Low-Dose Angiotensin II Increases Free Isoprostane Levels in Plasma
Abstract—Chronic intravenous infusion of subpressor doses of angiotensin II causes blood pressure to increase progressively over the course of several days. The mechanisms underlying this response, however, are poorly understood. Because high-dose angiotensin II increases oxidative stress, and some compounds that result from the increased oxidative stress (eg, isoprostanes) produce vasoconstriction and antinatriuresis, we tested the hypothesis that a subpressor dose of angiotensin II also increases oxidative stress, as measured by 8-epi-prostaglandin F2α (isoprostanes), which may contribute to the slow pressor response to angiotensin II. To test this hypothesis, we infused angiotensin II (10 ng/kg per minute for 28 days via an osmotic pump) into 6 conscious normotensive female pigs (30 to 35 kg). We recorded mean arterial pressure continuously with a telemetry system and measured plasma isoprostanes before starting the angiotensin II infusion (baseline) and again after 28 days with an enzyme immunoassay. Angiotensin II infusion significantly increased mean arterial pressure from 121±4 to 153±7 mm Hg (P<0.05) without altering total plasma isoprostane levels (180.0±24.3 versus 147.0±29.2 pg/mL; P=NS). However, the plasma concentrations of free isoprostanes increased significantly, from 38.3±5.8 to 54.7±10.4 pg/mL (P<0.05). These results suggest that subpressor doses of angiotensin II increase oxidative stress, as implied by the increased concentration of free isoprostanes, which accompany the elevation in mean arterial pressure elevation. Thus, isoprostane-induced vasoconstriction and antinatriuresis may contribute to the hypertension induced by the slow pressor responses of angiotensin II.
The fast pressor effect of angiotensin II, induced by an intravenous bolus injection, is characterized by a rapid elevation of blood pressure, reaching a maximal increase of blood pressure in 1 to 2 minutes.1 2 This effect is due to an arterial vasoconstriction. However, the mechanism responsible for the progressive increase in blood pressure with long-term administration of subpressor doses of angiotensin II3 (slow pressor response) remains undefined.4 Several processes have been implicated in mediating this response, but none of them appears to completely account for this phenomenon.5 6 7 8 More recently, it has been shown that angiotensin can stimulate superoxidation (O2), which may quench nitric oxide (NO) with the formation of strong oxidative compounds such as peroxynitrite (ONOO−).9 10 Under these conditions, ONOO− could oxidize arachidonic acid, releasing isoprostanes.11 12 Among these substances, the 8-isoprostaglandin F2α is considered the most ubiquitous and reliable index of oxidative stress.
Isoprostanes are not only reliable markers of oxidative stress but also possess intrinsic vasoconstrictor and antinatriuretic properties via specific receptors,11 12 raising the possibility that they contribute to the hypertensive effects of angiotensin II. If subpressor doses of angiotensin II also induce oxidative stress, consequently increasing isoprostane levels, then it is possible that the increases in isoprostanes are responsible, at least in part, for the slow pressor responses to angiotensin II. Therefore, in the present study we determined whether swine also develop slow responses to subpressor doses of angiotensin II and whether these responses are accompanied by an increase in oxidative stress, as expressed by the elevation of plasma isoprostane F2α.
The present study, which had approval of the Institutional Animal Care and Use Committee, was performed in 6 female pigs with an average body weight of 35±3 kg. The major reason for conducting these studies in pigs resides in the anatomic similarity of the renal system with humans. The animals had free access to water and were fed chow containing 0.19% sodium. The pigs were fasted 16 hours before surgery and then anesthetized with 150 mg of xylazine and 1 g of ketamine; anesthesia was supplemented during surgery if necessary.
Blood Pressure Measurements
Blood pressure was monitored via a Physiotel telemetry implant (Data Sciences International) placed in the ventral aspect of the neck. Implants use a fluid-filled catheter, which refers pressure from the catheter tip, inserted into the carotid artery, to a sensor located in the body of the implant. The implant transmits the blood pressure data telemetrically to a receiver that converts the radiofrequency signal to a PC-based data collection system. This allowed for blood pressure monitoring from conscious, freely moving laboratory animals 24 hours a day for 7 control and 28 angiotensin II–infused days.
Angiotensin II Infusions
After 7 days of baseline blood pressure measurements, the pigs were anesthetized, and a blood sample was withdrawn from the jugular vein for plasma isoprostane levels. Under sterile conditions, an osmotic pump with 2-mL capacity and of 4 weeks’ duration was implanted subcutaneously in the ventral aspect of the neck (model 2 ML4, Alza Corp). A polyethylene catheter connected to 1 end of the osmotic pump flow modulator was inserted into the jugular vein. The pump was primed with 20 mg of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe acetate salt) (Sigma) in 2 mL of sterile isotonic saline. Infusion occurred at a constant rate of 2.5 μL/h, which delivers ≈14 ng/kg per minute at the beginning of the study and ≈8 ng/kg per minute at the end of the study (because of the growth of the pig). At the end of 28 days, the pigs were anesthetized, and a blood sample was withdrawn from the jugular vein for determination of plasma isoprostane levels. Isoprostanes circulate in 2 distinct forms, esterified in LDL phospholipids and as the free acid.13 14
Isoprostane Assay Procedure
Extraction and enzyme immunoassay procedures used to measure isoprostanes follow the methods supplied in the kit provided by Cayman Chemical with a few modifications. Plasma isoprostanes exist in circulation as either a free base or esterified to lipoproteins. To measure the esterified isoprostanes, a further extraction hydrolysis step is required for conversion to free bases that can then be measured by extraction and enzyme immunoassay. This measurement of all isoprostanes is referred to as the total. Samples are removed from −80°C storage and thawed on ice. Of the sample, 1.0 mL is used for measurement of free isoprostanes, while 0.5 mL is used to measure total isoprostanes. Absolute methanol is first added to all samples, followed by thorough mixing and centrifugation. For free isoprostane, the eluent is poured into a water/buffer solution and kept on ice, while the total eluent is poured in a solution of 15% KOH and incubated for 1 hour at 38°C. After incubation, a water/buffer solution is also added to the total samples, with a pH of 3.1±0.5 in all samples. Extraction is then performed on a Sep-Pak C18 column, with washes of water and hexane. The isoprostanes are eluted from the column with a solution of 99%/1% ethyl acetate/methanol, which is then dried off under nitrogen, and the samples are reconstituted into a 1.0-mL assay buffer. For the assay, standards and samples are first added in triplicate to the 96-well plate provided in the kit, followed by addition of tracer and antibody and then incubation overnight at room temperature. The next day, the plate is washed several times with wash buffer, followed by addition of Ellman’s reagent. After optimal development of color, the plate is read at 405 nm, and values of unknowns are expressed as picograms per milliliter.
Data are expressed in absolute values ±SEM. Paired t tests were used to examine whether the isoprostane and blood pressure levels were different before and after chronic infusion of Angiotensin II. The control mean arterial pressure (MAP) was the average of the last control day (day 7). The paired comparison was made with the 28th day of angiotensin II infusions, the same day the plasma isoprostane levels were determined. When the normality test failed, the Wilcoxon rank sum test was used instead. For all analyses, P<0.05 was considered significant.
The effects of subpressor angiotensin II infusion on MAP are depicted in Figure 1⇓. Infusion of angiotensin II increased MAP by day 2 of angiotensin II infusion. The increase plateaued on day 3 but appeared to increase again on day 15 and remained at an elevated level throughout the experiment. The MAP was 121±4 mm Hg on control day 7 and increased to 153±7 mm Hg (P<0.05) by day 28 of angiotensin II infusion. Figure 2⇓ summarizes the plasma free isoprostane data. Plasma free isoprostane increased from 38.3±5.8 pg/mL on control day 1 to 54.7±10.4 pg/mL (P<0.05) on day 28 of angiotensin II infusion. Total plasma isoprostane levels were 180.0±24.3 pg/mL in control and 147.0±29.2 pg/mL on day 28 (P=NS).
Slow Responses to Angiotensin II
The results of the present study show that chronic intravenous administration of subpressor doses of angiotensin II produces, in swine, a progressive elevation of MAP that resembles all the characteristics of the slow pressor response. In fact, preliminary studies in 5 pigs showed that acute infusion of 10 ng/kg per minute angiotensin II for 1 hour increased plasma levels of free isoprostanes by 28% (P<0.09) and total isoprostanes by 29% (P<0.07), accompanied by a 4-mm Hg increase in MAP. This elevation of MAP is much smaller than the total increase of 32±3 mm Hg recorded after 28 days of infusion of 10 ng/kg per minute during chronic infusion. Another characteristic of the slow response to angiotensin II is that it takes at least 3 to 4 days to develop completely.15 Our results show that from day 1 of the infusion, there was a continuous increase of MAP from 121 to 135 mm Hg. On day 3, the blood pressure plateaued up to day 13. However, on day 15, there was a further increase of MAP, reaching 153±7 mm Hg on day 28. It should be noticed that on day 28, there was no indication that the increase of blood pressure had reached a plateau. The experiment was stopped at this time, however, because a new osmotic pump would have to be implanted to continue with the infusion of angiotensin. These results allow us to conclude that the continuous administration of 10 ng/kg per minute of angiotensin II, which does not increase MAP when given as an acute infusion (J.C. Romero et al, unpublished data, 1999), is capable of producing a marked increase in blood pressure of 32.5 mm Hg after 28 days of infusion. It remains undefined whether an infusion of 10 ng/kg per minute of angiotensin would have produced a greater increase in blood pressure if given for a longer period of time. These results are comparable to those obtained in rabbits,3 rats,1 2 7 8 and dogs.5 15
The mechanism underlying the slow response to angiotensin II remains undefined. However, it has been linked by other investigators to the level of sodium intake,5 sympathetic activation at the central nervous system,3 and arteriolar hypertrophy.15 A high sodium diet potentiates significantly the development of the slow response to angiotensin.5 From these observations, it has been concluded that the level of angiotensin in plasma during continuous infusion may be inappropriate with respect to the levels of extracellular fluid volume. This possibility has been discarded by some authors because the slow responses to angiotensin II are triggered by levels of angiotensin II that are below those that are usually needed to stimulate steroidogenesis or thirst. However, since angiotensin II itself has powerful natriuretic actions5 or, alternatively, it may stimulate antinatriuretic compounds (eg, isoprostanes), it is still possible that an alteration in the balance between the extracellular fluid volume and angiotensin levels may contribute to the hypertension.
The second possibility is that angiotensin permeates the central nervous system, stimulating sympathetic activity at the level of the fourth ventricle (such as the area postrema or nucleus of the tractus solitarius). However, sympathectomy in the rat with the use of 6-hydroxydopamine does not prevent the slow pressor effect in rats.16 There are no signs of sympathetic overactivity during angiotensin II infusion in dogs17 or men.18 Taken together, these findings argue against a significant role for the central nervous system in the slow pressor responses of angiotensin II.
The ability of angiotensin to stimulate oxidative stress was first recognized by Rajagopalan et al.19 However, these investigators used a very large amount of angiotensin (≈0.7 mg per animal) to produce hypertension in rats. If angiotensin indeed stimulates oxidant production, there is a likelihood for oxygen to quench NO, reducing its concentration at the level of smooth muscle in peripheral resistance vessels. Furthermore, the binding of oxygen to NO could form potent oxidant substances such as peroxynitrites, which could oxidize arachidonic acid with the formation of isoprostanes. This substance has been reported to be one of the most sensitive markers in reflecting increases in oxidative stress. Furthermore, these compounds are powerful vasoconstrictors of isolated vascular smooth muscle cells14 and particularly of the preglomerular vasculature (leading to decreased glomerular filtration rate) and also stimulate tubular sodium reabsorption.20 These vasoconstrictor and antinatriuretic effects are both frankly prohypertensinogenic, suggesting that they may be potentially important in generating and/or sustaining hypertension during chronic angiotensin infusion.
The present study reveals that low doses of angiotensin II stimulate oxidative stress and consequently increase the free isoprostane levels in circulating blood. Both free and total isoprostanes are markers of oxidative stress. Isoprostanes are initially formed in vivo esterified to tissue lipids, and subsequently free isoprostanes are hydrolyzed and released into the systemic circulation. Thus, it is tempting to speculate that they may be involved in the slow pressor responses of angiotensin II. Further studies are needed to establish a causative role rather than merely a correlation between these compounds and the slow pressor responses to angiotensin II. Finally, it should be noted that oxidation also produces other forms of isoprostanes such as isoprostane D and A2, which are also vasoconstrictors. However, the F2α isoprostane isoform is one of the more abundant isoforms in the circulation, suggesting that it may be biologically important. However, it is important to note that local concentrations may be quite different.
In conclusion, in the present study we found that infusion of subpressor doses of angiotensin II into pigs causes blood pressure to rise progressively. This increase in blood pressure is accompanied by an increase in the circulating levels of free isoprostanes. Whether isoprostane-induced vasoconstriction and antinatriuresis play a role in the hypertensive responses in this model remains to be established.
This study was supported by National Institutes of Health grant HL-16496 and by the Mayo Foundation. The authors would like to thank John M. Stulak and Michael P. Stulak for technical assistance and Kristy J. Zodrow for secretarial assistance.
Reprint requests to J. Carlos Romero, MD, Department of Physiology, Mayo Clinic, Rochester, MN 55905.
- Received May 10, 1999.
- Revision received June 4, 1999.
- Accepted July 14, 1999.
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