1. Introduction
Angiotensin II (Ang II), the main product and mediator of the Renin-Angiotensin System (RAS), is recognized as a pleiotropic agent involved in numerous physiological actions, including its significant role in elevating blood pressure (hypertension), by acting on the AT1 receptor (AT1R) [1] [2] [3] [4] . The AT1R responds to Ang II stimulation provoking pressor effects and growth of cardiac myocytes, and vascular smooth muscle cells, as well as aldosterone secretion, renal tubular Na+ reabsorption, thirst, activation of sympathetic nervous system, cardiac ionotropic and chronotropic actions and cardiovascular inflammation, hypertrophy and fibrosis [4] . Thus, diminution of Ang II synthesis by inhibitors of the angiotensin-converting enzyme, or AT1R antagonism leads to the decrease of blood pressure and reversion of cardiac hypertrophy [4] .
Previous studies have demonstrated that Ang II upregulates the expression of α1-adrenergic receptors (α1-ARs), particularly α1D-AR, promoting growth in rat vascular smooth muscle cells [5] , and contributing to cardiac hypertrophy and increased aorta contraction in the AHR−/− null mouse [2] [6] . Continuous Ang II exposure has been reported to induce aortic vascular hypertrophy in the rats, which could be prevented and reverted by the α1D-AR antagonist BMY-7378 [3] . This phenomenon was associated with an enhanced contractile response to the α1-AR agonist, phenylephrine, and correlated with aorta hypertrophy, and a reduction in both mRNA and protein of the α1D-AR [3] , suggesting that Ang II desensitized the α1D-AR in vivo, following the hypertrophic process, without significantly affecting α1A- or α1B-ARs [3] . Furthermore, Godínez et al. reported that captopril diminished the expression and function of the α1D-AR in young, pre-hypertensive SHR [7] ; whereas Rodríguez et al. showed that cardiac hypertrophy observed in the aged SHR was reverted by captopril and by BMY-7378, suggesting the interplay between ACE/AT1R and α1D-AR during heart hypertrophy [4] [8] . It is not clear if the increase in blood pressure and cardiovascular hypertrophy are due solely to Ang II acting on AT1R, or if it is added to noradrenergic action on α1D-AR [3] [8] . Our recent findings indicate that endogenous norepinephrine (NE) desensitizes α1D-AR when the aorta is cultured 24 h in DMEM, whereas the α1D-AR antagonist, BMY-7378 protects the α1D-AR from desensitization [9] . Consequently, this study aims to elucidate the influence of Ang II on the expression and function of α1-adrenergic receptors in rat aorta and vascular smooth muscle cells.
2. Materials and Methods
2.1. Animals and Ethical Statement
Male Wistar rats, aged 3 months and weighing 250 - 300 g, were housed under pathogen-free conditions with controlled parameters (40% - 60% humidity, 22˚C ± 2˚C, and a 12 h light/dark cycle), in our vivarium. They had ad libitum access to food and water. All animal care and experimental procedures were conducted in accordance with the Mexican Regulations of Animal Care and Use (NOM-062-ZOO-1999, SAGARPA, Mexico), and were consistent with the Guide for the Care and Use of Laboratory Animals, as promulgated by the U.S. National Institutes of Health [10] . The Institutional Ethics Committee of FES Iztacala, UNAM, approved all procedures (Protocol 1497).
2.2. Procedures
2.2.1. Incubation Conditions
Rats were euthanized, and the thoracic aortas were carefully dissected and cleaned of surrounding adipose tissue. In a laminar flow hood, the isolated aortas were sectioned into rings measuring 4 - 5 mm in length. To exclude the influence of endothelium-derived factors on the contractile response, the endothelium was gently removed with a rugged metal. The effectiveness of the endothelium removal was verified by the absence of relaxation to carbachol (1 × 10−6 M) [11] . Subsequently, the arterial rings were immersed in 3 ml of Dulbecco’s Modified Eagle Medium (DMEM), within a 6-well culture plate. These plates were incubated in a CO2 incubator at 37˚C (model BB 150, Thermo Scientific, Waltham, MA, USA), maintaining an atmosphere of 95% air and 5% CO2, for 24 h [9] .
2.2.2. Concentration-Response Curves (CRC)
The arterial rings were placed in 10 ml organ chambers filled with Krebs-Henseleit solution, maintained at 37˚C and pH 7.4. The solution had the following composition (in mM): NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25; glucose, 11.1 [3] . It was continuous bubbling with a gas mixture of 95% O2 and 5% CO2. Each arterial ring was connected to an isometric FT03E Grass force displacement transducer (Astro-Med, Inc., West Warwick, RI, USA). This transducer, in turn, was connected to a MP100A data acquisition system (Biopac Systems Inc., Santa Barbara, CA, USA), which recorded the isometric tension response. The aortic rings were adjusted to an optimal tension of 3 g [3] [12] .
2.2.3. α1-Adrenergic Receptor Stimulation in Aorta Exposed to Angiotensin II
Upon completion 24 h of incubation in DMEM, the aortic rings were transferred to the recording chamber. They were then exposed to norepinephrine (1 × 10−7 M) in the presence of rauwolscine (1 × 10−7 M) and propranolol (1 × 10−7 M), to antagonize α2- and β-adrenergic receptors, respectively. This solution was changed every 30 min over a 2 h period to allow for stabilization. Subsequently, a reproducible cumulative concentration-response curve (CRC) to norepinephrine was established, with concentrations ranging from 1 × 10−10 M to 1 × 10−4 M, increasing in half logarithm increments to establish a control curve.
In a parallel set of experiments, aortic rings were incubated in DMEM, supplemented with a constant concentration of Ang II (1 × 10−5 M) for 24 h. After this period, the aortic rings were transferred to the recording chamber and subjected to incremental half-logarithm concentrations of norepinephrine from 1 × 10−10 M to 1 × 10−4 M [3] [7] .
2.2.4. α1-Adrenergic Receptor Antagonism
To evaluate the effect of Ang II on α1-AR-mediated response, aortic rings were first incubated in DMEM for 24 h. Subsequent to this incubation, the rings were exposed to selective α1-ARs antagonists prior to being challenged with escalating concentrations of norepinephrine. The antagonists employed were 5-Methylurapidil for α1A-AR, AH11110A for α1B-AR, and BMY-7378 for α1D-AR [13] [14] [15] . The purpose of this protocol was to identify the specific α1-AR contributing to the contractile response to norepinephrine following 24 h incubation with Ang II (1 × 10−5 M).
2.2.5. Isolation and Culture of Aorta Smooth Muscle Cells
The aorta was obtained as described in section 2.2.1, followed by the removal of the endothelium via gently rubbing. The arterial segments were treated with collagenase II (2 mg/ml) during 15 min at 37˚C to facilitate the mechanical removal the adventitia layer, under a stereoscope (Zeiss Stemi 2000-C; Carl Zeiss, Oberkochen, Baden-Württemberg, Germany). Subsequently, smooth muscle cells were disaggregated using a combination of collagenase II and elastase (5 mg/ml and 0.1 mg/ml, respectively). Afterwards, the cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 U/ml) (Gibco), at maintained at 37˚C in a humidified 5% CO2 atmosphere. The medium was replenished every two days until the cells attained 90% - 95% confluency. The cells were kept quiescent in DMEM without FBS, after which they were exposed to Ang II (1 × 10−7 M) for various durations: 0.5, 1, 2, 4, 8, 12, and 24 h [4] . Smooth muscle cell morphology was verified through immunofluorescence using α-actin as a marker [16] .
2.2.6. Angiotensin II Influence on α1-ARs Protein Expression in Smooth Muscle Cells
The expression of α1-ARs proteins was detected by Western Blot analysis following the exposure of smooth muscle cells to Ang II (1 × 10−7 M), using specific antibodies (kindly provided by Dr. JA García-Sáinz) (10 μg per sample) were resolved on 10% SDS-PAGE under denaturing conditions, and subsequently transferred to a PDVF membrane using a Semi-Dry Transfer Blot system (Bio-Rad Labs., Hercules, CA, USA).
Blocking of non-specific binding was achieved with 5% non-fat milk dissolved in TBST. The membranes were incubated overnight at 4˚C with rabbit polyclonal antibodies to each α1-AR or to β-actin (Santa Cruz Biotechnology), at dilutions of 1:3000 and 1:1000, respectively, in non-fat milk. After thorough washing, membranes were exposed to goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Zymed Laboratories Inc., San Francisco, CA, USA) at a dilution of 1:1000 for 1 h at room temperature, followed by extensive washing. Detection was conducted using chemiluminescence with Luminol and captured on Hyperfilm (Amersham Biosciences, GE Healthcare, Buckinghamshire, UK). Densitometry was performed on bands corresponding to α1A-AR and α1D-AR (~72 kDa) and α1B-AR (~60 kDa) using a FLA-5000 scanner (Fujifilm).
2.2.7. Materials
All reagents were prepared either in Krebs-Henseleit solution or distilled water. Solutions were freshly prepared for every experiment. The compounds used, including Angiotensin II, (±)-Norepinephrine-HCl, (±) Propranolol-HCl, Rauwolscine-HCl, Carbachol-HCl, 5-Methylurapidil (5-MU, 5-Methyl-6[[3- [4-(2- methoxyphenyl)-1-piperazinyl]propyl]amino]-1,3-dimethyluracil), AH11110A (AH, 1-[Biphenyl-2-yloxy]-4-imino-4-piperidin-1-yl-butan-2-ol hydrochloride), BMY-7378 (BMY, 8-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro [4.5]decane-7,9-dione dihydrochloride), collagenase II, elastase, dithiotreitol, were obtained from Sigma-Aldrich (St. Louis, MO, USA). DMEM, fetal bovine serum, penicillin and streptomycin were purchased from Gibco (Thermo Fisher Scientific). All other reagents were of analytical grade and were obtained from local sources.
2.2.8. Statistical Analysis
Values for pD2 (-log EC50) were derived using nonlinear regression, while pA2 values were determined through Schild analysis, or pKB [17] [18] . Data are expressed as means ± standard error of the mean (SEM), based on observation from 8 rats per experimental group. Statistical evaluations were conducted by analysis of variance (ANOVA) followed by Bonferroni’s or Dunnett’s post hoc test, with differences statistically significant set at p < 0.05.
3. Results
To assess the viability of aortic rings after incubation of 24 h at 37˚C in DMEM, contractions were induced using high KCl (80 mM), which depolarizes the membrane, promoting Ca2+ entry into muscle cells and thus inducing contraction independent of receptor activation [19] . Figure 1 demonstrates that high KCl induced contraction in aortic rings following a 24 incubation at both 37˚C and 4˚C in DMEM, indicating that the incubation conditions did not modify tissue responsiveness.
The concentration-response curve (CRC) for norepinephrine and the α1-ARs antagonism was explored; Figure 2 displays control curve response of aortic rings incubated with different α1-ARs antagonists. Figure 2A shows the norepinephrine CRC and the rightward shift by the α1A-AR antagonist 5-Methylurapidil (5-MU), suggesting the presence of multiple receptor populations as inferred from a non-unitary slope in Schild analysis; the pKB was subsequently calculated to be 8.2. Figure 2B shows a rightward CRC shift in response to the α1B-AR antagonist AH11110A, with a pKB of 6; while Figure 2C demonstrates that BMY-7378 (α1D-AR antagonist) caused a rightward CRC shift with a pA2 of 8.9, whereas the average pD2 for norepinephrine was 8.3 ± 0.1.
In an attempt to identify which α1-AR was involved in the desensitization, aortic tissue was incubated for 24 h in separate assays, with RS-100329 (1 × 10−8.5 M, a highly selective α1A-AR antagonist, pA2 = 9.2/pKi = 9.6, [20] ), prazosin (1 × 10−9 M, nonselective α1-ARs antagonist, pA2 = 9.2, [21] ), or BMY-7378 (1 × 10−7 M, a highly selective α1D-AR antagonist, pA2 = 8.9/pKi = 9.4, [14] ). All three α1-ARs antagonists protected, in a different pattern the α1-ARs from desensitization; where BMY-7378 avoided desensitization, followed by partial protection by RS-100329, and by prazosin (Figure 3).
![]()
Figure 1. Time-course of aortic contraction induced by high KCl (80 mM) following incubation for 24 h at 4˚C (○) and 24 h at 37˚C (●), both in DMEM. n = 8 rats.
![]()
Figure 2. Concentration-Response Curves to norepinephrine (NE) in the aorta, and the displacement due to α1-ARs antagonists. The control curve represents NE-induced contraction (●), while the curves with (○, △, ▼) depict NE-induced contraction in the presence of (A) 5-Methylurapidil, (B) AH11110A, and (C) BMY-7378. n = 8 rats.
![]()
Figure 3. Desensitization of α1-ARs and protection by antagonists: Concentration-Response Curves to norepinephrine in the aorta incubated at 37˚C in DMEM at zero time (Control, ●), or for 24 h alone (○), or with added RS-100329 (1 × 10−8.5 M, g), prazosin (1 × 10−9 M, △), or BMY-7378 (1 × 10−7 M, ▼). n = 8 rats.
After a 24 h incubation at 37˚C in DMEM, a rightward shift of the norepinephrine CRC and reduction in maximal contraction were observed, indicative of α1-ARs desensitization. The calculated pD2 for noradrenaline under these conditions was 6.6 ± 0.1 vs. 8.3 ± 0.1, while the maximal effect was reduced to 2.5 ± 0.2 vs. 3.5 ± 0.1 in the non-incubated arteries.
Aortic arteries incubated for 24 h at 37˚C in DMEM with Ang II (1 × 10−5 M), exhibited a rightward CRC shift to norepinephrine and a reduced maximal contraction (pD2 = 6.6 ± 0.1), demonstrating desensitization to the catecholamine. The presence of Ang II further decreased the maximal norepinephrine response (Emax, 3.0 ± 0.3 g vs. 2.2 ± 0.2 g, Figure 4), without changing the pD2 for norepinephrine (6.7 ± 0.1). The α1-ARs antagonism did not produce further CRC shifts to norepinephrine, with pKB values of 6.5 for 5-MU and 7.4 for AH11110A, which unexpectedly caused a leftward shift; while BMY-74378 showed a pKB of 7.3 (Figures 5A-C), confirming the modulatory effect of Ang II on α1-ARs function.
This result prompted us to evaluate the action of Ang II on smooth muscle cells derived from rat aorta. As observed in Figure 5, Ang II (1 × 10−7 M) diminished the protein expression of α1A-AR between 1 and 4 h, restoring the expression at the basal value from 8 to 24 h (Figure 6A). α1B-AR expression remained unchanged over a 0.5 to 24-hour incubation period (Figure 6B), whereas α1D-AR was upregulated from 0.5 to 2 h reaching basal values afterwards (Figure 6C). Additional experimentation revealed that inhibiting protein synthesis with cycloheximide (CHX 10 μg/ml), as well as antagonizing the AT1 receptor with losartan (1 × 10−5 M), diminished Ang II-induced α1D-AR expression below basal value, with a more pronounced effect observed with CHX (Figure 6D).
4. Discussion
Angiotensin II, a pleiotropic agent, is implicated in various pathologies, including cardiovascular hypertrophy, hypertension, renal damage, among other pathologies [1] [2] [3] [4] [6] [7] [8] , and has been reported to upregulate α1-ARs in vascular smooth muscle cells and tissue, and in aryl hydrocarbon receptor (AHR−/−) null mouse aorta [5] [6] [7] . Contrary to our expectations that Ang II would enhance α1-ARs function in aorta after 24 h incubation in DMEM, however,
![]()
Figure 4. Desensitization of α1-ARs due to incubation at 37˚C in DMEM with Ang II (○) or without (Control, ●). n = 8 rats.
![]()
Figure 5. Concentration-Response Curves to norepinephrine (NE) in aorta incubated at 37˚C in DMEM 24 h with Ang II (1 × 10−5 M), and the displacement due to α1-ARs antagonists. The control curve represents NE-induced contraction (●), while the curves with (○, △, ▼) depict NE-induced contraction in the presence of (A) 5-Methylurapidil (5 MU), (B) AH11110 (AH), and (C) BMY-7378 (BMY). n = 8 rats.
![]()
Figure 6. Effect of Ang II on α1-ARs protein expression in aorta smooth muscle cells culture. Upper panels represent the action of Ang II on α1A-, α1B-, and α1D-ARs protein expression along 24 h. ANOVA followed by Bonferroni’s multiple comparison test: f p < 0.05 compared to 0.5 hr and *p < 0.05 compared to control. The lower panel represents the effect of losartan (LOS, AT1R antagonist) or Cycloheximide (CHX, protein translation inhibitor), on the Ang II-induced increase of α1D-AR protein expression. ANOVA followed by Dunnett’s multiple comparison post hoc test: * p < 0.05 compared to control. The sample size was n = 8 rats.
we observed two phenomena: incubation per se decreased both maximal effect and affinity of α1-ARs in vascular tissue [9] , and Ang II addition decreased further the contractile maximal response to norepinephrine without affecting affinity.
Previous reported contrasting results showed that an increase in circulating Ang II, either through AHR−/− knockout, or continuous infusion, provoked augmented maximal contractions to phenylephrine or noradrenaline in isolated aorta, suggesting that in vivo, constant Ang II exposure integer a whole animal’s response versus what is observed in isolated aorta [3] [6] .
The absence of a significant shift with α1A- and α1D-ARs antagonists in Ang II-treated tissue suggests that norepinephrine-induced contraction might be mediated by α1B-AR activation. However, competitive antagonism of α1B-AR with AH11110A resulted in a leftward CRC shift, indicating that α1B-AR might be modulating the action of norepinephrine on the other α1-ARs. This hypothesis could be supported with previous findings of no response to norepinephrine with the α1B-AR alkylating antagonist, chloroethyl clonidine (CEC), described previously [9] and confirmed in this study (not shown).
These discrepancies prompted us to evaluate Ang II action on the α1-ARs expression in isolated smooth muscle cells. Hu et al. reported that Ang II (1 × 10−7 M) increased α1-ARs RNA up to 70% above basal, in a time-dependent manner with a maximal effect at 8 h in vascular smooth muscle cells [5] . Similarly, they observed a significant transient increase in α1A/D-AR (currently identified as α1D-AR) expression after Ang II exposure (~2.5 fold above basal at 2 h after treatment), which returned to baseline by 24 h [5] . Our results show a similar pattern at the earlier times, the α1D-AR was overexpressed 30 min after Ang II treatment followed by a time-dependent decrease until basal values, suggesting that the peptide effects on the α1D-AR occur soon after its interaction with AT1R.
Furthermore, Hu et al. showed that blocking α1-ARs with the irreversible antagonist phenoxybenzamine (PBZ), significantly reduced α1-ARs, (~6 times; from 70 to 12 fmol/mg protein, control vs. PBZ), yet Ang II was able to increase eight times α1-ARs after PBZ treatment (from 12 to 96 fmol/mg protein). In line with this, our study reveals that both losartan, an AT1R antagonist, and cycloheximide (CHX), a protein synthesis inhibitor, acting on different targets diminished the action of Ang II on the α1D-AR expression below basal value [5] . This suggests that AT1R blockade leads to downregulation of α1D-AR expression and that prevention of protein translation inhibits the expression of α1D-AR. It is not clear at what step of signal amplification these two pathways interact, but it is known that receptor heterodimerization occurs between AT1R and α1D-AR [22] ; then it would be interesting to define if these receptors’ interaction promotes α1D-AR activation in the absence of catecholamines, that leads to muscle growth. It is important to mention that integration of hormone signaling between two pathways, i.e., RAS and α1-adrenergic, the so-called cross-talk, with physiology or pathophysiology leads to a better understanding of how the neural and cardiovascular systems work to keep body homeostasis.
5. Conclusion
Angiotensin II exerts a biphasic action on α1-ARs, at early times it increases α1D-AR, diminishes α1A-AR, and has no effect on α1B-AR; while at longer times it adds to incubation-induced desensitization on maximal aorta contraction. This initial increase of α1D-AR may trigger later effects on cellular machinery that promotes growth; so, it is interesting to block enzymatic steps downstream of signal amplification, in order to identify those steps involved in the gene expression, both of receptors and of proteins related to muscle growth.
These observations highlight the necessity for further studies to elucidate the apparently different actions of Ang II on cells vs. aortic tissue, specifically in terms of α1-ARs expression and functionality.
Acknowledgements
This study was supported in part by grants IN210222 (to RV-M) and IN221123 (to IAG-O) provided by PAPIIT, DGAPA, UNAM. The authors extend their gratitude to MVZ Leticia Flores, Anayántzin P. Heredia, PhD, and Biól. Tomás Villamar for their assistance in the care and maintenance of animals.