Clare J. Ray

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

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 (PGI2), or by an NO donor. After diclofenac, the A1 receptor agonist CCPA evoked no vasodilatation: we previously showed that A1, but not A2A, 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 A1 antagonist DPCPX, the remainder being attenuated by the A2A 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 A1, but not A2A, receptors caused a release of PGI2 from rat aorta, assessed by radioimmunoassay of its stable metabolite, 6‐keto PGF1α, that was abolished by diclofenac. These results suggest that during systemic hypoxia, adenosine acts on endothelial A1 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.

The cellular mechanisms by which adenosine evokes release of nitric oxide from rat aortic endothelium

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.

Measurement of nitric oxide release evoked by systemic hypoxia and adenosine from rat skeletal muscle in vivo.

It is accepted that NO plays a role in hypoxic vasodilatation in several tissues. For rat hindlimb muscle there is evidence that during systemic hypoxia endogenously released adenosine acts on endothelial A1 receptors to evoke dilatation in a NO‐dependent fashion, implying requirement for, or mediation by, NO. We tested in vivo whether systemic hypoxia and adenosine release NO from muscle. In anaesthetized rats, arterial blood pressure (ABP) and femoral blood flow (FBF) were recorded allowing computation of femoral vascular conductance (FVC). Blood samples taken from femoral artery and vein allowed electrochemical measurement of plasma [NO] after reduction of NO3− and NO2−. Systemic hypoxia and adenosine infusion for 5 min each, evoked an increase in FVC that was attenuated by the NO synthase (NOS) inhibitor l‐NAME (Group 1, n= 8) and adenosine A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, Group 2, n= 6). Concomitant systemic hypoxia and adenosine infusion evoked increases in venous–arterial [NO] difference ([NO]v‐a) from −1.4 ± 0.85 to 6.6 ± 1.6 and 2.3 ± 0.78 to 8.4 ± 1.8 nmol l−1, respectively (mean ±s.e.m), which were abolished by l‐NAME (−0.72 ± 0.90 to −0.87 ± 0.74 and 0.72 ± 0.85 to −0.97 ± 1.1 nmol l−1, respectively). DPCPX also abolished the hypoxia‐evoked increase in [NO]v‐a (control −4.2 ± 1.8 to 12.5 ± 3.7 nmol l−1, with DPCPX −0.63 ± 2.6 to 3.3 ± 2.9 nmol l−1) and decreased the adenosine‐evoked increase in [NO]v‐a (control 1.1 ± 1.5 to 24 ± 14, with DPCPX −0.43 ± 2.9 to 12 ± 5.9 nmol l−1). These results allow the novel conclusion that the muscle vasodilatation of systemic hypoxia is partly mediated by adenosine acting at endothelial A1 receptors to stimulate synthesis and release of NO, which then induces dilatation.

Elucidation in the rat of the role of adenosine and A2A-receptors in the hyperaemia of twitch and tetanic contractions.

Adenosine is implicated in playing a role in blood flow responses to situations where O2 delivery is reduced (hypoxia) or O2 consumption is increased (exercise). Strong isometric contractions have been shown to limit vasodilatation, potentially leading to a greater mismatch between and than during twitch contractions. Thus, we hypothesized that adenosine makes a greater contribution to the hyperaemia associated with isometric tetanic than isometric twitch contractions and aimed to elucidate the adenosine‐receptor subtypes involved in the response. In four groups of anaesthetized rats, arterial blood pressure (ABP), femoral blood flow (FBF) and tension in the extensor digitorum longus muscle were recorded; isometric twitch and tetanic contractions were evoked by stimulation of the sciatic nerve for 5 min at 4 Hz and 40 Hz, respectively. Groups 1 (twitch) and 3 (tetanic) were time controls for Groups 2 and 4, which received the selective A2A‐receptor antagonist ZM241385 before the third and 8‐sulphophenyltheophylline (8‐SPT; a non‐selective adenosine receptor antagonist) before the fourth contraction. Time controls showed consistent tension and hyperaemic responses: twitch and tetanic contractions were associated with a 3‐fold and 2.5‐fold increase in femoral vascular conductance (FVC, FBF/ABP) from baseline, respectively. ZM241385 reduced these responses by 14% and as much as 25%, respectively; 8‐SPT had no further effect. We propose that, while twitch contractions produce a larger hyperaemia, adenosine acting via A2A‐receptors plays a greater role in the hyperaemia associated with tetanic contraction. These results are considered in relation to the A1‐receptor‐mediated muscle dilatation evoked by systemic hypoxia.

Glycogen metabolism protects against metabolic insult to preserve carotid body function during glucose deprivation

The carotid body has been proposed to be an acute sensor of hypoglycaemia, although conflicting data exist regarding the ability of hypoglycaemia to stimulate the carotid body directly. The reason for these discrepancies is not known. In an in vitro, freshly isolated, intact rat carotid body preparation, chemoafferent function was unaffected and protected against metabolic injury for 30 min exposure to glucose deprivation. Glycogen granules and glycogen conversion enzymes were identified in type I cells and targeting of glycogenolysis or functional glycogen depletion both caused a more rapid run‐down of glycolysis during glucose deprivation. This shows that glycogen maintains carotid body sensory neuronal output in periods of restricted glucose delivery to protect the metabolic integrity of the organ during hypoglycaemia. The preservation of energetic status may account for the variation in the reported capacity of the carotid body to sense physiological glucose concentrations.

Adrenaline release evokes hyperpnoea and an increase in ventilatory CO2 sensitivity during hypoglycaemia: a role for the carotid body

Hypoglycaemia is counteracted by release of hormones and an increase in ventilation and CO2 sensitivity to restore blood glucose levels and prevent a fall in blood pH. The full counter‐regulatory response and an appropriate increase in ventilation is dependent on carotid body stimulation. We show that the hypoglycaemia‐induced increase in ventilation and CO2 sensitivity is abolished by preventing adrenaline release or blocking its receptors. Physiological levels of adrenaline mimicked the effect of hypoglycaemia on ventilation and CO2 sensitivity. These results suggest that adrenaline, rather than low glucose, is an adequate stimulus for the carotid body‐mediated changes in ventilation and CO2 sensitivity during hypoglycaemia to prevent a serious acidosis in poorly controlled diabetes.

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

Exercise hyperaemia is partly mediated by adenosine A2A‐receptors. Adenosine can evoke nitric oxide (NO) release via endothelial A2A‐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 A2A‐receptor inhibition with ZM241385 on femoral vascular conductance (FVC) and hindlimb O2 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 A2A‐receptors on the vascular smooth muscle to cause vasodilatation.

Ecto‐5′‐nucleotidase (CD73) regulates peripheral chemoreceptor activity and cardiorespiratory responses to hypoxia

Carotid body dysfunction is recognized as a cause of hypertension in a number of cardiorespiratory diseases states and has therefore been identified as a potential therapeutic target. Purinergic transmission is an important element of the carotid body chemotransduction pathway. We show that inhibition of ecto‐5′‐nucleotidase (CD73) in vitro reduces carotid body basal discharge and responses to hypoxia and mitochondrial inhibition. Additionally, inhibition of CD73 in vivo decreased the hypoxic ventilatory response, reduced the hypoxia‐induced heart rate elevation and exaggerated the blood pressure decrease in response to hypoxia. Our data show CD73 to be a novel regulator of carotid body sensory function and therefore suggest that this enzyme may offer a new target for reducing carotid body activity in selected cardiovascular diseases.

Contribution of non‐endothelium‐dependent substances to exercise hyperaemia: are they O2 dependent?

Abstract  This review considers the contributions to exercise hyperaemia of substances released into the interstitial fluid, with emphasis on whether they are endothelium dependent or O2 dependent. The early phase of exercise hyperaemia is attributable to K+ released from contracting muscle fibres and acting extraluminally on arterioles. Hyperpolarization of vascular smooth muscle and endothelial cells induced by K+ may also facilitate the maintained phase, for example by facilitating conduction of dilator signals upstream. ATP is released into the interstitium from muscle fibres, at least in part through cystic fibrosis transmembrane conductance regulator‐associated channels, following the fall in intracellular H+. ATP is metabolized by ectonucleotidases to adenosine, which dilates arterioles via A2A receptors, in a nitric oxide‐independent manner. Evidence is presented that the rise in arterial achieved by breathing 40% O2 attenuates efflux of H+ and lactate, thereby decreasing the contribution that adenosine makes to exercise hyperaemia; efflux of inorganic phosphate and its contribution may likewise be attenuated. Prostaglandins (PGs), PGE2 and PGI2, also accumulate in the interstitium during exercise, and breathing 40% O2 abolished the contribution of PGs to exercise hyperaemia. This suggests that PGE2 released from muscle fibres and PGI2 released from capillaries and venular endothelium by a fall in their local act extraluminally to dilate arterioles. Although modest hyperoxia attenuates exercise hyperaemia by improving O2 supply, limiting the release of O2‐dependent adenosine and PGs, higher O2 concentrations may have adverse effects. Evidence is presented that breathing 100% O2 limits exercise hyperaemia by generating O2−, which inactivates nitric oxide and decreases PG synthesis.

Is Carotid Body Physiological O2 Sensitivity Determined by a Unique Mitochondrial Phenotype

The mammalian carotid body (CB) is the primary arterial chemoreceptor that responds to acute hypoxia, initiating systemic protective reflex responses that act to maintain O2 delivery to the brain and vital organs. The CB is unique in that it is stimulated at O2 levels above those that begin to impact on the metabolism of most other cell types. Whilst a large proportion of the CB chemotransduction cascade is well defined, the identity of the O2 sensor remains highly controversial. This short review evaluates whether the mitochondria can adequately function as acute O2 sensors in the CB. We consider the similarities between mitochondrial poisons and hypoxic stimuli in their ability to activate the CB chemotransduction cascade and initiate rapid cardiorespiratory reflexes. We evaluate whether the mitochondria are required for the CB to respond to hypoxia. We also discuss if the CB mitochondria are different to those located in other non-O2 sensitive cells, and what might cause them to have an unusually low O2 binding affinity. In particular we look at the potential roles of competitive inhibitors of mitochondrial complex IV such as nitric oxide in establishing mitochondrial and CB O2-sensitivity. Finally, we discuss novel signaling mechanisms proposed to take place within and downstream of mitochondria that link mitochondrial metabolism with cellular depolarization.