Hossam A. Shaltout

Nuclear angiotensin II type 2 (AT2) receptors are functionally linked to nitric oxide production

Expression of nuclear angiotensin II type 1 (AT(1)) 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 (125)I-[Sar(1),Thr(8)]-ANG II ((125)I-sarthran) with the AT(1) antagonist losartan (LOS) or the AT(2) antagonist 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 (> or =70%) and PM (> or =80%) sites while LOS competition predominated in medullary NUC (> or =75%) and PM (> or =70%). Immunodetection with an AT(2) antibody revealed a single approximately 42-kDa band in both NUC and PM extracts, suggesting a mature molecular form of the NUC receptor. Autoradiography for receptor subtypes localized AT(2) in the tubulointerstitium, AT(1) in the medulla and vasa recta, and both AT(1) and AT(2) in 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 AT(2) receptors 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.

Leptin Impairs Cardiovagal Baroreflex Function at the Level of the Solitary Tract Nucleus

Circulating leptin is elevated in some forms of obesity-related hypertension, associated with impaired baroreflex function. Leptin receptors are present on vagal afferent fibers and neurons within the solitary tract nucleus, providing an anatomic distribution consistent with baroreflex modulation. Although solitary tract nucleus microinjection of 144 fmol/60 nL of leptin had no significant effect on baroreflex sensitivity for control of the heart rate in urethane/chloralose-anesthetized Sprague-Dawley rats, 500 fmol of leptin impaired baroreflex sensitivity for bradycardia in response to increases in pressure (1.15±0.04 versus 0.52±0.12 ms/mm Hg; P<0.01). Transgenic ASrAOGEN rats with low brain angiotensinogen have an upregulation of the leptin receptor and p85 &agr; mRNA in the dorsal medulla relative to Sprague-Dawley rats. Consistent with these observations, the response to leptin was enhanced in ASrAOGEN rats, because both the 144-fmol (1.46±0.08 versus 0.75±0.10 ms/mm Hg; P<0.001) and 500-fmol (1.36±0.32 versus 0.44±0.06 ms/mm Hg; P<0.05) leptin microinjections impaired baroreflex sensitivity. At these doses, leptin microinjection had no effect on resting pressure, heart rate, or the tachycardic response to decreases in pressure in Sprague-Dawley or ASrAOGEN rats. Thus, exogenous leptin at sites within the solitary tract nucleus impairs the baroreflex sensitivity for bradycardia induced by increases in arterial pressure, consistent with a permissive role in mediating increases in arterial pressure. Baroreflex inhibition was enhanced in animals with evidence of increased leptin receptor and relevant signaling pathway mRNA.

Alterations in Circulatory and Renal Angiotensin-Converting Enzyme and Angiotensin-Converting Enzyme 2 in Fetal Programmed Hypertension

Antenatal betamethasone treatment is a widely accepted therapy to accelerate lung development and improve survival in preterm infants. However, there are reports that infants who receive antenatal glucocorticoids exhibit higher systolic blood pressure in their early adolescent years. We have developed an experimental model of programming whereby the offspring of pregnant sheep administered clinically relevant doses of betamethasone exhibit elevated blood pressure. We tested the hypothesis as to whether alterations in angiotensin-converting enzyme (ACE), ACE2, and neprilysin in serum, urine, and proximal tubules are associated with this increase in mean arterial pressure. Male sheep were administered betamethasone (2 doses of 0.17 mg/kg, 24 hours apart) or vehicle at the 80th day of gestation and delivered at term. Sheep were instrumented at adulthood (1.8 years) for direct conscious recording of mean arterial pressure. Serum and urine were collected and proximal tubules isolated from the renal cortex. Betamethasone-treated animals had elevated mean arterial pressure (97±3 versus 83±2 mm Hg; P<0.05) and a 25% increase in serum ACE activity (48.4±7.0 versus 36.0±2.7 fmol/mL per minute) but a 40% reduction in serum ACE2 activity (18.8±1.2 versus 31.4±4.4 fmol/mL per minute). In isolated proximal tubules, ACE2 activity and expression were 50% lower in the treated sheep with no significant change in ACE or neprilysin activities. We conclude that antenatal steroid treatment results in the chronic alteration of ACE and ACE2 in the circulatory and tubular compartments, which may contribute to the higher blood pressure in this model of fetal programming–induced hypertension.

Update on the Angiotensin Converting Enzyme 2-Angiotensin (1–7)-Mas Receptor Axis: Fetal Programing, Sex Differences, and Intracellular Pathways

The renin-angiotensin-system (RAS) constitutes an important hormonal system in the physiological regulation of blood pressure. Indeed, dysregulation of the RAS may lead to the development of cardiovascular pathologies including kidney injury. Moreover, the blockade of this system by the inhibition of angiotensin converting enzyme (ACE) or antagonism of the angiotensin type 1 receptor (AT1R) constitutes an effective therapeutic regimen. It is now apparent with the identification of multiple components of the RAS that the system is comprised of different angiotensin peptides with diverse biological actions mediated by distinct receptor subtypes. The classic RAS can be defined as the ACE-Ang II-AT1R axis that promotes vasoconstriction, sodium retention, and other mechanisms to maintain blood pressure, as well as increased oxidative stress, fibrosis, cellular growth, and inflammation in pathological conditions. In contrast, the non-classical RAS composed of the ACE2-Ang-(1–7)-Mas receptor axis generally opposes the actions of a stimulated Ang II-AT1R axis through an increase in nitric oxide and prostaglandins and mediates vasodilation, natriuresis, diuresis, and oxidative stress. Thus, a reduced tone of the Ang-(1–7) system may contribute to these pathologies as well. Moreover, the non-classical RAS components may contribute to the effects of therapeutic blockade of the classical system to reduce blood pressure and attenuate various indices of renal injury. The review considers recent studies on the ACE2-Ang-(1–7)-Mas receptor axis regarding the precursor for Ang-(1–7), the intracellular expression and sex differences of this system, as well as an emerging role of the Ang1-(1–7) pathway in fetal programing events and cardiovascular dysfunction.

Glucocorticoid-Induced Fetal Programming Alters the Functional Complement of Angiotensin Receptor Subtypes Within the Kidney

We examined the impact of fetal programming on the functional responses of renal angiotensin receptors. Fetal sheep were exposed in utero to betamethasone (BMX; 0.17 mg/kg) or control (CON) at 80 to 81 days gestation with full-term delivery. Renal nuclear and plasma membrane fractions were isolated from sheep age 1.0 to 1.5 years for receptor binding and fluorescence detection of reactive oxygen species (ROS) or nitric oxide (NO). Mean arterial blood pressure and blood pressure variability were significantly higher in the BMX-exposed adult offspring versus CON sheep. The proportion of nuclear AT1 receptors sensitive to losartan was 2-fold higher (67±6% vs 27±9%; P<0.01) in BMX compared with CON. In contrast, the proportion of AT2 sites was only one third that of controls (BMX, 25±11% vs CON, 78±4%; P<0.01), with a similar reduction in sites sensitive to the Ang-(1-7) antagonist D-Ala7-Ang-(1-7) with BMX exposure. Functional studies revealed that Ang II stimulated ROS to a greater extent in BMX than in CON sheep (16±3% vs 6±4%; P<0.05); however, NO production to Ang II was attenuated in BMX (26±7% vs 82±14%; P<0.05). BMX exposure was also associated with a reduction in the Ang-(1-7) NO response (75±8% vs 131±26%; P<0.05). We conclude that altered expression of angiotensin receptor subtypes may be one mechanism whereby functional changes in NO- and ROS-dependent signaling pathways may favor the sustained increase in blood pressure evident in fetal programming.

Angiotensin-(1-12) Requires Angiotensin Converting Enzyme and AT1 Receptors for Cardiovascular Actions within the Solitary Tract Nucleus

The novel peptide, angiotensin (ANG)-(1-12), elicits a systemic pressor response and vasoconstriction. These effects are blocked by ANG converting enzyme (ACE) inhibitors or AT(1) receptor antagonists, suggesting a role as an ANG II precursor. However, ANG-(1-12) can serve as a substrate for either ANG II or ANG-(1-7) formation, depending on the local tissue enzymes. Although levels of ANG-(1-12) are higher than ANG I or ANG II in brain, the role and processing of this peptide for autonomic control of heart rate (HR) has yet to be considered. Thus we examined the effects of nucleus tractus solitarii (NTS) microinjection of ANG-(1-12) on baroreflex sensitivity for control of HR, resting arterial pressure (AP) and HR, and indexes of sympathovagal balance in urethane/chloralose anesthetized Sprague-Dawley rats. NTS injection of ANG-(1-12) (144 fmol/120 nl) significantly impaired the evoked baroreflex sensitivity to increases in AP [n = 7; 1.06 +/- 0.06 baseline vs. 0.44 +/- 0.07 ms/mmHg after ANG-(1-12)], reduced the vagal component of spontaneous baroreflex sensitivity and HR variability, and elicited a transient depressor response (P < 0.05). NTS pretreatment with an AT(1) receptor antagonist or ACE inhibitor prevented ANG-(1-12)-mediated autonomic and depressor responses. ANG-(1-12) immunostaining was observed in cells within the NTS of Sprague-Dawley rats, providing a potential intracellular source for the peptide. However, acute NTS injection of an ANG-(1-12) antibody did not alter resting baroreflex sensitivity, AP, or HR in these animals. Collectively, these findings suggest that exogenous ANG-(1-12) is processed to ANG II for cardiovascular actions at AT(1) receptors within the NTS. The lack of acute endogenous ANG-(1-12) tone for cardiovascular regulation in Sprague-Dawley rats contrasts with chronic immunoneutralization in hypertensive rats, suggesting that ANG-(1-12) may be activated only under hypertensive conditions.

Acute AT1-receptor blockade reverses the hemodynamic and baroreflex impairment in adult sheep exposed to antenatal betamethasone

To accelerate lung development and protect neonates from other early developmental problems, synthetic steroids are administered maternally in the third trimester, exposing fetuses that are candidates for premature delivery to them. However, steroid exposure at this point of gestation may lead to elevated blood pressure [mean arterial pressure (MAP)] during adolescence. We hypothesize that fetal exposure to steroids activates the renin-angiotensin system, inducing an elevation in blood pressure and attenuation of baroreflex sensitivity (BRS) that is angiotensin II dependent in early adulthood. To test this hypothesis, fetal sheep were exposed to betamethasone (Beta) or vehicle (control) administered to ewes at day 80 of gestation and delivered at full term. At 1.8 yr of age, male offspring were instrumented for conscious recording of MAP, heart rate, and measurement of BRS [as low-frequency-alpha, high-frequency-alpha, sequence (seq) UP, seq DOWN, and seq TOTAL]. Beta-exposed sheep (n = 6) had higher MAP than control sheep (n = 5) (93 + or - 2 vs. 84 + or - 2 mmHg, P < 0.01). Acute blockade of angiotensin type 1 receptors with candesartan (0.3 mg/kg iv) normalized MAP in Beta-exposed sheep (85 + or - 4 mmHg), with no effect in control sheep (82 + or - 3 mmHg). Before angiotensin type 1 blockade, BRS maximum gain was significantly lower in Beta-exposed vs. control sheep (11 + or - 3 vs. 26 + or - 3 ms/mmHg, P < 0.0.01). However, 45 min after candesartan injection, BRS was increased in Beta-exposed (21 + or - 5 ms/mmHg) and control (35 + or - 4 ms/mmHg) sheep. Heart rate variability (HRV) and blood pressure variability (BPV) revealed lower HRV (SD of beat-to-beat interval and root mean square of successive beat-to-beat differences in R-R interval duration) and higher BPV (SD of MAP, systolic arterial pressure in low-frequency range) in Beta-exposed sheep. Candesartan partially restored HRV in Beta-exposed sheep and fully corrected BPV. Thus, in utero exposure to synthetic glucocorticoids causes long-lasting programming of the cardiovascular system via renin-angiotensin system-dependent mechanisms.

Angiotensin peptides and central autonomic regulation.

Aging, hypertension, and fetal-programmed cardiovascular disease are associated with a functional deficiency of angiotensin (Ang)-(1-7) in the brain dorsomedial medulla. The resulting unrestrained activity of Ang II in brainstem regions negatively impacts resting mean arterial pressure, sympathovagal balance, and baroreflex sensitivity for control of heart rate. The differential effects of Ang II and Ang-(1-7) may be related to the cellular sources of these peptides as well as different precursor pathways. Long-term alterations of the brain renin-angiotensin system may influence signaling pathways including phosphoinositol-3-kinase and mitogen-activated protein kinase and their downstream mediators, and as a consequence may influence metabolic function. Differential regulation of signaling pathways in aging and hypertension by Ang II versus Ang-(1-7) may contribute to the autonomic dysfunction accompanying these states.

Angiotensin-(1-7) Deficiency and Baroreflex Impairment Precede the Antenatal Betamethasone Exposure-Induced Elevation in Blood Pressure

Betamethasone is administered to accelerate lung development and improve survival of premature infants but may be associated with hypertension later in life. In a sheep model of fetal programming resulting from exposure at day 80 of gestation to Betamethasone (Beta-exposed), adult sheep at 6 to 9 months or 1.8 years of age have elevated mean arterial pressure (MAP) and attenuated spontaneous baroreflex sensitivity (sBRS) for control of heart rate compared to age-matched controls associated with imbalances in angiotensin (Ang) II vs Ang-(1-7) tone. At 6 weeks of age, evoked BRS is already low in the Beta-exposed animals. In this study, we assessed the potential contribution of the renin-angiotensin system to the impaired sBRS. Female lambs (6 weeks old) with Beta exposure in utero had similar MAP to control lambs (78±2 vs 77±2 mm Hg, n=4–5 per group), but lower sBRS (8±1 vs 16±3 ms/mm Hg; P<0.05) and impaired heart rate variability. Peripheral AT1 receptor blockade using candesartan lowered MAP in both groups (≈10 mm Hg) and improved sBRS and heart rate variability in Beta-exposed lambs to a level similar to control. AT7 receptor blockade by infusion of D-ala Ang-(1-7) (700 ng/kg/min for 45 minutes) reduced sBRS 46%±10% in Beta-exposed vs in control lambs (P<0.15) and increased MAP in both groups (≈6±2 mm Hg). Our data reveal that Beta exposure impairs sBRS and heart rate variability at a time point preceding the elevation in MAP via mechanisms involving an imbalance in the Ang II/Ang-(1-7) ratio consistent with a progressive loss in Ang-(1-7) function.

Exaggerated sympathetic mediated responses to behavioral or pharmacological challenges following antenatal betamethasone exposure.

Glucocorticoid administration to women at risk for preterm delivery is standard practice to enhance neonatal survival. However, antenatal betamethasone exposure (β-exposure) increases mean arterial pressure (MAP) in adult sheep (1.8 yr old) and results in impaired baroreflex sensitivity (BRS) for control of heart rate (HR). In the current studies we tested the hypothesis that enhanced sympathetic nervous system and hypothalamo-pituitary-adrenal (HPA) axis-mediated responses are evident at an early age in β-exposed sheep. Pregnant ewes were administered betamethasone (0.17 mg/kg twice over 24 h) or vehicle (Veh-control) on the 80th day of gestation, and offspring were delivered at full term. Female β-exposed and control offspring instrumented at age 42 ± 3 days for conscious continuous recording of MAP and HR had similar resting values at baseline. However, BRS was ~45% lower in β-exposed offspring. β-Exposed lambs allowed to suckle for 10 min had a greater elevation in MAP than Veh-control lambs (19 ± 1 vs 12 ± 2 mmHg; n = 4-5, P < 0.05). MAP was reduced by 20% from baseline via sodium nitroprusside infusion (SNP) over 10 min, which triggered a rebound increase in MAP only in β-exposed lambs. HR increased with the reduction in MAP during SNP infusion in Veh-control lambs, whereas there was no change in HR with the reduction in MAP in β-exposed lambs. Combined vasopressin-CRF injection caused greater increases in MAP in the β-exposed lambs. Cortisol and ACTH responses were higher in response to SNP hypotension in the β-exposed lambs. The data reveal enhanced sympathetic and HPA axis responses associated with impaired BRS preceding differences in resting MAP in preweanling female lambs exposed in utero to glucocorticoids. The consequences of these alterations at an early age include eventual development of higher blood pressure in this ovine model of fetal programming.