Regarding the different mechanisms that control Ca²⁺ homeostasis in skeletal fibers, Ohlendieck et al., (2003) (73), revealed that chronic alcohol administration increased SERCA1 and Ca²⁺-ATPase protein levels. In turn, Cofan et al. (1995) (74) showed a depletion of intracellular Ca²⁺ when muscle was exposed to ethanol (20–200 mM). Studies on RyR1 modulation by ethanol, however, are scarce. More importantly, the available studies differ in methodology, experimental conditions and results, making it difficult to reach a definitive conclusion on ethanol-mediated regulation of RyR1 function. For example, acute administration of ethanol (2–20 mM), increased Ca²⁺release from heavy SR fractions isolated from rabbit skeletal muscle (41). Moreover, preliminary data from our laboratory showed that ethanol (50–100 mM) was able to increase the steady-state activity of recombinant RyR1 reconstituted into artificial phospholipid bilayers (75). These data indicate that RyR1 is a pharmacological target of ethanol at concentrations reached in blood during alcohol intoxication. The contribution of this alcohol action to skeletal fiber contractility, however, remains to be determined. In contrast to the activatory effects of alcohol on RyR1 considered above, pre-treatment of bullfrog SR vesicles with 2.2–217 mM ethanol had no effect by itself, yet Ca²⁺ release increased significantly in the presence of both 2.2 mM ethanol and caffeine. Moreover, Cofan et al. (1995) (74) showed that acute exposure to ethanol (20–200 mM) depleted the intracellular Ca²⁺ concentration of resting cultured rat myocytes while chronic exposure failed to do so, indicative of “ethanol tolerance.”

Studies on the alcohol-related skeletal muscle atrophy are predominantly focused on males. However, the deleterious outcomes of ethanol consumption on skeletal muscle function in women are more severe, despite ingesting lower concentrations of ethanol (7, 9–11, 68). It was shown that women at early stages of chronic alcohol abuse possessed decreased levels of titin and nebulin proteins and displayed diminished cross-sectional area of muscle fibers when compared to their male counterparts (11). This sex-sensitive mechanism of skeletal muscle atrophy has been investigated further; there is some evidence of a connection between the proto-oncogene cMyc and skeletal muscle atrophy (76). Indeed, chronic ethanol administration resulted in the upregulation of c-myc expression. This was proposed to be a downstream effect of ethanol-induced corticosteroid expression, which impairs catabolism in skeletal muscle (76). However further investigation needs to be done to confirm these findings and to establish the subcellular mechanisms by which ethanol disrupts skeletal muscle contraction.

Episodic consumption of large quantities of alcohol such as during “binge-drinking” can cause the onset of cardiac arrhythmias, the most common of which is atrial fibrillation (AF) (20, 57, 78, 79). In fact, it has been widely observed that otherwise healthy individuals often developed AF or other arrhythmias after indulging in binge drinking during vacations, holidays or weekends. This observation led to the coinage of the terms “Holiday Heart Syndrome” (HHS) or “Party Heart Syndrome” (20, 78, 79). HHS symptoms include chest pain, fainting and shortness of breath, though some affected individuals may be asymptomatic (78).

The overarching hypothesis that chronic consumption of low to moderate levels of alcohol may play a cardioprotective role, however, has been often termed the “French Paradox” (7, 15, 78, 80–84). This term was derived from the observation that, compared to their counterparts in other developed societies, equivalent French populations presented lower mortality rates, despite the presence of risk factors for developing cardiovascular disease (elevated cholesterol, diabetes, hypertension, etc.,). The French, however, are known for regular (daily) consumption of low amounts of ethanol, usually in the form of red wine (rather than episodic drinking of beer) (7, 78, 83). While the relationship between alcohol intake and risk of cardiovascular events does follow a “J” shape, suggesting cardiovascular protection at low ethanol concentrations (7), a major contention is centered around the exact components of alcohol involved in its cardioprotective effects. Some studies have indicated that ethanol itself is the critical component in providing cardioprotection (80, 84) while others point to the importance of anti-oxidant compounds, more abundant in red wines, such as resveratrol and polyphenols (81, 82). However, the methodology used in collecting these epidemiological data, such as insufficient randomization of the studies, has been criticized (85). Furthermore, recent studies have shown that even low doses of alcohol can increase the risk for developing AF (86).

Several changes at cellular and subcellular levels occur in response to acute and chronic alcohol consumption. At the cellular level, alcohol-induced cardiac dysfunction presents as impaired proteostasis (i.e., altered protein homeostasis), diminished intracellular Ca²⁺ handling and signaling, increased oxidative stress and increased apoptosis, all of which contributing to reduced cardiac contractility (87–89). However, the overall impairment of cardiac function, usually referred to as alcohol-induced cardiomyopathy, involves not only cardiac muscle components but also endothelial, neural and circulating factors (15, 16, 77).

Nevertheless, alcohol is indeed able to exert negative inotropism on cardiac muscle, independently of endothelial, neural, metabolic or circulating factors (16, 56, 87, 89, 90). Some of the biochemical players involved in mediating ethanol-induced negative ionotropic events in cardiac muscle include ROS (H₂O₂, O₂ ⁻), reactive nitrogen species (nitric oxide: NO^•), ion channels and associated proteins, such as SERCA, RyR2 and phospholamban (PLB), and acetaldehyde itself (88, 91–96). These molecular players all serve to disrupt Ca²⁺ signaling, which in turn disrupts E-C coupling and thus leads to reduced cardiac contractility (10, 56, 57). The molecular mechanisms used by these entities to mediate ethanol-induced disruption of E-C coupling are described below.

The oxidation of ethanol is carried out by three main enzymes: alcohol dehydrogenase, catalase, and CYP-2E1 (91–95). Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase 2 family member (ALDH₂) both oxidize alcohols to aldehydes and ketones (91, 95), catalase breaks down H₂O₂ to water and oxygen (96) and CYP-2E1 converts ethanol to acetaldehyde (93,95). These enzymes have been linked to the negative inotropic effects of ethanol via the production of ROS, which are critical biochemical players in the disruption of skeletal (13, 18, 66, 70, 71), cardiac (56, 88, 94, 96) and smooth muscle function (24, 97, 98).

Mitochondria play an important role in Ca²⁺ sequestration and EC-coupling and are the main source of ROS production (99). Ethanol has been shown to target the mitochondria, where it disturbs the structure and function of the mitochondrial membrane and its overall function as an organelle. One of the critical mitochondrial enzymes involved in ethanol metabolism is ALDH₂, which is highly expressed in cardiac myocytes and serves to metabolize acetaldehyde (99, 100). The downstream consequences of ethanol-induced mitochondrial dysfunction are increased apoptosis and necrosis (99) thus leading to a decrease in the contractile tissue mass.

The acute exposure of ALDH₂-knockout mice to ethanol led to significantly increased acetaldehyde production, impaired mitochondrial function, and decreased myocyte contractility when compared to wild type mice (101). In the case of chronic ethanol administration, ALDH₂ transgenic mice exhibited improved Ca²⁺ handling and homeostasis, increased cell shortening, and decreased apoptosis, with apoptosis signal-regulating kinase 1 (ASK-1) and CREB activity also being implicated in this phenotype (102). Likewise, Brandt et al. (2016) (103) showed that acetaldehyde upregulated NADPH oxidase-2 (NOX2), which has been linked to the onset of heart failure via augmentation of ROS production. However, in stark contrast to the preceding findings, low levels of acetaldehyde were found to play a cardioprotective function via a mechanism involving ALDH₂ (100).

Catalase is expressed in the myocardium, albeit in lower quantities in comparison to other organs. Still, it serves to metabolize the harmful and unstable H₂O₂ (96). Using ventricular myocytes from transgenic mice, Zhang et al., (2003) (96) showed that catalase overexpression diminished the negative inotropic events induced by acute ethanol administration. Moreover, RyR expression was upregulated and myocardial E-C coupling was improved owing to enhanced Ca²⁺ handling. These authors also found that protein expression of the Na⁺/Ca²⁺ exchanger (NCX) which removes intracellular Ca²⁺, was upregulated. There was no change however, in SERCA, PLB or DHPR expression/activity. Furthermore, AKT signaling was also increased, revealing a possible cardioprotective role of this pathway (104).

Following acute ethanol exposure, transgenic mice expressing the ADH gene, experienced enhanced inotropic events, such as aberrant Ca²⁺ handling and decreased cell shortening, as compared to wt FVB mice (91, 105). These results indicated the involvement of increased acetaldehyde levels in the onset of negative inotropic events and the dysregulation of cardiac contractility. Additionally, it was shown that simultaneous inhibition of catalase and ADH ablated the negative inotropic effects induced by acute ethanol consumption in female rats, further underscoring the involvement and importance of these enzymes to the deleterious effects of ethanol and acetaldehyde on cardiac muscle (95).

NO^• is a gaseous signaling molecule produced endogenously from the breakdown of L-arginine by NO^• synthetases (NOS). There are three NOS isoforms: endothelial (eNOS), neuronal (nNOS) and inducible (iNOS), which regulate the activity of many proteins involved in cardiac Ca²⁺ homeostasis and E-C coupling, such as L-type Ca²⁺ channels (DHPR), PLB, phosphodiesterase and RyRs. Therefore, NOS and NO^• are important regulators of cardiac contractility (106). Deng and Dietrich (2007) (97) showed the effect of NO^• production by iNOS on cardiac contractility. Ethanol was shown to bind and inhibit iNOS activity which then augmented cardiac contractility. iNOS also plays an important role in age-related ethanol-induced cardiac dysregulation (106). Acute ethanol administration induced negative inotropic effects in young mouse hearts but positive inotropic effects in the hearts of senescent mice. Senescent mice exhibited increased iNOS activity, hence ethanol -mediated inhibition of iNOS activity is more pronounced in old mice and may offer a cardioprotective effect (106).

Another pathway involved in acute consumption of heavy to moderate concentrations of alcohol is the c-Jun NH (2)-terminal kinase (JNK2) signaling pathway, which is normally activated in response to cellular stress (107). High concentrations of ethanol were shown to increase susceptibility to AF. Ethanol-induced upregulation of JNK2 activity in human and rabbit hearts resulted in amplified phosphorylation of CaMKII. Under physiological conditions, activated CaMKII phosphorylates PLB, causing its dissociation from SERCA, thereby facilitating Ca²⁺-leak from the SR (62). Upregulation of CaMKII activity, therefore, increases SERCA-mediated Ca²⁺-leak, ultimately leading to abnormal Ca²⁺ waves and disruption of E-C coupling (107). Moreover, CYP-2E1 inhibition also revealed the involvement of the JNK and ASK-1 pathways as mediators of the negative inotropic events induced by chronic alcohol administration to mice (88).

Direct exposure of human atrial muscle strips and mouse ventricular myocytes to ethanol, whether acute or chronic, resulted in the development of the typical hallmarks of negative inotropy, such as perturbed Ca²⁺ homeostasis (56, 87, 90, 107). Human atrial cardiomyocytes acutely exposed to 1–6% ethanol displayed severe Ca²⁺ leak from SR stores, decreased amplitudes of Ca²⁺-transients, decreased myofilament Ca²⁺-sensitivity, and increased NCX activity and SERCA-mediated Ca²⁺ reuptake into the SR. Altogether, these changes led to aberrant E-C coupling and negative inotropism by alcohol (56). In addition, the PI3K/Akt pathway was shown to exert some influence over the oxidative stress induced by acute alcohol consumption (108). It was also postulated that ethanol disturbed intracellular Ca²⁺ homeostasis by favoring RyR2-mediated Ca²⁺-leak, thus reducing the amount of Ca²⁺ to be released from the SR upon stimulation (56). Additionally, the NOX2 pathway and CAMKII activity were shown to be involved in the ethanol-induced upregulation of ROS production in the heart (56, 103, 109, 110). Previous findings from our lab indicated that RyR2 had an ethanol-sensing region and thus intoxicating concentrations of ethanol (18–100 mM) inhibited RyR2 activity (65). The contribution of this ethanol action to alcohol-induced depression of cardiac contractility, however, remains to be determined.

Lastly, it is important to underscore that the morphological and contractile effects of acute ethanol administration on human-induced pluripotent stem cell-derived cardiomyocytes (Hi-PSC-CMs) mimicked many of the effects seem in animal models (111).

Depressed cardiac contractility is also observed after chronic ethanol consumption (87, 88, 93, 104, 112). In FVB mice for example, chronic administration of 4% alcohol caused the classic hallmarks of negative inotropism. However, SERCA levels were decreased (104), in contrast to the upregulated SERCA activity observed after acute ethanol administration (56). Moreover, the protein levels of CYP-2E1, iNOS and PLB increased while NCX levels were downregulated (88). However, these changes in protein levels, along with the aberrant Ca²⁺-handling were significantly ablated in FVB mice expressing an IGF-1 transgene (104). Furthermore, CYP-2E1 and the JNK2 and ASK-1 signaling pathways were implicated in the ethanol-induced upregulation of ROS production after chronic ethanol exposure (88).

The data discussed so far clearly underscores the negative effects of acute and chronic alcohol consumption on cardiac muscle contraction. However, there seems to be a transition period between these two stages of AUD when considering cardiac muscle function (113). Using rat ventricular myocytes, the cardiac effects of ethanol exposure were monitored for different intervals (acute exposure = 10–20 min vs. chronic exposure = 1 and 3 months). Results revealed that the negative inotropic effects of chronic ethanol exposure were biphasic between 1- and 3-months. Both acute and chronic alcohol, however, caused the development of events in ventricular myocytes leading to negative inotropy, i.e., decrease in Ca²⁺ transient amplitude, Ca²⁺ rate of rise and decay of Ca²⁺transients. However, at the 1-month timepoint, indicators of positive inotropy such as increased cell shortening and the augmentation of Ca²⁺ amplitude were observed. In turn, authors concluded that ethanol-mediated decrease in SR load was the main determinant of the sustained negative inotropy in response to chronic alcohol consumption (113).

Acetaldehyde is the first product of ethanol metabolism (10, 87, 94). Like ethanol, acetaldehyde impairs E-C coupling in cardiac muscle via two main mechanisms. Firstly, acetaldehyde binds proteins to form “protein adducts” which are unstable, non-functional and immunogenic, and are therefore degraded via the ubiquitin proteasome pathway or autophagy (10, 87, 94). It has been noted that persons suffering from cardiac disorders produce antibodies against these acetaldehyde-associated protein adducts, as well as functional proteins (10, 87, 94). Secondly, acetaldehyde is known to induce negative inotropic events in the heart and is considered to be more potent than ethanol itself, owing to its enhanced bioreactivity with other compounds (91, 100–103). Increased acetaldehyde production disrupts the delicate balance between oxidants and antioxidants. For example, acetaldehyde can be further metabolized to form ROS through the activity of superoxide dismutase, aldehyde oxidase or xanthine oxidase to produce the highly unstable O₂ ⁻ (99).

Like ethanol, acetaldehyde also attenuates cardiac contractility (7, 87). For example, O₂ ⁻ reacts with NO^• to form the highly reactive ROS peroxynitrite which has been shown to damage contractile proteins and many enzymes critical to mitochondrial function (94, 97). Various animal studies have shown that the overexpression and knockdown of ALDH₂ served to attenuate and augment the effects of both acetaldehyde and O₂ ⁻ respectively. Acetaldehyde also modulates PLB protein levels (93) and intracellular Ca²⁺ handling (16,87,101).

Men and women exhibit differences in both ethanol metabolism and the pathogenesis of cardiac myopathies (10, 11, 114). Compared to men, women are more vulnerable to the toxic effects of ethanol abuse (10, 11). Subsequent to acute exposure, Duan et al., (2003) (10) observed that female mice were more sensitive to acetaldehyde-induced hypo-contractility than males. This increased sensitivity is believed to be closely related to estrogen (10, 115). Though there is limited data on the cross reactivity of estrogen and acetaldehyde-induced cardiac activity, estrogen is known to modulate the metabolism of acetaldehyde and increases the production of NO^•. Cardiac function is heavily modulated by NO^• and the release of this ROS augments both ethanol and acetaldehyde-induced cardiac hypercontractility (10).

In the epithelial cells of vascular muscle, estrogen increases NO^• production which then augments ethanol-induced effects, therefore this may be the case for cardiac muscle. Females may produce more NO^•, and may therefore be more prone than males to the deleterious effects of alcohol consumption. These conclusions indicate that both ethanol and its metabolite acetaldehyde attenuate cardiac contractility and these effects can be more potent in women than men (114).

There are many diverse pathways and components involved in the development of cardiomyopathies as a result of ethanol consumption. While the exact mechanisms involved are still unknown, there are many intriguing lines of research that can be explored.

In the aorta, as in several other vessels (see above), ethanol has been reported to evoke SM contraction and relaxation and thus, vasoconstriction and vasodilation respectively, depending upon species and experimental conditions. Rat aorta strips are constricted by ethanol through a PKC- and calmodulin-dependent mechanism (127). Likewise, acute ethanol administration (1–800 mM) leads to contraction of aortic SM cells, in this case via production and release of ROS (O₂ ⁻ and H₂O₂) from the vessel walls. ROS release in turn, increased the intracellular Ca²⁺concentration via a mechanism that is independent of endothelium and involves the cyclooxygenase (COX) pathway (98).

However, the presence of the endothelium cannot solely explain the differential effects of ethanol on aortic diameter and SM tone. For example, in rat aortic rings pre-contracted with either KCl or phenylephrine, Ru et al., (2008) (132) showed that acute administration of 0.1–7% ethanol evoked dilation in both intact and endothelium-denuded thoracic aorta rings; with this effect being more potent in the latter (132). These authors also reported that 2-APB and dantrolene (inhibitors of IP₃R and RyR respectively), both caused a significant decrease in ethanol-induced aortic dilation, underscoring a possible link between the release of Ca²⁺ from SR stores and ethanol-induced SM relaxation in the aorta. In contrast, Tirapelli et. al. (2006) (133), showed that phenylephrine-induced contraction was augmented in rat aortic rings isolated after chronic administration of 20% (v/v) ethanol. In turn, ethanol levels reflective of mild drinking (2–25 mM) were shown to evoke dilation of both intact and denuded rat aortic rings. This alcohol effect was mediated by ROS-dependent activation of NO^• as a consequence of eNOS upregulation by ethanol (24). This study also showed that rat aortic SM cells (VSMCs) could themselves produce NO^•, which in turn caused vasodilation via the cGMP pathway. Furthermore, catalase (a H₂O₂ scavenger), Tiron (an O₂ ⁻ scavenger), and L-NAME (a nonselective NOS inhibitor) attenuated this ethanol-induced aortic relaxation (24). Likewise, withdrawal from chronic ethanol exposure depressed the contractility of endothelium-denuded aortic rings from rats (134). This reduction in SM tone was independent of O₂ ⁻ and H₂O₂ signaling, yet it was suggested that the COX-2 pathway could be involved, in a mechanism that was not endothelium-dependent (24).

It is possible to advance that the signaling molecules that participate in ethanol-induced dilation of the aorta, may act upon ion channels which regulate VSMC membrane excitability and thus SM tone. Some voltage-dependent K⁺ channels and ATP-sensitive K⁺ channels are indeed involved in ethanol-induced aortic dilation (54, 135). Regarding SM BK channels, critical determinants of vascular myogenic tone and diameter (54), acute exposure to ethanol (10–100 mM) of native (136) or recombinant bslo1 isoform (137) channels from bovine aortic SM reconstituted into planar lipid bilayers led to a powerful decrease in channel activity, an ethanol action that would lead to increased SM tone and aortic constriction. However, the contribution of ethanol inhibition of BK channel activity to aortic SM tone and diameter is yet to be determined.

With regards to coronary arteries, K_V channels have been demonstrated to regulate arterial tone and their activity is modulated by the MAPK signaling pathway (27, 135). Ethanol has been shown to upregulate MAPK activity and decrease K_V channel currents, leading to vasoconstriction (27). In addition, inhibition of the MAPK pathway ameliorated ethanol-induced vasoconstriction, thereby positioning the MAPK signaling pathway as a possible therapeutic target in AUD (27).

Both ethanol and acetaldehyde have been shown to evoke dilation of intact superior mesenteric arteries (SMA) (28,134,138). Acetaldehyde-mediated vasodilation, however, was more potent than that of ethanol (28). A central role for the endothelium in ethanol-induced dilation of mesenteric arteries is underscored by data from Yuui et al. (2019) (138) who showed that chronic administration of moderate levels of ethanol to rats enhanced the activity of an endothelium-dependent hyperpolarizing factor (EDHF) pathway, thereby promoting vascular relaxation. In addition, Jin et al. (2019) (28) showed that ethanol dilation of intact SMA may be mediated through the activity of NO^• and guanylyl cyclase, the latter being a main target of EDHF. However, relaxation of SMA in response to ethanol was also evoked in de-endothelialized vessels (129, 132).

In contrast to the findings described in the previous paragraph, pre-treatment of intact mesenteric resistance arteries from mice with either ethanol or acetaldehyde leads to increased efficacy of the vasopressor phenylephrine (i.e., favoring mesenteric artery constriction) (139). The molecular mechanisms underlying this ethanol action remain to be established.

Chronic ethanol consumption has been shown to impair carotid artery relaxation via upregulation of the potent vasoconstrictor endothelin-1 (ET-1) (133). Of note, ET-1 is involved in pro-inflammatory and mitogenic processes, and its dysregulation has been implicated in several disorders of the cardiovascular system (140, 141). The study by Tirapelli et al. (2006) (133) showed that chronic administration of 20% (v/v) ethanol led to enhanced ET-1 production in endothelium-intact rat carotid rings, which in turn increased carotid artery constriction. Additionally, phenylephrine-induced contraction was not enhanced further in the presence of ethanol. The exact mechanism by which ethanol influences ET-1 activity is unknown. However, while neither the pre- nor post-transcription production of ET-1 was unchanged, the protein levels of the ETB receptor which regulates dilation in carotid arteries was significantly decreased (133).

As described and referenced in the beginning of this section, constriction of cerebral arteries in response to acute exposure to toxicologically relevant concentrations of ethanol (10–100 mM) is widespread, and is observed across different species and vessel types (e.g., cortical vessels, parenchymal arterioles, etc.). This drug action is largely mediated by ethanol itself rather than its vasoactive metabolites. It is important to underscore that heavy alcohol consumption has been linked to the induction of brain hypoperfusion in humans (142), systemic arterial hypertension (the main risk factor for stroke), and cerebral events including cerebral infarction and/or hemorrhage (23).

As outlined previously, Ca²⁺ plays a critical role in the regulation of smooth muscle contraction and vascular tone, including that of cerebral arteries (25, 123, 125, 143). Thus, it is not surprising that most studies pursuing a mechanism(s) to explain alcohol-induced cerebrovascular constriction have focused on ion channels and signaling molecules that control Ca²⁺ homeostasis in these vessels. For example, Yang et al. (2001) (143) observed that ethanol-induced constriction of canine basilar arteries was modulated by both SR Ca²⁺-release (via InsP₃ or RyR) and extracellular Ca²⁺ influx via voltage-gated Ca²⁺-channels. Ca²⁺ release from SR stores was transient while extracellular Ca²⁺ influx into the cytoplasm was prolonged, with both events mediating basilar artery constriction. While activation of voltage-gated Ca²⁺-channels would lead to cerebral artery constriction, there is no such evidence from available literature. In fact, our laboratory demonstrated that ethanol at concentrations that constricted MCA (50 mM) failed to modify voltage-gated Ca²⁺-channel activity in MCA SM (23).

In cerebral artery SM, however, Ca²⁺/voltage-gated K⁺ of big conductance (BK) channels play a key role in controlling Ca²⁺ homeostasis, myogenic tone and cerebral artery response to vasomodulators (23, 52, 54, 61). BK channels are activated by membrane depolarization and/or local vasodilatory Ca²⁺ signals (termed “sparks”) that are released from activated SR RyR channels. Eventually, activated BK channels generate spontaneous transient outward currents (STOCS) which lead to repolarization of the membrane, inactivation of VDCCs and inhibition of external Ca²⁺ influx. The cumulative results of these events are the blunting of SM contraction while relaxation and vasodilation are favored (52, 54, 144).

Using freshly isolated myocytes from rat MCA and isolated vessel segments, our laboratory demonstrated that inhibition of MCA STOCS and eventual MCA constriction by intoxicating levels of ethanol (50 mM) did not involve alcohol metabolites and was independent of the endothelium. Rather, this effect was due to ethanol-induced inhibition of both RyR-generated sparks (23, 65) and β₁ subunit (encoded by KCNMB1)-containing BK channels (23, 125). Indeed, KCNMB1-/- mice exhibited a significant reduction in ethanol-induced inhibition of BK-induced STOCs and its resulting vasoconstriction (125). These results identified the BK β₁ subunit as a possible therapeutic target to counteract alcohol-induced inhibition of SM BK channels and its associated cerebrovascular constriction, a proof-of-principle being obtained both in vitro and in vivo data with celastrol, a neuroprotective agent (145).

Remarkably, IP₃-mediated Ca²⁺ waves were not affected by toxicologically relevant levels of ethanol (23), therefore underscoring the selectivity of alcohol actions towards SR RyR and BK channels. In follow-up studies (65), we also documented that toxicologically relevant ethanol concentrations reduced the steady-state activity of recombinant RyR2 (the isotype that prevails in MCA SM (146). Equivalently, this effect was also shown using an RyR2 truncation mutant consisting only of the channel functional core and its activation domain (75). These results suggest that RyR2s present a delimited region that senses the presence of ethanol.

Alcohol, however, interacts with many endogenous compounds to exert its effects on cerebral arteries. Thus, some endogenous molecules may actually protect against the ethanol-induced increase in SM contraction and its associated cerebral artery constriction. Cholesterol (CLR), for example, alleviated ethanol-mediated contraction of myocytes isolated from MCA and eventual vessel constriction in mice through a mechanism that was not dependent on BK β₁ subunits (25). This CLR-mediated protection was further confirmed using mice that were maintained on a high fat diet; when both statins and 50 mM ethanol were co-administered, CLR levels were pointedly decreased in excised MCAs. This CLR-mediated vascular effect was accompanied by significantly increased arterial constriction (123, 147), and shown to be associated with PKC signaling pathway(s).

However, the enhancement of SM tone and cerebrovascular constriction by alcohol, is not limited to RyR, BK channels and their interconnecting signaling. The transient receptor potential cation channel subfamily V member 1 (TRPV1) is widely expressed in arterial blood vessels and is another ion channel that modulates ethanol-induced cerebrovascular activity (124). Ethanol and the stimulant caffeine are usually consumed together and it has been demonstrated that TRPV1 participates in the actions of both caffeine and ethanol in MCA. Their co-administration revealed the protective effect of caffeine against ethanol-induced MCA constriction, which was suggested to occur via a mechanism involving NO^•-mediated activation of TRPV1 (124, 148). Taken together, our findings underscore the central role of TRPV1 in the vasoactive properties of two of the most widely consumed recreational drugs in the world: caffeine and alcohol.

Pre-and peri-natal alcohol consumption increases the risk of impaired fetal cognitive functions and the development of Fetal alcohol syndrome (FAS) (149). However, a critical part of pregnancy is the remodeling of the uterine SM arteries, which facilitate the delivery of nutrients and gases to the developing fetus. In a healthy mother, this remodeling is characterized by the enlargement of the CSA of uterine arteries and a decrease in the media:lumen ratio (26, 150, 151). Chronic ethanol consumption restricts this uterine remodeling and increases myogenic constriction via unknown mechanisms. These developments are indicative of artery remodeling (26, 150, 151) and increase the risk of fetal undernutrition, miscarriage, low birth weight and the development of FAS (26). Furthermore, impaired ROS signaling is a mediator of ethanol-induced dysregulation of muscle contractility, and increased iNOS and NO^• production have been implicated in the development of FAS (97).

Alcohol abuse has many deleterious effects on the entire gastrointestinal tract. Some of these outcomes include; duodenal hemorrhage, and damage to the mucosal lining thus causing impaired gut permeability and increased endotoxin levels (152). In turn, these processes upregulate inflammatory mediators such as macrophages, resulting in a pro-inflammatory milieu. Acute alcohol consumption is associated with improper absorption of glucose, amino acids, deficiencies in vitamins such as B12, B6, vitamins C, A, D, E and K, and an overload of intracellular iron stores. In addition to these effects, chronic alcohol consumption causes malabsorption of macronutrients (carbohydrates, proteins and lipids), as well as increased absorption of H₂O and Na⁺ in the small intestine resulting in diarrhea.

The interplay between ethanol and different reactive nitrogen intermediates has been of particular interest to researchers (153, 154). The vast majority (∼95%) of the ethanol that reaches the liver is oxidized, thereby allowing its elimination form the body (9, 12, 13, 153). Ethanol metabolism also occurs in the stomach, where O₂ ⁻ and NO^• interact to produce the potent oxidizing agent peroxynitrite. The nitrosation of ethanol by peroxynitrite, produces the potent vasodilator ethyl nitrite (153, 154). Using gastric fundus strips, Gago et al., (2008) (154) showed that administration of nitrite (NO₂ ⁻), the precursor of NO^•, caused minor vasodilation. In contrast, acute ethanol exposure resulted in minor vasoconstriction. However, co-administration of the two compounds resulted in a significant increase in vasodilation owing to the formation of ethyl nitrite. The authors therefore, concluded that their findings demonstrated the protective effect of wines and brandy in the gut, when consumed in moderation (154). For further discussion of the vascular effects of ethanol on the GI or other body systems, please see the section on vascular smooth muscle.

It has also been demonstrated that ethanol causes both relaxation and constriction in a dose-dependent manner (155–157). Low concentrations of ethanol (25–100 mM) decreased smooth muscle contractility in both human, cat and canine esophageal muscle (155). This hypo-contractility was believed to be caused by the inhibition of Ca²⁺ influxes.

Conversely, data obtained in guinea pigs, showed that a higher ethanol concentration (342 mM) caused gastric smooth muscle constriction (156). The high ethanol concentration was reflective of the levels found in the gut due to the direct diffusion of ethanol across the mucosal surface, as compared to the lower ethanol levels found in blood. The authors advanced that this ethanol-induced constriction occurred through a phospholipase A2 (PLA₂)- mediated mechanism, since PLA₂ inhibitors decreased this effect. However, the specifics of this mechanism are still unknown. Likewise, 20–500 mM ethanol caused constriction of both longitudinal and circular smooth muscles of guinea pigs (156). This action was shown to be dependent on extracellular Ca²⁺ and believed to be mediated via a tyrosine kinase signaling pathway.

There is limited information about the mechanism(s) by which alcohol dysregulates smooth muscle contractility in the bladder since the relationship between alcohol and bladder cancer is the primary focus of research (158). Impaired detrusor contractility (IDC) is a condition which manifests as intermittent voiding (emptying of the bladder), increased bladder capacity, decreased volumes of released urine and increased residual volume (22, 159). Acute administration of ethanol to male rats caused impaired detrusor contractility and mimicked many of the effects observed in IDC (22). Likewise, alcohol has also been shown to decrease detrusor muscle contractility in response to different ligands (22, 160). However, chronic ethanol exposure of detrusor muscle strips from rat bladder caused muscle constriction, which was facilitated by the flux of Ca²⁺ from both intracellular stores and extracellular medium (160). These studies point to dysregulation of E-C coupling, resulting in impaired smooth muscle contractility, but the particular mechanisms involved remain unknown.

Chronic alcohol consumption increases the risk of developing pneumonia, lung infections and injury. In animal studies, chronic exposure to ethanol via inhalation caused cellular dysfunction and oxidative stress (161). Additionally, the phagocytic activity of alveolar macrophages was severely impaired leading to increased susceptibility and intensity of pneumonia. In addition, ethanol was also found to promote apoptosis in the epithelial cells of parenchymal tissue (161).

Regarding contractile events, ethanol has been shown to induce dilation within lung smooth muscle (162, 163). In lung parenchymal smooth muscle, chronic alcohol administration (via inhalation), was reported to decrease contractile force (163). The Rho-associated protein kinase (ROCK) pathway mediates “Ca²⁺ sensitization”, which is the increase in contractile force by signaling pathways that are independent of supplementary increases in intracellular Ca²⁺ (164). Ethanol attenuated this ROCK-mediated Ca²⁺ sensitization mechanism, which led to reduced contraction in lung parenchymal smooth muscle. Finally, in cultured rat airway smooth muscle cells, acute exposure of 100 mM ethanol caused relaxation via a cGMP/PKG mechanism (162).

The link between alcohol consumption and erectile dysfunction (ED) has been studied for years (165–170). The corpus cavernosum (CC) of the vas deferens are cavernous spaces which fill with blood to facilitate tumescence. In addition to the constriction of penile arteries, this increase in intracorporeal pressure is also dependent on the relaxation of non-vascular smooth muscle cells (169, 170). The chronic exposure of rats to ethanol (5–20% in diet) caused morphological changes in the smooth muscle myocytes, such as a decreased number of elastic fibers and collagen type 4 (170), and decreased smooth muscle area along with increased expression of Caspase 3 therefore increased apoptosis (169). As a result, these factors affected CC smooth muscle contractility and have been implicated in alcohol-induced ED.

Another protein which modulates the contraction of CC trabecular smooth muscle cells is endothelin1 (ET-1) along with other members of the endothelin pathway. ET-1 binds to ET_A and ET_B receptors which regulate contraction and relaxation respectively. Increased levels of these pathway constituents is associated with the development of ED. ET-1 causes vasoconstriction by binding to ET_A which activates NAD(P)H, resulting in the production O₂ ⁻ which promotes vasoconstriction. O₂ ⁻ is very unstable so is quickly converted by superoxide dismutase (SOD) and catalase to H₂O₂ which has vasodilation properties (171).

Chronic ethanol consumption has been shown to increase ET-1 expression and upregulate ET_A activity while also decreasing SOD activity and increases CAT activity (166, 168, 171). The cumulative result of this ethanol-induced effect is increased O₂ ⁻ production, which promotes vasoconstriction and decreased production of the vasodilatory H₂O_2. leading to increased CC contraction (166). Cyclooxygenase (COX) pathway mediators such as prostanoids also play a pivotal role in mediating dilation in trabecular smooth muscle cells and promote vasodilation through the endothelin pathway. Chronic ethanol consumption impairs COX pathway activity resulting in decreased prostanoid production which alleviates the vasodilatory influence on the endothelin pathway leading to CC contraction. There is also some evidence that ET-1 -mediated vasoconstriction may involve the ROCK pathway (168).

With respect to ethanol-induced modulation of E-C coupling, it was shown that acute and heavy alcohol consumption in peri-adolescent male Wistar rats caused decreased Ca²⁺ signaling in the vas deferens (172). A similar ethanol-induced impairment of E-C coupling was reported for the prostate and epididymis (167, 172). This impaired ethanol-induced Ca²⁺ influx was found to be mediated primarily by DHPR with little involvement of IP₃R- and RyR-controlled SR Ca²⁺ stores (167).