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Published ahead of print on May 12, 2005, doi:10.1165/rcmb.2005-0137OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 153-160, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0137OC

Spontaneous Propagating Calcium Waves Underpin Airway Peristalsis in Embryonic Rat Lung

Neil C. Featherstone*, Edwin C. Jesudason*, Marilyn G. Connell, David G. Fernig, Susan Wray, Paul D. Losty and Theodor V. Burdyga

Division of Child Health, Royal Liverpool Children's Hospital (Alder Hey), School of Biological Sciences, and The Physiological Laboratory, University of Liverpool, Liverpool, United Kingdom

Correspondence and requests for reprints should be addressed to Neil C. Featherstone, Medical Research Council Clinical Training Fellow, Division of Child Health, School of Reproductive and Developmental Medicine, University of Liverpool, Liverpool L69 3BX, UK. E-mail: N.C.Featherstone{at}Liverpool.ac.uk


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Prenatal airways from diverse species exhibit spontaneous peristaltic contractions (airway peristalsis). These contractile waves appear coupled to and may function to regulate prenatal lung growth. They are unaffected by atropine or tetrodotoxin but abolished by nifedipine. Nevertheless, the mechanisms by which these contractile waves are generated, regulated, and propagated remain obscure. Using calcium imaging and whole embryonic lung organ culture, we demonstrate for the first time that peristalsis of the embryonic airway is driven by spontaneous, regenerative, temperature-sensitive calcium (Ca2+) waves. These Ca2+ waves propagate between individual airway smooth muscle cells coupled via gap junctions, are likely to be action potential–mediated, and are dependent on not only extracellular calcium entry via L-type voltage-gated channels but also intracellular Ca2+ stores. Thus, if airway peristalsis regulates lung growth, these findings mean that airway smooth muscle Ca2+ waves in turn regulate prenatal lung morphogenesis.

Key Words: calcium waves • airway smooth muscle • lung branching morphogenesis


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Prenatal airway smooth muscle (ASM) exhibits spontaneous phasic contractility (airway peristalsis [AP]) immediately ex vivo and in vitro (1). ASM has been proposed to lack physiologic function and merely contribute to pathology (e.g., bronchial hyperreactivity) (2). Alternatively, ASM (via AP) may have a central role in normal lung development (1). Inhibition or augmentation of peristalsis frequency is associated with parallel changes in cultured lung growth (3, 4). Likewise, alteration of cultured lung growth is mirrored by change in peristalsis frequency (3). Numerous observations similarly relate other mechanical factors (fetal breathing movements, lung liquid stretch, stretched pulmonary cell cultures, cytoskeletal tension) with key aspects of lung development (5–8).

Spontaneous airway contractions were first observed in chick embryo explants (9) and subsequently in rabbit (1), guinea pig (10, 11), and human (12). Periodic luminal narrowing propels lung liquid and transiently distends the fluid-filled end buds of the embryonic lung (1). Prenatal ASM contractility is augmented by acetylcholine or depolarizing K+ solutions (13), unaffected by atropine or tetrodotoxin, and abolished by Ca2+ antagonists (e.g., nifedipine) (12). Airway peristalsis appears therefore to require extracellular Ca2+ influx. Lung morphogenesis appears similarly Ca2+ dependent: it is disrupted by Ca2+ antagonists in vitro and transgenic pulmonary expression of a calmodulin inhibitor protein in vivo (14). Despite these indications that Ca2+ participates in AP and pulmonary growth, direct observations of Ca2+ dynamics in peristalsing lung have not been described. How Ca2+ signaling underpins AP therefore remains unknown. For example, (1) Where within the lung primordia do putative Ca2+ waves originate and propagate? (2) Which embryonic cell type(s) are involved? (3) How is the putative Ca2+ wave transmitted? (4) Are both extracellular and intracellular sources of Ca2+ involved in the generation and regulation of Ca2+ waves?

To answer these questions, we developed a novel technique that employs confocal and photometric Ca2+ imaging of cultured whole lung primordia. In the first such observations, we show that AP is underpinned by spontaneous, regenerative, temperature-sensitive Ca2+ waves that use gap junctions to propagate through ASM cells. These results, together with the measured spatiotemporal characteristics of the Ca2+ waves, strongly support an action potential–mediated mechanism. We also demonstrate that spontaneous phasic Ca2+ waves require Ca2+ entry (via L-type Ca2+ channels) and intracellular Ca2+ release from the sarcoplasmic reticulum (using ryanodine [RyR] and inositol triphosphate [InsP3R] receptor channels). If AP regulates lung growth (3), our findings mean that prenatal lung morphogenesis is regulated by ASM Ca2+ entry, intracellular Ca2+ release, and the resulting Ca2+ waves.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Embryonic Lung Culture
In accordance with UK legislation, embryos from Sprague-Dawley rats (Charles River Ltd, Margate, UK) were harvested on Day 13.5 of gestation (vaginal plug = Day 0, term = Day 22.5) as previously described (15). This is 24 h after the lung anlage forms as a diverticulum from the foregut. Upon retrieval, embryos were transferred to an isotonic saline bath cooled on ice. Microdissected from their extra-embryonic membranes, embryos were secured in a lateral position using cranial and caudal entomology pins. Using a Leica (Leica Microsystems [UK] Ltd., Milton Keynes, UK) MZ6 stereomicroscope and microsurgical instruments, a thoracic incision was performed to expose the embryonic heart and lungs. The heart was teased from the lung rudiments before the esophagus was freed carefully from the primitive carina. The trachea was then sectioned and the pulmonary complex transferred by pipette into serum-free culture medium (DMEM/F12 1:1, Gibco; Invitrogen Life Technologies, Paisley, UK). Lung rudiments were positioned on translucent polytetrafluorethylene membrane culture-dish inserts (Millicell; Millipore Corp., Bedford, MA) with serum-free culture media incorporating penicillin (100 IU/ml) and streptomycin (100 µg/ml; GibcoBRL, Life Technologies, Paisley, UK). Lung primordia were incubated at 37°C in 5% CO2 for periods up to 78 h. Using brightfield microscopy, specimens were inspected daily for histologic integrity, viability, and the periodic motion of luminal lung fluid. Culture media were changed every 48 h. Spontaneous airway contractions are seen in 100% of explants by 48 h in vitro using this established culture technique (3).

Loading Lung Explants with Ca2+ Sensitive Fluorophores
Lungs were cultured for 54 or 78 h before 4 h incubation at 37°C with the membrane-permeant form of Fluo-4 AM (15 µM; Molecular Probes, Invitrogen Life Technologies) with Pluronic F (for confocal imaging), or 2 h incubation at 37°C with the membrane-permeant forms of Indo-1 AM or Fura-2 AM (15 µM; Molecular Probes) with Pluronic F (for ratiometric imaging of [Ca2+]i). Explants were then allowed to equilibrate for 30 min in physiologic saline at 37°C.

Confocal and Photometric Measurements
For confocal and photometric studies, lung primordia were secured in a 200-µl custom-made perfusion chamber by nylon mesh (Plastok, Birkenhead, UK) that holds the lung periphery against the coverslip without compressing principal airways.

Confocal imaging was used to determine spatiotemporal characteristics of intercellular Ca2+ waves and performed on an Ultraview LCI spinning Nipkow disc, widefield Olympus IX70 inverted microscope (Perkin-Elmer Confocal Microscope System, Cambridge, UK) with an ORCA ER-cooled CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). This permits image acquisition from a large area (from 2.1 x 1.6 mm with 4x objective to 160 x 110 µm with 60x objective) at 20–30 frames per second with relatively low intensity laser illumination and at good signal to noise ratio. Fluo-4 loaded explants were viewed at low power (4x objective, numerical aperture [NA] 0.13; 10x objective, NA 0.17) or high power magnification (60x water immersion objective, NA 0.9) and excited by argon/krypton laser at 488 nm. Resulting fluorescence was measured at 510 nm and is expressed as a function of maximum compared with basal values (F/F0). To reduce photobleaching, Ca2+ transients from each lung were observed for not more than 90 s. Propagation speeds of the Ca2+ and contractile waves were measured using the Perkin-Elmer Microscope System. Regions of interest were defined in the airways. Pixel counts between regions of interest were then converted to distance (µM) using calibration measurements for each objective and pixel binning option. Measurement of the interval (s) between initiation of the Ca2+ transient (or ASM contraction) at one region of interest and the next allowed propagation speed to be quantified.

To study intracellular Ca2+ waves and effects of their modulation by temperature and pharmacologic agents, calcium signaling was also measured photometrically using two ratiometric Ca2+-sensitive indicators, Indo-1 or Fura-2. The optical system has been described previously (16). Briefly, when Indo-1 was used, the photometric system consisted of an Olympus IX50 inverted microscope (Olympus UK Ltd, London, UK) and xenon lamp with an excitation wavelength of 340 nm. Light emitted by Indo-1 loaded explants at 400 nm and 500 nm was detected via photo-multiplier tubes and digitally recorded. The ratio of the fluorescence signal (F400/500; 400 nm:500 nm emission) was used as an indicator of [Ca2+]i. Photometric measurements of Fura-2–loaded explants employed a Cairn (Cairn Research Ltd., Faversham, UK) monochromator with dual excitation (340 and 380 nm) and the fluorescent signals were measured at 510 nm (F340/380). With both indicators, Ca2+ signals were recorded using a 20x objective (N.A. 0.75) from the second bronchial division of the right lung (previously reported origin for most mechanical contractions) (3). For each lung primordia Ca2+ transients were observed for at least 10 min. Temporal characteristics of the Ca2+ waves (duration of fast and slow phases, time to achieve 50% of peak amplitude, amplitude, plateau duration, relaxation half-time [T50] and duration at 50% relaxation) were determined from at least three representative transients per lung.

Solutions
Unless otherwise stated, lung explants were superfused at 37°C with buffered physiologic saline (pH 7.4) containing (mM): 120 NaCl, 5.6 KCl, 0.12 MgSO4, 2 CaCl2, 11.7 glucose, 10.9 Hepes. Zero Ca2+ solution used in certain experiments consists of physiologic saline with CaCl2 omitted and 2 mM EGTA added. Other additions to standard superfusate that were investigated include: nifedipine (10 µM), Bay-K 8644 (1 µM), 18-ß glycyrrhetinic acid (18-ßGA, 40 µM), tetraethylammonium (TEA, 10 mM), cyclopiazonic acid (CPA, 20 µM), caffeine (1 mM–10 mM), ryanodine (20 µM), carbachol (10–100 µM), 2-aminoethoxy-diphenylborate (2-APB, 50 µM), and high K+ solution (120 mM). Carbachol and ryanodine were dissolved in distilled water; CPA, 2-APB, and 18-ßGA were dissolved in dimethylsulfoxide (DMSO); and nifedipine and Bay-K 8644 were dissolved in ethanol (ETOH). Calcium wave generation and airway contractility were unaffected by applications of vehicle alone (DMSO or ETOH). Ryanodine was obtained from Calbiochem (Merck Biosciences Ltd., Nottingham, UK). Sigma (Poole, Dorset, UK) supplied all other chemicals.

Immunohistochemistry
Cultured lungs were preserved after 78 h in 4% paraformaldehyde (0.1 M phosphate-buffered saline [PBS], pH 7.4), rinsed in PBS, cryoprotected with 20% (wt/vol) sucrose, gelatine embedded (7.5% [wt/vol] gelatin, 15% [wt/vol] sucrose in PBS) before being covered in Cryo-M-Bed (Bright, Huntingdon, UK), and snap-frozen at –40°C. Lung sections were taken at 7 µm and mounted on chrome alum gel slides for storage at –40°C.

Slides were rinsed in PBS and incubated with {alpha}-smooth actin antibody (monoclonal anti–{alpha}-SMA Clone No.1A4, mouse; Sigma, Gillingham, UK) at 1:500 dilution with 0.1% BSA in PBS overnight at 4°C. They were then rinsed in PBS and the primary antibody labeled with FITC-labeled goat anti-mouse IgG (Sigma, Gillingham, UK) at 1:100 dilution. Slides were then mounted with Fluorescent mounting medium (S3023; Dako, Ely, UK).

Statistical Analysis
Data processing was performed using Origin 6.0 software (OriginLab Corporation, Northampton, MA). The SPSS v12.0 package (SPSS UK Ltd., Woking, UK) was used for statistical analysis. Parametric data are reported as mean ± SEM and compared using Student's t test. Non-parametric data are reported as medians (interquartile ranges) and compared with Mann-Whitney U test. Statistical significance was taken at P < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Spontaneous Ca2+ Waves Propagate via Smooth Muscle Cells before Contraction
Confocal imaging of Fluo-4 loaded embryonic lungs (n = 16) with low-power magnification (4x or 10x) at 37°C, revealed intercellular Ca2+ waves propagating longitudinally through principal airways (Figure 1A). The propagating Ca2+ waves originated from the trachea (Region 1, red box; Figures 1A, 1B, and 1D) or the second bronchial division of the right lung rudiment (Region 2, black box; Figures 1A, 1C, and 1D). Infrequently, separate intercellular Ca2+ waves emanated simultaneously from two foci (data not shown). The initial phase of the Ca2+ transient differed between sites of Ca2+ wave initiation and sites of propagation (Figure 1D). Irrespective of the site of origin and direction of travel, Ca2+ waves propagated with an average speed of 219 ± 14 µm/s (n = 4 lung explants, 10 individual Ca2+ waves), and after a short delay (349 ± 30 ms, n = 4 lung explants), were followed by the mechanical contractile wave (recorded as the decrease in the airway luminal diameter) (Figure 2). Contractile ASM waves invariably propagated from the Ca2+ wave origin at a similar speed: 177 ± 32 µm/s (n = 4 lungs, 8 contractile waves). Using high-power magnification (60x objective), calcium waves were also recorded in individual smooth muscle cells (Figures 1E and 1F). Calcium transients recorded photometrically or confocally exhibited similar time courses and four distinct phases: (1) fast upstroke, (2) slow attainment of peak, (3) plateau, and (4) relaxation (Figures 3A and 3B). Using a 60x objective, developing ASM cells were observed lying circumferentially around the developing airway with their long axes perpendicular to that of the bronchi (Figure 1E). The diameters of these cells were ~ 8–14 µm. Histology and {alpha}-actin immunohistochemistry (n = 10) confirmed this typical ASM orientation and characteristic gene expression (Figure 1E) (17).



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  		Figure 1. Intercellular Ca2+ waves in embryonic lung ASM. (A) Transmitted light photomicrograph of a whole lung explant after 54 h in vitro: the dashed box contains the proximal airways shown in B and C (T = Trachea; R = Right and L = Left lung rudiments; Marked regions 1 and 2 are the sites of recording of the calcium transients in D). (B and C) Stacks of pseudo-color confocal images of Fluo-4–loaded airways showing propagating Ca2+ waves with the initiation site (marked by an asterisk) in the trachea (B) and right lung rudiment (C). (D) Ca2+ transients measured in regions 1 and 2 (see A). The calcium wave originates in the trachea (region 1; top) or the right lung (region 2; bottom). (E) Left-hand panel shows immunohistochemistry for {alpha}-smooth actin on a 7-µm section of a cultured lung explant. Smooth muscle cells expressing {alpha}-smooth actin are arranged perpendicular to the long axis of the airway (T = Trachea; R = Right and L = Left lung rudiments). Right-hand panel shows greyscale image of Fluo-4–loaded individual smooth muscle cells (outlined by dashed line) surrounding embryonic airway at 60x objective magnification. The arrow demonstrates the long axis of the airway. Marked regions i (black) and ii (red) are the sites of recording of the calcium transients in F. (F) Ca2+ transients recorded in areas 1 and 2 (marked on cells at 60x objective magnification in E).

 


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  		Figure 2. Relationship between Ca2+ waves and ASM mechanical contractility. (A) Transmitted light image of a cultured lung (the dashed box contains the region shown at high magnification in B). T = Trachea; R = Right and L = Left lung rudiments. (B) Stack of grayscale transmitted light images showing airway contractility. The dashed arrow gives the direction of the contractile wave. The small arrows demonstrate the airway lumen. (C) Change in [Ca2+]i (top) and lumen diameter (bottom) with time at box in (A).

 


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  		Figure 3. Effects of gap junction blockade and cooling on lung Ca2+ oscillations. (A) Transmitted light image of the proximal airways of an embryonic lung. The dashed box shows the region where Ca2+ transients were recorded. (B) Superimposed normalized traces of a typical Ca2+ transient recorded photometrically (black trace) and confocally (red trace) from the region in A. (C) Inhibition of Ca2+ oscillations by 18-ß-glycyrrhetinic acid (40 µM). (D) Airway Ca2+ oscillations at 37°C and 27°C.

 
Role of Gap Junctions
The propagation speed (18–20) and temporal phases (21) (above) of ASM intercellular Ca2+ waves are consistent with an action potential-mediated mechanism. Propagation of the action potential in smooth muscle tissue is mediated by gap junctions (20, 22), To test whether propagating Ca2+ waves in the present experiments required gap junction integrity, we studied the effects of the gap junction uncoupler 18-ß glycyrrhetinic acid (40 µM) on the Ca2+ waves. 18-ß glycyrrhetinic acid abolished both propagating Ca2+ waves and coordinated peristalsis after an initial brief excitatory phase (n = 4) (Figure 3C).

Effects of Moderate Cooling
Airway peristalsis and excitation–contraction coupling are temperature dependent (9, 21). We therefore compared the temporal characteristics and frequency of ASM Ca2+ transients at 37°C and 27°C. Amplitude of the spontaneous Ca2+ transients is conventionally expressed as a percentage of peaks obtained with depolarizing 120 mM K+ solution or 100 µM carbachol. Amplitude of spontaneous Ca2+ transients (% of high K+ peak) was 27 ± 2% (n = 17 lungs) at 37°C and 22 ± 2% (n = 11 lungs) at 27°C (P = NS). In contrast, as a percentage of carbachol peak, amplitude of spontaneous Ca2+ transients was 28 ± 4% (n = 5 lungs) at 37°C and 15 ± 2% (n = 7 lungs) at 27°C (P = 0.01). Cooling to 27°C had no effect on time to 50% of peak amplitude of the Ca2+ transient, whereas plateau phase, T50, and duration at 50% amplitude were significantly prolonged (Table 1). There was a trend toward higher frequency Ca2+ oscillations at 37°C (0.81 ± 0.06 per minute, n = 23) compared with 27°C (0.7 ± 0.06 per minute, n = 22). Figure 3D highlights this trend.


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  		TABLE 1. Temporal characteristics of ca2+ oscillations recorded at 37°c and 27°c

 
Effects of Ca2+ and K+ Channel Modulators
Ca2+-free solution (with 2 mM EGTA) decreased baseline intracellular calcium [Ca2+]i and rapidly abolished both intracellular Ca2+ transients and peristalsis (n = 4) (Figure 4A). Readmission of Ca2+ quickly restored Ca2+ oscillations and mechanical activity. These data indicate that Ca2+ entry is required for the generation of propagating Ca2+ waves. Blockade of L-type Ca2+ channels with nifedipine (10 µM) rapidly abolished spontaneous Ca2+ transients and peristalsis while reducing baseline [Ca2+]i (n = 4) (Figure 4B). In contrast, the L-type Ca2+ channel agonist Bay-K 8644 (1 µM; n = 4) raised [Ca2+]i, and increased Ca2+ wave amplitude (from 27 ± 5% to 46 ± 5% of peak obtained with depolarizing 120 mM K+ solution; P = 0.045) and frequency (from 0.53 ± 0.14 to 2.57 ± 0.61 per minute; P = 0.017) (Figure 4C). The potassium channel blocker TEA (10 mM; n = 4) produced a trend toward increased Ca2+ wave amplitude (from 13 ± 4% to 23 ± 7% of peak with high K+ solution; P = NS) and frequency (from 0.65 ± 0.23 to 1.63 ± 0.46 per minute; P = NS) (Figure 4D). These data suggest that amplitude, duration, and frequency of the Ca2+ transients are controlled by L-type Ca2+ channels and potentially by TEA sensitive K+ channels (voltage-gated [Kv] or large conductance [BK] channels or both).



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  		Figure 4. Effects of Ca2+ channel and K+ channel modulators on Ca2+ oscillations in embryonic lung ASM. (A) Reversible inhibition of Ca2+ oscillations by removal of external Ca2+ (0 Ca2+ solution with 2 mM EGTA). (B) Abolition of Ca2+ oscillations by L-type Ca2+ channel blocker (nifedipine (10 µM). (C) Increased frequency of Ca2+ oscillations with L-type Ca2+ channel agonist Bay-K 8644 (1 µM). (D) K+ channel blocker (TEA, 10 mM) increases Ca2+ oscillation frequency.

 
Role of the Sarcoplasmic Reticulum
Calcium handling by the sarcoplasmic reticulum is vital in smooth muscle excitation–contraction coupling. Intracellular calcium release is mediated by SR channels, ryanodine receptors (RyR), and inositol triphosphate receptors (InsP3R). Removal of released Ca2+ from the cytoplasm occurs primarily via the SR Ca2+-ATPase (SERCA pump).

Caffeine facilitates Ca2+-induced Ca2+ release (CICR) properties of RyR (23). Carbachol activates InsP3R-mediated SR Ca2+ release through inositol triphosphate (IP3) production. Their effects were studied after initial application of high K+ solution (to generate a 100% reference for [Ca2+]i) and then, to eliminate plasmalemmal Ca2+ entry, 2 min superfusion with Ca2+-free solution (which decreased [Ca2+]i). Application of 20 mM caffeine then transiently increased [Ca2+]i (15% ± 7% of high K+ response; n = 4) (Figure 5A) with brief tonic airway contraction. Subsequent application of carbachol (100 µM) for 1 min generated high-amplitude Ca2+ transients (120 ± 27% of high K+ response; n = 4) (Figure 5A) and sustained tonic contraction. Therefore embryonic ASM cells appear to possess a sarcoplasmic Ca2+ store that is releasable via both RyR and InsP3R channels.



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  		Figure 5. Sarcoplasmic calcium regulates Ca2+ oscillations in embryonic ASM. (A) Agonist-induced SR Ca2+ release: [Ca2+]i recording showing the effects of high K+, caffeine (20 mM), and carbachol (100 µM). Caffeine and carbachol were applied after 2 min exposure to a 0 Ca2+ solution. Abolition of Ca2+ oscillations by (B) CPA (20 µM), (C) caffeine (1 mM), (D) 2-APB (50 µM), and (E) Ryanodine (20 µM).

 
To examine the role of the SR in regulation of Ca2+ waves in embryonic airway smooth muscle, we studied the effects on the Ca2+ transients of (1) blocking the SERCA pump with CPA (24), (2) activating CICR via RyRs with caffeine (23), (3) blocking RyR channels with ryanodine (25), or (4) blocking InsP3Rs with 2-APB (26).

CPA (20 µM) (n = 4) markedly increased baseline cytosolic [Ca2+]i and gradually abolished Ca2+ transients (Figure 5A). Similarly phasic mechanical contractions were progressively replaced by tonic airway contraction. Removal of CPA from the bathing solution reversed its effects. Caffeine's effects on [Ca2+]i were dose dependent: caffeine (1 mM) initially stimulated and then abolished Ca2+ waves and contractility (n = 4) (Figure 5B). Caffeine (10 mM) increased baseline [Ca2+]i and basal tone with abolition of Ca2+ waves (n = 4) (data not shown). Variation in caffeine's effects may be due to complex sequelae of CICR that may include activation of depolarizing chloride channels and/or hyperpolarizing K+ channels (27, 28). Blockade of the latter by TEA (10 mM) prevented caffeine's action and suggests that caffeine-induced CICR does mediate K+ channel opening (n = 4; data not shown). Ryanodine (20 µM) also abolished spontaneous Ca2+ waves and contractility (n = 4) (Figure 5C). InsP3R blockade by 20-min application of 2-aminoethoxy-diphenylborate (2-APB; 50 µM) increased baseline [Ca2+]i and gradually abolished both Ca2+ waves and contractility (n = 4) (Figure 5D) (29, 30). Collectively these data demonstrate that sarcoplasmic calcium uptake via the SERCA pump and release via RyR and InsP3R channels are required for spontaneous propagating ASM Ca2+ waves and consequent peristalsis.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Postnatal ASM function is under study due to the morbidity of asthma. Now prenatal ASM activity is subject to increasing scrutiny because of accumulating evidence that prenatal airway peristalsis (AP) regulates early lung growth (3). Altering either AP frequency or lung growth yields parallel changes in the other (3). Taking a specific example, nifedipine (L-type Ca2+ antagonist) not only abolishes AP (12, 13) but also inhibits lung morphogenesis and pulmonary cell proliferation in vitro (3, 4). Moreover, transgenic pulmonary epithelial expression of an inhibitor of the Ca2+-binding protein calmodulin disrupts lung morphogenesis in vivo (14). Given these observations, we postulated that Ca2+ signaling represents a promising mechanistic link between AP and lung growth. However, to the best of our knowledge, no direct observations of Ca2+ dynamics in embryonic lung have been described.

We report the first demonstration of prenatal pulmonary Ca2+ waves, determination of their spatiotemporal characteristics, and their relationship to consequent peristaltic airway contraction. We achieved this technically exacting task by Ca2+-sensitive indicator loading of whole embryonic lung (rather than single cell preparations). Given that AP involves the entire airway, whole organ Ca2+ imaging has been invaluable. We examined Ca2+ waves with confocal imaging to measure spatiotemporal propagation, and photometric techniques for extended measurements and pharmacologic manipulation. Finding both techniques yielded similar Ca2+ wave morphology reinforces the validity of our approach.

Using high-power magnification, we provide the first evidence that prenatal pulmonary Ca2+ waves propagate through typically arranged ASM cells rather than epithelium. Our data show that Ca2+ waves are transmitted via cells whose longitudinal axis is perpendicular to the airway. Furthermore the recordings demonstrate that these same cells then rapidly and invariably contract. Smooth muscle {alpha}-actin immunohistochemistry confirms that these cells are arranged in a typical circular fashion around the airway and are indeed ASM (17). Together this strongly indicates that ASM Ca2+ wave propagation causes airway peristalsis.

We also elucidated how ASM Ca2+ waves propagate. Calcium-dependent action potentials have been documented in a wide variety of smooth muscle. Their unequivocal demonstration requires micro-electrode recording. However, small preparations such as embryonic lung present considerable difficulties: contractile activity readily dislodges micro-electrodes. Addressing this problem by pharmacologic abolition of contractility may simultaneously extinguish Ca2+ waves. Instead, we can contend that ASM Ca2+ waves are likely to be action potential–mediated after demonstrating their following typical features: (1) high wave speed (18–20), (2) capacity for multidirectional propagation, (3) characteristic four-phase morphology of transients (21), (4) gap junction–dependent propagation (20, 22), and (5) apparent regulation by membrane potential. Observations that support the latter point include: delaying repolarization with 10 mM TEA increases Ca2+ wave frequency and amplitude; depolarization with high-K+ solution entrains a Ca2+ wave and contractility (data not shown); voltage-gated Ca2+ channel activity is required for and regulates ASM Ca2+ waves.

We have also demonstrated key mechanisms for generation and regulation of spontaneous ASM Ca2+ waves. Initially, we compared the temporal characteristics of ASM Ca2+ transients at 37°C and 27°C. Consistent with passive influx of extracellular Ca2+, temperature reduction affected neither the early sharp rise in intracellular Ca2+ nor response to high K+ (21). In contrast, plateau phase, T50 and duration at 50% amplitude were significantly prolonged, consistent with a requirement for active removal of cytosolic Ca2+ (21). These data implicate a plurality of calcium transport processes in the generation of airway Ca2+ transients.

We first established that extracellular Ca2+ entry via L-type channels is necessary for, and regulates, spontaneous regenerative ASM Ca2+ waves. Intracellular Ca2+ waves were abolished by removal, and then reinstated by reintroduction of external Ca2+. Abolition of airway peristalsis by nifedipine was accompanied by cessation of periodic Ca2+ oscillations. The L-type Ca2+ channel agonist Bay-K 8644 increased Ca2+ wave frequency and baseline [Ca2+]I, whereas zero Ca2+ solution or nifedipine prevented this.

However, extracellular Ca2+ influx alone is insufficient for generation of ASM Ca2+ waves; Ca2+ uptake/release from the sarcoplasmic reticulum is required for, and regulates, ASM Ca2+ waves. Thus, cyclopiazonic acid, which empties the SR (24), abolished spontaneous Ca2+ waves and peristaltic activity while increasing basal tone. Similar effects have been reported in gravid rat myometria (31). Second, inhibiting SR Ca2+ release via RyR channels, or InsP3R channels abolished Ca2+ wave generation and peristalsis (22, 32, 33). Third, we observed initial stimulation and subsequent abolition of Ca2+ waves when facilitating Ca2+-induced intracellular Ca2+ release with caffeine. This biphasic response may result from activation of not only depolarizing Ca2+-activated chloride channels (with positive feedback and increased contractile frequency), but also large-conductance (BK) Ca2+-activated K+ channels (which produce hyperpolarization and cessation of periodic mechanical activity) (27, 28). Because TEA blockade of Kv and BK channels prevented the effects of 1 mM caffeine, the latter mechanism is suggested.

In this first study to demonstrate prenatal pulmonary Ca2+ oscillations, we have provided clear evidence that peristaltic contractions of the prenatal airway are produced by spontaneous, regenerative, temperature-dependent Ca2+ oscillations of myogenic origin that require extracellular Ca2+ influx, sarcoplasmic Ca2+ uptake, intracellular Ca2+ release via RyR and InsP3R channels, and propagate via ASM cells in a gap junction–dependent manner that is likely to be action potential–mediated. Two striking consequences of these novel findings deserve immediate discussion. First, if as recent data suggest, AP regulates prenatal lung growth (3), then from the present study we can assert that intercellular Ca2+ oscillations in turn regulate antenatal lung development. Calcium signaling certainly participates in diverse roles ranging from transcriptional regulation and cell cycle kinetics (34) to modulation of cellular tension and contractility. Based on the present findings, we now propose that propagation of intercellular Ca2+ oscillations throughout a complex developing organ may be integral to, and help coordinate, morphogenesis.

Second, our demonstration of regular spontaneous Ca2+ waves and phasic contractility in embryonic ASM contrasts sharply with infrequent spontaneous Ca2+ oscillations and tonic contractility in adult ASM (35, 36). The stimulus for transition between ASM phenotypes is unknown. Indeed, although birth may be assumed to mark the phenotypic change, this has not been unequivocally demonstrated. ASM phenotype may be transformed by the same influences that modulate uterine contractility at the onset of labor. Alternatively descending neural control may change ASM behavior: while AP persists beyond structural development of the pulmonary nervous system, our studies show how muscarinic activation and InsP3R-mediated Ca2+ release could induce tonic contractility. Perhaps muscarinic activation of InsP3R-mediated Ca2+ release is incomplete prenatally. Developmental maturation of this pathway might then explain the transition between ASM phenotypes. Clearly further investigation of both pre- and postnatal ASM will be required to test these possibilities.

In summary, we have shown that prenatal airway peristalsis is driven by spontaneous regenerative myogenic Ca2+ oscillations that require and are regulated by extracellular calcium influx and intracellular Ca2+ release. These temperature-sensitive intercellular Ca2+ waves propagate via ASM in a gap junction–dependent manner that is highly likely to be action potential–mediated. Future studies are directed to exploring the relationship of Ca2+ oscillations to lung growth and understanding the differences between pre- and postnatal ASM activity. Integrating Ca2+ physiology and developmental biology should allow us to appreciate hitherto unrealized dynamics of human lung development and function.


   Acknowledgments
 
The authors thank Nicola P. Smith for technical assistance.


   Footnotes
 
Sources of support: Medical Research Council, The Royal College of Surgeons of England, The Academy of Medical Sciences, The Health Foundation, and Birth Defects Foundation.

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

* Joint first authors. Back

Received in original form April 13, 2005

Received in final form May 11, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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