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(Circulation Research. 2006;99:537.)
© 2006 American Heart Association, Inc.

Integrative Physiology

Impaired Endothelium-Derived Hyperpolarizing Factor-Mediated Dilations and Increased Blood Pressure in Mice Deficient of the Intermediate-Conductance Ca²⁺-Activated K⁺ Channel

Han Si^*, Willm-Thomas Heyken^*, Stephanie E. Wölfle, Marcin Tysiac, Rudolf Schubert, Ivica Grgic, Larisa Vilianovich, Günter Giebing, Tanja Maier, Volkmar Gross, Michael Bader, Cor de Wit, Joachim Hoyer, Ralf Köhler

From the Department of Internal Medicine-Nephrology (H.S., W.-T.H., M.T., I.G., G.G., T.M., J.H., R.K.), Philipps-University, Marburg; Department of Physiology (S.E.W., C.d.W.), University of Lübeck; Institute of Physiology (R.S.), University of Rostock; and Max-Delbrück-Centrum for Molecular Medicine (L.V., V.G., M.B.), Berlin-Buch, Germany.

Correspondence to R. Köhler, Department of Nephrology, Philipps-University, Baldingerstrasse, 35033 Marburg, Germany. E-mail rkoehler{at}med.uni-marburg.de

	Abstract

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The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca²⁺-activated K⁺ channels (K_Ca), leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. In the present study, we tested the functional role of the endothelial intermediate-conductance K_Ca (IK_Ca/K_Ca3.1) in endothelial hyperpolarization, in EDHF-mediated dilation, and in the control of arterial pressure by targeted deletion of K_Ca3.1. K_Ca3.1-deficient mice (K_Ca3.1^–/–) were generated by conventional gene-targeting strategies. Endothelial K_Ca currents and EDHF-mediated dilations were characterized by patch-clamp analysis, myography and intravital microscopy. Disruption of the K_Ca3.1 gene abolished endothelial K_Ca3.1 currents and significantly diminished overall current through K_Ca channels. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine was reduced in K_Ca3.1^–/– mice. Acetylcholine-induced dilations were impaired in the carotid artery and in resistance vessels because of a substantial reduction of EDHF-mediated dilation in K_Ca3.1^–/– mice. Moreover, the loss of K_Ca3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial K_Ca3.1 is a fundamental determinant of endothelial hyperpolarization and EDHF signaling and, thereby, a crucial determinant in the control of vascular tone and overall circulatory regulation.

Key Words: hypertension • endothelium • EDHF • intermediate-conductance Ca²⁺-activated K⁺ channel • K_Ca3.1^–/– mice

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The vascular endothelium plays a key role in the control of organ perfusion and contributes to the regulation of arterial blood pressure by releasing vasoactive factors that modulate the contractile state of the underlying smooth muscle.¹ In response to classical agonists, such as acetylcholine (ACh) and bradykinin, as well as to hemodynamic stimuli,² the endothelium produces, in principle, 3 vasodilating factors, nitric oxide (NO),^1,3 prostacyclin (PGI₂),⁴ and an endothelium-derived hyperpolarizing factor (EDHF).⁵ Although the modes of action of NO and PGI₂ became clear 20 years ago, the nature of the latest identified factor, EDHF, is still controversial and debated thoroughly since its discovery in the late eighties.^5,6

Regarding the relative contribution of NO and EDHF within the vascular tree, it appears that EDHF becomes more important when vessel diameter decreases, whereas NO seems to be predominant in large arteries.⁷ Importantly, defects in the NO system and also in EDHF signaling, as demonstrated more recently, are pathologically relevant in disease states such as hypertension,^8,9 diabetes,¹⁰ and renal insufficiency.¹¹ Studies in transgenic mice deficient in either endothelial NO synthase (eNOS)¹² or the PGI₂-generating cyclooxygenase-1 (COX-1),^13,14 or in both enzymes,¹⁴ revealed that endothelium-dependent dilation is still preserved, which was attributed to a compensatory effect by the respective other dilator and supposedly by EDHF.

Despite considerable progress in recent years, the identity of EDHF and the cellular mechanisms underlying EDHF signaling upstream of smooth muscle hyperpolarization and relaxation remains elusive. Several chemical factors among them, K⁺ ions and cytochrome P450-generated metabolites of arachidonic acid, have been suggested to serve as EDHF or contribute to EDHF signaling by intraendothelial mechanisms.^5,6,15 In addition, a nonchemical mechanism characterized by the direct spread of hyperpolarization from the endothelium to the subjacent smooth muscle via myoendothelial gap junctions was proposed to transfer the hyperpolarization directly, without the requirement for a factor, and account for the EDHF-related vasodilator principle.⁶ In fact, in most vessels in different species including humans, the membrane hyperpolarization that occurs initially in the endothelium seems to be a prerequisite for the initiation of the EDHF signal.^5,16 This endothelial hyperpolarization is in many tissues because of the activation of small-conductance Ca²⁺-activated K⁺ channels (K_Ca2.3, according to the new International Union of Basic and Clinical Pharmacology nomenclature; also known as SK_Ca3, KCNN3, SK3) and of intermediate-conductance Ca²⁺-activated K⁺ channels (K_Ca3.1; also known as IK1, KCNN4, IKCa1).^5,16–27 These 2 K_Ca channels are commonly known as endothelial IK_Ca and SK_Ca. As the combined and specific blockade of these channels abrogates endothelial hyperpolarization and the EDHF-type response, their activation is considered a hallmark herein.^{5,16,17,21,23,26,28} However, in most studies, a combined blockade of K_Ca3.1 channels and of K_Ca2.3 was performed to study EDHF-type responses. Interestingly, in the human mesenteric endothelium, K_Ca3.1 seems to be of special importance because endothelial hyperpolarization is almost completely blocked by inhibitors of this channel,²⁹ suggesting that the 2 K_Ca channels have specific functions, at least in certain vascular beds. However, the exact role of K_Ca3.1 as opposed to K_Ca2.3 to mediate endothelial hyperpolarization and its contribution to the control of vascular tone, EDHF signaling, and, possibly, the regulation of arterial pressure remain unclear.

To dissect the relative roles of K_Ca3.1 and K_Ca2.3, we generated mice with a targeted disruption of the K_Ca3.1^–/– gene. Here, we show that deletion of the K_Ca3.1^–/– gene and thus loss of K_Ca3.1 functions results in an impaired EDHF-mediated dilation and elevated systemic blood pressure.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Detailed methods for generation of K_Ca3.1^–/– mice,³⁰ Southern blot analysis, routine genotyping, RT-PCR, Western blot analysis, patch-clamp electrophysiology,^23,29,31 membrane-potential measurements, pressure myography,²³ intravital microscopy,³² and tail-cuff and telemetric blood pressure measurements³³ are included in the online data supplement, available at http://circres.ahajournals.org.

Statistics
Data are given as mean±SE or SEM as indicated. The Student’s t test was used to assess differences between groups. Probability values of P<0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of K_Ca3.1^–/– Mice
Mice deficient in K_Ca3.1^–/– were generated by homologous recombination in embryonic stem cells using conventional techniques (Figure 1A). This strategy deleted exon 4 of the K_Ca3.1 gene, which encodes the channel pore. Successful gene targeting of K_Ca3.1 was verified by Southern blot and PCR analysis (Figure 1B and 1C). Loss of K_Ca3.1 gene expression in K_Ca3.1^–/– was confirmed by RT-PCR in total mRNA extracts from spleen (Figure 1D). The lack of the protein was proven by Western blot analysis (Figure 1E) of erythrocyte ghost membranes known to contain the K_Ca3.1 protein (also referred to as "Gardos" channel).

View larger version (21K): [in this window] [in a new window]   Figure 1. Generation of KCa3.1–/– mice. A, Schematic representation of the gene targeting strategy. In the targeted allele, exon 4 of the KCa3.1 gene was replaced by the neomycin resistant gene (neor). P indicates the probe used for Southern blot; FP (forward primer) and RP1/2 (reverse primer), the positions of PCR primers used for genotyping. B, Genotyping of the KCa3.1–/– mice. Southern blot analysis of BglII-digested genomic DNA (10 µg/lane). The 4.1- and the 3.3-kb bands display the presence of the targeted and the wild-type allele, respectively. C, Electrophoretic analysis of PCR products derived from genomic DNA. The 320- and the 160-bp bands were amplified from the targeted and from wild-type allele, respectively. D, Electrophoretic analysis of RT-PCR products amplified from total spleen mRNA of KCa3.1–/– mice and wild-type littermates. Wild-type mice showed the expected 605- (exon 3 to exon 5) and 231-bp (exon 3 to exon 4) bands, whereas KCa3.1 mRNA was not detectable in KCa3.1–/– mice. E, Western blot analysis of KCa3.1 protein expression in erythrocyte ghost membranes from wild-type and KCa3.1–/– mice.

Heterozygous K_Ca3.1^–/+ breeding pairs produced offspring in a Mendelian manner: 25% were homozygous wild-type (+/+), 52% heterozygous (–/+), and 23% homozygous K_Ca3.1^–/–. Homozygous K_Ca3.1^–/– were viable and fertile. Mating of K_Ca3.1^–/– males with K_Ca3.1^–/– females produced normal offspring and litter sizes, similar to a different K_Ca3.1^–/– strain.³⁴

Deficiency of Endothelial K_Ca3.1 and Diminished Endothelial Hyperpolarization in K_Ca3.1^–/– Mice
To evaluate whether the K_Ca3.1 gene deletion results in diminished functional K_Ca currents in the endothelium of K_Ca3.1^–/– mice, we performed whole-cell patch-clamp experiments in freshly isolated aortic endothelial cells (AEC) of K_Ca3.1^–/– (n=8) and K_Ca3.1^+/+ mice (n=7) and in carotid artery endothelial cells (CAEC) in situ of these mice (n=3 each). For the activation of K_Ca channels, cells were dialyzed with a pipette solution containing 3 µmol/L Ca²⁺. To verify the lack of K_Ca3.1 currents, whole-cell patch-clamp experiments were conducted first in the presence of the K_Ca2.3 blocker UCL1684 (100 nmol/L)³⁵ to unmask K_Ca3.1 currents from K_Ca2.3 currents in AEC and CAEC in situ. K_Ca3.1 currents were not detectable in AEC and CAEC of K_Ca3.1^–/– mice, whereas K_Ca3.1 currents were present in AEC and in CAEC of K_Ca3.1^+/+ littermates. Original current traces and mean K_Ca current standardized to cell capacitance are shown in Figure 2A and 2B. TRAM-34, a selective blocker of K_Ca3.1 channels,³⁶ completely blocked this current (n=10; Figure 2), demonstrating that the UCL1684-insensitive current is indeed mediated by K_Ca3.1. Moreover, this indicates that K_Ca3.1 together with K_Ca2.3 confers the K_Ca currents in wild-type AEC,^24,37 as in rat EC, as shown previously.²¹ In a second set of experiments, we determined mixed K_Ca3.1/K_Ca2.3-current densities in AEC and in CAEC in situ of K_Ca3.1^+/+ mice and K_Ca2.3-current densities in AEC and in CAEC in situ of K_Ca3.1^–/– mice. Compared with the composite K_Ca3.1/K_Ca2.3-current density in EC of wild-type mice, the K_Ca2.3-current density in K_Ca3.1^–/– mice was significantly smaller (Figure 2B). K_Ca2.3-current densities (determined in the presence of TRAM-34) were similar in AEC of both genotypes (K_Ca3.1^–/–: 14±2 pA/pF, n=5; wild type: 10±3 pA/pF, n=13). Also, cell capacitance was not different between genotypes in AEC (K_Ca3.1^–/–: 9±1; wild type: 8±1 pF; n=21 and 25, respectively) and in CAEC in situ (K_Ca3.1^–/–: 8±1; wild type: 9±1 pF; n=8 and 12, respectively). These results indicate that deletion of K_Ca3.1 gene reduced the overall endothelial K_Ca-current density by &50%.

View larger version (14K): [in this window] [in a new window]   Figure 2. KCa3.1 currents in AEC. A, Current-voltage relationship of KCa3.1 in AEC from wild-type mice (top). Currents reversed at &–80 mV, which was close to the equilibrium potential for K+, and were inhibited by TRAM-34 (100 nmol/L). KCa3.1 currents were not detectable in AEC from KCa3.1–/– mice (bottom). Experiments were conducted in the presence of 100 nmol/L UCL 1684 to unmask KCa3.1 currents from KCa2.3 currents. VM indicates membrane potential. B, Mean KCa current densities (IKCa) standardized to cell capacitance (pA/pF) in AEC and CAEC in situ from KCa3.1–/– mice and KCa3.1+/+ mice. Numbers (n) in the chart refer to number of experiments. Values are given as mean±SE. *P<0.05, **P<0.01 (Student’s t test).

To test whether the loss of endothelial K_Ca3.1 currents and the reduced overall K_Ca currents in K_Ca3.1^–/– mice led to an impaired agonist-induced endothelial hyperpolarization, we conducted membrane-potential measurements in AEC clusters (>20 cells) and in the endothelium of carotid artery in situ (CAE). Capacitance values of >150 pF indicated intact electrical coupling in AEC clusters and in EC of the carotid artery (CA) of K_Ca3.1^–/– and K_Ca3.1^+/+ mice. As shown in Figure 3, the amplitude of the hyperpolarization in response to ACh (100 nmol/L) was significantly smaller in both preparations studied in K_Ca3.1^–/– (at 100 nmol/L ACh: &–10 mV versus &–25 mV in wild type). Moreover, the ACh-induced hyperpolarization was completely reversed by the K_Ca2.3 blocker UCL1684 (100 nmol/L) in K_Ca3.1-deficient tissues, whereas in wild-type tissues, a combination of UCL1684 and TRAM-34 (1 µmol/L) was required to fully reverse the hyperpolarization response. Similarly, membrane hyperpolarization in response to the K_Ca3.1/K_Ca2.3 opener DC-EBIO (10 µmol/L)³⁸ was significantly reduced in AEC clusters obtained from K_Ca3.1^–/– mice (Figure 3). Nevertheless, the endothelial resting potential did not differ between the genotypes (Figure 3B) and was likewise not altered in the presence of 1 or both K_Ca blockers, indicating that K_Ca3.1 and K_Ca2.3 do not contribute considerably to the setting of the resting potential under these conditions in these cells.

View larger version (16K): [in this window] [in a new window]   Figure 3. Impaired membrane hyperpolarization on ACh in endothelium of KCa3.1–/– mice. A, Representative membrane potential recordings in the endothelium of carotid arteries (CAE) show hyperpolarization in response to ACh (100 nmol/L) in wild-type (top) and KCa3.1–/– mice (bottom). Hyperpolarization was reversed by the combination of UCL1684 (100 nmol/L) and TRAM-34 (1 µmol/L, n=4) in wild-type but by UCL1684 alone (n=4) in KCa3.1–/– mice. B, Quantitative analyses of resting membrane potentials (RP) and maximal hyperpolarization in response to ACh and DC-EBIO in AEC and in CAE from wild-type (n=3) and KCa3.1–/– (n=3) mice. Numbers (n) in the chart refer to number of experiments. Values are given as mean±SE. *P<0.05, **P<0.01 vs wild type (Student’s t test).

Membrane potential measurements in smooth muscle of CAs as performed by using conventional sharp electrode technique (from the adventitial side) revealed that smooth muscle hyperpolarization to ACh (100 nmol/L) was blunted in CAs (n=6) of K_Ca3.1^–/– (–44±2 mV [&–5 mV] versus –58±3 mV [&–15 mV] in wild-type CA; n=6; P<0.01). Resting potentials were similar (–39±2 mV in K_Ca3.1^–/– versus –43±2 mV in wild-type mice; P=NS).

Together, these results suggest that the loss of endothelial K_Ca3.1 leads to an impaired endothelial and smooth muscle hyperpolarization in response to agonists.

Impaired EDHF-Mediated Dilation in K_Ca3.1^–/– Mice
To test whether the lower K_Ca-current densities and the reduced endothelial hyperpolarization response in K_Ca3.1^–/– led to impaired endothelial function and especially a defective EDHF-mediated dilation, we determined ACh- and DC-EBIO-induced dilator responses in CAs of K_Ca3.1^–/– and wild-type mice. In the presence of the NO synthase blocker N^G-nitro-L-arginine (L-NNA) (100 µmol/L) and the COX inhibitor INDO (10 µmol/L), ACh-induced dilations were significantly reduced in K_Ca3.1^–/– mice. Original traces and concentration-response curves are shown in Figure 4A. At 100 nmol/L ACh, the dilation of CAs of K_Ca3.1^–/– mice was approximately half of the dilatory response observed in wild-type mice. The EDHF-type dilation was partially suppressed by UCL1684 (100 nmol/L) and completely abrogated by the combination of UCL1684 and TRAM-34 (1 µmol/L) in wild-type mice. In contrast, UCL1684 was sufficient to effectively block EDHF-type dilations in K_Ca3.1^–/– mice, and TRAM-34 had no further inhibitory effects (Figure 4A, lower right). Notably, UCL1684 alone or in combination with TRAM-34 did not reduce vessel diameter in wild-type or K_Ca3.1^–/– mice, suggesting that these channels do not initiate or exert a tonic endothelial dilator effect in this preparation.

View larger version (31K): [in this window] [in a new window]   Figure 4. Impaired EDHF-mediated dilation in KCa3.1–/– mice. A, top, Original traces showing increases in CA diameter in response to increasing concentrations of ACh and to DC-EBIO in wild-type (left) and KCa3.1–/– mice (right). A, bottom, Concentration-response curves of ACh-induced EDHF-type dilation (in the presence of L-NNA [100 µmol/L] and INDO [10 µmol/L]) in wild-type and KCa3.1–/– mice. A, bottom right, Inhibitory effects of UCL1684 (100 nmol/L, n=4 each) and TRAM-34 (1 µmol/L, n=4 each) on ACh-induced (1 µmol/L) EDHF-type dilation in wild-type and KCa3.1–/– mice. B, Overall ACh-induced dilation (in the absence of blockers) in CAs from wild-type and KCa3.1–/– mice. C, Dilation in response to intraluminal application of DC-EBIO (10 µmol/L) in wild-type and KCa3.1–/– mice. Values are given as mean±SE. *P<0.05, **P<0.01 vs wild type (Student’s t test).

In the absence of blockers of NOS and COX, endothelium-dependent dilation was larger overall in wild-type and K_Ca3.1^–/– mice. This is in line with the notion that, particularly, NO synthesis and release contribute significantly to ACh-induced dilation in this artery (Figure 4B). Prostacyclin did not contribute to this enhanced dilator response in wild-type and K_Ca3.1^–/– mice because INDO alone had no inhibitory effect (also see Figure I in the online data supplement).

However, when compared with wild-type mice, the overall larger ("total") ACh-induced dilation was still reduced in K_Ca3.1^–/– mice (Figure 4B). Because inhibition of NOS reduced ACh-induced dilation to a similar extent in wild-type and in K_Ca3.1^–/– mice, it seems that the reduction of the total endothelium-dependent dilation in K_Ca3.1^–/– mice reflects an impaired EDHF-type response in these mice. Moreover, the persisting impairment of the ACh-induced dilation in K_Ca3.1^–/– mice in the presence of intact NO synthesis indicates that the diminished EDHF-type dilation is not compensated by an enhanced NO or prostacyclin release.

Dilation in response to direct pharmacological activation of both endothelial K_Ca by the potent K_Ca3.1/K_Ca2.3 opener DC-EBIO (10 µmol/L) was also significantly diminished in CAs of K_Ca3.1^–/– mice (Figure 4C), which shows that the loss of K_Ca3.1 also blunts pharmacologically induced EDHF-type dilation.

Vasoconstriction of CAs in response to phenylephrine (PE) (1 µmol/L) did not differ between K_Ca3.1^–/– mice and wild-type littermates. PE contracted CAs of K_Ca3.1^+/+ mice by 114±8 µm (from 572±9 to 458±10 µm; n=20) and CAs of K_Ca3.1^–/– mice by 125±9 µm (from 568±8 to 448±10 µm; n=23). Endothelium-independent dilation in response to sodium nitroprusside (SNP) (1 µmol/L) was also unchanged in K_Ca3.1^–/– mice. SNP dilated PE-precontracted CAs of K_Ca3.1^+/+ mice by 106±8 µm and CAs of K_Ca3.1^–/– mice by 119±10 µm (P=NS). Likewise, pressure-induced vasoconstriction (myogenic tone), as determined in myogenically active small arteries (gracilis artery, &100 µm in diameter), was not altered in K_Ca3.1^–/– mice (data not shown). These results indicate that deficiency of K_Ca3.1 does not alter the tonic dilatory effect of the endothelium on myogenic tone or tone initiated by ₁-adrenergic receptor stimulation using PE. In this regard, it is noteworthy that K_Ca2.3 have been proposed to a play role herein²⁴ (for additional discussion, see the online data supplement).

Collectively, these data from pressure myograph experiments show that the lack of K_Ca3.1 impairs ACh-induced dilation predominantly by affecting the EDHF-type dilation.

In addition, the contribution of K_Ca3.1 to endothelium-dependent dilations was investigated in resistance-sized vessels in the cremaster microcirculation in the presence of inhibitors of NOS and COX. The maximal diameter of the arterioles investigated ranged from 16 to 75 µm and was similar in wild-type and K_Ca3.1^–/– mice (36±1 versus 38±1 µm, respectively; n=80 arterioles in 8 mice, each genotype). The arteriolar dilation in response to ACh was significantly reduced in K_Ca3.1^–/– mice, which was most pronounced at intermediate concentrations (Figure 5). In contrast, the dilation in response to SNP (10 µmol/L) was similar in both genotypes (wild-type: 63±3%; K_Ca3.1^–/–: 66±3% of maximum dilation; P=NS). Thus, the K_Ca3.1 channel is also required in arterioles for an intact EDHF-type dilation.

View larger version (18K): [in this window] [in a new window]   Figure 5. Impaired EDHF-mediated dilation in resistance-sized vessels in the cremaster microcirculation of KCa3.1–/– mice. Concentration-response curves of ACh-induced EDHF-type dilation (in the presence of L-NNA [30 µmol/L] and INDO [3 µmol/L]) in wild-type and KCa3.1–/– mice (n=80 arterioles in 8 mice, each genotype). Values are given as mean±SEM. **P<0.001 vs wild type (Student’s t test).

Elevated Blood Pressure in K_Ca3.1^–/– Mice
Because deficiency in K_Ca3.1^–/– resulted in reduced K_Ca-current density, decreased hyperpolarization responses, and an impaired EDHF-type dilation in conducting and resistance vessels, we questioned whether this has functional consequences on the overall circulation. We tested this hypothesis by measuring systolic blood pressure (SBP) and diastolic blood pressure (DBP) using tail-cuff plethysmography. Compared with wild-type littermates, SBP and DBP were significantly increased by &13 mm Hg and &12 mm Hg in K_Ca3.1^–/– mice, respectively. The calculated mean arterial pressure was increased by &14 mm Hg (Table). In smaller sets of animals (n=4, per group), we conducted 24-hour pressure measurements by telemetry which revealed a comparable increase of the mean pressure in K_Ca3.1^–/– mice (115±2 versus 108±1 mm Hg; P<0.05) and confirmed the tail-cuff measurements. However, heart rate was unchanged (K_Ca3.1^–/–: 637±22; wild-type: 633±17 bpm). Interestingly, we also found a slight increase in heart weight and a larger cross-sectional area of the left ventricle on histological examination in K_Ca3.1^–/– (Table). This might be indicative of mild left-ventricular hypertrophy, presumably resulting from increased blood pressure because the cross-sectional area of the right ventricle was not altered in K_Ca3.1^–/– mice.

View this table: [in this window] [in a new window]   Table 1. Cardiovascular Parameters in Wild-Type and KCa3.1–/– Mice

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we show that the targeted disruption of the K_Ca3.1 gene abolished K_Ca3.1 functions and greatly reduced endothelial K_Ca currents. This reduction of K_Ca currents was associated with an impaired endothelial hyperpolarization and EDHF-type dilation in conducting and resistance vessels in response to the classical endothelial agonist ACh. In addition, systemic blood pressure was elevated in K_Ca3.1^–/– mice. Thus, by using a conventional gene-targeting approach, we demonstrate that the K_Ca3.1, on its own, is an important determinant of the capability of the endothelium to hyperpolarize on stimulation and, thereby, apparently supports EDHF-type dilation. Moreover, we show for the first time that targeted disruption of a crucial endothelial component of the EDHF-signaling pathway, ie, K_Ca3.1, has an impact on systemic blood pressure regulation.

In response to agonist stimulation, endothelial hyperpolarization is considered to be a prerequisite for the initiation of the EDHF-signaling pathway.⁵ The endothelial K_Ca channels mediating this initial endothelial hyperpolarization were identified by electrophysiologic, pharmacological, and molecular approaches as K_Ca3.1 and K_Ca2.3 channels in arteries from mice,²⁴ rats,^21,23 pigs,²² and humans.²⁹ These 2 K_Ca channels are not expressed in mature contractile vascular smooth muscle cells of CAs of mice (also see supplemental Figure II) and of rats.^23,39 In mice as in rats, endothelial K_Ca3.1 and K_Ca2.3 seem to contribute almost equally to the composite endothelial K_Ca currents, whereas in human EC in intact mesenteric artery preparations, endothelial K_Ca currents and hyperpolarization are largely mediated by K_Ca3.1.²⁹ In the present study, we found that deficiency of K_Ca3.1 caused a reduction of K_Ca currents in AEC and CAEC in response to ACh and a pharmacological activator of K_Ca. The amplitude of the residual K_Ca current in K_Ca3.1^–/– mice, which was mediated by K_Ca2.3, was approximately half of the amplitude of the composite K_Ca3.1/K_Ca2.3 current observed in wild-type mice, which suggests that K_Ca3.1 and K_Ca2.3 channels contribute equally to endothelial hyperpolarization. Moreover, the current in K_Ca3.1^–/– mice had roughly the same amplitude as the hyperpolarizing current in wild-type EC after pharmacological inhibition of K_Ca3.1. This important finding suggests that the lack of K_Ca3.1 is not compensated for by a higher activity and/or expression level of the K_Ca2.3.

The functional expression of both channels in the same cell implies that they have different functions or that their combined activation is needed to generate a complete hyperpolarization after endothelial stimulation. This latter could be attributable to a necessity to counteract concomitant depolarizing currents through second-messenger-gated cation channels of the transient receptor potential family after stimulation of G protein-coupled receptors.^26,28,40 Assuming a tight electrical coupling of the endothelium and smooth muscle via myoendothelial gap junctions,⁶ the endothelium may also be subjected to a depolarizing effect of the smooth muscle. In any case, the present study revealed that the loss of K_Ca3.1 dampened the endothelial hyperpolarization response to ACh as well as to the potent K_Ca3.1/K_Ca2.3 opener DC-EBIO. This highlights the requirement of this channel, which adds to endothelial hyperpolarization on stimulation to achieve its full amplitude. Whether the K_Ca3.1 is simply another source of hyperpolarizing current or acts as an amplifier cannot be clearly decided by the present study. However, it should be noted that &50% of the amplitude of the hyperpolarization response, which is mediated by K_Ca2.3 was preserved.

Because the endothelial hyperpolarization is critical to induce EDHF-type dilation and may be important for NO-mediated dilation as well, we assessed endothelium-dependent responses in arteries of K_Ca3.1-deficient mice. In the carotid artery of wild-type mice, NO mediated nearly half of the dilation at intermediate concentrations of ACh (100 nmol/L). Because prostacyclin does not contribute in these vessels, which was verified by the lack of effect of the COX inhibitor INDO, the other half of the response was NO- and prostacyclin-independent and thus apparently attributable to an EDHF-type dilation. This large contribution of EDHF to endothelium-dependent dilation in this artery differs from earlier findings of a lower, or even no, contribution of EDHF in carotid arteries of rats (&20% EDHF)^11,23 and mice,⁴¹ respectively. Likely explanations for this discrepancy are the use of different experimental approaches (pressure [this study] versus wire myography) or different compounds and concentrations used for precontraction such as phenylephrine (this study), prostaglandins, or thromboxane A₂ mimetics, ie, U46619. Especially the latter was shown to interfere with EDHF signaling in a negative fashion.²⁵ However, the loss of K_Ca3.1 reduced the overall endothelium-dependent dilation in the carotid artery. This reduction was not caused by an attenuation of the NO-mediated part of the response because inhibition of NO synthase was equally effective in both genotypes. This suggests an alteration of the EDHF-type dilation, as verified after blockade of NO and prostaglandin synthesis. Importantly, EDHF cannot be compensated for by NO and/or prostaglandins because the overall endothelium-dependent dilation is blunted. Although it is widely accepted that endothelial hyperpolarization mediated by K_Ca3.1 or K_Ca2.3 is required for generating an EDHF-mediated dilation, the specific contribution of either K_Ca3.1 or K_Ca2.3 is still unclear. Depending on vessel type, species, and experimental approach, a single blocker or only the combination of both blockers is effective in suppressing endothelial hyperpolarization, smooth muscle hyperpolarization, and EDHF-mediated dilation.^23,26,28,42 In the present study on carotid artery of wild-type mice, blockade of both K_Ca3.1 and K_Ca2.3 was required to suppress EDHF-type dilation, which is reflected also by the complete inhibition of endothelial hyperpolarization, and thus both endothelial K_Ca channels are important in initializing a full EDHF dilation.

This hypothesis was further verified by the gene-targeting approach in this present study, as K_Ca3.1^–/– mice exhibit an impaired EDHF-mediated dilation in the CA, which is most likely attributable to the reduced capability of the endothelium to hyperpolarize. The important role of K_Ca3.1 is not restricted to conducting arteries. Also, in resistance vessels of the microcirculation, K_Ca3.1 is critical to initiate a full EDHF-type dilation and its role appears to be prominent at lower concentrations of ACh. Because NO and prostaglandins do not contribute substantially to dilator responses in this preparation,^32,43 we have not assessed the role of NO in this study. However, a considerable portion of the EDHF-mediated dilation was preserved in both vessel types. This remaining dilation relied on the activation of K_Ca2.3 because the inhibitor of K_Ca2.3 almost completely blocked the EDHF-type dilation in K_Ca3.1-deficient mice.

The physiological importance of the impaired endothelial hyperpolarizing capability and diminished EDHF-mediated dilation in K_Ca3.1^–/– mice is reflected by the considerable increase in arterial pressure. This may reflect the importance of endothelial hyperpolarizations to coordinate vascular behavior along the vessel wall through gap junctions.^32,44 It is noteworthy, that a similar association of impaired endothelial K_Ca3.1 (and K_Ca2.3) expression and diminished EDHF-mediated dilation is present in uremic rats¹¹ and in restenosis disease,²¹ which supports the notion that a loss of endothelial K_Ca3.1 is relevant in cardiovascular diseases. With respect to the K_Ca2.3 channel, a recent study using transgenic K_Ca2.3 mice showed that suppression of K_Ca2.3 expression increased myogenic and phenylephrine-induced tone and systemic blood pressure, thus highlighting the functional role of this channel in endothelial control of vascular tone (for additional discussion, please see the online data supplement).

In conclusion, the present study demonstrated that the endothelial K_Ca3.1 contributes considerably to endothelial hyperpolarization and EDHF-mediated dilations. Importantly, the loss of K_Ca3.1 cannot be compensated for by enhanced activity of K_Ca2.3 and/or NO and, therefore, has an impact on overall circulatory function as reflected by the elevation of systemic blood pressure. Thus, this study presents first evidence that genetic deletion of one major component of the EDHF-signaling pathway significantly impairs cardiovascular functions. Conversely, selective openers of endothelial K_Ca3.1 may provide a novel therapeutic approach for the treatment of vascular diseases characterized by endothelial dysfunction and hypertension.

	Acknowledgments

 
Sources of Funding

This work was supported by Deutsche Forschungsgemeinschaft grants FOR 341/7, HO 1103/2-4, KO-1899/10-1, GRK 276, GRK 865 (to M.B., J.H., and R.K.); SCHU 805/7-1 (to R.S.); and WI 2071/1-1 (to C.d.W.).

Disclosures

None.

	Footnotes

 
*Both authors contributed equally to this study.

Original received June 9, 2006; revision received July 12, 2006; accepted July 17, 2006.

	References

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