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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-co, 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. spectral analysis; fetal programming; heart rate variability; blood pressure variability; renin-angiotensin system; hypertension doi: 10.1152/ajpheart.00100.2010.

Angiotensin-converting enzyme-2 (ACE2) converts angiotensin II (ANG II) to angiotensin-(1-7) [ANG-(1-7)], and this enzyme may serve as a key regulatory juncture in various tissues. Although the heart expresses ACE2, the extent that the enzyme participates in the cardiac processing of ANG II and ANG-(1-7) is equivocal. Therefore, we utilized the Langendorff preparation to characterize the ACE2 pathway in isolated hearts from male normotensive Sprague-Dawley [[Tg.sup.(-)]] and hypertensive [mRen2]27 [[Tg.sup.(+)]] rats. During a 60-rain recirculation period with 10 nM ANG II, the presence of ANG-(1-7) was assessed in the cardiac effluent. ANG-(1-7) generation from ANG II was similar in both the normal and hypertensive hearts [[Tg.sup(-)]: 510 [+ or -] 55 pM, n = 20 vs. [Tg.sup.(+)]: 497 [+ or -] 63 pM, n = 14] with peak levels occurring at 30 rain after administration of the peptide. ACE2 inhibition (MLN-4760, 1 [micro]M) significantly reduced ANG-(1-7) production by 83% (57 [+ or -] 19 pM, P < 0.01, n = 7) in the [Tg.sup.(-)] rats, whereas the inhibitor had no significant effect in the [Tg.sup.(-)] rats (285 [+ or -] 53 pM, P > 0.05, n = 10). ACE2 activity was found in the effluent of perfused [Tg.sup.(-)] and [Tg.sup.(+)] hearts, and it was highly associated with ACE2 protein expression (r = 0.78). This study is the first demonstration for a direct role of ACE2 in the metabolism of cardiac ANG II in the hypertrophic heart of hypertensive rats. We conclude that predominant expression of cardiac ACE2 activity in the [Tg.sup.(+)] may be a compensatory response to the extensive cardiac remodeling in this strain. angiotensin II; hypertension; isolated heart doi:10.1152/ajpheart.01198.2006.

Endogenous angiotensin (ANG) II and ANG-(1-7) act at the nucleus tractus solitarius (NTS) to differentially modulate neural control of the circulation. The role of these peptides endogenous to NTS on cardiovascular reflex function was investigated in transgenic rats with low brain angiotensinogen (Aogen) due to glial overexpression of an antisense to Aogen (ASrAOGEN) and in Sprague-Dawley (SD) rats. Arterial baroreceptor reflex sensitivity (BRS) for control of heart rate (HR) in response to increases in mean arterial pressure (MAP) was tested before and after bilateral microinjection of the angiotensin type 1 (A[T.sub.1]) receptor blocker candesartan or the ANG-(1-7) receptor blocker (D-[Ala.sup.7])-ANG-(1-7) into the NTS of urethane-chloralose-anesthetized ASrAOGEN and SD rats. Baseline MAP was higher in ASrAOGEN than in SD rats under anesthesia (P < 0.01). Injection of candesartan or (D-[Ala.sup.7])-ANG-(1-7) decreased MAP (P < 0.01) and HR (P < 0.05) in ASrAOGEN, but not SD, rats. The BRS at baseline was similar in ASrAOGEN and SD rats. Candesartan increased BRS by 41% in SD rats (P < 0.01) but was without effect in ASrAOGEN rats. In contrast, the reduction in BRS after (D-[Ala.sup.7])-ANG-(1-7) administration was comparable in SD (31%) and ASrAOGEN rats (34%). These findings indicate that the absence of glia-derived Aogen is associated with 1) an increase in MAP under anesthesia mediated via A[T.sub1] and ANG-(1-7) receptors within the NTS, 2) the absence of an endogenous ANG II contribution to tonic inhibition of BRS, and 3) a continued contribution of endogenous ANG-(1-7) to tonic enhancement of BRS. baroreceptors; solitary tract nucleus; brain renin-angiotensin system; transgenic rats

Despite the evidence that angiotensin-converting enzyme (ACE)2 is a component of the reninangiotensin 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 [[sup.125]I]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 [NH.sub.2] terminal-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 [+ or -] 17 fmol*[mg.sup.-1]*[min.sup.-1]) and ACE2 [ANG II to ANG-(1-7): 253 [+ or -] 11 fmol*[mg.sup.-1]*[min.sup.-1]], but lower neprilysin activity [ANG II to ANG-(1-4): 119 [+ or -] 24 fmol*[mg.sup.-1]*[min.sup.-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). ANG-(1-7); ANG-(1-5); neprilysin; ACE; ANG-(1-9)

The generation of the Lew.Tg(mRen2) congenic hypertensive rat strain, developed through a backcross of the hypertensive (mRen2)27 transgenic rat with normotensive Lewis rats, provides a new model by which primary hypertension can be studied without the genetic variability found in the original strain. The purpose of this study was to characterize the Lew.Tg(mRen2) rats by dually investigating the effects of type 1 angiotensin II (ANG II) receptor (ATe) blockade and angiotensin-converting enzyme (ACE) activity inhibition on the ANG-(1-7)/ACE2 axis of the renin-angiotensin system in this new hypertensive model. The control of blood pressure elicited by 12-day administration of either lisinopril (mean difference change = 92 [+ or -] 2, P < 0.05) or losartan (mean difference change = 69 [+ or -] 2, P < 0.05) was associated with 54% and 33% increases in cardiac ACE2 mRNA and 54% and 43% increases in cardiac ACE mRNA, respectively. Lisinopril induced a 3.1-fold (P < 0.05) increase in renal cortical expression of ACE2, whereas losartan increased ACE2 mRNA 3.5-fold (P < 0.05). Both treatment regimens increased renal ACE mRNA 2.6-fold (P < 0.05). The two therapies augmented ACE2 protein activity, as well as increased cardiac and renal [AT.sub.1] receptor mRNAs. ACE inhibition reduced plasma ANG 13 levels (81%, P < 0.05) and increased plasma ANG-(1-7) (265%, P < 0.05), whereas losartan had no effect on the peptides. In contrast with what had been shown in normotensive rats, ACE inhibition decreased renal ANG II excretion and transiently decreased ANG-(1-7) excretion, whereas losartan treatment was associated with a consistent decrease in ANG-(1-7) urinary excretion rates. In response to the treatments, the expression of both renal cortical renin and angiotensinogen mRNAs was significantly augmented. The paradoxical effects of blockade of ANG II synthesis and activity on urinary excretion rates of the peptides and plasma angiotensins levels suggest that, in Lew.Tg(mRen2) congenic rats, a failure of compensatory ACE2 and ANG-(1-7)-dependent vasodepressor mechanisms may contribute both to the development and progression of hypertension driven by increased formation of endogenous ANG II. lisinopril; losartan; angiotensin-converting enzyme 2; angiotensin-(1-7)

We established a new congenic model of hypertension, the mRen(2).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 [+ or -] 2 vs. 107 [+ or -] 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 [sup.125]I-sarthran. Isolated CORT nuclei exhibited a high density (B.sub.max] >200 fmol/mg protein) and affinity for the sarthran ligand ([K.sub.D]95%) were the A[T.sub.1] receptor subtype. CORT ANG II receptor [B.sub.max] and [K.sub.D] values 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 [+ or -] 4 vs. mRen2.Lewis: 21 [+ or -] 5 fmol/mg protein) and affinity (Lewis: 0.31 [+ or -] 0.1 vs. 0.69 [+ or -] 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 A[T.sub.1] sites in the mRen2.Lewis. The downregulation of A[T.sub.1] sites in the mRen2.Lewis rats may reflect a compensatory response to dampen the elevated levels of intrarenal ANG II. ANG II type 1 receptor; hypertension; intrarenal angiotensin; renal cortex; renal medulla; ANG II

This study evaluated the contribution of angiotensin peptides acting at various receptor subtypes to the arterial pressure and heart rate of adult 9-wk-old male conscious salt-depleted spontaneously hypertensive rats (SHR). Plasma ANG II and ANG I in salt-depleted SHR were elevated sevenfold compared with peptide levels measured in sodium-replete SHR, whereas plasma ANG-(1-7) was twofold greater in salt-depleted SHR compared with salt-replete SHR. Losartan (32.5 [micro]mol/kg), PD-123319 (0.12 [micro]mol.[kg.sup.-1] [min.sup.-1]), [D-[Ala.sup.7]]ANG-(1-7) (10 and 100 [micro]mol/min), and a polyclonal ANG II antibody (0.08 mg/min) were infused intravenously alone or in combination. Combined blockade of A[T.sub.2] and A[T.sub.(1-7)] receptors significantly increased the blood pressure of losartan-treated SHR (+ 15 [+ or -] 1 mmHg; P < 0.01); this change did not differ from the blood pressure elevation produced by the sole blockade of A[T.sub.(1-7)] receptors (15 [+ or -] 4 mmHg). On the other hand, sole blockade of A[T.sub.2] receptors in losartan-treated SHR increased mean arterial pressure by 8 [+ or -] 1 mmHg (P < 0.05 vs. 5% dextrose in water as vehicle), and this increase was less than the pressor response produced by blockade of A[T.sub.(1-7)] receptors alone or combined blockade of A[T.sub.(1-7)] and A[T.sub.2] receptors. The ANG II antibody increased blood pressure to the greatest extent in salt-depleted SHR pretreated with only losartan (+ 11 [+ or -] 2 mmHg) and to the least extent in salt-depleted SHR previously treated with the combination of losartan, PD-123319, and [D-[Ala.sup.7]]ANG-(1-7) (+7 [+ or -] 1 mmHg; P < 0.01). Losartan significantly increased heart rate, whereas other combinations of receptor antagonists or the ANG II antibody did not alter heart rate. Our results demonstrate that ANG II and ANG-(1-7) act through non-A[T.sub.1] receptors to oppose the vasoconstrictor actions of ANG II in salt-depleted SHR. Combined blockade of A[T.sub.2] and A[T.sub.(1-7)] receptors and ANG II neutralization by the ANG II antibody reversed as much as 67% of the blood pressure-lowering effect of losartan. angiotensin II; angiotensin-(1-7); angiotensin receptor antagonists; hypertension; losartan; PD-123319; salt depletion; spontaneously hypertensive rats

Receptor blocking studies were undertaken to identify the angiotensin receptor responsible for the attenuation of the baroreceptor reflex at the level of the tractus solitarius nucleus. The blocking experiments were carried out in anesthetized normotensive and spontaneously hypertensive rats. Results reveal that the type 1 receptor is primarily responsible for the activity of angiotensin at the tractus solitarius nucleus.

The losartan (AT1) receptor subtype regulates angiotensin II-induced (ANG) bradycardia and hypotension in dorsal medulla. The cardioinhibitory effects of ANG II on the dorsal medial solitary tract nucleus (NTS) involve neural mechanisms within the NTS. Cardiovascular effects are not due to peptide diffusion into the underlying dorsal motor nucleus of the vagus.

The significance of endogenous angiotensin II (ANG II) in the ventrolateral medulla (VLM) of spontaneously hypertensive rats (SHR) was investigated by microinjection of an ANG II antagonist either in the rostral orcaudal VLM. Rostrally, the antagonist elicited depressor and bradicardic effects while the caudal microinjection induced the opposite effects. These results suggest a tonic excitatory effect function for endogenous ANG II on VLMvasomotor neurons. Further, ANG II blood pressure responses is much higher in SHRs than in normotensive rats.

Ischemic stimulation of cardiac receptors evokes excitatory sympathetic reflexes. Although the nucleus of the solitary tract (NTS) is an important site for integration of visceral afferents, its involvement in the cardiac-renal sympathetic reflex remains to be fully defined. This study examined the role of glutamate receptor subtypes in the commissural NTS in the sympathetic responses to stimulation of cardiac receptors. Renal sympathetic nerve activity (RSNA) was recorded in anesthetized rats. Cardiac receptors were stimulated by epicardial application of bradykinin (BK; 10 [micro]g/ml). Application of BK significantly increased the mean arterial pressure from 78.2 [+ or -] 2.2 to 97.5 [+ or -] 2.9 mmHg and augmented RSNA by 38.5 [+ or -] 2.5% (P [is less than] 0.05). Bilateral microinjection of 10 pmol of 6-cyano-7-nitroquinoxaline-2,3-dione, a non-N-methyl-D-aspartate (NMDA) antagonist, into the commissural NTS eliminated the pressor and RSNA responses to BK application in 10 rats. However, microinjection of 2-amino-5-phosphonopentanoic acid (0.1 and 1 nmol, n = 8), an NMDA-receptor antagonist, or [Alpha]-methyl-4-carboxyphenylglycine (0.1 and 1 nmol, n = 5), a glutamate metabotropic receptor antagonist, failed to attenuate significantly the pressor and RSNA responses to stimulation of cardiac receptors with BK. Thus this study suggests that non-NMDA, but not NMDA and glutamate metabotropic, receptors in the commissural NTS play an important role in the sympathoexcitatory reflex response to activation of cardiac receptors during myocardial ischemia. cardiac nociceptors; sympathetic nervous system; cardiovascular reflexes; bradykinin; N-methyl-D-aspartate receptors; myocardial ischemia