D. Ganten

Opposing Actions of Angiotensin-(1-7) and Angiotensin II in the Brain of Transgenic Hypertensive Rats

Lack of specific antagonists to the amino-terminal heptapeptide angiotensin-(1-7) [Ang-(1-7)] prompted us to evaluate the central effects of delivering a specific affinity-purified Ang-(1-7) antibody on the blood pressure and heart rate of 12-week-old conscious homozygous female rats (n = 12) expressing the mouse submandibular Ren-2d gene [(mRen-2d)27] in their genome. Another group of transgenic hypertensive and strain-matched Sprague-Dawley controls were injected with a specific Ang II monoclonal antibody (KAA8). Cerebroventricular administration of the affinity-purified Ang-(1-7) antibody in conscious transgenic hypertensive rats caused significant dose-related elevations in blood pressure associated with tachycardia. The hypertensive response was augmented in transgenic rats studied 7 to 10 days after cessation of lisinopril therapy. Neutralization of Ang II with the Ang II antibody caused a hemodynamic response opposite to that obtained with the Ang-(1-7) antibody. All doses of the Ang II antibody produced hypotension and bradycardia. The magnitude of the depressor response was significantly augmented in transgenic rats weaned off lisinopril therapy. In contrast, central administration of either the Ang-(1-7) or Ang II antibodies had no effect on normotensive rats. Central injections of an affinity-purified IgG fraction were ineffective in both control and transgene-positive rats. These data suggest that in the brain of transgenic hypertensive rats, Ang-(1-7) opposes the action of Ang II on the central mechanism or mechanisms that contribute to the maintenance of this model of hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

NK1 Receptor Antagonist Blocks Angiotensin II Responses in Renin Transgenic Rat Medulla Oblongata

Angiotensin (Ang) II increases substance P (SP) efflux from perfused slices of medulla oblongata, and a peptide antagonist of SP, [Leu11,psiCH2NH10-11]SP, blocks the acute hypotension and bradycardia caused by Ang II injected into the nucleus tractus solitarii (nTS) of Harlan Sprague-Dawley (SD) rats. We investigated whether the same relationships exist in (mRen2)27 renin transgenic (TG) rats, which have chronic elevations of medullary tissue Ang II levels. Ang II increased SP efflux (48% above control; P<0.01) from slices of medulla prepared from 8- to 12-week old male TG rats. Injections of Ang II (250 fmol in 30 nL) into the nTS of chloralose-urethane anesthetized TG rats produced a significant increase in pressure of 7+/-2 mm Hg before a 13+/-3 mm Hg fall in pressure. Ang II induced similar depressor responses in Hannover SD rats but no increase in pressure. After nTS injection of the NK1-selective SP antagonist CP-96,345 (30 pmol in 60 nL), Ang II-induced hypotension was blocked in both groups, as was the pressor component in hypertensive rats. Hypotensive and bradycardic effects of glutamate (0.6 nmol in 30 nL) injected into the nTS were not altered by CP-96,345. In vitro receptor autoradiography showed that the SP antagonist (10 or 100 microM) did not compete for 125I-Ang II binding in the dorsal medulla, a result suggesting that it did not interact directly with Ang II receptors. Thus, the nTS cardiovascular effects of Ang II are mediated by SP in both normotensive rats and a model of hypertension with altered endogenous levels of Ang II. These findings link Ang II-induced effects on SP release from brain slices of the medulla oblongata to acute cardiovascular actions of the peptide through an NK1 receptor.

Role of Area Postrema in Transgene Hypertension

Transgenic [Tg(+)] rats carrying the mouse Ren-2d gene [(mRen-2d)27] are a newly established monogenetic form of experimental hypertension. To determine whether the area postrema contributes to the development of hypertension in mRen-2 Tg(+) rats, this circumventricular organ in the fourth ventricle was removed from 5-week-old Tg(+) rats. From weeks 4 through 9, systolic blood pressure was measured weekly by tail-cuff plethysmography in area postrema-lesioned and sham-lesioned Tg(+) rats. Although systolic blood pressure rose markedly in sham-lesioned Tg(+) rats, the increase in systolic blood pressure was significantly attenuated in area postrema-lesioned Tg(+) rats. At 9 weeks of age, a femoral artery was cannulated for the measurement of arterial pressure in awake rats. Mean arterial pressure (MAP) in area postrema-lesioned Tg(+) rats was significantly (P < .01) lower than that in sham-lesioned rats: 171 +/- 7 and 132.+/- 5 mm Hg, respectively. Baroreceptor reflex was evaluated by intravenous infusion of sodium nitroprusside. There was no significant difference in baroreceptor reflex sensitivity between the two groups. Intravenous pentolinium (5 mg/kg), used to produce sympathetic ganglionic block, caused significant decreases in MAP in both groups. However, the reduction of MAP in the sham-lesioned group was significantly (P < .05) greater than that in the area postrema-lesioned group: -73 +/- 4 and -48 +/- 6 mm Hg, respectively. The ratio of left ventricular weight to body weight in sham-lesioned Tg(+) rats was significantly larger than that of area postrema-lesioned rats. These results suggest that ablation of the area postrema markedly attenuates the development of hypertension in mRen-2d Tg(+) rats, and this attenuation may be attributed to decrease in sympathetic outflow.

Chronic estrogen treatment in female transgenic (mRen2)27 hypertensive rats augments endothelium-derived nitric oxide release.

Postmenopausal estrogen replacement therapy is associated with a reduction in cardiovascular events in women, but the mechanisms for this protection are unclear, especially in hypertensive subjects. In this study we investigated the effects of 17beta-estradiol (E2) treatment on blood pressure and endothelial function of transgenic [(mRen2)27] hypertensive and normotensive rats. Thirty female transgenic negative [Tg(-)] and hypertensive positive [Tg(+)] rats were ovariectomized and received either E2 (1.5 mg/rat, subcutaneously, for 3 weeks) or placebo. Chronic 17beta-estradiol treatment lowered mean blood pressure in both Tg hypertensive (159 +/- 4 v 145 +/- 4 mm Hg, P < .05, placebo v E2) and normotensive rats (119 +/- 4 v 108 +/- 2 mm Hg, P < .05, placebo v E2). Pressor responses to intravenous injection of phenylephrine were augmented in the Tg(+) as compared with Tg(-) rats. With chronic E2 treatment the pressor responses to phenylephrine were attenuated in both groups. Isometric tension of aortic rings was measured in vitro in organ chambers. The acetylcholine (Ach)-induced endothelium-dependent vascular relaxation was less potent in Tg(+) versus Tg(-) rats. E2 treatment significantly enhanced the Ach-induced relaxation of both Tg(+) and Tg(-) groups (ED50: 55.5 +/- 11.7 v 10.3 +/- 2.6; 23.8 +/- 6.5 v 5.1 +/- 1.2 nmol/L, placebo v E2 in Tg(+) and Tg(-), respectively). After E2 treatment the ED50 response in Tg(+) rats was no different from Tg(-) rats. However, the maximum vasodilation elicited by Ach was attenuated in Tg(+) as compared with Tg(-) rats. The calcium ionophore (A23187)-induced endothelium-dependent relaxation was less potent in Tg(+) as compared to Tg(-) rats and was enhanced by E2 treatment only in Tg(+) animals. There were no differences in the vasodilator responses elicited by sodium nitroprusside. Removal of endothelium and blockade of NO production abolished the endothelium-dependent vasodilation. The selective NO synthase inhibitor, N(G)-monomethyl-L-arginine (LMMNA), was used to evaluate indirectly the basal contribution of NO in vascular rings. The response to LMMNA was attenuated in untreated Tg(+) as compared to Tg(-) rats. E2 treatment augmented the contraction response to NOS inhibition in both Tg(+) and Tg(-) rats, resulting in a response in Tg(+) rats that was no different from Tg(-) rats. These results indicate that untreated, surgically ovariectomized hypertensive rats show deficiencies in endothelial function, which can be improved by estrogen replacement.

Depressor role of angiotensin AT2 receptors in the (mRen-2)27 transgenic rat

The (mRen-2)27 transgenic rat (Tg+), a hypertensive model dependent on increased expression of the renin angiotensin system, was used to explore the role of angiotensin AT2 receptors in the control of cardiovascular and renal excretory function. Experiments tested the effect of blockade of AT2 receptors on basal blood pressure and the pressor, renal excretory, and vasopressin (VP) responses to intravenous hypertonic saline (HS). Chronically catheterized male Tg+ and normotensive Sprague-Dawley rats (Tg-) were housed in metabolic cages. PD123319 (AT2 antagonist) or 0.9% NaCl was given by intravenous bolus (3 mg/kg) followed by infusion (50 microg/kg/ min). Blockade of AT2 receptors both in Tg+ and Tg- rats produced no change in basal mean arterial pressure (MAP). The pressor response to intravenous HS (10% NaCl; 325 microL/100 g body weight) was significantly greater in Tg+ than in Tg- rats. PD123319 did not affect the peak rise in MAP but extended the time course of the response only in Tg+ rats. MAP was increased 39+/-4 and 36+/-3 mm Hg in Tg+ rats with and without the antagonist as compared to 20+/-2 and 24+/-2 mm Hg in Tg- rats. In the antagonist-treated Tg+ rats, MAP remained elevated for 60 min as compared to 5 min for Tg+ control or Tg- control or antagonist-treated rats. Hypertonic saline caused similar increases in plasma Na, VP, and in the natriuretic and diuretic responses in both Tg+ and Tg- rats, with no effect of antagonist treatment. These results demonstrate that Tg+ rats are sensitive to the effects of peripheral osmotic stimulation showing an increased pressor response, not attributed to greater secretion of VP or diminished natriuresis. These data also suggest that angiotensin AT2 receptors play a depressor role in the sodium-induced pressor response in this model.

Endothelial Injury in Transgenic (mRen-2)27 Hypertensive Rats

Transgenic [(mRen-2)27] rats develop severe hypertension as the result of transfection with the mouse Ren-2 gene. This study tested the hypothesis that hypertensive [(mRen-2)27] rats have increased endothelial dysfunction by examining the extent of vascular endothelial cell injury and turnover within the thoracic aorta of age-matched female transgene positive [Tg(+)] and transgene negative [Tg(-)] littermates. Transgenic hypertensive rats had arterial pressures significantly higher than Tg(-) animals, but no differences in heart rate or body weight. The extent of endothelial cell injury was estimated in Haütchen preparations of thoracic aorta endothelium by counting cells immunostained for the presence of cytoplasmic immunoglobulin G (IgG) at sites with or without intercostal artery branches. Both Tg(+) and Tg(-) littermates had a greater percentage of injured endothelial cells at branch sites than at nonbranch aorta (P < .01). However, the number of vascular endothelial cells staining positively for IgG was significantly higher in hypertensive rats both at sites away from (P < .05) and in the immediate vicinity of (P < .1) the orifices of intercostal arteries. En face preparations of the thoracic aorta were also examined for cells incorporating 5-bromo-2 -deoxyuridine (BrdU) to estimate the percentage of endothelial cells undergoing replication. There was no difference in endothelial cell replication at either branch or nonbranch sites between hypertensive and normotensive rats. However, the percentage of endothelial cells undergoing replication at branch sites in both Tg(+) and Tg(-) rats was significantly greater than at nonbranch sites (P < .01). These data provide the first demonstration for the effects of high blood pressure on the vascular endothelium of a monogenetic model of hypertension produced by increased activity of the renin-angiotensin system. The divergent effects of this form of hypertension on vascular endothelial injury and endothelial turnover suggest that the decrease in the reparative capacity of the vascular endothelium induced by the combination of hypertension and associated angiotensinemia may contribute to the endothelial dysfunction accompanying vascular remodeling.

Altered Stress Responses in Rats with low Glial Angiotensinogen

Rats with low glial-derived angiotensinogen (ASrAogen) have higher AT1 receptors in subfornical organ and paraventericular nucleus, brain regions important in stress responses, compared to Sprague-Dawley (SD) rats at 48 and 68 weeks of age consistent with decreased tissue angiotensins (Ang). However, neuronal expression of Ang II and Ang-(1–7) in these nuclei is similar between strains. We measured systolic blood pressure (SBP) and baroreflex sensitivity (BRS; bradycardia in response to increase in pressure) in conscious 68 week old rats with intracerebroventricular (i.c.v.) cannulae. SBP averaged 151xa0mm Hg in SD and 122xa0mm Hg in ASrAogen rats, confirming their lower resting SBP ( P

Regulation of Brain Neprilysin and ACE2 in Transgenic Rats During Aging

Angiotensinogen (Aogen), the precursor to angiotensin (Ang) II and Ang-(1–7) is expressed in astrocytes and neurons. Neprilysin and angiotensin-converting enzyme 2 (ACE2) are potential enzymes for Ang-(1–7) formation from Ang I and Ang II respectively. We assessed their regulation in brain during aging in transgenic rats (ASrAogen) with an Aogen antisense behind a glial-promoter. These exhibit a 90% reduction in brain Aogen levels and low blood pressure that is maintained during aging at 16 and 70 weeks in hypothalamus (HYPO) and medulla (MED). HYPO ACE2 mRNA was 45% higher in ASrAogen than control SD rats and higher in older ASrAogen vs. younger rats. Neprilysin mRNA tended to increase during aging and values were 80% higher in ASrAogen vs. SD. In MED, mRNA of both enzymes during aging tended to decrease with no difference between strains. Immunocytochemistry revealed ACE2-staining in both strains localized to circumventricular organs and plexus choroideus. Thus, the ACE2 distribution suggests the enzyme contributes to Ang-(1–7) in cerebrospinal fluid but not neural pathways. Up regulation of mRNA for both enzymes in HYPO of ASrAogen rats may represent compensation for loss of glial-derived peptides. Lack of differences in MED between strains may reflect distinct sources (neural vs. glial) of peptides responsible for enzyme regulation in the two brain areas.