Angiotensin II Increases cGMP Content Via Endothelial Angiotensin II AT1 Subtype Receptors in the Rat Carotid Artery
Abstract Angiotensin II (Ang II) has been reported to modulate cGMP formation in various types of cells. To acquire direct information on the intracellular transduction involved in this mechanism, we tested the effects of Ang II on vascular tone and on cGMP content of in vitro isolated carotid arteries from 12-week-old Wistar-Kyoto rats. Segments of carotid artery 20 mm long (n=8 for each group) maintained at a transmural pressure of 100 mm Hg were immersed in a bath (38°C) containing oxygenated Tyrode’s solution. At the end of each experiment, the vessel diameter was measured, and the wall cGMP content was determined by enzyme immunoassay. Under basal conditions, mean diameter was 968±19 μm, and mean cGMP carotid artery content was 38.9±3.5 fmol/mg tissue. Incubation for 20 minutes with Ang II (10−5 mol/L) significantly increased cGMP wall content, twofold above the basal content (P<.01), and constricted the vessel (60±2.2% of the control diameter, P<.001). After preincubation with a nonselective antagonist of Ang II receptors, saralasin ([Sar1,Val5,Ala8]Ang II, 5×10−5 mol/L), or with a specific antagonist of Ang II AT1 receptor subtype, losartan (5×10−5 mol/L), carotid diameter and cGMP content were no longer affected by Ang II. Exposure of carotid arteries to a specific antagonist of Ang II AT2 receptor, PD 123319 (10−7 mol/L), modified neither Ang II–induced diameter decrease nor cGMP content increase. Constriction of the vessel with KCl (26±3%, P<.001) did not modify the basal cGMP wall content. Endothelium removal or incubation with NG-nitro-l-arginine methyl ester (10−3 mol/L) reduced the cGMP content (22±9%, P<.05 and 20±11%, P<.05, respectively); Ang II further decreased the diameter (P<.001) but no longer increased the cGMP content under these experimental conditions. The present study shows that Ang II constricts the carotid artery and increases cGMP level specifically via the Ang II AT1 receptor subtype in in vitro intact rat carotid artery. The mechanism underlying this increase in cGMP is thought to be mediated through endothelial NO synthase stimulation by Ang II.
- Received April 4, 1995.
- Accepted August 4, 1995.
Ang II, the biologically active component of the renin-angiotensin system, is classically involved in inducing potent contractile responses in different vascular preparations by direct stimulation of specific Ang II receptors on vascular smooth muscle cells and activation of the inositol phosphate pathway.1 Moreover, Ang II is known to regulate various physiological functions in target tissues, and several recent attempts have been made to determine whether such a broad diversity of actions may result from Ang II receptor heterogeneity, as well as a corresponding multiplicity of intracellular actions.2 In this regard, it has been described that Ang II can modulate cyclic nucleotide production, phosphoinositide hydrolysis, and ionic fluxes in many of its target cells.3 4 In particular, Ang II has been reported to modify cGMP metabolism. Buonassisi and Venter5 reported that in an endothelial cell line from rabbit aorta, Ang II significantly increases cGMP content of endothelial cells. In the same way, Chaki and Inagami6 reported that Ang II stimulated cGMP formation in neuroblastoma cultured cells. Conversely, it has been reported that the cGMP cellular level decreased in neuronal cultures from rat brain after Ang II stimulation.7 Therefore, the in vivo vascular effect of Ang II could be a combination of a direct vasoconstrictor effect of the peptide on the smooth muscle cells and an indirect vasomotion related to changes in cGMP resulting from stimulation of endothelial cells. However, no information is available concerning the effects of Ang II on the entire vessel in terms of cGMP production/degradation and of smooth muscle vasomotion. Thus, the aim of this study was to determine the action of Ang II in in vitro intact rat carotid artery on the vascular tone and on the tissue cGMP content simultaneously and to determine the intracellular transduction pathway involved in this mechanism.
Twelve-week-old normotensive rats (Wistar-Kyoto, n=101) weighing 310±15 g were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg), intubated, and ventilated (model 680, Harvard Apparatus). A midsternal thoracotomy and cervicotomy were performed, and the roots of both left and right carotid arteries were carefully dissected and exposed. The distal end of each carotid artery was first ligated and then catheterized with a 9-gauge catheter and connected via a three-way stopcock to a reservoir filled with Tyrode’s solution containing albumin (4%)8 and placed 130 cm above the animal. The presence of albumin in incubating and flushing solutions maintained a physiological osmotic pressure gradient across the arterial wall and preserved the endothelium9 integrity and functions. A second catheter closed with a three-way stopcock, pointing distally, was then inserted into the proximal ligated end of the carotid artery. By this procedure, a pressure of 130 cm H2O (≈100 mm Hg) was continuously maintained within an isolated segment of carotid artery (20 to 23 mm long). The two catheters were then clamped into a crossbar with two adjustable clamps that maintained the artery at its in vivo length and prevented shortening upon excision (Fig 1⇓). Removed carotid artery segments were then immersed in a bath containing Tyrode’s solution (pH 7.4, 95% O2/5% CO2, 38°C). A glass tube 30 cm long and 0.5 cm in diameter was filled with albumin–Tyrode’s solution and connected to a manometer pressurized to 100 mm Hg and to the still pressurized artery via a three-way stopcock. Once the stopcock was opened, the luminal pressure of the carotid equilibrated with that of the manometer. The artery was then perfused discontinuously for 30 seconds every 20 minutes. To change the intraluminal solution while keeping the artery pressurized, the stopcock was closed toward the artery and opened toward the glass tube. The solution to be tested was introduced into the glass tube, the connection was reestablished between the artery and the glass tube, and the carotid artery was then perfused by opening of the second stopcock. All experiments were performed with IBMX (10−4 mol/L), a nonselective inhibitor of phosphodiesterase, added to the intraluminal albumin–Tyrode’s solution.
At the end of each experiment, the integrity of the carotid endothelium was tested by flushing Evans blue (0.03%) albumin solution into the vessel. Absence of blue staining of the luminal surface indicated that the endothelium remained unaltered. Experiments with damaged endothelium were discarded.
In a series of experiments, the endothelium was removed by gentle mechanical stripping with a blunt catheter guide (2F, 0.66 mm) introduced into the vessel. The carotid artery was then flushed to wash away the scraped endothelial cells. In preliminary experiments, we verified that after removal of endothelium, (1) the medial layer was not damaged, as shown by ensuring that phenylephrine-induced contraction (10−6 mol/L) of the carotid before and after deendothelialization was unchanged, and (2) acetylcholine-induced dilation (10−6 mol/L) was abolished.
Measurement of the Arterial Diameter
Changes in the carotid artery diameter were determined with an ultrasonic microdimensiometer (Application Electronique Montreuil), which allowed continuous measurement of the arterial diameter. A cylindrical transducer 6 mm in diameter and 2.5 cm long, containing a 12-MHz piezoelectric element placed 45° to the longitudinal axis of the probe, was used to avoid multiple reflections of the ultrasonic beam from the sides of the bath (Fig 1⇑).10 The arterial diameter was determined from the transit time of the pair of echoes given by the proximal and distal walls. It was then easy to place the arterial segment in position to determine its diameter; the longitudinal axis of the artery was perpendicular to the ultrasonic beam, so the best position was that which gave the maximum time period between the pair of wall echoes, and this corresponded to the maximum amplitudes of the echoes. This method allows us to measure arterial diameter from 500 to 2000 μm with an accuracy of measurement better than 10 μm. In preliminary experiments (n=5), we tested the stability and reproducibility of our experimental model. Under control conditions, 20 minutes after installation of the segment of carotid artery into the experimental system, the diameter of the carotid artery maintained at a transmural pressure of 100 mm Hg remained unchanged for at least 2 hours, ie, longer than the duration of the experiments. Moreover, we made sure that under all experimental conditions, the diameter remained stable for at least 20 minutes. Therefore, the effects of the tested pharmacological agents and of the mechanical deendothelialization cannot be interpreted as a time effect on the experimental preparation.
Determination of the Carotid cGMP Content
After measurement of the diameters, carotid arteries were quickly frozen in liquid nitrogen and stored at −80°C. Frozen arteries were then cut into strips and homogenized in ice-cold 6% trichloroacetic acid with a Potter glass homogenizer at 4°C. The homogenated samples were centrifuged at 2000g for 15 minutes at 4°C. Supernatant fractions were extracted four times with 5 vol water-saturated diethyl ether, lyophilized, and assayed for cGMP content by enzyme immunoassay (Amersham kit). The assay is based on the competition between unlabeled cGMP and a fixed quantity of peroxidase-labeled cGMP for a limited number of binding sites on a cGMP-specific antibody. With fixed amounts of antibody and peroxidase-labeled cGMP, the amount of peroxidase-labeled ligand bound by the antibody will be inversely proportional to the concentration of added unlabeled ligand. cGMP was measured by acetylation of standards and unknowns to obtain higher sensitivity. The standard curves ranged from 2 to 512 fmol per well.
Experiments were performed in parallel on left and right pressurized (100 mm Hg) carotid arteries of the same rat; the effects of the tested agents were examined in one carotid artery (with albumin–IBMX–Tyrode’s intraluminal solution containing the tested agents), and the contralateral preparation served as a control (with albumin–IBMX–Tyrode’s intraluminal solution as control solution).
Concentration-Response Curves to Ang II
Intact arteries were exposed intraluminally for 20 minutes to albumin–IBMX–Tyrode’s solution in the absence (control carotid artery) or in the presence of one concentration of Ang II (from 10−10 to 10−4 mol/L; n=5 for each concentration). Changes in carotid artery diameters were recorded throughout the experiment; at the end of the exposure to Ang II (or to control solution), the preparations were quickly frozen into liquid nitrogen for further determination of the wall content of cGMP.
Effect of Ang II Receptor Antagonists
Carotid artery diameter and cGMP content were determined after a 20-minute intraluminal exposure to Ang II (10−5 mol/L) or control solution, incubated for 15 minutes to either a nonselective Ang II receptor antagonist, saralasin (5×10−5 mol/L) (Sar+Ang II, n=8); a specific Ang II AT1 receptor antagonist, losartan (5×10−5 mol/L) (Los+Ang II, n=8); or a specific Ang II AT2 receptor antagonist, PD 123319 (10−7 mol/L) (PD 123319+Ang II, n=8).
The direct effects of a 15-minute exposure to saralasin (Sar, 5×10−5 mol/L, n=8), to losartan (Los, 5×10−5 mol/L, n=8), or to PD 123319 (PD 123319, 10−7 mol/L, n=8) were also examined in separate experiments and compared with a 15-minute incubation with control solution.
The carotid artery diameter and the cGMP wall content were also determined after a 15-minute exposure to an 80 mmol/L potassium solution (equimolar substitution of KCl by NaCl in albumin–IBMX–Tyrode’s solution) (KCl, n=8) in the presence of prazosin (10−5 mol/L) and propranolol (10−6 mol/L) to antagonize α- and β-adrenoreceptors, respectively.
Effect of Endothelium
The artery diameter and cGMP content were determined after a 20-minute intraluminal exposure to Ang II (10−5 mol/L) or control solution; this was done after endothelium removal (Endo−+Ang II, n=8) and in intact preparations exposed to an inhibitor of NO synthesis, L-NAME (10−3 mol/L) for 15 minutes before the addition of the peptide (L-NAME+Ang II, n=8).
The direct effects on the vessel diameter and on the cGMP content of endothelium removal (Endo−, n=8) and of a 15-minute exposure to L-NAME (10−3 mol/L) (L-NAME, n=8) were also examined in separate experiments and compared with control solution incubation.
Solutions and Drugs
Tyrode’s solution contained the following (mmol/L): NaCl 137, KCl 2.68, CaCl2 1.8, MgCl2 0.526, NaHCO3 11.9, NaH2PO4 0.333, glucose 5.56. Losartan was supplied by DuPont-Merck Pharmaceuticals. PD 123319 was synthesized by IdRS. Purified bovine serum albumin, phenylephrine, acetylcholine, L-NAME, saralasin, prazosin, propranolol, and IBMX were purchased from Sigma Chemical Co.
Results are given as mean±SEM and expressed as percent values of the basal conditions (albumin–Tyrode’s solution containing IBMX). The experimental model allowed us to use analysis of variance with repeated measurements to provide evidence of differences related to treatment. Comparisons were made with basal conditions for each group. Differences between groups were evaluated by Bonferroni t test.11 Values of P<.05 were considered significant.
Under basal conditions (albumin–Tyrode’s solution containing IBMX), at 100 mm Hg, mean isolated carotid artery diameter was 968±18.6 μm, and mean cGMP wall content was 38.9±3.5 fmol/mg tissue (n=101).
Ang II Concentration-Response Curves in Carotid Artery With Intact Endothelium: Effects on Diameter and cGMP Content
As shown in Fig 2⇓, significant decreases in diameter were observed at 0.5×10−6 mol/L (85±1.4% of the control diameter, P<.01), at 10−6 mol/L (75±4% of the control diameter, P<.01), and 10−5 mol/L (60±2.2% of the control diameter, P<.001) Ang II. The maximum contraction of the carotid artery by Ang II plateaued at a concentration of 10−5 mol/L. Fig 3⇓ presents an example of a typical continuous recording of carotid artery diameter after a 20-minute intraluminal exposure to Ang II (10−5 mol/L). Ang II significantly increased the cGMP wall content at a concentration of 0.5×10−6 mol/L (176±23%, P<.01) (Fig 2⇓). Increases in cGMP content reached a maximum at 10−6 mol/L Ang II concentration (210±34%, P<.01).
Effects of Ang II Receptor Blockade on Carotid Artery Diameter and cGMP Wall Content
Incubation of the carotid artery with saralasin alone modified neither the vessel diameter (981±3.4 μm compared with the mean control diameter, 985±8.2 μm) nor the cGMP wall content (32±7.4 fmol/mg artery compared with the mean control cGMP content, 31±5.6 fmol/mg artery). Pretreatment of the carotid artery with saralasin abolished the Ang II–induced contraction as well as tissue cGMP production (Sar+Ang II conditions, Fig 4⇓). When losartan was incubated alone, no modification in cGMP wall content was observed (30±4.3 fmol/mg artery compared with mean control cGMP content, 31±4 fmol/mg artery); by contrast, the carotid diameter was significantly increased (1080±16.8 μm compared with the mean control diameter, 970±10.7 μm; P<.001). Addition of Ang II after exposure to losartan (Los+Ang II conditions, Fig 4⇓) did not decrease the carotid diameter, and the tissue cGMP level was not modified compared with the control values. Incubation of carotid artery with PD 123319 alone modified neither the diameter (985±5.5 μm compared with the mean control diameter, 987±7 μm) nor the cGMP wall content (30.5±3.4 fmol/mg artery compared with the mean control cGMP content, 31±4.4 fmol/mg artery). When Ang II was added to the solution containing PD 123319 (PD 123319+Ang II conditions, Fig 4⇓), the diameter decreased by 40±2.1% (P<.001), and the cGMP wall content increased by 135±29% (P<.01) compared with the PD 123319–treated vessels. The effects of Ang II on the vessel diameter and on the cGMP tissue content were not different in the presence or absence of PD 123319.
After incubation with KCl, the carotid artery diameter was significantly decreased (P<.001), but basal cGMP content was not modified.
Role of Endothelium in the Effects of Ang II
Fig 5⇓ shows that endothelium removal significantly increased the diameter of the artery (P<.001) and decreased the cGMP content (P<.05). Addition of Ang II to the lumen of deendothelialized carotid artery (Endo−+Ang II conditions) still constricted the carotid artery by 41±1.3% compared with the deendothelialized carotid artery diameter (Endo− conditions) (P<.001) but did not induce any change in the cGMP wall content compared with the endothelium removal conditions.
To determine whether the endothelial NO synthase pathway was involved in this mechanism of Ang II–induced cGMP production, isolated carotid arteries were pretreated with L-NAME. L-NAME induced a slight but significant contraction of the vessel, by 8±3.8% (P<.01), and significantly decreased the tissue cGMP content (P<.05) compared with the control conditions. After addition of Ang II (L-NAME+ANG II conditions), the carotid artery diameter decreased further, by 40.4±3% compared with the diameter obtained after L-NAME exposure (P<.001), without significant change in tissue cGMP content compared with the cGMP content after incubation with L-NAME alone.
No difference in percent diameter reduction was found when Ang II was added to carotid artery exposed or not exposed to L-NAME (40.4±3% of the diameter obtained after L-NAME exposure and 40±2% of the control diameter).
The present work was performed to assess the simultaneous changes in diameter and cGMP content of in vitro intact carotid artery kept at its in vivo length and maintained at a physiological transmural pressure. The use of IBMX in the intraluminal Tyrode’s solution allowed the exclusion of the potential interaction of the tested drugs on phosphodiesterase activity and therefore allowed us to focus the study on the activity of guanylate cyclase.
Ang II–Induced cGMP Production in Isolated Carotid Artery
Our present results show that Ang II simultaneously decreased the vessel diameter and increased the level of cGMP in the carotid artery, extending our previous observations described in a preliminary report.12 The main finding of this study is the opposing dose-dependent actions of Ang II: constriction of the carotid artery and a concurrent elevation of the carotid wall cGMP content, the second messenger classically involved in vasodilation. Both cGMP production and carotid artery constriction reached their maximum values when Ang II was applied intraluminally at a concentration of 10−5 mol/L. It is likely that such a high dose of Ang II was necessary to be effective on the diameter and on cGMP wall content because Ang II was applied intraluminally only. Indeed, a previous study by Falloon et al,13 using small arteries, clearly indicates that the profile of constriction was different when Ang II was applied intraluminally or extraluminally. One possible interpretation is that higher doses of Ang II are required to overcome endothelial cell peptidase degradation to reach the underlying smooth muscle cells when the peptide is applied intraluminally. Ang II–induced modulation of cGMP levels has already been reported and depends on the cell type studied: Ang II has been reported to stimulate intracellular production of cGMP in several cell types, including vascular endothelial cells5 and neuroblastoma cells.14 15 In addition, some studies in primary neuronal cultures from rat brain reported a decrease in cGMP content in response to Ang II.16 However, the originality of our experimental model compared with the cultured cells is that it allows the simultaneous assessment of both vascular tone and cGMP content in an isolated vessel exposed to intraluminal vasoactive agents. To make sure that Ang II–induced cGMP production was not simply dependent on the contraction of the vessel, we verified that contraction induced by depolarization of the membrane with KCl did not modify the basal cGMP content. This suggests that the increase in artery cGMP content in response to Ang II is not contraction-dependent but depends on specific mechanisms related to Ang II.
Under our experimental conditions, Ang II–induced cGMP production and vasoconstriction were inhibited by the nonselective, high-affinity antagonist saralasin, while the exposure of vessels to saralasin alone modified neither the diameter nor the cGMP content, suggesting that saralasin (5×10−5 mol/L) had no agonistic properties in isolated carotid artery. The selective AT1 receptor antagonist losartan, incubated alone, did not cause any change in cGMP wall content. As described in our previous study,17 losartan significantly increased the carotid artery diameter, suggesting that in the absence of exogenous substrates of the renin-angiotensin system, the vessel wall was capable of generating Ang II, whose action can be inhibited by a specific Ang II AT1 receptor antagonist. In addition, we can rule out a nonspecific effect of losartan on prostacyclin synthesis,18 since inhibition of Ang II formation with an angiotensin-converting enzyme inhibitor induced the same increase in diameter.17 In the present study, Ang II no longer caused a decrease in diameter when vessels were exposed to losartan, suggesting that Ang II–induced vasoconstriction is mediated by activation of the AT1 receptor subtype. This interpretation is in good agreement with previous studies. Indeed, it has been reported that the AT1 receptor subtype, which is the main receptor subtype classically described in the vasculature of adult animals,19 20 is responsible for the stimulation of phosphoinositide hydrolysis and the attendant mobilization of intracellular Ca2+.21 The new finding in the present study is that the AT1 receptor subtype involved in smooth muscle cell contraction is simultaneously involved in Ang II–induced cGMP production, since neither the Ang II–induced vessel constriction nor the increase in cGMP wall content was affected by blockade of Ang II AT2 receptors with a specific antagonist of AT2 receptors, PD 123319.22
Role of Endothelium in Ang II–Induced cGMP Production
We reported earlier that endothelium removal in rat isolated carotid artery results in increases in compliance and diameter, suggesting a predominantly vasoconstrictor function of the carotid endothelium under basal conditions.17 23 In the present study, removal of the endothelium also increased the diameter, thus confirming previous studies. In the present experimental conditions, the same entire isolated carotid artery was used to continually monitor the diameter both before and after deendothelialization. This is an advantage of our experimental model compared with isolated vessel rings, in which the role of the endothelium on the basal vasomotor tone cannot be determined, since the endothelium is removed before the ring is suspended in the organ chamber. The fact that incubation of intact carotid artery with L-NAME, an inhibitor of NO synthase, significantly decreased the diameter and the cGMP content suggested a basal release of NO under the present experimental conditions. In summary, it seems that in intact carotid artery, endothelium predominantly releases vasoconstrictor substances but also NO.
In the present study, the mechanism by which Ang II increases cGMP levels appears to be via the endothelium, since the effect was abolished after endothelium removal. In support of this result, Buonassisi and Venter5 reported that in rabbit aortic endothelial cells, Ang II significantly increases cGMP level. A previous study also reported that Ang II can act on cultured human vascular endothelial cells by stimulating prostaglandin release.24 A study using porcine pulmonary arterial and aortic endothelial cells demonstrated the presence of angiotensin receptors by binding of 125I-Ang II.25 These observations suggest that Ang II may have direct endothelium-dependent effects. Distribution of the AT1 receptor subtype both on the smooth muscle cells and on endothelial cells could simultaneously result in direct vasoconstriction and endothelium-mediated cGMP production.
Thus, the vascular actions of Ang II can be a combined effect on the AT1 receptor subtype at the surface of both cell types. Agonist-induced production of cGMP can be mediated by the activation of either particulate or soluble forms of guanylate cyclase. NO activates the intracellular soluble guanylate cyclase of subendothelial vascular smooth muscle and elevates cGMP to induce dephosphorylation of myosin light chain, leading to smooth muscle relaxation.26 27 To determine whether the intracellular formation of NO might be involved in Ang II–induced stimulation of guanylate cyclase, L-NAME was used to inhibit the endothelial NO synthase activity. Preincubation of the carotid artery with L-NAME completely inhibited the augmentation of cGMP elicited by Ang II. However, L-NAME had no significant effect on the Ang II–induced constriction: the percent decrease in vessel diameter caused by Ang II was comparable between control preparations and vessels exposed to L-NAME. Thus, Ang II is probably responsible for mobilization of intracellular Ca2+, activating the endothelial constitutive NO synthase via the endothelial AT1 receptor subtype. The absence of significant differences between the Ang II–induced contraction with basal or with inactivated endothelial NO pathway could be due to the potent direct contractile effect of Ang II on the smooth muscle, completely overcoming the putative vasomotion modulation of NO. However, the Ang II–induced carotid reduction in diameter was larger in deendothelialized than in intact arteries (442±22 and 386±23 μm, respectively), suggesting a possible modulation of the contracting effect of Ang II by its endothelium-dependent relaxing actions. Endothelial dysfunction reported in hypertension, atherosclerosis, and aging could impair this modulating effect of Ang II on the endothelium and increase the hypertensive action of Ang II.
In conclusion, our results suggest a specific receptor-mediated effect of Ang II on endothelial cells. Ang II constricts the carotid artery and increases cGMP levels specifically via the Ang II AT1 receptor subtype in the in vitro intact rat carotid artery. The mechanism underlying this increase in cGMP is thought to be mediated through endothelial NO synthase stimulation by Ang II. Ang II–dependent NO production by the endothelial cells could modulate the peptide-induced smooth muscle cell contraction. The in vivo functional significance of Ang II receptor–mediated increases in cGMP wall content and whether these changes in cGMP contribute to Ang II vascular effect need further investigation. Ang II–dependent NO production by the endothelial cells could modulate the peptide-induced smooth muscle cell contraction. Furthermore, contractile and trophic vascular effects of Ang II could be exaggerated under pathological conditions, when endothelial dysfunction can occur.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|saralasin||=||[Sar1,Val5, Ala8]Ang II|
This work was supported in part by MSD-INSERM grant 31 2004 and by SERVIER-INSERM grant 94 0930.
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