The physiology of smooth muscle and endothelial cells from different vascular beds in the same species differs from each other. Differences are also observed for the same vascular bed in different species. This is a result of the relative contribution of different receptors to vasoconstrictors and vasodilators and to the repartition of ionic channels incorporated in the cell membrane.1 In particular, the process of endothelium-dependent relaxation depends on the relative contribution of the release of NO, prostacyclin, or the phenomenon called endothelium-derived hyperpolizing factor (EDHF).2–4 To build up a pertinent hypothesis and to correctly interpret experimental results, as well as to compare different experimental models, it is crucial to know the particular reactivity of the studied model.
In this context, the physiology of pulmonary artery was extensively explored in sheep,5–7 pig,8,9 rabbit,10 and rat.11,12 However, the mouse has more recently become an interesting model, mainly because of the experimental possibilities offered by transgenic animals, but basic physiological data on murine pulmonary artery are lacking.
An important endogenous neurotransmitter implicated in the regulation of pulmonary vascular tone is acetylcholine (ACh). However, cholinergic blockade has no effect on basal pulmonary vascular tone.13 Indeed, the effects of ACh on the pulmonary artery are tone dependent.14–16 ACh induces endothelium-dependent relaxation of constricted vessels and constriction of relaxed vessels. In preconstricted rat pulmonary artery, ACh induces an endothelium-dependent relaxation, which is mediated by NO plus EDHF, relaxing the smooth muscles by hyperpolarizing them.17 However, Streudel et al18 showed that ACh-induced dilation of preconstricted pulmonary artery rings was completely absent in mice with congenital deficiency of nitric oxide synthase 3. This suggests that endothelial-derived nitric oxide (NO) is the major mechanism of ACh-induced vasodilation in murine pulmonary arteries. In addition, NO by itself hyperpolarizes the smooth muscle cells from some vascular beds.19,20 Our aim was to determine the mechanism of endothelium-dependent vasodilatation evoked by ACh in the murine main pulmonary artery.
Male mice C57BL/6J and CDI Crl were obtained from Iffa Credo (Lyon, France). They were studied from 8 to 12 weeks old. All experimental procedures were approved and carried out in accordance with the Swiss Veterinarian Animal Care guidelines. Mice were anesthetized with 2-bromo-2-chloro-1,1,1-trifluoroethane (halothane; Sigma, St Louis, MO) and killed by neck disruption. The chest was opened and the main pulmonary artery was cut from the right ventricle to its bifurcation into right and left pulmonary arteries. Care was taken to avoid damaging the endothelium.
Isometric Force and Membrane Potential Measurement
The isolated pulmonary artery ring was placed in modified Krebs–Ringer bicarbonate solution [(in mmol/L): NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, and glucose 11.1; aerated with 95% O2–5% CO2; pH 7.4)]. The vessel ring was opened to form a strip 1- to 2-mm wide and 1.5- to 2-mm long. The strip was incubated in a 100-μL bath continuously superfused (1.25 mL/min) with oxygenated Krebs solution at 37°C and pinned to the bottom of the chamber covered with a silicon surface (Sylgard, Dow Corning, Midland, MI) with 2 to 4 stainless steel insect needles at one extremity with its adventitial side facing up. The other extremity was put on an acupuncture needle fixed on isometric force transducer (Grass FT03C, Quincy, MA), and the force developed by the circular smooth muscles was measured.
The mechanical tension developed by the strip and the membrane potential of vascular smooth muscle or endothelial cells were simultaneously recorded. The membrane potential was measured with a glass microelectrode (WPI, Sarasota, FL) filled with 3 mol/L KCl solution (tip resistance, 70–100 MΩ). The electrical signal was amplified (Intra 767 amplifier, WPI) from recordings obtained from the adventitial side of the vessel for smooth muscle cells and from the intimal face for endothelial cells.21 The membrane potential was monitored continuously using an oscilloscope (Type1425, Gould, UK) and data were recorded on a chart recorder. The criteria for accepting a record were a sharp penetration and rise to 0 mV when the electrode was withdrawn from the recorded cell.
To penetrate an endothelial cell, the electrode was approached slowly toward the intimal face of the arterial wall. We considered that an endothelial cell was recorded only when the electrode penetrated a cell at the first contact with the tissue.21,22 To demonstrate the efficiency of this approach, endothelial cells impaled by this method were occasionally microiontophoretically injected with the fluorescent dye Lucifer yellow. The tip of the electrode was filled with Lucifer yellow dilithium, 5% diluted in water (Sigma) and was backfilled with 150 mmol/L LiCl. The injection was done by passing a current of 0.35 nÅ through the electrode for 2 min. After dye injection, the tissue was immediately fixed with 4% paraformaldehyde in phosphate buffer (pH 7.4) at room temperature for a few minutes. It was then examined with a fluorescent microscope with an excitation wavelength of 450 to 490 μm (Leitz Othoplan) and photographed with a digital camera (Nikon Coolpix).21,22
Isolated Vessel Tension Studies
C57BL/6J mice were administrated a lethal dose of pentobarbital (1 g/kg intraperitoneally) and the main pulmonary artery (PA) was immediately harvested, resulting in a ring of 1.5- to 2.0-mm length and 0.5- to 1.0-mm diameter. The vessel ring was suspended in organ chamber filled with 10 mL of modified Krebs–Ringer bicarbonate solution (mmol/L: 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, and 11.1 glucose) maintained at 37°C and aerated with 95% O2–5% CO2 (pH 7.4).5 Each ring was suspended by 2 stirrups (0.1-mm diameter) passed through the lumen. One stirrup was anchored to the bottom of the organ chamber, the other was connected to a strain gauge (PowerLab/8SP, ADInstruments, Colorado Springs, CO) for the measurement of isometric force. Vessels were brought to their optimal resting tension after 2 stretches of 0.5 g. After equilibration, indomethacin (10−5 mol/L) was added to exclude possible interference of prostanoid production. The vessels were then contracted with phenylephrine (10−5 mol/L) at a level corresponding at least to the maximal response to potassium (100 mmol/L KCl). Finally, a cumulative dose–response of pulmonary artery to acetylcholine (10−8–10−4 mol/L) was evaluated in the absence or presence of N G-nitro-L-arginine (NLA; 10−4 mol/L), an inhibitor of NOS activity.23 Change in tension induced by ACh was expressed as the percentage of the initial contraction induced by phenylephrine. During preliminary experiments, bradykinin and histamine were also tested because in many arteries, these molecules induce substantial endothelium-dependent vasodilatation.24,25
For ultrastructural examination, freshly dissected arteries were fixed with 2% glutaraldehyde solution in phosphate-buffered saline. Subsequently, the tissue was postfixed in 2% osmium tetroxide, embedded in Epon, and processed for conventional electron microscopy according to standard procedures. After staining with uranyl acetate and lead citrate, thin sections were examined with a Philips electron microscope (EM 410).26
The drugs used were phenylephrine (PE), acetylcholine (ACh), bradykinin, histamine, NLA (all from Sigma, St. Louis, MO), and nitroglycerin (Ng) as nitronal-A (G. Pohl-Boskamp, Germany).
Data are expressed as mean±SEM. Statistical significance was tested using Student t-test on paired data or unpaired data for isolated vessel tension studies; P<0.05 was regarded as significant; n represents the number of animals examined.
Isolated Vessel Tension Studies
Bradykinin (10−10–10−5 mol/L) and histamine (from 10−9–10−4 mol/L) did not relax pulmonary arterial rings in our experimental conditions (data not shown). ACh induced a dose-dependent relaxation of pulmonary arterial rings, with a maximal relaxation of 36±3% (n=15; mean±SEM; Fig. 1). The pattern of the pharmacological response observed in pulmonary arteries is caused by the tone-dependent effects of ACh. Low doses of ACh induced relaxation of precontracted arteries, whereas higher doses resulted in constriction of the relaxed vessels. The ACh-induced vasorelaxation of murine pulmonary arteries was completely inhibited in the presence of the NOS inhibitor nitro-L-arginine (10−4 mol/L; Fig. 1).
Smooth Muscle Cell Membrane Potential
The resting membrane potential of smooth muscle cells recorded in isolated arterial wall strip from C57BL/6J mice was −59±3 mV (n=5; mean±SEM). Stimulation with 10−5 mol/L PE contracted the strip by 0.18±0.03 nmol/L (n=4; mean±SEM). A depolarization of smooth muscle cells of 7±1 mV (n=5, mean±SEM) was observed when the membrane potential was continuously recorded during the contraction caused by PE (Fig. 2A). The membrane potential recorded in the presence of PE was −48±2 mV (n=11; mean±SEM). The addition of 4×10−6 mol/L ACh relaxed the strip by 23%±4% (n=4; mean±SEM). ACh did not significantly change the membrane potential of smooth muscle cells when the membrane potential was continuously recorded during the relaxation (Fig. 2A). The change was −1±1 mV (n=9; mean±SEM). A concentration of 15×10−6 mol/L nitroglycerin relaxed the strip by 48%±10% (n=4; mean±SEM). Nitroglycerin did not significantly change the membrane potential of smooth muscle cells when the membrane potential was continuously recorded during the relaxation (Fig. 2A). The change was −0.5±1 mV (n=9; mean±SEM).
Interestingly, in 1 of 11 observations, membrane potential oscillations were produced in the presence of 10−5 mol/L PE when nitroglycerin was removed after a first application. In that case also, nitroglycerin did not change the mean membrane potential but inhibited the oscillations (Fig. 2B). When using another mouse strain, CDI Crl, instead of C57BL/6J, the application of catecholamine α-agonist always (6 observations) produced a depolarization and a regular sinusoidal oscillation of the membrane potential with an amplitude of 7.3±1.3 mV (n=6; mean±SEM) and a frequency of 33±2 oscillations per minute (n=6; mean±SEM; Fig. 3A). In that case, nitroglycerin and ACh did not hyperpolarize the membrane potential of the smooth muscle cells, but they inhibited the oscillations (Fig. 3B).
Endothelial cells were identified by their shape following microiontophoretical injections with Lucifer yellow (5 observations). The endothelial cells consist of a monolayer of oval cells coupled in cluster. They unambiguously differ from the multiple layers of elongated smooth muscle cells (3 observations; Fig. 4). Heterocellular dye coupling between the endothelial cells and the smooth muscle cells was never observed.
Endothelial Cell Membrane Potential
The efficiency of our approach to record the membrane potential of the endothelial cells was demonstrated by the dye-coupling experiments. It is important to note that it is difficult to penetrate and stay in endothelial cells in this type of tissue (by comparison with porcine coronary arteries).22 Nevertheless, 5 endothelial cells were recorded during the application of ACh. The resting membrane potential of endothelial cells recorded in isolated arterial wall strip from C57BL/6J mice, in the presence of 10−5 mol/L PE was −43±2 mV (n=5; mean±SEM). We observed that ACh hyperpolarized the membrane potential of endothelial cells by 26±2 mV (n=4; mean±SEM) when the membrane potential was continuously recorded during its application (Fig. 5). In contrast, nitroglycerin did not modify endothelial membrane potential (Fig. 5).
The endothelium and the internal elastic membrane of mouse and rat pulmonary arteries were observed by transmission electron microscopy. Interruption in the internal elastic membrane was rarely observed in the rat pulmonary artery. Such interruptions were more frequent in mouse pulmonary artery. However, these interruptions are often filled with extracellular material and thus are not myoendothelial bridges. Myoendothelial bridges were rarely observed in pulmonary arteries in both species, but heterocellular gap junctions were never observed (Fig. 6).
Pulmonary vascular tone is regulated by an intricate group of mechanisms, including production of various vasoconstricting and vasorelaxing agents. The relative contribution of these agents varies within the lung and from species to species.1 Pulmonary vascular tone regulation was already studied in several animal models, such as sheep, pig, rabbit, and rat.5–12 However, little is known about the basic physiology of the murine pulmonary artery, despite the increasing importance of this animal model, mainly because of the experimental possibilities offered by transgenic animals.
ACh, bradykinin, and histamine act as endothelium-dependent dilators in many arteries. The vasorelaxation induced by these molecules is generally caused by NO plus EDHF.24,25 Although all of these molecules should have been good candidates to test mice pulmonary arteries for EDHF, we did not observe endothelium-dependent relaxation with bradykinin and histamine in this type of artery. ACh induced an endothelium-dependent relaxation of isolated murine pulmonary arteries, which was completely inhibited in the presence of the NOS inhibitor nitro-L-arginine, suggesting that NO production is necessary to allow relaxation of these vessels by ACh. Therefore, complete inhibition of ACh-induced relaxation of pulmonary artery in the presence of the NOS inhibitor NLA suggests that EDHF does not play a role in this type of vessel. This is in agreement with the observation that EDHF-like responses were not observed in rings of aorta, carotid, coronary, and mesenteric arteries from NOS knockout mice stimulated with ACh.27 Indeed, the residual relaxation observed in different types of vessels in the presence of ACh and a NOS inhibitor is generally attributed to EDHF.28–32
In the present study, measurement of isometric force developed by the strips of pulmonary arteries was used as a control to verify that the pharmacological molecules were working during electrophysiological recordings. Because the portion of the strip where microelectrodes were impaled was immobilized with 4 insect pins, the force developed by the strip is given as a guide and cannot be compared to the force developed by rings in isolated vessel tension studies.
ACh acts as an endothelium-dependent relaxing agent on different vascular beds. This process has been linked to hyperpolarization of endothelial cells.28–30 Our results are in agreement with these observations. However, in the murine pulmonary artery, ACh did not hyperpolarize smooth muscle cells in the presence of an intact endothelium, despite the large hyperpolarization observed in the endothelial cells. These observations suggest that hyperpolarization of endothelial cells by an endothelium-dependent vasodilator is not a sufficient condition for EDHF expression. The results obtained in isolated murine pulmonary arteries are in agreement with this conclusion.
Because ACh relaxed the murine pulmonary artery by the release of NO, without changing the smooth muscle cell membrane potential, our results imply that this molecule should not hyperpolarize smooth muscle cells. This is in agreement with our observation that an exogenous NO donor, nitroglycerin, did not change the membrane potential of the smooth muscle cells, whereas it relaxed the pulmonary artery strip.
Interestingly, with the strain of mouse we used (C57BL/6J), sinusoidal oscillations of membrane potential were rarely observed. However, such oscillations were commonly observed when another strain, CDI Crl, was used. Therefore, the ability of pulmonary artery to develop membrane potential oscillations appears to depend on the strain of mouse.
In small vessels, EDHF results from the electrotonic spreading of the hyperpolarization produced in the endothelial cell to the smooth muscle cells through gap junctions.33,34 This concept is well-documented in the rat mesenteric arteries.35 Moreover, there is an inverse correlation between the magnitude of NO- and gap junction-dependent relaxations evoked by ACh in resistance arteries of different sizes.36 Electron microscopic observations did not allow demonstration of the presence of myoendothelial gap junction plates in mouse main pulmonary artery. However, extensive Lucifer yellow coupling is shown between smooth muscle cells in large arteries when no gap junctions can be demonstrated by transmission electron microscopy.26 Therefore, the lack of observation of myoendothelial gap junctions does not demonstrate an absence of electrical coupling. Moreover, the absence of heterocellular dye coupling between the endothelial and the smooth muscle cells is not a proof of the absence of electrical coupling. Indeed, in porcine coronary artery, heterocellular electrical communication has been observed between the endothelial and the smooth muscle cells when no dye coupling can be shown.22,37 As a matter of fact, an absence of heterocellular dye coupling between the endothelial and the smooth muscle cells was observed in rat middle cerebral artery. In spite of these observations, heterocellular electrical coupling and the implication of gap junctions were demonstrated in this artery.38
Taken together, our results show that in murine pulmonary artery, ACh induces an endothelium-dependent relaxation and a hyperpolarization of endothelial cells. However, ACh did not cause an endothelium-dependent hyperpolarization of the smooth muscle cells in the mouse pulmonary artery. In summary, we observed that in mouse pulmonary artery, NO alone is responsible for ACh-induced endothelium-dependent vasodilatation, whereas the phenomenon called EDHF is not present.
The authors thank Francoise Gribi for expert technical assistance.
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