Interactions of adenosine, prostaglandins and nitric oxide in hypoxia-induced vasodilatation: in vivo and in vitro studies

Abstract

Adenosine, prostaglandins (PG) and nitric oxide (NO) have all been implicated in hypoxia-evoked vasodilatation. We investigated whether their actions are interdependent. In anaesthetised rats, the PG synthesis inhibitors diclofenac or indomethacin reduced muscle vasodilatation evoked by systemic hypoxia or adenosine, but not that evoked by iloprost, a stable analogue of prostacyclin (PGI(2)), or by an NO donor. After diclofenac, the A(1) receptor agonist CCPA evoked no vasodilatation: we previously showed that A(1), but not A(2A), receptors mediate the hypoxia-induced muscle vasodilatation. Further, in freshly excised rat aorta, adenosine evoked a release of NO, detected with an NO-sensitive electrode, that was abolished by NO synthesis inhibition, or endothelium removal, and reduced by ~50 % by the A(1) antagonist DPCPX, the remainder being attenuated by the A(2A) antagonist ZM241385. Diclofenac reduced adenosine-evoked NO release by ~50 % under control conditions, abolished that evoked in the presence of ZM241385, but did not affect that evoked in the presence of DPCPX. Adenosine-evoked NO release was also abolished by the adenyl cyclase inhibitor 2',5'-dideoxyadenosine, while dose-dependent NO release was evoked by iloprost. Finally, stimulation of A(1), but not A(2A), receptors caused a release of PGI(2) from rat aorta, assessed by radioimmunoassay of its stable metabolite, 6-keto PGF(1alpha), that was abolished by diclofenac. These results suggest that during systemic hypoxia, adenosine acts on endothelial A(1) receptors to increase PG synthesis, thereby generating cAMP, which increases the synthesis and release of NO and causes muscle vasodilatation. This pathway may be important in other situations involving these autocoids.

Figure 1. Calibration of the NO electrode

A, original trace showing recording of NO electrode calibration. Arrows represent concentration of NO generated on the addition of NaNO₂ to 0.1 m KI and 0.1 m H₂SO₄ solution. B, linear regression analysis of relationship between NO generated and change in voltage output of electrode: y= 0.11x+ 0.14, R²= 0.99, electrode sensitivity 0.11 mV nm⁻¹ or 1.10 pA nm⁻¹.

Figure 4. Responses evoked by adenosine in the absence (A) and presence (B) of EHNA and NBTI

A, original trace showing the NO release evoked by 1 mm adenosine. B, original trace showing NO release evoked by cumulative concentrations of adenosine (indicated by arrows) before (upper trace) and 30 min after l-NAME (100 μM, lower trace) in the presence of EHNA (adenosine deaminase inhibitor, 10 μM) and NBTI (adenosine uptake inhibitor, 10 μM).

Figure 2. Differential effects of diclofenac on muscle vasodilator responses evoked by different stimuli

Each panel, from left to right, shows mean (±s.e.m.) ABP (A) and FVC (B) at time 0 and after five 1 min intervals of breathing 8 % O₂ (hypoxia, left-hand panel) or infusion of agonist as indicated above panels, before (▪) and after (•) diclofenac. Diclofenac (1 mg kg⁻¹i.v.) reduced muscle dilator response evoked by hypoxia (n= 8) and adenosine (n= 6), but not that evoked by SNP (n= 7) or iloprost (n= 5). †P < 0.05 vs. control, Student's paired t test on integral of change in variable.

Figure 3. Effects of diclofenac followed by adenosine receptor blockade on cardiovascular responses evoked by systemic hypoxia

Each graph shows mean values (±s.e.m.) recorded at time 0 and at five 1 min intervals of breathing 8 % O₂ before (▪) and after diclofenac (•; 1 mg kg⁻¹i.v.), and after subsequent administration of the adenosine receptor antagonist, 8-SPT (▴; 10 mg kg⁻¹i.v., n= 5). Variables are indicated by ordinates. The muscle vasodilatation (increase in FVC) evoked by hypoxia was reduced by diclofenac and further reduced by 8-SPT, but there was no effect on the other variables. †P < 0.05 vs. control, *P < 0.05 vs. after diclofenac, Student's paired t test on integral of change in variable.

Figure 5. Adenosine evokes dose-dependent release of NO from endothelial surface of thoracic aorta

A, non-cumulative dose-response curve to adenosine: 1 mm adenosine evoked maximum NO release (n= 8). B, cumulative dose-response curve to adenosine in the absence (•) and presence of l-NAME (100 μM, ♦), both curves being obtained in the presence of EHNA (adenosine deaminase inhibitor, 10 μM) and NBTI (adenosine uptake inhibitor, 10 μM). Adenosine (10-100 μM) evoked a peak release of NO, which was significantly attenuated by l-NAME. In both A and B responses are shown as mean change in NO output ±s.e.m.; **P < 0.01, ANOVA followed by Fisher's test.

Figure 6. Nitric oxide release evoked by adenosine from rat aorta is mediated partly by A₁ and partly by A_2A receptors

Diclofenac reduces NO release evoked from rat aorta by adenosine when A₁ receptors are functionally active, but not when they are blocked. A, the response evoked by 1 mm adenosine was reduced by the A₁ receptor antagonist DPCPX (100 nm) and further reduced after subsequent addition of the A_2A receptor antagonist ZM241385 (100 nm, n= 5). B, the control response to adenosine (1 mm) was reduced by ≈50 % by diclofenac (1 μM, n= 8). C, diclofenac (1 μM) had no effect on the response to adenosine (1 mm) evoked in the presence of DPCPX (100 nm, n= 5), but (D) attenuated that evoked in the presence of ZM241385 (100 nm, n= 6). Columns show mean change in NO output (±s.e.m.); **P < 0.01, *P < 0.05, ANOVA followed by Fisher's test.

Figure 7. Iloprost causes dose-dependent release of NO from rat aorta

Data are shown as mean change in NO output (±s.e.m.). n= 5.

Figure 8. The activation of adenyl cyclase is required in order that adenosine acting at A₁ and A_2A receptors can evoke NO release

NO release evoked by adenosine (1 mm) in the presence of the A₁ receptor antagonist DPCPX (A) and the A_2A receptor antagonist ZM241385 (B) was significantly attenuated by adenyl cyclase inhibition with DDA (50 μM, n= 6). Columns show mean increase in NO output (±s.e.m.); **P < 0.01, *P < 0.05, ANOVA followed by Fisher's test.

Figure 9. Adenosine increases the generation of 6-keto PGF_1α by rat aorta by stimulating A₁ receptors but not A_2A receptors

Columns show 6-keto PGF_1α (mean ±s.e.m.) assayed in supernatant under control conditions (Group A, filled columns), in the presence of DPCPX (Group B, open columns), and in the presence of ZM241385 (Group C, hatched columns). Symbols below the chart show the presence (+) or absence (-) of vehicle for adenosine, adenosine, vehicle for diclofenac, diclofenac, DPCPX and ZM241385, in the assay tube. Within groups: ** significantly different from basal 6-keto PGF_1α generation (first column); δδ significantly different from adenosine-evoked 6-keto PGF_1α generation (second column). P < 0.01 in both cases. NS indicates no significant difference and †† indicates significant difference (P < 0.01) between groups, as indicated by brackets.