protein expression of BK, SK3 (a SKCa isoform) and IK1 (also known as IKCa) channels were performed to better characterize possible differences in the expression of the KCa channels isoforms, between WKY and SHRSP arteries. SK3 isoform was chosen because this isoform is present in the vascular endothelium (22).
No differences were found in BK protein levels between the groups (Figure 7B). IK1 protein expression was increased in mesenteric arteries from SHRSP, when compared to WKY arteries (Figure 7B). On the other hand, protein expression levels of SK3 in mesenteric arteries from SHRSP were reduced compared with those in WKY values (Figure 7B). Although IKCa channel was increased in arteries of SHRSP, it seems that this up-regulation is not sufficient to restore impaired EDHF-mediated relaxation in SHRSP.
Because REST was shown to negatively regulate IKCa channel expression (17), we evaluated protein levels for this transcription factor. Anti-REST antibody resulted in a predicted band size of 121kDa and an additional band was observed at 50kDa position. Mesenteric arteries from SHRSP displayed reduced REST expression, when compared with arteries from WKY (Figure 8A–B).
We took advantage from a pharmacological approach to inhibit IKCa channel, and determined whether blockade of these channels could lead to changes in REST or IK1 expression. After 24 hours of incubation with TRAM-34 (10µM) or vehicle (DMSO), proteins were extracted from mesenteric vessels from normotensive and hypertensive rats and expression of both IK1 and REST was determined. REST expression was drastically reduced after TRAM-34 incubation, both in arteries from WKY and SHRSP (Figure 8A–B). The difference in IK1 expression between arteries from normotensive and hypertensive animals was abolished after 24 hours of IKCa channel blockade. In order to control for changes in protein expression due to the 24 hour-incubation period, we compared IK1 and REST expression in freshly harvested mesenteric arteries and in arteries incubated for 24 hours with DMSO. No significant differences were found in IK1 and REST expression between freshly isolated and incubated arteries from control or hypertensive animals, indicating that changes in protein expression between WKY and SHRSP vessels are not due to the incubation procedure. Collectively, these results demonstrate that modifications in the activity of IKCa channel resulted in changes of REST and IKCa channel expression, reinforcing that REST negatively modulates IK Ca channel in vascular cells.
The endothelium is a major regulator of vascular tone, releasing vasoactive substances such as EDHF, NO, cyclooxygenase metabolites, endothelin-1 and other endothelium-derived constrictor factors (23). In most models of experimental hypertension, the endothelium-dependent relaxation is impaired. However, this endothelial dysfunction presents different characteristics depending on the experimental model studied (24). Konishi and Su first showed that endothelium-dependent relaxation is impaired in arteries from SHR (23). Similar results have been reported in the mesenteric artery from SHRSP (25, 26). Although ACh-induced relaxation was slightly impaired in small mesenteric vessels from SHRSP animals, in the present study we observed pronounced impairment in EDHF-mediated responses.
The IKCa and SKCa channels occur in abundance in endothelial cells and their activation results in EDHF-like hyperpolarization of these cells (10–15, 27, 28). The resulting endothelial hyperpolarization spreads via myoendothelial gap junctions to result in the EDHF-induced hyperpolarization and relaxation of the smooth muscle (2). EDHFs-mediated responses exhibit exquisite sensitivity to the combination of charybdotoxin and apamin (29)? showing the importance of both SKCa and IKCa, respectively, to EDHFs-mediated relaxation. Accordingly, experiments conducted in a transgenic mouse (SK3T/T), in which SK3 expression levels are suppressed with dietary doxycycline, demonstrated that decreased SK3 expression results in a pronounced and reversible elevation of blood pressure, around 30mmHg (30). In addition, K(Ca)3.1(−/−) [or IK1(−/−)] mice display reduced endothelial and smooth muscle hyperpolarization in response to acetylcholine and significant increase in arterial blood pressure, around 30 mmHg (31). Furthermore, IK1(−/−)/SK(T/T) mice treated with dietary doxycycline, which results in combined IK1/SK3 deficiency, display impaired acetylcholine-induced EDHF-mediated relaxation dilation in conduit arteries and in resistance arteries as well as elevated arterial blood pressure (32). These results indicate that the endothelial SKCa and IKCa are fundamental regulators of blood pressure and it seems likely that EDHF, through vascular dependent mechanisms, can influence blood pressure levels.
We have examined the relative contributions of SKCa, IKCa and BKCa to EDHF-mediated response, in SHRSP small mesenteric arteries. Our results demonstrate that the mechanisms underlying EDHF-mediated responses of small mesenteric arteries from SHRSP and WKY rats are distinct. EDHF-mediated relaxation in small mesenteric arteries from WKY rats was prevented by the blockade of SKCa channels, with UCL-1684.
In SHRSP, although SKCa contributes to EDHF-meditate relaxation, this was mainly mediated by IKCa channels. These results are in agreement with a previous report showing that EDHF-mediated relaxation is impaired in SHRSP and that KCa channels have a differential contribution in hypertensive and normotensive animals (26). In addition, the response to NS309 was decreased in arteries from SHRSP, further reinforcing that SKCa and IKCa function is impaired in mesenteric arteries from SHRSP. Unfortunately, no selective agonist for these channels, SKCa and IKCa, are available and although NS309-induced relaxation seems to be abolished in conditions where endothelium is removed (33, 34), it is important to consider that NS309 has been shown also to inhibit L-type voltage-dependent Ca2+-channel (35).
In agreement with our functional data, a differential expression of SKCa and IKCa channels was observed between the groups. Small arteries from SHRSP, when compared with WKY arteries, showed decreased levels of SKCa channel, whereas IKCa channel expression was increased.
Induction of IKCa plays an important role in the vasculature. The gene encoding IKCa, KCNN4, contains a functional REST binding site that is repressed by REST (17). REST has been considered a dynamic factor in smooth muscle cells. Cheong et al (2006) showed that REST gene expression declines when the cells proliferate in culture. Furthermore, they showed that loss of REST is a switch enabling KCNN4 expression (16). Indeed, some reports speculate that REST may be a common factor helping to suppress vascular diseases (16). Here, we showed for the first time that REST expression is closely associated with IKCa expression in arteries from hypertensive animals
Given that IKCa channels are important modulators of EDHF-induced relaxation in small mesenteric vessels from SHRSP, we have hypothesized that REST modulation of IKCa channel expression is decreased in hypertension, as a compensatory mechanism in response to impaired EDHF-mediated relaxation. Our main finding was that REST is down-regulated whereas IKCa channels are over expressed in arteries from SHRSP, when compared with WKY. Furthermore, we showed that the blockade of IKCa channels, by using TRAM-34 for 24 hours, induces vascular down-regulation of REST expression, and also abolishes the differences in IKCa channels expression between arteries from SHRSP and WKY. It is possible that continued inhibition of IKCa channels with TRAM-34 resulted in a compensatory tentative of the vascular tissue to restore this channel function. We speculate that this phenomenon, decreased REST expression and consequently, augmented IKCa channels expression, is a compensatory mechanism in the vasculature, to counter balance impaired EDHF-mediated relaxation.
It has been previously shown that TRAM-34 treatment modulates IK1 and REST expression. Tharp and colleagues (2008) demonstrated that coronary balloon injury robustly increased IK1 and decreased REST expression 2 hours postangioplasty. Interestingly, they showed that TRAM-34 delivered via balloon catheter blocked not only IK1 upregulation, but also the suppression of REST, which is in close opposition to the results presented here. However, one needs to consider that a different experimental model (an in vivo model for coronary injury), different TRAM-34 concentration and treatment duration may partially explain the differential results. In addition, our results point to the important observation that IK1 activity is able to modulate REST expression. REST expression probably is modulated by various factors, but the lack of information on REST expression regulation precludes further speculations.
Although KCa channels seem to play a major role in EDHFs-mediated relaxation (29), endothelial cell hyperpolarization can also result from endothelial to vascular smooth muscle cells interactions through myo-endothelial gap junctions (36, 37), as well as activation of inwardly K+ channels and Na+-K+-ATPase (38, 39). Other important point is that alterations in the vessel structure and vascular remodeliling, as a consequence of prolonged hypertension, could contribute to impaired EDHF-response in small-mesenteric arteries from SHRSP. It seems likely that increases in the number of SMC in the media may have an impact on EDHF ability to hyperpolarize the smooth muscle (40). These are important points that also need to be further evaluated in order to completely understand EDHF-dependent mechanisms involved in vascular relaxation during physiological and pathophysiological conditions.
In conclusion, we have shown that small-mesenteric arteries from SHRSP display significant impairment in EDHF-mediated relaxation. In addition, SKCa channels importantly contribute to this phenomenon both in normotensive and hypertensive animals, but IKCa channel plays a greater and important role in the EDHF-mediated relaxation in arteries from SHRSP. Protein levels of SKCa and IKCa channels were altered in arteries from hypertensive and REST seems to be a negative modulatory mechanism, controlling the augmented levels of IKCa channels in the SHRSP vasculature.
This study was supported by grants from the National Institutes of Health (HL-71138 and HL-74167). F.R.C.G. and F.S.C. are supported by doctoral fellowships from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP - São Paulo, Brazil). The authors declared no disclosures.
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