Differences in oxidative stress status and expression of MKP-1 in dorsal medulla of transgenic rats with altered brain renin-angiotensin system

Abstract

ANG II-stimulated production of reactive oxygen species (ROS) through NADPH oxidase is suggested to activate MAPK pathways, which are implicated in neurally mediated pressor effects of ANG II. Emerging evidence suggests that ANG-(1–7) up regulates MAPK phosphatases to reduce MAPK signaling and attenuate actions of ANG II. Whether angiotensin peptides participate in long-term regulation of these systems in the brain is not known. Therefore, we determined tissue and mitochondrial ROS, as well as expression and activity of MAPK phosphatase-1 (MKP-1) in brain dorsal medullary tissue of hypertensive transgenic (mRen2)27 rats exhibiting higher ANG II/ANG-(1–7) tone or hypotensive transgenic rats with targeted decreased glial expression of angiotensinogen, ASrAOGEN (AS) exhibiting lower ANG II/ANG-(1–7) tone compared with normotensive Sprague-Dawley (SD) rats that serve as the control strain. Transgenic (mRen2)27 rats showed higher medullary tissue NADPH oxidase activity and dihydroethidium fluorescence in isolated mitochondria vs. SD or AS rats. Mitochondrial uncoupling protein 2 was lower in AS and unchanged in (mRen2)27 compared with SD rats. MKP-1 mRNA and protein expression were higher in AS and unchanged in (mRen2)27 compared with SD rats. AS rats also had lower phosphorylated ERK1/2 and JNK consistent with higher MKP-1 activity. Thus, an altered brain renin-angiotensin system influences oxidative stress status and regulates MKP-1 expression. However, there is a dissociation between these effects and the hemodynamic profiles. Higher ROS was associated with hypertension in (mRen2)27 and normal MKP-1, whereas the higher MKP-1 was associated with hypotension in AS, where ROS was normal relative to SD rats.

reactive oxygen species (ros) not only influence cardiovascular physiology but are also implicated in neuropathogenesis of hypertension (27, 43). ANG II, a potent vasoconstrictor hormone of the renin-angiotensin system (RAS), has prooxidant properties, and many of its deleterious cardiovascular actions are thought to occur via increased generation of reactive oxygen species (ROS) and activation of redox-sensitive signaling pathways in brain (58, 59). ANG II activates NADPH oxidase, the major source of superoxide (O₂^·−) in many tissues, including the central nervous system (CNS) (26, 53, 59), and stimulates mitochondrial ROS production as another source of cellular ROS (18, 35). ANG II blockade, in rodent models of hypertension not only attenuates oxidant production, but also improves mitochondrial function (15).

ANG II-induced ROS are reported to activate MAPK signaling pathways, which are implicated in both the short- and long-term pressor effects of the peptide in brain (8, 9). MAPK activity is tightly regulated by the coordinated action of several protein kinases and phosphatases. Emerging evidence shows that ANG-(1–7), an alternative product of the RAS with vasodilatory and antihypertensive actions, upregulates the expression of regulatory phosphatases to attenuate ANG II-stimulated MAPK signaling pathways (12, 20, 22, 25, 48, 52).

An imbalance between ANG II and ANG-(1–7) over the long term that favors ANG II is implicated in several models of hypertension and aging; however, the mechanisms for these effects are not well understood (16, 46). Transgenic (mRen2)27 rats overexpressing the murine renin 2 gene are hypertensive (39) and have insulin resistance with evidence of reduced endogenous ANG-(1–7) actions in brain dorsal medullary tissue from an early age compared with the normotensive Hannover Sprague-Dawley (SD) control strain (16, 29, 30). By contrast, transgenic ASrAOGEN rats (AS) with low-brain angiotensinogen, resulting from glial overexpression of an antisense oligonucleotide to angiotensinogen, have mild hypotension and lower body weight, along with enhanced insulin and leptin sensitivity (29, 30, 49). These animals also exhibit loss of endogenous ANG II attenuation of the baroreflex sensitivity at the level of the nucleus of the solitary tract in the dorsal medulla, while maintaining endogenous actions of ANG-(1–7) for facilitation of baroreflex sensitivity (47). Young (mRen2)27 rats have higher phosphoinositol 3-kinase (PI3K) mRNA in dorsal medulla compared with SD rats that contributes to the hypertension (38). Thus, long-term alterations in the brain RAS may modulate signaling pathways involved in cardiometabolic diseases. In the present study, we compared NADPH oxidase activity and mitochondrial ROS in brain dorsal medulla of these animals. Furthermore, MKP-1 and phosphorylated MAPKs (substrates for MKP-1 activity), which are involved in leptin, insulin, and RAS signaling, were also measured.

MATERIALS AND METHODS

Animals.

Experiments were performed in 20- to 30-wk-old male rats from all three lines [(mRen2)27, AS and SD (average age of all rats used in three strains is 25 ± 1 wk)] obtained from the Hypertension and Vascular Research Center Colony, Wake Forest University School of Medicine, Winston-Salem, NC. The Hannover SD rats are the parent line for the two transgenic rat strains. Animals were bred and housed in humidity- and temperature-controlled rooms in group cages (12:12-h light-dark cycle) with free access to standard rat chow and water. All experimental protocols were approved by the Animal Care and Use Committee of Wake Forest University Health Sciences.

Blood pressure measurement.

Conscious blood pressure was measured in all groups of animals using the tail-cuff method (4). At least five determinations were made in each animal and averaged for a single determination per session. Rats were trained to the tail cuff procedures and were handled by the same investigator.

Tissue removal and sample collection.

Rats were decapitated, and the brain dorsal medulla, hearts, and blood (serum) samples were collected at approximately the same time of day (8 AM–10 AM) for all animals. Hearts (whole and left ventricle) were weighed, and brain dorsal medulla was either used fresh (for NADPH oxidase assay and mitochondria isolation) or frozen (for mRNA and protein analysis) on dry ice for later use.

Glucose, insulin, and leptin measurements.

Glucose was measured in the serum of each animal using a Freestyle glucose monitor, as described previously (29). Insulin and leptin were measured using radioimmunoassays specific for rat, according to the manufacturer's protocol (Linco, Lincoln, NE), as previously described (29).

NADPH oxidase activity.

Freshly isolated brain dorsal medullary tissues were washed with ice-cold PBS and homogenized in cold lysis buffer (20 mmol/l KH₂PO₄, pH 7.0, 1 mmol/l EGTA, containing Sigma protease inhibitor cocktail). The homogenate was centrifuged at 1,000 g for 10 min at 4°C. The pellet was resuspended in a lysis buffer containing protease inhibitors and manually homogenized on ice. NADPH oxidase activity was measured by a luminescence assay in a 50 mmol/l phosphate buffer, pH 7.0, containing 1 mmol/l EGTA, 150 mmol/l sucrose, 5 μmol/l dark-adapted lucigenin, 9,9′-bis(N-methylacridinium nitrate) (Sigma) as the electron acceptor, and 100 μmol/l NADPH (Sigma) as the substrate in a final volume of 180 μl. The reaction was started by the addition of 20 μl of homogenate (1 mg/ml protein concentration), and luminescence measurements in relative luminescence units (RLUs) were obtained every 38 s for 15 min using a luminometer (BMG Fluostar Optima, BMG Labtech, Cary, NC), as described previously (28, 32). Background-corrected values were then normalized to the control SD strain and expressed as a percentage change. Superoxide anion production is expressed as RLU/20 μg protein (% normalized to SD).

mRNA and protein analysis.

Total RNA was isolated from frozen dorsal rat medulla using TRIzol reagent (GIBCO Invitrogen, Carlsbad, CA) and first-strand cDNA was synthesized using avian myeloblastosis virus reverse transcriptase. The resultant cDNA was added to TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) with the appropriate gene-specific primer/probe set (Applied Biosystems), and amplification was performed on an ABI 7000 Sequence Detection System. All reactions were performed in triplicate. 18S ribosomal RNA served as an internal control. Results were quantified as Ct values, where Ct is defined as the threshold cycle at which the amplified product is initially detected. The data are expressed as relative gene expression (ratio of target/18S rRNA control). For protein expression, the dorsal medulla was excised immediately and homogenized in a lysis buffer containing protease and phosphatase inhibitors [250 mmol/l sucrose, 0.5 mmol/l EDTA, 50 mmol/l NaF, and 10 mmol/l Tris (pH 7.4) containing 0.01 mmol/l NaVO₄, 0.1 mmol/l PMSF, and 0.6 μmol/l leupeptin]. SDS-PAGE and Western blot hybridization of proteins transferred to PVDF membranes were carried out as described previously (40). Antibodies were obtained from the following sources: uncoupling protein 2 (UCP2) (Calbiochem, San Diego, CA); MKP-1, phospho-specific ERK1/2, JNK-1, and p38 MAP kinases (Cell Signaling, Beverly, MA); and β-actin (Sigma, St. Louis, MO). The optical density (OD) of the immunoreactive bands was normalized to the OD of anti-β-actin immunoreactive bands to account for variation in the protein loading. The data from multiple gels were combined by normalizing the OD values from each strain to the SD control strain run on the same gel.

Isolation of rat brain dorsal medullary mitochondria.

Mitochondria were isolated from fresh brain dorsal medulla by discontinuous Percoll gradient, as described previously (24, 45). Tissue was homogenized in mitochondrial isolation buffer [MIB: 250 mmol/l sucrose, 0.5 mmol/l EDTA and 10 mmol/l Tris, pH 7.4] using a 7-ml all-glass Dounce homogenizer (Kontes Glass, Vineland, NJ). The homogenate was centrifuged for 3 min at 500 g, and then the supernatant was collected and resuspended in equal amounts of 24% Percoll (Amersham Biosciences, Piscataway, NJ) in MIB. This suspension was layered onto a discontinuous Percoll gradient (24% and 40% in MIB). The gradient was centrifuged for 5 min at 28,000 g, and the layer between the 24% and 40% Percoll suspensions containing the purified mitochondria was collected. The preparation was washed in MIB and centrifuged for 12 min at 15,000 g. The structural integrity and purity of the isolated mitochondria were determined by Western blot hybridization (Fig. 3A). Antibodies were obtained from following sources: translocase of outer mitochondrial membrane (TOMM40; Abcam, Cambridge, MA); cytochrome c (BD Pharmingen, Franklin Lakes, NJ), complex IV, subunit III, COX IV (Invitrogen); manganese-dependent superoxide dismutase (Mn-SOD) or SOD2 (BD Biosciences); and nucleoporin p62 (BD Transduction Laboratories, San Jose, CA).

Fig. 3.

MKP-1 mRNA and protein are significantly lower in dorsal medulla of hypertensive (mRen2)27 compared with hypotensive AS rats. Top: MKP-1 relative gene expression and densitometry analyses of protein level normalized to β-actin. Bottom: representative Western blot. Values are expressed as means ± SE (n = 6–8 per group for relative gene expression by RT-PCR; n = 4–6 per group for relative protein expression by Western blot hybridization); *P < 0.05 vs. SD; †P < 0.05 vs. (mRen2)27 [mRen] rats.

Dihydroethidium (HEt) fluorescence.

ROS generation in isolated dorsal medullary mitochondria was visualized by dihydroethidium (Invitrogen) fluorescence imaging, as described previously (31). Briefly, isolated mitochondria were loaded with HEt (5 μmol/l) in MIB for 30 min at 37°C. After washing the mitochondria with MIB, the mitochondria from each set of animals were plated in duplicate on an eight-chambered glass bottom dish (Lab Tek II; Thermo Fisher Scientific, Waltham, MA) and spun at low speed (3000 g for 5 min) to ensure settling of mitochondria on the glass dish. HEt was excited by Argon laser at 488 nm, and the fluorescence emission was imaged through a 560-nm long-pass filter using a LSM 510 laser-scanning microscope system with a 63X C-Apochromat water immersion objective with N.A. of 1.2 (Zeiss, Jena, Germany). Four images per chamber were acquired (i.e., total eight images per animal). For an n of 3 per group, a total of 24 images per group were analyzed for ROS levels in isolated mitochondria. HEt fluorescence was quantified by selecting groups of 8–10 mitochondria identified on a differential contrast image using ImageJ software (NIH) and expressed as relative fluorescence units.

Statistical analyses.

Comparisons of baseline blood pressure, body and tissue weights, biochemical measurements, NADPH oxidase activity, mRNA and protein quantification, and mitochondrial ROS levels in the three animal lines were performed using one-way ANOVA and Student-Newman-Keuls post hoc tests. The criterion for statistical significance was P < 0.05, and all tests were performed using Prism 5.0 and InStat 3 (GraphPad Software, San Diego, CA). Numerical values are presented as means ± SE.

RESULTS

Profiles of (mRen2)27, Sprague-Dawley, and ASrAOGEN rats.

Profiles of hypertensive (mRen2)27, normotensive SD, and hypotensive AS rats are shown in Table 1. Systolic blood pressures and body weights of (mRen2)27 rats were significantly higher than either the SD or AS rats at ∼25 wk of age. Although both (mRen2)27 and AS rats had significantly higher heart-to-body weight ratio compared with SD rats, only the hypertensive strain showed signs of left ventricular hypertrophy. No significant differences in serum glucose and insulin levels were observed for the three groups, although there was a trend for lower insulin and significantly lower leptin in AS rats.

Table 1.

Profiles of (mRen2)27, Sprague-Dawley, and ASrAOGEN rats at ∼25 wk

	(mRen2)27	SD	AS
Systolic blood pressure, mmHg	211 ± 4*	138 ± 2	120 ± 3*†
Body weight, BW, g	551 ± 7*	477 ± 14	325 ± 4*†
Heart to BW ratio, %	0.335 ± 0.02*	0.270 ± 0.01	0.320 ± 0*
Left Ventricle to BW ratio, %	0.206 ± 0.02*	0.160 ± 0	0.185 ± 0
Serum glucose, mmol/l	106 ± 5	105 ± 4	102 ± 4
Serum insulin, ng/ml	1.05 ± 0.32	0.93 ± 0.25	0.6 ± 0.20
Serum leptin, ng/ml	9.3 ± 2.45	7.9 ± 0.30	3.6 ± 0.33*†

Values are expressed as mean ± SE (n = 4–6 per group). SD, Sprague-Dawley; AS, ASrAOGEN;

^†P < 0.05 vs. (mRen2)27;

^*P < 0.05 vs. SD.

NADPH oxidase activity.

NADPH oxidase activity in brain dorsal medulla was ∼42% higher in (mRen2)27 (142 ± 18) compared with SD (100 ± 5), while the AS (93 ± 9) did not differ from SD rats (Fig. 1). Pretreatment of the tissue extracts with diphenyleneiodonium (DPI) essentially eliminated the enzyme activity in all groups, showing the specificity of the assay for NADPH-dependent oxidase activity.

Fig. 1.

NADPH oxidase activity is higher in brain dorsal medullary tissue extracts of transgenic (mRen2)27 rats. NADPH oxidase activity was measured by luminescence assay using 5-μM lucigenin as an electron acceptor and 100 μM NADPH as a substrate in (mRen2)27 [mRen], Sprague-Dawley (SD), and ASrAOGEN (AS) rats. NADPH oxidase inhibitor diphenyleneiodonium (DPI; 10 μM) was used for specificity of the reaction. Values are expressed as means ± SE (n = 4 per group). *P < 0.05 vs. AS.

Mitochondrial ROS levels and uncoupling protein 2 expression.

Isolated brain dorsal medullary mitochondria were subjected to Western blot hybridization to test for structural integrity using antibodies targeted to different compartments of the mitochondria (Fig. 2A). The mitochondrial preparation was found to be free of cytosolic and nuclear contamination, as detected by lack of immunoreactivity toward β-actin and nucleoporin p62, respectively (Fig. 2A). Isolated mitochondria were loaded with HEt, a ROS indicator, which upon oxidation, binds with the DNA to emit a bright red fluorescence. HEt fluorescence intensity was ∼16% higher in (mRen2)27 compared with either SD or AS rats, suggesting higher ROS levels in mitochondria of the hypertensive strain [(mRen2)27: 83 ± 4 arbitrary units, AU; SD: 70 ± 4; AS: 68 ± 3; P < 0.05; Fig. 2, B and C]. UCP2 mRNA was significantly higher in (mRen2)27 compared with SD and AS rats [(mRen2)27: 1.75 ± 0.09 AU; SD: 1.04 ± 0.04; AS: 1.37 ± 0.11; P < 0.05], while UCP2 protein was unchanged in (mRen2)27 vs. the SD rats but lowered in the AS rats [(mRen2)27: 1.12 ± 0.13 AU; SD: 1 ± 0.1; AS: 0.52 ± 0.15; P < 0.05; Fig. 2, D and E].

Fig. 2.

Mitochondrial reactive oxygen species (ROS) and uncoupling protein 2 (UCP2) in the three rat strains. Brain dorsal medullary mitochondria were isolated using a discontinuous Percoll gradient as described in materials and methods. A: isolated mitochondria from (mRen2)27 [mRen], SD, and AS rats were subjected to Western blot hybridization. The mitochondrial preparation did not show immunoreactivity for β-actin or nucleoporin p62, suggesting no cytosolic or nuclear contamination. Lane 1 =20 μg of whole brain (dorsal medulla) extract and lanes 2 to 4 =10 μg of medullary mitochondria. B: isolated mitochondria were loaded with 5 μM dihydroethidium (HEt), a ROS-sensitive probe, and the red fluorescence by oxidized HEt was visualized by confocal microscopy. Differential contrast (DIC) and merged images show that the red fluorescence is specific to mitochondria. Images were adjusted for brightness/contrast using the Zeiss LSM image browser similar for all three strains and represent a slice thickness of 5 μM. C: HEt fluorescence intensity was quantified by ImageJ software and cumulative data are presented in a bar graph. D–F: mRNA (RT-PCR) and protein expression (densitometry analyses of protein level normalized to β-actin and representative Western blot, respectively) for UCP2 in brain dorsal medullary tissue extracts. Values in each panel are presented as means ± SE (n = 3 for HEt fluorescence; n = 6–8 for UCP2 mRNA and protein). *P < 0.05 vs. SD; †P < 0.05 vs. AS; ‡P < 0.05 vs. (mRen2)27. TOMM40, translocase of outer membrane 40; COX IV, cytochrome-c oxidase subunit IV; Mn-SOD, manganese superoxide dismutase.

MKP-1 expression and activity.

MKP-1 (dual specificity phosphatase, DUSP-1) negatively regulates MAPK pathway (6, 19, 42). MKP-1 mRNA [(mRen2)27: 0.99 ± 0.06 relative gene expression; SD: 1.05 ± 0.06; AS: 2.28 ± 0.14; P < 0.05] and protein [(mRen2)27: 0.80 ± 0.1 AU; SD: 1 ± 0.1; AS: 1.5 ± 0.2; P < 0.05] expression followed a similar trend and were higher for AS rats compared with both (mRen2)27 and SD rats (Fig. 3). Differences in MKP-1 protein expression were consistent with MKP-1 activity, as determined by quantifying phosphorylated protein expression for ERK 1/2 and JNK-1 (substrates for MKP-1 activity; Fig. 4). AS animals had significantly lower phosphorylated ERK1/2 [(mRen2)27: 1.1 ± 0.1 AU; SD: 1 ± 0.2; AS: 0.15 ± 0.1; P < 0.05] compared with (mRen2)27 and SD rats and JNK-1 [(mRen2)27: 1.6 ± 0.3 AU; SD: 1 ± 0.1; AS: 0.4 ± 0.1; P < 0.05] compared with (mRen2)27 rats. No significant differences were observed in levels of total ERK1/2 or phosphorylated p38 MAPK among the three strains.

Fig. 4.

Phosphorylated ERK1/2 and JNK-1 MAP kinases are significantly reduced in transgenic AS rats compared with (mRen2)27 and SD rats. ERK1/2, JNK-1, and p38 MAP kinase activities were measured by Western blot hybridization using phospho-specific antibodies in brain dorsal medullary tissues from (mRen2)27 [mRen], SD, and AS rats. Top: densitometry analyses of phoshorylated protein levels normalized to β-actin. Bottom: representative Western blots. Values are means ± SE (n = 3 per group). *P < 0.05 vs. SD; †P < 0.05 vs. (mRen2)27 rats.

DISCUSSION

Our results show basal differences in NADPH oxidase activity, mitochondrial ROS levels, and MKP-1 expression, and activity, as determined by phosphorylated MAPKs in transgenic animals with altered brain RAS. The major finding of the present study is that hypertensive (mRen2)27 rats that exhibit enhanced ANG II actions relative to ANG-(1–7) in brain medulla show higher NADPH oxidase activity and mitochondrial ROS in brain dorsal medulla compared with SD and AS rats (16, 50). However, there were no differences in expression or activity of MKP-1 in the hypertensive strain. On the other hand, AS rats that have enhanced ANG-(1–7) actions relative to ANG II in brain medulla exhibit higher MKP-1 expression and activity, but no differences in NADPH oxidase activity or mitochondrial ROS compared with SD rats (2, 46). This is the first study to report basal ROS generation, mitochondrial ROS levels, and MKP-1 expression and activity in brain dorsal medulla of hemodynamically well-characterized transgenic (mRen2)27 and AS rats. However, the direct contribution of these imbalances to the cardiovascular and metabolic profiles of these transgenic strains and the protection from age-related decline in the AS rats remains to be determined (2, 29, 30, 46). The overall pattern reveals that increased oxidative stress within the dorsal medulla is independent of the changes in MAPK pathways and vice versa in the (mRen2)27 rats (Fig. 5).

Fig. 5.

Proposed signaling mechanisms regulating mean arterial pressure (MAP) and baroreflex function (BRS) within the brain dorsal medulla of transgenic rats with long-term alterations in brain renin-angiotensin system. A: oxidative stress but not MAPK signaling may be associated with hypertension and impaired autonomic function in transgenic (mRen2)27 rats with higher ANG II actions in the brain. ANG II produces reactive oxygen species (ROS) through NADPH oxidase and mitochondria. Both ANG II and ROS can activate MAPKs, which can further stimulate ROS production through activation of NADPH oxidase. Increased ROS or MAPK signaling can cause higher MAP and impaired BRS directly or through altered redox signaling and downstream gene expression changes leading to hypertension. ROS pathway is predominant in (mRen2)27 rats in the dorsal medullary region. B: reduced MAPK signaling may contribute to lower resting MAP and enhanced BRS in transgenic ASrAOGEN rats with higher ANG-(1–7) actions in the brain. ANG-(1–7) through upregulation of MAP kinase phosphatase (MKP-1) negatively regulates MAPK signaling. Whether unchanged levels of ROS in ASrAOGEN rats is through direct actions of ANG-(1–7) on NADPH oxidase or mitochondria, or through reduced MAPK signaling is not known (?). Reduced MAPK signaling within the brain dorsal medulla of ASrAOGEN rats may contribute to lower MAP and enhanced BRS through altered redox signaling and downstream gene expression changes.

Blood pressure and body weight differences in the transgenic and control animals were consistent with earlier studies (29, 30). AS rats exhibit lower resting blood pressures and body weight than the other two strains. Both the (mRen2)27 and AS rats had significantly higher heart-to-body weight ratio compared with SD rats. However, only the (mRen2)27 rats showed a higher left ventricle-to-body weight ratio, which was evident in spite of the higher body weight in this group, indicating signs of left ventricular hypertrophy. The organ weight measures in the AS may be influenced more by the lower body weight, resulting from less fat mass rather than overt changes in organ weights (7). AS rats have similar serum insulin levels as SD rats, with a trend for lower values than (mRen2)27 as reported (29). AS rats at ∼25 wk of age in the present study have significantly lower serum leptin (an appetite suppressor) relative to the other two strains, consistent with the higher food intake and lower sympathetic nervous system outflow reported for the AS animals (29). Interestingly, while comparisons in younger animals (14–17 wk of age) showed similar leptin levels among strains, AS rats had lower serum leptin than the other groups at 43–48 wk of age, primarily reflecting elevated serum leptin concentrations in the older SD and (mRen2)27 rats (29). Thus, our present findings provide new data showing that the onset of the age-related increase in leptin is as early as 25 wk of age in the SD and (mRen2)27 rats relative to the healthy aging AS rats.

Activation of ROS-generating enzymes, dysregulation of ROS scavenging systems, or mitochondrial disruption may lead to an overall increase in oxidative stress. Higher NADPH oxidase activity and mitochondrial ROS in the (mRen2)27 are consistent with studies that demonstrate a critical role of NADPH oxidase-derived ROS in various tissues, including the CNS in ANG II-induced hypertension (14, 59). In addition, ANG II signaling in neurons is proposed to involve a ROS mechanism, where NADPH oxidase-derived ROS induces mitochondrial-oxidative stress through impairment of mitochondrial electron transport chain complexes (11, 41). Increased mitochondrial ROS (particularly H₂O₂) may also stimulate NADPH oxidase expression and activity in a feed-forward fashion (21, 37). We note that total ROS levels or the predominant ROS species were not determined in the present study, nor the extent of antioxidant enzyme activities [cytosolic and mitochondrial superoxide dismutase and catalase]; future studies will address whether differences in these parameters are apparent in the three rat strains.

Among the three main isoforms of UCP expressed in brain, UCP2 is the most extensively characterized. UCP2 has neuroprotective effects under various toxic insults and reduces ROS production in the brain under pathological, but not physiological conditions (3, 10, 54). We found significantly higher UCP2 mRNA in medulla of hypertensive (mRen2)27 rats compared with normotensive SD and hypotensive AS rats. UCP2 protein levels were, however, not different between (mRen2)27 and SD rats but were lower in the AS relative to other strains. The lack of higher UCP2 protein in (mRen2)27 rats suggests an inability to compensate against oxidative stress and may lead to higher ROS in these rats.

MKPs are a family of protein phosphatases that inactivate MAPKs through dephosphorylation of threonine and/or tyrosine residues in the kinase activation domain (33). At present, 13 dual-specificity MKPs are identified, which are further categorized into four groups based on their structure and functional characteristics; these dephosphorylate all three major MAPKs (ERK1/2, JNK, and p38) in mammalian cells (19, 34). MKP-1 is an early inducible protein localized in the nucleus and expressed constitutively at low levels in most tissues, including the brain (5). The physiological role of MKP-1 under pathological conditions is not well understood. We found significantly higher MKP-1 mRNA and protein in AS vs. (mRen2)27 and SD animals. Moreover, the differences in MKP-1 expression corresponded with changes in activity, as measured by phosphorylated MAPK expression [ERK1/2 and JNK-1, but not p38]. The expression of total ERKs was similar among strains, suggesting that the differences in phosphorylated products may reflect changes in MAPK activity.

Modulation of MKP-1 expression by oxidative stress in animals has not been studied, although its regulation in several cell lines has been reported (36, 44, 56). In vitro studies suggest that ROS may induce MKP-1 gene expression in the acute settings; however, long-term effects of ROS on gene expression are not known. It is known that oxidative damage of the protein may affect the net activity of the phosphatase (13). MKP-1 can be induced by acute hemodynamic stress through either restraint or ANG II infusion in the aorta but not other tissues of Wistar rats (55).

MKP-1 is higher in the AS rats that have reportedly high ANG-(1–7) tone associated with lower resting systolic pressure and enhanced baroreflex sensitivity (BRS) (16, 17). We recently reported that exogenous ANG-(1–7) expression via gene transfer reduces high blood pressure, improves BRS, and increases MKP-1 mRNA in (mRen2)27 rats (23). ANG II-mediated PI3K pathway signaling pathways are also activated in hypertensive rats, contributing to the increase in arterial pressure and suppression of the BRS (51, 57). Inhibition of protein tyrosine phosphatase 1b (PTP1b, a negative regulator of PI3K pathway) decreased BRS in SD and AS rats (1), while inhibition of PI3K improved BRS in (mRen2)27 rats (38). Blockade of PI3K does not alter blood pressure or BRS in SD rats, suggesting that PI3K has no major role in regulating BRS or blood pressure under normotensive conditions and may only be relevant in hypertensive rats (38, 51). These studies suggest that a balance of kinases and phosphatases is important in regulating BRS and blood pressure. Our results suggest that MKP-1 expression may be associated with a lower MAPK activity in the medulla and lower systolic blood pressure and higher BRS. However, dorsal medullary MKP-1 and MAPKs were similar between (mRen2)27 and SD, suggesting no association with hypertension or increased oxidative stress within this brain area. The lack of increased MAPK activity in the (mRen2)27 despite higher ANG II actions and elevated ROS levels contrasts with studies by Chan et al. (8, 9) that reported higher ROS following ANG II infusion. This discrepancy may reflect differences in 1) the site of investigation within the brain (dorsal vs. the ventral medulla), 2) the type of animal models used [the transgenic (mRen2)27 vs. ANG II infusion in normotensive rats], or 3) the site and duration of ANG II stimulation (acute microinjection in the ventrolateral medulla or 7-day intracerebroventricular infusion). Whether differences in the brain RAS regarding ANG II and ANG-(1–7) affect MKP-1 expression is not known at this time; however, higher baseline levels of the phosphatase might mitigate responses to acute activation of the kinases in the AS animals.

Perspectives and Significance

Our findings suggest a role for brain RAS in the regulation of oxidative stress and MKP-1 expression. However, the dissociation of MKP-1 and MAPKs relative to either hypertension or oxidative stress illustrates distinct regulatory factors. Because impaired BRS, oxidative stress, and mitochondrial dysfunction are implicated in cardiovascular diseases, further understanding these interrelationships is warranted. Increasing MKP-1 by altering the ANG II to ANG-(1–7) ratio may be a potential therapeutic strategy to improve BRS, but whether this will offset oxidative damage remains to be determined.

GRANTS

Consortium for Southeastern Hypertension Control Warren Trust (to M. Nautiyal), American Heart Association Postdoctoral Fellowship (to M. Nautiyal), Farley Hudson Foundation (Jacksonville, NC), and National Institutes of Health (Grant HL-51952 to D. I. Diz, M. C. Chappell, E. A. Tallant, and P. E. Gallagher, Grant HL-56973 to D. I. Diz, M. C. Chappell, and P. E. Gallagher; and Grants HL-077731, HL-030260, HL-093554, and HL-065380 to D. W. Busija) provided support for this project.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: M.N., P.V.K., and P.E.G. performed experiments; M.N., P.V.K., and P.E.G. analyzed data; M.N., P.V.K., P.E.G., E.A.T., M.C.C., and D.I.D. interpreted results of experiments; M.N. and P.V.K. prepared figures; M.N. drafted manuscript; M.N., P.V.K., D.W.B., P.E.G., E.A.T., M.C.C., and D.I.D. edited and revised manuscript; P.V.K., D.W.B., P.E.G., E.A.T., M.C.C., and D.I.D. approved final version of manuscript; E.A.T., M.C.C., and D.I.D. conception and design of research.