Endothelial cationic amino acid transporter-1 overexpression can prevent oxidative stress and increases in arterial pressure in response to superoxide dismutase inhibition in mice

Acta Physiol 2014, 210, 845–853

Endothelial cationic amino acid transporter-1 overexpression can prevent oxidative stress and increases in arterial pressure in response to superoxide dismutase inhibition in mice G. Konstantinidis,1,2 G. A. Head,1 R. G. Evans,2 T.-P. Nguyen-Huu,1 K. Venardos,1 K. D. Croft,3 T. A. Mori,3 D. M. Kaye1 and N. W. Rajapakse1,2 1 Baker IDI Heart and Diabetes Institute, Melbourne, Vic., Australia 2 Department of Physiology, Monash University, Melbourne, Vic., Australia 3 School of Medicine and Pharmacology, Royal Perth Hospital Unit, University of Western Australia, Perth, WA, Australia

Received 2 July 2013, revision requested 30 August 2013, revision received 12 November 2013, accepted 11 December 2013 Correspondence: N. W. Rajapakse, Heart Failure Research Group, Baker IDI Heart and Diabetes Institute, 75, Commercial Road, Melbourne, Vic. 3004, Australia. E-mail: niwanthi.rajapakse @bakeridi.edu.au

Abstract Aim: Oxidative stress may play an important role in the pathogenesis of hypertension. The aim of our study is to examine whether increased expression of the predominant endothelial L-arginine transporter, cationic amino acid transporter-1 (CAT1), can prevent oxidative stress-induced hypertension. Methods: Wild-type mice (WT; n = 9) and endothelial CAT1 overexpressing (CAT+) mice (n = 6) had telemetry probes implanted for the measurement of mean arterial pressure (MAP), heart rate (HR) and locomotor activity. Minipumps were implanted for infusion of the superoxide dismutase inhibitor diethyldithiocarbamic acid (DETCA; 30 mg kg 1 day 1; 14 days) or its saline vehicle. Baseline levels of MAP, HR and locomotor activity were determined before and during chronic DETCA administration. Mice were then killed, and their plasma and kidneys collected for analysis of F2-isoprostane levels. Results: Basal MAP was less in CAT+ (92  2 mmHg; n = 6) than in WT (98  2 mmHg; n = 9; P < 0.001). During DETCA infusion, MAP was increased in WT (by 4.2  0.5%; P < 0.001) but not in CAT+, when compared to appropriate controls (PDETCA*genotype = 0.006). DETCA infusion increased total plasma F2-isoprostane levels (by 67  11%; P = 0.05) in WT but not in CAT+. Total renal F2-isoprostane levels were greater during DETCA infusion in WT (by 72%; P < 0.001), but not in CAT+, compared to appropriate controls. Conclusion: Augmented endothelial L-arginine transport attenuated the prohypertensive effects of systemic and renal oxidative stress, suggesting that manipulation of endothelial CAT1 may provide a new therapeutic approach for the treatment of cardiovascular disease associated with oxidative stress. Keywords cationic amino acid transporter-1, hypertension, L-arginine transport, nitric oxide, oxidative stress.

Hypertension remains a major risk factor for a number of cardiovascular diseases. It has been shown that the activity of the enzyme superoxide dismutase (SOD) is markedly reduced in hypertensive patients when

compared with normotensive controls (Pedro-Botet et al. 2000). Direct renal medullary infusion of the SOD inhibitor, diethyldithiocarbamic acid (DETCA) increased renal interstitial superoxide concentration

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and produced sustained hypertension in Sprague– Dawley (SD) rats (Makino et al. 2002). Collectively, these data indicate that renal SOD plays a critical role in scavenging superoxide and regulating the long-term set point of arterial pressure. An extensive body of experimental data supports a role for antioxidants in reducing blood pressure (F€ orstermann 2010, Ahmeda & Johns 2012, Haines et al. 2012, Ahmeda et al. 2013, Carlstr€ om et al. 2013, Vaneckova et al. 2013). However, large-scale clinical trials failed to demonstrate beneficial effects of the antioxidants vitamins C and E in hypertension and related cardiovascular complications (F€ orstermann 2010). In this context, there is considerable evidence that the amino acid L-arginine has antioxidant properties (Rajapakse & Mattson 2009), in addition to its well-known effects in increasing nitric oxide (NO). For example, it has been shown that L-arginine can blunt increases in urinary H2O2, 8-isoprostane and thromboxane B2 excretion in Dahl S rats placed on a high-salt diet (Fujii et al. 2003). L-arginine has also been shown to blunt the up-regulation of gp91phox and p47phox subunits of NADPH oxidase in the renal cortex of these rats (Fujii et al. 2003). In addition, L-arginine can act as a scavenger of reactive oxygen species and blunt the release of oxygen free radicals from endothelial cells (Wascher et al. 1997). Of note, L-arginine can produce NO via a nonenzymatic pathway by reacting with hydrogen peroxide (Nagase et al. 1997). Both y+ and y+L transport mechanisms can mediate L-arginine transport in endothelial cells (Rajapakse & Mattson 2009). System y+ consists of various cationic transporters encoded as CAT1, CAT2A, CAT2B and CAT3 (Rajapakse & Mattson 2009), while the y+L mechanism observed in endothelial cells is associated with CD/984F2 heavy chain (Rajapakse & Mattson 2009). Cationic amino acid transporter 1 (CAT1) is the predominant L-arginine transporter expressed in endothelial cells (Rajapakse & Mattson 2013). The precise roles of other transporters remain to be determined. We have previously shown that L-arginine transport is impaired in hypertensive individuals as well as individuals genetically predisposed to hypertension (Schlaich et al. 2004). These data suggest that impairments in L-arginine transport are causative in the development of hypertension. Oral administration of L-arginine has not been shown to be beneficial in hypertensive patients (Rajapakse & Mattson 2009), perhaps reflecting the need to correct impairments in L-arginine transport in order to reveal its antioxidant and NO increasing effects (Rajapakse & Mattson 2009). While there are multiple lines of evidence that increase in L-arginine transport can increase NO availability (Chin-Dusting et al. 2007, Rajapakse & 846

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Mattson 2009), the antioxidant properties of augmented L-arginine transport has not been investigated. In the present study, we therefore aimed to determine whether increased endothelial CAT1 expression can prevent SOD inhibition–induced oxidative stress, particularly within the kidney, and related elevations in arterial pressure. We therefore examined, in both wild-type (WT) mice and mice with endothelial cationic amino acid transporter-1 (CAT1) overexpression (CAT+ mice), the effects of chronic SOD inhibition on arterial pressure. We also assessed plasma and renal tissue F2-isoprostanes as markers of oxidative stress.

Methods Animals Transgenic mice with overexpression of CAT1 under the control of the Tie2 promoter (Yang et al. 2007) and WT mice were used in the present study. All animals were allowed access to standard mouse chow and water ad libitum. All experiments were approved by the Alfred Medical Research Education Precinct Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for the Use of Animals for Scientific Purposes.

Surgical preparation With the use of a sphygmomanometer, calibration of telemetry probes was verified to be accurate within 1 mmHg before implantation. Radiotelemetry transmitters (model TA11PA-C10; Data Sciences International, St Paul, MN, USA) were implanted in mice under isoflurane anaesthesia (Fluothane, 5% v/v induction and 2% v/v maintenance; AstraZeneca, Macclesfield, UK). The catheter was inserted into the left carotid artery and the transmitter body was implanted along the right flank as previously described (Butz & Davisson 2001). A blood sample (approx. 0.2 mL) was collected from the tail of the mouse, and plasma was stored at 80 °C in the presence of butylated hydroxytoluene (BHT), for later measurement of F2-isoprostanes. Mice were housed in a room, which was completely isolated from surrounding vibrations and noise from the environment, and were exposed to light over 01:00–13:00 hours, thus maintaining a 12 : 12-h light/dark cycle. A 10-day period was allowed for recovery after which mice were housed individually in cages for the remainder of the study. Fourteen days after implanting the telemetry probe, a minipump (Alzet Model 2004; Alzet Corporation, Cupertino, CA, USA) was inserted subcutaneously into the upper back of the mouse under isoflurane anaesthesia.

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Data acquisition

Plasma total F2-isoprostanes

Pulsatile arterial pressure and gross locomotor activity were monitored continuously and were sampled at 1000 Hz with the use of an analog-to-digital data acquisition card (8024E; National Instruments, Austin, TX, USA) during 72-h recording sessions. Heart rate (HR) and beat-to-beat mean arterial pressure (MAP) were detected online and analysed using a program written in LABVIEW (Head et al. 2001). MAP was calculated by the computer software, which detected systolic blood pressure, diastolic blood pressure, interheartbeat interval and instantaneous HR. All haemodynamic parameters were saved onto disk in ASCII format. A file with values averaged over 60-s was saved and used for analysis.

Plasma samples were hydrolysed to liberate bound F2isoprostanes and then purified using an affinity sorbent column (Cayman Chemical, Ann Arbor, MI, USA). Total F2-isoprostanes in plasma were determined using a commercially available enzyme immunoassay kit from Cayman Chemical (Cat number 516351) that predominantly measures 8-isoprostane which is one isomer of the F2-isoprostane family of compounds. This assay kit has a detection limit of 2.7 pg mL 1, which is well below the plasma 8-isoprostane concentrations detected in our samples. Hence, this kit is suitable to measure plasma 8-isoprostane levels in mice.

Experimental protocol Commencing 10 days after implantation of telemetry probes, MAP, HR and locomotor activity were measured continuously in unrestrained mice for three consecutive days. Once these basal measurements had been obtained, mice were anaesthetized again to allow implantation of minipumps to administer DETCA. We also conducted preliminary experiments to establish an effective systemic dose of DETCA to induce hypertension in mice. These studies demonstrated that systemic doses of 60–200 mg kg 1 day 1 caused burn-like scars in the skin of mice, so administration of these doses was discontinued (data not shown). Thus, we chose to administer DETCA at a dose of 30 mg kg 1 day 1 as this was the highest dose which did not have untoward side effects. DETCA (30 mg kg 1 day 1; n = 6–9 per group; Sigma, Sydney, NSW, Australia) or its saline vehicle (n = 5 per genotype) was administered for a period of 14 days. Our pilot data indicated that the effects of DETCA on MAP plateaued from day 7 onwards. Therefore, in subsequent experiments, we measured MAP from day 8 to 10 after commencement of DETCA infusion. Commencing 7 days after minipump implantation, MAP, HR and locomotor activity were measured over a 3-day period. Mice were then killed and a blood sample (approx. 0.2 mL) was collected via heart puncture. Plasma samples were stored at 80 °C in the presence of 0.005% BHT for later analysis of total plasma F2-isoprostane levels. Kidneys were snap-frozen in liquid nitrogen and stored at 80 °C for analysis of total renal F2-isoprostane levels. Telemetry probes were recalibrated at the end of the study to determine the drift, which was then compensated for in the results using a regression equation.

F2-isoprostane levels in the kidney Renal tissue samples were powdered under liquid nitrogen, and lipids were extracted into chloroform/ methanol (2 : 1 containing BHT). The organic phase was hydrolysed with KOH in methanol, acidified and applied to prewashed Certify II cartridges (Varian). F2-isoprostanes were quantified by stable isotope dilution capillary gas chromatography/electron-capture negative ionization mass spectrometry as previously described (Mori et al. 1999).

Data analysis Files with 60-s averages were used for data analysis. Hourly averages of MAP over a 24-h period were calculated by averaging respective 60-s averages collected over a period of 72 h. Hourly averages of MAP were used to determine the effects of genotype on baseline MAP, HR and locomotor activity. Average MAP over a 72-h period was used to determine whether the effects of DETCA on MAP, HR and activity differed between genotypes.

Statistical analysis Data are presented as mean  SEM. Analysis of variance (GraphPad Prism, version 5; GraphPad Software, Inc., La Jolla, CA, USA) was used for determining (i) whether variables changed systematically over a 24-h time period (PTime), (ii) the effects of treatment (DETCA/saline) (PTreatment) or genotype (PGenotype), (iii) whether effects of genotype differed over a 24-h time period (PTime*Genotype) and (iv) whether effects of the treatments differed in CAT+ compared with WT mice (PTreatment*genotype). Unpaired t-tests were used for single measurements to compare between mice genotypes. Two-tailed P ≤ 0.05 was considered statistically significant.

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respectively, in WT mice compared with CAT+ mice (Fig. 3b).

Results Baseline measurements in WT and CAT+ mice Baseline MAP was greater by 4, 6 and 6% during day 1, 2 and 3, respectively, in WT mice compared to CAT+ mice (Fig. 1). Baseline levels of MAP, HR and locomotor activity were 98  2 mmHg, 557  12 bpm, and 1.2  0.3 units, respectively, when averaged across all nine WT mice over a 72-h period. In CAT+ mice, the average baseline levels of MAP, HR and activity were 92  2 mmHg, 554  17 bpm and 0.8  0.1 units, respectively. Baseline MAP and activity were greater (by 5 and 22%, respectively) in WT mice compared to CAT+ mice (Fig. 2a).

Relationship between locomotor activity and MAP The slope of the linear relationship between log activity and MAP was not significantly different between WT and CAT+ mice (P = 0.12; Fig. 2b), but the Y intercept was greater in the former genotype compared to the latter (P = 0.03; Fig. 2b), suggesting that MAP was greater in WT mice than in CAT+ mice regardless of the level of locomotor activity. Of note, MAP was greater in WT mice than in CAT+ mice during the inactive phase of mice (01:00– 13:00 hours). During this phase, there was no significant difference in locomotor activity between WT and CAT+ mice (Fig. 3a). The differences in MAP and activity were most prominent during the active phase (13:00–01:00 hours; Fig. 3b), and during this time, MAP and activity were greater by 7 and 34%,

Effects of DETCA on baseline haemodynamics In WT mice, 72-h averages of MAP increased (by 4.2  0.5%; P < 0.001) during the period 8–10 days after commencing DETCA infusion when compared with respective levels of MAP during the pretreatment period (Fig. 4). In contrast, 72-h averages of HR and locomotor activity remained relatively stable during DETCA infusion (P ≥ 0.53). In WT mice, 72-h averages of MAP, HR and locomotor activity were 101  2 mmHg, 557  9 bpm and 0.9  0.1 units, respectively, during DETCA infusion. In CAT+ mice, 72-h averages of MAP, HR and locomotor activity remained relatively stable across the course of the DETCA infusion (P ≥ 0.4). In these mice, 72-h averages of MAP, HR and locomotor activity were 91  3 mmHg, 562  16 bpm and 0.69  0.03 units respectively, during the period 8–10 days after commencing DETCA infusion. During DETCA administration, MAP in CAT+ mice was less by 9 mmHg when compared with respective levels of MAP in WT mice (P = 0.01; Fig. 4).

Effects of saline on baseline haemodynamics In both WT and CAT+ mice, baseline levels of MAP and locomotor activity were not significantly different during the period 8–10 days after commencing saline vehicle infusion when compared with respective levels of these variables during the pretreatment period

Figure 1 Baseline arterial pressure during days 1, 2 and 3 in WT and CAT+ mice before diethyldithiocarbamic acid (DETCA) administration. Symbols and error bars represent the mean  SEM of 1-h periods in WT mice (open circles, n = 9) and CAT+ mice (open squares, n = 6). Mice were housed in a room, which was completely isolated from surrounding vibrations and noise from the environment, and were exposed to light over 01:00–13:00 hours, thus maintaining a 12 : 12-h light/dark cycle. P-values are the outcomes of two-way analysis of variance. MAP, mean arterial pressure; WT, wild-type mice; CAT+, endothelial CAT1 overexpressing mice.

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(a)

Figure 2 (a) Baseline arterial pressure and locomotor activity averaged over three consecutive days in WT and CAT+ mice before diethyldithiocarbamic acid (DETCA) administration. Symbols and error bars represent the mean  SEM of 1-h periods in WT mice (open circles, n = 9) and CAT+ mice (open squares, n = 6). Mice were housed in a room, which was completely isolated from surrounding vibrations and noise from the environment, and were exposed to light over 01:00–13:00 hours, thus maintaining a 12 : 12-h light/dark cycle. P-values are the outcomes of two-way analysis of variance. (b) Relationship between MAP and log activity for each minute over 72 h in WT (top, grey lines, n = 5) and CAT+ mice (lower black lines, n = 5). Thin lines represent individual regression lines and thick lines represent average regression with r = 0.47 for WT mice and r = 0.40 for CAT+ mice.*P < 0.05 vs. WT mice. P-value was derived from an unpaired t-test. MAP, mean arterial pressure; WT, wild-type mice; CAT+, endothelial CAT1 overexpressing mice.

(b)

(PSaline within genotype ≥ 0.5). However in WT mice, HR decreased by 10  2% (PSaline within genotype < 0.001) during saline infusion when compared with levels of this variable during the pretreatment period (data not shown).

Plasma F2-isoprostane levels in WT and CAT+ mice Prior to administration of DETCA, basal plasma concentrations of total F2-isoprostanes were 39  4 and 42  4 pg mL 1 in WT and CAT+ mice, respectively (P = 0.73). In WT mice, plasma F2-isoprostane levels increased by 67  11% following DETCA administration (PDETCA within genotype = 0.05). In contrast, plasma F2-isoprostane levels did not significantly increase in response to DETCA administration in CAT+ mice (PDETCA within genotype = 0.62). Fourteen days after commencing DETCA treatment, plasma concentrations of total F2-isoprostanes were greater in WT mice

when compared with respective levels of F2-isoprostanes in CAT+ mice (P = 0.04; Fig. 5).

Renal F2-isoprostane levels in WT and CAT+ mice In WT and CAT+ mice administered saline vehicle, renal F2-isoprostane levels averaged 13  1 and 15  3 pg mg 1 tissue, respectively (P = 0.49). F2-isoprostane levels in the kidney were greater by 72% in WT mice administered DETCA compared to WT mice administered saline vehicle (P < 0.001). In contrast, F2-isoprostane levels did not significantly differ between CAT+ mice administered DETCA or saline (P = 0.13; Fig. 6).

Discussion There are three major novel findings from this study. First, we found that the SOD inhibitor DETCA

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(a)

Figure 3 Baseline arterial pressure and locomotor activity during (a) daytime and (b) night-time in wild-type (WT) and CAT+ mice before diethyldithiocarbamic acid (DETCA) administration. Symbols and error bars represent the mean  SEM of 1-h periods in WT mice (open circles, n = 9) and CAT+ mice (open squares, n = 6). Mice were housed in a room, which was completely isolated from surrounding vibrations and noise from the environment, and were exposed to light over 01:00– 13:00 hours, thus maintaining a 12 : 12h light/dark cycle. P-values and abbreviations are as for Figure 1. CAT+, endothelial CAT1 overexpressing mice.

(b)

increased plasma and renal F2-isoprostane levels in WT mice. In contrast, DETCA failed to increase plasma and renal F2-isoprostane levels in CAT+ mice. Second, and consistent with the blunting of oxidative stress in CAT+ mice, we found that DETCA increased MAP in WT but not CAT+ mice. Third, and consistent with our previous finding that baseline NO availability is greater in CAT+ mice compared to WT mice (Yang et al. 2007), our present data indicate that baseline MAP is lower in CAT+ mice compared to WT mice. This difference in MAP between the two genotypes of mice was most prominent during the night-time when mice were active, suggesting that the effects of CAT1 overexpression vary diurnally. These data are consistent with the proposition that endothelial CAT1 exerts antioxidant effects that can blunt elevations in arterial pressure in states of oxidative stress. There is extensive experimental evidence that increased formation of reactive oxygen species can elevate blood pressure (Schulz et al. 2011). In addition, there is strong evidence that most forms of experimental and human hypertension are associated with 850

increased levels of oxidative stress (Ceriello 2008). Consistent with this, we found that the SOD inhibitor DETCA increased plasma and renal F2-isoprostane levels and arterial pressure in WT mice, as has been previously reported in SD rats (Makino et al. 2002). Collectively, these data provide direct evidence that SOD inhibition can increase oxidative stress and arterial pressure. Superoxide can induce vasoconstriction in the systemic circulation and also has important effects within the kidney, particularly in the medulla (Evans et al. 2010, Ahmeda & Johns 2012, Haines et al. 2012, Ahmeda et al. 2013, Carlstr€ om et al. 2013, Vaneckov a et al. 2013). In support of a role for the renal medulla in DETCA-induced hypertension, it has previously been shown that direct renal medullary infusion of a relatively low dose of DETCA (7.5 mg kg 1 day 1) increased renal O2 concentrations by approximately eightfold in SD rats (Makino et al. 2002). In addition, these authors reported a 20-mmHg increase in MAP during renal medullary DETCA infusion (Makino et al. 2002) compared with the 4-mmHg increase in MAP observed during a much

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MAP (mmHg)

Total renal F2 isoprostanes (pg mg–1 tissue)

Saline DETCA

Total plasma F2-isoprostanes (pg mL–1)

Figure 4 Seventy-two hour averages of MAP in WT and endothelial CAT1 overexpressing mice before and during DETCA administration (30 mg kg 1 day 1; n = 6 per genotype). P-values are the outcomes of two-way analysis of variance #, indicates P ≤ 0.05 vs. levels of MAP during preDETCA infusion within the same genotype. MAP, mean arterial pressure; DETCA, diethyldithiocarbamic acid; CAT+, endothelial CAT1 overexpressing mice; WT, wild-type mice.

80 60 40 20 0

WT

CAT+

Figure 5 Effects of superoxide dismutase (SOD) inhibitor DETCA on total plasma F2-isoprostane levels in WT and endothelial CAT1 overexpressing mice. DETCA (30 mg kg 1 day 1; n = 5 per genotype) was administered via a subcutaneously implanted minipump for a period of 14 days. P-values are the outcomes of two-way analysis of variance. DETCA, diethyldithiocarbamic acid; CAT+, endothelial CAT1 overexpressing mice; WT, wild-type mice.

higher dose of DETCA (30 mg kg 1 day 1; iv) infusion in the current study. In the current study, we found that renal F2-isoprostane concentrations were 72% greater in WT mice administered DETCA compared to those administered saline. This suggests the presence of renal oxidative stress in WT mice treated with DETCA in our current study. However, the levels of renal oxidative stress were likely much smaller than that achieved by direct renal medullary infusion. Nevertheless, these data provide evidence that renal

30

20

10

0

WT

CAT+

Figure 6 Effects of superoxide dismutase (SOD) inhibitor DETCA on total renal F2-isoprostane levels in WT and endothelial CAT1 overexpressing mice. DETCA (30 mg kg 1 day 1; n = 5 per genotype) or saline vehicle (n = 5 per genotype) was administered via a subcutaneously implanted minipump for a period of 14 days. P-values are the outcomes of two-way analysis of variance. DETCA, diethyldithiocarbamic acid; CAT+, endothelial CAT1 overexpressing mice; WT, wild-type mice.

oxidative stress plays a major role in SOD-inhibitioninduced increases in arterial pressure. We also conducted preliminary experiments to establish an effective systemic dose of DETCA to induce hypertension in mice. Our data indicate that systemic doses of 60–200 mg kg 1 day 1 caused burn-like scars in the skin of mice, so administration of these doses was discontinued (data not shown). Collectively, these data suggest that the renal medulla is a likely site at which the SOD inhibitor DETCA acted in our current experiment to increase MAP in WT mice. Previous reports indicate that superoxide can increase sodium reabsorption in the thick ascending limb of the kidney (Oritz & Gavin 2002) and reduce renal medullary perfusion (Makino et al. 2002), and both these actions can elevate arterial pressure (Cowley et al. 2003). Accordingly, interventions that reduce oxidative stress in the renal medulla are likely to protect against the development of hypertension. We hypothesized that increase in endothelial CAT1 expression can reduce oxidative stress and thus prevent elevations in arterial pressure induced by pro-oxidant stimuli. Our current data are in strong agreement with this hypothesis. Locomotor activity in WT and CAT+ mice changed little during DETCA administration; hence, we can be confident that the effects of DETCA on MAP were not confounded by concomitant changes in locomotor activity. To assess the potential mechanisms underlying the antihypertensive effects of endothelial CAT1 overexpression, we examined total F2-isoprostane levels in plasma as well as renal F2-isoprostanes before and

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after DETCA or saline infusion in both genotypes of mice. Our data indicate that total plasma F2-isoprostane levels increased in response to DETCA infusion in WT but not in CAT+ mice, suggesting that the latter, but not former, genotype is resistant to developing oxidative stress in response to SOD inhibition. Consistent with this, renal F2-isoprostanes were greater in WT, but not CAT+ mice, administered DETCA compared to those that received saline. Thus, the ability of CAT+ mice to be resistant to the development of oxidative stress, particularly within the kidney, is likely to contribute to the antihypertensive effects of endothelial CAT1 overexpression. One limitation of our current study arises from the fact that we did not assess NO bioavailability during DETCA infusion. Thus, while we can conclude that CAT-1 provides protection against hypertension and oxidative stress, we have no evidence to directly link this effect to the increased NO production and bioavailability characteristic of CAT+ mice (Yang et al. 2007). Another limitation arises from locomotor activity and arterial pressure being greater in WT mice than in CAT+ mice. Therefore, greater arterial pressure in WT mice compared to CAT+ mice may in part arise from differences in locomotor activity between the two genotypes. However, our data indicate that the slope of the linear relationship between log activity and MAP was not significantly different between WT and CAT+ mice, but the Y intercept was greater in the former genotype compared to the latter. These data suggest that MAP was greater in WT mice than in CAT+ mice regardless of the level of locomotor activity. Therefore, we can be confident that greater baseline MAP in WT mice compared to CAT+ mice was not due to differences in locomotor activity between genotypes. Importantly, our data also indicate that baseline MAP was greater in WT mice compared to CAT+ mice during the inactive phase when there was no significant difference in locomotor activity between the two genotypes. Collectively, these data provide strong evidence that endothelial CAT1 overexpression can lower baseline arterial pressure. We conclude that SOD inhibition can increase oxidative stress and induce increases in arterial pressure. Endothelial CAT1 overexpression can prevent the development of oxidative stress and increases in MAP. Accordingly, the development of approaches that provide a sustained increase in intracellular L-arginine concentration may be highly beneficial in pathological states associated with increased oxidative stress such as hypertension.

Conflict of interest The authors declare no conflicts of interest. 852

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Authors thank Ms Ouda Khammy for performing the F2-isoprostane assay in plasma samples. This work was funded by the National Health and Medical Research Council, Australia (CJ Martin fellowship ID 384299 to NR, Program grant ID 1036352 to DK) and by the National Heart Foundation, Australia (PF09M4668 to NR; PF09M4698 to KV). The present study was also supported in part by the Victorian Government’s Operational Infrastructure Support Program.

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