Interstitial Adenosine Concentration Research Articles

Adenosine reuptake inhibition reduces diabetes-induced glomerular hyperfiltration via the adenosine A2a receptor.

Diabetes-induced glomerular hyperfiltration is an early alteration in kidney function in diabetes. Previous studies have shown that reduced adenosine A2a receptor signaling contributes to diabetes-induced glomerular hyperfiltration. The present study investigated the effects of enhanced interstitial adenosine concentration by inhibition of cellular adenosine reuptake, thereby promoting endogenous adenosine signaling. Insulinopenic diabetes was induced by streptozotocin in adult male Sprague-Dawley rats. Two weeks after diabetes induction, kidney function in terms of glomerular filtration rate, and total, cortical, and medullary renal blood flows were evaluated under thiobutabarbital anesthesia during baseline and after renal artery infusion of two doses of the adenosine reuptake inhibitor dilazep. Dilazep did not affect mean arterial pressure indicating that the effects of the interventions were intrarenal. Diabetics had increased glomerular filtration rate compared with controls and dilazep dose-dependently decreased glomerular filtration rate in diabetics, whereas it had no significant effect in controls. Dilazep increased cortical renal blood flows in controls, whereas medullary blood flow was not significantly changed. Dilazep did not affect total renal blood flow in any of the groups but decreased cortical blood flow in diabetics, resulting in decreased filtration fraction by dilazep in diabetics. Pretreatment with the adenosine A2a antagonist ZM241385 prevented intrarenal dilazep-mediated effects on glomerular filtration rate and filtration fraction in diabetics. In conclusion, enhancing intrarenal adenosine signaling by dilazep normalizes diabetes-induced glomerular hyperfiltration at least in part by activation of adenosine A2a receptors.

Inhibition of Nitric Oxide Synthesis Eliminates Interstitial Adenosine Production During Systemic Hypoxia in Rat Skeletal Muscle

The purpose of this study was to examine the relationship between nitric oxide (NO) synthesis and adenosine (Ado) production in the interstitial space of skeletal muscle during systemic hypoxia. In order to accomplish this, microdialysis fibers were inserted into the hindlimb of anesthetized rats and perfused with the following solutions; 1) Ringer's (control), 20 mM SNP (NO donor), 10 mM L‐NAME (NO inhibitor), 500 μM AOPCP (Ado inhibitor) and 10 mM L‐NAME+ 500 μM AOPCP during baseline (100% O2) and hypoxia (10.5% O2). Ringer's perfusion did not result in any changes in interstitial NO concentrations during either baseline or hypoxia. The perfusion of L‐NAME, AOPCP and L‐NAME+AOPCP inhibited (p<0.05) NO production below detection limits, while SNP perfusion increased interstitial NO levels beyond detection limits throughout the experiment. During Ringer's perfusion, interstitial Ado concentrations were increased (p<0.05) during hypoxia (0.07±0.01) and decreased (P<0.05) during recovery (0.05±0.01 μM) as compared to baseline (0.04±0.01 μM). The perfusion of L‐NAME, AOPCP and L‐NAME+AOPCP all inhibited adenosine production to the point where interstitial Ado levels were below detection limits (p<0.05) during baseline, hypoxia and recovery. Surprisingly, the perfusion of SNP also dramatically decreased (p<0.05) the interstitial adenosine concentrations below detectable limits during hypoxia (undetectable) and recovery as compared to baseline. These data demonstrate that interstitial Ado production is very closely linked to NO availability and even under conditions when interstitial Ado production is stimulated (hypoxia), blocking NO availability completely eliminates all interstitial Ado production. These data further indicate that interstitial Ado production is tightly regulated within a narrow range of NO concentrations and a static pool of NO is required for normal interstitial Ado production.Support or Funding InformationSupported by NSERCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FSCPX, a Chemical Widely Used as an Irreversible A₁ Adenosine Receptor Antagonist, Modifies the Effect of NBTI, a Nucleoside Transport Inhibitor, by Reducing the Interstitial Adenosine Level in the Guinea Pig Atrium.

Based on in silico results, recently we have assumed that FSCPX, an irreversible A1 adenosine receptor antagonist, inhibits the action of NBTI that is apparent on E/c curves of adenosine receptor agonists. As a mechanism for this unexpected effect, we hypothesized that FSCPX might modify the equilibrative and NBTI-sensitive nucleoside transporter (ENT1) in a way that allows ENT1 to transport adenosine but impedes NBTI to inhibit this transport. This assumption implies that our method developed to estimate receptor reserve for agonists with short half-life such as adenosine, in its original form, overestimates the receptor reserve. In this study, therefore, our goals were to experimentally test our assumption on this effect of FSCPX, to improve our receptor reserve-estimating method and then to compare the original and improved forms of this method. Thus, we improved our method and assessed the receptor reserve for the direct negative inotropic effect of adenosine with both forms of this method in guinea pig atria. We have found that FSCPX inhibits the effects of NBTI that are mediated by increasing the interstitial concentration of adenosine of endogenous (but not exogenous) origin. As a mechanism for this action of FSCPX, inhibition of enzymes participating in the interstitial adenosine production can be hypothesized, while modification of ENT1 can be excluded. Furthermore, we have shown that, in comparison with the improved form, the original version of our method overestimates receptor reserve but only to a small extent. Nevertheless, use of the improved form is recommended in the future.

The effects of adenine nucleotide perfusion on interstitial adenosine production in rat skeletal muscle.

The purpose of the present study was to utilize the microdialysis technique in rat skeletal muscle to perfuse varying concentrations of AMP, ADP, and ATP into the interstitium to examine the effects that these adenine nucleotides have on the production of adenosine in the interstitial space. Interstitial adenosine production appears to be related to the type (ATP, ADP, or AMP) and concentration (2-60 μmol/L) of the adenine nucleotide perfused. Interstitial adenosine levels increased (P < 0.05) from baseline (0.18 ± 0.02 and 0.22 ± 0.02 μmol/L) to 0.23 ± 0.02 and 0.41 ± 0.05 μmol/L following 5 and 30 μmol/L AMP perfusion, respectively. Similarly, perfusion with 30 μmol/L ADP and 30, 40, and 60 μmol/L ATP resulted in an increase (P < 0.05) in interstitial adenosine concentration from baseline (0.25 ± 0.02, 0.26 ± 0.02, 0.19 ± 0.03, and 0.14 ± 0.02 μmol/L) to 0.30 ± 0.02, 0.32 ± 0.02, 0.36 ± 0.04, and 0.33 ± 0.04 μmol/L, respectively. Interestingly, the most prominent increase in interstitial adenosine production occurred during the perfusion of 60 μmol/L ATP (126% increase from baseline). These data strongly suggest that interstitial ATP may play a more potent role in stimulating interstitial adenosine production as compared with ADP or AMP. In addition, interstitial adenosine production can occur independent of muscle contraction (voluntary or involuntary) or hypoxia when adequate concentrations of adenine nucleotides are available.

Is there such as a thing as non-ischaemic cardiac pain?

Over time, in the minds of clinicians and laypeople alike, cardiogenic pain is intimately associated with angina pectoris, the characteristic syndrome which accompanies myocardial ischaemia. However, in conditions such as...

The effect of adenosine deaminase inhibition on the A1 adenosinergic and M2 muscarinergic control of contractility in eu- and hyperthyroid guinea pig atria.

The A1 adenosine and M2 muscarinic receptors exert protective (including energy consumption limiting) effects in the heart. We investigated the influence of adenosine deaminase (ADA) inhibition on a representative energy consumption limiting function, the direct negative inotropic effect elicited by the A1 adenosinergic and M2 muscarinergic systems, in eu- and hyperthyroid atria. Furthermore, we compared the change in the interstitial adenosine level caused by ADA inhibition and nucleoside transport blockade, two well-established processes to stimulate the cell surface A1 adenosine receptors, in both thyroid states. A classical isolated organ technique was applied supplemented with the receptorial responsiveness method (RRM), a concentration estimating procedure. Via measuring the contractile force, the direct negative inotropic capacity of N6-cyclopentyladenosine, a selective A1 receptor agonist, and methacholine, a muscarinic receptor agonist, was determined on the left atria isolated from 8-day solvent- and thyroxine-treated guinea pigs in the presence and absence of 2′-deoxycoformycin, a selective ADA inhibitor, and NBTI, a selective nucleoside transporter inhibitor. We found that ADA inhibition (but not nucleoside transport blockade) increased the signal amplification of the A1 adenosinergic (but not M2 muscarinergic) system. This action of ADA inhibition developed in both thyroid states, but it was greater in hyperthyroidism. Nevertheless, ADA inhibition produced a smaller rise in the interstitial adenosine concentration than nucleoside transport blockade did in both thyroid states. Our results indicate that ADA inhibition, besides increasing the interstitial adenosine level, intensifies the atrial A1 adenosinergic function in another (thyroid hormone-sensitive) way, suggesting a new mechanism of action of ADA inhibition.

Extracellular NAADP affords cardioprotection against ischemia and reperfusion injury and involves the P2Y11-like receptor

Background and purposeExtracellular nucleotides may play important regulatory roles within the cardiovascular system and notably in cardioprotection. We aimed to look for a possible pharmacological preconditioning effect of extracellular NAADP ([NAADP]e) against ischemia/reperfusion injury. [NAADP]e has been recently reported to be a full agonist of the P2Y11 receptor. Therefore, we characterized the involvement of the P2Y11-like receptor in mediating ischemic/reperfusion tolerance induced by [NAADP]e. Experimental approachThe cardioprotective effects of [NAADP]e were evaluated in a model of ischemia/reperfusion carried out on Langendorff perfused rat hearts. This model was also instrumented with a microdialysis probe. Furthermore, using isolated cardiomyocytes, we assessed cAMP, inositol phosphate accumulation and prosurvival protein kinases activation induced by [NAADP]e pretreatement. ResultsPretreatment with 1μM [NAADP]e induced cardioprotective effects with regards to functional recovery, necrosis and arrhythmogenesis (p<0.05). These effects were completely suppressed with NF157, an antagonist of the P2Y11 receptor. Moreover, global ischemia induced a time-dependent increase in interstitial concentration of adenosine, NAADP and UTP. In cardiomyocyte cultures, NF157 suppressed cAMP and inositol phosphate accumulation induced by [NAADP]e. [NAADP]e induced phosphorylation of ERK 1/2, AKT and its downstream target GSK-3β (p<0.05). These activations were also suppressed by NF157. ConclusionsEvidence suggests that NAADP signalling at the P2Y11-like receptor affords significant cardioprotection against ischemia/reperfusion injury. Besides adenosine and UTP, microdialysis study supports a potential endogenous role of [NAADP]e.

Traditional Acupuncture Triggers a Local Increase in Adenosine in Human Subjects

Acupuncture is a form of Eastern medicine that has been practiced for centuries. Despite its long history and worldwide application, the biological mechanisms of acupuncture in relieving pain have been poorly defined. Recent studies in mice, however, demonstrate that acupuncture triggers increases in interstitial adenosine, which reduces the severity of chronic pain through adenosine A1 receptors, suggesting that adenosine-mediated antinociception contributes to the clinical benefits of acupuncture. We asked here whether acupuncture in human subjects is also linked to a local increase in interstitial adenosine concentration. We collected microdialysis samples of interstitial fluid before, during, and after delivering 30 minutes of conventional acupuncture in the Zusanli point in human subjects. The interstitial adenosine concentration increased significantly during acupuncture and remained elevated for 30 minutes after the acupuncture. Acupuncture-mediated adenosine release was not observed if acupuncture was not delivered in the Zusanli point or if the acupuncture needle was inserted, but not rotated. This study strengthens the role of adenosine in acupuncture-mediated antinociception by directly providing such evidence in humans. PerspectiveThis article presents further evidence of the role of adenosine in acupuncture-mediated antinociception by demonstrating that local adenosine concentrations increase in the acupoint in human subjects receiving traditional acupuncture.

Impaired formation of vasodilators in peripheral tissue in essential hypertension is normalized by exercise training

This study examined vascular function and the adenosine system in skeletal muscle of patients diagnosed with essential hypertension (n = 10) and of normotensive (n = 11) patients, before and after aerobic training. : Before and after 8 weeks of aerobic training, the patients completed experiments in which leg blood flow was determined during infusion of adenosine, acetylcholine and during exercise (20 W); muscle interstitial fluid and femoral venous plasma were sampled via microdialysis probes during baseline conditions, exercise and adenosine infusion and resting muscle biopsies were obtained from muscle vastus lateralis. Before training, leg vascular conductance in response to arterial adenosine infusion was similar in the hypertensive and normotensive groups and the individual vascular response was positively correlated to that of both acetylcholine infusion (r = 0.66, P < 0.001) and exercise (r = 0.72, P < 0.001). Before training, interstitial adenosine concentrations during exercise and prostacyclin (PGI2) concentrations after adenosine infusion were lower in the hypertensive than the normotensive group (P < 0.05). In the hypertensive group, training did not affect the vasodilatory response to arterially infused adenosine but increased the formation of interstitial adenosine and PGI2 and lowered blood pressure. In the normotensive group, training resulted in lower (P < 0.05) leg vascular conductance in response to arterial adenosine infusion. The present data suggest that essential hypertension is associated with a reduced capacity to form adenosine and PGI2 at the skeletal muscle microcirculatory level, which is likely to contribute to the increased peripheral vascular resistance related to the disease. This impairment in vasodilator formation can be normalized by aerobic training.

Cilostazol Increases Tissue Blood Flow in Contracting Rabbit Gastrocnemius Muscle

The mechanisms underlying the ability of cilostazol to improve walking distance in patients with intermittent claudication (IC) are not fully understood, but may be related to its phosphodiesterase type 3 (PDE3) and adenosine uptake inhibition. In the present study the effect of cilostazol on blood flow and interstitial adenosine concentration was compared with that of the PDE3 inhibitor, milrinone, and the adenosine uptake inhibitor, draflazine. Rabbit gastrocnemius muscle blood flow was measured under resting, contracting and ischemic conditions. Interstitial adenosine was sampled by microdialysis. None of the drugs affected tissue blood flow at rest. Blood flow in electrically stimulated muscle was 2- to 3-fold higher in vehicle-, milrinone- and draflazine-treated animals. However, cilostazol caused an 8-fold increase. Ligation of the femoral artery decreased blood flow in the stimulated muscle in all groups to a similar degree. Cilostazol and draflazine increased the dialysate adenosine concentration during the first 10 min of muscle contraction, but had no effect during ischemia, most likely because of the high AMP deaminase activity in skeletal muscle. Cilostazol increases blood flow in the gastrocnemius muscle during contraction and it is this effect that may be partially responsible for the improved walking distance in IC patients. (Circ J 2010; 74: 181 - 187).

Ischaemic Preconditioning and Postconditioning do not Affect Adenosine A1 and A2A Receptor Sensitivity

Ischaemic Preconditioning and Postconditioning do not Affect Adenosine A1 and A2A Receptor Sensitivity

Attenuation of Chronic Pulmonary Inflammation in A2B Adenosine Receptor Knockout Mice

Pharmacologic evidence suggests that activation of A(2B) adenosine receptors results in proinflammatory effects relevant to the progression of asthma, a chronic lung disease associated with elevated interstitial adenosine concentrations in the lung. This concept has been challenged by the finding that genetic removal of A(2B) receptors leads to exaggerated responses in models of acute inflammation. Therefore, the goal of our study was to determine the effects of A(2B) receptor gene ablation in the context of ovalbumin-induced chronic pulmonary inflammation. We found that repetitive airway allergen challenge induced a significant increase in adenosine levels in fluid recovered by bronchoalveolar lavage. Genetic ablation of A(2B) receptors significantly attenuated allergen-induced chronic pulmonary inflammation, as evidenced by a reduction in the number of bronchoalveolar lavage eosinophils and in peribronchial eosinophilic infiltration. The most striking difference in the pulmonary inflammation induced in A(2B) receptor knockout (A(2B)KO) and wild-type mice was the lack of allergen-induced IL-4 release in the airways of A(2B)KO animals, in line with a significant reduction in IL-4 protein and mRNA levels in lung tissue. In addition, attenuation of allergen-induced transforming growth factor-beta release in airways of A(2B)KO mice correlated with reduced airway smooth muscle and goblet cell hyperplasia/hypertrophy. In conclusion, genetic removal of A(2B) adenosine receptors in mice leads to inhibition of allergen-induced chronic pulmonary inflammation and airway remodeling. These findings are in agreement with previous pharmacologic studies suggesting a deleterious role for A(2B) receptor signaling in chronic lung inflammation.

Mysteries of Renal Autoregulation

Autoregulation is an important renal regulatory mechanism that provides an important protective role in glomerular hemodynamics.1 The phenomenon of autoregulatory behavior has been recognized for many decades, but a clear understanding of the underlying mechanisms involved in mediating resistance adjustments remains to be established. As illustrated in Figure 1, pressure-mediated autoregulatory behavior operates by recognizing a change in renal perfusion pressure, or renal vascular transmural pressure, and initiating induction of autoregulatory resistance changes intended to stabilize renal blood flow and/or glomerular filtration rate. The net result is that renal blood flow and glomerular filtration rate remain relatively stable over a wide range of renal perfusion pressures. Autoregulatory behavior is exquisitely sensitive in healthy normal kidneys but significantly less efficient in pathological settings, such as diabetes mellitus and some forms of hypertension. Complete loss of autoregulatory control results in renal blood flow that behaves more passively, thus increasing variability and rendering flow more directly determined by renal perfusion pressure.1,2 Figure 1. Outline of the pressure-induced signaling scheme postulated for autoregulatory adjustments in the afferent arteriolar diameter. Whole kidney autoregulation reflects regulatory input from the myogenic and tubuloglomerular feedback mechanisms at the very least.3–7 Recently, contributions of a third and possibly a fourth mechanism have been suggested.6,7 Consequently, with continued study, the mechanisms underlying renal autoregulatory resistance adjustments are slowly becoming clearer. In addition to the P1 receptor hypothesis put forth to explain signal transduction for tubuloglomerular feedback-dependent resistance changes, we have explored the hypothesis that pressure-mediated autoregulatory responses are mediated by extracellular ATP and activation of P2X1 receptors.3,8,9 Conclusive identification of the signaling molecules that mediate autoregulation remains a point of debate, but it appears clear that the release of a purine-based moiety is required for autoregulatory adjustments in afferent arteriolar resistance.3–5,8–10 This …

Adenosine A1 receptor expression during the transition from compensated pressure overload hypertrophy to heart failure

Myocardial adenosine is increased in pressure-overload hypertrophy (POH) and exerts important cardioprotective effects that delay transition to left ventricular failure. Adenosine-mediated signaling is attenuated in POH, but whether this depends on receptor or postreceptor defects is unknown. We therefore examined left ventricular adenosine A1-receptor gene and protein expression in experimental POH. Six week-old Sprague-Dawley rats were subjected to abdominal aortic banding (group B) or sham operation (group S). Echocardiography and left ventricular catheterization were performed 10 weeks later under ketamine anesthesia. Left ventricular and lung weight indices were obtained postmortem. A1-Receptor mRNA and protein expression were measured in samples from left ventricular, right ventricular and aortic arch tissue. Group B rats were subgrouped as having compensated or decompensated hypertrophy according to the absence or presence of lung congestion (lung weight index below or above mean +/- 2SD compared with group S rats). Both mRNA and protein A1-receptor expression were significantly increased in compensated group B versus group S rats (by, respectively, 37 and 77%; both P < 0.01). This was not observed in decompensated group B rats. No consistent gene or receptor expression changes were observed in right ventricular or aortic tissues. In compensated POH, increased interstitial adenosine concentrations are accompanied by increased expression of the specific receptor mediating the major cardioprotective effects of this autacoid. Such overexpression is no longer detectable once the transition from POH to left ventricular failure has occurred. These observations may have pathophysiological and, in perspective, therapeutic relevance to the course of hypertensive heart disease.

Post-Reperfusion Myocardial Infarction: Long-Term Survival Improvement Using Adenosine Regulation With Acadesine

The purpose of this study was to assess the safety and efficacy of the adenosine regulating agent (ARA) acadesine for reducing long-term mortality among patients with post-reperfusion myocardial infarction (MI). No prospectively applied therapy exists that improves long-term survival after MI associated with coronary artery bypass graft (CABG) surgery-a robust model of ischemia/reperfusion injury. Pretreatment with the purine nucleoside autocoid adenosine mitigates the extent of post-ischemic reperfusion injury in animal models. Therefore, we questioned whether use of the ARA acadesine-by increasing interstitial adenosine concentrations in ischemic tissue-would improve long-term survival after post-reperfusion MI. At 54 institutions, 2,698 patients undergoing CABG surgery were randomized to receive placebo (n = 1,346) or acadesine (n = 1,352) by intravenous infusion (0.1 mg/kg/min; 7 h) and in cardioplegia solution (placebo or acadesine; 5 microg/ml). Myocardial infarction was prospectively defined as: 1) new Q-wave and MB isoform of creatine kinase (CK-MB) elevation (daily electrocardiography; 16 serial CK-MB measurements); or 2) autopsy evidence. Vital status was assessed over 2 years, and outcomes were adjudicated centrally. Perioperative MI occurred in 100 patients (3.7%), conferring a 4.2-fold increase in 2-year mortality (p < 0.001) compared with those not suffering MI. Acadesine treatment, however, reduced that mortality by 4.3-fold, from 27.8% (15 of 54; placebo) to 6.5% (3 of 46; acadesine) (p = 0.006), with the principal benefit occurring over the first 30 days after MI. The acadesine benefit was similar among diverse subsets, and multivariable analysis confirmed these findings. Acadesine is the first therapy proven to be effective for reducing the severity of acute post-reperfusion MI, substantially reducing the risk of dying over the 2 years after infarction.

Inhibition of adenosine kinase by phosphonate and bisphosphonate derivatives

The enzyme adenosine kinase (AK) plays a central role in regulating the intracellular and interstitial concentration of the purine nucleoside adenosine (Ado). In view of the beneficial effects of Ado in protecting tissues from ischemia and other stresses, there is much interest in developing AK inhibitors, which can regulate Ado concentration in a site- and event-specific manner. The catalytic activity of AK from different sources is dependent upon the presence of activators such as phosphate (Pi). In this work we describe several new phosphorylated compounds which either activate or inhibit AK. The compounds acetyl phosphate, carbamoyl phosphate, dihydroxyacetone phosphate and imidodiphosphate were found to stimulate AK activity in a dose-dependent manner comparable to that seen with Pi. In contrast, a number of phosphonate and bisphosphonate derivatives, which included clodronate and etidronate, were found to inhibit the activity of purified AK in the presence of Pi. These AK inhibitors (viz. clodronate, etidronate, phosphonoacetic acid, 2-carboxyethylphosphonic acid, N-(phosphonomethyl)-glycine and N-(phosphonomethyl)iminodiacetic acid), at concentrations at which they inhibited AK, were also shown to inhibit the uptake of (3)H-adenosine and its incorporation into macromolecules in cultured mammalian cells, indicating that they were also inhibiting AK in intact cells. The drug concentrations at which these effects were observed showed limited toxicity to the cultured cells, indicating that these effects are not caused by cellular toxicity. These results indicate that the enzyme AK provides an additional cellular target for the clinically widely used bisphosphonates and related compounds, which could possibly be exploited for a new therapeutic application. Our structure-activity studies on different AK activators and inhibitors also indicate that all of the AK activating compounds have a higher partial positive charge (delta(+)) on the central phosphorous atom in comparison to the inhibitors. This information should prove helpful in the design and synthesis of more potent inhibitors of AK.

Adenosine concentration in the porcine coronary artery wall and A2A receptor involvement in hypoxia‐induced vasodilatation

We tested whether hypoxia-induced coronary artery dilatation could be mediated by an increase in adenosine concentration within the coronary artery wall or by an increase in adenosine sensitivity. Porcine left anterior descendent coronary arteries, precontracted with prostaglandin F(2alpha) (10(-5) M), were mounted in a pressure myograph and microdialysis catheters were inserted into the tunica media. Dialysate adenosine concentrations were analysed by HPLC. Glucose, lactate and pyruvate were measured by an automated spectrophotometric kinetic enzymatic analyser. The exchange fraction of [(14)C]adenosine over the microdialysis membrane increased from 0.32 +/- 0.02 to 0.46 +/- 0.02 (n = 4, P < 0.01) during the study period. At baseline, interstitial adenosine was in the region of 10 nM which is significantly less than previously found myocardial concentrations. Hypoxia (P(O(2)) 30 mmHg for 60 min, n = 5) increased coronary diameters by 20.0 +/- 2.6% (versus continuous oxygenation -3.1 +/- 2.4%, n = 6, P < 0.001) but interstitial adenosine concentration fell. Blockade of adenosine deaminase (with erythro-9-(2-hydroxy-3-nonyl-)-adenine, 5 microM), adenosine kinase (with iodotubericidine, 10 microM) and adenosine transport (with n-nitrobenzylthioinosine, 1 microM) increased interstitial adenosine but the increase was unrelated to hypoxia or diameter. A coronary dilatation similar to that during hypoxia could be obtained with 30 microM of adenosine in the organ bath and the resulting interstitial adenosine concentrations (n = 5) were 20 times higher than the adenosine concentration measured during hypoxia. Adenosine concentration-response experiments showed vasodilatation to be more pronounced during hypoxia (n = 9) than during normoxia (n = 9, P < 0.001) and the A(2A) receptor antagonist ZM241385 (20 nM, n = 5), attenuated hypoxia-induced vasodilatation while the selective A(2B) receptor antagonist MRS1754 (20 nM, n = 4), had no effect. The lactate/pyruvate ratio was significantly increased in hypoxic arteries but did not correlate with adenosine concentration. We conclude that hypoxia-induced coronary artery dilatation is not mediated by increased adenosine produced within the artery wall but might be facilitated by increased adenosine sensitivity at the A(2A) receptor level.

Intravenous adenosine protects the myocardium primarily by activation of a neurogenic pathway

Endogenous adenosine is a trigger for ischemic myocardial preconditioning (IPC). Although intravascular administration of adenosine has been used to further unravel the mechanism of protection by IPC, it is questionable whether adenosine and IPC employ the same signaling pathways to exert cardioprotection. We therefore investigated whether the active metabolic barrier of the endothelium prevents an increase in myocardial interstitial adenosine concentrations by intravenous adenosine, using microdialysis, and also the role of NO and activation of a neurogenic pathway in the cardioprotection by adenosine. In pentobarbital-anesthetized rats, area at risk and infarct size (IS) were determined 120 min after a 60-min coronary artery occlusion (CAO), using trypan blue and nitro-blue-tetrazolium staining, respectively. IPC with a single 15-min CAO and a 15-min adenosine infusion (ADO, 200 microg min(-1) i.v.) limited IS to the same extent (IS = 41 +/- 6% and IS = 40 +/- 4%, respectively) compared to control rats (IS = 63 +/- 3%, both P < 0.05). However, IPC increased myocardial interstitial adenosine levels seven-fold from 4.3 +/- 0.7 to 27.1 +/- 10.0 microM (P < 0.05), while ADO had no effect on interstitial adenosine (4.1 +/- 1.2 microM), or any of the other purines. The NO synthase inhibitor N(omega)-nitro-L-arginine (LNNA), which did not affect IS (IS = 62 +/- 3%), attenuated the protection by ADO (IS = 56 +/- 3%; P < 0.05 vs ADO, P = NS vs LNNA). The ganglion blocker hexamethonium, which had also no effect on IS (IS = 66 +/- 3%), blunted the protection by ADO (IS = 55 +/- 4%; P < 0.05 vs ADO and vs hexamethonium). These observations demonstrate that cardioprotection by ADO is dependent on NO, and is primarily mediated by activation of a neurogenic pathway.

In vivo evidence against a role for adenosine in the exercise pressor reflex in humans

The pressor response to exercise is of great importance in both physiology and pathophysiology. Whether endogenous adenosine is a trigger for this reflex remains controversial. Muscle interstitial adenosine concentration can be determined by microdialysis. However, there are indications that local muscle cell damage by the microdialysis probe confounds these measurements in exercising muscle. Therefore, we used the nucleoside uptake inhibitor dipyridamole as pharmacological tool to bypass this confounding. We used microdialysis probes to measure endogenous adenosine in forearm skeletal muscle of healthy volunteers during two cycles of 15 min of intermittent isometric handgripping. During the second contraction, dipyridamole (12 microg.min(-1).dl forearm(-1)) was administered into the brachial artery. Dipyridamole potentiated the exercise-induced increase in dialysate adenosine from 0.30 +/- 0.08 to 0.48 +/- 0.10 micromol/l (n = 9, P < 0.05), but it did not potentiate the exercise-induced increase in blood pressure. A time-control study without dipyridamole revealed no difference in exercise-induced increase in adenosine between both contractions (n = 8). To exclude the possibility that the dipyridamole-induced increase in dialysate adenosine originates from extravasation of increased circulating adenosine, we simultaneously measured adenosine with microdialysis probes in forearm muscle and antecubital vein. In a separate group of nine volunteers, simultaneous intrabrachial infusion of 100 microg.min(-1).dl(-1) dipyridamole and 5 microg.min(-1).dl(-1) adenosine increased dialysate adenosine from the intravenous but not the interstitial probe, indicating preserved endothelial barrier function for adenosine. We conclude that dipyridamole significantly inhibits uptake of interstitial adenosine without affecting the pressor response to exercise, suggesting that interstitial adenosine is not involved in the pressor response to rhythmic isometric exercise.

Synergy between A2B adenosine receptors and hypoxia in activating human lung fibroblasts.

Chronic inflammatory airway diseases, such as asthma, chronic obstructive pulmonary disease and pulmonary fibrosis, are associated with subepithelial fibroblast activation, myofibroblast hyperplasia, hypoxia, and increase in interstitial adenosine concentrations. The goal of this study was to determine the effect of adenosine and its receptors on activation of human lung fibroblasts under normoxia (21% O2) and hypoxia (5% O2). Under the normoxic condition, adenosine and its stable analog, 5'-(N-ethylcarboxamido)-adenosine, via activation of A2B adenosine receptors, increased the release of interleukin (IL)-6 by 14-fold and induced the differentiation of human lung fibroblasts to myofibroblasts. This latter effect of 5'-(N-ethylcarboxamido)-adenosine was abolished by an IL-6-neutralizing antibody. Hypoxia increased the release of IL-6 by 2.8-fold, and there was a synergy between hypoxia and activation of A2B adenosine receptors to increase the release of IL-6 and to induce differentiation of fibroblasts into myofibroblasts. Hypoxia increased the expression of A2B adenosine receptors by 3.4-fold. Altogether, these data suggest that hypoxia amplifies the effect of adenosine on the release of IL-6 and cell differentiation by upregulating the expression of A2B adenosine receptors. Our findings provide a novel mechanism whereby adenosine participates in the remodeling process of inflammatory lung diseases.