OUP user menu

Time-Course of Neuroendocrine Changes and Its Correlation with Hypertension Induced by Ethanol Consumption

Andreia Lopes da Silva, Silvia G. Ruginsk, Ernane Torres Uchoa, Carlos C. Crestani, America A. Scopinho, Fernando Morgan A. Correa, Bruno Spinosa De Martinis, Lucila Leico Kagohara Elias, Leonardo B. Resstel, Jose Antunes-Rodrigues
DOI: http://dx.doi.org/10.1093/alcalc/agt040 495-504 First published online: 2 June 2013


Ethanol (ETOH) consumption has been associated with endocrine and autonomic changes, including the development of hypertension. However, the sequence of pathophysiological events underlying the emergence of this effect is poorly understood. Aims: This study aimed to establish a time-course correlation between neuroendocrine and cardiovascular changes contributing to the development of hypertension following ETOH consumption. Methods: Male adult Wistar rats were subjected to the intake of increasing ETOH concentrations in their drinking water (first week: 5%, second week: 10%, third and fourth weeks: 20% v/v). Results: ETOH consumption decreased plasma and urinary volumes, as well as body weight and fluid intake. Furthermore, plasma osmolality, plasma sodium and urinary osmolality were elevated in the ETOH-treated rats. ETOH intake also induced a progressive increase in the mean arterial pressure (MAP), without affecting heart rate. Initially, this increase in MAP was correlated with increased plasma concentrations of adrenaline and noradrenaline. After the second week of ETOH treatment, plasma catecholamines returned to basal levels, and incremental increases were observed in plasma concentrations of vasopressin (AVP) and angiotensin II (ANG II). Conversely, plasma oxytocin, atrial natriuretic peptide, prolactin and the hypothalamus–pituitary–adrenal axis components were not significantly altered by ETOH. Conclusions: Taken together, these results suggest that increased sympathetic activity may contribute to the early increase in MAP observed in ETOH-treated rats. However, the maintenance of this effect may be predominantly regulated by the long-term increase in the secretion of other circulating factors, such as AVP and ANG II, the secretion of both hormones being stimulated by the ETOH-induced dehydration.


Ethanol (ETOH) consumption is known to induce functional changes in the central nervous system (CNS), resulting in neuroendocrine, behavioral and cardiovascular changes (Clarck, 1985; Resstel et al., 2006, 2008). It has been previously demonstrated that acute ETOH intake in humans promotes diuresis, while repeated doses induce a reduction in urinary volume (Eggleton, 1942). Moreover, chronic ETOH intake was shown to induce hyperosmolality (Millerchoen and Riggs, 1969; Collins et al., 1992) as well as increased renin activity, in parallel with a decreased urinary volume and sodium excretion (Hsieh et al., 1992; Addolorato et al., 1999).

Several studies performed both in human and in animal experimental models have demonstrated that chronic ETOH intake is associated with hypertension without affecting heart rate (HR) (Clarck, 1985; Abdel-Rahman and Wooles, 1987), this mild hypertensive state being observed at an early phase of ETOH consumption (Resstel et al., 2006). In fact, a positive correlation between the extent of ETOH consumption and the development of hypertension has been reported (Abdel-Rahman and Wooles, 1987), suggesting that the period of ETOH exposure is the major factor contributing for the development of hypertension. Several mechanisms have been proposed to be involved with the hypertensive effect of long-term ETOH consumption, such as neuroendocrine changes, sympathetic nervous system activation and alterations in the contractility of vascular smooth muscle. However, the sequence of pathophysiological events underlying the emergence of this effect is poorly understood, since most studies conducted so far have only evaluated hypertension after it has been installed. Therefore, this study aimed to establish a time-course correlation between neuroendocrine and cardiovascular changes contributing to the development of hypertension following ETOH exposure.


ETOH treatment

All of the procedures employed in this study were previously approved by the institutional Ethics Committee for Animal Use of the School of Medicine of Ribeirao Preto, University of Sao Paulo (065/2009), and are in accordance with the international guidelines for animal use and welfare. Male adult Wistar rats (250–300 g) were housed either individually in metabolic cages (for the evaluation of behavioral responses) or collectively in plastic cages (for all other experiments) for 3–5 days of habituation with free access to standard food and water. All the animals were kept under standard conditions of the light/dark cycle (lights on at 6 AM, lights off at 6 PM) for habituation (3–5 days) at the Animal Care Unit of the Department of Physiology of the School of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil. After, rats were divided into two groups: the first group received tap water (control group) and the second group received increasing concentrations (v/v) of ETOH (Labsynth, Diadema, SP, Brazil) in their drinking water for 4 weeks (first week: 5%, second week: 10%, third and fourth weeks: 20%). For brain and blood samples collection, animals were euthanized by decapitation.

Weight gain, food and liquid intake

Weight gain was estimated by a digital scale after each week of treatment and values represent the mean obtained for each experimental group. Food and liquid intakes were assessed by daily measures performed either indirectly (by weighing food containers) or directly (evaluating liquid intake in a graduated drinking cylinder).

ETOH measurement in the peripheral blood

ETOH analyses were carried out by gas chromatography (Agilent GC 7890 A gas chromatograph, Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector (model 1177, Varian Inc., Walnut Creek, CA, USA) and a Carbowax fused silica capillary column (30 m × 0.25 mm of internal diameter, film thickness 0.25 µm; Chrompack Instrumentos Cientificos, Sao Paulo, SP, Brazil). The method used was the headspace with an automatic dispenser (CombiPal, CTC Analytics, Switzerland). Whole blood samples were put in a vial with standard solution (isobutanol, 100 mg dl−1). Samples were heated at 80°C for 10 min and shaken at 250 rpm before the injection in the chromatographic column. Injection mode was splitless for 30 s. The column temperature was initially set at 50°C for 2 min and then increased at a rate of 20°C/min until it reached 105°C and at a rate of 40°C/min until it reached 180°C The temperatures of injector and detector were set at 220 and 300°C, respectively. Nitrogen was used as a carrier gas with linear velocity set at 5 ml s−1.

Arterial pressure and HR recordings

The mean arterial pressure (MAP), diastolic arterial pressure (DAP), systolic arterial pressure (SAP) and HR of the animals used in this experimental protocol were recorded after 1, 2, 3 or 4 weeks of ETOH treatment. The animals were submitted to the cannulation of the femoral artery under anesthesia (tribromoethanol, 250 mg kg−1, intraperitonially) 24 h before the recordings. On the day of the experiment, rats were placed in individual cages, and an arterial catheter was flushed with a small volume (100 µl) of saline containing heparin and connected to a pressure transducer equipped with a signal amplifier (HP7754-A, Hewlett Packard, Houston, TX, USA). Data were collected using a computerized data acquisition system (MP-100, Biopac Systems, Inc., Goleta, CA, USA) and both MAP and HR were recorded for a total period of 30 min.

Osmolality, sodium and potassium concentrations and urinary volume

After collecting urinary samples in plastic graduated tubes, their volumes were measured. The concentrations of sodium and potassium were assessed in urine and serum samples (obtained from trunk blood in non-heparinized tubes) using flame photometry (model b262, Micronal, Sao Paulo, SP, Brazil). Finally, plasma and urinary osmolalities were evaluated by the freezing point method (model 3250, Advanced® Single-Sampler Osmometer, Norwood, MA, USA).

Blood collection and immunoassays

After the treatment, the rats were placed in individual plastic cages and decapitated between 8 AM and 10 AM after 1, 2, 3 or 4 weeks of ETOH treatment. After decapitation, the trunk blood was collected in chilled plastic tubes containing heparin or EDTA with protease inhibitors. The blood was then centrifuged (3000 rpm, 20 min at 4°C), and the plasma was separated and stored at −20°C for hormone determination. The hematocrit was obtained by blood collection into capillaries, which were subjected to centrifugation (model 211, FANEM, Sao Paulo, SP, Brazil), and the total plasma protein concentrations were assessed by the Bradford method (Bio-Rad, Hercules, CA, USA).

All the primary antibodies employed in radioimmunoassays, with the exception of anti-atrial natriuretic peptide (anti-ANP) and anti-corticosterone, were purchased from Peninsula Laboratories Incorporation (San Carlos, CA, USA). For the measurement of vasopressin (AVP) and oxytocin (OT) plasma concentrations, plasma was extracted with acetone and petroleum ether, as previously described (Haanwinckel et al., 1995). The assay sensitivity and the intra- and inter-assay coefficients of variation were 0.8 pg ml−1, 7.7% and 11.9% for AVP (rabbit anti-AVP, #T4563) and 0.9 pg ml−1, 7% and 12.6% for OT (rabbit anti-OT, #T4084), respectively. For the measurement of plasma concentrations of ANP (antibody raised in rabbit, a kind donation of Dr Jolanta Gutkowska) and angiotensin II (ANG II) (rabbit anti-ANG II, #T4007), plasma was extracted with activated octadecylsilane cartridges (Sep-Column, Peninsula Laboratories, Inc.), as previously described (Gutkowska et al., 1984). The assay sensitivity and the intra- and inter-assay coefficients of variation were 0.7 pg ml−1 and 0.5 pg ml−1, 4.8% and 10.9%, and 10.0% and 18.3%, for ANP and ANG II, respectively. Plasma angiotensin I (ANG I) concentrations were determined by an in-house developed immunoassay (rabbit anti-ANG I, #T4166), where the sensitivity, intra-assay and ED50 coefficients were 1.2 pg ml−1, 11.2% and 39.6 pg ml−1, respectively. Corticosterone (rabbit anti-corticosterone, #C8784, Sigma Aldrich, St. Louis, MI, USA) was extracted from plasma with ethanol as previously described (Vecsei, 1979). The assay sensitivity and the intra- and inter-assay coefficients were 0.4 µg dl−1, 8.0% and 19.0%, respectively. Prolactin (PRL) radioimmunoassay was performed using NIDDK RIA reagents (NIH, Bethesda, MD, USA), the assay sensitivity, intra- and inter-assay coefficients of variation were 0.4 ng ml−1, 5.0% and 11.7%, respectively. For measurements of the adrenaline and noradrenaline concentrations, plasma was mixed with sodium metabisulfite and stored at −20°C. Samples were extracted with alumina and then catecholamines were isolated from the eluents with a high-performance liquid chromatograph (LC-7A, Shimadzu Instruments) using a 5-µm Spherisorb ODS-2 reversed-phase column (Sigma Aldrich, St. Louis, MI, USA). Catecholamines and internal standards were quantified with an electrochemical detector (LC-ECD-6A, Shimadzu Scientific Instruments, Inc., Columbia, MD, USA). Glucose was measured in plasma samples using a commercially available kit (Glucox 500, Doles Reagents, Goiania, GO, Brazil).

Tissue sample collection, RNA extraction and semi-quantitative analysis of mRNA expression by real-time PCR

After 1, 2, 3 or 4 weeks of treatment, the animals were placed in individual cages and euthanized between 8 AM and 10 AM. After decapitation, the brains were quickly removed and collected under RNAse-free conditions. To determine the relative mRNA expression of ANP and C-type natriuretic peptide (CNP), the medial basal hypothalamus was immediately dissected out from an area 2 mm lateral to the midline that was delimited anteriorly by the anterior border of the optic chiasm and posteriorly by the anterior border of the mammillary bodies. The depth of the hypothalamic fragments was ∼1 mm and included the nuclei located at the anterior ventral wall of the third cerebral ventricle, as well as the paraventricular (PVN), supraoptic (SON), suprachiasmatic (SCN) and arcuate (ARC) nuclei. The relative expression of pro-opiomelanocortin (POMC) mRNA was determined from anterior pituitaries that were freshly collected and placed directly into sterile plastic tubes. To determine the relative AVP, OT and corticotrophin releasing factor (CRF) mRNA expression, microdissections of the PVN or SON were performed on 1.200 μm-thick cryostat sections using a stainless steel punch needle of 1.5 mm diameter, corresponding to the coordinates 0.8–2.0 mm posterior to bregma, in accordance with the Paxinos and Watson rat brain atlas (Paxinos and Watson, 1997). All tissue samples were kept in the RNAlater reagent (Ambion, Austin, TX, USA) and maintained at −80°C until extraction. Total RNA was isolated from each tissue sample using TRIzol reagent (Invitrogen, Auckland, New Zealand), according to the manufacturer's instructions. The RNA concentration was determined using a UV spectrophotometer, and 500 ng of RNA was used for complementary DNA synthesis using the high-capacity complementary DNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Semi-quantitative real-time PCR was performed using the Step One Plus Real-Time PCR System (Applied Biosystems). PCR reactions were performed in triplicate and water was used as a negative control. The quantitative expression of AVP, OT, CRF, POMC, ANP and CNP mRNAs, as well as two housekeeping genes, was determined for each cDNA sample by specific assays (TaqMan®, Applied Biosystems). For each sample, the threshold cycle (Ct) was measured and normalized to the housekeeping genes (ΔCt = CtUnknownCtHousekeeping genes). The fold change of mRNA in the unknown sample relative to the control group was determined by 2−ΔΔCt, where ΔΔCt = ΔCtUnknown − ΔCtControl. Therefore, data are presented as mRNA expression relative to the control group.

Statistical analysis

Two-way analysis of variance (treatment versus time) followed by Bonferroni's post hoc test was used. When a positive interaction was observed, data were compared using Student's t-test. The correlation between hormones and MAP was performed through a linear regression analysis. Results were considered statistically significant if P-value <0.05.


The time-course effects of ETOH ingestion on ETOH concentrations in the peripheral circulation are shown in Table 1. It can be observed that ETOH concentrations in the blood increase in parallel with the increment in the ETOH percentage offered in the drinking water, except for the fourth week of treatment.

View this table:
Table 1.

Time-course of diary ETOH ingestion and ETOH concentrations in the blood of animals receiving ETOH in increasing concentrations (first week: 5%, second week: 10%, third and fourth weeks: 20%, v/v.)

GroupETOH concentration (g l−1)Quantity of ingested ETOH (g kg−1 day−1)
1st weekn = 5n = 8
5% ETOH0.310 ± 0.165.9
2nd weekn = 6n = 8
10% ETOH0.584 ± 0.099.1
3rd weekn = 6n = 8
20% ETOH1.418 ± 0.4816.2
4th weekn = 7n = 7
20% ETOH0.612 ± 0.1714.8
  • Values are expressed as means ± SEM.

  • ND, nondetectable.

The results also demonstrate that ETOH induces a progressive increase in MAP (F1,36 = 126.9; P < 0.0001, Table 2). This effect began at the first week of treatment and was maintained during the entire evaluation period. Both the SAP (F1,37 = 101.4; P < 0.0001, Table 2) and DAP (F1,37 = 113.8; P < 0.0001, Table 2) showed a progressive increase during ETOH treatment. Moreover, no significant changes in the HR were observed in ETOH-treated animals (Table 2).

View this table:
Table 2.

Time-course of MAP, HR, SAP and DAP in animals receiving water (control group) or ETOH in increasing concentrations (first week: 5%, second week: 10%, third and fourth weeks: 20%, v/v)

GroupMAP (mmHg)HR (bpm)SAP (mmHg)DAP (mmHg)
1st weekn = 5–7n = 5–7n = 5–7n = 5–7
Control100.4 ± 2.5335.2 ± 13.8122.8 ± 1.581.0 ± 2.9
5% ETOH108.7 ± 1.7*354.0 ± 5.7132.4 ± 1.3*91.4 ± 0.9*
t = 2.823t = 1.416t = 4.737t = 3.908
2nd weekn = 5–6n = 5–6n = 5–6n = 5–6
Control97.8 ± 2.1343.7 ± 6.9123.7 ± 2.883.5 ± 1.7
10% ETOH112.0 ± 1.3*349.8 ± 7.7137.6 ± 1.2*94.6 ± 0.9*
t = 5.434t = 1.049t = 5.766t = 4.813
3rd weekn = 6n = 6n = 6n = 6
Control99.8 ± 1.6337.7 ± 8.4124.5 ± 1.884.7 ± 1.7
20% ETOH118.0 ± 1.7*331.7 ± 12.9143.5 ± 1.7*100.8 ± 1.7*
t = 7.658t = 0.39t = 4.483t = 6.634
4th weekn = 5n = 5n = 5n = 5
Control98.2 ± 1.9333.0 ± 12.4124.2 ± 2.881.0 ± 3.3
20% ETOH117.6 ± 1.9*349.2 ± 11.8146.2 ± 4.0*102.8 ± 2.2*
t = 7.084t = 0.95t = 3.8t = 5.548
  • Values are expressed as the means ± SEM. Two-way ANOVA.

  • *P < 0.05, n = 4–10.

ETOH treatment also induced a decrease in weight gain (F1,192 = 125.2; P < 0.0001) and liquid intake (F1,363 = 342.2; P < 0.0001) compared with the control group (Fig. 1A and B, respectively). This effect appeared during the first week and was maintained for the entire period of evaluation. During ETOH treatment, a decrease in urinary volume (F1,364 = 315.8; P < 0.0001, Fig. 1C) and an increase in urinary osmolality (F1,349 = 129.5; P < 0.0001, Fig. 1D) were also observed. The excretion of sodium (UNa+.V; F1,360 = 902.6; P < 0.0001, Fig. 1E) and potassium (UK+.V; F1,360 = 559.7; P < 0.0001, Fig. 1F) was decreased in ETOH-treated animals.

Fig. 1.

Time-course of body weight gain (A), liquid ingestion (B), urinary volume (C), urinary osmolality (D), sodium (E) and potassium (F) excretion in animals receiving water (control group) or ETOH in increasing concentrations (first week: 5%, second week: 10%, third and fourth weeks: 20%, v/v). Values are expressed as means ± SEM. Two-way ANOVA, *P < 0.05 representing the whole period, n = 7–13.

Plasma osmolality (F1,84 = 72.04; P < 0.0001) (Fig. 2A), plasma protein (F1,67 = 14.47; P < 0.0005) (Fig. 2C) and hematocrit (F1,95 = 20.68; P < 0.0001) (Fig. 2D) were increased by ETOH, these effects being detected after the second week of treatment. An increase in plasma sodium concentrations was also observed in ETOH-treated animals (F1,86 = 10.39; P < 0.01), and this effect occurred during the first, second and third weeks of treatment (Fig. 2B).

Fig. 2.

Time-course of plasma osmolality (A), plasma sodium (B), plasma protein (C) and hematocrit (D) in animals receiving water (control group) or ETOH in increasing concentrations (first week: 5%, second week: 10%, third and fourth weeks: 20%, v/v). Values are expressed as means ± SEM. Two-way ANOVA, *P < 0.05, n = 6–12.

Table 3 shows the effects of ETOH treatment on plasma concentrations of potassium, glucose, noradrenaline and adrenaline. We observed that plasma potassium concentrations did not change after ETOH ingestion (F1,87 = 1.804; P > 0.05). Furthermore, plasma glucose levels were altered by treatment (F1,87 = 5.062; P < 0.05) and time of exposure to ETOH (F3,87 = 27.97; P < 0.0001), although a significant increase in this parameter was only detected after 2 weeks of treatment, compared with the corresponding control group. ETOH treatment increased plasma noradrenaline concentrations (F1,39 = 11.33; P < 0.01) at the first and second weeks of treatment, whereas adrenaline concentrations were elevated only after the first week of ETOH intake (F1,39 = 27.18; P < 0.0001).

View this table:
Table 3.

Time-course of plasma concentrations of potassium, glucose, adrenaline and noradrenaline in animals receiving water (control group) or ETOH in increasing concentrations (first week: 5%, second week: 10%, third and fourth weeks: 20%, v/v)

GroupPotassium (mEq l−1)Glucose (mg dl−1)Adrenaline (ng ml−1)Noradrenaline (ng ml−1)
1st weekn = 12n = 12n = 8–10n = 8–9
Control4.513 ± 0.157149.6 ± 2.72513.91 ± 1.05.299 ± 0.6
5% ETOH4.449 ± 0.088154.1 ± 2.18617.91 ± 0.6*7.566 ± 0.6*
t = 0.350t = 1.286t = 3.559t = 2.719
2nd weekn = 11–12n = 11–12n = 10n = 9
Control4.380 ± 0.112154.2 ± 2.50014.39 ± 1.45.022 ± 0.7
10% ETOH4.458 ± 0.143163.0 ± 2.922*15.14 ± 0.97.629 ± 0.8*
t = 0.417t = 2.277t = 0.46t = 2.439
3rd weekn = 12n = 12n = 5–8n = 9–10
Control4.260 ± 0.115136.0 ± 1.76814.79 ± 0.75.134 ± 0.4
20% ETOH4.185 ± 0.096141.4 ± 2.80314.56 ± 0.96.500 ± 0.9
t = 0.490t = 1.636t = 0.187t = 1.262
4th weekn = 12n = 12n = 10n = 9–10
Control4.597 ± 0.157142.7 ± 2.24113.21 ± 0.84.805 ± 0.5
20% ETOH4.162 ± 0.153139.7 ± 2.38312.64 ± 1.24.923 ± 0.5
t = 2.370t = 0.931t = 0.4t = 0.156
  • Values are expressed as means ± SEM. Two-way ANOVA.

  • *P < 0.05.

ETOH-treated animals also exhibited increased concentrations of AVP after 2 weeks of treatment (F1,83 = 22.97; P < 0.0001), this effect being maintained during the remaining period of evaluation (t = 2.717; P < 0.01) (Fig. 3A). AVP mRNA expression in the SON was also increased after 2 weeks of ETOH treatment (F1,66 = 66.19; P < 0.0001) and remained elevated during the entire experiment (t = 4.123; t = 6.396 and t = 5.789, for second, third and fourth weeks, respectively; P < 0.05) (Fig. 3B). This response was similarly observed in the PVN, which showed increased AVP mRNA expression (F1,56 = 26.56; P < 0.0001) at the third (t = 3.403; P < 0.05) and fourth (t = 5.384; P < 0.05) weeks following exposure to ETOH (Fig. 3C).

Fig. 3.

Time-course of plasma concentrations of vasopressin (AVP, A), relative expression of AVP mRNA in the supraoptic (SON, B) and paraventricular nucleus of hypothalamus (PVN, C) in animals receiving water (control group) or ETOH in increasing concentrations (first week: 5%, second week: 10%, third and fourth weeks: 20%, v/v). Values are expressed as means ± SEM. Two-way ANOVA, *P < 0.05, n = 6–12.

The results demonstrate that ANG I increased after ETOH treatment (F1,77 = 28.11; P < 0.0001) (Fig. 4A), this effect being observed at the first week and maintained until the end of the evaluation (t = 2.388; t = 3.57; t = 3.726 and t = 2.633, for first, second, third and fourth weeks, respectively; P < 0.05). Similar to ANG I, plasma ANG II also increased in ETOH-treated groups (F1,73 = 24.03; P < 0.0001) at the second, third and fourth weeks of treatment (t = 2.255; t = 3.158, t = 2.979, respectively; P < 0.05) (Fig. 4B). The linear regression analysis showed a positive correlation between MAP and AVP (r = 0.68, df = 20), MAP and ANG II (r = 0.53, df = 20), MAP and noradrenaline (r = 0.62, df = 20), osmolality and AVP (r = 0.44, df = 46) and osmolality and ANG II (r = 0.61, df = 46).

Fig. 4.

Time-course of plasma concentrations of angiotensin I (A) and angiotensin II (B) in animals receiving water (control group) or ETOH in increasing concentrations (first week: 5%, second week: 10%, third and fourth weeks: 20%, v/v). Values are expressed as means ± SEM. Two-way ANOVA, *P < 0.05, n = 7–13.

Table 4 shows the effects of ETOH administration on plasma PRL and OT concentrations. The results show that plasma PRL was not altered by ETOH (F1,72 = 2.468; P > 0.05). Furthermore, OT secretion was not changed in ETOH-treated animals compared with the corresponding control groups (F1,84 = 1.929; P > 0.05). We also detected no changes on OT mRNA expression in the SON of ETOH-treated rats (F1,68 = 2.109; P > 0.05), although a significant increase in this parameter was found in the PVN of the ETOH-treated groups after the fourth week of treatment (F1,64 = 0.612; P > 0.05).

View this table:
Table 4.

Time-course of plasma concentrations of PRL and OT and the relative expression of OT mRNA in the supraoptic (SON) and paraventricular nucleus of hypothalamus (PVN) in animals receiving water (control group) or ETOH in increasing concentrations (first week: 5%, second week: 10%, third and fourth weeks: 20%, v/v)

GroupOxytocin (pg ml−1)OT—SON (mRNA)OT—PVN (mRNA)Prolactin (ng ml−1)
1st weekn = 10–12n = 8–10n = 8–9n = 10–11
Control5.535 ± 0.6660.922 ± 0.0951.016 ± 0.064.039 ± 0.586
5% ETOH6.124 ± 0.7760.788 ± 0.0340.882 ± 0.0653.468 ± 0.553
t = 0.381t = 1.208t = 1.115t = 0.489
2nd weekn = 11–12n = 8–10n = 8–10n = 8–10
Control4.697 ± 0.7440.997 ± 0.0560.97 ± 0.0424.421 ± 0.541
10% ETOH8.673 ± 2.0940.769 ± 0.0590.94 ± 0.0596.622 ± 0.742
t = 2.637t = 2.049t = 0.259t = 1.736
3rd weekn = 12n = 9–10n = 9–10n = 10–12
Control5.872 ± 0.8640.960 ± 0.0911.015 ± 0.0454.429 ± 0.515
20% ETOH4.940 ± 1.0120.892 ± 0.0830.948 ± 0.1426.858 ± 1.474
t = 0.632t = 0.635t = 0.595t = 2.123
4th weekn = 11–12n = 10–11n = 9n = 9–10
Control4.092 ± 0.7040.946 ± 0.0731.023 ± 0.084.818 ± 0.718
20% ETOH4.653 ± 0.5841.063 ± 0.0931.436 ± 0.09*4.538 ± 0.598
t = 0.372t = 1.135t = 3.56t = 0.228
  • Values are expressed as means ± SEM. Two-way ANOVA.

  • *P < 0.05.

The effects of ETOH on plasma ANP and on the mRNA expression for the natriuretic peptides in the medium basal hypothalamus (MBH) are shown in Table 5. The results demonstrate that ANP secretion was not altered by chronic ETOH ingestion (F1,80 = 0.587; P > 0.05). An increase in mRNA expression for ANP, but not for CNP, was observed after 4 weeks in ETOH-treated animals (F1,81 = 5.401; P < 0.05).

View this table:
Table 5.

Time-course of plasma concentrations of atrial natriuretic peptide (ANP) and the relative expression of ANP and CNP mRNAs in the MBH in animals receiving water (control group) or ETOH in increasing concentrations (first week: 5%, second week: 10%, third and fourth weeks: 20%, v/v)

1st weekn = 10–12n = 9–12n = 10–12
Control53.51 ± 5.031.006 ± 0.041.00 ± 0.03
5% ETOH51.27 ± 3.660.987 ± 0.020.96 ± 0.03
t = 0.410t = 0.411t = 1.002
2nd weekn = 8–12n = 10–12n = 10–12
Control57.10 ± 4.581.003 ± 0.030.974 ± 0.024
10% ETOH53.27 ± 4.021.003 ± 0.040.923 ± 0.032
t = 0.658t = 0.002t = 1.158
3rd weekn = 10–13n = 10–13n = 10–13
Control47.78 ± 3.670.99 ± 0.0330.902 ± 0.023
20% ETOH46.00 ± 2.860.94 ± 0.0280.924 ± 0.037
t = 0.333t = 1.033t = 0.531
4th weekn = 10–13n = 10–13n = 10–13
Control49.51 ± 3.170.94 ± 0.0470.929 ± 0.029
20% ETOH48.94 ± 3.780.80 ± 0.018*0.872 ± 0.029
t = 0.107t = 3.26t = 1.312
  • Values are expressed as means ± SEM. Two-way ANOVA.

  • *P < 0.05.

Table 6 summarizes the effects of chronic ETOH treatment on corticosterone secretion and on the mRNA expression for CRF in the PVN and POMC in the anterior pituitary (AP). The results show that chronic ETOH ingestion increased corticosterone secretion (F1,82 = 7.399; P < 0.01). However, this effect was only statistically different from the control group after 4 weeks of treatment. We also observed a decrease in the CRF mRNA expression after 3 weeks of ETOH ingestion (F1,68 = 9.180; P < 0.01). In contrast, no significant changes were induced by ETOH treatment in the POMC relative mRNA expression in the AP.

View this table:
Table 6.

Time-course of plasma concentrations of corticosterone and the relative expression of POMC and CRF in the CNS of animals receiving water (control group) or ETOH in increasing concentrations (first week: 5%, second week: 10%, third and fourth weeks: 20%, v/v)

Corticosterone (µg dl−1)CRF—PVN (mRNA)POMC—AP (mRNA)
1st weekn = 10–12n = 10n = 6
Control1.950 ± 0.7091.013 ± 0.051.007 ± 0.051
5% ETOH1.894 ± 0.4600.792 ± 0.100.971 ± 0.121
t = 0.068t = 2.128t = 0.263
2nd weekn = 11n = 9–10n = 9–10
Control1.759 ± 0.5200.951 ± 0.0961.055 ± 0.07
10% ETOH2.931 ± 0.8560.971 ± 0.0850.832 ± 0.06
t = 1.170t = 0.185t = 2.083
3rd weekn = 11–12n = 9–10n = 9–10
Control1.388 ± 0.3000.98 ± 0.0691.137 ± 0.086
20% ETOH3.049 ± 0.8560.60 ± 0.066*1.146 ± 0.086
t = 1.897t = 3.572t = 0.083
4th weekn = 11–12n = 9n = 7–10
Control0.874 ± 0.2710.978 ± 0.0621.147 ± 0.087
20% ETOH2.772 ± 0.701*0.914 ± 0.0521.044 ± 0.069
t = 2.612t = 0.586t = 0.895
  • Values are expressed as means ± SEM. Two-way ANOVA.

  • PVN, paraventricular nucleus of hypothalamus; AP, anterior pituitary.

  • *P < 0.05.


This study demonstrated for the first time that hypertension appears early during treatment with low concentrations of ETOH (i.e. during the first week ETOH 5% v/v). In this regard, the literature had only reported the development of hypertension after 28 days of treatment with 6.7% v/v ETOH (Silva et al., 2004) and none of the articles was able to correlate hypertension and dehydration. Furthermore, this manuscript is the first to demonstrate the sequence of neuroendocrine events happening during ETOH consumption and establish a possible correlation between the ETOH-induced dehydration and the development of hypertension. We demonstrated that ETOH intake does not affect HR but induces a progressive increase in the MAP, which could be observed just after the first week of treatment with low percentage of ETOH (5% v/v). This increase is gradual and follows the increment of ETOH content in the circulation. Accordingly, an increase in MAP induced by treatment with 20% ETOH solution (v/v) for a longer period of time was also described (Husain et al., 2008; Resstel et al., 2008). These studies showed a positive correlation of quantity, duration of ETOH intake and development of hypertension. However, similar to other reports in the literature, they did not aim to demonstrate when hypertension begins. In this regard, we showed here for the first time that the increase in MAP is gradual and appears early during ETOH consumption.

This study also confirmed that ETOH ingestion causes dehydration because treated animals exhibited increased hematocrit and plasma protein concentrations, which reflect a decrease in plasma volume, as well as increased plasma osmolality and plasma sodium concentrations. Figure 2 clearly shows that plasma sodium concentrations slightly fluctuate along the experiment. This response is likely to be derived from compensatory mechanisms at renal level. The main candidates for mediating this response are ANGII (through its direct effects on sodium reabsorption or acting indirectly, stimulating aldosterone secretion) and AVP (which induces water reabsorption, in an attempt to decrease hyperosmolality). Since ANGII secretion slightly changes from the third to the fourth week of ETOH treatment (Fig. 4), we can speculate that the decrease in plasma sodium concentrations observed at the fourth week may be driven by the increased water reabsorption induced by the high levels of AVP observed at this time point (Fig. 3). Indeed, the pattern of AVP secretion oppositely matches plasma sodium concentrations: when AVP increases, plasma sodium concentrations slightly decrease (second and fourth weeks of treatment). However, this effect of AVP in increasing water reabsorption may not have impacts on plasma osmolality, since ETOH metabolites also account for the hyperosmolality observed in ETOH-treated animals.

It is well established that both plasma hyperosmolality and decreased circulating volume are potent stimuli for AVP secretion. Our data show an increase in AVP plasma concentrations after the second week of treatment (10% v/v ETOH), which runs in parallel with the increase in plasma osmolality. Furthermore, we demonstrated that ETOH decreases urinary volume and increases urinary osmolality, effects that could be associated with an increase in AVP secretion and in the renal excretion of ETOH and its metabolites, such as acetaldehyde. It has been proposed that the diuresis resulting from acute ETOH intake could promote free water loss enough to increase plasma osmolality and, therefore, AVP secretion. The final effect would be the stimulation of water reabsorption with the consequent decrease in diuresis, which would overcome the direct stimulatory effect of ETOH on renal water loss (Eggleton, 1942; Millerchoen and Riggs, 1969; Bisset and Chowdrey, 1988). Therefore, our data are in agreement with previous reports demonstrating that chronic ETOH consumption is associated with antidiuretic effects (Garcia-Delgado et al., 2004) and increased AVP circulating levels (Resstel et al., 2008). This study also confirmed previous findings obtained by our group, showing that the relative mRNA expression for AVP in the SON and PVN was increased by ETOH (Resstel et al., 2008), this effect being directly related with the increased demand for hormone secretion. Taken together, these data showed that the clear state of dehydration caused by ETOH intake can elicit AVP secretion, and reinforce the hypothesis that AVP may regulate circulating volume and vascular tonus in response to prolonged ETOH ingestion.

It is well established that dehydration also stimulates the renin–angiotensin–aldosterone system (RAAS) and induces thirst (Di Nicolantonio and Mendelshohn, 1986), and it has been demonstrated that acetaldehyde, a metabolite of ETOH, can directly activate the RAAS, increasing ANG I in plasma without renin (Thevananther and Brecher, 1994). Accordingly, our results show that ETOH-treated animals exhibit increased circulating levels of ANG I and ANG II, as well as a decrease in renal sodium and water excretion, an effect that may be potentially related to an increase in aldosterone secretion and, to a lesser extent, to a direct effect of ANG II in renal tubules (Finberg et al., 1978).

Besides having their actions on sodium and water balance, ANG II and AVP could also act in vessel vasculature to cause vasoconstriction, thereby contributing to the increase in MAP as well as to the maintenance of the hypertensive state. However, the increased secretion of both hormones occurs only after the second week of treatment, which does not explain the onset of hypertension. In fact, we observed that plasma noradrenaline is higher in ETOH-treated animals after the first and second weeks of treatment, whereas adrenaline is higher only after the first week of ETOH ingestion. Noradrenaline concentration is provided by the sympathetic nervous system and the adrenal medulla, whereas the adrenal medulla is the only source of plasma adrenaline. This could explain the temporal differences found in our model. As a result of noradrenaline action in α-adrenergic receptors of peripheral vessels, vasoconstriction would be induced (Charkoudian and Rabbitts, 2009). Indeed, the literature shows that the vasodilatory effect of ETOH is completely suppressed by increased sympathetic activity, which could be the main cause of hypertension in alcoholics (Russ et al., 1991; Randin et al., 1995). Accordingly, Altura and Altura (1987) showed that ETOH stimulates catecholamines release. Therefore, we hypothesize that, in our experimental model, the increase in plasma catecholamines could trigger the ETOH-induced hypertension.

Counterbalancing the effects of the sympathetic, angiotensinergic and vasopressinergic systems on the control of cardiovascular function is the group of natriuretic peptides, among them ANP and CNP. It has been demonstrated that ANP lowers blood pressure by increasing renal sodium excretion. Furthermore, both ANP and CNP are produced locally in several regions of the CNS related to cardiovascular control (Gibson et al., 1986). Although increased blood pressure and plasma osmolality are strong stimuli for the secretion of ANP, we did not observe any significant changes in plasma concentrations of this peptide or in the relative expression of ANP and CNP mRNAs in the MBH, suggesting that alterations in the natriuretic system are not likely to contribute to the generation of hypertension in ETOH-treated rats.

Another plausible cause of hypertension in ETOH-treated animals would be the activation of the HPA axis. The POMC system is functionally integrated with the HPA axis, which have central roles in the mediation of behavioral and neuroendocrine responses induced by ETOH (Spencer and McEwen, 1997). It has been demonstrated that ACTH and corticosterone, besides their correlation with stress responses, can also induce hypertension in experimental animals (Lou et al., 2001; Singh et al., 2007; Lorenz et al., 2008). However, no significant changes were found by the present study in the AP expression of POMC mRNA. We found a slight increase of corticosterone secretion at the fourth week of ETOH treatment, whereas CRF mRNA expression was significantly reduced in the PVN after 3 weeks. Therefore, the HPA axis seems not to be consistently implicated in the generation or in the maintenance of hypertension in the present study, although decreased levels of CRF have been associated with more intense craving for alcohol in chronic experimental models (Kiefer and Wiedemann, 2004).

This study also investigated the participation of other circulating factors, such as OT and PRL, in the development of the ETOH-induced hypertension. It is already known that both OT and PRL may be used as stress markers in rodents (Lang et al., 1983). In this regard, we found that OT mRNA expression in the PVN was only altered by ETOH at the end of the experiment, although no significant alterations in OT secretion were observed. This apparent dissociative effect between the OT hormone release and mRNA expression could be correlated with the recruitment of diverse PVN subpopulations for OT production and release to the circulation or to other CNS areas, including those related to the control of autonomic function (Swanson and Sawchenko, 1983). PRL, similar to OT, was not altered in response to ETOH. Therefore, considering that both OT and PRL seem to play an important role in stress responses, we could not observe a clear relationship between the secretion of these hormones and a stress-mediated response elicited by ETOH intake.

We also demonstrated that ETOH consumption decreases body weight and fluid intake. We found that both effects appear early during ETOH treatment and progressively increase with time and in parallel with ETOH percentage, especially the fall in body weight gain, which may occur due to a decrease in food and/or liquid intake. In this regard, Gill and colleagues (Gill et al., 1996) had previously stated that there is no direct association between total food intake and total alcohol intake, since no compensation for the extra calories ingested in the form of alcohol via a reduction in total food intake was observed. Furthermore, they also showed that there were no significant differences between water and ETOH in terms of their distribution in relation to food. However, in the present study, we could not clearly dissociate water and ETOH intakes because the solutions containing the increasing percentages of ETOH were the only source of water for the animals. It has been assumed here that the decrease in ETOH intake is not likely to be related to an aversive behavior, as previously reported by Vetter et al (Vetter et al., 2007). These authors have demonstrated that the consumption of ETOH solutions generally yielded positive preference scores compared with water.

Regarding effects on energy homeostasis, it has been already demonstrated that ETOH may cause deficient absorption and synthesis of essential nutrients (Morgan, 1982; Lieber, 1991; Bode and Bode, 1997). Considering that ETOH only produces energy when oxidized by alcohol dehydrogenase and that there is a limitation for the action of this enzyme, Gruchow and colleagues (Gruchow et al., 1985) observed that, despite their higher alcohol intake, drinkers were no more obese than were nondrinkers and concluded that alcohol calories may be less efficiently utilized. It has been proposed that ETOH can activate other mechanisms, such as thermogenesis (Sonko et al., 1994; Murgatroyd et al., 1996), which would consume its caloric content, resulting in energy expenditure instead of energy production (Pirola and Lieber, 1976; Lands and Zakhari, 1991). Aguiar et al. (2004) showed that well-nourished rats cannot utilize ETOH-derived calories and, therefore, lose weight. In this regard, animal studies are consistent in reporting a decrease in the body weight of rats receiving ETOH solutions as the only source of liquids with concentrations as low as 5% or as high as 40% (v/v) (Laure-Achagiotis et al., 1990; Macieira et al., 1997).

In conclusion, our results suggest that increased sympathetic activity may contribute to the early increase in MAP observed in ETOH-treated rats. The maintenance of this mild hypertensive state, however, is likely to involve a long-term increase of vasoactive peptides such as AVP and ANG II, as this effect is potentially induced by the dehydration observed after ETOH exposure (Fig. 5). Moreover, these results also indicate that the HPA axis, PRL and the natriuretic systems may not participate in the generation of the altered responses induced in the early phase of ETOH consumption.

Fig. 5.

Schematic time-course diagram of cardiovascular and neuroendocrine changes induced by ETOH consumption [first week: 5%, second week: 10%, third and fourth weeks: 20%, v/v]. The ETOH-induced dehydration (characterized in the present study by the increase in hematocrit and plasma protein concentrations, hyperosmolality and decreased urinary volume) initially causes an increase in plasma catecholamines (adrenaline-Adr and noradrenaline-Nor), an effect that is temporally correlated with the increase in MAP. The maintenance of this hypertensive state, however, is likely to be mediated by the dehydration-induced increase in vasopressin (AVP) and ANG II secretion.


This work was financially supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (individual PhD grant for Andreia Lopes da Silva 2009/51676-9, main researcher grant for Jose Antunes Rodrigues 2008/50611-8) and Conselho Nacional de Desenvolvimento Tecnologico (CNPq, grant number 301827/2011/7). Conflict of interest statement The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.


View Abstract