Inhibition of mucin release from airway goblet cells by polycationic peptides

Kwang Ho Ko1,2, Choong Jae Lee1,3, Chan Young Shin1, Mijeong Jo1,3, and K. Chul Kim3

3 Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201; and 1 Department of Pharmacology, College of Pharmacy, Seoul National University, Seoul; and 2 Center for Biofunctional Molecules, Postech, Korea


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we investigated whether polycationic peptides affect mucin release from cultured airway goblet cells. Confluent primary hamster tracheal surface epithelial cells were metabolically radiolabeled with [3H]glucosamine for 24 h and chased for 30 min in the presence of varying concentrations of either poly-L-arginine (PLA) or poly-L-lysine (PLL) to assess the effects on [3H]mucin release. Possible cytotoxicity by the polycations was assessed by measuring lactate dehydrogenase release, 51Cr release, and cell exfoliation. The results were as follows: 1) both PLA and PLL inhibited mucin release in a dose-dependent fashion; 2) there was no significant difference in either lactate dehydrogenase release, 51Cr release, or the number of floating cells between control and treatment groups; 3) the effects of both PLA and PLL on mucin release were completely blocked by neutralizing the positive charges either by pretreatment with heparin or by N-acetylation of the polycations; and 4) both PLA and PLL completely masked the stimulatory effect of ATP on mucin release. We conclude that these polycationic peptides can inhibit mucin release from airway goblet cells without any apparent cytotoxicity, and the inhibitory effect seems to be attributable to their positive charges. These are the first nonsteroidal agents, to the best of our knowledge, that have been shown to inhibit mucin release from airway goblet cells.

polycations; epithelial cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ASTHMA IS DEFINED as an inflammatory disease characterized by mucus hypersecretion as one of its major manifestations (4). Asthmatic airways are affected with hyperresponsiveness or hyperreactivty resulting from various stimuli including allergens, airborne irritants, low temperature, exercise, viral respiratory infections, and emotional stress (1). Hypereosinophilia in the blood, bronchoalveolar lavage fluid, and sputum is a remarkable characteristic of asthmatic patients (8, 24), and eosinophils have been suggested to play a key role in the pathogenesis of airway hyperresponsiveness in asthma (6, 7). Eosinophils have cytoplasmic granules that contain basic granule proteins (7), of which the major basic protein of eosinophils (MBP) has been shown to provoke bronchial hyperreactivity after direct instillation into intact tracheae in animals (9). Coyle et al. (2) reported that the synthetic cationic homopolypeptides poly-L-arginine (PLA) and poly-L-lysine (PLL) can also induce airway hyperreactivity in intact animals, suggesting that the hyperreactivity provoked by MBP is due to its high positive charge. Because mucus hypersecretion in the airway is one of the major manifestations associated with asthma (4), we hypothesized that mucus hypersecretion may be caused by eosinophil basic proteins. In this report, we examined the effect of the polycationic peptides on airway mucin release using a primary hamster tracheal surface epithelial (HTSE) cell culture, an in vitro model for goblet cell metaplasia (25). Mucins are multimillion-dalton glycoproteins that are mainly responsible for the physicochemical property of mucus and thus have been used as a biochemical marker for mucus hypersecretion. Here we report that polycationic peptides can inhibit mucin release in a dose-dependent fashion without any noticeable cytotoxicity of the epithelial cells. To the best of our knowledge, these are the first nonsteroidal agents that have ever been reported to inhibit mucin release by direct action on airway mucin-secreting cells.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. All of the chemicals and reagents used in this experiment were purchased from Sigma (St. Louis, MO) unless otherwise specified.

Primary HTSE cell culture. Tracheae were obtained from male Golden Syrian hamsters 7-8 wk of age (Harlan Sprague Dawley, Indianapolis, IN). HTSE cells were harvested and cultured on a thick collagen gel as previously reported (16).

Metabolic labeling of mucins and treatment of cultures. Mucins were metabolically radiolabeled for 24 h by incubating confluent cultures (24-well plates, 5 × 105 cells/well) with 0.2 ml/well of a "complete" medium containing 10 µCi/ml of [3H]glucosamine as previously described (15). The complete medium was prepared by supplementing a mixture of medium 199-Dulbecco's modified Eagle's medium (1:1) with 5 µg/ml of insulin, 5 µg/ml of transferrin, 12.5 ng/ml of epidermal growth factor, 0.1 µM hydrocortisone, 0.01 µM sodium selenite, 0.1 µM retinoic acid, and 5% fetal bovine serum (HyClone, Logan, UT). At the end of the 24-h incubation, the spent medium (the pretreatment sample) was collected, and the labeled cultures were washed twice with Dulbecco's PBS without Ca2+ and Mg2+ before being chased for 30 min in the presence of varying concentrations of PLA (average mol wt 8,900) or PLL (average mol wt 9,600). The chased medium was referred to as the treatment sample. Low-molecular-weight heparin (LMWH; average mol wt 6,000; from porcine intestinal mucosa) was added 5 min before the cultures were chased for 30 min in the presence of the polycationic peptides. In an experiment designed to see whether the polycationic peptides can inhibit ATP-induced mucin release, ATP was added with and without the polycationic peptides during the 30-min chase period. PLA, PLL, ATP, and LMWH were prepared in PBS, and the final pH values of these solutions were between 7.0 and 7.4. PBS solutions within this pH range did not affect mucin release from HTSE cells (15). Floating cells and cell debris were removed by centrifugation of the samples at 12,000 g for 5 min. Fifty microliters of the treatment samples were used for lactate dehydrogenase (LDH) assay, and the remaining samples were stored at -80°C until assayed for their [3H]mucin content.

Quantitation of [3H]mucins. High-molecular-weight glycoconjugates, excluded after Sepharose CL-4B (Pharmacia, Uppsala, Sweden) gel-filtration column chromatography and resistant to hyaluronidase, were defined as mucins (17) and measured by column chromatography as previously described (18).

Cytotoxicity tests. Cultures were treated with either PLA or PLL for 30 min. The LDH assay was conducted with an LDH assay kit according to the manufacturer's directions. 51Cr release was carried out as previously described (18). The degree of exfoliation was measured by counting the floating cells at the end of the 24-h posttreatment period. Briefly, floating cells were collected as a pellet from the spent medium by centrifugation at 200 g for 5 min at 4°C. The resulting cell pellet was suspended in a 0.05% trypsin-0.02% EDTA solution and incubated at 37°C for 10 min before dissociated cells were counted with a hemacytometer.

N-acetylation of the polycationic peptides. N-acetylation was performed through the peracetylation reaction. Briefly, both PLA and PLL were dissolved in water to 10-4 M and then neutralized to pH 7.0 with sodium acetate. Five microliters of acetic anhydride were added to 100 µl of this solution followed by immediate vortexing, and the resulting solution was incubated for 10 min at room temperature. After the procedures were repeated five times, the reaction mixtures were heated to 100°C for 2 min followed by cooling in ice. For purification of N-acetyl polycationic peptides, the reaction mixtures (200 µl) were applied to CM-Sepharose CL-6B cation-exchange columns (0.7 × 5 cm; Pharmacia) preequilibrated with 0.05 M phosphate buffer, pH 7.2. The columns were eluted first with the same buffer and then with 2 M NaCl, with fractions of 320 µl collected. Absorbance at 210 nm was measured for each fraction with an ultraviolet spectrophotometer. Follow-through fractions containing N-acetylated polycationic peptides and the salt-eluted fractions containing the original (unacetylated) polycationic peptides were collected separately and used for the mucin release experiment (see Fig. 4).

Statistics. Means of individual groups were converted to percent control value and are expressed as means ± SE. The difference between groups was assessed with Student's t-test for unpaired samples, and P < 0.05 was considered significantly different.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of PLA and PLL on mucin release. Treatment of HTSE cells with varying concentrations of PLA resulted in a dose-dependent decrease in the amount of released mucins, reaching an 85% "inhibition" at 10-5 M (Fig. 1). PLL showed a similar pattern of effect, with an 87% inhibition at 10-5 M (Fig. 1).


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Fig. 1.   Effect of poly-L-arginine (PLA) and poly-L-lysine (PLL) on mucin release. Confluent hamster tracheal surface epithelial (HTSE) cells (16-mm wells) were metabolically radiolabeled with [3H]glucosamine for 24 h and chased for 30 min in presence of varying concentrations of either PLA or PLL. Amount of [3H]mucins in spent medium was measured as described in METHODS. cont, Control. Values are means ± SE from 4 culture wells. * Significantly different from respective control value, P < 0.05.

Effects of PLA and PLL on LDH release, 51Cr release, and cell exfoliation. Treatment of HTSE cells with either 10-5 M PLA or 10-5 M PLL for 30 min caused no significant increases in LDH release (Fig. 2). Likewise, the same concentration of PLA and PLL caused no significant change in 51Cr release from HTSE cells (100 ± 4% for control cells, 102 ± 11% for PLA, and 103 ± 10% for PLL). The number of floating cells per well during the 24-h posttreatment period was also not significantly different among these groups (1,992 ± 132 control cells, 2,154 ± 84 cells for 10-5 M PLA, and 1,920 ± 70 cells for 10-5 M PLL). There was no apparent microscopic difference between control and treated HTSE cells, at least based on light microscopy, either at the end of the 30-min treatment period or at the end of the 24-h posttreatment period (data not shown).


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Fig. 2.   Effect of PLA and PLL on lactate dehydrogenase (LDH) release. Confluent HTSE cells (16-mm wells) were treated with varying concentrations of either PLA or PLL for 30 min, and aliquots of spent medium were collected for LDH assay as described in methods. Values are means ± SE from 4 culture wells. There was no significant difference among different concentration groups, P > 0.05.

Inhibition of the inhibitory effect of the polycationic peptides by LMWH. The inhibitory effects of 10-5 M PLA and PLL on mucin release were completely blocked by pretreatment of HTSE cells with 2 × 10-5 M LMWH (Fig. 3).


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Fig. 3.   Inhibition of inhibitory effect of polycationic peptides (P) by low-molecular-weight heparin (LMWH; H). Confluent HTSE cells (16-mm wells) were metabolically radiolabeled with [3H]glucosamine for 24 h and pretreated with 2 × 10-5 M LMWH for 5 min before being chased for 30 min in presence of either 10-5 M PLA or 10-5 M PLL. Amount of [3H]mucins in spent medium was measured as described in methods. Values are means ± SE from 4 culture wells. * Significantly different from respective control value, P < 0.05.

Cation-exchange column chromatography of PLA and PLL following N-acetylation. After N-acetylation of PLA and PLL, the reactant was subjected to the cation-exchange column chromatography. Figure 4 shows that, after N-acetylation, the PLA peak (fractions 13-17) originally retained in the column was completely eluted before the salt solution was applied. The same chromatographic pattern was obtained with PLL (data not shown). The size of the follow-through peak (fractions 3-8) was virtually identical to that of the retained (salt-elutable) peak, indicating complete N-acetylation of PLA. Both of the peak fractions were separately pooled and then dialyzed against PBS before being tested for their ability to inhibit mucin release.


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Fig. 4.   CM-Sepharose cation-exchange column chromatography of polycationic peptides after N-acetylation. PLA (10-4 M) was N-acetylated with acetic anhydride and subjected to CM-Sepharose cation-exchange column chromatography as described in methods. Original PLA (unacetylated) was retained in column (top), whereas N-acetylated PLA eluted as follow-through fractions (bottom). Yield of N-acetylated PLA was >95%.

Effects of N-acetylated PLA and PLL on mucin release. The inhibitory effects of 10-5 M PLA and PLL were completely abolished after N-acetylation of these polycationic peptides (Fig. 5). There was no significant difference between the effect of the "original" (without column purification) PLA or PLL and that of the column-purified PLA and PLL (Fig. 4), indicating that the inhibitory effect of the original PLA and PLL was not due to any possible contaminants in the preparation. The purity of the original preparation was also apparent based on the profile of the cation-exchange column chromatography (Fig. 4).


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Fig. 5.   Effect of N-acetylated PLA and PLL on mucin release. Confluent HTSE cells (16-mm wells) were metabolically radiolabeled with [3H]glucosamine for 24 h and chased for 30 min in presence of 10-5 M polycationic peptides. Amount of [3H]mucins in spent medium was measured as described in methods. P, original (without column purification) unacetylated polycationic peptides; N-AcP, N-acetylated polycationic peptides (column purified; see Fig. 4); P-P, unacetylated polycationic peptides (column purified; see Fig. 4). Values are means ± SE from 4 culture wells. * Significantly different from respective control value, P < 0.05.

Inhibition of ATP-induced mucin release by PLA and PLL. ATP (2 × 10-4 M) caused an increase in mucin release by 90% (Fig. 6). The stimulatory effect of ATP was completely blocked in the presence of either 10-5 M PLA or 10-5 M PLL.


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Fig. 6.   Effect of polycationic peptides on ATP-induced mucin release. Confluent HTSE cells (16-mm wells) were metabolically radiolabeled with [3H]glucosamine for 24 h and chased for 30 min in presence of a combination of ATP (A; 2 × 10-4 M) and polycationic peptides (10-5 M). Amount of [3H]mucins in spent medium was measured as described in methods. Values are means ± SE from 4 culture wells. * Significantly different from respective control value, P < 0.05.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

As seen in Fig. 1, both PLA and PLL caused a dose-dependent decrease in the amount of mucins present in the spent medium of HTSE cell cultures, suggesting the possible inhibition of mucin release by these polycationic peptides. The decrease in the amount of mucins was neither due to the degradation of mucins during the treatment period nor due to the interference with the mucin assay (gel-filtration column chromatography) by the polycationic peptides (data not shown). These possibilities were examined with purified [3H]mucins that had been prepared according to a previous study by Kim et al. (17). A possible contribution of the pH of the medium was also ruled out because pH of the 10-4 M PLA and 10-4 M PLL solutions prepared in PBS was 7.4. Kim et al. (15) have previously shown that mucin release from HTSE cells is not affected by this pH. Having ruled out all these possible factors, we concluded that both PLA and PLL have an ability to inhibit the basal secretion of mucins from primary HTSE cells.

A large number of positive charges within these polycationic peptides may cause some cytotoxicity in HTSE cells. It was recently shown (3) that both PLA and PLL caused LDH release in rabbit neutrophils, whereas polycations such as protamine failed to cause LDH release in glomerular epithelial cells (11). In the present experiment, the possible cytotoxicity of these polypeptides was assessed by four different methods: 1) LDH release, 2) 51Cr release, 3) cell exfoliation, and 4) light microscopy. There was no significant difference between the control and treated groups (10-5 M) in all these measurements, which were taken immediately after the 30-min treatment period as well as after the 24-h posttreatment period. Therefore, in primary HTSE cells, both PLA and PLL do not appear to be toxic at the concentrations that showed the inhibitory effect on mucin release. The absence of cell exfoliation by these polycationic peptides is of great interest. It was originally thought that airway hyperreactivity, which is a characteristic of the late phase of asthma, might be induced by eosinophil basic proteins that cause epithelial cell exfoliation or desquamation (5, 6, 8, 10, 19). Coyle et al. (2), however, showed that the cationic proteins such as MBP, PLL, PLA, platelet factor 4, and cathepsin G could induce airway hyperreactivity in animals without epithelial damage. Our present result seems to be consistent with the finding of Coyle et al.

In the next experiments, we examined whether the inhibitory effect of PLA or PLL is due to its positive charges. To this end, both pharmacological and chemical approaches were taken. In the first experiment, LMWH, a highly negatively charged polysaccharide, was added to neutralize the polycationic peptides, and in the second experiment, the polycationic peptides were N-acetylated to block the positive charges. Figure 3 shows that LMWH completely blocked the inhibitory effect by the polycationic peptides. Likewise, N-acetylation of the polycationic peptides also resulted in a complete loss of their ability to inhibit mucin release (Fig. 5). These results indicate that the presence of positive charges is necessary for the inhibitory effect of the polycationic peptides. Similar results were previously reported (2, 23) for airway hyperreactivity induced by these polycationic peptides.

Finally, to see whether these polycationic peptides can also block the "stimulated" or "regulated" (12) mucin release, we treated the cells with ATP, the most potent mucin secretagogue for airway goblet cell mucins that has ever been identified (13), in the presence and absence of the polycationic peptides. Figure 6 shows that the stimulatory effect of ATP was completely blocked in the presence of both polycationic peptides. Interestingly, however, these polycationic peptides not only blocked the stimulated release by ATP but also further inhibited the release almost to the levels that would be expected in the absence of ATP. This "super" inhibition might be either due to an excess of positive charges to negative charges or due to a nonspecific nature of the inhibition by these polycationic peptides. Based on the above mechanistic studies, albeit preliminary, the biological activities of these polycationic peptides, such as airway hyperreactivity and the inhibitory effect on mucin release, seem to require the presence of positive charges of these molecules. It has been suggested that the interaction of the positive charges of the polycationic peptides with the negative charges on the cell membrane may affect the cellular functions by structural alterations of the cell membrane (20-22). For example, an increase in the permeability of cultured epithelial monolayers by protamine, a cationic protein, was shown to be completely blocked by pretreatment with negatively charged molecules such as heparin and dextran sulfate (20). The detailed molecular mechanism of action by these polycationic peptides, however, remains to be investigated.

The present finding that these polycationic peptides can control the hypersecretion of mucins without any apparent cytotoxicity could be of great value from both the scientific and practical viewpoints. To the best of our knowledge, these are the first nonsteroidal agents that have an ability to suppress mucin release by acting directly on airway mucin-secreting cells (14). Their lack of apparent cytotoxicity, combined with their metabolism into endogenous amino acids, seems to make these polycationic peptides quite attractive as possible candidates for a mucin-controlling drug.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: K. C. Kim, Dept. of Pharmaceutical Sciences, Univ. of Maryland School of Pharmacy, 20 N. Pine St., Rm 446, Baltimore, MD 21201 (E-mail: kkim{at}umaryland.edu).

Received 4 March 1998; accepted in final form 2 June 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 277(4):L811-L815
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