Am J Physiol Cell Physiol AJP: Lung Cellular and Molecular Physiology
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Am J Physiol Cell Physiol 275: C285-C292, 1998;
0363-6143/98 $5.00
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Vol. 275, Issue 1, C285-C292, July 1998

Ca2+ and protein kinase C activation of mucin granule exocytosis in permeabilized SPOC1 cells

C. E. Scott, Lubna H. Abdullah, and C. William Davis

Cystic Fibrosis/Pulmonary Research and Treatment Center and the Department of Physiology, University of North Carolina, Chapel Hill, North Carolina 27599-7248

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mucin secretion by airway goblet cells is under the control of apical P2Y2, phospholipase C-coupled purinergic receptors. In SPOC1 cells, the mobilization of intracellular Ca2+ by ionomycin or the activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) stimulates mucin secretion in a fully additive fashion [L. H. Abdullah, J. D. Conway, J. A. Cohn, and C. W. Davis. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L201-L210, 1997]. This apparent independence between PKC and Ca2+ in the stimulation of mucin secretion was tested in streptolysin O-permeabilized SPOC1 cells. These cells were fully competent to secrete mucin when Ca2+ was elevated from 100 nM to 3.1 µM for 2 min following permeabilization; the Ca2+ EC50 was 2.29 ± 0.07 µM. Permeabilized SPOC1 cells were exposed to PMA or 4alpha -phorbol at Ca2+ activities ranging from 10 nM to 10 µM. PMA, but not 4alpha -phorbol, increased mucin release at all Ca2+ activities tested: at 10 nM Ca2+ mucin release was 2.1-fold greater than control and at 4.7 µM Ca2+ mucin release was maximal (3.6-fold increase). PMA stimulated 27% more mucin release at 4.7 µM than at 10 nM Ca2+. Hence, SPOC1 cells possess Ca2+-insensitive, PKC-dependent, and Ca2+-dependent PKC-potentiated pathways for mucin granule exocytosis.

lung; airways; mucus; goblet cells; cellular regulation

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CALCIUM AND protein kinase C (PKC), the active effectors of the phospholipase C (PLC) signal transduction system, have been implicated widely in the control of regulated exocytosis. In contrast to the apparently universal activation of the exocytotic mechanism by Ca2+, the effects of PKC vary by cell type: stimulatory, inhibitory, and modulatory effects on exocytosis have been reported (see Ref. 31). For mucin-secreting cells of the gastrointestinal tract (18-20) and the airways (1, 16), exocytosis is reported to be activated independently by Ca2+ and PKC; we have tested this apparent independence in a permeabilized cell model using SPOC1 cells, a mucin-secreting cell line from the airways (2, 46).

Native airway goblet cells secrete mucin in response to the interaction of purinergic agonists [ATP, UTP, adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S)] with P2Y2 receptors (= P2U) as do primary cultures of airway epithelial cells and SPOC1 cells (for review, see Ref. 15). Consistent with the known coupling of this receptor class through heterotrimeric G proteins to PLC (10), primary cultures of airway epithelial cells comprised predominantly of mucin-secreting cells release inositol phosphates on stimulation (28). Although intracellular Ca2+ levels have yet to be determined in goblet cells, in SPOC1 cells agents known to elevate intracellular Ca2+ (ionomycin, thapsigargin) also stimulate mucin secretion (1). Similarly, activation of PKC by phorbol 12-myristate 13-acetate (PMA) elicits mucin secretion in several cultured airway cell models (24, 27, 35, 52, 53) and in SPOC1 cells (Ref. 1; see also Refs. 15, 16). Notably, the mucin secretory response to ionomycin and PMA was fully additive at maximal doses in SPOC1 cells. Downregulation of PKC by overnight exposure to a half-maximal dose of PMA abolished the ability of SPOC1 cells to respond to maximal doses of either PMA or UTP, but they responded maximally to ionomycin (1). These results suggest that Ca2+ and PKC are independent in their actions, and, in the experiments reported here, we test whether PMA is effective in promoting mucin secretion in permeabilized cells where Ca2+ is controlled by an exogenous Ca2+ buffer system.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Culture medium was purchased from GIBCO BRL (Gaithersburg, MD), and the supplements were from Collaborative Research (Bedford, MA). Nucleotides were purchased from Boehringer Mannheim (Indianapolis, IN), streptolysin O (SLO) was from Murex Diagnostics (Norcross, GA), and TO-PRO was from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma Chemical (St. Louis, MO).

SPOC1 cell culture and mucin secretion. SPOC1 cells, passages 7-15, were seeded at densities of 18,000 cells/well in 48-well cluster plates (Costar, Cambridge, MA) and were grown in a DMEM-F12-based culture medium described previously (26, 46). Briefly, the medium was supplemented with 30 mM HEPES, 6.5 mM L-glutamine, 10 µg/ml insulin, 0.1 µg/ml hydrocortisone, 0.1 µg/ml cholera toxin, 5 µg/ml transferrin, 50 µM phosphoethanolamine, 80 µM ethanolamine, 25 ng/ml epidermal growth factor, 1% vol/vol bovine pituitary extract, 1 mg/ml bovine serum albumin (essentially globulin-free, Sigma no. A7638), 50 U/ml penicillin, and 50 µg/ml streptomycin. Except for cells grown solely for passaging, the medium also contained 10 nM retinoic acid. Culture media were changed daily, and the cultures were used for experiments 6-12 days postconfluence.

SPOC1 cell mucin secretion and enzyme-linked lectin assay. Before all experiments, SPOC1 cells were removed from the incubator, washed twice in DMEM-F12, and incubated at 35°C for 30 min; this procedure was repeated twice for a total equilibration period of 90 min. To study the response of intact cells to nucleotide agonist challenges, SPOC1 cells were subsequently exposed to UTP or ATPgamma S over a wide range of concentrations, each dose in triplicate, during a single 30-min incubation. Other details of these experiments are given in RESULTS.

Samples of media removed from the wells of the cluster plate were assessed for mucin content by enzyme-linked lectin assay (2): 100-µl samples were bound to 96-well high-binding microtiter plates (Costar no. 3590) with an overnight or a 2-h incubation at 4 or 37°C, respectively. The plates were washed (Dynatech MR5000; Chantilly, VA) with PBS containing 0.05% Tween 20 and 0.02% thimerosal, blocked with gelatin, and incubated with 1-5 µg/ml horseradish peroxidase-conjugated soybean agglutinin for 1 h at 37°C. After plates were washed and developed (incubation in 0.04% wt/vol of the substrate, O-phenylenediamine in 0.0175 M citrate-phosphate buffer, pH 5.0, containing 0.01% hydrogen peroxide), the reaction was stopped by the addition of 4 M sulfuric acid and optical density was determined at 490 nm (Dynatech model MR5000 microtiter plate reader). Optical density was converted to nanograms mucin from a standard curve using known amounts of purified SPOC1 mucin (2); mucin standards were applied to each microtiter plate assessed.

Cell permeabilization and Ca2+ buffering system. Differentiated SPOC1 cells were permeabilized, as epithelial sheets in the bottom of 48-well cluster plates, by SLO (22). SLO was resuspended at 3 U/ml in intracellular buffer (Bufi), which had a final composition (in mM) of 130 potassium glutamate, 20 PIPES, 1.0 MgATP, and 3.0 total EGTA; the pH was adjusted to 6.8, and pCa was adjusted to a desired activity by the addition of CaEGTA. Free Ca2+ levels were calculated with the aid of the computer program Chelator (49); although these activities were calculated in log units of pCa, for convenience, they are expressed in nanomolar or micromolar in the RESULTS and DISCUSSION. Stock solutions of EGTA and CaEGTA, nominally 50 mM, were made according to the Ca2+-buffering system of Gomperts and Tatham (22). EGTA concentrations were determined by titration (38), using a Ca2+ solution made from a freshly opened bottle of CaCl2 · 2H2O; aliquots of these stocks were stored at -20°C. Rapid solution changes during the cell permeabilization procedure were facilitated with a Finnpipette multistepper pipetter (Needham Heights, MA; using 6 of the 8 tips) and a six-tip vacuum manifold fabricated from Delrin and disposable, plastic 100-µl pipette tips. With these tools, the medium in a 48-well cluster plate could be removed and replaced, consistently, in <10 s.

After equilibration (described above), the cells were washed in rapid succession, twice in PBS (400 µl) and twice in pCa 7.0 Bufi (400 µl). Unless stated otherwise, the cells were then permeabilized by a 30-s incubation in pCa 7.0 Bufi containing 1 U/ml SLO (150 µl), subsequently washed twice in pCa 7.0 Bufi (150 µl), and then incubated in a solution for a time appropriate to the experiment (described in RESULTS). In most experiments, this medium was then removed and assessed for mucin content. Last, the degree of cell permeabilization following each experiment was assessed using the membrane-impermeant, DNA-staining dye TO-PRO: the cells were washed with PBS before and after a 10-min incubation in TO-PRO (10 µM in PBS), fixed in 3% formaldehyde in PBS for 10 min, washed, and covered with 200 µl of PBS-glycerol (1:1). Nuclear fluorescence was viewed by video microscopy (Hamamatsu C5985 charge-coupled device camera mounted on a Leica IM/DRB microscope), using a ×5 objective to visualize the central portion of each well. In experiments characterizing the permeabilization procedure, fluorescence intensity was quantified by acquiring an image of each well at constant camera gain and integration period (determined for the brightest well on the plate) and determining the full-frame integrated pixel intensity of each image using a MetaMorph image analysis workstation (Universal Imaging, West Chester, PA). In all other experiments, each well in a plate was checked by fluorescence microscopy to confirm that the cells had been permeabilized, as expected.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

SPOC1 cells grown in 48-well cluster plates. The spatial variation in mucin secretion and production by intact SPOC1 cells grown on 48-well plates was tested by determining the quantity of mucin released during 40-min basal and agonist-stimulated periods (100 µM ATPgamma S) and that in the remaining intracellular pool (lysis in hypotonic buffer: 1 mM CaCl2, 1 mM MgCl2, 20 mM TES, pH 7.4). The mucins released during the basal and stimulated periods and the total cellular mucin (= basal + stimulated + lysis) are shown for three SPOC1 cell passages in Table 1. Consistent with previous results (2), the cells responded to ATPgamma S with a 3.2-fold increase in mucin secretion; this mucin represented 31.1% of the total cellular mucin pool. Well-to-well variation in mucin release and content, determined as the "coefficient of variation," was low for total mucin production (7.1%), moderately higher for the mucins secreted during purinergic stimulation (12.1%), and highest for basal secretion (18%).

                              
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  		Table 1.   Variation in mucin production and secretion by SPOC1 cells grown in 48-well cluster plates

The behavior of SPOC1 cells grown in 48-well cluster plates was also tested by determining the effects of two purinergic agonists on mucin secretion. Dose-response curves for UTP and ATPgamma S were constructed from single plates of SPOC1 cells, with each dose tested in triplicate. As shown in Fig. 1, these curves were sigmoidal; the EC50 was 3-4 µM, and the mucin secretory response saturated above 30 µM, consistent with previous results (2). Together, these two studies show that the cells grown in 48-well cluster plates possess reasonably uniform well-to-well cellular mucin pools and agonist responses, making them good candidates for permeabilization experiments.


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  		Fig. 1.   Purinergic agonist activation of mucin secretion in intact SPOC1 cells. Cells were grown on two 48-well cluster plates and after equilibration were exposed to the indicated doses of agonist for 40 min, with each dose tested in triplicate. Quantity of mucin released into the medium was determined by enzyme-linked lectin assay; mean values for each dose are presented.

SLO permeabilization. In extensive experiments not shown, various SLO permeabilization protocols were tested on SPOC1 cells. These attempts included a SLO exposure and wash at 4°C, followed by the cells being rewarmed such that the permeabilization step occurred after unbound SLO and potential contaminants in the material provided by the manufacturer were removed from the medium (34). The only procedure attempted that produced a consistent activation of mucin release by high Ca2+, however, was a brief exposure to SLO in pCa 7.0 Bufi (100 nM Ca2+) at 35°C followed by an immediate wash. Figure 2 depicts the 35°C dose-permeabilization effects of SLO on SPOC1 cells, including videomicrographs of TO-PRO-stained cells at selected doses. The low molecular weight (MW) dye, TO-PRO (MW 645), was chosen as a marker of permeabilization because it is similar to EGTA (MW 380) in size and its fluorescence excitation and emission spectra are similar to fluorescein. As shown in Fig. 2, SLO effectively permeabilized the cells to TO-PRO at doses above 0.1 U/ml. The clustering pattern of nuclear fluorescence that is observed at 0.3 and 1 U/ml SLO is consistent with the pattern of SPOC1 cell differentiation that occurs in culture (2, 17, 46). That is, the cells grow as extensive patches of multilayered cells, with cells in the outermost layers containing mucin secretory granules. The more uniform staining observed at 3 U/ml reflects the permeabilization of cells in areas of the well occupied by those cells growing as a single layer in contact with the plastic substratum. These cells, and their adjacent counterparts in the multilayered areas, resemble the basal cells of pseudostratified airway epithelia.


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  		Fig. 2.   Streptolysin O (SLO) permeabilization of SPOC1 cells. Cells were exposed to indicated doses of SLO in pCa 7.0 intracellular buffer (Bufi; 100 nM) at 35°C for 10 min and then stained with TO-PRO. Fluorescence from the central portion of each well in 48-well cluster plates was imaged by a ×5 objective, acquired by a cooled charge-coupled device video camera, and quantified as the full-frame, integrated pixel intensity. Insets: sample images for selected SLO doses. Each dose was tested in triplicate on each plate, and data are presented as means ± SE (n = 3 SPOC1 cell passages).

The time courses of SLO permeabilization to TO-PRO at 1 and 3 U/ml are shown in Fig. 3. TO-PRO fluorescence increased rapidly with increasing SLO exposure time at both doses to a pseudo-plateau between 30 and 60 s at 70-80% of maximal. Longer exposures resulted in only moderately higher levels of TO-PRO fluorescence.


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  		Fig. 3.   Time course of SLO permeabilization of SPOC1 cells. Cells were incubated at 1 or 3 U/ml SLO in pCa 7.0 Bufi (100 nM), in triplicate, at 35°C for time periods indicated and then washed and incubated for 10 min in TO-PRO in the same buffer. After fixation, cellular fluorescence was quantified as described in Fig. 2. Data are presented, normalized to the maximal fluorescence on each plate, as means ± SE (n = 3 SPOC1 cell passages).

Mucin release in permeabilized SPOC1 cells. In preliminary experiments, Ca2+-dependent mucin release was maximal following a 30-s exposure of SPOC1 cells to 1 U/ml SLO; a higher SLO dose (3 U/ml) or a longer exposure time (60 s) resulted in a reduced amount of mucin released to the bath (data not shown). Figure 4 shows the results of experiments designed to establish two important parameters related to permeabilization with a 30-s, 1 U/ml SLO protocol, namely, the postpermeabilization period of time necessary to achieve maximal mucin release and recovery and the period of time SPOC1 cells were capable of supporting Ca2+-dependent mucin release following permeabilization. The data show first that a 10- to 15-min incubation in high Ca2+ (3.2 µM) was necessary to achieve a maximal release of mucin to, and recovery from, the medium (Fig. 4A). Second, the permeabilized cells were fully competent to secrete mucin in response to a high Ca2+ stimulus for a minimum of 2 min. When the cells were held at 100 nM Ca2+ for longer periods, they exhibited a reduced response to the subsequent high Ca2+ stimulus such that after 10 min the cells released ~50% of the amount of mucin released by cells stimulated by high Ca2+ immediately following permeabilization. This 2-min window of full secretory competency for permeabilized SPOC1 cells is narrower than that observed in other permeabilized cell models (see DISCUSSION); however, it was sufficiently broad for the purposes of the experiments reported herein. That the cells were capable of maximal secretion for 2 min following permeabilization, and yet 10-15 min were required to achieve a maximal recovery of mucin after stimulation by high Ca2+, suggests that the relatively longer time requirement for mucin recovery resulted from a need for secreted mucins to hydrate, temper, and then solubilize into the medium (see Refs. 12, 51).


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  		Fig. 4.   Temporal parameters for Ca2+-dependent mucin release in permeabilized SPOC1 cells. In one-half of each plate (A), immediately following the pCa 7.0 (100 nM) postpermeabilization wash, Ca2+ was elevated to pCa 5.5 (3.2 µM); cells were incubated for the indicated time intervals, and then the media were harvested for mucin determinations. Cells in the other half of each plate (B) were washed after permeabilization and held at pCa 7.0 (100 nM) for the indicated time intervals, and then pCa was elevated to 5.5 (3.2 µM) for a constant 10-min incubation. Data were normalized to the maximal amount of mucin released on each plate and are presented as means ± SE (n = 3 SPOC1 cell passages).

As expected from the presence of P2Y2 receptors on SPOC1 cell apical membranes, interpretation of the data on mucin release from SLO-permeabilized cells was complicated by the millimolar levels of ATP contained in Bufi. Figure 5 shows the results of an experiment in which SPOC1 cells were exposed to 0.1-3 U/ml SLO followed by a Bufi incubation with Ca2+ ranging from 100 nM to 3.2 µM. Note that mucin release was highest in the cells exposed to the lowest dose of SLO; because the cells were mostly intact at 0.1 U/ml SLO (Fig. 2), mucin release at this dose was due to the activation of P2Y2 receptors by ATP (Fig. 1; Ref. 2), with the subsequent activation of PLC, release of Ca2+ from internal stores, and the production of diacylglycerol (DAG). At 0.3 U/ml SLO, at which an intermediate degree of permeabilization occurs (Fig. 2), the mucins released at all Ca2+ activities were reduced relative to the 0.1 U/ml dose of SLO. Mucin release with 0.3 U/ml SLO was the lowest at the lower Ca2+ activities (100 and 316 nM), thereby indicating some degree of Ca2+ specificity in the overall secretory response. At the full levels of permeabilization produced by 1 and 3 U/ml SLO, SPOC1 cells exhibited a clear Ca2+-dependent mucin-secretory response: at low Ca2+ (100 and 316 nM) mucin release was greatly diminished, and at high levels of Ca2+ it was stimulated. Presumably, the high degree of permeabilization achieved with these doses of SLO allowed efficient intracellular buffering of Ca2+, such that the cells responded to the free Ca2+ controlled by the EGTA-buffer system rather than to ATP activation of P2Y2 receptors. Last, note in Fig. 5 that SPOC1 cells permeabilized with 1 U/ml SLO achieved a release of mucin at 3.2 µM Ca2+, which was 82.9% of that secreted by the agonist-stimulated cells (0.1 U/ml SLO), showing that the permeabilization procedure had relatively minor effects on the ability of the cells to secrete.


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  		Fig. 5.   Mucin release by SPOC1 cells permeabilized with different doses of SLO. After a 30-s permeabilization at pCa 7.0 (100 nM) with indicated doses of SLO, cells were washed and incubated at the indicated Ca2+ activities for 15 min; mucins released to the bath were then quantified. Data were normalized to the maximal amount of mucin on each plate and are presented as means ± SE (n = 3 SPOC1 cell passages). Note that mucin release at all Ca2+ activities was highest at the lowest doses of SLO (see text for explanation).

Ca2+-dependent and PKC-dependent mucin release. The effects of Ca2+ on mucin release in permeabilized SPOC1 cells was investigated in a fine-grain, Ca2+ dose-response study on cells grown in single 48-well cluster plates over four separate passages. Figure 6, depicting an individual result, shows that relative to control of 100 nM Ca2+ mucin release by permeabilized SPOC1 cells incubated at 10 nM Ca2+ was moderately inhibited (36 ± 12%, n = 4). This apparent reduction in mucin release at lower than normal Ca2+ activities, however, was not always observed. In other experiments, there was no difference between the mucins released at 10 and 100 nM Ca2+ (e.g., see Figs. 5 and 7). Mucin release in these studies was slightly increased at Ca2+ activities between 100 nM and 1 µM Ca2+, strongly stimulated above 1 µM Ca2+, and saturated above 10 µM Ca2+, with a maximal 2.49 ± 0.55-fold increase over control. The Ca2+ EC50 for the mucin secretory response was 2.29 ± 0.07 µM.


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  		Fig. 6.   Ca2+-dependent mucin release in permeabilized SPOC1 cells. Result is shown of an individual experiment in which SPOC1 cells were permeabilized with 1 U/ml SLO (30 s), washed, and incubated for 15 min in Bufi containing the indicated Ca2+ activities. Data are presented as mean values of triplicate wells, normalized to the amount of mucins released at pCa 7.0 (100 nM). Ca2+-dependent waveform depicted is typical of 4 experiments; summary data are given in RESULTS.

To test whether PKC activation of mucin granule exocytosis is dependent on or independent of Ca2+, SPOC1 cells grown in pairs of 48-well plates were permeabilized and incubated at Ca2+ activities ranging from 10 nM to 10 µM. One-half of the cells on one plate was exposed to a maximal dose of PMA (300 nM; Ref. 1), whereas one-half of the cells on the other plate was exposed to the same concentration of 4alpha -phorbol, an inactive phorbol ester. PMA stimulated mucin release in permeabilized SPOC1 cells over the entire range of Ca2+ activities (Fig. 7). At 10 nM Ca2+, which is approximately one-tenth of basal intracellular Ca2+ levels in most cells, mucin release was stimulated 2.1-fold over the 100 nM Ca2+ control. This stimulation by PMA, in fact, was as strong as the maximal Ca2+-dependent response, a 2.1-fold stimulation at 10 µM Ca2+. As Ca2+ stimulated mucin granule exocytosis at activities >1 µM, the PMA response was potentiated. At 4.7 µM Ca2+, PMA-stimulated cells released 27% more mucin relative to their paired controls than at 10 nM Ca2+ (P < 0.05, paired t-test); the Ca2+ EC50 for cells exposed to PMA was 1.0 µM compared with 2.2 µM for the paired control. The PMA response at 2.1 µM Ca2+ appeared to be diminished relative to the maximal levels recorded at 4.7 and 10 µM, but it was still significantly higher than its paired control. Because 4alpha -phorbol had no discernible effect at any Ca2+ activity, the PMA effects appeared to be specific and were presumably due to PKC activation.


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  		Fig. 7.   Effects of phorbol 12-myristate 13-acetate (PMA) on mucin release in permeabilized SPOC1 cells. After permeabilization, one-half of the SPOC1 cells on each 48-well cluster plate was treated with 300 nM 4alpha -phorbol (black-triangle) or PMA (bullet ) and the other half served as control (open symbols). Cells were exposed to the indicated range of Ca2+ activities, in triplicate, for a 15-min incubation. Mucin released in each well was normalized to that released under control conditions (pCa 7.0 or 100 nM), and data are presented as means ± SE (n = 4 SPOC1 cell passages). For clarity, SE bars not shown for the 4alpha -phorbol control data. PMA values are all different from control, P < 0.05, paired t-test. Dotted line indicates a 2-fold stimulation over the pCa 7.0 (100 nM) controls.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Regulated exocytosis by neurons and secretory cells has been the focus of intense investigation over the past 20 years (recent reviews in Refs. 4, 41, 57). These efforts were aided substantially by the development of permeabilization techniques pioneered by Baker et al. (7, 30) and Gomperts et al. (8, 21, 25), which allowed access to and control of the intracellular milieu (see also Refs. 3, 9, 39). Many permeabilized secretory cell models have been subsequently described for study of the control of exocytosis by intracellular Ca2+, PKC, nucleotides, and specific proteins (e.g., see Refs. 11, 22, 23, 34). Pancreatic acini, however, represent the only other permeabilized epithelial cell model so developed, and this study with SPOC1 cells represents the first model developed for an epithelium studied in a polarized configuration. The bacterial toxin SLO was chosen as the permeabilization agent for these studies to ensure efficient intracellular Ca2+ buffering by EGTA. This toxin binds plasma membrane cholesterol and then polymerizes to form ring-shaped 24- to >30-nm pores that render the plasma membrane permeable to organic molecules as large as lactate dehydrogenase (MW 140,000; Refs. 3, 50). Use of ionophores to control intracellular Ca2+ is impractical because of the difficulties in controlling the intracellular quantities of permeant EGTA or related buffers. Use of alpha -toxin for this purpose is also questionable because of restricted EGTA diffusivity through its 2- to 3-nm pores. In alpha -toxin-permeabilized gonadotropes, for instance, 30 mM CaEGTA buffers, or a 20-min preequilibration with 10 mM CaEGTA buffers at 0°C, were required to effectively demonstrate Ca2+-activated luteinizing hormone release (6). Because efficient Ca2+ buffering was required in these studies with SPOC1 cells to test the effects of PKC activation at very low levels of intracellular Ca2+, we chose to use SLO.

Permeabilization was most efficient with short luminal SLO exposures of SPOC1 cells at 35°C, followed by a rapid wash in Bufi. These cells were fully competent to secrete mucin in response to an elevation in Ca2+ for 2 min following permeabilization (Fig. 4). In other SLO-permeabilized cells, the period of secretory competency is tens of minutes in duration (e.g., see Ref. 29, 55), and secretory activity in rundown cells can be restored through the addition to the medium of cytosol (14, 47) or purified proteins (37, 43, 44). Under optimal conditions (30-s exposure to 1 U/ml SLO; Figs. 3 and 5), and following a maximal activation by Ca2+, permeabilized SPOC1 cells released a quantity of mucin only ~20% less than that secreted by intact cells following purinergic stimulation (Fig. 5). Thus, although brief, the window of secretory competency for permeabilized SPOC1 cells was sufficiently broad and the secretory response was sufficiently robust to allow the experiments necessary to test for Ca2+- and PKC-activated exocytotic release of mucin.

Permeabilized SPOC1 cells exhibited a graded mucin secretory response to increases in Ca2+ activity (Figs. 5-7), with the initial responses occurring above 320 nM. In preliminary measurements with intact SPOC1 cells, we have determined basal Ca2+ activities to be 80-100 nM (data not shown). Consequently, these cells are similar to virtually every other secretory cell studied in possessing a secretory pathway activated by Ca2+ at suprabasal levels. The Ca2+ EC50 of this response in SPOC1 cells, 2.29 ± 0.07 µM, was in the same low micromolar range described for most other permeabilized cells (e.g., see Refs. 13, 29, 33, 36, 40, 48). These data are consistent with the positive effects of ionomycin and thapsigargin on intact SPOC1 cells (1), and together they lend strong support for a role of Ca2+ in mediating agonist responses in airways mucin secreting cells (see also Ref. 16; cf. Ref. 32).

The effects of PKC activation in secretory cells are more varied than the effects of Ca2+. At Ca2+ activities below the nominal 100 nM basal intracellular Ca2+ levels, some cells are not affected by PMA or other PKC-activating reagents (i.e., pancreatic acini, Ref. 29; chromaffin cells, Ref. 30); however, most secretory cells exhibit some degree of PKC-activated exocytosis (e.g., gonadotropes, Ref. 36; PC-12 cells, Ref. 48). At 10 nM Ca2+, SPOC1 cells were powerfully stimulated by PMA; the amount of mucin released in response to PMA under these conditions was the same as that released maximally by micromolar levels of Ca2+ (Fig. 7). Indeed, in the robustness of this PMA response at subbasal Ca2+ levels, SPOC1 cells stand out from all other secretory cells studied.

In most other secretory cells, PMA has been shown to potentiate Ca2+-dependent responses (e.g., PC-12 cells, Ref. 48; mast cells, Ref. 40). In SPOC1 cells, PMA had slight synergism with Ca2+, with the PMA-related increase in Ca2+-dependent secretion being 27% greater than the effects of PMA at subbasal Ca2+ (Fig. 7). Given the apparent additivity of ionomycin and PMA in intact SPOC1 cells (1), this minor degree of synergism between PKC and Ca2+ is not surprising.

PKC has been shown in recent years to be a family of at least 11 isoforms that may be categorized into 3 or 4 subfamilies (for review, see Ref. 42). Pertinent to this discussion are those isoforms activated by phosphatidylserine and DAG or PMA, that is, the conventional or cPKC isoforms (which are Ca2+-dependent) and the novel or nPKC isoforms (which are Ca2+-insensitive) (PMA does not activate the atypical isoforms or PKCµ). Because secretion in SPOC1 and other cells is activated by PMA at subbasal Ca2+ levels, a likely possibility is that nPKC isoforms will prove to be responsible for this effect. Recent data in fact support this notion: nPKC isoforms have been implicated in the agonist regulation of secretion for colonic cell lines (24), pancreatic acini (45), and lachrymal glands (56), and overexpression of nPKCepsilon in GH4 cells leads to a selective increase in basal prolactin secretion rates (5). In other secretory cells, however, cPKC isoforms have been implicated in modulating secretion (e.g., RBL cells, Ref. 11). Hence, the PKC isoforms active in activating and/or modulating secretion are likely cell-type dependent.

In conclusion, the purinergic regulation of mucin secretion in SPOC1 cells appears to possess Ca2+-independent, PKC-activated, and PKC-potentiated Ca2+-dependent pathways; in this regard, they are similar to many other nonepithelial secretory cells but not to pancreatic acini. The identities of the PKC isoforms responsible for Ca2+-independent mucin secretion and the degree of independence between these two pathways at the molecular level require further investigation. For the latter topic, a major question is whether multiple exocytotic mechanisms exist in a given secretory cell type or, alternately, whether the apparent independence between Ca2+ and PKC lies with one or more rate-limiting steps (e.g., cortical microfilaments; Ref. 54) situated proximal to exocytotic docking sites.

    ACKNOWLEDGEMENTS

We gratefully thank Drs. Peter Tatham and Bastien Gomperts for critical advice and encouragement during these studies.

    FOOTNOTES

This work was supported by a research grant from Glaxo Wellcome.

Address for reprint requests: C. W. Davis, 6009 Thurston-Bowles, CB 7248, Univ. of North Carolina, Chapel Hill, NC 27599.

Received 15 September 1997; accepted in final form 6 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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