Robin R. Socci

Alterations in Aldosterone and Angiotensin II Levels in Salt-Induced Hypertension

Several studies have demonstrated that plasma renin-angiotensin activity is reduced in rats administered a high salt diet. We evaluated changes in plasma and tissue levels of aldosterone (ALDO) and angiotensin II (A-II), as well as the reduced-to-oxidized glutathione ratio. Male Dahl salt-sensitive (SS) rats were placed on either a high-salt (8% NaCl; HS) or a normal-salt (0.3% NaCl; NS) diet for 3 weeks. Prior to and weekly on the diets, systolic blood pressure was measured by tail cuff plethysmography. Levels of A-II and ALDO in plasma, heart, and kidney were analyzed by enzyme immunoassay. Reduced and oxidized gluthatione were simultaneously measured by HPLC fluorescence detection. Heart and kidney tissues were prepared for histological analysis. Systolic blood pressure in animals on a HS diet was significantly elevated above that of those on a NS diet. High salt caused a reduction in both plasma A-II and ALDO levels; while their levels in the heart and kidney were increased. Exposure to a high-salt diet led to the enlargement of both heart and kidney. The reduced-to-oxidized glutathione ratio in plasma, heart and kidney was lowered by exposure to a HS diet. Kidneys from animals on a high-salt diet showed fibroid necrosis associated with wrinkling and thickening of the glomerular capillary wall, while hearts were hypertrophic. Taken together, high dietary salt induces inappropriate activation of the local renin-angiotensin-aldosterone systems. Tissue levels of angiotensin II and aldosterone may be more reflective of the severity of vascular maladaptations than are plasma levels, and may play a greater role in the maintenance of hypertension.

1A-779 attenuates angiotensin-(1-7) depressor response in salt-induced hypertensive rats.

Chronic infusion of angiotensin-(1-7) [Ang-(1-7)] lowers blood pressure in salt-induced and spontaneously hypertensive (SHR) rats. In the present study, we have examined the acute effect of Ang-(1-7) in salt-induced hypertension using Dahl salt-sensitive rats placed on low (0.3%) or high (8.0% NaCl) salt diets for 2 weeks. Rats fed a high salt diet showed a greater rise in BP than those fed a low salt diet. Ang-(1-7) (24 microg/kg) reduced mean arterial pressure (MAP), enhanced the release of prostacyclin and nitric oxide, and suppressed thromboxane A(2) levels. A-779 (48 microg/kg, i.v), a selective Ang-(1-7) antagonist, partially blocked these effects of Ang-(1-7). The Ang-(1-7)-induced depressor response observed in these animals was related to an increase in vasodilatory prostanoids, a decrease in the constrictor prostanoid thromboxane A(2), and an increase in nitric oxide levels in both plasma and isolated aortic rings.

Acute inhibition of glutathione biosynthesis alters endothelial function and blood pressure in rats

The cardiovascular and biochemical responses during acute oxidative stress induced by D,L-buthionine-(S,R)-sulfoximine (BSO) were investigated in Sprague-Dawley rats. Mean arterial pressure, heart rate and vascular reactivity were measured after subcutaneous injection of BSO (4 mmol/kg). Control rats received saline. Levels of GSH and GSSG in blood and tissues as well as renal superoxide were determined. Nitric oxide, prostacyclin and thromboxane A(2) in plasma and aorta, and isoprostane in plasma were also measured. Blood pressure was elevated at 24 h (121+/-2 vs. 104+/-2 mm Hg), with increased reactivity to phenylephrine (by a 59+/-4 vs. 45+/-2 mm Hg change), and impaired response to sodium nitroprusside (by a -35+/-2 vs. -63+/-2 mm Hg change), P<0.05. The GSH:GSSG ratio was reduced at 8 and 24 h in blood (4.1+/-0.6 and 5.1+/-0.3, respectively, vs. 8.5+/-0.2), and at 8 h in the aorta (1.0+/-0.2 vs. 2.9+/-0.5), heart (1.6+/-0.3 vs. 2.3+/-0.1) and kidney (2.1+/-0.2 vs. 3.7+/-0.4), P<0.05. Superoxide fluorescence was increased at 24 h via NADH (4131+/-194 vs. 2853+/-199), NADPH (2874+/-272 vs.1479+/-257) and succinate (2475+/-133 vs. 1594+/-2150), P<0.05. Plasma prostacyclin was reduced at 8 and 24 h (36+/-4 and 52+/-13, respectively, vs. 310+/-44 pg/ml), P<0.001, whereas nitric oxide was reduced at 24 h (6.4+/-1 vs. 22+/-2 microM), P<0.01. Also at 24 h, thromboxane A(2) was increased both in plasma (374+/-154 vs. 61+/-10 pg/ml) and the aorta (174.4+/-38 vs. 27+/-3.4 pg/mg), P<0.05. Thus, acute BSO-induced oxidative stress alters blood pressure and endothelial function by mechanisms involving increased plasma levels and aortic release of thromboxane A(2) and reduced nitric oxide and prostacyclin.

Gender differences in the attenuation of salt-induced hypertension by angiotensin (1-7).

Chronic infusion of angiotensin (1-7) [Ang-(1-7)] lowers blood pressure in spontaneously hypertensive rats (SHR). To assess the role of Ang-(1-7) in salt-induced hypertension, Ang-(1-7) (24 microg/kg/hr) or saline was administered chronically via osmotic minipump into the jugular vein of 5-6 wk-old male (M) and female (F) Dahl salt-sensitive rats placed on a high-salt (8% NaCl) diet for 2 weeks. Blood pressure (BP) and heart rate were measured prior to the start of the diet and weekly thereafter. Ang-(1-7) significantly attenuated the BP increase after 1 wk on the diet in both M and F rats, but after 2 weeks only in F rats. Enhanced release of prostacyclin, (6-keto PGF1 alpha), following Ang-(1-7) treatment was observed in both M and F rats. In addition, significant increases in aortic blood flow and plasma levels of nitric oxide were observed in the F rats following Ang-(1-7) treatment. These findings demonstrate that the reduction in BP is due to both prostacyclin and NO and that there is a gender difference in the attenuation of salt-induced hypertension by Ang-(1-7).

High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

The role of gender in salt-induced hypertension.

To evaluate gender differences in salt-induced hypertension, female and male Dahl salt-sensitive rats were fed high (8.0% NaCl, HS) and low (0.3% NaCl, LS) salt diets. During a 3-week treatment period, blood pressure was significantly elevated in both female and male HS groups compared to their respective LS groups. The blood pressure and 4 week mortality rate of the female HS group, however, were significantly lower than those of the male HS group. Renal and aortic blood flows were reduced in male rats on HS diet compared to the LS group, while, in females, renal blood flow was elevated and aortic flow was maintained while on HS diet. Plasma prostaglandin E2 and prostacyclin levels were higher in females than males and unaffected by diet. In contrast, plasma nitric oxide levels were reduced by HS, regardless of gender. In isolated aortic rings, HS diet caused a smaller elevation in the stimulated norepinephrine release ratio in female rats than in males. Thus, salt-induced hypertension is associated with a reduction in levels of nitric oxide regardless of gender. Plasma prostaglandin E2 and prostacyclin levels were higher in females. Taken together, the higher plasma prostaglandin levels and reduced sympathetic activity in females may be contributing factors in their lower blood pressure and reduced mortality.

Cardiovascular responses to simulated microgravity in Sprague-Dawley rats.

Microgravity is known to induce orthostatic intolerance and baroreflex impairment in astronauts. Cardiovascular responses observed in 30° head-down tilt rat models, whether 24 hr whole body suspension (WBS) or 7 day tail-suspension (TS), mimic observations made during exposure to microgravity. We evaluated the cardiovascular effects of simulated microgravity and the subsequent post-suspension in rats using the above models. Mean arterial pressure (MAP) of both WBS and TS rats did not change during suspension. In both models, MAP decreased post-suspension and this response lasted for 6 hrs. Salt-loaded animals did not show a post-suspension reduction in MAP. Plasma ionized calcium was decreased at 2 hr of WBS, with no change in sodium, potassium, magnesium, glucose, or hematocrit. Body weight changes were similar for all animals whether under suspension or control conditions. Both rat models demonstrate post-suspension hypotension and these results support the notion that salt-loading may have some beneficial effects in ameliorating this hypotension.

L-NAME, A NITRIC OXIDE SYNTHASE INHIBITOR, AS A POTENTIAL COUNTERMEASURE TO POST-SUSPENSION HYPOTENSION IN RATS

A large number of astronauts returning from spaceflight experience orthostatic hypotension. This hypotension may be due to overproduction of vasodilatory mediators, such as nitric oxide (NO) and prostaglandins. To evaluate the role of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) as a countermeasure against the post-suspension reduction in mean arterial pressure (MAP), we assessed the cardiovascular responses and vascular reactivity to 7-day 30° tail-suspension and a subsequent 6 hr post-suspension period in conscious rats. After a pre-suspension reading, direct MAP and heart rate (HR) were measured daily and every 2 hrs post-suspension. The NO synthase inhibitor L-NAME (20 mg/kg, i.v.), or saline, were administered after the 7th day reading prior to release from suspension and at 2 and 4 hrs post-suspension. At 6 hrs post-suspension, vascular reactivity was assessed. While MAP did not change during the suspension period, it was reduced post-suspension. Heart rate was not significantly altered. L-NAME administration reversed the post-suspension reduction in MAP. In addition, the baroreflex sensitivity for heart rate was modified by L-NAME. Thus, the post-suspension reduction in MAP may be due to overproduction of NO and altered baroreflex activity.

7-OH-DPAT-Induced Inhibition of Norepinephrine Release in PC12 Cells

The purpose of this study was to investigate mechanisms of suppression of norepinephrine release by 7-OH-DPAT, a dopamine D₂/D₃ receptor agonist, in PC12 cells pretreated with nerve growth factor (NGF). 7-OH-DPAT caused inhibition of basal and K⁺-evoked norepinephrine release, which could be blocked by pretreatment with raclopride, a D₂/D₃ receptor antagonist. Moreover, dopamine D₂ and D₃receptors were identified by immunocytochemistry. Expression of D₂, D₃, and D₄ mRNAs and their proteins were detected using RT-PCR and immunoblotting. Furthermore, 7-OH-DPAT produced no change in cGMP levels; however, 7-OH-DPAT inhibited forskolin-stimulated cAMP accumulation that was antagonized by pretreatment with raclopride. In addition, 7-OH-DPAT inhibited carbachol-induced Ca²⁺ transient, conversely, 7-OH-DPAT had no effect on 4-aminopyridine-induced Ca²⁺ transient. Taken together, suppression of cAMP accumulation and calcium mobilization by 7-OH-DPAT is involved in the inhibition of norepinephrine release through activation of dopamine D₂/D₃ receptors.

NOS II inhibition attenuates post-suspension hypotension in Sprague-Dawley rats.

The reduction in mean arterial pressure observed in astronauts may be related to the impairment of autonomic function and/or excessive production of endothelium‐derived relaxing factors. Here, we examined the role of a nitric oxide synthase II (NOS II) inhibitor AMT (2‐amino‐dihydro‐6‐methyl‐4H‐1,3‐thiazine) against the post‐suspension reduction in mean arterial pressure (MAP) in conscious male Sprague‐Dawley rats. Direct MAP and heart rate were determined prior to tail‐suspension, daily during the 7‐day suspension and every 2 hrs post‐suspension. Prior to release from suspension and at 2 and 4 hrs post‐suspension, AMT (0.1 mg/kg), or saline, were administered intravenously. During the 7‐day suspension, MAP was not altered, nor were there significant changes in heart rate. The reduction in MAP post‐suspension in saline‐treated rats was associated with significant increases in plasma nitric oxide and prostacyclin. 2‐Amino‐dihydro‐6‐methyl‐4H‐1,3‐thiazine reduced plasma nitric oxide levels, but not those of prostacyclin, attenuated the observed post‐suspension reduction in MAP and modified the baroreflex sensitivity for heart rate. Thus, the post suspension reduction in mean arterial pressure is due, in part, to overproduction of nitric oxide, via the NOS II pathway, and alteration in baroreflex activity.