The endothelium plays an important role in the control of vascular tone.1 Exposure to endothelium-dependent vasodilators causes the production by endothelial cells of at least 1 of these 3 vasoactive factors, depending on the blood vessel: nitric oxide (NO),2 prostacyclin (PGI2),3,4 and endothelium-derived hyperpolarizing factor (EDHF).5-7 The production of these endothelium-derived vasodilators is triggered by an increasing cytosolic free calcium ([Ca2+]i) in the endothelial cells.8-12
Acetylcholine (ACh) was the first endothelium-dependent vasodilator described.1 The receptors of ACh divided into 2 classes: the nicotinic receptors that are ionotropic and the muscarinic receptors that are metabotropic. The muscarinic receptors are responsible for the endothelium-dependent vasodilatation.
Five subtypes of muscarinic receptors named M1, M2, M3, M4, and M5 are described. The endothelial cells from the vascular system express M1, M2, and M3 receptors, but not M4 or M5 subtypes.13 M1 and M3 receptors are coupled to the phospholipase C through a G protein. When activated, these receptors induce the inositol-1,4,5-trisphosphate (IP3) and diacylglycerol pathway. IP3 released in the cytoplasm acts on its receptor on the surface of the endoplasmic reticulum to release calcium in the cytosol. M2 receptor inhibits adenylate cyclase through G protein and opens inwardly rectifying potassium channels inducing a hyperpolarization.14
Different approaches were used to determine the subtype of muscarinic receptors responsible for the endothelium-dependent vasodilatation. Subtypes of muscarinic receptors present on arterial endothelial cells were determined in freshly isolated and cultured bovine aortic or pulmonary arterial endothelial cells by radioligand binding assay.15-17 Functional M1, M2, M3, or even unknown receptors responsible for endothelium-dependent relaxation were determined using a pharmacological approach in many arteries issued from different species.15-34 The M5 receptor is detected by RT-PCR and in situ hybridization in endothelial cells of cerebral arteries from mouse and human.35,36 In addition, endothelium-dependent vasodilatation of cerebral arteries is abolished in M5 knockout (KO) mice.37 In summary, in the periphery, M1, M2, and M3 receptors were detected in endothelial cells, whereas M5 receptor was shown in the endothelium of cerebral arteries.
This apparent discrepancy could be explained by the different arteries as well as the different species used. It could also arise from the difficulty to dispose of specific agonists and antagonists for the muscarinic receptors. In addition, endothelium-dependent relaxations can be caused by at least 3 different vasodilators: NO, EDHF, and PGI2. Consequently, activation of distinct receptor subtypes could be responsible for the response to these different vasodilators.
An alternative approach to determine the cholinergic receptor responsible for the endothelium-dependent vasodilatation could be to test the effect of ACh on isolated rings of arteries from mice that are KO for the different candidate muscarinic receptor genes. Indeed, endothelium-dependent vasodilatation was abolished in the aorta from M3 KO mice.38
Many studies using fluorescent calcium indicators, such as Fura-2, have demonstrated that endothelium-dependent vasodilators, acetylcholine and adenosine 5′-triphosphate (ATP), elevate the intracellular levels of [Ca2+]i in endothelial cells.8,12,39-41 Actually, we already showed that endothelial cells responding to distinct endothelium-dependent vasodilators were not homogeneously distributed in intact murine thoracic aorta.42 Therefore, besides the pharmacological approach cited above, endothelial cells responding to ACh by an increase in [Ca2+]i could allow testing for the presence of functional cholinergic receptors on these cells. Laser scanning confocal microscopy, in combination with sensitive fluorescent dyes, have been shown to be both powerful means to monitor [Ca2+]i changes within endothelial cells of the vascular wall.43-51 Therefore, calcium imaging using confocal microscopy of the endothelium of mouse thoracic aorta could be used to observe the proportion of cells responding to ACh using KO mice for the different muscarinic receptors.
Overall, the goal of the present study was to specify the muscarinic receptor subtype responsible for endothelium-dependent relaxation of arteries in the mouse. In this context, the molecules (NO, EDHF, prostanoids) responsible for the endothelium-dependent relaxation were determined by a pharmacological approach. For this purpose, wild-type (WT) and KO mice for the M1, M2, and M3 muscarinic receptors were used as an alternative approach to pharmacological studies to determine unambiguously the receptors responsible for the endothelium-dependent relaxation of mouse femoral artery and thoracic aorta. In addition, calcium imaging of thoracic aorta intima was used to visualize the responding endothelial cells in these different KO mice.
MATERIALS AND METHODS
KO Mice Elaboration
The generation of KO mice for M1, M2, and M3 genes was described elsewhere.52-54 The compound mutant mice for M1 and M2 genes were generated by crossing the M1 and M2 mutant mice. The mice used in this study were backcrossed with C57BL/6JJcl (CLEA, Japan) for at least 10 generations. The WT mice used were C57BL/6JJcl. The mice used for the experiments were 7 to 11 weeks old.
All animal procedures were further followed in accordance with institutional guidelines established by both “L'Académie suisse des Sciences médicales” and “La Société helvétique des Sciences naturelles: animal experimentation authorization 31.10.1008/3185/0”.
Mice were anesthetized by exposure to 2-bromo-2chloro-1,1,1-trifluoroethane (halothane) and sacrificed by neck disruption. The thoracic aorta and both femoral arteries were removed and placed immediately in Krebs-Ringer buffer of the following composition (mM): NaCl 118.7, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 24.8, D-glucose 10.1, pH 7.4 and bubbled with 95% O2 and 5% CO2 gas mixture. Then, the arteries were cleared from all adherent fat and connective tissue surrounding the adventitia. In some experiments, the endothelium of aorta was destroyed. To remove the endothelium, the aorta was perfused for 6 minutes with dry air. The absence of functional endothelium was proved in each experiment by the abolishment of endothelium-dependent relaxation caused by ACh (10−5 M). However, it was not possible to do the same in femoral artery because of its frailty. To assess the role of the endothelium in this artery, the synthesis of the potential endothelium-dependent vasodilators NO and prostanoids was inhibited by using NG-nitro-L-arginine (L-NA, 10−4 M) and indomethacin (10−5 M), respectively.
Isometric Tension Measurement
These experiments were done with femoral artery and thoracic aorta. In femoral artery, the endothelium-dependent relaxations are relatively important in comparison to that of the aorta. However, we did not succeed in performing calcium imaging with the too small femoral arteries. Consequently, calcium imaging was done only on thoracic aorta, and we verified by tension measurement that the same muscarinic receptor was responsible for the endothelium-dependent relaxation in both arteries.
Changes in tension were measured isometrically by using a Mulvany microvessel Myograph (Multimyograph, model 610 M; Danish Myo Technology A/S, Aarhus, Denmark). An approximately 2-mm-wide ring was cut from the proximal extremity of the right and left femoral arteries. Care was taken not to damage the endothelium. The same procedures were also applied to 2 adjacent rings from a thoracic aorta. These 2 rings from the aorta or from the 2 femoral arteries, providing from the same animal, were mounted separately for isometric tension recording in a 5-ml stainless steel organ bath with a U-shaped stainless steel hook slid into each extremity. One extremity was connected to the lever of a force displacement transducer. The bath contained warmed (37°C) and oxygenated (95% O2, 5% CO2) Krebs-Ringer buffer. In some experiments, indomethacin (10−5 M) and L-NA (10−4 M) were added separately or together to the Krebs-Ringer incubation medium to inhibit the production of prostanoids or NO or both in order to unravel the role of NO, prostanoids or EDHF, respectively.
Recorded data were digitized with an analog-digital interface (PowerLab, AD instruments) and then stored on the hard disk of a Macintosh computer. These data were analyzed using a data acquisition software (PowerLab System, AD instruments).42
A passive resting tension of 0.5 mN was initially applied stepwise, and the rings were allowed to stabilize for 1 hour. The applied tension was readjusted regularly to 0.5 mN at 15 minutes intervals.
Immediately after this equilibration period of 1 hour, arterial rings were continuously incubated with U 46619 (a stable analog of thromboxane A2, 3 × 10−7 M) to achieve strong tonic contraction. When a stable contraction plateau was reached, endothelium-dependent relaxations of the rings were tested in response to noncumulative application of 1, 10, and 100 nM and 1, 10, and 1000 μM ACh.
Cytosolic Free Calcium Measurement
The thoracic aorta was used for calcium imaging since it was difficult to open femoral arteries without damaging the endothelium. We verified that the same muscarinic receptors were responsible for ACh-induced endothelium-dependent relaxation in the aorta.
Loading of Endothelial Cells
The method has been previously described42. Briefly, the thoracic aorta was opened longitudinally to form a strip (≈10 mm in length), and washed with Krebs-Ringer buffer to remove the remaining blood clots. A particular attention was paid to avoid damaging the endothelium.
The endothelial cells were loaded with the acetoxymethyl (AM) ester form of both fluorescent calcium indicator dyes Fura-red and Fluo-4. The thoracic aorta were incubated at 37°C for 30 minutes in a Hepes (N-2-hydroxyethylpiperazine-N'-2 ethane-sulfonic acid) buffered saline solution medium (NaCl 145 mM, KCl 5 mM, CaCl2 1 mM, MgSO4 0.5 mM, NaH2PO4 1 mM, Hepes 20 mM, D-glucose 10.1 mM, pH 7.4) containing: 1) Fura-red/AM and Fluo-4/AM at concentrations of 20 μM and 40 μM, respectively; and 2) pluronic acid F-127 (2% v/v). After concomitant loading with both Fura-red/AM and Fluo-4/AM, a segment of the thoracic aorta wall was pinned with cactus spines in Petri dish lined with silicon rubber (Sylgard), the intimal side facing up. The use of dual indicators to study intracellular calcium dynamics minimizes the contribution of artifactual changes in the fluorescence signal unrelated to changes in [Ca2+]i. We applied this principle here to study [Ca2+]i dynamics in endothelium arterial wall using the combination of Fluo-4 and Fura-red. In this manner, an increase in [Ca2+]i will simultaneously increase the fluorescence of Fluo-4 and reduce that of Fura-red.
Laser Scanning Confocal Microscopy
Dye-loaded aortic endothelial cells were examined using an Axiovert 200 M MAT right microscope coupled to an LSM 510 laser scanning unit (LSM 510 META; Carl Zeiss, Oberkochen, Germany). An Achroplan 20X/0.50 ω Ph2 Carl-Zeiss microscope objective was used. All experiments were performed at room temperature. The preparation was excited at a wavelength of 488 nm. The emitted fluorescence of Fura-Red and Fluo-4 were split with a dichroïc mirror (NFT 635). The 510-nm light was then filtered (BP 505 to 530) and recorded on a photomultiplicator. The 650 nm was simultaneously recorded using a “meta detector” (636 to 750 nm). Images resulting from the scanning were recorded as RGB pictures in time series (1 image/s).
Data of isometric tension measurement are presented as mean ± SEM. Relaxation responses are expressed as percentage of aortic rings contraction to U 46619 (3 × 10−7 M). The number of samples is 4, each sample coming from 1 distinct animal.
The EC50 of the agonists were calculated by fitting the results with a sigmoidal concentration-response curve (4 parameter logistic equation), using the computer program GraphPad Prism 4 (San Diego, CA). Student t test was used to compare EC50. The results were considered as significant when P value was less than 0.05.
Measurements of fluorescence intensities were obtained from the data set of images using the MetaMorph Series 6.1 software (Universal Imaging Corporation). The color channels were split from RGB images in order to separate the 510-nm and the 650-nm images, and the ratio between emitted fluorescence recorded at 510 and 650 nm was then calculated for all frames.50 The processed images were represented as pseudo-colored images according to an intensity scale ranging from 0 to 255, the lowest to highest intensities being in black to blue to green and to red, respectively. ATP was used to test the responsiveness of the endothelial cells. Almost, all endothelial cells responded to ATP by a large tonic increase in [Ca2+]i. Only occasionally, few dead cells did not respond to ATP because they are already concentrated in [Ca2+]i before the application of any stimulation. We expressed the proportion of cell stimulated by ACh in percentage of cells responding to ATP.
ACh, ATP, 2-bromo-2chloro-1,1,1-trifluoroethane (halothane), and indomethacin were obtained from Sigma Chemical (Buchs, Switzerland). Pluronic acid F-127 was obtained from Calbiochem (San Diego, CA). Fura-red and Fluo-4 were purchased from Molecular probes (Eugene, OR). U 46619 (9,11-dideoxy-9,11-methanoepoxyprostaglandin F2) was obtained from Cayman (Ann Arbor, MI), and L-NA was obtained from Alexis (Lausen, Switzerland).
Vasomotor Reactivity WT Mouse Femoral Artery Ring
Endothelium-dependent relaxations induced by ACh were measured for 10−9 M to 10−5 M ACh on rings of femoral arteries from WT mice contracted by 3 × 10−7 M U 46619. The rings of these arteries started to relax from 10−8 M ACh and reached a maximal relaxation from 10−6 M ACh (Figure 1 and 2A). The EC50 values of ACh were 7.5 × 10−8 M (standard error of the log EC50 0.15). The presence of indomethacin did not change ACh-induced relaxation (EC50 6.3 × 10−8 M; standard error of the log EC50 0.15). L-NA alone or in combination with indomethacin abolished the ACh-induced relaxation (Figure 2A). However, an NO donor, DEA/NO (10−4 M), relaxed the rings by 77 ± 13 % (n = 4) in the presence of indomethacin plus L-NA.
Muscarinic receptors could be present as well on smooth muscle cells and mediate ACh contraction. This would interfere with the effect on the endothelium. We were unable to remove the endothelium in these small arteries; therefore, we tested the possible contractile effect of ACh (10−9 to 10−5M) on WT mice in a medium containing L-NA (10−4 M) and indomethacin (10−5 M). ACh did not contract the femoral artery rings incubated with these inhibitors (Figure 2A).
Endothelium-Dependent Relaxation of Femoral Artery Rings
Endothelium-dependent relaxations induced by ACh were obtained over the concentration range of 10−9 M to 10−5 M. The rings of femoral arteries from WT M1 + M2, M1, and M2 KO mice contracted by U 46619 (3 × 10−7 M) started to relax from 10−8 M ACh and reached a maximal relaxation from 10−6 M ACh. The EC50 values of ACh were 7.5 × 10−8 M for WT mice, 6 × 10−8 M for M1 + M2 (standard error of the log EC50 0.06), 11 × 10−8 M for M1 (standard error of the log EC50 0.16), and 10 × 10−8 M for M2 KO animals (standard error of the log EC50 0.15). All these values were not significantly different. The maximal relaxation of ring from M1 + M2 KO mice was 68 ± 4%, 56 ± 9% for M2 KO mice, and 52 ± 8 % for M1 KO mice, as compared to the maximal relaxation of WT femoral rings, which was 62 ± 6% (Figure 3A). These values were not significantly different.
On the contrary, all tested concentrations of ACh had almost no effect on the force developed by rings of femoral arteries from M3 KO mice. The force was maximally inhibited by 3 ± 2% after the application of 10−6 M ACh (Figure 3A).
It is commonly admitted that EDHF7 is implicated in an endothelium-dependent relaxation resistive to NO and prostanoid synthesis inhibition. To unravel a possible role of EDHF, we evaluated the response of femoral arteries to ACh in presence of L-NA (10−4 M) and indomethacin (10−5 M). In WT mice, endothelium-dependent relaxation to ACh (10−6 M) was inhibited by 89% and 88% in presence of L-NA (10−4 M) and indomethacin (10−5 M), respectively. In M1 + M2 KO mice, ACh (10−6 M) induced a maximal relaxation of 8 ± 3% (Figure 3A). We consider this relaxation too little for EDHF to play a significant role in femoral artery relaxation.
Vasomotor Reactivity of WT Mouse Aortic Ring
Muscarinic receptors could be present as well on smooth muscle cells and mediate ACh contraction interfering with the effect on the endothelium. We therefore tested a possible contractile effect of ACh (10−9 to 10−5 M) on WT mice without endothelium. ACh did not contract the aorta rings without endothelium (Figure 2B).
Rings of aortas contracted with 3 × 10−7 M U 46619 started to relax from about 10−8 M ACh and reached a maximal relaxation from 10−6 M ACh. The EC50 values of ACh were 8.6 × 10−8 M (standard error of the log EC50 0.05). The presence of indomethacin did not change ACh-induced relaxation, and the EC50 values of ACh were 7.3 × 10−8 M (standard error of the log EC50 0.08). L-NA alone or in combination with indomethacin abolished ACh-induced relaxation. Similarly, rings without endothelium contracted by U 46619 (3 × 10−7 M) were not relaxed by 10−9 M to 10−5 M ACh (Figure 2B). However, the NO donor, DEA/NO (10−4 M), relaxed these rings without endothelium by 80 ± 4% (n = 4).
Endothelium-Dependent Relaxation Thoracic Aorta Rings
Endothelium-dependent relaxations induced by ACh were obtained over the concentration range of 10−9 M to 10−5 M (Figure 3B). The concentrations of ACh of 10−9 M and 10−8 M has no effect on the force developed by rings of aorta from WT and M1 + M2 KO mice contracted by U 46619 (3 × 10−7 M). The rings of these aortas started to relax from about 10−7 M ACh and reached a maximal relaxation from 10−6 M ACh. The EC50 values of ACh were 8.6 × 10−8 M for WT mice and 7.3 × 10−8 M (standard error of the log EC50 0.18) for M1 + M2 KO animals. These 2 values are not significantly different. The maximal relaxation of rings from M1 + M2 KO mice was 27 ± 4% and 27 ± 0.3% for WT. These values are not significantly different (Figure 3B).
All the tested concentration of ACh had almost no effect on the force developed by rings of thoracic aorta rings from M3 KO mice (Figure 3B). They were maximally relaxed by 2 ± 0.2% in response to 10−5 M ACh.
In WT mice, the inhibition of cyclooxygenase and NO synthase by L-NA (10−4 M) plus indomethacin (10−5 M) inhibited by 89% and 85% the endothelium-dependent relaxations caused by 10−6 and 10−5 M ACh, respectively. In M1 + M2 KO mice, ACh (10−5 to 10−6 M) induced a maximal relaxation of 4 ± 1%, we considered this relaxation too little for EDHF to play a significant role in thoracic aorta relaxation.
Calcium Imaging of Thoracic Aorta Endothelium
Almost all endothelial cells responded to ATP by a large tonic increase in [Ca2+]i. Only occasionally, a few dead cells did not respond to ATP because they were already concentrated in [Ca2+]i before the application of any stimulation. Therefore, we expressed our results as the number of cells responding to 10−5 M ACh in the percent of cells responding to 10−4 M ATP (all living endothelial cells). As already observed, only a fraction of 20 ± 7% (n = 6) of aortic endothelial cells respond to ACh by an increase in cytosolic free calcium (Figure 4, A and B). Similarly, 23 ± 7% of endothelial cells from M1 + M2 KO mice responded. At the opposite, only 4 ± 1% of aortic endothelial cells responded to ACh by an increase in cytosolic free calcium in M3 KO mice (Figure 4B).
Our results show that ACh do not cause smooth muscles contraction of mice aorta and femoral artery. Therefore, hypothetical contractions induced by ACh would not interfere with the endothelium-dependent relaxations we observed. We also show that ACh-induced relaxation of aorta and femoral artery is only caused by an endothelium-dependent phenomenon. In addition, the only endothelium-relaxing factor present in both arteries is NO. This observation was already demonstrated for the aorta in NO synthase KO mice.55 Interestingly, femoral artery was more sensitive to the endothelium-dependent relaxation induced by ACh than the aorta because it is maximally relaxed by 62% by comparison with aorta, which is only relaxed by 26%.
Our results show also that only the mice with NO M3 receptors but expressing M1 and M2 receptors had no endothelium-dependent relaxations in response to ACh. Mice without M1 and M2 receptors but expressing only M3 receptors presented a similar endothelium-dependent relaxation as WT animals. These observations unambiguously indicate that the M3 is cholinergic muscarinic receptor responsible for the endothelium-dependent relaxation of the femoral artery and the thoracic aorta of the mouse. In addition, the suppression of endothelium-dependent relaxation of femoral artery and thoracic aorta expressing only the M3 receptors in response to ACh when the synthesis of NO and prostacyclin are inhibited shows that NO without significant contribution of EDHF causes these endothelium-dependent relaxations of femoral and thoracic aorta. Moreover, inhibition of NO and prostacyclin synthesis in thoracic aorta and femoral artery of M3 KO mice resulted in abolition of endothelium-dependent relaxation, suggesting that NO could induce relaxation without EDHF. Indeed, an endothelium-dependent relaxation resistive to NO and prostanoid inhibition is considered as EDHF.7 A recording of the membrane potential of smooth muscle cells with an absence of hyperpolarization would confirm this observation.5
To explain the specificity of the M3 receptor, it would be possible that the three receptors are expressed in the endothelium but only the stimulation of M3 receptors would be responsible for the production of NO. Alternatively, the stimulation of M1, M2 or M3 receptors could results in NO production but only M3 receptors would be expressed on the endothelial cells. To resolve this alternative, the presence of functional cholinergic receptors were studied by evaluating the increase in [Ca2+]i in endothelial cells from thoracic aorta in response.
The increase in [Ca2+]i in endothelial cells is triggered by NO production and was used to visualize the cells activated by Ach.8-12 We observed that one fifth of endothelial cells of the intima of thoracic aorta responded to ACh by a cytosolic free calcium increase when M3 receptors only were expressed. Curiously, when ATP, another neurotransmitter, was applied, most of endothelial cells responded as already published.42 Amazingly, in thoracic aorta ring, the maximal endothelium-dependent vasodilatation caused by ACh was larger than that caused by ATP.56 In thoracic aorta from mice where the M1 and M2 receptors are expressed but not the M3, 4% of the cells still responded to ACh. This indicates that M1 and/or M2 receptors are expressed on the endothelial cells of M3 KO animals. However, the femoral artery and the thoracic aorta of these mice did not present endothelium-dependent relaxation in response to ACh. The reason could be either that ACh activates not enough endothelial cells (4%) or that these particular cells cannot synthesize NO. In addition we do not know whether this 4% of functional endothelial cells with M1 and M2 receptors is a consequence of the M3 KO or whether WT animals also express few M1 or M2 receptors in addition to M3 receptors.
These observations are consistent with our pharmacological experiments. Muscarinic receptors M1, M2, and M3 could be present on arterial endothelial cells, but M3 is expressed predominantly on many cells as compared to M1 and M2. In other words, arterial tissues is a mosaic of individually different cells and the specificity of one receptor for an artery is dependent on the proportion of cells that express this particular receptor.
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