Role of alcohol oxidative metabolism in its cardiovascular and autonomic effects

Abstract

Several review articles have been published on the neurobehavioral actions of acetaldehyde and other ethanol metabolites as well as in major alcohol-related disorders such as cancer and liver and lung disease. However, very few reviews dealt with the role of alcohol metabolism in the adverse cardiac and autonomic effects of alcohol and their potential underlying mechanisms, particularly in vulnerable populations. In this chapter, following a brief overview of the dose-related favorable and adverse cardiovascular effects of alcohol, we discuss the role of ethanol metabolism in its adverse effects in the brain stem and heart. Notably, current knowledge dismisses a major role for acetaldehyde in the adverse autonomic and cardiac effects of alcohol because of its low tissue level in vivo. Contrary to these findings in men and male rodents, women and hypertensive individuals are more sensitive to the adverse cardiac effects of similar amounts of alcohol. To understand this discrepancy, we discuss the autonomic and cardiac effects of alcohol and its metabolite acetaldehyde in a model of hypertension, the spontaneously hypertensive rat (SHR) and in female rats. We present evidence that enhanced catalase activity, which contributes to cardioprotection in hypertension (compensatory) and in the presence of estrogen (inherent), becomes detrimental due to catalase catalysis of alcohol metabolism to acetaldehyde. Noteworthy, studies in SHRs and in estrogen deprived or replete normotensive rats implicate acetaldehyde in triggering oxidative stress in autonomic nuclei and the heart via: (i) the Akt/extracellular signal-regulated kinases (ERK)/nitric oxide synthase (NOS) cascade, and (ii) estrogen receptor-alpha (ERα) mediation of the higher catalase activity, which generates higher ethanol-derived acetaldehyde in female heart. The latter is supported by the ability of ERα blockade or catalase inhibition to attenuate alcohol-evoked myocardial oxidative stress and dysfunction. More mechanistic studies are needed to further understand the mechanisms of this public health problem.

2. Introduction

Alcohol is the most commonly used mood-altering drug worldwide. The proportions of pleasant and unpleasant effects of alcohol experienced by any individual depend largely on the amount of alcohol consumed and the pattern of alcohol use (1). Studies have shown that alcohol exerts both favorable and detrimental cardiovascular effects (figure 1), possibly contributing to the J- or U-shaped association between alcohol consumption and overall disease and cardiovascular risks. Specifically, mild-to-moderate alcohol consumption is associated with protection against coronary artery disease, and reduced incidence of heart failure, atherosclerosis, and peripheral vascular disease. The beneficial cardiovascular effects of mild/moderate alcohol consumption have been attributed to increased high-density lipoprotein cholesterol, improved endothelial function, anti-inflammatory effect, and decreased platelet aggregation (2–4).

Figure 1.

Beneficial and harmful cardiovascular effects of alcohol and contributing etiologies.

On the other hand, excessive or binge drinking is one of the leading causes of higher cardiovascular morbidity and mortality worldwide (5–7). Alcohol-induced cardiomyopathy is characterized by cardiac hypertrophy, interstitial fibrosis, and compromised myocardial contractile capacity (8–10). The cardiodepressant effect of alcohol often results from chronic and excessive exposure to alcohol, e.g. a daily alcohol dose that exceeds 40 g (11). It is characterized by the loss of contractile capacity, cardiomegaly, derangement of myofibrillary architecture, and increased incidence of heart failure, stroke and hypertension (8–10, 12). Cardiac arrhythmia is another consequence of excessive alcohol consumption, which may not be related to other common alcohol-related co-morbidities such as liver disease, hypertension, diabetes, renal failure, peripheral vascular disease (12–14).

In addition to cardiomyopathy, a tight association exists between alcohol consumption and elevated arterial pressure. Most of habitual alcohol users exhibit higher systolic blood pressures and hypertension compared with nondrinkers or light to moderate drinkers (7, 15–17). In such cases, alcohol withdrawal or at least reduced intake is one of the clinical recommendations for hypertension management (7, 18). The relationship between alcohol intake and blood pressure appears to be independent of any other confounding pathological variables such as diabetes mellitus, coronary heart disease, age, cigarette smoking and dyslipidemia. Reported experimental findings confirmed epidemiological evidence that alcohol use positively correlates with the incidence of hypertension. Several underlying mechanisms have been implicated in the hypertensive action of alcohol such as impairment of arterial baroreceptor activity, vascular endothelial dysfunction, oxidative stress, cardiovascular inflammation, sympathetic and renin-angiotensin-aldosterone system hyperactivities, and increases in cortisol levels and vascular reactivity (7, 19–22).

3. Peripheral and central metabolism of alcohol

Accumulating evidence suggests that at least 95% of alcohol is eliminated via metabolism, with the remaining fraction being excreted unchanged through exhalation, sweating, or urinary excretion (23). As depicted in figure 2, alcohol is mainly metabolized by hepatic oxidative degradation into acetaldehyde and acetate by cytosolic alcohol dehydrogenase (ADH) enzyme and mitochondrial aldehyde dehydrogenase (ALDH) enzymes, respectively. Acetaldehyde, the first oxidative product of ethanol, is a highly toxic molecule and is probably 10 times more toxic than alcohol itself (24). In the liver, the conversion of ethanol into acetaldehyde is catalyzed by ADH, cytochrome P450 2E1 (CYP2E1), and, to a lesser extent, by catalase (25). The two latter enzymes (CYP2E1 and catalase) constitute what is known as the non-ADH pathway, which contributes minimally to the hepatic alcohol metabolism. Nevertheless, the metabolic role of this pathway is magnified under circumstances of excessively high blood alcohol levels or chronic alcohol exposure (26) and under settings of increased catalase activity, which might explain the higher estrogen-related acetaldehyde level in women (27).

Figure 2.

Scheme of alcohol oxidative metabolism in liver and brain.

Ethanol, but not acetaldehyde, can easily reach the brain after crossing the blood-brain barrier. The cellular components of the blood-brain barrier such as endothelial cells and oligodendrocites contain ALDH (28, 29), which breaks down circulating acetaldehyde of peripheral origin to acetate and limits its passage into brain tissues (30). Nonetheless, acetaldehyde can be formed locally in the brain from alcohol that has crossed the blood brain barrier. That said, the enzymatic pattern of central ethanol metabolism is different from the peripheral one. As mentioned above, the hepatic oxidative metabolism of ethanol into acetaldehyde is catalyzed mainly by ADH (23, 25, 31). This enzymatic profile of ethanol metabolism is not visualized in the brain tissues where catalase, and to a lesser extent CYP2E1, serve as the principal metabolizing enzymes (32, 33) (Figure 2). Protein expression studies showed that catalase is expressed in all neural cells of the brain (30). Moreover, immunohistochemical catalase-positive staining is particularly prominent in brain areas containing aminergic neuronal bodies (34).

Conflicting data are reported on the role of acetaldehyde in the neurobiological and epigenetic changes caused by ethanol. Numerous studies have implicated acetaldehyde in behavioral, reinforcing, and neurotoxic effects of ethanol. For example, microinjection of the catalase knockdown (shRNA) in ventral tegmental area of rats virtually abolishes the voluntary consumption of alcohol, suggesting that central metabolism of ethanol into acetaldehyde is necessary for generating reward and reinforcement (35). Ethanol and acetaldehyde cause similar disruption of cellular differentiation and growth, with subsequent abnormalities in fetal development (36). Locomotor stimulation induced by alcohol administration into the hypothalamic arcuate nucleus is abolished after pharmacologic catalase inhibition (37). By contrast, evidence obtained from other studies failed to establish such causal relationship between ethanol effects and its oxidative product acetaldehyde or at least reached a conclusion that the two materials produce similar actions, but the underlying cellular mechanisms are probably different (38, 39). Although acetaldehyde is considered the prime perpetrator for ethanol-induced organ damage, other products of ethanol metabolism, e.g. fatty acid ethyl esters, may also contribute to the onset and progression of alcoholic organ injury. The transport of these fatty acids from intracellular sites to mitochondrial membranes causes mitochondrial injury and loss of its energy generation capacity (40–42).

Several review articles on the role of acetaldehyde and other oxidative products in the neurobehavioral actions of ethanol have been published over the last few years (38) (43). Similarly, reviews on the involvement of oxidative products and enzymes of ethanol in major alcohol-related disorders such as cancer (44–47), liver disease (48, 49), lung disease (50), and alcoholism and addiction (51, 52) are also available. Yet, little or no reviews have been recently published that summarize reported findings on the role of acetaldehyde and synthesizing and degrading enzymes in cardiovascular complications induced by alcoholism and potential cellular and molecular mechanisms of these interactions.

4. Role of central metabolizing enzymes in alcohol-induced hypertension

4.1. Alcohol-induced hypertension

Epidemiological evidence supports a strong association between alcohol use and hypertension (7, 22, 53, 54). Experimentally, ethanol elicits hypertension after its acute, parenteral or oral administration (19, 55). Gender, route of administration, rat strain, and arousal state are considerable factors that modify the blood pressure response elicited by alcohol (55–60). However, the effect of ethanol on blood pressure is not dependent on obesity, cigarette smoking, or physical activity (53, 61) and is observed in both normotensive as well as in hypertensive patients (54, 62). The ethanol-induced pressor effect is reversible because it disappears when ethanol intake is stopped (54, 62).

Specifically, the role of sympathoexcitation in the ethanol-evoked hypertension is evidenced by the rises in plasma norepinephrine (63) and efferent sympathetic neural activity (64). In a previous report from our laboratory (65), we employed microinjection and electrochemical protocols to determine neuronal substrates in the brainstem that underlie the hypertensive action of ethanol. The rostral ventrolateral medulla (RVLM) is the brainstem pressor region from which bulbospinal sympathetic neurons descend to the intermediolateral cell column of the spinal cord (66, 67) and is a major site for the sympathoexcitatory action of ethanol (68). The C1 neurons of the RVLM contain essential NE synthesizing enzymes such as tyrosine-hydroxylase, dopamine hydroxylase, and phenylethanolamine-N-methyltransferase (69). Indeed, the quantity of the norepinephrine (NE)-containing neurons in the RVLM positively correlates with sympathetic neural activity (70). Measurements of NE and its metabolites in the RVLM neurons by microdialysis or electrochemistry denote NE neuronal activity (68, 70–72).

The unilateral administration of ethanol (1–10 μg) into the RVLM causes dose-dependent increases in norepinephrine electrochemical signal and blood pressure in SHRs in contrast to much smaller pressor effects in Wistar Kyoto rats (WKYs) (65). Similarly greater increases in blood pressure and RVLM norepinephrine are observed in SHRs after systemic ethanol administration (68). Considering that the RVLM contains noradrenergic nerve terminals that originate from other brain areas (66, 69, 73) and that the activity of these neurons is proportionally related to the sympathetic neural activity (66, 68), these findings suggested a primary role for norepinephrine released from RVLM noradrenergic neurons in the sympathoexcitatory and pressor actions of ethanol in SHRs (65).

The impairment of arterial baroreceptor activity is a plausible mechanism for the sympathoexcitatory and the subsequent hypertensive actions of ethanol (19, 20, 63, 74, 75). The arterial baroreflex function is one of the most rapidly acting homeostatic mechanisms for the regulation of blood pressure. Baroreceptor function is impaired in human hypertensives (76) and precedes the development of hypertension in some experimental models including the ethanol-induced hypertension (64, 77–79). The cardiovascular nuclei of the brainstem such as the nucleus tractus solitarius are important neuroanatomical targets for the baroreflex depressant effect of ethanol (21, 80). The ethanol enhancement of γ-aminobutyric acid–mediated (GABAergic) (81) and attenuation of glutamatergic (82) neurotransmission in the brainstem have been implicated in baroreflex dysfunction induced by ethanol.

4.2. Acetaldehyde mediates the pressor effect of intra-RVLM alcohol

As indicated above, the local synthesis of acetaldehyde from alcohol in the brain is catalyzed mainly by catalase and CYP2E1. Catalase is present in all neural cells and its expression is particularly evident in aminergic neuronal bodies (30, 34). Further, central CYP2E1 expression is region-specific and is found in peroxisomes and mitochondria (83). Although the role of acetaldehyde in the biological effects of ethanol has been extensively investigated, the specific involvement of the oxidative products of alcohol in ethanol-induced hypertension has only been recognized in recent reports. Further, considering a key role of brain catalase in the central ethanol disposition (84) (33) is challenging because reported findings on catalase activity in experimental models of genetic hypertension are limited and inconsistent. For example, a higher striatal (85), but not renal (86), catalase activity was reported in SHRs compared with WKYs.

Although the oxidative product acetaldehyde has been implicated in the behavioral effects of ethanol (38, 43, 87, 88), scarce data exist on acetaldehyde contribution to the ethanol-evoked pressor response. Integrative and electrochemical studies revealed heightened pressor and sympathoexcitatory effects of intra-RVLM ethanol in conscious SHRs compared with WKYs (65, 68). In a series of recent and novel reports from our laboratory, we established compelling evidence that implicated acetaldehyde in this hypertension-specific pressor effect of ethanol and identified the molecular underpinnings of this effect (89–91). We first demonstrated that local catalase-mediated oxidation, into acetaldehyde, accounted for the hypertensive response elicited by intra-RVLM ethanol in SHRs. It is not surprising, therefore, that an imbalance between ethanol-derived acetaldehyde and its further oxidation to acetate (via ALDH) results in acetaldehyde accumulation and a subsequent RVLM neuronal oxidative stress serve as underlying mechanisms for the heightened increases in central sympathetic tone and blood pressure in SHRs (92, 93).

This premise is further supported by: (i) compared to control WKYs, the SHR RVLM exhibits higher catalase activity, and (ii) the pressor effect of ethanol was dramatically reduced in SHRs pretreated systemically with 3-amino-1,2,4-triazole (3-AT, catalase inhibitor) (89). Further, the finding that similar increases in blood pressure caused by acetaldehyde (2 μg) or ethanol (10 μg) microinjection into the RVLM supports the premise that acetaldehyde is the principal mediator of ethanol-evoked hypertension in SHRs (89). In this circumstance, the blood pressure rises caused by acetaldehyde were similar, in duration and magnitude, to that caused by intra-RVLM ethanol. Considering the importance of discrete areas of the brainstem such as the RVLM in blood pressure control (66, 94), these findings suggested a centrally-mediated effect of acetaldehyde on blood pressure. Because systemic 3-AT caused significant drop in blood pressure, it is possible that 3-AT counterbalanced the pressor effect of intra-RVLM ethanol perhaps via physiological antagonism. This possibility seems unlikely because under the same conditions, systemic 3-AT failed to alter the pressor response elicited by intra-RVLM acetaldehyde (89), and produced variable effects on blood pressure that include decreases (95), increases (96), or no changes (97).

Another evidence for the acetaldehyde hypothesis are the findings in normotensive (WKY) rats pretreated with an ALDH inhibitor. Whereas intra-RVLM acetaldehyde failed to alter blood pressure in WKY rats, substantial increases in blood pressure were seen when acetaldehyde was microinjected in the same rat strain pretreated with the ALDH inhibitor cyanamide (89). It is plausible, therefore, that ALDH rapidly oxidizes acetaldehyde into acetate, thus preventing the accumulation of this neurotoxic aldehyde in the RVLM of the WKY. Conceivably, ALDH inhibition appears to have created RVLM environment conducive to acetaldehyde accumulation and the unraveling of its pressor effect in WKY rats. While these findings argue against a significant role for acetate, the next step in ethanol metabolism (98), in the pressor effect of ethanol or acetaldehyde, it is imperative to note that acetate contributes to other neurobiological/behavioral effects of ethanol (99, 100).

Recent neurochemical data reveal substantially higher catalase, but similar ALDH, activity in the RVLM of SHRs and WKY rats (89). Such enzymatic profile is anticipated to provoke greater formation and accumulation of the ethanol-derived acetaldehyde in the RVLM of in the SHRs. Studies on catalase activity in other brain areas of hypertensive rats demonstrate inconsistent effects. For example, the SHRs exhibit higher and lower catalase activity in the striatum and the whole brain homogenate, respectively, compared with normotensive controls (85, 86). As discussed earlier, intra-RVLM acetaldehyde elevates blood pressure in SHRs, but not in WKY rats, despite the similar RVLM ALDH activity (89). It is likely, therefore, that this ethanol metabolizing enzymes profile or other intrinsic non-enzymatic mechanisms (38) contribute to such strain-dependent blood pressure effect of ethanol or its first metabolite, acetaldehyde.

It is noteworthy that the pressor response elicited by intra-RVLM ethanol or acetaldehyde is associated with significant decreases in the heart rate. While the pressor effect of ethanol relates to the enhancement of central sympathetic tone (65, 68), the mechanism of the associated bradycardia has not been elucidated. Speculatively, the bradycardic action of ethanol might erupt as a reflex response due to the activation of arterial baroreceptors in the aortic arch and carotid sinus (101–103). However, this possibility is contradicted by the findings that intra-RVLM acetaldehyde elicited pressor responses in cyanamide-pretreated WKY rats or in 3-AT pretreated SHRs without affecting HR. Therefore, more studies are needed to characterize the underlying mechanism of the bradycardic effect of intra-RVLM ethanol (89).

It is important to comment on the clinical relevance of the microinjected alcohol dose. Given the similarity of the cardiovascular effects produced by systemic (1 g/kg) (20, 21, 74, 81) and intra-RVLM (10 μg) (65, 68) alcohol in SHRs, it is plausible that these two alcohol regimens would produce comparable levels of the drug in the RVLM. This premise is supported by the study by Robinson et al. (104), which showed similar blood and brain levels of ~25 mM following alcohol (1 g/kg; i.v). It is conceivable, therefore, that the 10 μg intra-RVLM dose of alcohol used in our studies might lead to tissue alcohol concentration of approximately 25 mM, which is consistent with blood levels achieved following social alcohol consumption (63, 105). Moreover, the use of the 2 μg dose of acetaldehyde for intra-RVLM studies was based on reported relative potencies of acetaldehyde and ethanol in cardiovascular (89) and behavioral studies (38). A higher intra-RVLM dose of acetaldehyde (4 μg) increased blood pressure to levels that were not different from those produced by the 2 μg dose (91).

Apart from neurohumoral pathways, a potential role for osmolality changes in the pressor effect of ethanol is likely because clinical and experimental studies showed that hyperosmolality increases sympathetic neural activity and blood pressure through the stimulation of central osmoreceptors (106). These osmotically mediated effects have been attributed to the activation of vasopressinergic and glutamatergic projections to the RVLM sympathetic neurons (107–109). Given that ethanol is osmotically active and that osmolarity modulation accounts for the ethanol-evoked changes in secretory (110) and disease states (111), future studies are warranted to investigate whether osmotic changes in RVLM neurons contribute to the sympathetic and blood pressure responses elicited by ethanol.

Notably, the reported pressor and sympathoexcitatory effects of acetaldehyde appear to be at odds with its direct effects on vascular reactivity. In vitro studies have shown that acute or chronic acetaldehyde exposure relaxes vascular smooth muscle and reduce responsiveness to vasoconstrictor stimuli (112–114). Ren et al. (115) reported that aortic relaxations induced by acetaldehyde are dependent upon the endothelium and blood pressure states. Compared with normotensive counterparts, acetaldehyde-induced vasorelaxations are diminished and augmented in SHR aortas with intact and denuded endothelium, respectively (115). Electrophysiological evidence suggests a vital role for the inhibition of voltage-dependent Ca²⁺ currents in the attenuating action of acetaldehyde on contractions induced by potassium depolarization in vascular smooth muscle cells (114). High blood acetaldehyde concentrations seen in some Asians with a genetically low ALDH activity (116, 117) have been implicated in hypotension, flushing, and palpitation associated with alcohol use. It is likely, therefore, that the blood pressure response elicited by ethanol signifies the net of its vascular and neurohumoral effects at central and peripheral sites.

4.3. ALDH2 polymorphism modulates alcohol-evoked hypertension

As discussed above, acetaldehyde is detoxified into acetate by ALDH2, which subsequently enters into the Krebs cycle to generate CO₂ and water as the end-products of ethanol oxidation (118, 119). Clinical data demonstrate elevated circulating acetaldehyde levels following acute or chronic alcohol use (120, 121). Impairment or genetic mutations (polymorphism) of ALDH result in elevated circulating levels of acetaldehyde in alcoholics (120). Compared to blood acetaldehyde concentrations of ~5 μM in Asians with intact ALDH2 activity, individuals with one mutant ALDH2 allele exhibit acetaldehyde levels of 30–125 μM, and develop severe organ injury following alcohol intake (122–124).

The generation of free radicals and oxidative stress are the main mechanisms responsible for cytotoxicity induced by alcohol and acetaldehyde. Further, the oxidation of acetaldehyde into acetate may produce oxidative free radicals such as superoxide, acetyl, hydroxyl, and methyl radicals (125) through a number of subcellular enzymes and structures including aldehyde oxidase, xanthine oxidase, mitochondria and microsomes (126). ALDH2 mutation is linked to increased prevalence of hypertensive states including that induced by alcohol (127–129). It should be remembered, however, that the influence of ALDH2 genotype on blood pressure is rather complex and depends on the amount of alcohol consumed, the timing of blood pressure measurement, and environmental and genetic factors. Collectively, while Iwai and colleagues identified a tie between the ALDH2*1/*1 genotype and the prevalence of hypertension (130), the precise role of ALDH2 polymorphism in the regulation of blood pressure, particularly in alcoholics remains largely unclear.

4.4. Central MAPK signaling contributes to ethanol/acetaldehyde-evoked hypertension

Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine kinases that transfer extracellular stimuli into a wide range of cellular responses (131, 132). Conventional MAPKs comprise ERK1/2, c-Jun amino (N)-terminal kinases (JNK1/2/3), and p38 (131). The pathogenic intermediary roles of MAPKs in serious adverse effects of alcohol such as hepatotoxicity, pancreatitis, neurotoxicity, and increased cancer risk have been recognized (133–135). MAPKs phosphorylation might be facilitated or inhibited by ethanol depending on factors such as the duration and dose of alcohol exposure, cell type, and particular MAPK isoform under consideration (133). In-vitro studies showed that vascular contractions and elevations in intracellular calcium caused by alcohol in cerebral smooth muscle cells are mediated via activation of ERK1/2 and p38 (136). The alcohol-induced contractions of isolated aortas are suppressed after MEK1/2 inhibition, thereby implicating ERK1/2 in the alcohol effect (137). In whole animal studies, pharmacologic inhibition of RVLM ERK1/2 attenuates the pressor response caused by the microinjection of angiotensin II into the same neuroanatomical area (138).

Evidence implicates MAPKs within the RVLM neurons in the pressor effect of alcohol or its metabolite acetaldehyde in SHRs (91) because: (i) the RVLM area, which controls blood pressure and central sympathetic activity and is neuroanatomical site for mediating the pressor effect of alcohol or its metabolite, acetaldehyde (65, 68, 89); (ii) RVLM MAPKs signaling is crucial for blood pressure control in normotensive and hypertensive states (138); (iii) activation of RVLM ERK1/2 and p38 account for the higher sympathetic activity in stroke-prone SHRs (139) and in models of heart failure (140). A causal role for enhanced ERK2 signaling in the pressor effect of intra-RVLM alcohol was supported by the observations that alcohol microinjection caused significant increases in local ERK2 phosphorylation and that prior RVLM ERK1/2 inhibition by PD98059 virtually abolished the pressor action of alcohol (91).

Unlike MAPK-ERK1/2, conflicting data are obtained regarding the roles of p38 and JNK signaling in alcohol-induced pressor effect. Whereas RVLM p38 phosphorylation is upregulated by alcohol, the associated pressor response is preserved after pharmacological inhibition of p38 (SB203580) (91), denoting no role for the increase in RVLM p38 activity in the pressor effect of alcohol. On the other hand, despite the lack of change in neuronal p-JNK expression in alcohol-treated SHRs, JNK inhibition (SP600125) compromises the pressor effect of alcohol. While these findings preclude a direct interaction of alcohol with JNK, the possibility that alcohol interacts with downstream effectors of JNK signaling cannot be overlooked. For instance, the immediate early gene c-jun and/or the transcription factor activator protein-1 (141, 142) might contribute to the alcohol-evoked pressor response. Interestingly, a causal link exists between increases in c-Jun or its message, c-jun, in the RVLM and sympathetic activity (143, 144).

Like alcohol, acetaldehyde has no effect on phosphatase activity, but increases RVLM p-ERK2 level and caused p-ERK2, but not p38, dependent elevation in blood pressure. These data further support a critical role for acetaldehyde in the molecular and cardiovascular effects of alcohol. Notably, the effects of alcohol and acetaldehyde are not usually identical. One important difference is the dependence of pressor effect of alcohol, but not acetaldehyde, on enhanced RVLM JNK2/3 phosphorylation (91). Thus, it is possible that while enhancement of JNK2/3 and ERK2 phosphorylation underlie alcohol-evoked pressor response, ERK2 phosphorylation plays the major role in the pressor effect of acetaldehyde. Our molecular findings are in consistent with the observations that (i) enhanced RVLM ERK1/2 signaling mediates increases in sympathetic activity and blood pressure caused by the activation of RVLM angiotensin AT1 receptors (139), and (ii) ERK1/2 inhibition in the REVLM causes hypotension and reduces the pressor response caused by intra-RVLM angiotensin II (138).

Based on the individual molecular profiles of alcohol and acetaldehyde, it is possible that JNK2/3 and ERK2 are phosphorylated consecutively in the signaling pathway leading to the acetaldehyde-dependent pressor effect of alcohol. Evidence suggests a facilitatory role for alcohol dehydrogenase in the alcohol-evoked JNK phosphorylation (145) in addition to its established role in alcohol metabolism (38). Notably, the oxidative metabolism of alcohol into acetaldehyde by alcohol dehydrogenase/catalase mediates the pressor action of intra-RVLM alcohol in SHRs (89). Unlike alcohol, the pressor effect of acetaldehyde involved the direct activation of ERK, thereby bypassing JNK and the alcohol metabolic pathway. These cellular mechanisms along with the proposed cascade of neuronal substrates involved in the MAPKs-related pressor effect of alcohol /acetaldehyde are illustrated in figure 3.

Figure 3.

Schematic paradigm of neuronal events in the RVLM that lead to the increase in central sympathetic outflow and elevation in blood pressure caused by intra-RVLM administration of ethanol or acetaldehyde in spontaneously hypertensive rats (SHRs). ADH, alcohol dehydrogenase; IEGs, immediate early genes; AP-1, activator protein-1; MEK1/2, mitogen-activated protein kinase kinase; p-ERK2, phosphorylated extracellular signal-regulated kinases.

Protein phosphatases are enzymes that dephosphorylate amino acid residues of protein substrates including MAPKs and are involved in multiple regulatory processes such as DNA replication, metabolism, transcription, and development (131, 146). Studies have shown that the inhibition of phosphatase activity in cell culture contributes to alcohol-evoked increase in p-JNK level (147) and that enhanced MAPKs signaling in the RVLM increases sympathetic activity (139, 140). With this in mind, it would be expected that the inhibition of RVLM phosphatases, might have contributed to the ethanol-evoked hypertension and enhancement of ERK1/2 phosphorylation was evaluated. Such argument seems unlikely because the measurement of neuronal phosphatase activity in RVLM tissues of rats treated with alcohol or its metabolite acetaldehyde revealed no differences from control tissues. Even though, similar to alcohol, the inhibition of neuronal ser/thr phosphatase activity by intra-RVLM okadaic acid increased blood pressure and p-ERK2 expression (91). It appears therefore that RVLM phosphatases serve tonically to limit the buildup of neuronal phosphorylated MAPKs, which is believed to enhance sympathetic activity (139, 140). Collectively, the increases in blood pressure and RVLM p-ERK2 caused by okadaic acid lend credence to the concept that higher levels of phosphorylated kinases in the RVLM mediate, at least partly, the pressor effect of intra-RVLM alcohol in SHRs.

One potential limitation of microinjection studies of alcohol relates to the possibility that the microinjected alcohol dose into brainstem neurons might cause neuronal damage and nonspecific effects. The microinjected dose of alcohol (10 μg) has been used in brainstem microinjection studies in our laboratory (21, 68) and by others (74, 81) to discern the role of these brain areas in the cardiovascular effects of alcohol. Alcohol, microinjected into brainstem nuclei, impairs arterial baroreflexes via interaction with specific subsets of central GABA and glutamate receptors, and these effects disappeared within 1 to 2 hours (21, 81). The findings that RVLM ERK and JNK, but not p38, contributed to pressor effect of alcohol (91) reflect its selectivity, and along with the reversibility in the observed effects, made it unlikely that the alcohol-evoked cardiovascular and molecular responses could be attributed to nonspecific neuronal damage.

4.5. Brainstem phosphatases dampen alcohol-induced hypertension

As discussed above, the microinjection of ethanol or acetaldehyde into the RVLM only modestly increased local ERK phosphorylation and blood pressure in conscious normotensive, WKY, rats (65, 89). To understand the underlying mechanisms that dampen these effects, studies were undertaken in conscious normotensive rats to test the hypothesis that RVLM phosphatases act tonically to dampen ethanol or acetaldehyde evoked ERK phosphorylation and subsequent increases in blood pressure. As is the case in SHRs (91), the ERK1/2 inhibitor PD98059 abolished the modest pressor effect caused by intra-RVLM ethanol in WKY rats, suggesting a key role for ERK1/2 phosphorylation in alcohol-evoked increases in blood pressure response and sympathetic activity (65). More importantly, the simultaneous intra-RVLM administration of ethanol and okadaic acid (nonselective inhibitor of all ser/thr phosphatase isoforms, PP1 through PP6) or fostriecin (selective inhibitor of PP1 and PP2A) (148) caused exaggerated inhibition of phosphatase activity along with greater and more sustained elevations in blood pressure.

Similar exacerbation of the pressor response and local phosphatases inhibition are seen when acetaldehyde is combined with okadaic acid or fostriecin (90). The data highlight a preferential role for phosphatases of the PP1 and PP2A types in dephosphorylating ERK1/2 and the dampening of acetaldehyde-dependent pressor effect of intra-RVLM alcohol in normotensive rats. In addition to its selective phosphatase inhibitory effect, fostriecin also offers a better experimental tool for phosphatase inhibition because of the reported cytotoxicity associated with the use of okadaic acid (149, 150). Collectively, our observations reinforce a restraining influence of local RVLM phosphatases on alcohol-evoked pressor response (91).

5. Alcohol metabolism contributes to its sex-dependent cardiovascular effects

5.1. Estrogen provokes cardiodepressant and hypotensive effects of alcohol

Clinical and experimental findings demonstrate that the net effect of alcohol on blood pressure follows a J-shaped relationship due probably to the complex effects of alcohol on cardiovascular functions under different settings. Acute alcohol increases (19), decreases (151) or has no effect (81, 152) on blood pressure while chronic alcohol increases (64) or decreases (153–155) blood pressure. The mechanisms by which alcohol elevates blood pressure include increase in sympathetic activity as indicated by the rise in plasma norepinephrine levels (63, 156, 157). The observation that sympathetic neural activity is elevated in alcohol-fed, compared with pair-fed control, rats provides more direct evidence for the involvement of the sympathetic nervous system in alcohol-induced hypertension (64). The attenuation by alcohol of the gain in arterial baroreceptor activity may also contribute to the increases in sympathetic activity and the subsequent pressor effect (19, 75).

On the other hand, ethanol elicits other cardiovascular actions that may counterbalance its sympathoexcitatory effects and result in a net drop in blood pressure such as direct myocardial depression (40, 158), reduction in cardiac output (159), vasodilation (160), and α-adrenoceptor blockade (105). Alcohol use results in an increased incidence of cardiac morbidity and mortality. As recently reviewed (8), the alcohol-evoked cardiomyopathy manifests as ventricular dilation, reduced ventricular wall thickness, myofibrillary disarray, interstitial fibrosis, hypertrophy and contractile dysfunction. The underlying mechanisms include ethanol/acetaldehyde toxicity (161), mitochondrial production of reactive oxygen species (162), oxidative injury, apoptosis (163), impaired myofilament Ca²⁺ sensitivity (164), and abnormalities in fatty acid deposition (165).

Experimental reports suggest fundamental roles for ovarian hormones in the sex-specific cardiovascular derangements caused by alcohol. Alcohol reduces cardiac output, stroke volume, and blood pressure in female rats during proestrus, which exhibits the highest level of circulating estrogen (55, 159). Indices of myocardial contractility such as left ventricular pressure over time (dP/dt_max) and left ventricular developed pressure are also significantly reduced by alcohol. These hemodynamic effects of alcohol are minimal or absent in male rats and in ovarian hormones deprived (ovariectomized) rats, but are restored following estrogen replacement in both preparations (166–168). Because reductions in myocardial contractility indices and blood pressure are tightly related, it is concluded that myocardial depression is largely responsible for the developed hypotension in these reported studies (166–168). These findings have clinical implications because alcohol lowers blood pressure in young, but not in older women (169). The measurements of left ventricular developed pressure (LVDP), dP/dt_max (170–173) and sympathovagal control of the heart (174, 175) provide more direct assessment of cardiac contractility and autonomic control. Our reported findings implicated cardiac vagal dominance in the estrogen-dependent chronic hypotensive effect of alcohol in female rats (176, 177). At the molecular level, facilitation of the myocardial PI3K/Akt/nNOS signaling cascade contributes, at least partly, to these effects (178).

Studies in proestrus rats provided evidence for a causal role for autonomic dysregulation in the sex-specific myocardial oxidative stress and dysfunction. Specifically, ethanol-evoked reductions in cardiac output (159, 176), blood pressure and indices of myocardial contractility, LVDP and dP/dt_max were associated with prolongation of the left ventricular isovolumic relaxation constant Tau (166), which is a measure of cardiac diastolic function (179, 180). Further, the power spectral analysis of heart rate variability revealed a shift towards vagal dominance in the presence of ethanol in estrogen replete rats. Most compelling are the findings that pharmacological interruption of cardiac vagal innervation attenuated the estrogen-dependent myocardial oxidative stress/dysfunction. These findings suggest a pivotal role for enhanced vagal dominance in the cardiodepressant effect of alcohol in female rats (177, 178).

Pharmacological (iNOS inhibition) and molecular studies implicate vascular iNOS upregulation in the sex/estrogen-dependent hypotensive effect of alcohol (181). The latter is associated with an adaptive increase in the gene expression of the immediate early gene c-jun in the RVLM (154), which reflects an increase in sympathetic neural activity to counterbalance the primary decreases in blood pressure. Notably, the estrogen dependence of the hypotensive effect of alcohol may be explained in view of the similarity of the vascular effects of the alcohol and estrogen because both inhibit calcium influx (160, 182), promote endothelial nitric oxide activity (183, 184), and reduce α-adrenoceptor responsiveness (185, 186). It is possible, therefore, that ethanol may interact synergistically with estrogen to elicit vascular changes that trigger vasodilatation and subsequent falls in blood pressure.

The role of estrogen receptors (ER) in the estrogen-dependent hypotensive and myocardial depressant actions of alcohol has been investigated. The three ERs, ERα, ERβ, and G protein-coupled ER (GPER), are distributed throughout the cardiovascular system. They act as important regulators of myocardial function by genomic and non-genomic signaling mechanisms (188, 189). The observations that non-genomic effects are involved in the cardiovascular effects of alcohol in female (167) and male (190) rats might link one or more of ER subtypes to the acute estrogen-dependent myocardial depressant effect of alcohol. It was also imperative to identify the ER subtype implicated in estrogen enhancement of the activity of two myocardial redox enzymes, catalase and ALDH2, which confer cardioprotection (191, 192) and also catalyze alcohol oxidation to acetaldehyde and acetate, respectively (25, 26). Pharmacological loss-of-function studies using highly selective antagonists showed that compared with ERβ (PHTPP) or GPER (G15) blockade, ERα blockade (MPP) greatly attenuated the reductions in myocardial contractility indices (dP/dt_max and left ventricular developed pressure) and blood pressure produced by ethanol (168). However, because PHTPP did reduce the cardiac, but not hypotensive, effect of alcohol during the last 30 min of the study, it is plausible that functional ERβ and GPER might be required for the demonstration of the delayed effects of alcohol on the heart. These findings are consistent with the interplay of ER subtypes in regulating cardiac functions (2, 3, 6). The preserved hypotensive effect of ethanol in ERβ-blocked rats might be related to ERα/GPER-mediated enhancement of nitric oxide-dependent vasodilation (193, 194). Discrepancies in the modulation of eNOS and nNOS by ER subtypes in the myocardium and vasculature (195–197) might account for the preservation of ethanol-evoked hypotension in ERβ-blocked rats. Mechanistically, our findings implicated the Akt/ERK1/2/p38 pathway to the estrogen-dependent myocardial depressant effect of ethanol (168, 178), which is also causally related to oxidative stress (198–200). ERα and GPER, but not ERβ, contribute to these molecular events. These pharmacological and biochemical findings support a pro-oxidant role for estrogen, in the presence of ethanol, mediated via MAPK phosphorylation (166). These findings were extended by pharmacological gain-of-function studies using highly selective ER subtype agonists in the absence or presence of alcohol in ovariectomized rats (201). The latter study showed that compared with ERα or ERβ, GPER activation exhibited a lesser ability in uncovering the adverse hemodynamic effects of alcohol. Accordingly, replacement with selective GPER agonists might present a safer therapeutic alternative for estrogen in women with habitual alcohol consumption.

5.2. Enhanced alcohol metabolism underlies its heightened cardiodepressant effect in females

In peripheral tissues, alcohol is metabolized via alcohol dehydrogenase (ADH) and catalase to acetaldehyde, which is subsequently eliminated by aldehyde dehydrogenase (ALDH2) (23, 24, 38). Acetaldehyde is largely responsible for myocardial dysfunction induced by alcohol and underlying cellular mechanisms (187, 202). Because estrogen enhances alcohol metabolism to acetaldehyde (203), which is known to upregulate several molecular entities along the oxidative stress signaling (8), recent studies interrogated the role of acetaldehyde accumulation in the female myocardium in the exacerbated estrogen-dependent myocardial depressant effect of alcohol (166). The elegant studies by Ren et. al. (187) showed higher sensitivity of the isolated female cardiomyocytes to the myocyte contractile depressant effect of ethanol-derived acetaldehyde. These in vitro (187) and the aforementioned in vivo (55, 159) findings might explain the higher sensitivity of women to alcohol-induced cardiomyopathy. While a major role for locally generated acetaldehyde in the adverse effects of alcohol in peripheral tissues is debated, there are many findings that support this premise in the heart, particularly in the presence of estrogen. Unfortunately, direct measurements of acetaldehyde levels in the heart and linking such levels to cardiac function are rather challenging. Nonetheless, many alternative approaches support this argument starting with the findings that acetaldehyde being far more toxic and reactive than ethanol and is largely blamed for alcohol-induced cardiac damage (191). The heart obtained from alcohol-treated rats exhibited higher ADH, but not ALDH2, activity reflecting higher myocardial acetaldehyde levels after alcohol administration (166). These biochemical findings coincided with the (i) reductions in left ventricular pressure, dP/dt_max, and blood pressure, (ii) declining concentrations of blood alcohol (178), and (iii) increased cardiac accumulation of cardiotoxic aldehydes such as malondialdehyde and 4-hydroxy-2-nonenal adducts (166, 178). The latter products are cardiotoxic and potent inducers of cardiac pathology and oxidative damage (24, 204). Under this circumstance the preservation of ALDH2 activity would be expected to facilitate the detoxification of acetaldehyde as well as other cardiotoxic aldehydes. Li et al. (191) suggested a therapeutic potential of ALDH2 in alcoholic complications because ALDH2 overexpression effectively alleviates acetaldehyde-induced injury in cardiomyocytes through an ERK1/2 and SPAK/JNK-dependent mechanism. The study by Budas et al. (192) has reached a similar conclusion and proposed that ALDH2 activation might be exploited to preserve the cardioprotective effect of ethanol while minimizing the side effects associated with alcohol consumption. Together, the worsened cardiac profile seen in alcohol-treated rats despite the preservation of ALDH2 activity may be attributed to the enhanced ADH activity and subsequent elevations in the cardiac levels of toxic acetaldehydes, which exceed the detoxification capacity of ALDH2.

The presumption that acetaldehyde mediates myocardial depression triggered by alcohol in proestrus rats receives support from earlier reports, which demonstrated that acetaldehyde is responsible for the higher female propensity to alcohol-induced myocardial dysfunction (187, 202, 205). Also, similar amounts of alcohol produced higher acetaldehyde levels in premenopausal women and in women on oral contraceptives, compared to men (27). Additionally, the overproduction of acetaldehyde that follows ADH overexpression accelerated cardiac dysfunction in female, more than in male, cardiomyocytes (187, 206). Notably, contrary to our recent report (166), none of these reported studies linked the molecular events in the myocardium to the alcohol-evoked myocardial dysfunction at the integrative level. Our data have also shown that myocardial acetaldehyde accumulation (albeit indirectly assessed) triggers molecular events conducive to the generation of oxidative state. Alcohol caused remarkable increases in myocardial Akt/ERK1/2 and nicotinamide adenine dinucleotide phosphate oxidase (NADPHox) activation and reactive oxygen species production (figure 4). Tempol, a superoxide dismutase mimetic with antioxidant activity, abrogated myocardial oxidative stress induced by alcohol and preserved myocardial function (166).

Figure 4.

Cardiac and vascular signaling events involved in the estrogen-dependent myocardial depressant and hypotensive effects of alcohol in female rats. ADH, alcohol dehydrogenase; ALDH2, alcohol dehydrogenase 2; eNOS, endothelial nitric oxide synthase; ROS, reactive oxygen species; PI3K, Phosphoinositide 3-kinase; GC, Guanylyl cyclase; GTP, guanosine triphosphate; MAPK-ERK1/2, mitogen-activated protein kinase-extracellular signal-regulated kinases.

These biochemical findings lend credence to earlier findings that alcohol elicits acetaldehyde-dependent activation of NADPHox (187, 207) and ERK1/2 (191) in other tissues, and PI3K/Akt signaling mediates NADPHox activation and reactive oxygen species (ROS) production in macrophages (208). Moreover, these findings support and extend our earlier findings that implicated PI3K/Akt activation in the alcohol-evoked reductions in cardiac output and blood pressure in proestrus female rats (176, 209). As illustrated in figure 4, p-Akt may also enhance ROS generation via endothelial nitric oxide synthase (eNOS) uncoupling (210, 211), which could be exacerbated by the accumulation of the cardiotoxic aldehyde adducts.

5.3. Inhibition of alcohol metabolism attenuates its cardiovascular toxicity

Recent pharmacological evidence further supports acetaldehyde contribution to estrogen-dependent myocardial oxidative stress and dysfunction because the latter were: (i) partially reduced following ADH/CYP2E1 (4-methylpyrazole; 4-MP) or catalase (3-AT) inhibition, and (ii) virtually abolished following combined enzyme inhibition (4-MP plus 3-AT) (84). The oxidative stress caused by alcohol in cardiac tissues (ROS generation) and concomitant increases in ERK1/2 phosphorylation were ameliorated in similar fashions after inhibition of the oxidative metabolism of alcohol to acetaldehyde (84). The partial attenuation of the alcohol effects by 3-AT or 4-MP might be explained by the ability of the functional enzyme(s) to compensate for the inhibited one. Also, the elevations in blood alcohol levels that appeared after enzyme inhibition might have produced direct cardiotoxicity (212). Alternatively, the accumulation of non-oxidative alcohol metabolites such as fatty acid ethyl ester in blood (213) and heart (214) that follows the inhibition of alcohol oxidative metabolism do not seem to contribute to the alcohol effects because the latter were virtually abolished after combined ADH/ALDH inhibition (84).

Further support for ADH- or catalase-mediated acetaldehyde production in the adverse cardiovascular effects of alcohol include the involvement of the enhanced acetaldehyde production, via ADH, in alcohol-evoked cardiomyocyte contractile dysfunction, autophagy, mitochondrial damage and apoptosis (215–217). A role for a catalase-based oxidation in the estrogen-dependent cardiovascular and oxidative damage caused by alcohol in the female population is also likely because (i) estrogen enhances catalase catalytic activity in the female rat heart (166–168, 218), (ii) alcohol increases cardiac catalase activity and causes myocardial oxidative stress and dysfunction in ovariectomized rats treated with the ERα agonist propylpyrazole triol (201), and (iii) catalase inhibition by 3-AT attenuates the alcohol-evoked left ventricular dysfunction (84). More pharmacologic and molecular studies are necessary to identify the precise roles of estrogen receptor subtypes in alcohol metabolism and related cardiovascular anomalies.

6. ALDH2-mediated cardioprotection dampens alcohol-evoked cardiotoxicity

ALDH2 is an important mitochondrial Redox enzyme that combats oxidative stress via facilitating the oxidation of toxic aldehydes into less toxic acids. By reducing the cellular “aldehyde load”, ALDH2 may contribute to the cardioprotective effect of alcohol and to the protection against cardiovascular disease. ALDH2*2 is an inactive variant of ALDH2 with no capacity for metabolizing acetaldehyde (219, 220). Approximately of 40% of East Asians carry at least one copy of the gene encoding the defective ALDH2*2 variant. In people who carry two copies of the defective gene (i.e. homozygous for ALDH2*2/*2), the activity of the enzyme is reduced by > 95% compared with people who are homozygous for the normal, active form of the enzyme (i.e., ALDH2*1). On the other hand, people who carry only one copy of the mutant gene and one copy of the normal gene (i.e., who are heterozygous ALDH2*1/*2) the activity of the resulting enzyme is about 40% of the normal ALDH2 (221). ALDH2*2 carriers exhibited higher levels of troponin I and malondialdehyde and hydroxynonenal adducts after coronary artery bypass grafting (222). Further, the intensive care unit time and postoperative hospitalization were longer in ALDH2*2 carriers (222). In the elegant study by Chen et al. (220). the administration of Alda-1, an ALDH2 activator, to rats prior to an ischemic event increased the catalytic activity of ALDH2 by 2-fold and reduced infarct size by 60% through probably its inhibitory effect on the formation of cytotoxic aldehydes. Similarly, Alda-1 attenuates the cardiotoxic effect of ethanol in rats (223).

Credible evidence supports a key role for ADH/ALDH2 imbalances in the modulation of alcohol-induced cardiomyopathy. Our findings that left ventricular dysfunction induced by alcohol is coupled with elevated ADH, but not ALDH2, activity signify the involvement of myocardial acetaldehyde accumulation in the developed cardiomyopathy (166). Similar observations are reported by others (215, 216). Genetic manipulations showed that opposite effects ALDH2 knockout (accentuation) (224) and overexpression (inhibition) (191) of ethanol-induced cardiac depression. Based on these reports, a therapeutic potential for ALDH2 in alcoholic complications has been proposed. For example, the transfection of fetal human cardiac myocytes with ALDH2 attenuates ROS generation, apoptosis and phosphorylation of ERK1/2 and SAPK/JNK caused by alcohol or acetaldehyde (191). Because our previous studies implicated the same molecular stressors in cardiac (84, 166, 168, 178) and blood pressure anomalies (65, 89) caused by alcohol, the utilization of pharmacological or molecular strategies that boost ALDH2 abundance and/or activity could be viable therapeutic options for correcting alcohol-evoked cardiac adverse effects.

7. Conclusions

In this chapter we discussed mounting experimental evidence that supports scarce clinical findings on the adverse cardiac and autonomic effects of ethanol. While the mechanisms of a potential cardioprotective effect of mild/moderate alcohol consumption are beyond the scope of this book chapter, it is important to note that some recent clinical findings question this dogma. Our discussion focused on less appreciated adverse cardiac effects produced by alcohol doses (blood concentrations) that usually have minimal cardiac effects in health men and male rats. In the latter, limited metabolism of alcohol to acetaldehyde, and compensatory mechanisms including increased sympathetic tone and induction of ALDH2 seem to counterbalance the adverse cardiac effects of alcohol. By marked contrast, under less studied sex- (ovarian hormones/estrogen) or pathological (hypertension) conditions, the changes in these homeostatic mechanisms seem to favor the generation and accumulation of the alcohol cardiotoxic metabolite acetaldehyde and the exacerbation of the adverse cardiac effect of alcohol. Specifically, we discussed findings that implicated enhanced catalase activity in the brain stem of hypertensive rats and in the hearts of estrogen replete rats in exaggerated neurotoxic and cardiotoxic effects of the same dose(s) of alcohol, compared to normotensive and ovarian hormones deprived rats, respectively. Localized microinjections of alcohol or acetaldehyde into brain stem areas, the use of selective alcohol metabolizing enzyme inhibitors as well as MAPK and phosphatase inhibitors and other pharmacological interventions supported the premise that enhanced alcohol metabolism to acetaldehyde is pivotal for the induction of autonomic dysregulation and cardiac dysfunction. The reviewed findings implicated the Akt-ERK1/2-NOS signaling cascade in the neurotoxic and cardiotoxic effects of alcohol. Interestingly, while direct cardiac effects of alcohol or its metabolite acetaldehyde produce cardiotoxicity, pharmacological evidence supports the shift of cardiac autonomic regulation towards vagal dominance significantly contributes to and might explain the sex/estrogen dependent exaggeration of alcohol-evoked myocardial dysfunction. More studies are needed to further understand the cellular mechanisms that underlie the increased sensitivity of cardiac myocytes in the presence of estrogen and brain stem neurons under hypertension conditions to the toxic effects of alcohol and acetaldehyde. These future studies will help identify new targets for the development of potential therapeutics to mitigate the adverse autonomic and cardiac effects of alcohol in more vulnerable populations, hypertensives as well as premenopausal and post or surgical menopausal women receiving estrogen replacement therapy.