Background

Ascorbic acid is an essential vitamin and a powerful antioxidant. Many studies have highlighted the benefits of ascorbic acid for chronic cardiovascular diseases such as hypertension in which angiotensin II (Ang II) plays an significant role. We therefore hypothesized that ascorbic acid could modify the pharmacological properties of the AT₁ receptor for Ang II

Methods

Binding studies and Ca²⁺ mobilization studies were performed with HEK293 cells stably expressing the AT₁ receptor for Ang II. Smooth muscle contraction studies were performed with rabbit aorta strips that endogenously express the AT₁ receptor.

Results

Scatchard analysis revealed that ascorbic acid decreased the binding affinity of the AT₁ receptor without modifying its maximal binding capacity. Ascorbic acid did not modify the binding affinity of the AT₂ receptor for Ang II or of the UT receptor for urotensin II. In single-cell Ca²⁺ imaging assays, ascorbic acid reduced the frequency of intracellular Ca²⁺ oscillations induced by a low dose of Ang II. In functional assays, ascorbic acid significantly diminished the contraction of rabbit aorta pre-contracted with Ang II but not those pre-contracted with urotensin II.

Conclusions

Ascorbic acid decreases the binding affinity of the AT₁ receptor. These results offer a mechanistic explanation for the reported blood pressure lowering effect of ascorbic acid.

Methods

Materials. Dulbecco's modified Eagle's medium, fetal bovine serum, and penicillin–streptomycin were purchased from Gibco Life Technologies (Gaithersburg, MD). Fugene 6 was purchased from Roche Diagnostics (Indianapolis, IN). ¹²⁵I-Ang II (1000 Ci/mmol) was prepared using Iodogen (Pierce Chemical, Rockfield, IL) as previously described and purified by high-performance liquid chromatography on a C-18 column.¹⁸ Bovine serum albumin and bacitracin were purchased from Sigma (St. Louis, MO). Ascorbic acid was purchased from Fisher Scientific (Nepean, Ontario, Canada).

Cell cultures and transfections. HEK-293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 50 IU/ml of penicillin, and 50 g/ml of streptomycin. At 70% confluency the cells were transfected in serum-free Dulbecco's modified Eagle's medium containing 4 g of DNA and 8 l of Fugene 6. Forty-eight hours post-transfection, the cells were used for binding experiments. The HEK-293 cell line stably expressing the human AT₁ receptor was developed in our laboratory as previously described.¹⁹

Ang II binding experiments. Cells were frozen in liquid nitrogen, thawed in ice-cold phosphate-buffered saline and broken by five cycles of aspiration–expulsion with a 10 ml serological pipette tightly apposed to the bottom of the dish. Broken cells were centrifuged at 2500g for 15 min at 4 °C and resuspended in binding buffer (25 mmol/l Tris–HCl pH 7.4, 100 mmol/l NaCl, 5 mmol/l mgCl₂, 0.1% bovine serum albumin, and 0.01% bacitracin). Broken cells were incubated for 1 h at room temperature in binding buffer containing increasing concentrations of ¹²⁵I-Ang II, in the presence or absence of ascorbic acid. Bound and free ligands were separated by filtration through GF/C filters presoaked for 2 h in binding buffer. Nonspecific binding was measured in the presence of 1 mol/l unlabeled Ang II.

Dynamic video imaging of cytosolic Ca²⁺. HEK-293 cells stably expressing the AT₁ receptor were grown for 24 h on glass coverslips (no. 1) in complete Dulbecco's modified Eagle's medium. They were washed twice with HEPES-buffered physiological saline (Hank's-balanced salt solution: 20 mmol/l HEPES pH 7.4, 120 mmol/l NaCl, 5.3 mmol/l KCl, 0.8 mmol/l mgSO₄, 1.8 mmol/l CaCl₂, and 11.1 mmol/l dextrose) and incubated with 0.2 mol/l fura 2-acetoxymethyl ester for 20 min in the presence or absence of 100 mmol/l ascorbic acid. The cells were then washed and bathed in fresh Hank's-balanced salt solution for 20 min in the presence or absence of 100 mmol/l ascorbic acid to ensure complete hydrolysis of the fura 2-acetoxymethyl ester before mounting the Teflon chamber onto the stage of a Axiovert inverted microscope (Carl Zeiss, Thornwood, NY) fitted with an Attofluor Digital Imaging and Photometry System (Attofluor, Rockville, MD). Fluorescence was monitored at excitation wavelengths of 334 and 380 nm and emission wavelength of 510 nm. Data acquisition was typically at 3-s intervals and lasted for 30 min.

Rabbit aorta strip contraction. New Zealand White rabbits (1.5–2.5kg) were anesthetized by intramuscular injection of a mixture of ketamine (48 mg/kg) and xylazine (7 mg/kg). All experimental procedures conformed to Canadian Council on Animal Care guidelines and were approved by the Ethics Committee for Animal Research of the University of Sherbrooke Medical School. Helical strips of aorta devoid of endothelium were prepared as previously described.²⁰ The tissues were suspended in 10-ml organ baths containing warm (37 °C) oxygenated (95% O₂/5% CO₂) Krebs' (118 mmol/l NaCl, 4.7 mmol/l KCl, 2.5 mmol/l CaCl₂, 1.2 mmol/l KH₂PO₄, 1.2 mmol/l mgSO₄, 25 mmol/l NaHCO₃, and 5.5 mmol/l dextrose). The aorta strips were stretched at an initial tension of 2 g and were allowed to equilibrate for 1–2 h. Isometric contractions were recorded using a Grass Instrument Model 7D polygraph.

Results

Ascorbic acid decreases AT₁ receptor binding affinity

HEK-293 cells stably expressing the AT₁ receptor were treated with increasing concentrations of ascorbic acid and their Ang II binding properties were analyzed. Figure 1a shows that ascorbic acid decreased the specific binding of ¹²⁵I-Ang II in a concentration-dependent manner. Binding was decreased by 20% with 1 mol/l ascorbic acid. The maximal inhibitory effect (50%) was obtained with 100 mol/l ascorbic acid. The inhibitory effect of ascorbic acid was still observed for at least 4 h after washing the broken cells with the binding buffer (Figure 1b). To better define the effect of ascorbic acid, ¹²⁵I-Ang II saturating binding isotherms were performed in the absence or presence of 100 mol/l ascorbic acid (Figure 1c). Scatchard analysis of the binding data revealed that untreated broken cells had a high binding affinity (K _d of 0.8 0.2 nmol/l) whereas ascorbic acid–treated broken cells had a significantly lower K _d of 1.8 0.5 nmol/l. The treatment with ascorbic acid did not significantly affect the maximal binding capacity of the broken cells (2.4 0.6 pmol/mg of protein vs. 2.2 0.7 pmol/mg of protein for the untreated and the ascorbic acid–treated cells, respectively). These results clearly show that ascorbic acid decreases the binding affinity of the AT₁ receptor. Like other GPCRs, the AT₁ receptor can adopt a low affinity state when it is uncoupled from its cognate G protein.²¹ To verify whether the low affinity state induced by treatment with ascorbic acid was due to the uncoupling of the AT₁ receptor from its G protein, we evaluated the binding properties in the presence of the well-known G protein uncoupling agent GTPS. After treatment with ascorbic acid, the incubation of broken cells with 1.0 nmol/l ¹²⁵I-Ang II yielded a specific binding value of 7259 470 c.p.m. Under the same conditions, GTPS diminished ¹²⁵I-Ang II binding to a value of 5287 276 c.p.m., which represents a significant (P < 0.05) decrease of 37 6% (mean of three independent experiments). These results show that GTPS produced an uncoupling effect on ascorbic acid–treated cells, implying that the AT₁ receptor was still coupled to its G protein after the treatment with ascorbic acid and therefore the mechanism by which ascorbic acid decreases the binding affinity of the AT₁ receptor is not related to G protein coupling.

Figure 1.

Ascorbic acid decreases the binding affinity of the AT₁ receptor. (a) Broken HEK-293 cells stably expressing the AT₁ receptor (25 g of protein) were incubated with 1 nmol/l ¹²⁵I-angiotensin II (Ang II) for 1 h at room temperature in the presence of increasing concentrations of ascorbic acid. (b) Broken HEK-293 cells stably expressing the AT₁ receptor (10 g of protein) were incubated at room temperature with 1 nmol/l ¹²⁵I-Ang II in the presence of 100 nmol/l ascorbic acid. After 30 min, the broken cells were centrifuged and resuspended in binding buffer without ascorbic acid and incubated for different periods of time. Results are expressed as percentage of binding to untreated broken cells. The histograms represent the mean s.d. of values obtained in three independent eriments. (c) Broken HEK-293 cells stably expressing the AT₁ receptor (10 g of protein) were incubated (filled symbols) or not (open symbols) with 100 mol/l ascorbic acid for 60 min at room temperature in the presence of increasing concentrations of ¹²⁵I-Ang II. Scatchard analysis of the binding data is shown in the inset. Nonspecific binding was assessed in the presence of 1 mol/l unlabeled Ang II. Each data point represents the mean s.d. of triplicate values (representative of three independent experiments).

Full figure and legend (23K)

The effect of ascorbic acid is specific to the AT₁receptor

Ang II specifically interacts with the AT₁ and the AT₂ receptors. We verified whether ascorbic acid could also modulate the affinity of the AT₂ receptor transiently expressed in HEK-293 cells. Scatchard analysis of ¹²⁵I-Ang II saturation binding isotherms showed that ascorbic acid did not modify the K _d of the AT₂ receptor (1.5 0.4 nmol/l vs 1.8 0.4 nmol/l for the untreated and the ascorbic acid–treated cells, respectively). As observed for the AT₁ receptor, Figure 2a shows that ascorbic acid did not modify the maximal binding capacity of the AT₂ receptor (7.6 1.1 vs. 6.9 0.3 pmol/mg of protein for the untreated and the ascorbic acid–treated cells, respectively). Similarly, Figure 2b shows that ascorbic acid did not modify the K _d (4.3 0.1 nmol/l vs. 3.9 1.7 nmol/l for the untreated cells and the ascorbic acid–treated cells, respectively) or the maximal binding capacity (4.4 0.1 vs. 4.2 0.5 pmol/mg of protein for the untreated and the ascorbic acid–treated cells, respectively) of the UT receptor for urotensin II, another GPCR involved in the regulation of blood pressure. These results show that ascorbic acid modified the binding properties of the AT₁ receptor but did not affect the binding properties of two other GPCRs expressed in the cardiovascular system.

Figure 2.

The effect of ascorbic acid is specific to the AT₁ receptor. Broken HEK-293 cells transiently expressing the AT₂ receptor or the urotensin (UT) receptor (10 g of protein) were incubated (filled symbols) or not (open symbols) with 100 mol/l ascorbic acid for 60 min at room temperature in the presence of increasing concentrations of ¹²⁵I-Ang II or ¹²⁵I-UII. Scatchard analysis of the binding data is shown for (a) the AT₂ receptor and the (b) UT receptor. Nonspecific binding was assessed in the presence of 1 mol/l unlabeled peptide. Each data represents the mean s.d. of triplicate values (representative of three independent experiments). Ang II, angiotensin II; UTR, urotensin II receptor.

Full figure and legend (15K)

Ascorbic acid decreases Ang II–induced Ca²⁺responses

We previously showed that low concentrations of Ang II induce intracellular Ca²⁺ oscillations in HEK-293 cells stably expressing the AT₁ receptor.¹⁹Figure 3a shows a typical trace where a low concentration of Ang II (0.1 nmol/l) elicited repetitive Ca²⁺ oscillations with a frequency of 33 5 oscillations/h (mean s.d. of 215 cells, representative of three independent experiments) while a maximal dose of Ang II (1 mol/l) produced a high amplitude Ca²⁺ transient. Under these conditions, only a small proportion of cells (6%) did not respond to Ang II. Figure 3b shows a typical trace where, after a 30-min treatment with ascorbic acid (100 mol/l), Ang II (0.1 nmol/l) elicited repetitive Ca²⁺ oscillations with a frequency of 22 4 oscillations/h (mean s.d. of 224 cells, representative of three independent experiments) while a maximal dose of Ang II (1 mol/l) produced a high amplitude Ca²⁺ transient that was similar to that obtained with untreated cells. After the treatment with ascorbic acid, the proportion of cells that did not respond to 0.1 nmol/l Ang II increased to 15%. These results are consistent with a decreased binding affinity of the AT₁ receptor caused by ascorbic acid and suggest that the integrity of the Ca²⁺ signaling machinery is unaffected by ascorbic acid.

Figure 3.

Ascorbic acid slows Ca²⁺ oscillations in single cells. HEK-293 cells stably expressing the AT₁ receptor were loaded with fura 2-acetoxymethyl ester and their intracellular Ca²⁺ concentration was monitored as described in "Methods." (a) Untreated cells and (b) cells treated with 100 mol/l ascorbic acid were stimulated at the indicated times with different concentrations of Ang II (white and black arrows). These typical traces showing variations of the fluorescence ratio (F ₃₃₄/F ₃₈₀) within single cells are representative of more than 200 different cells for each condition. Panel (c) shows the quantitative analysis of Ca²⁺ oscillatory rates (*P < 0.05 compared with respective untreated cells). Ang II, angiotensin II.

Full figure and legend (23K)

Ascorbic acid decreases the Ang II–induced contraction of smooth muscle

The AT₁ receptor is endogenously expressed in the smooth muscle of rabbit aorta. Figure 4a shows a classic experiment where Ang II elicited a sustained contraction of the rabbit aorta strip. In a typical experiment, the force of contraction elicited by Ang II (1 nmol/l) was 9.1 2.6 mN (mean s.d.). At the peak of the contraction, the addition of 100 mol/l ascorbic acid relaxed the tissue by 35 10% (mean s.d. of three experiments). Like the results obtained with the binding experiments, ascorbic acid had no effect on the urotensin II–induced contraction of the rabbit aorta (Figure 4b). Furthermore, ascorbic acid had no significant effect on the basal tension of rabbit aorta strips (data not shown). These results show that ascorbic acid decreased the contraction induced by the AT₁ receptor but not the contraction induced by the UT receptor. Figure 4c shows that ascorbic acid did not modify the maximal response, but caused a rightward shift (from 1.8 0.1 nmol/l to 2.6 0.3 nmol/l, P < 0.05) of the concentration–response curve for Ang II–induced contraction of the rabbit aorta. Together, these results suggest that all the components of the signaling pathway downstream from the UT and the AT₁ receptors are not affected by ascorbic acid. Also, these results are consistent with a direct effect of ascorbic acid on the AT₁ receptor resulting in a decreased binding affinity.

Figure 4.

Ascorbic acid decreases the angiotensin II (Ang II)-induced isometric contraction of rabbit aorta. Typical traces showing the effect of ascorbic acid on the contractile retsponse of isolated endothelium-denuded rabbit aorta strips stimulated with (a) Ang II or with (b) UII. The compounds were added as indicated by arrows. These typical traces are representative of at least six independent experiments. Panel (c) shows the concentration–response curves for Ang II–induced rabbit aorta strip contraction in the absence (open symbols) or the presence of ascorbic acid (filled symbols). Each point within the two curves is the average of data issued from eight independent experiments. AII, angiotensin II; UII, urotensin II.

Full figure and legend (9K)

Discussion

This study provides evidence that ascorbic acid dose-dependently decreases the binding affinity of the AT₁ receptor without modifying its level of expression at the cell surface. This is not a common effect of ascorbic acid on GPCRs because the AT₂ receptor and the UT receptor were not affected. Ascorbic acid is a well-known cofactor for several enzymes involved in many metabolic pathways in the body.² Ascorbic acid is also one of the most effective antioxidants in biological fluids.³ In humans, the antioxidant activity is involved in the regulation of lipid, DNA, and protein oxidation.² The binding affinity of the AT₁ receptor can be regulated by redox mechanisms. Reducing agents such as -mercaptoethanol and dithiothreitol have been used to discriminate between the AT₁ and the AT₂ receptor because they decrease the binding affinity of the AT₁ receptor^14,15 but do not affect the AT₂ receptor.²² These effects are related to the disruption of critical intramolecular disulfide bridges within the AT₁ receptor that are essential for Ang II binding. Another study reported that N-acetylcysteine, another antioxidant, decreases Ang II binding in vascular smooth muscle cells.²³ The authors reported that N-acetylcysteine affect receptor density at the cell surface without affecting the binding affinity. The authors suggested that N-acetylcysteine mimicked the effect of dithiothreitol by disrupting a disulfide bridge in the AT₁ receptor. However, our results indicate that ascorbic acid only decreased the binding affinity of the AT₁ receptor without affecting the receptor density at the cell surface. The AT₁ receptor contains two disulfide bridges whose disruption causes a complete loss of binding activity.²⁴ However it was shown that mild reducing conditions do not abolish the binding properties of the AT₁ receptor but cause a significant decrease in binding affinity.¹⁴ These results imply that one disulfide bridge in the AT₁ receptor is more labile than the other and that the disruption of this labile disulfide bridge decreases the binding affinity. It is thus likely that ascorbic acid acts by reducing a labile disulfide bridge within the AT₁ receptor. Other determinants of the AT₁ receptor can be regulated by a redox mechanism. We previously demonstrated that the affinity of the AT₁ receptor can be decreased by S-nitrosylation of one of its endogenous transmembrane cysteine residues (Cys²⁸⁹).¹⁶ However, a mutant receptor whose Cys²⁸⁹ was replaced by an Ala remained sensitive to ascorbic acid (data not shown), suggesting that another determinant of the AT₁ receptor is involved in the ascorbic acid effect. Further studies are warranted to identify the exact molecular mechanism by which ascorbic acid modulates AT₁ receptor binding affinity.

The AT₁ receptor is a G_q-coupled GPCR that can raise the intracellular Ca²⁺ concentration. We previously showed that low concentrations of Ang II increased the frequency of Ca²⁺ oscillations within spontaneously oscillating HEK-293 cells, whereas high concentrations of Ang II–induced a high amplitude Ca²⁺ transient within these cells.¹⁹ The frequency of Ca²⁺ oscillations is therefore dependent (up to a certain point) on the amount of receptors occupied by the agonist. In the present study, we show that ascorbic acid slowed the rate of Ca²⁺ oscillations induced by a low dose of Ang II but did not affect the amplitude of the Ca²⁺ transient induced by a high dose of Ang II. These results are consistent with the notion that ascorbic acid decreases the binding affinity of the AT₁ receptor. Because the maximal Ca²⁺ response can still be obtained with high concentrations of Ang II, these results also suggest that the Ca²⁺ signaling machinery downstream from the AT₁ receptor is not affected by ascorbic acid.

We also show that ascorbic acid decreased the Ang II–induced but not the urotensin II–induced isometric contraction of the rabbit aorta strip. These results suggest that ascorbic acid may be involved in the regulation of vascular tone. Regulation of vascular tone by ascorbic acid has been inferred from several epidemiological studies that demonstrated that there is an inverse correlation between the plasma concentration of ascorbic acid and mean blood pressure.^5,6,7,8,9 In a randomized, double-blind, placebo-controlled study, Duffy et al. ¹⁰ showed that a dietary supplement of ascorbic acid lowers the blood pressure of hypertensive patients,¹⁰ with the plasma concentration of ascorbic acid reaching 100 mol/l after 1 month of daily intake. Another study showed that a 1-month treatment with a daily dose of 500 mg of ascorbic acid lowers the brachial blood pressure and arterial stiffness of type 2 diabetes patients,¹¹ with the plasma concentration of ascorbic acid reaching 80 mol/l.

The mechanism by which ascorbic acid regulates blood pressure is not well understood. In blood vessels, endothelial cells constitutively produce nitric oxide, a vasodilator that is inactivated by complexing with superoxide, a product of normal oxidative metabolism.^25,26 Several studies have suggested that ascorbic acid scavenges superoxide, thus increasing the availability of nitric oxide in blood vessels.^27,28 A recent study showed that Ang II attenuated endothelium-dependent forearm vasodilation in healthy young men. Because the effect was reversed by ascorbic acid, the authors suggested that Ang II acted by increasing the production of superoxide anion.²⁹ However, it appears that millimolar concentrations of ascorbic acid are required to scavenge superoxide.³⁰ These concentrations are much higher than those required to lower blood pressure. It is interesting to note that the concentration at which ascorbic acid modulates the binding affinity of the AT₁ receptor is approximately 100 mol/l, which is well within the range of concentrations at which ascorbic acid is reported to reduce blood pressure. In summary, the data presented here indicate that ascorbic acid is a negative modulator of the AT₁ receptor affinity. This effect is specific to the AT₁ receptor. These results offer a mechanistic explanation for the reported blood pressure lowering effect of ascorbic acid.

DISCLOSURE

The authors declared no conflict of interest.

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Acknowledgments

This work is part of the Ph.D. thesis of P.C.L. and was supported by grants from the Canadian Institutes of Health Research. E.E. is the recipient of a J.C. Edwards Chair in cardiovascular research. R.L. and F.G. Jr. are the recipients of scholarships from the Fonds de la Recherche en Santé du Québec (FRSQ). P.C.L. and C.D.P. are the recipients of studentships from the Heart and Stroke Foundation of Canada. G.A. is the recipient of a studentship from the Natural Sciences and Engineering Research Council of Canada. S.B. is the recipient of a studentship from the FRSQ.