INTRODUCTION

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MICE WITH DISRUPTED ₁ SUBUNIT (BK₁) gene for the large-conductance, Ca²⁺-activated K⁺ (BK) channel are hypertensive (2, 14). Targeted deletion of BK₁ increased mean arterial blood pressure by 14 mmHg and 20 mmHg in mice with C57BL/6 and 129 svj genetic background, respectively. This increase in systemic blood pressure was accompanied by cardiac hypertrophy (2), as seen in many humans after long-standing essential hypertension. The reason for the development of elevated arterial blood pressure in both / BK₁ mice strains is unclear. Aortic rings from / BK₁ mice responded to agonists and elevated KCl with an increased contractility (14), and isolated pressurized cerebral arteries of / BK₁ mice were more constricted than +/+ BK₁ arteries of 129 svj mice (2). Coupling of Ca²⁺ sparks to BK channels was ineffective in / BK₁ arterial smooth muscle cells of both strains (2, 14), which was suggested to be responsible for increased arterial tone and elevation of blood pressure in mice lacking BK₁.

However, little is known about the physiological levels of global intracellular Ca²⁺ concentration ([Ca²⁺]_i) and its regulation by Ca²⁺ spark/BK channels in the smooth muscle cells of / and +/+ BK₁ small mouse arteries. Whereas the function of Ca²⁺ sparks/spontaneous transient outward currents has been studied extensively in rat cerebral arteries (1, 4, 6, 7, 11-13), information on the role of Ca²⁺ sparks/BK channels in mouse arteries is meager by comparison because previous studies on isolated mouse arteries have involved recordings of diameter alone (2). In particular, Brenner et al. (2) observed an increased myogenic small arterial tone in / BK₁ mice compared with +/+ BK₁ mice. In their study, the diameter of / BK₁ arteries was not affected by iberiotoxin (100 nM), a blocker of BK channels, whereas +/+ arteries were constricted by this toxin by 10 µm. This finding suggests impaired BK channel function in regulating diameter of / BK₁ cerebral arteries. However, Brenner et al. did not measure [Ca²⁺]_i; therefore, it is unknown whether the effects are mediated through changes in global [Ca²⁺]_i. In addition, although Nelson and co-workers (8, 12) have suggested that release of sarcoplasmic reticulum (SR) Ca²⁺ has no impact on global [Ca²⁺]_i in their experiments on rat cerebral arteries, other groups have reported physiological nonlocalized Ca²⁺ release from the peripheral SR by Ca²⁺ influx (Ca²⁺-induced Ca²⁺ release) (11-14). Moreover, because the influence of the genetic background is unknown, the analysis of the Ca²⁺ spark/BK channel negative-feedback control mechanism may require comparison of both / and +/+ arteries with the same genetic background (but see Ref. 2).

Whether local Ca²⁺ release originating from ryanodine receptor (RyR) channels (Ca²⁺ sparks) in the SR of arterial smooth muscle cells limits myogenic tone in mouse cerebral arteries through activation of BK channels, which limit in turn voltage-dependent Ca²⁺ influx and [Ca²⁺]_i through tonic hyperpolarization, is not sufficiently known. It is unknown whether deletion of BK₁ disrupts this negative feedback mechanism, leading to increased arterial tone through an increase in [Ca²⁺]_i.

We tested the hypothesis that the BK₁ subunit of the BK potassium channel operates as the molecular sensor of local Ca²⁺ transients, i.e., sparks released by RyR Ca²⁺-release channels of the SR, to limit global [Ca²⁺]_i and myogenic vasoconstriction. We utilized a BK₁ knockout C57BL/6 mouse model and studied the effects of RyR and BK channel inhibitors on the global arterial wall [Ca²⁺]_i and diameter of small pressurized (60 mmHg) cerebral arteries (110-130 µm) from both +/+ and / BK₁ mice with identical C57BL/6 genetic background by means of digital fluorescence video imaging.

	MATERIALS AND METHODS

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Basilar and posterior cerebral arteries were obtained from adult, age-matched (6-8 wk) +/+ and / BK₁ C57BL/6 mice genotyped before the experiment (14). After decapitation, the brain was removed and quickly transferred to cold (4°C) oxygenated (95% O₂-5% CO₂) physiological salt solution (PSS) of the following composition (in mM): 119 NaCl, 4.7 KCl, 25 NaHCO₃, 1.2 KH₂PO₄, 1.6 CaCl₂, 1.2 MgSO₄, 0.03 EDTA, and 11 glucose. Thereafter, the cerebral arteries were dissected from the brain and were placed in cold PSS. Connective tissue was removed. For measurements of arterial wall [Ca²⁺]_i, the vessels were incubated with the Ca²⁺-sensitive indicator fura 2-AM (5 µM) and pluronic acid (0.005%; wt/vol) for 45 min at room temperature in oxygenated PSS (for details, see Refs. 4, 6, and 7). After loading with fura 2-AM, the arteries were cannulated in a chamber. Glass cannulas were introduced into both ends, allowing the application of hydrostatic pressure to the vessel. Calibration of arterial wall [Ca²⁺]_i was done in pressurized arteries at an intravasal pressure of 60 mmHg (for details, see Ref. 7). Arterial wall [Ca²⁺]_i was calculated using the equation [Ca²⁺]_i = K_D × (R  R_min)/(R_max  R), with an apparent K_D of 147 nM (4, 7). K_D for fura 2-AM was determined using an in situ titration of [Ca²⁺]_i in fura 2-loaded arteries (pressurized to 60 mmHg). Elevated Ca²⁺ permeability of the vascular smooth muscle cells was induced by 10 µM ionomycin added to the bath solution containing (in mM) 140 KCl, 20 NaCl, 5 HEPES, 5 EGTA, 1 MgCl₂, and 5 nigericin, at pH 7.4 (adjusted with KOH) for 10 min (7). [Ca²⁺]_i and diameter were measured simultaneously by using a conventional spectrometer (dm3000, SPEX, Edison, NJ) and a videomicroscopic system (Nikon Diaphot, Duesseldorf, Germany) connected to a personal computer with appropriate software for detection of changes of vessel diameter (TSE, Bad Homburg, Germany) (for calibration and details, see Refs. 4, 6, 7). All experiments were performed at 37°C. All experiments were done by experimenters blinded to the study conditions.

Fura 2-AM was purchased from Molecular Probes (Eugene, OR). Stock solutions (0.25 mM) of fura 2-AM were made using DMSO as the solvent. All salts and drugs were obtained from Sigma-Aldrich (Deisenhofen, Germany) or Merck (Darmstadt, Germany). High external potassium solutions were made by isosmotic substitution of NaCl with KCl in the PSS. All values are given as means ± SE. For group comparisons, paired and unpaired Student's t-tests or nonparametric Wilcoxon tests were used as appropriate. A value of P < 0.05 was considered statistically significant; n represents the number of arteries tested.

	RESULTS

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We first measured arterial wall [Ca²⁺]_i and vessel diameter of isolated cerebral arteries from +/+ and / mice at an intravasal pressure of 10 mmHg. Figure 1 shows that resting [Ca²⁺]_i (~120 nM) and diameter (~100 µm) were not different (P > 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Comparison of resting arterial wall intracellular Ca2+ concentration ([Ca2+]i) and vessel diameter in / and +/+ BK1 cerebral arteries. Parameters were not significantly different (n = 8 +/+ and / basilar arteries, respectively; n = 8 +/+ and / posterior cerebral arteries, respectively). Intravasal pressure was 10 mmHg. BK1 refers to the 1-subunit gene for the large-conductance Ca2+-activated K+ (BK) channel.

+/+ BK₁ arteries. We next examined electromechanical coupling in +/+ cerebral arteries. We increased intravasal pressure from 10 to 60 mmHg in +/+ cerebral arteries and applied high concentrations of external K⁺ (60 mM). Figure 2A shows that 60 mM K⁺, which would depolarize to approximately 20 mV (4), evoked an increase in [Ca²⁺]_i to ~500 nM and reduced the vessel diameter from ~100 to 60 µm (P < 0.05, n = 14). These effects were completely reversible on washout of 60 mM K⁺. Inhibitors of RyR channels (ryanodine) or BK channels (iberiotoxin) have been shown to depolarize (by ~9 mV) and constrict (by 20-30%) pressurized (60 mmHg) rabbit and rat cerebral arteries in a nonadditive manner (1, 6, 8, 12). Figure 2A illustrates the increase in arterial wall [Ca²⁺]_i and vasoconstriction of pressurized +/+ mouse cerebral arteries caused by ryanodine and iberiotoxin and shows that the effects of these agents were not additive. The results of the experiments are summarized in Fig. 3. Ryanodine (10 µM) increased arterial wall [Ca²⁺]_i by 78 ± 15 nM and constricted the +/+ arteries by 10 ± 4 µm. Addition of iberiotoxin (100 nM) to +/+ arteries bathed in ryanodine (10 µM) did not result in an additional effect (arterial wall [Ca²⁺]_i and diameter under these conditions was increased by 8% and reduced by ~1 µm, respectively). In the presence of ryanodine and iberiotoxin, the arteries were able to increase [Ca²⁺]_i further, because the elevation of external potassium to 60 mM increased [Ca²⁺]_i to ~500 nM and reduced the vessel diameter from ~100 to 60 µm (n = 6). Removal of external Ca²⁺ induced reduced [Ca²⁺]_i to ~50 nM and increased the vessel diameter to ~150 µm (data not shown). In line with previous data on fura 2-AM-unloaded BK₁ +/+ arteries (2), iberiotoxin (100 nM) constricted the vessels by 10 ± 7 µm (n = 4). This effect was accompanied by an increase in arterial wall [Ca²⁺]_i by 81 ± 18 nM (n = 4).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effects of ryanodine and iberiotoxin on arterial wall [Ca2+]i and diameter of +/+ (A) and / (B) BK1 cerebral arteries. Arterial wall [Ca2+]i and vessel diameter were measured simultaneously. Intravasal pressure was increased to 60 mmHg. The presence of 60 mM KCl, ryanodine (10 µM), and iberiotoxin (100 nM) is indicated by horizontal lines.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Changes in arterial wall [Ca2+]i and vessel diameter of / and +/+ BK1 cerebral arteries induced by ryanodine (A; 10 µM) and the combination of ryanodine (B: 10 µM) and iberiotoxin (100 nM). Changes are expressed as changes of steady-state arterial wall [Ca2+]i (nM) and vessel diameter (µm) obtained with 60 mmHg before and after administration of the agents (n  8). For comparison of data and statistical significance, see RESULTS.

/ BK₁ arteries. In contrast to +/+ arteries, ryanodine and iberiotoxin had no significant effect on diameter of pressurized / arteries (P < 0.05). Figure 2B illustrates the effects of these agents. The results of the experiments are summarized in Fig. 3. In pressurized / arteries, ryanodine (10 µM) induced an increase in arterial wall [Ca²⁺]_i that was smaller than in +/+ arteries (P < 0.05). This increase in arterial wall [Ca²⁺]_i was not significant to reduce diameter (arterial wall [Ca²⁺]_i and diameter were increased by 52 ± 12 nM and decreased by 1 µm, respectively; n = 8, P > 0.05). Addition of iberiotoxin (100 nM) to arteries bathed in ryanodine (10 µM) did not result in an additional effect (arterial wall [Ca²⁺]_i and diameter under these conditions was increased by 5% and reduced by ~1 µm, respectively, P > 0.05). In the presence of ryanodine and iberiotoxin, the arteries were able to increase [Ca²⁺]_i further, because the elevation of external potassium to 60 mM increased [Ca²⁺]_i to ~500 nM and reduced the vessel diameter from ~100 to 60 µm (n = 8). Removal of external Ca²⁺ induced reduced [Ca²⁺]_i to ~50 nM and increased the vessel diameter to ~150 µm (data not shown).

	DISCUSSION

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This study provides the first direct information on the regulation of arterial wall [Ca²⁺]_i in mouse cerebral arteries by ryanodine-sensitive (RyR) Ca²⁺-release channels and Ca²⁺-activated K⁺ (BK) channels. This study provides further evidence that RyR channels in the SR regulate cerebral artery diameter through changes in BK channel activity at the level of the intact artery (12). The study indicates that the BK₁ operates as the molecular sensor of Ca²⁺ sparks to limit myogenic vasoconstriction through a decrease of global [Ca²⁺]_i.

Mouse strains. We utilized a BK₁ knockout C57BL/6 mouse model and studied the effects of RyR and BK channel inhibitors on the arterial wall [Ca²⁺]_i and diameter of small pressurized (60 mmHg) mouse cerebral arteries (100-130 µm) by means of digital fluorescence video imaging. We observed that ryanodine (an inhibitor of RyR channels) and iberiotoxin (an inhibitor of BK channels) increased arterial wall [Ca²⁺]_i by ~80 nM and constricted +/+ BK₁ wild-type, (pressurized to 60 mmHg) arteries with myogenic tone by 10 ± 4 µm. In contrast, ryanodine and iberiotoxin had no effect on diameter of / BK₁-deficient, myogenic (pressurized to 60 mmHg) cerebral arteries of C57BL/6 mice. These observations are consistent with the findings of Brenner et al. (2), who observed a similar (10 µm) reduction of vessel diameter by iberiotoxin in +/+ BK₁ wild-type, (pressurized to 60 mmHg) cerebral arteries but not in / BK₁ cerebral arteries that are more constricted (by 10 µm) at this pressure. Because Brenner et al. utilized mice with another genetic (129 svj) background, we conclude that the diameter of pressurized cerebral arteries is similarly regulated by BK channels in both mouse strains.

Ca²+ sparks/BK channels regulate arterial tone in +/+ but not / BK₁ arteries. Our results are consistent with the idea that local Ca²⁺ release, originating from RyR channels (Ca²⁺ sparks) in the SR of arterial smooth muscle cells, activate BK channels to limit myogenic tone in cerebral arteries (12). Our data indicate that these channels limit myogenic tone by lowering voltage-dependent Ca²⁺ influx and [Ca²⁺]_i in +/+ wild-type mouse cerebral arteries. However, these channels exhibited reduced function in / BK₁ mice. We showed that deletion of BK₁ leads to increased arterial tone through an increase in [Ca²⁺]_i (Fig. 4). The following observations support the idea that Ca²⁺ sparks regulate the diameter of pressurized mouse cerebral arteries through BK channel activity, with BK₁ being the molecular sensor for Ca²⁺ sparks. Ca²⁺ sparks activate BK channels in cerebral artery myocytes (2, 13) but not in cells lacking BK₁ (2, 14). Inhibitors of Ca²⁺ sparks, such as ryanodine, elevate arterial wall [Ca²⁺]_i and constrict pressurized (denuded) +/+ cerebral arteries but not / arteries lacking BK₁ (present study). Iberiotoxin is without effects in the presence of ryanodine in both +/+ BK₁ arteries (Fig. 2) and / BK₁ arteries (present study). In contrast, in nontreated +/+ BK₁ arteries, iberiotoxin induces vasoconstriction (2) that is accompanied by an elevation of arterial wall [Ca²⁺]_i (present study). Because iberiotoxin was without effect in the presence of ryanodine and inhibitors of Ca²⁺ sparks (ryanodine) and BK channels (iberiotoxin) were unable to elevate arterial wall [Ca²⁺]_i to constrict / BK₁ arteries, these results suggest that BK channels do not contribute significantly to membrane conductance of the smooth muscle cells in intact pressurized mouse cerebral arteries in the absence of BK₁ subunits or Ca²⁺ sparks.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Proposed model for the regulation of steady-state arterial wall [Ca2+]i and diameter of mouse cerebral arteries by BK1. Graded increase in intravasal pressure depolarizes the smooth muscle cells in the arterial wall, which increases the steady-state open probability of voltage-dependent Ca2+ channels. This leads to an elevation in global, steady-state arterial wall [Ca2+]i, which activates myosin light chain kinase leading to steady force development, and maintained vasoconstriction (myogenic tone). The depolarization-induced increase in steady-state activity of individual Ca2+ channels in caveolae (11), at least in part, would increase open probability of multiple, clustered ryanodine receptors (RyRs) to release a Ca2+ spark. Increased Ca2+ spark frequency would increase the activity of BK channels (increased spontaneous transient outward current frequency; Refs. 8, 12), with BK1 being the sensor for Ca2+ sparks, causing a tonic hyperpolarization to oppose the depolarization in response to pressure. Smaller RyR Ca2+ release events (e.g., hypothetical "Ca2+ quarks" released by single, individual RyRs) and global, steady-state [Ca2+]i are unable to induce sufficient BK channel activity to regulate membrane potential, global, steady-state [Ca2+]i and arterial tone (6). Inhibition of Ca2+ sparks (e.g., by ryanodine) or BK channels (e.g., by iberiotoxin) would induce membrane depolarization, increase global [Ca2+]i and reduce the vessel diameter. In BK1-deficient arteries, inhibition of Ca2+ sparks or BK channels would have no effect on membrane potential, global [Ca2+]i, and arterial diameter because BK1 provides effective coupling of Ca2+ sparks to BK channels (2, 14). This proposed mechanism accounts for previous observations on the regulation of rat arterial diameter by BK channels (1, 7, 8, 12), on caveolae-depleted cells (11) and is supported by our new results in this study using direct measurements of arterial wall [Ca2+]i and diameter in BK1-deficient mouse arteries.

Nonlocalized Ca²+ release from the SR. Although Nelson and co-workers (8, 12) have suggested that release of SR Ca²⁺ has no impact on global [Ca²⁺]_i in their experiments on rat cerebral arteries, we observed an increase in global intracellular arterial wall [Ca²⁺]_i by ryanodine in / BK₁ mouse cerebral arteries. Although this effect was relatively small and not sufficient to induce vasoconstriction, it suggests that nonlocalized Ca²⁺ release from the peripheral SR (Ca²⁺-induced Ca²⁺ release), originating from RyR channels in the SR of smooth muscle cells, may contribute to bulk [Ca²⁺]_i in some circumstances, as reported for arteries of other species (3, 5, 9, 10). It is unlikely that the ryanodine effect on global arterial wall [Ca²⁺]_i is mediated by BK channels because these channels are nonfunctional in / arteries. Further studies should determine the nature of these SR Ca²⁺ signals.