Interstitial and Plasma Adenosine Stimulate Nitric Oxide and Prostacyclin Formation in Human Skeletal Muscle
One major unresolved issue in muscle blood flow regulation is that of the role of circulating versus interstitial vasodilatory compounds. The present study determined adenosine-induced formation of NO and prostacyclin in the human muscle interstitium versus in femoral venous plasma to elucidate the interaction and importance of these vasodilators in the 2 compartments. To this end, we performed experiments on humans using microdialysis technique in skeletal muscle tissue, as well as the femoral vein, combined with experiments on cultures of microvascular endothelial versus skeletal muscle cells. In young healthy humans, microdialysate was collected at rest, during arterial infusion of adenosine, and during interstitial infusion of adenosine through microdialysis probes inserted into musculus vastus lateralis. Muscle interstitial NO and prostacyclin increased with arterial and interstitial infusion of adenosine. The addition of adenosine to skeletal muscle cells increased NO formation (fluorochrome 4-amino-5-methylamino-2′,7-difluorescein fluorescence), whereas prostacyclin levels remained unchanged. The addition of adenosine to microvascular endothelial cells induced an increase in NO and prostacyclin levels. These findings provide novel insight into the role of adenosine in skeletal muscle blood flow regulation and vascular function by revealing that both interstitial and plasma adenosine have a stimulatory effect on NO and prostacyclin formation. In addition, both skeletal muscle and microvascular endothelial cells are potential mediators of adenosine-induced formation of NO in vivo, whereas only endothelial cells appear to play a role in adenosine-induced formation of prostacyclin.
Skeletal muscle blood flow is closely regulated to match O2 delivery to the metabolic demand of the contracting muscle.1 This precise regulation of muscle blood flow is believed to be regulated by a balance between vasoconstrictor activity and locally derived vasoactive substances.2 Three such vasoactive compounds shown to be of importance for muscle blood flow regulation are adenosine, NO, and prostacyclin (PGI2). Accordingly, it has been shown that blockade of adenosine receptors reduces exercise hyperemia by ≈15% to 20%3,4 and simultaneous inhibition of the enzymes NO synthase (NOS), which catalyzes the formation of NO, and cyclooxygenase (COX), which initiates the conversion of arachidonic acid to PGI2, reduces exercise hyperemia in both the forearm and leg by ≈30%.3,5–8 Importantly, these 3 vasoactive compounds show a close interaction in that the vasodilator effect of adenosine primarily appears to be mediated via the formation of NO and PGI2, as evidenced by markedly reduced vasodilation in response to arterially infused adenosine when NOS3,9 and COX3 are inhibited. Moreover, adenosine receptor blockade does not further reduce exercise hyperemia when combined with inhibition of NOS and COX,3 suggesting that the vasodilator effect of endogenous adenosine is NO and PGI2 dependent. However, because pharmacological inhibition does not discriminate between the vasodilator effect of interstitial and plasma adenosine and only provides indirect evidence for an adenosine-induced formation of NO and PGI2, actual in vivo measurements are still needed to determine whether adenosine in the muscle interstitium and plasma has a stimulatory effect on formation of these vasodilators. There also appears to be a redundancy between NO and PGI2 in that either of the systems appears to compensate for the other when that system is inhibited, thereby maintaining blood flow during exercise.3,6,10 In addition, intra-arterial coinfusion of the NOS inhibitor NG-monomethyl-l-arginine has been shown to blunt the vasodilation evoked by the PGI2 analog epoprostenol, suggesting that PGI2-induced vasodilation is partly mediated through NO.11 Taken together, this high degree of interdependency suggests that the interaction among these 3 vasodilators may be a major player in the regulation of muscle blood flow and vascular function. Additional knowledge regarding the role of adenosine and its interaction with NO and prostanoid formation is also important for the understanding of mechanisms underlying alterations in vascular function in cardiovascular disease and aging. Such information could also be of value for medical treatment of cardiovascular disease by, for example, adenosine. Furthermore, an interdependency of the 3 systems suggests that if one system is impaired, the effect of the other systems may also be altered.
Another major unresolved issue in muscle blood flow regulation is that of the role of circulating versus interstitial vasodilatory compounds. During muscle contraction, adenosine increases in the muscle interstitial fluid at a rate closely associated with the increase in blood flow,12 and this adenosine is likely to originate from AMP 5′nucleotidase located on the extracellular membrane of skeletal muscle and endothelial cells.13 Because pharmacological inhibitors affect both intravascular and interstitial systems, such designs cannot reveal the role or mechanisms of action of interstitial adenosine. Potentially, interstitial adenosine might induce vasodilation by acting directly on smooth muscle adenosine receptors14,15 or by acting on adenosine receptors located on endothelial cells16 and/or skeletal muscle cells, thereby inducing formation of NO and PGI2. The latter pathway may be more important in humans, as evidenced by the above-mentioned lack of additional reduction in exercise-induced blood flow when adenosine receptors and the NO and PGI2 systems are inhibited simultaneously.3 Moreover, the concentration of adenosine is also ≈35-fold higher in the interstitium than in the circulation during moderate-intensity exercise. Whether interstitial adenosine leads to formation of NO and PGI2 is not known, because direct measurements of these compounds in response to adenosine infusion have never been performed previously.
Although never tested in humans, the endothelium has been described to function as an effective barrier for adenosine in other species,17 suggesting that the vasodilator effect of plasma adenosine is mediated via formation of vasodilators released from vascular endothelial cells and not through a direct action on vascular smooth muscle cells. Given this scenario, an interesting aspect is to what extent circulating adenosine affects interstitial levels of NO and PGI2 at the capillary level. In theory, adenosine acting on capillary endothelial cells should enhance the release of NO and PGI2 in both directions unless the enzyme systems are located solely on the luminal side.
In the current study we hypothesized that both interstitial and intra-arterial adenosine stimulate the formation of NO and PGI2 and that microvascular endothelial cells and skeletal muscle cells release NO and PGI2 on adenosine exposure. To test these hypotheses, a combination of experiments was performed in human subjects using microdialysis in skeletal muscle and the femoral vein and in cultures of rat skeletal muscle cells and microvascular endothelial cells isolated from the rat skeletal muscle.
Ten moderately trained male subjects with a mean (±SD) age of 26±5 years, body weight of 76±11 kg, height of 183±6 cm, and V̇o2max relative to body mass of 56.9±6.9 mL · min−1 · kg−1 participated in the study. The purpose, nature, and potential risks were explained to the subjects before they gave their informed, written consent to participate in the study. The study was approved by the Ethics Committee of Copenhagen and Frederiksberg (H-KF 11 289201) and conducted in accordance with the guidelines of the Declaration of Helsinki. The subjects were informed to abstain from caffeine, alcohol, and exercise for 24 hours before the experiment. An expanded Methods section is available in the online Data Supplement (see http://hyper.ahajournals.org).
Experimental Protocol: Microdialysis
Before the experiment, the subjects visited the laboratory and were screened to ensure that they had a body mass index <25, were normotensive, nonsmokers, and not taking any medications. During this visit, the subjects also performed an incremental bicycle ergometer exercise test in which pulmonary maximal oxygen uptake (l min−1, V̇o2max) was determined online (Quark b2 system, Cosmed).
On the day of the experiment, the subjects arrived at the laboratory at 8:30 am after a light breakfast. After 30 minutes in the supine position, catheters were placed into the femoral artery and vein of the experimental leg under local anesthesia (Lidocaine, 20 mg · mL−1). One microdialysis probe (CMA Microdialysis AB) was inserted in the distal direction of the femoral vein of the experimental leg, and 2 microdialysis probes were inserted into the thigh muscle (musculus vastus lateralis) of the experimental leg. Thirty minutes after insertion of the probes, the subjects performed a 10-minute exercise bout at 10 W with the purpose of minimizing the tissue response to insertion trauma.18 To re-establish resting conditions, the subjects rested for another 30 minutes, and dialysate was then collected for 20 minutes during the following conditions: (1) rest (baseline); (2) infusion of a low dose of adenosine (ITEM development AB; 0.16±0.01 μmol · min−1 · kg of leg mass−1) into the femoral artery; and (3) infusion of a high dose of adenosine (0.31±0.01 μmol · min−1 · kg of leg mass−1) into the femoral artery. Infusion of the 2 doses of adenosine was separated by 10 minutes of rest. After 45 minutes of rest, dialysate from the probes inserted into the thigh muscle was collected for 20 minutes during rest (baseline) and infusion of adenosine (0.09±0.01 μmol · min−1) through the probes (interstitial adenosine infusion). Leg blood flow (LBF) was measured after 10 minutes of collection of dialysate for each of the above conditions and before infusion of the high dose of intra-arterial adenosine. Blood samples (1 to 5 mL) were drawn simultaneously from the femoral artery and vein of the experimental leg at the same time that LBF was measured.
Experimental Protocol: Cell Cultures
Before experiments were performed, the cells were washed once with PBS containing 5 mmol/L of glucose, and then PBS containing 100 μmol/L of l-arginine and 5 mmol/L of glucose was added. After 30 minutes of incubation, the cells were washed once with PBS containing 5 mmol/L of glucose, and then PBS containing 10 μmol/L of NO-sensitive fluorochrome 4-amino-5-methylamino-2′,7-difluorescein (DAF-FM) and 5 mmol/L of glucose was added.
To investigate the role of adenosine for microvascular endothelial and skeletal muscle cell NO and PGI2 formation, either adenosine (20 μmol/L, 100 μmol/L, or 1 mmol/L) or PBS containing 5 mmol/L of glucose (control) was added to culture medium. The adenosine concentration of 1 mmol/L was chosen because it has been shown previously to elicit the maximal adenosine-evoked release of NO from rat aortic endothelial cells.16 Medium for determination of DAF-FM fluorescence was collected after 5 minutes of incubation and transferred to microplates, and fluorescence was measured immediately with a fluorescence microplate reader (Fluoroskan Ascent, Thermo Labsystems) calibrated for excitation at 485 nm and emission at 520 nm. Medium for determination of PGI2 (6-keto PGF1α) was collected after 10 minutes of incubation and immediately stored in a freezer (−80°C) for later analysis.
A 1-way repeated-measures ANOVA was performed to test significance within trials in the microdialysis study. Effect of interstitial adenosine infusion was determined by a paired t test. Values from cells treated with adenosine were expressed as formation rate per milligram of protein. A 1-way ANOVA on ranks was used to test the effect of adenosine on cells. After a significant F test, pairwise differences were identified using the Tukey honestly significant difference post hoc procedure. The significance level was set at P<0.05, and data are mean±SE unless otherwise indicated.
Leg and Systemic Variables During Arterial and Interstitial Adenosine Infusion
Arterial adenosine infusion increased (P<0.05) LBF in a dose-dependent manner from ≈0.4 L · min−1 to 1.3±0.1 and 2.2±0.4 L · min−1 during the low and high dose of adenosine, respectively (Figure S1, available in the online Data Supplement at http://hyper.ahajournals.org). Mean arterial pressure remained unchanged during both doses of adenosine. Leg vascular conductance increased (P<0.05) from ≈4 mL · min−1 · mm Hg−1 to 15±1 and 24±4 mL · min−1 · mm Hg−1 during the low and high dose of adenosine, respectively. Leg arteriovenous oxygen difference decreased (P<0.05) during both adenosine infusions such that leg oxygen consumption remained unchanged. Interstitial adenosine infusion had no effect on LBF, mean arterial pressure, leg vascular conductance arteriovenous oxygen difference, or leg oxygen consumption.
Muscle Interstitial NO, 6-Keto PGF1α, and Adenosine Levels With Arterial Infusion of Adenosine
Muscle interstitial NO (NOx) was 27±6 μmol/L at baseline, and the level increased (P<0.05) 2- and 3-fold during the low and high dose of adenosine, respectively (Figure 1). Interstitial 6-keto PGF1α levels increased (P<0.05) 1.7- and 1.9-fold from a baseline value of 1570±635 pg · mL−1 during the low and high dose of adenosine, respectively. Interstitial adenosine was not affected by either of the 2 adenosine infusions.
Venous Plasma NOx and 6-Keto PGF1α Levels and Efflux With Arterial Infusion of Adenosine
At baseline, venous plasma NOx and 6-keto PGF1α were 43±3 μmol/L and 1329±571 pg · mL−1, respectively, and did not change during the 2 adenosine infusions (Figure S2). When accounting for plasma flow, venous plasma efflux of NOx and 6-keto PGF1α increased 6.4- and 5.4-fold, respectively, during the high dose of adenosine (P<0.05).
Muscle Interstitial NOx and 6-Keto PGF1α Levels With Interstitial Infusion of Adenosine
Interstitial NOx was 25±6 μmol/L at baseline, and the level increased (P<0.05) 2.6-fold during adenosine infusion (Figure 2). Similarly, interstitial 6-keto PGF1α was 675±133 pg · mL−1 at baseline and increased (P<0.05) 2.3-fold during adenosine infusion.
Standard Curve for DAF-FM
In the DAF-FM, standard curve fluorescence values were found to increase linearly with increasing concentrations of the NO donor s-nitroso-n-acetyl-penicillamine in the range from 5 to 100 nmol/L (r=0.99; P<0.001).
Effect of Adenosine on DAF-FM Fluorescence and 6-Keto PGF1α Levels
The addition of 20 μmol/L, 100 μmol/L, and 1 mmol/L of adenosine to microvascular endothelial cells induced an increase (P<0.05) in DAF-FM fluorescence, indicating NO formation, and the level of 6-keto PGF1α (Figures 3 and 4⇓). The addition of 100 μmol/L and 1 mmol/L of adenosine to skeletal muscle cells increased (P<0.05) DAF-FM fluorescence, whereas 6-keto PGF1α levels remained unchanged.
In the present study, novel information is provided on the mechanisms by which adenosine regulates skeletal muscle blood flow in humans. We demonstrate that both plasma and interstitial adenosine stimulate the formation of NO and PGI2 in muscle interstitium, which we propose to be of importance for vascular function and control of blood flow. Based on cell culture studies, we further show that both skeletal muscle cells and microvascular endothelial cells are potential mediators of adenosine-induced formation of NO in vivo, whereas only endothelial cells appear to play a role in adenosine-induced formation of PGI2.
It has been shown that the adenosine concentration increases in the exercising muscle interstitium at a rate closely associated with the magnitude of muscle blood flow,12 indicating that interstitial adenosine could be important for exercise hyperemia. Interstitial adenosine could potentially induce vasodilation through a direct interaction with vascular smooth muscle14,15 or via formation of NO and PGI2.3 To examine the latter possibility, adenosine was infused directly into the muscle interstitium. We hypothesized that infusion of adenosine would increase the interstitial concentrations of NO and PGI2. Our findings confirm that interstitial adenosine is a stimulator of NO and PGI2 formation in humans, supporting the suggestion that interstitial adenosine contributes to local vasodilation during exercise by stimulating the formation of these vasodilators.3
To determine which cells in the interstitium are responsible for the increase in NO and PGI2, experiments were performed on cell cultures of skeletal muscle myotubes and muscle microvascular endothelial cells. Both endothelial cells and skeletal muscle cells are potential sources of NO and PGI2, because both cell-types express adenosine receptors19 and contain NOS20 and COX.21 In congruence with our hypothesis, the microvascular endothelial cells and the myotubes were found to produce NO in response to adenosine application. The release of NO per milligram of protein from skeletal muscle cells was approximately one eight of that observed from microvascular endothelial cells, suggesting a minor role of skeletal muscle for adenosine-induced formation of NO. However, taking into account that the amount of skeletal muscle cell tissue in vivo is many-fold greater than that of endothelial cells, the interstitial adenosine-evoked release of NO from skeletal muscle may well be of similar importance as that from microvascular endothelial cells. Although it cannot be excluded that cell types other than muscle cells and endothelial cells contribute to muscle interstitial NO levels during contraction, we conclude from our data that these 2 cell types are major sources.
In contrast to our hypothesis, only microvascular endothelial cells were found to produce PGI2 in response to adenosine. The lack of release of PGI2 from myotubes is in agreement with the observation that PGI2 levels remain unaltered when myotubes are electrostimulated (n=9; M Nyberg and Y Hellsten, unpublished results). These observations suggest that the increase in interstitial PGI2 in response to plasma or interstitial adenosine, or in response to exercise,22,23 primarily originates from microvascular endothelial cells in the muscle tissue. By immunohistochemical methods, COX-1 and COX-2 have been found to be present in skeletal muscle cells from humans and rodents, but it has been proposed that the main product of skeletal muscle COX is prostaglandin E2,21 which, similar to PGI2, increases in the human muscle interstitium during exercise.23
Several findings have indicated an essential role for endothelial cells in contraction-induced dilation of skeletal muscle vasculature.24,25 The findings from the current study showing that interstitial adenosine stimulates the formation of endothelial NO and PGI2 suggests that this mechanism could constitute one of the endothelium-dependent regulatory pathways leading to changes in skeletal muscle blood flow. How interstitial adenosine activates endothelial cells at the arteriolar level that, in turn, communicate with adjacent smooth muscle cells to produce the dilatory response still needs to be determined. It is, however, unlikely that adenosine diffuses through all of the structures of the vessel wall, in particular, the tight basal lamina. Interestingly, stimulation of adenosine receptors on endothelial cells produces vasodilation that spreads to remote regions with a concomitant increase in endothelial Ca2+ observed along with this response.26 Because conducted vasodilation has been shown to be associated with increases in Ca2+ and release of NO and prostanoids,27 interstitial adenosine formed during muscle contraction may interact with endothelial cells located at the capillary level, thereby inducing a wave of NO and PGI2 release from endothelial cells located upstream at the arteriolar level (Figure 5).
In contrast to the present findings that indicate that the vasodilator effect of adenosine is mediated through formation of NO, it has been suggested previously that adenosine released during muscle contraction in the rat hindlimb does not depend on NO synthesis to produce vasodilation.28 This suggestion was based on the observation that adenosine made a substantial contribution to muscle vasodilation when NOS was inhibited. Because the vasodilatory effect of adenosine depends not only on NO formation but also on prostanoid formation,3 the vasodilatory effect of adenosine during NOS blockade in the study by Ray and Marshall28 may well reflect PGI2-mediated vasodilation.
The present results show that arterial infusion of adenosine increases the interstitial concentrations of NO and PGI2. Because significant increases in blood flow can only be obtained with substantial dilation of small arteries and arterioles,29 it may be speculated that plasma adenosine acts primarily on endothelial cells lining arterioles, thereby stimulating formation of NO and PGI2 that diffuses to adjacent smooth muscle cells to induce vasodilation. In this setting, the increase in interstitial NO and PGI2 during adenosine infusion suggests that these substances also cross the smooth muscle cell layer. Another, not mutually exclusive, possibility also exists, because plasma adenosine most likely also acts on capillary endothelial cells leading to the formation of NO and PGI2 that diffuses directly into the muscle interstitium.
It is well known that exposure of endothelial cells to laminar shear stress stimulates NO and PGI2 production.30 Consequently, the observed increase in the formation rate of NO and PGI2 during arterial adenosine infusion may have been caused by a direct effect of plasma adenosine on vascular smooth muscle cells, because this would have induced vasodilation and a concurrent increase in blood flow and shear stress. However, the current study does demonstrate that arterial infusion of adenosine, sufficient to elevate LBF ≈5-fold, does not increase the concentration of interstitial adenosine. Therefore, although it cannot be excluded that the release of NO and PGI2 was partly related to an increase in shear stress, a direct effect of plasma adenosine on smooth muscle cells appears improbable. Furthermore, an increase in luminal flow has been shown to induce endothelial NOS translocation from the basolateral membrane and cytoplasm to the apical membrane,31 suggesting that NO is primarily released into the luminal space on exposure to shear stress, which is in contrast to the observed increase in interstitial NO observed in the present study.
The concentration of adenosine used to stimulate NO and PGI2 formation in microvascular endothelial cells was 10- to 20-fold higher than that observed in vivo during contraction as adenosine increases in the human muscle interstitium to ≈1 to 2 μmol/L during light to heavy intensity exercise.12 This dose of adenosine was selected to ensure that not all of the adenosine was metabolized or taken up by cells during the time of incubation, because endothelial cells can take up adenosine via transporters and show a high activity of ectoenzymes that metabolize adenine nucleotides and adenosine.13 Thus, when the transport mechanism for adenosine and adenosine deaminase is blocked, the dose-response curve for adenosine in intact endothelium from aorta is shifted leftward by several orders of magnitude.16 Moreover, the concentrations of adenosine in close proximity to the cell membrane and, hence, adenosine receptors are likely to exceed the concentrations measured in microdialysate, because adenosine originates from ecto-AMP 5′nucleotidase located on the extracellular membrane of endothelial cells.13
Despite the increase in interstitial concentrations, venous plasma concentrations of NO and PGI2 remained unchanged. This discrepancy may be related to a methodological limitation, because the interstitial microdialysis probe allowed sampling in the immediate vicinity of microvascular endothelial cells, whereas the probe for plasma sampling was located within the femoral vein. Accordingly, the mean transit time from the arterial infusion site to the sampling site in the femoral vein has been found to be ≈15 to 10 seconds at similar LBFs, as observed during the adenosine infusions,32 thereby allowing time for substantial intravascular degradation and uptake of NO, PGI2, and their metabolites along the vascular tree by endothelial and red blood cells. Furthermore, because plasma flow was elevated during adenosine infusion, the venous plasma efflux was also determined to account for this diluting effect. These results show an increased luminal efflux of NO and PGI2 during the high dose of adenosine, which cannot solely be explained by the increase in plasma flow, because this increased ≈4.5-fold whereas the venous plasma efflux of NO and PGI2 increased ≈6.5- and 5.5-fold, respectively. Hence, the observed increase in plasma efflux suggests that NO and PGI2 are released into the intravascular space.
To reduce the effect of tissue damage and consequent inflammatory responses associated with insertion of microdialysis probes, subjects performed a 10-minute exercise bout at 10 W followed by a long resting period. This procedure has been shown to stabilize the tissue and minimize the response to damage.18 Moreover, the interventions were performed during resting conditions, and it is likely that the observed alterations in NOx and PGI2 in response to the infused adenosine were independent of damage.
It is well established that NO and PGI2 are involved in a wide variety of regulatory and protective mechanisms within the cardiovascular system, and physiological actions include antiproliferative and antithrombotic effects. The current finding that adenosine in plasma and the interstitium is stimulatory for NO and PGI2 formation suggests a close interdependency between these systems and also that, if the adenosine system is impaired, NO and PGI2 levels and thereby vascular function may be affected. Because changes in vascular function are a key early feature in the development of human vascular disease, the current findings raise the possibility of novel approaches to prevention and treatment of vascular dysfunction, for example, by stimulation of the adenosine system.
Karina Olsen is gratefully acknowledged for her isolation of microvascular endothelial cells and other excellent technical assistance.
Sources of Funding
This study was supported by a grant from the Lundbeck Foundation and the Danish Medical Research Council. S.P.M. was supported by a grant from the Copenhagen Hospital system.
- Received August 19, 2010.
- Revision received September 2, 2010.
- Accepted October 6, 2010.
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