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Am J Physiol Heart Circ Physiol 292: H719-H725, 2007. First published August 18, 2006; doi:10.1152/ajpheart.00593.2006
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INNOVATIVE METHODOLOGY

Evidence for the involvement of nitric oxide in A2B receptor-mediated vasorelaxation of mouse aorta

Habib R. Ansari,1 Ahmed Nadeem,1 M. A. Hassan Talukder,2 Shilpa Sakhalkar,1 and S. Jamal Mustafa1

1Department of Physiology and Pharmacology, Center for Interdisciplinary Research in Cardiovascular Sciences, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia, 2David Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio

Submitted 6 June 2006 ; accepted in final form 10 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have investigated the role of adenosine and its analogs on vasorelaxation of mouse aorta in intact endothelium with rank order of potency as follows: 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine > adenosine >> CGS-21680, which is consistent with the profile of A2B-adenosine receptor (A2BAR). In endothelium-intact tissues, acetylcholine produced relaxation ranging from 65 to 80% in phenylephrine (PE, 10–7 M)-precontracted mouse aorta, whereas no relaxation was observed in endothelium-denuded tissues. The A2BAR antagonist alloxazine (10–5 M) shifted concentration-response curve for NECA (EC50 = 0.005 x 10–5 M) to the right with an EC50 of 2.8 x 10–5 M, demonstrating that this relaxation is partially dependent on functional endothelium mediated predominantly via A2BAR in this tissue. This conclusion was further supported by the following findings: 1) in the endothelium-intact mouse aorta, the EC50 values for NECA and adenosine were found to be 0.05 and 1.99 x 10–4 M, respectively; however, in denuded endothelium, these values were 0.098 and 3.55 x 10–4 M, respectively; 2) NECA-induced relaxation was significantly blocked by NG-nitro-L-arginine methyl ester (L-NAME; 10–4 M) in endothelium-intact tissues, which was reversed by pretreatment with L-arginine (10–4 M), whereas no significant inhibition was found in endothelium-denuded tissues; 3) total nitrites and nitrates (NOx) in intact endothelium with L-NAME (10–4 M) alone and in combination with L-arginine were 59% (P < 0.05) and 96%, respectively, in comparison with control (PE + NECA); and 4) endothelial nitric oxide synthase gene expression was found to be 67% (P < 0.05) less in endothelium-denuded as opposed to endothelium-intact mouse aorta. Thus these data demonstrate that adenosine-mediated vasorelaxation is partially dependent on A2BAR in mouse aorta.

adenosine; endothelium

ADENOSINE HAS BEEN SHOWN to produce potent vasodilatory effects in majority of vascular beds of mammalian species in addition to its well-defined cardiac effects (13, 16, 28, 39). It is now well established that the vasodilatory effects of adenosine are mediated by A2 receptors (29, 30, 36, 37), and, in some cases, the subtypes involved have been characterized by the use of selective A2A and A2B agonists (7, 10). In the guinea pig, an adenosine A2A receptor antagonist identified adenosine A2 receptor in the coronary vessels as A2A and, on the other hand, an A2B receptor in aorta (25, 40). Similarly, the vascular effect of adenosine in aorta and coronary vessels of rat has been reported to be mediated by both A2A and A2B receptors (19, 20). Adenosine has been shown to cause totally endothelium-dependent, -independent, and/or partially endothelium-dependent relaxation of various isolated vessels (14, 31, 38, 44, 46, 47).

The endothelium exerts a modulatory effect on vascular smooth muscle function by releasing vasoactive factors. Nitric oxide (NO) is one of those relaxing factors that is released from the vascular endothelium. NO is synthesized from L-arginine by the action of NO synthase (NOS). Adenosine enhances NO production by arterial endothelial cells (21); therefore, it is possible that vascular responses to adenosine are blunted in the presence of endothelial dysfunction (8).

Although resistance vessels are physiologically related to the regulation of systemic blood pressure, different aortic preparations have also been routinely used to demonstrate the functional effects of vasodilator substances (20, 40) in vitro. Recently, potent vasodilatory effects of adenosine, acetylcholine, CGRP, sodium nitroprusside, and estrogen (6, 12, 33, 35, 41) have been reported in mouse aorta; however, there are no reports of adenosine receptor-mediated vascular effects through NO in this preparation. In the large conduit arteries that do not contribute to peripheral vascular resistance, any alteration in their endothelial functions may be critical for the occurrence of vascular disease and its complications since endothelium plays an important role as a source of vasodilator substances (21). In mouse aorta, acetylcholine-induced relaxation has been reported to be completely abolished by NOS inhibitor, suggesting that NO is the sole endothelial vasodilator substance for acetylcholine (6). In this study, we report an endothelium-dependent relaxation with adenosine and its analogs primarily through A2B receptor in the mouse aorta. Real-time PCR and biochemical data showed attenuated endothelial NOS (eNOS) gene expression and NOx levels, respectively, in endothelium-denuded compared with endothelium-intact tissues, suggesting the role of NO in endothelium-dependent relaxation. Our results indicate that A2B adenosine receptor (A2BAR) predominantly contributes to the observed 5'-N-ethylcarboxamidoadenosine (NECA)-induced relaxation response through NO production in mouse endothelium-intact aortic rings.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All the experimental protocols were performed according to a protocol approved by the Animal Care and Use Committee at West Virginia University (Morgantown, WV).

Preparation of isolated mouse aorta. Male Balb/C mice 8–10 wk old were used in this study. Mice were killed by deep anesthesia with pentobarbital sodium (100 mg/kg ip). After a thoracotomy was performed, the aorta was gently removed, cleaned of fat and connective tissues, and cut transversely into rings of 3–4 mm in length. Extreme care was taken not to damage the endothelium in the rings. In some rings, the endothelium was removed by placing a piece of wire in the lumen and rubbing the aortic ring gently over a wet blotting paper. The rings were mounted vertically between two stainless steel wire hooks over wet blotting paper. Two rings were suspended in 10-ml organ baths containing Krebs-Henseleit buffer. The Krebs-Henseleit buffer contained 118 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 11 mM glucose, and 2.5 mM CaCl2. The pH of the buffer was adjusted and maintained at 7.4 with 95% O2-5% CO2 at 37°C. Aortic rings were equilibrated for 90 min with a resting force of 1 g, with changes of the bathing solution at 15-min intervals.

Isometric force measurement. For measurement of isometric force response, the tissues were allowed to equilibrate for 90 min. The resting force of 1 g has been used in our laboratory and by others in mouse thoracic aorta (6, 12, 33, 42). At the end of equilibration period, tissues were contracted with 50 mM KCl to check the contractility of individual aortic rings, which was washed out with Krebs-Henseleit buffer. Aortic rings were then constricted with phenylephrine (PE) for relaxation studies, and changes in tension were monitored continuously with fixed-range precision-force transducer (model TSD 125C, Biopac Systems) connected to the differential amplifier (model DA 100B, Biopac Systems). Data were recorded using MP 100 WSW digital acquisition system (Biopac Systems) and were analyzed using Acknowledge 3.5.7 software (Biopac Systems) (33).

Relaxation experiments. The tissues were contracted with PE (10–7 M) to produce consistent submaximal (~90%) response in our experiments. After equilibration, the responsiveness and stability of individual rings were checked by successive administration of submaximally effective concentration of PE (10–7 M). The integrity of the vascular endothelium was assessed pharmacologically by acetylcholine to produce relaxation of PE-precontracted rings. Tissues that did not elicit reproducible and stable contraction with PE (10–7 M) and relax <50% with acetylcholine (10–7 M) were discarded from the study. Aortic rings were considered to be denuded of functional endothelium when there was no relaxation response to acetylcholine. Preparations were then washed several times with Krebs-Henseleit solution and allowed to equilibrate for 30 min before the experimental protocol began. In experiments where the involvement of endothelium and NOS was investigated, either the endothelium was removed by using a thin stainless steel wire or tissues were incubated with 10–4 M NG-nitro-L-arginine methyl ester (L-NAME) (26, 45) for 30 min. Reversal of this effect was observed by incubation with 10–4 M L-arginine (11, 23) for 30 min.

Experimental protocol. The concentration-response curves for adenosine and its analogs were obtained by cumulative addition of drugs (antagonists) in PE (10–7 M)-contracted tissues. In all cases, agonists were added to yield the next higher concentration only when the response to the earlier dose reached a steady state. In experiments where the effects of different antagonists were measured, the drugs were added 30 min before contraction of the tissue with PE and were present throughout the experiments. Relaxation responses were expressed as percent decrease in the contraction with respect to PE in response to each concentration of agonist used.

Total nitrates and nitrites (NOx) assay. Mouse aortic tissues with and without endothelium were prepared and divided into two halves, one half used for organ bath study and the other half for NOx assay as a measure of NO production. For NOx assay, tissues were equilibrated with 95% O2-5% CO2 in Krebs-Henseleit buffer for 60 min and then subsequently incubated with PE (10–7 M) alone or together with NECA (10–5 M) for 10 min at 37°C in the absence and presence of L-NAME (10–4 M) alone and in combination with L-arginine (10–4 M) for 30 min. The incubation solution was removed, tissues were homogenized in potassium phosphate buffer (300 mM, pH 7.4), and the supernatant was used for the NOx assay as described by Grisham et al. (15) with a slight modification. Briefly, all the nitrates in the aortic supernatant were converted into nitrites by using aspergillus nitrate reductase in the presence of FAD and NADPH. The incubation was carried out at 37°C for 2 h. Griess reagent [1:1 mixture of 1% sulfanilamide in 5% vol/vol orthophosphoric acid and 0.1% wt/vol N-(1-naphthyl)ethylene diamine] was added for the development of color, and readings were taken after 10 min in a 96-well plate at 540 nm on µQuant plate reader (BioTek Instruments). Standard curve was generated by using known concentrations of sodium nitrite. Results were expressed in nanomoles per milligram protein.

Real-time PCR. Total RNA was isolated from the mouse aortic tissue similar to our earlier report (27) with and without endothelium using the TRIzol reagent from Life Technologies/Invitrogen followed by DNase treatment to eliminate potential genomic DNA contamination. This was followed by conversion of 0.5 µg of total RNA into cDNA by using High Capacity cDNA archive kit (Applied Biosystems, Foster City, CA) according to the instructions of the manufacturer in a total volume of 100 µl. Real-time PCR was performed with the use of ABI PRISM 7300 Detection System (Applied Biosystems) using Taqman Universal Mastermix (Applied Biosystems, Branchburg, NJ) according to the instructions of the manufacturer. The reaction volume (25 µl) included 12.5 µl of 2x Taqman Universal Mastermix, 1 µl of cDNA, and 1.25 µl of 20x FAM-labeled Taqman gene expression assay master mix solution. For the real-time PCR of the eNOS gene, the Taqman inventoried gene expression product (Gen Bank accession no. NM_008713) was purchased from Applied Biosystems. 18S rRNA was used as an endogenous control. The fold difference in expression of target cDNA was determined by using the comparative threshold cycle (CT) method. The {Delta}CT value was determined in each experiment by subtracting the average 18S CT value from the corresponding average CT for eNOS in aorta with and without endothelium. The standard deviation was calculated using the formula

Formula
To set the relative unit to 1, {Delta}{Delta}CT was calculated by subtraction of the {Delta}CT calibrator value (eNOS {Delta}CT values with endothelium). The fold difference in gene expression of the target was calculated as the average value from 2–{Delta}{Delta}CT+s and 2–{Delta}{Delta}CT–s (22).

Drugs. Adenosine, 2-chloroadenosine (CAD), (–)-phenylephrine hydrochloride, acetylcholine, NG-nitro-L-arginine methyl ester (L-NAME), S-2-amino-5-[(aminoiminomethyl)amino] pentaenoic acid (L-arginine), 5'-N-ethylcarboxamidoadenosine (NECA), (2-[2-carboxyethy)] phenylethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), and alloxazine [benzo(g)pteridine-2,4(1H,3H)-dione] were purchased from Sigma Chemical (St. Louis, MO). All other chemicals were of the highest grade available and were purchased from Sigma Chemical. NECA, CGS-21680, and adenosine antagonists were dissolved in 100% DMSO as 10–2 M stock solution. The final concentration of DMSO (vehicle, 0.1%, ~ negligible) in 10 ml of organ bath chamber had no effect by itself on the aortic rings.

Data analysis. All values are presented as means ± SE (n = number of animals) Concentration-response curves for muscle relaxation were constructed by cumulative addition of the agonist in the organ bath. The data (including EC50) were analyzed by GraphPad Prism statistical software (version 3.0). Statistical significance between means was determined by using the Student-Newman-Keuls method. P < 0.05 was taken as significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Adenosine-receptor mediated relaxation-response curve for adenosine, NECA, CAD, and CGS-21680 in mouse aorta. Adenosine and its analogs NECA, CAD, and CGS-21680 evoked a concentration-dependent relaxation with rank order of potency as follows: NECA > CAD > adenosine >> CGS-21680 on PE (10–7 M)-precontracted isolated mouse aorta (Fig. 1). The maximal responses achieved by these agonists varied, and only CAD produced complete relaxation when compared with NECA, CGS-21680, and adenosine. EC50 values for NECA, CAD, and adenosine were 0.05 ± 0.002 x 10–4 M, 1.3 ± 0.079 x 10–4 M, and 1.99 ± 0.13 x 10–4 M, respectively. NECA, CAD, and adenosine at 10–4 M relaxed aortic rings by 70 ± 3%, 44 ± 2.6%, and 26 ± 3.2%, respectively (Fig. 1).


Figure 1
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  		Fig. 1. Concentration-dependent effects of CGS-21680, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloroadenosine (CAD), and adenosine (Ado) on percent relaxation in mouse aorta (intact endothelium). PE, phenylephrine. Values are expressed as means ± SE (n = 10 animals).

 
Role of functional endothelium on NECA and adenosine-induced relaxations. As can be seen in Fig. 2, in endothelium-intact aortic rings, NECA and adenosine produced a concentration-dependent relaxation with EC50 values of 0.05 ± 0.002 x 10–4 M and 1.99 ± 0.13 x 10–4 M, respectively. Maximum relaxation obtained with 10–5 M concentration of NECA and adenosine in endothelium-intact tissues was 46 ± 2% and 7 ± 0.5%, respectively, whereas, at 10–4 M concentrations, these values were 68 ± 2.3% and 26 ± 2.3%, respectively, (Fig. 2A). The EC50 values for endothelium-denuded aorta for NECA and adenosine were 0.098 ± 0.005 x 10–4 M and 3.55 ± 0.25 x 10–4 M, respectively, which were significantly different in comparison with endothelium-intact aorta (P < 0.05, Student's t-test). At 10–5 M, NECA- and adenosine-induced relaxation values were 20 ± 2.5% and 4.5 ± 0.09%, whereas they were 37 ± 2% and 12 ± 1.1%, respectively at 10–4 M for endothelium-denuded aorta (Fig. 2B). These results demonstrate that the order of potency for endothelium-dependent relaxation was NECA >> adenosine, which is consistent with the A2 adenosine receptor profile.


Figure 2
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  		Fig. 2. Concentration-relaxation response curves for NECA (A) and adenosine (B) in mouse aorta in intact (+ Endothelium) and denuded (– Endothelium). Each point represents the mean ± SE (n = 9). *P < 0.05 compared with + Endothelium.

 
Effect of L-NAME on PE-induced contraction response in intact and denuded endothelium aorta. To demonstrate the involvement of NO in NECA-induced relaxation by the endothelium, we investigated the effect of the NOS inhibitor L-NAME (10–4 M) on tension development by PE (10–7 M) in mouse aorta. Previously, L-NAME was employed by others who investigated the functional role of endothelium in mouse aorta and rat mesenteric artery (35, 43, 48). As can be seen from Fig. 3 in endothelium-intact tissues, PE increased the tension by 62% after L-NAME treatment compared with control (before L-NAME, P < 0.05). In endothelium-denuded aortic rings, PE (10–7 M) increased the tension by 55% compared with control (intact endothelium, before L-NAME). The developed tensions were similar to the tensions produced in endothelium-denuded and endothelium-intact tissues after L-NAME (52%). These data suggest that NO production, in part, is endothelium mediated in intact aortic rings.


Figure 3
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  		Fig. 3. Total tension before and after NG-nitro-L-arginine methyl ester (L-NAME) with PE (10–7 M) in endothelium-intact (+ E) and endothelium-denuded (– E) mouse aorta. The contraction is expressed as %tension in response to PE (10–7 M), and values are expressed as means ± SE (n = 12). *P < 0.05 compared with before L-NAME with + E.

 
eNOS gene expression by real-time PCR. Relative measurements of eNOS expression in endothelium intact (+E) and endothelium denuded (–E) mouse aorta were carried out by using real-time PCR (Fig. 4). The relative eNOS gene expression levels were 1.003 ± 0.12 for +E and 0.33 ± 0.09 for –E, respectively. These data show the suppression of eNOS gene expression by 67% in endothelium-denuded aorta compared with endothelium intact aorta (P < 0.05).


Figure 4
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  		Fig. 4. Relative endothelial nitric oxide synthase (eNOS) gene expression by real-time PCR in the intact and denuded endothelium mouse aorta. The eNOS gene expression is represented as means ± SE (n = 4). *P < 0.05 compared with + Endothelium.

 
Effects of L-NAME and L-arginine on NECA-induced relaxation in endothelium-intact and -denuded mouse aorta. The tissues were preincubated with L-NAME, and concentration-response curve for NECA was generated. L-NAME significantly inhibited NECA-induced relaxation by 40% and 52% at 10–6 and 10–5 M concentrations, respectively, in endothelium-intact aortic rings (Fig. 5A), whereas it did not affect the responses to denuded aortic rings (Fig. 5B). The inhibitory effect of L-NAME on NECA-induced relaxation in endothelium-intact rings was completely reversed by L-arginine. The reversal effect of L-arginine was not observed in endothelium-denuded aortic rings. These results demonstrate the involvement of endothelium in adenosine-mediated relaxation via NO production in aortic rings.


Figure 5
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  		Fig. 5. Concentration-relaxation response curves for NECA with endothelium-intact (A) and -denuded (B) mouse aorta and their modulation by L-NAME (10–4 M) alone and in combination with L-arginine (10–4 M). Each point represents the mean ± SE (n = 9). *P < 0.05 compared with control (+ Endothelium).

 
Effects of A2A and A2B adenosine receptor antagonists on NECA-induced relaxations. To investigate the role of A2 receptors (A2A and A2B) and their involvement in response to NECA-induced relaxation in mouse endothelium intact aorta, the A2A receptor antagonist ZM-241385 and the A2B antagonist alloxazine were used in this study. The ZM-241385 (10–5 M) inhibited NECA-induced relaxation minimally (15%) when compared with control (NECA alone; data not shown) that was not significant. In Fig. 6, we have shown the effect of alloxazine on NECA-induced relaxation in mouse aorta with intact endothelium. At 10–6 and 10–5 M, alloxazine inhibited NECA-induced (10–5 M) relaxation by 5% and 76%, respectively. These data demonstrate that A2B receptors are involved in NECA-induced relaxation in mouse aorta.


Figure 6
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  		Fig. 6. Effect of A2B receptor antagonist alloxazine on NECA-induced relaxation in endothelium-intact mouse aorta. Each point represents the mean ± SE (n = 8). *P < 0.05 compared with control.

 
NECA-induced total nitrates and nitrites (NOx) production. As can be seen in Table 1, PE (10–7 M)-precontracted intact endothelium aorta in the presence of NECA (10–5 M) had total nitrates and nitrites (NOx) levels with a value of 3.2 ± 0.25 (in nM/mg protein, 100%), whereas this value was significantly reduced to 59% (P < 0.05) in the presence of L-NAME (10–4 M) with NECA (10–5 M). Addition of L-arginine (10–4 M) with L-NAME (10–4 M) restored the NOx production back to control levels (96%, PE 10–7 M + NECA 10–5 M). In endothelium-denuded aorta, NOx production induced by PE (10–7 M) was found to be 1.85 ± 0.11 (in nM/mg protein), which was not affected in the presence of L-NAME (10–4 M) alone and in combination with L-arginine (10–4 M), respectively (data not shown). These results suggest that L-NAME exerted significant inhibitory effects on NECA-induced NOx production, whereas this effect was reversed by L-arginine in endothelium-intact aorta.


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  		Table 1. Total nitrates/nitrites (NOx) in intact mouse aorta

 
Effects of ZM-241385 and alloxazine on NECA-induced NOx production. The NECA-induced total nitrates and nitrites (NOx) were measured in endothelium-intact mouse aorta. The aortic rings were preincubated with A2A receptor antagonist ZM-241385 (10–5 M) and A2B receptor antagonist alloxazine (10–5 M) for 30 min each. The ZM-241385 suppressed the NOx level by 12.5% when compared with control (NECA, 10–5 M + PE 10–7 M), which was insignificant. In contrast, pretreatment with alloxazine at 10–5 M inhibited NECA-induced NOx production significantly by 56% when compared with control (NECA 10–5 M + PE 10–7 M; P < 0.05, Fig. 7). These data suggest that NECA-induced NOx production in endothelium-intact aorta was predominantly mediated through activation of A2B receptor subtype.


Figure 7
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  		Fig. 7. Effects of alloxazine (10–5 M) and ZM-241385 (10–5 M) on NOx production, induced by NECA (10–5 M) in mouse aorta with intact endothelium. Values are expressed as means ± SE (n = 9). *P < 0.05 compared with NECA.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of the present study are 1) in intact endothelium, adenosine and its analogs (NECA, CAD, and CGS-21680) produced a dose-dependent relaxation with rank order of potency favoring A2B receptor profile (NECA > CAD > adenosine >> CGS-21680; 2) treatment of aortic rings either with L-NAME and/or alloxazine significantly inhibited the NECA-induced relaxation responses as well as NOx levels; and 3) relative eNOS gene expression decreased 67% after denudation of mouse aortic endothelium compared with intact endothelium aorta. These findings, coupled with the observation that L-NAME had minimal inhibitory effects on NECA-induced relaxation and total nitrates and nitrites (NOx) production in mouse aorta with denuded endothelium, suggest that the adenosine receptor-mediated relaxation in this tissue may be, in part, through A2B receptor via endothelium-dependent NO release.

We found that removal of functional endothelium totally abolished the endothelium-dependent relaxation of acetylcholine but only partially attenuated NECA-induced relaxation. This information at least provides evidence that a portion of the relaxation produced by NECA involves activation of A2 receptors. Here it is noteworthy that A2B are the low-affinity receptors, whereas A2A are the high-affinity receptors (5). It is likely that higher concentrations of NECA are required to activate A2B receptors for a functional response as we have found in this study. Alloxazine, a relatively selective A2B receptor antagonist, inhibited the relaxation to NECA, and removal of functional endothelium also attenuated NECA-induced relaxation, suggesting that A2B receptors are involved in mediating the vasorelaxing effects via endothelium in mouse aorta. Similar studies have been reported earlier from our lab and others as well (1, 3, 17, 32, 41). In this study, the percent inhibition by alloxazine in mouse aortic preparation was similar to the one reported for National Institutes of Health 3T3 fibroblast membranes that showed selectivity of alloxazine for A2B receptors (4).

The relative potencies of CGS-21680 and NECA have been used to differentiate A2A from A2B receptors in many instances (10). Thus, to further explore the possibility of A2AAR involvement in vasorelaxation, we used specific agonist and antagonist of A2AAR, i.e., CGS-21680 and ZM-241385, respectively. Adenosine and its analogs CAD and NECA, which are capable of activating both A2A and A2B receptors, produced marked relaxation of mouse aorta, whereas CGS-21680, an A2A-selective agonist, was very weak in producing relaxation. On the other hand, ZM-241385, a specific and potent A2A receptor antagonist (34), caused insignificant inhibition of NECA-induced vasorelaxation in mouse aorta. The involvement of A2B receptor rather than A2A receptor was further supported by the inability of CGS-21680 to produce profound relaxation similar to that elicited by NECA since both these agonists are equally potent at A2A receptors, whereas NECA activates A2B receptors characteristically at concentrations in micromolar range (10), which was also observed in the present study.

We have reported previously that adenosine-induced relaxation is NO dependent in porcine coronary artery (1). In the present study, we have provided experimental evidence that suggests that mouse aortic relaxation is also endothelium dependent because the NOS inhibitor L-NAME attenuated the relaxation response in endothelium-intact, but not in endothelium-denuded, tissues to NECA. These data suggest that the relaxation responses produced by NECA in intact endothelium are due to endothelium-dependent NO production. L-Arginine, a substrate required for the synthesis of NO by NOS, reversed the effect of L-NAME on NECA-induced relaxation and NO production in endothelium-intact aorta, indicating the involvement of A2 receptors. Similar observations have been made from this laboratory (2, 32,), using human and porcine coronary endothelial cells, and by others in renal artery of rat where adenosine A2B receptors have been shown to be involved in endothelium-mediated release of NO (24).

As a further evidence to support a role for NO in our study, pretreatment of aortic rings with L-NAME increased the force generation in endothelium-intact aortic rings, whereas in denuded rings, there was no significant change in force generation with L-NAME (Fig. 3). These observations are further supported by the findings of Faraci et al. (9), who demonstrated that acetylcholine caused contraction in the aortic artery of eNOS–/– mice. To further confirm that the NO is released by endothelium, we performed the experiments for eNOS gene expression in intact and denuded endothelium mouse aorta by real-time PCR. The endothelium-denuded aortic rings showed 67% less eNOS expression when compared with endothelium-intact aorta. These findings support a role for endothelium in mediating the release of NO in this tissue, and similar findings have been reported by others in different tissues (18, 21 ).

The finding in the present study that an A2B receptor antagonist, alloxazine (10–5 M), inhibited NECA-induced relaxation response by 76% and NO production by 43% in comparison with NECA alone (100%) indicates that relaxation responses in this tissue were due to NO production mediated through A2B receptor activation. NO-associated vasorelaxation has been reported in a wide variety of tissues (see Introduction); however, there is little evidence to support A2B-mediated, endothelium-dependent NO production in intact mouse aorta. In the present work, we have shown that adenosine and its analog, NECA, induced a concentration-dependent relaxation of mouse aorta in intact and denuded endothelium. Our data in this study clearly indicate that, in this tissue, A2B receptors via endothelium play an important role in the regulation of vasorelaxation of mouse aorta, and the data further suggest that NO pathway could play an important regulatory role. These observations are consistent with the finding in porcine coronary artery reported previously from this laboratory showing that the removal of endothelium resulted in inhibition of adenosine-mediated vasorelaxation (1, 2). This conclusion is based on the findings that 1) adenosine-mediated vasorelaxation is inhibited by treatment with NG-monomethyl-L-arginine (L-NMMA); 2) pretreatment with L-arginine plus L-NMMA reversed the effect of L-NMMA (alone) on relaxation; and 3) there was a decrease in nitrite levels in denuded endothelium in comparison to intact endothelium with adenosine analog-treated tissues. Another report from our laboratory showing that the functional A2A and A2B receptor-mediated NO production is endothelium dependent in coronary artery (22) further supports the present findings. These findings along with our present data support the evidence that A2B receptors are linked to NO production in human, porcine, and mouse aortic endothelial cells.

In summary, the results presented here indicate that activation of A2B receptors plays an important role in endothelium-mediated relaxation of mouse aorta through NO release. Finally, endothelium-dependent responses and NO signaling pathways may play a significant role in the regulation of vascular tone by adenosine receptors that may be altered in cardiovascular complications.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work is supported by National Heart, Lung and Blood Institute Grant HL-027339.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. J. Mustafa, Dept. of Physiology and Pharmacology, Center for Interdisciplinary Research in Cardiovascular Sciences (CIRCS), Robert C. Byrd Health Science Center, West Virginia Univ., Morgantown, WV 26506 (e-mail: smustafa{at}hsc.wvu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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