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Published in final edited form as: Biochim Biophys Acta Mol Cell Biol Lipids. 2020 Dec 28;1866(4):158874. doi: 10.1016/j.bbalip.2020.158874

Cholesterol antagonism of alcohol inhibition of smooth muscle BK channel requires cell integrity and involves a protein kinase C-dependent mechanism(s)

Anna N Bukiya 1, Alex M Dopico 1
PMCID: PMC7870541  NIHMSID: NIHMS1658067  PMID: 33383194

Abstract

Alcohol constricts cerebral arteries via inhibition of voltage/calcium-gated, large conductance potassium (BK) channels in vascular myocytes. Using a rat model of high-cholesterol (high-CLR) diet and CLR enrichment of cerebral arteries in vitro, we recently showed that CLR protected against alcohol-induced constriction of cerebral arteries. The subcellular mechanism(s) underlying CLR protection against alcohol-induced constriction of the artery is unclear. Here we use a rat model of high-CLR diet and patch-clamp recording of BK channels in inside-out patches from cerebral artery myocytes to demonstrate that this diet antagonizes inhibition of BK currents by 50 mM ethanol. High-CLR-driven protection against alcohol inhibition of BK currents is reversed following CLR depletion in vitro. Similar to CLR accumulation in vivo, pre-incubation of arterial myocytes from normocholesterolemic rats in CLR-enriching media in vitro protects against alcohol-induced inhibition of BK current. However, application of CLR-enriching media to cell-free membrane patches does not protect against the alcohol effect. These different outcomes point to the involvement of cell signaling in CLR-alcohol interaction on BK channels. Incubation of myocytes with the PKC activators phorbol 12-myristate 13-acetate or 1,2-dioctanoyl-sn-glycerol, but not with the PKC inhibitor Gouml 6983, prior to patch excision precludes CLR enrichment from antagonizing alcohol action. Thus, PKC activation either disables the CLR target(s) or competes with elevated CLR. Favoring the latter possibility, 1,2-dioctanoyl-sn-glycerol protects against alcohol-induced inhibition of BK currents in patches from myocytes with naïve CLR. Our findings document that CLR antagonism of alcohol-induced BK channel inhibition requires cell integrity and is enabled by a PKC-dependent mechanism(s).

Keywords: MaxiK channel, vascular myocyte, cerebral artery, patch-clamp

INTRODUCTION

Episodic alcohol consumption with blood alcohol levels reaching 30–80 mM is a well-recognized factor for cerebrovascular disease (Altura and Altura, 1984; Puddey et al., 1999; Reynolds et al., 2003; Hvidtfeldt et al., 2008; Patra et al., 2010; Zhang et al., 2014). At these levels, alcohol constricts cerebral arteries in several experimental species and humans (Altura and Altura, 1984; Liu et al., 2004; Bukiya et al., 2009). Alcohol-induced constriction does not require neuronal network activity, bioactive factors from systemic circulation, or a functional endothelium because the alcohol effect is observed in isolated, de-endothelialized, and in vitro pressurized cerebral arteries (Liu et al., 2004). Data on subcellular factors that determine alcohol effects on cerebral artery diameter remain scarce.

Cholesterol (CLR) is a characteristic constituent of Western diets (Cordain et al., 2005). Increased dietary intake of CLR and the attendant eventual increase in blood CLR levels pose a significant risk for numerous systems and organs, including cerebral arteries (Hayakawa et al., 2007; Kitayama et al., 2007; Sasani et al., 2011). Despite the deleterious effect of high-CLR dietary intake on vascular function, elevated CLR exerts a protective effect against alcohol-induced constriction. Using a rat model of a high-CLR diet, we have recently shown that dietary CLR protects against alcohol-induced constriction of cerebral arteries in vivo (Bukiya et al., 2014b). Moreover, the phenomenon is fully replicated in vitro in pressurized arteries that are dissected from animals on a high-CLR diet when compared to animal donors on a control diet (rodent chow) (Bukiya et al., 2014b). Such protection has been proposed to arise from high-CLR chow-driven increases in blood CLR levels and accompanying buildup of CLR within cerebral artery smooth muscle (Bukiya et al., 2014b, Bisen et al., 2016). Yet, the mechanism enabling CLR antagonism of alcohol effect on cerebral artery diameter remains unknown.

Alcohol-induced constriction of cerebral arteries is largely driven by alcohol-induced inhibition of large conductance, calcium- and voltage-gated potassium (BK) channels in vascular smooth muscle (Liu et al., 2004). Smooth muscle BK channels are tetramers of channel-forming alpha subunits, which are accompanied by accessory proteins (Orio et al., 2002; Evanson et al., 2014). Upon depolarization-induced calcium entry, myocyte BK channels generate outward potassium currents that provide negative feedback to myocyte contraction (Jaggar et al., 1998; Brenner et al., 2000; Dopico et al., 2018). Consistently, BK current inhibition by alcohol results in vasoconstriction (Liu et al., 2004). Using in vitro CLR enrichment of rat and mouse cerebral artery myocytes, we recently demonstrated CLR antagonism of alcohol-induced inhibition of smooth muscle BK currents. Whether CLR accumulation in vivo via dietary intake protects against alcohol-induced inhibition of vascular smooth muscle BK channels remains unclear. Moreover, the subcellular mechanism(s) that drives CLR antagonism of alcohol effects on BK current is unknown.

MATERIAL AND METHODS

Ethical Aspects of Research.

Care of animals and experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center, which is an institution accredited by the Association for Assessment and Accreditation of Laboratory Animal Care international (AAALACi).

Manipulations with dietary CLR.

Male Sprague-Dawley rats (50 g) were placed on a high-CLR chow diet (2% CLR, Teklad) ad libitum. The control group received standard rodent chow ad libitum that was isocaloric to high-CLR food. Rats were receiving chow for 18–22 weeks. This time is sufficient to observe a statistically significant increase in blood and cerebral artery wall CLR levels compared to time-matched standard chow (Bukiya et al., 2014b).

Myocyte isolation from rat middle cerebral arteries (MCAs).

Rats were decapitated under deep anesthesia with isoflurane. MCA segments were dissected out from brain and placed into ice-cold dissociation media with the following composition (mM): 0.16 CaCl2, 0.49 EDTA, 10 HEPES, 5 KCl, 0.5 KH2PO4, 2 MgCl2, 110 NaCl, 0.5 NaH2PO4, 10 NaHCO3, 0.02 phenol red, 10 taurine, 10 glucose. Each artery was cut into 1- to 2-mm long rings (up to 30 rings/experiment). Individual myocytes were enzymatically isolated following a procedure described in detail in our earlier work (Bukiya et al., 2011). Briefly, rat arterial rings were put in 3 mL of dissociation media containing 0.03% papain, 0.05% bovine serum albumin, and 0.004% dithiothreitol at 37ºC for 9–15 min in a polypropylene tube and incubated in an agitating water bath at 37°C and 60 oscillations/min for 9–15 min. Then, the supernatant was discarded, and the tissue transferred to a polypropylene tube with 3 mL of dissociation media containing 0.06% soybean trypsin inhibitor, 0.05% bovine serum albumin, and 2% collagenase (26.6 units/mL). The tube was incubated again in an agitating water bath at 37ºC and 60 oscillations/min for 15 min. Finally, the tissue pellet was transferred into a tube with 3 mL of dissociation media containing 0.06% soybean trypsin inhibitor. Tissue-containing dissociation media was pipetted using a series of borosilicate Pasteur pipettes having fire-polished, diminishing internal diameter tips. The procedure rendered a cell suspension containing individual myocytes. The cell suspension was stored in ice-cold dissociation media supplemented with 0.06% soybean trypsin inhibitor and 0.06% bovine serum albumin. Cells were used for patch-clamp recordings up to 3 h after isolation.

In vitro modification of CLR levels in arteries and myocytes.

For CLR depletion, myocytes were incubated for 60 min in patch-clamp bath solution (see below) containing CLR carrier 5 mM methyl-β-cyclodextrin (MβCD). For CLR enrichment, bath solution contained 5 mM MβCD+0.625 mM CLR (8:1 molar ratio). To ensure MβCD saturation with CLR, the solution was vortexed and sonicated for 30 min at room temperature, then agitated at 37°C overnight (Zidovetzki and Levitan, 2007; Bisen et al., 2016). Myocytes and isolated membrane patches were incubated in MβCD+CLR complex for 5 or 20 min per experimental design (see Results).

Patch-clamp recording of BK channel activity from rat MCA myocytes.

Electrophysiology experiments on freshly isolated rat arterial myocytes were performed in excised, inside-out (I/O) membrane patches. Both bath and electrode solutions contained (mM) 130 KCl, 5 EGTA, 2.44 MgCl2, 15 HEPES, 1.6 HEDTA, 5.59 CaCl2 ([Ca2+]free≈30 μM), pH 7.35. Nominal free Ca2+ was calculated with MaxChelator Sliders (C. Patton, Stanford University, CA) and validated experimentally using Ca2+-selective electrodes (Corning Incorporated Science Products Division). Immediately before recording, the tip of each recording glass electrode was fire-polished on a microforge WPI MF-200 (World Precision Instruments) to give resistances of 5–9 MΩ when filled with electrode solution. An agar bridge with Cl− as the main anion was used as a ground electrode. Channel activity was recorded at [Ca2+]free=30 µM with transmembrane voltage range set between −80 and −20 mV. These values of transmembrane voltage and intracellular calcium include conditions that are faced by BK channels in the myocyte (Knot and Nelson, 1998; Pérez et al., 2001). Upon membrane patch excision, solutions were applied onto the patches using a computerized and pressurized OctaFlow system (ALA Scientific Instruments) via a micropipette tip with an internal diameter of 100 μm. BK channel basal activity was recorded for 1 min under perfusion with the patch-clamp bath solution, and then probed with 50 mM (230 mg/dL) ethyl alcohol. This alcohol level approximates that of human blood following moderate-to-heavy alcohol intake (Heatley and Crane, 1990). Alcohol was perfused onto the intracellular side of the excised patch for 10 min. This time frame is similar to alcohol-induced constriction of cerebral arteries (Bukiya et al., 2014b; Bisen et al., 2016). BK channel activity was recorded for the entire time of alcohol perfusion, and NPo was calculated for each minute of recording. In a separate set of membrane patches, time-matched perfusion was performed with a bath solution to account for possible fluctuations in BK channel activity over time. Experiments were carried out at room temperature (21°C). Ionic current was recorded with an EPC8 amplifier (HEKA) at 1 kHz using a low-pass, eight-pole Bessel filter (model 902LPF; Frequency Devices). Data were digitized at 5 kHz using Digidata 1320A and pCLAMP 8.0 (Molecular Devices).

Chemicals.

Alcohol (ethyl alcohol, 100% purity, 200 proof) was purchased from American Bioanalytical. CLR and 1,2-dioctanoyl-sn-glycerol were purchased from Avanti Lipids and Cayman Chemical, respectively. Gouml 6983 and phorbol 12-myristate 13-acetate (PMA) were purchased from Abcam and Alomone labs, respectively. All other chemicals were purchased from Sigma-Aldrich. Alcohol was freshly diluted in bath solution immediately before the experiment and delivered to the membrane patch via an OctaFlow system. Each membrane patch was only exposed to alcohol once to avoid reduced responsiveness to multiple applications. Upon arrival from manufacturer, 1,2-dioctanoyl-sn-glycerol stock in acetonitrile was dried under nitrogen atmosphere, and then 1,2-dioctanoyl-sn-glycerol was directly dissolved in patch-clamp solution at 20 µg/mL (Lum et al., 2016). Gouml 6983 was prepared as a 10 mM stock in dimethyl sulfoxide, and then further diluted to 1 µM in patch-clamp solution (Brakat et al., 2018; König et al., 2019). PMA was prepared as a 10 mM stock solution in dimethyl sulfoxide, and then further diluted to 0.6 µM in patch-clamp solution (Hepler et al., 1988; Regazzi et al., 1989).

Data analysis.

Electrophysiological data were analyzed with Clampfit 9.2 (Molecular Devices). In patch-clamp recordings, NPo was used as an index of steady-state channel activity, where NPo = N (number of channels in the patch) x Po (single channel open probability). The number of channels in the patch was determined after applying a depolarizing voltage step to +40 mV. At this voltage, and in the presence of 30 µM [Ca2+]free used in our experiments, BK channels reach near-maximal activation, with Po approaching 1. Thus, N could be determined from the maximal number of opening levels. Knowing N, NPo, and Po could be obtained using the built-in option in Clampfit 9.2 (Molecular Devices).

Further analysis, plotting, and fitting of data were conducted using Origin 2020 (OriginLab Corp.) and InStat 3.0 (GraphPad Software, Inc.). When the number of experimental data-points did not exceed ten, Gaussian distribution of data could not be established with certainty. Thus, data were analyzed using a non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Results were considered significant at an alpha level of 0.05. Data are expressed as the mean ± S.E.M., n=number of membrane patches, with each patch obtained from a different myocyte. The individual experimental groups contained data from three to nine rats.

RESULTS

High-CLR dietary intake in vivo protects cerebral artery smooth muscle BK channels against inhibition by physiologically relevant alcohol concentration.

Our earlier work demonstrated the protective effect of dietary CLR against alcohol-induced constriction of rat MCA in vivo and even in de-endothelialized arteries in vitro (Bukiya et al., 2014b). BK channels are major targets of alcohol within vascular smooth muscle, with their inhibition by alcohol underlying alcohol-induced constriction of cerebral arteries (Liu et al., 2004). Thus, we hypothesized that high-CLR dietary intake in vivo blunted alcohol-induced inhibition of cerebral artery myocyte BK channels. BK channel activity was recorded in excised, I/O patches from freshly isolated MCA myocytes from rats subjected to high-CLR dietary intake for 18–22 weeks as opposed to their littermates on regular rodent chow. Consistent with our earlier reports (Bukiya et al., 2011; Bisen et al., 2016), administration of 50 mM alcohol caused persistent inhibition of BK currents in MCA myocytes from rats on control diet. This inhibition reached up to 10–15% when compared to bath perfusion at corresponding time points (Fig. 1A–B). Alcohol-induced BK channel inhibition, however, vanished in MCA myocytes from rats on the high-CLR diet (Fig. 1C–D).

Figure 1. High-CLR dietary intake in vivo protects against alcohol-induced inhibition of rat cerebral artery myocyte BK currents.

Figure 1.

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(A) BK recordings from an I/O patch excised from an arterial myocyte of male Sprague-Dawley rats placed on control chow for 21 weeks. Channel activity is shown before and during exposure to either alcohol-free (bath) or 50 mM alcohol (EtOH) solution. Here and in all other figures, channel openings are shown as downward deflections, arrows indicate baseline (all channels are closed). Unless noted otherwise, Vm = −40 mV, [Ca2+]free = 30 μM. Wash was performed with bath solution. (B) Averaged changes in BK channel activity over time in MCA myocyte membrane patches from rats on control chow. Myocytes were isolated from MCAs of rats on control chow feeding that lasted 18–22 weeks. *Statistically significant difference between alcohol and bath (0.0015<P≤0.0374). Here and in all other figures, the dotted line underscores the level of activity during first minute of patch perfusion. (C) BK recordings from an I/O patch excised from an arterial myocyte of male Sprague-Dawley rat placed on high-CLR chow for 21 weeks. (D) Averaged changes in BK channel activity over time in MCA myocyte membrane patches from rats on high-CLR chow. Myocytes were isolated from MCAs of rats subjected to high-CLR feeding for 18–22 weeks.

In an earlier report, we documented that an 18–22 week-long feeding of rats with high-CLR chow resulted in a significant increase in the CLR level of de-endothelialized MCAs (Bukiya et al., 2014b). Moreover, CLR in vitro depletion restored alcohol sensitivity of de-endothelialized MCAs (Bukiya et al., 2014b). In our present work, we subjected MCA myocytes from rats on high-CLR chow to a 20 min-long CLR depletion in vitro. In earlier study we documented the efficiency of this treatment, as cholesterol levels in de-endothelialized cerebral arteries of rats were reduced by 50% (Bukiya et al., 2011). Here, CLR depletion in vitro reinstated BK channel inhibition by 50 mM alcohol in myocyte patches from rats on high-CLR chow (Fig. 2A–B, Fig. 3).

Figure 2. CLR in vitro depletion but not enrichment restores BK channel sensitivity to alcohol in myocytes from rats on high-CLR chow.

Figure 2.

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(A) BK recordings from an I/O patch excised from an arterial myocyte of male Sprague-Dawley rat placed on high-CLR chow for 21 weeks. Prior to patch excision, myocyte was subjected to a 20 min-long depletion of CLR in vitro. (B) Averaged changes in BK channel activity over time. Myocytes were isolated from MCAs of rats subjected to high-CLR feeding for 18–22 weeks. Prior to patch excision, myocyte was subjected to a 20 min-long depletion of CLR in vitro. *Statistically significant difference between alcohol and bath (P=0.049). (C) BK recordings from an I/O patch excised from an arterial myocyte of male Sprague-Dawley rat placed on high-CLR chow for 21 weeks. Prior to patch excision, myocyte was subjected to a 20 min-long enrichment of CLR in vitro. (D) Averaged changes in BK channel activity over time. Myocytes were isolated from MCAs of rats subjected to high-CLR feeding for 18–22 weeks. Prior to patch excision, myocyte was subjected to a 20 min-long enrichment of CLR in vitro.

Figure 3. In vivo dietary cholesterol protects against alcohol-induced BK channel inhibition via accumulation of CLR in cerebral artery myocytes.

Figure 3.

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Fold-change in BK channel NPo. *Statistically significant difference (P < 0.05).

Consistent with the critical role of elevated CLR in maintaining BK channels’ resistance to alcohol, 20 min-long CLR in vitro enrichment failed to modify the lack of alcohol sensitivity of cerebral artery myocyte BK channels from rats subjected to high-CLR dietary intake in vivo (Fig. 2C–D, Fig. 3). Yet, the protective effect of a high-CLR diet against alcohol-induced inhibition of BK channels could be mimicked in normocholesterolemic rats by mere CLR enrichment of MCAs in vitro (Bisen et al., 2016).

CLR blunting of alcohol-induced BK channel inhibition in vascular smooth muscle requires a cellular environment.

We have recently established a time-course for CLR in vitro enrichment of rat MCA using CLR complexed with MβCD (Slayden et al., 2020). It became apparent that CLR enrichment significantly increased CLR levels up to 30–50% within the first 5 min of incubation. Moreover, the increase in artery smooth muscle CLR level persisted for several hours after removal of CLR enriching media (Slayden et al., 2020). Thus, we incubated freshly isolated myocytes in CLR-enriching media for 5 min and immediately proceeded to patch excision and alcohol testing. As expected from successful CLR enrichment, the procedure rendered BK channels resistant to alcohol presence (Fig. 4A–B).

Figure 4. CLR in vitro enrichment of whole myocytes but not cell-free myocyte membranes renders BK channel resistant to alcohol-induced inhibition.

Figure 4.

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(A) BK recordings from an I/O patch excised from an arterial myocyte of male Sprague-Dawley rat, myocyte was subjected to a 5 min-long incubation in CLR-enriching media prior to patch excision. (B) Averaged changes in BK channel activity over time. Myocytes were isolated from MCAs of rats on control chow and subjected to a 5 min-long incubation in CLR-enriching media prior to patch excision. (C) BK recordings from an I/O patch excised from an arterial myocyte of male Sprague-Dawley rat, patch was subjected to a 5 min-long incubation in CLR-enriching media immediately after excision. (D) Averaged changes in BK channel activity over time. Myocytes were isolated from MCAs of rats on control chow, patches were subjected to a 5 min-long incubation in CLR-enriching media prior to excision from myocytes. *Statistically significant difference between alcohol and bath (0.0025<P≤0.0325). (E) Fold-change in BK channel NPo. *Statistically significant difference (P < 0.05).

Next, we set to determine whether CLR-driven protection against alcohol inhibition of BK channels required an intracellular environment or could be replicated in cell-free membranes. For this, we perfused intracellular leaflets of excised membrane patches from freshly isolated myocytes with CLR-enriching media. Successful enrichment of excised patches with cholesterol was confirmed by a decrease in BK channel activity (Supplementary Fig. 1; Crowley et al., 2003; Singh et al., 2012). Five min-long perfusion of CLR-enriching media to excised patches failed to evoke protection against alcohol-induced inhibition of BK channels (Fig. 4A–B vs. C–D, Fig. 5). Indeed, 50 mM alcohol at 6–9 min of application caused up to a 40% drop in BK channel NPo when compared to channel activity in patches that were perfused with bath solution (Fig. 4D). Thus, CLR blunting of alcohol-induced BK channel inhibition within vascular smooth muscle requires a cellular environment and cannot be replicated in cell-free membrane patches (Fig. 5).

Figure 5. Fold-change in BK NPo upon perfusion of CLR-rich patches with either bath or alcohol-containing solution.

Figure 5.

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*Statistically significant difference (P < 0.05).

CLR regulation of vascular smooth muscle BK channel sensitivity to alcohol is fine-tuned by a protein kinase C-dependent mechanism(s).

Considering that kinase activity represents one of the most common pathways of intracellular signal transduction, we set to determine whether a protein kinase-mediated pathway could underlie CLR modulation of the alcohol effect on BK channels. Thus, we performed patch-clamp recordings in membrane patches following myocyte enrichment with CLR in the presence of 1 µM Gouml 6983. The latter effectively inhibits protein kinase C (PKC) activity (Brakat et al., 2018; König et al., 2019). Myocyte incubation with 1 µM Gouml 6983 did not affect CLR ability to antagonize alcohol-induced inhibition of BK currents (Fig. 6A, C–D). However, CLR failed to protect BK currents from alcohol-induced inhibition when CLR enrichment of myocytes was performed in the presence of a PKC activator: either 06 µM PMA or 20 µg/mL 1,2-dioctanoyl-sn-glycerol (Fig. 6B, D) (Hepler et al., 1988; Lum et al., 2016). This outcome suggests that PKC activation either disables the CLR target(s) or competes with CLR for a common molecular pathway. If the latter is the case, then PKC activation would protect against alcohol-induced inhibition of BK currents even at naïve CLR level. Indeed, myocytes incubation in 1,2-dioctanoyl-sn-glycerol without manipulation with naïve CLR level resulted in protection against alcohol-induced inhibition of BK currents (Fig. 7). In contrast, myocytes incubation in Gouml 6983 did not modify alcohol effect (Fig. 7). In synthesis, CLR modification of alcohol sensitivity is enabled by a PKC-dependent mechanism(s).

Figure 6. PKC activation but not inhibition prevents CLR antagonism of alcohol effect on BK current.

Figure 6.

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(A) BK recordings from an I/O patch excised from an arterial myocyte of male Sprague-Dawley rat, myocyte was subjected to a 5 min-long incubation in CLR-enriching media in presence of PKC inhibitor 1 µM Gouml 6983 prior to patch excision. Vm = −30 mV. (B) BK recordings from an I/O patch excised from an arterial myocyte of male Sprague-Dawley rat, myocyte was subjected to a 5 min-long incubation in CLR-enriching media in presence of PKC activator 0.6 µM PMA prior to patch excision. Vm = −20 mV. (C) Averaged changes in BK channel activity over time. Each time-point represents mean±S.E.M. of NPo over time-matched control that is an averaged NPo at the same timepoint obtained during patch perfusion with bath solution. Myocytes were isolated from MCAs of rats on control chow and subjected to a 10 min-long incubation in either 1 µM Gouml 6983, 0.6 µM PMA or 20 µg/mL 1,2-dioctanoyl-sn-glycerol (DAG) prior to addition of CLR-enriching media for 5 min. (D). Scattered data highlighting fold-difference in BK NPo when comparing 6 throughout 9 minutes of perfusion by 50 mM alcohol with time-matched perfusion with bath. Averaged data for bath perfusion were obtained from 5, 3, 5, and 6 patches for CLR enrichment, CLR enrichment in presence of Gouml 6983, PMA, and 1,2-dioctanoyl-sn-glycerol, respectively. *Statistically significant difference from alcohol effect in membranes from CLR-enriched myocytes (P < 0.05); **Statistically significant difference from alcohol effect in membranes from CLR-enriched myocytes (P < 0.01); ##Statistically significant difference from alcohol effect in membranes from myocytes that were enriched with CLR in presence of PKC inhibitor Gouml 6983 (P < 0.01); ###Statistically significant difference from alcohol effect in membranes from myocytes that were enriched with CLR in presence of PKC inhibitor Gouml 6983 (P < 0.001).

Figure 7. PKC activation but not inhibition protects against alcohol effect on BK current at naïve CLR level.

Figure 7.

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(A) Averaged changes in BK channel activity over time. Each time-point represents mean±S.E.M. of NPo over time-matched control that is an averaged NPo at the same timepoint obtained during patch perfusion with bath solution. Myocytes were isolated from MCAs of rats on control chow and subjected to a 10 min-long incubation in either 1 µM Gouml 6983 or 20 µg/mL 1,2-dioctanoyl-sn-glycerol (DAG) prior to a 5 min-long incubation in bath solution to preserve naïve CLR level. (B). Scattered data highlighting fold-difference in BK NPo when comparing 6 throughout 9 minutes of perfusion by 50 mM alcohol with time-matched perfusion with bath. Averaged data for bath perfusion were obtained from 6, 5, and 6 patches for naïve CLR, Gouml 6983, and 1,2-dioctanoyl-sn-glycerol, respectively. *Statistically significant difference (P < 0.05).

DISCUSSION

In this work, we utilized patch-clamp electrophysiology to demonstrate reduced BK channel sensitivity to alcohol-induced inhibition in cerebral artery myocytes following high-CLR dietary intake in vivo. Moreover, we have established for the first time that the CLR-driven reduction in alcohol-induced BK channel inhibition requires a cellular environment and involves a PKC-dependent mechanism(s).

Alcohol-induced constriction of cerebral arteries and CLR-alcohol interactions have been linked to the activity of vascular-smooth muscle BK channels (Liu et al., 2004; Bisen et al., 2016; North et al., 2018). However, previous studies focused on acute CLR enrichment of cerebral arteries in vitro (Bisen et al., 2016; North et al., 2018). The ability of CLR enrichment in vitro to confer protection against alcohol-induced BK channel inhibition was further validated in the current work. More importantly, the current work demonstrates that a high-CLR dietary intake in vivo also decreases alcohol-induced BK channel inhibition in artery myocytes (Figs. 1, 3). This action is likely attributed to CLR buildup in cerebral artery myocytes. Indeed, in the course of high-CLR dietary intake, CLR buildup in the de-endothelialized cerebral arteries of rats has been demonstrated by our group (Bukiya et al., 2014b). Removal of excessive CLR restored alcohol-induced constriction of cerebral arteries (Bukiya et al., 2014b) and, as shown in the present work, restored alcohol-induced inhibition of BK currents (Fig. 2A–B, Fig. 3). One may argue that the observed effect is attributed to the CLR carrier MβCD, as cyclodextrins have been recently shown to modify Kv currents (Zakany et al., 2020). However, we previously reported that CLR delivery to cerebral arteries via alternative carrier- low-density lipoprotein also diminished alcohol-induced constriction of rat MCA (Bisen et al., 2016). Moreover, in the present work, both CLR in vitro depletion and enrichment utilized MβCD yet restored and failed to modify alcohol sensitivity of MCAs respectively from rats receiving high-CLR chow (Figs. 2–3). Therefore, it is unlikely that the observed effects are specific to the CLR delivery system rather than CLR itself.

A major finding of this study reveals the cell-dependent nature of CLR protection against alcohol-induced inhibition of BK channels (Figs. 4–5). This outcome was rather unexpected. The ability of elevated CLR to progressively antagonize BK channel response to alcohol has been demonstrated before in a cell-free system, i.e., artificial lipid bilayers (Crowley et al., 2003). That work was performed on a recombinant channel that lacked regulatory β1 and γ subunits, which are characteristic of the smooth muscle BK channel and abundant in cerebral artery myocytes (Brenner et al., 2000; Orio et al., 2002; Evanson et al., 2014). Noteworthy, β1 subunit may drastically alter the mode of BK channel modulation by CLR. Our most recent data demonstrate that subjecting cerebral artery myocytes to CLR enrichment increases the myocyte plasmalemmal fraction of β1 subunits and thus switches the effect of this steroid from inhibition to activation of BK channels with a corresponding enhancement of β1 subunit-specific features of the BK current (Bukiya et al., 2020). Thus, it could be argued that CLR antagonism of alcohol effect relies on BK channel accessory subunits. Our earlier work using BK β1 subunit-lacking KCNMB1 knock-out mice, however, ruled out the possible involvement of the BK β1 subunit in CLR antagonism of alcohol-induced BK channel inhibition and the resulting constriction of cerebral arteries (Bisen et al., 2016). The possible role of BK channel γ subunits in CLR-alcohol interaction remains to be explored and may explain the discrepancy between our current work and earlier studies using an artificial lipid bilayer. Moreover, the artificial lipid bilayer was only formed by two lipid species (1-palmitoyl-2-oleoyl phosphatidylethanolamine/1-palmitoyl-2-oleoyl phosphatidylserine), and thus was far from replicating the complexity and asymmetry of native plasma membranes (Dopico and Tigyi, 2007). As was reported in a study utilizing artificial lipid membranes, the lipid environment surrounding the BK channel had critical importance for CLR tuning of alcohol effect on BK current (Yuan et al., 2011). The advantage of our experimental approach lies in the use of native BK channels in their native cellular environment, in which BK accessory subunits, membrane lipids other than 1-palmitoyl-2-oleoyl phosphatidylethanolamine or 1-palmitoyl-2-oleoyl phosphatidylserine, and intracellular signaling may all contribute to CLR control over BK channels’ sensitivity to alcohol.

Indeed, our work documents the loss of CLR ability to antagonize alcohol effect on BK currents in patches excised from CLR-enriched myocytes that were pre-incubated with the PKC activators (Fig. 6D). PKC is one of several kinase types that control BK channel activity (Shipston and Armstrong, 1996; Schubert and Nelson, 2001; Zhu et al., 2009). In smooth muscle, inhibition of BK currents by PKC is reported in pulmonary arterial myocytes of the fawn-hooded rat model of pulmonary hypertension (Barman et al., 2004). Yet, the PKC activator phorbol myristate acetate increased BK currents in Sprague-Dawley rat pulmonary arterial myocytes (Barman et al., 2004). This effect was blocked by the specific antagonist of a cGMP-dependent protein kinase (Barman et al., 2004). In rabbit coronary artery myocytes, however, whole-cell BK currents were reported to be insensitive to PKC inhibition by bisindolylmaleimide I (Park et al., 2005). While species- and pathology-related differences may explain the diversity of reports pertaining to PKC effects on basal behavior of BK channels, this diversity also reflects multiple points of BK current regulation by PKC. Indeed, PKC phosphorylation sites at Ser695 and Ser1151 have been reported within the bovine tracheal smooth muscle BK channel-forming alpha subunit (Zhou et al., 2010). It is possible to hypothesize that PKC phosphorylation at these sites leads to conformational changes within BK channel-forming protein that disable CLR antagonism of alcohol-induced BK current inhibition. This hypothesis gains further plausibility considering that CLR-sensing Tyr450 (Singh et al., 2012) and alcohol-sensing Lys361 (Bukiya et al., 2014a) both reside in the vicinity of PKC phosphorylation sites. Moreover, an earlier report on GH3 pituitary tumor cells showed that the effect of alcohol on BK currents is mediated by PKC stimulation and phosphorylation of the channels (Jakab et al., 1997). However, direct activation of PKC by ethyl alcohol is unlikely, as it could not be detected in a number of in vitro assays as reviewed (Stubbs and Slater, 1999).

A proposed scenario of allosteric pre-conditioning of CLR- and/or alcohol-sensing site(s) by preceding phosphorylation by PKC should take into account our finding that the PKC activator 1,2-dioctanoyl-sn-glycerol protects against alcohol-induced inhibition of BK currents at naïve CLR level (Fig. 7). The ability of 1,2-dioctanoyl-sn-glycerol to protect against alcohol effect at naïve CLR level combined with 1,2-dioctanoyl-sn-glycerol ability to cancel CLR-alcohol antagonism upon CLR enrichment favors the possibility that 1,2-dioctanoyl-sn-glycerol and elevated CLR compete for a common pathway(s) that results in antagonizing alcohol effect on BK currents. While either 1,2-dioctanoyl-sn-glycerol or elevated CLR protects against alcohol-driven inhibition of BK current, the combination of 1,2-dioctanoyl-sn-glycerol and CLR fails to do so. Consistent with the physiological function of 1,2-dioctanoyl-sn-glycerol as PKC activator, CLR elevation and PKC activation may compete over controlling alcohol effect. Notably, elevation of CLR levels within cerebral artery is expected to increase PKC activity. Indeed, CLR-induced activation of PKC has been reported in cultured ascites tumor cells (Haeffner and Wittmann, 1994). Moreover, amplified PKC activation by natural trigger diacylglycerol was observed in the presence of physiologically relevant CLR levels within phosphatidylcholine/phosphatidylserine bilayers (Armstrong and Zidovetzki, 2008).

Unlike the PKC activators, the PKC inhibitor Gouml 6983 failed to modify CLR control over BK current’s sensitivity to alcohol (Figs. 6D, 7B). One explanation could be that PKC tonic level and/or activity does not exert a measureable effect on CLR-alcohol interaction at BK currents. In this scenario, blocking PKC would not modify CLR antagonism of alcohol sensitivity, as was indeed observed in our work (Figs. 6D, 7B). An alternative explanation, however, it that PKC activators 1,2-dioctanoyl-sn-glycerol and PMA target specific PKC isoforms when compared to Gouml 6983. The PKC activator PMA used in our study is rather non-selective (Radresa et al., 2014). Although bioactive lipid species of the diacylglycerol family differ in their efficiency, they all serve as activators of conventional (α, βII and γ) and novel (δ, ε, η and θ) PKC isoforms (Kamiya et al., 2016). While Gouml 6983 is often referred to as a pan- PKC inhibitor that targets PKCα, PKCβ, PKCγ, PKCδ and PKCμ, half-maximal inhibitory concentrations vary between 6 nM and 20 mM (Gschwendt et al., 1996). At 1 µM, as used in our study, Gouml 6983 is expected to inhibit the four major PKC isoforms. This analysis leaves the possibility that remaining PKC isoform(s) may still enable CLR-alcohol antagonism. The use of more selective pharmacological modulators in the future will help to delineate the possible differential contribution of PKC isoforms into CLR antagonism of BK channels’ alcohol sensitivity. Moreover, the potential involvement of proteins kinases other than PKC cannot be ruled out. Indeed, PKC phosphorylation of Ser1151 within the BK channel-forming alpha subunit prevented channel activation by protein kinase A but not protein kinase G (Zhou et al., 2010). In this regard, PKC sites may serve as plug-ins for cellular cascades that enable CLR antagonism of alcohol effects on BK current and go beyond the PKC pathway.

In summary, we have established a critical role for cellular PKC-sensitive mechanism(s) in mediating the protective effect of CLR enrichment against alcohol-induced BK channel inhibition. Further studies to identify the molecular underpinnings of CLR-alcohol interactions that control BK channel function and cerebral artery diameter will ultimately help to develop novel therapeutic approaches to counteract the negative consequences of episodic drinking on cerebral circulation.

Supplementary Material

1
2

Highlights.

  • Alcohol constricts cerebral arteries via myocyte BK channel inhibition

  • Dietary cholesterol protects against alcohol inhibition of BK current

  • Cholesterol protection against alcohol inhibition of BK current requires cell integrity

  • Cholesterol protection against alcohol is lost in presence of PKC activators

Acknowledgments.

The current work was supported by NIH grants R01 AA-023764 (AB), R01 HL-104631, and R37 AA-11560 (AD).

Abbreviations.

BK

calcium- and voltage-gated large conductance potassium (current or channel)

CLR

cholesterol

EtOH

ethanol

I/O

inside-out (patch)

MβCD

methyl-beta-cyclodextrin

MCA

middle cerebral artery

NPo

number of channels (N) x open probability of a single channel (Po)

PMA

phorbol 12-myristate 13-acetate

PKC

protein kinase C

Footnotes

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Conflict of interest. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Supplementary Materials

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2
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