Interstitial Adenosine Levels Research Articles

High Grade AV Block Associated with Dipyridamole Infusion

The use of dipyridamole for thallium imaging has proved very successful in demonstrating coronary arterial disease [1]. Dipyridamole causes coronary arteriolar vasodilation by increasing interstitial adenosine levels by inhibition of both adenosine deaminase and facilitated cellular adenosine uptake. Differential flow changes occur in coronary arteries if a significant luminal stenosis exists [2]. Intravenous infusion of dipyridamole is safe although transient noncardiac side effects are common. These side effects are mostly mild and not need to treat although can be treated with aminophylline [5]. High-grade AV block after dipyridamole has been described in few case reports and mostly was associated with transient myocardial ischemia [2-4]. We describe a case of long-lasting high grade AV block following dipyridamole infusion. In context of near normal baseline ECG. In our case the AV block occurred without evidence of ischemia in myocardial perfusion imaging and we think it can be an unexpected adverse effect of dipyridamole and clinicians should be aware of AV block as a possible adverse effect of dipyridamole.

A Comprehensive Review of the Pleiotropic Effects of Ticagrelor.

This review summarizes the findings of preclinical studies evaluating the pleiotropic effects of ticagrelor. These include attenuation of ischemia-reperfusion injury (IRI), inflammation, adverse cardiac remodeling, and atherosclerosis. In doing so, it aims to provide novel insights into ticagrelor's mechanisms and benefits over other P2Y12 inhibitors. It also generates viable hypotheses for the results of seminal clinical trials assessing ticagrelor use in acute and chronic coronary syndromes. A comprehensive review of the preclinical literature demonstrates that ticagrelor protects against IRI in the setting of both an acute myocardial infarction (MI), and when MI occurs while on chronic treatment. Maintenance therapy with ticagrelor also likely mitigates adverse inflammation, cardiac remodeling, and atherosclerosis, while improving stem cell recruitment. These effects are probably mediated by ticagrelor's ability to increase local interstitial adenosine levels which activate downstream cardio-protective molecules. Attenuation and augmentation of these pleiotropic effects by high-dose aspirin and caffeine, and statins respectively may help explain variable outcomes in PLATO and subsequent randomized controlled trials (RCTs). Most RCTs and meta-analyses have not evaluated the pleiotropic effects of ticagrelor. We need further studies comparing cardiovascular outcomes in patients treated with ticagrelor versus other P2Y12 inhibitors that are mindful of the unique pleiotropic advantages afforded by ticagrelor, as well as possible interactions with other therapies (e.g., aspirin, statins, caffeine).

CD73 induces GM-CSF/MDSC-mediated suppression of T cells to accelerate pancreatic cancer pathogenesis.

Metabolic alterations regulate cancer aggressiveness and immune responses. Given the poor response of pancreatic ductal adenocarcinoma (PDAC) to conventional immunotherapies, we investigated the link between metabolic alterations and immunosuppression. Our metabolic enzyme screen indicated that elevated expression of CD73, an ecto-5’–nucleotidase that generates adenosine, correlates with increased aggressiveness. Correspondingly, we observed increased interstitial adenosine levels in tumors from spontaneous PDAC mouse models. Diminishing CD73 by genetic manipulations ablated in vivo tumor growth, and decreased myeloid-derived suppressor cells (MDSC) in orthotopic mouse models of PDAC. A high-throughput cytokine profiling demonstrated decreased GM-CSF in mice implanted with CD73 knockdowns. Furthermore, we noted increased IFN-γ expression by intratumoral CD4+ and CD8+ T cells in pancreatic tumors with CD73 knockdowns. Depletion of CD4+ T cells, but not CD8+ T cells abrogated the beneficial effects of decreased CD73. We also observed that splenic MDSCs from Nt5e knockdown tumor-bearing mice were incompetent in suppressing T cell activation in the ex vivo assays. Replenishing GM-CSF restored tumor growth in Nt5e knockout tumors, which was reverted by MDSC depletion. Finally, anti-CD73 antibody treatment significantly improved gemcitabine efficacy in orthotopic models. Thus, targeting the adenosine axis presents a novel therapeutic opportunity for improving the anti-tumoral immune response against PDAC.

Why Not Dipyridamole: a Review of Current Guidelines and Re-evaluation of Utility in the Modern Era.

Dipyridamole is an old anti-platelet and coronary vasodilator agent that inhibits platelet phosphodiesterase and increases interstitial adenosine levels. Its use in coronary artery disease (CAD) has fallen out of practice in the modern era with the advent of new anti-platelet agents, and most modern guidelines on the management of CAD either neglect to comment on its utility or outright recommend against it. The majority of the studies used in these guidelines are outdated and took place in an era when high doses of aspirin were used and statins were not widely utilized. There is growing evidence in rat models of dipyridamole’s synergy with statins through adenosine modulation resulting in significant myocardial protection against ischemia–reperfusion injury and limitation of infract size. The data in human studies are limited but show a similar potential synergy between dipyridamole and statins. It would thus be prudent to reconsider the recommendations against the use of dipyridamole in CAD and to re-evaluate its possible role and potential benefits through well-designed randomized trials combining it with statins, low-dose aspirin, and/or other anti-platelet agents.

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.

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.

Sitagliptin attenuates sympathetic innervation via modulating reactive oxygen species and interstitial adenosine in infarcted rat hearts

We investigated whether sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, attenuates arrhythmias through inhibiting nerve growth factor (NGF) expression in post-infarcted normoglycemic rats, focusing on adenosine and reactive oxygen species production. DPP-4 bound adenosine deaminase has been shown to catalyse extracellular adenosine to inosine. DPP-4 inhibitors increased adenosine levels by inhibiting the complex formation. Normoglycemic male Wistar rats were subjected to coronary ligation and then randomized to either saline or sitagliptin in in vivo and ex vivo studies. Post-infarction was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Compared with vehicle, infarcted rats treated with sitagliptin significantly increased interstitial adenosine levels and attenuated oxidative stress. Sympathetic hyperinnervation was blunted after administering sitagliptin, as assessed by immunofluorescent analysis and western blotting and real-time quantitative RT-PCR of NGF. Arrhythmic scores in the sitagliptin-treated infarcted rats were significantly lower than those in vehicle. Ex vivo studies showed a similar effect of erythro-9-(2-hydroxy-3-nonyl) adenine (an adenosine deaminase inhibitor) to sitagliptin on attenuated levels of superoxide and NGF. Furthermore, the beneficial effects of sitagliptin on superoxide anion production and NGF levels can be reversed by 8-cyclopentyl-1,3-dipropulxanthine (adenosine A1 receptor antagonist) and exogenous hypoxanthine. Sitagliptin protects ventricular arrhythmias by attenuating sympathetic innervation via adenosine A1 receptor and xanthine oxidase-dependent pathways, which converge through the attenuated formation of superoxide in the non-diabetic infarcted rats.

Differential Effect of Phosphodiesterase-3 Inhibitors on Sympathetic Hyperinnervation in Healed Rat Infarcts

The effect of phosphodiesterase-3 (PDE-3) inhibitors on arrhythmia remains controversial, so the purpose of this study was to determine their differential effects on sympathetic hyperinnervation and the involved mechanisms in a rat model of myocardial infarction. After ligating the coronary artery, male Wistar rats were randomized to cilostazol or milrinone, chemically unrelated inhibitors of PDE-3, or vehicle for 4 weeks. The postinfarction period was associated with increased myocardial norepinephrine levels and oxidant release, as measured by myocardial superoxide level and dihydroethidine fluorescence staining. Infarcted rats in the milrinone- and cilostazol-treated groups had favorable ventricular remodeling with similar potency. Compared with milrinone, cilostazol significantly increased interstitial adenosine levels and reduced the production of myocardial cAMP and superoxide. Cilostazol significantly blunted sympathetic hyperinnervation, as assessed by immunofluorescent analysis of sympathetic innervation, and western blotting and real-time quantitative RT-PCR of nerve growth factor. Furthermore, the inhibitory effect of cilostazol on nerve growth factor was reversed by 8-cyclopentyl-1,3-dipropylxanthine, a selective A1 receptor antagonist, and enhanced by tempol administration. In spite of similar arrhythmic vulnerability during programmed stimulation in both the vehicle-and cilostazol-treated groups, cilostazol did not have proarrhythmic effects compared with milrinone. Unlike milrinone, cilostazol has therapeutic neutrality in arrhythmias because of adenosine uptake inhibition, which antagonizes the PDE-3-induced increase of sympathetic reinnervation via mediation of an adenosine A1 receptor-mediated antioxidation.

Abstract 144: Interstitial Formation of Adenosine and Inosine Is Proarrhythmic Through Adenosine A1 Receptor in the Developing Heart

We previously established that exogenous adenosine (ADO) induces transient arrhythmias in the developing heart via the ADO A1 receptor (A1AR) and downstream activation of NADPH oxidase/ERK and PLC/PKC pathways. We assessed the hypothesis that both endogenous ADO and its derived compound inosine (INO) can simultaneously act in an autocrine/paracrine manner and induce rhythm and conduction disturbances. The validated model of the spontaneously beating heart obtained from 4-day-old chick embryos was used. The basal ADO/INO ratio determined by HPLC was approximately 10. Blockade of Equilibrative Nucleoside Transporters (ENTs) by dipyridamole, which is known to increase interstitial ADO level, induced transient atrial dysrhythmias and atrio-ventricular blocks in 70% of the hearts (n=30) and, in parallel, increased ERK2 phosphorylation. When extracellular conversion of AMP to ADO by ecto-5’-nucleotidase/CD73 was inhibited by AOPCP, the dipyridamole-induced arrhythmias were prevented. The dipyridamole-induced arrhythmias and ERK2 phosphorylation were prevented by the specific antagonist of the A1AR (DPCPX) but not by antagonists of A2AAR ( SCH58261 ), A2BAR (MRS1754) or A3AR (MRS1523). Interestingly, inhibition by EHNA of the conversion of ADO to INO by adenosine deaminase was not proarrhythmic whereas exogenous INO (500 µM) induced arrhythmias which were prevented by DPCPX. In conclusion, these findings suggest that endogenous ADO and INO in the interstitial compartment of the developing heart can provoke transient pacemaking and conduction disturbances via A1AR and downstream ERK activation in an autocrine/paracrine manner. Overall, our results provide new insights into the mechanisms underlying cardiac dysfunction when a hypoxic-ischemic episode induces local overproduction of ADO and INO

Cilostazol protects the heart against ischaemia reperfusion injury in a rabbit model of myocardial infarction: Focus on adenosine, nitric oxide and mitochondrial ATP-sensitive potassium channels

1. The present study examined whether or not cilostazol reduces the myocardial infarct size, and investigated its mechanism in a rabbit model of myocardial infarction. 2. Japanese white rabbits underwent 30 min of coronary occlusion, followed by 48 h of reperfusion. Cilostazol (1 and 5 mg/kg) or vehicle was given intravenously 5 min before ischaemia. 8-p-sulfophenyl theophylline (8SPT; an adenosine receptor blocker, 7.5 mg/kg), Nω-nitro-L-arginine methylester (l-NAME; an NOS inhibitor, 10 mg/kg) or 5-hydroxydecanoic acid sodium salt (5-HD; a mitochondrial ATP-sensitive potassium (KATP) channel blocker, 5 mg/kg) was given intravenously 5 min before cilostazol injection. Infarct size was determined as a percentage of the risk area. 3. The myocardial interstitial levels of adenosine and nitrogen oxide (NOx) during ischaemia and reperfusion, and the intensity of myocardial dihydroethidium staining were determined. 4. Infarct size was significantly reduced in the cilostazol 1 mg/kg (38.4% (2.9%)) and cilostazol 5 mg/kg (30.7% (4.7%)) groups compared with that in the control group (46.5% (4.2%)). The infarct size-reducing effect of cilostazol was completely abolished by 8SPT (46.6% (3.5%)), L-NAME (49.0% (5.5%)), or 5HD (48.5% (5.1%)). 8SPT, L-NAME or 5HD alone did not affect the infarct size. Cilostazol treatment significantly increased myocardial levels of adenosine and NOx during ischaemia, and attenuated the intensity of dihydroethidium staining during reperfusion. 5. These findings show that cilostazol reduces the myocardial infarct size by increasing adenosine and NOx levels, attenuating superoxide production and opening the mitochondrial KATP channels. Cilostazol might provide a new strategy for the treatment of coronary heart disease.

Involvement of reperfusion injury salvage kinases in preconditioning depends critically on the preconditioning stimulus

Different preconditioning stimuli can activate divergent signaling pathways. In rats, adenosine-independent pathways (triple 3-min coronary artery occlusion [3CAO3]) and adenosine-dependent pathways (one 15-min coronary artery occlusion [ICAO15]) exist, both ultimately converging at the level of the mitochondrial respiratory chain. Furthermore, while 3CAO3, 1CAO15 and exogenous adenosine (ADO) are equally cardioprotective, only 1CAO15 increases interstitial myocardial adenosine levels. Reperfusion Injury Salvage Kinase (RISK) pathway kinases have been implicated in ischemic preconditioning, but not all preconditioning stimuli activate this pathway. Consequently, we evaluated in anesthetized rats the effects of three distinctly different preconditioning stimuli (3CAO3, 1CAO15 or ADO) on infarct size (IS), signaling pathways with a special emphasis on kinases belonging to the RISK pathway (phosphatidylinositol 3-kinase-Akt-nitric oxide synthase and extracellular signal-related kinase [ERK]) and mitochondrial respiration. All three stimuli increased state-2 respiration (using succinate as complex-II substrate), thereby decreasing the respiratory control index, which was accompanied by a limitation of IS produced by a 60-min coronary artery occlusion (CAO). Nitric oxide synthase inhibition abolished the mitochondrial effects and the cardioprotection by 3CAO3, 1CAO15 or ADO. In contrast, the PI3 kinase inhibitor, wortmannin, blocked protection by 1CAO15, but did not affect protection by 3CAO3 or ADO. Western blotting confirmed that phosphorylation of Akt and ERK were increased by 1CAO15 (which was inhibited by wortmannin), but not by 3CAO3 or ADO. In conclusion, while the three cardioprotective stimuli 3CAO3, 1CAO15 and ADO afford cardioprotection via nitric oxide-mediated modulation of mitochondrial respiration, only the 1CAO15 exerts its protection via activation of kinases belonging to the RISK pathway.

Interstitial nitric oxide concentrations are elevated in rat gastrocnemius muscle following recovery from femoral occlusion

The present study examined the effects of ischemia‐reperfusion on the interstitial levels of nitric oxide (NO) and adenosine (Ado). Eight anesthetized rats had two microdialysis fibers inserted into the gastrocnemius muscle of each leg and perfused with Ringers (control) or 500 μM AOPCP (5'ectonucleotidase inhibitor). Following baseline collections both femoral arteries were clamped for 60 min (samples at 20, 40 and 60 min) and then released (samples at 20 min). With Ringers perfusion there were no increases in interstitial NO (baseline = 4.2±0.4 μM) during ischemia but NO was elevated (P&lt;0.05) following reperfusion (5.4±0.5 μM). Prior to occlusion AOPCP treatment resulted in an increase (P&lt;0.05) in NO from 4.3±0.6 to 5.5±0.5 μM as compared to Ringers. No further increases in NO were observed with AOPCP during ischemia, but NO was further elevated (P&lt;0.05) following reperfusion (6.5±0.6 μM). Interstitial NO concentrations were higher (P&lt;0.05) during AOPCP treatment compared to Ringers at all time points. There were no changes in interstitial Ado during the study. These data show that NO and Ado are not altered during femoral occlusion, but NO is elevated during reperfusion and that NO is even higher during ischemia‐reperfusion when Ado production is inhibited. These findings suggest that NO plays an important role in interstitially mediating vasodialtion during skeletal muscle reperfusion.Supported by NSERC

Effect of Nucleoside Transport Blockade on the Interstitial Adenosine Level Characterized by a Novel Method in Guinea Pig Atria

Several accepted methods are available to estimate the adenosine (Ado) concentration of interstitial fluid ([Ado]ISF) in functioning heart, providing results spanning over nano- to micromolar concentrations. This extremely large range points to the necessity of novel approaches for estimating [Ado]ISF or at least the alteration from basal [Ado]ISF. In the present study, the change in [Ado]ISF was characterized following nucleoside transport (NT) blockade elicited by 10 micromol/L dipyridamole or 10 micromol/L nitrobenzylthioinosine in isolated guinea pig atria, by means of our novel procedure referred to as receptorial responsiveness method (RRM). The RRM provided an index of the change in [Ado]ISF under NT blockade, namely the concentration of N-cyclopentyladenosine (CPA; a relatively stable A1 Ado receptor agonist), which is equieffective with the change in [Ado]ISF regarding the contractility. Our results show that dipyridamole or nitrobenzylthioinosine produced an elevation in [Ado]ISF at the cardiomyocyte A1 Ado receptors equivalent to about 16 or 20 nmol/l CPA, respectively. In addition, nitrobenzylthioinosine was found more appropriate for selective NT blockade than dipyridamole.

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.

Role of adenosine A(1) receptor in angiotensin II- and norepinephrine-induced renal vasoconstriction.

We investigated the contributions of adenosine A(1) receptors to angiotensin II- and norepinephrine-induced renal vasoconstriction. Intrarenal administrations of angiotensin II (3, 10, and 30 ng) or norepinephrine (100 and 500 ng) produced dose-dependent renal vasoconstriction in anesthetized dogs. Under resting conditions, angiotensin II (30 ng) and norepinephrine (500 ng) significantly decreased renal blood flow by -43 +/- 3 and -19 +/- 2%, respectively (n = 21). Intra-arterial infusion of adenosine (5 microg/kg/min) significantly augmented renal blood flow responses to both angiotensin II and norepinephrine (-64 +/- 4 and -45 +/- 14%, n = 7). Renal blood flow responses to angiotensin II and norepinephrine were also augmented by inhibition of cellular uptake of adenosine with dipyridamole (10 microg/kg/min, n = 6). Blockade of adenosine A(1) receptors with 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902; 10 microg/kg/min) did not alter basal renal blood flow but significantly attenuated angiotensin II- and norepinephrine-induced renal vasoconstriction (-34 +/- 6 and -9 +/- 3%, n = 7). Furthermore, KW-3902 completely prevented augmentation of renal blood flow responses to angiotensin II and norepinephrine produced by adenosine or dipyridamole (n = 7 and 6, respectively). Administrations of angiotensin II (30 ng) or norepinephrine (500 ng) into the common carotid artery significantly decreased carotid blood flow by -20 +/- 5 and -41 +/- 10%, respectively; however, neither adenosine (5 microg/kg/min) nor KW-3902 (10 microg/kg/min) affected the carotid blood flow responses to angiotensin II and norepinephrine (n = 5, respectively). Adenosine concentrations in dialysates were not significantly changed by administrations of angiotensin II (from 19 +/- 3 to 24 +/- 4 nM, n = 6) or norepinephrine (from 16 +/- 3 to 19 +/- 3 nM, n = 6). These results suggest that basal interstitial adenosine levels influence both angiotensin II and norepinephrine-induced vasoconstriction via A(1) receptors in the kidney but not in the area drained by the common carotid artery. The responses of adenosine to angiotensin II- and norepinephrine-induced renal vasoconstriction may not be mediated through de novo intrarenal adenosine accumulation due to angiotensin II- and norepinephrine-induced renal vasoconstriction.

Potential roles of interstitial fluid adenosine in the regulation of renal hemodynamics

Adenosine exerts membrane receptor-mediated effects on renal hemodynamics and function. Endogenously produced adenosine is proposed to be formed primarily by renal tubular epithelial cells and approaches the vascular smooth cells of the renal vasculature from the extracellular space, i.e. the interstitium. Hence, it is important to determine the dynamics of adenosine levels in renal interstitial fluid. Indeed, many studies have indicated that the effects of adenosine on renal hemodynamics depend on its concentration in the interstitium. Furthermore, in vitro studies reveal that adenosine in the renal interstitial fluid binds to membrane receptors from the adventitial side and initiates renal microvascular responses. Recently, we developed a renal microdialysis method to monitor the concentration of adenosine in renal interstitial fluid. This method is well suited for in vivo studies investigating alterations in renal interstitial adenosine levels. In this review, we summarize the literature pertaining to alterations in renal interstitial fluid concentrations of adenosine in some pathophysiological conditions. Potential roles of interstitial adenosine for regulating renal hemodynamics were also discussed.

Elevated interstitial adenosine concentrations do not activate the muscle reflex.

The purpose of the present study was to examine the effects of adenosine perfusion of the isolated triceps surae muscle group in the decerebrate cat on interstitial adenosine concentrations as well as heart rate and blood pressure responses. In six male cats (6.0 +/- 0.21 kg), the triceps surae muscle group of both legs was perfused with an artificial blood solution containing no additives (control) and then with blood containing 20 mM or 100 microM adenosine for 10 min. An intact muscle reflex was confirmed by bolus injections of 50 mM phosphate and/or saturated KCl administered into the triceps surae muscle via the cannulated popliteal artery before and after adenosine blood perfusion. Microdialysis of the triceps surae muscle group during muscle perfusion revealed that interstitial adenosine was elevated (P < 0.05) from 0.9 +/- 0.3 microM during control blood perfusion to 2,421 +/- 547 microM during 20 mM adenosine perfusion. In addition, interstitial adenosine levels were increased (P < 0.05) from 1.1 +/- 0.3 microM during control blood perfusion to 4.1 +/- 1.2 microM during perfusion with 100 microM adenosine. Despite the large increases in interstitial adenosine levels, perfusion of the triceps surae muscle group with the two blood adenosine solutions resulted in no significant increases in heart rate or blood pressure. These data strongly suggest that elevated interstitial adenosine concentrations do not play a role in activating the muscle reflex and confirm our previous in vivo human findings (J Appl Physiol 83: 1045-1053, 1997).

Purine Uptake and Release in Rat C6 Glioma Cells

Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.

Inhibition of adenosine uptake and augmentation of ischemia-induced increase of interstitial adenosine by cilostazol, an agent to treat intermittent claudication.

Cilostazol (Pletal), a quinolinone derivative with a cyclic nucleotide phosphodiesterase type 3 (PDE3) inhibitory activity, was recently approved by the Food and Drug Administration for treatment of symptoms of intermittent claudication (IC). However, the underlying mechanisms of action are not entirely clear. In this study, we showed that cilostazol inhibited adenosine uptake into cardiac ventricular myocytes, coronary artery smooth muscle, and endothelial cells with a median effective concentration (EC50) approximately 10 microM. In vivo, cilostazol increased cardiac interstitial adenosine levels after a 2-min ischemia in rabbit hearts (329 +/- 92% increase vs. 102 +/- 29% ischemia alone). The combination of cilostazol and 2-min ischemia reduced infarction from subsequent 30-min regional ischemia and 3 h of reperfusion (infarct size was 18 +/- 4% vs. 53 +/- 3% in the hearts with 2-min ischemia alone or 48 +/- 2% in the hearts treated with cilostazol alone). In contrast, milrinone had no effect on either adenosine uptake or interstitial adenosine levels. These data show that cilostazol, unlike milrinone, inhibits adenosine uptake, and thus potentiates adenosine accumulation from a 2-min ischemia. Future studies are needed to investigate the role of adenosine in the treatment of IC by cilostazol.