ATP Infusion Research Articles

The ATP Level in the mPFC Mediates the Antidepressant Effect of Calorie Restriction.

Food deprivation can rescue obesity and overweight-induced mood disorders, and promote mood performance in normal subjects. Animal studies and clinical research have revealed the antidepressant-like effect of calorie restriction, but little is known about the mechanism of calorie restriction-induced mood modification. Previous studies have found that astrocytes modulate depressive-like behaviors. Inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) is the predominant isoform in mediating astrocyte Ca2+ signals and its genetic knockout mice are widely used to study astrocyte function in vivo. In this study, we showed that deletion of IP3R2 blocked the antidepressant-like effect induced by calorie restriction. In vivo microdialysis experiments demonstrated that calorie restriction induced an increase in ATP level in the medial prefrontal cortex (mPFC) in naïve mice but this effect disappeared in IP3R2-knockout mice, suggesting a role of astrocytic ATP in the calorie restriction-induced antidepressant effect. Further experiments showed that systemic administration and local infusion of ATP into the mPFC induced an antidepressant effect, whereas decreasing ATP by Apyrase in the mPFC blocked calorie restriction-induced antidepressant regulation. Together, these findings support a role for astrocytic ATP in the antidepressant-like effect caused by calorie restriction.

Reduction of prefrontal purinergic signaling is necessary for the analgesic effect of morphine

SummaryMorphine is commonly used to relieve moderate to severe pain, but repeated doses cause opioid tolerance. Here, we used ATP sensor and fiber photometry to detect prefrontal ATP level. It showed that prefrontal ATP level decreased after morphine injection and the event amplitude tended to decrease with continuous morphine exposure. Morphine had little effect on prefrontal ATP due to its tolerance. Therefore, we hypothesized that the analgesic effect of morphine might be related to ATP in the medial prefrontal cortex (mPFC). Moreover, local infusion of ATP partially antagonized morphine analgesia. Then we found that inhibiting P2X7R in the mPFC mimicked morphine analgesia. In morphine-tolerant mice, pretreatment with P2X4R or P2X7R antagonists in the mPFC enhanced analgesic effect. Our findings suggest that reduction of prefrontal purinergic signaling is necessary for the morphine analgesia, which help elucidate the mechanism of morphine analgesia and may lead to the development of new clinical treatments for neuropathic pain.

Involvement of P2X2 receptor in the medial prefrontal cortex in ATP modulation of the passive coping response to behavioral challenge.

P2X2 and P2X3 receptors are widely expressed in both the peripheral nervous system and the central nervous system and have been proven to participate in different peripheral sensory functions, but there are few studies on the involvement of P2X2 and P2X3 receptors in animal behaviors. Here we used P2X2 and P2X3 knockout mice to address this issue. P2X2 knockout mice showed normal motor function, exploratory behavior, anxiety-like behaviors, learning and memory behaviors and passive coping response to behavioral challenge. Nevertheless, the effect of ATP infusion in the medial prefrontal cortex (mPFC) on the passive coping response was blocked by P2X2 but not P2X3 receptor deletion. Additionally, no deficits in a wide variety of behavioral tests were observed in P2X3 knockout mice. These findings demonstrate a role of P2X2 receptor in the mPFC in adenosine-5'-triphosphate modulation of the passive coping response to behavioral challenge and show that the P2X2/P2X3 receptor is dispensable for behaviors.

Attenuation of exaggerated blood pressure responses to exercise: a byproduct of improved vascular function with passive heat therapy?

Attenuation of exaggerated blood pressure responses to exercise: a byproduct of improved vascular function with passive heat therapy?

Old cells die hard - sympatholysis and the ageing erythrocyte.

Old cells die hard - sympatholysis and the ageing erythrocyte.

Augmentation of endothelium-dependent vasodilatory signalling improves functional sympatholysis in contracting muscle of older adults.

The ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction (functional sympatholysis) is critical for maintaining blood flow during exercise-mediated sympathoexcitation. Functional sympatholysis and endothelial function are impaired with ageing, resulting in compromised blood flow and oxygen delivery to contracting skeletal muscle during exercise. In the present study, intra-arterial infusion of ACh or ATP to augment endothelium-dependent signalling during exercise attenuated α1 -adrenergic vasoconstriction in the contracting muscle of older adults. The vascular signalling mechanisms capable of functional sympatholysis are preserved in healthy ageing, and thus the age-related impairment in functional sympatholysis probably results from the loss of a functional signal (e.g. plasma [ATP]) as opposed to an intrinsic endothelial dysfunction. The ability of contracting skeletal muscle to attenuate sympathetic α-adrenergic vasoconstriction ('functional sympatholysis') is impaired with age. In young adults, increasing endothelium-dependent vasodilatory signalling during mild exercise augments sympatholysis. In the present study, we tested the hypothesis that increasing endothelium-dependent signalling during exercise in older adults can improve sympatholysis. In 16 older individuals (Protocol 1, n=8; Protocol 2, n=8), we measured forearm blood flow (Doppler ultrasound) and calculated changes in vascular conductance (FVC) to local intra-arterial infusion of phenylephrine (PE; α1 -agonist) during (i) infusion of an endothelium-dependent vasodilator alone (Protocol 1: ACh or Protocol 2: low dose ATP); (ii) mild handgrip exercise (5% maximum voluntary contraction; MVC); (iii) moderate handgrip exercise (15% MVC); and (iv) mild or moderate handgrip exercise+infusion of ACh or ATP to augment endothelium-dependent signalling. PE caused robust vasoconstriction in resting skeletal muscle during control vasodilator infusions (ΔFVC: ACh: -31±3 and ATP: -30±4%). PE-mediated vasoconstriction was not attenuated by mild or moderate intensity exercise (ΔFVC: 5% MVC: -30±9; 15% MVC: -33±8%; P>0.05vs. control ACh and ATP), indicative of impaired sympatholysis, and ACh or ATP infusion during mild exercise did not impact this response. However, augmentation of endothelium-dependent signalling via infusion of ACh or ATP during moderate intensity exercise attenuated PE-mediated vasoconstriction (ΔFVC: -13±1 and -19±5%, respectively; P<0.05vs. all conditions). Our findings demonstrate that, given a sufficient stimulus, endothelium-dependent sympatholysis remains intact in older adults. Strategies aimed at activating such pathways represent a viable approach for improving sympatholysis and thus tissue blood flow and oxygen delivery in older adults.

Insulin-induced membrane permeability to glucose in human muscles at rest and following exercise.

Increased insulin action is an important component of the health benefits of exercise, but its regulation is complex and not fully elucidated. Previous studies of insulin-stimulated GLUT4 translocation to the skeletal muscle membrane found insufficient increases to explain the increases in glucose uptake. By determination of leg glucose uptake and interstitial muscle glucose concentration, insulin-induced muscle membrane permeability to glucose was calculated 4h after one-legged knee-extensor exercise during a submaximal euglycaemic-hyperinsulinaemic clamp. It was found that during submaximal insulin stimulation, muscle membrane permeability to glucose in humans increases twice as much in previously exercised vs. rested muscle and outstrips the supply of glucose, which then becomes limiting for glucose uptake. This methodology can now be employed to determine muscle membrane permeability to glucose in people with diabetes, who have reduced insulin action, and in principle can also be used to determine membrane permeability to other substrates or metabolites. Increased insulin action is an important component of the health benefits of exercise, but the regulation of insulin action in vivo is complex and not fully elucidated. Previously determined increases in skeletal muscle insulin-stimulated GLUT4 translocation are inconsistent and mostly cannot explain the increases in insulin action in humans. Here we used leg glucose uptake (LGU) and interstitial muscle glucose concentration to calculate insulin-induced muscle membrane permeability to glucose, a variable not previously possible to quantify in humans. Muscle membrane permeability to glucose, measured 4h after one-legged knee-extensor exercise, increased ∼17-fold during a submaximal euglycaemic-hyperinsulinaemic clamp in rested muscle (R) and ∼36-fold in exercised muscle (EX). Femoral arterial infusion of NG -monomethyl l-arginine acetate or ATP decreased and increased, respectively, leg blood flow (LBF) in both legs but did not affect membrane glucose permeability. Decreasing LBF reduced interstitial glucose concentrations to ∼2mM in the exercised but only to ∼3.5mM in non-exercised muscle and abrogated the augmented effect of insulin on LGU in the EX leg. Increasing LBF by ATP infusion increased LGU in both legs with uptake higher in the EX leg. We conclude that it is possible to measure functional muscle membrane permeability to glucose in humans and it increases twice as much in exercised vs. rested muscle during submaximal insulin stimulation. We also show that muscle perfusion is an important regulator of muscle glucose uptake when membrane permeability to glucose is high and we show that the capillary wall can be a significant barrier for glucose transport.

Rapid-onset vasodilator responses to exercise in humans: Effect of increased baseline blood flow.

What is the central question of this study? What is the effect of an elevated baseline blood flow, induced by high-dose intra-arterial infusion of either adenosine or ATP, on the rapid-onset vasodilatory response to a single forearm muscle contraction? What is the main finding and its importance? The peak response to a single contraction is unaffected by augmented baseline blood flow, and thus, is likely to be attributable to a feedforward vasodilatory mechanism. The hyperaemic responses to single muscle contractions are proportional to exercise intensity, which, in turn, is proportional to tissue metabolic demand. Hence, we tested the hypothesis that the rapid-onset vasodilatory response after a single muscle contraction would be unaffected when baseline blood flow was increased via high-dose intra-arterial infusion of either adenosine (ADO) or ATP. Twenty-four healthy young participants (28±1years) performed a single forearm contraction (20% maximal voluntary contraction) 75min after commencement of a continuous infusion of ADO (n=6), ATP (n=8) or saline (control; n=10). Brachial artery diameter and blood velocity were measured using Doppler ultrasound. Resting forearm vascular conductance (FVC; in millilitres per minute per 100mmHg per decilitre of forearm volume) was significantly higher during ADO (33±17) and ATP infusion (33±17) compared with the control infusion (8±3; P<0.05). The peak FVCs post-contraction during ADO and ATP infusions were significantly greater than during the control infusion (P<0.05), but not different from one another. The peak change in FVC from baseline was similar in all three conditions (control, 14±1; ADO, 24±2; and ATP, 23±6; P=0.15). Total FVC (area under the curve) did not differ significantly between ADO and ATP (333±69 and 440±125); however, total FVC during ATP infusion was significantly greater compared with the control value (150±19; P<0.05). We conclude that the peak response to a single contraction is unaffected by augmented baseline blood flow and is therefore likely to be attributable to a feedforward vasodilatory mechanism.

Reduced skeletal-muscle perfusion and impaired ATP release during hypoxia and exercise in individuals with type 2 diabetes.

Plasma ATP is a potent vasodilator and is thought to play a role in the local regulation of blood flow. Type 2 diabetes is associated with reduced tissue perfusion. We aimed to examine whether individuals with type 2 diabetes have reduced plasma ATP concentrations compared with healthy control participants (case-control design). We measured femoral arterial and venous plasma ATP levels with the intravascular microdialysis technique during normoxia, hypoxia and one-legged knee-extensor exercise (10W and 30W) in nine participants with type 2 diabetes and eight control participants. In addition, we infused acetylcholine (ACh), sodium nitroprusside (SNP) and ATP into the femoral artery to assess vascular function and ATP signalling. Individuals with type 2 diabetes had a lower leg blood flow (LBF; 2.9 ± 0.1l/min) compared with the control participants (3.2 ± 0.1l/min) during exercise (p < 0.05), in parallel with lower venous plasma ATP concentration (205 ± 35 vs 431 ± 72nmol/l; p < 0.05). During systemic hypoxia, LBF increased from 0.35 ± 0.04 to 0.54 ± 0.06l/min in control individuals, whereas it did not increase (0.25 ± 0.04 vs 0.31 ± 0.03l/min) in the those with type 2 diabetes and was lower than in the control individuals (p < 0.05). Hypoxia increased venous plasma ATP levels in both groups (p < 0.05), but the increase was higher in control individuals (90 ± 26nmol/l) compared to those with type 2 diabetes (18 ± 5nmol/l). LBF and vascular conductance were lower during ATP (0.15 and 0.4μmolmin-1 [kg leg mass]-1) and ACh (100μgmin-1 [kg leg mass]-1) infusion in individuals with type 2 diabetes compared with the control participants (p < 0.05), whereas there was no difference during SNP infusion. These findings demonstrate that individuals with type 2 diabetes have lower plasma ATP concentrations during exercise and hypoxia compared with control individuals, and this occurs in parallel with lower blood flow. Moreover, individuals with type 2 diabetes have a reduced vasodilatory response to infused ATP. These impairments in the ATP system are both likely to contribute to the reduced tissue perfusion associated with type 2 diabetes. ClinicalTrials.gov NCT02001766.

PL - 026 Mismatch between skeletal muscle glucose delivery, interstitial concentration and membrane permeability may limit insulin sensitivity after exercise

Objective The relationship between skeletal muscle perfusion, interstitial glucose concentration and sarcolemmal permeability to glucose in exercise-induced increases in muscle insulin sensitivity is not well established. A single bout of exercise increases skeletal muscle insulin sensitivity through coordinated increases in insulin-stimulated microvascular perfusion and insulin signalling Reducing leg and muscle microvascular blood flow with local nitric oxide synthase (NOS) inhibition during a hyperinsulinaemic euglycaemic clamp reduces leg glucose uptake in a previously exercised, but not in a contralateral non-exercised leg, without affecting insulin signalling in either leg (Sjoberg et al. 2017). Therefore, it is possible that the reduction in muscle perfusion decreases muscle interstitial glucose concentration to a point that limits skeletal muscle insulin-stimulated glucose uptake following exercise. We examined this using microdialysis of vastus lateralis muscle.&#x0D; Methods Ten healthy males (Age: 27±1 yr., Weight: 77.7±2.3 kg, BMI 23.9±0.5, VO2 peak: 50.7±1.5 ml·kg-1·min-1) performed 60 min of 1-legged knee extensor exercise at 80% of 1-legged peak work load with three 5 min intervals at 100% 1-legged peak work load. Participants then rested for 4 hours and catheters were inserted into the femoral artery and vein of both legs for subsequent measurement of leg glucose uptake and for femoral artery infusion of the NOS inhibitor NG-monomethyl L-arginine acetate (L-NMMA) and the vasodilator ATP. Catheters were also placed in antecubital veins for infusion of insulin and glucose. Three microdialysis catheters, with a semi-permeable membrane the length of 30 mm and a molecular cut-off at 20,000 dalton, were inserted into the vastus lateral muscle of both legs. Glucose and D-[6-3H(N)]glucose were added to the perfusate. Four hours after discontinuing the exercise a 225 minute euglycaemic hyperinsulinaemic clamp was initiated (insulin infusion 1.4 mU-1kg-1min). Ninety min into the clamp L-NMMA was infused at a constant rate (0.4 mg·kg-1 leg mass·min-1) into both femoral arteries for 45 min. The insulin infusion was maintained for another 90 min and during the last 45 min ATP (0.3 μmol∙ml-1) was infused locally into both femoral arteries at a rate of 200-350 μl∙min-1 to obtain a leg blood flow that was double the blood flow during insulin only infusion. A second control protocol was undertaken that was identical in regards to exercise and recovery but no insulin, L-NMMA or ATP was infused.&#x0D; Results During the clamp leg glucose uptake and leg blood flow were higher (P&lt;0.05) in the previously exercised than the control leg whereas the interstitial glucose concentration decreased to lower (P&lt;0.05) values in the exercised (~3.1mM) than the control (~4.8mM) leg. Estimated sarcolemmal glucose permeability was twice as high (P&lt;0.05) in the exercised compared with the rested leg. The NOS inhibitor L-NMMA decreased LBF in both legs and interstitial glucose concentration dropped to ~2.3 mM in the exercised but only to ~3.7 mM in non-exercised muscle. This abrogated the augmented effect of insulin on LGU in the exercised leg while apparent sarcolemmal permeability to glucose remained unchanged with L-NMMA in both legs. Doubling leg blood flow by local infusion of ATP increased leg glucose uptake in both legs without any major change in interstitial glucose concentration or sarcolemmal permeability to glucose.&#x0D; Conclusions These findings suggest that during flow restriction due to L-NMMA, the interstitial glucose concentration becomes limiting for leg glucose uptake in exercised but not in non-exercised muscle. Therefore, the vasodilatory effect of insulin is an important component of the increased insulin sensitivity to stimulate glucose uptake following exercise by limiting the drop in the interstitial glucose concentration that occurs due to the increased sarcolemmal permeability to glucose.&#x0D; Reference&#x0D; Sjoberg, K. A., C. Frosig, R. Kjobsted, L. Sylow, M. Kleinert, A. C. Betik, C. S. Shaw, B. Kiens, J. F. P. Wojtaszewski, S. Rattigan, E. A. Richter, and G. K. McConell. Exercise Increases Human Skeletal Muscle Insulin Sensitivity via Coordinated Increases in Microvascular Perfusion and Molecular Signaling. Diabetes 66: 1501-10, 2017.

Muscle α-adrenergic responsiveness during exercise and ATP-induced vasodilation in chronic obstructive pulmonary disease patients.

-0.04 ± 0.01 l·min-1·kg leg mass-1, P > 0.05). Compared with age-matched healthy control subjects, the vasodilatory response to ATP is intact in COPD patients and their ability to blunt sympathetic vasoconstriction (functional sympatholysis) as evaluated by intra-arterial Tyramine during exercise or ATP infusion is maintained. NEW & NOTEWORTHY The ability to blunt sympathetic vasoconstriction in exercising muscle and ATP-induced dilation in chronic obstructive pulmonary disease patients remains unexplored. Chronic obstructive pulmonary disease patients demonstrated similar sympathetic vasoconstriction in response to intra-arterial Tyramine during exercise and ATP-induced vasodilation compared with age-matched healthy control subjects.

Sympatholytic effect of intravascular ATP is independent of nitric oxide, prostaglandins, Na+ /K+ -ATPase and KIR channels in humans.

Intravascular ATP attenuates sympathetic vasoconstriction (sympatholysis) similar to what is observed in contracting skeletal muscle of humans, and may be an important contributor to exercise hyperaemia. Similar to exercise, ATP-mediated vasodilatation occurs via activation of inwardly rectifying potassium channels (KIR ), and synthesis of nitric oxide (NO) and prostaglandins (PG). However, recent evidence suggests that these dilatatory pathways are not obligatory for sympatholysis during exercise; therefore, we tested the hypothesis that the ability of ATP to blunt α1 -adrenergic vasoconstriction in resting skeletal muscle would be independent of KIR , NO, PGs and Na+ /K+ -ATPase activity. Blockade of KIR channels alone or in combination with NO, PGs and Na+ /K+ -ATPase significantly reduced the vasodilatatory response to ATP, although intravascular ATP maintained the ability to attenuate α1 -adrenergic vasoconstriction. This study highlights similarities in the vascular response to ATP and exercise, and further supports a potential role of intravascular ATP in blood flow regulation during exercise in humans. Exercise and intravascular ATP elicit vasodilatation that is dependent on activation of inwardly rectifying potassium (KIR ) channels, with a modest reliance on nitric oxide (NO) and prostaglandin (PG) synthesis. Both exercise and intravascular ATP attenuate sympathetic α-adrenergic vasoconstriction (sympatholysis). However, KIR channels, NO, PGs and Na+ /K+ -ATPase activity are not obligatory to observe sympatholysis during exercise. To further determine similarities between exercise and intravascular ATP, we tested the hypothesis that inhibition of KIR channels, NO and PG synthesis, and Na+ /K+ -ATPase would not alter the ability of ATP to blunt α1 -adrenergic vasoconstriction. In healthy subjects, we measured forearm blood flow (Doppler ultrasound) and calculated changes in vascular conductance (FVC) to intra-arterial infusion of phenylephrine (PE; α1 -agonist) during ATP or control vasodilatator infusion, before and after KIR channel inhibition alone (barium chloride; n=7; Protocol 1); NO (l-NMMA) and PG (ketorolac) inhibition alone, or combined NO, PGs, Na+ /K+ -ATPase (ouabain) and KIR channel inhibition (n=6; Protocol 2). ATP attenuated PE-mediated vasoconstriction relative to adenosine (ADO) and sodium nitroprusside (SNP) (PE-mediated ΔFVC: ATP: -16±2; ADO: -38±6; SNP: -59±6%; P<0.05 vs. ADO and SNP). Blockade of KIR channels alone or combined with NO, PGs and Na+ /K+ -ATPase, attenuated ATP-mediated vasodilatation (∼35 and ∼60% respectively; P<0.05 vs. control). However, ATP maintained the ability to blunt PE-mediated vasoconstriction (PE-mediated ΔFVC: KIR blockade alone: -6±5%; combined blockade:-4±14%; P>0.05 vs. control). These findings demonstrate that intravascular ATP modulates α1 -adrenergic vasoconstriction via pathways independent of KIR channels, NO, PGs and Na+ /K+ -ATPase in humans, consistent with a role for endothelium-derived hyperpolarization in functional sympatholysis.

Evidence for a functional vasoconstrictor role for ATP in the human cutaneous microvasculature.

What is the central question of this study? In young adults, about half of the cold-related reduction in skin blood flow during cold exposure is mediated by noradrenaline, while the remainder is attributable to other substances co-released with noradrenaline that have yet to be identified. What is the main finding and its importance? Purinergic receptor blockade blunted the vasoconstriction response to whole-body cooling and to intradermal administration of tyramine. These results indicate that ATP is necessary to vasoconstrict blood vessels in the skin adequately and prevent heat loss in a cold environment. Noradrenaline is responsible for eliciting ∼60% of the reflex cutaneous vasoconstriction (VC) response in young adults, while the remainder is attributable to one or more unidentified co-released sympathetic adrenergic neurotransmitter(s). Inconsistent evidence has placed neuropeptide Y in this role; however, other putative cotransmitters have yet to be tested. We hypothesize that ATP contributes to the reflex cutaneous VC response. Two protocols were conducted in young adults (n=10); both involved the placement of three microdialysis probes in forearm skin and whole-body cooling (skin temperature=30.5°C). In protocol 1, the following solutions were infused: (i) lactated Ringer solution (control); (ii) 10mm l-NAME; and (iii) purinergic receptor blockade with 1mm suramin plus l-NAME. In protocol 2, the following solutions were infused: (i) lactated Ringer solution; (ii) suramin plus l-NAME; and (iii) suramin plus l-NAME plus adrenoreceptor blockade with 5mm yohimbine plus 1mm propranolol. Laser Doppler flux (LDF) was measured over each microdialysis site, and cutaneous vascular conductance (CVC) was calculated (CVC=LDF/MAP) and expressed as percentage changes from baseline (%ΔCVCBASELINE ). l-NAME was used to block the vasodilatory influence of ATP and unmask the P2 X-mediated VC response to exogenous ATP infusion (-21±6%ΔCVCBASELINE ). During cooling, the VC response (control, -39±8%ΔCVCBASELINE ) was attenuated at the suramin site (-21±4%ΔCVCBASELINE ) and further blunted with combined adrenoreceptor blockade (-9±3%ΔCVCBASELINE ; P<0.05). Compared with the control site (-22±5%ΔCVCBASELINE ), suramin inhibited pharmacologically induced VC to tyramine (-12±6%ΔCVCBASELINE ; P<0.05), which displaces adrenergic neurotransmitters from axon terminals. These data indicate that ATP contributes to the cutaneous VC response in humans.

Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature-dependent ATP release from human blood and endothelial cells.

New Findings What is the central question of this study? Skin and muscle blood flow increases with heating and decreases with cooling, but the temperature‐sensitive mechanisms underlying these responses are not fully elucidated. What is the main finding and its importance? We found that local tissue hyperaemia was related to elevations in ATP release from erythrocytes. Increasing intravascular ATP augmented skin and tissue perfusion to levels equal or above thermal hyperaemia. ATP release from isolated erythrocytes was altered by heating and cooling. Our findings suggest that erythrocytes are involved in thermal regulation of blood flow via modulation of ATP release. Local tissue perfusion changes with alterations in temperature during heating and cooling, but the thermosensitivity of the vascular ATP signalling mechanisms for control of blood flow during thermal interventions remains unknown. Here, we tested the hypotheses that the release of the vasodilator mediator ATP from human erythrocytes, but not from endothelial cells or other blood constituents, is sensitive to both increases and reductions in temperature and that increasing intravascular ATP availability with ATP infusion would potentiate thermal hyperaemia in limb tissues. We first measured blood temperature, brachial artery blood flow and plasma [ATP] during passive arm heating and cooling in healthy men and found that they increased by 3.0 ± 1.2°C, 105 ± 25 ml min−1 °C−1 and twofold, respectively, (all P < 0.05) with heating, but decreased or remained unchanged with cooling. In additional men, infusion of ATP into the brachial artery increased skin and deep tissue perfusion to levels equal or above thermal hyperaemia. In isolated erythrocyte samples exposed to different temperatures, ATP release increased 1.9‐fold from 33 to 39°C (P < 0.05) and declined by ∼50% at 20°C (P < 0.05), but no changes were observed in cultured human endothelial cells, plasma or serum samples. In conclusion, increases in plasma [ATP] and skin and deep tissue perfusion with limb heating are associated with elevations in ATP release from erythrocytes, but not from endothelial cells or other blood constituents. Erythrocyte ATP release is also sensitive to temperature reductions, suggesting that erythrocytes may function as thermal sensors and ATP signalling generators for control of tissue perfusion during thermal interventions.

Safety and clinical value of ATP-induced stress myocardial perfusion imaging in the diagnosis of coronary heart disease

Objective To assess the safety and clinical value of ATP-induced stress MPI (ATP-MPI) in the diagnosis of coronary heart disease (CHD). Methods A total of 83 patients (61 males, 22 females, age: 43-68(55.4±6.7) years) with suspected CHD were included. Rest MPI was performed 90 min post injection of 296 MBq 99Tcm-MIBI. ATP was infused intravenously 3 h later at a rate of 0.16 mg·kg-1·min-1 for 5 min. Then, a dose of 925 MBq 99Tcm-MIBI was injected intravenously 3 min post ATP infusion. ATP-MPI was performed 90 min later. All patients underwent CAG within 1 week after MPI to evaluate the diagnostic efficiency of ATP-MPI. The adverse reaction was observed. Results Ninety-nine diseased vessels were identified by CAG in 64 patients: 38 patients had one-vessel disease, 17 patients had two-vessel disease and 9 patients had three-vessel disease. ATP-MPI detected 91 diseased vessels (38 LAD, 29 LCX and 24 RCA) in 60 patients. In the diagnosis of CHD, ATP-MPI had a sensitivity of 85.9% (55/64), a specificity of 14/19, an accuracy of 83.1% (69/83), a positive predictive value of 91.7% (55/60) and a negative predictive value of 60.9% (14/23). The adverse reaction incidence of ATP stress test was 84.3%(70/83). The symptoms included mild hypotension, palpitations, shortness of breath, dizziness and facial flush, which all resolved spontaneously without any treatment. Conclusion ATP-MPI is a noninvasive, convenient and safe imaging modality for the diagnosis of CHD, and can be routinely used. Key words: Coronary disease; Tomography, emission-computed, single-photon; Adenosine triphosphate

Abstract 10412: The Effect of Positive Remodeling Area Index and Large Plaque Burden over Fractional Flow Reserve

Introduction: Coronary angiographic anatomical stenosis has not been well correlated with physiological fractional flow reserve (FFR), however, the mechanism of the discordance remains poorly understood. We focused on the patients who had no anatomical significant stenosis by coronary angiogram (CAG) but physiological significant stenosis by FFR (reverse mismatch) in the proximal or mid left anterior descending artery (LAD). We explored what coronary CT angiography (CCTA) findings of the target lesion of the LAD predict abnormal FFR. Methods: ECG-gated CCTA was performed using SOMATOM Definition AS+ (128 slice Siemens) and CAG underwent within 4 weeks. FFR was measured using a pressure guide wire (verrata, Volcano) during ATP infusion. Forty consecutive reverse mismatch (with no anatomical stenosis and FFR≦0.8) pts and 40 no mismatch (with no anatomical stenosis and FFR&gt;0.8) pts were selected. Results: There were no significant differences in mean age (72±10y vs 74±8y), gender (M/F 21/19 vs 28/12), coronary risk factors (DM (9 vs 17), HT(32 vs 30), dyslipidemia (23 vs 18), Smoking (5 vs 4)), angle of LAD and LCX (70±21vs71±20 deg.), proximal reference lumen area (0.14±0.14 vs 0.14±0.11 mm2) and vessel area (0.15±0.04 vs 0.15±0.06mm2), distal reference lumen area (0.11±0.12 vs 0.10±0.04mm2) and vessel area (0.14±0.13 vs 0.12±0.04mm2), grade of plaque calcification, and presence of low density plaque (8 vs 7 pts) between no mismatch and reverse mismatch groups. However, vessel area (0.21±0.07mm2, P=0.0004) and positive remodeling area index (1.40±0.26, P&lt;0.0001) at the minimum lumen area (MLA) in reverse mismatch group were higher than those (0.16±0.05mm2, 1.01±0.18) in no mismatch groups, respectively. AUC of remodeling area index was 0.924 for diagnosis of reverse mismatch on ROC analysis. Sensitivity was 82.5%, specificity was 84.6% when the remodeling area index was 1.13. Conclusions: Even if anatomical significant stenosis is not observed, FFR was depressed in patients with large vessel area and positive remodeling area index derived from CCTA at MLA. Large plaque at MLA may be one of the mechanisms of reverse mismatch.

Intradermal administration of ATP augments methacholine-induced cutaneous vasodilation but not sweating in young males and females.

Acetylcholine released from cholinergic nerves is a key neurotransmitter contributing to heat stress-induced cutaneous vasodilation and sweating. Given that sympathetic cholinergic nerves also release ATP, ATP may play an important role in modulating cholinergic cutaneous vasodilation and sweating. However, the pattern of response may differ between males and females given reports of sex-related differences in the peripheral mechanisms governing these heat loss responses. Cutaneous vascular conductance (CVC, laser-Doppler perfusion units/mean arterial pressure) and sweat rate (ventilated capsule) were evaluated in 17 young adults (8 males, 9 females) at four intradermal microdialysis skin sites continuously perfused with: 1) lactated Ringer (Control), 2) 0.3 mM ATP, 3) 3 mM ATP, or 4) 30 mM ATP. At all skin sites, methacholine was coadministered in a concentration-dependent manner (0.0125, 0.25, 5, 100, 2,000 mM, each for 25 min). In both males and females, CVC was elevated with the lone infusion of 30 mM ATP (both P < 0.05), but not with 0.3 and 3 mM ATP compared with control (all P >0.27). However, 0.3 mM ATP induced a greater increase in CVC compared with control in response to 100 mM methacholine infusion in males (P < 0.05). In females, 0.3 mM ATP infusion resulted in a lower concentration of methacholine required to elicit a half-maximal response (EC50) (P < 0.05). In both males and females, methacholine-induced sweating was unaffected by any concentration of ATP (all P > 0.44). We demonstrate that ATP enhances cholinergic cutaneous vasodilation albeit the pattern of response differs between males and females. Furthermore, we show that ATP does not modulate cholinergic sweating.

The ubiquitous ATP molecule: could it be the elusive thermal mediator igniting skin perfusion and sweating in the heat-stressed human?

Skin blood flow and sweating are two key thermoregulatory responses that help regulate body temperature in heat-stressed human beings. Adjustments in skin blood flow and sweating in response to thermal stimuli are mediated by local and reflex neural mechanisms (Johnson et al. 2014). Local thermosensitive mechanisms are important, as the application of heating to a particular area of the skin results in a localized elevation of blood flow and sweating in the heated skin. However, the precise pathways underpinning heat loss responses to heat stress, particularly those involving ATP as a mediator signal, are not fully established or understood. Heat stress-induced hyperthermia is associated with increases in intravascular ATP and accompanied by vasodilatation of the human skin and deep limb tissue vasculatures (Pearson et al. 2011; Heinonen et al. 2011). This supports a mechanistic coupling between the rise in intravascular ATP and the concomitant increase in local perfusion (Kalsi & Gonzalez-Alonso, 2012). However, sympathetic cholinergic nerves, which are known to modulate heat loss responses, co-release ATP with acetylcholine. These sensory nerves are therefore potential sources of ATP in the dermal interstitium. This raises the possibility that ATP may act as a thermal mediator signal, involved in the regulation of vasomotor and sudomotor activity in conditions of hyperthermia. In support of this idea, intradermal administration of ATP has been shown to induce cutaneous vasodilatation in human beings in vivo (Wingo et al. 2010), but the downstream mediators and its effect on sweating have not been investigated. Mechanistically, ATP can bind to P2Y purinergic receptors located in the cutaneous vasculature, and the sweat gland, and trigger cutaneous vasodilatation and increase sweating via at least three pathways: (i) activation of nitric oxide synthase (NOS), (ii) activation of cyclooxygenase (COX) and/or (iii) via the action of the ATP degradation compound adenosine. In this issue of The Journal of Physiology, Fujii et al. (2015) report the findings from an outstanding series of integrative physiology studies in human beings. The authors systematically investigated the roles of NOS and COX in the cutaneous vascular and sweating responses to intradermal administration of ATP, as well as the contribution of adenosine to ATP-induced skin vasodilatation in vivo. A major finding was that intradermal ATP infusion at a rate of 120 and 1200 nmol min−1 causes significant cutaneous vasodilatation. Co-infusion of the NOS inhibitor Nω–nitro-l-arginine (l-NNA) blunted about one-fourth of the ATP-evoked vasodilator response, implicating NOS as a mediator. The study also excluded two other potential regulatory pathways; blockade of COX or adenosine with co-infusion of ketorolac or theophylline did not alter the vasodilator response to intradermal ATP infusion. Lastly, none of the study protocols increased sweating. These observations collectively advance the basic physiological understanding of the control of skin blood flow and sweating. By showing that part of the vasodilator response to intradermal ATP administration is mediated by NOS and by providing conclusive evidence that ATP does not directly increase sweating in vivo, these data suggest that skin blood flow and sweating are not regulated by the same thermal mediator signal. The physiological relevance of interstitial ATP on thermal hyperaemia was not addressed in the study of Fujii et al. (2015). ATP needs to accumulate in the dermal interstitium for this ubiquitous molecule to exert its cutaneous vasodilator effects directly by binding to interstitial purinergic receptors. However, recent evidence suggests that local heating does not result in an accumulation of ATP in the interstitial space of human skin, and thus such accumulation is not necessary for local thermal hyperaemia to occur (Gifford et al. 2012). This observation and the additional finding by Fujii and coworkers that the intradermal administration of ATP at low infusion rates (0.12–12 nmol min−1) does not increase skin blood flow indicate that under physiological conditions ATP-mediated cutaneous vasodilatation is more likely to occur via the activation of purinergic receptors residing in the vascular endothelium. Although further research is needed to establish the physiological relevance of intravascular versus interstitial ATP in thermoeffector activity, the timely investigation of Fujii et al. (2015) highlights the capacity of ATP to be the elusive thermal mediator igniting skin perfusion in heat-stressed human beings, but not the signal that increases sweating.

Cutaneous vascular and sweating responses to intradermal administration of ATP: a role for nitric oxide synthase and cyclooxygenase?

In humans in vivo, the mechanisms behind ATP-mediated cutaneous vasodilatation along with whether and how ATP increases sweating remains uncertain. Recent work has implicated nitric oxide synthase (NOS), cyclooxygenase (COX) and/or adenosine in the modulation of cutaneous vasodilatation and sweat production during both local (i.e. localized heating) and whole-body heat stress (i.e. exercise-induced heat stress). We evaluated whether ATP-mediated cutaneous vasodilatation and sweating is mediated via NOS, COX and/or adenosine. We show that in humans in vivo, intradermal administration of ATP induces pronounced vasodilatation which is partially mediated by NOS, but neither COX nor adenosine influences ATP-mediated vasodilatation, and ATP alone does not induce an increase in sweating. These findings advance our basic physiological knowledge regarding control of skin blood flow and sweating, and provide insight into the mechanisms governing thermoeffector activity, which has major implications for whole-body heat exchange and therefore core temperature regulation in humans during heat stress. In humans in vivo, the mechanisms behind ATP-mediated cutaneous vasodilatation and whether and how ATP increases sweating remain uncertain. We evaluated whether ATP-mediated cutaneous vasodilatation and sweating is mediated via nitric oxide synthase (NOS), cyclooxygenase (COX) and/or adenosine-dependent mechanisms. Cutaneous vascular conductance (CVC, laser Doppler perfusion units/mean arterial pressure) and sweat rate (ventilated capsule) were evaluated at intradermal microdialysis forearm skin sites, each receiving pharmacological agents (two separate protocols). In Protocol 1 (n = 12), sites were perfused with: (1) lactated Ringer solution (Control), (2) 10 mm N(ω) -nitro-l-arginine (l-NNA, a NOS inhibitor), (3) 10 mm ketorolac (Ketorolac, a COX inhibitor) or (4) a combination of 10 mm l-NNA + 10 mm ketorolac (l-NNA + Ketorolac). In Protocol 2 (n = 8), sites were perfused with: (1) lactated Ringer solution (Control) or (2) 4 mm theophylline (Theophylline, an adenosine receptor inhibitor). At all sites, ATP was simultaneously perfused at 0.12, 1.2, 12, 120 and 1200 nm min(-1) (each for 20 min). Relative to CVC at the Control site with ATP infused at 120 nm min(-1) (71 ± 9% of max CVC), CVC at the Ketorolac site was comparable (64 ± 13% of max CVC, P = 0.407), but lower at l-NNA (51 ± 15% of max CVC, P = 0.040) and l-NNA + Ketorolac (51 ± 13% of max CVC, P = 0.049) sites. Conversely, across the four skin sites at any other ATP infusion rate (all P > 0.174), no differences in CVC were observed. Theophylline did not influence CVC at any ATP infusion rate (all P > 0.234). Furthermore, no ATP infusion rate elicited an increase in sweating from baseline at any skin site (all P > 0.235). We show that NOS, but neither COX nor adenosine receptors, modulates ATP-mediated cutaneous vasodilatation, whereas ATP does not directly increase sweating.

ATP Antagonism Attenuates the Reflex Cutaneous Vasoconstriction Response in Humans

Norepinephrine is responsible for ~60% of the reflex cutaneous VC response in young individuals, whereas the remainder is due to a coreleased sympathetic adrenergic neurotransmitter(s) that has yet to be identified. There is conflicting evidence supporting neuropeptide Y in this role; however, other putative cotransmitters have yet to be tested in human skin. We hypothesize that ATP contributes to the reflex cutaneous VC response to whole‐body cooling (Tsk = 30.5 oC). Three microdialysis (MD) fibers were placed in the forearm skin of 7 (25 ± 1 years; 4 males, 3 females) individuals for infusion of 1) lactated Ringer's solution (control), 2) 10 mM L‐NAME, and 3) purinergic receptor blockade with 1mM suramin + L‐NAME. Laser Doppler flux (LDF) was measured over each MD site and cutaneous vascular conductance (CVC) was calculated as CVC = LDF/mean arterial pressure and expressed as a percent change from baseline (%ΔCVC). L‐NAME was used to block the vasodilatory influence of ATP and unmask the P2X‐mediated VC response to exogenous ATP infusion (‐18 ± 2 %ΔCVC). This response was blocked in the suramin‐pretreated site (‐2 ± 1 %ΔCVC). During whole body cooling (Tsk = 30.5 oC), the VC response in the suramin site was attenuated (‐24 ± 3 %ΔCVC; p &lt; 0.05) compared to the control site (‐35 ± 4 %ΔCVC). These data suggest that ATP has a contributory role in the reflex cutaneous VC response in humans during whole‐body cooling.**Funded by Iowa Osteopathic &amp; Education Research Grant (IOER #03‐14‐01)