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This Article Abstract Full Text (PDF) All Versions of this Article: 90/4/607    most recent expphysiol.2005.030015v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cholewa, B. C Articles by Mattson, D. L Search for Related Content PubMed PubMed Citation Articles by Cholewa, B. C Articles by Mattson, D. L Related Collections Cardiovascular control

¹ Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA

	Abstract

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The present experiments were performed to determine the influence of intravenous administration of renin substrate on plasma angiotensin II levels and mean arterial blood pressure in conscious C57BL/6J mice. Mice with chronic indwelling femoral arterial and venous catheters were acutely or chronically administered intravenous doses of a synthetic peptide corresponding to the 14 amino acids on the N-terminal of angiotensinogen. A dose-dependent increase in arterial blood pressure was observed as the intravenous bolus dose of the renin substrate was increased from 0.18 to 180 nmol kg^–1 with a maximal increase in pressure of 40 ± 3 mmHg achieved following administration of the 18 nmol kg^–1 bolus (n = 11). Additional experiments demonstrated that a sustained intravenous infusion of the renin substrate led to a long-term increase in arterial blood pressure. The continuous infusion of renin substrate at 0.05 nmol kg^–1 min^–1 for 3 days did not alter arterial blood pressure from the control level of 119 ± 5 mmHg (n = 5); however, arterial blood pressure significantly increased to 129 ± 6 mmHg with an infusion rate of 0.5 nmol kg^–1 min^–1 and further increased to 141 ± 3 mmHg when the renin substrate infusion was increased to 5.0 nmol kg^–1 min^–1. Finally, the infusion of renin substrate at 5.0 nmol kg^–1 min^–1 resulted in a significant increase in plasma angiotensin II concentration from 34 ± 6 pg ml^–1 in vehicle-infused mice to 288 ± 109 pg ml^–1. These results demonstrate that modulation of the circulating level of angiotensinogen can alter the plasma angiotensin II level and arterial blood pressure in normal animals.

(Received 21 January 2005; accepted after revision 12 April 2005; first published online 15 April 2005)
Corresponding author D. L. Mattson: Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, PO Box 26509, Milwaukee, WI 53226-0509, USA. Email: dmattson{at}mcw.edu

	Introduction

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A role for angiotensinogen (AGT) in hypertension has been recognized from genetic studies in humans and animals. A variant of the AGT gene has been found to associate with high blood pressure (Jeunemaitre et al. 1992) and blood pressure-associated phenotypes in humans (Pereira et al. 2003). Furthermore, a positive correlation between blood pressure and genetic loci near the AGT gene was identified in recombinant inbred strains of rats (Pravenec et al. 1995). Genetic association data in both humans and animals therefore indicate that AGT may be important in the development of hypertension.

Genetic manipulation studies in mice have also demonstrated an important role for AGT in the regulation of blood pressure. Gene-duplication and gene-targeting techniques have produced mice with zero, one, two, three or four copies of the mouse AGT gene (Kim et al. 1995). In these mice, steady-state levels of AGT and arterial blood pressure increased progressively with gene copy number (Kim et al. 1995). More recently, transgenic mice that express different variants of the human (Cvetkovic et al. 2002) or rat (Kimura et al. 1992) AGT gene have demonstrated the importance of different AGT haplotypes on arterial blood pressure.

Although the modulation of plasma renin is generally considered to be the primary regulator of the renin–angiotensin system (RAS), experiments in laboratory animals have demonstrated that the circulating or tissue level of AGT may also be important in the regulation of the RAS. Studies in the spontaneously hypertensive rat demonstrated that antisense inhibition of an AGT mRNA-stabilizing protein leads to a reduction in arterial blood pressure (Klett et al. 2004). Moreover, intrarenal AGT mRNA and protein (Kobori et al. 2001a; Kobori et al. 2001b) levels are increased in experimental angiotensin (ANG) II-induced hypertension in rats, indicating both a role of AGT in hypertension and a potential feed-forward effect of ANG II on AGT. Recent studies from our laboratory in normal rats and mice demonstrated that circulating AGT levels increased as dietary sodium intake was reduced, indicating that circulating AGT is physiologically modulated during changes in sodium intake (Cholewa et al. 2005). In addition, modulation of AGT levels appears to be important to maintain normal plasma ANG II levels in mice. Plasma renin concentration (PRC) in mice is markedly elevated, though plasma renin activity (PRA) and circulating ANG II levels in mice (Yan et al. 1998; Catanzaro et al. 1999; Mazzolai et al. 2000; Cholewa & Mattson, 2001; Cholewa et al. 2005) are similar to the levels of PRA and ANG II observed in other species (Rieder et al. 1997; Gross et al. 1998; Cholewa et al. 2005). The ability of mice to maintain normal ANG II levels in the face of a high PRC appears to be due to the 8- to 10-fold reduction in circulating AGT levels when compared to other species. Together, these data indicate that plasma AGT levels are physiologically modulated in the feedback pathways which regulate both the RAS and the long-term level of arterial blood pressure.

Despite these intriguing observations, which indicate that circulating AGT levels are critical in the regulation of blood pressure, experiments to directly test the importance of circulating AGT on plasma ANG II levels and the chronic regulation of arterial blood pressure have not been performed. In the present experiments, we hypothesized that acute and chronic elevations in the concentration of renin substrate will lead to increased plasma ANG II concentration and increased mean arterial pressure (MAP) in conscious mice. To test this hypothesis, we examined the acute and chronic influence of intravenous infusion of renin substrate on MAP in conscious C57BL/6J mice. Concomitant pharmacological studies and biochemical measurements of PRC and plasma ANG II concentration were made to document modulation of the RAS during the administration of renin substrate.

	Methods

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Experiments were performed on 6- to 8-week old male C57BL/6J mice (25–30 g) obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Normal food and tap water were provided ad libitum. The Medical College of Wisconsin Animal Care Committee approved all animal procedures.

Separate groups of animals were prepared for the protocols detailed below. The chronic catheterization of the femoral artery and the femoral vein was performed as we previously described (Cholewa & Mattson, 2001; Cholewa et al. 2005). Mice were pre-anaesthetized with isoflurane, and sodium pentobarbital (50 mg kg^–1, I.P.) was administered to induce anaesthesia. Supplemental anaesthesia was administered as needed. Using aseptic techniques, catheters were placed in the femoral artery for the measurement of arterial pressure and blood sampling and in the femoral vein for infusions. The catheters were tunnelled subcutaneously and exteriorized through a lightweight tethering spring at the back of the neck. During the surgical procedure and recovery from anaesthesia, the animals were kept warm on a heated surgical table. The animals were allowed to recover for 3–5 days before the experimental protocol. Any animal exhibiting pain or distress was killed with an overdose of pentobarbital I.V. or I.P. Arterial pressures (systolic, diastolic and mean) were measured as previously described for 2–3 h at the same time each day (Cholewa & Mattson, 2001; Cholewa et al. 2005).

All mice were housed individually in metabolic cages within a specially designed, chronic rodent haemodynamic monitoring facility. Arterial pressure was recorded using solid-state pressure transducers (Argon Medical Technologies, Athens, TX, USA). The output of the analog pressure signals was amplified (StemTec, GPA-4: Quintron, Menominee Falls, WI, USA), low-pass filtered (30 s^–1, 4 pole) and sampled at 360 s^–1 (Data Translation, Marlboro, MA, USA). The frequency response of the entire analog and digital system (catheter, transducer, amplifier, A/D converter and computer) was evaluated and found to be of second order with a damping ratio of 0.4, a roll off frequency of >16 s^–1, and an average amplitude ratio of 0.993 over the range of 0–35 s^–1. The pulsatile blood pressure signals were reduced to periodic averages of mean arterial blood pressure and heart rate.

Protocol 1: influence of acute intravenous administration of synthetic renin substrate on MAP in conscious mice

Following the surgical recovery period, the blood pressure response to intravenous doses of renin substrate, the amino terminal tetradecapeptide of AGT, was measured. An intravenous bolus doses of synthetic rat renin substrate (0, 0.18, 1.8, 18 and 180 nmol (kg body weight)^–1) was administered to the mice in 100 µl of saline. Preliminary studies demonstrated that blood pressure was equally sensitive to both synthetic mouse renin substrate (H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Tyr-His-Asn-Y) (Hatae et al. 1994) and synthetic rat renin substrate (H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-Y) (Hatae et al. 1994). The commercially available, synthetic rat renin substrate (American Peptide Company, Sunnyvale, CA) was used in the present experiments to ensure accurate administration of the purified peptide. Prior to the administration of a bolus of the renin substrate, a 10-min control MAP was recorded. The synthetic renin substrate bolus was then administered, and MAP was continuously recorded until it returned to the control levels (average control MAP ± 1 S.D.). These steps were repeated for each successive dose of the renin substrate. The change in MAP was calculated by subtracting the control value from the peak increase of MAP following the administration of the bolus. Additional experiments were performed to document the response to synthetic renin substrate using a pharmacological inhibitor of the RAS. The pressor response to 1.8 nmol kg^–1 of the renin substrate was measured before and 1 h after intravenous administration of the angiotensin AT₁ receptor blocker losartan (100 mg kg^–1, I.V.).

Protocol 2: influence of long-term intravenous infusion of renin substrate on MAP in conscious mice

Mice were prepared as described above and allowed to recover from surgery while maintained on normal chow and tap water. The mice received a continuous, intravenous infusion of isotonic saline (3 ml day^–1) for the initial 3 days of the protocol; the synthetic renin substrate was then added to the intravenous infusate to continuously deliver 0.05 nmol kg^–1 min^–1 for 3 days. The synthetic renin substrate infusion rate was then increased to 0.5 nmol kg^–1 min^–1 for an additional 3 days, and was increased to 5.0 nmol kg^–1 min^–1 for the final 3 days of the experimental protocol.

Plasma samples were obtained for the measurement of PRC and plasma ANG II concentration after the vehicle infusion period and at the end of the final experimental period in which the synthetic renin substrate was infused at 5.0 nmol kg^–1 min^–1. As previously described (Cholewa & Mattson, 2001), a relatively large amount of blood was required for the assays (25 µl for PRC and 400 µl for ANG II concentration). The withdrawal of these combined amounts of whole blood leads to a marked hypotensive response in conscious mice. To minimize the disturbances of blood withdrawal on systemic haemodynamics and to permit repeated sampling from the same mouse, the blood withdrawn from the femoral artery was simultaneously replaced with an equal volume of donor blood infused into the femoral venous catheter. The donor blood consisted of red blood cells obtained from littermate donor mice suspended in artificial plasma. All assays were performed as we previously described (Cholewa & Mattson, 2001; Cholewa et al. 2005). PRC was measured by radioimmunoassay (RIA) using a modification of previously published methods (Sealey & Laragh, 1977) with nephrectomized rat plasma used as substrate for PRC. Plasma ANG II concentration was measured by RIA following HPLC separation of ANG I, ANG II and the primary angiotensin metabolites, as previously described (Rieder et al. 1997; Cholewa et al. 2005).

Statistical analysis

All data are expressed as mean ± S.E.M. Data were analysed with either a paired t test or a repeated-measures one-way ANOVA with Tukey's post hoc test. P < 0.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Protocol 1: influence of acute intravenous administration of renin substrate on MAP in conscious mice

The influence of acute intravenous bolus injection of synthetic renin substrate on arterial blood pressure and heart rate in conscious C57BL/6J mice is illustrated in Fig. 1. The baseline level of MAP and heart rate (HR) averaged 113 ± 4 mmHg and 612 ± 26 beats min^–1, respectively. A dose-dependent increase in MAP was observed as the bolus dose of the synthetic renin substrate was increased from 0.18 to 180 nmol kg^–1 with a maximal significant increase in pressure of 40 ± 3 mmHg achieved following administration of the 18 nmol kg^–1 bolus (n = 11). Concurrently, HR was significantly decreased when the renin substrate was administered. The timing of the response was also assessed for the 18 nmol kg^–1 bolus dose of the tetradecapeptide. The maximal increase in blood pressure following the administration of the bolus was achieved in 33 ± 3 s and blood pressure returned to within one standard deviation of the control MAP in 5 ± 1 min after the administration of the peptide.

View larger version (12K): [in this window] [in a new window]   Figure 1.  Effect of acute intravenous bolus administration of synthetic renin substrate on the change in mean arterial blood pressure and heart rate in conscious C57BL/6J mice n = 11; *P < 0.05 versus control.

Protocol 2: influence of long-term intravenous infusion of renin substrate on MAP in conscious mice

The influence of sustained intravenous administration of synthetic renin substrate to conscious C57BL/6J mice is illustrated in Fig. 2. The MAP value for each dose of the substrate is represented as the mean of the values from the 3 days of infusion. The infusion of renin substrate at 0.05 nmol kg^–1 min^–1 for 3 days did not alter MAP from the control level of 119 ± 5 mmHg (n = 5), but MAP was significantly increased to 129 ± 6 mmHg with 0.5 nmol kg^–1 min^–1 and further increased to 141 ± 3 mmHg when the infusion was increased to 5.0 nmol kg^–1 min^–1 for 3 days. During the infusion of the renin substrate (5.0 nmol kg^–1 min^–1), PRC was suppressed from 1643 ± 667 to 756 ± 144 ng ANG I ml^–1 h^–1. In contrast, plasma ANG II concentration significantly increased from 35 ± 5 pg ml^–1 in vehicle-infused mice to 288 ± 94 pg ml^–1 following administration of the renin substrate (5.0 nmol kg^–1 min^–1).

View larger version (12K): [in this window] [in a new window]   Figure 2.  Effect of sustained intravenous administration of synthetic renin substrate on mean arterial blood pressure and heart rate in conscious C57BL/6J mice Each dose was administered for 3 consecutive days. n = 5; *P < 0.05 versus control.

	Discussion

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These results are in general agreement with observations made in genetically manipulated mice, where MAP was elevated as AGT gene copy number increased (Kim et al. 1995). The data are also qualitatively in agreement with studies performed in Sprague-Dawley rats that demonstrated that MAP was acutely increased following bolus injections of AGT, though the maximal response observed in the rats did not occur for 30–40 min following the injection (Klett & Granger, 2001). Unlike the pressor response in conscious rats to bolus injections of AGT, the blood pressure response in the conscious C57BL/6J mice occurred within a shorter period of time, with detectable increases in MAP beginning within 30 s of the administration of the renin substrate. An explanation for the difference in the timing of the response between rats and mice is not apparent, though one potential reason for this discrepancy is that mice have a PRC significantly greater than the PRC in other species, including Sprague-Dawley rats. The increased level of plasma renin in mice may permit a more rapid conversion of the renin substrate to ANG II and therefore a more rapid pressor effect. A second possibility is a difference in the reaction kinetics between renin and AGT in comparison to the tetradecapeptide. It has been demonstrated that human renin cleaves AGT more slowly in vitro than the synthetic human tetradecapeptide substrate (Cumin et al. 1987; Stammers et al. 1987). If mouse renin reacts more readily in vivo with the tetradecapeptide than with AGT, this could explain the rather rapid pressor response observed in the present experiment.

Previous studies from our laboratory have demonstrated that MAP in the normal laboratory mouse is sensitive to an acute bolus administration of ANG II, with maximal increases in MAP observed following the administration of approximately 0.25 nmol kg^–1 of ANG II (Cholewa & Mattson, 2001). Our present studies showed a similar dose–response relationship to bolus injections of synthetic renin substrate, though the concentration of synthetic renin substrate necessary to elicit the maximal increase in MAP was 18 nmol kg^–1, approximately 72-fold greater in comparison with the response to acute bolus injections of ANG II (Cholewa & Mattson, 2001). This difference is likely to be due to the relatively slow conversion rate of renin substrate to ANG I. In separate studies, we have observed that the increases in MAP in response to ANG I in conscious C57BL/6J mice are nearly the same as those of ANG II (authors' unpublished observations), indicating that the conversion of ANG I to ANG II is not involved in limiting the rate of this reaction.

In summary, the present data demonstrate that MAP in conscious mice is sensitive to both acute and chronic intravenous infusion of synthetic renin substrate. These results demonstrate that elevating the available levels of AGT can increase circulating ANG II levels and ultimately MAP in normal mice. The data support the hypothesis that alterations in AGT levels can play an important role in the short- and long-term regulation of arterial blood pressure.

	References

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	Acknowledgements

 
This work was partially supported by National Institutes of Health Grants HL-29587 and DK-62803.

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This Article Abstract Full Text (PDF) All Versions of this Article: 90/4/607    most recent expphysiol.2005.030015v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cholewa, B. C Articles by Mattson, D. L Search for Related Content PubMed PubMed Citation Articles by Cholewa, B. C Articles by Mattson, D. L Related Collections Cardiovascular control