Endothelin-induced contraction of bronchiole and pulmonary arteriole smooth muscle cells is regulated by intracellular Ca2+ oscillations and Ca2+ sensitization

Jose F. Perez-Zoghbi, Michael J. Sanderson

Abstract

Endothelin-1 (ET) induces increases in intracellular Ca2+ concentration ([Ca2+]i), Ca2+ sensitization, and contraction of both bronchiole and pulmonary arteriole smooth muscle cells (SMCs) and may play an important role in the pathophysiology of asthma and pulmonary hypertension. However, because it remains unclear how changes in [Ca2+]i and the Ca2+ sensitivity regulate SMC contraction, we have studied mouse lung slices with phase-contrast and confocal microscopy to correlate the ET-induced contraction with the changes in [Ca2+]i and Ca2+ sensitivity of bronchiole and arteriole SMCs. In comparison with acetylcholine (ACh) or serotonin (5-HT), ET induced a stronger and long-lasting contraction of both bronchioles and arterioles. This ET-induced contraction was associated with prominent asynchronous Ca2+ oscillations that were propagated as Ca2+ waves along the SMCs. These Ca2+ oscillations were mediated by cyclic intracellular Ca2+ release and required external Ca2+ for their maintenance. Importantly, as the frequency of the Ca2+ oscillations increased, the extent of contraction increased. ET-induced contraction was also associated with an increase in Ca2+ sensitivity. In “model” slices in which the [Ca2+]i was constantly maintained at an elevated level by pretreatment of slices with caffeine and ryanodine, the addition of ET increased bronchiole and arteriole contraction. These results indicate that ET-induced contraction of bronchiole and arteriole SMCs is regulated by the frequency of Ca2+ oscillations and by increasing the sensitivity of the contractile machinery to Ca2+.

  • confocal microscopy
  • blood vessels
  • airways
  • lung slices
  • acetylcholine
  • serotonin

the presence of endothelin-1 (ET) within the lung and its ability to induce SMC contraction (6, 16, 25), SMC proliferation (23, 39), and proinflammatory cytokine release (13) has led to the suggestion that ET contributes to the development of a variety of lung diseases (14). These include pulmonary arterial hypertension, which results from an increase of pulmonary vascular resistance and leads to heart failure (37), and asthma, which results from excessive contraction of airway SMCs and is associated with inflammation (10).

Although ET within the lung is mainly synthesized and secreted by vascular endothelial cells of blood vessels (33, 54), the epithelial cells of the major airways (tracheal and bronchi) (15, 30, 46) and the airway SMCs themselves (15, 51) also produce ET. With respect to asthma, these sources may be particularly important, because ET synthesis and release is increased in response to challenges with histamine and cytokines that are typically associated with airway hyperresponsiveness (6, 14). Other sources of ET are alveolar type II cells (8) and macrophages (27).

The intracellular signaling cascade induced by ET to activate SMC contraction is not fully understood. Briefly, ET can act at the cell membrane via one or both of two G protein-coupled ET receptor subtypes (ETA and ETB) to increase phospholipase C (PLC) activity (34). This, in turn, produces inositol 1,4,5-trisphosphate (IP3) to stimulate an elevation of intracellular Ca2+ (11, 43) that leads to the activation of myosin light chain kinase (MLCK) to initiate actin-myosin cross-bridge cycling and SMC contraction.

Although previous studies have shown that ET induces Ca2+ increases in isolated SMCs, these Ca2+ increases occurred as simple biphasic signals consisting of an initial transient increase in Ca2+ followed by a sustained plateau (26, 36). By contrast, we and others have recently found, with the advantages of high temporospatial resolution provided by confocal microscopy, that G protein-coupled receptor agonists such as serotonin (5-HT), acetylcholine (ACh), and ATP induce repetitive Ca2+ oscillations rather than static increases in Ca2+ in airways (4, 28, 42, 44, 45) and arterioles (35, 41). Because the frequency of these Ca2+ oscillations is dependent on both agonist type and concentration and correlates with SMC contraction, we initially believed that the frequency of these Ca2+ oscillations regulates the magnitude of bronchiole and arteriole SMC contraction (41, 42). However, our subsequent studies clearly indicate that SMC contraction in lung slices (3) can be also regulated by a mechanism known as Ca2+ sensitivity (18, 50). In this study, we investigate the relative contribution of Ca2+ oscillations and Ca2+ sensitivity to SMC contraction induced by ET in living lung slices. This understanding of the mechanisms regulating ET-initiated SMC contraction will significantly facilitate our search for therapeutic approaches to treat lung diseases.

MATERIALS AND METHODS

All the materials and methods used have been previously described in detail (42); only a brief outline is given. Reagents were obtained either from Invitrogen Life Technologies-GIBCO (Carlsbad, CA) or Sigma (St. Louis, MO). ET (E-7764) was obtained from Sigma. Hanks’ balanced salt solution was supplemented with 20 mM HEPES buffer (sHBSS). Hanks’ 0-Ca2+ solution was prepared by supplementing sHBSS without Ca2+ and Mg2+ with 0.9 mM MgSO4 and 1 mM Na2H2-EGTA. Bosentan sodium salt was a gift from Actelion Pharmaceuticals (Allschwil, Switzerland). A stock solution of bosentan was prepared at 1 mM in water on the same day of use.

Lung slices.

Lung slices were prepared from BALB/C mice between 7 and 10 wk old. The trachea was cannulated with an intravenous catheter, and after the chest cavity was opened, the collapsed lungs were reinflated with 1.3 ± 0.1 ml of 2 % agarose (low-gelling temperature) in sHBSS, followed by the injection of 0.2 ± 0.1 of air to flush the agarose-sHBSS out of the airways and into the distal alveolar space. Subsequently, a warm (37°C) solution of gelatin (type A, porcine skin, 300 bloom, 6% in sHBSS) was perfused through the intrapulmonary blood vessels via the pulmonary artery by injecting ∼0.3 ml into the right ventricle. The warm agar and gelatin were gelled with cold sHBSS. A single lung lobe was removed and cut into serial sections of ∼130 μm thick with a vibratome at ∼4 °C, starting at the lung periphery. Slices were maintained in DMEM (Invitrogen) at 37°C and 10% CO2 for up to 3 days. Serum was not used, because it contains 5-HT and growth factors (1). At 37°C, the gelatin in the blood vessel lumen dissolved, leaving the blood vessel lumen empty. Only lung slices that had both a bronchiole and an accompanying arteriole with a well-defined lumen were selected for the experiments. No significant changes in the contractility of bronchioles and arterioles in response to stimulation with 1 μM 5-HT were detected during the first 48 h of culture; therefore, for these experiments we used slices maintained for <48 h.

Measurement of the contractile response of bronchioles and arterioles induced by agonists.

Lung slices were mounted in a custom-made perfusion chamber and held in place with a small sheet of nylon mesh. A second cover glass edged with silicone grease was placed over the lung slice. Perfusion of the lung slice was performed using a gravity-fed perfusion system. The volume of the chamber was ∼100 μl with a perfusion rate of ∼800 μl/min. For phase-contrast microscopy, the lung slice was observed with an inverted microscope with a ×10 objective, and images were recorded using a charge-coupled device camera and image acquisition software (Video Savant). Digital images were recorded in time lapse (30 frames/min). The area of the bronchiole and arteriole lumen was calculated, from each image, by pixel summing using custom-written software. Experiments were performed at room temperature.

Measurements of intracellular Ca2+.

Approximately 10–12 lung slices were incubated in 2 ml of 20 μM Oregon green 488 BAPTA-1 AM, 100 μM sulfobromophthalein (an inhibitor temporarily used to prevent dye extrusion through anion exchangers), and 0.2% Pluronic F-127 for 50 min at 30°C, followed by an additional 50 min at 30°C in sHBSS containing 100 μM sulfobromophthalein. Lung slices were mounted in the perfusion chamber as described. Fluorescence imaging was performed using a video-rate confocal microscope (47). Briefly, a 488-nm laser was used as the excitation wavelength. The resultant fluorescence (>510 nm) was detected by a photomultiplier tube and a frame capture board to form images that were recorded at 15 Hz. Changes in fluorescence intensity were analyzed by selecting regions of interest (ROI) of ∼7 × 7 pixels. Average fluorescence intensities of an ROI were obtained, frame by frame, using custom-written software that allowed the tracking of the ROI within a SMC as it moved with contraction. Final fluorescence values are expressed as a fluorescence ratio (F/F0) normalized to the initial fluorescence (F0). Line-scan analysis of images was performed by extracting a line of pixels from each image and placing them sequentially, as a time sequence, in a single image.

Preparation of caffeine-ryanodine model slices.

“Model” lung slices were prepared by exposing lung slices to 20 mM caffeine with 50 μM ryanodine for 3–4 min, followed by a washout with sHBSS. This treatment locks the ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR) of bronchiole and arteriole SMCs in an open state, which depletes the intracellular Ca2+ stores, and this, in turn, increases the Ca2+ influx across the plasma membrane (3). Therefore, in caffeine-ryanodine model slices, the [Ca2+]i of bronchiole and arteriole SMCs is dependent on the extracellular Ca2+ concentration ([Ca2+]o) and can be controlled by adjusting the [Ca2+]o. Because the [Ca2+]i in these model SMCs does not change with the addition of agonists, the model slices are used to study the effect of agonists on the Ca2+ sensitivity of bronchiole and arteriole contraction.

Data and statistical analysis.

Statistical values are means ± SE. A Student's t-test was used to test for significant differences between means.

Online supplemental material.

Videos consisting of sequences of phase-contrast or confocal fluorescence images were produced with Video Savant and are provided as supplemental material. Supplemental videos for this article are available online at the American Journal of Physiology-Lung Cellular and Molecular Physiology website.

RESULTS

Contractile potencies of ET, ACh, and 5-HT in bronchioles and arterioles.

To establish the relative potency of ET as a contractile agonist for both the bronchioles and arterioles, we simultaneously compared the contractile responses of the bronchioles and arterioles in a lung slice sequentially stimulated with maximal concentrations of ACh and 5-HT (1 μM) and with ET (10 nM) (Fig. 1 and Supplemental Video 1). The agonists ET, ACh, and 5-HT induced bronchiole and arteriole contraction with different potencies and kinetics (Fig. 1; Table 1). In the bronchiole, the contraction (measured after 8 min of stimulation) induced by ET was similar to or bigger than the contraction induced by ACh or 5-HT, respectively (Table 1). In the arteriole, ET and 5-HT induced contractions of similar magnitude (Fig. 1; Table 1), whereas ACh had no effect. However, the contraction of bronchioles and arterioles induced by 10 nM ET was slower and had a longer onset time than the contraction induced by ACh or 5-HT (Table 1). In contrast to the fast relaxation of bronchioles and arterioles following ACh and 5-HT removal, the relaxation following the washout of ET was extremely slow (Fig. 1B; Table 1). This suggests a slower dissociation rate for ET than for ACh or 5-HT from their receptors.

Fig. 1.

Contractile response of bronchioles and arterioles to acetylcholine (ACh), serotonin (5-HT), and endothelin-1 (ET). A: phase-contrast images showing the appearance of a bronchiole and arteriole in a lung slice before (1, resting) and after 8 min of stimulation (arrows in B) with 1 μM ACh (2), 1 μM 5-HT (3), and 10 nM ET (4). B: change in cross-sectional area of the lumen of the bronchiole (solid line) and arteriole (shaded line) with respect to time in response to ACh, 5-HT, and ET as indicated. ACh induced contraction of the bronchiole but not the arteriole. The apparent increase in arteriole lumen size resulted from passive stretching by the contraction of the airway. 5-HT and ET induced contraction of both the bronchiole and arteriole. After washout of ACh and 5-HT with Hanks’ balanced salt solution supplemented with 20 mM HEPES buffer (sHBSS), the contraction of the bronchiole and the arteriole was quickly relaxed; however, after washout of ET, the bronchiole only relaxed very slowly, whereas the arteriole showed no relaxation. A representative experiment selected from 6 different slices from 3 mice. A movie of these data is shown in Supplemental Video 1.

View this table:
Table 1.

Potency and kinetics of contractile responses of bronchioles and arterioles to ET, 5-HT, and ACh

Concentration dependence of bronchiole and arteriole contraction on ET.

To determine the relative sensitivity of bronchioles and arterioles to ET, we measured the changes in lumen area in response to different concentrations of ET (Fig. 2). However, because the contractile response of bronchioles and arterioles to ET was essentially irreversible, we used different lung slices for each ET concentration. To reduce experimental variation associated with different lung slices, ET stimulation was preceded by a calibration stimulation with 1 μM 5-HT, and only slices responding to 5-HT with a reduction in bronchiole lumen area of 40–60 % and arteriole lumen area of 60–80 % were analyzed.

Fig. 2.

Concentration dependence of ET-induced bronchiole and arteriole contraction. A–C: representative experiments showing the contractile responses of bronchioles and arterioles in single lung slices stimulated consecutively with 1 μM 5-HT and 1 nM ET (A), 5 nM ET (B), or 50 nM ET (C) as indicated. Contraction of the bronchioles and arterioles was faster and larger with increasing concentrations (1 to 50 nM) of ET. D: summary of the ET-induced bronchiole and arteriole contraction with respect to ET concentration. Contraction was measured as the decrease in lumen area after 8 min of ET exposure. Each point represents the mean ± SE from at least 4 different experiments on different slices from at least 2 mice. The contractility data were fitted with logistic function curves. The estimated effective concentration (EC50) was 1.2 nM in bronchioles and 0.7 nM in arterioles.

Bronchiole and arteriole contraction in response to 1 nM ET was characterized by a delayed onset (∼3 min after solution change) and a slow rate (maximal rate <1% lumen area/s) (Fig. 2A). The reduction in lumen area after 8 min of stimulation was 22 ± 11% in bronchioles and 52 ± 7% in arterioles (Fig. 2D). Increasing concentrations of ET from 1 to 50 nM reduced the delay before contraction began and increased the rate of the contraction of both bronchioles and arterioles (Fig. 2, A–C). The magnitude of the bronchiole and arteriole contraction increased with ET concentration from 1 to 10 nM but was maximal at concentrations >10 nM. At all concentrations of ET, the arteriole contraction was greater than the bronchiole contraction (Fig. 2D). Collectively, the induction of strong and maximal contraction by low ET concentrations, the reduction of the onset time to contraction by increasing ET concentrations, and the slow relaxation upon ET removal indicated that ET stimulates contraction by a cumulative binding with high affinity to its receptors.

Ca2+ signaling in bronchiole and arteriole SMCs induced by ET.

To characterize the Ca2+ signaling underlying the ET-induced bronchiole and arteriole contraction, we used confocal microscopy to examine the changes in the [Ca2+]i of the SMCs in response to ET. Bronchiole and arteriole SMCs responded to stimulation with 10 nM ET with an increase in [Ca2+]i followed by Ca2+ oscillations (Figs. 3B and Figs. 4B; Supplemental Videos 2 and 3). However, these changes in [Ca2+]i occurred after a delay of 45–90 s. Line-scan plots obtained from linear ROIs, orientated either across the bronchiole wall (Fig. 3A) or along the arteriole wall (Fig. 4A) to include a few adjacent SMCs, show that the onset of the Ca2+ response did not occur simultaneously in each SMC. Instead, each SMC initiated a Ca2+ increase after a varying delay (Figs. 3C and 4C; Supplemental Videos 2 and 3). The simultaneous measurements of the changes in [Ca2+]i in SMCs with the changes in the luminal area of the bronchiole and arteriole show that the initiation of the Ca2+ oscillations in bronchiole and arteriole SMCs correlated with the onset of the contraction (Figs. 3B and 4B). A similar correlation is indicated by the line-scan plots (Figs. 3C and 4C). However, it is important to point out that bronchiole or arteriole contraction is the result of the cooperative action of multiple SMCs, whereas the Ca2+ measurements pertain to single SMCs. After the initiation the Ca2+ response, the subsequent Ca2+ oscillations occurred asynchronously with respect to neighboring SMCs.

Fig. 3.

Ca2+ signaling in bronchiole smooth muscle cells (SMCs) induced by ET. A: a fluorescence confocal image showing part of a bronchiole in a lung slice loaded with Oregon green. Epithelial cells (EPCs) are observed lining the bronchiole lumen, and the SMCs lie underneath the EPCs. B: simultaneous recordings of Ca2+ signaling and contraction in bronchiole SMCs during ET (10 nM) stimulation. Fluorescence changes of the intracellular Ca2+ concentration ([Ca2+]i) indicator (top trace) were measured in a small region of interest (ROI; ∼7 × 7 pixels) defined within a single SMC and plotted as a ratio (F/F0) with respect to time. Stimulation with 10 nM ET was followed by an interval of ∼60 s with no change in [Ca2+]i, followed by a subsequent increase in baseline [Ca2+]i and Ca2+ oscillations. Bronchiole contraction (bottom trace) was estimated from the change in the imaged bronchiole lumen area (measured in each fluorescence image, see image in A) normalized to the lumen area before ET stimulation. C: a line-scan plot with respect to time from a region across the bronchial wall spanning several adjacent SMCs (indicated by dashed line C in A) showing the asynchronous Ca2+ oscillations (white vertical lines) in different SMCs. Arrows indicate the first Ca2+ increase observed in each SMC. D: a line-scan plot with respect to time from the longitudinal axis of a single SMC (indicated by dotted line D in A) showing the propagation of the Ca2+ oscillations as Ca2+ waves (bright white lines) in a single bronchiole SMC. This line-scan plot was obtained after 5 min of ET stimulation. Representative data are from 9 experiments from different slices from 4 mice. A movie of the effect of ET on Ca2+ signaling in bronchiole SMCs is shown in Supplemental Video 2.

Fig. 4.

Ca2+ signaling in arteriole SMCs induced by ET. A: a fluorescence confocal image of an arteriole with an oblique orientation (illustrated by inset) with endothelial cells (ENCs) and SMCs oriented in parallel or perpendicular to the long axis of the arteriole, respectively. B: fluorescence changes in a small ROI within a single arteriole SMC (top trace) showing the Ca2+ oscillations in response to 10 nM ET. The decrease in arteriole lumen (bottom trace) accompanied the Ca2+ oscillations and contraction of arteriole SMCs. C: a line-scan plot (dashed line C in A) showing the asynchronous Ca2+ oscillations (white vertical lines) in different SMCs. Arrows indicate the first Ca2+ increase observed in each SMC. D: a line-scan plot from the longitudinal axis of a single SMC (dotted line D in A) showing the propagation of the Ca2+ oscillations as Ca2+ waves (white lines) along the arteriole SMC. Representative data are from at least 6 experiments from different slices from 4 mice. A movie of the effect of ET on Ca2+ signaling in arteriole SMCs is shown in Supplemental Video 3.

Line-scan plots from linear ROIs orientated along the longitudinal axis of single bronchiole and arteriole SMCs demonstrate that the individual Ca2+ oscillations consisted of repetitive Ca2+ waves that propagated along the length of the SMC (Figs. 3D and 4D; Supplemental Videos 2 and 3). Although the Ca2+ waves within single bronchiole or arteriole SMCs were generally observed to initiate from the same region of the cell, the initiation site and propagation direction could change with time. The average velocity of the Ca2+ waves in bronchiole SMCs was 33.5 ± 0.9 μm/s (9 cells from 7 slices of 3 animals), but in arteriole SMCs, the average velocity was slower at a rate of 11.7 ± 0.5 μm/s (14 cells from 6 slices of 3 animals) (Figs. 3D and 4D). The differences in the propagation velocity of the Ca2+ waves between bronchiole and arteriole SMCs suggest differences in the mechanism of Ca2+ wave propagation between bronchiole and arteriole SMCs.

Relationship between the frequency of Ca2+ oscillations and contraction.

When lung slices were stimulated with 10 nM ET, both the frequency of the Ca2+ oscillations and the contraction of bronchiole and arteriole SMCs increased as a function of time (Fig. 5, A and B). In bronchioles, the frequency of ET-induced Ca2+ oscillations increased from 8.9 ± 4.3 cycles/min, at the beginning of the Ca2+ response, to a sustained frequency of 26.5 ± 3.0 cycles/min at 3 min after the onset of the Ca2+ response (Fig. 5A). The contraction of the bronchioles increased during 2–3 min until it began to plateau at ∼50% (Fig. 5B). The simultaneous correlation of the Ca2+ oscillation frequency with contraction indicates that as the frequency of the Ca2+ oscillations increased, there was a concomitant increase in bronchiole contraction (Fig. 5C).

Fig. 5.

Relationship between Ca2+ oscillation frequency and contraction of bronchioles and arterioles during stimulation with ET. The frequency of the Ca2+ oscillations and the contraction in response to 10 nM ET were calculated during the first 3 min after the onset of the Ca2+ response from experiments similar to those shown in Figs. 3 and Figs. 4. A: frequency of the Ca2+ oscillations in bronchiole SMCs (n = 8 SMCs from 6 experiments) and arteriole SMCs (n = 9 SMCs from 5 experiments) as a function of time. B: contraction of the bronchioles and arterioles as a function of the time. C: relationship between Ca2+ oscillation frequency and contraction from data in A and B. Bronchiole data were fitted with a single straight line (r2 = 0.90). Arteriole data were fitted with 2 straight lines: phase 1 (r2 = 0.97) correlates the data points in A and B for the first minute of ET stimulation; phase 2 (r2 = 0.81) correlates the subsequent data points.

The frequency of the Ca2+ oscillations in arteriole SMCs was significantly lower than in bronchiole SMCs and increased from 4.3 ± 2.0 cycles/min at the beginning of the Ca2+ response to 8.9 ± 2.0 cycles/min at 3 min after the onset of the Ca2+ response (Fig. 5A). In contrast to the frequency of Ca2+ oscillations, the contraction was significant larger in arterioles than in bronchioles. In addition, arteriole contraction increased quickly during the first minute of ET-stimulation before reaching a plateau with ∼80 % contraction (Fig. 5B). The simultaneous correlation of the Ca2+ signaling and contraction in arteriole SMCs showed two phases (Fig. 5C). In phase 1, there was a concomitant increase in the frequency of the Ca2+ oscillations and the contraction (up to ∼50%). Subsequently, in phase 2, the arteriole continued to contract an additional ∼25% even though the frequency of Ca2+ oscillations slowed before resuming with a small increase in frequency.

This temporal correlation between the Ca2+ oscillation frequency and the changes in bronchiole or arteriole lumen area suggests that bronchiole and arteriole SMC contraction is, in part, regulated by the frequency of the Ca2+ oscillations. However, the relationship between Ca2+ oscillation frequency and contraction was different for the bronchiole and arteriole SMCs. Over the same contraction range, the frequency of Ca2+ oscillations was, in general, about three times faster in bronchiole SMCs than in arteriole SMCs (Fig. 5C). These findings that bronchiole SMCs require faster Ca2+ oscillation frequencies than arteriole SMCs to sustain ET-stimulated contraction and that arteriole contraction occurs in two phases suggests that the contraction is regulated by additional mechanisms besides just the frequency of Ca2+ oscillations (see below).

Role of extracellular Ca2+ in Ca2+ signaling and contraction.

To investigate and compare the mechanisms of ET-induced Ca2+ oscillations in bronchiole and arteriole SMCs, we studied the effect of removing the extracellular Ca2+. In lung slices prestimulated with ET, the removal of the extracellular Ca2+ induced bronchiole and arteriole relaxation (Fig. 6A) and the cessation of ongoing Ca2+ oscillations (within 1 min) in bronchiole (Fig. 6B) and arteriole SMCs (Fig. 6C). In addition, removal of extracellular Ca2+ also reduced the basal [Ca2+]i associated with the Ca2+ oscillations. Note that the bronchioles relaxed faster than arterioles after removal of extracellular Ca2+ (Fig. 6A). After 3 min in zero extracellular Ca2+, the bronchioles were ∼ 90% relaxed, whereas the arterioles were ∼15 % relaxed. Simultaneous recordings of Ca2+ and lumen area changes indicate that bronchiole and arteriole relaxation coincided with the inhibition of Ca2+ oscillations and a reduction in the basal [Ca2+]i (Fig. 6, B and C). However, the rate of relaxation was again slower in the arterioles than in the bronchioles.

Fig. 6.

Effect of the absence of extracellular Ca2+ on the contraction and Ca2+ oscillations of bronchiole and arteriole SMCs induced by ET. A: effect of the removal of extracellular Ca2+ (0 Ca2+) on the contraction induced by 10 nM ET in a bronchiole and an arteriole in the same lung slice. Both the bronchiole and the arteriole contracted in response to ET in the presence of extracellular Ca2+ but relaxed in the absence of extracellular Ca2+. The bronchiole relaxed faster than the arteriole. The subsequent readdition of extracellular Ca2+ induced a fast recontraction of both the bronchiole and the arteriole. B and C: simultaneous recordings of changes in [Ca2+]i in single SMCs (top traces) and contraction (bottom traces) of a bronchiole (B) and arteriole (C) during stimulation with ET in the presence and absence of extracellular Ca2+ as indicated. Removal of extracellular Ca2+ resulted in the cessation of ET-induced Ca2+ oscillations with a decrease in [Ca2+]i and relaxation of the bronchiole and arteriole. Representative traces are from at least 5 experiments from different slices of 3 mice.

In a reverse series of experiments, with lung slices preincubated (1 min) and maintained in Ca2+-free medium, ET triggered Ca2+ oscillations and contraction in both bronchiole and arteriole SMCs. However, the Ca2+ oscillations were not maintained (abolished after 2 min), and the bronchioles and arterioles relaxed (4 experiments from 3 mice; data not shown).

Role of intracellular Ca2+.

To test the idea that Ca2+ oscillations are mediated by intracellular Ca2+ release, we examined the effect of inhibiting the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) with cyclopiazonic acid (CPA). Addition of 10 μM CPA to lung slices stimulated with ET resulted in a progressive increase in the basal [Ca2+]i with a consequent reduction in the amplitude of the Ca2+ oscillations until a sustained elevated [Ca2+]i was attained in both bronchiole (Fig. 7A) and arteriole SMCs (Fig. 7B). Simultaneous recordings of Ca2+ and contraction indicated that although CPA inhibit the dynamic Ca2+ signaling, the sustained Ca2+ increase continued to induced an additional small increase in contraction in both bronchioles and arterioles (Fig. 7). The subsequent removal of extracellular Ca2+ reduced the [Ca2+]i to a level lower than that of the unstimulated cell, and this was accompanied by a fast relaxation of bronchioles but a slower relaxation of arterioles. These changes in [Ca2+]i and contraction were reversed by the readdition of Ca2+ to the extracellular medium (Fig. 7). Collectively, these results and those from experiments in Ca2+-free medium (Fig. 6) suggest that ET-induced Ca2+ oscillations in both bronchiole and arteriole SMCs resulted from Ca2+ release from SR but required some Ca2+ influx to sustain their activity.

Fig. 7.

Role of internal Ca2+ stores in Ca2+ oscillations and contraction of bronchioles and arterioles induced by ET. A and B: simultaneous recordings of changes in Ca2+ in single SMCs (top traces) and contraction (bottom traces) of a bronchiole (A) and an arteriole (B) during stimulation with ET and the subsequent addition of 10 μM cyclopiazonic acid (CPA) in the presence and absence of extracellular Ca2+ as indicated. In both bronchiole and arteriole SMCs, the addition of CPA stopped the Ca2+ oscillations, leaving an elevated sustained level of [Ca2+]i. Under theses conditions, the bronchiole and arteriole remained contracted. Removal of extracellular Ca2+ in the presence of ET and CPA induced a decrease in [Ca2+]i and relaxation of the bronchiole and arteriole. The subsequent addition of extracellular Ca2+ resulted in an increase in [Ca2+]i and recontraction of both the bronchiole and the arteriole. Representative traces are from at least 4 experiments from different slices of 3 mice.

Ca2+ sensitization induced by ET.

Because we found that bronchiole and arteriole contractility did not appear to be simply proportional to the Ca2+ oscillation frequency, we investigated the effect of ET on the Ca2+ sensitivity of the bronchiole and arteriole contraction with caffeine-ryanodine model slices. Figure 8 shows the changes in [Ca2+]i and contraction of the bronchiole and arteriole in response to control stimulation with 5-HT during the establishment of the caffeine-ryanodine model slice and the subsequent responses to ET.

Fig. 8.

Effect of ET on Ca2+ sensitivity of bronchiole and arteriole SMCs. A and B: changes in [Ca2+]i signaling in single SMCs (top traces) and contraction (bottom traces) of a bronchiole (A) and an arteriole (B) during stimulation with 1 μM 5-HT, 20 mM caffeine plus 50 μM ryanodine (Caff+Ry), and 10 nM ET in the presence or absence of extracellular Ca2+ as indicated. The fluorescence was recorded at 15 frames/s during 5-HT stimulation to resolve the fast Ca2+ oscillations and thereafter at 1 frame/s to prevent photobleaching. The possibility of fast Ca2+ changes during this latter time period was ruled out in separate experiments by recording at 15 frames/s for 5 min after the addition of each compound. 5-HT induced Ca2+ oscillations and contraction in bronchiole and arteriole SMCs. Caff+Ry induced a transient increase in [Ca2+]i and transient contraction of the bronchiole and arteriole. The elevated [Ca2+]i that persisted after the washout of Caff+Ry caused slight bronchiole contraction and a pronounced arteriole contraction. Subsequent stimulation with ET did not change [Ca2+]i but induced a strong bronchiole contraction and an additional arteriole contraction. Washout of extracellular Ca2+ reduced the [Ca2+]i and induced relaxation of the bronchiole and arteriole. C: simultaneous contraction of a bronchiole and an arteriole in the same lung slice showing the differences in their Ca2+ sensitivity. Washout of Caff+Ry was followed by a slight contraction of the bronchiole and a strong contraction of the arteriole. The subsequent addition of ET induced a strong contraction of the bronchiole and further contraction of the arteriole. Representative traces from at least 4 experiments from different slices of 3 mice.

In response to 5-HT, the bronchiole and arteriole showed typical Ca2+ oscillations and contraction (Fig. 8, A–C). Addition of caffeine plus ryanodine induced a transient increase in [Ca2+]i followed by a sustained increase in [Ca2+]i in both bronchiole (Fig. 8A) and arteriole SMCs (Fig. 8B). This response is consistent with the expected initial Ca2+ release from internal Ca2+ stores followed by a sustained influx of Ca2+ (3). However, the bronchiole and arteriole only contracted transiently in response to caffeine plus ryanodine (Fig. 8, A–C). This is consistent with the relaxing effect of caffeine on SMCs. After washout of the caffeine and ryanodine, the [Ca2+]i remained elevated (Fig. 8, A and B), but the contractile response to the sustained [Ca2+]i elevation was significantly different between bronchioles and arterioles (Fig. 8C). The bronchioles either remained relaxed or only showed a small recontraction (6 ± 4% of their initial area), whereas the arterioles strongly recontracted (55 ± 9% of their initial area). The subsequent addition of ET to model slices did not induce a change in [Ca2+]i but initiated a strong contraction of the bronchioles (66 ± 9%) and a further contraction of the arterioles (41 ± 7%) (Fig. 8, A–C). Bronchiole and arteriole contractions were abolished by subsequent removal of extracellular Ca2+ (Fig. 8, A and B). These results demonstrate that arterioles are inherently much more sensitive to Ca2+ than bronchioles and that although ET increases the Ca2+ sensitivity of both bronchiole and arteriole SMCs, ET-induced Ca2+ sensitization is essential for bronchiole contraction.

Type of ET receptors.

To investigate the type of the ET receptors that mediates the ET-induced contraction of bronchioles and arterioles, we compared the inhibitory effects of the selective ETA and ETB receptor antagonists BQ-123 and BQ-788, respectively, and the nonselective ET receptor antagonist bosentan on the contractile response to ET (Fig. 9) In all experiments, a prestimulation with 5-HT was used to calibrate the bronchiole and arteriole responses in different slices. Compared with the control stimulation with 5 nM ET (Fig. 9A), all antagonists inhibited the ET-induced contractile response of bronchioles and arterioles by delaying the onset, slowing the rate, and reducing the magnitude of the contraction (Fig. 9, B–D). However, each antagonist inhibited the ET-induced contraction with a different potency. The rightward shift and decreased maxima of the concentration-contraction response (Fig. 9, E and F) indicate that BQ-788 and bosentan produced the strongest inhibitory effects on bronchioles and arterioles, respectively. By comparing the maximal contraction (Δareamax) and the concentration of ET inducing one-half of Δareamax (SC50) for each antagonist (Table 2), we established the following orders of potency: for bronchioles, BQ-788 > bosentan > BQ-123; for arterioles, bosentan > BQ-788 = BQ-123. These results suggest that ET-induced contraction of bronchioles is mediated by both ETA and ETB receptors with a preponderance of ETB receptors, whereas ET-induced contraction of the arterioles involves an equal participation of ETA and ETB receptors. These results are consistent with reported results for mouse bronchioles (38) and rat arterioles (17, 32).

Fig. 9.

Type of receptor involved in ET-induced contraction of bronchioles and arterioles. A–D: representative traces showing the contraction of bronchioles (solid lines) and arterioles (shaded lines) in single lung slices stimulated sequentially with 1 μM 5-HT and 5 nM ET in the absence (A) or presence of the ET receptor blockers 1 μM BQ-123 (B), 1 μM BQ-788 (C), and 1 μM bosentan (D) as indicated. ET-induced bronchiole and arteriole contraction was inhibited by ET receptor blockers. E and F: summary of ET-induced bronchiole (E) and arteriole contraction (F) in the absence or presence of ET receptor blockers. Contraction was measured as the decrease in lumen area (5 min after ET stimulation) and normalized to the calibration contraction (measured after 8 min of 5-HT stimulation) in each experiment. Each point represents the mean ± SE from at least 3 different experiments on different slices from at least 2 mice. The contractility data were fitted with logistic function curves, or data points were joined by a straight line.

View this table:
Table 2.

Potency of antagonists on ET-induced contraction

DISCUSSION

The anatomical proximity of the small bronchioles and arterioles in the lung allows the simultaneous measurements of bronchiole and arteriole contraction in lung slices using phase-contrast microscopy. This direct comparison of bronchiole and arteriole contraction is important for an integrative understanding of the effect of agonists such as ET. A unique advantage of thin lung slices is that agonist-induced contraction can also be simultaneously measured with the changes in [Ca2+]i in individual bronchiole and arteriole SMCs. This simultaneous determination of Ca2+ and contraction is crucial to understand how the dynamic changes in [Ca2+]i in SMCs regulate the contraction of bronchioles and arterioles during agonists stimulation. In previous studies, we examined in detail the regulation of bronchiole and arteriole contraction by changes in Ca2+ induced by ACh and 5-HT (41, 42). In this study, we extended these studies to include ET, because ET is recognized as a potent endogenous constrictor producing long-lasting contraction of pulmonary blood vessels (7, 31) and tracheal or bronchial airways (2, 52).

The contraction of the bronchioles and arterioles induced by ET was significantly different from the contraction induced by ACh and 5-HT. First, ET induced contractions at low concentrations; 10 nM ET produced a similar or stronger contraction than that induced by 1 μM ACh or 5-HT. Second, ET induced contractions that had a delayed onset and occurred at a slow rate. Finally, ET induced long-lasting contractions that were resistant to reversal by washout. These properties of ET-induced contractions observed in small bronchioles and arterioles were similar to those observed in other pulmonary and nonpulmonary vascular beds (14) and are consistent with results from binding studies of ET to SMC membrane preparations from several SMC types (53) reporting that ET binds to its receptor with relative slow association and dissociation kinetic constants, resulting in a half-dissociation time (t1/2) of ∼165 h. Therefore, ET is known to be a pseudo-irreversible ligand of ET receptors (11).

With confocal microscopy, we found that stimulation of lung slices with ET induced persistent and asynchronous Ca2+ oscillations in both bronchiole and arteriole SMCs. These Ca2+ oscillations correspond to repetitive intracellular Ca2+ waves that originated at one point of the cell and propagated along the longitudinal axis of the SMC. However, this dynamic Ca2+ signaling differs from the ET-induced Ca2+ signaling observed in other studies using different airway and pulmonary artery preparations and other Ca2+ detection techniques. For most tissue preparations, including strips of trachea (22), isolated rat trachea SMCs (36), cultured SMCs from human bronchi (40), rat intrapulmonary arteries (49), or isolated SMCs from rabbit intrapulmonary arteries (26), ET induced an initial transient increase in [Ca2+]i that was usually followed by a sustained increase or plateau in [Ca2+]i. The failure to resolve Ca2+ oscillations in these studies was most likely due to the slow sampling speeds used to monitor the Ca2+ changes. This is emphasized by the fact that when higher sampling speeds were used, ET was reported to induced a Ca2+ transient followed by three to five oscillations of decreasing amplitude in rat SMCs isolated from the trachea (20), pulmonary artery, and intrapulmonary arteries (19). We believe our ability to observe sustained Ca2+ oscillations within the SMCs with a maximal frequency of ∼25 per minute results from two fundamental advantages: we sampled at a rate of 15 images/s, and, perhaps more importantly, we used a lung slice preparation that preserves many of the in situ characteristics of the lung tissue.

Although the spatial aspects of the ET-induced Ca2+ oscillations in the bronchiole and arteriole SMCs were similar to the Ca2+ oscillations induced by ACh and 5-HT (41, 42), the temporal characteristics were different. In bronchiole and arteriole SMCs, ACh or 5-HT induced Ca2+ oscillations that occurred with a steady frequency. However, ET induced Ca2+ oscillations in bronchiole and arteriole SMCs that had a frequency that was initially low but progressively increased for ∼3 min before a sustained frequency was attained. This difference in Ca2+ signaling may be explained by the difference in the kinetics of the agonists binding to their receptors. In contrast to ACh and 5-HT, ET binds to its receptors with slower kinetics but higher affinity (11). Consequently, the perfusion of lung slices with a low concentration of ET would be predicted to progressively increase the number of activated ET receptors.

The progressive increase in the Ca2+ oscillation frequency in bronchiole SMCs was correlated with a concomitant increase in bronchiole contraction. A similar correlation between the increase in Ca2+ oscillation frequency and bronchiole contraction was observed during stimulation of lung slices with increasing concentrations of ACh or 5-HT (42). This finding, together with data from other studies, has led to the hypothesis that contraction is regulated by the frequency of the Ca2+ oscillations in airway SMCs (4, 28, 42), pulmonary vascular SMCs (41), and systemic vascular SMCs (29, 35, 48).

The frequency of ET-induced Ca2+ oscillations was approximately three times slower in arteriole (∼10 cycles/min) compared with bronchiole SMCs (∼30 cycles/min). A similar threefold difference in the Ca2+ oscillation frequency between arteriole and bronchiole SMCs was observed for stimulation with 5-HT (41). An explanation for the difference in the Ca2+ oscillation frequency between SMCs types could be due to differences in receptor activation or the signal transduction mechanism linking receptor activation to the generation of Ca2+ oscillations. The similarity of the Ca2+ oscillation frequencies of arteriole and bronchiole SMCs in response to two different agonists (ET and 5-HT) suggests that the difference lies with the coupling of receptor activation to the generation of Ca2+ oscillations. However, the identity of this difference is beyond the scope of the present study.

The most likely mechanism utilized for the generation of ET-induced Ca2+ oscillations is agonist activation of G protein-coupled ET receptors, which in turn stimulate PLC activity to synthesize IP3 (11, 43). Sensitization of IP3 receptors by IP3 is followed by Ca2+-induced activation and inactivation of IP3 receptors (5). Ca2+ release from the SR is accompanied by Ca2+ reuptake by SERCA pump. Ca2+ oscillations are themselves generated by continuous cycles of Ca2+ release and Ca2+ reuptake from the ER. In support of this mechanism, our data show that blockage of Ca2+ reuptake by the SR with CPA completely inhibits the ET-induced Ca2+ oscillations. Similar results were obtained by blocking SERCA pump with thapsigargin (not shown). In addition to the dependence of intracellular Ca2+ release for generation of Ca2+ oscillations, our data also indicate that an influx of Ca2+ through membrane Ca2+ channels is required for the maintenance of Ca2+ oscillations and the contraction of bronchiole and arteriole SMCs.

Our data also illustrate an important facet of the concept of frequency regulation of contraction. When the intracellular Ca2+ stores were neutralized with CPA, the Ca2+ oscillations were abolished and replaced with a sustained elevation of [Ca2+]i without changes in the agonist-induced bronchiole and arteriole contraction. This demonstrates that the lower mean [Ca2+]i associated with Ca2+ oscillations is equal, in terms of contractility, to a higher mean [Ca2+]i associated with a sustained Ca2+ increase. This has the advantage for the living cell; Ca2+ oscillations can maintain contraction while the toxic effects of sustained high [Ca2+]i can be avoided.

We also have investigated the possibility that ET regulates bronchiole and arteriole contraction by increasing the sensitivity of the contractile apparatus to Ca2+. To test this hypothesis, we used caffeine-ryanodine model slices (3). By simultaneously monitoring the [Ca2+]i and contraction, we found that ET induced a sustained contraction of bronchioles and arterioles at constant high [Ca2+]i, strongly suggesting that ET induces Ca2+ sensitization of bronchiole and arteriole SMC contraction.

ET-induced Ca2+ sensitization was particularly important in bronchioles, where a sustained high [Ca2+]i itself did not produce sustained bronchiole contraction, but subsequent stimulation with ET induced the full bronchiole contraction (same magnitude as ET induced in normal slices) but with no change in [Ca2+]i. ET has been shown to induce Ca2+ sensitization of canine (21, 55) and porcine (9) α-toxin-permeabilized tracheal strips. However, in these studies, high [Ca2+]i itself induced a strong airway contraction. This result, although contrary to our observations, could be related to differences in the contractile properties between trachea and bronchial airways or between species. Other possible explanation may related to the loss of intracellular components during the membrane permeabilization with α-toxin. In our model slices, the Ca2+ permeability of the plasma membrane was increased by emptying the Ca2+ stores, but the integrity of the membrane was preserved. A similar absence of bronchiole contraction induced by high [Ca2+]i has been observed in a prior study in our laboratory, and sensitization of bronchiole SMC contraction to Ca2+ was observed with methacholine (3). This study concluded that the lack of a sustained contraction of bronchioles in presence of high [Ca2+]i was the result of a Ca2+-induced relaxation (Ca2+ desensitization) that is antagonized by stimulation with the agonist (Ca2+ sensitization).

In contrast to the bronchioles, high [Ca2+]i within the arterioles produced sustained arteriole contraction in model slices, and the subsequent addition of ET induced an additional arteriole contraction without changes in [Ca2+]i, indicating increased Ca2+ sensitization. ET-induced Ca2+ sensitization was also observed in α-toxin-permeabilized rat pulmonary artery rings (12). Although the mechanism of ET-induced Ca2+ sensitization is beyond the scope this work, a likely explanation for the increase in contraction at a constant [Ca2+]i is an increase in phosphorylation of the myosin light chain (MLC) due to ET-induced inactivation of the MLC phosphatase. In support of this hypothesis, increases in force at constant [Ca2+]i were correlated with increases in MLC phosphorylation in α-toxin-permeabilized rat pulmonary artery rings during ET-stimulation (12) and in canine tracheal strips during ACH stimulation (24).

In conclusion, ET is a powerful agonist in the lung, inducing a potent and sustained contraction of intrapulmonary bronchioles and arterioles. ET-induced contraction is mediated by activation of both ETA and ETB receptors to generate Ca2+ oscillations. The frequency of the Ca2+ oscillations, in combination with Ca2+ sensitization of SMC contraction, induced by ET serves as a mechanism to regulate SMC contraction. This understanding of how Ca2+ signaling regulates SMC contraction is essential to the search for therapeutic approaches to asthma and hypertension.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-71930 (to M. J. Sanderson).

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

REFERENCES

View Abstract