Analyzing Angiotensin II Receptor Type 1 Clustering in PC12 Cells in Response to Hypoxia Using Direct Stochastic Optical Reconstruction Microscopy (dSTORM).

Angiotensin II (Ang II) is a hormone that plays a major role in maintaining homeostasis. The Ang II receptor type 1 (AT1R) is expressed in acute O2 sensitive cells, including carotid body (CB) type I cells and pheochromocytoma 12 (PC12) cells, and Ang II increases cell activity. While a functional role for Ang II and AT1Rs in increasing the activity of O2 sensitive cells has been established, the nanoscale distribution of AT1Rs has not. Furthermore, it is not known how exposure to hypoxia may alter the single-molecule arrangement and clustering of AT1Rs. In this study, the AT1R nanoscale distribution under control normoxic conditions in PC12 cells was determined using direct stochastic optical reconstruction microscopy (dSTORM). AT1Rs were arranged in distinct clusters with measurable parameters. Across the entire cell surface there averaged approximately 3 AT1R clusters/µm2 of cell membrane. Cluster area varied in size ranging from 1.1 × 10-4 to 3.9 × 10-2 µm2. Twenty-four hours of exposure to hypoxia (1% O2) altered clustering of AT1Rs, with notable increases in the maximum cluster area, suggestive of an increase in supercluster formation. These observations could aid in understanding mechanisms underlying augmented Ang II sensitivity in O2 sensitive cells in response to sustained hypoxia.

Risk of incident obstructive sleep apnoea in patients with type 1 diabetes: a population-based retrospective cohort study.

AIMS/HYPOTHESIS: People with type 2 diabetes are at increased risk of developing obstructive sleep apnoea. However, it is not known whether people with type 1 diabetes are also at an increased risk of obstructive sleep apnoea. This study aimed to examine whether people with type 1 diabetes are at increased risk of incident obstructive sleep apnoea compared with a matched cohort without type 1 diabetes. METHODS: We used a UK primary care database, The Health Improvement Network (THIN), to perform a retrospective cohort study between January 1995 and January 2018 comparing sleep apnoea incidence between patients with type 1 diabetes (exposed) and without type 1 diabetes (unexposed) (matched for age, sex, BMI and general practice). The outcome was incidence of obstructive sleep apnoea. Baseline covariates and characteristics were assessed at the start of the study based on the most recent value recorded prior to the index date. The Cox proportional hazards regression model was used to estimate unadjusted and adjusted hazard ratios, based on a complete-case analysis. RESULTS: In total, 34,147 exposed and 129,500 matched unexposed patients were included. The median follow-up time was 5.43 years ((IQR 2.19-10.11), and the mean BMI was 25.82 kg/m2 (SD 4.33). The adjusted HR for incident obstructive sleep apnoea in patients with type 1 diabetes vs those without type 1 diabetes was 1.53 (95% CI 1.25, 1.86; p<0.001). Predictors of incident obstructive sleep apnoea in patients with type 1 diabetes were older age, male sex, obesity, being prescribed antihypertensive or lipid-lowering drugs, atrial fibrillation and depression. CONCLUSIONS/INTERPRETATION: Individuals with type 1 diabetes are at increased risk of obstructive sleep apnoea compared with people without diabetes. Clinicians should suspect obstructive sleep apnoea in patients with type 1 diabetes if they are old, have obesity, are male, have atrial fibrillation or depression, or if they are taking lipid-lowering or antihypertensive drugs.

ß-Adrenoceptor blockade prevents carotid body hyperactivity and elevated vascular sympathetic nerve density induced by chronic intermittent hypoxia.

Carotid body (CB) hyperactivity promotes hypertension in response to chronic intermittent hypoxia (CIH). The plasma concentration of adrenaline is reported to be elevated in CIH and our previous work suggests that adrenaline directly activates the CB. However, a role for chronic adrenergic stimulation in mediating CB hyperactivity is currently unknown. This study evaluated whether beta-blocker treatment with propranolol (Prop) prevented the development of CB hyperactivity, vascular sympathetic nerve growth and hypertension caused by CIH. Adult male Wistar rats were assigned into 1 of 4 groups: Control (N), N + Prop, CIH and CIH + Prop. The CIH paradigm consisted of 8 cycles h-1, 8 h day-1, for 3 weeks. Propranolol was administered via drinking water to achieve a dose of 40 mg kg-1 day-1. Immunohistochemistry revealed the presence of both ß1 and ß2-adrenoceptor subtypes on the CB type I cell. CIH caused a 2-3-fold elevation in basal CB single-fibre chemoafferent activity and this was prevented by chronic propranolol treatment. Chemoafferent responses to hypoxia and mitochondrial inhibitors were attenuated by propranolol, an effect that was greater in CIH animals. Propranolol decreased respiratory frequency in normoxia and hypoxia in N and CIH. Propranolol also abolished the CIH mediated increase in vascular sympathetic nerve density. Arterial blood pressure was reduced in propranolol groups during hypoxia. Propranolol exaggerated the fall in blood pressure in most (6/7) CIH animals during hypoxia, suggestive of reduced sympathetic tone. These findings therefore identify new roles for ß-adrenergic stimulation in evoking CB hyperactivity, sympathetic vascular hyperinnervation and altered blood pressure control in response to CIH.

A student practical to conceptualize the importance of Poiseuille's law and flow control in the cardiovascular system.

The laboratory practical reported here is based in Poiseuille's law and utilizes low-cost laboratory consumable items, thereby making it easy to deploy in any teaching laboratory. In the practical, students take an experimental approach of individually changing physical variables and measuring fluid flow rates. Plotting these results allows them to discuss the effect each variable has on flow. Furthermore, students enhance their appreciation of experimental errors and variability by making repeat measurements. In the follow-up teaching session, the students are encouraged to apply their experimental findings to the cardiovascular system and the control of blood flow both at rest and under conditions of altered cardiac output, such as during exercise. By tackling the topic of flow control as a core concept, it allows the students to then apply their understanding in wider physiological contexts, such as airflow in the respiratory system.

G-Protein-Coupled Receptor (GPCR) Signaling in the Carotid Body: Roles in Hypoxia and Cardiovascular and Respiratory Disease.

The carotid body (CB) is an important organ located at the carotid bifurcation that constantly monitors the blood supplying the brain. During hypoxia, the CB immediately triggers an alarm in the form of nerve impulses sent to the brain. This activates protective reflexes including hyperventilation, tachycardia and vasoconstriction, to ensure blood and oxygen delivery to the brain and vital organs. However, in certain conditions, including obstructive sleep apnea, heart failure and essential/spontaneous hypertension, the CB becomes hyperactive, promoting neurogenic hypertension and arrhythmia. G-protein-coupled receptors (GPCRs) are very highly expressed in the CB and have key roles in mediating baseline CB activity and hypoxic sensitivity. Here, we provide a brief overview of the numerous GPCRs that are expressed in the CB, their mechanism of action and downstream effects. Furthermore, we will address how these GPCRs and signaling pathways may contribute to CB hyperactivity and cardiovascular and respiratory disease. GPCRs are a major target for drug discovery development. This information highlights specific GPCRs that could be targeted by novel or existing drugs to enable more personalized treatment of CB-mediated cardiovascular and respiratory disease.

Cuff inflation time significantly affects blood flow recorded with venous occlusion plethysmography.

PURPOSE: We tested whether the values of limb blood flow calculated with strain-gauge venous occlusion plethysmography (VOP) differ when venous occlusion is achieved by automated, or manual inflation, so providing rapid and slower inflation, respectively. METHOD: In 9 subjects (20-30 years), we calculated forearm blood flows (FBF) values at rest and following isometric handgrip at 70% maximum voluntary contraction (MVC) when rapid, or slower inflation was used. RESULT: Rapid and slower cuff inflation took 0.23 ± 0.01 (mean ± SEM) and 0.92 ± 0.02 s, respectively, reflecting the range reported in published studies. At rest, FBF calculated from the 1st cardiac cycle after rapid and slower inflation gave similar values: 10.5 ± 1.4 vs. 9.6 ± 1.3 ml dl- 1 min- 1, respectively (P > 0.05). However, immediately post-contraction, FBF was ~ 40% lower with slower inflation: 54.6 ± 5.1 vs. 33.8 ± 4.2 ml dl- 1 min- 1 (P < 0.01). The latter value was similar to that calculated over the 3rd cardiac cycle following rapid inflation: 2nd cardiac cycle: 40.5 ± 4.5; 3rd cycle: 32.6 ± 4.5 ml dl- 1 min- 1. Regression analyses of FBFs recorded at intervals post-contraction showed those calculated over the 1st, 2nd, or 3rd cardiac cycles with rapid inflation correlated well with those from the 1st cardiac cycle with manual inflation (r = 0.79, 0.82, 0.79; P < 0.01). However, only the slope for the 3rd cycle with rapid inflation vs. slower inflation was close to unity (2.07, 1.34, and 0.94, respectively). CONCLUSION: These findings confirm that the 1st cardiac cycle following venous occlusion should be used when calculating FBF using VOP and, but importantly, indicate that cuff inflation should be almost instantaneous; just ≥ 0.9 s leads to substantial underestimation, especially at high flows.

Ecto-5'-nucleotidase (CD73) regulates peripheral chemoreceptor activity and cardiorespiratory responses to hypoxia.

KEY POINTS: 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. ABSTRACT: Augmented sensory neuronal activity from the carotid body (CB) has emerged as a principal cause of hypertension in a number of cardiovascular related pathologies, including obstructive sleep apnoea, heart failure and diabetes. Development of new targets and pharmacological treatment strategies aiming to reduce CB sensory activity may thus improve outcomes in these key patient cohorts. The present study investigated whether ecto-5'-nucleotidase (CD73), an enzyme that generates adenosine, is functionally important in modifying CB sensory activity and cardiovascular respiratory responses to hypoxia. Inhibition of CD73 by α,ß-methylene ADP (AOPCP) in the whole CB preparation in vitro reduced basal discharge frequency by 76 ± 5% and reduced sensory activity throughout graded hypoxia. AOPCP also significantly attenuated elevations in sensory activity evoked by mitochondrial inhibition. These effects were mimicked by antagonism of adenosine receptors with 8-(p-sulfophenyl) theophylline. Infusion of AOPCP in vivo significantly decreased the hypoxic ventilatory response (Δ V̇E control 74 ± 6%, Δ V̇E AOPCP 64 ± 5%, P < 0.05). AOPCP also modified cardiovascular responses to hypoxia, as indicated by reduced elevations in heart rate and exaggerated changes in femoral vascular conductance and mean arterial blood pressure. Thus we identify CD73 as a novel regulator of CB sensory activity. Future investigations are warranted to clarify whether inhibition of CD73 can effectively reduce CB activity in CB-mediated cardiovascular pathology.

Caffeine, gravity, and baroreceptor function: the integration of diet and cardiovascular control.

We describe a simple, cost-effective experiment to demonstrate cardiovascular integration of heart rate and blood pressure to accommodate the environmental and dietary factors of gravity and caffeine. Specific learning objectives associated with this include understanding the effects of posture on blood pressure and heart rate, coupled with the role of caffeine in modifying this response. Inclusion of ECG measurements, coupled with heart rate variability analysis, added a demonstration of the contribution made by the autonomic nervous system under these conditions. We clearly demonstrate that the cardiac work, estimated as rate-pressure product, necessary to undertake the transition from supine to standing, is fixed for a given group of subjects. However, the individual contribution of heart rate and systolic pressure to the cardiac workload is subject to the external factors of gravity and caffeine. Such an activity also demonstrates additional benefits, including unstructured teaching opportunities to augment classroom learning associated with integrative physiology and also the discussion of ethical issues with regard to human experimentation.

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

KEY POINTS: 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. ABSTRACT: Hypoglycaemia in vivo induces a counter-regulatory response that involves the release of hormones to restore blood glucose levels. Concomitantly, hypoglycaemia evokes a carotid body-mediated hyperpnoea that maintains arterial CO2 levels and prevents respiratory acidosis in the face of increased metabolism. It is unclear whether the carotid body is directly stimulated by low glucose or by a counter-regulatory hormone such as adrenaline. Minute ventilation was recorded during infusion of insulin-induced hypoglycaemia (8-17 mIU kg(-1)  min(-1) ) in Alfaxan-anaesthetised male Wistar rats. Hypoglycaemia significantly augmented minute ventilation (123 ± 4 to 143 ± 7 ml min(-1) ) and CO2 sensitivity (3.3 ± 0.3 to 4.4 ± 0.4 ml min(-1)  mmHg(-1) ). These effects were abolished by either ß-adrenoreceptor blockade with propranolol or adrenalectomy. In this hypermetabolic, hypoglycaemic state, propranolol stimulated a rise in P aC O2, suggestive of a ventilation-metabolism mismatch. Infusion of adrenaline (1 µg kg(-1)  min(-1) ) increased minute ventilation (145 ± 4 to 173 ± 5 ml min(-1) ) without altering P aC O2 or pH and enhanced ventilatory CO2 sensitivity (3.4 ± 0.4 to 5.1 ± 0.8 ml min(-1)  mmHg(-1) ). These effects were attenuated by either resection of the carotid sinus nerve or propranolol. Physiological concentrations of adrenaline increased the CO2 sensitivity of freshly dissociated carotid body type I cells in vitro. These findings suggest that adrenaline release can account for the ventilatory hyperpnoea observed during hypoglycaemia by an augmented carotid body and whole body ventilatory CO2 sensitivity.

Mild Chronic Intermittent Hypoxia in Wistar Rats Evokes Significant Cardiovascular Pathophysiology but No Overt Changes in Carotid Body-Mediated Respiratory Responses.

Models of chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnoea (OSA), have demonstrated dysregulation of the cardiovascular and respiratory systems resulting in hypertension, cardiac hypertrophy and alterations in the hypoxic ventilatory response (HVR) due to changes in sympathetic and respiratory control by the carotid body. In the UK, treatment of OSA is only offered to patients with an apnoea-hypopnoea index (AHI) >15, we investigated whether mild CIH produced significant pathophysiological changes, which might inform treatment guidelines.Rats were exposed to CIH (6 h(-1), 8 h day(-1), 5 % O(2) nadir) for 2 weeks and then arterial blood pressure (ABP), heart rate (HR) and ventilation were recorded in these and normoxic control rats (N) under Alfaxan anaesthesia, at baseline and in response to Dejours test, graded hypoxia and hypercapnia. Hearts were analysed post-mortem.CIH induced significant increases in baseline ABP (142 ± 5 vs 122 ± 2 mmHg), HR (448 ± 9 vs 412 ± 5 bpm) and cardiac mass (3.5 ± 0.1 vs 2.7 ± 0.1 g kg body mass(-1)) as a result of a selective left ventricular hypertrophy (1.6 ± 0.1 vs 1.3 ± 0.08 g kg body mass(-1); FCSA 464 ± 32 µm(2) vs 314 ± 9 µm(2)). There was no significant difference between N and CIH in baseline respiration or the response to Dejours test, graded hypoxia and hypercapnia.These results demonstrate that mild CIH can induce the significant cardiovascular changes associated with OSA without overt changes in respiratory function. Given evidence that CIH changes carotid body sensory activity, a possible explanation for these results is that there is differential integration of chemoreceptor input with respiratory and cardiac sympathetic outputs.

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

The view that the carotid body (CB) type I cells are direct physiological sensors of hypoglycaemia is challenged by the finding that the basal sensory neuronal outflow from the whole organ is unchanged in response to low glucose. The reason for this difference in viewpoint and how the whole CB maintains its metabolic integrity when exposed to low glucose is unknown. Here we show that, in the intact superfused rat CB, basal sensory neuronal activity was sustained during glucose deprivation for 29.1 ± 1.2 min, before irreversible failure following a brief period of excitation. Graded increases in the basal discharge induced by reducing the superfusate PO2 led to proportional decreases in the time to the pre-failure excitation during glucose deprivation which was dependent on a complete run-down in glycolysis and a fall in cellular energy status. A similar ability to withstand prolonged glucose deprivation was observed in isolated type I cells. Electron micrographs and immunofluorescence staining of rat CB sections revealed the presence of glycogen granules and the glycogen conversion enzymes glycogen synthase I and glycogen phosphorylase BB, dispersed throughout the type I cell cytoplasm. Furthermore, pharmacological attenuation of glycogenolysis and functional depletion of glycogen both significantly reduced the time to glycolytic run-down by ∼33 and 65%, respectively. These findings suggest that type I cell glycogen metabolism allows for the continuation of glycolysis and the maintenance of CB sensory neuronal output in periods of restricted glucose delivery and this may act as a key protective mechanism for the organ during hypoglycaemia. The ability, or otherwise, to preserve energetic status may thus account for variation in the reported capacity of the CB to sense physiological glucose concentrations and may even underlie its function during pathological states associated with augmented CB discharge.

Contribution of non-endothelium-dependent substances to exercise hyperaemia: are they O(2) dependent?

This review considers the contributions to exercise hyperaemia of substances released into the interstitial fluid, with emphasis on whether they are endothelium dependent or O(2) 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 A(2A) receptors, in a nitric oxide-independent manner. Evidence is presented that the rise in arterial achieved by breathing 40% O(2) 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), PGE(2) and PGI(2), also accumulate in the interstitium during exercise, and breathing 40% O(2) abolished the contribution of PGs to exercise hyperaemia. This suggests that PGE(2) released from muscle fibres and PGI(2) 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 O(2) supply, limiting the release of O(2)-dependent adenosine and PGs, higher O(2) concentrations may have adverse effects. Evidence is presented that breathing 100% O(2) limits exercise hyperaemia by generating O(2)(-), which inactivates nitric oxide and decreases PG synthesis.

Are Multiple Mitochondrial Related Signalling Pathways Involved in Carotid Body Oxygen Sensing?

It is generally acknowledged that the carotid body (CB) type I cell mitochondria are unique, being inhibited by relatively small falls in PaO2 well above those known to inhibit electron transport in other cell types. This feature is suggested to allow for the CB to function as an acute O2 sensor, being stimulated and activating systemic protective reflexes before the metabolism of other cells becomes compromised. What is less clear is precisely how a fall in mitochondrial activity links to type I cell depolarisation, a process that is required for initiation of the chemotransduction cascade and post-synaptic action potential generation. Multiple mitochondrial/metabolic signalling mechanisms have been proposed including local generation of mitochondrial reactive oxygen species (mitoROS), a change in mitochondrial/cellular redox status, a fall in MgATP and an increase in lactate. Although each mechanism is based on compelling experimental evidence, they are all not without question. The current review aims to explore the importance of each of these signalling pathways in mediating the overall CB response to hypoxia. We suggest that there is unlikely to be a single mechanism, but instead multiple mitochondrial related signalling pathways are recruited at different PaO2s during hypoxia. Furthermore, it still remains to be determined if mitochondrial signalling acts independently or in partnership with extra-mitochondrial O2-sensors.

Mitochondrial Succinate Metabolism and Reactive Oxygen Species Are Important but Not Essential for Eliciting Carotid Body and Ventilatory Responses to Hypoxia in the Rat.

Reflex increases in breathing in response to acute hypoxia are dependent on activation of the carotid body (CB)-A specialised peripheral chemoreceptor. Central to CB O2-sensing is their unique mitochondria but the link between mitochondrial inhibition and cellular stimulation is unresolved. The objective of this study was to evaluate if ex vivo intact CB nerve activity and in vivo whole body ventilatory responses to hypoxia were modified by alterations in succinate metabolism and mitochondrial ROS (mitoROS) generation in the rat. Application of diethyl succinate (DESucc) caused concentration-dependent increases in chemoafferent frequency measuring approximately 10-30% of that induced by severe hypoxia. Inhibition of mitochondrial succinate metabolism by dimethyl malonate (DMM) evoked basal excitation and attenuated the rise in chemoafferent activity in hypoxia. However, approximately 50% of the response to hypoxia was preserved. MitoTEMPO (MitoT) and 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SKQ1) (mitochondrial antioxidants) decreased chemoafferent activity in hypoxia by approximately 20-50%. In awake animals, MitoT and SKQ1 attenuated the rise in respiratory frequency during hypoxia, and SKQ1 also significantly blunted the overall hypoxic ventilatory response (HVR) by approximately 20%. Thus, whilst the data support a role for succinate and mitoROS in CB and whole body O2-sensing in the rat, they are not the sole mediators. Treatment of the CB with mitochondrial selective antioxidants may offer a new approach for treating CB-related cardiovascular-respiratory disorders.

Prostaglandin contribution to postexercise hyperemia is dependent on tissue oxygenation during rhythmic and isometric contractions.

The role of prostaglandins (PGs) in exercise hyperemia is controversial. We tested their contributions in moderate intensity forearm exercise, whether their release is oxygen (O2 )-dependent or affected by aging. A total of 12 young (21 ± 1 years) and 11 older (66 ± 2 years) recreationally active men performed rhythmic and isometric handgrip contractions at 60% maximum voluntary contraction for 3 min during air breathing after placebo, after cyclooxygenase (COX) inhibition with aspirin, while breathing 40% O2 and during their combination (aspirin + 40% O2 ). Forearm blood flow (FBF) was recorded with venous occlusion plethysmography (forearm vascular conductance (FVC): FBF/mean arterial pressure). Venous efflux of PGI2 and PGE2 were assessed by immunoassay. Postcontraction increases in FVC were similar for rhythmic and isometric contractions in young and older men, and accompanied by similar increases in efflux of PGI2 and PGE2 . Aspirin attenuated the efflux of PGI2 by 75%-85%, PGE2 by 50%-70%, (p < .05 within group; p > .05 young versus. older), and postcontraction increases in FVC by 22%-27% and 17%-21% in young and older men, respectively (p < .05 within group and young versus. older). In both age groups, 40% O2 and aspirin + 40% O2 caused similar inhibition of the increases in FVC and efflux of PGs as aspirin alone (p < .05 within group). These results indicate that PGs make substantial contributions to the postcontraction hyperemia of rhythmic and isometric contractions at moderate intensities in recreationally active young and older men. Given PGI2 is mainly released by endothelium and PGE2 by muscle fibers, we propose PG generation is dependent on the contraction-induced falls in O2 at these sites.

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 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.

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 O(2) delivery is reduced (hypoxia) or O(2) 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 A(2A)-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 A(2A)-receptors plays a greater role in the hyperaemia associated with tetanic contraction. These results are considered in relation to the A(1)-receptor-mediated muscle dilatation evoked by systemic hypoxia.

Adrenaline activation of the carotid body: Key to CO2 and pH homeostasis in hypoglycaemia and potential pathological implications in cardiovascular disease.

Ventilatory and neuroendocrine counter-regulatory responses during hypoglycaemia are essential in order to maintain glycolysis and prevent rises in PaCO2 leading to systemic acidosis. The mammalian carotid body has emerged as an important driver of hyperpnoea and glucoregulation in hypoglycaemia. However, the adequate stimulus for CB stimulation in hypoglycaemia has remained controversial for over a decade. The recent finding that adrenaline is a physiological activator of CB in hypoglycaemia raises the intriguing possibility that CB stimulation and hyperpnoea may be necessary to maintain pH in other adrenaline-related hypermetabolic states such as exercise. This review will therefore focus on 1) The important functional contribution of the CB in the counter-regulatory and ventilatory response to hypoglycaemia, 2) the proposed mechanisms that cause CB stimulation in hypoglycaemia including hormonal activation by adrenaline and direct low glucose sensing and 3) the possible pathological consequences of repetitive CB activation by adrenaline that could potentially be targeted to reduce CB-mediated cardiovascular disease.

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.

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.