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

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.

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.

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.

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.

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.

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.

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.