JOURNAL OF CELLULAR PHYSIOLOGY 147:13%148 (1991)
Growth and Differentiation of Primary Tracheal Epithelial Cells in Culture: Regulation by Extracellular Calcium WALTER R. MARTIN, CHRIS BROWN, YAO JUANZHANG, AND REEN WU* Pulmonary and Critical Care Medicine, Department of lnternal Medicine, School of Medicine (W.R.M., R. W.) and California Primate Research Center (W.R.M., C.B., Y.).Z., R. W.), University of California, Davis, California 956 16 Growth and differentiation of primary monkey tracheal epithelial (MTE) cells maintained on collagen gel substrata were studied in a defined serum-free culture medium containing 0.03 to 3.0 m M extracellular calcium. Cell attachment efficiency (40-60%) was not altered by different calcium levels. Growth of primary MTE cells on collagen gel substrata, which was vitamin Adependent, was enhanced 50% in the medium supplemented with high calcium (>0.3 mM). High calcium medium also increased cell-cell interactions, formation of desmosomes, and multi-cell layering. The relative content of mucous cells, which were identified by a mucin-specific monoclonal antibody and the presence of mucussecreting granules at the ultrastructural level, was greater in the high-calcium medium. Furthermore, the secretion of mucin into the medium, determined either by an ELISA or by the incorporation of 'H-glucosamine into mucous glycoprotein fractions, was also increased more than 5-fold in media containing high calcium content (>0.6 mM). In contrast, MTE cells cultured in low calcium medium ( O X mM), while a one-to-two cell thickness layer was observed in low calcium medium containing vitamin A (Fig. 4). The intercellular spaces were also wider in low calcium medium (Fig. 4A,B) than were observed in high calcium medium (Fig. 4C,D). This phenomenon was further examined at the ultrastructural level. More cell-cell interactions and
143
EFFECTS OF CALCIUM ON CULTURED MTE CELLS
membrane interdigitation were observed in cultures with high calcium medium (Fig. 5B,C). In low calcium medium, microvilli were extensively developed (Fig. 5A), but cell-cell interactions were greatly reduced. Prominent tonofilament bundles were frequently observed in cells of the basal cell layer of low calcium cultures even in the presence of vitamin A (Fig. 6A). However, in the basal cell layer region of high calciumsupplemented cultures, many desmosomes but no tonofilament bundles were found (Fig. 6B). Re ardless of the calcium concentration in the medium, t icker and more abundant tonofilament bundles were observed in cells maintained in vitamin A-depleted medium (data not shown). It was possible to demonstrate the presence of conspicuous mucus-secreting granules in the apical region of cells that were maintained in high calcium and vitamin A-supplemented medium (Fig. 6C), an infreauent observation in cells maintained in low calcium medium. Effects of calcium on mucous cell differentiation. Effects of calcium on the expression of mucous cell functions in culture were examined by both immunological and biochemical methods. Monoclonal antibody 17B1, specific to humanimonkey tracheal mucin and mucus-secreting granules (St. George et al., 1985; Lin et al., 1989), was used to identify mucous cell populations in culture and to quantify mucin secreted in the culture medium. In the high calcium medium, more cells were stained with the monoclonal antibody and some of the stains were in a granular pattern (Fig. 7B). In contrast, few cells were stained in low calcium cultures (Fig. 7A). The amount of mucin secreted into the culture medium, as determined by the ELISA method (Lin et al., 1989), was also dependent on the calcium concentration (Fig. 8). Cells cultured in high calcium medium (>0.6 mM) secreted 5 times more mucin than those maintained in medium containing 0.1 mM calcium or less. To further evaluate the effects of calcium concentration in the medium on mucin synthesis, 3H-glucosamine was used to label the mucin-like glycoproteins synthesized in culture. Previous studies have demonstrated that the 3H-glucosamine-labeled macromolecules isolated from the Vo fractions of a Sepharose CL-2B column are a mixture of hyaluronate and mucin-like molecules (Kim et al., 1985; Wu et al., 1985a,b, 1990). In order to quantify the incorporation into mucin-like molecules, the media and cell extracts were treated with hyaluronidase, and the results were compared with those obtained from samples without the hyaluronidase treatment. As shown in Figure 9, the total incorporation of 3H-glucosamine into the Vo fractions was greatly enhanced in medium from cultures containing 0.15 mM calcium as compared with 0.06 mM,
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Fig. 5. Electron micrographs of MTE cells grown on a collagen gel substratum in medium containing various concentrations of calcium. Cells were maintained in retinol-supplemented medium with various concentrations of calcium for 14 days. One to 2 cell thickness layers were found in low calcium cultures (A: 0.06 mM; B: 0.15 mM CaCI,). Up to eight cell thickness layers were found in cultures maintained in 1.5 mM calcium (C). Mucus secreting granules (as indicated by an arrow and MI were found in cells of the apical region. Bars = 2 pm. The collagen gel substrate layer is indicated as CG and further indicated by the arrowheads. N: nucleus.
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while there was a decreased total incorporation in media containing 0.6 or 1.5 mM calcium as compared with 0.15 mM calcium. Hyaluronidase degraded 70 to 90% of the total radioactivity incorporated into Vo fractions in cultures supplemented with low calcium concentrations (Fig. 9a,b). In contrast, more than 90% of the radioactivity in the Vo fractions in high calcium cultures was not degraded by hyaluronidase (Fig. 9c,d). In cell extracts, the total incorporation of radioactivity into the Vo fractions was proportional to the concentration of calcium in the medium (Fig. 10).However, in contrast to the results observed in the medium, about 70% of the total cell-associated radioactivity that eluted in the Vo fractions was degraded by hyaluronidase regardless of the calcium concentration in the medium. DISCUSSION We have observed that the calcium concentration in the culture medium has profound effects on MTE cells grown on a collagen gel substratum. A similar result was observed in human tracheobronchial epithelial cells (data not shown). An important difference between the current study and previous ones (Lechner et al., 1982, 1986) is that the current system allows airway epithelial cells to express mucous cell differentiation, while previous culture conditions only allowed squamous properties to be expressed. We have shown that MTE cells cultured on collagen gel substrata are able to express mucous cell differentiation like those previously described for hamster (Wu et al., 1985b) and human tracheobronchial epithelial cells (Wu et al., 1990). Evidence of mucous cell differentiation in MTE cultures is provided by the immunohistochemical staining of cultured cells (Fig. 7), by mucin ELISA assay of culture medium (Fig. S), by the presence of mucous granules at the EM level (Fig. 6), and by the biochemical analysis of the incorporation of 3H-glucosamine into hyaluronidase-resistant Vo fractions (Figs. 9, 10). All of these activities are consistently low in cells cultured in medium containing low concentrations of calcium ion (0.6 mM) do not have filament bundles as a prominent feature. Cell-cell interactions are greatly enhanced, as evidenced by membrane interdigitation and desmosome formation. Four to 10 cell layers are observed under these culture conditions and, interestingly, mucous granules are observed only in cells of the apical layer. These observations of cell-cell interactions are consistent with previous studies that were performed under culture conditions that allowed only squamous cell differentiation to be expressed (Hennings and Holbrook, 1983; O'Keefe et al., 1987). Our morphological observation of mucus-secreting granules in cells cultured in high calcium medium is consistent with the analysis of secretion products. We have shown that the majority of the products secreted by cells cultured in high calcium medium is mucin-like, i.e., resistant to hyaluronidase, while hyaluronate is the major secretory product for cultures maintained in low calcium medium. Hyaluronate secretion has been implicated as one of the markers associated with lung injury (Sahu and Ulsamer, 1980), and as a marker for fibroblast proliferation (Tomida et al., 1975). We have
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5000 I 5000 . A
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Fig. 9. Effects of calcium concentration on the incorporation of 'H-glucosamine into a high molecular weight glycoconjugate fraction. Primary MTE cells were maintained in the serum-free hormonesupplemented medium containing various concentrations of calcium (A: 0.06 mM; B: 0.15 mM; C: 0.6 mM; D: 1.5 mM) a s described in Figure 1. At day 13, 3H-glucosamine (20 pCiimli was added. Culture media were harvested the next day (16 hr). An aliquot of medium equivalent to 2.5 x lo5 cells treated with hyaluronidase overnight ( 0 ) or without the treatment (o),was applied to a Sepharose CL-2B column (1x 50 cm) chromatographed as described in the text. Arrows indicate the void volume (Vo) fractions.
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Fig. 10. Effects of calcium concentration on the cell-associated incorporation of 3H-glucosamine into large molecular weight glycoconjugates. Experiments were carried out as in Figure 9. Cells, including the collagen gel substrate, were extracted with keratinextraction buffer (Huang et al., 1989). After centrifugation, the supernatants were either treated with hyaluronidase (e) or were not treated (0).The profiles in the figure represent 2 x lo5 cells equivalent. A 0.06 mM; B: 0.15 mM; C: 0.6 mM; D: 1.5 mM calcium.
extracellular calcium concentration may enhance the intracellular calcium level, which favors the expression of epithelial differentiated function. Secretion is demonstrated previously the synthesis of hyaluronate thought to be initiated by a rise in cytosolic Ca+2 in human TBE and nasal epithelial cells grown on a activity (Chandler, 1978; Douglas, 1978; Rubin, 1982). tissue culture plastic surface (Wu et al., 1985a; Wu, It is not yet clear whether influx of extracellular 1986). Both human TBE and nasal epithelial cells calcium or release of calcium from an intracellular site cultured on a tissue culture plastic surface do not have is responsible for this event. In primary keratinocyte any features resembling mucus-secreting cell types, cultures, an increase in the extracellular calcium consuch as the presence of mucus-secreting granules or centration from 0.05 to 1.2 mM induces a rapid and mucin-like glycoprotein synthesis. These results fur- sustained increase in intracellular calcium concentrather support the hypothesis that media containing high tion from 30 to 70 nM (Fairley et al., 1988; Hennings calcium concentrations mediate mucous cell differenti- et al., 1989),a phenomenon thought to be characteristic ation, while media low in calcium concentration pro- of cells actively proliferating. The prolonged increase of mote the proliferation of basal cell types. the intracellular calcium level coincides with the exThe mechanism whereby extracellular calcium influ- pression of terminal squamous cell differentiation. This ences mucous cell differentiation is not known. In observation is consistent with in vivo reports that physiologic s stems, cytosolic calcium concentration is progressively higher levels of intracellular and extraapproximate1yy three to four orders of magnitude less cellular calcium are associated with keratinocyte difthan the extracellular calcium concentration (Tsien ferentiation (Menon et al., 1985; Forslind, 1986). It is et al., 1984; Murphy et al., 1988). An increase in possible that a similar mechanism of sustained in-
EFFECTS OF CALCIUM ON CULTURED MTE CELLS
crease in intracellular calcium concentration in airway epithelial cells cultured in high calcium medium may trigger their commitment to mucous cell differentiation. In agreement with this hypothesis, it has been demonstrated that calcium is found in high concentration inside secretory granules of mucus-secreting granules (Roomans et al., 1982; Izutsu et al., 1985; Verdugo et al., 1987) and is elevated in the mucus of cystic fibrosis patients (Potter et al., 1967).
ACKNOWLEDGMENTS The authors thank the California Primate Research Center and personnel for their assistance in obtaining the tissues used in this study, and Viviana Wong for her help with the electron microscopy. Authors also thank Drs. Jerold Last and Karen Reiser for their critical review of the manuscript prior to its submission. This work was supported in part by grants from NIH HL 35635 and ES 00628, Cystic Fibrosis Foundation, California Lung Association, and The Council for Tobacco Research-U.S.A., Inc. LITERATURE CITED Becci, P.J., McDowell, E.M., and Trump, B.F. (19781 The respiratory epithelium. IV. Histogenesis of epidermoid metaplasia and carcinoma in situ in the hamster. J.N.C.I., 61:577-586. Boren, H.G., and Paradise. L.J. (1978) Cytokinetics of lung. In: Pathogenesis and Therapy of Lung Cancer. C.C. Harris, ed. Dekker, New York, pp. 369-418. Chandler, D.E. (1978) Control of pancreatic enzyme secretion: a critique on the role of calcium. Life Sci., 23:323-334. Douglas, W.W. (1978) Respiratory Tract Mucus, Ciba Foundation Symposium 54. Elsevier, Amsterdam, pp. 61-90. Fairley, J.A., Ewing, N.M., and Keng, P.C. (1988) Extracellular calcium-induced terminal differentiation of keratinocytes is accompanied by an increase in intracellular free calcium. J . Invest. Dermatol., 90t556 (abs.). Forslind, B. (1986) Particle probe analysis in the study of skin physiology. Scan. Electron Microsc., 111:1007-1014. Ham, R.G. (1965) Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc. Natl. Acad. Sci. U.S.A., 53.288293. Harris, C.C., Sporn, M.B., Kaufman, U.G., Smith, J.M., Baker, M.S., and Saffioti U. (1972) Histogenesis of squamous metaplasia in the hamster tracheal epithelium caused by vitamin A deficiency or benzo(a)oyrene-ferricoxide. J.N.C.I.,48t743-761. Hennings, H., and Holbrook, K. (1983) Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. Exp. Cell Res., 143: 127-142. Hennings, H., Michael, D., Chengs, C., Steinert, P., Holbrook, K., and Yuspa, S.H. (1980)Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell, 19: 245-254. Hennings, H., Holbrook, K.A., and Yuspa, S.H. (1983) Potassium mediation of calcium-induced terminal differentiation of eoidermal cells in culture. J. Invest. Dermatol., 80:50s55s. Hennings, H., Kroszewski, F.H., Yuspa, S.H., and Tucker, R.W. (1989) Intra
Growth and Differentiation of Primary Tracheal Epithelial Cells in Culture: Regulation by Extracellular Calcium WALTER R. MARTIN, CHRIS BROWN, YAO JUANZHANG, AND REEN WU* Pulmonary and Critical Care Medicine, Department of lnternal Medicine, School of Medicine (W.R.M., R. W.) and California Primate Research Center (W.R.M., C.B., Y.).Z., R. W.), University of California, Davis, California 956 16 Growth and differentiation of primary monkey tracheal epithelial (MTE) cells maintained on collagen gel substrata were studied in a defined serum-free culture medium containing 0.03 to 3.0 m M extracellular calcium. Cell attachment efficiency (40-60%) was not altered by different calcium levels. Growth of primary MTE cells on collagen gel substrata, which was vitamin Adependent, was enhanced 50% in the medium supplemented with high calcium (>0.3 mM). High calcium medium also increased cell-cell interactions, formation of desmosomes, and multi-cell layering. The relative content of mucous cells, which were identified by a mucin-specific monoclonal antibody and the presence of mucussecreting granules at the ultrastructural level, was greater in the high-calcium medium. Furthermore, the secretion of mucin into the medium, determined either by an ELISA or by the incorporation of 'H-glucosamine into mucous glycoprotein fractions, was also increased more than 5-fold in media containing high calcium content (>0.6 mM). In contrast, MTE cells cultured in low calcium medium ( O X mM), while a one-to-two cell thickness layer was observed in low calcium medium containing vitamin A (Fig. 4). The intercellular spaces were also wider in low calcium medium (Fig. 4A,B) than were observed in high calcium medium (Fig. 4C,D). This phenomenon was further examined at the ultrastructural level. More cell-cell interactions and
143
EFFECTS OF CALCIUM ON CULTURED MTE CELLS
membrane interdigitation were observed in cultures with high calcium medium (Fig. 5B,C). In low calcium medium, microvilli were extensively developed (Fig. 5A), but cell-cell interactions were greatly reduced. Prominent tonofilament bundles were frequently observed in cells of the basal cell layer of low calcium cultures even in the presence of vitamin A (Fig. 6A). However, in the basal cell layer region of high calciumsupplemented cultures, many desmosomes but no tonofilament bundles were found (Fig. 6B). Re ardless of the calcium concentration in the medium, t icker and more abundant tonofilament bundles were observed in cells maintained in vitamin A-depleted medium (data not shown). It was possible to demonstrate the presence of conspicuous mucus-secreting granules in the apical region of cells that were maintained in high calcium and vitamin A-supplemented medium (Fig. 6C), an infreauent observation in cells maintained in low calcium medium. Effects of calcium on mucous cell differentiation. Effects of calcium on the expression of mucous cell functions in culture were examined by both immunological and biochemical methods. Monoclonal antibody 17B1, specific to humanimonkey tracheal mucin and mucus-secreting granules (St. George et al., 1985; Lin et al., 1989), was used to identify mucous cell populations in culture and to quantify mucin secreted in the culture medium. In the high calcium medium, more cells were stained with the monoclonal antibody and some of the stains were in a granular pattern (Fig. 7B). In contrast, few cells were stained in low calcium cultures (Fig. 7A). The amount of mucin secreted into the culture medium, as determined by the ELISA method (Lin et al., 1989), was also dependent on the calcium concentration (Fig. 8). Cells cultured in high calcium medium (>0.6 mM) secreted 5 times more mucin than those maintained in medium containing 0.1 mM calcium or less. To further evaluate the effects of calcium concentration in the medium on mucin synthesis, 3H-glucosamine was used to label the mucin-like glycoproteins synthesized in culture. Previous studies have demonstrated that the 3H-glucosamine-labeled macromolecules isolated from the Vo fractions of a Sepharose CL-2B column are a mixture of hyaluronate and mucin-like molecules (Kim et al., 1985; Wu et al., 1985a,b, 1990). In order to quantify the incorporation into mucin-like molecules, the media and cell extracts were treated with hyaluronidase, and the results were compared with those obtained from samples without the hyaluronidase treatment. As shown in Figure 9, the total incorporation of 3H-glucosamine into the Vo fractions was greatly enhanced in medium from cultures containing 0.15 mM calcium as compared with 0.06 mM,
!i
Fig. 5. Electron micrographs of MTE cells grown on a collagen gel substratum in medium containing various concentrations of calcium. Cells were maintained in retinol-supplemented medium with various concentrations of calcium for 14 days. One to 2 cell thickness layers were found in low calcium cultures (A: 0.06 mM; B: 0.15 mM CaCI,). Up to eight cell thickness layers were found in cultures maintained in 1.5 mM calcium (C). Mucus secreting granules (as indicated by an arrow and MI were found in cells of the apical region. Bars = 2 pm. The collagen gel substrate layer is indicated as CG and further indicated by the arrowheads. N: nucleus.
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while there was a decreased total incorporation in media containing 0.6 or 1.5 mM calcium as compared with 0.15 mM calcium. Hyaluronidase degraded 70 to 90% of the total radioactivity incorporated into Vo fractions in cultures supplemented with low calcium concentrations (Fig. 9a,b). In contrast, more than 90% of the radioactivity in the Vo fractions in high calcium cultures was not degraded by hyaluronidase (Fig. 9c,d). In cell extracts, the total incorporation of radioactivity into the Vo fractions was proportional to the concentration of calcium in the medium (Fig. 10).However, in contrast to the results observed in the medium, about 70% of the total cell-associated radioactivity that eluted in the Vo fractions was degraded by hyaluronidase regardless of the calcium concentration in the medium. DISCUSSION We have observed that the calcium concentration in the culture medium has profound effects on MTE cells grown on a collagen gel substratum. A similar result was observed in human tracheobronchial epithelial cells (data not shown). An important difference between the current study and previous ones (Lechner et al., 1982, 1986) is that the current system allows airway epithelial cells to express mucous cell differentiation, while previous culture conditions only allowed squamous properties to be expressed. We have shown that MTE cells cultured on collagen gel substrata are able to express mucous cell differentiation like those previously described for hamster (Wu et al., 1985b) and human tracheobronchial epithelial cells (Wu et al., 1990). Evidence of mucous cell differentiation in MTE cultures is provided by the immunohistochemical staining of cultured cells (Fig. 7), by mucin ELISA assay of culture medium (Fig. S), by the presence of mucous granules at the EM level (Fig. 6), and by the biochemical analysis of the incorporation of 3H-glucosamine into hyaluronidase-resistant Vo fractions (Figs. 9, 10). All of these activities are consistently low in cells cultured in medium containing low concentrations of calcium ion (0.6 mM) do not have filament bundles as a prominent feature. Cell-cell interactions are greatly enhanced, as evidenced by membrane interdigitation and desmosome formation. Four to 10 cell layers are observed under these culture conditions and, interestingly, mucous granules are observed only in cells of the apical layer. These observations of cell-cell interactions are consistent with previous studies that were performed under culture conditions that allowed only squamous cell differentiation to be expressed (Hennings and Holbrook, 1983; O'Keefe et al., 1987). Our morphological observation of mucus-secreting granules in cells cultured in high calcium medium is consistent with the analysis of secretion products. We have shown that the majority of the products secreted by cells cultured in high calcium medium is mucin-like, i.e., resistant to hyaluronidase, while hyaluronate is the major secretory product for cultures maintained in low calcium medium. Hyaluronate secretion has been implicated as one of the markers associated with lung injury (Sahu and Ulsamer, 1980), and as a marker for fibroblast proliferation (Tomida et al., 1975). We have
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Fig. 9. Effects of calcium concentration on the incorporation of 'H-glucosamine into a high molecular weight glycoconjugate fraction. Primary MTE cells were maintained in the serum-free hormonesupplemented medium containing various concentrations of calcium (A: 0.06 mM; B: 0.15 mM; C: 0.6 mM; D: 1.5 mM) a s described in Figure 1. At day 13, 3H-glucosamine (20 pCiimli was added. Culture media were harvested the next day (16 hr). An aliquot of medium equivalent to 2.5 x lo5 cells treated with hyaluronidase overnight ( 0 ) or without the treatment (o),was applied to a Sepharose CL-2B column (1x 50 cm) chromatographed as described in the text. Arrows indicate the void volume (Vo) fractions.
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Fig. 10. Effects of calcium concentration on the cell-associated incorporation of 3H-glucosamine into large molecular weight glycoconjugates. Experiments were carried out as in Figure 9. Cells, including the collagen gel substrate, were extracted with keratinextraction buffer (Huang et al., 1989). After centrifugation, the supernatants were either treated with hyaluronidase (e) or were not treated (0).The profiles in the figure represent 2 x lo5 cells equivalent. A 0.06 mM; B: 0.15 mM; C: 0.6 mM; D: 1.5 mM calcium.
extracellular calcium concentration may enhance the intracellular calcium level, which favors the expression of epithelial differentiated function. Secretion is demonstrated previously the synthesis of hyaluronate thought to be initiated by a rise in cytosolic Ca+2 in human TBE and nasal epithelial cells grown on a activity (Chandler, 1978; Douglas, 1978; Rubin, 1982). tissue culture plastic surface (Wu et al., 1985a; Wu, It is not yet clear whether influx of extracellular 1986). Both human TBE and nasal epithelial cells calcium or release of calcium from an intracellular site cultured on a tissue culture plastic surface do not have is responsible for this event. In primary keratinocyte any features resembling mucus-secreting cell types, cultures, an increase in the extracellular calcium consuch as the presence of mucus-secreting granules or centration from 0.05 to 1.2 mM induces a rapid and mucin-like glycoprotein synthesis. These results fur- sustained increase in intracellular calcium concentrather support the hypothesis that media containing high tion from 30 to 70 nM (Fairley et al., 1988; Hennings calcium concentrations mediate mucous cell differenti- et al., 1989),a phenomenon thought to be characteristic ation, while media low in calcium concentration pro- of cells actively proliferating. The prolonged increase of mote the proliferation of basal cell types. the intracellular calcium level coincides with the exThe mechanism whereby extracellular calcium influ- pression of terminal squamous cell differentiation. This ences mucous cell differentiation is not known. In observation is consistent with in vivo reports that physiologic s stems, cytosolic calcium concentration is progressively higher levels of intracellular and extraapproximate1yy three to four orders of magnitude less cellular calcium are associated with keratinocyte difthan the extracellular calcium concentration (Tsien ferentiation (Menon et al., 1985; Forslind, 1986). It is et al., 1984; Murphy et al., 1988). An increase in possible that a similar mechanism of sustained in-
EFFECTS OF CALCIUM ON CULTURED MTE CELLS
crease in intracellular calcium concentration in airway epithelial cells cultured in high calcium medium may trigger their commitment to mucous cell differentiation. In agreement with this hypothesis, it has been demonstrated that calcium is found in high concentration inside secretory granules of mucus-secreting granules (Roomans et al., 1982; Izutsu et al., 1985; Verdugo et al., 1987) and is elevated in the mucus of cystic fibrosis patients (Potter et al., 1967).
ACKNOWLEDGMENTS The authors thank the California Primate Research Center and personnel for their assistance in obtaining the tissues used in this study, and Viviana Wong for her help with the electron microscopy. Authors also thank Drs. Jerold Last and Karen Reiser for their critical review of the manuscript prior to its submission. This work was supported in part by grants from NIH HL 35635 and ES 00628, Cystic Fibrosis Foundation, California Lung Association, and The Council for Tobacco Research-U.S.A., Inc. LITERATURE CITED Becci, P.J., McDowell, E.M., and Trump, B.F. (19781 The respiratory epithelium. IV. Histogenesis of epidermoid metaplasia and carcinoma in situ in the hamster. J.N.C.I., 61:577-586. Boren, H.G., and Paradise. L.J. (1978) Cytokinetics of lung. In: Pathogenesis and Therapy of Lung Cancer. C.C. Harris, ed. Dekker, New York, pp. 369-418. Chandler, D.E. (1978) Control of pancreatic enzyme secretion: a critique on the role of calcium. Life Sci., 23:323-334. Douglas, W.W. (1978) Respiratory Tract Mucus, Ciba Foundation Symposium 54. Elsevier, Amsterdam, pp. 61-90. Fairley, J.A., Ewing, N.M., and Keng, P.C. (1988) Extracellular calcium-induced terminal differentiation of keratinocytes is accompanied by an increase in intracellular free calcium. J . Invest. Dermatol., 90t556 (abs.). Forslind, B. (1986) Particle probe analysis in the study of skin physiology. Scan. Electron Microsc., 111:1007-1014. Ham, R.G. (1965) Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc. Natl. Acad. Sci. U.S.A., 53.288293. Harris, C.C., Sporn, M.B., Kaufman, U.G., Smith, J.M., Baker, M.S., and Saffioti U. (1972) Histogenesis of squamous metaplasia in the hamster tracheal epithelium caused by vitamin A deficiency or benzo(a)oyrene-ferricoxide. J.N.C.I.,48t743-761. Hennings, H., and Holbrook, K. (1983) Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. Exp. Cell Res., 143: 127-142. Hennings, H., Michael, D., Chengs, C., Steinert, P., Holbrook, K., and Yuspa, S.H. (1980)Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell, 19: 245-254. Hennings, H., Holbrook, K.A., and Yuspa, S.H. (1983) Potassium mediation of calcium-induced terminal differentiation of eoidermal cells in culture. J. Invest. Dermatol., 80:50s55s. Hennings, H., Kroszewski, F.H., Yuspa, S.H., and Tucker, R.W. (1989) Intra
