Biological and Pharmaceutical Bulletin
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A Comparative Study of Vasorelaxant Effects of ATP, ADP, and Adenosine on the Superior Mesenteric Artery of SHR
Shun WatanabeTakayuki MatsumotoMakoto AndoShota KobayashiMaika IguchiKumiko TaguchiTsuneo Kobayashi
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2016 Volume 39 Issue 8 Pages 1374-1380

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Abstract

We investigated superior mesenteric arteries from spontaneously hypertensive rats (SHR) to determine the relaxation responses induced by ATP, ADP, and adenosine and the relationship between the relaxant effects of these compounds and nitric oxide (NO) or cyclooxygenase (COX)-derived prostanoids. In rat superior mesenteric artery, relaxation induced by ATP and ADP but not by adenosine was completely eliminated by endothelial denudation. In the superior mesenteric arteries isolated from SHR [vs. age-matched control Wistar Kyoto rats (WKY)], a) ATP- and ADP-induced relaxations were weaker, whereas adenosine-induced relaxation was similar in both groups, b) ATP- and ADP-induced relaxations were substantially and partly reduced by NG-nitro-L-arginine [a NO synthase (NOS) inhibitor], respectively, c) indomethacin, an inhibitor of COX, increased ATP- and ADP-induced relaxations, d) ADP-induced relaxation was weaker under combined inhibition by NOS and COX, and e) adenosine-induced relaxation was not altered by treatment with these inhibitors. These data indicate that levels of responsiveness to these nucleotides/adenosine vary in the superior mesenteric arteries from SHR and WKY and are differentially modulated by NO and COX-derived prostanoids.

Extracellular nucleotides and their metabolites including ATP, ADP, and adenosine play pivotal roles in (patho)physiological processes, including immune responses, inflammation, cardiac fibrosis, atherosclerosis, hypertension, and diabetes.110) They also exert a variety of effects on the vasculature, such as platelet activation and alteration in smooth muscle contraction and relaxation, by activating plasmalemmal receptors (e.g., purinoceptors).1,2,8,11,12) Purinoceptors are classified into two subtypes, namely P1 and P2, based on their pharmacological properties and molecular cloning characteristics.11,12) Adenosine and its phosphates ATP and ADP are endogenous ligands for P1 and P2 receptors, respectively. Several studies have suggested that the expression and signaling responses of these receptors is altered under disease conditions.4,11,1317) Moreover, these receptors can mediate a variety of responses to several nucleotides, resulting in the existence of multiple receptors with overlapping ligand preferences.11,18) Therefore, it is difficult to understand the effect of nucleotides on vascular function under pathophysiological conditions.

Hypertension is a leading risk factor for cardiovascular-associated morbidity and mortality. An important and contributory element in sustained hypertension is an increase in vascular tone, potentially resulting from concurrently reduced relaxation and augmented contraction induced by various endogenous ligands.1926) Hypertension-associated vascular dysfunction is partly regulated by endothelium-derived factors.2124) There is a fine balance between endothelium-derived relaxing and contracting factors, a disturbance of this balance can contribute to changes in blood pressure.1921,24,27,28) Nitric oxide (NO) is one of the most greatest important factors derived from endothelial cells. Moreover, other factors such as endothelium-derived hyperpolarizing factor (EDHF) and cyclooxygenase (COX)-mediated substances (prostanoids) are important in the regulation of vascular tone.1921,24) Indeed, several studies have suggested that 1) vascular responses to extracellular nucleotides/nucleosides are altered in the vasculature of hypertensive individuals,11,16,29,30) and 2) these endogenous ligand-induced responses are mediated by arterial endothelium-derived factors under various conditions.13,14,30,31) However, very few studies have investigated the vasorelaxant activity of ATP/ADP/adenosine along with the modulative effects of NO and COX-derived substances on the same arteries in hypertensive individuals.

In this present study, we aimed to compare vasorelaxant activities of ATP, ADP, and adenosine in superior mesenteric arteries isolated from spontaneously hypertensive rats (SHR). Furthermore, we investigated the effects of NO synthase (NOS) and/or COX inhibitions on the relaxation induced by ATP, ADP, and adenosine using pharmacological strategies.

MATERIALS AND METHODS

Reagents

The following reagents were used: phenylephrine (PE), NG-nitro-L-arginine (L-NNA), indomethacin, ATP, ADP, adenosine (Sigma-Aldrich, St. Louis, MO, U.S.A.), and acetylcholine (ACh) chloride (Daiichi-Sankyo Pharmaceuticals, Tokyo, Japan).

Animals and Experimental Design

This study was approved by the Hoshi University Animal Care and Use Committee, and all studies were conducted in accordance with the “Guide for the Care and Use of Laboratory Animals,” published by the U.S. National Institutes of Health, and “Guide for the Care and Use of Laboratory Animals,” adopted by the Committee on the Care and Use of Laboratory Animals of Hoshi University. Four-week-old male SHR and Wistar Kyoto rats (WKY) were obtained from Hoshino Laboratory Animals, Inc. (Ibaraki, Japan). Male Wistar rats were purchased from Japan Laboratory Animals, Inc. (Tokyo, Japan). All animals were fed and watered ad libitum in a controlled environment (temperature: 21–22°C, humidity: 50±5% with a 12-h : 12-h light–dark cycle) until they were 15 or 16 weeks old. The systolic blood pressure (SBP) of 13- or 14-week-old rats was measured, as previously described.22,25) In brief, SBP was measured employing the tail-cuff method using a blood pressure analyzer (BP-98A; Softron, Tokyo, Japan) after the rat had been in a constant-temperature box at 37°C for at least 5 min. Surgical procedures were performed under anesthetic cover of isoflurane as previously described.22,25,3234)

Functional Studies

Vascular isometric force was essentially recorded as described in our previous studies.13,17,22,25,26,28,3234) Concentration–response curves to ATP (10−8–10−3 M), ADP (10−8–10−4 M), or adenosine (10−8–10−3 M) were plotted for experiments performed in the superior mesenteric arteries of SHR and WKY and precontracted with equieffective concentrations of PE. These experiments were performed in the absence or presence of 10−4 M L-NNA (NOS inhibitor), 10−5 M indomethacin (COX inhibitor), or a combination of both. Arteries were pretreated with the inhibitors for 20 min prior to PE application. To investigate the effect of endothelium on relaxations, we analyzed concentration–response curves in the presence/absence of endothelium of superior mesenteric arteries from Wistar rat at 17 weeks of age. Endothelial denudation was achieved by infusing a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solution (CHAPS; 0.1%, for 1 min), which was subsequently flushed out with modified Krebs–Henseleit solution; the inability of ACh to relax these segments confirmed the success of this procedure, as reported previously.13)

Data Analysis

Data are expressed as the mean±standard error (S.E.). Relaxation responses are shown as a percentage of PE-induced precontraction, and individual concentration–response curves were analyzed using a nonlinear regression curve fitting relaxation–drug concentration relationships to determine the maximal response generated by the relaxant drugs (Emax) and a negative logarithm of EC50, which is the concentration of the agonist producing 50% Emax (pEC50). Statistical evaluations were performed using Student’s t-test between two groups and one-way ANOVA followed by Bonferroni’s test for comparisons in more than three groups. Statistical evaluations of concentration–response curves were performed using two-way ANOVA with repeated measures followed by a Bonferroni post-hoc test. All data analysis was performed using GraphPad Prism (ver. 5.0 for Mac, San Diego, CA, U.S.A.). p<0.05 was considered statistically significant.

RESULTS

Body Weight and SBP

The measured SBP was significantly higher in SHR (189±4 mmHg, n=20, p<0.001) than in WKY (112±3 mmHg, n=19). The body weight at the point of experimentation (15–16 weeks of age) was also noted to be lower in SHR (342.6±5.4 g, n=20, p<0.05) than in WKY (358.4±4.9 g, n=19).

Effects of Endothelium on ATP-, ADP-, and Adenosine-Induced Relaxations in Superior Mesenteric Arteries

Firstly, to investigate the effect of endothelium on ATP-, ADP-, and adenosine-induced relaxation in superior mesenteric arteries, we evaluated the concentration–response curves of ATP (Fig. 1A), ADP (Fig. 2A), and adenosine (Fig. 3A) for the presence/absence of the endothelium of superior mesenteric arteries obtained from Wistar rats. In endothelium intact arteries, all ligands led to concentration-dependent relaxation. ATP- and ADP-induced relaxations were completely eliminated by endothelium denudation, whereas adenosine-induced relaxation was unaffected by endothelial denudation.

Fig. 1. ATP-Induced Relaxation in Superior Mesenteric Arteries

(A) Concentration–response curves for ATP in the absence (−EC) and presence (Intact) of endothelium in the arteries obtained from Wistar rats. (B–E) Concentration–response curves for ATP in the isolated rings of superior mesenteric arteries in the absence (B) and presence of inhibitors [L-NNA, 10−4 M (C), indomethacin, 10−5 M (D), and L-NNA (10−4 M) plus indomethacin (10−5 M) (E)] obtained from SHR and WKY. Data are representative of the mean±S.E. n=3–9. * p<0.05 vs. intact (A) or WKY (B).

Fig. 2. ADP-Induced Relaxation in Superior Mesenteric Arteries

(A) Concentration–response curves for ADP in the absence (−EC) and presence (Intact) of endothelium in the arteries obtained from Wistar rats. (B–E) Concentration–response curves for ADP in the isolated rings of superior mesenteric arteries in the absence (B) and presence of inhibitors [L-NNA, 10−4 M (C), indomethacin, 10−5 M (D), and L-NNA (10−4 M) plus indomethacin (10−5 M) (E)] obtained from SHR and WKY. Data are representative of the mean±S.E. n=3–8. * p<0.05 vs. intact (A) or WKY indo L-NNA (E).

Fig. 3. Adenosine-Induced Relaxation in Superior Mesenteric Arteries

(A) Concentration–response curves for adenosine in the absence (−EC) and presence (Intact) of endothelium in the arteries obtained from Wistar rats. (B–E) Concentration–response curves for adenosine in the isolated rings of superior mesenteric arteries in the absence (B) and presence of inhibitors [L-NNA, 10−4 M (C), indomethacin, 10−5 M (D), and L-NNA (10−4 M) plus indomethacin (10−5 M) (E)] obtained from SHR and WKY. Data are representative of the mean±S.E. n=3–6.

Relaxation Induced by ATP, ADP, and Adenosine and Effects of NOS and COX Inhibitors in Superior Mesenteric Arteries of SHR and WKY

Next, we compared these ligand-induced relaxations of superior mesenteric arteries obtained from SHR and WKY and investigated the effects of NOS and COX inhibitors on these relaxations (Figs. 1–3, Table 1). As shown in Fig. 1B and Table 1, ATP-induced relaxation was reduced in SHR compared with WKY. In both SHR and WKY groups, the ATP-induced relaxations were greatly blocked by the NOS inhibitor L-NNA (Fig. 1C, Table 1). In contrast, ATP-induced relaxation was slightly increased by the COX inhibitor indomethacin (Fig. 1D, Table 1). Treatment with each inhibitor abolished the differences of ATP-induced relaxations between the SHR and WKY groups. The non-NO and non-prostanoids-mediated relaxations induced by ATP were small and similar between SHR and WKY (Fig. 1E, Table 1).

Table 1. Maximal Responses and EC50 Values for ATP-, ADP-, and Adenosine-Induced Relaxations of Superior Mesenteric Arteries from SHR and WKY
Agonist/TreatmentWKYSHR
EmaxpEC50EmaxpEC50
ATP
No treatment70.9±7.5 (8)5.4±0.2 (8)56.6±6.0 (8)5.5±0.3 (8)
L-NNA25.4±10.2 (8)*4.3±0.3 (8)15.8±4.6 (8)*4.2±0.4 (8)*
Indomethacin79.7±6.6 (9)5.8±0.1 (9)83.2±4.8 (9)5.4±0.1 (9)
L-NNA+indomethacin31.5±7.9 (9)*4.5±0.3 (9)22.3±6.4 (8)*5.2±0.3 (8)
ADP
No treatment77.2±3.6 (7)5.9±0.1 (7)63.8±4.8 (8)5.8±0.1 (8)
L-NNA38.5±8.5 (7)*5.7±0.1 (7)26.9±5.6 (8)*5.2±0.3 (8)
Indomethacin90.9±2.4 (7)5.8±0.2 (7)86.2±2.3 (8)*6.0±0.1 (8)
L-NNA+indomethacin69.1±4.8 (6)5.5±0.1 (6)45.4±5.0 (6)5.1±0.2 (6)
Adenosine
No treatment100.0±0.03 (6)4.6±0.1 (6)95.9±1.8 (6)4.5±0.2 (6)
L-NNA100.0±0 (6)4.4±0.2 (6)99.7±0.3 (6)4.3±0.3 (6)
Indomethacin100.0±0 (4)4.9±0.04 (4)100.0±0 (4)4.6±0.2 (4)
L-NNA+indomethacin100.0±0 (6)5.0±0.2 (6)99.5±0.3 (6)4.9±0.2 (6)

Values are expressed as the mean±S.E. The number of experiments is shown within parentheses. Emax, maximal agonist-induced relaxation expressed as % of precontraction; pEC50, −log (corresponding agonist) required to produce 50% of the maximal relaxation response. * p<0.05 vs. corresponding no treatment.

The ADP-induced relaxation was slightly reduced in SHR than in WKY (Fig. 2B, Table 1). L-NNA significantly but not completely suppressed the ADP-induced relaxation in both SHR and WKY groups (Fig. 2C, Table 1), whereas indomethacin enhanced the relaxation in both groups (Fig. 2D, Table 1). The non-NO and non-prostanoids-mediated relaxations induced by ADP were significantly impaired in SHR than in WKY (Fig. 2E).

As shown in Fig. 3 and Table 1, adenosine-induced relaxations were similar between SHR and WKY groups. Neither L-NNA (Fig. 3C) and indomethacin (Fig. 3D) nor a combination of L-NNA and indomethacin (Fig. 3E) affected the adenosine-induced relaxations in both SHR and WKY groups.

DISCUSSION

Extracellular nucleotides such as ATP and ADP along with adenosine play a role in vascular functions.1,2,7,8,11,12,35) Previous studies have reported that the application of these substances results in vasoconstriction or vasodilatation and that the extent of these responses is altered in hypertensive arteries.1,2,11,29) However, few studies have compared the vasorelaxant effect of these three substances in the same region of arteries from hypertensive subjects. Furthermore, endothelium-derived factors such as NO, COX-derived prostanoids, and EDHF can modulate extracellular nucleotide and adenosine-induced responses in various arteries.1,2,11,29,35,36) Therefore, we investigated the role of these factors on ATP/ADP/adenosine-induced relaxation in superior mesenteric arteries from SHR and WKY. Here, we observed the differences of responses induced by these ligands between SHR and WKY and the participation of NO and prostanoids on the relaxations.

ATP-induced vasodilatation results from the activation of purinergic P2Y receptors (P2Y1 and P2Y2) located on endothelial cells, while ATP-induced vasoconstriction results from the activation of P2X receptors located on smooth muscle.1,2,11) In addition, vasodilator responses induced by ATP occur due to the release of NO.35) Stanford et al.36) suggested that ATP-induced dilatation occurs in response to not only the presence of NO but also the release of EDHF in rat mesenteric arterial beds. In the present study, we found that 1) ATP-induced relaxation was completely blocked by endothelial denudation, 2) the relaxation was reduced in the SHR group (vs. WKY group), 3) the relaxation was largely suppressed by NOS inhibitors, 4) indomethacin presence augmented relaxation, and 5) ATP-induced relaxation was almost abolished with the preservation of the EDHF component after cotreatment with both NOS and COX inhibitors. These results suggest that ATP-induced relaxation is largely mediated by NO (but not by EDHF) and is suppressed by COX-derived substances including prostaglandins and thromboxane A2 (TXA2).

ADP-induced relaxation was slightly impaired in the superior mesenteric arteries of SHR. ADP-induced relaxation was reduced by the NOS inhibition in both SHR and WKY, with relaxation further impaired in the arteries of SHR. Furthermore, pretreatment with indomethacin abolished the difference of the relaxation between SHR and WKY. Under NOS and COX inhibition, ADP-induced non-NO non-prostanoid relaxation, namely EDHF-type relaxation because the ADP-induced relaxation was completely eliminated by endothelial denudation, was reduced in SHR compared with WKY. These results suggest that impaired ADP-induced relaxation in SHR is attributable to increased COX-derived vasoconstrictor prostanoids (e.g., TXA2) as well as reduced EDHF-mediated signaling.

In the present study, adenosine-induced relaxation was not impaired in the superior mesenteric arteries from SHR compared with WKY and was not affected by the blockade of NOS or COX, either alone or in combination. Moreover, the adenosine-induced relaxation was unaffected by the endothelial denudation. These results suggest that adenosine-induced relaxation is largely dependent on the activation of purinoceptors in smooth muscle cells. The preservation of adenosine-induced relaxation in the arteries of SHR might represent a compensatory mechanism resulting from sustained hypertension; however, the establishment of a role for adenosine-induced relaxation in combination with the effects of nucleotides/adenosine on the development of vascular dysfunction requires further experimentation focusing on, for example, time–course alterations in hypertension.

The effect of extracellular concentrations of nucleotides/nucleosides on the plasma membrane of various cells is regulated by a fine balance their release from cells and hydrolysis by extracellular ectonucleotidases.1,2,10,11) These nucleotides/adenosine have auto- and/or paracrine effects on cell surface purineceptors.1,2,11) It may be impossible to clearly define the bioavailability of these nucleotides and adenosine for the purpose of regulating vascular tone as their production, ectonucleotidase activity, and functional purinoceptor expression may change under hypertension. Indeed, the activity of ectonulcleotidases are known to be altered under hypertensive conditions.37,38) At present, mechanisms underlying the vasorelaxant response to ATP/ADP/adenosine and the pathophysiological roles of these substances in the development of hypertension-associated vascular complications remain unclear. Further studies are therefore needed to address these issues.

In summary, the present study provides pharmacological evidence indicating differences in the extent of vasorelaxant responsiveness to ATP, ADP, and adenosine and the varying effects of NO, COX-derived prostanoids, and EDHF on arterial responsiveness in SHR.

Acknowledgments

We thank H. Sashikubi, A. Suwa, M. Takeuchi, M. Takahashi, M. Nagata, S. Hotozuka, J. Nomoto, and M. Majima for technical assistance. This study was supported in part by JSPS KAKENHI (Grant Numbers 26460107, 15K21419, and 15K07975) and by the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES
 
© 2016 The Pharmaceutical Society of Japan
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