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Human Isolated Bronchial Smooth Muscle Contains Functional Ryanodine/Caffeine–sensitive Ca-Release Channels JEAN-MARC HYVELIN, CÉCILE MARTIN, ETIENNE ROUX, ROGER MARTHAN, and JEAN-PIERRE SAVINEAU Laboratoire de Physiologie Cellulaire Respiratoire INSERM (E 9937), Université Bordeaux 2, Bordeaux, France; and Molecular Endocrinology, Molecular Medicine Center, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom

Human bronchial smooth muscle (HBSM) contraction is implicated in a variety of respiratory diseases, including asthma. Yet, the presence of an operative calcium-induced calcium release (CICR) mechanism, identified in various smooth muscles, has not been established in HBSM. We therefore studied Ca-releasing mechanisms in HBSM obtained at thoracotomy with special attention to ryanodine-sensitive receptor channels (RyRs). In freshly isolated bronchial myocytes, ryanodine (0.5 to 50 ␮M) and caffeine (1 to 25 mM) induced transient increases in the cytoplasmic calcium concentration ([Ca2⫹]i). Higher ryanodine concentrations (⬎ 100 ␮M) inhibited the caffeine-induced [Ca2⫹]i response, which was also blocked in the presence of tetracaine (300 ␮M) or ruthenium red (200 ␮M), two potent CICR inhibitors. In HBSM strips, caffeine induced a transient contraction which, likewise, was inhibited by ryanodine and tetracaine. However, ryanodine (200 ␮M) modified neither the [Ca2⫹]i response nor the contraction induced by K⫹rich (110 mM) solution. Reverse transcriptase/polymerase chain reaction (RT-PCR) and RNase protection assay performed in HBSM have revealed the existence of mRNAs encoding only the type 3 RyR. We also characterized acetylcholine-induced [Ca2⫹]i and contractile responses. None of these responses was altered by ryanodine or by tetracaine. These results demonstrate, for the first time, the existence of functional RyRs in HBSM cells which, owing to the type of isoform or the amount of protein expressed, are not involved, under physiologic conditions, in depolarization- or agonist-induced contraction.

Bronchial smooth muscle is one of the main effectors of bronchial reactivity, and excessive contraction of this muscle is, at least in part, responsible for respiratory diseases such as asthma. Whereas cellular and molecular mechanisms leading to the contraction of striated muscle are now well established (1), such is not the case for smooth muscle. For example, the characterization of extracellular and intracellular sources of activator calcium, as well as the type of intracellular Ca-release channels [i.e., inositol 1,4,5-trisphosphate (InsP3)- and ryanodine-sensitive channel receptor] involved in the contractile response vary from one smooth muscle to another (2). In human bronchial smooth muscle (HBSM), the intracellular source of calcium plays a critical role in contractile response to cholinergic agonists (3) which are very potent bronchoconstrictors (4). InsP3-sensitive calcium store has been clearly identified in cultured HBSM cells (5) and production of InsP3 in response

(Received in original form November 4, 1999 and in revised form February 24, 2000) Supported by grants from “Ministère de L’Environnement” and “Agence De l’Environnement et de la Maîtrise d’Energie” (ADEME) (97034) and “Conseil Régional d’Aquitaine” (960301117 and 980301115). J. M. Hyvelin is an ADEME studentship recipient. Correspondence and requests for reprints should be addressed to R. Marthan, M.D., Ph.D., Laboratoire de Physiologie Cellulaire Respiratoire, INSERM (E 9937), Université Bordeaux, 2, 146, rue Léo Saignat, 33076 Bordeaux, France. E-mail: [email protected] Am J Respir Crit Care Med Vol 162. pp 687–694, 2000 Internet address: www.atsjournals.org

to stimulation by agonists, including acetylcholine (ACh) has been demonstrated (6), indicating that InsP3-induced calcium release (IICR) is a potent physiologic mechanism triggering the human bronchoconstrictor response. In contrast, very little is known about the existence of ryanodine Ca-release channels (RyRs) and the associated calcium-induced calcium release (CICR) mechanism in this muscle. Such a mechanism linking calcium influx through transmembrane Ca channels and/or IICR to additional Ca release from the sarcoplasmic reticulum, plays an important role in the contraction of not only the cardiac but also a variety of smooth muscles (7–9). Whereas, in human bronchial muscle, both the presence of L-type voltage-dependent Ca channels and depolarizationinduced contraction, using K-rich solution, have long been demonstrated (10–12), to the best of our knowledge there is no available information about CICR mechanism in this preparation. This issue, which may be important in the understanding of the physiology and pathophysiology of bronchial responsiveness, has been difficult to investigate so far because, in airway multicellular preparations, the usual pharmacologic tool that activates RyRs, i.e., caffeine, often displays an inhibitory effect on tension (13). We therefore designed the present study to examine the existence and the function of RyRs in isolated HBSM using a combination of molecular biology [reverse transcriptase/polymerase chain reaction (RT-PCR) and ribonuclease (RNase) protection], indo-1 microspectrofluorimetry in single myocytes, and measurement of isometric contraction in multicellular strips. We demonstrate, for the first time, that HBSM cells contain ryanodine- and caffeine-sensitive receptor-channels, which, owing to the type of isoform and/or the amount of protein expressed are involved in neither depolarization-induced nor agonist-induced contraction under physiologic conditions.

METHODS Collection of Human Tissue Human lung was obtained, as previously described (3, 10), at thoracotomy from patients undergoing resection for lung tumor. Specimens were rapidly transferred to the laboratory in cool Krebs-Henseleit (KH) solution (composition in mM: 118.4 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11 D-glucose, pH 7.4). Characteristics of the 29 patients whose lungs were used in the present study are given in Table 1. As in previous studies, specimens were selected from patients whose lung function was within a normal range, i.e., whose forced expiratory volume in one second (FEV1) and total lung capacity (TLC) were more than 80% of predicted. Further, analysis of medical records of the patients revealed that none of them had a history of atopy. Whereas lung mechanics was used as a criterion to select lung specimens, such was not the case for blood gas values (Table 1). We did collect lung specimens from hypoxemic patients on the basis that hypoxemia was often caused by ventilation–perfusion mismatch related to the presence of the tumor. From all of the previously described specimens, segments of bronchi (third and fourth generations, 30 to 40 mm in length, 4 to 5 mm in internal diameter) were

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carefully dissected from surrounding parenchyma in a macroscopically tumor-free part of the lungs.

RNA Isolation Segments of bronchi were opened and the epithelium was carefully scraped. Tissues were then washed to remove all traces of blood, cut into small fragments, directly frozen in liquid nitrogen, and stored at ⫺70⬚ C for later RNA preparation. Total RNA from bronchi was isolated as described (14), resuspended in RNase-free H2O, and stored at ⫺70⬚ C. All samples had intact 18 S and 28 S RNAs as judged by ethidium bromide staining after agarose gel electrophoresis. Human cerebellum and skeletal muscle were handled in the same manner to provide positive control RNA for RT-PCR and RNase protection assay experiments.

Oligonucleotide Primers, Reverse Transcription, and PCR Amplification In the following, ryanodine- and InsP3-receptor genes are abbreviated as ryr and InsP3R, respectively, ryr1-3 and InsP3RI RT-PCR methodology, with nondegenerate sense and antisense primers (Oswel DNA Service, Southampton, UK), has been optimized and previously described (15). Amplified products were analyzed directly by electrophoresis on 1% (wt/vol) agarose gels and, after transfer onto membrane (Hybond-N; Herst Amersham, UK), by Southern hybridization analysis

RNase Protection Assay The quality of the RNA samples was verified by gel electrophoresis before the RNase protection assays (RPA). RPA were performed using the HybSpeed RPA kits (Ambion Inc., Abingdon, UK) according to the manufacturer’s instructions. Human ryr1-3 and InsP3RI partial complementary DNA (cDNA) clones were linearized and transcribed in vitro using radioactive phosphorus–uridine triphosphate ([␣-32P]UTP) (800 Ci/mmol; Amersham). A human actin complementary RNA (cRNA) (pTRI-␤-actin; Ambion) was also synthesized and used as an TABLE 1 CLINICAL CHARACTERISTICS OF STUDY POPULATION Patient No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Histologic type

Sex (M/F)

Age (yr)

Smoking (pack-yr)

FEV1 (%)

sGaw (%)

TLC (%)

PaO2 (mm Hg)

BP SqCC SqCC AdC SqCC AdC AdC AdC BP BP AdC UnC SqCC SqCC BP AdC AdC AdC AdC SqCC AdC SqCC SqCC SqCC SqCC AdC SqCC SqCC AdC

F M M M M M M M M F F M M M M M F M M M M M M M M F M M M

55 58 59 69 65 50 50 68 25 58 77 59 67 65 62 66 50 56 57 74 64 69 48 74 67 57 75 71 63

0 30 60 0 0 45 20 40 14 0 0 0 60 80 80 25 20 25 0 0 75 60 56 30 40 30 25 0 0

108 106 85 100 93 113 99 80 82 113 105 86 82 82 96 80 109 85 80 88 80 81 97 80 92 85 94 97 82

90 156 139 309 198 — 113 100 75 60 151 102 180 114 228 103 176 100 94 95 62 121 389 157 83 111 140 58 53

113 97 86 95 89 119 124 80 97 118 122 103 84 106 88 99 97 111 102 95 126 91 103 114 109 107 87 80 112

87 43 68 64 75 89 105 72 75 85 87 62 67 83 66 83 90 96 62 82 74 77 84 65 63 94 65 75 76

Definition of abbreviations: AdC ⫽ adenocarcinoma; BP ⫽ benign pathology; PaO2 ⫽ arterial oxygen partial pressure; % ⫽ percentage of predicted values; sGaw ⫽ specific airway conductance; SqCC ⫽ squamous cell carcinoma; UnC ⫽ undifferentiated carcinoma.

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internal control for assay variability. Negative controls contained yeast RNA instead of human RNA. After purification by passage over Nick columns (Pharmacia, Herts, UK), cRNA probes (9 ⫻ 105 counts per minute [cpm]) were hybridized to total human bronchus RNA (50 ␮g) and whole brain RNA (25 ␮g) in hybridization buffer (Ambion) for 10 min at 68⬚ C. Free cRNA (nonhybridized) was removed by digestion with 1/25 dilution of a mixture of RNase A (1 mg/ml) and RNase T1 (20,000 units/ml) for 30 min at 37⬚ C. Samples were ethanol-precipitated and resuspended in loading buffer for separation on a 4% or 5% (wt/vol) polyacrylamide gel containing 8 M urea. The gels were dried and exposed to X-ray film (Dupont, Stevenage, UK) at ⫺70⬚ C for up to 2 wk using intensifying screens.

Preparation of Isolated HBSM Cells Bronchial segments were cut longitudinally and, under binocular control, the epithelium was mechanically removed. Epithelium-free muscular strips were cut into several pieces (1 ⫻ 1 mm) and incubated for 15 min in Ca2⫹- and Mg2⫹-free physiologic saline solution (PSS, composition in mM: 130 NaCl, 5.6 KCl, 11.1 D-glucose, 10 Hepes, pH 7.4 with NaOH). The tissue was then incubated in Ca2⫹-free PSS containing 1.0 mg ⭈ ml⫺1 collagenase, 0.7 mg ⭈ ml⫺1 pronase, 0.06 mg ⭈ ml⫺1 elastase, and 3 mg ⭈ ml⫺1 bovine serum albumin at 37⬚ C for two successive periods of 25 min. After this time, the solution was removed and the muscle pieces were incubated again in a fresh enzyme-free solution and triturated with a fire-polished Pasteur pipette to release cells. Cells were stored for 1 to 3 h to attach on coated polymerase-L-lysine glass coverslips at 4⬚ C in PSS containing 0.8 mM Ca2⫹ and used on the same day.

Fluorescence Measurement and Estimation of [Ca2⫹]i Changes in [Ca2⫹]i were monitored fluorimetrically using the Ca2⫹sensitive probe into-1. Freshly isolated cells were loaded with indo-1 by incubation in PSS containing 1.2 ␮M indo-1 penta-acetoxymethyl ester (indo-1-AM), the cell-permeable acetoxy-methylderivative of indo-1, for 25 min at room temperature (20 to 22⬚ C). Coverslips containing indo-1-loaded cells were then washed for 25 min with fresh PSS to remove extracellular indo-1-AM. Coverslips were then mounted in a perfusion chamber and continuously superfused at room temperature. [Ca2⫹]i was estimated from the indo-1 fluorescence ratio using single-wavelength excitation (360 ⫾ 10 nm) and dual emission (405 ⫾ 10 and 480 ⫾ 10 nm). The recording system included a Nikon Diaphot inverted microscope fitted with epifluorescence (Nikon France, Charenton-le-Pont, France). The intensities of transmitted light from a window that was manually adjusted to be slightly larger than the examined cell were simultaneously recorded by two photometers (P100; Nikon), and single photon currents were converted to voltage signals. Signals at each wavelength were digitized and stored on an IBM-PC using a PC-Lab Card 812PG interface (Advantech, Nanterre, France). Sampling was at 17 Hz. The fluorescence ratio (405/480) was calculated on-line and displayed with the two voltage signals on a monitor. [Ca2⫹]i was estimated from the 405/480 ratio (16) using a calibration for indo-1 determined within bronchial smooth muscle cells as previously described (17). Experiments were performed at room temperature.

Application of Agonists Agonists were applied to the recorded cell by pressure ejection from a glass pipette located close to the cell for the period indicated on the records. It was verified, in control experiments, that no change in [Ca2⫹]i was observed during test ejection of PSS. When ruthenium red had to be applied intracellularly, we used the conventional patchclamp technique in the whole-cell current recording mode as previously described (10). Borosilicate patch pipettes of 3 to 7 M⍀ resistance were obtained with a vertical puller (Narishige, Tokyo, Japan). To assess the dynamic changes in [Ca2⫹]i of individual bronchial myocytes, indo-1 was also present in the patch pipette. To ensure that indo-1 and ruthenium red entered correctly into the cell, recording was started a few minutes after establishment of the whole-cell-recording mode. For each of these experiments, at a holding membrane potential of ⫺60 mV, an ACh-elicited macroscopic membrane current was first visualized (not shown) using an IBM-PC computer (pCLAMP system; Axon Instruments Inc., Foster City, CA). Recording of this current ensured that ruthenium red, present in the patch pipette solu-

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Hyvelin, Martin, Roux, et al.: Ca-release Channels in Human Bronchus tion, continuously entered the cell after correct establishment of the whole-cell-recording mode. The patch pipettes were left in place throughout the duration of the experiment. We verified that the presence of the pipette in the absence of ruthenium red did not alter the ACh-induced Ca2⫹ response.

Isometric Contraction Measurement From segments of human bronchi, small epithelium-free strips (approximately 500 ␮m wide, 6 to 8 mm length) were cut transversely and carefully transferred to an experimental chamber described previously (3). Isometric tension was measured using a highly sensitive force transducer (Akers, AE 801; SensoNor, Horten, Norway). The chamber was filled with physiologic solution (composition in mM: 130 NaCl, 5.6 KCl, 1 MgCl2, 2.5 CaCl2, 11 D-glucose buffered by Tris-HCl to pH 7.4 at 30⬚ C). Tissues were set at optimal length by equilibration against a passive load of 40 mg as determined previously for this type of preparation (18). At the beginning of each experiment, K⫹-rich (110 mM) solution, was repeatedly applied to obtain at least two contractions similar in both amplitude and kinetics. This contraction served as a reference response that was used to normalize subsequent contractile responses.

Chemicals and Drugs Collagenase (type CLS1) was from Worthington Biochemical Corp. (Freehold, NJ). Pronase (type E), elastase (type 3), bovine serum albumin, ACh, caffeine, neomycin, papaverine, ryanodine, tetracaine, ruthenium red, and thapsigargin were purchased from Sigma (Saint Quentin Fallavier, France). Indo-1-AM was from Calbiochem (France Biochem, Meudon, France). Indo-1-AM and ryanodine were dissolved in dimethyl sulfoxide (DMSO). the maximal concentration of DMSO used in our experiments was less than 0.1% and had no effect per se on either contraction or increase in [Ca2⫹]i induced by agonists (data not shown).

Data Analysis and Statistics

tion. InsP3RI RT-PCR amplification products were easily detectable on ethidium bromide–stained agarose gels, suggesting that the InsP3RI gene is highly expressed in human bronchus (Figure 1A). Furthermore, no major difference in the intensity of the 476 bp product was observed between the different bronchus samples studied, and the level of expression of InsP3RI mRNA was comparable between human cerebellum and human bronchus. Primers specific for each ryr subtype were used for RT-PCR on total RNA from human bronchus. Although ryr1 and ryr2 mRNA amplification products from human skeletal muscle or cerebellar RNA were easily detected on ethidium bromide–stained agarose gels, no products were detected from human bronchus even after Southern blotting (Figure 1A). ryr3 mRNA were detected in human bronchus, but at a much lower level than in human cerebellum (Figure 1A). Therefore, ryr3 mRNA is the only ryr mRNA isoform expressed in human bronchus. Moreover, RNA probes complementary to InsP3RI mRNA and ryr3 mRNA were used in an RNase protection assay together with a human ␤-actin cRNA to control for assay variability. Both InsP3RI mRNA and ryr3 mRNA can be detected by this method, but considerable variation in the level of expression of ryr3 mRNA was seen between bronchus samples (Figure 1B), possibly dependent on the amount of smooth muscle and non–smooth muscle tissues (connective and adipose tissues) present in the sample. RPA showed that the amount of InsP3RI and ryr3 mRNAs detected in human bronchus was at a much lower level than in human cerebellum. Effect of Ryanodine and Caffeine on [Ca2⫹]i in Freshly Isolated Myocytes

To assess the function of these RyRs in human bronchus, we examined the effect of ryanodine and caffeine, two compounds

Results of [Ca2⫹]i are expressed as the mean ⫾ SEM with n the number of cells originating from different lung specimens. In each pulmonary specimen, a mean value for both control and experimental conditions was calculated to be representative of that pulmonary specimen. Each experiment was replicated in 3 to 6 different lung specimens and statistical comparisons were carried out using two-tailed Student’s paired t-tests. Results were considered significant at p ⬍ 0.05. When concentration–response curves were constructed, the mean [Ca2⫹]i values calculated on n cells for the various concentration of agonists were fitted by a sigmoidal Boltzmann’s equation and a mean EC50 (the concentration of drug giving the half-maximal response) was derived. In contraction experiments, the contractile response of each muscle strip was expressed as a percentage of the control contraction evoked by K⫹-rich (110 mM) solution in the same strip. For each pulmonary specimen 2 to 3 muscular strips were studied and the mean values of contractile responses were calculated to be representative of that pulmonary specimen. Each experiment was replicated on 2 to 4 different human lungs, and statistical comparisons were carried out using Student’s paired t tests. For the noncumulative concentration–response curve to caffeine, the mean contractile values calculated on n muscle strips for the various concentrations of caffeine were fitted by a sigmoidal Boltzmann’s equation. Because experiments were performed pairwise, statistical comparisons of paired mean cumulative concentration response curve (CCRC) were made using an analysis of variance (two-way analysis of variance).

RESULTS ryr Messenger RNA (mRNA) Subtypes in Human Bronchus

The presence of mRNA encoding ryr receptor subtypes in human bronchus total RNA was determined by RT-PCR. Although not quantitative, this technique gave a clear indication about the presence of the mRNA in the sample tested. Total RNA from human cerebellum [which expressed all of the receptors (15)] and skeletal muscle were used as a positive control for the PCR reaction. RT-PCR of InsP3RI mRNA was used to validate the technique and to test the quality of the RNA prepara-

Figure 1. (A) RT-PCR analysis of InsP3RI and ryr1-3 isoforms in human bronchus samples. The InsP3RI and ryr3 products were detected in human bronchus (lanes 1–3) and in positive control, i.e., human cerebellum (lane 4). Products were only visible for the positive control for ryr1 (human skeletal muscle) and for ryr2 (human cerebellum). No product was detected in a PCR reaction containing no RNA (lane 5). RNA samples without reverse transcriptase were used to check for genomic DNA contamination (results not shown). The nucleic acid markers are indicated (M). (B) Autoradiographs of RNase protection analysis of InsP3RI and ryr3 mRNA expression in three different samples of human bronchus RNA (50 ␮g). 50 ␮g of yeast total RNA was used as a negative control, and human cerebellum RNA was used as a positive control (results not shown). The autoradiographs were exposed for approximately 2 h at room temperature for ␤-actin, and for 7 d at ⫺70⬚ C for InsP3RI and ryr3.

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Figure 2. Effect of ryanodine on cytosolic Ca2⫹ concentration ([Ca2⫹]i) in freshly isolated HBSM cells. (A) Typical [Ca2⫹]i responses revoked by short (30-s) ejection of ryanodine (0.5 to 10 ␮M) near the cell. (B) Relationship between ryanodine concentration and the amplitude of the [Ca2⫹]i peak (a) or the percentage of responding cells (b). In (a) each symbol represents the mean value of [Ca2⫹]i rise calculated from 15 to 30 cells; vertical lines indicate standard error of the mean (SEM).

known to open these channels in various tissues. In freshly isolated human bronchial myocytes, the mean resting concentration of [Ca2⫹]i was 92 ⫾ 7 nM (n ⫽ 180). Short (20-s) microejection of ryanodine induced a concentration-dependent transient rise in [Ca2⫹]i (Figure 2A). The concentration–response curve exhibited a bell shape (Figure 2B). Between 0.5 and 10 ␮M ryanodine, the amplitude of the peak (i.e., the maximal increase in [Ca2⫹]i above baseline), as well as the percentage of responding cells gradually increased to reach a maximal value for 10 ␮M (168 ⫾ 28 nM and 68%, respectively, n ⫽ 30). For higher ryanodine concentrations, both the amplitude of peak and the percentage of responding cells decreased and no response was observed for a concentration equal or above 200 ␮M ryanodine (Figure 2B). Caffeine (0.01 to 25 mM) induced a concentration-dependent transient rise in [Ca2⫹]i (Figure 3A). Both the amplitude of the peak, and the percentage of responding cells gradually increased with the caffeine concentration and were maximal for 10 mM (300 ⫾ 67 nM, 96%, n ⫽ 50) and remained consistent for higher concentrations (Figure 3B). The EC50 for the caffeine-induced [Ca2⫹]i was 1.02 mM. Removal of external Ca2⫹ ions in the presence of 0.4 mM ethyleneglycol-bis-(␤-aminoethyl ether)-N,N⬘tetraacetic acid (EGTA) for 15 min induced a slight decrease in resting [Ca2⫹]i (85 ⫾ 6 nM, n ⫽ 80), but did not alter the [Ca2⫹]i response to caffeine (10 mM) (275 ⫾ 42 nM versus 296 ⫾ 56 nM in the absence and in the presence of extracellular Ca2⫹, respectively, n ⫽ 50; p ⬎ 0.05) (Figure 3Ba). In contrast, pretreatment of myocytes with 1 ␮M thapsigargin, a specific inhibitor of the sarcoplasmic reticulum Ca2⫹ pump (SERCA) (19), including in airway smooth muscle (20), fully abolished the caffeine (10 mM)-induced [Ca2⫹]i transient response (Figure 3C). Effect of Modulators of Signal Transduction Pathways on the Caffeine-induced [Ca2⫹]i Response

To further characterize the caffeine-induced [Ca2⫹]i response, we examined the effect of a variety of substance known to

Figure 3. Effect of caffeine (CAF) on [Ca2⫹]i in HBSM cells. (A) Typical [Ca2⫹]i responses evoked by short (30-s) ejection of CAF (0.01 to 25 mM). (B) Relationship between [CAF] (abscissa scale) and both the amplitude of the [Ca2⫹]i response in the presence (filled squares) and in the absence (open circles) of extracellular Ca2⫹ (a), and the percentage of responding cells (b). In (a) each symbol represents the mean value of [Ca2⫹]i rise calculated from 20 to 50 cells; vertical lines indicate SEM. (C) Effect of thapsigargin (TG) on the CAF-induced [Ca2⫹]i response. Time in parentheses indicates the duration of exposure to TG before CAF ejection. Each trace was recorded from different cells and is typical of approximately 20 cells.

interfere with signaling pathways leading to calcium release from intracellular stores. Caffeine-induced [Ca2⫹]i response was not altered in the presence of neomycin (1 ␮M), an inhibitor of phosphoinositide–phospholipase C (21) (300 ⫾ 10 nM versus 322 ⫾ 15 nM in control cells, n ⫽ 20; p ⬎ 0.05) (Figure 4A). In contrast, pretreatment of human bronchial myocytes with tetracaine (200 ␮M, n ⫽ 30), ryanodine (200 ␮M, n ⫽ 20), or ruthenium red (200 ␮M, applied intracellularly via a patch pipette [see METHODS], n ⫽ 10)—three potent inhibitors of the CICR mechanism (22)—inhibited the caffeine (10 nM)-induced [Ca2⫹]i response (Figure 4B). Characteristics of ACh-induced [Ca2⫹]i Response

We then compared the RyR-dependent [Ca2⫹]i response to that obtained with a membrane receptor agonist known to activate the InsP3-dependent pathway in human bronchus, i.e., ACh. Stimulation of isolated bronchial myocytes by ACh caused a rapid increase in [Ca2⫹]i. Generally, the peak was followed by a plateau phase which remained above baseline values as long as the stimulation persisted (Figure 5A). Amplitude of the peak and of the plateau phase, as well as the percentage of cells that responded, increased with increasing ACh concentration (0.01 to 10 ␮M) (Figure 5B). For 10 ␮M ACh, the [Ca2⫹]i peak and the percentage of responding cells reached a maximal value: 355 ⫾

Hyvelin, Martin, Roux, et al.: Ca-release Channels in Human Bronchus

Figure 4. Effect of neomycin (Ab), tetracaine (Bb), ryanodine (Bc), and ruthenium red (Bd) on the CAF-induced [Ca2⫹]i response. In A–B the first record (a) is the control response obtained in the absence of drugs. Time in parentheses indicates the duration of exposure to drugs before CAF ejection. Each trace was recorded from different cells and is typical of 10 to 30 cells. Ruthenium red was applied intracellularly using a patch pipette.

49 nM and 95%, respectively (n ⫽ 90) (Figure 5B). The EC50 for ACh-induced [Ca2⫹]i rise in responding cells was 0.18 ␮M. Removal of extracellular Ca2⫹ for 15 min did not significantly alter the pattern and the amplitude of the response to either a low (0.1 ␮M) or a high (10 ␮M) ACh concentration (101 ⫾ 10 nM and 322 ⫾ 42 nM in Ca2⫹-free solution versus 103 ⫾ 13 nM and 341 ⫾ 30 nM in Ca2⫹-containing solution for 0.1 ␮M ACh [n ⫽ 50] and 10 ␮M ACh [n ⫽ 60], respectively, p ⬎ 0.05). Pretreatment of the cells with thapsigargin (1 ␮M) for 15 min suppressed the ACh-induced [Ca2⫹]i response (n ⫽ 45) (not shown). However, in contrast to the caffeine-induced [Ca2⫹]i response, the ACh-induced response was progressively suppressed by neomycin (1 ␮M, n ⫽ 40) (Figure 6A), and not altered by tetracaine (300 ␮M) (267 ⫾ 16 nM versus 282 ⫾ 16 nM, in the presence and in the absence of tetracaine, respectively, n ⫽ 45, p ⬎ 0.05), ryanodine (200 ␮M) (269 ⫾ 15 nM versus 283 ⫾ 10 nM, in the presence and in the absence of ryanodine, respectively, n ⫽ 20, p ⬎ 0.05), or ruthenium red (200 ␮M, applied intracellularly via a patch pipette) (340 ⫾ 32 nM versus 326 ⫾ 26 nM, in the presence and in the absence of ruthenium red, respectively, n ⫽ 10, p ⬎ 0.05) (Figure 6B). Role of RyRs in Excitation–contraction Coupling in Human Airway Muscle

Both molecular biology results and microspectrofluorimetry experiments revealed the existence of functional RyRs in this muscle. We then tried to examine the involvement of CICR mechanism in excitation–contraction (E-C) coupling triggered by either the depolarizing agent KCl (electromechanical coupling) or ACh, an activator of the pharmacomechanical coupling. In isolated cells, short (30 s) microejection of KCl (60 to 110 mM) near freshly isolated cells, induced a concentrationdependent transient rise of [Ca2⫹]i (110 ⫾ 4 nM to KCl 60 mM, n ⫽ 30, and 224 ⫾ 15 nM to KCl 110 mM, n ⫽ 30) (Figures 7A and 7B). Incubation of the cells with 1 ␮M verapamil, a potent inhibitor of voltage-dependent Ca2⫹ channels, abolished the

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Figure 5. Effect of ACh on the [Ca2⫹]i in freshly isolated HBSM cells. (A) Typical [Ca2⫹]i responses evoked by short (30-s) ACh (0.01 to 10 ␮M) stimulation. Each trace was recorded from different cells and is representative of 30 to 90 cells. (B) Relationship between the ACh concentration and both the amplitude of [Ca2⫹]i rise (A) and the percentage of responding cell (b). In (a) each symbol represents the mean value of [Ca2⫹]i rise calculated from 30 to 90 cells; vertical lines indicate SEM.

KCl (110 mM)-induced [Ca2⫹]i response (Figure 7B). As expected, pretreatment of cells with 1 ␮M thapsigargin had no effect on the KCl-induced [Ca2⫹]i response (225 ⫾ 20 nM, n ⫽ 20, Figure 7C). However, as for ACh (Figure 6Bc), 200 ␮M ryanodine had no effect on the KCl-induced [Ca2⫹]i response (195 ⫾ 22 nM, n ⫽ 20) (Figure 7C). In multicellular strips, we first determined the experimental conditions required to investigate the calcium-dependent contractile effect of caffeine. Caffeine (1 to 40 mM) evoked a biphasic mechanical response consisting of a transient contraction followed by relaxation (Figure 8A). The amplitude of both the peak of contraction and that of relaxation was concentration-dependent and reached a maximal value in response to 20 mM caffeine (34.9 ⫾ 4.7% and ⫺22.6 ⫾ 5% of the maximal K⫹-rich [110 mM]induced contractile response, n ⫽ 12). After the washout of caffeine, baseline tension was progressively restored. In the presence of papaverine (100 ␮M), a potent smooth muscle relaxant (23), the baseline tension decreased to a level similar to that previously reached with the maximal caffeine concentration (⫺24.4 ⫾ 5.6% versus ⫺22.6 ⫾ 5%, n ⫽ 8, p ⬎ 0.05) (Figure 8Ab). Under these conditions, caffeine (1 to 40 mM) induced only a transient contraction the amplitude of which was slightly enhanced (48.6 ⫾ 5.8% versus 34.9 ⫾ 4.7%, n ⫽ 6, p ⬎ 0.05) (Figure 8Ac). We then examined the effect of CICR inhibitors, high ryanodine concentration (⬎ 50 ␮M), and tetracaine (300 ␮M) on the human isolated bronchial strip contraction. Whereas ryanodine fully abolished the caffeine-induced contraction (n ⫽ 5), it altered neither the KCl (60 –110 mM)-induced nor the ACh (0.01 – 10 ␮M)-induced contraction (n ⫽ 5, Figure 8B). Likewise, pretreatment of the strip with tetracaine (300 ␮M) fully abolished

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Figure 6. Effect of neomycin (A), tetracaine (Bb), ryanodine (Bc), and ruthenium red (Bd) on the ACh-induced [Ca2⫹]i response. In A–B the first record (a) is the control response obtained in the absence of drugs. In Bd ruthenium red was directly applied into the cell using a patch pipette. Time in parentheses indicates the duration of exposure to drugs before muscarinic stimulation. Each trace was obtained from different cells and is typical of 10 to 45 cells.

the caffeine-induced contraction (n ⫽ 10), but was without effect on both the amplitude and the kinetics of the ACh (0.01–10 ␮M)induced contraction (n ⫽ 6) (Figure 8C).

DISCUSSION To the best of our knowledge, this is the first study that has examined the distribution and function of RyRs in HBSM. The main result of our study is that, although RyRs are expressed and functional in HBSM, they are not implicated in E-C coupling of this muscle under physiologic conditions. The lack of operative CICR mechanism could be ascribed to the distribution of RyRs isoforms in the human bronchus in which only the type 3 RyR is expressed and/or the amount of protein expressed. Ryanodine- and Caffeine-sensitive Ca-release Channels

The measurement of both [Ca⫹]i and mechanical activity in human isolated bronchus has demonstrated the presence of functional Ca-release from internal store activated by ryanodine and caffeine. The bell shape of the concentration-[Ca2⫹]i– response curve for ryanodine (Figure 1B) is consistent with the known action of ryanodine on its receptor channel protein in other tissues including smooth muscles: i.e., activation of RyR leading the channel in a semi-opened state for low ryanodine concentrations, and inhibition of RyR impairing the opening of the channel for high ryanodine concentrations (24). Moreover, the fact that the caffeine-induced [Ca2⫹]i re-

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Figure 7. Effect of K⫹-rich solutions on the [Ca2⫹]i in HBSM cells. (A) Typical [Ca2⫹]i responses evoked by short (30-s) stimulation with K⫹rich (60 to 110 mM) solutions. Each trace was recorded from different cells and is typical of 30 cells. (B) Relationship between K⫹-rich solution and amplitude of the [Ca2⫹]i rise in the absence or in the presence of verapamil (1 ␮M). (C) Effect of thapsigargin (TG, 1 ␮M) and ryanodine (200 ␮M) on the amplitude of the K⫹-rich (110 mM) solution-induced [Ca2⫹]i response. Each column represents the mean value of [Ca2⫹]i rise calculated from 20 cells; vertical lines indicate SEM.

sponse (1) remained unaltered in absence of external Ca2⫹ but disappeared in the presence of thapsigargin, and (2) was not modified by neomycin but was blocked in the presence of high ryanodine concentration and in the presence of tetracaine and ruthenium red, two other substances known to interact with RyR (22) further supports the idea that, upon appropriate stimulation, RyR could release a substantial amount of Ca2⫹ in these cells. Finally, the observation that caffeine-induced contraction was inhibited by high ryanodine concentration and tetracaine demonstrates that HBSM cells contain a ryanodine-sensitive Ca-release mechanism able to provide sufficient calcium ions to activate the contractile apparatus. In this connection, the amplitude of [Ca2⫹]i peak induced by ryanodine or caffeine is close to that required to activate the contractile apparatus as determined from the Ca2⫹ concentration–response relationship that we have previously characterized in human permeabilized bronchial smooth muscle (25). A recent work of Iizuka and colleagues (26) has also shown caffeine-induced contraction in permeabilized human bronchial strips. Interestingly, as in a variety of smooth muscle including airways (13, 18), caffeine displayed a biphasic action (contraction and relaxation) in the human isolated bronchial muscle. This action can be ascribed to the methylxanthine-related inhibitory effect of caffeine on cyclic nucleotide phosphodiesterases. This hypothesis is supported by the following lines of evidence: (1) papaverine, another potent inhibitor of cyclic nucleotide phosphodiesterases (23), also relaxes human bronchial strips and impairs the subsequent relaxing effect of caffeine; (2) both adenosine 3⬘,-5⬘-cyclic monophosphate (cyclic AMP) and cyclic guanosine 3⬘,5⬘-monophosphate (cyclic GMP) antagonize Ca-activated contraction in permeabilized human bronchial smooth muscle (25).

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feine, i.e., via another type of Ca release channel: the InsP3receptor. In this connection, it has been previously demonstrated that ACh induces production of InsP3 in human bronchial smooth muscle (6) and we have observed the expression of mRNA for InsP3RI in human bronchus (Figure 1). Implication of RyRs in the E-C Coupling of the Human Isolated Bronchus

Figure 8. Role of RyRs in contraction of isolated HBSM. (A) Caffeine (CAF, 5 to 20 mM) induced a dual effect (contraction followed by relaxation) in bronchial strips (a). After pretreatment of the strip with papaverine (100 ␮M), CAF (5 to 40 mM) induced only a contractile effect (b). Concentration–response curves for the contractile effect of CAF in the absence (䊏) and in the presence of papaverine (O) (c). Each data point is the mean ⫾ SE of 6 to 12 experiments. (B) Ryanodine (50 ␮M) fully abolished CAF (40 mM)-induced contraction but had no effect on KCl (60 –110 mM)-induced and acetylcholine (ACh, 0.1–10 ␮M)-induced contraction. (C) Tetracaine (300 ␮M) fully abolished CAF-induced contraction but had no effect on ACh-induced contraction.

ACh-sensitive Ca-release Mechanism

Similar to the ryanodine- or caffeine-sensitive Ca release mechanism, we have also observed an ACh-sensitive Ca-release mechanism in human isolated bronchial smooth muscle. In the present study, ACh-induced [Ca2⫹]i response appears essentially due to a release from an internal store because it was not modified in the absence of external Ca but was inhibited by thapsigargin. This observation is in agreement with previous studies showing that the contractile response to ACh in human bronchus is primarily dependent on internal calcium (3). Moreover, the concentration–response curve for ACh effect on both the rise in [Ca2⫹]i and the percentage of responding cells (Figure 4B) is in close agreement with the concentration– response curve for ACh-induced contraction (3). However, in contrast to the caffeine-induced [Ca2⫹]i response, the AChinduced [Ca2⫹]i response was not modified in the additional presence of ryanodine, tetracaine, or intracellularly applied ruthenium red, but was inhibited after pretreatment of cells with neomycin, a potent inhibitor of phosphoinositide–phospholipase C (21). Moreover, ACh-induced contraction was not impaired by ryanodine or tetracaine. These functional results strongly suggest that ACh releases Ca2⫹ from an internal store via a mechanism different from that activated by caf-

In striated as well as in a variety of smooth muscles, RyRs are directly implicated in E-C coupling. In human bronchus, we have examined the involvement of RyRs in E-C coupling triggered by the depolarizing agent KCl (electromechanical coupling), or ACh (pharmacomechanical coupling). KCl-induced [Ca2⫹]i and contractile responses were not significantly modified by high concentrations of ryanodine, a compound which block the caffeine-induced response. Similarly, ACh-induced [Ca2⫹]i and contractile response were not altered by the CICR inhibitors, ryanodine and tetracaine, in agreement with the findings of Wang and Kotlikoff (27) who showed that ryanodine blockade does not alter cholinergic-induced calcium and current response. These results suggest that, under physiologic conditions, RyRs are involved neither in depolarizationinduced nor in agonist-induced contractile response of the human bronchial muscle. Our findings can be related to that of a recent study showing that two novel endogenous Ca releasers, i.e., adenosine diphosphate ribose (cADPR) and adenine dinucleotide phosphate (NAADP), presumably acting at the level of type 2 RyR (22) do not alter the carbachol-induced contraction in permeabilized human isolated bronchial smooth muscle (26). The issue of RyR isoform distribution in HBSM should thus be addressed. In the present study, using RT-PCR and RNase protection assays we have demonstrated, for the first time in HBSM, the presence of ryr mRNA. However, in contrast to the situation in human cerebellum and skeletal muscle, only the ryr3 mRNA isoform is expressed in the bronchus. This is an original feature of HBSM because alternative distributions of RyRs have been characterized in a number of other animal smooth muscles, i.e., porcine trachea, rat myometrium, rat vessels (9, 28, 29) or even human smooth muscles, i.e., bladder (30). This specificity in the isoform distribution may have important functional consequences. Whereas type 1 and type 2 RyR have been clearly implicated in E-C coupling in skeletal and cardiac muscle, respectively (1), the function of type 3 RyR, which is present at a low level in a variety of tissues, is still poorly understood. In particular, studies using transgenic mice homologous for a targeted disruption of ryr gene have shown that, whereas mutation lacking RyR2 is lethal (31) and mutation lacking RyR1 lost normal E-C coupling in skeletal muscle (32), mutation lacking RyR3 is accompanied by no major physiologic abnormality (33). Differences in structure [RyR3 is devoid of the amino acid sequence 1303–1406, which is critical for the E-C coupling in striated muscles (34, 35)] or in the level of expression between RyRs isoforms could explain the apparent lack of physiologic role of type 3 RyR. In skeletal muscle where RyR3 is coexpressed with RyR1, only RyR1 participates in normal E-C coupling, and RyR3 could represent a Ca-release channel with a higher threshold (36). Nevertheless, it has been suggested that RyR3 may have a role in the normal morphogenesis of this muscle (37). Whereas we observed that, in the adult normal bronchial smooth muscle, the RyR isoform expressed is type 3 and consistently is not involved in E-C coupling, we believe that expression and function of RyRs may vary according to the site along the tracheobronchial tree and to the disease status. For example, in the porcine trachealis, it has been shown that RyRs are necessary

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to maintain ACh-induced Ca oscillations (9). This functional role of RyRs was associated with the expression of the three isoforms in this muscle (9). Whether the difference between this latter study and ours is to be ascribed to site or species differences, or to both, remains to be established. More importantly, in cardiac muscle including from human, it has been shown that RyR expression changes during development of cardiac hypertrophy (38). Hypertrophy and hyperplasia of bronchial smooth muscle are observed in asthma-induced airway remodeling (39). It is therefore important to determine if a change in RyR expression and function could contribute to bronchial hyperresponsiveness in asthma or immunologic sensitization (40). Acknowledgment : The authors are grateful to Dr. R. H. Ashley (Membrane Biology Group, University of Edinburgh) for fruitful discussions about Carelease channels, and to Dr. K. E. Chapman (Molecular Endocrinology, University of Edinburgh) for helpful advice and discussions regarding molecular biology techniques.

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