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 |
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 |
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 |
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 |
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 |
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.
 |
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