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Calcium signaling in human airway goblet cells following purinergic activation

¹Department of Cell and Molecular Physiology, ²Michael Hooker Microscopy Facility, and ³Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina

Submitted 7 March 2006 ; accepted in final form 17 August 2006

	ABSTRACT

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Despite the general importance of Ca²⁺ signaling in signal transduction, and of goblet cell mucin hypersecretion in inflammatory pulmonary diseases, measurement of airway goblet cell intracellular Ca²⁺ (Ca) has not been reported. In this article, we describe the results of experiments measuring Ca in primary cultures of human bronchial goblet cells after stimulation with the purinergic agonist adenosine 5'-O-(3-thiotriphosphate) (ATPS) and phorbol 12-myristate 13-acetate (PMA). Ca²⁺ signaling in human goblet cells after purinergic stimulation follows the classic paradigm of a Ca transient from a basal activity of 110 nM to a peak response of 260.1 ± 41.2 nM within 2 min, followed by a long superbasal plateau (155.3 ± 0.2 nM) between 10 and 15 min. The rise in Ca appears to result from a mobilization of intracellular stores, because the transient was nearly abolished by inhibition of PLC with the phosphatidylinositol-specific PLC inhibitor U-73122, and it was not affected significantly by removal of extracellular Ca²⁺. Loading goblet cells with BAPTA inhibited the ATPS-induced Ca²⁺ transient by 86.0 ± 13.1%, relative to control. Finally, in contrast to the massive effects of high doses of PMA (300 nM) on mucin secretion from goblet cells, phorbol ester stimulated a small (27.1 ± 7% of the ATPS control peak), brief rise in Ca. This diminutive signal likely denotes a local Ca²⁺ gradient, which may be associated with the mucin granule exocytotic process.

mucus; exocytosis; purinergic signaling; P2Y₂

P2Y₂ purinoceptors represent a major G protein-coupled receptor (GPCR) pathway regulating mucin secretion from airway goblet cells (3, 9, 11, 22). Consistent with a coupling of P2Y₂ receptors to PLC (18), mucin secretion is stimulated by the diacylglycerol mimic phorbol 12-myristate 13-acetate (PMA) (2, 11, 22, 26) and by maneuvers that mobilize intracellular Ca²⁺ (Ca) [ionomycin, inositol 1,4,5-trisphosphate (IP₃), thapsigargin] (2, 11, 22, 39), and it is inhibited by sequestering Ca(BAPTA) (22). Beginning with the demonstration in 1965 that synaptic transmission is Ca²⁺ dependent (21), we now know that Ca regulates numerous critical processes leading to the exocytotic release of neurotransmitters and secreted proteins from neurons and secretory cells. In secretory cells, numerous studies have suggested that Ca²⁺ is the trigger for exocytosis during at least three distinct stages: 1) disassembly of the actin cytoskeleton (42) to permit secretory granules to access exocytotic sites on the plasma membrane, 2) priming of the docked granule (24), and 3) triggering of the fusion event between secretory granule and plasma membranes (4, 15, 41) to allow the release of secretory granule contents. In this article, we offer the results of the first experiments to measure Ca in airway goblet cells, after activation by purinergic agonists, with the goal of defining the fundamental Ca²⁺ signaling mechanisms in this important cell type preparatory to more advanced studies.

	MATERIALS AND METHODS

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Materials. Chemicals were obtained from the following sources: adenosine 5'-O-(3-thiotriphosphate) (ATPS) from Roche Applied Science (Indianapolis, IN); PMA from Calbiochem (La Jolla, CA); BAPTA-AM, fura-2 AM, and a Ca²⁺ calibration kit from Molecular Probes (Eugene, OR); U-73122 and D-609 from Biomol (Plymouth Meeting, PA); HEPES from Media Tech (Herndon, VA), MnCl₂, NaCl, and Na₂HPO₄ from Mallinckrodt (Hazelwood, MO); and digitonin, CaCl₂, EGTA, MgCl₂, KCl, and -tubulin monoclonal antibody from Sigma Chemical (St. Louis, MO). Dermabond, a nontoxic cyanoacrylate tissue adhesive, was obtained from Ethicon Incorporated (Somerville, NJ), and Sylgard 184 was obtained from Dow Corning (Midland, MI).

Human bronchial epithelial cell culture and staining procedure. Human bronchial epithelial (HBE) cells were obtained in accordance with Institutional Review Board-approved protocols, as described previously (29, 30, 36), from normal human bronchi. Briefly, HBE cells were isolated and grown on plastic culture dishes in bronchial epithelial cell growth medium (BEGM) (17) and passaged at 80% confluence, and first-passage cells were seeded onto 12-mm Transwell-Col supports (TCols; Costar) at 250,000 cells per support. After confluence was obtained, the cells were maintained under air-liquid interface (ALI) conditions in ALI culture medium (BEGM modified per Refs. 29, 36), which was changed at the basolateral surface three times a week. HBE cell cultures were used for experiments 4–6 wk after confluence, a time when the columnar cells are well differentiated as ciliated or goblet cells.

Preparations were stained for cilia and mucin identification (see Fig. 1). HBE cell cultures on TCols, derived from five different donors, were gently washed to remove luminal mucus, fixed from the luminal side in 4% paraformaldehyde for 5 min, permeabilized with cold methanol for 3 min, stained for mucin with the periodic acid biotin-hydrazide (PABH) procedure (11), and then immunostained for -tubulin with a rat-derived monoclonal antibody and standard procedures.

View larger version (54K): [in this window] [in a new window]   Fig. 1. Experimental preparation and human bronchial epithelial (HBE) cell culture. A: schematic of perfusion chamber used for HBE cell cultures, incorporating a Bioptechs (heating) 35-mm culture dish. Note that the culture's luminal aspect faces the objective of an inverted microscope and is perfused with warmed medium; the serosal bath is static. After assembly, the chamber is fitted into a holder on the stage of the microscope, which holds the components together. WD, working distance. B: HBE cell culture, 4 wk after confluence, stained for mucin [periodic acid biotin-hydrazide (green); Ref. 11] and -tubulin (red). The image represents a maximum projection of a z-series of 24 x-y optical sections acquired by a Zeiss 510 confocal microscope at a z-axis interval of 1.95 µm. Scale bar = 20 µm.

Microscopy and Ca_i²⁺ measurements. A Nikon (Tokyo, Japan) TE-2000 inverted microscope and x60 water immersion objective (1.0 numerical aperture, 2.0-mm working distance) were used to view HBE cells by simultaneous differential interference contrast (DIC) and fluorescence microscopy, with a Nikon dual image module, under the control of Simple PCI software (Compix, Cranberry Township, PA). A DVC-1312 cooled charge-coupled device camera (DVC, Austin, TX) collected red transmitted light (605–615 nm) for DIC imaging, and a DVC-1412 Intensicam (Gen III intensifier; 1,392 x 1,040 pixels x 12 bits) was used to collect fura-2 fluorescence (510 ± 40 nm). Fura-2 was excited alternatively at 340 ± 5 and 380 ± 5 nm with a 175-W xenon arc lamp integral to a DG-4 rapid-switching illuminator (Sutter Instruments, Novato, CA). Fura-2 fluorescence at the two excitation wavelengths and the DIC images were collected every 3 s, using acquisition times for excitation at 340 and 380 nm of 250 and 200 ms, respectively. The DIC images were used to distinguish between ciliated cells and goblet cells (see Fig. 1B) as well as to generally monitor the appearance of the preparation.

At the end of an experiment, a solution containing digitonin (20 µM) and MnCl₂ (2 mM) was used to quench the dye. Background fluorescence images were obtained at each excitation wavelength, which then were subtracted from the images at each wavelength collected during the experiment. Simple PCI software was used to define a region of interest containing 8–10 goblet cells in each preparation for offline analysis. Ca activities were calculated and reported in nanomoles per liter only for the experiment depicted in Fig. 2; an external Ca²⁺ calibration kit (Molecular Probes) was used for this purpose. For all other experiments, the data are presented as the ratio of fura-2 emission at 510 nm with 340-nm and 380-nm excitation.

View larger version (15K): [in this window] [in a new window]   Fig. 2. ATP-induced Ca2+ response in HBE cultures. Fura-2-loaded HBE cultures were perfused apically with buffer. The solution was changed after 5 min to expose the cultures to adenosine 5'-O-(3-thiotriphosphate) (ATPS; 100 µM) for 20 min. The fluorescence at 510 nm, elicited by alternate excitation at 340 and 380 nm, was collected at 3-s intervals, and ratios were calculated offline after subtracting background fluorescence obtained after permeabilization of the cells with digitonin (20 µM) and quenching fura-2 fluorescence with 2 mM manganese. Ca2+ activities for this experiment were calculated with the Kd of fura-2 (127 nM), as determined by external calibrations. In this and all other experiments, the 3-s interval data from 5 preparations are presented as means ± SE. Generally, the symbol representing the mean is displayed in white so that it shows up against the dark envelope formed by the tightly spaced error bars.

	RESULTS

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HBE cell cultures grown on TCols were readily imaged at fair resolution in the perfusion chamber. Ciliated cells were easily identified by their incessantly beating cilia, and a short video clip was recorded at the beginning of each experiment to aid in their identification during offline analysis. We were unable to clearly visualize mucin secretory granules in the living, nonciliated, columnar cells in the cultures, but we were usually able to observe the accumulation of mucus on their luminal surfaces following ATPS stimulation, indicating they were goblet cells. Unfortunately, the mucus could not be imaged clearly because it is optically transparent. We observed it to appear on the luminal surface when the cells were stimulated, however, as clear blobs that grew in size with time after it initially appeared and that could be washed away. To test the veracity of this identification, we stained several intact HBE cell cultures for cilia, using an antibody against -tubulin, and for mucin, using the PABH stain (11). Figure 1B shows a typical result: the columnar cells visible at the luminal aspect of the culture stained either for axonemal tubulin or for mucin. In the five cultures derived from different patients so assessed the columnar cells observed stained for tubulin or for mucin, but none stained for both. To minimize the possible inclusion of data derived from an occasional nonciliated, nongoblet cell, we routinely measured the Ca²⁺ responses from 8–10 nonciliated cells in each preparation and averaged the results.

ATPS-induced calcium signals in human airway goblet cells. ATP, UTP, and ATPS are approximately equipotent in the stimulation of mucin secretion from airway goblet cells (3, 11, 22), which indicates a lack of a significant expression of P2Y₁₁ receptors (10, 35). Hence, to stimulate P2Y₂ receptors fairly specifically, we used ATPS as an agonist, taking advantage of its poor hydrolyzability to diphosphate nucleotides. Figure 2 depicts the Ca response of HBE goblet cells to ATPS over a time course of 20 min. Immediately subsequent to this experiment, a Ca²⁺ calibration procedure was performed to allow the data to be expressed directly as activities. Basal Ca was 110.7 ± 0.3 nM, and ATPS triggered a rise to a peak of 260.1 ± 41.2 nM, after which Ca spontaneously declined to a sustained plateau lasting for the duration of the experiment of 155.3 ± 0.2 nM (measured between 10 and 25 min). Hence, HBE goblet cells appear to respond to a PLC-coupled GPCR agonist like most cells, with a classic peak-and-plateau type of Ca response (13, 18, 31).

To determine whether sequential stimuli could be used with these cells to gain the advantage of internally controlled experiments, HBE cell cultures were exposed to ATPS twice, 5 min each, separated by a 15-min washout. ATPS triggered a peak Ca response in each instance (Fig. 3A) that was similar to the peak elicited in the previous experiment, and there was no significant difference between the Ca peaks elicited by the first and second exposures to ATPS (Fig. 3B). These results suggest that successive agonist challenges, bracketing a 15-min washout period, elicit statistically similar responses.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Intracellular Ca2+ (Ca) response to successive ATPS exposures. HBE cultures were exposed to ATPS (100 µM) for 5 min, perfused with buffer for 15 min (washout period), and then exposed to ATPS a second time. A: time course of the 2 Ca responses. B: peak changes () in Ca, with the data plotted as absolute values of the changes in fura-2 fluorescence ratio or normalized to control (first peak). Note that in this and other similar plots the y-axis represents changes in both the 340-to-380 ratio and the normalized data (n = 5).

Dependence of ATPS-induced Ca increase on PLC and extracellular Ca²⁺.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Effect of PLC inhibition on the ATPS-induced Ca2+ peak response. HBE cells were exposed to ATPS (5 min) and then either to D-609, a phosphatidylcholine-specific PLC inhibitor (10 µM; A), or U-73122, a phosphatidylinositol-specific PLC inhibitor (30 µM; B), during the wash period and during the second exposure to ATPS. C: quantification of the peak Ca2+ response in the presence of D-609 and U-73122, showing the absolute and normalized data (*P < 0.005, n = 5).

View larger version (18K): [in this window] [in a new window]   Fig. 5. Effect of extracellular Ca2+ (Cao2+) on the ATPS-induced Ca2+ response. A: HBE cultures were exposed to ATPS (100 µM) in buffer containing 2 mM Ca2+ and then to buffer lacking Ca2+ and containing EGTA (1 mM) for 10 min. ATPS in the nominally free Ca2+ solution was then applied for 5 min. B: quantification of the peak Cai2+ response with and without Cao2+. There was no significant difference between the ATPS-induced peak Ca2+ response in the presence and absence of Cao2+ (n = 5).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Effect of Ca buffering by BAPTA on the ATPS-induced Ca2+ response. A: HBE cultures were loaded with BAPTA-AM (250 µM for 1 h) and then exposed to ATPS for 5 min. In this experiment, the controls were cultures derived from the same patients as the BAPTA-loaded cells. B: quantitation of the peak Ca2+ response without and with BAPTA loading, normalized to the ATPS-induced peak Ca2+ response of the paired controls (*P < 0.005, n = 5).

Effects of PMA on Ca.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Effects of phorbol 12-myristate 13-acetate (PMA) on Ca. HBE cultures were exposed to PMA (300 nM) for 5 min, after exposure to ATPS (100 µM; 5 min) and a 10-min recovery period. Note that both the peak of the small PMA-induced Ca response (0.271 ± 0.082 relative to the peak ATPS Ca response) and the slope of its onset were significantly different from the pre-PMA baseline (*P < 0.05, n = 5).

	DISCUSSION

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ATPS-induced Ca²⁺ response in HBE cells.

Important to our experimental strategy, successive 5-min exposures to ATPS, separated by a 15-min washout, elicited comparable responses (Fig. 3), suggesting that the cells were capable of generating a full Ca²⁺ response shortly after a previous exposure. In previous work (e.g., Ref. 11), we found that long initial exposures to saturating amounts of agonist lead to poor mucin secretory responses on rechallenge. This poor second response is likely due to goblet cell stores being depleted over the first 20–30 min of initial agonist exposure (see Ref. 22). Hence, in the present studies we kept the exposures to agonist brief to avoid excessive depletion of mucin stores.

P2Y₂ receptors typically couple to PLC (18), and as therefore expected Ca²⁺, IP₃, and PMA all stimulate mucin secretion (1, 2, 11, 22, 26, 39). Thus we tested whether PLC inhibition affects the ATPS-induced Ca²⁺ response in human airway goblet cells and found that the phosphatidylinositol-specific PLC inhibitor U-73122 nearly abolished ATPS-induced Ca²⁺ response in goblet cells (Fig. 4, B and C). The phosphatidylcholine-specific PLC inhibitor D-609 had no effect on Ca(Fig. 4, A and C), a result that stands in contrast to its recently reported inhibitory effects on ATP-induced mucin secretion from HBE cells (22). Other studies with PLC inhibitors in airway epithelia generally support the notion that U-73122 inhibits cellular responses to ATP, including inhibition of Ca in rabbit tracheal epithelial cells (14) and of mucin secretion in human tracheobronchial epithelial cells (9). Hence, the Ca²⁺ response caused by exposure of HBE goblet cells to ATPS most likely activates PLC, with a subsequent, sequential liberation of IP₃ to the cytosol and mobilization of Ca²⁺ from the intracellular stores.

Requirement for Ca. The requirement for Ca²⁺ in regulated exocytosis can be traced back to original work on the neuromuscular junction, where it was shown that removal of Ca inhibited synaptic transmission (21). The Ca²⁺ dependence of regulated exocytosis was later confirmed in nonexcitable secretory cells subjected to various membrane permeabilization procedures (e.g., Refs. 5, 6), but it was only studies enabled by the development of Ca²⁺-sensitive fluorescent dyes that revealed that these cells secreted in response to agonist, in the absence of Ca(e.g., Refs. 16, 20). Hence, our findings that removal of Ca had no significant effect on the response of goblet cells to ATPS (Fig. 5) is consistent with work on regulated exocytosis in other secretory cells. It is interesting, however, that a recent report suggests that Ca²⁺ entry across the apical membrane of HT-29 cells influences the kinetics of mucin granule exocytosis: removing Ca caused a delay in the Ca transient and strongly attenuated the peak change in membrane capacitance (7, 27). Because increases in membrane capacitance equate to exocytotic events only during the initial phase of a secretory response, i.e., before significant amounts of membrane are lost to endocytic retrieval, the dependence of HT-29 cell mucin secretion on Ca over exposures longer than the first 30 s or so is uncertain. Our results with HBE cells (Fig. 4) suggest that any dependence of a full mucin secretory response on external Ca²⁺ is likely a modest one.

PMA-related Ca²⁺ release. Previous studies in our laboratory (2, 11) showed that PMA elicits mucin secretion in SPOC1 and HBE cells. In SPOC1 cells, PMA likely activates PKC and other proteins to stimulate mucin secretion: PMA caused the translocation of PKC and small increases in mucin secretion at doses <30 nM, whereas the major effects of PMA to stimulate mucin secretion occurred at higher doses, between 100 and 1,000 nM (1). Initial studies using EGTA to probe the relationship between PMA and Ca²⁺ (ionomycin) effects in SPOC1 cells permeabilized with streptolysin O suggested that PMA-induced mucin secretion in SPOC1 cells was independent of Ca²⁺ (40). Using the faster binding kinetics of BAPTA, however, we later found (39) that PMA-induced mucin secretion in SPOC1 cells was dependent on local Ca²⁺ gradients. Therefore, we monitored the Ca response following exposure to maximal doses of PMA in goblet cells and found that the PMA-related Ca²⁺ increase was small (27.1 ± 7%), compared with the ATPS-induced Ca²⁺ response (Fig. 7). The change was too small to reflect a full, agonist-like effect on Ca mobilization. One explanation for this diminutive change in Ca²⁺ is that PMA triggers generation of localized Ca²⁺ gradients at the apical membrane that are barely detectable above the basal, whole cell fluorescence collected by wide-field microscopy. Interestingly, our recent permeabilized SPOC1 cell studies (39) provided evidence for such a local Ca²⁺ release: PMA stimulated mucin granule exocytosis that depended on local, IP₃-independent Ca²⁺ gradients. The Ca²⁺ transient induced by PMA in the present study (Fig. 7) was observed regardless of whether Ca was removed or PLC was inhibited, suggesting that Ca²⁺ is released from an intracellular store, independent of IP₃. Interestingly, there is precedence for local Ca²⁺ release from yeast vacuoles occurring before the final stage of exocytotic membrane fusion (34), which suggests that an IP₃-independent mechanism may exist to trigger local Ca²⁺ release from vesicles that serves to trigger exocytosis (see Ref. 39). However, it is also possible that PMA stimulates a local Ca²⁺ release from elements of the endoplasmic reticulum in close juxtaposition to the granule and plasma membranes (33).

In summary, Ca²⁺ signaling in human goblet cells after purinergic stimulation follows the classic paradigm of a Ca transient with a peak response of 2.4-fold over a basal activity of 110 nM, followed by a long superbasal plateau. The rise in Ca appears to result from a mobilization of intracellular stores, because the transient was nearly abolished by inhibition of PLC and was not affected significantly by removal of Ca. Finally, in contrast to the massive effects of PMA on mucin secretion, phorbol ester stimulates but a small, brief rise in Ca. This effect of PMA on Ca may relate more directly to the exocytotic mechanism than to a direct effect of the compound on a Ca²⁺ release mechanism.

	GRANTS

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The studies reported were supported by Grant HL-063756 from the National Heart, Lung, and Blood Institute.

	ACKNOWLEDGMENTS

 
We thank Dr. Patrick Sears for valuable discussions over the course of this work.

	FOOTNOTES

 

Address for reprint requests and other correspondence: C. W. Davis, CF/Pulmonary Research & Treatment Center, 6009 Thurston Bowles, Univ. of North Carolina, Chapel Hill, NC 27599-7248 (e-mail: cwdavis{at}med.unc.edu)

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

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