Microvascular Research 98 (2015) 16–22

Contents lists available at ScienceDirect

Microvascular Research journal homepage: www.elsevier.com/locate/ymvre

Hypoxanthine uptake by skeletal muscle microvascular endothelial cells from equilibrative nucleoside transporter 1 (ENT1)-null mice: Effect of oxidative stress D.B.J. Bone 1, M. Antic, D. Quinonez, J.R. Hammond 2,⁎ Department of Physiology and Pharmacology, Western University, London, Ontario N6A 5C1, Canada

a r t i c l e

i n f o

Article history: Accepted 17 November 2014 Available online 22 November 2014 Keywords: Adenosine Hypoxanthine Vasculature Endothelium Ischemia Oxidative stress Nucleoside Nucleobase Transporters

a b s t r a c t Adenosine is an endogenous regulator of vascular tone. This activity of adenosine is terminated by its uptake and metabolism by microvascular endothelial cells (MVEC). The predominant transporter involved is ENT1 (equilibrative nucleoside transporter subtype 1). MVEC also express the nucleobase transporter (ENBT1) which is involved in the cellular flux of adenosine metabolites such as hypoxanthine. Changes in either of these transport systems would impact the bioactivity of adenosine and its metabolism, including the formation of oxygen free radicals. MVEC isolated from skeletal muscle of ENT1+/+ and ENT1−/− mice were subjected to oxidative stress induced by simulated ischemia/reperfusion or menadione. The functional activities of ENT1 and ENBT1 were assessed based on zero-trans influx kinetics of radiolabeled substrates. There was a reduction in the rate of ENBT1-mediated hypoxanthine uptake by ENT1+/+ MVEC treated with menadione or after exposure to conditions that simulate ischemia/reperfusion. In both cases, the superoxide dismutase mimetic MnTMPyP attenuated the loss of ENBT1 activity, implicating superoxide radicals in the response. In contrast, MVEC isolated from ENT1−/− mice showed no reduction in ENBT1 activity upon treatment with menadione or simulated ischemia/ reperfusion, but they did have a significantly higher level of catalase activity relative to ENT1+/+ MVEC. These data suggest that ENBT1 activity is decreased in MVEC in response to the increased superoxide radical that is associated with ischemia/reperfusion injury. MVEC isolated from ENT1−/− mice do not show this reduction in ENBT1, possibly due to increased catalase activity. © 2014 Published by Elsevier Inc.

Introduction Microvascular endothelial cells (MVEC) play a critical role in the local regulation of vascular tone via the production and release of vasoactive agents such as nitric oxide and prostacyclin (Tune, 2007). A key factor that stimulates the release of these agents is the endogenous

Abbreviations: ECGS,endothelialcellgrowth supplement; EDTA,ethylenediaminetetraacetic acid; ENBT1, equilibrative nucleobase transporter 1; ENT, equilibrative nucleoside transport; ENT1, equilibrative nucleoside transporter subtype 1; ENT2, equilibrative nucleoside transporter subtype 2; MnTMPyP, manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin; NBMPR, nitrobenzylmercaptopurine riboside; MVEC, microvascular endothelial cells; NMG, N-methyl-glucamine; PBS,phosphate buffered saline; ROS, reactive oxygen species; sI/R, simulated ischemia/reperfusion; SOD, superoxide dismutase; TBHP, tert-butyl hydroperoxide ⁎ Corresponding author at: Department of Pharmacology, 9-70 Medical Sciences Building, University of Alberta, Edmonton, AB T6G 2H7, Canada. E-mail addresses: [email protected] (D.B.J. Bone), [email protected] (D. Quinonez), [email protected] (J.R. Hammond). 1 Current address: Molecular Signaling Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA. 2 Current address: Department of Pharmacology, 9-70 Medical Sciences Building, University of Alberta, Edmonton, AB T6G 2H7, Canada.

http://dx.doi.org/10.1016/j.mvr.2014.11.005 0026-2862/© 2014 Published by Elsevier Inc.

nucleoside adenosine (Nyberg et al., 2010). This ‘retaliatory metabolite’ (Newby et al., 1985) can be released from cells under biological stress, such as that which occurs during ischemic conditions, and acts in the vasculature through a family of G-protein coupled receptors expressed in endothelial and smooth muscle cells (Headrick et al., 2011; Ryzhov et al., 2007). Activation of adenosine receptors has been shown to be cardioprotective (Cohen and Downey, 2008; McIntosh and Lasley, 2012). Furthermore, adenosine inhibits platelet aggregation, has anti-inflammatory activities, and is a potent vasodilator (Riksen and Rongen, 2012). It has also been implicated in the phenomenon known as ischemic preconditioning, whereby a short period of ischemia/reperfusion attenuates the damage caused by a subsequent ischemia/reperfusion event (Headrick and Lasley, 2009; Yang et al., 2010). In the vasculature, adenosine concentrations are regulated by the concerted activities of a number of enzymes and membrane transporters (Arch and Newsholme, 1978; Deussen, 2000; Deussen et al., 2006). Under normal physiological conditions, extracellular adenosine (produced primarily by 5′-ecto-nucleotidase-mediated metabolism of adenine nucleotides) is taken up rapidly into cells by specific plasma membrane transporters (Loffler et al., 2007). In both human (Bone and Hammond, 2007) and mouse (Bone et al., 2010) MVEC, adenosine is accumulated and released primarily via the equilibrative nucleoside

D.B.J. Bone et al. / Microvascular Research 98 (2015) 16–22

transporter subtype 1 (ENT1). Once inside the cells, adenosine is used immediately to maintain the cellular adenine nucleotide pool via the adenosine kinase pathway or, once the kinase pathway is saturated, metabolized by adenosine deaminase to inosine. Hence, intracellular adenosine concentrations are typically very low, maintaining an inward adenosine gradient across the plasma membrane, with much of the extracellular adenosine arising from metabolism of adenine nucleotides via 5′-ecto-nucleotidase. However, under conditions of cellular stress, adenosine formed from the intracellular breakdown of adenine nucleotides can be released from cells via ENT1 and acts on extracellular Gprotein coupled adenosine receptors. In the absence of ENT1 activity, this excess of intracellular adenosine does not exit the cells and is thus metabolized to inosine via adenosine deaminase and then hypoxanthine via purine nucleoside phosphorylase. Elevated intracellular hypoxanthine levels result in an increased generation of reactive oxygen species (ROS) (via xanthine oxidase-mediated metabolism of hypoxanthine, and subsequently from xanthine metabolism to uric acid) of a magnitude that is dependent on level of xanthine oxidase activity and the ability of the cell to extrude the excess hypoxanthine (Baudry et al., 2008; Berry and Hare, 2004). Generation of ROS in the vasculature is known to underlie the vascular dysfunction that is a hallmark of ischemia/reperfusion injury (Beetsch et al., 1998; Verma et al., 2002). We have shown previously that MVEC from ENT1−/− mice do not compensate for this deletion by up-regulating any of the other transporters responsible for the plasma membrane translocation of purine nucleosides (Bone et al., 2010). As a consequence, ENT1−/− derived MVEC are severely compromised in their ability to take up and release adenosine, compared to those isolated from wild-type mice. The ENT1−/− mice do, however, show a significant up-regulation of adenosine deaminase, presumably in response to elevated intracellular adenosine levels (Bone et al., 2010). This might be expected to lead to enhanced generation of ROS via xanthine oxidase which, in turn, may result in cellular compensation in antioxidant mechanisms, or in the processes by which cells can regulate hypoxanthine levels such as via the equilibrative nucleobase transporter (ENBT1) (Bone and Hammond, 2007). We have recently shown that simulated ischemia/reperfusion (sI/R) and the generation of intracellular superoxide with menadione results in a decrease in ENBT1 activity in primary human cardiac microvascular endothelial cells (Bone et al., 2014). Based on the defined roles of ENT1 in regulating adenosine bioactivity in the vasculature (reviewed by Loffler et al., 2007), and previous work showing that hearts from ENT1−/− mice are resistant to damage caused by ischemia/reperfusion (Rose et al., 2010), we hypothesized that MVEC isolated from ENT1−/− mice have an altered response to ischemia/reperfusion induced cellular stress. In the present work, we studied the effects of sI/R and pharmacologically-induced oxidative stress on ENT1- and ENBT1mediated nucleoside and nucleobase transport, respectively, in MVEC isolated from skeletal muscle of ENT1+/+ mice and ENBT1-mediated nucleobase transport in MVEC from ENT1−/− mice. We show that ENBT1 transport activity in MVEC from wild-type mice, but not ENT1−/− mice, is decreased by oxidative stress, and that this difference may be, in part, due to increased catalase activity in the ENT1−/− MVEC. Materials and methods Materials 2-Chloro[8- 3 H]adenosine (16 Ci mmol - 1 ), [2,8- 3 H]hypoxanthine (28.7 Ci mmol − 1 ), and [3 H]water (1 Ci mmol − 1 ) were purchased from Moravek Biochemicals (Brea, CA). Non-radiolabeled 2-chloroadenosine, hypoxanthine, dipyridamole (2,6-bis (diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]pyrimidine), adenine, collagenase, trypsin, bovine serum albumin, tert-butyl hydroperoxide (TBHP) and menadione were from Sigma-Aldrich (St. Louis, MO). Manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin

17

(MnTMPyP) was from Calbiochem (Mississauga, ON). All cell culture media, fetal bovine serum (FBS), culture grade Dulbecco's phosphate buffered saline (PBS), trypsin-EDTA, antibiotic/antimycotic (penicillin, streptomycin, and amphotericin B), and heparin were purchased from GIBCO/BRL (Burlington, ON, Canada). Endothelial cell growth supplement (ECGS) was supplied by Beckton Dickinson (Oakville, ON, Canada). The Superoxide Dismutase (SOD) and Catalase Assay Kits were purchased from Cayman Chemicals via Cedarlane Labs (Burlington, ON, Canada). ENT1−/− mice were obtained from Dr D.S. Choi (Mayo Clinic, Mn) and backcrossed with C57BL/6 mice. The mouse colony was maintained through the breeding of heterozygous animals to obtain wild-type (ENT1+/+) and knockout (ENT1−/−) littermates. Mice were housed in standard cages and maintained on a 12-hour light/dark cycle, with rodent chow and water available ad libitum.

MVEC isolation/culture MVEC were isolated as from ENT1+/+ and ENT1−/− mice as described previously (Bone et al., 2010). Each isolation involved pooling of tissue from 2–3 mice. The extensor digitorum longus muscles of female mice (~ 3 months of age) were removed from the hind limbs under anesthesia by pentobarbital sodium (42 mg kg−1 ip). After careful removal and disposal of tissue containing larger blood vessels, the isolated muscle was sliced into ∼ 0.5-mm pieces and digested for 30–45 min in 0.84 mg/ml collagenase, 0.12 mg ml−1 trypsin and dispase, and 1.62 mg ml− 1 bovine serum albumin in 50 ml Krebs– Ringer solution containing (in mM) 127 NaCl, 4.6 KCl, 1.1 MgSO4, 1.2 KH2PO4, 8.3 D-glucose, 24.8 NaHCO3, 2 pyruvate, 11.4 creatinine, 20 taurine, 5 D-ribose, 2 L-asparagine, 2 L-glutamine, 1 L-arginine, and 0.5 uric acid in a 37 °C water-jacketed organ bath bubbled with 95% O2–5% CO2. The enzymatic digest was filtered through 100-μm nylon mesh to remove undigested fragments, and the dissociated cells were collected by centrifugation and washed in 37 °C DMEM-F12 media. Cells were then suspended with ∼ 1 × 106 Bandeiraea simplicifolia (BSI-B4; Sigma) coated Dynabeads (M-450 Epoxy, Dynal, Lake Success, NY) and rotated on a spindle for 10–15 min. Endothelial cells bound to the magnetic beads were collected using a magnetic particle collector (MPC-1, Dynal), washed twice, and transferred to six-well plates in DMEM-F12 media supplemented with 20% FBS, 100 U ml−1 penicillin G, 100 μg ml− 1 streptomycin sulfate, 0.25 μg ml−1 amphotericin B, 2 mM L-glutamine, 0.025 IU ml−1 heparin, and 125 μg ml−1 ECGS and cultured at 37 °C in a 5% CO2–95% room air humidified atmosphere. The media was changed routinely every 3 to 4 days, and the cells were passaged using 0.05% trypsin in 0.53 mM EDTA upon reaching confluence. After passage 3, the FBS and ECGS levels were reduced to 10% and 75 μg ml−1, respectively. In general, six passages were required to obtain sufficient cells to undertake the transport assays described herein. Previous studies from our laboratory (Bone et al., 2010) have established that cells isolated using this procedure were over 95% endothelial in phenotype based on the expression of von Willebrand factor VIII and BSI-B4 antigens. Cell morphology and proliferation rate was monitored closely and cultures were terminated if any change in these parameters were noted. In preparation for experimental assays, MVEC (passages 6–12) were trypsinized for 5 min at 37 °C and then diluted with DMEM-F12 media + 10% FBS and collected by centrifugation. The cells were washed once by suspension/centrifugation in PBS containing (in mM) 137 NaCl, 6.3 Na2HPO4, 2.7 KCl, 1.5 KH2PO4, 0.9 CaCl2·2H2O, and 0.5 MgCl2·6H2O (pH 7.4) and then suspended in PBS for immediate use. For low oxygen exposure, culture flasks were placed in hypoxia chambers (Billups-Rothenberg). Chambers were flushed with 1% O2 for 5 min and expelled air was measured with an O2 detector to confirm low oxygen levels. Chambers were then sealed and placed in a 37 °C incubator for 2 h. Following hypoxia, flasks were removed from the chambers and harvested for substrate uptake as described above.

18

D.B.J. Bone et al. / Microvascular Research 98 (2015) 16–22

Simulated ischemia/reperfusion (sI/R) Based on a method described by Meldrum and colleagues (Meldrum et al., 2001) and used previously by our laboratory in a study on human MVEC (Bone et al., 2014), cells were incubated under a layer of mineral oil to simulate the conditions attained in vivo during ischemia. Briefly, cells were harvested and re-suspended in fresh complete medium, placed in a 37 °C 5% CO2–95% air humidified atmosphere for 1 h to equilibrate, collected by centrifugation, and the medium aspirated leaving approximately 200 μl to cover the cell pellet. In some cases, the SOD mimetic MnTMPyP (100 μM) was added to the media at this point; this concentration has been shown by others to be effective in reversing free radical effects in other cell models (Day et al., 1997; Gonzalez-Polo et al., 2004). The cell pellet was re-suspended by tapping the side of the tube gently. Simulated ischemia was initiated by layering mineral oil over the cell suspension, creating a gas exchange barrier. Mineral oil over-laid cell suspensions were incubated at 37 °C for 2 h. Control cells were left in complete medium with no oil-overlay and incubated in parallel at 37 °C. Cells were mixed gently every 30 min, taking care not to disrupt the oil barrier. Cells were then diluted 200-fold (with disruption of the oil barrier) with 37 °C PBS and incubated for a further hour at 37 °C (‘reperfusion’). Following treatment, cells were washed thrice with PBS by centrifugation and then suspended in PBS for the [3H]substrate uptake assays. Treatment with ROS generators Menadione (100 mM) was prepared from powder in warmed (37 °C) PBS just prior to use. Immediately after harvest, cells were suspended in PBS alone (control) or PBS containing 100 μM TBHP or 100 μM menadione and incubated for 30 min in a 37 °C 5%CO2–95% air humidified atmosphere. After the incubation period, cells were washed twice with room temperature PBS and used immediately for the substrate uptake assays as shown below. This wash procedure would be expected to remove excess endogenous nucleosides or nucleobases that may compete with the added [3H]substrates in transport assays described below. [3H]nucleoside/nucleobase uptake Uptake was initiated by the addition of suspended cells (~750,000 cells assay−1) to 2-chloro[3H]adenosine (nucleoside) or [3H]hypoxanthine (nucleobase) layered over 200 μl of silicone/mineral oil (21:4 vol vol− 1) in 1.5-ml microcentrifuge tubes. After a defined incubation time, uptake was terminated by centrifugation for 10 s (~ 12,000 g). Non-mediated uptake of 2-chloro[3H]adenosine or [3H]hypoxanthine was defined by the inclusion of 5 μM NBMPR/5 μM dipyridamole or 1 mM adenine, respectively, in the assay. We showed previously (Bone and Hammond, 2007) that 1 mM adenine is sufficient to inhibit all ENBT1-mediated [3H]hypoxanthine influx in MVEC. Aqueous substrate and oil layers were removed by aspiration, and pelleted cells were digested in 1 M sodium hydroxide overnight (12–16 h). A sample of the digest was analyzed for 3H content using standard liquid scintillation counting techniques. Uptake data are presented as picomoles per microliter of intracellular volume after correction for the amount of extracellular 3H in the cell pellet. Total volume was determined by incubating cells with 3H2O for 3 min and processed as above. Extracellular water space was estimated by extrapolation of the linear time course of nonmediated uptake to zero time. Catalase and SOD activity measurements Enzyme activity was assessed following suppliers' instructions. MVEC isolated from ENT1+/+ and ENT1−/− mice were harvested from T175 flasks using a cell scraper. For catalase activity assays, cells were pelleted by centrifugation at 4 °C and then sonicated on ice in 2 ml of

ice-cold buffer (50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA). The lysed cells were collected by centrifugation at 10,000 g for 15 min at 4 °C, and the supernatant removed and stored at −80 °C for subsequent analysis within 7 days. Measurement of catalase activity was based on its reaction with methanol in the presence of H2O2 to produce formaldehyde. The formaldehyde is measured colorimetrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazol as the chromogen in units of nmol of formaldehyde formed min−1 mg cell lysate protein−1. For SOD activity assays, cells were pelleted by centrifugation at 4 °C and then sonicated on ice in 2 ml of ice-cold buffer (20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose). The lysed cells were then collected by centrifugation at 1500 g for 5 min at 4 °C, and the supernatant removed and stored at −80 °C for subsequent analysis within 7 days. The measurement of SOD activity is based on the conversion of tetrazolium salt to formazan dye by superoxide radicals generated by xanthine oxidase and hypoxanthine. This assay measures all three types of SOD (cytosolic Cu/Zn-SOD, mitochondrial MnSOD, and extracellular SOD). Results are expressed as units (U) of SOD mg cell lysate protein−1, where one unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Statistics All data are presented as mean ± SEM of results obtained from at least 4 independent experiments. Non-linear curves were fitted to data using the Michaelis–Menten relationship with the aid of GraphPad Prism v.5 (GraphPad Software Inc., La Jolla, CA). Significant differences were determined by using the two-tailed Student's t-test for paired or unpaired samples (P b 0.05). Paired analyses were used for comparison of individual data points when data was obtained from treated and untreated cells in parallel using the same cell population. Kinetic parameters (Km, Vmax) derived from Michaelis–Menten curve fits were treated as unpaired data sets. Results We reported previously that MVEC isolated from mouse skeletal muscle expressed mainly the ENT1 subtype of equilibrative nucleoside transporter, with minimal contribution of ENT2 to the total nucleoside uptake (Bone et al., 2010). We also showed that MVEC displayed high levels of activity of ENBT1, a purine-selective nucleobase transporter, and that ENBT1 activity in human cardiac microvascular endothelial cells is decreased in response to sI/R (Bone et al., 2014). We now extend these findings to a comparison of the responses of ENBT1 in ENT1+/+ and ENT1−/− MVEC to sI/R. The Km values for transport of [3H]hypoxanthine and [3H]2-chloroadenosine averaged 101 ± 14 μM and 40 ± 8 μM, respectively, and neither were affected significantly by the treatments employed in this study. sI/R resulted in a significant reduction (~ 40%) in the amount of ENBT1-mediated [3H]hypoxanthine transport in the ENT1+/+ MVEC (Vmax of 15 ± 3 and 8 ± 1 pmol μl−1 s−1 for control and treated cells, respectively; Fig. 1A) but had no effect on ENBT1-mediated transport in the ENT1−/− MVEC (Vmax for [3H]hypoxanthine uptake in the control and sI/R treated cells of 14 ± 2 and 16 ± 2 pmol μl−1 s−1 respectively) (Fig. 1B). This effect of sI/R on ENT1+/+ cells was attenuated by including 100 μM MnTMPyP in the media during the ischemia phase of the treatment (Fig. 1C). MnTMPyP itself had no effect on [3H]hypoxanthine transport in cells that were not subjected to sI/R, nor did MnTMPyP affect hypoxanthine uptake by ENT1−/− MVEC (Fig. 1D). In contrast to that seen for ENBT1, the influx of [3H]2-chloroadenosine by ENT1 in ENT1+/+ MVEC was not affected by the sI/R (Fig. 2). The ability of the SOD mimetic MnTMPyP to reverse the effects of sI/ R on hypoxanthine transport by MVEC implicated the involvement of ROS in this process. Treatment of MVEC isolated from ENT1+/+ mice with 100 μM TBHP (an extracellular hydrogen peroxide generator)

D.B.J. Bone et al. / Microvascular Research 98 (2015) 16–22

19

Fig. 1. Effect of sI/R on ENBT1 activity in the presence or absence of the superoxide dismutase mimetic MnTMPyP. MVEC isolated from ENT1+/+ (Panels A, C) or ENT1−/− (Panels B, D) mice were subjected to sI/R, in the absence (Panels A, B) or presence (Panels C, D) of 100 μM MnTMPyP, by incubation under a mineral oil barrier for 2 h followed by 1 h incubation in oxygenated buffer at 37 °C (sI/R). Control cells were incubated in parallel for the same time period in 5% CO2–95% air at 37 °C. Cells (Control and sI/R) were then incubated with increasing concentrations of [3H]hypoxanthine for 10 s in the presence (nonmediated uptake) or absence (total uptake) of 1 mM adenine. Results are presented as the mean ± SEM initial rate of transporter-mediated uptake (pmol μl−1 s−1) calculated as the difference between the total and nonmediated uptake components (n = 5). Significant differences (*) were defined using Student's t-test for paired samples (P b 0.05).

had no significant effect on either [3H]2-chloroadenosine transport by ENT1 or [3H]hypoxanthine transport by ENBT1 (Fig. 3). This concentration of TBHP has been shown to generate significant levels of hydrogen peroxide in other models (Dhanya et al., 2014; Zhang et al., 2013). Treatment with menadione (an intracellular superoxide radical generator), on the other hand, resulted in a significant reduction of [3H]hypoxanthine transport, from a Vmax of 11 ± 1 to 6 ± 1 pmol μl−1 s−1 (Fig. 4A) This effect was not observed in MVEC isolated from ENT1−/− mice (Fig. 4B). Interestingly, menadione treatment also had a significant (65 ± 18%) depressant effect on the Vmax of transport of [3H]2-chloroadenosine by the MVEC from ENT1+/+ mice (Fig. 4C).

Based on previous studies, this sI/R model would be expected to lead to a decrease in PO2 in the cells under the mineral oil layer (Meldrum et al., 2001). There was no significant difference in [3H]hypoxanthine transport in cells incubated with 1% O2 (hypoxic) compared with those maintained for 2 h in a 5% O2 environment (Fig. 5A). The transport of [3H]2-chloroadenosine by ENT1 was, however, increased significantly (68%) upon exposure to 1% O2 for 2 h (Vmax of 4.6 ± 0.6 and 2.8 ± 0.4 pmol μl−1 s− 1 in 1% O2 versus control conditions, respectively; Fig. 5B). Incubation of the cells with 100 μM cobalt chloride to stabilize HIF1α (Saini et al., 2010; Vengellur and LaPres, 2004) had no effect on the transport of [3H]hypoxanthine or [3H]2-chloroadenosine (data not shown). On the basis of the observation that MVEC isolated from the ENT1−/− mice were insensitive to the effects of ROS, we compared the activities of the antioxidant enzymes SOD and catalase in the two cell populations. There was no significant difference in the level of SOD activity between the MVEC isolated from ENT1+/+ (9.5 ± 2.8 U mg protein−1) and ENT1−/− (7.8 ± 0.9 U mg−1) mice. However, catalase activity was more than double in the MVEC from the ENT1−/− mice (23.7 ± 3.9 nmol min−1 mg protein−1) relative to the ENT1+/+ MVEC (9.5 ± 2.0 nmol min−1 mg−1) (Fig. 6). Discussion

Fig. 2. Effect of sI/R on ENT1 activity in MVEC. MVEC isolated from ENT1+/+ mice (Control and sI/R treated) were incubated with a range of concentrations of [3H]2-chloroadenosine for 10 s at room temperature in the presence or absence of 5 μM dipyridamole/ nitrobenzylthioinosine to define the rate of transporter-mediated uptake. Data are plotted as the concentration of 2-chloroadenosine (μM) versus the initial rate of substrate influx (pmol μl cell water−1 s−1) to obtain substrate affinity and maximum velocity of transport as reported in the text (n = 4).

Previous work established that hearts from ENT1−/− mice are resistant to damage caused by ischemia/reperfusion (coronary occlusion for 30 min followed by reperfusion for 2 h) (Rose et al., 2010). We have also shown that MVEC isolated from skeletal muscle of ENT1−/− mice are severely compromised in their ability to transport nucleosides, but they do retain the capacity to transport nucleobases via ENBT1 (Bone et al., 2010). In addition, MVEC isolated from ENT1−/− mice have a higher level of adenosine deaminase activity than MVEC from ENT1+/+ mice

20

D.B.J. Bone et al. / Microvascular Research 98 (2015) 16–22

Fig. 3. Effect of TBHP on ENBT1- and ENT1-mediated substrate uptake by MVEC isolated from ENT1+/+ mice. Cells were incubated in parallel in the absence (Control) or presence of 100 μM TBHP for 30 min at 37 °C. Cells were then washed with room temperature buffer and used immediately for the analysis of the rate of uptake (10 s incubation) of [3H]hypoxanthine in the presence or absence of 1 mM adenine (Panel A) or [3H]2chloroadenosine in the presence or absence of 5 μM dipyridamole/nitrobenzylthioinosine (Panel B). Data are shown as the mean ± SEM of the initial rate (pmol μl−1 s−1) of adenine-sensitive hypoxanthine uptake or dipyridamole/nitrobenzylthioinosine-sensitive 2-chloroadenosine uptake (n = 4).

(Bone et al., 2010). On the basis of these studies, we hypothesized that ENT1−/− mice may have developed mechanisms to counter the enhanced production of ROS that accompany an increased metabolism of adenosine down the xanthine oxidase pathway to uric acid. Such compensations may contribute to the relative insensitivity of ENT1−/− cardiac tissue to myocardial infarction. In the present study, we show that sI/R leads to a significant decrease in ENBT1-mediated hypoxanthine transport activity in MVEC isolated from ENT1+/+ mice. In contrast, the same treatment had no effect on ENT1-mediated 2-chloroadenosine transport. This indicates that the model of sI/R used in this work does not have a general nonspecific impact on membrane transport proteins. The finding that menadione, an intracellular superoxide generator (but not TBHP, an extracellular hydrogen peroxide generator) could mimic the effects of sI/R on MVEC from ENT1+/+ mice, and incubation of these cells in a hypoxic environment for 2 h had no effect on hypoxanthine transport, suggests that the effect of the sI/R on hypoxanthine transport is due to the generation of intracellular superoxide radicals (Comporti, 1989). This conclusion is further supported by the results showing that the SOD mimetic MnTMPyP (Day et al., 1997; Liang et al., 2009) can attenuate the effects of sI/R on hypoxanthine transport in MVEC. Intracellular hypoxanthine can be metabolized by hypoxanthineguanine phosphoribosyl transferase (HGPRT) to inosine monophosphate which then enters the purine nucleotide pool. Thus, decreased ENBT1mediated efflux of hypoxanthine from MVEC during periods of cellular stress may be beneficial in maintaining intracellular nucleotide pools in the face of reduced cellular nucleoside availability. It is noteworthy that, in this study, ENT1-mediated 2-chloroadenosine transport was enhanced by the 2 h hypoxia treatment. Previous studies have shown that hypoxia down-regulates ENT1-mediated transport in HL-1 cardiomyocytes (Chaudary et al., 2004) and macrovascular

Fig. 4. Effect of menadione on ENBT1- and ENT1 activity in MVEC isolated from ENT1 +/+ and ENT1 −/− mice. Cells were incubated in parallel in the absence (Control) or presence of 100 μM menadione for 30 min at 37 °C. Cells were then washed with room temperature buffer and used immediately for the analysis of the rate of uptake (10 s incubation) of [3H]hypoxanthine in the presence or absence of 1 mM adenine (Panels A and B) or [ 3 H]2-chloroadenosine in the presence or absence of 5 μM dipyridamole/nitrobenzylthioinosine (Panel C). Data are shown as the mean ± SEM of the initial rate (pmol μl− 1 s−1) of adenine-sensitive hypoxanthine uptake (A, B) (n = 5) or dipyridamole/nitrobenzylthioinosine sensitive 2-chloroadenosine uptake (C) (n = 6). Significant differences (*) were defined using Student's t-test for paired samples (P b 0.05).

endothelial cells (Casanello et al., 2005). These data suggest that microvascular endothelial cells respond differently from macrovascular endothelial cells to hypoxia in terms of ENT1 regulation. In contrast to that seen using ENT1+/+ MVEC, cells isolated from ENT1−/− mice were resistant to the effects of both sI/R and menadione with respect to hypoxanthine transport by ENBT1. These results are consistent with the hypothesis that cells from ENT1−/− mice are relatively resistant to the effects of oxidative stress. This resistance may be due, at least in part, to an up-regulation of catalase activity. Metabolism of hypoxanthine by xanthine oxidase results in the formation of superoxide radical (Berry and Hare, 2004), which is removed by the concerted activities of SOD and catalase (Halliwell, 1978). SOD converts superoxide into water and hydrogen peroxide; SOD activity is not different between the ENT1+/+ and ENT1−/− derived MVEC. Catalase converts hydrogen peroxide to water and oxygen (Bannister et al., 1987;

D.B.J. Bone et al. / Microvascular Research 98 (2015) 16–22

21

Likewise, there are several studies showing that an increase in catalase expression/activity can protect hearts and vasculature against ischemia/reperfusion injury (Li et al., 1997; Pendergrass et al., 2011; Whyte et al., 2011). Recent studies show that the livers (Zimmerman et al., 2013) and kidneys (Grenz et al., 2012) of ENT1−/− mice are also relatively resistant to the effects of ischemia/reperfusion. The ischemia/ reperfusion injury protection seen in these studies was attributed to activation of the A2b adenosine receptor by the increased interstitial adenosine levels in ENT1−/− tissues. In this regard, it is noteworthy that activation of A2b receptors has been shown to inhibit superoxide production in cardiomyocytes (Yang et al., 2011). In summary, we have shown that MVEC isolated from skeletal muscle of ENT1−/− mice are resistant to oxidative stress produced either indirectly (via sI/R) or directly (via menadione treatment), when measured in terms of the effect of this stress on hypoxanthine transport by ENBT1. We also presented evidence that this resistance is due, at least in part, to increased activity of catalase. We propose that this represents a developed compensatory mechanism to chronic superoxide exposure in these cells that arise from the altered purine metabolism concomitant with the loss of the major adenosine transporter ENT1. Acknowledgments

Fig. 5. Effect of hypoxia (1% O2) on the activity of ENBT1 and ENT1: Culture flasks containing MVEC isolated from ENT1+/+ mice were placed in hypoxia chambers (Billups-Rothenberg) and flushed with 1% O2 for 5 min then sealed and placed in a 37 °C incubator for 2 h. Cells of the same passage were incubated in parallel at 37 °C in a normal 5% CO2–95% room air humidified atmosphere (Control). Following hypoxia, flasks were removed from the chambers and processed for substrate transport assays using the oil-stop method as described in the text. Data are shown as the mean ± SEM of the initial rate (pmol μl−1 s−1) of adeninesensitive hypoxanthine uptake (A) (n = 4), or dipyridamole/nitrobenzylthioinosinesensitive 2-chloroadenosine uptake (B) (n = 5). Significant differences (*) were defined using Student's t-test for paired samples (P b 0.05).

We wish to thank Dr Tim Regnault (Physiology and Pharmacology, Western University, London, Canada) for the use of his hypoxic cell culture chambers, and Dr Doo-Sup Choi (Mayo Clinic, Rochester, Mn) for providing the ENT1−/− breeding pairs to establish the mouse colony at Western University. This work was supported by a grant (T-7275) to JRH from the Heart and Stroke Foundation of Ontario. DBJB thanks the Schulich School of Medicine and Dentistry at Western University for financial support during the course of this work. DBJB was also the recipient of a PhD Studentship Award from the Canadian Institutes for Health Research. References

−/−

Goyal and Basak, 2010). The increase in catalase activity in the ENT1 mice would enhance the removal of hydrogen peroxide, which plays a key role in both physiological and pathological processes in endothelial cells (Vara and Pula, 2014). This may, in part, explain why hearts from these mice are relatively protected against myocardial infarction induced by acute coronary occlusion (Rose et al., 2010). It is well established that vascular ischemia followed by reperfusion results in the formation of ROS that contribute to the endothelial dysfunction associated with this pathology (Forstermann, 2010; Munzel et al., 2010).

Fig. 6. Superoxide dismutase and catalase activities. MVEC isolated from ENT1+/+ or ENT1−/− mice were lysed by sonication in hypotonic buffer solutions and then centrifuged to obtain the supernatant for analysis of superoxide dismutase (SOD) and catalase activity. For SOD, results are expressed as units of SOD per mg cell lysate protein. For catalase, results are expressed as nmol of formaldehyde formed/min/mg cell lysate protein. Each bar represents the mean ± SEM from 4 experiments conducted in duplicate using 3 independent cell isolations. Significant differences (*) were defined using Student's t-test for unpaired samples (P b 0.05).

Arch, J.R.S., Newsholme, E.A., 1978. The control of the metabolism and the hormonal role of adenosine. Essays Biochem. 14, 82–123. Bannister, J.V., Bannister, W.H., Rotilio, G., 1987. Aspects of the structure, function, and applications of superoxide dismutase. CRC Crit. Rev. Biochem. 22, 111–180. Baudry, N., Laemmel, E., Vicaut, E., 2008. In vivo reactive oxygen species production induced by ischemia in muscle arterioles of mice: involvement of xanthine oxidase and mitochondria. Am. J. Physiol. Heart Circ. Physiol. 294, H821–H828. Beetsch, J.W., Park, T.S., Dugan, L.L., Shah, A.R., Gidday, J.M., 1998. Xanthine oxidasederived superoxide causes reoxygenation injury of ischemic cerebral endothelial cells. Brain Res. 786, 89–95. Berry, C.E., Hare, J.M., 2004. Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications. J. Physiol. 555, 589–606. Bone, D.B., Hammond, J.R., 2007. Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: characterization of a novel nucleobase transporter. Am. J. Physiol. Heart Circ. Physiol. 293, H3325–H3332. Bone, D.B., Choi, D.S., Coe, I.R., Hammond, J.R., 2010. Nucleoside/nucleobase transport and metabolism by microvascular endothelial cells isolated from ENT1−/− mice. Am. J. Physiol. Heart Circ. Physiol. 299, H847–H856. Bone, D.B., Antic, M., Vilas, G., Hammond, J.R., 2014. Oxidative stress modulates nucleobase transport in microvascular endothelial cells. Microvasc. Res. 95C, 68–75. Casanello, P., Torres, A., Sanhueza, F., Gonzalez, M., Farias, M., Gallardo, V., Pastor-Anglada, M., San, M.R., Sobrevia, L., 2005. Equilibrative nucleoside transporter 1 expression is downregulated by hypoxia in human umbilical vein endothelium. Circ. Res. 97, 16–24. Chaudary, N., Naydenova, Z., Shuralyova, I., Coe, I.R., 2004. Hypoxia regulates the adenosine transporter, mENT1, in the murine cardiomyocyte cell line, HL-1. Cardiovasc. Res. 61, 780–788. Cohen, M.V., Downey, J.M., 2008. Adenosine: trigger and mediator of cardioprotection. Basic Res. Cardiol. 103, 203–215. Comporti, M., 1989. Three models of free radical-induced cell injury. Chem. Biol. Interact. 72, 1–56. Day, B.J., Fridovich, I., Crapo, J.D., 1997. Manganic porphyrins possess catalase activity and protect endothelial cells against hydrogen peroxide-mediated injury. Arch. Biochem. Biophys. 347, 256–262. Deussen, A., 2000. Metabolic flux rates of adenosine in the heart. Naunyn Schmiedebergs Arch. Pharmacol. 362, 351–363. Deussen, A., Brand, M., Pexa, A., Weichsel, J., 2006. Metabolic coronary flow regulation— current concepts. Basic Res. Cardiol. 101, 453–464.

22

D.B.J. Bone et al. / Microvascular Research 98 (2015) 16–22

Dhanya, R., Arun, K.B., Syama, H.P., Nisha, P., Sundaresan, A., Santhosh Kumar, T.R., Jayamurthy, P., 2014. Rutin and quercetin enhance glucose uptake in L6 myotubes under oxidative stress induced by tertiary butyl hydrogen peroxide. Food Chem. 158, 546–554. Forstermann, U., 2010. Nitric oxide and oxidative stress in vascular disease. Pflugers Arch. 459, 923–939. Gonzalez-Polo, R.A., Soler, G., Rodriguezmartin, A., Moran, J.M., Fuentes, J.M., 2004. Protection against MPP+ neurotoxicity in cerebellar granule cells by antioxidants. Cell Biol. Int. 28, 373–380. Goyal, M.M., Basak, A., 2010. Human catalase: looking for complete identity. Protein Cell 1, 888–897. Grenz, A., Bauerle, J.D., Dalton, J.H., Ridyard, D., Badulak, A., Tak, E., McNamee, E.N., Clambey, E., Moldovan, R., Reyes, G., Klawitter, J., Ambler, K., Magee, K., Christians, U., Brodsky, K.S., Ravid, K., Choi, D.S., Wen, J., Lukashev, D., Blackburn, M.R., Osswald, H., Coe, I.R., Nurnberg, B., Haase, V.H., Xia, Y., Sitkovsky, M., Eltzschig, H.K., 2012. Equilibrative nucleoside transporter 1 (ENT1) regulates postischemic blood flow during acute kidney injury in mice. J. Clin. Invest. 122, 693–710. Halliwell, B., 1978. Biochemical mechanisms accounting for the toxic action of oxygen on living organisms: the key role of superoxide dismutase. Cell Biol. Int. Rep. 2, 113–128. Headrick, J.P., Lasley, R.D., 2009. Adenosine receptors and reperfusion injury of the heart. Handb. Exp. Pharmacol. 189–214. Headrick, J.P., Peart, J.N., Reichelt, M.E., Haseler, L.J., 2011. Adenosine and its receptors in the heart: regulation, retaliation and adaptation. Biochim. Biophys. Acta 1808, 1413–1428. Li, G., Chen, Y., Saari, J.T., Kang, Y.J., 1997. Catalase-overexpressing transgenic mouse heart is resistant to ischemia–reperfusion injury. Am. J. Physiol. 273, H1090–H1095. Liang, H.L., Hilton, G., Mortensen, J., Regner, K., Johnson, C.P., Nilakantan, V., 2009. MnTMPyP, a cell-permeant SOD mimetic, reduces oxidative stress and apoptosis following renal ischemia–reperfusion. Am. J. Physiol. Renal Physiol. 296, F266–F276. Loffler, M., Morote-Garcia, J.C., Eltzschig, S.A., Coe, I.R., Eltzschig, H.K., 2007. Physiological roles of vascular nucleoside transporters. Arterioscler. Thromb. Vasc. Biol. 27, 1004–1013. McIntosh, V.J., Lasley, R.D., 2012. Adenosine receptor-mediated cardioprotection: are all 4 subtypes required or redundant? J. Cardiovasc. Pharmacol. Ther. 17, 21–33. Meldrum, K.K., Meldrum, D.R., Hile, K.L., Burnett, A.L., Harken, A.H., 2001. A novel model of ischemia in renal tubular cells which closely parallels in vivo injury. J. Surg. Res. 99, 288–293. Munzel, T., Gori, T., Bruno, R.M., Taddei, S., 2010. Is oxidative stress a therapeutic target in cardiovascular disease? Eur. Heart J. 31, 2741–2748. Newby, A.C., Worku, Y., Holmquist, C.A., 1985. Adenosine formation. Evidence for a direct biochemical link with energy metabolism. Adv. Myocardiol. 6, 273–284. Nyberg, M., Mortensen, S.P., Thaning, P., Saltin, B., Hellsten, Y., 2010. Interstitial and plasma adenosine stimulate nitric oxide and prostacyclin formation in human skeletal muscle. Hypertension 56, 1102–1108.

Pendergrass, K.D., Varghese, S.T., Maiellaro-Rafferty, K., Brown, M.E., Taylor, W.R., Davis, M.E., 2011. Temporal effects of catalase overexpression on healing after myocardial infarction. Circ. Heart Fail. 4, 98–106. Riksen, N.P., Rongen, G.A., 2012. Targeting adenosine receptors in the development of cardiovascular therapeutics. Expert. Rev. Clin. Pharmacol. 5, 199–218. Rose, J.B., Naydenova, Z., Bang, A., Eguchi, M., Sweeney, G., Choi, D.S., Hammond, J.R., Coe, I.R., 2010. Equilibrative nucleoside transporter 1 plays an essential role in cardioprotection. Am. J. Physiol. Heart Circ. Physiol. 298, H771–H777. Ryzhov, S., McCaleb, J.L., Goldstein, A.E., Biaggioni, I., Feoktistov, I., 2007. Role of adenosine receptors in the regulation of angiogenic factors and neovascularization in hypoxia. J. Pharmacol. Exp. Ther. 320, 565–572. Saini, Y., Kim, K.Y., Lewandowski, R., Bramble, L.A., Harkema, J.R., LaPres, J.J., 2010. Role of hypoxia-inducible factor 1{alpha} in modulating cobalt-induced lung inflammation. Am. J. Physiol. Lung Cell. Mol. Physiol. 298, L139–L147. Tune, J.D., 2007. Control of coronary blood flow during hypoxemia. Adv. Exp. Med. Biol. 618, 25–39. Vara, D., Pula, G., 2014. Reactive oxygen species: physiological roles in the regulation of vascular cells. Curr. Mol. Med. 14, 1103–1125. Vengellur, A., LaPres, J.J., 2004. The role of hypoxia inducible factor 1alpha in cobalt chloride induced cell death in mouse embryonic fibroblasts. Toxicol. Sci. 82, 638–646. Verma, S., Fedak, P.W., Weisel, R.D., Butany, J., Rao, V., Maitland, A., Li, R.K., Dhillon, B., Yau, T.M., 2002. Fundamentals of reperfusion injury for the clinical cardiologist. Circulation 105, 2332–2336. Whyte, J.J., Samuel, M., Mahan, E., Padilla, J., Simmons, G.H., Arce-Esquivel, A.A., Bender, S.B., Whitworth, K.M., Hao, Y.H., Murphy, C.N., Walters, E.M., Prather, R.S., Laughlin, M.H., 2011. Vascular endothelium-specific overexpression of human catalase in cloned pigs. Transgenic Res. 20, 989–1001. Yang, X., Cohen, M.V., Downey, J.M., 2010. Mechanism of cardioprotection by early ischemic preconditioning. Cardiovasc. Drugs Ther. 24, 225–234. Yang, X., Xin, W., Yang, X.M., Kuno, A., Rich, T.C., Cohen, M.V., Downey, J.M., 2011. A2B adenosine receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes via a mechanism sensitive to Pertussis toxin. Br. J. Pharmacol. 163, 995–1006. Zhang, T., Liang, X., Shi, L., Wang, L., Chen, J., Kang, C., Zhu, J., Mi, M., 2013. Estrogen receptor and PI3K/Akt signaling pathway involvement in S-(-)equol-induced activation of Nrf2/ARE in endothelial cells. PLoS One 8, e79075. Zimmerman, M.A., Tak, E., Ehrentraut, S.F., Kaplan, M., Giebler, A., Weng, T., Choi, D.S., Blackburn, M.R., Kam, I., Eltzschig, H.K., Grenz, A., 2013. Equilibrative nucleoside transporter (ENT)-1-dependent elevation of extracellular adenosine protects the liver during ischemia and reperfusion. Hepatology 58, 1766–1778.