Essential role for smooth muscle BK channels in alcohol-induced cerebrovascular constriction

Abstract

Binge drinking is associated with increased risk for cerebrovascular spasm and stroke. Acute exposure to ethanol at concentrations obtained during binge drinking constricts cerebral arteries in several species, including humans, but the mechanisms underlying this action are largely unknown. In a rodent model, we used fluorescence microscopy, patch-clamp electrophysiology, and pharmacological studies in intact cerebral arteries to pinpoint the molecular effectors of ethanol cerebrovascular constriction. Clinically relevant concentrations of ethanol elevated wall intracellular Ca²⁺ concentration and caused a reversible constriction of cerebral arteries (EC₅₀ = 27 mM; E_max = 100 mM) that depended on voltage-gated Ca²⁺ entry into myocytes. However, ethanol did not directly increase voltage-dependent Ca²⁺ currents in isolated myocytes. Constriction occurred because of an ethanol reduction in the frequency (–53%) and amplitude (–32%) of transient Ca²⁺-activated K⁺ (BK) currents. Ethanol inhibition of BK transients was caused by a reduction in Ca²⁺ spark frequency (–49%), a subsarcolemmal Ca²⁺ signal that evokes the BK transients, and a direct inhibition of BK channel steady-state activity (–44%). In contrast, ethanol failed to modify Ca²⁺ waves, a major vasoconstrictor mechanism. Selective block of BK channels largely prevented ethanol constriction in pressurized arteries. This study pinpoints the Ca²⁺ spark/BK channel negative-feedback mechanism as the primary effector of ethanol vasoconstriction.

Moderate–heavy episodic alcohol intake, such as in binge drinking, remains a major public health problem (1, 2). Moderate–heavy drinking is associated, independently of any other factor, with an increased risk for stroke and deaths from ischemic stroke (3, 4). Binge drinkers are significantly predisposed to brain hemorrhage, cerebrovascular spasm, and stroke (3, 5).

Cerebrovascular disease associated with moderate–heavy alcohol intake is independent of beverage type and alcohol metabolism but linked to ethanol (EtOH) itself (6, 7). Strong evidence for a dose–response relationship between EtOH intake and risk for stroke suggests causality (8). EtOH cerebral artery constriction is considered responsible for cerebral vasospasm, ischemia, and stroke in moderate–heavy drinkers (6, 9). Acute EtOH at legally intoxicating (≥20 mM) blood levels in naive subjects constricts cerebral arteries in several species, including humans (7, 9).

Rats are excellent models to study EtOH cerebral artery constriction and stroke (7, 10, 11). Evidence from this and other species indicates that EtOH constricts cerebral arteries by acting primarily on the smooth muscle (7, 11, 12). However, the molecular mechanisms mediating EtOH cerebral artery constriction remain largely unidentified.

In cerebrovascular smooth muscle, an elevation in global intracellular Ca²⁺ ([Ca²⁺]_ic) leads to contraction (13). Ca²⁺ mobilization in response to EtOH may result from direct potentiation of mechanisms leading to Ca²⁺ influx/release from internal organelles and/or impairment of negative-feedback mechanisms that reduce global [Ca²⁺]_ic and, thus, cerebrovascular tone.

In resistance-size cerebral arteries from several species, including humans, activation of smooth-muscle, large-conductance, Ca²⁺-activated K⁺ (BK) channels is a fundamental mechanism that opposes constriction (13). In cerebrovascular myocytes, BK channel activity is evidenced primarily by spontaneous transient outward currents (STOCs) (14, 15). These currents occur because of BK channel activation by subsarcolemmal Ca²⁺ transients (Ca²⁺ sparks) that result from Ca²⁺ release from the sarcoplasmic reticulum (SR) (13–15). STOCs hyperpolarize the myocyte membrane potential and counteract contraction by reducing voltage-dependent Ca²⁺ channel activity (13, 15).

EtOH decreases the activity of aortic smooth muscle BK channels reconstituted into lipid bilayers (16) and BK pore-forming bslo subunits expressed in oocytes (17). However, EtOH inhibition of BK channels is not universal, because both channel activation (18, 19) and refractoriness (20) were observed. EtOH action on BK channels varies amongst slo subunit isoforms (21), cell domains (20), and bilayer lipid species (19). Thus, EtOH action on channels heterologously expressed may differ dramatically from drug action on the native channel studied in intact cerebrovascular myocytes. In particular, EtOH modulation of BK channel activity is a function of internal [Ca²⁺] (17, 18). Thus, it becomes critical to determine any possible modulatory role of EtOH on BK channels in intact myocytes where Ca²⁺ sparks regulate [Ca²⁺] near the BK channel and, thus, channel activity. In addition, EtOH studies on BK channels using expression or reconstitution systems are unable to address the impact of EtOH-targeting of this channel type on arterial contractility.

In the present study, we used patch-clamp electrophysiology to test whether acute exposure to [EtOH] found in circulation after binge drinking inhibits STOCs in freshly isolated rat cerebral artery myocytes. To gain mechanistic insights, we probed EtOH action on voltage-dependent Ca²⁺ currents (VDCC) in myocytes and BK single channel activity in isolated membrane patches. We used confocal microscopy to study EtOH action on Ca²⁺ sparks and waves, two major vasoregulatory Ca²⁺ signals in cerebral artery smooth muscle. Pharmacological studies in pressurized cerebral arteries addressed the contribution of BK channels to EtOH increase in arterial wall [Ca²⁺]_ic and constriction.

Methods

Artery Isolation and Diameter Measurement. Male Sprague–Dawley rats (≈250 g) were decapitated with a guillotine. Brains were removed and placed in ice-cold (4°C) bicarbonate solution (119 mM NaCl/4.7 mM KCl/24 mM NaHCO₃/1.2 mM KH₂PO₄/1.6 mM CaCl₂/1.2 mM MgSO₄/0.023 mM EDTA/11 mM glucose, gassed with 95% O₂/5% CO₂, pH 7.4). Both middle cerebral arteries (100- to 200-μm diameter) were removed and cleaned of connective tissue. Then, each artery was cut perpendicular to its main axis into 1- to 2-mm-long segments that were kept in ice-cold bicarbonate solution until use.

Artery pressurization and diameter measurement were performed as described elsewhere (22). After a 10-min equilibration in physiological saline solution (PSS) (112 mM NaCl/4.8 mM KCl/26 mM NaHCO₃/1 mM CaCl₂/1.2 mM MgSO₄/1.2 mM KH₂PO₄/5 mM glucose,gassed with 74% N₂/21% O₂/5% CO₂,pH 7.4;37°C), intravascular pressure was increased from 10 to 60 mmHg,which is the estimated half systolic pressure of rat cerebral arteries in situ (23) and depolarizes the membrane potential from approximately –60 to –40 mV (13). When appropriate, at 60 mmHg, the artery was exposed to a high K⁺ solution (63.7 mM NaCl/60 mM KCl/1.2 mM KH₂PO₄/1.6 mM CaCl₂/1.2 mM MgSO₄/0.023 mM EDTA/11 mM glucose/24 mM NaHCO₃, pH 7.4), which allowed us to establish, for each segment, a reproducible degree of contraction to which EtOH constriction could be compared (Fig. 1A). At the end of each experiment, maximal artery relaxation was evaluated by exposing the vessel to Ca²⁺-free medium (119 mM NaCl/4.7 mM KCl/1.2 mM KH₂PO₄/1.2 mM MgSO₄/2 mM EGTA/11 mM glucose/24 mM NaHCO₃, pH 7.4).

Fig. 1.

EtOH constricts pressurized cerebral arteries. (A) Arterial diameter trace showing that 60 mM KCl and 50 mM EtOH reversibly constrict an isolated, intact artery. (B) EtOH constriction is concentration-dependent, with maximal constriction being obtained with 100 mM EtOH. *, Different from 3 mM EtOH (P < 0.001); #, different from 3, 10, 25, or 50 mM EtOH (P < 0.001); n is shown in parentheses. (Inset) Logit-log plot of change in diameter (d) as a function of [EtOH]. Abscissa intercept gives the EC₅₀ (27.3 mM); d_max, maximal diameter. (C) Endothelium removal abolishes carbachol vasodilation but fails to modify Na⁺ nitroprusside vasodilation. EtOH constriction is identical in vessels with vs. those without endothelium. *, Different from dilation in arteries with endothelium (P < 0.001; n = 4).

Endothelium was removed by passing an air bubble into the vessel lumen for 1 min. The absence of a functional endothelium was confirmed by comparing the vasodilation evoked by 1 μM carbachol with that caused by 1 μM Na⁺ nitroprusside in PSS (24).

For details on drug application, see supporting information, which is published on the PNAS web site.

Simultaneous Arterial Wall [Ca²⁺]_ic and Diameter Measurements. Before cannullation, the arterial segment was incubated with 3 μM fura-2AM and 0.06% pluronic F-127 in PSS for 45 min at room temperature. The artery was cannulated and kept for 10–15 min in the dark. Fura-2 was alternately excited at 340 or 380 nm by using a PC-driven hyperswitch (Ionoptix, Milton, MA). The artery was also illuminated with 655-nm incident light to obtain an image of the arterial wall by using a charge-coupled device camera (Ionoptix). A 585LP dichroic mirror split fura-2 emission to a photomultiplier tube and incident red light to the camera. Arterial wall [Ca²⁺]_ic and diameter were determined as described (22, 25).

Measurement and Analysis of Ca²⁺ Sparks and Waves. Arterial segments on rectangular glass cannulae were placed for 60 min into Hepes-buffered PSS (134 mM NaCl/6 mM KCl/2 mM CaCl₂/1 mM MgCl₂/10 mM Hepes/10 mM glucose, pH 7.4), which contained 10 μM fluo-4AM and 0.05% pluronic F-127. Segments were then placed into Hepes-buffered PSS for 30 min to allow indicator deesterification.

Ca²⁺ sparks in myocytes within intact arterial segments were measured in an extracellular solution containing 30 mM K⁺. This [K⁺] depolarizes smooth muscle cells to approximately –40 mV, which is similar to the membrane potential of cerebral arteries pressurized to 60 mmHg (14).

Images were obtained by using laser scanning confocal microscopy as described (25). For imaging myocytes in arteries, at least two different areas of the same arterial segment were scanned for ≥10 s under each condition. In a few experiments, data were obtained by scanning the same arterial segment area. Using our specifications, imaging the same smooth muscle cells twice for 10 s did not alter spark frequency: second acquisition frequency was 104 ± 12% of first acquisition (n = 6).

Ca²⁺ sparks were detected by using software written by M. Nelson and A. Bonev (University of Vermont, Burlington). Automated and manual detection of Ca²⁺ sparks were performed as described in supporting information. Ca²⁺ waves were detected by using 4.84-μm² boxes in individual myocytes and refer to a change in F/F₀ > 1.2 that propagated for at least 20 μm (26).

Cell Isolation. Individual myocytes were isolated from rat basilar and middle cerebral arteries. Arteries were cleaned of connective tissue and endothelium and enzymatically digested (supporting information). Cells were used up to 6 h after being isolated.

STOC Measurement and Analysis. STOCs and their modulation by EtOH were measured by using perforated-patch recordings. Pipettes contained 110 mM K⁺ aspartate, 30 mM KCl, 10 mM NaCl, 1 mM MgCl₂, 10 mM Hepes, and 0.05 mM EGTA (pH 7.2). Amphotericin B was dissolved in DMSO and diluted into pipette solution to a final concentration of 250 μg/ml. The bath solution contained 134 mM NaCl, 6 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM Hepes, and 10 mM glucose (pH 7.4). Currents were recorded at a membrane potential of –40 mV. The threshold for defining a STOC was three times the BK channel unitary current (15). Bath solution containing either EtOH or urea isosmotically replacing EtOH (control) was perfused with a pressurized, DAD-12 superfusion system (ALA).

Single Channel Recordings. BK channel recordings were obtained in outside-out and inside-out patches by using standard techniques (18). Data were acquired and analyzed as described in supporting information. Channel steady-state activity (NP_o) in the presence of EtOH was compared to that in control (18). Both control and EtOH-containing solutions were applied to the patch surface facing the bath solution by using a pressurized system. EtOH was diluted in bath solution immediately before recordings.

Statistics. Student's t tests or ANOVA followed by the Bonferroni or Tukey–Kramer test were conducted according to experiment design. Normal distribution of data were tested with a Kolmogorov–Smirnov test. Data plotting and fitting were performed by using origin 7 (OriginLab, Northampton, MA). Data are expressed as mean ± SEM.

Chemicals. Deionized, 100% pure EtOH was purchased from American Bioanalytical, iberiotoxin (IbTX) from Alomone (Jerusalem), tetraethylammonium (TEA) from Alfa Aesar (Ward Hill, MA), and Fura-4, Fura-2AM, and pluronic F-127 from Molecular Probes. All other chemicals were purchased from Sigma.

Results

Alcohol Constricts Pressurized Cerebral Arteries by a Direct Action on Smooth Muscle. The EtOH blood level constituting legal intoxication in the U.S. is 20 mM, and ≥100 mM is usually lethal in naive subjects (27). After myogenic tone development at 60 mmHg, artery segments were exposed to PSS containing 3–200 mM EtOH. Acute EtOH caused cerebral artery constriction, which was fully reversible and concentration-dependent (Fig. 1 A and B). Because EtOH constriction occurred in isolated vessels, the mechanism underlying EtOH action is independent of circulating vasoactive substances. Given the almost immediate start of, and vessel recovery from, EtOH action (Fig. 1A), data suggest that constriction of arteries is caused not by alcohol metabolites but by EtOH itself.

Maximal constriction was evoked by 100 mM EtOH (13 ± 1% diameter reduction), with 200 mM rendering no further decrease in diameter (P > 0.05) (Fig. 1B). A ceiling effect in diameter responses to EtOH allowed us to construct a logit–log plot (Fig. 1B Inset) (18), from which we obtained the EC₅₀ (27.3 mM). In summary, EtOH produces a reversible, dose-dependent cerebrovascular constriction at concentrations found during legal intoxication.

We then focused on 50 mM EtOH to address mechanistic aspects of EtOH action. This [EtOH] falls within the range obtained in circulation during moderate–heavy alcohol intake and of alcoholic patients with stroke-like episodes (4, 27, 28). In contrast to the EtOH effect, application of PSS containing either dextrose or urea (50 mM) consistently failed to evoke constriction [diameter: 105.8 ± 2.2% of controls (n = 4) and 98.4 ± 0.7% of controls (n = 4)]. Therefore, EtOH cerebrovascular constriction cannot be explained by an osmotic effect, but it should involve distinct targets.

Endothelial factors regulate cerebral artery tone (29). Thus, we determined whether EtOH targets could be located in/modulated by the cerebrovascular endothelium. EtOH constriction was similar in intact arteries and those without functional endothelium (Fig. 1C; P > 0.05). Thus, a smooth muscle target(s) is not only necessary but also sufficient for EtOH action. A direct EtOH action on rat cerebrovascular smooth muscle is consistent with data reported in cerebral arteries from other species (12, 30) suggesting conserved mechanisms of drug action. Our data contrast with those from rat aorta, where endothelial mechanisms are involved in EtOH vasoactive properties (31), stressing the importance of regional differences in vessel responses to alcohol.

Voltage-Dependent Ca²⁺ Entry Is a Major Contributor to EtOH Cerebrovascular Constriction. We investigated whether EtOH constriction was related to an increase in arterial smooth muscle [Ca²⁺]. EtOH readily and reversibly increased arterial wall Ca²⁺ in pressurized arteries (Fig. 2A Upper). This result was replicated in five other arteries, rendering an average increase of 33 ± 7 nM (P < 0.001) (Fig. 2B) and a simultaneous constriction of 21 ± 3 μm(P < 0.001; n = 6) (Fig. 2 A Lower and C). Data support the idea that EtOH increases in wall [Ca²⁺] and contraction are causally related.

Fig. 2.

EtOH cerebrovascular constriction is paralleled by increases in arterial wall Ca²⁺.(A) Time courses of ratiometric measurement of arterial wall [Ca²⁺] and diameter in pressurized cerebral arteries. Both 60 mM KCl and 50 mM EtOH readily and reversibly increased arterial wall Ca²⁺ and synchronously decreased diameter. This result was replicated in five other arteries, and the averages are given in B (wall [Ca²⁺]) and C (diameter). B and C show that, in the presence of 50 μM diltiazem, EtOH fails to modify arterial wall [Ca²⁺] and diameter (P > 0.05; n = 5). *, Different from controls (P < 0.01; n = 6).

Because voltage-dependent Ca²⁺ channels are the main Ca²⁺ entry pathway in cerebrovascular smooth muscle (13), we determined whether these channels are involved in EtOH action. Diltiazem (50 μM) decreased arterial wall [Ca²⁺] by 87 ± 23 nM (P < 0.05) and increased diameter by 55 ± 11 μm (P < 0.05). In the presence of diltiazem, EtOH failed to change both arterial wall [Ca²⁺] and diameter (n = 5; Fig. 2 B and C), which indicates that Ca²⁺ entry through voltage-dependent Ca²⁺ channels is involved in EtOH constriction. The simplest explanation for this result is that EtOH directly potentiates VDCC. However, EtOH mildly inhibited VDCC in freshly isolated cerebral artery myocytes at all potentials tested (supporting information). Maximal inhibition occurred at 0 mV: peak inward currents reached 61.6 ± 9.4 vs. 43.6 ± 5.8 pA in the absence and presence of EtOH (P < 0.05, n = 6). This result is consistent with those obtained in several preparations, including vascular smooth muscle (32).

In cerebrovascular myocytes, depolarization can be effectively achieved by inhibition of BK channel-mediated outward currents, leading to Ca²⁺ entry (13). Thus, we tested whether EtOH reduced BK currents.

EtOH Blocks STOCs and Ca²⁺ Sparks. In intact cerebrovascular myocytes, BK channel activity is evidenced by the presence of STOCs, which occur because of the opening of several BK channels (14, 15). EtOH action on STOCs was studied with the myocyte membrane voltage clamped at –40 mV, which mimics the in vivo condition (13, 14). Confirming that these outward currents were caused by BK channel activity, time-averaged STOC decreased to 12 ± 3% of control (P < 0.001; n = 4) after being exposed to 100 nM IbTX, a selective BK channel blocker, for 5–10 min.

Acute EtOH caused a sustained reduction in STOC frequency and amplitude (Fig. 3A) to 47.3 ± 4% and 67.8 ± 10.6% of controls, respectively (P < 0.05; n = 4) (Fig. 3B). Inhibition of STOCs was sustained throughout EtOH perfusion (5–15 min) and reversible upon washout (Fig. 3A).

Fig. 3.

EtOH (50 mM) blocks STOCs in voltage-clamped cerebral artery smooth muscle cells. Bar indicates duration of EtOH application. (A) STOC recording at –40 mV illustrating reversible block by 50 mM EtOH. (B) Mean relative effects of EtOH (50 mM) on STOC frequency and amplitude. *, Different from controls (P < 0.05; n = 4).

In cerebral artery smooth muscle, STOCs occur because of Ca²⁺ sparks (13, 14). Ca²⁺ sparks raise [Ca²⁺] in the vicinity of BK channels to micromolar levels and, thus, increase BK channel activity up to 10⁶-fold, opposing constriction (15). Conceivably, EtOH may reduce STOCs via Ca²⁺ spark inhibition. Indeed, acute EtOH decreased Ca²⁺ spark frequency to 50.3% of controls: 0.7 ± 0.09 vs. 0.35 ± 0.06 (P < 0.05; n = 4) (Fig. 4). In contrast, EtOH did not change Ca²⁺ spark amplitude: control, 1.68 ± 0.03; EtOH, 1.76 ± 0.06 Hz (P > 0.05). EtOH decrease in Ca²⁺ spark frequency was similar to the decrease in STOC frequency, supporting the idea that the latter largely depends on the former.

Fig. 4.

EtOH inhibits Ca²⁺ sparks in myocytes of intact cerebral arteries. (A) Average fluo-4 fluorescence (100 of 600 images) over 10 s of the same 56.3 × 52.8 μm areas of the same artery in control and after 50 mM EtOH. The locations of Ca²⁺ sparks that occurred during 10 s are indicated by white boxes (1.54 μm × 1.54 μm), some of them labeled with lowercase letters. Representative localized F/F₀ changes over time are illustrated below the respective images and labeled accordingly (B) Average relative effects of EtOH on Ca²⁺ spark frequency and amplitude. *, Different from control (P < 0.05; n = 7 arteries).

In contrast to its modulation of Ca²⁺ sparks and STOCs, EtOH failed to modify Ca²⁺ waves (supporting information), another major Ca²⁺ signal that regulates cerebrovascular contractility. Ca²⁺ sparks and waves were determined in the same myocytes, which highlights the selectivity of EtOH action on one Ca²⁺ signal over the other. Importantly, the lack of EtOH effect on Ca²⁺ waves distinguishes EtOH from many other vasoconstrictors (26).

In vascular smooth muscle, Ca²⁺ spark frequency is regulated by SR Ca²⁺ load, which modulates ryanodine receptor (RyR) channel activity (25). Thus, we tested next whether acute EtOH modifies SR Ca²⁺ load in cerebrovascular myocytes. Data demonstrate that EtOH does not reduce, but elevates SR Ca²⁺ load (supporting information), as expected for a RyR channel blocker (25). Thus, data strongly suggest that EtOH inhibits Ca²⁺ sparks by blocking RyR channels, although the molecular underpinnings of such EtOH action remains to be determined. In summary, EtOH significantly reduces Ca²⁺ spark frequency, this pathway being the one explanation for the EtOH reduction in STOC frequency.

STOC amplitude is determined by BK channel activity that, in turn, is primarily controlled by Ca²⁺ sparks (15, 25). Because EtOH did not alter Ca²⁺ spark amplitude (Fig. 4B), the reduction in STOC amplitude (Fig. 3) could be caused by EtOH-inhibition of BK channels. We tested this by using single-channel recordings. Current events under study showed all typical characteristics of BK unitary currents (33), including block by external TEA or IbTX (supporting information).

We evaluated EtOH action on channel function in inside-out patches under conditions that mimicked those in the vicinity of the BK channels during a Ca²⁺ spark (V =–40 mV and [Ca²⁺] ≈ 10 μM) (15). EtOH caused a transient (1–2 min) increase in channel NP_o, followed by a sustained, significant reduction to 56 ± 12% of control (P < 0.05; n = 4). This marked inhibition of activity was fully reversible upon wash, NP_o reaching 94 ± 5% of control (Fig. 5). In contrast, EtOH failed to modify unitary conductance (control, 252; EtOH, 248 pS). Data indicate that EtOH reduces BK currents by altering channel gating transitions without major modifications in permeant ion conduction. EtOH reduced channel NP_o in the absence of nucleotides and >15 min after patch excision. Thus, this EtOH action neither requires the continuous presence of cytosolic mediators nor can be attributed to cell metabolism of EtOH. Rather, it reflects a direct interaction between EtOH and the BK channel or its immediate proteolipid environment.

Fig. 5.

EtOH causes sustained reduction in BK channel activity in excised patches from cerebral artery myocytes. (A) Single BK channel activity (NP_o) recorded before (Top), during the second (Upper Middle) or fifth (Lower Middle) minute of EtOH (50 mM) application, and after wash in bath solution (Bottom), obtained from the same inside-out patch (V = –40 mV; free [Ca²⁺]_ic = 10 μM). Channel NP_o was recorded 10–15 min after patch excision. Openings are shown as downward deflections. NP_o values were obtained from ≥20 s of continuous recordings. (B) Acute EtOH (50 mM) causes a transient (<2 min) increase in BK channel NP_o followed by a sustained reduction that is fully reversible upon wash. *, Different from controls (P < 0.05; n = 4). A dotted line highlights the control level.

In summary, data shown in Figs. 3, 4, 5 demonstrate that the two major determinants of BK channel currents in cerebrovascular myocytes, Ca²⁺ spark frequency and BK channel NP_o, are significantly reduced by [EtOH] obtained in circulation during binge drinking.

BK Channels Are Critical Contributors to EtOH Cerebrovascular Constriction. Bath application of either EtOH or TEA (1 mM) reduced the mean diameter of pressurized (60 mmHg) artery segments by 7.5 ± 0.5 and 17.4 ± 2.5%, respectively. In contrast, EtOH applied in the continued presence of TEA only decreased diameter by 4.6 ± 0.3% (Fig. 6A). Similarly, application of TEA in the continued presence of EtOH decreased diameter by only 3.3 ± 0.6%. The lack of major additivity in EtOH and TEA contractile effects strongly suggests that EtOH primarily targets TEA-sensitive channels to produce constriction. Because 1 mM TEA blocks BK channels in cerebrovascular myocytes rather selectively (34), these results support the notion that BK channels play a major role in EtOH cerebrovascular constriction.

Fig. 6.

BK channels are the primary effectors of EtOH cerebrovascular constriction. Panels show traces of artery diameter in response to drugs as a function of time. After artery pressurization and development of myogenic tone, application of submaximal contractile [EtOH] (50 mM) and [TEA] that block BK channels (1 mM) fails to evoke contractile synergism in cerebral arteries (A). In addition, coapplication of EtOH (50 mM) and 100 nM IbTX fails to evoke significant contractile synergism (B). An amplified trace (C) highlights this result. In contrast, application of 50 mM EtOH in the presence of 1 mM 4-aminopyridine, a blocker of K_V channels other than BK, evokes a contractile response that is identical to that caused by EtOH alone (D).

It is possible that EtOH augmentation of cerebrovascular tone involves TEA-sensitive channels known to control tone other than BK (29). However, IbTX (100 nM) caused a reduction in artery diameter of 7.3 ± 0.4%. When applied alone, EtOH reduced diameter by 7.5 ± 0.5% (Fig. 1B), which is quantitatively similar to IbTX action (P > 0.05) and consistent with the idea that both peptide and EtOH produce cerebrovascular constriction through targeting common effectors. Although neither IbTX nor EtOH evoked maximal artery constriction (compare with larger constriction caused by KCl), EtOH in the presence of IbTX only decreased diameter by an additional 3.1 ± 0.2% (Fig. 6 B and C). These data show the lack of synergism between a very selective BK channel blocker and EtOH. In contrast, in the presence of 1 mM 4-aminopyridine (which blocks most K_V but not BK channels) (34), EtOH decreased diameter by an additional 8.6 ± 0.8% (Fig. 6D), which is identical to the constriction caused by EtOH alone in the same artery (n = 5). Together, data indicate that BK channel currents are the primary, rather selective effectors of EtOH cerebrovascular constriction.

Discussion

Data demonstrate that EtOH at concentrations obtained in circulation during binge drinking produces a powerful but reversible constriction of rat cerebral arteries that is independent of circulating vasoactive agents and a functional endothelium. Rather, EtOH action results from direct contraction of smooth muscle. Supporting earlier findings (11), EtOH cerebrovascular constriction cannot be explained by an osmotic load to the vessel. On the contrary, EtOH action results from modulation of specific molecular mechanisms in vascular smooth muscle: our study pinpoints the Ca²⁺ spark–BK channel negative-feedback loop as the primary effector of alcohol cerebral artery constriction.

Molecular Mechanisms. The role of BK channels as regulators of cerebrovascular tone has been demonstrated by using a variety of approaches: pharmacological block of this channel leads to cerebrovascular constriction (Fig. 6 B and C) (13), enhanced or attenuated Ca²⁺ spark–STOC coupling leads to cerebrovascular relaxation or constriction, respectively (13, 35), and BK β₁-subunit gene knockout leads to increased myogenic tone and increased occurrence of stroke-like events (36). Consistent with the key role of myocyte BK channels in limiting the degree of cerebrovascular constriction, we demonstrate that EtOH cerebral artery constriction is caused by inhibition of smooth muscle BK currents.

EtOH inhibition of BK currents occurs via both “indirect” and “direct” mechanisms. The former constitutes an EtOH decrease in STOC frequency caused by a reduction in Ca²⁺ spark frequency (Figs. 3 and 4), whereas the latter constitutes a reduction in BK channel NP_o, which occurs in the absence of cytosolic mediators and cell integrity (Fig. 5).

Modulation of Ca²⁺ spark frequency is a fundamental mechanism by which vasoactive agents regulate cerebrovascular contractility (13). Ca²⁺ sparks per se do not contribute sufficient Ca²⁺ to directly increase global [Ca²⁺] in cerebrovascular smooth muscle (13, 14). However, Ca²⁺ spark inhibition causes a membrane potential depolarization of ≈ 10 mV in pressurized cerebral arteries, which leads to VDCC activation, elevation in global [Ca²⁺]_ic, and vasoconstriction (13). Our data demonstrate that EtOH not only dramatically reduces Ca²⁺ spark frequency but also elevates global [Ca²⁺] to levels sufficient to cause cerebrovascular constriction.

Ca²⁺ signaling and buffering in smooth muscle cells complex and involve a variety of targets that could contribute to an EtOH-induced global [Ca²⁺]_ic elevation. RyR-mediated Ca²⁺ mobilization generates Ca²⁺ sparks and can contribute to propagating Ca²⁺ waves, the latter Ca²⁺ modality also being activated by inositol (1,4,5) trisphosphate (IP3)-mediated Ca²⁺ mobilization (26, 37). We show that EtOH does not alter Ca²⁺ waves (supporting information) but inhibits Ca²⁺ sparks. Because IP3-mediated Ca²⁺ release would not have been blocked by IbTX, it is unlikely that IP3-sensitive Ca²⁺ channels contribute to the EtOH-induced raise in global [Ca²⁺]. In contrast, EtOH readily impaired the Ca²⁺ spark/STOC negative feedback loop that opposes depolarization and constriction (13). Supporting a key role for voltage-dependent Ca²⁺ entry, the EtOH constriction and rise in global Ca²⁺ were both abolished by a voltage-dependent Ca²⁺ channel blocker (Fig. 2). Because EtOH failed to directly activate VDCC, the increase in voltage-dependent Ca²⁺ entry is attributed to synergistic inhibition of BK currents through the indirect and direct pathways mentioned above. Conceivably, EtOH could reduce Ca²⁺ sparks and STOCs in part by VDCC inhibition (38), at least at depolarized membrane potentials. In rat cerebral arteries, however, the entire range of potentials from low to high pressures is from approximately –63 to –23 mV (39). Within this voltage range, I/V plot data show that EtOH inhibition of VDCC is minimal (supporting information). Thus, under physiological conditions, EtOH inhibition of STOCs and the resulting membrane depolarization likely predominate over mild inhibition of VDCCs by EtOH, leading to a net increase in Ca²⁺ entry through voltage-gated Ca²⁺ channels.

A direct interaction between EtOH and the BK channel or its immediate proteolipid environment leads to a sustained reduction in channel NP_o (Fig. 5) and, thus, STOC amplitude (Fig. 3) (40). Although EtOH caused a transient increase in BK channel NP_o, this could have failed to transiently increase STOC amplitude due to simultaneous transient reduction in Ca²⁺ spark amplitude. EtOH-induced transient BK activation followed by inhibition was also observed after heterologous expression of mslo subunits (A.M.D. and S. Treistman, unpublished data); the molecular mechanisms require investigation.

EtOH is a more effective inhibitor of bslo channels in excised patches as internal [Ca²⁺] is increased (17). In addition, EtOH activates mslo channels at submicromolar internal [Ca²⁺] but inhibits activity when [Ca²⁺]_ic is ≥3 μM (18). These results have been interpreted as EtOH impairing slo channel activation by micromolar levels of internal Ca²⁺. This Ca²⁺–EtOH antagonism is abolished by mutating the Ca²⁺ bowl,§ a slo subunit domain that senses [Ca²⁺]_ic in the low micromolar range (41). Here, we demonstrate that EtOH inhibits cerebrovascular BK channels in excised patches with internal Ca²⁺ at 10 μM and also in the intact myocyte during Ca²⁺ spark activity when [Ca²⁺]_ic is in the micromolar range (15). Thus, impairment of micromolar internal [Ca²⁺] sensing in slo appears to be a critical mechanism that could contribute to EtOH-inhibition of BK channels during Ca²⁺ spark activity.

Clinical Implications. Stroke is the third leading cause of death and the leading cause of serious, long-term disability in the U.S. Moderate–heavy alcohol drinking represents an independent major risk factor for stroke and acute brain infarction (3, 4), and alcohol abuse represents one of the three most common risk factors among stroke patients (42, 43). The majority (88%) of strokes are ischemic, basically resulting from impaired vasodilation and/or enhanced constriction of cerebral arteries (3, 43).

The vast majority of studies that addressed the alcohol–cerebral vessel interaction have focused on chronic EtOH exposure (10, 44–46). However, binge drinking, in which cerebral vessels are acutely exposed to moderate–heavy [EtOH], is the dominant style of drinking in Western societies (47) and most closely associated with deaths from ischemic stroke (3, 42, 43). Here, we used an in vitro model to demonstrate that acute exposure to [EtOH] obtained in blood during moderate–heavy alcohol intake (27) produces a reversible cerebral artery constriction that is caused by targeting of myocyte BK channels. The constriction caused by 50 mM EtOH (–10% in diameter) is sufficient to decrease cerebral blood flow (CBF) by ≈30%, because minor changes in artery diameter result in large changes in CBF (48). This [EtOH] is similar to those reported to cause spasm in cortical arterioles and venules in dogs and is found in human circulation during alcohol-induced stroke-like episodes (3, 4, 28).

In conclusion, we have identified the major molecular effectors leading to cerebral artery constriction in response to [EtOH] found in circulation after acute, moderate–heavy drinking. Our data underscore the importance of the functional communication between Ca²⁺ sparks and BK channels in alcohol action.