Published online before print April 20, 2009, doi: 10.1161/CIRCULATIONAHA.108.846634
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(Circulation. 2009;119:2323-2332.)
© 2009 American Heart Association, Inc.
Hypertension |
Genetic Deficit of SK3 and IK1 Channels Disrupts the Endothelium-Derived Hyperpolarizing Factor Vasodilator Pathway and Causes Hypertension
From the Department of Internal Medicine/Nephrology, Philipps University, Marburg, Germany (S.B., A.K., C.B., B.P.K., M.K., A.H., I.G., J.H., R.K.); Department of Physiology, University of Lübeck, Lübeck, Germany (V.J.S., S.E.W., C.d.W.); Division of Nephrology, Albert Einstein College of Medicine, Bronx, NY (H.S.); Vollum Institute, Oregon Health and Science University, Portland (C.T.B., J.P.A.); and Department of Pharmacology, University of California, Davis (H.W.).
Correspondence to Ralf Köhler, PhD, Biomedical Research Center, Hans-Meerweinstrasse, 35037 Marburg, Germany. E-mail rkoehler{at}med.uni-marburg.de
Received December 22, 2008; accepted March 3, 2009.
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Abstract |
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Background— It has been proposed that activation of endothelial SK3 (KCa2.3) and IK1 (KCa3.1) K+ channels plays a role in the arteriolar dilation attributed to an endothelium-derived hyperpolarizing factor (EDHF). However, our understanding of the precise function of SK3 and IK1 in the EDHF dilator response and in blood pressure control remains incomplete. To clarify the roles of SK3 and IK1 channels in the EDHF dilator response and their contribution to blood pressure control in vivo, we generated mice deficient for both channels.
Methods and Results— Expression and function of endothelial SK3 and IK1 in IK1–/–/SK3T/T mice was characterized by patch-clamp, membrane potential measurements, pressure myography, and intravital microscopy. Blood pressure was measured in conscious mice by telemetry. Combined IK1/SK3 deficiency in IK1–/–/SK3T/T (+doxycycline) mice abolished endothelial KCa currents and impaired acetylcholine-induced smooth muscle hyperpolarization and EDHF-mediated dilation in conduit arteries and in resistance arterioles in vivo. IK1 deficiency had a severe impact on acetylcholine-induced EDHF-mediated vasodilation, whereas SK3 deficiency impaired NO-mediated dilation to acetylcholine and to shear stress stimulation. As a consequence, SK3/IK1-deficient mice exhibited an elevated arterial blood pressure, which was most prominent during physical activity. Overexpression of SK3 in IK1–/–/SK3T/T mice partially restored EDHF- and nitric oxide-mediated vasodilation and lowered elevated blood pressure. The IK1-opener SKA-31 enhanced EDHF-mediated vasodilation and lowered blood pressure in SK3-deficient IK1+/+/SK3T/T (+doxycycline) mice to normotensive levels.
Conclusions— Our study demonstrates that endothelial SK3 and IK1 channels have distinct stimulus-dependent functions, are major players in the EDHF pathway, and significantly contribute to arterial blood pressure regulation. Endothelial KCa channels may represent novel therapeutic targets for the treatment of hypertension.
Key Words: blood pressure • endothelium-derived factors • ion channels • KCa3.1 protein, mouse
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Introduction |
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The arterial endothelium modulates the contractile state of the underlying vascular smooth muscle and thus blood pressure by releasing the vasodilating autacoids nitric oxide (NO)1 and prostacyclin (prostaglandinI2 [PGI2]).2 Besides NO and PGI2, a third factor or signaling pathway termed endothelium-derived hyperpolarizing factor (EDHF)3 adds to endothelium-dependent vasodilation by causing hyperpolarization and relaxation of vascular smooth muscle. Although EDHF has been proposed to be as powerful3–5 as NO and PGI2 in controlling vascular tone and blood pressure, direct evidence for this is missing. Moreover, the molecular identities of EDHF and the EDHF-signaling pathways are still a matter of ongoing debate. Indeed, EDHF dilatory responses have been attributed to a variety of diffusible EDHFs such as cytochrome P450–generated metabolites of arachidonic acid,6,7 lipoxygenase products,8 NO itself,9 hydrogen peroxide,10 and C-type natriuretic peptide,11 as well as to electrotonic coupling12 of endothelium and smooth muscle (for extensive review, see References 3 and 5). Whatever EDHF might be, activation of the Ca2+-activated K+ channels SK3 (KCa2.3)13 and IK1 (KCa3.1),14 located in the endothelium,15–18 has been proposed to initiate EDHF signaling in a variety of vascular beds and species.17–20 On intracellular Ca2+ mobilization in response to receptor stimulation by pharmacological stimuli, SK3 and IK1 mediate K+ efflux and produce membrane hyperpolarization.16,18 This endothelial hyperpolarization and/or the concomitant K+ efflux17 causes subsequent smooth muscle hyperpolarization, leading to the closure of voltage-activated Ca2+ channels and finally relaxation. However, the concept that IK1 and SK3 are required to initiate the EDHF dilator response is based mainly on studies of isolated vessels and pharmacological evidence.3,5,17,18 Recently, we reported that IK1-deficient mice (IK1–/–) exhibit a partial defect in EDHF signaling and a moderately elevated blood pressure.20 Genetic modulation of SK3 expression in mice21 has also been shown to alter blood pressure.22 In these SK3T/T mice, expression levels of SK3 are controlled by site-specific insertion of a tetracycline-based genetic switch.21 Dietary doxycycline administration dramatically diminished SK3 expression, whereas the channel is overexpressed
3-fold in the absence of doxycycline.21 Although SK3 suppression by doxycycline treatment causes a substantial elevation of blood pressure,22 it is unknown whether the higher blood pressure in SK3T/T (+doxycycline) mice is related to defects in EDHF signaling. Thus, the understanding of the specific roles of SK3 and IK1 in the EDHF dilator response and the contribution of EDHF to endothelial control of vascular tone, and ultimately blood pressure in vivo, is incomplete.3,5 To elucidate the roles of SK3 and IK1, we generated mice deficient for both channels (IK1–/–/SK3T/T+doxycycline) and studied signaling related to EDHF as well as arterial blood pressure in these mice. The present study identifies endothelial SK3 and IK1 channels as major effectors in EDHF signaling in vivo and reveals their significant impact on systemic blood pressure control.
Clinical Perspective on p 2332
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Methods |
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IK1–/–SK3T/T animals were generated by interbreeding SK3T/T mice and IK1–/– mice and genotyped by polymerase chain reaction. For suppression of SK3, mice received doxycycline in the drinking water (2 mg/mL, 2% sucrose) for at least 6 days. SK3/IK1 expression was detected by Western blot analysis of brain (SK3) and liver extracts (IK1). Vascular SK3/IK1 expression was detected by immunohistochemistry on carotid arteries (CA). Whole-cell IK1/SK3 currents were recorded in freshly isolated CA endothelial cells with the use of patch-clamp and smooth muscle membrane potentials in CA by sharp-electrode techniques. Endothelium-dependent and -independent vasodilation was studied by pressure myography in CA and intravital microscopy of resistance arterioles in the cremaster muscle in vivo. Arterial blood pressure, heart rate, and locomotor activity were measured telemetrically in freely moving mice for at least 3 consecutive days in the presence or absence of doxycycline or 24 hours before and after injection of the IK1-opener SKA-31.
Statistical Analysis
Data are given as mean±SEM. One-way ANOVA tests followed by Newman-Keuls or Bonferroni post hoc tests or paired Student t test were used as indicated to assess differences between groups.
For a detailed description of Methods, see the online-only Data Supplement.
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Results |
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Generation of IK1–/–/SK3T/T Mice
IK1–/– and SK3T/T mice were generated as described previously20,21 and interbred to generate IK1–/–/SK3T/T (Figure 1A). IK1–/–/SK3T/T offspring were viable, fertile, and had no overt behavioral or neurological defects with or without doxycycline administration. In IK1X/SK3T/T mice, dietary doxycycline for at least 6 days virtually abolished SK3 expression in brain (Figure 1B). In IK1–/–/SK3X, loss of IK1 protein was confirmed in liver (Figure 1B). Immunohistochemistry demonstrated loss of endothelial SK3 and IK1 from IK1–/–/SK3T/T+doxycycline and overexpression of SK3 in IK1–/–/SK3T/T–doxycycline (Figure 1C).
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-tubulin demonstrates equal loading. Bottom, IK1 in liver from IK1+/+/SK3X mice. C, Immunohistochemistry of SK3 (top) and IK1 (bottom) expression in endothelium of CA. Note the absence of specific IK1 and SK3 staining in endothelium of IK1–/–/SK3T/T+doxycycline and the intense SK3 staining in IK1–/–/SK3T/T–doxycycline compared with WT. Me indicates media; Lu, lumen; and En, endothelium.
Combined IK1 and SK3 Suppression Abolishes Endothelial KCa Currents and Reduces Endothelium-Mediated Smooth Muscle Hyperpolarization
Endothelial IK1/SK3 functions were determined by whole-cell patch-clamp experiments20 on freshly isolated CA endothelial cells (Figure 2A through 2G). On Ca2+ dialysis, wild-type (WT) CA endothelial cells showed a K+ current that was abolished by a combination of the IK1 blocker TRAM-3423 (1 µmol/L) and the SK blocker UCL168424 (1 µmol/L). After suppression of SK3 by dietary doxycycline, the amplitude of this K+ current was reduced by 50% in SK3T/T mice, and the remaining K+ current was inhibited by TRAM-34. TRAM-34 unmasked a small UCL1684-sensitive current, indicating that the doxycycline treatment reduced SK3 expression by 90%.22 The total K+ current was reduced to a similar extent in IK1–/– mice, and the residual current was abolished by UCL1684. In doxycycline-administered IK1–/–/SK3T/T mice, the K+ current was virtually abolished, and the very small residual K+ current was sensitive to UCL1684. In contrast, the UCL1684-sensitive K+ current was 10-fold larger in untreated IK1–/–/SK3T/T mice compared with the composite K+ current in WT CA endothelial cells, which reflects the overexpression of SK3. In doxycycline-administered IK1+/+/SK3T/T mice, the IK1-opener SKA-31(naphtho[1,2-d]thiazol-2-ylamine)25 (500 nmol/L) enhanced the TRAM-34–sensitive K+ currents by 2- to 3-fold, thus strongly potentiating IK1 currents, without an appreciable effect on the residual UCL1684-sensitive SK3 current.
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To study endothelial IK1/SK3-mediated hyperpolarization of smooth muscle to acetylcholine (100 nmol/L), we performed sharp-electrode measurements of smooth muscle membrane potentials in pressurized (80 mm Hg) and preconstricted (phenylephrine, 1 µmol/L) CA, as shown in Figure 3. In WT, acetylcholine elicited a robust hyperpolarization from a resting membrane potential of –40±1 to –61±4 mV. Suppression of SK3 (SK3T/T+doxycycline) slightly reduced the hyperpolarization to acetylcholine (from –40±1 to –57±1 mV; P<0.05 versus WT). In IK1–/–, the hyperpolarization response was markedly reduced (from –40±1 to –51±1 mV; P<0.01 versus WT and SK3T/T+doxycycline). In CA from IK1–/–/SK3T/T+doxycycline, the hyperpolarization response was attenuated even further (from –39±1 to –45±1 mV; P<0.01 versus other groups). However, in CA that lacked IK1 and overexpressed SK3 (IK1–/–/SK3T/T –doxycycline), we observed a restoration (from –39±1 to –58±1 mV), and the response was not different from WT. Interestingly, potentiation of IK1 functions by SKA-31 in IK1+/+/SK3T/T+doxycycline strongly enhanced acetylcholine-induced hyperpolarization (from –41±2 to –78±1 mV; P<0.01).
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MP) from different groups. Dox indicates doxycycline. Values are given as mean±SE; numbers (n) in charts refer to number of CA. *P<0.05, **P<0.01 vs WT, 1-way ANOVA followed by Newman-Keuls post hoc test; #P<0.01 vs IK1+/+/SK3T/T+doxycycline.
Loss of Endothelial IK1 and SK3 Channels Disrupts the EDHF Dilator Response
We next examined EDHF-type vasodilation in pressurized and preconstricted CA20 using a "classic" EDHF protocol3 in which we blocked NO synthase using N
-nitro-L-arginine (L-NNA) (300 µmol/L) and cyclooxygenase using indomethacin (10 µmol/L) to eliminate NO-mediated and, although unlikely in these murine vessels, prostacyclin-mediated vasodilations.20 Figure 4A shows representative tracings of acetylcholine-induced EDHF-type vasodilations for WT, IK1–/–, IK1–/–/SK3T/T+doxycycline, and IK1–/–/SK3T/T– doxycycline. For concentration-response curves from all groups, see Figure 4B. Intraluminally applied acetylcholine (100 nmol/L) produced a robust EDHF-type vasodilation in WT. In contrast, in CA from IK1–/–/SK3+/+, vasodilation to 100 nmol/L acetylcholine was reduced by 75% (P<0.001), and in CA from IK1–/–/SK3T/T+doxycycline, 100 nmol/L acetylcholine induced virtually no dilation (99% reduction; P<0.001; Figure 4B, left panel). EDHF-type vasodilation to 100 nmol/L acetylcholine was not significantly altered in IK1+/+/SK3T/T+doxycycline as well as in SK3-overexpressing IK1+/+/SK3T/T–doxycycline (Figure 4B, right panel). However, in CA from mice lacking IK1–/– and overexpressing SK3, the EDHF-type vasodilation was restored. Potentiation of IK1 functions by SKA-31 in SK3T/T/IK1+/++doxycycline caused a significant increase of amplitude and duration of EDHF-type vasodilation to 10 and 100 nmol/L acetylcholine (Figure 4C), which reflects a left shift of the concentration-response curve.
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We performed a second set of experiments in the absence of L-NNA and indomethacin to evaluate combined NO- and EDHF-mediated vasodilation to acetylcholine (Figure 4D). Under these conditions, acetylcholine (100 nmol/L) produced a 1.5-fold larger vasodilation in WT because of the contribution of NO. With intact NO synthesis, the vasodilation to 100 nmol/L acetylcholine was equally reduced by 30% in CA from IK1–/– (P<0.05) and IK1–/–/SK3T/T+doxycycline (P<0.01). Interestingly, the deficit of SK3 and IK1 impaired the NO-mediated vasodilation at a lower acetylcholine concentration of 10 nmol/L (P<0.05; insert in Figure 4D, left panel). A similar impairment was caused by the lack of SK3 alone in SK3T/T+doxycycline (P<0.05; insert in Figure 4D, right panel), which suggests an additional role for SK3 in NO production elicited by low levels of circulating vasoactive factors. In CA from IK1X/SK3T/T–doxycycline, which overexpressed SK3, vasodilation was similar to WT. Endothelium-independent vasodilations to sodium nitroprusside (10 µmol/L), vasoconstriction on phenylephrine (1 µmol/L), and elevated K+ (60 mmol/L) were similar in all genotypes (Figure 4A and Figure I in the online-only Data Supplement).
Because EDHF has been reported to play a prominent role in the microcirculation, we next investigated the contribution of IK1 and SK3 in EDHF-type and NO/EDHF-mediated vasodilation in resistance-sized arterioles of the cremaster microcirculation in vivo by intravital microscopy.26,27 This in vivo technique allows monitoring of arteriolar diameters during superfusion of vasoactive compounds. In the presence of inhibitors of NO synthase/cyclooxygenase, acetylcholine (1 µmol/L) produced significant vasodilation in WT, which was abolished in IK1–/–/SK3T/T+doxycycline (P<0.01) (Figure 4E). With intact NO synthase/cyclooxygenase, acetylcholine induced a comparable dilation in WT (Figure 4E, left panel), which was also reduced by 50% in IK1–/–/SK3T/T+doxycycline. At low concentrations of acetylcholine (100 nmol/L) only, dilations were mainly NO mediated in WT, which was virtually absent in IK1–/–/SK3T/T+doxycycline, indicating a contribution of endothelial KCa channels to NO formation.
In a second set of experiments, we tested the contribution of the individual KCa channels to EDHF-mediated vasodilation (Figure 4E, right panel). The deficit of IK1 in IK1–/– strongly reduced EDHF dilator responses to acetylcholine (P<0.05), whereas the deficit of SK3 in IK1+/+/SK3T/T+doxycycline did not reduce the dilation. The response at 3 µmol/L acetylcholine was apparently larger than in WT. Overexpression of SK3 in IK1-deficient mice (IK1–/–/SK3T/T–doxycycline) increased dilations to WT levels or enhanced them even further (at 10 µmol/L, P<0.05). Acetylcholine dilations obtained during intact NO synthase and cyclooxygenase were comparable to pure EDHF-type responses in these genotypes (Figure IV in the online-only Data Supplement), again demonstrating the insignificant role of NO and prostaglandins in the absence of either endothelial KCa channel. Endothelium-independent vasodilations in response to sodium nitroprusside (10 µmol/L) and dilations to adenosine (1 and 10 µmol/L) were similar in all groups (data not shown). These findings in a conduit artery and in the cremaster microcirculation suggest that IK1 channels are of major importance for EDHF-mediated dilations to acetylcholine and that SK3 only contributes to the response if IK1 channels are absent.
Loss of Endothelial SK3 but Not of IK1 Channels Impairs Wall Shear Stress-Induced Dilator Responses
Endothelial ion channels have been suggested to contribute to wall shear stress-induced arterial vasodilation and thus to mechanisms of endothelial mechanotransduction of altered hemodynamics.28 However, the specific contribution of either SK3 or IK1 channels is unknown. To elicit wall shear stress-mediated dilation in different groups, we increased the viscosity of the perfusion medium by adding 5% dextran. In CA of WT, the increase in viscosity elicited a small but appreciable EDHF-type dilator response of 14% (Figure 5A, presence of L-NNA/indomethacin). In SK3T/T+doxycycline, this wall shear stress-induced dilation was reduced to 2% (P<0.01) and was attenuated to a similar extent in IK1–/–/SK3T/T+doxycycline (5%; P<0.05). In contrast, CA of IK1–/– displayed dilations similar to that observed in WT CA. Overexpression of SK3 in IK1X/SK3T/T–doxycycline did not affect this dilation. Similarly, wall shear stress-induced dilation elicited by increasing flow from 30 to 600 µL/min was impaired in IK1X/SK3T/T+ doxycycline, whereas it remained intact in IK1–/– and IK1X/SK3T/T–doxycycline (Figure IIA in the online-only Data Supplement). In the absence of inhibitors of NO/prostacyclin synthesis, WT CA responded to wall shear stress stimulation by high viscosity (Figure 5B) as well as by an increase of flow (Figure IIB in the online-only Data Supplement) with more pronounced dilations of 23% and 25%, respectively, due to the contribution of NO.28 Again, this response was severely impaired in IK1X/SK3T/T+doxycycline, whereas it remained intact in IK1–/– and IK1X/SK3T/T–doxycycline. In addition, the IK1-opener SKA-31 had no effect on wall shear stress-induced dilation, elicited by either high viscosity or flow, in SK3T/T+ doxycycline (Figure IIC in the online-only Data Supplement). These findings suggest that SK3 channels are particularly crucial for EDHF/NO-mediated dilations of CA to wall shear stress stimulation.
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Combined IK1 and SK3 Suppression Increases Arterial Blood Pressure
We next assessed whether the loss of both endothelial KCa channels and thus of EDHF signaling mediated by IK1 and SK3 alters systemic blood pressure. For this purpose, we conducted continuous telemetric blood pressure measurements for at least 3 days in mice of both genders after doxycycline treatment (>6 days) (Figure 6A). WT had a mean arterial pressure of 100±1 mm Hg, which was unaffected by doxycycline administration (99±2; n=5). SK3T/T– doxycycline (n=5) had a comparable mean arterial pressure of 102±1 mm Hg, whereas SK3T/T+doxycycline had an elevated mean arterial pressure of 106±1 mm Hg (P<0.01 versus WT; P<0.05 versus SK3T/T–doxycycline). Likewise, IK1–/– had a higher mean arterial pressure of 108±1 mm Hg (P<0.01 versus WT), similar to a previous report.20 Doxycycline administration did not alter mean arterial pressure in IK1–/– (107±5; n=3). However, IK1–/–/SK3T/T+ doxycycline exhibited an even higher mean arterial pressure of 110±1 mm Hg (P<0.001 versus WT; P<0.05 versus SK3T/T+doxycycline, but not significantly different from IK1–/– [P=0.15]). Doxycycline removal (>2 weeks) lowered mean arterial pressure to 105±1 mm Hg in these animals (n=5; P<0.05 versus IK1–/–/SK3T/T+doxycycline), but mean arterial pressure was still higher than in the WT (P<0.05). In addition, in IK1–/–/SK3T/T, which never received doxycycline, mean arterial pressure was lower (104±1 mm Hg) than in the doxycycline-administered group (P<0.05) but also higher than in the WT (P<0.05; Figure 6A). This indicated that overexpression of SK3 in IK1–/–/SK3T/T–doxycycline reduced mean arterial pressure but was not able to return it to normotensive levels. In contrast, pharmacological potentiation of IK1 in IK1+/+/SK3T/T+ doxycycline (n=4) by a single injection of the IK1-opener SKA-31 (30 mg/kg IP) lowered mean arterial pressure to normotensive levels over 24 hours (Figure 6B).
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Heart rate was similar in all genotypes (Figure 6A, second panel). Pulse pressure (Figure 6A, third panel) was comparable in SK3T/T+doxycycline and WT but elevated in IK1–/–, IK1–/–/SK3T/T+doxycycline, and IK1–/–/SK3T/T–doxycycline. Locomotor activity (Figure 6A, fourth panel) was similar in SK3T/T+doxycycline and WT and tended to be higher in IK1–/– and IK1–/–/SK3T/T+doxycycline (P=0.08 and P=0.09 versus WT, respectively). SKA-31 injections in IK1+/+/SK3T/T+ doxycycline did not significantly alter heart rate, pulse pressure, and locomotor activity (Figure III in the online-only Data Supplement), although a trend toward lower values was observed.
Further analysis of circadian variation of blood pressure revealed that the enhancement of mean arterial pressure in the SK3T/T+doxycycline, IK1–/–, and IK1–/–/SK3T/T+ doxycycline was mainly caused by a higher mean arterial pressure in the dark period (Figure 6C, left) and during locomotor activity (Figure 6C, right).
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Discussion |
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Endothelial IK1 and SK3 channels have been hypothesized to contribute to EDHF signaling mainly on the basis of pharmacological evidence. However, the contribution of the EDHF dilator system to arterial tone in vivo and to systemic blood pressure control has not been elucidated previously. Therefore, we generated IK1–/–/SK3T/T mice in which activation of a tetracycline-based genetic switch21 by doxycycline treatment effectively abolished SK3 expression, resulting in deficiency of both endothelial KCa channels. This approach enabled us to study the effect of deficiency of both channels on endothelium-dependent dilation and blood pressure control in vivo as well as the potential rescuing effects of SK3 overexpression and of pharmacological potentiation of IK1 activity in mice lacking the respective other channel. We demonstrate that KCa currents are mediated by SK3 and IK1 (Figure 2). However, despite their equal ability to generate endothelial KCa currents, the 2 channels make distinct contributions to acetylcholine-induced EDHF-mediated smooth muscle hyperpolarization. The activation of IK1 seems to be more important because its absence impaired the hyperpolarization of smooth muscle more severely than the loss of SK3 alone (Figure 3). However, SK3 channels also contribute to smooth muscle hyperpolarization as demonstrated by the fact that lack of SK3 alone slightly reduces hyperpolarization and further impairs the reduced hyperpolarization response in IK–/–. In keeping with this finding, strong overexpression of SK3 in IK1–/–/SK3T/T–doxycycline (enhancing the endothelial KCa currents >10-fold) restored the acetylcholine-induced smooth muscle hyperpolarization to near-normal values despite the lack of IK1. Conversely, potentiation of IK1 activity by the IK1-opener SKA-31 strongly enhanced hyperpolarization in IK1-expressing and SK3-deficient mice to above the level seen in WT, thus further demonstrating a key role of IK1 in mediating acetylcholine-induced smooth muscle hyperpolarization.
The different roles of IK1 and SK3 became further apparent in the EDHF dilator responses in the different genotypes (Figure 4A). The loss of IK1 alone resulted in a pronounced effect at intermediate acetylcholine concentrations. However, the additional deletion of SK3 in IK1–/–/SK3T/T+doxycycline further impaired dilations, highlighting the importance of both channels for generating EDHF-type dilations. In contrast to IK1 deficiency, the suppression of SK3 in IK1-expressing mice was without appreciable effects. However, the impaired dilation in IK1 deficiency could be augmented by overexpression of SK3 but hardly reached WT levels. Moreover, overexpression of SK3 in IK1 WT did not produce a larger EDHF response, which suggests that the importance of SK3 is only demonstrated in the absence of IK1. In contrast, potentiation of IK1 by SKA-31 clearly enhanced the EDHF response in SK3-deficient mice (shown here), as well as in WT, although not in IK1–/–, as shown recently by us.25 Importantly, the present experiments in the microcirculation clearly validate the requirement of both IK1 and SK3 for the EDHF dilator response in resistance-sized arterioles in vivo. Only at high concentrations of acetylcholine was a remaining small dilation observed, which was comparable to responses in conduit arteries. Moreover, the findings in mice lacking either IK1 or SK3 or overexpressing SK3 during IK1 deficiency are in agreement with the findings in the conduit artery and further underscore a major role of IK1 in the EDHF response to acetylcholine, whereas SK3 contributes only in the absence of IK1.
Thus, these findings support the notion that IK1 in particular is of pivotal importance for the EDHF response elicited by stimulation of G-protein-coupled receptors (by acetylcholine) and subsequent Ca2+ release from the endoplasmic reticulum. The distinct roles may be related to the recently suggested spatial separation of the 2 channels within in the endothelium: SK3 at endothelial cell junctions29 (and perhaps in caveolae30) and IK1 at endothelial projections through the holes in the elastic lamina (also the sites of myoendothelial gap junctions29) and in close proximity to the endoplasmic reticulum.31 Thus, acetylcholine-triggered Ca2+ release events (recently termed Ca2+ pulsars31) could preferentially activate colocalized IK1 channels.
Depending on the type of vessel, the contribution of NO and prostaglandins in endothelium-dependent dilations varies substantially.4,18,32 In the conduit vessels used here, NO contributes considerably to acetylcholine-induced dilation.20 At maximal acetylcholine stimulation and with unimpeded NO synthesis, the dilation of the CA remains intact despite the lack of KCa channels (Figure 4D). However, at lower acetylcholine concentrations, dilations were reduced in SK3- and IK1/SK3-deficient mice despite intact NO synthase, suggesting that KCa channels, in particular SK3, are required for NO synthesis. In contrast to CA, the lack of KCa channels substantially reduces dilations in the murine microcirculation with a functioning NO system (Figure 4E), highlighting the special importance of the EDHF-dependent dilation in arterioles, the minor role of NO-dependent mechanisms,32 and the inability of NO to compensate for the lack of KCa channels. This suggests that, at least in the macrocirculation, an endothelial hyperpolarization contributes to the activation of NO synthase possibly by providing the driving force for Ca2+ influx, as suggested previously.33 Interestingly, SK3 channels were found to be more important in this phenomenon because both SK3- and IK1/SK3-deficient mice exhibited reduced vasodilations to low concentrations of acetylcholine, whereas IK1-deficient mice did not show impairment.
The differential roles of the 2 endothelial KCa channels are further highlighted by the finding that SK3 deficiency but not IK1 deficiency impaired dilations of CA in response to shear stress stimulation by increasing either viscosity or flow. The attenuation in animals lacking SK3 was evident in the presence as well as in the absence of NO, suggesting that SK3 is required not only for the EDHF-mediated but also for the larger NO-mediated flow-induced dilation. Overexpression of SK3 in either IK1-lacking or IK1-expressing mice failed to further increase shear stress-induced dilation, suggesting either that basal SK3 expression levels are sufficient to produce hyperpolarization or that the ensuing vasodilation simply abrogates the mechanical stimulus.34 Potentiation of IK1 functions by SKA-31 in SK3-deficient mice was ineffective to restore the response, which further supports the notion that IK1 is not activated by shear stress-induced [Ca2+]i increases. Thus, SK3, unlike IK1, seems to be crucial for adequate endothelial mechanotransduction in response to shear stress stimulation and may therefore be important for tonic EDHF activity and endothelial NO formation under continuous blood flow in vivo.
Taken together, our data suggest that activation of IK1 and SK3 in a stimulus-dependent fashion constitutes the major electric trigger for EDHF dilator responses. The importance of the SK3/IK1-initiated EDHF system for blood pressure control is supported by our finding of a significant increase in blood pressure in mice lacking 1 or both channels. Intriguingly, the defect in the SK3/IK1 EDHF dilator system increased blood pressure mainly during locomotor activity, suggesting that the EDHF system is especially required to ensure adequate vasoregulation and thereby blood pressure control during physical activity. Interestingly, pulse pressure was elevated in mice lacking IK1 but not in SK3-lacking animals (for additional discussion, see the online-only Data Supplement). Because the combined loss of SK3 and IK1 had few additive effects on blood pressure, the 2 channels exert distinct but essential roles in blood pressure control. This hypothesis is supported by the inability of SK3 overexpression to completely compensate for the loss of IK1 and to restore normal blood pressure. This may be related to a more significant role of IK1 in EDHF-type dilation, which is underscored by the ability of the IK1-opener SKA-31 to return the elevated blood pressure in SK3-deficient and IK1-expressing mice back to normotensive levels. Additionally, SKA-31 lowered blood pressure in WT mice but not the elevated blood pressure in IK1-deficient mice,25 which emphasizes its 10-fold higher potency for IK1 over SK3. Alternatively, both channels may use distinct downstream EDHF signaling pathways, ie, electrotonic coupling mechanisms (SK3) and/or K+ release (IK1), as suggested recently.3,5,12,17,27,29 Moreover, SK3 may add to blood pressure control by exerting a tonic influence on vascular tone,22 possibly by its role in NO dilator responses to low levels of vasoactive factors and flow alterations.
In summary, the present study demonstrates that EDHF-mediated dilations are virtually absent in SK3/IK1-deficient mice and that these mice exhibit enhanced blood pressure. The analysis of these animals further provides substantial evidence that SK3 and IK1 play distinct roles in vessel physiology despite their overlapping function as endothelial KCa channels. We suggest that SK3/IK1-deficient mice represent an "EDHF knockout," which may provide a useful model to study the physiological actions of putative EDHFs4,5,11 in the circulation.
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Acknowledgments |
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Sources of Funding
This work was supported by grants of the Deutsche Forschungsgemeinschaft (project A11 of SFB 593 and KO1899/10-1 [Drs Köhler and Hoyer] and WI2071/2-1 [Dr de Wit]) and by National Institutes of Health grants MH081860 (Dr Adelman) and NS052165 (Dr Wulff).
Disclosures
None.
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Footnotes |
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*The first 2 authors contributed equally to this article.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.846634/DC1.
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Circulation 2009 119: 2295-2296.
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