Much of the research investigating the chronic effects of ethanol on the cardiovascular system has addressed vascular responsiveness to vasoconstrictor agents. In this regard, enhanced vascular reactivity to vasoconstrictor agents or impairment of vascular relaxation is described to contribute to the cardiovascular complications associated with chronic ethanol consumption. The initial studies in this field showed enhanced vascular reactivity to α₁-adrenoceptor agonists in different arteries from ethanol-fed rats. Pinardi et al[36] found that chronic ethanol consumption significantly enhanced the contractile response induced by phenylephrine of endothelium-intact aortic rings. Noradrenaline-induced contraction of the superior mesenteric artery was shown to be greater in rings from ethanol-treated rats[37]. Likewise, there was an ethanol-associated increase in the maximal contractile response to phenylephrine, a selective α₁-adrenoceptor agonist, in endothelium-denuded aortic rings[38]. Later, Ladipo et al[39] demonstrated that chronic ethanol consumption increased the sensitivity of rat aortic rings to noradrenaline. At this point, although it was well established that chronic ethanol consumption enhanced α₁-induced contraction, the mechanisms underlying this response were poorly understood. Moreover, the experiments designed to study the vascular effects of chronic ethanol consumption on α₁-induced contraction used only one period of treatment[21,28,29]. Based on these observations, we proposed a study to investigate the time-course of changes in vascular reactivity to phenylephrine in aortas from chronically ethanol-treated rats as well as to evaluate in detail the mechanisms underlying the effects of long-term ethanol consumption on α₁-induced contraction. Chronic ethanol consumption produced an increased responsiveness to phenylephrine in aortas, although there was no relationship between the period of treatment (2, 6 and 10 wk) and the magnitude of the enhancement of α₁-induced contraction[40]. Importantly, the increased responsiveness to phenylephrine was also observed after endothelial denudation, further suggesting that the increased sensitivity to α₁-adrenergic agonists was not dependent on the presence of the endothelium. The enhanced vascular response to phenylephrine observed in the aorta of ethanol-treated rats was maintained by two mechanisms: an increased release of thromboxane A2, a vascular smooth muscle-derived vasoconstrictor prostanoid, and an increased extracellular Ca²⁺ influx. One interesting finding of this study was that the increased response to phenylephrine was not the result of a nonspecific increase in rat aorta reactivity induced by chronic ethanol intake, as the contractile responses to endothelin-1 or KCl were not affected by the ethanol treatment. In fact, while studying the effect of ethanol consumption on the reactivity of rat carotids to endothelin-1, we found an increase in endothelin-1-induced contraction in this artery with no change in the contraction induced by phenylephrine[41,42]. The hyperactivity to endothelin-1 in the rat carotid was not different among the three periods of treatment (2, 6 and 10 wk) used in our study. The potentiation of endothelin-1-induced contraction in the rat carotid was caused by reduced expression of pro-relaxation endothelial endothelin receptor type B (ET_B) receptors (Table 2).

Most of the experiments designed to study the relationship between alterations in vascular functionality and increases in blood pressure induced by ethanol consumption used conduit vessels, such as the aorta. However, while the aorta does not offer substantial resistance to blood flow, the contribution made by vessels of smaller diameter to peripheral vascular resistance is much greater. In rats, the mesenteric circulation receives approximately one-fifth of the cardiac output[43], and thus, regulation of this bed provides a significant contribution to the regulation of systemic blood pressure. To further analyze this aspect, we evaluated whether alterations in the reactivity of the mesenteric arterial bed could account for the hypertensive state associated with ethanol consumption[31]. Chronic ethanol consumption produced an endothelium-dependent increased responsiveness to phenylephrine in a perfused mesenteric arterial bed isolated from rats treated with ethanol for 6 wk but not from rats treated for 2 wk. However, increased blood pressure was observed in ethanol-treated animals after 2 wk, whereas altered responsiveness to phenylephrine was only observed in rats treated for 6 wk. These observations supported the notion that the altered responsiveness of resistance arteries was not the cause, but rather the consequence, of the increased blood pressure associated with ethanol intake[31,32]. The increased vascular response to phenylephrine observed in the mesenteric arterial bed was maintained by two mechanisms: an increased release of endothelial-derived vasoconstrictor prostanoids and a reduced modulatory action of endothelial nitric oxide (NO); the latter is likely associated with a reduced expression of the enzyme eNOS (endothelial NO synthase)[32].

Impairment of vascular relaxation may also contribute to the cardiovascular complications associated with chronic ethanol consumption. Long-term ethanol consumption significantly reduced acetylcholine-induced relaxation in the aortic rings from rats treated with ethanol for 12 wk[44] and 8 wk[45]. In the rat carotid, the relaxation induced by IRL1620, a selective endothelin ET_B receptor agonist, was reduced after treatment with ethanol; this effect was mediated by a mechanism involving the downregulation of endothelial ET_B receptors[41]. More recently, we found that chronic ethanol consumption reduced the endothelium-dependent relaxation induced by the peptide adrenomedullin in the rat aorta[46].

In resistance arteries, Hatton et al[37] showed an increased response of mesenteric arteries to noradrenaline in rats treated with ethanol for 18 wk. The finding that the relaxation induced by acetylcholine, but not by sodium nitroprusside, was reduced in the mesenteric arterial bed from ethanol-treated rats indicated that chronic ethanol consumption decreased the action of NO or its endothelial cell receptor-stimulated production/release[32]. Similarly, ethanol consumption was also found to reduce the endothelium-dependent relaxation induced by adrenomedullin in the rat mesenteric arterial bed[33]. The vascular relaxation induced by adrenomedullin in the rat mesenteric arterial bed is endothelium-dependent and involves the activation of the NO-cyclic guanosine monophosphate pathway[47]. In our study, no differences in adrenomedullin-induced relaxation were detected in control and ethanol-exposed tissues after incubation with L-nitro-arginine methyl ester, a NOS inhibitor, suggesting that the reduced adrenomedullin responsiveness of the mesenteric arterial bed from ethanol-treated rats was due to an impaired modulation of adrenomedullin-induced relaxation by NO[33].

The vascular endothelium and vascular smooth muscle cells are important targets for the effects of ethanol consumption. These effects are complex, and the identification of biochemical/molecular mechanisms that could explain such effects is warranted. A number of mechanisms have been postulated to explain the pathogenesis of high-dose ethanol toxicity in the vasculature. These mechanisms include an increase in intracellular Ca²⁺ levels with a subsequent increase in vascular reactivity, oxidative stress and a reduction in NO bioavailability. These processes will be discussed in the following sections.

One of the mechanisms by which chronic ethanol consumption leads to alterations in vascular responsiveness is by increasing the intracellular Ca²⁺ levels in vascular smooth muscle cells. Ca²⁺ is a cation of critical importance for many cellular control mechanisms, including muscle contraction. During excitation, the intracellular Ca²⁺ concentration increase by either (1) Ca²⁺ entry through the plasma membrane through voltage- or ligand-gated ion channels, or (2) release from intracellular stores (sarcoplasmic reticulum or mitochondria).

Some studies have provided evidence that ethanol consumption increases the intracellular Ca²⁺ concentration. This response may result from a direct effect of ethanol on plasma membrane permeability, Na⁺ transport and Na⁺-Ca²⁺ exchange, and/or impaired Ca²⁺ transport due to a secondary abnormality, such as Mg²⁺ depletion, which is described in alcoholics[48]. Increased Ca²⁺ influx results in increased vascular contractility and reactivity, and those responses increase vascular tone and peripheral vascular resistance, thereby elevating blood pressure[49]. Tirapelli et al[40] described an increased phenylephrine-induced contractility of arteries from ethanol-treated rats. SQ29548, a potent and selective thromboxane A2 receptor antagonist, reduced the maximal CaCl₂ response of aortic rings from ethanol-treated rats, suggesting that the enhanced response to extracellular Ca²⁺ was modulated by PGH₂/TXA₂. Based on these results, it was concluded that prostanoids mediate the enhanced reactivity to phenylephrine by mechanisms that alter the mobilization of or sensitivity to extracellular Ca^2+[40].

The effect of chronic ethanol administration on blood pressure and its relation to Ca²⁺ were also investigated by Hsieh et al[50] in 7-wk-old Wistar rats that had received 15% ethanol in their drinking water. The blood pressure in ethanol-treated rats was significantly higher than in the controls. The extracellular fluid volume was increased in ethanol-treated rats, and the blood pressure significantly correlated with increases in the intracellular Ca²⁺ concentration. These results suggest that increased intracellular Ca²⁺ and augmented body fluid volume contributed to the development of ethanol-induced hypertension. It was also suggested that these responses were partly mediated by Mg²⁺ depletion and suppressed Na⁺ pump activity[50]. In fact, these factors appear to be all-important in the etiology of hypertension[51].

In 2008, Tirapelli et al[52] reported an increased responsiveness to KCl of arteries from female rats chronically treated with ethanol. Because KCl-induced contraction depends almost exclusively on Ca²⁺ influx through the activation of voltage-sensitive channels[53], it was suggested that ethanol consumption increases the Ca²⁺ influx through these channels. Vasdev et al[54] observed that ethanol consumption (10% ethanol in drinking water-6 wk) increased systolic blood pressure and that this response was associated with an increased Ca²⁺ uptake by aortas from ethanol-treated animals. These findings suggested that increases in cytosolic free Ca²⁺ and in Ca²⁺ uptake in the vasculature are associated with ethanol-induced hypertension. Two years later, these authors reported that verapamil, a Ca²⁺ channel blocker, reversed the increase in systolic blood pressure and aortic Ca²⁺ uptake induced by chronic ethanol consumption. In addition to the effects observed previously, the authors observed smooth muscle cell hyperplasia in small arteries and in renal arterioles from ethanol-treated rats[25].

In a clinical study, it was demonstrated that both systolic and diastolic blood pressures were significantly higher in individuals drinking 275 g ethanol per week[55]. In these subjects, increased plasma Ca²⁺ levels were correlated with increased diastolic blood pressure. An increment in diastolic pressure of 6.9 mmHg correlated with increments of 0.1 mmol/L in plasma Ca²⁺ concentration. Those findings suggested that regular ethanol consumption predisposes to hypertension by facilitating Ca²⁺ accumulation in cells involved in blood pressure regulation[55]. Taken together, the above-mentioned studies suggest a role for Ca²⁺ in ethanol-induced hypertension. In this scenario, ethanol consumption would alter Ca²⁺ influx/permeability in the vasculature with a consequent increase in vascular contractility and peripheral resistance, which in turn would be responsible for the increase in blood pressure associated with ethanol consumption.

Reactive oxygen species (ROS) are reactive chemical entities produced as intermediates in reduction-oxidation (redox) reactions. Perturbations of the balance between ROS production and scavenging by antioxidant systems result in oxidative stress and presumably in pathophysiologic changes. Oxidative stress is a common mediator of pathogenicity in cardiovascular diseases, such as hypertension[56,57]. ROS have an important pathophysiological role in inflammation (by influencing platelet aggregation and migration of monocytes), hypertrophy, proliferation, fibrosis, angiogenesis, processes that are involved in cardiovascular remodeling and endothelial dysfunction[58-61].

The role of ROS in the pathophysiology of hypertension is well established[62-64]. The causal relationship between ethanol, ROS and hypertension most likely occurs at the vascular level, where ethanol promotes oxidative stress, endothelial dysfunction, vascular inflammation, increased vascular reactivity and structural remodeling. Together, these responses lead to increased peripheral resistance and therefore to increased blood pressure[65,66].

It is known that ROS modulate specific cellular pathways (redox signaling), leading to changes in gene transcription and in functional oxidative modifications of cellular proteins that cause cellular dysfunction[56,67,68]. Thus, oxidative stress not only causes direct and irreversible oxidative damage to macromolecules, but it also affects redox-dependent signaling in the vasculature[69]. ROS generation by ethanol is important to its pathophysiology in the cardiovascular system, as ethanol is extensively metabolized into acetaldehyde in the liver, mainly by the enzyme alcohol dehydrogenase[70]. Acetaldehyde, in turn, is oxidized to acetate by acetaldehyde dehydrogenase, which results in the generation of ROS and decreased NO levels[71].

In addition to the ROS generated during ethanol metabolism, some studies have shown the involvement and contribution of the nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidases to dysfunctions promoted by chronic ethanol consumption in several tissues[72-76]. Increased vascular oxidative stress induced by ethanol consumption is related to the activation of the enzyme NAD(P)H oxidase, and this mechanism is involved in the increased blood pressure caused by chronic ethanol consumption. NAD(P)H oxidase is the main source of ROS in endothelial and smooth muscle vascular cells[65], and it is considered a key factor in the vascular dysfunctions induced by ethanol. Husain et al[77] demonstrated that chronic ethanol consumption leads to an increased NAD(P)H oxidase activity and ROS generation that leads to membrane lipid peroxidation. The authors also observed increased phenylephrine-induced contraction and reduced acetylcholine-induced relaxation in aortas from ethanol-treated rats[77]. These data suggest that the initial step in the cardiovascular dysfunction associated with chronic ethanol consumption involves the formation of ROS, and this process can be mediated by the enzyme NAD(P)H oxidase. Moreover, this enzyme has been implicated in the activation of xanthine oxidase and the uncoupling of eNOS, which leads to ROS overproduction[78].

The antioxidant enzymes are the first line of defense against ROS-induced oxidative tissue injury. In vascular tissue, the enzymatic antioxidant system mainly consists of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), thioredoxins and peroxiredoxins. The non-enzymatic antioxidants include ascorbate, tocopherol, glutathione, bilirubin and uric acid[79,80]. The antioxidant mechanisms antagonizing the consequences of chronic ethanol consumption have particularities related mainly to the type of tissue studied, the duration of treatment and the concentration of ethanol used. Das and Vasudevan[81] showed that ethanol consumption increased SOD activity and decreased CAT activity in a time- and dose-dependent manner[81]. Husain et al[82] demonstrated increased SOD activity in the liver of rats treated with ethanol[82]. It is known that SOD activity is modulated by increased ROS generation and by lipid peroxidation[83,84]. In rats, chronic ethanol treatment led to increased CAT activity and impaired the maintenance of the glutathione redox cycle in renal tissue, with an increase in GPx activity and a decrease in GSH (reduced glutathione) levels[84].

In clinical studies, increased plasma activity of SOD and GPx was observed in subjects who regularly consume ethanol[85,86]. Husain et al[87] demonstrated that chronic ethanol consumption by rats significantly depressed both cytosolic CuZn-SOD and mitochondrial Mn-SOD activities in the plasma, indicating an inability of the cells to scavenge superoxide anion. Moreover, plasma CAT and GPx activities were also significantly decreased in ethanol-treated rats. The inhibition of these enzymes may increase superoxide anion availability, which can react with NO to form peroxynitrite[87].

The role of oxidative stress in ethanol-induced hypertension is complex and may involve increases in ROS generation or reductions in antioxidant systems. The increase in oxidative stress promoted by ethanol is associated with endothelial dysfunction, vascular inflammation and increased vascular reactivity. These processes may contribute directly or indirectly to increased peripheral resistance and therefore to increased blood pressure.

In 1980, Furchgott et al[88], in classic study, discovered that endothelial cells produce an endothelium-derived relaxing factor (EDRF) in response to stimulation by acetylcholine. In 1987, Palmer et al[89] and Ignarro et al[90] identified EDRF as NO, a free radical that diffuses to underlying smooth muscle to induce vasodilatation[89,90]. These findings marked the beginning of a major worldwide expansion of research into the role of NO in vascular physiology and pathophysiology.

The endothelium plays a pivotal role as a sensor, transducer, and integrator of signaling processes regulating vascular homeostasis, and it is known that vascular diseases, including hypertension, are characterized by impaired endothelium-derived NO bioactivity. The effect of ethanol on the function of the endothelial is complex[91]. Appreciating the importance of NO in the maintenance of vascular tone, some studies have examined the mechanisms underlying the impairment of NO-mediated vasodilatation by chronic ethanol consumption[92]. In theory, such a decrease in NO bioactivity could result from reduced NO production or from the inactivation of NO[93]. NO is produced by NOS (nitric oxide synthase) via one of three isoforms: the neuronal NOS (nNOS/NOS1), inducible NOS (iNOS/NOS2), and the endothelial NOS (eNOS/NOS3)[94]. Ethanol exerts different effects on these isoforms in a variety of cells and tissues. Tirapelli et al[52] demonstrated that chronic ethanol consumption reduced the vascular expression of eNOS in female rats. Conversely, iNOS expression in arteries from ethanol-treated rats was significantly increased compared with control tissues. This response could be the result of a compensatory mechanism, where increased iNOS expression could induce a substantial and sustained release of NO to compensate for the reduction of eNOS expression[52]. In the rat liver, ethanol decreased eNOS expression and activity[95]. Krecsmarik et al[96] demonstrated that chronic ethanol consumption induced an increase in iNOS activity and a decrease in nNOS expression in the rat gastrointestinal tract[96]. Moreover, chronic ethanol treatment reduced the eNOS-dependent relaxation of cerebral arterioles in rats[97].

The effect of ethanol on endothelial NO bioavailability appears to be related to the dose of ethanol. In this sense, it was shown that low concentrations of ethanol induced an increased release of endothelial NO due to the activation and expression of NOS[98,99]. Utkan et al[28] described that chronic ethanol consumption potentiates endothelium-dependent relaxation in aortic rings, most likely through interference with the synthesis and/or release of NO or adaptive alterations in muscarinic receptors on the endothelial cells[28].

While low concentrations of ethanol are described to increase endothelial NO production, the chronic consumption of high doses of ethanol impairs endothelial function in association with reduced NO bioavailability. Husain et al[44,100] described down-regulation of the NO-generating system, leading to impaired vasorelaxation and hypertension. Male Fisher rats orally administered 20% ethanol (4 g/kg - 12 wk) showed increased systolic and diastolic blood pressures and impaired vascular relaxation compared with controls. The expression of eNOS in the thoracic aorta isolated from ethanol-fed rats was down-regulated, leading to a depletion of aortic NO. This process may alter resistance vessel architecture, reducing its dilatory capacity[44,100]. In 2004, Kuhlmann et al[101] reported that high concentrations of ethanol decreased NO synthesis in and proliferation of endothelial cells from human umbilical veins.

The concentration of plasma asymmetric dimethylarginine (ADMA) in alcoholics is higher than in non-alcoholic subjects[102]. ADMA is an endogenous inhibitor of NO production, which is generated from the methylation of arginine residues by arginine methyltransferases and subsequent proteolysis. In this sense, increased ADMA levels could also contribute to the reduced bioavailability of NO in alcoholics.

NO, which is constantly formed, readily reacts with reactive molecules, such as superoxide anion[103,104]. Most of the cytotoxicity attributed to NO is due to peroxynitrite, which is produced from the reaction between NO and superoxide anion[105]. This loss of NO that occurs in the reaction with superoxide anion deprives vascular smooth muscle cells of NO. Ethanol reduces the bioavailability of NO through both the inhibition of eNOS and through the formation of peroxynitrite, which can lead to cellular damage[106].