Selective restoration of calcium coupling to muscarinic M3 receptors in contractile cultured airway myocytes

Richard W. Mitchell, Andrew J. Halayko, Sibel Kahraman, Julian Solway, and Mark E. Wylam

Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, Illinois 60637; Department of Pediatrics, Mayo Clinic, Rochester, Minnesota 55905; and Department of Anesthesiology and Reanimation, Hacettepe University Medical School, Ankara, Turkey


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We previously demonstrated that after several days of serum deprivation about one-sixth of confluent cultured canine tracheal myocytes acquire an elongated, structurally and functionally contractile phenotype. These myocytes demonstrated significant shortening on ACh exposure. To evaluate the mechanism by which these myocytes acquire responsiveness to ACh, we assessed receptor-Ca2+ coupling using fura 2-AM fluorescence imaging and muscarinic receptor expression using Western analysis. Cells were grown to confluence in 10% fetal bovine serum and then maintained for 7-13 days in serum-free medium. A fraction of serum-deprived cells exhibited reproducible intracellular Ca2+ mobilization in response to ACh that was uniformly absent from airway myocytes before serum deprivation. The Ca2+ response to 10-4 M ACh was ablated by inositol 1,4,5-trisphosphate (IP3) receptor blockade using 10-6 M xestospongin C but not by removal of extracellular Ca2+. Also, 10-7 M atropine or 10-7 M 4-diphenylacetoxy-N-methylpiperidine completely blocked the response to ACh, but intracellular Ca2+ mobilization was not ablated by 10-6 M pirenzepine or 10-6 M methoctramine. In contrast, 10-5 M bradykinin (BK) was without effect in these ACh-responsive myocytes. Interestingly, myocytes that did not respond to ACh demonstrated robust increases in intracellular Ca2+ on exposure to 10-5 M BK that were blocked by removal of extracellular Ca2+ and were only modestly affected by IP3 receptor blockade. Serum deprivation increased the abundance of M3 receptor protein and of BK2 receptor protein by two- to threefold in whole cell lysates within 2 days of serum deprivation, whereas M2 receptor protein fell by >75%. An increase in M3 receptor abundance and restoration of M3 receptor-mediated Ca2+ mobilization occur concomitant with reacquisition of a contractile phenotype during prolonged serum deprivation. These data demonstrate plasticity in muscarinic surface receptor expression and function in a subpopulation of airway myocytes that show mutually exclusive physiological and pharmacological diversity with other cells in the same culture.

canine tracheal smooth muscle; fura 2-acetoxymethyl ester; acetylcholine; bradykinin; airway remodeling


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ABOUT ONE-SIXTH OF CANINE TRACHEAL smooth muscle cells in culture acquire a structurally and functionally contractile phenotype after prolonged (>7-day) serum deprivation as recently reported by us (2) and by Ma et al. (11). These contractile myocytes are distinguished by their elongated, wormlike morphology; their abundant cables of myofilaments replete with smooth muscle alpha -actin, smooth muscle myosin, and SM22; their expression of immunoreactive surface muscarinic M3 receptors; and their high-velocity shortening in response to electrical field stimulation or acetylcholine (ACh) exposure. The remaining myocytes instead become flattened and circular, express little contractile protein, and do not contract in response to agonists (2, 11).

In this study, we sought to identify the mechanism through which serum-deprived cultured airway myocytes reacquire contractile responsiveness to ACh. Specifically, we tested the hypotheses that the reacquisition of ACh responsiveness is associated with the restoration of ACh coupling to intracellular Ca2+ mobilization and increased abundance of muscarinic M3 receptor protein. In our previous study, immunocytochemistry suggested that muscarinic M3 receptors are present in a perinuclear distribution within confluent canine tracheal myocytes before serum deprivation, even though they do not contract in response to ACh stimulation (2). Myocytes that became elongated during serum deprivation showed greater immunoreactivity for muscarinic M3 receptors, and these receptors appeared localized in punctate clusters at the cell surface. Thus it is uncertain whether the restoration of ACh-induced contraction in this subpopulation of serum-deprived cells is associated with greater abundance of M3 receptors, improved coupling of the receptor to Ca2+ mobilization, or both. To address these questions and the signaling pathways through which ACh elevates intracellular Ca2+ concentration ([Ca2+]i) in these cells, we evaluated responses to ACh or bradykinin (BK) in confluent canine tracheal smooth muscle cultures using the Ca2+-sensitive dye fura 2-acetoxymethyl ester (AM) before and after 7-13 days of serum deprivation and quantified muscarinic and BK receptor abundance using Western analysis.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Canine tracheal smooth muscle primary cultures. Tracheae were obtained from adult mongrel dogs, and primary airway smooth muscle cell cultures were established as described previously (2). Briefly, cleaned tracheal muscle was obtained by dissection and minced with scissors. Myocytes were enzymatically dispersed for 60 min at 37°C in buffered saline containing 600 U/ml collagenase, 10 U/ml elastase, and 2 U/ml Nagarse protease. Isolated cells were seeded on uncoated glass coverslips or in uncoated plastic culture plates at a density of 5-10 × 103 cells/cm2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids (NEAA), 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were grown at 37°C in a humidified incubator under 5% CO2. For passage of cultures at confluence, cells were lifted using 0.05% trypsin and 0.5 mM EDTA and reseeded into three new culture plates per confluent dish. Cells from passage 1 or 2 were used in the studies described.

To induce the contractile phenotype, cultured myocytes were grown to confluence and then serum-containing growth medium was replaced with serum-free Ham's F-12 medium supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 0.1 mM NEAA, 50 U/ml penicillin, and 50 µg/ml streptomycin. Fresh serum-free medium was provided every 48-72 h thereafter.

Measurement of intracellular free Ca2+ responses. To assess calcium responses, cells were incubated in buffered saline, pH 7.4 (in mM: 130 NaCl, 5 KCl, 1 CaCl2, 1 MgSO4, 10 HEPES, and 10 dextrose), with 0.1% BSA containing 5 µM fura 2-AM for 30 min at room temperature. Cells were then washed twice with fresh buffer and incubated for another 30 min to allow for complete hydrolysis of the ester. The coverslip was then transferred to an inverted Nikon Diaphot microscope equipped with a Princeton Instruments RTE/CCD 12-bit digital camera (Trenton, NJ) controlled through a PC workstation. A Nikon ×20/0.75 objective lens was used, and image size was set to 720 × 540 pixels. At this setting, pixel intensities ranged between 25 and 2,500 gray levels. A fluorescent light excited the cells alternately at 340 and 380 nm with a Lambda 10 filter wheel (Sutter Instruments; Novato, CA). Emitted fluorescence (510 nm) was acquired for 300 ms at each excitation wavelength and used to calculate calcium concentrations (in nM) at each pixel from an in vitro calibration curve of known free Ca2+ (0-1.35 µM) and pentapotassium fura 2 (50 µM) (17). Calcium responses within individual cells were determined using Metamorph software (Jandel Scientific; Santa Barbara, CA) by circumscribing single myocytes and spatially averaging fura 2 fluorescence within the borders of each cell. Intracellular free Ca2+ was recorded for 20 s to establish a baseline and for at least 220 s to characterize peak and plateau responses after addition of either ACh or BK.

Coverslips were placed in a modified Sykes-Moore chamber (with a large glass coverslip forming the bottom of the open chamber) containing 400 µl of BSA-containing HEPES buffer (maximum volume 1 ml). For the addition of agonists, an equal volume (400 µl) of buffer containing twice the target concentration was pipetted into the chamber to ensure rapid mixing for the whole volume. For antagonists, cells were equilibrated with buffer containing the receptor blocker for at least 10 min before addition of agonist.

Intracellular free Ca2+ responses to ACh and BK. Two coverslips of tracheal myocytes were prepared as described in Measurement of intracellular free Ca2+ responses. Cells were exposed to either ACh (10-4 M final concentration) or BK (10-5 M final concentration) in random order, and responses of individual cells were recorded. Myocytes were then washed with three volumes of buffer. After 5 min of equilibration, basal [Ca2+]i again was recorded, the other agonist was added to the chamber, and [Ca2+]i response was measured. ACh and BK responses were compared in up to 50 individual cells per coverslip.

Assessment of long axis-to-short axis ratio in ACh- and BK-responsive tracheal myocytes. Cells were grown to confluence and serum-deprived as described in Measurement of free Ca2+ responses. On day 12 of serum deprivation, myocytes were loaded with fura 2-AM and peak [Ca2+]i responses to ACh or BK exposure were recorded as described. Three coverslips of cells were exposed to 10-7 M BK and 10-6 M ACh sequentially, and Ca2+ mobilization was recorded. Care was taken not to disturb the visual field between agonist exposures as BK was washed out from the chamber. Using Metamorph, we subtracted the baseline 380-nm image from the 380-nm exposure obtained at peak agonist response to yield images that reflect peak calcium responses. Peak responses to BK or to ACh stimulation were pseudocolored green and red, respectively, to allow for identification of BK-responsive, ACh-responsive, or dually responsive myocytes. Using Metamorph software, we measured the long axis and the perpendicular short axis at the widest point (usually at or near the midpoint of the long axis of each cell) of 15 individual ACh-responsive and 15 individual BK-responsive myocytes on each of these three coverslips (45 myocytes per agonist total) and calculated the long axis-to-short axis ratio as an index of cell elongation. Areas of overlap of red and green images appeared yellow when the two peak response images from an individual coverslip were superimposed.

Reproducibility of calcium mobilization to ACh. Coverslips of tracheal myocytes were prepared as described in Measurement of intracellular free Ca2+ responses; ACh (10-4 M final concentration) was added to the chamber and responses of individual cells were recorded. Myocytes that mobilized calcium in response to ACh were noted, and the circumference of each responsive myocyte was demarcated using Metamorph software. Cells were then washed with three volumes of buffer. After 5 min of equilibration, basal [Ca2+]i again was recorded, ACh (10-4 M final concentration) again was added to the chamber, and the [Ca2+]i response was measured. First and second responses of individual ACh-responsive myocytes were compared.

Assessment of heterogeneity of [Ca2+]i responses to ACh among individual myocytes. Basal (preagonist), peak, and plateau (postagonist) [Ca2+]i responses to dual ACh exposures were recorded. Unlike the reproducibility study, two different concentrations of ACh were employed during these measurements. First, myocytes were exposed to ACh at a final concentration ranging from 10-12 M to 10-4 M. The second [Ca2+]i response was elicited by exposure to 10-4 M ACh, to provide a near maximal stimulus. Borders of cells that responded to 10-4 M ACh were circumscribed, and calcium concentration responses to first and second stimulations were determined. Comparison of the first to the second [Ca2+]i responses among individual myocytes revealed the fraction of potentially ACh-responsive myocytes that mobilized intracellular Ca2+ on submaximal ACh stimulation.

Effect of muscarinic receptor antagonists on [Ca2+]i response to ACh. Basal and peak [Ca2+]i responses were recorded in individual myocytes stimulated with 10-4 M ACh. Myocytes were washed with two volumes of buffer and then bathed in 400 µl of buffer containing either no antagonist, 10-6 M atropine (nonspecific muscarinic receptor antagonist), 10-6 M pirenzepine (M1 receptor specific), 10-6 M methoctramine (M2 receptor specific), or 10-6 M 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; M3 receptor specific). After 10 min of equilibration, basal and peak [Ca2+]i levels in response to 10-4 M ACh were again recorded and compared with preantagonist responses.

Calcium pool utilization during Ca2+ mobilization in ACh- and BK-responsive myocytes. Basal and peak [Ca2+]i responses were recorded as described during exposure to 5 mM caffeine. In additional experiments, we assessed the influence of 10-6 M xestospongin C [inositol 1,4,5-trisphosphate (IP3) receptor antagonist] or 10-5 M ryanodine (in greater concentration blocks ryanodine receptors) on ACh- or BK-induced calcium mobilization in individual myocytes exposed to 10-6 M ACh or 10-7 M BK before and during incubation with the above agents. Finally, the role of extracellular Ca2+ was explored by recording [Ca2+]i responses to ACh or BK in the presence and then nominal absence of extracellular Ca2+.

Western analysis of receptor protein expression after serum deprivation. Temporal changes in receptor protein composition of canine tracheal myocytes in culture were assessed by Western analysis. After 0-7 days of serum deprivation, cultures were washed with PBS and then total protein homogenates were prepared in extraction buffer [0.3% sodium dodecyl sulfate (SDS), 50 mM Tris, pH 7.6, 0.6 M beta -mercaptoethanol, 20 µg/ml leupeptin, 250 µM phenylmethylsulfonyl fluoride, and 50 mg/ml soybean trypsin inhibitor]. Protein from whole cell lysates (15 µg/lane) was size-fractionated by SDS-polyacrylamide gel electrophoresis and then was electroblotted onto nitrocellulose membranes using semidry transfer. Blots were blocked overnight at 4°C with 3% nonfat milk in Tris-buffered saline, pH 7.4, containing 0.1% Tween 20 (TBST). Blots were then incubated at room temperature for 2-4 h in primary antibodies diluted 1:800 for M1, 1:1,000 for M2, 1:1,000 M3, and 1:1,000 for BK2 receptor protein in TBST with 1% dry milk. Blots were incubated for 40 min in a secondary antibody, sheep anti-mouse IgG diluted 1:1,000 in TBST. Streptavidin-horseradish peroxidase in TBST was used (1:5,000) for the tertiary incubation. Immunoreactive bands were detected on Hyperfilm-ECL using enhanced chemiluminescence reagents (Amersham Life Sciences). To assess the change with time in the relative abundance of individual receptor proteins from the resulting chemilumigrams, a Hewlett-Packard scanner with Scanplot software was used (15).

Materials. Collagenase, purified from Clostridium histolyticum, was purchased from GIBCO BRL, Life Technologies (Grand Island, NY). Caffeine, elastase type IV, and Nagarse protease type XXVII were purchased from Sigma (St. Louis, MO). Insulin-transferrin-selenium culture medium supplement was obtained from Collaborative Biomedical Products (Bedford, MA). Nonessential amino acid nutrient supplement was purchased from GIBCO BRL, Life Technologies. Fura 2-AM used for measurement of intracellular Ca2+ concentration was purchased from Molecular Probes (Eugene, OR). Ryanodine and xestospongin C were purchased from Calbiochem-Novabiochem (La Jolla, CA). Primary antibodies for Western blotting included monoclonal anti-BK2 receptor (clone 20) from Transduction Laboratories (Lexington, KY) and polyclonal rabbit antibodies for muscarinic M1, M2, or M3 receptors from Research and Diagnostic Antibodies (Berkeley, CA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular Ca2+ responses to ACh and BK. Figure 1 shows representative pseudocolor images derived from analysis of fura 2-AM fluorescence within cultured canine tracheal myocytes; these images were obtained at the time of peak intracellular Ca2+ mobilization responses to stimulation with ACh or BK. Confluent myocytes before serum deprivation (day 0) exhibited virtually no intracellular Ca2+ mobilization after stimulation with ACh (10-4 M), but BK (10-5 M) exposure induced a brisk and substantial rise in [Ca2+]i.


View larger version (80K):
[in this window]
[in a new window]
 
Fig. 1.   Pseudocolor images of fura 2 fluorescence in response to bradykinin (BK) and acetylcholine (ACh) in 0- and 12-day serum-deprived tracheal myocytes. In canine tracheal myocyte cultures at confluence (day 0 of serum deprivation, a-c) and before stimulation, intracellular Ca2+ concentration ([Ca2+]i) was uniformly low (~200 nM) in all cells. These cells (a, 380-nm excited image) exhibited no [Ca2+]i elevation on ACh exposure (10-6 M; c) but uniformly increased peak [Ca2+]i substantially in response to 10-7 M BK (b). In contrast, after 12 days of serum deprivation, cultures of tracheal myocytes (d) demonstrated brisk [Ca2+]i responses to ACh (f) and also to BK (e). Pseudocolor scales represent [Ca2+]i in nM.

In contrast to the day 0 cells, many but not all myocytes in 12-day serum-deprived cultures (Fig. 1) demonstrated substantial increases in [Ca2+]i after ACh (10-6 M). In addition, many but not all myocytes exhibited brisk increases in [Ca2+]i after BK (10-7 M) stimulation. These results were typical of cultures deprived of serum for 7 or more days, and, therefore, data from cultures studied at days 7-13 of serum deprivation are considered together.

Mutually exclusive BK or ACh responsiveness in serum-deprived myocytes. To determine whether the ACh-responsive and BK-responsive myocytes in serum-deprived cultures represent the same or different populations, we recorded peak intracellular Ca2+ responses in additional 12-day serum-deprived cells on three coverslips sequentially exposed to 10-7 M BK and then to 10-6 M ACh. Figure 2 shows these peak calcium responses, with responses to BK pseudocolored green and responses to ACh pseudocolored red. Overlay of these two images from each coverslip demonstrates that two distinct subpopulations of cells are present and suggests that these two subpopulations are mutually exclusive in their responsiveness to ACh or BK (Fig. 2). The only overlap between BK and ACh responses (seen as yellow in the overlay image of Fig. 2, middle row) reflects divergent responses in physically overlapping myocytes. To evaluate further whether BK or ACh responses occur in different subpopulations, we quantified calcium responses to both agonists in additional 13-day serum-deprived myocytes. As shown in Fig. 3, there is considerable heterogeneity of BK and ACh responses among serum-deprived myocytes. Furthermore, myocytes that exhibit substantial intracellular Ca2+ mobilization in response to ACh exposure have virtually no response to BK (ACh responsive, Fig. 4), and myocytes that exhibit great intracellular Ca2+ elevations during BK exposure have virtually no response to ACh (BK responsive, Fig. 4). Thus ACh-responsive and BK-responsive myocytes comprise different subpopulations of serum-deprived cells. It is also interesting to note that serum-deprived ACh-responsive cells maintained a significantly elevated plateau [Ca2+]i after ACh exposure (Figs. 3 and 4). Not until ACh was washed out of the cell chamber did [Ca2+]i return to baseline. This sustained response is in sharp contrast to serum-deprived BK-responsive myocytes, which demonstrate a transient peak response to BK without a sustained plateau (Figs. 3 and 4).


View larger version (68K):
[in this window]
[in a new window]
 
Fig. 2.   Individual serum-deprived tracheal myocytes demonstrate exclusive responsiveness to ACh or BK but not both. Three coverslips of cells in disparate states of cell density (a, e, and i) were exposed to 10-7 M BK (c, g, and k) and 10-6 M ACh (b, f, and j) sequentially, and Ca2+ mobilization was noted. Images were created in Metamorph by subtracting the 380-nm image at peak agonist response from baseline. Peak [Ca2+]i mobilization to ACh is artificially displayed in red, whereas peak [Ca2+]i responses to BK are shown in green. ACh and BK peak response images are overlaid in d, h, and l and show essentially no overlap (which would appear as yellow) in ACh or BK responsiveness.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 3.   Serum deprivation induces heterogeneous responses to contractile agonists. These figures represent the course of Ca2+ mobilization in response to 10-4 M ACh or 10-5 M BK in confluent cultures at day 0 and day 13 of serum deprivation. On day 0 of serum deprivation (A), tracheal myocytes were homogeneous in their response to BK and ACh. Each of 25 myocytes responded to BK and none demonstrated Ca2+ mobilization in the presence of ACh. After 13 days of serum deprivation (B), [Ca2+]i responses measured in 50 tracheal myocytes were variable, with 2 distinct populations evident, those that responded to BK and those that mobilized Ca2+ in response to ACh.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Responses to BK and ACh are mutually exclusive in serum-deprived tracheal myocytes. We studied 40-50 canine tracheal myocytes from 2 coverslips. Some serum-deprived myocytes exhibited increased peak [Ca2+]i on stimulation with BK (P < 0.001 vs. basal) but had no response to ACh (BK responsive). In contrast, other serum-deprived myocytes increased [Ca2+]i 4-fold on ACh stimulation (P < 0.001 vs. basal) and maintained an elevated [Ca2+]i plateau 120 s (see also Fig. 3) after agonist exposure (P < 0.005 vs. basal). However, ACh-responsive elongated myocytes did not respond to BK.

To evaluate whether the subpopulations identified above correspond to the elongated (contractile) or nonelongated (noncontractile) phenotype we previously described (2), we quantified the cell shape of ACh- or BK-responsive myocytes shown in Fig. 2. As shown in Fig. 5, the long axis-to-short axis ratio of ACh-responsive myocytes was 2.6-fold greater (P < 0.001, t-test) than that of BK-responsive cells. Thus ACh responsiveness is a property of elongated serum-deprived myocytes, and BK responsiveness occurs in more nonelongated myocytes.


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 5.   Ratio of long axis to short axis in ACh- and BK- responsive serum-deprived tracheal myocytes. Ninety canine tracheal myocytes from the 3 coverslips depicted in Fig. 2 were studied. Length and perpendicular width at widest point of 15 ACh-responsive and 15 BK-responsive cells from each of the 3 coverslips were measured using Metamorph calibration software. Ratio of long axis to short axis was significantly greater (P < 0.001) in ACh- vs. BK-responsive myocytes.

Pharmacological characterization of muscarinic ACh receptors on elongated serum-deprived myocytes. We tested the reproducibility of [Ca2+]i responses of individual ACh-responsive elongated myocytes to two sequential exposures to 10-4 M ACh. Intracellular Ca2+ responses to the second ACh exposure were systematically reduced compared with those observed after the initial ACh exposure, averaging 71.7 ± 3.1% of the first response. The two responses were closely correlated (r2 = 0.920; Fig. 6), and all cells that responded to the first ACh activation exhibited a reduced but clear-cut response to the second ACh exposure. This finding allowed us to evaluate the influence of various muscarinic receptor antagonists on ACh-induced Ca2+ mobilization by administering dual ACh challenges in the absence and presence of muscarinic antagonist. As shown in Table 1, ACh-responsive serum-deprived myocytes demonstrated robust increases in [Ca2+]i that were completely blocked in a second ACh exposure by 10-6 M atropine (nonspecific muscarinic receptor antagonist) or 10-6 M 4-DAMP (M3 specific) but were unaffected by 10-6 M pirenzepine (M1 specific antagonist). Methoctramine (10-6 M), an M2 receptor antagonist with partial anti-M3 receptor activity, caused partial inhibition of [Ca2+]i responses to ACh. Thus ACh acts through muscarinic M3 receptors to mobilize intracellular Ca2+ in serum-deprived elongated tracheal myocytes.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 6.   Reproducibility of [Ca2+]i response to ACh in serum-deprived elongated tracheal myocytes. Peak [Ca2+]i in response to 2 serial 10-4 M ACh exposures are shown. There was reproducible partial reduction of second response vs. first response. On average, the second peak [Ca2+]i was 71.7 ± 3.1% of the first; r2 = 0.920. All cells that responded to first ACh exposure also responded to second.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of muscarinic receptor antagonists on intracellular Ca2+ mobilization during second 10-4 M ACh exposure

Heterogeneous and graded activation of contractile phenotype myocytes by ACh. To evaluate whether individual elongated myocytes in serum-deprived cultures exhibit a graded intracellular Ca2+ response to increasing ACh concentrations, we exposed cells sequentially first to ACh at 10-12 M, 10-10 M, 10-8 M, 10-6 M, or 10-4 M and second to 10-4 M ACh. For each cell that exhibited a clear-cut rise in [Ca2+]i during the second ACh stimulation, we determined the rise over baseline [Ca2+]i induced by the first ACh exposure. Because there is no widely accepted standard for the magnitude of [Ca2+]i increase that constitutes a "response," we calculated the fraction of ACh-responsive myocytes whose peak [Ca2+]i increment over baseline during the first ACh exposure exceeded 100 nM, 200 nM, and 300 nM. Figure 7 demonstrates that 1) maximal stimulation with 10-4 M ACh results in a wide range of peak [Ca2+]i elevations in elongated, serum-deprived myocytes; 2) submaximal stimulation with lesser ACh concentrations results in graded peak [Ca2+]i elevations in these cells and there is substantial heterogeneity of response among individual cells; and 3) the fraction of ACh-responsive myocytes that exhibit [Ca2+]i responses increases with ACh concentration whether the threshold for response was set at 100, 200, or 300 nM.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 7.   Concentration dependence of [Ca2+]i responses to ACh exposure in elongated serum-deprived tracheal myocytes. A: peak increment in [Ca2+]i over basal (peak increment = peak [Ca2+]i - basal [Ca2+]i) vs. ACh concentration during initial ACh exposure. Wide range of [Ca2+]i responses is evident at all ACh concentrations >10-12 M. B: percent of cells whose peak incremental [Ca2+]i response exceeds 100, 200, or 300 nM vs. ACh concentration during initial ACh exposure. Increasing fraction of elongated contractile serum-deprived myocytes responds to increasing concentrations of ACh.

Mechanisms of intracellular Ca2+ coupling. Neither ACh-responsive elongated nor BK-responsive nonelongated 13-day serum-deprived myocytes mobilized Ca2+ in response to 5 mM caffeine. Also, both subpopulations failed to respond to ryanodine alone, and in the presence of 50 µM ryanodine, second responses to 10-7 M BK or 10-6 M ACh were unaffected in BK-responsive and ACh-responsive myocytes, respectively (Fig. 8). However, 10 µM xestospongin C, a blocker of IP3 receptors on the sarcoplasmic reticulum (SR), almost completely inhibited Ca2+ mobilization in response to a second ACh exposure in ACh-responsive myocytes (Fig. 8). Xestospongin C also partially reduced Ca2+ response to a second BK exposure in BK-responsive cells to 50% of that observed during the first exposure to agonist (Fig. 8). The simultaneous presence of ryanodine had no additive effect on the reduction of Ca2+ mobilization caused by xestospongin C in either ACh-responsive or BK-responsive myocytes.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 8.   Mechanisms of intracellular Ca2+ coupling. Pretreatment (15 min) with ryanodine (50 µM) did not significantly alter subsequent agonist responses. However, similar pretreatment with 10 µM xestospongin C (an inhibitor of inositol 1,4,5-trisphosphate-mediated sarcoplasmic reticulum Ca2+ release) almost completely inhibited ACh responses in elongated myocytes that had previously responded to this agonist. Xestospongin C also significantly decreased subsequent BK responses but to a lesser extent than in ACh-responsive myocytes. In contrast, BK-responsive, serum-deprived cells failed to respond to agonist in absence of extracellular Ca2+ ([Ca2+]ec), whereas removal of [Ca2+]ec did not alter Ca2+-evoked fluorescence in ACh-responsive myocytes. * Significantly less than 100%, P < 0.05 (by t-test).

Removal of extracellular Ca2+ had a profound effect on the second BK response in BK-responsive cells. In the absence of extracellular Ca2+, intracellular Ca2+ mobilization by BK was completely blocked in serum-deprived cells (Fig. 8). However, removal of extracellular Ca2+ did not affect the second peak Ca2+ mobilization caused by ACh exposure in serum-deprived ACh-responsive myocytes. Thus initial intracellular Ca2+ coupling mechanisms that determine peak calcium responses differ importantly in the two subpopulations of serum-deprived myocytes. Elongated myocytes initially mobilize calcium from the SR on ACh stimulation through downstream activation of IP3 receptors, whereas nonelongated myocytes respond to BK both through initial influx of extracellular Ca2+ and an intracellular IP3 receptor-dependent mechanism.

Western analysis of receptor expression during contractile phenotype modulation. Western analysis of whole culture lysates using anti-muscarinic M3 receptor antibody revealed a single immunoreactive band of ~70 kDa, which was detected in confluent serum-fed (day 0) myocyte cultures but progressively increased in abundance after 2 or 7 days of serum deprivation (Fig. 9). In contrast, M2 receptor abundance (detected as a ~70- kDa immunoreactive band with anti-muscarinic M2 receptor antibody) decreased significantly with prolonged serum deprivation (Fig. 9). Muscarinic M1 receptors were not detectable at any time by Western analysis (data not shown). Immunoblots using BK2 receptor-specific antibody revealed a protein doublet at ~42 kDa (Fig. 9). In whole culture lysates, BK2 receptor abundance increased significantly within 2 days of serum deprivation but tended to decrease somewhat thereafter.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 9.   Western analysis of receptor expression during serum deprivation in confluent cultures of tracheal myocytes. A: typical Western blots. B: quantification of relative receptor protein abundance. Antibody for M3 receptor (M3R) protein produced immunoblot at ~70 kDa. Muscarinic M3 receptor was detected at day 0 of serum deprivation and increased significantly with time after serum deprivation. On day 2, M3 receptor protein increased to 250 ± 15% relative to that on day 0 (n = 3, P = 0.001 vs. day 0, ANOVA and Tukey's test) and to 328 ± 12% by day 7 (n = 3, P < 0.0005 vs. day 0, P = 0.015 vs. day 2). In contrast, M2 receptor (M2R) protein decreased with prolonged serum deprivation. Antibody for M2 receptor protein produced an immunoblot at ~70 kDa; M2 receptor decreased significantly to 25 ± 14% compared with that in day 0 cultures (n = 3, P = 0.040 vs. day 0). Cultures deprived of serum for 7 days demonstrated similar quantities of M2 receptor protein remaining at 32 ± 15% relative to that in day 0 cultures (n = 3, P < 0.058 vs. day 0, P = 0.949 vs. day 2). Immunoblots using BK2 receptor (BK2R)-specific antibody revealed protein doublet at ~42 kDa. BK2 receptor protein increased significantly after serum deprivation, by 273 ± 18% at day 2 (n = 3, P = 0.005 vs. day 0). There was a trend toward diminished BK2 protein by day 7. Receptor protein decreased to 211 ± 18% by day 7 relative to that in day 0 cultures (n = 3, P < 0.038 vs. day 0, P = 0.241 vs. day 2). Muscarinic M1 receptor protein was not detectable at any time (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cultured airway smooth muscle typically loses many structural and functional characteristics of smooth muscle cells within intact tissue (1, 6, 7, 14, 16). When maintained under growth-promoting conditions, airway myocytes assume a nonelongated spindle shape, contain sparse contractile apparatus proteins, and exhibit virtually no ability to contract. However, we (2) and Ma et al. (11) recently reported that prolonged serum deprivation of confluent cultured airway myocytes promotes a monolayer culture and restores contractile structure and function to a subset of cells. Myocytes that acquire the contractile phenotype accumulate abundant contractile apparatus proteins, exhibit an elongated wormlike morphology, and shorten substantially on electrical field stimulation (11) or ACh exposure (2). The current study was performed to identify mechanisms through which ACh-induced contraction is restored in these contractile cultured cells.

We found that ACh stimulates intracellular Ca2+ mobilization within elongated, serum-deprived, contractile airway myocytes but does not do so in serum-fed confluent myocytes or in serum-deprived cells that fail to elongate (Figs. 1-5). Conversely, BK elicited intracellular Ca2+ responses in serum-fed confluent myocytes and in nonelongated serum-deprived cells but not in elongated serum-deprived myocytes. Thus responsiveness to ACh and responsiveness to BK were mutually exclusive in our culture system (Figs. 2 and 4). Pharmacological studies demonstrate clearly that, as in intact trachealis tissue (14), muscarinic M3 receptors mediate ACh-induced calcium responses in elongated, contractile myocytes (Table 1). Furthermore, increasing ACh concentration, which increases force generation by intact tissue in a graded fashion (13), also increases overall calcium mobilization in cultured contractile airway myocytes both by increasing the number of cells exhibiting any response and by increasing the magnitude of response within individual myocytes (Figs. 6 and 7).

Interestingly, elongated, ACh-responsive, serum-deprived myocytes and nonelongated, BK-responsive cells mobilize calcium in response to agonist by separate mechanisms. The ACh-responsive phenotype mobilizes calcium from a pool that is almost totally inhibited by xestospongin C pretreatment; peak fluorescence from these elongated myocytes during ACh exposure was not affected by removal of extracellular Ca2+ (Fig. 8). Thus calcium from the SR is mobilized in the presence of ACh, and its release is mediated through IP3 receptors on the SR. These results are in striking contrast with serum-deprived, nonelongated, BK-responsive cells. Xestospongin C only partially inhibited calcium mobilization in BK-responsive cells, whereas removal of extracellular Ca2+ totally inhibited fluorescence in response to agonist (Fig. 8). Thus extracellular Ca2+ may be the major source of the fluorescence response in the BK-responsive subpopulation, and calcium-stimulated calcium release may operate in these cells. Furthermore, the lack of response to caffeine or ryanodine in either subpopulation suggests that canine tracheal myocytes in culture may not express ryanodine receptors on the SR or that this mechanism of calcium release is not predominant in canine tracheal myocytes in culture (Fig. 8).

These findings have several implications. In elongated, contractile myocytes, muscarinic M3 receptors are found in clusters at the cell surface (2). In contrast, M3 receptors localize to the perinuclear region in serum-fed confluent or serum-deprived nonelongated airway myocytes (2), neither of which mobilize calcium on ACh exposure (Fig. 1). Thus translocation of muscarinic M3 receptors to the cell membrane appears to be one mechanism through which M3 receptor-calcium coupling is restored in elongated myocytes during serum deprivation. It may also be that an increase in total M3 receptor number during serum deprivation contributes to the restoration of ACh responsiveness (Fig. 9), although the relative importance of each mechanism remains uncertain.

Second, airway smooth muscle cells exhibit plasticity of cell surface receptor expression and function. In intact canine trachealis tissue, ACh but not BK elicits contraction (R. Mitchell, unpublished observations). Culture and passage of canine tracheal myocytes provoke the disappearance of functionally coupled M3 receptors and the emergence of BK-induced calcium mobilization. Yet, during prolonged serum deprivation, myocytes that reacquire the elongated contractile phenotype also regain the spectrum of agonist responsiveness present within intact tissue. The present study did not address the mechanisms by which M3 or BK receptor abundance is controlled in individual myocytes. However, the observed increase in total M3 receptor protein (and accompanying decrease in M2 receptor protein) suggests that either increased de novo synthesis or diminished degradation of muscarinic M3 receptors occurs during serum deprivation (Fig. 9). Because the muscarinic M3 receptor gene promoter has not been cloned, the nuclear mechanisms controlling M3 receptor gene transcription remain unknown.

Third, elongated, contractile cultured airway myocytes exhibit a wide range of responses to ACh as reflected in their heterogeneous intracellular Ca2+ responses to high concentrations of ACh and their heterogeneous responses to threshold concentrations (Fig. 7). Perhaps this variation among individual elongated myocytes reflects greater or lesser progression toward a "fully" contractile phenotype, but it also resembles the dispersion of other characteristics recently identified among individual myocytes acutely isolated from intact airway muscle (3-5). If this variation among cultured contractile airway myocytes reflects smooth muscle behavior in vivo, then the graded contractile responses to increasing ACh concentration characteristic of intact airway muscle strips (10, 12, 13) may stem both from increasing activation of individual myocytes and from recruitment of increasing numbers of myocytes participating in contraction. To our knowledge, this possibility has not yet been tested directly in intact airway smooth muscle or acutely isolated myocytes.

Previously, Yang and co-workers studied canine tracheal myocytes in culture that had been growth arrested by reduction of serum in the maintenance medium (18, 19, 21). They found that growth arrest increased the number of ligand-binding surface muscarinic receptors (18), that both M2 and M3 receptors were present (20), and that pharmacological activation of these receptors increased IP3 production (19-21). Interestingly, calcium was mobilized in these cultures both by carbachol and by BK in Yang's studies (20-25). Importantly, all cells in culture were treated as a single population rather than as discrete subpopulations of contractile vs. noncontractile (i.e., elongated vs. nonelongated) myocytes. Our studies confirm and extend Yang's work by demonstrating that serum deprivation induces heterogeneity of myocytes in culture and that only the contractile, elongated phenotype demonstrates M3 receptor-mediated calcium mobilization in response to cholinergic agonist. Thus restoration of ACh-mediated calcium signaling is not a general consequence of serum deprivation. Our data also show that the functional coupling of muscarinic M3 receptors to calcium mobilization is mediated through IP3 receptors on the SR in serum-deprived, cultured airway myocytes.

There are limitations to the interpretation of our data. M3 receptor protein was detected before serum deprivation but was apparently internalized (2) and not functionally coupled to Ca2+ mobilization. Prior studies of cultured confluent human airway myocytes suggest that muscarinic M2 receptors are associated with the sarcolemma because isoproterenol-induced cAMP formation is inhibited by carbachol and muscarinic M2 receptor antagonists block this inhibition (16). We also found that methoctramine, an M2 receptor antagonist, reduced calcium mobilization in response to ACh in elongated serum-deprived myocytes (Table 1); this partial antagonism in part may be due to low-affinity binding of methoctramine to M3 receptors (20). Furthermore, we could not quantify the distribution of M3 receptor protein among elongated vs. nonelongated serum-deprived myocytes. Thus it is conceivable that the increase in overall M3 receptor protein observed during serum deprivation occurred in part within the nonelongated, noncontractile subpopulation. Similarly, we suspect, but cannot prove, that the increase in BK2 receptor protein evident from analysis of whole culture lysates was localized primarily to the noncontractile myocytes. Also, we could not detect any significant response in either elongated or nonelongated myocytes to caffeine and ryanodine. However, Janssen et al. (8, 9) have noted that freshly dissociated canine tracheal smooth muscle cells rarely contract to these agents, and in only 3 of 7 cells tested (8) was there some transient calcium mobilization in response to concentrations of ryanodine similar to those used in our studies. This finding limits precise interpretation of the lack of a response to caffeine and ryanodine in our serum-deprived myocytes, which thus may indicate a possible continued perturbation in excitation-contraction coupling in the elongated, contractile myocytes. Finally, we cannot be sure that every phenotypically contractile, elongated serum-deprived myocyte does respond to ACh and does not respond to BK. However, we selected visual fields in which both elongated and flattened nonelongated myocytes were present so that we could directly compare [Ca2+]i responses to both ACh and BK. Quantification of cell shape (Fig. 5) confirms that ACh-responsive and BK-responsive subpopulations correspond to the contractile and noncontractile phenotypes we characterized previously (2), and the differences in [Ca2+]i responses and the mechanisms of calcium mobilization to ACh and BK between elongated tracheal myocytes and nonelongated cells in the same image field were striking.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Specialized Center of Research Grants HL-56399 and HL-64095, Inspiraplex, Merck-Frosst Canada, Sprague Memorial Institute, and Blowitz-Ridgeway Foundation.


    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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. Solway, Section of Pulmonary and Critical Care, Dept. of Medicine, Univ. of Chicago, 5841 S. Maryland Ave., MC 6026, Chicago IL 60637 (E-mail: jsolway{at}medicine.bsd.uchicago.edu).

Received 5 April 1999; accepted in final form 7 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Chamley-Campbell, JH, Campbell GR, and Ross R. Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens. J Cell Biol 89: 379-383, 1981[Abstract].

2.   Halayko, AJ, Camoretti-Mercado B, Forsythe SM, Vieira JE, Mitchell RW, Wylam ME, Hershenson MB, and Solway J. Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes. Am J Physiol Lung Cell Mol Physiol 276: L197-L206, 1999[Abstract/Free Full Text].

3.   Halayko, AJ, Rector E, and Stephens NL. Characterization of molecular determinants of smooth muscle cell heterogeneity. Can J Physiol Pharmacol 75: 917-929, 1997[ISI][Medline].

4.   Halayko, AJ, Rector E, and Stephens NL. Airway smooth muscle cell proliferation: characterization of subpopulations by sensitivity to heparin inhibition. Am J Physiol Lung Cell Mol Physiol 274: L17-L25, 1998[Abstract/Free Full Text].

5.   Halayko, AJ, Salari H, Ma XF, and Stephens NL. Markers of airway smooth muscle cell phenotype. Am J Physiol Lung Cell Mol Physiol 270: L1040-L1051, 1996[Abstract/Free Full Text].

6.   Hall, IP, and Kotlikoff M. Use of cultured airway myocytes for study of airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 268: L1-L11, 1995[Abstract/Free Full Text].

7.   Hirst, SJ. Airway smooth muscle cell culture: application to studies of airway wall remodeling and phenotype plasticity in asthma. Eur Respir J 9: 808-820, 1996[Abstract/Free Full Text].

8.   Janssen, LJ, Betti PA, Netherton SJ, and Walters DK. Superficial buffer barrier and preferentially directed release of Ca2+ in canine airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 276: L744-L753, 1999[Abstract/Free Full Text].

9.   Janssen, LJ, and Sims SM. Ca2+-dependent Cl- current in canine tracheal smooth muscle cells. Am J Physiol Cell Physiol 269: C163-C169, 1995[Abstract/Free Full Text].

10.   Koenig, SM, Mitchell RW, Kelly E, White SR, Leff AR, and Popovich KJ. beta -Adrenergic relaxation of dog trachealis: contractile agonist-specific interaction. J Appl Physiol 67: 181-185, 1989[Abstract/Free Full Text].

11.   Ma, X, Wang Y, and Stephens NL. Serum deprivation induces a unique hypercontractile phenotype of cultured smooth muscle cells. Am J Physiol Cell Physiol 274: C1206-C1214, 1998[Abstract/Free Full Text].

12.   Madison, JM, Jones CA, Tom-Moy M, and Brown JK. Affinities of pirenzepine for muscarinic cholinergic receptors in membranes isolated from bovine tracheal mucosa and smooth muscle. Am Rev Respir Dis 135: 719-724, 1987[ISI][Medline].

13.   Mitchell, RW, Koenig SM, Popovich KJ, Kelly E, Tallet J, and Leff AR. Pertussis toxin augments beta-adrenergic relaxation of muscarinic contraction in canine trachealis. Am Rev Respir Dis 147: 327-331, 1993[ISI][Medline].

14.   Tom-Moy, M, Madison JM, Jones CA, de Lanerolle P, and Brown JK. Morphologic characterization of cultured smooth muscle cells isolated from the tracheas of adult dogs. Anat Rec 218: 313-328, 1987[ISI][Medline].

15.   Vincent, SG, Cunningham PR, Stephens NL, Halayko AJ, and Fisher JT. Quantitative densitometry of proteins stained with Coomassie blue using a Hewlett Packard scanjet scanner and Scanplot software. Electrophoresis 18: 67-71, 1997[ISI][Medline].

16.   Widdop, S, Daykin K, and Hall IP. Expression of muscarinic M2 receptors in cultured human airway smooth muscle cells. Am J Respir Cell Mol Biol 9: 541-546, 1993[ISI][Medline].

17.   Wylam, ME, Gungor N, Mitchell RW, and Umans JG. Eosinophils, major basic protein, and polycationic peptides augment bovine airway myocyte Ca2+ mobilization. Am J Physiol Lung Cell Mol Physiol 274: L997-L1005, 1998[Abstract/Free Full Text].

18.   Yang, CM. Muscarinic receptor expression in the primary culture of tracheal smooth muscle cells. J Recept Res 10: 235-247, 1990[ISI][Medline].

19.   Yang, CM, and Chou S-P. Primary culture of canine tracheal smooth muscle cells in serum-free medium: effects of insulin-like growth factor I and insulin. J Recept Res 13: 943-960, 1993[ISI][Medline].

20.   Yang, CM, Chou SP, and Sung TC. Muscarinic receptor subtypes coupled to generation of different second messengers in isolated tracheal smooth muscle cells. Br J Pharmacol 104: 613-618, 1991[Abstract].

21.   Yang, CM, Chou SP, Sung TC, and Chien HJ. Regulation of functional muscarinic receptor expression in tracheal smooth muscle cells. Am J Physiol Cell Physiol 261: C1123-C1129, 1991[Abstract/Free Full Text].

22.   Yang, CM, Hsia H-C, Chou S-P, Ong R, Hsieh J-T, and Luo S-F. Bradykinin-stimulated phosphoinositide metabolism in cultured canine tracheal smooth muscle cells. Br J Pharmacol 111: 21-28, 1994[Abstract].

23.   Yang, CM, Hsia HC, Hsieh JT, Ong R, and Luo SF. Bradykinin-stimulated calcium mobilization in cultured canine tracheal smooth muscle cells. Cell Calcium 16: 59-70, 1994[ISI][Medline].

24.   Yang, CM, Hsu MC, Tsao HL, Chiu CT, Ong R, Hsieh JT, and Fan LW. Effects of cAMP elevating agents on carbachol-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells. Cell Calcium 19: 243-254, 1996[ISI][Medline].

25.   Yang, CM, Yo YL, and Wang YY. Intracellular calcium in canine cultured tracheal smooth muscle cells is regulated by M3 muscarinic receptors. Br J Pharmacol 110: 983-988, 1993[Abstract].


Am J Physiol Lung Cell Mol Physiol 278(5):L1091-L1100
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society