The cellular mechanisms by which adenosine evokes release of nitric oxide from rat aortic endothelium
1 Department of Physiology, The Medical School, University of Birmingham, Birmingham B15 2TT, UK
Abstract
Adenosine and nitric oxide (NO) are important local mediators of vasodilatation. The aim of this study was to elucidate the mechanisms underlying adenosine receptor-mediated NO release from the endothelium. In studies on freshly excised rat aorta, second-messenger systems were pharmacologically modulated by appropriate antagonists while a NO-sensitive electrode was used to measure adenosine-evoked NO release from the endothelium. We showed that A1-mediated NO release requires extracellular Ca2+, phospholipase A2 (PLA2) and ATP-sensitive K+ (KATP) channel activation whereas A2A-mediated NO release requires extracellular Ca2+ and Ca2+-activated K+ (KCa) channels. Since our previous study showed that A1- and A2A-receptor-mediated NO release requires activation of adenylate cyclase (AC), we propose the following novel pathways. The K+ efflux resulting from A1-receptor-coupled KATP-channel activation facilitates Ca2+ influx which may cause some stimulation of endothelial NO synthase (eNOS). However, the increase in [Ca2+]i also stimulates PLA2 to liberate arachidonic acid and stimulate cyclooxygenase to generate prostacyclin (PGI2). PGI2 acts on its endothelial receptors to increase cAMP, so activating protein kinase A (PKA) to phosphorylate and activate eNOS resulting in NO release. By contrast, the K+ efflux resulting from A2A-coupled KCa channels facilitates Ca2+ influx, thereby activating eNOS and NO release. This process may be facilitated by phosphorylation of eNOS by PKA via the action of A2A-receptor-mediated stimulation of AC increasing cAMP. These pathways may be important in mediating vasodilatation during exercise and systemic hypoxia when adenosine acting in an endothelium- and NO-dependent manner has been shown to be important.
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Adenosine is an important mediator of vasodilatation in the coronary, cerebral and skeletal muscle circulations in a number of conditions including hypoxia and exercise (Berne et al. 1983). For many years it was accepted that adenosine evoked dilatation by stimulating A2 receptors (particularly the A2A subtype) on the vascular smooth muscle (VSM) via an increase in cAMP (see Olsson & Pearson, 1990); however, more recent evidence shows the A1 receptor subtype can also mediate dilatation (Merkel et al. 1992; Nakhostine & Lamontagne, 1993; Danialou et al. 1997; Bryan & Marshall, 1999a). In addition, a number of studies have now shown that adenosine can evoke dilatation in an endothelium-dependent fashion that is also nitric oxide (NO) dependent (Rose'Meyer & Hope, 1990; Prentice & Hourani, 2000).
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In our recent studies on lengths of endothelium-intact rat aorta, adenosine acting via A1and A2A adenosine receptors evoked dose-dependent NO release (measured by an NO-sensitive electrode) from the endothelium (Ray et al. 2002). Furthermore, A1-receptor stimulation evoked NO release that was attenuated by a cyclooxygenase (COX) inhibitor and release of prostacyclin (PGI2) from the endothelium, as assessed by radioimmunoassay. Iloprost, an analogue of PGI2, also evoked endothelial NO release, raising the possibility of an interaction between adenosine, NO and PGI2 in mediating dilatation (Ray et al. 2002). An additional finding of this study was that both A1- and A2A-mediated NO release were dependent on an increase in cAMP, as both responses were attenuated by adenylate cyclase (AC) inhibition.
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These findings led us to propose that both A1- and A2A-mediated NO release are dependent on an increase in cAMP. However, we proposed that A2A-receptor activation might directly increase cAMP (since A2A receptors are considered to be positively coupled to AC; Londos et al. 1980). In contrast, we proposed that stimulation of A1 receptors – which are considered to be negatively coupled to AC (Londos et al. 1980) – increases the synthesis of PGI2 which acts on endothelial cells in an autocrine fashion to stimulate AC-linked prostacyclin receptors (IP receptors; Moncada & Vane, 1979), so leading to an increase in intracellular cAMP (Ray et al. 2002). Both proposals accord with recent evidence that NO release can be stimulated by protein kinase A (PKA)-mediated phosphorylation of endothelial NO synthase (eNOS; Zhang & Hintze, 2001).
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However, our proposals clearly leave many questions unanswered as to exactly how adenosine stimulates NO release via its A1 and A2A receptors. Elucidating these mechanisms was the objective of the present study.
In Chinese hamster ovary (CHO) cells, stimulation of transfected A1 receptors augmented the increase in phospholipase A2 (PLA2) activity induced by ACh and thrombin (Akbar et al. 1994; Dickenson & Hill, 1997). PLA2 is the enzyme that cleaves cell membrane phospholipids to yield arachidonic acid (AA), the precursor for prostaglandins (PGs) generated by COX.
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Both the action of PLA2 and activation of eNOS have been associated with, or shown to be dependent on, an increase in intracellular Ca2+ (Busse & Mülsch, 1990; Balsinde et al. 1999). Ca2+ can be released from intracellular stores by inositol 1,4,5-trisphosphate (IP3) which is formed by the action of phospholipase C (PLC). In cultured CHO cells and rabbit airway smooth muscle, activation of A1 receptors stimulated PLC (Abebe & Mustafa, 1998; Dickenson & Hill, 1998). Furthermore, in isolated astrocytes, the increase in [Ca2+]i evoked by the non-selective adenosine analogue (1-6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuronamide) (NECA), was completely blocked by a PLC inhibitor, and was attenuated by an A2 receptor antagonist (Pilitsis & Kimelberg, 1998). Therefore, both A1 and A2 adenosine receptors can be functionally coupled to PLC, at least in some cell types.
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Intracellular Ca2+ can also be raised in endothelial cells by influx of extracellular Ca2+. Activation of K+ channels leads to K+ efflux, membrane hyperpolarization and a membrane potential-dependent driving force for the influx of extracellular Ca2+ (Busse et al. 1988; Lückhoff & Busse, 1990). There is already evidence that in cultured coronary endothelial cells, adenosine can cause membrane hyperpolarization via the activation of Ca2+-activated K+ (KCa) channels, but the adenosine receptor subtype responsible for this response was not tested (Mehrke & Daut, 1990). Furthermore, there is pharmacological evidence linking both A1 and A2A adenosine receptors and ATP-sensitive K+ (KATP) channels in cultured VSM cells, endothelium-intact porcine coronary microvessels, isolated rabbit heart coronary arteries and rat diaphragmatic arterioles (Dart & Standen, 1993; Nakhostine & Lamontagne, 1993; Kleppisch & Nelson, 1995; Danialou et al. 1997; Hein et al. 2001). Further, the NO-dependent vasodilatation evoked in rat hindlimb by A1 receptor stimulation was attenuated by KATP-channel inhibition (Bryan & Marshall, 1999b).
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In view of this evidence, we have used selective pharmacological agents to investigate whether NO released from the endothelial cells of freshly excised rat aorta by A1 and/or A2A receptor stimulation is dependent on extracellular Ca2+, PLC, PLA2, KCa and KATP channels. The results obtained allow us to propose different pathways for A1- and A2A-receptor-evoked stimulation of NO release and to suggest how the mechanisms uncovered may play a role in physiological situations in which adenosine levels are increased.
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Methods
The release of NO from lengths of rat thoracic aorta was recorded continuously with an NO-sensitive electrode (ISO-NOP, WPI, FL, USA) with a 2 mm diameter tip, connected to a meter (ISO-NO Mark II, WPI), as previously described (Ray et al. 2002). The electrode is highly selective for NO, it has a lower detection limit of 1 nM and can measure NO in solution over a linear range of 1 nM to 20 μM (Broderick & Taha, 1995). Briefly, lengths (9.78 ± 1.56 mm) of thoracic aorta were removed from male Wistar rats immediately after they were killed, in accordance with the UK Animals (Scientific Procedures) Act 1986, by cervical dislocation under anaesthesia (3.5% halothane in O2). Each length of aorta was opened longitudinally, pinned endothelial surface uppermost in a Petri dish and bathed in 10 ml Krebs solution (pH 7.4, containing (mM): 118 NaCl. 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4, 1.1 MgSO4, 10 Hepes and 5.6 glucose; Ray et al. 2002). With the aid of a micromanipulator, the NO electrode was advanced as close as possible to the endothelial surface of the vessel, and the output was recorded via a data acquisition system (MacLab/2e, AD Instruments Ltd, Oxfordshire, UK) connected to a computer (Power Macintosh 6100/60). The experimental apparatus was placed in a grounded Faraday cage to reduce electrical noise and interference. The electrode was calibrated by chemical generation of NO on each experimental day according to the following equation:
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It has been previously demonstrated that this experimental system allows reproducible measurement of NO release (Guo et al. 1996; Simonsen et al. 1999; Ray et al. 2002).
Protocols
Group 1. The NO release evoked by adenosine was tested in the presence and absence of Ca2+. The response to 1 mM adenosine was tested, the bathing medium of the vessel was then replaced with Ca2+-free Krebs containing Ca2+ chelators EDTA and EGTA (1 mM; Michel et al. 1997), and the response to 1 mM adenosine was tested after 30 min. Finally, Ca2+-containing Krebs solution was restored to the vessel and the response to 1 mM adenosine was retested after 30 min.
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It should be noted that 1 mM is a relatively high agonist concentration; however, it is comparable with the dose used in numerous studies (e.g. Mian & Marshall, 1991; Danialou et al. 1997; Rubin et al. 2000), and in our previous studies adenosine evoked a dose-dependent increase in NO release, with a consistent maximal response being achieved with a dose of 1 mM (Ray et al. 2002). Exogenous adenosine has two fates, rapid uptake by the adenosine transporter or deamination to inosine by adenosine deaminase (Olsson & Pearson, 1990). Our own experiments using adenosine deaminase and uptake inhibitors (erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) and nitrobenzylthioinosine (NBTI), respectively, suggest that these mechanisms substantially reduce the effective concentration of adenosine at the receptors. Thus, in the rat aorta preparation, concentrations of adenosine in the range 10–9 to 10–4M, evoked dose-dependent NO release when adenosine transport and deamination were inhibited. Therefore, we have argued that using a standard dose of 1 mM adenosine to study its mechanisms of action on endothelial cells is reasonable (see Ray et al. 2002 for further discussion).
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Group 2. (a) The NO release evoked by 1 mM adenosine was tested before and 10 and 30 min after addition of the specific PLC inhibitor U73122 (1 μM). This concentration of U73122 abolished agonist-induced increases in [Ca2+]i in cultured endothelial cells (Lee & Wu, 1999) and relaxation of preconstricted rat mesenteric arteries in vitro (Parenti et al. 2000). (b) As a control, the NO response to adenosine (1 mM) was tested before and 10 and 30 min after addition of U73343 – the inactive structural analogue of U73122 (1 μM; see Lee & Wu, 1999).
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Group 3. (a) The NO release evoked by 1 mM adenosine was tested before and 10, 30 and 60 min after the addition of the inhibitor of PLA2, AACOCF3 (10 μM). AACOCF3 binds to and inhibits PLA2 (Street et al. 1993) and at this concentration inhibited endothelin-1-induced release of radiolabelled AA from rat tail artery segments in vitro (Trevisi et al. 2002). To differentiate between a role for PLA2 in A1- and A2A-mediated NO release, the NO response to 1 mM adenosine was tested before and after (b) the addition of the A1 adenosine receptor antagonist DPCPX (100 nM) or (c) the A2A adenosine receptor antagonist ZM241385 (100 nM), and then after the subsequent addition of AACOCF3 (10 μM). These concentrations of DPCPX and ZM241385 are those used in our previous study to effectively block A1 and A2A receptors, respectively (Ray et al. 2002).
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Group 4. (a) The NO release evoked by adenosine (1 mM) was tested before and 10 and 30 min after the addition of iberiotoxin (100 nM) a specific inhibitor of large-conductance KCa (BKCa) channels. Iberiotoxin at this concentration has been shown to inhibit BKCa-channel activation in endothelial cells using the patch-clamp technique in response to various agonists (Faehling et al. 2001; Kuhlmann et al. 2003) and to attenuate significantly agonist-induced coronary arteriolar dilatation in vitro (Hein et al. 2000). At this concentration, iberiotoxin does not affect small-conductance (SKCa) channels (Paolocci et al. 2001). To differentiate between a role for BKCa channels in A1- and A2A-mediated NO release the NO response evoked by adenosine (1 mM) was tested before and after the addition of (b) DPCPX (100 nM) or (c) ZM241385 (100 nM), and then after the subsequent addition of iberiotoxin (100 nM).
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Group 5. (a) The NO release evoked by adenosine (1 mM) was tested before and 10 and 30 min after the addition of apamin (500 nM), a specific inhibitor of SKCa channels. Concentrations of apamin in this range effectively blocked the hyperpolarization (as assessed by direct measurement of membrane potential) and resultant relaxation of isolated mesenteric arteries in response to ACh (Chen & Cheung, 1997). In addition, as in Groups 3 and 4, NO release in response to 1 mM adenosine was tested before and after the addition of (b) DPCPX (100 nM) or (c) ZM241385 (100 nM), and then after the subsequent addition of apamin (500 nM).
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Group 6. (a) The NO release evoked by 1 mM adenosine was tested before and 10, 30 and 60 min after the addition of glibenclamide (3 μM), an inhibitor of KATP channels. This concentration of glibenclamide attenuated the relaxation of rat aortic rings in vitro in response to the NO donors S-nitroso-glutathione and S-nitroso-N-acetylcystein (Ceron et al. 2001), and in response to adenosine (Harada et al. 2001) and hyperpolarization of human umbilical vein endothelial cells evoked by a high concentration of D-glucose (Flores et al. 2003). The NO response evoked by adenosine (1 mM) was tested before and after the addition of (b) DPCPX (100 nM) or (c) ZM241385 (100 nM) and then 10, 30 and 60 min after the subsequent addition of glibenclamide (3 μM).
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Statistical analyses
Responses were measured as the maximum change in the NO output signal for each dose of adenosine before and after the various pharmacological agents (see Ray et al. 2002). All results are expressed as means ±S.E.M. All responses were analysed by ANOVA followed by Scheffé's post hoc test when appropriate. P < 0.05 was considered significant. In all cases, n is the number of animals.
Results
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We have previously shown that the application of 1 mM adenosine to endothelium-denuded aortic lengths evoked no measurable output of NO (Ray et al. 2002). Thus, any NO release measured in the present study can be assumed to be the result of stimulation of the endothelial cells. Tests performed at the end of the experiments of Groups 1–5 showed that the addition of the pharmacological agents used in the present study to the tissue bath, in the absence of arterial tissue, evoked no detectable change in the output of the NO electrode. Moreover, the addition of all pharmacological inhibitors to endothelium-intact lengths of aorta during experimental protocols evoked no measurable change in the output of NO.
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Group 1. We have previously described the NO response to 1 mM adenosine (Ray et al. 2002): a rapid increase in NO release, reaching a peak within 10 s, remaining at this level for the next 1–3 min and gradually returning towards baseline over the following 3–6 min. The NO response to adenosine (34.3 ± 4.68 nM; n= 7) was significantly attenuated by the removal of extracellular Ca2+ (4.69 ± 1.24 nM), but the full response returned (37.7 ± 6.23 nM) when Ca2+ was restored by changing the bathing medium to standard Krebs solution.
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Group 2. The NO response to 1 mM adenosine (29.1 ± 4.44 nM; n= 7) was not affected by (a) PLC inhibition with U73122 at 10 (29.3 ± 5.34 nM) or 20 min (30.5 ± 5.50 nM), or by (b) the structural analogue of U73343 (23.5 ± 3.80 nM before U73343, and 21.4 ± 4.04 and 21.1 ± 3.64 nM at 10 and 20 min after U73343, respectively).
Group 3. (a) The NO response evoked by adenosine (1 mM) was attenuated to similar extents 10, 30 and 60 min after inhibition of PLA2 with AACOCF3 (n= 6; Fig. 1A). (b) As expected (Ray et al. 2002) the NO response to 1 mM adenosine was reduced in the presence of the A1 receptor antagonist DPCPX: the remaining NO response was not affected 10 min after the addition of AACOCF3 (n= 10; Fig. 1B). (c) The NO release evoked by 1 mM adenosine was reduced by the A2A receptor antagonist ZM241385 (Ray et al. 2002). This NO response was abolished 10 min after the addition of AACOCF3 (n= 10; Fig. 1C).
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The NO response evoked by 1 mM adenosine (filled bars) was: significantly reduced 10, 30 and 60 min after the addition of the PLA2 inhibitor AACOCF3 (n= 6) (A); reduced in the presence of the A1 receptor antagonist DPCPX but unaffected by the further addition of AACOCF3 (n= 10) (B); reduced in the presence of the A2A receptor antagonist ZM241385 and was completely abolished after the addition of AACOCF3 (n= 10) (C). Columns show mean (±S.E.M.) NO release. P < 0.0001; P < 0.001; P < 0.01; P < 0.05; as indicated by brackets.
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Group 4. (a) The NO response to 1 mM adenosine was significantly attenuated 10 and 30 min after the inhibition of BKCa channels with iberiotoxin (n= 10; Fig. 2A). (b) The NO release evoked by adenosine (1 mM) in the presence of DPCPX was attenuated 10 min after BKCa-channel inhibition with iberiotoxin (n= 8; Fig. 2B). (c) In contrast, the NO response to adenosine in the presence of ZM241385 was not altered 10 min after the subsequent addition of iberiotoxin (n= 8; Fig. 2C).
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The NO response evoked by 1 mM adenosine (filled bars) was: significantly attenuated 10 and 30 min after the addition of the BKCa-channel inhibitor iberiotoxin (n= 10) (A); reduced by the A1 receptor antagonist DPCPX and further attenuated by iberiotoxin (n= 8) (B); reduced by the A2A receptor antagonist ZM241385 but unchanged by the further addition of iberiotoxin (n= 8) (C). Columns show mean (±S.E.M.) NO output. P < 0.0001; P < 0.01; as indicated by brackets.
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The NO response evoked by 1 mM adenosine (filled bars) was: significantly attenuated 10 and 30 min after the addition of the SKCa-channel inhibitor apamin (n= 10) (A); reduced by the A1 receptor antagonist DPCPX and further attenuated by the administration of apamin (n= 9) (B); reduced by the A2A receptor antagonist ZM241385 but unchanged by the further addition of apamin (n= 9) (C). Columns show mean (±S.E.M.) NO output. P < 0.0001, as indicated by brackets.
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Group 6. (a) The NO response to adenosine (1 mM) was significantly attenuated 10, 30 and 60 min after the addition of the KATP-channel inhibitor glibenclamide; the extent of the inhibition appeared to develop with time (n= 8; Fig. 4A). (b) The NO release evoked by 1 mM adenosine in the presence of DPCPX was not affected 10 and 30 min after KATP-channel inhibition with glibenclamide, but was significantly attenuated 60 min after glibenclamide addition (n= 10; Fig. 4B). (c) In contrast, the NO response to adenosine in the presence of ZM241385 was substantially attenuated 10 min after glibenclamide but there was no further attenuation at 30 and 60 min (n= 10; Fig. 4C).
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The NO response evoked by 1 mM adenosine (filled bars) was: significantly attenuated 10, 30 and 60 min after the addition of the KATP-channel inhibitor glibenclamide (n= 8) (A); reduced by the A1 receptor antagonist DPCPX, was unaffected 10 and 30 min after the further addition of glibenclamide, but was significantly reduced 60 min after the administration of glibenclamide (n= 10) (B); reduced by the A2A receptor antagonist ZM241385 and further attenuated 10, 30 and 60 min after the administration of glibenclamide (n= 10) (C). Columns show mean (±S.E.M.) NO output. P < 0.0001; P < 0.001; P < 0.05; as indicated by the brackets.
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Discussion
The results of the present study indicate for the first time that the NO release evoked by adenosine from the endothelium of rat aorta is dependent on extracellular Ca2+, but not on stimulation of PLC. They also demonstrate that the adenosine-evoked response is dependent on stimulation of PLA2 and the action of large- and small-conductance KCa channels and of KATP channels. The experiments performed in the presence of the A2A adenosine receptor antagonist ZM241385, or the A1 adenosine receptor antagonist DPCPX, indicate, respectively, that A1-mediated NO release is dependent on PLA2 and activation of KATP channels, whereas A2A-mediated NO release is dependent on activation of small- and large-conductance KCa channels.
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Before discussing our results more fully, the potential limitations of the study should be considered, Firstly, the standard dose of adenosine used in the present study was high (1 mM). As indicated in the Methods, although 1 mM adenosine appears high, endothelial cells exhibit avid metabolism of adenosine, and in our previous experiments we showed that in the presence of adenosine deaminase and uptake inhibitors, adenosine evoked NO release over the full range of concentrations from 10–9 to 10–4M. Thus, it can be argued that the effective concentration of adenosine at the receptors when a 1 mM dose of adenosine is applied is physiological (see Ray et al. 2002 for further discussion). This is supported by the fact that in our previous and present studies, nanomolar concentrations of the adenosine receptor antagonists DPCPX and ZM241385 significantly reduced the NO response evoked by 1 mM adenosine.
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Secondly, there was variation in the mean concentration of NO released by 1 mM adenosine between experimental groups. Previous studies have shown that the sensitivity of the NO electrode does not alter over the course of an individual experiment although the sensitivity does alter from day to day (Simonsen et al. 1999). Further, we have observed that changing the NO-permeable membrane, which covers the NO electrode, also affects the sensitivity of NO measurement (C. J. Ray & J. M. Marshall, unpublished observations). Thus, in the present study, as in previous studies (Simonsen et al. 1999; Ray et al. 2002), these problems were avoided by daily calibration of the electrode, and by conducting all the experiments for each experimental group using only one NO-permeable membrane. This allowed accurate within-group comparisons of NO release to be performed but any comparisons between different experimental groups should be performed with caution.
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The question then arises as to whether the concentrations of NO released in response to adenosine in the present study are sufficient to produce vasodilatation. In fact, the NO concentrations measured in the present study were similar to those measured using an NO-sensitive electrode in rat mesenteric arteries in response to ACh (21 ± 6 nM; Simonsen et al. 1999) and porcine coronary artery rings in response to bradykinin (105 ± 19.6 nM; Ge et al. 2000) when vasodilatation was also recorded. The in vivo measurement of NO has proven technically difficult due to its half-life of 3–5 s under physiological conditions (Ignarro, 1990). However, our own experiments in which the change in the arterial–venous difference in plasma nitrate levels across hindlimb muscle was used as an index of NO release in response to systemic hypoxia and adenosine infusion, suggested that an increase in the plasma concentration of NO of 20–40 nM causes vasodilatation (Ray & Marshall, 2005). These results agree with those obtained in the coronary circulation of conscious exercising dogs where NO levels, estimated from blood NOx by chemiluminescence, in the range of 40–130 nm were accompanied by an increase in coronary blood flow (Bernstein et al. 1996). Therefore, it is reasonable to assume that the NO concentrations measured in the present study are within the physiologically relevant range and would be able to mediate vasodilatation.
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Requirement for extracellular Ca2+ in adenosine-evoked NO release
In the present study, the removal of extracellular Ca2+ from the medium bathing the rat aorta severely attenuated, and in most cases abolished, adenosine-evoked release of NO from the endothelium; the response to adenosine returned when extracellular Ca2+ was restored. Thus, adenosine-induced release of NO is critically dependent on influx of extracellular Ca2+. From our previous study on rat aorta (Ray et al. 2002) and the present study, we know that 50% of adenosine-evoked NO release is A1 mediated and 50% is A2A mediated. The dramatic effect of Ca2+ removal on the response to adenosine therefore indicated that both A1- and A2A-stimulated NO release is critically dependent on Ca2+ influx. This is not surprising as the established view is that agonist-stimulated release of NO from endothelial cells is mediated predominantly via a Ca2+–calmodulin (CaM)-dependent mechanism (Busse & Mülsch, 1990) and requires sustained influx of extracellular Ca2+ to stimulate eNOS (Lückhoff & Busse, 1990). However, influx of extracellular Ca2+ can stimulate the release of Ca2+ from intracellular stores (Ca2+-induced Ca2+ release). Indeed, when [Ca2+]i and NO release were monitored using fluorescent techniques, ATP evoked a biphasic response attributable to: (i) a rapid, transient increase in [Ca2+]i due to Ca2+ release from IP3-sensitive stores and (ii) a sustained plateau in [Ca2+]i due to the Ca2+ influx from the extracellular space (Holda et al. 1998). Therefore, changes in [Ca2+]i leading to eNOS stimulation can be mediated in a number of different ways, including via the PLC–IP3 pathway.
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In the present study, NO released by adenosine was not affected by the PLC inhibitor U73122. Therefore, it seems that release of intracellular Ca2+ by IP3 and/or by diacylglycerol did not play a significant role in the NO release evoked by either A1 or A2A stimulation, but that influx of Ca2+ was responsible. Agonist-evoked endothelial cell NO production is mainly controlled by myristoylated, membrane-associated eNOS, which is sensitive to Ca2+ influx via store-operated Ca2+ (SOC) channels (Lin et al. 2000). Thus, adenosine-evoked NO release from rat aortic endothelium may be mediated by influx of Ca2+ via SOC channels. However, Ca2+ influx could also occur via agonist-activated non-selective Ca2+-permeable cation channels, or cyclic nucleotide-activated non-selective cation channels (Nilius & Droogmans, 2001); there are no voltage-operated Ca2+ channels present on the endothelium (Nilius & Droogmans, 2001).
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Involvement of PLA2
AACOCF3 which inhibits PLA2, significantly attenuated the NO released by adenosine in the rat aorta indicating that adenosine normally stimulates PLA2 to release NO. In fact, AACOCF3 inhibited the NO released by adenosine in the presence of ZM241385, but not in the presence of DPCPX, indicating that A1, but not A2A, receptor stimulation, involves activation of PLA2. This result is in complete agreement with our previous findings that A1- but not A2A-receptor stimulation leads to PGI2 production and release (Ray et al. 2002), for PLA2 is the key enzyme in the COX pathway that liberates AA from cell membranes (Lands, 1979; Smith, 1992). There is evidence that A1 receptors are coupled to PLA2 in a number of different cell types (Akbar et al. 1994; Schachter et al. 1995; Dickenson & Hill, 1997).
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Involvement of K+ channels
The role of KCa channels in the NO response evoked by adenosine was investigated in the present study by using iberiotoxin (an inhibitor of BKCa channels), and apamin (an inhibitor of SKCa channels). Both iberiotoxin and apamin attenuated the NO released by adenosine to the same extent, suggesting that both BKCa and SKCa channels are present on rat aortic endothelium and are involved in the adenosine-evoked NO response. However, the amount of NO released by adenosine in the presence of ZM241385 was not affected by iberiotoxin or apamin, whereas that released in the presence of DPCPX, was substantially attenuated by both iberiotoxin and by apamin. Thus, these results suggest that both BKCa and SKCa channels are coupled to A2A adenosine receptors, but not to A1 receptors.
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In some previous studies, the application of iberiotoxin and apamin in combination potentiated the effect that each inhibitor had when used alone (Chen & Cheung, 1997; Petersson et al. 1997). In contrast, other studies demonstrated no potentiation when the drugs were applied together (Frieden et al. 1999). In the present study, iberiotoxin and apamin administered separately, each produced more or less complete inhibition of the NO release evoked by A2A adenosine receptor stimulation. Thus, in rat aortic endothelium, it seems the blockade of one of these subtypes of KCa channel, somehow affects the ability of adenosine to stimulate NO release by opening the other subtype of KCa channel or indeed, by stimulation of other pathways. In fact, activation of a KCa channel is apparently essential for A2A-stimulated NO release.
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Various agonists including bradykinin, ATP and ACh have been shown to activate endothelial KCa channels (Rusko et al. 1992; Fulton et al. 1994; Ishida et al. 1999). Indeed, the NO-dependent relaxation of porcine coronary arteries evoked by bradykinin was attenuated by KCa-channel inhibition (Ge et al. 2000) and vasorelaxation evoked in the isolated preconstricted rat mesenteric bed by direct activation of KCa channels with 1-ethyl-2-benzimidazolinone, was abolished by NOS inhibition with L-NAME (Adeagbo, 1999), implicating KCa channels in NO-dependent dilatation. The present results indicate that adenosine acting via A2A receptors can be added to this list of agonists and receptors for which activation of KCa channels is linked to NO release.
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The KATP-channel inhibitor, glibenclamide, also attenuated the NO response to adenosine. However, in contrast to apamin and iberiotoxin, the effect of glibenclamide on NO release was present at 10 min after application, and was greater at 30 and 60 min. This suggests that glibenclamide affects NO release by more than one mechanism. The experiments with selective A1- or A2A-receptor blockade allowed these effects to be differentiated. The NO response evoked by adenosine acting at the A2A receptors was not affected at 10 and 30 min after glibenclamide administration, but was moderately attenuated at 60 min. The most likely explanation for these results is that A2A adenosine receptors are not functionally coupled to KATP channels, at least as far as NOS stimulation and NO release is concerned. Rather, it is likely that the inhibition of KATP channels on endothelial cells caused by glibenclamide affected L-arginine transport. Thus, L-arginine, the substrate for NO synthesis, is transported into endothelial cells via a system y+ cationic amino acid transporter (Bogle et al. 1991, 1996). Accordingly, L-arginine transport is sensitive to membrane potential (Sobrevia et al. 1997; Zharikov et al. 1997; Zharikov & Block, 1998): it is stimulated by drugs that cause membrane hyperpolarization, and inhibited by those that cause membrane depolarization (Zharikov & Block, 1998). Thus, endothelial cell depolarization caused by glibenclamide may have depleted the pool of L-arginine available for eNOS such that A2A-receptor-stimulated NO release was attenuated at 60 min after glibenclamide administration.
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By contrast, the finding that NO released by A1 receptor stimulation was attenuated at 10 min, suggests that these receptors are functionally coupled to KATP channels. The fact that there was no time-dependent effect of glibenclamide on the A1-mediated NO response, can be explained if the NO response was already attenuated to such an extent at 10 min, that limitation of L-arginine transport did not further affect the ability of A1 receptor stimulation to generate small amounts of NO.
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Previous studies on rat diaphragmatic arterioles in vitro and hindlimb muscle in vivo, have demonstrated that the NO-dependent dilatation evoked by a specific A1 adenosine receptor agonist was attenuated by KATP-channel inhibition with glibenclamide (Danialou et al. 1997; Bryan & Marshall, 1999a,b). In those studies no differentiation was possible between A1 receptors and KATP channels on the endothelium or VSM cells. The results of the present study provide direct evidence that, at least in the aorta, endothelial A1 receptors are coupled to KATP channels and that their activation is essential for A1-mediated NO release.
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A possible pathway for A1-mediated NO release
The results of our previous in vitro studies (Ray et al. 2002) showed that A1-stimulated NO release from the endothelium of rat aorta is dependent on PGI2 synthesis. The present results substantiate these findings by showing that A1-mediated NO release is dependent on Ca2+ influx, PLA2 and KATP-channel activation. Taken together, these results allow the proposal that, in contrast to their classical inhibitory coupling to AC (Londos et al. 1980), the A1 receptors are functionally coupled to KATP channels (event 1 in Fig. 5), so that when A1 receptors are stimulated, they initiate KATP-channel opening and the resulting K+ efflux facilitates Ca2+ influx (event 2 in Fig. 5), so increasing [Ca2+]i. This increase in [Ca2+]i may lead to some direct activation of eNOS via the classical Ca2–CaM pathway (event 3), but would not explain the involvement of PLA2. In isolated porcine microvessels and canine coronary arteries, forskolin and isoproterenol (both of which increase intracellular cAMP) released NO, as assessed by the Greiss reaction, in a manner that was dependent on PKA (Kudej et al. 2000; Zhang & Hintze, 2001; Zhang & Leffler, 2002). Moreover, experiments on cultured endothelial cells showed that agonist-induced stimulation of eNOS can involve activation of a cAMP-dependent protein kinase and phosphorylation of eNOS independent of an increase in [Ca2+]i (Butt et al. 2000). However, the NO release evoked by adenosine in the present study was virtually abolished when extracellular Ca2+ was removed. Thus, in view of the dependence of A1-stimulated NO release on PGI2 synthesis, we propose that an increase in [Ca2+]i evoked by activation of KATP channels is important in stimulating PLA2 (event 4), so leading to the liberation of AA and stimulation of the COX (event 5) pathway to generate PGI2 (event 6). The PGI2 is then presumably released from the cell and acts on its endothelial IP receptors (event 7), which are linked to AC, to increase intracellular cAMP, activate PKA (event 8) and so phosphorylate and activate eNOS (event 9). Although this pathway may seem surprisingly complex, it may be that it is functionally necessary because, in the absence of a PGI2-stimulated increase in cAMP, the direct inhibitory effect of A1 receptor stimulation on cAMP (see Introduction; Londos et al. 1980) would mean that the phosphorylation state of eNOS would be too low for it to be activated by the increase in Ca2+ that results from A1-stimulated KATP-channel opening.
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The bold numbers (1–9) on the endothelial cell on the left and the bold letters (A–G) on the endothelial cell on the right represent the possible order of events in the proposed pathways for A1 and A2A receptor stimulation, respectively.
A possible pathway for A2A-mediated NO release
As indicated in the Introduction, the results of our previous study (Ray et al. 2002) suggest, in agreement with many previous studies, that the A2A receptors present on the endothelial cells of rat aorta are positively coupled to AC and that it is by this mechanism that A2A receptor stimulation leads to NO release. A2A-receptor-stimulated NO release is therefore apparently dependent on an increase in intracellular concentration of cAMP (event A in Fig. 5). The increase in cAMP (event B) may directly mediate the phosphorylation and activation of eNOS (event C) as described above. However, agonists that increase intracellular cAMP can also stimulate PKA and lead to the phosphorylation and activation of KCa channels (Schubert et al. 1997). Thus, in view of the present evidence that KCa channels are involved in A2A-mediated NO release, we suggest a pathway in which, in addition to any potential direct coupling between A2A receptors and KCa channels, PKA stimulation by increased intracellular cAMP leads to KCa-channel activation (event D): K+ efflux through KCa channels (event E) then causes endothelial membrane hyperpolarization, so facilitating Ca2+ influx (see above; event F) and providing a preferential source of Ca2+ for eNOS activation and subsequent NO synthesis and release (event G). Phosphorylation of eNOS arising from an A2A-receptor-stimulated increase in cAMP may facilitate this process (event C).
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Adenosine-receptor-mediated NO release – a physiological role
There is substantial evidence in the literature that adenosine arising from skeletal muscle fibres plays an important role in the vasodilatation evoked by exercise and that endothelial-derived adenosine is involved in the dilatation evoked by systemic hypoxia (Marshall, 2000; Radegran & Hellsten, 2000). Moreover, there is significant evidence that NO also evokes dilatation under these conditions (Marshall, 2000; Radegran & Hellsten, 2000). Therefore it is possible that the mechanisms of adenosine-evoked NO release may have an important role to play in these situations. For example, the adenosine-mediated component of skeletal muscle vasodilatation evoked by systemic hypoxia is mediated by locally produced adenosine acting at endothelial A1 receptors in an NO- and PG-dependent manner that involves activation of KATP channels (Marshall et al. 1993; Bryan & Marshall, 1999b; Ray et al. 2002). Thus, it seems reasonable to propose that the pathway we have described above on the basis of our in vitro studies is functionally important during systemic hypoxia in skeletal muscle. A1 receptor agonists caused vasodilatation of isolated rings of porcine coronary artery (Merkel et al. 1992) and arterioles of rat diaphragm (Danialou et al. 1997), that appeared to be in part mediated via the modulation of potassium channels (Merkel et al. 1992), particularly KATP channels (Danialou et al. 1997); this raises the possibility that the pathway uncovered in the present study may have physiological relevance in a variety of circulations.
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A2A adenosine receptors are implicated in locally mediated vasodilatation in the coronary, pulmonary, cerebral and skeletal muscle circulations (Palmer & Ghai, 1982; Collis & Brown, 1983; Mustafa & Askar, 1985; Oei et al. 1988; McCormack et al. 1989; Poucher, 1996). Thus, the pathway we have identified may be important in causing NO-mediated vasodilatation in these tissues in situations where adenosine levels are increased sufficiently to stimulate the A2A receptors which have an affinity constant of 2–20 μM for adenosine; that is approximately three orders of magnitude less than that of the A1 receptors (affinity constant, 10–100 nM; Daly et al. 1981).
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Our findings have wide-reaching implications for several reasons. Firstly, they show the coupling of adenosine receptors to second-messenger signalling pathways other than the classically recognized AC–cAMP system; these pathways may underlie the mechanisms by which adenosine signals in tissues other than blood vessels. Secondly, the results of this study extend recent findings that show alternative ways of activating eNOS rather than simply via the Ca2–CaM pathway. As adenosine, PGs and NO are mediators involved in vasodilatation in all circulations and under various different physiological and pathological conditions, the findings of the present study and the pathways they allow us to propose, may have relevance to many situations in which one or other of these mediators are implicated. Finally, the release of NO by mechanisms other than a simple Ca2+-dependent pathway, has far-reaching consequences for the stimulation of eNOS and the release of NO by agonists that not only increase intracellular Ca2+, but are known to modulate other intracellular pathways.
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Abstract
Adenosine and nitric oxide (NO) are important local mediators of vasodilatation. The aim of this study was to elucidate the mechanisms underlying adenosine receptor-mediated NO release from the endothelium. In studies on freshly excised rat aorta, second-messenger systems were pharmacologically modulated by appropriate antagonists while a NO-sensitive electrode was used to measure adenosine-evoked NO release from the endothelium. We showed that A1-mediated NO release requires extracellular Ca2+, phospholipase A2 (PLA2) and ATP-sensitive K+ (KATP) channel activation whereas A2A-mediated NO release requires extracellular Ca2+ and Ca2+-activated K+ (KCa) channels. Since our previous study showed that A1- and A2A-receptor-mediated NO release requires activation of adenylate cyclase (AC), we propose the following novel pathways. The K+ efflux resulting from A1-receptor-coupled KATP-channel activation facilitates Ca2+ influx which may cause some stimulation of endothelial NO synthase (eNOS). However, the increase in [Ca2+]i also stimulates PLA2 to liberate arachidonic acid and stimulate cyclooxygenase to generate prostacyclin (PGI2). PGI2 acts on its endothelial receptors to increase cAMP, so activating protein kinase A (PKA) to phosphorylate and activate eNOS resulting in NO release. By contrast, the K+ efflux resulting from A2A-coupled KCa channels facilitates Ca2+ influx, thereby activating eNOS and NO release. This process may be facilitated by phosphorylation of eNOS by PKA via the action of A2A-receptor-mediated stimulation of AC increasing cAMP. These pathways may be important in mediating vasodilatation during exercise and systemic hypoxia when adenosine acting in an endothelium- and NO-dependent manner has been shown to be important.
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Adenosine is an important mediator of vasodilatation in the coronary, cerebral and skeletal muscle circulations in a number of conditions including hypoxia and exercise (Berne et al. 1983). For many years it was accepted that adenosine evoked dilatation by stimulating A2 receptors (particularly the A2A subtype) on the vascular smooth muscle (VSM) via an increase in cAMP (see Olsson & Pearson, 1990); however, more recent evidence shows the A1 receptor subtype can also mediate dilatation (Merkel et al. 1992; Nakhostine & Lamontagne, 1993; Danialou et al. 1997; Bryan & Marshall, 1999a). In addition, a number of studies have now shown that adenosine can evoke dilatation in an endothelium-dependent fashion that is also nitric oxide (NO) dependent (Rose'Meyer & Hope, 1990; Prentice & Hourani, 2000).
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In our recent studies on lengths of endothelium-intact rat aorta, adenosine acting via A1and A2A adenosine receptors evoked dose-dependent NO release (measured by an NO-sensitive electrode) from the endothelium (Ray et al. 2002). Furthermore, A1-receptor stimulation evoked NO release that was attenuated by a cyclooxygenase (COX) inhibitor and release of prostacyclin (PGI2) from the endothelium, as assessed by radioimmunoassay. Iloprost, an analogue of PGI2, also evoked endothelial NO release, raising the possibility of an interaction between adenosine, NO and PGI2 in mediating dilatation (Ray et al. 2002). An additional finding of this study was that both A1- and A2A-mediated NO release were dependent on an increase in cAMP, as both responses were attenuated by adenylate cyclase (AC) inhibition.
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These findings led us to propose that both A1- and A2A-mediated NO release are dependent on an increase in cAMP. However, we proposed that A2A-receptor activation might directly increase cAMP (since A2A receptors are considered to be positively coupled to AC; Londos et al. 1980). In contrast, we proposed that stimulation of A1 receptors – which are considered to be negatively coupled to AC (Londos et al. 1980) – increases the synthesis of PGI2 which acts on endothelial cells in an autocrine fashion to stimulate AC-linked prostacyclin receptors (IP receptors; Moncada & Vane, 1979), so leading to an increase in intracellular cAMP (Ray et al. 2002). Both proposals accord with recent evidence that NO release can be stimulated by protein kinase A (PKA)-mediated phosphorylation of endothelial NO synthase (eNOS; Zhang & Hintze, 2001).
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However, our proposals clearly leave many questions unanswered as to exactly how adenosine stimulates NO release via its A1 and A2A receptors. Elucidating these mechanisms was the objective of the present study.
In Chinese hamster ovary (CHO) cells, stimulation of transfected A1 receptors augmented the increase in phospholipase A2 (PLA2) activity induced by ACh and thrombin (Akbar et al. 1994; Dickenson & Hill, 1997). PLA2 is the enzyme that cleaves cell membrane phospholipids to yield arachidonic acid (AA), the precursor for prostaglandins (PGs) generated by COX.
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Both the action of PLA2 and activation of eNOS have been associated with, or shown to be dependent on, an increase in intracellular Ca2+ (Busse & Mülsch, 1990; Balsinde et al. 1999). Ca2+ can be released from intracellular stores by inositol 1,4,5-trisphosphate (IP3) which is formed by the action of phospholipase C (PLC). In cultured CHO cells and rabbit airway smooth muscle, activation of A1 receptors stimulated PLC (Abebe & Mustafa, 1998; Dickenson & Hill, 1998). Furthermore, in isolated astrocytes, the increase in [Ca2+]i evoked by the non-selective adenosine analogue (1-6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuronamide) (NECA), was completely blocked by a PLC inhibitor, and was attenuated by an A2 receptor antagonist (Pilitsis & Kimelberg, 1998). Therefore, both A1 and A2 adenosine receptors can be functionally coupled to PLC, at least in some cell types.
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Intracellular Ca2+ can also be raised in endothelial cells by influx of extracellular Ca2+. Activation of K+ channels leads to K+ efflux, membrane hyperpolarization and a membrane potential-dependent driving force for the influx of extracellular Ca2+ (Busse et al. 1988; Lückhoff & Busse, 1990). There is already evidence that in cultured coronary endothelial cells, adenosine can cause membrane hyperpolarization via the activation of Ca2+-activated K+ (KCa) channels, but the adenosine receptor subtype responsible for this response was not tested (Mehrke & Daut, 1990). Furthermore, there is pharmacological evidence linking both A1 and A2A adenosine receptors and ATP-sensitive K+ (KATP) channels in cultured VSM cells, endothelium-intact porcine coronary microvessels, isolated rabbit heart coronary arteries and rat diaphragmatic arterioles (Dart & Standen, 1993; Nakhostine & Lamontagne, 1993; Kleppisch & Nelson, 1995; Danialou et al. 1997; Hein et al. 2001). Further, the NO-dependent vasodilatation evoked in rat hindlimb by A1 receptor stimulation was attenuated by KATP-channel inhibition (Bryan & Marshall, 1999b).
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In view of this evidence, we have used selective pharmacological agents to investigate whether NO released from the endothelial cells of freshly excised rat aorta by A1 and/or A2A receptor stimulation is dependent on extracellular Ca2+, PLC, PLA2, KCa and KATP channels. The results obtained allow us to propose different pathways for A1- and A2A-receptor-evoked stimulation of NO release and to suggest how the mechanisms uncovered may play a role in physiological situations in which adenosine levels are increased.
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Methods
The release of NO from lengths of rat thoracic aorta was recorded continuously with an NO-sensitive electrode (ISO-NOP, WPI, FL, USA) with a 2 mm diameter tip, connected to a meter (ISO-NO Mark II, WPI), as previously described (Ray et al. 2002). The electrode is highly selective for NO, it has a lower detection limit of 1 nM and can measure NO in solution over a linear range of 1 nM to 20 μM (Broderick & Taha, 1995). Briefly, lengths (9.78 ± 1.56 mm) of thoracic aorta were removed from male Wistar rats immediately after they were killed, in accordance with the UK Animals (Scientific Procedures) Act 1986, by cervical dislocation under anaesthesia (3.5% halothane in O2). Each length of aorta was opened longitudinally, pinned endothelial surface uppermost in a Petri dish and bathed in 10 ml Krebs solution (pH 7.4, containing (mM): 118 NaCl. 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4, 1.1 MgSO4, 10 Hepes and 5.6 glucose; Ray et al. 2002). With the aid of a micromanipulator, the NO electrode was advanced as close as possible to the endothelial surface of the vessel, and the output was recorded via a data acquisition system (MacLab/2e, AD Instruments Ltd, Oxfordshire, UK) connected to a computer (Power Macintosh 6100/60). The experimental apparatus was placed in a grounded Faraday cage to reduce electrical noise and interference. The electrode was calibrated by chemical generation of NO on each experimental day according to the following equation:
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It has been previously demonstrated that this experimental system allows reproducible measurement of NO release (Guo et al. 1996; Simonsen et al. 1999; Ray et al. 2002).
Protocols
Group 1. The NO release evoked by adenosine was tested in the presence and absence of Ca2+. The response to 1 mM adenosine was tested, the bathing medium of the vessel was then replaced with Ca2+-free Krebs containing Ca2+ chelators EDTA and EGTA (1 mM; Michel et al. 1997), and the response to 1 mM adenosine was tested after 30 min. Finally, Ca2+-containing Krebs solution was restored to the vessel and the response to 1 mM adenosine was retested after 30 min.
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It should be noted that 1 mM is a relatively high agonist concentration; however, it is comparable with the dose used in numerous studies (e.g. Mian & Marshall, 1991; Danialou et al. 1997; Rubin et al. 2000), and in our previous studies adenosine evoked a dose-dependent increase in NO release, with a consistent maximal response being achieved with a dose of 1 mM (Ray et al. 2002). Exogenous adenosine has two fates, rapid uptake by the adenosine transporter or deamination to inosine by adenosine deaminase (Olsson & Pearson, 1990). Our own experiments using adenosine deaminase and uptake inhibitors (erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) and nitrobenzylthioinosine (NBTI), respectively, suggest that these mechanisms substantially reduce the effective concentration of adenosine at the receptors. Thus, in the rat aorta preparation, concentrations of adenosine in the range 10–9 to 10–4M, evoked dose-dependent NO release when adenosine transport and deamination were inhibited. Therefore, we have argued that using a standard dose of 1 mM adenosine to study its mechanisms of action on endothelial cells is reasonable (see Ray et al. 2002 for further discussion).
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Group 2. (a) The NO release evoked by 1 mM adenosine was tested before and 10 and 30 min after addition of the specific PLC inhibitor U73122 (1 μM). This concentration of U73122 abolished agonist-induced increases in [Ca2+]i in cultured endothelial cells (Lee & Wu, 1999) and relaxation of preconstricted rat mesenteric arteries in vitro (Parenti et al. 2000). (b) As a control, the NO response to adenosine (1 mM) was tested before and 10 and 30 min after addition of U73343 – the inactive structural analogue of U73122 (1 μM; see Lee & Wu, 1999).
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Group 3. (a) The NO release evoked by 1 mM adenosine was tested before and 10, 30 and 60 min after the addition of the inhibitor of PLA2, AACOCF3 (10 μM). AACOCF3 binds to and inhibits PLA2 (Street et al. 1993) and at this concentration inhibited endothelin-1-induced release of radiolabelled AA from rat tail artery segments in vitro (Trevisi et al. 2002). To differentiate between a role for PLA2 in A1- and A2A-mediated NO release, the NO response to 1 mM adenosine was tested before and after (b) the addition of the A1 adenosine receptor antagonist DPCPX (100 nM) or (c) the A2A adenosine receptor antagonist ZM241385 (100 nM), and then after the subsequent addition of AACOCF3 (10 μM). These concentrations of DPCPX and ZM241385 are those used in our previous study to effectively block A1 and A2A receptors, respectively (Ray et al. 2002).
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Group 4. (a) The NO release evoked by adenosine (1 mM) was tested before and 10 and 30 min after the addition of iberiotoxin (100 nM) a specific inhibitor of large-conductance KCa (BKCa) channels. Iberiotoxin at this concentration has been shown to inhibit BKCa-channel activation in endothelial cells using the patch-clamp technique in response to various agonists (Faehling et al. 2001; Kuhlmann et al. 2003) and to attenuate significantly agonist-induced coronary arteriolar dilatation in vitro (Hein et al. 2000). At this concentration, iberiotoxin does not affect small-conductance (SKCa) channels (Paolocci et al. 2001). To differentiate between a role for BKCa channels in A1- and A2A-mediated NO release the NO response evoked by adenosine (1 mM) was tested before and after the addition of (b) DPCPX (100 nM) or (c) ZM241385 (100 nM), and then after the subsequent addition of iberiotoxin (100 nM).
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Group 5. (a) The NO release evoked by adenosine (1 mM) was tested before and 10 and 30 min after the addition of apamin (500 nM), a specific inhibitor of SKCa channels. Concentrations of apamin in this range effectively blocked the hyperpolarization (as assessed by direct measurement of membrane potential) and resultant relaxation of isolated mesenteric arteries in response to ACh (Chen & Cheung, 1997). In addition, as in Groups 3 and 4, NO release in response to 1 mM adenosine was tested before and after the addition of (b) DPCPX (100 nM) or (c) ZM241385 (100 nM), and then after the subsequent addition of apamin (500 nM).
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Group 6. (a) The NO release evoked by 1 mM adenosine was tested before and 10, 30 and 60 min after the addition of glibenclamide (3 μM), an inhibitor of KATP channels. This concentration of glibenclamide attenuated the relaxation of rat aortic rings in vitro in response to the NO donors S-nitroso-glutathione and S-nitroso-N-acetylcystein (Ceron et al. 2001), and in response to adenosine (Harada et al. 2001) and hyperpolarization of human umbilical vein endothelial cells evoked by a high concentration of D-glucose (Flores et al. 2003). The NO response evoked by adenosine (1 mM) was tested before and after the addition of (b) DPCPX (100 nM) or (c) ZM241385 (100 nM) and then 10, 30 and 60 min after the subsequent addition of glibenclamide (3 μM).
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Statistical analyses
Responses were measured as the maximum change in the NO output signal for each dose of adenosine before and after the various pharmacological agents (see Ray et al. 2002). All results are expressed as means ±S.E.M. All responses were analysed by ANOVA followed by Scheffé's post hoc test when appropriate. P < 0.05 was considered significant. In all cases, n is the number of animals.
Results
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We have previously shown that the application of 1 mM adenosine to endothelium-denuded aortic lengths evoked no measurable output of NO (Ray et al. 2002). Thus, any NO release measured in the present study can be assumed to be the result of stimulation of the endothelial cells. Tests performed at the end of the experiments of Groups 1–5 showed that the addition of the pharmacological agents used in the present study to the tissue bath, in the absence of arterial tissue, evoked no detectable change in the output of the NO electrode. Moreover, the addition of all pharmacological inhibitors to endothelium-intact lengths of aorta during experimental protocols evoked no measurable change in the output of NO.
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Group 1. We have previously described the NO response to 1 mM adenosine (Ray et al. 2002): a rapid increase in NO release, reaching a peak within 10 s, remaining at this level for the next 1–3 min and gradually returning towards baseline over the following 3–6 min. The NO response to adenosine (34.3 ± 4.68 nM; n= 7) was significantly attenuated by the removal of extracellular Ca2+ (4.69 ± 1.24 nM), but the full response returned (37.7 ± 6.23 nM) when Ca2+ was restored by changing the bathing medium to standard Krebs solution.
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Group 2. The NO response to 1 mM adenosine (29.1 ± 4.44 nM; n= 7) was not affected by (a) PLC inhibition with U73122 at 10 (29.3 ± 5.34 nM) or 20 min (30.5 ± 5.50 nM), or by (b) the structural analogue of U73343 (23.5 ± 3.80 nM before U73343, and 21.4 ± 4.04 and 21.1 ± 3.64 nM at 10 and 20 min after U73343, respectively).
Group 3. (a) The NO response evoked by adenosine (1 mM) was attenuated to similar extents 10, 30 and 60 min after inhibition of PLA2 with AACOCF3 (n= 6; Fig. 1A). (b) As expected (Ray et al. 2002) the NO response to 1 mM adenosine was reduced in the presence of the A1 receptor antagonist DPCPX: the remaining NO response was not affected 10 min after the addition of AACOCF3 (n= 10; Fig. 1B). (c) The NO release evoked by 1 mM adenosine was reduced by the A2A receptor antagonist ZM241385 (Ray et al. 2002). This NO response was abolished 10 min after the addition of AACOCF3 (n= 10; Fig. 1C).
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The NO response evoked by 1 mM adenosine (filled bars) was: significantly reduced 10, 30 and 60 min after the addition of the PLA2 inhibitor AACOCF3 (n= 6) (A); reduced in the presence of the A1 receptor antagonist DPCPX but unaffected by the further addition of AACOCF3 (n= 10) (B); reduced in the presence of the A2A receptor antagonist ZM241385 and was completely abolished after the addition of AACOCF3 (n= 10) (C). Columns show mean (±S.E.M.) NO release. P < 0.0001; P < 0.001; P < 0.01; P < 0.05; as indicated by brackets.
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Group 4. (a) The NO response to 1 mM adenosine was significantly attenuated 10 and 30 min after the inhibition of BKCa channels with iberiotoxin (n= 10; Fig. 2A). (b) The NO release evoked by adenosine (1 mM) in the presence of DPCPX was attenuated 10 min after BKCa-channel inhibition with iberiotoxin (n= 8; Fig. 2B). (c) In contrast, the NO response to adenosine in the presence of ZM241385 was not altered 10 min after the subsequent addition of iberiotoxin (n= 8; Fig. 2C).
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The NO response evoked by 1 mM adenosine (filled bars) was: significantly attenuated 10 and 30 min after the addition of the BKCa-channel inhibitor iberiotoxin (n= 10) (A); reduced by the A1 receptor antagonist DPCPX and further attenuated by iberiotoxin (n= 8) (B); reduced by the A2A receptor antagonist ZM241385 but unchanged by the further addition of iberiotoxin (n= 8) (C). Columns show mean (±S.E.M.) NO output. P < 0.0001; P < 0.01; as indicated by brackets.
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The NO response evoked by 1 mM adenosine (filled bars) was: significantly attenuated 10 and 30 min after the addition of the SKCa-channel inhibitor apamin (n= 10) (A); reduced by the A1 receptor antagonist DPCPX and further attenuated by the administration of apamin (n= 9) (B); reduced by the A2A receptor antagonist ZM241385 but unchanged by the further addition of apamin (n= 9) (C). Columns show mean (±S.E.M.) NO output. P < 0.0001, as indicated by brackets.
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Group 6. (a) The NO response to adenosine (1 mM) was significantly attenuated 10, 30 and 60 min after the addition of the KATP-channel inhibitor glibenclamide; the extent of the inhibition appeared to develop with time (n= 8; Fig. 4A). (b) The NO release evoked by 1 mM adenosine in the presence of DPCPX was not affected 10 and 30 min after KATP-channel inhibition with glibenclamide, but was significantly attenuated 60 min after glibenclamide addition (n= 10; Fig. 4B). (c) In contrast, the NO response to adenosine in the presence of ZM241385 was substantially attenuated 10 min after glibenclamide but there was no further attenuation at 30 and 60 min (n= 10; Fig. 4C).
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The NO response evoked by 1 mM adenosine (filled bars) was: significantly attenuated 10, 30 and 60 min after the addition of the KATP-channel inhibitor glibenclamide (n= 8) (A); reduced by the A1 receptor antagonist DPCPX, was unaffected 10 and 30 min after the further addition of glibenclamide, but was significantly reduced 60 min after the administration of glibenclamide (n= 10) (B); reduced by the A2A receptor antagonist ZM241385 and further attenuated 10, 30 and 60 min after the administration of glibenclamide (n= 10) (C). Columns show mean (±S.E.M.) NO output. P < 0.0001; P < 0.001; P < 0.05; as indicated by the brackets.
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Discussion
The results of the present study indicate for the first time that the NO release evoked by adenosine from the endothelium of rat aorta is dependent on extracellular Ca2+, but not on stimulation of PLC. They also demonstrate that the adenosine-evoked response is dependent on stimulation of PLA2 and the action of large- and small-conductance KCa channels and of KATP channels. The experiments performed in the presence of the A2A adenosine receptor antagonist ZM241385, or the A1 adenosine receptor antagonist DPCPX, indicate, respectively, that A1-mediated NO release is dependent on PLA2 and activation of KATP channels, whereas A2A-mediated NO release is dependent on activation of small- and large-conductance KCa channels.
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Before discussing our results more fully, the potential limitations of the study should be considered, Firstly, the standard dose of adenosine used in the present study was high (1 mM). As indicated in the Methods, although 1 mM adenosine appears high, endothelial cells exhibit avid metabolism of adenosine, and in our previous experiments we showed that in the presence of adenosine deaminase and uptake inhibitors, adenosine evoked NO release over the full range of concentrations from 10–9 to 10–4M. Thus, it can be argued that the effective concentration of adenosine at the receptors when a 1 mM dose of adenosine is applied is physiological (see Ray et al. 2002 for further discussion). This is supported by the fact that in our previous and present studies, nanomolar concentrations of the adenosine receptor antagonists DPCPX and ZM241385 significantly reduced the NO response evoked by 1 mM adenosine.
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Secondly, there was variation in the mean concentration of NO released by 1 mM adenosine between experimental groups. Previous studies have shown that the sensitivity of the NO electrode does not alter over the course of an individual experiment although the sensitivity does alter from day to day (Simonsen et al. 1999). Further, we have observed that changing the NO-permeable membrane, which covers the NO electrode, also affects the sensitivity of NO measurement (C. J. Ray & J. M. Marshall, unpublished observations). Thus, in the present study, as in previous studies (Simonsen et al. 1999; Ray et al. 2002), these problems were avoided by daily calibration of the electrode, and by conducting all the experiments for each experimental group using only one NO-permeable membrane. This allowed accurate within-group comparisons of NO release to be performed but any comparisons between different experimental groups should be performed with caution.
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The question then arises as to whether the concentrations of NO released in response to adenosine in the present study are sufficient to produce vasodilatation. In fact, the NO concentrations measured in the present study were similar to those measured using an NO-sensitive electrode in rat mesenteric arteries in response to ACh (21 ± 6 nM; Simonsen et al. 1999) and porcine coronary artery rings in response to bradykinin (105 ± 19.6 nM; Ge et al. 2000) when vasodilatation was also recorded. The in vivo measurement of NO has proven technically difficult due to its half-life of 3–5 s under physiological conditions (Ignarro, 1990). However, our own experiments in which the change in the arterial–venous difference in plasma nitrate levels across hindlimb muscle was used as an index of NO release in response to systemic hypoxia and adenosine infusion, suggested that an increase in the plasma concentration of NO of 20–40 nM causes vasodilatation (Ray & Marshall, 2005). These results agree with those obtained in the coronary circulation of conscious exercising dogs where NO levels, estimated from blood NOx by chemiluminescence, in the range of 40–130 nm were accompanied by an increase in coronary blood flow (Bernstein et al. 1996). Therefore, it is reasonable to assume that the NO concentrations measured in the present study are within the physiologically relevant range and would be able to mediate vasodilatation.
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Requirement for extracellular Ca2+ in adenosine-evoked NO release
In the present study, the removal of extracellular Ca2+ from the medium bathing the rat aorta severely attenuated, and in most cases abolished, adenosine-evoked release of NO from the endothelium; the response to adenosine returned when extracellular Ca2+ was restored. Thus, adenosine-induced release of NO is critically dependent on influx of extracellular Ca2+. From our previous study on rat aorta (Ray et al. 2002) and the present study, we know that 50% of adenosine-evoked NO release is A1 mediated and 50% is A2A mediated. The dramatic effect of Ca2+ removal on the response to adenosine therefore indicated that both A1- and A2A-stimulated NO release is critically dependent on Ca2+ influx. This is not surprising as the established view is that agonist-stimulated release of NO from endothelial cells is mediated predominantly via a Ca2+–calmodulin (CaM)-dependent mechanism (Busse & Mülsch, 1990) and requires sustained influx of extracellular Ca2+ to stimulate eNOS (Lückhoff & Busse, 1990). However, influx of extracellular Ca2+ can stimulate the release of Ca2+ from intracellular stores (Ca2+-induced Ca2+ release). Indeed, when [Ca2+]i and NO release were monitored using fluorescent techniques, ATP evoked a biphasic response attributable to: (i) a rapid, transient increase in [Ca2+]i due to Ca2+ release from IP3-sensitive stores and (ii) a sustained plateau in [Ca2+]i due to the Ca2+ influx from the extracellular space (Holda et al. 1998). Therefore, changes in [Ca2+]i leading to eNOS stimulation can be mediated in a number of different ways, including via the PLC–IP3 pathway.
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In the present study, NO released by adenosine was not affected by the PLC inhibitor U73122. Therefore, it seems that release of intracellular Ca2+ by IP3 and/or by diacylglycerol did not play a significant role in the NO release evoked by either A1 or A2A stimulation, but that influx of Ca2+ was responsible. Agonist-evoked endothelial cell NO production is mainly controlled by myristoylated, membrane-associated eNOS, which is sensitive to Ca2+ influx via store-operated Ca2+ (SOC) channels (Lin et al. 2000). Thus, adenosine-evoked NO release from rat aortic endothelium may be mediated by influx of Ca2+ via SOC channels. However, Ca2+ influx could also occur via agonist-activated non-selective Ca2+-permeable cation channels, or cyclic nucleotide-activated non-selective cation channels (Nilius & Droogmans, 2001); there are no voltage-operated Ca2+ channels present on the endothelium (Nilius & Droogmans, 2001).
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Involvement of PLA2
AACOCF3 which inhibits PLA2, significantly attenuated the NO released by adenosine in the rat aorta indicating that adenosine normally stimulates PLA2 to release NO. In fact, AACOCF3 inhibited the NO released by adenosine in the presence of ZM241385, but not in the presence of DPCPX, indicating that A1, but not A2A, receptor stimulation, involves activation of PLA2. This result is in complete agreement with our previous findings that A1- but not A2A-receptor stimulation leads to PGI2 production and release (Ray et al. 2002), for PLA2 is the key enzyme in the COX pathway that liberates AA from cell membranes (Lands, 1979; Smith, 1992). There is evidence that A1 receptors are coupled to PLA2 in a number of different cell types (Akbar et al. 1994; Schachter et al. 1995; Dickenson & Hill, 1997).
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Involvement of K+ channels
The role of KCa channels in the NO response evoked by adenosine was investigated in the present study by using iberiotoxin (an inhibitor of BKCa channels), and apamin (an inhibitor of SKCa channels). Both iberiotoxin and apamin attenuated the NO released by adenosine to the same extent, suggesting that both BKCa and SKCa channels are present on rat aortic endothelium and are involved in the adenosine-evoked NO response. However, the amount of NO released by adenosine in the presence of ZM241385 was not affected by iberiotoxin or apamin, whereas that released in the presence of DPCPX, was substantially attenuated by both iberiotoxin and by apamin. Thus, these results suggest that both BKCa and SKCa channels are coupled to A2A adenosine receptors, but not to A1 receptors.
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In some previous studies, the application of iberiotoxin and apamin in combination potentiated the effect that each inhibitor had when used alone (Chen & Cheung, 1997; Petersson et al. 1997). In contrast, other studies demonstrated no potentiation when the drugs were applied together (Frieden et al. 1999). In the present study, iberiotoxin and apamin administered separately, each produced more or less complete inhibition of the NO release evoked by A2A adenosine receptor stimulation. Thus, in rat aortic endothelium, it seems the blockade of one of these subtypes of KCa channel, somehow affects the ability of adenosine to stimulate NO release by opening the other subtype of KCa channel or indeed, by stimulation of other pathways. In fact, activation of a KCa channel is apparently essential for A2A-stimulated NO release.
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Various agonists including bradykinin, ATP and ACh have been shown to activate endothelial KCa channels (Rusko et al. 1992; Fulton et al. 1994; Ishida et al. 1999). Indeed, the NO-dependent relaxation of porcine coronary arteries evoked by bradykinin was attenuated by KCa-channel inhibition (Ge et al. 2000) and vasorelaxation evoked in the isolated preconstricted rat mesenteric bed by direct activation of KCa channels with 1-ethyl-2-benzimidazolinone, was abolished by NOS inhibition with L-NAME (Adeagbo, 1999), implicating KCa channels in NO-dependent dilatation. The present results indicate that adenosine acting via A2A receptors can be added to this list of agonists and receptors for which activation of KCa channels is linked to NO release.
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The KATP-channel inhibitor, glibenclamide, also attenuated the NO response to adenosine. However, in contrast to apamin and iberiotoxin, the effect of glibenclamide on NO release was present at 10 min after application, and was greater at 30 and 60 min. This suggests that glibenclamide affects NO release by more than one mechanism. The experiments with selective A1- or A2A-receptor blockade allowed these effects to be differentiated. The NO response evoked by adenosine acting at the A2A receptors was not affected at 10 and 30 min after glibenclamide administration, but was moderately attenuated at 60 min. The most likely explanation for these results is that A2A adenosine receptors are not functionally coupled to KATP channels, at least as far as NOS stimulation and NO release is concerned. Rather, it is likely that the inhibition of KATP channels on endothelial cells caused by glibenclamide affected L-arginine transport. Thus, L-arginine, the substrate for NO synthesis, is transported into endothelial cells via a system y+ cationic amino acid transporter (Bogle et al. 1991, 1996). Accordingly, L-arginine transport is sensitive to membrane potential (Sobrevia et al. 1997; Zharikov et al. 1997; Zharikov & Block, 1998): it is stimulated by drugs that cause membrane hyperpolarization, and inhibited by those that cause membrane depolarization (Zharikov & Block, 1998). Thus, endothelial cell depolarization caused by glibenclamide may have depleted the pool of L-arginine available for eNOS such that A2A-receptor-stimulated NO release was attenuated at 60 min after glibenclamide administration.
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By contrast, the finding that NO released by A1 receptor stimulation was attenuated at 10 min, suggests that these receptors are functionally coupled to KATP channels. The fact that there was no time-dependent effect of glibenclamide on the A1-mediated NO response, can be explained if the NO response was already attenuated to such an extent at 10 min, that limitation of L-arginine transport did not further affect the ability of A1 receptor stimulation to generate small amounts of NO.
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Previous studies on rat diaphragmatic arterioles in vitro and hindlimb muscle in vivo, have demonstrated that the NO-dependent dilatation evoked by a specific A1 adenosine receptor agonist was attenuated by KATP-channel inhibition with glibenclamide (Danialou et al. 1997; Bryan & Marshall, 1999a,b). In those studies no differentiation was possible between A1 receptors and KATP channels on the endothelium or VSM cells. The results of the present study provide direct evidence that, at least in the aorta, endothelial A1 receptors are coupled to KATP channels and that their activation is essential for A1-mediated NO release.
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A possible pathway for A1-mediated NO release
The results of our previous in vitro studies (Ray et al. 2002) showed that A1-stimulated NO release from the endothelium of rat aorta is dependent on PGI2 synthesis. The present results substantiate these findings by showing that A1-mediated NO release is dependent on Ca2+ influx, PLA2 and KATP-channel activation. Taken together, these results allow the proposal that, in contrast to their classical inhibitory coupling to AC (Londos et al. 1980), the A1 receptors are functionally coupled to KATP channels (event 1 in Fig. 5), so that when A1 receptors are stimulated, they initiate KATP-channel opening and the resulting K+ efflux facilitates Ca2+ influx (event 2 in Fig. 5), so increasing [Ca2+]i. This increase in [Ca2+]i may lead to some direct activation of eNOS via the classical Ca2–CaM pathway (event 3), but would not explain the involvement of PLA2. In isolated porcine microvessels and canine coronary arteries, forskolin and isoproterenol (both of which increase intracellular cAMP) released NO, as assessed by the Greiss reaction, in a manner that was dependent on PKA (Kudej et al. 2000; Zhang & Hintze, 2001; Zhang & Leffler, 2002). Moreover, experiments on cultured endothelial cells showed that agonist-induced stimulation of eNOS can involve activation of a cAMP-dependent protein kinase and phosphorylation of eNOS independent of an increase in [Ca2+]i (Butt et al. 2000). However, the NO release evoked by adenosine in the present study was virtually abolished when extracellular Ca2+ was removed. Thus, in view of the dependence of A1-stimulated NO release on PGI2 synthesis, we propose that an increase in [Ca2+]i evoked by activation of KATP channels is important in stimulating PLA2 (event 4), so leading to the liberation of AA and stimulation of the COX (event 5) pathway to generate PGI2 (event 6). The PGI2 is then presumably released from the cell and acts on its endothelial IP receptors (event 7), which are linked to AC, to increase intracellular cAMP, activate PKA (event 8) and so phosphorylate and activate eNOS (event 9). Although this pathway may seem surprisingly complex, it may be that it is functionally necessary because, in the absence of a PGI2-stimulated increase in cAMP, the direct inhibitory effect of A1 receptor stimulation on cAMP (see Introduction; Londos et al. 1980) would mean that the phosphorylation state of eNOS would be too low for it to be activated by the increase in Ca2+ that results from A1-stimulated KATP-channel opening.
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The bold numbers (1–9) on the endothelial cell on the left and the bold letters (A–G) on the endothelial cell on the right represent the possible order of events in the proposed pathways for A1 and A2A receptor stimulation, respectively.
A possible pathway for A2A-mediated NO release
As indicated in the Introduction, the results of our previous study (Ray et al. 2002) suggest, in agreement with many previous studies, that the A2A receptors present on the endothelial cells of rat aorta are positively coupled to AC and that it is by this mechanism that A2A receptor stimulation leads to NO release. A2A-receptor-stimulated NO release is therefore apparently dependent on an increase in intracellular concentration of cAMP (event A in Fig. 5). The increase in cAMP (event B) may directly mediate the phosphorylation and activation of eNOS (event C) as described above. However, agonists that increase intracellular cAMP can also stimulate PKA and lead to the phosphorylation and activation of KCa channels (Schubert et al. 1997). Thus, in view of the present evidence that KCa channels are involved in A2A-mediated NO release, we suggest a pathway in which, in addition to any potential direct coupling between A2A receptors and KCa channels, PKA stimulation by increased intracellular cAMP leads to KCa-channel activation (event D): K+ efflux through KCa channels (event E) then causes endothelial membrane hyperpolarization, so facilitating Ca2+ influx (see above; event F) and providing a preferential source of Ca2+ for eNOS activation and subsequent NO synthesis and release (event G). Phosphorylation of eNOS arising from an A2A-receptor-stimulated increase in cAMP may facilitate this process (event C).
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Adenosine-receptor-mediated NO release – a physiological role
There is substantial evidence in the literature that adenosine arising from skeletal muscle fibres plays an important role in the vasodilatation evoked by exercise and that endothelial-derived adenosine is involved in the dilatation evoked by systemic hypoxia (Marshall, 2000; Radegran & Hellsten, 2000). Moreover, there is significant evidence that NO also evokes dilatation under these conditions (Marshall, 2000; Radegran & Hellsten, 2000). Therefore it is possible that the mechanisms of adenosine-evoked NO release may have an important role to play in these situations. For example, the adenosine-mediated component of skeletal muscle vasodilatation evoked by systemic hypoxia is mediated by locally produced adenosine acting at endothelial A1 receptors in an NO- and PG-dependent manner that involves activation of KATP channels (Marshall et al. 1993; Bryan & Marshall, 1999b; Ray et al. 2002). Thus, it seems reasonable to propose that the pathway we have described above on the basis of our in vitro studies is functionally important during systemic hypoxia in skeletal muscle. A1 receptor agonists caused vasodilatation of isolated rings of porcine coronary artery (Merkel et al. 1992) and arterioles of rat diaphragm (Danialou et al. 1997), that appeared to be in part mediated via the modulation of potassium channels (Merkel et al. 1992), particularly KATP channels (Danialou et al. 1997); this raises the possibility that the pathway uncovered in the present study may have physiological relevance in a variety of circulations.
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A2A adenosine receptors are implicated in locally mediated vasodilatation in the coronary, pulmonary, cerebral and skeletal muscle circulations (Palmer & Ghai, 1982; Collis & Brown, 1983; Mustafa & Askar, 1985; Oei et al. 1988; McCormack et al. 1989; Poucher, 1996). Thus, the pathway we have identified may be important in causing NO-mediated vasodilatation in these tissues in situations where adenosine levels are increased sufficiently to stimulate the A2A receptors which have an affinity constant of 2–20 μM for adenosine; that is approximately three orders of magnitude less than that of the A1 receptors (affinity constant, 10–100 nM; Daly et al. 1981).
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Our findings have wide-reaching implications for several reasons. Firstly, they show the coupling of adenosine receptors to second-messenger signalling pathways other than the classically recognized AC–cAMP system; these pathways may underlie the mechanisms by which adenosine signals in tissues other than blood vessels. Secondly, the results of this study extend recent findings that show alternative ways of activating eNOS rather than simply via the Ca2–CaM pathway. As adenosine, PGs and NO are mediators involved in vasodilatation in all circulations and under various different physiological and pathological conditions, the findings of the present study and the pathways they allow us to propose, may have relevance to many situations in which one or other of these mediators are implicated. Finally, the release of NO by mechanisms other than a simple Ca2+-dependent pathway, has far-reaching consequences for the stimulation of eNOS and the release of NO by agonists that not only increase intracellular Ca2+, but are known to modulate other intracellular pathways.
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