Nitric oxide (NO) does not contribute to the generation or action of adenosine during exercise hyperaemia in rat hindlimb

Abstract

Exercise hyperaemia is partly mediated by adenosine A(2A)-receptors. Adenosine can evoke nitric oxide (NO) release via endothelial A(2A)-receptors, but the role for NO in exercise hyperaemia is controversial. We have investigated the contribution of NO to hyperaemia evoked by isometric twitch contractions in its own right and in interaction with adenosine. In three groups of anaesthetized rats the effect of A(2A)-receptor inhibition with ZM241385 on femoral vascular conductance (FVC) and hindlimb O(2) consumption at rest and during isometric twitch contractions (4 Hz) was tested (i) after NO synthase inhibition with l-NAME, and when FVC had been restored by infusion of (ii) an NO donor (SNAP) or (iii) cell-permeant cGMP. Exercise hyperaemia was significantly reduced (32%) by l-NAME and further significantly attenuated by ZM241385 (60% from control). After restoring FVC with SNAP or 8-bromo-cGMP, l-NAME did not affect exercise hyperaemia, but ZM241385 still significantly reduced the hyperaemia by 25%. There was no evidence that NO limited muscle during contraction. These results indicate that NO is not required for adenosine release during contraction and that adenosine released during contraction does not depend on new synthesis of NO to produce vasodilatation. They also substantiate our general hypothesis that the mechanisms by which adenosine contributes to muscle vasodilatation during systemic hypoxia and exercise are different: we propose that, during muscle contraction, adenosine is released from skeletal muscle fibres independently of NO and acts directly on A(2A)-receptors on the vascular smooth muscle to cause vasodilatation.

Figure 1. Involvement of NO and adenosine in the response to isometric twitch contractions

A–D show mean (±s.e.m.) EDL tension, FBF, ABP and FVC for control stimulation 2 (▪, continuous line), stimulation after l-NAME (○, dashed line) and stimulation after l-NAME + ZM241385 (▴, dotted line) in the 1 min before (baseline), 5 min of (S1–5) and 7 min after (R1–7) sciatic nerve stimulation (4 Hz). *P < 0.05 vs control 2 and §P < 0.05 vsl-NAME at time points indicated. n= 10.

Figure 3. Effect of restoring baseline FVC with 8-bromo-cGMP on the involvement of NO and adenosine in the response to isometric twitch contractions

A–D show mean (±s.e.m.) EDL tension, FBF, ABP and FVC for control stimulation 2 (▪, continuous line), stimulation after l-NAME (○, dashed line), stimulation after l-NAME + cGMP (▴, dotted line) and stimulation after l-NAME + cGMP + ZM241385 (♦, dashed and dotted line) in the minute before (baseline), 5 min of (S1–5) and 7 min after (R1–7) sciatic nerve stimulation (4 Hz). *P < 0.05 vs control 2, §P < 0.05 vsl-NAME and †P < 0.05 vsl-NAME + cGMP at time points indicated. n= 8.

Figure 4. Involvement of NO and adenosine in the response to isometric twitch contraction: influence on change in integrated tension (ΔInt Tension/Tension Time Index (TTI)) and the change in integrated FVC (ΔInt FVC)

Left hand panels show mean (±.s.e.m.) ΔInt tension and right hand panels show mean (±s.e.m.) ΔInt FVC for: Group 1, A and B, for control stimulation 2 (filled), stimulation after l-NAME (diagonal lines) and stimulation after l-NAME + ZM241385 (chequered); Group 2, C and D, for control stimulation 2 (filled), stimulation after l-NAME (diagonal lines), stimulation after l-NAME + SNAP (cross-hatched) and stimulation after l-NAME + SNAP + ZM241385 (chequered); Group 3, E and F, for control stimulation 2 (filled), stimulation after l-NAME (diagonal lines), stimulation after l-NAME + cGMP (dotted) and stimulation after l-NAME + cGMP + ZM241385 (chequered). *P < 0.05 vs control 2, §P < 0.05 vsl-NAME and †P < 0.05 vsl-NAME + SNAP or l-NAME + cGMP as indicated. NS, no significant difference between bars indicated.

Figure 2. Effect of restoring baseline FVC with SNAP on the involvement of NO and adenosine in the response to isometric twitch contractions

A-D show mean (±s.e.m.) EDL tension, FBF, ABP and FVC for control stimulation 2 (▪, continuous line), stimulation after l-NAME (○, dashed line), stimulation after l-NAME + SNAP (▴, dotted line) and stimulation after l-NAME + SNAP + ZM241385 (♦, dashed and dotted line) in the minute before (baseline), 5 min of (S1–5) and 7 min after (R1–7) sciatic nerve stimulation (4 Hz). *P < 0.05 vs control 2, §P < 0.05 vsl-NAME and †P < 0.05 vsl-NAME + SNAP at time points indicated. n= 12.

Figure 5. Influence of NO and adenosine onand ER before (baseline) and during (contraction) isometric twitch contractions in Group 1

A shows ; B, ; and C, O₂ extraction ratio (mean ±s.e.m.) for control stimulation 2 (filled), stimulation after l-NAME (diagonal lines) and stimulation after l-NAME + ZM241385 (chequered). *^,†P < 0.05 vs control 2 and l-NAME, respectively, and δP < 0.05 baseline vs contraction for each condition as indicated.