SESSION
10 200
Nucleotides and Mucin Release from Cultured Airway Epithelial cells· K. Chul Kim, Ph.D.; Andrew K. Wilson, B.S.; and Bong C. Lee, M.D.
M
ucus lining the aiIway luminal surface plays an important role in host defense against airborne particles and chemicals. The protective function of mucus is due mainly to the physicochemical properties of constituent mucous glycoproteins (mucins). Therefore, abnormalities in either the quality or the quantity of airway mucins not only affects removal of respired particles and bacteria, but may also impair host defenses leading to further pathologic fin~ings. In humans, airway mucins are derived from 2 different cell types; goblet cells of surface epithelium and mucous cells of the submucosal gland. Recentl}; we made an observation that mechanical strain of cultured airway epithelial secretory cells can stimulate mucin release.! Since mechanical strain on plasma membrane may activate some membrane proteins,2.3 and since activation of GTP-binding proteins in the plasma membrane resulted in exocytosis in some secretory cells,4.s we intended to examine whether GTP binding proteins were involved in the regulation of mucin secretion from cultured primary hamster tracheal surface epithelial (HTSE) cells. In this communication, we report that mucin release from cultured airway goblet cells can be stimulated not only by GTP but also by ATP via a P2 receptor-mediated mechanism. These observations provide, for the first time, a secretory con ~rol mechanism for airway goblet cell mucins that could be physiologic in nature and likely involves a cell surface receptor.
.. '0 ~
c 0
RESULTS AND DISCUSSION
Confluent cultures which had been treated with 5 Vlml streptolysin ()9 responded to 200 ...M GTP'YS by increasing their mucin release by 50%, and the effect of GTP'YS was dose-dependent. However, the effect of 200 ... M GTP'YS was ·From the Department of Pharmacology and Toxicology, University of Maryland School of Pharmacy, Baltimore. This study was supported in part by NIH grant HL-47125 and the Cystic Fibrosis Foundation.
88S
150
u
~
• '•i I)
II ~
100 80
+
GDP"S
+
GTP-yS
+ +
+ GTP
1. Effect of GDP~S, GTP..,S, and GTP on 3H-mucin release. Confluent cultures (16 mm) which had been pretreated with streptolysin 0 were pulsed with 38-glucosamine for 24 h and chased for 30 min in the presence of the nucleotides (200 ....M GTP..,S, 200 ....M GDP~S, and 200 ....M GTP). In panel A, GDP~S was added immediately before GTP..,S. Data represent means ± SE of 4 culture dishes. Mucin release by GTP..,S was not significantly (p>0.05) different in the presence or absence of GDP~S. Based on Student's t-test for unpaired samples. FIGURE
not blocked by 200 ...M CDPpS which seems to suggest that GTP binding proteins may not be responsible for the GTP'YSinduced secretion (Fig IA). When GTF: a hydrolyzable form, was tested, its mucin-releasing effect was equipotent with GTP'YS (Fig IB). Thus, this result rules out the involvement of GTP binding proteins and suggests the involvement of
METHODS
Tracheas were obtained from male golden Syrian hamsters 8 to 10 weeks of age. The HTSE cells were harvested and cultured as previously described. fi Dissociated cells were plated on a thick collagen. gel prepared inside 24 well tissue culture dishes (Falcon) as previously described. 6 Mucins were metabolically radioactively labeled by incubating confluent cultures with 3H-glucosamine, and effects of nucleotides on mucin release were measured as previously described. 6 Mucins were defined as high molecular weight glycoconjugates excluded from sepharose CL-4B and resistant to proteoglycan-digesting enzymes7 •H and were assayed accordingly. 6 The presence or absence of cytoplasmic leak due to cell membrane damage following treatments was identified by measuring la~tic acid dehydrogenase in the culture medium using a commercial LDH assay kit as previously described. 6
B
A
300
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250
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200
Q)
en
as
Q)
...
150
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100 80
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20
200
2000
FIGURE 2. Effect of An AD~ AMP and adenosine on 3H-mucin release. Confluent cultures (16 mm) were pulsed with 3H-glucosamine for 24 h and chased for 30 min in the presence of the adenine analogues. Data represent means ± SE of four culture dishes.
34th Annual Thomas L Petty Aspen Lung Conference
purinergic receptors. The All: a P2 purinoceptor agonist, 10 caused mucin release in a dose-dependent fashion with an apparent ECSO of 20 fLM, whereas adenosine, a PI purinoceptor agonist,IO did not inHuence the rate of mucin release even at 2 mM concentration (Fig 2), indicating the involvement of P2 purinergic mechanism. The effects of adenine analogues on mucin release are compared in Figure 2. These results indicate that mucin release from HTSE cells can be enhanced by a mechanism involving P2 purinoceptors. The P2 purinoceptor has recently been shown to be involved in the secretion of phospholipids from cultured type II pneumocytes, another type of airway epithelial cells. 11 None of the above compounds at 2 mM concentrations showed any detectable cytotoxicity based on LDH release. We conclude that mucin release from cultured airway goblet cells can be stimulated by ATP via a P2 receptor-mediated mechanism. At present, we do not know whether or not P2 purinoceptors are involved in the mechanical strain-induced mucin release from cultured HTSE cells. Nevertheless, this may be a physiologic mechanism involved in the regulation of mucin release by airway goblet cells in vivo. 12 Understanding of the pharmacology of the P2-purinoceptor-induced mucin release may provide useful strategies for development of new drugs controlling airway mucin secretion. REFERENCES 1 Kim KC, Brody JS. Gel contraction causes mucin release in primary hamster tracheal epithelial ceUs growing on a collagen gel. J CeU Bioi 1987; 105:158a 2 Lansman JB, Hallam TJ, Rink TJ. Single stretch-activated ion channels in vascular endothelial ceUs as mechanotransducers? Nature 1987; 325:811-13 3 Olesen SE Clapham DE, Davies PF. Haemodynamic shear stress activates a K current in vascular endothelial ceUs. Nature 1988; 331:168-70 4 Barrowman MM, Cockcroft S, Gomperts BD. Two roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils. Nature 1986; 319:504-07 5 Burgoyne RD. Control of exocytosis. Nature 1987; 328:112-13 6 Kim KC, Nassiri J, Brody JS. Mechanisms of aitway goblet cell mucin release: studies with cultured tracheal surface epithelial cells. Am J Respir CeU Mol Bioi 1989; 1:137-43 7 Kim ICC, Rearick JI, Nettesheim E Jetten AM. Biochemical characterization of mucous glycoproteins synthesized and secreted by hamster tracheal epithelial ceUs in primary culture. J Bioi Chern 1985; 260:4021-27 8 Kim KC, Opaskar-Hincman H, Bhaskar KR. Secretions from primary hamster tracheal surface epithelial ceUs in culture: mucin-like glycoproteins, proteoglycans, and lipids. Exp Lung Res 1989; 15:299-314 9 Stuch6.eld J, Cockcroft S. Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytotic secretion from HL60 cells permeabilized with streptolysin o. Biochem J 1988; 250:375-82 10 Burnstock G. A basis for distinguishing two types of purinergic receptor. In: Straub ~ et al. Cell membrane receptors for drugs and hormones: a multidisciplinary approach. New York: Raven Press, 1978: 107-18 11 Rice WR, Singleton FM. P2-purinoceptors regulate surfactant secretion from rat isolated alveolar type II ceUs. Br J Phannacol 1986; 89:485-91 12 Kim KC. Biochemistry and pharmacology of mucin-like glycoproteins produced by cultured airway epithelial cells. Exp Lung Res 1991; 17:533-45
Regulation of Ciliary Beat Frequency in Respiratory Tract cells· Michael J Sanderson~ Ph.D.; Alison B. lAnsley, Ph.D.; and Ellen R. Di,.ksen~ Ph. D.
A
major function of respiratory tract (RT) epithelial cells is the maintenance of unobstructed airways, a goal achieved by mucociliary clearance. Ciliary activity provides the driving force for mucus propulsion, but the nature of the stimulus-response coupling that regulates ciliary beat frequency is not well understood. The possibility that mechanical interaction between the mucus and ciliated cell may act as a specific control signal was evaluated by examining the effect of mechanical stimulation on cultured RT cells while monitoring ciliary beat frequency with a photoelectronic technique. I Mechanical stimulation of the apical surface of a single ciliated cell in culture with a glass microprobe elevated the ciliary beat frequency, not only ofthe stimulated cell, but also ofadjacent cells. This resulted in a wave of increased beat frequency spreading across the culture. 2,3 The increase in beat frequency of each cell occurred after a lag-phase that was proportional to the distance of the cell from the stimulated cell. Stimulation ofa nonciliated cell also initiated an increase in beat frequency of adjacent ciliated cells. Preliminary evidence suggested that these responses were mediated by elevations in [Ca2 + l.. 2 Consequently, video imaging techniques were used to monitor the fluorescence of the Ca2 +specific dye fura-2 to quantitate the spatial-temporal changes in [Ca2+ ],.4 A single mechanical stimulus to either a ciliated or nonciliated cell immediately induced an increase in [Ca2 +], at the site of contact that then spread throughout the cell. After a short delay of about 0.5 s, a wave of increasing [Ca2 +], occurred in adjacent cells, spreading from one end of the cell to the other. This increase in [Ca2 + ], or Ca2 + wave, continued to spread through the cell culture in a cell-by-eell manner travelling across 4 to 7 cells in all directions (Fig 1, a). The increase in [Ca2 +], was correlated with, but always preceded, the increase in ciliary beat frequency. Mechanical stimulation deforms the cell membrane and may be transduced by opening stretch-activated channels. Ca2 + -conducting, stretch-activated channels have been identified with patch-clamp techniques in enzymatically-isolated RT cellsS. Although Ca2 + may enter the cell through stretchactivated channels, external Ca2 + is not essential for the transduction of mechanical stimulation. Under Ca2 + -free conditions, an increase in [Ca2 + l. does not occur in the stimulated cell but does occur in adjacent cells. The important implications of these results is that an increase in [Ca2 +], in the stimulated cell is not essential for the propagation of a Ca2 + wave to the first adjacent cells, and that the *From the Department of Anatomy and CeU Biology, VCLA School of Medicine, Los Angeles. This study was supported by the Smokeless Tobacco Research Council, Inc, the NIH, the Cystic Fibrosis Foundation, and by the Cigarette and Tobacco Surtax Fund of the State of California through the tobacco-related disease research program of the V Diversity of California. Reprint requests: Dr. Sanderson~ Department of Anatomy and CeU Biology~ UCLA School of Medicine~ ws Angeles 90024 CHEST I 101 I 3 I MARCH. 1992 I Supplement
695
10 200
Nucleotides and Mucin Release from Cultured Airway Epithelial cells· K. Chul Kim, Ph.D.; Andrew K. Wilson, B.S.; and Bong C. Lee, M.D.
M
ucus lining the aiIway luminal surface plays an important role in host defense against airborne particles and chemicals. The protective function of mucus is due mainly to the physicochemical properties of constituent mucous glycoproteins (mucins). Therefore, abnormalities in either the quality or the quantity of airway mucins not only affects removal of respired particles and bacteria, but may also impair host defenses leading to further pathologic fin~ings. In humans, airway mucins are derived from 2 different cell types; goblet cells of surface epithelium and mucous cells of the submucosal gland. Recentl}; we made an observation that mechanical strain of cultured airway epithelial secretory cells can stimulate mucin release.! Since mechanical strain on plasma membrane may activate some membrane proteins,2.3 and since activation of GTP-binding proteins in the plasma membrane resulted in exocytosis in some secretory cells,4.s we intended to examine whether GTP binding proteins were involved in the regulation of mucin secretion from cultured primary hamster tracheal surface epithelial (HTSE) cells. In this communication, we report that mucin release from cultured airway goblet cells can be stimulated not only by GTP but also by ATP via a P2 receptor-mediated mechanism. These observations provide, for the first time, a secretory con ~rol mechanism for airway goblet cell mucins that could be physiologic in nature and likely involves a cell surface receptor.
.. '0 ~
c 0
RESULTS AND DISCUSSION
Confluent cultures which had been treated with 5 Vlml streptolysin ()9 responded to 200 ...M GTP'YS by increasing their mucin release by 50%, and the effect of GTP'YS was dose-dependent. However, the effect of 200 ... M GTP'YS was ·From the Department of Pharmacology and Toxicology, University of Maryland School of Pharmacy, Baltimore. This study was supported in part by NIH grant HL-47125 and the Cystic Fibrosis Foundation.
88S
150
u
~
• '•i I)
II ~
100 80
+
GDP"S
+
GTP-yS
+ +
+ GTP
1. Effect of GDP~S, GTP..,S, and GTP on 3H-mucin release. Confluent cultures (16 mm) which had been pretreated with streptolysin 0 were pulsed with 38-glucosamine for 24 h and chased for 30 min in the presence of the nucleotides (200 ....M GTP..,S, 200 ....M GDP~S, and 200 ....M GTP). In panel A, GDP~S was added immediately before GTP..,S. Data represent means ± SE of 4 culture dishes. Mucin release by GTP..,S was not significantly (p>0.05) different in the presence or absence of GDP~S. Based on Student's t-test for unpaired samples. FIGURE
not blocked by 200 ...M CDPpS which seems to suggest that GTP binding proteins may not be responsible for the GTP'YSinduced secretion (Fig IA). When GTF: a hydrolyzable form, was tested, its mucin-releasing effect was equipotent with GTP'YS (Fig IB). Thus, this result rules out the involvement of GTP binding proteins and suggests the involvement of
METHODS
Tracheas were obtained from male golden Syrian hamsters 8 to 10 weeks of age. The HTSE cells were harvested and cultured as previously described. fi Dissociated cells were plated on a thick collagen. gel prepared inside 24 well tissue culture dishes (Falcon) as previously described. 6 Mucins were metabolically radioactively labeled by incubating confluent cultures with 3H-glucosamine, and effects of nucleotides on mucin release were measured as previously described. 6 Mucins were defined as high molecular weight glycoconjugates excluded from sepharose CL-4B and resistant to proteoglycan-digesting enzymes7 •H and were assayed accordingly. 6 The presence or absence of cytoplasmic leak due to cell membrane damage following treatments was identified by measuring la~tic acid dehydrogenase in the culture medium using a commercial LDH assay kit as previously described. 6
B
A
300
o...
250
.., c:
o o
il ...,
200
Q)
en
as
Q)
...
150
Q)
100 80
o
2
20
200
2000
FIGURE 2. Effect of An AD~ AMP and adenosine on 3H-mucin release. Confluent cultures (16 mm) were pulsed with 3H-glucosamine for 24 h and chased for 30 min in the presence of the adenine analogues. Data represent means ± SE of four culture dishes.
34th Annual Thomas L Petty Aspen Lung Conference
purinergic receptors. The All: a P2 purinoceptor agonist, 10 caused mucin release in a dose-dependent fashion with an apparent ECSO of 20 fLM, whereas adenosine, a PI purinoceptor agonist,IO did not inHuence the rate of mucin release even at 2 mM concentration (Fig 2), indicating the involvement of P2 purinergic mechanism. The effects of adenine analogues on mucin release are compared in Figure 2. These results indicate that mucin release from HTSE cells can be enhanced by a mechanism involving P2 purinoceptors. The P2 purinoceptor has recently been shown to be involved in the secretion of phospholipids from cultured type II pneumocytes, another type of airway epithelial cells. 11 None of the above compounds at 2 mM concentrations showed any detectable cytotoxicity based on LDH release. We conclude that mucin release from cultured airway goblet cells can be stimulated by ATP via a P2 receptor-mediated mechanism. At present, we do not know whether or not P2 purinoceptors are involved in the mechanical strain-induced mucin release from cultured HTSE cells. Nevertheless, this may be a physiologic mechanism involved in the regulation of mucin release by airway goblet cells in vivo. 12 Understanding of the pharmacology of the P2-purinoceptor-induced mucin release may provide useful strategies for development of new drugs controlling airway mucin secretion. REFERENCES 1 Kim KC, Brody JS. Gel contraction causes mucin release in primary hamster tracheal epithelial ceUs growing on a collagen gel. J CeU Bioi 1987; 105:158a 2 Lansman JB, Hallam TJ, Rink TJ. Single stretch-activated ion channels in vascular endothelial ceUs as mechanotransducers? Nature 1987; 325:811-13 3 Olesen SE Clapham DE, Davies PF. Haemodynamic shear stress activates a K current in vascular endothelial ceUs. Nature 1988; 331:168-70 4 Barrowman MM, Cockcroft S, Gomperts BD. Two roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils. Nature 1986; 319:504-07 5 Burgoyne RD. Control of exocytosis. Nature 1987; 328:112-13 6 Kim KC, Nassiri J, Brody JS. Mechanisms of aitway goblet cell mucin release: studies with cultured tracheal surface epithelial cells. Am J Respir CeU Mol Bioi 1989; 1:137-43 7 Kim ICC, Rearick JI, Nettesheim E Jetten AM. Biochemical characterization of mucous glycoproteins synthesized and secreted by hamster tracheal epithelial ceUs in primary culture. J Bioi Chern 1985; 260:4021-27 8 Kim KC, Opaskar-Hincman H, Bhaskar KR. Secretions from primary hamster tracheal surface epithelial ceUs in culture: mucin-like glycoproteins, proteoglycans, and lipids. Exp Lung Res 1989; 15:299-314 9 Stuch6.eld J, Cockcroft S. Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytotic secretion from HL60 cells permeabilized with streptolysin o. Biochem J 1988; 250:375-82 10 Burnstock G. A basis for distinguishing two types of purinergic receptor. In: Straub ~ et al. Cell membrane receptors for drugs and hormones: a multidisciplinary approach. New York: Raven Press, 1978: 107-18 11 Rice WR, Singleton FM. P2-purinoceptors regulate surfactant secretion from rat isolated alveolar type II ceUs. Br J Phannacol 1986; 89:485-91 12 Kim KC. Biochemistry and pharmacology of mucin-like glycoproteins produced by cultured airway epithelial cells. Exp Lung Res 1991; 17:533-45
Regulation of Ciliary Beat Frequency in Respiratory Tract cells· Michael J Sanderson~ Ph.D.; Alison B. lAnsley, Ph.D.; and Ellen R. Di,.ksen~ Ph. D.
A
major function of respiratory tract (RT) epithelial cells is the maintenance of unobstructed airways, a goal achieved by mucociliary clearance. Ciliary activity provides the driving force for mucus propulsion, but the nature of the stimulus-response coupling that regulates ciliary beat frequency is not well understood. The possibility that mechanical interaction between the mucus and ciliated cell may act as a specific control signal was evaluated by examining the effect of mechanical stimulation on cultured RT cells while monitoring ciliary beat frequency with a photoelectronic technique. I Mechanical stimulation of the apical surface of a single ciliated cell in culture with a glass microprobe elevated the ciliary beat frequency, not only ofthe stimulated cell, but also ofadjacent cells. This resulted in a wave of increased beat frequency spreading across the culture. 2,3 The increase in beat frequency of each cell occurred after a lag-phase that was proportional to the distance of the cell from the stimulated cell. Stimulation ofa nonciliated cell also initiated an increase in beat frequency of adjacent ciliated cells. Preliminary evidence suggested that these responses were mediated by elevations in [Ca2 + l.. 2 Consequently, video imaging techniques were used to monitor the fluorescence of the Ca2 +specific dye fura-2 to quantitate the spatial-temporal changes in [Ca2+ ],.4 A single mechanical stimulus to either a ciliated or nonciliated cell immediately induced an increase in [Ca2 +], at the site of contact that then spread throughout the cell. After a short delay of about 0.5 s, a wave of increasing [Ca2 +], occurred in adjacent cells, spreading from one end of the cell to the other. This increase in [Ca2 + ], or Ca2 + wave, continued to spread through the cell culture in a cell-by-eell manner travelling across 4 to 7 cells in all directions (Fig 1, a). The increase in [Ca2 +], was correlated with, but always preceded, the increase in ciliary beat frequency. Mechanical stimulation deforms the cell membrane and may be transduced by opening stretch-activated channels. Ca2 + -conducting, stretch-activated channels have been identified with patch-clamp techniques in enzymatically-isolated RT cellsS. Although Ca2 + may enter the cell through stretchactivated channels, external Ca2 + is not essential for the transduction of mechanical stimulation. Under Ca2 + -free conditions, an increase in [Ca2 + l. does not occur in the stimulated cell but does occur in adjacent cells. The important implications of these results is that an increase in [Ca2 +], in the stimulated cell is not essential for the propagation of a Ca2 + wave to the first adjacent cells, and that the *From the Department of Anatomy and CeU Biology, VCLA School of Medicine, Los Angeles. This study was supported by the Smokeless Tobacco Research Council, Inc, the NIH, the Cystic Fibrosis Foundation, and by the Cigarette and Tobacco Surtax Fund of the State of California through the tobacco-related disease research program of the V Diversity of California. Reprint requests: Dr. Sanderson~ Department of Anatomy and CeU Biology~ UCLA School of Medicine~ ws Angeles 90024 CHEST I 101 I 3 I MARCH. 1992 I Supplement
695
