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【摘要】
Objective- Nitric oxide (NO)-induced vasorelaxation involves activation of large conductance Ca 2+ -activated K + channels (BK). A regulatory BKß1 subunit confers Ca 2+, voltage, and NO/cGMP sensitivity to the BK channel. We investigated whether endothelial function and NO/cGMP signaling is affected by a deletion of the ß1-subunit.
Methods and Results- Vascular superoxide in BKß1 -/- was measured using the fluorescent dye hydroethidine and lucigenin-enhanced chemiluminescence. Vascular NO formation was analyzed using electron paramagnetic resonance (EPR), expression of NADPH oxidase subunits, the endothelial NO synthase (eNOS), the soluble guanylyl cyclase (sGC), as well as the activity and expression of the cyclic GMP-dependent kinase I (cGK-I) were assessed by Western blotting technique. eNOS, sGC, cGK-I expression and acetylcholine-induced NO production were unaltered in Bkß1 -/- animals, whereas endothelial function was impaired and the activity of the cGK-I was reduced. Vascular O 2 - and expression of the NADPH oxidase subunits p67 phox and Nox1 were increased. Endothelial dysfunction was normalized by the NADPH oxidase inhibitor apocynin. Potassium chloride- and iberiotoxin-induced depolarization mimicked the effect of BKß1-deletion by increasing vascular O 2 - in an NADPH-dependent fashion.
Conclusions- The deletion of BKß1 causes endothelial dysfunction by increasing O 2 - formation via increasing activity and expression of the vascular NADPH oxidase.
Endothelium-dependent relaxation is compromised in mice lacking the ß1 subunit of the large conductance, Ca 2+ -activated K + channel. Expression of the NADPH oxidase subunit p67 phox and aortic superoxide are increased, but endothelial NO production is normal. Thus, BKß1 deletion induces endothelial dysfunction, at least in part, via NADPH oxidase-mediated ROS production.
【关键词】 arterial tone BK Ca channel subunit ß NADPH oxidase NO/cGMP pathway vascular dysfunction
Introduction
Large conductance calcium-activated potassium channels (BK) are important for the control of arterial smooth muscle tone. They are activated by localized increases of intracellular calcium concentration (Ca 2+ sparks) and by membrane depolarization, and their activation results in membrane hyperpolarization as well as a decrease in vascular tone. 1,2 BK channels are composed of pore-forming BK and auxiliary BKß subunits, which confer an increased sensitivity to changes in membrane potential and [Ca 2+ ] i. BK is widely expressed, whereas the ß1 subunit appears to be exclusively expressed in smooth muscle and mesangial cells. 3-6 Deletion of the ß1 subunit in mice functionally uncouples Ca 2+ sparks from the activation of BK channels, leading to membrane potential depolarization as well as increased vascular myogenic tone and hypertension. 7,8 In angiotensin-induced hypertension and in spontaneously hypertensive rats a marked downregulation of the ß1 subunit has been associated with the reduced functional uncoupling of BK channels from Ca 2+ sparks and with vascular dysfunction. 9 Furthermore, a gain-of-function BKß1 variant is associated with a low prevalence of diastolic hypertension, 10 highlighting the importance of the ß1 subunit of the BK channel in the regulation of blood pressure. A previous report has demonstrated a significantly impaired endothelium-dependent (ACh-elicited) relaxation of isolated mouse aorta in the presence of iberiotoxin, a specific inhibitor of BK Ca channels, pointing to a crucial role of the BK Ca in the modulation of endothelial function. 11
Endothelium-derived nitric oxide (NO) is an important endogenous modulator of vascular tone that is generated by the endothelial NO-synthase (eNOS) from L-arginine. 12 NO stimulates soluble guanylyl cyclase (sGC) in subjacent vascular smooth muscle cells, increases cGMP levels, and activates cGMP-dependent protein kinase 1 (cGK-I) 13. Vascular relaxation results after the phosphorylation of cGK-I substrates, such as the inositol trisphosphate (inositol trisphosphate [IP 3 ])-receptor associated cGK-I substrate protein (IRAG), 14 which interferes with agonist-induced intracellular Ca 2+ mobilization. cGMP-induced relaxation is also mediated in part by activation of BK channels 15 and cGK-I phosphorylates BK channels directly, increasing the open probability of the channels, 16 and may also activate BK channels indirectly by increasing both the frequency and amplitude of Ca 2+ sparks. 1 The ß1 subunit was shown to be required for NO/cGMP-dependent activation of BK Ca channels. 17
Hypertension is associated with increased oxidative stress in the vessel wall, 18,19 and increased levels of vascular superoxide (O 2 - ) inactivate the NO formed by eNOS and therefore impair NO/cGMP-mediated vasorelaxation. 20 Among the different vascular sources of O 2 -, the NADPH oxidase activity and expression has been shown to increase in hypertension. 20 In vascular smooth muscle cells, the functional NADPH oxidase consists of two membrane-spanning subunits, the gp91 phox homologue Nox1 and p22 phox, as well as the associated cytosolic factors p47 phox, p67 phox, and the small GTPase rac1. 19 Some reports have suggested that cell depolarization might activate vascular NADPH oxidases. 21,22
Therefore, the present study was designed to examine to what extent BKß1 deficiency may influence vascular NO production, the sensitivity of the vasculature to exogenously administered NO, vascular superoxide production, and sGC/cGK-I signaling.
Materials and Methods
All experiments were performed in mice carrying a targeted deletion of the ß1 subunit of the BK channel (BKß1 -/- ) and their wild-type littermates (BKß1 +/+ ), in accordance with national guidelines for the care and use of research animals. The generation of the BKß1 -/- has been described in detail previously. 8 All mice were genotyped after the experiments.
Vascular Reactivity Studies
Mice were anesthetized using CO 2, the thoracic aortas were rapidly removed, cut into ring segments of 4 mm length and mounted in organ chambers for isometric tension recording, as described previously. 23 In some experiments, vessels were pre-incubated with apocynin (1 mmol/L) for 30 minutes.
Immunoblotting
Western blots of aortic protein were performed as described. 18,24 For extended protocol see supplementary information.
Spin Tapping of NO
NO was measured by an EPR-based method using colloid Fe(II)-diethyldithiocarbamate (Fe(DETC) 2 ) as a spin trap. The vascular NO production was quantified using NO-Fe(MGD) 2 standard and expressed in pmol/(mg dry weight x h). 25 For extended protocol see supplementary information.
Oxidative Fluorescent Microtopography and NADPH Oxidase Activity
Dihydroethidine (DHE) was used to detect the in situ formation of superoxide according to the oxidative fluorescent microtopography, as described recently. 26 For extended protocol see supplementary information.
NADPH-oxidase activity in aortic tissue was measured as described by Iwai et al 27 using cytochrome c (Sigma Aldrich) and 100 µmol/L NADPH. NADPH-oxidase activity was expressed as initial O 2 - formation rate . Heart membrane fractions were prepared as recently described 28 and superoxide formation was measured as previously published. 29 For extended protocol see supplementary information.
Statistical Analysis
Results are expressed as mean±SEM. EC 50 s were calculated from a logit transformation of relaxation data (Sigma Plot). A Scheffe post-hoc test was used to examine differences between groups, when significance was indicated. All other data were analyzed by 1-way ANOVA. P <0.05 were considered significant.
Results
Vascular Reactivity Studies
The endothelium-dependent vasodilator acetylcholine (ACh) elicited the concentration-dependent relaxation of prostaglandin F 2 -preconstricted isolated aortic rings of both, BKß1 +/+ and BKß1 -/- mice ( Figure 1 A). Aortic rings of BKß1 -/- had a markedly impaired endothelium-dependent relaxation as compared to BKß1 +/+ mice ( Figure 1 A and Table I in supplementary information). Importantly, the impaired response of BKß1 -/- vessels to ACh was normalized by pre-incubation with the NADPH oxidase inhibitor apocynin ( Figure 1 A and Table I in supplementary information). The potency and efficacy in response to the endothelium-independent vasodilator nitroglycerin (NTG) was not significantly different between BKß1 +/+ and BKß1 -/- mice ( Figure 1 B and Table I in supplementary information).
Figure 1. Effects of deletion of the BKß1 subunit on endothelium-dependent relaxation by ACh (A) and endothelium-independent relaxation by nitroglycerin (NTG) (B) in isolated, prostaglandin F 2 -constricted aortic rings from mice. The effects of NADPH oxidase inhibition on impaired ACh-elicited relaxation in BKß1 -/- mice was tested by apocynin. Data are mean±SEM from 9 to 24 experiments.
Vascular NO/cGMP Signaling: Expression of eNOS, sGC, cGK-I, VASP, and P-VASP
Expression levels of eNOS, sGC, and cGK-I in mouse aorta were assessed by using the Western blotting technique. P-VASP represents the phosphorylated vasodilator stimulated phosphoprotein at serine 239, a specific phosphorylation target of cGK-I and as shown previously reflects cGK-I activity. 30 Expression of eNOS, sGC and cGK-I was not changed in BKß1 -/- mice (Figure I in supplementary information). In contrast, basal activity of cGK-I, as detected by the P-VASP/total VASP ratio, was found to be markedly reduced in BKß1 -/- mice ( Figure 2A,2 B). In vessels of BKß1 +/+ mice, ACh significantly increased P-VASP ( Figure 2C, 2 D), whereas in BKß1 -/- mice already basal levels of P-VASP were significantly decreased as compared with wild type and ACh evoked no significant changes ( Figure 2C,2 D).
Figure 2. Effects of deletion of the BKß1 subunit on the expression of P-VASP and VASP under basal conditions (A,B) and after stimulation with ACh (C,D) in mice. A,C, Original blots. B,D, Bar diagram of a densitometric evaluation. Data are mean±SEM from 11 to 12 experiments. P-VASP data in (B) are expressed as P-VASP/total VASP ratios, in (D) as % of basal P-VASP of BKß1+/+ mice. * P <0.05 BKß1 -/- vs BKß1 +/+; P <0.05 ACh vs basal.
Vascular NO Production
As shown in Figure 3, the ACh (1 µmol/L)-induced generation of NO in aortic rings from BKß1 +/+ mice (34.6±0.9 pmol NO per mg dry weight per hour; n=3) was not significantly different in BKß1 -/- mice (40.9±9.0 pmol/; n=3). The Ca 2+ -ionophore A23187 (10 µmol/L)-stimulated NO production in BKß1 +/+ mice was also not significantly different from that recorded in BKß1 -/- mice (113.4±4.8 versus 111.6±6.0 pmol/; n=4). These results indicate that agonist-stimulated generation of NO is not impaired in aortae from BKß1 -/- mice.
Figure 3. Detection of NO by electron paramagnetic resonance (EPR) spin trapping in isolated aortae from BKß1 -/- and BKß1 +/+ mice. Aortae were incubated in the presence of ACh (1 µmol/L) and colloid Fe(DETC) 2 (200 µmol/L) at 37°C for 60 minutes. A, Original EPR recordings; the intensity (I) of the characteristic triplet EPR signal with g av =2.035 reflects the absolute amount of NO-Fe(DETC) 2 formed in the sample during incubation. B, Bar diagrams of NO production after quantification and normalization of the EPR signal intensities (mean±SEM of 3 experiments).
Determination of Superoxide in the Aortic Wall
A main factor limiting the bioavailability of NO is the superoxide anion radical (O 2 - ). 12 We therefore assessed O 2 - formation in the aortic wall by means of DHE-derived fluorescence. Staining of aortic sections with DHE revealed a marked increase in vascular O 2 - in BKß1 -/- compared with levels in tissue from BKß1 +/+ mice. This increase was observed primarily in the media, but also to a lower degree in the intima and adventitia ( Figure 4, upper panel). Importantly, increased DHE staining in vessels from BKß1 -/- animals was markedly decreased by incubation of the vessels with apocynin, a NADPH oxidase inhibitor ( Figure 4, right panel). To mimic the effect of ß1 deletion on O 2 - production we blocked the BK-channel by acute application of 200 nmol/L iberiotoxin (Ibtx) or depolarized vascular tissue from wild-type mice by addition of 80 mmol/L KCl. As indicated in Figure 4 (left and medium panel), these maneuvers increased markedly O 2 - formation in wild-type vessels, all of which was normalized by pre-incubation with the NADPH oxidase inhibitor apocynin. O 2 - formation in the BKß1 -/- vessels was also decreased by chelerythrine (1 µmol/L), suggesting an involvement of protein kinase C (PKC) in mediating this phenomenon ( Figure 4, right panel).
Figure 4. In situ detection of superoxide in mouse aorta. Fluorescent photomicrographs of microscopic sections of aortas from BKß1 +/+ mice (left and middle) and BKß1 -/- mice (right). Vessels were labeled with dihydroethidine dye, which produces red fluorescence when oxidized to ethidine by superoxide. Effects of the NADPH oxidase activity inhibitor apocynin in BKß1 -/- aorta and on KCl-induced and iberiotoxin (Ibtx)-induced depolarization in BKß1 +/+ aorta. The effects of the PKC inhibitor chelerythrine were also determined. The green auto-fluorescence corresponds to the basal laminae. Data are representative of n=4 experiments. E indicates endothelium.
NADPH Oxidase Activity and Subunit Nox1 and p67 phox Expression
Inhibitory effects of apocynin on vascular O 2 - production in BKß1 -/- animals suggested an involvement of the NADPH oxidase in being responsible for increased oxidative stress in these animals. Therefore, we assessed the NADPH oxidase activity in aorta and heart membrane fractions of both mouse strains. Superoxide formation was significantly increased in aortic and heart membrane fractions from BKß1 -/- mice ( Figure 5A, 5 B). The signal in heart membranes was decreased below control levels by the inhibitor of flavin-dependent oxidoreductases diphenyleneiodonium chloride (DPI) and normalized by the NADPH oxidase inhibitors phenylarsine oxide (PAO) and apocynin ( Figure 5 B).
Figure 5. Effects of deletion of the BKß1 subunit on the NADPH oxidase activity in aortic (A) and heart membrane fractions (B), and the expression of the NADPH oxidase subunit p67 phox (C) and Nox1 (D) in aortic tissue. NADPH oxidase activity was measured by the cytochrome C assay and lucigenin (5 µmol/L)-enhanced chemiluminescence. The effect of NADPH oxidase inhibition by DPI, PAO (each 150 µmol/L), and apocynin (1 mmol/L) was also determined in heart membrane fractions. Data are the mean±SEM of 4 (A) and 7 to 18 (B) experiments. Representative blots for p67 phox and Nox1 expression and densitometric evaluation (mean±SEM) from n=4 to 6 experiments. * P <0.05 BKß1 -/- vs BKß1 +/+ and P <0.05 vs BKß1 -/-.
We determined expression of the oxidase subunit Nox1 that harbors the catalytic center and the cytosolic subunit p67 phox in aortic tissue from both mouse strains. In aortae from BKß1 -/- mice, expression of the p67 phox and Nox1 subunits, determined by immunoblotting, were almost twice that detected in BKß1 +/+ mice (n=4) ( Figure 5 C, D).
Discussion
The results of the present investigation show that disruption of the BKß1 subunit leads to an impaired endothelium-dependent (ACh-elicited) relaxation of the aorta, reduced basal and stimulated activity of cGK-I, increased vascular O 2 - production and increased NADPH oxidase activity as well as enhanced expression of the NADPH oxidase subunits, p67 phox and Nox1. The effects of ß1 deletion on the NADPH oxidase activity could be mimicked in vessels from wild-type mice by membrane depolarization with KCl or blocking the BK-channel with iberiotoxin. Importantly, inhibition of BK Ca channel by iberiotoxin has been shown to impair ACh-induced, endothelium-dependent relaxation. 11 The results of the present study indicate that a depolarization in response to iberiotoxin and high dose KCl of the membrane stimulates vascular NADPH-oxidase-driven O 2 - production. Therefore it is tempting to speculate that a deletion of BKß1 may lead to more depolarized membrane potentials in vascular smooth muscle cells, all of which may contribute to the depressed vascular responses to endothelium-dependent vasodilators.
We also established a marked degree of endothelial dysfunction in vessels from BKß1 -/- mice. In addition, we observed a reduction in the basal and stimulated activity of the cGK-I phosphorylation target, the vasodilatory-stimulated phosphoprotein (VASP). Importantly, this parameter had been previously established to reflect vascular NO bioavailability after reaction of NO with superoxide. 18,30-32 Because eNOS expression and agonist-stimulated NO production as assessed by EPR were not impaired in aortae from Bkß1 -/- mice, it seems unlikely that the uncoupling of NOS may significantly contribute to this phenomenon. It should be noted that in our model, the site of NO production/NO trapping was spatially dissociated from the primary site of O 2 - superoxide production (intima and media, respectively). As it has been shown previously, this method for detection of NO in isolated vessels is rather resistant to elevated extracellular O 2 - levels and therefore reflects true NO production by eNOS, rather than vascular NO bioavailability. 25,33 Taking these considerations into account, it is not surprising that elevated levels of O 2 - in aortas of BKß1 -/- mice, did not affect the NO signal.
We also observed that the expression of the sGC and cGK-I were not modified in vessels of Bkß1 -/- mice. These observations point to a reduced vascular bioavailability of endothelium-derived NO, caused by increased vascular O 2 - production. To test for the underlying source of O 2 -, vessels from Bkß1 -/- were incubated with the NADPH oxidase inhibitor apocynin. Because DHE staining was markedly reduced after incubation we hypothesized an involvement of this enzyme in being responsible for increased O 2 - production in the Bkß1 -/- animals. We found increased NADPH-driven O 2 - production in aortic and heart membrane fractions, which was blocked by inhibitors of the NADPH oxidase such as PAO, apocynin and DPI. In addition, we established increased expression of the NADPH oxidase subunits p67 phox and Nox1 (smooth muscle specific) in vessels from BKß1 -/- mice (see Figure 5 ).
To address the contribution of increased vascular NADPH oxidase activity to endothelial dysfunction, vessels from BKß1 -/- vessels were challenged with the endothelium-dependent vasodilator ACh in the presence and absence of the NADPH oxidase inhibitor apocynin. Importantly, apocynin almost completely corrected endothelial dysfunction in vessel from BKß1 -/-, suggesting a major contribution of the oxidase in causing endothelial dysfunction in this particular animal model. Previous studies have indicated an involvement of protein kinase C in activating the oxidase. 18,32,34 To address this issue, vessels from BKß1 -/- mice were stained with DHE in the presence and absence of the PKC inhibitor chelerythrine. Importantly, chelerythrine was able to wipe out increased superoxide levels pointing to crucial role of PKC in activating the enzyme.
Endothelial dysfunction in these animals is also associated with hypertension, which averages 10 mm Hg increase in systolic blood pressure (BP). 7,8 Thus, the question arises, whether this mild increase in BP may activate the NADPH oxidase or whether the increase in BP may be secondary to increased vascular NADPH-oxidase-mediated O 2 - and therefore decreased vascular NO bioavailability. Indeed, hypertension per se has been shown to trigger adaptive responses in smooth muscle cells, such as proliferation and vascular remodeling. 35 These responses have been shown to be linked to increased rac1 activity and superoxide-dependent signaling for altered gene expression. 36 In addition, acute and chronic hypertension 37,38 increase O 2 - formation in arteries by stimulating the NADPH oxidase. It is therefore conceivable that in the animal model studied, hypertension may have contributed (at least in part) to the increased O 2 - formation and that this is at least in part caused by an activation of the NADPH oxidase.
To address the role of channel blocking and depolarization per se on vascular O 2 - production, vessels from wild-type animals were incubated with 200 nmol/L Ibtx and 80 mmol/L KCl, and DHE staining was performed in the presence and absence of the NADPH oxidase inhibitor apocynin. Interestingly, Ibtx as well as KCl markedly increased DHE staining in particular in the media, all of which was strikingly reduced by apocynin, pointing to a role of the NADPH oxidase as an important vascular O 2 - source in the depolarized state. These findings go along with previous observations with cultured endothelial cells, where depolarization of these cells led to increased O 2 - production, which was driven mainly by the NADPH oxidase. 21
The vascular NADPH-oxidase plays an important pathogenetic role in vascular dysfunction. 19 For example, endothelial dysfunction in hypercholesterolemic rabbits 39 as well as in vascular tissue from patients with diabetes mellitus 40 was consistently found to be associated with an increased NADPH-oxidase-mediated vascular O 2 - production. Recent studies indicate that vascular BK channels are inhibited directly by reactive oxygen species, 41 which target a cysteine residue of the BK subunit. 42 Thus, a direct inhibition of the BK subunit by O 2 - may further aggravate vascular dysfunction in a positive feedback fashion.
The results of the present studies provide new insight into the vascular consequences of a disruption of the ß1 subunit of the BK channel. Mice lacking the ß1 subunit develop a moderate degree of hypertension, vascular dysfunction, and exhibit an increased aortic O 2 - production throughout the vessel wall, which is to a large extent mediated by the vascular NADPH oxidase. These changes were associated with a marked inhibition of cGK-I activity, whereas endothelial eNOS-mediated NO formation remained unaltered. Taken together these findings point to an important consequence of disturbed physiological regulation of the BK channel on endothelium-dependent regulation of tone in large arteries.
Acknowledgments
We appreciate the technical support by Kornelia Anders, Hartwig Wiebold, Jörg Schreiner, and Claudia Kuper.
Sources of Funding
This study was supported by the Deutsche Forschungsgemeinschaft: Mu 1079/4-1 (to T.M.) and SFB553/C17 (to A.D.), Eh 109/12-1 (to HE), and Po 137/33-1 (to OP) and by the European Vascular Genomic Network, a Network of Excellence supported by the European Community?s sixth Framework Program (Contract LSHM-CT-2003-503254).
Disclosures
None.
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作者单位：From II. Medizinische Klinik (M.O., A.W., P.W., A.L.K., M.C., U.H., A.D., T.M.), Johannes Gutenberg-Universität, Mainz, Germany; Institut für Vegetative Physiologie und Pathophysiologie (J.F., H.E.), Universitätsklinikum Eppendorf, Hamburg, Germany; Institut für Neurale Signalver