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(Circulation. 2005;112:60-68.)
© 2005 American Heart Association, Inc.

Hypertension

Elevated Blood Pressure Linked to Primary Hyperaldosteronism and Impaired Vasodilation in BK Channel–Deficient Mice

Matthias Sausbier, PhD^*; Claudia Arntz, PhD^*; Iancu Bucurenciu, MD; Hong Zhao, MD, PhD; Xiao-Bo Zhou, MD; Ulrike Sausbier, PhD; Susanne Feil, PhD; Simone Kamm; Kyrill Essin, PhD; Claudia A. Sailer, PhD; Usamah Abdullah; Peter Krippeit-Drews, PhD; Robert Feil, PhD; Franz Hofmann, MD; Hans-Günther Knaus, MD; Chris Kenyon, MD; Michael J. Shipston, PhD; Johan F. Storm, MD, PhD; Winfried Neuhuber, MD; Michael Korth, MD; Rudolf Schubert, MD; Maik Gollasch, MD; Peter Ruth, PhD

From Pharmakologie und Toxikologie, Pharmazeutisches Institut der Universität Tübingen, Tübingen, Germany (M.S., C.A., I.B., H.Z., U.S., U.A., P.K.-D., P.R.); Institut für Pharmakologie für Pharmazeuten, Universitätsklinikum Hamburg-Eppendorf, Hamburg-Eppendorf, Germany (X.Z., M.K.); Institut für Pharmakologie und Toxikologie der Technischen Universität München, München, Germany (S.F., S.K., R.F., F.H.); Helios Franz-Volhard-Klinik, Med Klinik für Nephrologie und Intensivmedizin, MDC für Molekulare Medizin, Humboldt Universität Berlin, Berlin, Germany (K.E., M.G.); Institut für Biochemische Pharmakologie, Universität Innsbruck, Innsbruck, Austria (C.A.S., H.K.); Endocrinology Unit, School of Molecular and Clinical Medicine, Molecular Medicine Centre, Western General Hospital, Edinburgh, Scotland (C.K.); Membrane Biology Group, Division of Biomedical Science, University of Edinburgh, Edinburgh, Scotland (M.J.S.); Department of Physiology and Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, Norway (J.F.S.); Anatomisches Institut, Universität Erlangen-Nürnberg, Erlangen-Nürnberg, Germany (W.N.); and Institut für Physiologie der Universität Rostock, Rostock, Germany (R.S.).

Correspondence to Dr Peter Ruth, Pharmakologie und Toxikologie, Pharmazeutisches Institut der Universität Tübingen, Tübingen, Germany. E-mail peter.ruth{at}uni-tuebingen.de

Received August 4, 2004; revision received October 12, 2004; accepted October 25, 2004.

	Abstract

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Background— Abnormally elevated blood pressure is the most prevalent risk factor for cardiovascular disease. The large-conductance, voltage- and Ca²⁺-dependent K⁺ (BK) channel has been proposed as an important effector in the control of vascular tone by linking membrane depolarization and local increases in cytosolic Ca²⁺ to hyperpolarizing K⁺ outward currents. However, the BK channel may also affect blood pressure by regulating salt and fluid homeostasis, particularly by adjusting the renin-angiotensin-aldosterone system.

Methods and Results— Here we report that deletion of the pore-forming BK channel subunit leads to a significant blood pressure elevation resulting from hyperaldosteronism accompanied by decreased serum K⁺ levels as well as increased vascular tone in small arteries. In smooth muscle from small arteries, deletion of the BK channel leads to a depolarized membrane potential, a complete lack of membrane hyperpolarizing spontaneous K⁺ outward currents, and an attenuated cGMP vasorelaxation associated with a reduced suppression of Ca²⁺ transients by cGMP. The high level of BK channel expression observed in wild-type adrenal glomerulosa cells, together with unaltered serum renin activities and corticotropin levels in mutant mice, suggests that the hyperaldosteronism results from abnormal adrenal cortical function in BK^–/– mice.

Conclusions— These results identify previously unknown roles of BK channels in blood pressure regulation and raise the possibility that BK channel dysfunction may underlie specific forms of hyperaldosteronism.

Key Words: blood pressure • ion channels • vasoconstriction • vasodilation • hyperaldosteronism

	Introduction

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Increased arterial tone is a hallmark of elevated blood pressure. During hypertension, pressure-induced depolarization of vascular smooth muscle cells contributes to increased vascular tone by increasing Ca²⁺ influx through voltage-dependent Ca²⁺ channels.^1,2 In vitro evidence suggests that the unique large-conductance, voltage- and Ca²⁺-activated K⁺ (BK) channel limits Ca²⁺ entry and thereby arterial contraction by repolarizing smooth muscle cells and closing voltage-dependent Ca²⁺ channels previously opened by pressure or vasoconstrictors.³ BK channels also mediate steady hyperpolarization and vasorelaxation as a result of transient outward currents carried by BK channels spontaneously activated by local release of Ca²⁺ from intracellular stores via ryanodine receptor channels.⁴ The vascular BK channel consists of 4 subunits that form the ion-conducting pore and 4 auxiliary ß1 subunits. The ß1 subunits, which are restricted to smooth muscle, maintain the normal high voltage and Ca²⁺ sensitivity of the pore-forming subunit.⁵ The role of the BK channel auxiliary ß₁ subunit in blood pressure regulation has been tested previously by deleting its gene in mice. The resulting loss of this subunit impaired the coupling between Ca²⁺ release and the activation of hyperpolarizing BK currents, leading to systemic hypertension.^6,7 Recent studies raise the possibility that changes in ß1 subunit expression contribute to the development of hypertension in rat⁸ and that gain of function mutation in the same subunit decreases the prevalence of diastolic hypertension in humans.⁹ However, even in the absence of functional ß1 subunits, the subunit can still form functional channels, which might be activated at physiological potentials if their voltage and Ca²⁺ sensitivity are increased by other factors such as endothelial factors^10,11 and/or phosphorylation.^12,13 Thus, functional BK channels may be operative in blood vessels even when the ß1 subunit is lacking. In addition, BK channels in tissues other than vasculature, such as the adrenal gland,¹⁴ may also influence blood pressure regulation. Therefore, we used mice lacking the BK channel subunit (BK^–/–15 to evaluate the global impact of BK channels on blood pressure regulation.

	Methods

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Details are given in the online-only Data Supplement.

Mice
BK^–/– mice were generated as described.¹⁵ Wild type (WT) and BK^–/– mice with the hybrid SV129/C57BL6 background (always F2 generation) were used. Either litter- or age-matched animals were randomly assigned to the experimental procedures undertaken in accordance with the German legislation on protection of animals.

Immunohistochemistry of Adrenal Gland
For immunofluorescence, on-slide 5-µm cryostat slices from nonfixed WT and BK^–/– adrenal glands were incubated with anti-BK_(674–1115). BK expression was analyzed with a confocal laser-scanning microscope (Biorad MRC1000 attached to Nikon Diaphot 300 and equipped with a krypton-argon laser). For peroxidase/DAB detection, 10-µm cryosections of WT and BK^–/– adrenal glands were perfused with 4% paraformaldehyde and incubated with anti-BK_(674–1115). Data analysis was performed with a Zeiss Axioplan 2 microscope equipped with a Zeiss Axio Cam digital camera.

Determination of Renin Activity, Corticotropin, and Cortisterone Levels From Serum
Plasma renin concentration was measured as the generation of angiotensin I (ng/mL per hour) when plasma samples were incubated with excess renin substrate (plasma with no intrinsic renin activity from a binephrectomized rat). Angiotensin I was measured by radioimmunoassay as previously described.¹⁶ For the determination of corticotropin in plasma, we used a 2-site solid-phase immunoradiometric assay (IRMA) kit (Euria-acth IRMA kit) from Euro-Diagnostica AB. This assay measures intact corticotropin (1–39); in the assay used, the limit of detection was 5 pg/mL and the intra-assay variation <5%. Serum corticosterone was analyzed with an in-house specific radioimmunoassay, as described previously, and modified for microtiter plate scintillation proximity assay.¹⁷

Serum Electrolytes and Serum Aldosterone Levels
Serum was separated from nonheparinized blood collected by heart puncture from WT and BK^–/– mice euthanized by inhalation of carbon dioxide. The serum concentrations of Na⁺ and K⁺ were measured by flame photometry. Serum concentrations of aldosterone were measured by radioimmunoassay.

Electrophysiology of Tibial Artery and Aortic Smooth Muscle Cells
For cell isolation, tibial artery, a fourth-order branch of the aorta, or aorta was incubated in Ca²⁺-free physiological saline solution (PSS) containing papain at 37°C for 30 minutes. Then the solution was exchanged for PSS containing Ca²⁺, collagenase type H, and hyaluronidase, and digestion was continued for another 10 minutes at 37°C. For measuring of outward membrane currents (whole-cell mode), the free Ca²⁺ concentration in the pipette solution was 300 nmol/L. The holding potential was –50 mV (arterial cells) and –20 mV (aortic cells), and test pulses of 300-ms duration were applied every 5 seconds. For measuring membrane potentials (whole-cell perforated patch), the pipette solution contained nystatin. For recording of macroscopic Ca²⁺ channel currents, cells were voltage-clamped at a holding potential of –60 mV, and the potential was stepped, for 300 ms every 5 seconds, in 10-mV increments up to 50 mV. The inward current was measured as peak inward current with reference to zero current.

Luminal Diameter Analysis of Small Arteries by Videomicroscopy
Tibial small arteries were equilibrated in buffer, and an intravascular pressure of 80 mm Hg was applied under nonflow conditions. The chamber was continuously perfused at a rate of 2 mL/min with buffer at 37.0±0.5°C. The small artery was allowed to equilibrate under videomicroscopic recording until a stable myogenic tone spontaneously developed after 15 to 20 minutes. At the end of each experiment, Ca²⁺-free buffer was applied to determine maximal vessel diameters. No significant difference in maximal vessel diameter was detected between the 2 genotypes (WT, 69±3 µm; BK^–/–, 63±2 µm). All compounds were administered to the adventitial side of the pressurized small arteries. To exclude prostaglandin effects, the buffer contained 1 µmol/L diclofenac.

[Ca²⁺]_i Measurements in Aortic Smooth Muscle Cells
Single aortic smooth muscle cells (prepared as above) were loaded with 2.5 µmol/L fura 2-AM. For [Ca²⁺]_i measurement, cells were transferred to a glass coverslip coated with 0.01% poly-L-lysine solution and continuously superfused with PSS at 36°C at a flow rate of 2 to 4 mL/min. [Ca²⁺]_i measurements were performed with the use of the dual-wavelength microfluorescence technique. Two [Ca²⁺]_i transients were elicited consecutively with a 15-minute interval in between to allow refilling of intracellular Ca²⁺ stores.

Spontaneous Transient Outward Current and Ca²⁺ Spark Analysis
Cerebral arteries were placed in Ca²⁺-free Hanks’ solution supplemented with papain for 15 minutes at 36°C. The segments were then placed in Hanks’ solution containing collagenase type F and H (ratio 30% and 70%, respectively) and 0.1 mmol/L CaCl₂ for 6 minutes at 36°C. After several washes in Ca²⁺-free Hanks’ solution, single cells were isolated. Spontaneous transient outward currents (STOCs) were measured in the perforated patch mode. Holding potential was set at –60 mV. The pipette solution contained 250 µg/mL amphotericin. To measure Ca²⁺ sparks, cells were incubated with fluo 3-AM for 30 minutes at room temperature in Ca²⁺-free Hanks’ solution. Ca²⁺ sparks were measured as local fractional fluorescence increases (F/F₀) by confocal fluorescence microscopy at room temperature. The baseline fluorescence (F₀) was determined by averaging line-scan images in the absence of Ca²⁺ sparks.

Long-Term Radiotelemetric Blood Pressure Analysis
Mean arterial blood pressure (MAP), heart rate (HR), and physical activity were analyzed in conscious male WT and BK^–/– mice (n=7 for each genotype). Mice (aged 3 to 4 months), either litter- or age-matched, did not significantly differ in body weight (26.2±1.3 and 23.5±0.7 g, respectively). Mice were anesthetized with isoflurane. A ventral midline incision was performed before careful isolation of the left common carotid artery. For ligation and retraction, 2 silk ligatures were passed under the vessel, one 0.8 cm caudal to the bifurcation of the interior and exterior carotid artery and another 0.5 cm rostral from the caudal ligature. A tiny incision in the carotid artery was made for insertion of the catheter. The inserted catheter tip was advanced to the thoracic aorta and fixed with suture. A subcutaneous pocket was performed along the right flank for placing the transmitter body. After subcutaneous fixation of the transmitter, the incision was closed with 6-0 silk. MAP, HR, and physical activity were recorded at days 5 to 7 after surgery, when mice have regained normal locomotor activity. Radiotelemetric signals were sampled for 1 minute at 5-minute intervals.

	Results

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Increased Myogenic Tone and Attenuated cGMP-Dependent Vasorelaxation in BK^–/– Small Arteries
In smooth muscle cells from the tibial artery of WT mice, depolarization elicited large, iberiotoxin-sensitive outward K⁺ currents, but no such currents were detected in cells from BK^–/– cells (Figure 1a). Furthermore, BK^–/– cells did not exhibit spontaneous hyperpolarizations and showed a more depolarized mean membrane potential (–31.0±2.3 mV) than WT cells (–41.0±3.9 mV). Depolarization was also observed in WT cells when BK channels were specifically blocked by iberiotoxin (Figure 1b). The BK channel has recently been found to be physically and functionally associated with the L-type Ca²⁺ channel.¹⁸ In the arterial cells, however, the expression of nifedipine-sensitive L-type Ca²⁺ channels was not changed, as revealed by almost identical current densities in BK^–/– and WT arterial cells, thereby excluding a compensatory downregulation of this channel in BK^–/– arteries (Figure 1c). It is conceivable that the sustained depolarization of BK^–/– arterial cells compared with WT cells could produce a sustained rise in cytosolic Ca²⁺ because of a "window current" caused by incomplete inactivation of L-type Ca²⁺ channels.¹⁹ Interestingly, the steady state calcium window in smooth muscle cells was maximal at –30 mV,¹⁹ which corresponds closely to the membrane potential in BK^–/– cells.

View larger version (27K): [in this window] [in a new window]   Figure 1. Resting membrane potential and current density of L-type Ca2+ current in tibial artery smooth muscle cells. a, Current-voltage relationship of K+ outward currents in cells from 3 WT (blue circles) and 3 BK–/– (red squares) tibial arteries. Whole-cell currents were measured at 300 nmol/L [Ca2+]i from a holding potential of –50mV without (blue circles, red squares) and with 300 nmol/L iberiotoxin (respective data points are covered by the red squares); n=11 (WT) and n=15 (BK–/–) cells. b, Membrane potential recordings from WT and BK–/– tibial artery cells with and without iberiotoxin and mean±SEM membrane potential statistics (13 and 18 cells from WT and BK–/– mice, respectively); *P<0.05. c, Amplitudes of voltage-gated Ca2+ channel currents in WT and BK–/– cells do not differ. Currents were measured in the whole-cell configuration, and barium was used as charge carrier. Inward currents were evoked by step depolarizations (300-ms duration) of increasing amplitude from a holding potential of –60 mV up to +50 mV in 10-mV increments. Current-voltage relationships of peak inward currents are shown (n=11 from 3 WT and n=7 from 3 BK–/– mice). Mean±SEM current densities are plotted against the respective test potential. Inset, Averaged current tracings before (ctr) and after superfusion of a WT cell with 1 µmol/L nifedipine. Inward currents were activated by step depolarizations from –60 to +10 mV and maintained for 300 ms.

Thus, the more depolarized membrane potential in BK^–/– cells may influence small-artery tone, which is obligatorily dependent on pressure-evoked depolarization triggering Ca²⁺ entry through voltage-gated Ca²⁺ channels.²⁰ In fact, at physiological relevant pressures (80 and 120 mm Hg), tibial arteries from BK^–/– mice showed increased myogenic tone compared with arteries from WT mice (Figure 2a). However, the myogenic response, ie, the change in myogenic tone induced by pressure steps, was not significantly different in BK^–/– versus WT tibial arteries (Figure 2b), suggesting that BK channels do not determine the myogenic response per se. Apparently, membrane depolarization and increases in global [Ca²⁺]_i plus local Ca²⁺ sparks in response to transmural pressure are not sufficient to evoke a BK channel–mediated negative feedback regulation of pressure-induced contraction. Rather, BK channels seem to mediate essentially steady dilatation over a large pressure range, inasmuch as their deletion produced an almost parallel upward shift of the pressure-tone relationship (Figure 2a).

View larger version (22K): [in this window] [in a new window]   Figure 2. Increased myogenic tone and attenuated cGMP-dependent vasorelaxation in BK–/– small arteries. a, Effect of transmural pressure on myogenic tone in tibial artery. Myogenic tone was calculated as (1–d/dmax), where d is the vessel diameter at a certain pressure and dmax the diameter in Ca2+-free buffer at that pressure. b, Effect of pressure steps on myogenic tone change (myogenic tone, ie, tone at end pressure minus tone at initial pressure of the step); n=4 WT and 4 BK–/– arteries. c, cGMP- and adenosine-induced relaxation of myogenic tone of tibial artery. Relaxation was calculated as drug-induced change of inner vessel diameter as a percentage of maximal diameter in Ca2+-free buffer; n=6 arteries per genotype. All data are mean±SEM; *P<0.05; **P<0.01.

It is well established that the myogenic tone can be decreased by activation of the NO/cGMP/protein kinase G (PKG) and cAMP/protein kinase A (PKA) pathways.^21,22 Application of 8-pCPT-cGMP, a cell-permeable cGMP analogue that preferentially activates PKG, produced significantly less relaxation of BK^–/– arteries than WT arteries (Figure 2c). In contrast, adenosine, which raises [cAMP]_i via A₂ receptors,²³ was equally potent in WT and BK^–/– arteries, suggesting that the cGMP/PKG, but not the cAMP/PKA, pathway requires BK channel activation to promote arterial relaxation (Figure 2c). The partial relaxation produced by 8-pCPT-cGMP in BK^–/– arteries may involve other PKG substrates or cross-activation of PKA at high agonist concentrations.

Blood vessel relaxation by cGMP/PKG has been ascribed to suppression of Ca²⁺ transients.²⁴ To test this hypothesis, aortic smooth muscle cells from WT and BK^–/– mice, basically exhibiting the same differences in membrane potential as cells from tibial artery (Data Supplement Figure), were stimulated with the ₁-adrenergic receptor agonist phenylephrine, and the resulting Ca²⁺ transients (due to Ca²⁺ release and influx via Ca²⁺ channels²⁵) were measured in the absence of 8-Br-cGMP. The area under the curve (AUC) of the Ca²⁺ transients was almost identical in BK^–/– (n=76) and WT cells (n=64) (2.1±0.1 and 2.2±0.2 arbitrary units, respectively). Additionally, the ratio between consecutively elicited Ca²⁺ transients (AUC 2/AUC 1) was not significantly different in BK^–/– and WT cells (87±8% and 103±7%, respectively) (Figure 3). Preincubation with 8-Br-cGMP diminished the second transients in WT cells to 38±7% of control, whereas BK^–/– cells were less affected (72±11%). The attenuated suppression in BK^–/– cells suggests that BK channel activation via cGMP/PKG is important for relaxation, probably involving hyperpolarization-induced inhibition of voltage-gated Ca²⁺ channels. The small residual cGMP-induced suppression of Ca²⁺ transients in BK^–/– smooth muscle cells may be caused by cGMP-mediated inhibition of Ca²⁺ release from IP₃-sensitive stores.²⁶

View larger version (14K): [in this window] [in a new window]   Figure 3. Reduced suppression of Ca2+ transients by cGMP in BK–/– aortic smooth muscle cells. Left, Consecutive [Ca2+]i transients (first and second transients are shown), elicited by phenylephrine (10 µmol/L for 1 minute, arrows) at 15-minute intervals, in WT (blue) and BK–/– (red) aortic cells, after preincubation with or without 8-Br-cGMP (1 mmol/L, 5 minutes). Right, [Ca2+]i transients were quantified as AUC (40 to 63 cells per genotype). All data are mean±SEM; *P<0.05; **P<0.01.

The amount of Ca²⁺ in intracellular stores of aortic cells was apparently not changed by the absence of the BK channel. Caffeine at 10 mmol/L stimulated Ca²⁺ release from the sarcoplasmic reticulum to a similar extent in BK^–/– (3.6±0.2 arbitrary units of AUC; n=108) and WT cells (3.4±0.2; n=92).

Lack of STOCs in BK^–/– Arterial Cells Did Not Affect Ca²⁺ Sparks
Transient activation of outward hyperpolarizing currents (STOCs), carried by BK channels, has been proposed as a mechanism for the paradoxical Ca²⁺ spark–mediated relaxation of small arteries.²⁷ Furthermore, STOCs have been proposed to provide a negative feedback mechanism to control Ca²⁺ spark dynamics through regulation of voltage-dependent calcium entry and resultant changes in sarcoplasmic reticulum Ca²⁺ loading and ryanodine receptor sensitization. We examined STOCs and Ca²⁺ spark activity in BK^–/– cells. As expected, STOCs were completely absent in BK^–/– cerebral arterial cells, even at depolarized membrane potentials that promote Ca²⁺ influx via voltage-gated Ca²⁺ channels (Figure 4a), implicating that STOCs are carried by BK channels. However, Ca²⁺ spark parameters were not affected in BK^–/– cells, arguing against a feedback between STOCs and Ca²⁺ sparks via Ca²⁺ entry (Figure 4b). In summary, 2 mechanisms important for determining vascular tone were absent in BK^–/– small arteries: (1) BK channel activity as a major effector of cGMP/PKG-mediated relaxation and (2) the steady hyperpolarizing effect in response to Ca²⁺ sparks. Hence, pathophysiological consequences for systemic blood pressure were expected in BK^–/– mice.

View larger version (38K): [in this window] [in a new window]   Figure 4. Lack of STOCs but presence of normal Ca2+ sparks in BK–/– cerebral artery smooth muscle cells. a, STOC activity in WT and BK–/– cerebral arterial cells recorded at increasing membrane potentials and STOC frequency at –20 mV from 5 to 7 WT and BK–/– cells. b, Confocal line scans of fluo 3–loaded WT and BK–/– cells and time course of corresponding Ca2+ sparks. Spark amplitudes were measured as local fractional fluorescence increases (F/F0; F0 is baseline). Spark duration was measured at half-maximal amplitude; n=5 to 12 cells per genotype. All data are mean±SEM; *P<0.05; **P<0.01.

Elevated Blood Pressure and Hyperaldosteronism in BK^–/– Mice
MAP in the carotid artery, HR, and locomotor activity were measured by radiotelemetry in male mice. BK^–/– mice were significantly less active during the dark phase as a result of motor impairment¹⁵ (Figure 5a). Because physical activity affects MAP, we measured it during an interval (12 AM to 6 PM) when the 2 genotypes showed similar activity (Figure 5a, 5b). The BK^–/– mice showed a significantly (5.4 mm Hg) higher MAP than WT mice (Figure 5b). Additionally, diastolic and systolic blood pressures were significantly elevated in the mutants, whereas HR did not differ. This excludes the possibility that higher sympathetic tone and HR caused MAP elevation. Analysis of the MAP of BK^–/– and WT mice as a function of locomotor activity suggested that for any activity the MAP was shifted upward by 6.1 mm Hg in the mutants (Figure 5c). However, BK^–/– mice did not show high-range locomotor activity because of their ataxia.¹⁵ In contrast to BKß₁ knockouts,^6,7 the increase in blood pressure in BK^–/– mice was not accompanied by significant cardiac hypertrophy in 4- to 6-month-old animals, as indicated by heart/body weight ratio (WT, 4.6±0.2 mg/g; BK^–/–, 4.8±0.1 mg/g) (Figure 5d). Nevertheless, the elevated MAP observed under resting conditions in BK^–/– mice exhibiting HRs similar to those of WT mice supports the hypothesis that vascular BK channels may be important for the control of vascular tone and systemic blood pressure in vivo, mainly because of their effector role for both cGMP signaling and Ca²⁺ sparks.

View larger version (24K): [in this window] [in a new window]   Figure 5. Elevated arterial blood pressure in BK–/– mice. a, Locomotor activity recorded by radiotelemetry for 24 hours at days 5, 6, and 7 after surgery. Bar indicates period of MAP and HR measurement shown below; open bar indicates dark phase. b, Mean values of MAP, HR, and activity from telemetric blood pressure analysis are calculated hour by hour between 12 AM and 6 PM, the period when WT and BK–/– mice exhibited comparable locomotor activity; n=7 per genotype. c, Correlations between locomotor activity and MAP. Linear regression lines were y=4.56x+97.5 (R2=0.85) (WT) and y=4.44x+103.6 (R2=0.69) (BK–/–). d, Absence of cardiac hypertrophy in BK–/– mice. Statistics of heart weight (Hw)/body weight (Bw) (mg/g) from 10 to 11 mice per genotype are shown. All data are mean±SEM; *P<0.05; **P<0.01.

However, blood pressure regulation also involves essential endocrine mechanisms such as the renin-angiotensin-aldosterone system, which might also be affected by BK channels.²⁸ Analysis of the serum electrolytes revealed a gender-independent decrease in the K⁺ concentration ([K⁺]_serum) in mutants compared with WT (male WT 8.3±0.3 mmol/L versus male BK^–/– 6.5±0.8 mmol/L; female WT 9.2±0.2 mmol/L versus female BK^–/– 7.5±0.4 mmol/L). In general, [K⁺]_serum values of WT mice were in agreement with previously published [K⁺]_serum values of mice with a genetic background similar to the BK^–/– mice.²⁹ However, [Na⁺]_serum did not differ between the 2 genotypes (male WT 140±4 mmol/L versus male BK^–/– 137±2 mmol/L; female WT 137±2 mmol/L versus female BK^–/– 141±2 mmol/L) (Figure 6a). The lower [K⁺]_serum of BK^–/– mice was associated with markedly increased serum aldosterone levels in both genders (male WT 185±24 pg/mL versus male BK^–/– 573±108 pg/mL; female WT 236±33 pg/mL versus female BK^–/– 597±132 pg/mL) (Figure 6b). Consistent with this phenotype, we found high levels of BK channel subunit expression in the zona glomerulosa (Figure 6c). Here, these channels may control aldosterone production and/or release by influencing the membrane potential and hence the open probability of voltage-gated Ca²⁺ channels (L- and/or T-type) via the membrane potential.^30,31 In contrast to glomerulosa cells, cortisol-synthesizing cells from zona fasciculata were very weakly stained with the BK channel antibody (Figure 6c). In agreement herewith is the finding that corticosterone levels in the serum were not altered between WT and BK^–/– mice (Figure 6d). A potential causative role of the renin/angiotensin system and/or the pituitary for the observed increase in aldosterone was also considered. The determination of serum corticotropin and serum renin activity, however, revealed no significant changes of these 2 parameters between both genotypes, suggesting that the hyperaldosteronism found in BK^–/– mice was of a primary nature. Thus, elevation of blood pressure in BK^–/– mice may be attributed to both vascular and hormonal dysfunctions.

View larger version (61K): [in this window] [in a new window]   Figure 6. Primary hyperaldosteronism in BK–/– mice and BK channel expression in adrenal glomerulosa cells. a, Statistics of serum electrolyte concentrations from 6 to 9 male (M) and female (F) mice per genotype (WT, blue; BK–/–, red). b, Statistical analysis of serum aldosterone from 6 to 9 mice per genotype and gender. c, Left, Immunohistochemical detection of BK channels in the adrenal gland by peroxidase/DAB staining. Prominent staining is found in the zona glomerulosa (G), very weak staining in the zona fasciculata (F) and reticularis (R), and intermediate staining in the adrenal medulla (M). Sections from BK–/– adrenal were not stained under identical conditions. Right, Confocal single optical section demonstrates specific BK immunofluorescence on the surface (dashed line) of zona glomerulosa cells (arrows) of adrenal cortex (bar=100 µm). BK–/– sections showed no staining (not shown). d, Statistical analysis of serum renin activity, corticosterone, and corticotropin (ACTH) levels from 8 to 16 mice per genotype. All data are mean±SEM; *P<0.05; **P<0.01.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The deletion of the BK channel subunit permitted the identification of physiological functions of this unique channel in regulating arterial blood pressure. BK^–/– mice exhibit a moderate increase in blood pressure that was traced back to vascular and endocrine abnormalities. Our data indicate that the vascular abnormalities arise from lack of spontaneous outward currents that contribute to the mean resting membrane potentials in small and large vessels (Figure 1b; Data Supplement Figure, panel b). Thus, the lack of BK channels may account for the observed membrane depolarization (Figure 1b), which will tend to increase myogenic tone, ie, vessel tone in response to intravascular pressure (Bayliss effect; Figure 2). The pathophysiology of an increased vascular tone in BK^–/– mice apparently includes several mechanisms. First, we found that relaxation of myogenic tone by cGMP is impaired in BK^–/– vessels. This finding indicates that the vascular BK channel is an important effector of the cGMP/cGMP kinase pathway. Previous studies, in which several mouse models with genetic ablations were used, have shown that this pathway contributes to basal blood pressure regulation.^24,32,33 By contrast, cAMP-mediated relaxation of small arteries involves effectors other than the BK channel, although cAMP kinase has also been shown to control BK channel activity in vascular smooth muscle cells.³⁴

Second, BK channel deficiency abolished the transient outward K⁺ currents that are induced by local Ca²⁺ release from internal stores or, alternatively, by Ca²⁺ influx through T-type Ca²⁺ channels, a mechanism that recently was proposed to operate in coronary vessels.³⁵ Regardless of the Ca²⁺ source, the lack of transient K⁺ currents should increase the open probability of voltage-gated Ca²⁺ channels, thus contributing to vasoconstriction of small arteries. In such arteries, myogenic tone in response to intravascular pressure was absent when L-type Ca²⁺ channels of the Cav1.2 type were inactivated specifically in smooth muscle.³⁶ This suggests that the increased myogenic tone in BK^–/– arteries is mediated by enhanced Ca²⁺ influx through Cav1.2 channels, presumably because of the less negative membrane potential of arterial muscle cells and an attendant increase of the Cav1.2 "window current."¹⁹ The subsequent increase of global [Ca²⁺]_i, however, seems to be insufficient for stimulating the frequency or intensity of Ca²⁺ sparks evoked from intracellular stores (Figure 4b). Apart from these vascular dysfunctions, we found a marked hyperaldosteronism that probably contributes to the blood pressure elevation in BK^–/– mice. Aldosterone, via the mineralocorticoid receptor, is the major regulator of ENaC expression and activity in the cortical collecting duct, thereby providing renal Na⁺ reabsorption, which also necessitates increased water reabsorption to maintain Na⁺ concentration at or near 140 mmol/L. Several rare forms of inherited hypertension are based on monogenetic defects associated with increased mineralocorticoid synthesis or dysfunctional aldosterone signaling (for a review, see Lifton et al³⁷). We also found that BK channel protein is localized in the cortices of adrenal glands and is highest in zona glomerulosa cells therein. However, the physiological contribution of BK channels to aldosterone secretion from this cell layer is controversial.^28,38 Both T-type and L-type voltage-dependent Ca²⁺ channels are expressed in glomerulosa cells and have been implicated in steroidogenesis and aldosterone secretion.^30,31,39,40 Interestingly, both types have been described to be functionally associated with BK channels in other cell types.^18,35 Thus, loss of control of Ca²⁺ influx through Ca²⁺ channels in glomerulosa cells may be a plausible mechanism for the observed hyperaldosteronism in BK^–/– mice. In support of this idea is the finding that neither the renin/angiotensin pathway nor pituitary hormones seem to evoke the hyperaldosteronism.

Considering that the BK^–/– mice showed several synergistic mechanisms that all tend to increase blood pressure, it seems surprising that their blood pressure elevation was not more severe. The relative mildness of their blood pressure elevation was confirmed by the absence of significant cardiac hypertrophy in 4- to 6-month-old mutants (Figure 5d). We hypothesize that other peripheral or central regulators that are able to dampen blood pressure may be more active in BK^–/– than in WT mice. This idea is supported by the finding that deletion of the less widespread regulatory BK channel ß1 subunit caused a more severe blood pressure elevation^6,7 (and consequently also significant cardiac hypertrophy in mutant mice⁶) than the deletion of the BK subunit in the present study. The predominant expression of the BKß₁ subunit in smooth muscle⁵ suggests that BKß₁^–/– mice rather than BK^–/– mice represent the more selective "vascular" BK channel deletion, although lack of the BKß₁ subunit leaves intact subunits that can be still activated at physiological voltages by high local Ca²⁺, cGMP/cGMP kinase, and additional pathways. Presumably, a selective deletion of only the vascular BK subunit would produce an even more severe blood pressure elevation than BKß₁^–/– deletion. However, in terms of revealing the basis of genetically anchored diseases and identifying susceptibility genes contributing to hypertension, the condition of a naturally occurring loss of function mutation of the BK gene is best reflected by the general BK channel knockout as presented here.

In conclusion, the results of this study indicate that the systemic blood pressure phenotype of complete BK channel deletion is relatively mild—milder than expected—at least under resting conditions. We hypothesize that the renovascular role of the BK channel, which often acts as an "emergency brake",^41,42 may become more evident when studying microvasculature function and organ perfusions in BK^–/– animals that are challenged with pathophysiological conditions such as ischemia.

	Acknowledgments

 
We thank Dan-Yang Huang and Dr Volker Vallon for assisting us in flame photometry analysis, Isolde Breuning and Janina Smykowsky for excellent technical assistance, and Deutsche Forschungsgemeinschaft, Fonds zur Förderung der Wissenschaftlichen Forschung, The Wellcome Trust, Research Council of Norway, Thyssen-Stiftung and Schilling Foundation for financial support.

	Footnotes

 
*Drs Sausbier and Arntz contributed equally to this work.

The online-only Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/01.CIR.0000156448.74296.FE/DC1.

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