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(Circulation Research. 1997;80:189-195.)
© 1997 American Heart Association, Inc.

Articles

In Vitro Modulation of a Resistance Artery Diameter by the Tissue Renin-Angiotensin System of a Large Donor Artery

Daniel Henrion, Joelle Benessiano, Bernard I. Levy

the Institut National de la Sante et de la Recherche Medicale (INSERM) Unit 141, IFR Circulation Lariboisiere, Universite Paris (France) VII.

Correspondence to D. Henrion, PhD, INSERM U 141, Hopital Lariboisiere, 41 Bd de la Chapelle, 75475 Paris, cedex 10, France. E-mail levy@infobiogen.fr

	Abstract

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A local renin-angiotensin system (RAS) is present in the vasculature and might have an important role in the control of vascular resistance. In order to assess its functional role in the control of vasomotor tone, we investigated the effect of the RAS of a donor vessel (rat carotid artery) on the diameter of a recipient rat mesenteric resistance artery. Arteries were perfused in series in an arteriograph at a rate of 100 µL/min, under a pressure of 100 mm Hg. The two vessels were superfused in separate organ chambers to which drugs were added. Recipient artery internal diameter was measured continuously. Phenylephrine (0.1 µmol/L) was present in the organ baths throughout the experiments, ensuring a preconstriction of the recipient artery (236±4 to 174±3 µm, n=65 arterial segments from 34 rats). The angiotensin I–converting enzyme inhibitors (ACEIs) cilazapril (1 µmol/L) and captopril (10 µmol/L) inhibited phenylephrine-induced constriction by 30±12% (n=7, P<.001) and 20±8% (n=5, P<.01), respectively. Addition of cilazapril (1 µmol/L) or captopril (10 µmol/L) to the donor vessel chamber further inhibited the constriction by 8±3% (n=7, P<.01) and 31±10% (n=5, P<.05), respectively. The angiotensin II receptor (AT₁) antagonist losartan (10 µmol/L) prevented, in part, the relaxation due to the ACEI. The association of losartan (10 µmol/L) with the bradykinin B₂ receptor antagonist HOE 140 (1 µmol/L) totally prevented the relaxation due to the ACEI. Finally, angiotensin II was measured in the perfusate of the carotid artery and was found to be released at a rate of 11.9±2.2 pg in 60 minutes (n=8), which was significantly decreased to 1.4±0.4 pg in 60 minutes (n=4) by cilazapril (1 µmol/L). This study provides functional evidence that tissue-generated angiotensin II and bradykinin, produced locally and in upstream arteries, control the diameter of a resistance mesenteric artery.

Key Words: vascular reactivity • angiotensin II • angiotensin I–converting enzyme inhibitor • captopril • cilazapril

	Introduction

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The renin-angiotensin system was first described as a circulating hormone system participating in the homeostasis of blood pressure.¹ More recent studies have also reported a tissue (local) renin-angiotensin system located in the blood vessel wall.^{2 3 4 5} Findings of several experiments support this hypothesis: angiotensin is still present 48 hours after nephrectomy,⁶ venous concentration of angiotensin I is higher than arterial concentration,⁷ and renin is present in the vessel wall.⁸ In addition, molecular biology has shown that all the elements of the renin-angiotensin system are present in cultured vascular endothelial and smooth muscle cells.^{9 10 11} Finally, vessel wall renin is due to uptake from the circulation.^{12 13 14 15} We have previously shown that local production of angiotensin II is responsible for an active tone in the rat carotid artery perfused and pressurized in vitro.¹⁶ Furthermore, a local production of angiotensin II has been shown in the rat cremaster muscle microcirculation in vivo.¹⁵

Our hypothesis is that angiotensin I–converting enzyme activity in a large artery wall, through its effect on the local renin-angiotensin system, might affect smooth muscle function in downstream resistance arteries. Thus, in order to investigate the existence of a functional tissue or local renin-angiotensin system in a resistance mesenteric artery and to determine the ability of a donor vessel to produce angiotensin II that will influence a resistance artery tone, a segment of rat mesenteric resistance artery and a segment of rat carotid artery were mounted in cascade in an arteriograph and perfused under a pressure of 100 mm Hg and a flow rate of 100 µL/min, which are physiological conditions for resistance mesenteric arteries.¹⁷ In addition, we measured the amount of angiotensin II released by the isolated carotid artery.

	Materials and Methods

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Isolated Arteries
Twelve-week-old male Wistar Kyoto rats (Iffa-Credo, Lyon, France) weighing 300 g were used in the present study. After anesthesia (pentobarbital 50 mg/kg IV), the left carotid artery and a mesenteric artery segment (internal diameter, 200 µm) were isolated and cannulated at both ends and mounted in a video-monitored perfusion system.¹⁸ The arteries were bathed in two independent 5-mL organ baths (Fig 1) containing PSS of the following composition (mmol/L): NaCl 135.0, NaHCO₃ 15.0, KCl 4.6, CaCl₂ 1.5, MgSO₄ 1.2, glucose 11.0, and HEPES 5.0. The solution was bubbled with 95% O₂/5% CO₂. The pH of the solution was 7.4. The arteries were superfused at a rate of 4 mL/min. Perfusion of the arteries with a PSS of the same composition was set at a rate of 100 µL/min. The arteries were perfused in series, with the perfusion solution flowing from the carotid (donor vessel) to the mesenteric artery (recipient vessel) within 5 to 6 seconds. The perfusate and the superfusate were not recirculated. The pressure in the proximal end of the carotid artery segment was monitored using a pressure transducer and controlled by a servoperfusion system (Living System Instrumentation Inc). Arterial diameter was recorded using a video monitoring system (Living System Instrumentation Inc). Equilibrium diameter changes were measured when intraluminal pressure was set at 100 mm Hg. PSS containing PE (0.1 µmol/L) was present in the perfusate and the superfusate of both the mesenteric and the carotid arteries in order to ensure preconstriction. At the end of each experiment, the artery was perfused and superfused with a Ca²⁺-free PSS containing EGTA (2 mmol/L) and SNP (100 µmol/L) in order to determine the passive diameter of the vessels, ie, diameter in the absence of smooth muscle tone.

View larger version (63K): [in this window] [in a new window]   Figure 1. Schematic representation of the experimental setup used to measure the internal diameter of a small resistance mesenteric artery in vitro. A resistance mesenteric artery (art. mes.) and a carotid artery were mounted in two separate organ chambers and perfused in cascade at a rate of 100 µL/min and a pressure (P) of 100 mm Hg. Drugs could be added to each organ chamber separately. The perfusate was flowing from the carotid artery to the mesenteric resistance artery within 5 to 6 seconds.

Pressure and diameter measurements were collected by a computerized data acquisition system (Biopac MP 100), recorded, and analyzed on a Macintosh Quadra computer (Apple) using Acqknowledge software (Biopac). Results are given in micrometers for artery diameters or as changes in diameter (in micrometers).

Integrity of the endothelium was assessed in each mesenteric arterial segment by its ability to dilate to acetylcholine (1 µmol/L) after preconstriction with PE (0.1 µmol/L). Because diameter measurements could be performed on only one vessel, ie, the mesenteric artery in the present study, integrity of the carotid artery endothelium was assessed indirectly. After preconstriction of the mesenteric artery with PE (0.1 µmol/L) added to the superfusate, the further addition of PE (0.1 µmol/L) to the superfusate of the carotid artery induced a dilation of the mesenteric artery due to NO as it was blocked by L-NNA (10 µmol/L). Diffusion of PE into the lumen of the carotid artery and, consequently, to the lumen of the mesenteric artery had no more contracting effect, because the mesenteric artery was already preconstricted with PE (0.1 µmol/L). In addition, the integrity of the endothelial cells was verified as previously described.¹⁹ Briefly, the carotid and the mesenteric artery were opened longitudinally at the end of each experiment. Segments were washed and examined for bound Evans blue. No bound dye means that the endothelium surface is intact.¹⁹ Experiments in which the endothelium was stained at the end of the experiment were rejected.

In preliminary experiments, we determined that PE (0.1 µmol/L) added to the bath of the carotid artery induced a decrease in the diameter of the carotid artery (1125±160 to 1050±80 µm, n=4, P<.05). In another series of experiments, the addition of PE (0.1 µmol/L) to the bath of the carotid artery induced a decrease in diameter of the mesenteric artery (218±17 to 175±14 µm, n=5, P<.01) after 2 to 3 minutes. This experiment showed that drugs added to the organ bath of the carotid artery diffuse into the lumen of the vessel and are carried downstream to the mesenteric artery. As a consequence, we used an experimental protocol taking into account this diffusion of drugs through the vessel wall (see below). Diameters of carotid and mesenteric arteries could not be measured simultaneously. Thus, in all the experiences described below, only mesenteric arterial segment diameter was continuously measured. Indeed, the presence of a functional renin-angiotensin system in the carotid artery has been shown in a previous study.¹⁶ This is also our reason for using the carotid artery as a donor vessel.

Because substances added into the bath of the carotid artery diffuse through the vessel wall and then flow to the mesenteric artery, we first assessed the direct effect of adding ACEI to the recipient artery chamber; the additional effects of adding ACEI to both the donor and the recipient artery baths were attributed to ACE inhibition in the donor vessel.

Experimental Protocols
In the first group of experiments, the NO synthesis inhibitor L-NNA (10 µmol/L) was added into the chamber of the donor artery for 10 minutes, and then L-NNA was added to the chamber of the mesenteric artery ("recipient" vessel) for an additional 10-minute duration (protocol A, Fig 2). In other experiments, L-NNA was added to the recipient vessel and then to the donor vessel.

View larger version (33K): [in this window] [in a new window]   Figure 2. Experimental protocol used to study the effect of L-NNA, the ACEIs (cilazapril or captopril), losartan, and HOE 140 on the internal diameter of a small resistance mesenteric artery. A segment of resistance mesenteric artery (right) and a segment of carotid artery (left) were mounted in two separate organ chambers. They were perfused with PSS in series, from the carotid to the mesenteric artery, at a rate of 100 µL/min and a pressure of 100 mm Hg. Drugs could be added to each organ chamber separately. When several drugs were added consecutively, 1 designates the first drug added; 2, the second; and 3, the third.

In a second group of experiments, the ACEI captopril or cilazapril (0.1 to 10 µmol/L) was added to the organ chamber of the recipient vessel (protocol B, Fig 2). Each concentration was left for 20 minutes.

In a third group of experiments, the ACEI was added to the organ chamber of the donor artery for 20 minutes. The ACEI was then added to the organ chamber of the recipient artery for an additional period of 20 minutes (protocol C, Fig 2).

In a fourth group of experiments, the ACEI was added to the organ chamber of the recipient vessel for 20 minutes and was then added to the organ chamber of the donor vessel for an additional period of 20 minutes (protocol D, Fig 2).

In a fifth group of experiments, the angiotensin II receptor blocker losartan (10 µmol/L)²⁰ was added to the organ chamber of the recipient vessel 20 minutes before the addition of the ACEI to the organ chamber of the donor vessel and/or of the recipient artery (protocol E, Fig 2).

In a sixth group of experiments, losartan was added to the organ chamber of the donor vessel for 20 minutes (protocol F, Fig 2).

Finally, HOE 140 (1 µmol/L), a specific bradykinin B₂ receptor blocker,²¹ was added to the organ chamber of the recipient vessel after the addition of cilazapril (1 µmol/L) to the organ chamber of the recipient artery and/or to the organ chamber of the donor vessel (protocol G, Fig 2). In some experiments, HOE 140 (1 µmol/L) was added to the organ chamber of the recipient artery after the addition of losartan and cilazapril (1 µmol/L) to the donor vessel or to the bath of the recipient vessel.

Care and euthanasia of the study animals were in accordance with the European Community standards on the care and use of laboratory animals (Ministere de l'agriculture, France, authorization nb 006422).

Determination of Angiotensin II in the Perfusate
In a separate series of experiments, carotid arteries were mounted in the arteriograph as described above. They were then perfused at a rate of 100 µL/min and under a pressure of 100 mm Hg. After 1 hour of equilibration, the perfusate was collected for 60 minutes and frozen before angiotensin II determination. In another series of experiments, carotid arteries were bathed in PSS containing cilazapril (1 µmol/L).

Angiotensin II was measured according to the method of Simon et al.²² Briefly, angiotensin peptides were extracted from the perfusate by reversible adsorption to bonded phase silica (Bond Elut, Analytichem). Angiotensin II in the solid-phase extract was then measured by a direct radioimmunoassay method with an immobilized monoclonal antibody, anti–angiotensin II (ERIA Diagnostics Pasteur).

Statistical Analysis
Results are expressed as mean±SEM. Significance of the effect of a drug on the mesenteric arterial diameter was determined after one-factor ANOVA for repeated measures, and means were compared using the Bonferroni test. Values of P<.05 were considered to be significant.

Drugs
HEPES, L-NNA, captopril, and EGTA were purchased from Sigma Chemical Co. Cilazapril was obtained from Lederle, and HOE 140 was from Hoechst-France.

	Results

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Sixty-five segments of mesenteric artery and 65 segments of carotid artery were isolated from 34 rats (mean carotid arterial pressure, 115±8 mm Hg). In Ca²⁺-free PSS containing EGTA (2 mmol/L) and SNP (100 µmol/L), passive diameter of the mesenteric arterial segments was 236±4 µm. In a PSS containing PE (0.1 µmol/L), the mesenteric artery developed tone that reduced the vessel diameter to 174±3 µm. These arterial segments were divided into 13 groups (n=4 to 7). There was no significant difference in passive diameter or diameter under PE between the different groups.

Only recipient vessels responding to acetylcholine (1 µmol/L) by dilation were included in an experimental protocol. Acetylcholine (1 µmol/L) induced a significant dilation (169±3 to 217±4 µm, n=65, P<.001) of PE-induced constriction (0.1 µmol/L, 230±4 to 169±3 µm) in recipient vessels. Similarly, only donor vessels able to dilate a recipient vessel preconstricted by PE (0.1 µmol/L) after the addition of PE (0.1 µmol/L) to the donor vessel were included in the study. Recipient arteries preconstricted with PE (0.1 µmol/L, 234±5 to 175±4 µm) were significantly dilated when PE (0.1 µmol/L) was added in the bath of the donor vessel (175±4 to 189±3 µm, n=65, P<.001).

Fig 3 shows typical recordings of experiments in which L-NNA (10 µmol/L) was added to the donor vessel chamber. L-NNA (10 µmol/L) induced two successive constrictions of the recipient vessel in addition to the constriction induced by PE (Fig 3, upper trace). This was observed in three of five vessels. In Fig 3 (lower trace), the recipient artery was pretreated with L-NNA (10 µmol/L), and the further addition of L-NNA (10 µmol/L) to the donor vessel chamber induced only one constriction of the recipient vessel. The failure to see a second decrease in diameter suggests that the second decrease in diameter seen in the upper trace (Fig 3) may be due to L-NNA transport to the recipient vessel.

View larger version (20K): [in this window] [in a new window]   Figure 3. Typical recording showing the effect of L-NNA (10 µmol/L) on the internal diameter of a mesenteric resistance artery. A resistance mesenteric artery (recipient) and a carotid artery (donor) were mounted in two separate organ chambers and perfused in cascade. L-NNA was added in the donor vessel (top recording) or to the recipient and then to the donor artery (bottom recording).

When added to the donor artery, L-NNA (10 µmol/L) induced a significant constriction (15±6% increase of PE-induced constriction) of the recipient artery. A further significant constriction (7±3% increase of PE-induced constriction) was observed when L-NNA (10 µmol/L) was added to the recipient vessel (Fig 4, right panel). In another series of experiments, L-NNA significantly constricted the recipient vessel when added successively to the recipient vessel (32±7% increase of PE-induced constriction) and to the donor vessel (9±2% increase of PE-induced constriction) (Fig 4, left panel).

View larger version (37K): [in this window] [in a new window]   Figure 4. Effect of L-NNA (10 µmol/L) on the diameter of mesenteric resistance arterial segments preconstricted with PE (0.1 µmol/L). L-NNA was added in the recipient vessel and then to the donor vessel (left, n=5) or in the donor vessel and then in the recipient vessel (right, n=4). Values are mean±SEM. *P<.01 by one-factor ANOVA for repeated measures.

Fig 5 shows a typical recording of an experiment in which cilazapril (1 µmol/L) was added first to the recipient vessel chamber and induced a dilation. When cilazapril (1 µmol/L) was added to both the donor and the recipient vessel baths, a second dilation of the recipient vessel was observed. This dilation was partly counteracted when HOE 140 (1 µmol/L) was added to the recipient vessel chamber. Finally, the passive diameter was determined by the addition of a Ca²⁺-free, SNP (10 µmol/L)–containing, and EGTA (2 mmol/L)–containing PSS.

View larger version (24K): [in this window] [in a new window]   Figure 5. Typical recording showing the effect of the ACEI cilazapril (CIL) and of the bradykinin B2 receptor blocker HOE 140 on the diameter of mesenteric resistance arterial segments preconstricted with PE (0.1 µmol/L). The resistance mesenteric artery (recipient) and a carotid artery (donor) were mounted in two separate organ chambers and perfused in cascade. CIL (1 µmol/L) was added to the recipient vessel and then to the donor vessel. This was followed by the addition of HOE 140 (1 µmol/L) to the recipient vessel. Finally, the passive diameter of the artery was determined by the replacement of the bathing solution for a solution containing 0 Ca2+, SNP (10 µmol/L), and EGTA (2 mmol/L).

Cilazapril (0.1, 1, and 10 µmol/L) caused a concentration-dependent dilation of PE-induced constriction in the recipient vessel: 11±3% (n=4, P<.05), 35±10% (n=4, P<.05), and 33±8% (n=4, P<.05) dilation, respectively. In another group of experiments, cilazapril (1 µmol/L) induced a dilation (30±12% dilation of PE-induced constriction, n=7, P<.001) when added to the recipient vessel chamber and a further dilation (8±3% dilation of PE-induced constriction, n=7, P<.001) when added to the donor vessel (Fig 6, left panel). Cilazapril (1 µmol/L) also caused cumulative dilations of the recipient vessel when added successively to the donor vessel (11±4% dilation of PE-induced constriction, n=7, P<.001) and the recipient vessel (12±5% dilation of PE-induced constriction, n=7, P<.001) (Fig 6, right panel).

View larger version (38K): [in this window] [in a new window]   Figure 6. Effect of cilazapril (CIL, 1 µmol/L) on the diameter of mesenteric resistance arterial segments preconstricted with PE (0.1 µmol/L). The resistance mesenteric artery (recipient) and a carotid artery (donor) were mounted in two separate organ chambers and perfused in cascade. CIL was added to the recipient vessel and then to the donor vessel (left) or to the donor vessel and then to the recipient vessel (right). Values are mean±SEM (n=5 or 6 per group). *P<.001 by one-factor ANOVA for repeated measures.

After losartan (10 µmol/L)–induced dilation of the recipient vessel (35±11% dilation of PE-induced constriction, n=5, P<.01), the addition of cilazapril (1 µmol/L) to the recipient vessel caused a further dilation (7±3% dilation of PE-induced constriction, n=5, P<.05), whereas the addition of cilazapril (1 µmol/L) to the donor vessel caused no more significant dilation (Fig 7). Angiotensin II (10 nmol/L)–induced constriction (218±11 to 133±9 µm, n=4, P<.001) was abolished by losartan (10 µmol/L) in the rat resistance mesenteric artery (228±13 µm, not significantly different from 225±12 µm, n=5).

View larger version (44K): [in this window] [in a new window]   Figure 7. Effect of losartan (LOS, 10 µmol/L) and cilazapril (CIL, 1 µmol/L) on the diameter of mesenteric resistance arterial segments preconstricted with PE (0.1 µmol/L). The resistance mesenteric artery (recipient) and a carotid artery (donor) were mounted in two separate organ chambers and perfused in cascade. After the addition of LOS in the donor vessel, CIL was added to the recipient vessel and then to the donor vessel. Values are mean±SEM (n=6 per group). *P<.01 by one-factor ANOVA for repeated measures.

After losartan (10 µmol/L)–induced dilation of the recipient vessel (24±8% dilation of PE-induced constriction, n=6, P<.01), the addition of cilazapril (1 µmol/L) to the donor vessel or to the recipient vessel caused a further and significant dilation (8±4% dilation of PE-induced constriction, n=6, P<.05), which was reversed by the addition of HOE 140 (1 µmol/L) to the recipient vessel (Fig 8, left panel). When added to the recipient vessel after losartan (10 µmol/L), HOE 140 (1 µmol/L) prevented cilazapril (1 µmol/L)–induced dilation (Fig 8, right panel). HOE 140 (1 µmol/L) prevented bradykinin (1 µmol/L)–induced dilation of the recipient vessel (4±2.5% dilation of PE-induced constriction [n=4 with HOE 140] versus 46±1% [n=5 without HOE 140], P<.01).

View larger version (42K): [in this window] [in a new window]   Figure 8. Effect of losartan (LOS, 10 µmol/L), cilazapril (CIL, 1 µmol/L), and HOE 140 (1 µmol/L) on the diameter of mesenteric resistance arterial segments preconstricted with PE (0.1 µmol/L). The resistance mesenteric artery (recipient) and a carotid artery (donor) were mounted in two separate organ chambers and perfused in cascade. LOS was added in the recipient vessel, and then CIL was added to the donor vessel (left) or to the recipient vessel (right). HOE 140 was then added to the recipient vessel. Values are mean±SEM (n=6 per group). *P<.01 by one-factor ANOVA for repeated measures.

The cumulative addition of captopril (0.1, 1, and 10 µmol/L) to the recipient vessel induced a dilation of the preconstricted recipient vessel (235±14 to 172±11 µm, n=4, P<.001) by 2±1.5% (n=4, P=NS), 14±4% (n=4, P<.05), and 24±6% (n=4, P<.01), respectively. In another group of experiments, captopril (10 µmol/L) induced a significant dilation (183±42 to 197±41 µm, n=6, P<.01) of the recipient vessel when added to the recipient vessel and a further significant dilation (197±41 to 214±47 µm, n=6, P<.05) when added to the donor vessel. Captopril (10 µmol/L) also caused cumulative dilations of the recipient when added successively to the donor vessel (190±46 to 210.5±44 µm, n=5, P<.05) and then to the recipient vessel (210.5±44 to 222±46 µm, n=5, P<.05). Losartan (10 µmol/L) induced a significant dilation (18±6% dilation of PE-induced constriction, n=6, P<.01) of the recipient vessel when added to the recipient vessel. Further addition of captopril (10 µmol/L) to the recipient vessel caused an additional dilation (13±5% dilation of PE-induced constriction, n=6, P<.01), whereas the further addition of captopril (10 µmol/L) to the donor vessel caused no more significant dilation.

Determination of Angiotensin II in the Perfusate of the Carotid Artery
Angiotensin II was measured in the perfusate flowing through the lumen of the carotid artery for 60 minutes. The amount of angiotensin II released was 11.9±2.2 pg (n=8, P<.01 versus blank). It was significantly decreased to 1.4±0.5 (n=4, P<.01) by cilazapril (1 µmol/L). The "blank" measurement was performed with a PSS that did not perfuse the artery (0.77 pg). The limit of detection of the technique was 0.76 pg per essay, and the range of the calibration curve was 1.5 to 60 pg.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major new finding of the present study is that the local tissue renin-angiotensin and kallikrein-kinin systems of a large artery can significantly modulate smooth muscle tone in a downstream resistance artery. Thus, we showed that angiotensin I–converting enzyme activity in a large artery wall, through putative effects on the local renin-angiotensin system, not only was a regulator of the local smooth muscle tone but also affected smooth muscle function in a downstream resistance artery.

The angiotensin I–converting enzyme catalyzes the generation of the vasoconstrictor angiotensin II and the degradation of the vasodilator bradykinin. The antihypertensive action of ACEIs can be attributed to both the inhibition of the synthesis of angiotensin II and the inhibition of the degradation of bradykinin. The vasorelaxant effect of bradykinin is enhanced by ACEI,^{23 24 25 26} and plasma kinins increase in patients treated with ACEI.²⁷ Functionally, it has been shown that angiotensin II potentiates vessel responses to vasoconstrictor agonists in vitro^{28 29 30 31 32 33} and that endogenous angiotensin II potentiates PE-induced constriction in vivo.³⁴

In a previous study,¹⁶ we have shown that the carotid artery under physiological conditions of length, flow, and pressure possesses a basal tone that is decreased by ACEI and by the angiotensin II receptor blocker losartan, whereas HOE 140, the bradykinin B₂ receptor blocker,²¹ has no effect. Thus, we used the carotid artery as a donor vessel in the present study because this vessel possesses the ability to produce angiotensin II locally. We used the mesenteric resistance artery as in a previous study,³⁴ and we have shown in vivo that endogenous angiotensin II modulates mesenteric arterial vascular tone.

The arteriograph used in the present study allowed the perfusion and the pressurization of two isolated arteries mounted in cascade: one was a large "donor" vessel, and one was a small resistance artery, the "recipient" vessel. Transit time of the intraluminal physiological solution between donor and recipient vessels was 5 to 6 seconds. The release of NO by isolated vessels has already been widely documented,^{35 36} and in perfused arteries, NO is most probably released upon stimulation by shear stress due to flow.^{37 38 39 40}

In the present study, we demonstrated that angiotensin II produced by the wall of an artery was able to influence the diameter of another artery located downstream. Under control conditions, angiotensin II produced by the carotid artery increased mesenteric arterial tone. This was antagonized by the addition of an ACEI to the bath of the donor vessel and by the addition of losartan to the bath of the recipient vessel. Furthermore, angiotensin II produced by the carotid artery in the perfusate could be measured and was significantly decreased by the ACEI. The amount of angiotensin II released in 60 minutes by the carotid artery is of the same order as that found in a previous study in an organ culture model of rabbit aorta.⁴¹

We also found that a small mesenteric resistance artery is probably capable of producing angiotensin II (since ACEI relaxed the vessel) and that losartan can partially prevent this effect. This is the demonstration of a local production of angiotensin II in a resistance artery. This result is consistent with our previous studies performed in the carotid artery,¹⁶ in large mesenteric arteries,³⁴ and in the microcirculation.¹⁵ Other studies have also shown that angiotensin I is produced by the arterial wall⁷ and that renin is present in the vessel wall⁸ after extraction from the circulation.^{12 13 14 15} To avoid nonspecific effects, we used two different ACEIs, captopril and cilazapril, and we found a concentration-dependent effect of each ACEI.

Losartan blocked approximately two thirds of the ACEI-induced dilation of the mesenteric resistance artery when ACEIs were added either to the donor or to the recipient vessel. The proportion of the ACEI-induced dilation that was not blocked by losartan most probably was due to bradykinin, since HOE 140 suppressed this dilation. This is the first functional evidence that in addition to angiotensin II, bradykinin is produced locally in the carotid artery and in the mesenteric artery and is able to modulate mesenteric resistance arterial tone. Previous studies have shown that ACEIs potentiate bradykinin-induced dilation both in large^{23 24} and small²⁶ blood vessels. In another study, it has been shown that in rat skeletal muscle arterioles, bradykinin contributes to basal tone but not to PE-induced tone.⁴² In the present study, vascular tone was mainly due to PE, and both ACEI and HOE 140 were efficient in modulating this tone. In addition, in large mesenteric arteries precontracted with PE³⁴ as well as in carotid arteries with spontaneous tone,¹⁶ HOE 140 does not seem to have an effect. This most likely demonstrates that the repartition between angiotensin II and bradykinin in the control of vascular tone and in the effect of ACEI depends on the vascular region and on the type of tone involved.

In conclusion, we confirmed in the present work that isolated mesenteric resistance arterial tone is influenced by angiotensin II and bradykinin produced locally. More important, we show for the first time that angiotensin II and bradykinin produced by a large vessel (the carotid artery) could influence arterial tone in a resistance artery (mesenteric) located downstream.

	Selected Abbreviations and Acronyms

 

ACEI = angiotensin I–converting enzyme inhibitor L-NNA = N-nitro-L-arginine PE = phenylephrine PSS = physiological salt solution SNP = sodium nitroprusside

	Acknowledgments

 
This study was supported in part by a grant from Whyeth-Lederle (Paris, France) and by the Fondation pour la Recherche Medicale (Paris, France).

Received May 31, 1996; accepted November 12, 1996.

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