Adenosine mediates relaxation of human small resistance-like coronary arteries via A_2B receptors

Abstract

1. The receptor subtype and mechanisms underlying relaxation to adenosine were examined in human isolated small coronary arteries contracted with the thromboxane A₂ mimetic, 1,5,5-hydroxy-11α, 9α-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619) to approximately 50% of their maximum contraction to K⁺ (125 mM) depolarization (F_max). Relaxations were normalized as percentages of the 50% F_max contraction.

2. Adenosine caused concentration-dependent relaxations (pEC₅₀, 5.95±0.20; maximum relaxation (R_max), 96.7±1.4%) that were unaffected by either combined treatment with the nitric oxide inhibitors, N^G-nitro-L-arginine (L-NOARG; 100 μM) and oxyhaemoglobin (HbO; 20 μM) or the ATP-dependent K⁺ channel (K_ATP) inhibitor, glibenclamide (10 μM). The pEC₅₀ but not R_max to adenosine was significantly reduced by high extracellular K⁺ (30 mM). Relaxations to the adenylate cyclase activator, forskolin, however, were unaffected by high K⁺ (30 mM).

3. Adenosine and a range of adenosine analogues, adenosine, 2-chloroadenosine (2-CADO), 5′-N-ethyl-carboxamidoadenosine (NECA), R(−)-N⁶-(2-phenylisopropyl)-adenosine (R-PIA), S(+)-N⁶-(2-phenylisopropyl)-adenosine (S-PIA), N⁶-cyclopentyladenosine (CPA), 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide (IB-MECA), 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamido adenosine hydrochloride (CGS 21680), relaxed arteries with a rank order of potency of NECA=2-CADO>adenosine=IB-MECA=R-PIA= CPA>S-PIA)>CGS 21680.

4. Sensitivity but not R_max to adenosine was significantly reduced approximately 80 and 20 fold by the non-selective adenosine receptor antagonist, 8-(p-sulphophenyl)theophylline (8-SPT) and the A₂ receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX). By contrast, the A₁-selective antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) had no effect on pEC₅₀ or R_max to adenosine.

5. These results suggest that A_2B receptors mediate relaxation to adenosine in human small coronary arteries which is independent of NO but dependent in part on a K⁺-sensitive mechanism.

Introduction

Adenosine is an endogenous vasodilator and is believed to play important roles in the regulation of coronary blood flow under normal physiological conditions and also during hypoxia and reperfusion after ischaemia (Berne, 1980; Collis & Hourani, 1993; Bouchard & Lamontagne, 1996; Feoktistov & Biaggioni, 1997; Minamino et al., 1998). Whilst it is established that adenosine A_2A receptors mediate vasodilatation to adenosine throughout the vasculature (Hussain et al., 1996; Belardinelli et al., 1996; Ongini & Fredholm, 1996; Conti et al., 1997; Feoktistov & Biaggioni, 1997), less is known of the roles of A_2B receptors. Thus, from limited functional studies, it remains controversial as to whether any vasodilator effects of A_2B receptors are endothelium-dependent or -independent and mediated by nitric oxide (NO) or hyperpolarization, particularly via the opening of ATP-dependent K⁺ channels (K_ATP) (Feoktistov & Biaggioni, 1997). Furthermore, there is no information as to the identity of receptor subtypes underlying vasodilatation to adenosine in the human coronary vasculature, although both A_2A and A_2B receptors have been found on isolated human endothelial cells (Iwamoto et al., 1994; Feoktistov & Biaggioni, 1997). Therefore, we investigated both the receptor type and the mechanisms involved in relaxation of human, resistance-like coronary arteries to adenosine in vitro. Our data demonstrate that A_2B receptors mediate relaxation to adenosine in these clinically relevant arteries independent of nitric oxide (NO) and the endothelium but partly dependent upon the activation of non-K_ATP K⁺ channels.

Methods

Tissue source

Small coronary arteries were obtained from the discarded tip of the right atrial appendage from patients (64.4±1.7 years; 31 male, 12 female) undergoing mitral valve (one patient), aortic valve (four patients) or coronary bypass graft surgery (38 patients). Immediately following surgical removal, the atrial appendages were placed in cold, carboxygenated Krebs' solution and transported back to the laboratory. The segments of atrial appendage were viewed with a dissecting-light microscope under which the small coronary arteries were carefully freed of surrounding tissue.

Mounting of vessels in myograph

Coronary arteries were cut into 2 mm lengths and mounted horizontally on two parallel stainless steel wires (40 μm) in small vessel Mulvany-Halpern myographs (JP Trading, Denmark) to record changes in isometric force as previously described (Kemp & Cocks, 1997). Vessels were maintained in physiological Krebs' solution at 37°C and continuously gassed with carbogen (95% 0₂, 5% CO₂). The Krebs' solution was composed of (in mM): Na⁺ 143.1, K⁺ 5.9, Ca²⁺ 2.5, Mg²⁺ 1.2, Cl⁻ 128.7, HCO₃⁻ 25, SO₄²⁻ 1.2, H₂PO₄⁻ 1.2 and glucose 11, pH 7.4.

Normalization

After a 30 min equilibration period, vessel rings were maximally relaxed with sodium nitroprusside (SNP; 10 μM), and then set to passive tensions equivalent to that required to produce 90% of their internal circumference when exposed to a transmural pressure of 100 mmHg (Kemp & Cocks, 1997). In brief, a passive length-tension curve was constructed for each vessel. From this curve, the effective transmural pressure was calculated and the vessel set at a tension equivalent to that generated at 0.9 times the diameter of the vessel at 100 mmHg (D₁₀₀).

Experimental protocol

Following normalization, arteries were contracted with isotonic, high K⁺ Krebs' solution (124 mM KCl; KPSS) to determine their maximum levels of active force (F_max; Kemp & Cocks, 1997). When the tissues reached F_max, the bathing solution was replaced with normal Krebs' and the tissues allowed to return to their resting levels of passive force. Regardless of treatment, all arteries were then contracted to ∼50% F_max with titrated concentrations of the thromboxane A₂ mimetic, 1,5,5-hydroxy-11α, 9α-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619; 1–30 nM) and in some cases acetylcholine (ACh; 1–3 μM), which in this tissue only causes endothelium-independent contractions (Angus et al., 1991). Once these contractions reached steady plateaus, cumulative concentration-relaxation curves to adenosine, adenosine analogues or forskolin were constructed. All concentration-response curves were obtained in the presence of indomethacin (3 μM) to inhibit prostanoid synthesis and SNP (10 μM) to inhibit characteristic spontaneous contraction of these arteries since in preliminary studies we found relaxation to adenosine to be independent of NO. Furthermore, any masking of the vasodilator effects of endothelial adenosine receptors by SNP was unlikely since both endothelial A_2A and A_2B receptors are coupled via Gs to increase levels of cyclic AMP (Iwamoto et al., 1994). In a previous study examining endothelium- and NO-dependent relaxations to bradykinin in similar tissue, however, we used isoprenaline to control spontaneous activity and thus avoid such functional block of responses to endothelial NO (Kemp & Cocks, 1997). Thus, isoprenaline was substituted for SNP in the three experiments in which we examined the response to bradykinin in adjacent rings of artery used for the adenosine studies.

Responses to adenosine were obtained in the absence and presence of the NO inhibitors, N^G-nitro-L-arginine (L-NOARG, 100 μM) and oxyhaemoglobin (HbO, 20 μM), high K⁺ (30 mM) isotonic Krebs' solution, the ATP-sensitive potassium channel (K_ATP) antagonist, glibenclamide (10 μM), and the adenosine receptor antagonist, 8-(p-sulphophenyl)theophylline (8-SPT, 100 μM), 3,7-dimethyl-1-propargylxanthine (DMPX, 100 μM) and 1,3-dipropyl-8-cyclopentyl-xanthine (DPCPX, 10 nM). These antagonists were added 30 min prior to precontraction with U46619 and ACh. Forskolin relaxations were obtained in the absence and presence of high extracellular K⁺ (30 mM).

Drugs

U46619 ([1,5,5-hydroxy-11α, 9α-(epoxymethano)prosta-5Z, 13E-dienoic acid], Upjohn, Kalamazoo, MI, U.S.A.); ACh, indomethacin, N^G-nitro-L-arginine (L-NOARG), adenosine, bovine haemoglobin (Sigma, U.S.A.); 5′-N-ethyl-carboxamidoadenosine (NECA), 2-p-(2-carboxyethyl)phenethylamino-5′ N-ethylcarboxamido adenosine hydrochloride (CGS 21680), 2-chloroadenosine (2-CADO), 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino] -9H -purin-9-yl]-N-methyl-β-D-ribofuranuronamide (IB-MECA), R(−)-N⁶-(2-phenylisopropyl)-adenosine (R-PIA), S(+)-N⁶-(2-phenylisopropyl)-adenosine (S-PIA), N⁶-cyclopentyladenosine (CPA), 8-(p-sulphophenyl)theophylline (8-SPT), 3,7-dimethyl-1-propargylxanthine (DMPX), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), forskolin (RBI, U.S.A.); bradykinin triacetate (Fluka, U.K.); sodium nitroprusside (DBL., Australia). Stock solutions of U46619 (1 mM) were made up in absolute ethanol, L-NOARG (100 mM) in 1 M NaHCO₃, and indomethacin (100 mM) in 1 M Na₂CO₃. Adenosine, S-PIA, R-PIA, NECA, 2-CADO, CGS 21680, CPA and DPCPX were made up as stock solutions (100 mM) in dimethyl sulphoxide (DMSO). Haemoglobin was dissolved in 0.9% NaCl to make up a 1 mM stock solution. The stock solution was subsequently reduced to oxyhaemoglobin (HbO) by the addition of a small amount (<0.1 g) of sodium dithionite. Excess sodium dithionite was extracted by running the solution through a sephadex (PD-10) column equilibrated with 0.9% NaCl. All subsequent dilutions of stock solutions were in distilled water and all other drugs were made up in distilled water.

Statistical analysis

Responses were expressed as percentage reversal of the U46619 and ACh contraction (Kemp & Cocks, 1997). Contractile responses were measured as a percentage of the maximum contraction to KPSS (F_max). The individual relaxation curves were fitted (Graphpad Prism, version 1.00) to the sigmoidal logistic equation,

where X=the logarithm of the agonist concentration and Y=the response. BOTTOM=the lower response plateau, TOP=the upper response plateau and pEC₅₀ is the X value when the response is halfway between BOTTOM and TOP. The variable HILLSLOPE controls the slope of the curve. From this relationship, computer estimates of pEC₅₀ values were determined and expressed as −log M.

Differences between mean pEC₅₀ and maximum relaxation (R_max) values were tested either with a two-tailed Student's unpaired t-test or one way analysis of variance (ANOVA) with comparisons to control or between all experimental groups made via Dunnett's and Tukey's modified t-tests, respectively. Results are expressed as mean±s.e.mean and statistical significance was accepted at the P<0.05 level. n values refer to number of rings from separate patients.

Results

The effect of L-NOARG, HbO and high K⁺ on responses to adenosine

Adenosine caused concentration-dependent relaxations (R_max; 96.7±1.4%, pEC₅₀; 5.95±0.20) in small coronary arteries with diameters (D₁₀₀) of 207.3±6.2 μm (n=20). The response to adenosine was unaffected by a combination of HbO (20 μM) and L-NOARG (100 μM) (Figure 1). By contrast, in separate arteries from the same patients, endothelium-dependent relaxation to bradykinin, as we previously reported (Kemp & Cocks, 1997), was markedly attenuated by this treatment (n=3, data not shown). Raising the extracellular concentration of K⁺ to 30 mM, however, caused a significant 12 fold (P<0.05) decrease in the sensitivity (pEC₅₀; 4.88±0.32, n=4) to adenosine with no change in R_max (92.5±2.1%, n=4) (Figure 1). Glibenclamide (10 μM) had no effect on either the sensitivity (pEC₅₀, 6.07±0.21, n=5) or R_max; (92.5±2.1%, n=5) to adenosine (Figure 1) and relaxations to forskolin (pEC₅₀; 7.43±0.22, R_max, 79.7±5.1%, n=4) were unchanged in the presence of high K⁺ (30 mM; Figure 1).

Figure 1.

Relaxations in human isolated small coronary arteries to (A) adenosine in normal Krebs' (control), high K⁺ (30 mM KCl) and a combination of oxyhaemoglobin (HbO) and L-NOARG (n=4–5), (B) adenosine in the presence of normal Krebs' (control, n=5) or glibenclamide (n=5) and (C) forskolin in normal Krebs' (control, n=4) and high K⁺ (n=4). Values are mean±s.e.mean. (^*) indicates pEC₅₀ value significantly different from control (P<0.05, Dunnett's modified t-statistic).

Responses to adenosine receptor agonists and antagonists

All the adenosine analogues used caused concentration-dependent relaxation in human small coronary arteries with a rank order of potency (pEC₅₀ values in parentheses; n=4–5) of NECA (6.98±0.20)=2-CADO (6.98±0.20)>adenosine (6.07±0.21)=IB-MECA (5.93±0.09)=R-PIA (5.72±0.10) =CPA (5.63±0.09)>S-PIA (5.27±0.09)>CGS 21680 (<5.00) (Figure 2). All analogues caused near maximal relaxation except CGS 21680 (30 μM) which reversed the level of contraction by 32.3±14.3%.

Figure 2.

Relaxations to (A) the adenosine agonists; NECA, 2-CADO, adenosine, IB-MECA, R-PIA, CPA, S-PIA and CGS 21680 (n=4–5) and (B) adenosine in the absence (control) and presence of the adenosine antagonists, DPCPX, DMPX and 8-SPT (n=4–5) in human isolated small coronary arteries. Values are mean±s.e.mean. (^*) indicates pEC₅₀ value significantly different from control (P<0.01, Dunnett's modified t-statistic).

The response to adenosine was unchanged in the presence of the selective A₁ adenosine receptor antagonist, DPCPX (10 nM) (pEC₅₀, 5.89±0.21; R_max; 94.0±1.9%, n=4; Figure 2). By contrast, the A₂ adenosine receptor antagonist, DMPX (100 μM), and the non-selective adenosine receptor antagonist, 8-SPT (100 μM), caused significant 20 (P<0.01) and 80 fold (P<0.01) decreases respectively in the sensitivity to adenosine, with no change in R_max (Figure 2).

Discussion

This study is the first to demonstrate that adenosine mediates relaxation of human small isolated coronary arteries via A_2B receptors. Furthermore, the response to adenosine in these clinically important arteries appeared to be independent of NO and thus the endothelium, but partly mediated by a K⁺-sensitive mechanism.

Four subtypes of adenosine receptors, A₁, A_2A, A_2B and A₃ have been cloned (Collis & Hourani, 1993; Tucker & Linden, 1993; Burnstock, 1995; Feoktistov & Biaggioni, 1997) and adenosine and its analogues display characteristic orders of potencies at each of these receptors. In the present study, the adenosine analogue, NECA, was significantly more potent than both CPA and R-PIA suggesting that A₂ rather than A₁ receptors were involved (Collis & Hourani, 1993). Also, the >100 fold higher potency of NECA compared with CGS 21680 indicated the presence of A_2B rather than A_2A receptors since these agonists are equipotent at A_2A receptors (Feoktistov & Biaggioni, 1997). Thus, the rank order of potency of adenosine and its analogues in the present study was NECA=2-CADO>adenosine=IB-MECA=R-PIA= CPA>S-PIA>CGS 21680, which is characteristic of A_2B receptors (Collis & Hourani, 1993; Feoktistov & Biaggioni, 1997). Furthermore, the potency ratios relative to NECA for NECA, adenosine, CPA, R-PIA, S-PIA and CGS21680 observed here were 1, 0.15, 0.06, 0.07, 0.02 and <0.01 respectively. These compare favourably with values from two separate studies with similar adenosine analogues at A_2B receptors in the guinea-pig aorta. Thus, Martin (1992) found values of 1, 0.16, 0.03, and <0.001 for NECA, adenosine, R-PIA and CGS21860 respectively, whilst in the same tissue, Gurden et al. (1993) reported values of 1, 0.03, 0.03, 0.003 and 0.002 for NECA, CPA, R-PIA, S-PIA and CGS21860 respectively.

It is unlikely that A₃ receptors mediated the response to adenosine (Hannon et al., 1995) in the human small coronary artery given the low potency of the A₃-selective agonist, IB-MECA, compared with its affinity of 1 nM at rat A₃ receptors (Jacobson et al., 1995) and A₃ receptors are resistant to inhibition by the A₁, A₂ non-selective antagonist, 8-SPT (10 μM; Hannon et al., 1995). 8-SPT, however, antagonized the response to adenosine with an estimated affinity of 5.9 which is similar to that reported for 8-SPT at A_2B receptors in the guinea-pig aorta (pK_B=5.3; Hargreaves et al., 1991). Furthermore, the A₂ antagonist, DMPX, inhibited the relaxation curve to adenosine in an apparent competitive manner yet in contrast the selective A₁ antagonist, DPCPX, failed to have any effect. Therefore, our findings indicate that A_2B receptors mediate relaxation to adenosine in human small coronary arteries. This is in contrast to substantial published literature which demonstrates that adenosine mediates most of its vasodilator responses via A_2A receptors (Ongini & Fredholm, 1996; Feoktistov & Biaggioni, 1997; Shryock & Belardinelli, 1997). For example, A_2A receptors appear to mediate relaxation to adenosine in large coronary arteries of the pig (Conti et al., 1997), cow (Conti et al., 1993) and vasodilation in the isolated perfused guinea-pig heart (Vials & Burnstock, 1993; Poucher et al., 1995; Belardinelli et al., 1996, 1998; Shryock et al., 1998). Also, although an undefined A₂ receptor has been reported in human large coronary arteries, it was not further subtyped as either A_2A or A_2B (Ramagopal et al., 1988).

In the present study, we confirmed our earlier finding in similar human small coronary arteries that endothelium-dependent relaxation to bradykinin was largely blocked by combined L-NOARG and HbO treatment (Kemp & Cocks, 1997). Therefore, the inability of the same NO inhibitors to block relaxation to adenosine in adjacent, similar-sized arteries suggests that adenosine did not mediate its effect via either NO or the endothelium. Also, it was unlikely that any endothelial A_2A-mediated responses were masked by functional antagonism caused by the presence of SNP to inhibit spontaneous activity since CGS 21680 was still virtually inactive in tissues treated with isoprenaline instead of SNP to control spontaneous activity (Kemp & Cocks, unpublished data). Similar endothelium-independent vasodilator responses to adenosine in the rat mesenteric bed as those described here have also been reported to be mediated by A_2B receptors (Rubino et al., 1995). The direct smooth muscle relaxing effect of adenosine, however, appeared to involve K⁺ channels and thus a hyperpolarization-dependent mechanism given that it was partially inhibited by high extracellular K⁺. In coronary vascular preparations where K_ATP have been implicated in relaxation to adenosine via hyperpolarization (Randall, 1995; Bouchard & Lamontagne, 1996; Nakae et al., 1996; Mutafovayambolieva & Keef, 1997), the opening of these channels is thought to be regulated by changes in intracellular levels of cyclic AMP (Miyoshi & Nakaya, 1993; Kleppisch & Nelson, 1995; Feoktistov & Biaggioni, 1997). Our data, however, indicate that cyclic AMP-regulated K_ATP do not underlie relaxation to adenosine in the human small coronary arteries since the response was refractory to glibenclamide and relaxation to forskolin was unaffected by high extracellular K⁺. A role for other K⁺ channels such as inward rectifying (K_IV), voltage-dependent (K_V) and Ca²⁺-activated K⁺ channels (K_Ca), however, cannot be excluded since Cabell et al. (1994) found that tetraethylammonium (TEA) and the more selective inhibitor of large-conductance K_Ca, iberiotoxin, blocked adenosine-induced relaxation in canine coronary arteries. Thus, adenosine may cause smooth muscle relaxation, in part, via the opening of K_Ca channels as has been reported in large coronary arteries of the pig and human (Olanrewaju et al., 1995) and small coronary arteries of the dog (Cabell et al., 1994).

In conclusion, the present study provides strong circumstantial evidence that relaxation of human small resistance-like coronary arteries by adenosine is mediated by A_2B receptors which are coupled to K⁺ channels to mediate part of the response. A more definitive demonstration that A_2B receptors mediate coronary vasodilatation to adenosine in humans, however, awaits the development of selective A_2B receptor antagonists.

Abbreviations

ACh

 

A₁

 

A_2A

 

A_2B

 

A₃

 

ANOVA

 

2-CADO

 

CGS 21680

 

CPA

 

D₁₀₀ DMPX

 

DMSO

 

DPCPX

 

F_max

 

Hb0

 

IB-MECA

 

K_ATP

 

K_Ca

 

K_IV

 

K_V

 

KPSS

 

L-NOARG

 

NECA

 

pEC₅₀

 

pK_B

 

R_max

 

R-PIA

 

SNP

 

S-PIA

 

8-SPT

 

TEA

 

U46619