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Elevated serum uric acid increases the risk of hypertension, and individuals born preterm have higher blood pressure (BP) and uric acid, but the mechanisms remain unclear. Preclinical studies demonstrate uric acid increases BP via increased renin–angiotensin system (RAS) expression, especially angiotensin (Ang) II, but the association of uric acid with Ang-(1–7) is unknown. Ang-(1–7), an alternative RAS product, counteracts Ang II by stimulating sodium excretion, vasodilation, and nitric oxide, thus contributing to lower BP. Plasma Ang-(1–7) is lower in preterm-born adolescents. We hypothesized uric acid is associated with a higher ratio of Ang II to Ang-(1–7) in plasma, especially in preterm-born adolescents. We measured BP, serum uric acid, and plasma RAS components in a cross-sectional analysis of 163 14-year olds (120 preterm, 43 term). We estimated the associations between uric acid and the RAS using generalized linear models adjusted for sex, obesity, sodium intake, and fat intake, stratified by birth status. Uric acid was positively associated with Ang II/Ang-(1–7) (adjusted ß(aß): 0.88?mg/dl, 95% CI 0.17–1.58), plasma renin activity (aß: 0.32?mg/dl, 95% CI 0.07–0.56), and aldosterone (aß: 1.26?mg/dl, 95% CI 0.18–2.35), and inversely with Ang-(1–7) (aß: -1.11?mg/dl, 95% CI -2.39 to 0.18); preterm birth did not modify these associations. Higher Ang II/Ang-(1–7) was associated with higher uric acid in adolescents. As preterm birth is associated with higher BP and uric acid, but lower Ang-(1–7), the imbalance between uric acid and Ang-(1–7) may be an important mechanism for the development of hypertension.

•Mindfulness and sham mindfulness meditation reduced pain during noxious heat.•Mindfulness and sham mindfulness meditation increased heart rate variability (HRV).•Mindfulness-based pain relief was associated with higher HRV.•Higher HRV during sham mindfulness meditation was associated with higher pain.

Background:Antenatal corticosteroid (ANCS) treatment hastens fetal lung maturity and improves survival of premature infants, but the long-term effects of ANCS are not well-described. Animal models suggest that ANCS increases the risk of cardiovascular disease through programmed changes in the renin-angiotensin (Ang)-aldosterone system (RAAS). We hypothesized that ANCS exposure alters the RAAS in adolescents born prematurely.Methods:A cohort of 173 adolescents born prematurely was evaluated, of whom 92 were exposed to ANCS. We measured plasma and urine Ang II and Ang-(1–7) and calculated Ang II/Ang-(1–7) ratios. We used general linear regression models to estimate the difference in the RAAS between the ANCS-exposed and unexposed groups, adjusting for confounding variables.Results:In unadjusted analyses, and after adjustment for sex, race, and maternal hypertension, ANCS exposure was associated with increased urinary Ang II/Ang-(1–7) (estimate 0.27 (95% CI 0.03, 0.5), P = 0.03), increased plasma Ang-(1–7) (0.66 (0.26, 1.07), P = 0.002), and decreased plasma Ang II/Ang-(1–7) (−0.48 (−0.91, −0.06), P = 0.03).Conclusion:These alterations indicate an imbalance in the urinary RAAS, promoting the actions of Ang II at the expense of Ang-(1–7), which over time may increase the risk of renal inflammation and fibrosis and ultimately hypertension and renal disease.

Introduction:Secondary injury due to neuroinflammation poses a target in ICH research. We have demonstrated elevated serum IL-12 levels in rodents with cardiometabolic syndrome with hypertension compared with normotensive rodents. As hypertension is a risk factor in ICH, it is critical to understand whether there are also local cerebral changes in pro-inflammatory cytokines to explore targets for therapeutics.Methods:A pilot study of autologous-injection ICH was induced in (mREN2)27 (n=7) and control Hanover Sprague-Dawley (SD) (n=7) rats. Animals underwent tail-cuff blood pressure measurement, pre- and post-ICH neurobehavioral testing, and serum cytokine measurements. Sacrifice with brain harvest was performed at 24h and 7h post-ICH. Homogenization of the brain with quantitative measurement of hemoglobin and IL-12 concentrations was performed.Results:IL-12 peaked in the ipsilateral hemisphere in both SD and mREN rats at 24h post-ICH. There was a significant difference in local IL-12 levels between the ipsilateral and contralateral hemispheres at 72h, with minimal IL-12 elevation in the contralateral hemisphere (p=0.05) (Figure 1). Additionally, there was high correlation between hematoma size and IL-12 local expression. While there was no difference in cerebral IL-12 levels between strains, there was a significant elevation in baseline IL-12 serum levels in mREN compared to SD rats, and a trend towards serum elevation at 72h post-ICH.Conclusions:Cerebral IL-12 levels ipsilateral to ICH are significantly elevated compared to contralateral levels and peak 24h post-ICH. While serum IL-12 levels are elevated in rats with cardiometabolic syndrome, this is not seen in brain tissue following ICH. With the high correlation between hematoma size and IL-12 expression, these findings implicate macrophage recruitment from the hematoma as a driver of neuroinflammation and potential therapeutic target to augment surgical interventions for ICH.

Hypotension after trauma is most commonly assumed to be hemorrhagic, or hypovolemic, in origin. However, hypotension may occur in pediatric patients with isolated head injury, challenging accepted tenets of trauma care. We sought to quantify the contribution of head injury to the development of hypotension after pediatric trauma.

As they age, Sprague–Dawley (SD) rats develop elevated systolic blood pressure associated with impaired baroreflex sensitivity (BRS) for control of heart rate. We previously demonstrated in young hypertensive (mRen2)27 rats that impaired BRS is restored by CB1cannabinoid receptor blockade in the solitary tract nucleus (NTS), consistent with elevated content of the endocannabinoid 2-arachidonoylglycerol (2-AG) in dorsal medulla relative to normotensive SD rats. There is no effect of CB1receptor blockade in young SD rats. We now report in older SD rats that dorsal medullary 2-AG levels are 2-fold higher at 70 versus 15 weeks of age (4.22 ± 0.61 vs. 1.93 ± 0.22 ngmg tissue; P< 0.05). Furthermore, relative expression of CB1receptor messenger RNA is significantly lower in aged rats, whereas CB2receptor messenger RNA is significantly higher. In contrast to young adult SD rats, microinjection of the CB1receptor antagonist SR141716A (36 pmole) into the NTS of older SD rats normalized BRS in animals exhibiting impaired baseline BRS (0.56 ± 0.06 baseline vs. 1.06 ± 0.05 msmm Hg after 60 minutes; P< 0.05). Therefore, this study provides evidence for alterations in the endocannabinoid system within the NTS of older SD rats that contribute to age-related impairment of BRS.

We hypothesized that orthostatic intolerance (OI) is associated with gastric dysrhythmias, nausea, and abdominal pain, which improves using fludrocortisone to treat OI.

We hypothesized that orthostatic intolerance (OI) is associated with gastric dysrhythmias, nausea, and abdominal pain, which improves using fludrocortisone to treat OI. Patients (n = 16, girls) with OI completed questionnaires before and after fludrocortisone treatment (age 14.8 ± 2.8 years). Ten patients underwent electrogastrograms (EGGs) before fludrocortisone. All EGGs showed gastric dysrhythmias. Fludrocortisone reduced mean scores as follows: nausea, 3.1 ± 0.8 to 2.1 ± 1.1 (P= 0.016); dizziness, 3.0 ± 1.0 to 2.2 ± 1.1 (P= 0.0371); abdominal pain, 2.8 ± 1.3 to 1.9 ± 1.4 (P= 0.0063); flushing, 2.3 ± 1.2 to 1.5 ± 1.4 (P= 0.0476); and missing school, 2.2 ± 1.5 to 1.2 ± 1.5 (P= 0.0078). Chronic nausea and abdominal pain accompany OI and improve with OI treatment.

Ouabain (Oua)-induced hypertension in rodents provides a model to study cardiovascular changes associated with human hypertension. We examined vascular function in rats after a long-term treatment with Oua. Systolic blood pressure was measured by tail-cuff plethysmography in male Sprague–Dawley rats treated with Oua (∼25 µgd) or placebo for 8 weeks. Blood pressure increased in Oua-treated animals, reaching 30 above baseline systolic blood pressure after 7 weeks. At the end of treatment, vascular responses were studied in mesenteric resistance arteries (MRAs) by wire myography. Contraction to potassium chloride in intact and denuded arteries showed greater sensitivity in Oua-treated animals. Contraction to phenylephrine and relaxation to acetylcholine were similar between groups with a lower response to sodium nitroprusside in Oua-treated arteries. Sensitivity to endothelin-1 was higher in Oua-treated arteries. Na-KATPase activity was decreased in MRAs from Oua-treated animals, whereas protein expression of the Na-KATPase 2isoform was increased in heart and unchanged in mesenteric artery. Preincubation with indomethacin (10−5M) or N-nitro-L-arginine methyl ester (10−4M) abolished the differences in potassium chloride response and Na-KATPase activity. Changes in MRAs are consistent with enhanced vascular smooth muscle cell reactivity, a contributor to the increased vascular tone observed in this model of hypertension.

Long-term systemic blockade of the renin–angiotensin system (RAS) with either an angiotensin (Ang) II type 1 receptor antagonist or an angiotensin-converting enzyme inhibitor attenuates age-related cardiac remodeling and oxidative damage, and improves myocardial relaxation. However, the role of the brain RAS in mediating the development of diastolic dysfunction during aging is not known. We hypothesized that low brain RAS protects against the development of age-related diastolic dysfunction and left ventricular remodeling.

Transgenic (mRen2)27 rats are hypertensive with impaired baroreflex sensitivity for control of heart rate compared with Hannover Sprague–Dawley rats. We assessed blood pressure and baroreflex function in male hemizygous (mRen2)27 rats (30–40 weeks of age) instrumented for arterial pressure recordings and receiving into the cisterna magna either an Ang-(1-7) fusion protein or a control fusion protein (CTL-FP). The maximum reduction in mean arterial pressure achieved was −38 ± 7 mm Hg on day 3, accompanied by a 55 enhancement in baroreflex sensitivity in Ang-(1-7) fusion protein-treated rats. Both the high-frequency alpha index (HF-) and heart rate variability increased, suggesting increased parasympathetic tone for cardiac control. The mRNA levels of several components of the renin–angiotensin system in the dorsal medulla were markedly reduced including renin (−80), neprilysin (−40), and the AT1areceptor (−40). However, there was a 2-fold to 3-fold increase in the mRNA levels of the phosphatases PTP-1b and dual-specificity phosphatase 1 in the medulla of Ang-(1-7) fusion protein-treated rats. Our finding that replacement of Ang-(1-7) in the brain of (mRen2)27 rats reverses in part the hypertension and baroreflex impairment is consistent with a functional deficit of Ang-(1-7) in this hypertensive strain. We conclude that the increased mRNA expression of phosphatases known to counteract the phosphoinositol 3 kinase and mitogen-activated protein kinases, and the reduction of renin and AT1areceptor mRNA levels may contribute to the reduction in arterial pressure and improvement in baroreflex sensitivity in response to Ang-(1-7).

Despite positive metabolic effects, genetic deletion of protein phosphatase 1b (PTP1b) results in sympathetically mediated elevations in arterial pressure (AP) in mice. Because several PTP1b-regulated peptides also impair the baroreflex sensitivity (BRS) for control of heart rate (HR), we hypothesized that PTP1b in the solitary tract nucleus (NTS) participates in the maintenance of resting baroreflex function. To test this hypothesis, we performed acute bilateral microinjection of an allosteric PTP1b inhibitor (100 nM120 nL) in the NTS of urethanechloralose anesthetized Sprague-Dawley rats and assessed the BRS, responses to cardiac vagal chemosensitive fiber activation, and resting AP and HR before and after the injection. PTP1b inhibition impaired the BRS for bradycardia (n = 6; 0.93 ± 0.14 baseline vs. 0.48 ± 0.04 at 10 minutes vs. 0.49 ± 0.04 millisecondmm Hg at 60 minutes; P< 0.01), with no significant effect on the BRS for tachycardia (0.30 ± 0.16 baseline vs. 0.24 ± 0.08 at 10 minutes vs. 0.24 ± 0.12 millisecondmm Hg at 60 minutes). The reduced BRS for bradycardia was associated with a significant decrease in alpha-adrenergic responsiveness to phenylephrine at 60 minutes after PTP1b inhibition. Injection of the PTP1b inhibitor in the NTS elicited transient decreases in AP and HR in these animals. However, there was no effect of the inhibitor on depressor or bradycardic responses elicited by activation of cardiac vagal chemosensitive fibers, which converge with baroreceptor afferents in the NTS. These results suggest that PTP1b within the NTS may be a novel molecular mechanism for preservation of resting baroreflex function and provides further evidence for deleterious cardiovascular effects associated with PTP1b inhibition.

The phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway in brain of spontaneously hypertensive rats, but not Wistar–Kyoto (WKY) rats, contributes to elevated mean arterial pressure (MAP). The role of PI3K in the regulation of blood pressure or autonomic function in the nucleus tractus solitarii (NTS) is yet to be established in other Ang II–dependent models of hypertension. Thus, we microinjected PI3K inhibitors, wortmannin or LY294002, into the NTS, and measured MAP, baroreflex sensitivity (BRS) for heart rate (HR) control, and HR variability (HRV) in mRen2.Lewis congenic and (mRen2)27 transgenic rats. Bilateral NTS microinjections of wortmannin (100 nmol/L; 50 nL) reduced MAP in (mRen2)27 and mRen2.Lewis rats (33 ± 5 mm Hg, n = 7, and 32 ± 6 mm Hg, n = 9, respectively) for approximately 90 minutes. Spectral and sequence analysis showed improvements in spontaneous BRS and HRV (50%–100%) after treatment in both hypertensive strains. Injections of wortmannin into NTS of Hannover Sprague–Dawley or Lewis control rats failed to alter MAP, BRS, or HRV. In mRen2.Lewis, but not in control Lewis rats, LY294002 (50 μmole/L) reduced MAP and increased BRS and HRV similar to wortmannin. Thus, the pharmacologic blockade of the PI3K signaling pathway in NTS reveals an important contribution to resting MAP and BRS in rats with overexpression of the Ren2 gene.

We assessed the contribution of angiotensin-(1-7) [Ang-(1-7)] to captopril-induced cardiovascular protection in spontaneously hypertensive rats (SHRs) chronically treated with the nitric oxide synthesis inhibitor NG-nitro-l-arginine methyl ester (SHR-l). NG-nitro-l-arginine methyl ester (80 mg/L) administration for 3 weeks increased mean arterial pressure (MAP) from 196 ± 6 to 229 ± 3 mm Hg (P< 0.05). Treatment of SHR-l with Ang-(1-7) antagonist [d-Ala7]-Ang-(1-7) (A779; 744 μg·kg−1·d−1ip) further elevated MAP to 253 ± 6 mm Hg (P< 0.05 vs SHR-l or SHR). Moreover, A779 treatment attenuated the reduction in MAP and proteinuria by either captopril (300 mg/L in drinking water) or hydralazine (1.5 mg·kg−1·d−1ip). In isolated perfused hearts, the recovery of left ventricular function from global ischemia was enhanced by captopril or hydralazine treatment and was exacerbated with A779. The Ang-(1-7) antagonist attenuated the beneficial effects of captopril and hydralazine on cardiac function. Recovery from global ischemia was also improved in isolated SHR-l hearts acutely perfused with captopril during both the perfusion and reperfusion periods. The acute administration of A779 reduced the beneficial actions of captopril to improve recovery after ischemia. We conclude that during periods of reduced nitric oxide availability, endogenous Ang-(1-7) plays a protective role in effectively buffering the increase in blood pressure and renal injury and the recovery from cardiac ischemia. Moreover, Ang-(1-7) contributes to the blood pressure lowering and tissue protective actions of captopril and hydralazine in a model of severe hypertension and end-organ damage.

The kidney is an important target for the actions of the renin-angiotensin system (RAS) and this tissue contains a complete local RAS that expresses the bioactive peptides angiotensin II (ANG II) and Ang-(1–7). We find both angiotensin type 1 (AT1R) and type 2 (AT2R) receptors expressed on renal nuclei that stimulate reactive oxygen species and nitric oxide (NO), respectively. Since Ang-(1–7) also exhibits actions within the kidney and the Ang-(1–7)/Mas receptor protein contains a nuclear localization sequence, we determined the expression of Ang-(1–7) receptors in nuclei isolated from the kidneys of young adult sheep. Binding studies with 125I-[Sar1Thr8]-ANG II revealed sites sensitive to the Ang-(1–7) antagonist [d-Ala7]-Ang-(1–7) (DALA, A779), as well as to AT2and AT1antagonists. Incubation of Ang-(1–7) [10−15to 10−9M] with isolated cortical nuclei elicited a dose-dependent increase in the fluorescence of the NO indicator [4-amino-5-methylamino-2′,7′]-difluorofluorescein diacetate. The NO response to Ang-(1–7) was abolished by the NO inhibitor N-nitro-l-arginine methyl ester and DALA, but not the AT1antagonist losartan or the AT2blocker PD123319. Immunofluorescent studies utilizing the Ang-(1–7)/Mas receptor antibody revealed immunolabeling of the proximal tubules but not staining within the glomerulus in cortical sections of the sheep kidney. In the nuclear fraction of isolated proximal tubules, immunoblots revealed the precursor angiotensinogen and renin, as well as functional activity for ACE, ACE2, and neprilysin. We conclude that renal nuclei express Ang-(1–7)/Mas receptors that are functionally linked to NO formation. The marked sensitivity of the intracellular NO response to Ang-(1–7) implicates a functional role of the Ang-(1–7) axis within the nucleus. Moreover, evidence for the precursor and enzymatic components of the RAS within the nuclear compartment of the proximal tubules provides a potential pathway for the intracellular generation of Ang-(1–7).

Expression of nuclear angiotensin II type 1 (AT1) receptors in rat kidney provides further support for the concept of an intracellular renin-angiotensin system. Thus we examined the cellular distribution of renal ANG II receptors in sheep to determine the existence and functional roles of intracellular ANG receptors in higher order species. Receptor binding was performed using the nonselective ANG II antagonist 125I-[Sar1,Thr8]-ANG II (125I-sarthran) with the AT1antagonist losartan (LOS) or the AT2antagonist PD123319 (PD) in isolated nuclei (NUC) and plasma membrane (PM) fractions obtained by differential centrifugation or density gradient separation. In both fetal and adult sheep kidney, PD competed for the majority of cortical NUC (≥70%) and PM (≥80%) sites while LOS competition predominated in medullary NUC (≥75%) and PM (≥70%). Immunodetection with an AT2antibody revealed a single ∼42-kDa band in both NUC and PM extracts, suggesting a mature molecular form of the NUC receptor. Autoradiography for receptor subtypes localized AT2in the tubulointerstitium, AT1in the medulla and vasa recta, and both AT1and AT2in glomeruli. Loading of NUC with the fluorescent nitric oxide (NO) detector DAF showed increased NO production with ANG II (1 nM), which was abolished by PD and N-nitro-l-arginine methyl ester, but not LOS. Our studies demonstrate ANG II receptor subtypes are differentially expressed in ovine kidney, while nuclear AT2receptors are functionally linked to NO production. These findings provide further evidence of a functional intracellular renin-angiotensin system within the kidney, which may represent a therapeutic target for the regulation of blood pressure.

Endogenous angiotensin (Ang)-(1-7) enhances, while Ang II attenuates, baroreceptor sensitivity (BRS) for reflex control of heart rate (HR) in Sprague-Dawley (SD) rats. In (mRen2)27 renin transgenic rats [(mRen2)], there is overexpression of the mouse Ren2 gene in brain, leading to elevated Ang II and reduced Ang-(1-7) in brain medullary, and associated with hypertension and impaired BRS.

Despite the evidence that angiotensin-converting enzyme (ACE)2 is a component of the renin-angiotensin system (RAS), the influence of ACE2 on angiotensin metabolism within the kidney is not well known, particularly in experimental models other than rats or mice. Therefore, we investigated the metabolism of the angiotensins in isolated proximal tubules, urine, and serum from sheep. Radiolabeled [125I]ANG I was hydrolyzed primarily to ANG II and ANG-(1–7) by ACE and neprilysin, respectively, in sheep proximal tubules. The ACE2 product ANG-(1–9) from ANG I was not detected in the absence or presence of ACE and neprilysin inhibition. In contrast, the proximal tubules contained robust ACE2 activity that converted ANG II to ANG-(1–7). Immunoblots utilizing an NH2terminal-directed ACE2 antibody revealed a single 120-kDa band in proximal tubule membranes. ANG-(1–7) was not a stable product in the tubule preparation and was rapidly hydrolyzed to ANG-(1–5) and ANG-(1–4) by ACE and neprilysin, respectively. Comparison of activities in the proximal tubules with nonsaturating concentrations of substrate revealed equivalent activities for ACE (ANG I to ANG II: 248 ± 17 fmol·mg−1·min−1) and ACE2 [ANG II to ANG-(1–7): 253 ± 11 fmol·mg−1·min−1], but lower neprilysin activity [ANG II to ANG-(1–4): 119 ± 24 fmol·mg−1·min−1; P< 0.05 vs. ACE or ACE2]. Urinary metabolism of ANG I and ANG II was similar to the proximal tubules; soluble ACE2 activity was also detectable in sheep serum. In conclusion, sheep tissues contain abundant ACE2 activity that converts ANG II to ANG-(1–7) but does not participate in the processing of ANG I into ANG-(1–9).

During aging increases in body weight, insulin resistance, and elevated systolic pressure contribute to the development of metabolic syndrome. Long-term systemic blockade of the renin-angiotensin system (RAS) with either an angiotensin (Ang) II type 1 (AT1) receptor antagonist or angiotensin converting enzyme inhibitor improves insulin sensitivity and decreases risk of new onset (type II) diabetes. However, the role of the brain RAS in mediating development of insulin insensitivity during aging is not known. Therefore, we compared responses to an oral glucose load in transgenic rats with selective antisense suppression of brain angiotensinogen (ASrAogen); (mRen2)27 rats with high brain angiotensin II; and control Hannover Sprague-Dawley (SD) rats, at wk 16 and 68 of age. ASrAogen animals had lower body weight than either SD or (mRen2) 27 rats at both ages (p<0.001). The oral glucose tolerance test at 16 wk in (mRen2)27 animals revealed a higher glucose-insulin index (154,421±11,231 untits; p<0.05) and a lower glucose-insulin index in ASrAogen rats (41,580±10,923 units, p<0.05) compared to SD rats (97,134±19,822 units), suggesting insulin resistance in the (mRen2)27 and enhanced insulin sensitivity in the ASrAogen relative to SD rats. At 68 wk, the glucose-insulin index remained low in the ASrAogen rats as evidence of maintained insulin sensitivity during aging compared with either SD or (mRen2)27 (p<0.05). SD animals do not differ from (mRen2)27 rats at 68 wk indicating the development of a state of relative insulin resistance with increased age in the SD rats. Moreover, there was a positive correlation (r=0.44; p<0.05) between body weight and the glucose-insulin index in SD, but not ASrAogen or (mRen2)27 rats. The relationships between insulin and leptin, insulin and glucose, and leptin and body weight observed in SD rats were absent in ASrAogen and (mRen2)27 rats. We conclude that the glial RAS plays a role in development of insulin resistance as well as influencing weight gain associated with early aging.

We established a new congenic model of hypertension, the mRen2.Lewis rat and assessed the intracellular expression of angiotensin peptides and receptors in the kidney. The congenic strain was established from the backcross of the (mRen2)27 transgenic rat that expresses the mouse renin 2 gene onto the Lewis strain. The 20-wk-old male congenic rats were markedly hypertensive compared with the Lewis controls (systolic blood pressure: 195 ± 2 vs. 107 ± 2 mmHg, P< 0.01). Although plasma ANG II levels were not different between strains, circulating levels of ANG-(1–7) were 270% higher and ANG I concentrations were 40% lower in the mRen2.Lewis rats. In contrast, both cortical (CORT) and medullary (MED) ANG II concentrations were 60% higher in the mRen2.Lewis rats, whereas tissue ANG I was 66 and 84% lower in CORT and MED. For both strains, MED ANG II, ANG I, and ANG-(1–7) were significantly higher than CORT levels. Intracellular ANG II binding distinguished nuclear (NUC) and plasma membrane (PM) receptor using the ANG II radioligand 125I-sarthran. Isolated CORT nuclei exhibited a high density (Bmax>200 fmol/mg protein) and affinity for the sarthran ligand (KD<0.5 nM); the majority of these sites (>95%) were the AT1receptor subtype. CORT ANG II receptor Bmaxand KDvalues in nuclei were 75 and 50% lower, respectively, for the mRen2.Lewis vs. the Lewis rats. In the MED, the PM receptor density (Lewis: 50 ± 4 vs. mRen2.Lewis: 21 ± 5 fmol/mg protein) and affinity (Lewis: 0.31 ± 0.1 vs. 0.69 ± 0.1 nM) were lower in the mRen2.Lewis rats. In summary, the hypertensive mRen2.Lewis rats exhibit higher ANG II in both CORT and MED regions of the kidney. Evaluation of intracellular ANG II receptors revealed lower CORT NUC and MED PM AT1sites in the mRen2.Lewis. The downregulation of AT1sites in the mRen2.Lewis rats may reflect a compensatory response to dampen the elevated levels of intrarenal ANG II.

Transgenic rats with targeted decreased glial expression of angiotensinogen (ASrAogen rats) did not show an increase in systolic pressure compared with Sprague-Dawley (SD) rats during aging (15–69 wk of age). ASrAogen animals had lower body weights throughout the study, similar to reports for animals with systemic knockout of angiotensinogen or treated long term with renin-angiotensin system (RAS) blockers. Further characterization of indexes of growth and metabolism in ASrAogen rats compared with (mRen2)27 and SD rats, which express elevated versus normal brain and tissue angiotensin II levels, respectively, revealed that serum leptin was 100–200% higher in SD and (mRen2)27 rats at 46 wk and 69 wk of age. Consistent with low serum leptin, ASrAogen rats had higher food intake (73%) compared with SD or (mRen2)27 rats. (mRen2)27 rats had higher resting insulin levels than ASrAogen rats at all ages. Insulin levels were constant during aging in ASrAogen rats, whereas an increase occurred in SD rats, leading to higher insulin levels at 46 and 69 wk of age compared with ASrAogen rats. IGF-1 was comparable among strains at all ages, but (mRen2)27 rats had longer and ASrAogen rats had shorter tail lengths versus SD rats at 15 wk of age. In conclusion, reduced expression of glial angiotensinogen blunts the age-dependent rise in insulin levels and weight gain, findings that mimic the effects of long-term systemic blockade of the RAS or systemic knockout of angiotensinogen. These data implicate glial angiotensinogen in the regulation of body metabolism as well as hormonal mechanisms regulating blood pressure.

The brain renin-angiotensin system (RAS) is important in fluid balance and blood pressure regulation. In this study, we compared angiotensin (Ang) receptor density in the subfornical organ (SFO) and paraventricular nucleus (PVN) of a) brain angiotensinogen deficient rats (ASrAogen); b) those with high levels of brain Ang II [(mRen2)27]; c) Hannover Sprague Dawley (SD) rats at 48 and 68 wks of age. Since there was no difference between the two ages in any of the three strains, the data from the 48 and 68 wk time points were combined. There was a significantly higher level of AT1 receptors in the SFO and PVN of ASrAogen animals compared to both the SD and (mRen2)27 rats. This suggests that the brain RAS is important in regulating receptor density and that the differences may be explained by lower levels of the peptide locally. These higher levels of receptors suggest that the ASrAogen animals in adulthood and early aging would be more sensitive to either circulating or endogenous brain Ang II than the SD animals of similar age. In contrast, the similar receptor density in the (mRen2)27 and SD rats suggest that previous reports of reduced responses in the (mRen2)27 rats may result from differences in post receptor mechanisms such as intracellular signaling. Moreover, our data reveal that functional assessments are necessary in addition to receptor density levels to understand the consequences of long-term alterations in brain tissue peptides.

In the kidney, angiotensin-(1–7) Ang-(1–7) exhibits diuretic and natriuretic properties associated with an increase in prostaglandin production. The prohypertensive effects of Ang II are attenuated in rats infused with Ang-(1–7), consistent with recent work showing that Ang-(1–7) downregulates AT1receptors in Chinese hamster ovary–AT1Aor vascular smooth muscle cells. To determine whether exposure to Ang-(1–7) reduces AT1receptors in the kidney through an increase in prostaglandin production, kidney slices from Sprague-Dawley rats were incubated with 10 n M–1 MAng-(1–7) in the presence or absence of 5 Mmeclofenamate, a cyclooxygenase inhibitor. Following these treatments, the kidney slices were retrieved, frozen, and sectioned for determination of 125I-Ang II binding using in vitro receptor autoradiography. Greater than 90 of the specific binding was competed for by losartan, indicating that the majority of binding was to the AT1receptor. Incubation of kidney slices with 1 MAng-(1–7) caused a 20 reduction in 125I-Ang II binding (n 8) in the cortical tubulointerstitium, which was prevented when Ang-(1–7)-treated slices were incubated in the presence of 5 Mmeclofenamate (1 ± 2 increase, n 8; p < 0.05). Incubation with 5 Mmeclofenamate alone had no effect on 125I-Ang II binding (−3 ± 3). The decrease in 125I-Ang II binding with Ang-(1–7) was also blocked by the Ang-(1–7) antagonist d-Ala7-Ang-(1–7). Treatment with 1 Md-Ala7-Ang-(1–7) alone had no effect on 125I-Ang II binding (−3 ± 6 of control). Pretreatment with 1 MAng II caused a similar reduction in 125I-Ang II binding in the cortical tubulointerstitium. Neither Ang-(1–7) nor Ang II had any effect on 125I-Ang II binding in the glomeruli and the area of the vasa recta of the kidney. These original findings suggest that prior exposure to Ang-(1–7) or Ang II causes a modest decrease in the number of AT1receptors in the cortical tubulointerstitial area of the kidney. The reduction in Ang II binding by Ang-(1–7) was blocked by meclofenamate and d-Ala7-Ang-(1–7), suggesting that cyclooxygenase products released through activation of a novel receptor participate in this effect.

Angiotensin (Ang)-(1-7), the amino terminal heptapeptide fragment of Ang II, is an endogenous Ang peptide with vasodilatory and antiproliferative actions. Because Ang II causes vasoconstriction and promotes growth through activation of Ang type 1 (AT1) receptors, we investigated whether the actions of Ang-(1-7) are due to its regulation of these receptors. Studies were performed in CHO cells stably transfected with the AT1Areceptor. Ang-(1-7) competed poorly with 125I-Ang II for the AT1Abinding site and was ineffective at shifting the IC50for Ang II competition with 125I-Ang II for binding to the AT1Areceptor. However, if CHO-AT1Acells were pretreated with Ang-(1-7) and then treated with acidic glycine to remove surface-bound ligand, the heptapeptide caused a concentration-dependent reduction in Ang II binding, with a maximal inhibition to 67.8 ± 4.6 of total (p < 0.05) at 1 MAng-(1-7) compared with a reduction to 24 of total by 10 n MAng II. Ang-(1-7) pretreatment caused a small but significant decrease in the affinity of 125I-Ang II for the AT1Areceptor and a significant reduction in the total number of binding sites. The Ang-(1-7)-induced reduction in binding was rapid (occurring as early as 5 min after exposure to the peptide), was maintained for 30 min during continued exposure of the cells to Ang-(1-7), and rapidly recovered after removal of the heptapeptide. The AT1receptor antagonist L-158,809 reduced the Ang-(1-7)-induced downregulation of the AT1Areceptor, suggesting that interactions with AT1Areceptors mediate the regulatory events. Pretreatment with 1 Mor 10 MAng-(1-7) significantly reduced inositol phosphate production in response to 10 n MAng II. The decrease in binding and responsiveness of the AT1Areceptor after exposure to micromolar concentrations of Ang-(1-7) suggests that the heptapeptide downregulates the AT1Areceptor to reduce responses to Ang II. Because downregulation of the receptor only occurred at micromolar concentrations of the heptapeptide, our findings suggest that Ang-(1-7) is not a potent antagonist at the AT1Areceptor. However, when the balance between Ang II and Ang-(1-7) is shifted in favor of Ang-(1-7), such as during inhibition of Ang-converting enzyme, some contribution of this mechanism may come into play.

We studied the vasoconstrictor effects of the thromboxane A2(TxA2) analogue U46619 in the perfused hind limb of rats under constant flow before and after intravenous injection of irbesartan, an angiotensin II AT1receptor antagonist, to test whether irbesartan interacts in vivowith the thromboxane A2/prostaglandin endoperoxidase H2(TxA2/PGH2) receptor.

Two of the primary sites of actions for angiotensin (ANG)-(1—7) are the vasculature and the kidney. Because little information exists concerning the metabolism of ANG-(1—7) in these tissues, we investigated the hydrolysis of the peptide in rat lung and renal brush-border membrane (BBM) preparations. Radiolabeled ANG-(1—7) was hydrolyzed primarily to ANG-(1—5) by pulmonary membranes. The ANG-converting enzyme (ACE) inhibitor lisinopril abolished the generation of ANG-(1—5), as well as that of smaller metabolites. Kinetic studies of the hydrolysis of ANG-(1—7) to ANG-(1—5) by somatic (pulmonary) and germinal (testes) forms of rat ACE yielded similar values, suggesting that the COOH-domain is responsible for the hydrolysis of ANG-(1—7). Pulmonary metabolism of ANG-(1—5) yielded ANG-(3—5) and was independent of ACE but may involve peptidyl or dipeptidyl aminopeptidases. In renal cortex BBM, ANG-(1—7) was rapidly hydrolyzed to mono- and dipeptide fragments and ANG-(1—4). Aminopeptidase (AP) inhibition attenuated the hydrolysis of ANG-(1—7) and increased ANG-(1—4) formation. Combined treatment with AP and neprilysin (Nep) inhibitors abolished ANG-(1—4) formation and preserved ANG-(1—7). ACE inhibition had no effect on the rate of hydrolysis or the metabolites formed in the BBM. In conclusion, ACE was the major enzymatic activity responsible for the metabolism of ANG-(1—7) in the lung, which is consistent with the ability of ACE inhibitors to increase the half-life of circulating ANG-(1—7) and raise endogenous levels of the peptide. An alternate pathway of metabolism was revealed in the renal cortex, where increased AP and Nep activities, relative to ACE activity, promote conversion of ANG-(1—7) to ANG-(1—4) and smaller fragments.

The present study determined the effect of either occlusion of the left renal artery for 60 min (ischemia) or sham operation on angiotensin (ANG) receptors and tissue and urinary levels of ANG peptides between 24 and 72 h recovery in male Sprague-Dawley rats. At 24 h postischemia, urinary concentrations of ANG I and ANG-(1–7) rose by an average of 83 and 64%, respectively (P< 0.05) but had declined to control levels by 72 h. Tissue ANG II rose at 24 h in postischemic kidneys by an average of 63% compared with the contralateral nonischemic kidney (P< 0.05). Whereas the enzymatic activity of angiotensin-converting enzyme and neprilysin was reduced after ischemia, renal renin activity in ischemic kidneys rose by 74% compared with sham-operated kidneys. Receptor autoradiography using 125I-labeled [Sar1,Thr8]ANG II (125I-Sarthran) (0.8 nM) revealed a decreased apparent density of ANG receptors (>80% AT1) in ischemic kidneys with a trend for a decrease in the contralateral nonischemic kidneys compared with the kidneys from sham-operated rats. Twenty-four hours after ischemia, ANG II receptors decreased by 68% in glomeruli (P< 0.05), 49% in the outer cortical tubulointerstitial area (P< 0.05), and 48% in the inner cortical-outer medullary area of the vasa recta (P< 0.05). Medullary binding decreased ∼50% in both the ischemic kidney and the contralateral nonischemic kidney compared with sham. In all regions of the ischemic kidney, receptors recovered by 72 h to levels not different from sham control rats. The marked change in urinary ANG I and ANG-(1–7) at 24 h following occlusion indicates these peptides may be potential urinary markers for acute renal ischemia. The reduction of receptors in vascular and tubular regions of the ischemic kidney provides a mechanism for the loss of vasoconstrictor responses to ANG II following ischemia previously reported by others.

Prostaglandins are known to participate in the antihypertensive actions of angiotensin-converting enzyme (ACE) inhibition and angiotensin type I (AT1)-receptor antagonism. Because angiotensin-(1-7) Ang-(1-7) is markedly elevated after prolonged ACE-inhibitor treatment, we determined whether the antihypertensive effects of Ang-(1-7) were mediated by release of prostaglandins. Male spontaneously hypertensive rats (SHRs, 10 weeks) were treated for 9 days with either lisinopril (20 mg/kg) or losartan (10 mg/kg) or a combination of both drugs. Rats were implanted with catheters in the carotid artery and jugular vein to record blood pressure and to infuse drug solutions, respectively. Neutralization of circulating Ang-(1-7) by monoclonal antibody resulted in a dose-dependent increase in blood pressure in SHRs treated with either lisinopril or losartan. Administration of CGS 24592 to block Ang-(1-7) formation also resulted in an increase in blood pressure that was comparable to antibody infusion. However, Ang-(1-7) blockade evoked a greater elevation in blood pressure in the lisinopril and lisinopril/losartan-treated rats in comparison to those treated with losartan alone. Acute treatment with the cyclooxygenase (COX) inhibitor indomethacin increased blood pressure to a similar extent to that of CGS 24592, as well as blocked the increase in pressure with the neprilysin inhibitor in the lisinopril/losartan group. In the losartan-treated animals, however, indomethacin increased blood pressure by a larger extent than that of the Ang-(1-7) antibody or CGS 24592, and CGS 24592 did not abolish the subsequent pressor response to indomethacin in these animals. In contrast to the antibody or neprilysin inhibitor, administration of the Ang-(1-7) antagonist D-Ala7-Ang-(1-7) increased blood pressure to a similar extent in lisinopril or losartan treatments. Moreover, D-Ala7-Ang-(1-7) increased blood pressure to a comparable extent as indomethacin and blocked any further increase with the COX inhibitor in the losartan-treated SHRs. High-resolution emulsion autoradiography revealed 125I-Sarcosine1, Threonine8-Ang II (Sarthran) binding in the mesenteric artery and thoracic aorta in the presence of both LOS and the AT2antagonist PD123319. The non-AT1/non-AT2Sarthran binding was displaced by Ang-(1-7), DALA, or Ang II. These studies suggest that vasodilatory eicosanoids mediate the antihypertensive effects of endogenous Ang-(1-7) in both LIS and LIS/LOS therapies. Furthermore, in the presence of AT1-receptor blockade, Ang II may interact with a DALA-sensitive site to promote eicosanoid release.

Introduction:Spontaneous intracerebral hemorrhage (ICH) accounts for 10-15% of strokes and carries a 40% mortality rate within one month. Neuroinflammation plays a key role in secondary brain injury after ICH and inflammatory pathways may represent important therapeutic targets. A major obstacle in developing novel therapeutics is the lack of a translatable animal model that recapitulates the comorbidities of patient’s that suffer from ICH. Hypertension, diabetes, and obesity are components of the metabolic syndrome often seen in ICH patients, and may modulate the neuroinflammatory response to ICH. Thus, we aimed to characterize peripheral markers of systemic inflammation in a transgenic hypertensive (mRen2)27 rat model of metabolic syndrome.Methods:Serum proinflammatory cytokines IL-1β and IL-12 were measured using ELISA in naïve male and female (mRen2)27 (n=15) and healthy SD rats at 32 weeks of age (n=14). Systolic blood pressure (SBP) was measured by tail-cuff plethysmography. Statistical analyses of group differences were conducted using two-sample t-tests.Results:SBP (mm Hg) was significantly higher in the (mRen2)27 transgenic rats compared to SD rats, in both males (195 ± 4, n=8 vs. 125 ± 4, n=6, p< 0.05) and females (183±7, n=7 vs. 114±10, n=8, p< 0.05). Female (mRen2)27 rats had significantly higher serum concentrations (pg/mL) of IL-12 (1073±47, n=7 vs. 438±101, n=8, p< 0.05) and IL-1β (pg/mL) (11±3, n=7;. 4±1 , n=8, p< 0.05). Similarly, male (mRen2)27 rats had significantly higher levels of IL-12 compared to SD rats (825±58 , n=8 v. 582±90 , n=6, p< 0.05). IL-12 levels were higher in mRen female rats compared with males (1073±47, n=7 v. 825±58 , n=8, p< 0.05). Serum levels of IL-1β was not significantly different between male SD and (mRen2)27 rats.Conclusion:Compared to healthy SD rats, the pro-inflammatory cytokines IL-1β and IL-12 are elevated in a clinically-relevant animal model of spontaneous hypertension. These findings simulate human studies that have linked cardiometabolic disease with elevated levels of pro-inflammatory cytokines. Future studies using this animal model will seek to further characterize changes in perihematomal and peripheral levels of proinflammatory and inflammation-resolving cytokines following ICH.

Selective antagonists were used to determine the presence of angiotensin II (Ang II) receptor subtypes (ΑT1and AT2) in normal human renal cortex and renal cell carcinoma. Normal and tumor tissues were obtained from fresh radical nephrectomy specimens in 7 patients. All patients had a patent renal artery, and the mean preoperative serum creatinine level was 1.1mg./dl. Tissues were snap frozen and sectioned (14μm.) for in vitro autoradiography, then incubated in 125I-Ang II (0.3nM.), with or without unlabeled Ang II or subtype selective antagonists (1nM. to 1μΜ.), rinsed, air dried and apposed to SB-5 X-ray film for 3 to 21 days. In normal renal tissue, low densities of diffuse 125I-Ang II binding sites were observed in cortical areas containing tubules. Higher densities of binding sites occurred over glomeruli and large cortical vessels. Specific binding ranged between 60 and 90% depending on the area as determined by displacement with excess unlabeled Ang II. Specific binding in large cortical vessels was displaced by the two AT2selective antagonists PD123177 (1μΜ.) or CGP 42112A (0.01μΜ.), whereas these antagonists were less effective competitors for 125I-Ang II binding in glomeruli. In contrast, the AT1selective antagonists, DuP 753 and L-158,809 (0.1 and .01μΜ., respectively), were potent competitors for glomerular, but not extraglomerular, cortical vessel binding. In the normal cortical tubulointerstitium, both ΑΤ1and AT2antagonists caused partial displacement of specific binding (55 ± 12% AT1, 39 ± 12% AT2). Low density 125I-Ang II binding was present in all tumors. Specific binding averaged 59 ± 10% as defined by displacement with unlabeled Ang II (1μΜ.). As in the normal tubulointerstitial area, each of the selective antagonists produced partial displacement of the specific binding (60 ± 12% AT1, 31 ± 8% AT2).

Imidazoline/guanidinium receptive sites (IGRS) are shown to be present in the subfornical organ and hypothalamic arcuate nucleus by a derivative of cirazoline, 2-(3-amino-4-[125I]iodophenoxy)methylimidazoline ([125I]AMIPI). Because many of the nonpeptide angiotensin II (Ang II) receptor antagonists contain imidazole ring structures, they may interact with IGRS. Therefore, we studied competitive activity of Ang II and several nonpeptide Ang II receptor antagonists [DuP 753 (losartan), EXP 3174, CV11974, and PD123319] at IGRS in rat forebrain. The results showed specific binding of 944 ± 169 fmol/mg protein in the subfornical organ (n = 11) and of 367 ± 27 fmol/mg protein in the arcuate nucleus (n = 6) at 0.4 nM[125I]AMIPI, as defined by competition with 10 μMcirazoline. Specific [125I]AMIPI binding was competed for completely by 10 μMidazoxan or clonidine as further characterization of IGRS. Ang II and the nonpeptide AT1and AT2antagonists did not significantly compete for specific [125I]AMIPI binding in either brain region at concentrations of 10 μM(<20% competition with each compound), which is 10- to 100-fold higher than the concentration necessary to compete completely for their respective Ang II receptor subtypes. Only at the highest concentration (100 μM) did losartan compete significantly for binding (56 ± 8%). Therefore, Ang II receptor antagonists interact with IGRS in rat forebrain cardiovascular areas only at high concentrations.

Increasing evidence suggests an association between hypertension and abnormalities of glucose metabolism. Since components of the renin-angiotensin system exist in a variety of tissues consistent with paracrine actions of the peptide, we sought to determine whether the pancreas contains a local angiotensin system. We report the presence of angiotensinogen messenger (m) RNA, angiotensinogen protein, angiotensin II and high-affinity binding sites for angiotensin II in the canine pancreas. These novel findings establish a foundation for future studies to evaluate whether angiotensin acts as a paracrine regulator of endocrine and/or exocrine functions of the pancreas.