Shridhar N. Iyer

Converting Enzyme Determines Plasma Clearance of Angiotensin-(1–7)

We determined the mechanism accounting for the removal and metabolism of angiotensin-(1-7) [Ang-(1-7)] in 21 anesthetized spontaneously hypertensive (SHR), 18 age-matched normotensive Sprague-Dawley (SD), and 36 mRen-2 transgenic (TG+) rats. Animals of all 3 strains were provided with tap water or tap water containing losartan, lisinopril, or a combination of lisinopril and losartan for 2 weeks. On the day of the experiment, Ang-(1-7) was infused for a period of 15 minutes at a rate of 278 nmol . kg-1 . min-1. After this time, samples of arterial blood were collected rapidly at regular intervals for the assay of plasma Ang-(1-7) levels by radioimmunoassay. Infusion of Ang-(1-7) had a minimal effect on vehicle-treated SD rats but elicited a biphasic pressor/depressor response in vehicle-treated SHR and TG+ rats. In lisinopril-treated rats, Ang-(1-7) infusion increased blood pressure, whereas losartan treatment abolished the pressor component of the response without altering the secondary fall in arterial pressure. Combined treatment with lisinopril and losartan abolished the cardiovascular response to Ang-(1-7) in all 3 strains. In vehicle-treated SD, SHR and TG+ the half-life (t1/2) of Ang-(1-7) averaged 10+/-1, 10+/-1, and 9+/-1 seconds, respectively. Lisinopril alone or in combination with losartan produced a statistically significant rise in the half-life of Ang-(1-7) in all 3 strains of rats. Plasma clearance of Ang-(1-7) was significantly greater in the untreated SD rats compared with either the SHR or TG+ rat. Lisinopril treatment was associated with reduced clearance of Ang-(1-7) in all 3 strains. Concurrent experiments in pulmonary membranes from SD and SHR showed a statistically significant inhibition of 125I-Ang-(1-7) metabolism in the presence of lisinopril. These studies showed for the first time that the very short half-life of Ang-(1-7) in the circulation is primarily accounted for peptide metabolism by ACE. These findings suggest a novel role of ACE in the regulation of the production and metabolism of the two primary active hormones of the renin angiotensin system.

Evidence that Prostaglandins mediate the antihypertensive actions of angiotensin-(1-7) during chronic blockade of the renin-angiotensin system

Prostaglandins are known to participate in the antihypertensive actions of angiotensin-converting enzyme (ACE) inhibition and angiotensin type 1 (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 AT2 antagonist PD123319. The non-AT1/non-AT2 Sarthran 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.

Losartan Versus Gene Therapy: Chronic Control of High Blood Pressure in Spontaneously Hypertensive Rats

Interruption of the renin-angiotensin system by pharmacological manipulations attenuates high blood pressure (BP) in the spontaneously hypertensive rat (SHR). However, these agents, such as losartan, need to be administered daily to maintain effective BP control. Therefore, we have hypothesized that a genetic intervention in the expression of angiotensin type 1 receptor (AT1R) should attenuate development of hypertension on a long-term basis in SHR. A retroviral-mediated AT1R antisense cDNA gene delivery system (LNSV-AT1R-AS) was used to test this hypothesis and to compare its BP-lowering effects with those of losartan. Introduction of LNSV-AT1R-AS into 5-day-old Wistar-Kyoto rats and SHR resulted in a robust expression of AT1R antisense (AS) within 3 days and persisted for at least 30 days. This expression was associated with a selective attenuation of high BP in SHR by 25 to 30 mm Hg. Although basal lowering of BP was exclusive to SHR, the angiotensin II (Ang II) pressor response was significantly reduced in all LNSV-AT1R-AS-treated rats. The decreased response to Ang II was associated with a similar attenuation of Ang II-induced dipsogenic responses in both strains of rats. The BP-lowering effects of LNSV-AT1R-AS treatment and losartan treatment were similar and primarily observed in SHR. However, the antihypertensive effect lasted less than 24 hours in losartan-treated SHR compared with 90 days in LNSV-AT1R-AS-treated SHR. In addition, losartan was unable to further lower BP in LNSV-AT1R-AS-treated SHR. Collectively, these results suggest that both losartan and LNSV-AT1R-AS treatment produces an antihypertensive response selectively in SHR that is mediated by interruption of AT1R function. However, a single, acute genetic treatment with LNSV-AT1R-AS can result in long-term control of high BP at a similar level of effectiveness as losartan, without altering plasma Ang II levels.

Effect of Acute and Chronic Losartan Treatment on Glucose Tolerance and Insulin Sensitivity in Fructose-Fed Rats

The purpose of this study was to evaluate the role of angiotensin II (AII) in fructose-treated rats by assessing the effects of acute and chronic losartan treatment on glucose tolerance and insulin sensitivity. The effect of acute losartan treatment was assessed on blood glucose and serum insulin profiles during an intravenous glucose tolerance test (IVGTT) in control and fructose-fed rats. Acute losartan treatment was effective in lowering both blood glucose and plasma insulin profiles in fructose-treated rats during the IVGTT compared to the saline-injected fructose-treated rats. In a subsequent study, 2 weeks after chronic fructose and losartan treatment, IVGTT were performed. Chronic losartan treatment also improved both the glucose tolerance and insulin sensitivity in fructose-treated rats. Additionally chronic losartan treatment also was effective in blocking the hyperinsulinemia, hypertension, and cardiac hypertrophy observed in fructose-fed rats. In neither acute nor chronic studies were there any effects of losartan in control animals. These results strongly suggest that the renin-angiotensin system modulates the insulin resistance, hypertension, and cardiac hypertrophy in fructose-treated rats.

Vascular Reactivity to Phenylephrine and Angiotensin II in Hypertensive Rats Associated with Insulin Resistance

Previous reports suggest that when rats are fed a carbohydrate-enriched diet they develop hyperinsulinemia associated with elevated blood pressure. The purpose of this study was to assess the vascular reactivity of fructose-treated rats to various pressor agents. Male Sprague Dawley rats (n = 24) were used for this study and were divided into two equal groups. One of the groups was fed normal rat chow and served as the control group, whereas the other group was fed a fructose-enriched diet for four weeks. Mean blood pressure was elevated in the fructose-treated rats at the end of the second week of fructose treatment and remained elevated for the remainder of the study. At the end of the second and fourth weeks of fructose treatment, six rats from each group were used to assess both in vivo and subsequently in vitro vascular reactivity to various pressor agents. The jugular vein and carotid artery were cannulated under anesthesia. Twenty four hours after recovery from surgery pressor responses to angiotensin II (AII) and phenylephrine (PE) were determined. Twenty four hours later rats were decapitated and the thoracic aorta was removed, cleaned of adhering fat and cut into ring segments for vascular reactivity studies. Tissues were suspended in muscle baths containing physiological saline solution and maintained at 37 degrees C. Dose-response curves were generated in the aorta in response to potassium chloride (KCl), AII and PE. At the end of the second week of fructose treatment pressor response to AII was significantly increased in the fructose-treated rats compared to the controls whereas there was no significant difference in pressor response to PE. There was no significant difference in pressor response to AII and PE between the two groups at the end of the fourth week of fructose treatment. In vitro contractile response of the aorta to AII and PE were significantly greater in the fructose-fed rats compared to the controls at the end of the second week of fructose treatment; however, there was no change in the EC50 between the two groups. At the end of the fourth week of fructose treatment, the contractile responses to AII and PE were similar in both groups, although the response to AII tended to be lower in the fructose-fed rat. There was no significant difference in the contractile response to potassium chloride or in acetylcholine-induced relaxation throughout the study. These results strongly suggest that hypertension in fructose-treated rats is associated with increased in vitro vascular reactivity to AII and PE in the early stages of hypertension.

At1 Receptor Density Changes During Development of Hypertension in Hyperinsulinemic Rats

In a previous study we showed that the renin-angiotensin system (RAS) plays a role in the etiology of fructose-induced hypertension. To our knowledge, no previous study has evaluated changes in angiotensin II (Ang II) type I receptor (AT1) density in fructose-fed rats that are insulin resistant and hypertensive. The purpose of this study was to determine the changes in plasma Ang II and AT1 density associated with the elevation of blood pressure in fructose-treated rats. Male Sprague-Dawley rats were divided into two groups and were fed either normal rat chow or a 60% fructose-enriched diet for four weeks. Plasma Ang II and serum insulin levels of the fructose-treated rats were significantly elevated (p < 0.01) by the end of the second week of fructose treatment. Plasma Ang II levels of the fructose-fed rats returned to basal levels by the end of the fourth week of dietary treatment, whereas the serum insulin levels consistently remained elevated. Blood pressure was significantly elevated in the fructose-fed rats within two weeks of fructose treatment. Elevation of blood pressure was associated with left ventricular hypertrophy. Furthermore, there was a significant increase in AT1 receptor density in the ventricles and a significant decrease in AT1 receptor density in the aortas of fructose-fed rats at the end of fourth week. There were no significant changes in receptor density in the hypothalami or adrenal glands of fructose-treated rats. These results suggest that chronic fructose treatment activates the renin-angiotensin system, which is manifested by an increase in plasma Ang II, elevation of blood pressure, cardiac hypertrophy, and changes in AT1 receptor density.

Role of At1 and At2 Receptors in the Plasma Clearance of Angiotensin Ii

This study assessed the role of angiotensin (Ang) AT1 and AT2 receptors as modulators of the plasma clearance of Ang II. Groups of male spontaneously hypertensive rats (SHRs; n = 25) were given an intravenous injection of either saline, losartan, PD123319, losartan in combination with PD123319, or Sar1-Thr8-Ang II. One hour later, Ang II (0.5 microg/kg) was infused for 15 min into a vein. Immediately thereafter, arterial blood samples were collected at regular intervals for the assay of plasma Ang II levels by radioimmunoassay. The infusion of Ang II significantly increased baseline mean arterial pressure (MAP) in rats pretreated with either saline or PD123319 but not in those receiving losartan, losartan combined with PD123319, or Sar1-Thr8-Ang II. The plasma clearance of Ang II was significantly greater in rats injected with either PD123319, losartan combined with PD123319, or Sar1-Thr8-Ang II compared to those injected either saline or losartan. Furthermore, the half-life of Ang II in rats pretreated with saline or losartan was significantly greater than that measured in the other three groups. These results suggest that plasma clearance of Ang II in the SHRs is independent of an AT1 receptor, but plasma levels of the peptide are unexpectedly protected by an AT2 receptor-dependent mechanism.

Fructose feeding in rats is not associated with sodium retention.

Chronic fructose treatment in rats repeatedly has been shown to elevate blood pressure associated with insulin resistance and hyperinsulinemia. Activation of the sympathetic nervous system and the renin-angiotensin system, vascular hypertrophy, and sodium retention by the kidney tubules have been proposed to be some of the mechanisms by which insulin elevates blood pressure. The precise mechanism by which hypertension develops in fructose fed rats is still not known. The purpose of the current study was twofold. The first objective was to assess the effect of a fructose-enriched diet on urinary sodium excretion. The second objective was to investigate any changes in plasma volume and extracellular volume in fructose-fed rats. In both experiments, male Sprague-Dawley rats were divided into equal groups. Rats in the control group were fed Purina Laboratory Chow, whereas those in the experimental group were fed a 60% fructose diet. There was a significant elevation in the blood pressure of fructose-fed rats at the end of the second week of treatment, and it remained elevated for the remainder of the dietary intervention. In the first experiment, there was no significant difference in sodium, potassium or urine excretion throughout the 6 weeks of dietary treatment. At the end of this study, the serum insulin levels of fructose-fed rats were significantly greater than the levels in the control group. In the second experiment, which was a 4-week study, there was no significant difference in hematocrit, plasma volume, or extracellular fluid volume between control and fructose-fed animals at 2 or 4 weeks of dietary treatment. The results of these two in vivo studies are the first to document that elevation of blood pressure in fructose-fed rats does not occur directly via sodium retention or an increase in fluid volume.

Changes in Angiotensin AT1 Receptor Density During Hypertension in Fructose-Fed Rats

Feeding carbohydrate-enriched diets to normal rats has been shown to induce insulin resistance and hyperinsulinemia associated with an elevation of blood pressure. Previously we reported that the renin-angiotensin system (RAS) is likely to be involved in the elevation of blood pressure. The purpose of this study was to determine the changes in plasma angiotensin II (AII) and AII receptor density associated with the elevation of blood pressure in fructose-treated rats. Male Sprague-Dawley rats were divided into two groups and were fed either normal rat chow or a 60% fructose-enriched diet for four weeks. Plasma insulin of fructose-treated rats was significantly elevated (p < 0.05) by the end of first week of fructose treatment and remained elevated throughout the study. Plasma AII levels of fructose-fed rats was 3.5 fold greater than the controls at the end of second week and returned to basal levels at the end of the fourth week of dietary treatment. Blood pressure was significantly elevated in the fructose-fed rats within two weeks of fructose treatment. Elevation of blood pressure was associated with left ventricular hypertrophy. Angiotensin II type I receptor (AT1) density was determined in the left ventricle, aorta, adrenal gland and hypothalamus. There was a significant increase in AT1 receptor density in the ventricle at the end of third and fourth weeks of treatment, whereas there was a significant decrease in the receptor density in the aorta at the end of the fourth week of treatment. Receptor density in the adrenal gland and hypothalamus of fructose-fed rats was similar to their respective controls. The results of this study suggest that the RAS plays a role in the elevation of blood pressure of fructose-fed rats and also contributes to the ventricular hypertrophy observed in these rats.