Pediatric Pulmonary Division, University of Connecticut Health Center, Farmington, Connecticut 06030
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ABSTRACT |
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Tannin, isolated from cotton bracts, inhibits
chloride secretion in airway epithelium. In bovine tracheal epithelial
cells, tannin (25 µg/ml) blunted isoproterenol (Iso)-stimulated
adenosine 3',5'-cyclic monophosphate (cAMP) accumulation.
Inhibition was time and dose dependent, with 52 ± 5% (mean ± SE, n = 6) inhibition at 60 min and 82 ± 9% (n = 3) inhibition at 8 h.
Inhibition was reversible starting at 4 h.
Low-molecular-mass tannin (1,000-5,000 Da) had no
effect on Iso-stimulated cAMP accumulation, whereas N-acetylcysteine, which interacts with
cysteine residues, blocked the effects of tannin on Iso-stimulated cAMP
accumulation. Tannin exposure (25 µg/ml for 30 min) had no effect on
the dissociation constant
(Kd) for
[3H]dihydroalprenolol
(DHA) (0.41 ± 0.03 nM, n = 3) but
decreased maximal binding from 252 ± 32 to 162 ± 36 fmol/mg
protein. Using single-point analysis and
[3H]CGP-12177, we
determined that tannin (25 µg/ml for 4 h) decreased surface
-adrenergic receptor density from 26.4 ± 4.3 (n = 12) to 11.9 ± 3.0 fmol/mg
protein and that the decrease was dose dependent. Agonist binding
affinity by Iso displacement of DHA demonstrated a two-site model
(Kd values = 27 ± 9 and 2,700 ± 600 nM) and a ratio of high- to
low-affinity receptors of 1:1. Tannin (25 µg/ml) steepened the curve
and shifted it to the right, as did Gpp(NH)p. Gpp(NH)p had
no further effect on the shape or position of the displacement curve in
the presence of tannin. In contrast, when polymer length was decreased
by oxidation, tannin had no effect on the DHA displacement curve. These
data demonstrate that tannin reversibly desensitizes bovine tracheal
epithelial cells to Iso, decreases
-adrenergic receptor density, and
uncouples the receptor from its stimulatory G protein.
These data also suggest that the polymer length of tannin and its
interaction with cysteine residues are important for these effects.
These studies provide additional evidence for the role of tannin in the
occupational lung disease byssinosis.
byssinosis; chloride secretion; dihydroalprenolol; CGP-12177
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INTRODUCTION |
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INHALATION OF COTTON MILL dust by some textile workers results in the development of the occupational lung disease byssinosis, a disease characterized by symptoms of dyspnea, coughing, wheezing, and chest tightness that begin several hours after exposure to mill dust (1). These symptoms are accompanied by an across-shift reduction in lung ventilatory capacity due to reversible bronchoconstriction and are worse on Mondays or after prolonged absences from the mill (1, 22, 23, 27). Although the etiology of byssinosis is not known, endotoxin and tannin, isolated from cotton bracts, have been implicated as important etiologic agents (20, 21).
Endotoxin has no direct effect on the airway epithelium (8). In
contrast, tannin inhibits net chloride
(Cl) secretion in the
airway epithelium (8). This inhibition demonstrates specificity for the
apical membrane and is dose dependent and reversible. Signal
transduction pathways involved in
Cl
secretion in the airway
epithelium and affected by tannin include protein kinase C activity,
nonmetabolized arachidonic acid release, and intracellular calcium
release (5, 6). In addition, tannin has significant effects on the
adenylyl cyclase-
-adrenergic receptor pathway of
Cl
secretion. Increasing
concentrations of tannin inhibit basal and epinephrine-stimulated
adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in part by
decreasing maximum binding
(Bmax) without affecting the
dissociation constant
(Kd) (7). In
addition, when the
-adrenergic receptor is bypassed by
forskolin, tannin noncompetitively and reversibly inhibits
forskolin-stimulated adenylyl cyclase activity in a dose-dependent
manner (4). Thus tannin has profound effects on the
-adrenergic
receptor and on cAMP.
We have hypothesized that the unusually long tannin polymer, through an
affinity for cysteine residues, alters the tertiary configuration of
the -adrenergic receptor and thus affects the binding of
-agonists to the receptor and the coupling between the receptor and
its stimulatory G protein. To test this hypothesis, we examined the
effects of changes in tannin polymer length and the effects of
N-acetylcysteine, which interacts with
cysteine residues, on isoproterenol-stimulated cAMP accumulation. We
also examined the effect of tannin and polymer length on the coupling between the
-adrenergic receptor and its stimulatory G protein. In
these experiments, we demonstrate that tannin reversibly decreases cAMP
levels in response to isoproterenol, that
N-acetylcysteine inhibits the effects
of tannin on cAMP accumulation, that tannin uncouples the
-adrenergic receptor from its stimulatory G protein, and that
polymer length is important for isoproterenol-stimulated cAMP
accumulation and for receptor-stimulatory G protein uncoupling.
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MATERIALS AND METHODS |
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Bovine tracheae were obtained from a local slaughterhouse and placed in cold Hanks' buffered saline solution (HBSS). Cell suspensions were prepared by scoring, stripping, and cutting the bovine tracheal epithelium into small pieces using sharp dissection as previously described (4, 7). Cells were isolated by gently stirring the strips at room temperature for 2 h in 50 ml of 50% Dulbecco's modified Eagle's medium-50% Ham's F-12 medium (DMEM-F12, BioWhittaker) with 5% fetal calf serum containing dithiothreitol (5 mM; Sigma Chemical, St. Louis, MO), deoxyribonuclease I (100 mg/ml, Sigma), and 0.1% protease type XIV (Sigma). Cells were centrifuged, resuspended in media, and allowed to rest for 1 h at 37°C to remove any contaminating fibroblasts. Cells were then plated onto collagen-coated plastic culture dishes at 250,000 cells/cm2 and grown in culture medium consisting of DMEM-F12 supplemented with 5% fetal calf serum and (per ml) 80 µg gentamicin, 2.5 µg Fungizone, 100 U penicillin, and 100 µg streptomycin. After 3-4 days in culture, the culture medium was replaced with HBSS containing 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, pH 7.4) and various combinations of different compounds as described below.
Membrane fragments were prepared using airway epithelial cells scraped from the surface of bovine tracheae and placed in DMEM-F12 overnight (13). The suspension was centrifuged, and the pellet was washed twice with HBSS. The pellet was resuspended in lysis buffer [10 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris · HCl), 5 mM MgCl2, 2 mM dithiothreitol, and 10 mM phenylmethylsulfonyl fluoride, pH 7.4] and homogenized on ice using a Dounce homogenizer. After a low-speed spin (300 g) for 8 min at 4°C, the pellet was resuspended and rehomogenized. The rehomogenized pellet was combined with the previous supernatant and centrifuged for 8 min at 4°C at 2,000 g. The postnuclear fraction (supernatant) was resuspended and spun for 10 min at 4°C at 45,000 g, and the resulting pellet, consisting of both apical and basolateral membrane fragments, was used as the crude membrane fragment preparation.
Binding studies were performed at 30°C for 30 min using
[3H]dihydroalprenolol
([3H]DHA, 100 Ci/mmol,
NEN) at concentrations between
1010 and
10
7 M and crude membrane
fragments. Membrane fragments were separated from buffer by vacuum
filtration. The pellet was washed twice with 4 ml of buffer solution at
4°C to optimally separate free and bound
[3H]DHA and to reduce
nonspecific binding. The radioactivity in the pellet was counted using
liquid scintillation spectrophotometry (Beckman LS1801). Nonspecific
binding was determined by displacement of
[3H]DHA binding with
10
6 M propranolol. Data
were analyzed as a function of free ligand concentration using an
iterative nonlinear curve-fitting program (Ligand) and by Scatchard and
Hill analysis to derive
Kd and
Bmax, with the latter expressed in
terms of membrane protein content (15).
The effect of tannin on cell surface number was more precisely
determined using
[3H]CGP-12177 (38 Ci/mmol; Amersham, Arlington Heights, IL). Bovine tracheal epithelial
(BTE) cells in culture (~4 × 105 cells) were exposed to
5-25 µg/ml tannin for 4 h and then incubated in 1 ml of DMEM
containing 25 mM HEPES and 30 µg/ml bovine serum albumin (pH 7.4, Sigma) at 4°C for 3 h in the presence of a saturating concentration
(1 nM) of
[3H]CGP-12177. Cell
surface receptor density was calculated from one-point analysis in
which 106 M propranolol was
used to assess nonspecific binding. Results from tannin experiments
were compared with similar experiments using isoproterenol
(10
5 M for 3 h), which is
known to cause a rapid decrease in cell surface receptor number (18).
In other experiments, membrane fragments (~500 mg of protein) were
diluted in a 50 mM Tris · HCl (pH 7.5)-120 mM NaCl-5
mM KCl-3 mM MgCl2 (binding buffer)
solution and incubated with
[3H]DHA (2.5 nM) and
16 concentrations of ()-isoproterenol at 30°C for 30 min in
the presence or absence of the nonhydrolyzable GTP analog Gpp(NH)p
(5'-guanylylimidodiphosphate; 50 mM). The binding incubations were
terminated by the addition of 4 ml of ice-cold buffer solution and
poured over Whatman GF/C glass fiber filters under vacuum. The filters
were washed once with 4 ml of cold buffer solution and counted in a
liquid scintillation counter.
cAMP was measured using a radioimmunoassay kit (Amersham). The cells were then treated with 1 N NaOH to dissolve cellular protein, which was measured according to the method of Lowry et al. (15), using bovine serum albumin as the standard. cAMP levels were calculated as picomoles cAMP per milligram protein.
In some experiments, cells in culture were incubated for 6 h at 37°C with either 10 or 30 mM N-acetylcysteine (Sigma). The N-acetylcysteine was dissolved in cell media (DMEM-F12), and the pH was adjusted with NaOH before addition to the culture well. cAMP levels were measured under various conditions in the presence and absence of tannin.
Condensed tannins were isolated from the 1985 crop of bracts from Acala SJ-5 cotton grown in Texas utilizing sequential Amicon ultrafiltration and a modification of the procedure of Taylor et al. (26) as previously described (8). Stock solutions of the high-molecular-mass tannin [YM-10 retentate, molecular mass >10,000 Da] were prepared daily, immediately before use, by dissolving the tannin at a concentration of 19.2 mg/ml in water. This represented the tannin concentration in the cotton bract extract used in the original study of Cloutier and Rohrbach (8). Tannin concentrations are reported as micrograms per milliliter. In some experiments, the effects of stock solutions of low-molecular-mass tannin (YM-2 retentate, molecular mass 1,000-5,000 Da) on cAMP accumulation were examined. In other experiments, high-molecular-mass tannin was prepared in water and allowed to sit at room temperature for 72 h. This exposure to room air results in tannin oxidation and decreases polymer length (molecular mass 500-7,500 Da) (19).
Data were analyzed using analysis of variance and Student's t-test unless otherwise specified (2).
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RESULTS |
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Effects on cAMP accumulation.
In BTE cells grown in culture, cAMP accumulation after a 10-min
exposure to isoproterenol
(105 M) was inhibited in
cells exposed to tannin (25 µg/ml) for increasing times (5 min to 8 h). Inhibition began within 10 min and reached a maximum of 52 ± 5% (mean ± SE, n = 6) at 60 min
(Fig. 1). After an 8-h exposure to 25 µg/ml tannin, there was further inhibition (82 ± 9%,
P < 0.001) in
isoproterenol-stimulated cAMP accumulation. Inhibition was dose
dependent. Five micrograms per milliliter tannin inhibited
isoproterenol-stimulated cAMP accumulation by 17 ± 4% after 10 min
and by 33 ± 9% (n = 6, P < 0.001) after 8 h of exposure.
The inhibition of isoproterenol-stimulated cAMP accumulation was
reversible after 8 h of exposure to 25 µg/ml tannin. Reversibility
began within 4 h and approached baseline by 24 h.
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Effects on -adrenergic
receptor.
-Adrenergic receptor density was determined using saturation binding
with [3H]DHA and BTE
membrane fragments at 30°C. In preliminary experiments, we
demonstrated that binding equilibrium occurred by 30 min and that
between 0.1 and 5 nM DHA, specific binding was >75% of total binding. In control membrane fragments, the
Kd for
[3H]DHA was 0.41 ± 0.03 nM, with a Bmax of 252 ± 32 fmol/mg protein (n = 3). In
membranes incubated with tannin (25 µg/ml for 30 min), there was no
change in Kd
(0.26 ± 0.06 nM), whereas Bmax
decreased to 162 ± 36 fmol/mg protein
(P < 0.05), a 36% decrease in
receptor number (Fig. 3). Using
[3H]CGP-12177, we
determined that cell surface receptor density decreased 55% after
tannin (25 µg/ml for 4 h) as shown in Table 2. The decrease in cell surface receptor
density was tannin dose dependent. Isoproterenol
(10
5 M) exposure produced a
72% decrease in cell surface receptor density after a 3-h exposure.
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DISCUSSION |
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These experiments demonstrate that tannin blunts the response to
isoproterenol in bovine tracheal airway epithelial cells and that
polymer length and cysteine residues appear to be important for these
effects. Tannin also decreases -adrenergic receptor density and
uncouples the receptor from its stimulatory G protein, effects due to
polymer length. The tannin present in cotton mill dust is a polymer of
monoflavanoid subunits (19). Compared with other plant tannins, cotton
bract tannin has a longer polymer length (~9.4 monomer units) and an
unusual monoflavanoid composition (procyanidin and prodelphinidin in a
ratio of 2:3) (3). Cyanidins react strongly with cysteine residues
(11), and of the 15 cysteine residues present in the
-adrenergic
receptor, 4 are in the putative extracellular domain (14). These four
cysteines form disulfide bonds, which provide stability to the
receptor, result in high-affinity ligand binding (17), contribute to
the pharmacological specificity of agonists and antagonists, promote
coupling of the
-adrenergic receptor to stimulatory G proteins, and
play a major role in agonist-induced stimulation of adenylyl cyclase
(14, 17, 25). We have hypothesized that cotton bract tannin interacts
with extracellular cysteine residues and that this interaction affects
ligand binding, uncouples the receptor from its stimulatory G protein,
and inhibits cAMP accumulation. Results from experiments in which
polymer length was changed either by oxidation or through
ultrafiltration and results from experiments with
N-acetylcysteine support this
hypothesis.
N-acetylcysteine had no effect on
basal cAMP accumulation and no effect on isoproterenol-stimulated cAMP
accumulation. The increase in basal cAMP levels approached but did not
attain statistical significance. Higher concentrations of
N-acetylcysteine were not examined.
Both the low and the high
N-acetylcysteine concentrations were
equally effective in inhibiting the effects of tannin, irrespective of
their ability to stimulate basal cAMP accumulation. The mechanism for
this inhibition is not known, although it is possible that the
interaction of N-acetylcysteine with
cysteine residues blocks the interaction of tannin with these same
cysteine residues. N-acetylcysteine has been shown to activate a non-cystic fibrosis transmembrane conductance regulator Cl
conductance in airway epithelial cells (12), but the mechanism is
unknown. Data from our laboratory suggest that
N-acetylcysteine at low concentrations
(10 mM), however, has no effect on
Cl
secretion (unpublished
data); this is compatible with our current data.
Tannin also decreased -adrenergic receptor number. DHA measures both
intracellular and surface receptors, whereas CGP-12177 measures surface
receptors only. DHA binding studies suggested a decrease in
-adrenergic receptors after tannin exposure. This decrease is
secondary, at least in part, to a decrease in cell surface receptor
density, which is dose dependent. The effect of tannin on DHA binding
has been previously observed using epinephrine and whole cell airway
epithelial cell preparations, and studies revealed a 38% decrease in
receptor binding (7). Receptor number was, however, considerably
higher, probably secondary to the decreased specificity of epinephrine
to nonreceptor acceptor sites and to oxidation and enzyme metabolism
(25).
Epithelial cells release relaxing and contracting factors that play a
role in modulating airway smooth muscle tone (16). Through its effects
on the -adrenergic receptor, tannin could alter the balance between
relaxing and contracting factors and contribute to the across-shift
Monday changes in pulmonary function that occur with acute exposure to
cotton dust. The diminishing symptoms that occur during the remaining
weekdays are compatible with rapid desensitization of
-adrenergic
receptors with
-adrenergic receptor-stimulatory G protein
uncoupling. Partial recovery of receptor number and resensitization
during weeknights away from the mill could occur and contribute to some
continued symptoms with daily exposure. Baseline pulmonary function
would not be altered, but the responsiveness with repeated exposure
would be affected. A state of airway hyporesponsiveness after repeated exposure to cotton dust has been observed in mill workers with byssinosis (10). Such a mechanism of rapid desensitization has also
been proposed for patients with asthma taking
-adrenergic drugs,
except in asthma the stimulus is rarely continuous (9). Tannin
exposure, however, is one of the few instances in which exposures are
repetitive and frequent and occur over long periods of time.
In summary, tannin desensitizes the airway epithelium to -agonists,
decreases cell surface
-adrenergic receptor number, and uncouples
the
-adrenergic receptor from its stimulatory G protein. These
effects are related to polymer length and possibly to the interaction
of tannin with cysteine residues. These effects result in
Cl
secretion inhibition,
which could alter secondary water transport in the airway, mucus
secretion, and mucociliary transport. These effects could result in the
pathological pulmonary findings in patients with byssinosis. Decreases
in mucociliary transport would also result in secretion retention and
exposure of the epithelium to other substances in cotton dust, such as
endotoxin, for longer than normal periods of time. Thus tannin may
directly and indirectly contribute to the pathogenesis of byssinosis.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. Mekha Reza for technical support and to Ellen Berger for administrative services.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-28669 and a grant from the University of Connecticut Research Foundation.
Address for reprint requests: M. M. Cloutier, Pediatric Pulmonary Division, Connecticut Children's Medical Center, 282 Washington St., Hartford, CT 06106.
Received 7 May 1996; accepted in final form 11 November 1997.
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