Janice M. Marshall

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.

Correlation of the directly observed responses of mesenteric vessels of the rat to nerve stimulation and noradrenaline with the distribution of adrenergic nerves

1. The effects of nerve stimulation and of the topical application of noradrenaline on arteries, capillaries and veins of the mesentery of the anaesthetized rat were examined by direct observation under a microscope. The distribution of adrenergic nerves to the vessels of the mesentery was studied using the fluorescence histochemical method.

The influence of the sympathetic nervous system on individual vessels of the microcirculation of skeletal muscle of the rat

1. Direct observations have been made on the responses of individual vessels of the microcirculation of rat spinotrapezius muscle to stimulation of the sympathetic paravascular nerve fibres and to topically applied catecholamines.

Mechanisms of Raynaud's disease.

Raynauds phenomenon is due to transient cessation of blood flow to the digits of the hands or feet. An attack of Raynaud’s phenomenon is classically manifested as triphasic color changes. The white phase is due to excessive vasoconstriction and cessation of regional blood flow. This phase is followed by a cyanotic phase, as the residual blood in the finger desaturates. The red phase is due to hyperemia as the attack subsides and blood flow is restored. An attack is frequently associated with pain and/or paresthesia due to sensory nerve ischemia. Variants of Raynaud’s phenomenon include acrocyanosis and primary livedo reticularis, each of which is associated with reduced skin blood flow, exacerbated by cold or emotional upset. Raynaud’s phenomenon in the absence of other disorders is primary Raynaud’s phenomenon, or Raynaud’s disease. The mechanisms of Raynaud’s disease include increased activation of the sympathetic nerves, in response to cold or emotion; an impaired habituation of the cardiovascular response to stress may contribute. In addition, there appears to be a local fault, which is likely multifactorial. This local fault is due to an alteration in vascular function rather than vascular structure. The alteration in vascular function may be related to increased sensitivity to cold of the adrenergic receptors on the digital artery vascular smooth muscle. In some cases, locally released or systemically circulating vasoconstrictors may participate, including endothelin, 5-hydroxytryptamine and thromboxane. A deficiency or increased degradation of nitric oxide, possibly due to increased oxidative stress, may be involved in some cases. These recent pathophysiological insights may lead to new therapeutic options.

Adenosine receptor subtypes and vasodilatation in rat skeletal muscle during systemic hypoxia: a role for A1 receptors

1 In anaesthetized rats we tested responses evoked by systemic hypoxia (breathing 8% O2 for 5 min) and adenosine (i.a. infusion for 5 min) before and after administration of a selective adenosine A1 receptor antagonist DPCPX (8‐cyclopentyl‐1,3‐dipropylxanthine), or a selective adenosine A2A receptor antagonist ZM 241385. Arterial blood pressure, (ABP), heart rate (HR), femoral blood flow (FBF) and femoral vascular conductance (FVC: FBF/ABP) were recorded together with the K+ concentration in arterial blood ([K+]a) and in venous blood of hindlimb muscle ([K+]v) before and at the 5th minute of hypoxia or agonist infusion. 2 In 12 rats, DPCPX reversed the fall in ABP and HR and the increase in FVC evoked by the selective A1 agonist CCPA (2‐chloro‐N6‐cyclopentyladenosine; i.a. infusion for 5 min). DPCPX also reduced both the increase in FVC induced by hypoxia and that induced by adenosine; the control responses to these stimuli were comparable in magnitude and both were reduced by ∼50%. 3 In 11 rats, ZM 241385 reversed the fall in ABP and increase in FVC evoked by the selective A2A agonist CGS 21680 (2‐p‐(2‐carboxyethyl)‐phenethylamino‐5′‐N‐ethylcarboxamidoadenosine hydrochloride; i.a. infusion for 5 min). ZM 241385 also reduced the increase in FVC induced by adenosine by ∼50%, but had no effect on the increase in FVC induced by hypoxia. 4 In these same studies, before administration of DPCPX, or ZM 241385, hypoxia had no effect on the venous‐arterial difference for K+ ([K+]v‐a), whereas after administration of either antagonist, hypoxia significantly reduced [K+]v‐a suggesting an increase in hypoxia‐induced K+ uptake, or a reduction in K+ efflux. 5 These results indicate that both A1 and A2A receptors are present in hindlimb muscle and can mediate vasodilatation and that A1 and A2A receptors contribute equally to dilatation induced by infused adenosine. However, they suggest that endogenous adenosine released during systemic hypoxia induces dilatation only by acting on A1 receptors. Given previous evidence that adenosine can stimulate receptors on skeletal muscle fibres that are coupled to ATP‐sensitive K+ (KATP) channels so promoting K+ efflux, our results allow the proposal that KATP channels may be coupled to both A1 and to A2A receptors and may be stimulated to open by adenosine released during hypoxia, but indicate that, during systemic hypoxia, K+ efflux caused by either receptor subtype makes a very minor contribution to the muscle vasodilatation.

Cellular mechanisms by which adenosine induces vasodilatation in rat skeletal muscle: significance for systemic hypoxia

1 In anaesthetized rats, we recorded arterial blood pressure (ABP), heart rate (HR), femoral blood flow (FBF) and femoral vascular conductance (FVC). We tested the effects of the nitric oxide (NO) synthesis inhibitor l‐NAME (nitro‐l‐arginine methyl ester), or the ATP‐sensitive K+ (KATP) channel inhibitor glibenclamide, on responses evoked by systemic hypoxia (breathing 8% O2 for 5 min) or i.a. infusion for 5 min of adenosine, the NO donor sodium nitroprusside (SNP), the adenosine A1 receptor agonist CCPA (2‐chloro‐N6‐cyclopentyladenosine) or the adenosine A2A receptor agonist CGS 21680 (2‐p‐(2‐carboxyethyl)‐phenethylamino‐5′‐N‐ethylcarboxamidoadenosine hydrochloride). 2 l‐NAME (10 mg kg−1 i.v.) greatly reduced the increase in FVC induced by hypoxia or adenosine, as we have shown before, but had no effect on the increase in FVC evoked by SNP. In addition, l‐NAME abolished the increase in FVC evoked by CCPA and greatly reduced that evoked by CGS 21680. These results substantiate the view that muscle vasodilatation induced by systemic hypoxia and infused adenosine are largely NO dependent. They also indicate that muscle dilatation induced by A1 receptor stimulation is entirely NO dependent while that induced by A2A receptors is largely NO dependent; dilatation may also be induced by direct stimulation of A2A receptors on the vascular smooth muscle. 3 Glibenclamide (10 or 20 mg kg−1 i.v.) reduced the increase in FVC induced by hypoxia, preferentially affecting the early part (< 1 min). In addition, glibenclamide greatly reduced the increase in FVC induced by adenosine, but it had no effect on that evoked by SNP. Further, glibenclamide abolished the increase in FVC evoked by CCPA and greatly reduced that evoked by CGS 21680. These results substantiate the view that hypoxia‐induced muscle vasodilatation is initiated by KATP channel opening. They also indicate that NO does not induce muscle vasodilatation by opening KATP channels on the vascular smooth muscle, but indicate that the dilatation induced by adenosine and by A2A receptor stimulation is largely dependent on KATP channel opening, while that induced by A1 receptor stimulation is wholly dependent on KATP channel opening. 4 These results, together with previous evidence that hypoxia‐induced vasodilatation in skeletal muscle is largely mediated by adenosine acting on A1 receptors, lead us to propose that adenosine is released from endothelium during systemic hypoxia and acts on endothelial A1 receptors to open KATP channels on the endothelial cells and cause synthesis of NO, which then acts on the vascular smooth muscle to cause dilatation. During severe systemic hypoxia we propose that adenosine may also act on A2A receptors on the endothelium to cause dilatation by a similar process and may act on A2A receptors on the vascular smooth muscle to cause dilatation by opening KATP channels.

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.

Role of adenosine and its receptors in the vasodilatation induced in the cerebral cortex of the rat by systemic hypoxia

1 In anaesthetized rats, we have examined the role of adenosine in vasodilatation evoked in the cerebral cortex by systemic hypoxia (breathing 8 % O2). Red cell flux was recorded from the surface of the exposed parietal cortex (CoRCF) by a laser Doppler probe, cortical vascular conductance (CoVC) being computed as CoRCF divided by mean arterial blood pressure. All agonists and antagonists were applied topically to the cortex. 2 Systemic hypoxia or adenosine application for 5 or 10 min, respectively, induced an increase in CoRCF and CoVC. These responses were substantially reduced by 8‐phenyltheophylline (8‐PT), an adenosine receptor antagonist which is non‐selective between the adenosine A1 and A2A receptor subtypes. By contrast, the adenosine receptor antagonist 8‐sulphophenyltheophylline (8‐SPT) which is similarly non‐selective, but unlike 8‐PT, does not cross the blood‐brain barrier, reduced the increases in CoRCF and CoVC induced by adenosine, but had no effect on those induced by hypoxia. 3 The A2A receptor agonist CGS21680 produced a substantial increase in CoRCF and CoVC, but the A1 receptor agonist 2‐chloro‐N6‐cyclopentyladenosine had minimal effects. 4 The A2A receptor antagonist ZM241385 reduced the increase in CoRCF and CoVC induced by adenosine and reduced the increase in CoRCF induced by hypoxia. 5 We propose that exogenous adenosine that is topically applied to the cerebral cortex produces vasodilatation by acting on A2A receptors on the vascular smooth muscle. However, during systemic hypoxia, we propose that adenosine is released from endothelial cells and acts on endothelial A2A receptors to produce the major part of the hypoxia‐induced dilatation in the cerebral cortex.

The roles of adenosine and related substances in exercise hyperaemia

The role of adenosine in exercise hyperaemia has been controversial. Accumulating evidence now demonstrates that adenosine is released into the venous efflux of exercising muscle and that adenosine is responsible for 20–40% of the maintained phase of the muscle vasodilatation that accompanies submaximal and maximal contractions. This adenosine is mainly generated from AMP that is released from the skeletal muscle fibres and dephosphorylated by ecto 5′nucleotidase bound to the sarcolemma. During exercise, the concentration of ecto 5′nucleotidase may be increased by translocation from the cytosol, while release of AMP and affinity of ecto 5′nucleotidase for AMP are increased by acidosis. The adenosine so formed, acts on extraluminal A2A receptors on the vascular smooth muscle. In addition, ATP is released from red blood cells into the plasma during exercise, in association with the unloading of O2 from haemoglobin, while ATP and adenosine may be released from endothelium as a consequence of local hypoxia. It is unlikely that this intraluminal ATP, or adenosine, contributes significantly to exercise hyperaemia, for muscle vasodilatation induced by intraluminal ATP or adenosine is strongly nitric oxide dependent, while vasodilatation induced by adenosine in hypoxia is mediated by A1 receptors. Neither is a recognized feature of exercise hyperaemia.

Analysis of cardiovascular responses evoked following changes in peripheral chemoreceptor activity in the rat.

1. Comparisons have been made between rats anaesthetized with pentobarbitone and Saffan (Glaxovet), of respiratory and cardiovascular changes evoked by (1) brief stimulation of carotid body chemoreceptors (c.b.); (2) systemic hypoxia induced by N2 breathing for 5 s; (3) brief unloading of peripheral chemoreceptors with dopamine; and (4) O2 breathing for 10 s. The results are discussed in relation to responses reported in other species. 2. Under pentobarbitone, c.b. stimulation evoked hyperventilation, tachycardia, and vasoconstriction in hindlimb muscle and renal and mesenteric circulation. The effects of vagotomy and/or of holding ventilation constant indicated that the primary cardiac response to c.b. stimulation was bradycardia which could be overcome by tachycardia, due to a reflex mediated by pulmonary stretch receptors with vagal afferents and to other secondary effects of hyperventilation. However, reflex vasodilatation initiated by hyperventilation did not modulate the chemoreceptor‐induced peripheral vasoconstriction. 3. Under light pentobarbitone, N2 evoked a similar pattern of response to c.b. stimulation, except that the tachycardia apparently also reflected the known effects of increased central inspiratory drive and central nervous hypoxia on cardiac vagal and sympathetic activity. However, under deep pentobarbitone or after guanethidine, N2 induced generalized vasodilatation. It is proposed that these responses reflected the local vasodilator actions of hypoxia. 4. Under light Saffan anaesthesia, both c.b. stimulation and N2 evoked the autonomic components of the alerting stage of the defence response which includes tachycardia and vasodilatation in hindlimb muscle, which are not secondary to hyperventilation, with renal and mesenteric vasoconstriction, pupillary dilatation and exophthalmus. However, under deep Saffan anaesthesia, c.b. stimulation and N2 produced the patterns of response they each evoked under deep pentobarbitone. It is proposed that light Saffan anaesthesia allows chemoreceptor stimulation to activate the defence areas and that under such conditions the primary response to c.b. stimulation and direct effects of hypoxia may be overridden. 5. Under pentobarbitone or Saffan, the hypoventilation induced by I.V. dopamine and by O2 indicated that almost 50% of eupnoeic ventilation was due to drive from peripheral chemoreceptors. This drive apparently played no significant role in setting the baseline level of heart rate, but could account for 10% of total peripheral resistance and of the baseline level of arterial pressure under Saffan, rather less under pentobarbitone.