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Ethanol Consumption Alters the Expression and Reactivity of Adrenomedullin in the Rat Mesenteric Arterial Bed

Juliana T. Rocha, Ulisses V. Hipólito, Alisson Martins-Oliveira, Daniela P.C. Tirapelli, Marcelo E. Batalhão, Evelin C. Carnio, Regina H. Queiroz, Eduardo B. Coelho, Thiago M. Cunha, José E. Tanus-Santos, Carlos R. Tirapelli
DOI: http://dx.doi.org/10.1093/alcalc/agr141 9-17 First published online: 21 October 2011

Abstract

Aims: Adrenomedullin (AM) is a peptide that displays cardiovascular protective activity. We investigated the effects of chronic ethanol consumption on arterial blood pressure, vascular reactivity to AM and the expression of AM system components in the rat mesenteric arterial bed (MAB). Methods: Male Wistar rats were treated with ethanol (20% vol/vol) for 6 weeks. Systolic, diastolic and mean arterial blood pressure were monitored in conscious rats. Vascular reactivity experiments were performed on isolated rat MAB. Matrix metalloproteinase-2 (MMP-2) levels were determined by gelatin zymography. Nitrite and nitrate generation were measured by chemiluminescence. Protein and mRNA levels of pre-pro-AM, CRLR (calcitonin receptor-like receptor) and RAMP1, 2 and 3 (receptor activity-modifying proteins) were assessed by western blot and quantitative real-time polymerase chain reaction, respectively. Results: Ethanol consumption induced hypertension and decreased the relaxation induced by AM and acetylcholine in endothelium-intact rat MAB. Phenylephrine-induced contraction was increased in endothelium-intact MAB from ethanol-treated rats. Ethanol consumption did not alter basal levels of nitrate and nitrite, nor did it affect the expression of MMP-2 or the net MMP activity in the rat MAB. Ethanol consumption increased mRNA levels of pre-pro-AM and protein levels of AM in the rat MAB. Finally, no differences in protein levels or mRNA of CRLR and RAMP1, 2 and 3 were observed after treatment with ethanol. Conclusion: Our study demonstrates that ethanol consumption increases blood pressure and the expression of AM in the vasculature and reduces the relaxation induced by this peptide in the rat MAB.

INTRODUCTION

Chronic ethanol consumption elevates blood pressure, and this phenomenon is thought to be a mechanism underlying the increased risk that is associated with excessive ethanol intake (Klatsky et al., 1977). The exact mechanism(s) by which chronic ethanol consumption causes a related rise in blood pressure is not yet completely known. Current investigation in this field has centered on areas known to be involved in human hypertension. In this line, it has been suggested that enhanced vascular reactivity to vasoconstrictor agents or impairment of the vascular relaxation contribute to the cardiovascular complications associated with chronic ethanol consumption (Kahonen et al., 1999; Utkan et al., 2001). However, most of the experiments designed to study the relation between alterations in the vascular functionality and the increase in blood pressure induced by ethanol consumption use conduit vessels such as the aorta (Utkan et al., 2001; Tirapelli et al., 2006). While the aorta does not offer great resistance to flow, the contribution made by vessels of smaller diameter to peripheral vascular resistance is much greater. The mesenteric circulation of the rat receives approximately one-fifth of the cardiac output and, thus, regulation of this bed makes a significant contribution to the regulation of systemic blood pressure (Nichols et al., 1985). More recently, data from our laboratory showed that ethanol intake reduces the endothelium-dependent relaxation induced by acetylcholine in the rat mesenteric arterial bed (MAB), further indicating that ethanol consumption alters the reactivity of peripheral vascular resistance arteries (Tirapelli et al., 2008).

Some studies have demonstrated that paracrine/autocrine factors contribute to the alteration in vascular function induced by ethanol and in the pathogenesis of cardiovascular diseases associated with ethanol consumption. Among these factors, vasoactive peptides, such as endothelin-1 and angiotensin II were described to contribute to the cardiovascular dysfunction induced by ethanol (Nanji et al., 1994; Husain et al., 2008). On the other hand, ethanol injury up-regulates the expression of the peptide adrenomedullin (AM) and its receptor in the gastric mucosa of rats (Wang et al., 1999). Interestingly, the expression of AM can be increased during cardiovascular diseases where it may have a role in several cardiovascular protective actions (Ishimitsu et al., 1994; Nishikimi et al., 1995).

AM was initially isolated from human pheochromocytomas cells and induces a potent and long-lasting hypotensive effect (Kitamura et al., 1993). Circulating AM is primarily synthesized and secreted from vascular endothelial cells and vascular smooth muscle cells. AM has a wide range of autocrine, paracrine and endocrine mechanisms, including vasodilatory, anti-apoptotic, angiogenic and anti-fibrotic actions. AM is therefore considered an important regulatory peptide that helps to regulate cardiovascular homeostasis. AM levels present in plasma and cardiovascular tissues are elevated to compensate for changes during cardiovascular diseases, such as atherosclerosis, hypertension, heart failure, acute myocardial infarction and pulmonary hypertension (Gibbons et al., 2007; Yanagawa and Nagaya, 2007). The action of AM is mediated by the seven-transmembrane G-protein-coupled calcitonin receptor-like receptor (CRLR), which co-assembles with subtypes 2 and 3 of the receptor activity-modifying proteins (RAMPs) family, thus forming a receptor-co-receptor system (McLatchie et al., 1998; Poyner et al., 2002). In the rat MAB, the relaxation induced by AM is endothelium-dependent and mediated mainly by nitric oxide (NO) (Champion et al., 2001).

Although the expression of AM can be increased in individuals with cardiovascular diseases, to the best of our knowledge, no studies have evaluated the expression of this peptide in the vasculature of ethanol-treated rats. We hypothesized that ethanol consumption increases the expression of AM in the rat MAB and alters the vascular reactivity to this peptide. In the present study, we investigated the effect of ethanol consumption on the expression of AM system components in the rat MAB. We also investigated the effect of ethanol consumption on blood pressure and MAB reactivity to AM.

MATERIALS AND METHODS

Experimental design

Male Wistar were housed under standard laboratory conditions with free access to food and water. The housing conditions and experimental protocols were approved by the Ethical Animal Committee of the University of São Paulo—Campus of Ribeirão Preto (Protocol number 10.1.201.53.9). The rats initially weighing 250–300 g (50–70 days old) were randomly divided into two groups: control and ethanol. Control rats received water ad libitum. Rats from the ethanol group received 20% (vol/vol) ethanol in their drinking water (Tirapelli et al., 2006, 2008). To avoid a considerable loss of animals, the ethanol-treated group was submitted to a brief and gradual adaptation period. The animals received 5% ethanol in their drinking water in the first week, 10% in the second week and 20% in the third week. At the end of the third week, the experimental stage began. The rats were treated for 6 weeks and weighted weekly.

Blood ethanol measurements

Blood was collected from the aorta of anesthetized rats (ketamine/xylazine, 80/10 mg/kg, i.p.) and ethanol analysis was carried out using a CG-17A gas chromatographer (Shimadzu, Kyoto, Japan) as previously described (Tirapelli et al., 2006).

Cannulation procedures and blood pressure measurements

Thirty-six hours before the experiment, animals were anesthetized with ketamine/xylazine (80/10 mg/kg, i.p.) and a polyethylene catheter was implanted into the femoral artery for direct recording of mean arterial pressure (MAP), systolic arterial pressure (SAP) and diastolic arterial pressure (DAP). The catheters were tunneled subcutaneously and exteriorized at the back of the neck between the scapulae. The blood pressure of conscious freely moving rats was recorded using a Grass polygraph (Grass P122, USA) connected to a pressure transducer (Grass, P23XL-1). On the day of the experiment, the arterial catheter was connected to a pressure transducer for the measurement of blood pressure, as mentioned above. A period of at least 30 min was allowed at the beginning of the experiment for stabilization of blood pressure. Basal MAP, SAP and DAP were measured and expressed as mean ± SEM.

MAB perfusion

The rat MAB isolated and perfused in vitro was used as a model of resistance vascular territory as previously described (Tirapelli et al., 2008). In brief, the rats were anesthetized with ketamine/xylazine (80/10 mg/kg, i.p.), the abdominal cavity was opened and the intestinal loops were exposed. The superior mesenteric artery was dissected close to its origin in the abdominal aorta and cannulated with a PE-50 polyethylene catheter. The MAB was perfused with 1 ml of Krebs solution (in mmol/l): 120.0 NaCl, 4.7 KCl, 25.0 NaHCO3, 2.4 CaCl2 2H2O, 1.4 MgCl2 6H2O, 1.17 KH2PO4 and 11.0 glucose containing 500 IU of heparin. The intestinal loops were removed en bloc, the MAB was separated by cutting close to the intestinal loops, and the preparation was placed in a chamber at 37°C. The cannulated superior mesenteric artery was coupled to a perfusion pump (LKB 2215 Multiperpex pump, Broma, Sweden) and the MAB was perfused with Krebs solution bubbled with 95% O2 and 5% CO2, pH 7.4, at a constant flow of 4 ml/min. A pressure transducer (R 511A, Beckman Inst., Schiller Park, IL, USA) was coupled in a ‘y’ arrangement to the system for perfusion pressure recording. The pre-amplified and filtered outlet signal was coupled to the data acquisition system DATAQ DI-150 (Akron, OH, USA) connected to the RS 232 parallel port of a Pentium II personal computer (Intel, USA), and stored for later analysis with the Windaq software, version 2.5 (DATAQ).

Vascular reactivity to AM, acetylcholine, sodium nitroprusside and phenylephrine

The rat MAB was perfused with Krebs solution and left to rest for 15 min for the stabilization of the basal perfusion pressure, whose mean values were continuously recorded. Dose–response curves for AM (15–180 ng or 2.5–300 pmol), acetylcholine (0.5–50 μg or 2.8 nmol–0.3 µmol) and sodium nitroprusside (SNP, 0.5–50 μg or 1.7 nmol–0.2 µmol) were obtained in MAB pre-contracted with a dose of phenylephrine capable of increasing the basal perfusion pressure by 60 mmHg. To avoid a possible influence of the pre-contracting levels induced by phenylephrine on acetylcholine-induced relaxation, the increase in perfusion pressure was evoked with a dose of phenylephrine capable of increasing the basal perfusion pressure with similar magnitude in tissues from control or ethanol-treated rats. Dose–response curves for phenylephrine (0.5–80 μg or 2.5 nmol–0.4 µmol) were obtained by injecting increasing doses of the agonist with a 50 μl Hamilton syringe. The interval between injections was 5 min or the time needed for perfusion pressure to return to initial values. The dose–response curves for phenylephrine and AM were performed in endothelium-intact and -denuded MAB. The endothelium was removed with a solution of sodium deoxycholate (1 mg/ml–2 ml, in bolus). Endothelial integrity was qualitatively assessed by the degree of relaxation caused by acetylcholine in the presence of contractile tone induced by phenylephrine. For studies of endothelium-denuded MAB, the tissues were discarded if there was any degree of relaxation. Relaxation was expressed as the percent change from the phenylephrine-contracted levels. In another set of experiments, dose–response curves for AM were obtained in the presence of NG-nitro-L-arginine-methyl-ester (L-NAME, a non-selective NOS inhibitor, 100 µmol/l). The agonist concentration-response curves were fitted using a nonlinear interactive fitting program (GraphPad Prism 3.0; GraphPad Software Inc., San Diego, CA, USA). Agonist potencies and maximal responses were expressed as EC50 (concentration of agonist producing 50% of the maximal response) and Emax (maximum effect elicited by the agonist), respectively.

Measurement of nitrite and nitrate generation

The MAB was removed, cleaned of adherent connective tissues and frozen in liquid nitrogen. Nitrite and nitrate levels were measured in the supernatants of total MAB homogenates prepared under liquid nitrogen. Aliquots of 10 μl were injected into a Sievers chemiluminescence analyzer (model 280) and pelleted by centrifugation with vanadium trichloride and HCl (at 95°C) which act as reductants for nitrate, or NaI and acetic acid, which function as reductants for nitrite. The results were normalized for protein concentration, as assessed by the Bradford technique.

Measurement of matrix metalloproteinase-2 levels by gelatin zymography and gelatinolytic activity assay

Tissues were homogenized in buffer containing 20 mmol/l Tris–HCl (pH 7.4), 1 mmol/l 1,10-phenanthroline, 1 mmol/l phenylmethylsulfonyl fluoride (PMSF), 1 mmol/l N-ethylmaleimide and 10 mmol/l CaCl2. Briefly, tissue extracts (50 µg of protein) that were normalized for protein concentration were subjected to electrophoresis on 7% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) gels co-polymerized with gelatin (1%) as the substrate. After electrophoresis, the gels were incubated for 1 h at room temperature in a 2% Triton X-100 solution, washed two times with water and incubated at 37°C for 16 h in Tris–HCl buffer (pH 7.4), containing 10 mmol/l CaCl2. The gels were fixed with 30% methanol and 10% acetic acid, stained with 0.05% Coomassie Brilliant Blue G-250 and then destained with 30% methanol and 10% acetic acid. Evidence of gelatinolytic activity was distinguished by the presence of unstained bands against the background of the Coomassie blue-stained gelatin. The staining differences were assayed by densitometry using a Kodak Electrophoresis Documentation and Analysis System 290 (Kodak, Rochester, NY, USA). Intergel analysis was possible after the normalization of gelatinolytic activity with an internal standard (fetal bovine serum 2%) (Ceron et al., 2010). Results are reported as ratio between bands density and internal standard value. Drugs and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The forms of matrix metalloproteinase-2 (MMP-2) were identified as bands at 75 and 72 kDa.

Net MMP activity in the MAB homogenates was measured using a gelatinolytic activity kit (E12055; Molecular Probes, Eugene, OR, USA). The protein contents of MAB homogenates were determined by the Bradford method (Sigma). Briefly, 60 μg of freshly prepared MAB extract was added to each microplate well and all determinations were carried out in duplicate. Proteolytic activity was measured in Tris–CaCl2 buffer (50 mmol/l Tris, 10 mmol/l CaCl2 and 1 μmol/l ZnCl2), using 5 μg/ml dye-quenched gelatin (E12055; Molecular Probes) as the substrate. Gelatinolytic activity was measured with a microplate spectrofluorimeter (at λexcitation 495, λemission 515 nm; Gemini EM, Molecular Devices, Sunnyvale, CA, USA) after 30 min of incubation at 37°C. A standard curve of gelatinolytic activity was prepared as recommended by the manufacturer of the kit. Vehicle alone, 1 mmol/l phenanthroline and 1 mmol/l PMSF were used to confirm the MMP activity in the MAB homogenates (Ceron et al., 2010).

Quantitative real-time polymerase chain reaction

Total cellular RNA was extracted using Trizol® Reagent (Invitrogen, Carlsbad, CA, USA), and RNA was reverse-transcribed to single-stranded cDNA using a High Capacity Kit (Applied Biossystems, Foster City, CA, USA) according to manufacturer's protocol. For the quantitative analysis of the genes of interest, which consisted of pre-pro-AM (Rn 00562327_m1), CRLR (Rn 00562334_m1), RAMP1 (Rn 01427056_m1), RAMP2 (Rn 00824652_m1) and RAMP3 (Rn 00571815_m1), we used the commercially available TaqMan Assay-on-Demand System, which consists of oligonucleotides and probes (Applied Biosystems). Reverse transcription was performed using 1 μg total RNA for each sample in 20 μl of the total reaction mixture. The cDNA obtained was diluted 1:10 and 4.5 μl was used for each 10 μl of the RQ-PCR mixture using the TaqMan Master Mix (Applied Biosystems). All reactions were carried out in duplicate and analyzed with the 7500 Sequence Detection System apparatus (Applied Biosystems). Data were analyzed using the ABI-7500 SDS software. The total RNA absorbed was normalized on the basis of the Ct value for the GAPDH gene (Rn 01775763_m1). The variation in expression among samples was calculated by the Embedded Imagemethod, with the mean delta Ct value for a group of six samples from control used for calibration.

Immunobloting

Frozen tissue was homogenized in lysis buffer (50 mmol/l Tris/HCl, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate and 1%, SDS). Homogenates were centrifuged at 5000g for 10 min, the nuclei-rich pellet was then discarded and the supernatant fluid was stored at −80°C. Fifty micrograms of protein was separated by electrophoresis on a 10 or 15% polyacrylamide gel, and transferred onto a nitrocellulose membrane. The 15% polyacrylamide gel was used for AM analysis. Non-specific-binding sites were blocked with 5% skim milk in Tris-buffered saline solution with Tween-20 for 60 min at 24°C. The membranes were then incubated with the following specific antibodies (Santa Cruz Biotechnology) overnight at 4°C: AM (sc-16496, 1:250), CRLR (sc-18007, 1:1000), RAMP1 (sc-11379, 1:250), RAMP2 (sc-11380, 1:250) and RAMP3 (sc-11381, 1:250). Beta-actin (sc-1616, 1:2000) was used as an internal control. After incubation with labeled secondary antibodies, signals were revealed with chemiluminescence, visualized by autoradiography and quantified densitometrically.

Statistical analysis

Data are presented as means ± SEM. Statistically significant differences were calculated by Student's t-test or analysis of variance followed by Bonferroni's multiple comparison test. P < 0.05 was considered as statistically significant.

RESULTS

Body weight and blood ethanol levels

The body weights of the rats prior to treatment averaged 276 ± 3 g in the control group and 281 ± 4 g in the ethanol group. Animals receiving ethanol in the drinking water for 6 weeks showed reduced body weight (454 ± 12 g) in comparison with age-matched control rats (523 ± 21 g) (P < 0.05; Student's t-test). Blood ethanol levels in the ethanol-treated rats averaged 1.67 ± 0.11 mg/ml (∼36 mmol/l).

Effect of ethanol consumption on blood pressure

There was a significant increase in MAP, SAP and DAP after treatment with ethanol for 6 weeks (Table 1).

View this table:
Table 1.

Effect of chronic ethanol consumption in MAP, SAP and DAP

ControlEthanol
Mean arterial pressure (mmHg)94 ± 1.5124 ± 1.1*
Systolic arterial pressure (mmHg)120 ± 1.1155 ± 1.6*
Diastolic arterial pressure (mmHg)78 ± 1.9104 ± 1.7*
  • Values are means ± SEM of six animals per group.

  • *< 0.05 compared with control group; Student's t-test.

Effect of ethanol consumption on MAB reactivity

The baseline perfusion pressure values of MAB from 6-weeks ethanol-treated rats (19 ± 0.9 mmHg, n = 12) were not different from those found for control (18 ± 0.8 mmHg, n = 12). Ethanol consumption reduced the relaxation induced by AM in endothelium-intact MAB (Fig. 1). Endothelial denudation completely abolished AM-induced relaxation in the rat MAB (data not shown). L-NAME reduced AM-induced relaxation in endothelium-intact MAB from control but not ethanol-treated rats (Fig. 1).

Fig. 1.

Effect of chronic ethanol consumption on the relaxation induced by AM in endothelium-intact rat MAB. The relaxation induced by AM was determined in endothelium-intact rat MAB from control and ethanol-treated rats in the absence or presence of L-NAME (100 µmol/l). Values are means ± SEM of 5–7 independent preparations. *< 0.05 compared with control group; ANOVA followed by Bonferroni's multiple comparison test.

The endothelium-dependent relaxation induced by acetylcholine was significantly different between control (Emax: 55.5 ± 1.6%; EC50: 4.6 ± 0.9 µg, n = 9) and ethanol-treated rats (Emax: 29.9 ± 3.4%; EC50: 7.1 ± 1.9 µg, n = 7) (P < 0.05; Student's t-test). The endothelium-independent relaxation induced by SNP did not significantly differ between control (Emax: 42.4 ± 3.1%; EC50: 7.5 ± 2.9 µg, n = 7) or ethanol-treated rats (Emax: 50.6 ± 3.4%; EC50: 8.4 ± 1.6 µg, n = 6). The maximum increase in perfusion pressure induced by phenylephrine was significantly higher in endothelium-intact MAB from 6-weeks ethanol-treated rats (137.6 ± 1.7 mmHg; EC50: 4.6 ± 1.4 µg, n = 7) when compared with the tissues from control animals (106.3 ± 3.3 mmHg; EC50: 5.3 ± 0.9 µg, n = 9) (P < 0.05; Student's t-test). However, no differences in the Emax values for phenylephrine were found after endothelial denudation (control: 144.8 ± 3.9 mmHg, EC50: 3.1 ± 0.4 µg, n = 8; ethanol: 138.5 ± 5.8 mmHg, EC50: 3.0 ± 0.3 µg, n = 7) (Fig. 2).

Fig. 2.

Effect of chronic ethanol consumption on the MAB reactivity to acetylcholine, SNP and phenylephrine. Concentration-response curves for acetylcholine and SNP were determined in endothelium-intact and endothelium-denuded rat MAB, respectively. The contraction induced by phenylephrine was determined in both endothelium-intact (Endo+) and denuded (Endo−) rat MAB. Values are means ± SEM of 6–9 independent preparations. *< 0.05 compared with control group; Student's t-test.

Effect of ethanol consumption on nitrite and nitrate levels

No difference on the basal nitrite generation (μmol/l/mg protein) was found between tissues from control (2.4 ± 0.2, n = 10) and ethanol-treated rats (2.0 ± 0.1, n = 10). Similarly, the treatment with ethanol did not alter nitrate levels (μmol/l/mg protein) in the rat MAB (control: 13.6 ± 0.5, n = 7; ethanol: 14.1 ± 0.3, n = 7).

Effect of ethanol consumption on MMP-2 levels and MMPs activity

Gelatin zymograms were used to assess MMP levels in MAB extracts. Figure 3A shows a representative zymogram of MAB extracts showing molecular weights of the MMP-2 bands. MAB from ethanol-treated rats showed no differences in the levels of MMP-2 (75 and 72 kDa) compared with those from control animals (Fig. 3B and C). We assessed net MMP activity in the MAB using a gelatinolytic activity assay, which provides a quantitative determination of total MMP activity in tissue homogenates. Total MMP activity was not altered after treatment with ethanol (Fig. 3D).

Fig. 3.

Representative SDS–PAGE gelatin zymogram of MAB samples (A). Molecular weights of MMP-2 bands were identified after electrophoresis on 7% SDS–PAGE gel. Std, internal standard, C, control, E, ethanol. Densitometric analysis for MAB 75 and 72 kDa MMP-2 were represented in (B) and (C), respectively. FBS 2%, fetal bovine serum 2%. Net gelatinolytic activity of MMPs (C). Values are means ± SEM of seven MAB for each group.

Effect of ethanol consumption on mRNA expression of pre-pro-AM, CRLR and RAMP 1, 2 and 3 in the rat MAB

The results obtained by quantitative real-time polymerase chain reaction (qRT-PCR) show that ethanol consumption increased the mRNA levels (fold 2Embedded Image) of pre-pro-AM in the rat MAB. However, no differences in the mRNA levels of CRLR, RAMP1, RAMP2 or RAMP3 were detected between control and ethanol groups (Fig. 4).

Fig. 4.

Effect of ethanol consumption on mRNA levels of pre-pro-AM, CRLR and RAMP1, 2 and 3 in the rat MAB. The levels of were quantified by qRT-PCR. The results are presented as the expression of the individual mRNAs with normalization to the housekeeping gene GAPDH by using the Ct method. Values are means ± SEM of 5–7 independent preparations.*< 0.05 compared with control group; Student's t-test.

Effect of ethanol consumption on the expression of AM, CRLR and RAMP1, 2 and 3 in the rat MAB

Western immunoblotting assays showed that ethanol consumption increased the expression of AM in the rat MAB. On the other hand, no differences in the expression of CRLR, RAMP1, RAMP2 or RAMP3 were observed after treatment with ethanol (Fig. 5).

Fig. 5.

Representative western immunoblotting products of 50 µg total protein extracted from endothelium-intact rat MAB from control (closed bars) and ethanol-treated rats (open bars). The bar graphs show the relative absorbance values of AM, CLRL, RAMP1, RAMP2 and RAMP3 bands. Values were normalized by the corresponding β-actin bands, used as internal standard. Results are reported as means ± SEM and are representative of five experiments for each group. *< 0.05 compared with control group; Student's t-test.

DISCUSSION

Mild hypertension following chronic ethanol ingestion was observed in the present study. Moderate ethanol consumption is generally considered to be in the range of 1–3 drinks/day (Klatsky et al., 1992; Thun et al., 1997), giving rise to blood ethanol levels of ∼5–25 mmol/l. In alcoholics, blood ethanol levels can reach 100 mmol/l (Kalant, 1971). Thus, the concentrations of ethanol found in our study (∼36 mmol/l) are physiologically relevant. Our findings are consistent with previous results demonstrating a strong relationship between ethanol consumption and hypertension (Klatsky et al., 1977; Tirapelli et al., 2007, 2008).

The regulation of the mesenteric circulation has a significant contribution to the regulation of systemic blood pressure since it receives approximately one-fifth of the cardiac output (Nichols et al., 1985). Interestingly, we have previously described that increased blood pressure induced by chronic ethanol intake alters the vascular reactivity of rat MAB by a mechanism that includes endothelial dysfunction and down-regulation of endothelial NO synthase expression (Tirapelli et al., 2008). The vascular relaxation induced by AM in the rat MAB is endothelium-dependent and involves the activation of the NO-cyclic guanosine monophosphate pathway (Champion et al., 2001). Thus, we hypothesized that ethanol consumption could affect AM-induced relaxation in the rat MAB. Our results show that chronic ethanol consumption reduces the endothelium-dependent relaxation induced by AM in the rat MAB. Since no differences could be detected in the relaxation induced by AM between control and ethanol-exposed tissues after incubation with L-NAME, it is possible that the reduced responsiveness of MAB from ethanol-treated to AM was due to an impaired modulation of NO on AM-induced relaxation. The finding that the relaxation induced by acetylcholine, but not SNP, was reduced in vessels from ethanol-treated rats indicates that chronic ethanol consumption does not decrease the action of NO. This observation suggests that ethanol decreases the endothelial cell receptor-stimulated production/release of NO. Moreover, chronic ethanol consumption produced an increased responsiveness to phenylephrine in endothelium-intact, but not denuded MAB, further indicating a role for the endothelium in the ethanol-induced increased responsiveness to phenylephrine. Therefore, the decreased reactivity of the MAB from ethanol-treated rats to AM cannot be explained by a specific action in the vascular AM system but rather by an impairment of the endothelial activity. Moreover, ethanol consumption did not alter the baseline perfusion pressure values of MAB or the basal levels of nitrite and nitrate. These findings strength our initial hypotheses that ethanol consumption decreases the endothelial cell receptor-stimulated production/release of NO rather than its basal release. Of note, our data demonstrate that ethanol alter the vascular reactivity to AM in concentrations that are physiologically relevant.

MMP-2 cleave AM and are involved in the metabolism of this peptide (Lewisa et al., 1997). The cleavage of AM by MMP-2 produces a vasoconstrictor out of a vasodilator peptide (Martínez et al., 2004). Long-term ethanol consumption was described to up-regulate MMP-2 activity in the aorta (Partridge et al., 1999). Our findings show that chronic ethanol consumption did not alter the expression of MMP-2 in the rat MAB, further suggesting that MMPs are not related to the reduced relaxation response to AM observed in our study. This observation corroborates our initial proposal that the decreased reactivity of the MAB from ethanol-treated rats to AM is not related to a specific action in the vascular AM system.

The actions of AM are generally protective and beneficial to the cardiovascular system, suggesting that increased AM expression or activity could act as a compensatory response to cardiovascular injuries or dysfunction (Hinson et al., 2000). In fact, AM levels are increased in the context of hypertension and heart failure (Ishimitsu et al., 1994; Nishikimi et al., 1995). Pre-pro-AM gene codifies pre-pro-AM, the precursor peptide of AM that consists of 185 amino acids. AM is produced from pre-pro-AM by a two-step enzymatic pathway. In the first step, pre-pro-AM precursor is converted to C-terminal glycine-extended AM (AM-Gly), a 53-amino-acid peptide that is an inactive intermediate form of AM. Subsequently, inactive AM-Gly is converted to the active form of mature AM (Kitamura et al., 1998). In the cardiovascular system, AM exerts its biological activity through receptor complexes composed of the CRLR and specific RAMP (McLatchie et al., 1998). The RAMP family has three single-transmembrane protein members, RAMP 1, 2 and 3, which transport CRLR to the cell surface and determine the specificity and affinity of ligands. The combination of CRLR and RAMP2 or RAMP3 comprises receptors for AM, termed AM1 and AM2 receptors, respectively (Poyner et al., 2002). In this report, we show that MAB from ethanol-treated rats exhibits a 5-fold increase in the mRNA expression of pre-pro-AM, compared with control rats. Moreover, ethanol consumption increased the protein levels of AM in the vasculature. Ethanol was previously described to up regulate AM expression in gastric mucosa of rats (Wang et al., 1999), but to the best of our knowledge this is the first report describing the effect of ethanol consumption in the expression of pre-pro-AM and AM in the vasculature. In the cardiovascular system, AM plays protective actions against the noxious influences of angiotensin II, endothelin-1 and oxidative stress (Ishimitsu et al., 2006), which were previously described to be increased in animals treated chronically with ethanol (Nanji et al., 1994; Husain et al., 2008). Given the evidence for a key role of AM in vascular protection, stimulation by ethanol of AM production in vivo would be expected to result in increased antioxidant protection, vascular relaxation and decreased in blood pressure. However, while ethanol increased AM expression in the rat MAB, our data demonstrate increased blood pressure and reduction in the relaxation induced by AM after treatment with ethanol. Thus, increased AM expression could not show protective actions against endothelial dysfunction induced by ethanol in our model. Blood ethanol content is a potential explanation for this observation. In fact, it is well known that the effect of ethanol on the cardiovascular system is dose-dependent (Abdel-Rahman et al., 1985; Abdel-Rahman and Wooles, 1987; Resstel et al., 2006). Whether AM can prevent the vascular dysfunction induced by doses of ethanol lower than that found in our study remains to be determined.

The levels of CRLR, RAMP1, 2 and 3 are described to be increased in cardiovascular diseases (Ishimitsu et al., 2006). Although ethanol consumption increased mRNA levels of pre-pro-AM, we observed that mRNA levels of CRLR and RAMP1, 2 and 3 were not altered by ethanol consumption. In fact, ethanol consumption is associated with both increase and decrease of mRNA levels (Li et al., 2004; Cullen et al., 2005). The differential effect of ethanol on mRNA levels is related to its action in transcriptional factors such as nuclear factor κ-B and activating protein-1 (Cullen et al., 2005; Yeligar et al., 2009). Moreover, ethanol alters mRNA expression by activating different pathways. For example, ethanol-induced up-regulation of endothelin-1 in endothelial cells has been show to involve activation of NADPH oxidase and hypoxia-inducible factor-1α (Yeligar et al., 2009). Interestingly, these pathways also regulate AM gene expression (Ishimitsu et al., 2006), which was described to be increased in our study.

Ever since the discovery of AM, efforts have been made to clarify the role of this bioactive peptide in blood vessels and a substantial amount of basic and clinical data has been accumulated. Several studies suggest that AM functions as a protective factor for blood vessel, exerting various vascular actions, mostly inhibitory against vascular damage and remodeling. Considering the increased expression of AM in cardiovascular diseases, AM is recognized as a peptide regulating blood pressure and circulation in cardiovascular diseases. Thus, although increased expression of AM was detected in the vasculature of rats chronically treated with ethanol, the reduction in AM-induced relaxation described here could be one of the mechanisms by which ethanol predisposes individuals to vascular dysfunction and hypertension.

The major new finding of the present study is that ethanol consumption increases the expression of AM in the vasculature. Moreover, we found that ethanol consumption increases blood pressure and reduces the vascular relaxation induced by AM in the rat MAB.

Conflict of interest statement. None declared.

Funding

This study was supported by grants and funds from Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (Processes number: 06/60076-7 and 10/09962-1).

Acknowledgement

We thank Sonia Dreossi for her technical assistance.

REFERENCES

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