First Department of Internal Medicine, Tohoku University School of Medicine, Sendai 980-8574, Japan
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ABSTRACT |
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We investigated
the acute effects of erythromycin (EM) and its derivatives on ionic
currents in airway glands from feline tracheae. Therapeutic
concentrations of EM or clarithromycin (CAM) attenuated the whole cell
currents evoked by ACh in a competitive manner. The maximally
stimulated inward Cl
currents were reduced to 54 and 83% and the outward
K+ currents to 55 and 84% of
control values by EM and CAM, respectively, whereas the responses
induced by phenylephrine, norepinephrine, caffeine, or ionomycin were
unaffected by EM, CAM, or EM523, a synthetic derivative of EM.
K+ channels in excised outside-out
patches were not influenced by macrolides. Although therapeutic
concentrations of macrolides showed no effect on the baseline currents,
high concentrations of macrolides alone evoked currents mimicking the
ACh response, which were abolished completely by atropine. We concluded
that macrolides act as a partial agonist on cholinergic receptors, resulting in a reduction of
Cl
secretion at
pharmacological doses of the agents, which may exhibit a pronounced
effectiveness on hypertrophied and/or cholinergically sensitized
submucosal glands in pathological airways.
submucosal gland; chloride secretion; patch clamp; erythromycin; partial agonist
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INTRODUCTION |
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ERYTHROMYCIN (EM) and its derivatives are known to relieve the symptoms related to airway hypersecretion in such lung diseases as chronic bronchitis and diffuse panbronchiolitis (13, 23, 32). A variety of mechanisms have been reported to explain the basis of the clinical benefit of macrolides in the airways. For instance, EM attenuates the production of cytokines including interleukin (IL)-8 from Pseudomonas aeruginosa-stimulated human neutrophils (18) and from cultivated human bronchial epithelial cells induced by Haemophilus influenzae endotoxin (12). Neutrophil elastase is a strong secretagogue of the airway submucosal gland (30), and suppressing the pulmonary recruitment of neutrophils by reducing the production of neutrophil chemotactic factors such as IL-8 could expectedly result in a decrement of excessive airway secretion (4, 23). This neutrophil-associated hypersecretion appears to also be involved in goblet cell mucus discharge induced by the inhalation of Escherichia coli lipopolysaccharide, which was inhibited by macrolides in guinea pig in vivo (34).
On the other hand, the suppressive effect of EM on respiratory mucus
secretion was noticed more directly in an in vitro preparation of human
airways (7) in which EM reduced both spontaneous (baseline) and
stimulated respiratory glycoconjugate secretion (by either histamine or
cholinergic agonist) from airways in culture. In addition to the
reduction in mucus secretion, baseline
Cl transport across
cultured sheets of canine tracheal epithelium was suppressed by a
submucosal application of EM (33). In this context, macrolide
antibiotics are thought to act as modifiers of biological responses
rather than as antimicrobial agents. Although the possible mechanisms
underlying the direct inhibitory action of EM were discussed in these
reports, they remain unproven.
The airway submucosal gland is a mixed (mucous and serous) gland, and
the secretion of mucins and water (therefore, electrolytes) is known to
be regulated separately (1, 5, 6). Because human airway epithelium is
likely to be primarily absorptive (2, 35), a major fraction of the
airway fluid, which follows an active
Cl secretion from the
glandular acini (6, 26), seems to be derived from the submucosal gland
(21). It is therefore of interest to clarify whether macrolides affect
water secretion as well as mucus secretion from the gland. Sasaki et
al. (27) previously showed, using freshly isolated human
and feline airway gland acinar cells, that cholinergic and
-adrenergic agonists evoked a
Cl
current that is
dependent on Ca2+ released
intracellularly. In the present study, we investigated the acute effect
of macrolide antibiotics on the ionic currents induced by various
agents in feline tracheal submucosal gland using patch-clamp
techniques. Here we report that macrolide antibiotics attenuated the
acetylcholine (ACh)-evoked
Cl
current via an
inhibition of muscarinic receptors. Moreover, very high concentrations
of macrolides per se stimulated the ionic currents, and these were
abolished by atropine.
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MATERIALS AND METHODS |
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Cell preparation. Submucosal glands were isolated from the tracheae of cats (2-5 kg body wt) anesthetized with intramuscular ketamine hydrochloride and intravenous thiopental sodium (30 mg/kg) (see Ref. 29 for a detailed description of the isolation technique). The isolated glands were further dispersed enzymatically into single or clustered acinar cells by incubating them with an enzyme solution containing collagenase (200 U/ml), DL-dithiothreitol (0.31 mg/ml), trypsin inhibitor (1 mg/ml), and bovine serum albumin (0.5 mg/ml) for 30 min at 37°C. After dispersion and a wash with centrifugation at 180 g, the cells were resuspended in a standard extracellular solution (see Electrical recordings) until used.
Electrical recordings. Ionic currents
were measured with a patch-clamp amplifier (EPC9, HEKA Electronic),
low-pass filtered at 1.0 kHz, and monitored on both a built-in software
oscilloscope and a pen recorder (RECTI-HORIZ-8K, Nippondenki San-ei,
Tokyo, Japan). Patch pipettes were made with a vertical puller (PP-83, Narishige Scientific Instruments, Tokyo, Japan) from glass capillaries with an outer diameter of 1.5 mm and a tip resistance of 2-6 M. The junction potential between the patch pipette and the bath solution
was nulled by the amplifier circuitry. After a high-resistance (>1
G
) tight seal was established, the whole cell configuration was
obtained by rupturing the patch membrane with negative pressure applied
to the pipette tip. Membrane currents were monitored at two different
holding potentials (HPs) except where noted otherwise, i.e., 0 and
80 mV, each of which roughly corresponded to the Cl
equilibrium potential
and the K+ equilibrium potential,
respectively, under the present electrolyte conditions. This was
accomplished by applying 100-ms voltage pulses of
80 mV at a
frequency of 2 Hz to the pipette HP of 0 mV (24, 25, 27). The upward or
downward deflection of the current tracing represents outward or inward
current, respectively. In the experiments of excised patch-channel
recordings, the ionic currents were filtered at 300 Hz and analyzed
with Patch Analyst Pro software (TM Corporation, Tokyo, Japan). The
solutions employed were of the following compositions (in mM):
extracellular (bath) solution, 120 NaCl, 4.7 KCl, 1.13 MgCl2, 1.2 CaCl2, 10 glucose, and 10 HEPES;
and intracellular (pipette) solution, 120 KCl, 1.13 MgCl2, 0.5 EGTA, 1 Na2ATP, 10 glucose, and 10 HEPES.
The fluids were superfused over the cell(s) by hydrostatic
pressure-driven application (40-50
cmH2O) through polyethylene tubes.
All solutions were at pH 7.2, and all experiments were carried out at
room temperature (22-25°C).
Quantification procedure. The net electric charge movement across the cell membrane was quantified with a pen recorder chart by measuring the area circumscribed with the current trace and baseline with a digital planimeter (PLACOM KP-92N, Koizumi, Tokyo, Japan) and was converted to picocoulombs per second (pQ/s). The effects of macrolides on the agonist-stimulated responses were estimated by comparing the bidirectional electric charge movements for 20-s durations just before and after treatment with macrolides and are expressed as the percentage of pretreatment control values.
Reagents. HEPES was purchased from Dojin (Kumamoto, Japan). Collagenase was from Wako Pure Chemicals (Osaka, Japan). Clarithromycin (CAM) was a generous gift from Taisho Pharmaceutical (Osaka, Japan), and EM523 was a gift from Dr. A. Mizumoto (Gunma University, Gunma, Japan). All other chemicals used were purchased from Sigma (St. Louis, MO).
Statistics. The data are expressed as means ± SE; n is the number of experiments on different cells. Data were analyzed by paired t-test, and significance was accepted at P < 0.05.
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RESULTS |
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Effects of EM and its derivatives on baseline and
ACh-induced currents. Sasaki et al. (27) previously
showed that ACh induced bidirectional
Ca2+-dependent currents in
tracheal gland acinar cells. That is, the outward current recorded at a
HP of 0 mV was a [tetraethylammonium (TEA)-blockable]
K+ current, and the inward current
at 80-mV HP
(Ii) was
carried mainly by Cl
effluxed out of the cell. The ion species that carried
Ii was validated
by the findings that 1) the major
fraction of Ii
was abolished in the absence of
Cl
in both (bath and
pipette) solutions when replaced by
SO2
4 or gluconate and
2)
Ii was still
activated normally in the absence of
Na+ when replaced by
N-methyl-D-glucamine
(27).
Low concentrations (6-15 nM) of ACh stimulated oscillatory
currents in both directions. As shown in Fig.
1A,
the oscillatory inward Cl
current recorded at a
80-mV HP was abolished rapidly in the presence of 10
6 M EM; i.e.,
the net electric charge movement was reduced to 14 ± 5% of the
pretreatment control value (P < 0.05; n = 6). EM alone exhibited no effect on baseline currents within the concentration range
of 10
7 to
10
4 M (98 ± 2%
compared with control level; not significant;
n = 26). Interestingly, however, high
concentrations (>10
3 M)
of EM alone induced oscillatory (Figs.
1A and
2A)
or sustained currents (Fig. 2B) in
response to increasing doses of the agent (Fig.
2C). Airway gland cells still
responded to ACh after the withdrawal of high concentration EM (Fig.
1A), indicating the physiological
relevance of the EM-induced response. This agonistic action was found
also with EM523 (>10
4 M;
220 ± 44 pQ/s; n = 5), a synthetic
derivative of the macrolide (17). The stimulatory effect of high
concentrations of macrolides was significantly inhibited in the
presence of the muscarinic antagonist atropine. As shown in Fig.
3, the responses to 1-5 mM EM were
reduced to 32 ± 10% of control value in the presence of
10
6 to
10
4 M atropine
(P < 0.01;
n = 10).
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As exemplified in Fig. 1B, higher
concentrations (108 to
10
7 M) of ACh induced
oscillatory responses imposed on sustained currents, which were also
reduced in the presence of EM
(10
6 M; 25 ± 7% of
preceding control value; P < 0.01;
n = 12) and CAM
(10
6 to
10
5 M; 76 ± 5%;
P < 0.05;
n = 11), one of the derivatives of EM
(Fig. 1B). The dose-dependent
inhibitory effect of EM on the ACh (20 nM)-induced response is
summarized in Fig. 4. The
maximal inhibitory effect was observed at
10
5 M EM. This suppressive
effect of low-dose macrolides on the ACh-induced response was rapidly
reversible (Fig. 1).
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A maximal concentration of ACh
(106 M) stimulated large
and sustained currents without oscillation. As shown in Fig.
1C, both EM and CAM attenuated the
maximal stimulation of the currents. To quantify the effects of
macrolides on maximally stimulated inward and outward currents, we
compared the electric charge responses to three successive ACh
applications of short duration (~30 s), with 3-min intervals. Namely,
to avoid contamination from desensitization to maximal ACh stimuli,
mean ionic currents of the first and third responses without macrolide
were compared with the second response, which was in the presence of
macrolide (see Fig. 1C). The inward Cl
current was reduced to
54 ± 9% of the nontreatment control values by
10
5 M EM
(P < 0.05;
n = 7). The outward current was also
attenuated significantly to 55 ± 7% of the control value
(P < 0.05;
n = 7). CAM
(10
5 M) also suppressed the
ACh-induced currents significantly; i.e., the inward and outward
currents were reduced to 83 ± 4 (P < 0.05; n = 5) and 84 ± 3%
(P < 0.05;
n = 5) of control values, respectively.
Effect of macrolides on
K+-channel
activity in the excised membrane patch.
To examine whether macrolides act directly on ion channels in the
plasma membrane of acinar cells, excised outside-out patch experiments
were performed. As previously reported (27), a large-conductance K+ channel was the most frequent
channel at the basolateral aspect of this cell type. The channel
conductance was ~165 pS under
K+-rich saline on both sides of
the patch membrane, and the activity was eradicated by outside TEA, a
maxi-K+-channel inhibitor. This
channel was Ca2+ dependent because
1) in the inside-out excised-patch
experiment, the channel activity was totally abolished when
Ca2+ was removed from the
solution, with 1 mM EGTA added at the cytosolic aspect of the membrane,
even at positive membrane potentials, and
2) the outward current at a 0-mV HP
in the whole cell configuration was abolished in the presence of either
TEA or
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a membrane-permeable Ca2+
chelator. Thus the outward current at a 0-mV HP in the whole cell
configuration was carried by K+
released through the maxi-K+
channel (BK channel). In the present study, the activity of the maxi-K+ channel was unaffected by
either EM (105 and 3 × 10
3 M;
n = 5 each), CAM
(10
5 M;
n = 3), or EM523
(10
4 M;
n = 3; Fig.
5). The open-state probability of the
K+ channel was 0.133 ± 0.020 for control activities and 0.132 ± 0.044 after
10
5 M EM (not significant;
n = 5).
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DISCUSSION |
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In some exocrine glands including those from airways, ACh (via the
muscarinic receptor), phenylephrine, and norepinephrine (via the
-adrenergic receptor) activate
K+ and
Cl
currents through
Ca2+ as a common second messenger
(20, 27). These secretagogues activate, via a guanine
nucleotide-binding protein (G protein)-dependent mechanism, the plasma
membrane PLC that generates inositol 1,4,5-trisphosphate and
diacylglycerol, the former releasing
Ca2+ from intracellular pools and
initiating the Ca2+ signal. In the
present study, we showed that EM and its derivatives reversibly
attenuated the ACh-evoked but not the phenylephrine- and
norepinephrine-induced
Ca2+-dependent ionic currents in
freshly isolated feline tracheal glands. This ACh-specific inhibitory
effect of therapeutic levels of macrolides (16) indicates that the
point of action of the agents is upstream from the G protein linked to
the muscarinic receptor. Furthermore, high doses of macrolides per se
stimulated the ionic currents in a manner similar to that of ACh, and
these were abolished in the presence of atropine. This finding
indicates that macrolides have a certain affinity to muscarinic
receptors. The term "partial agonist" is defined as a drug that
produces submaximal tissue responses and competitively blocks the
effects of those with higher intrinsic efficacies (11). Although
decisive pharmacological analyses were not carried out, EM may act as a partial agonist with a certain threshold on airway gland electrolyte secretion.
The inhibitory action of macrolides on ACh-evoked currents has also
been reported in the guinea pig nasal gland (9), although secretagogues
other than ACh were not examined. These investigators found that the
ACh-evoked K+ and
Cl currents were attenuated
concurrently in the presence of macrolides as in the present study.
However, the authors attributed the effect of macrolides to their
direct inhibitory action on
Cl
conductance. If that
were true also in tracheal glands, it would be necessary for macrolides
to block the K+ channel directly
as well as the Cl
channel.
However, this possibility is remote in tracheal gland acinar cells
because the Ca2+-dependent
K+ channel was not sensitive to
macrolides in the present excised-patch experiments (Fig. 5). In vivo,
it might be possible that a membrane hyperpolarization due to a
Cl
-channel blockade could
induce an inhibition of K+
channels secondarily because the dominant
K+ channels on the tracheal gland
plasma membrane are activated by membrane depolarization as well as by
cellular Ca2+ (27). But this would
not be pertinent to the voltage-clamp experiments. Finally, the
possibility of a direct
Cl
-channel inhibition was
excluded in the tracheal gland preparation because macrolides were
without effect on the inward
Cl
currents stimulated by
adrenergic agonists and ionomycin (Fig. 6).
A direct effect of EM on respiratory mucus secretion has been
investigated in an in vitro preparation of human airways (7). In
contrast to the present results in electrolyte secretion, EM was found
to reduce both spontaneous (baseline) and stimulated glycoconjugate
secretion (by either histamine or the cholinergic agonist methacholine)
from airways in culture. It is known that the mucus and fluid
secretions from the submucosal gland are regulated individually. That
is, the mucus secretion is regulated largely by -adrenergic stimuli
via cAMP (5), whereas the fluid secretion is regulated mainly by
-aderenergic stimuli and cholinergic stimuli energize both
secretions (1). Additionally, it has recently been shown in porcine
bronchi that the ACh-induced liquid secretion could be uncoupled from
mucus secretion by pretreatment with blockers of both
Cl
and
HCO
3 transporters (10), which clearly demonstrated that different mechanisms underlie the two types of
secretions. Expectedly, methacholine in combination with EM yielded
almost the same mucus production compared with that in control cells
(7), and the inhibitory effect of EM on mucus secretion was more
specific to histamine. Their findings are consistent with ours in
regard to the anticholinergic effect. However, with respect to the
effect on histamine-induced mucus secretion, probably mediated by cAMP
through the activation of histamine
H2 receptors, a distinctive
pathway other than anticholinergic may exist, that is, a blockade of
PLA2, as they proposed (7).
In the present study, we could not find a common mechanism responsible for the reported clinical and experimental observations as a whole. This was probably because the present investigation focused on the acute effects of the agents, at most several tens of minutes. Macrolides exhibit not only a secretory inhibitory effect but also anti-inflammatory actions by inhibiting the release of cytokines and/or the migration of inflammatory cells (12, 18) to the extent that it results in a structural restoration of pathological lungs (8). It is notable, in this respect, that the clinical efficacy of macrolides on copious airway secretion has been shown to be attained very rapidly, even within a few days (13), whereas it takes months of treatment to achieve the anti-inflammatory effects (8, 18). Macrolides have at least two sites of action. One is a cell surface receptor, which is known as "motilide action," resulting from an interaction with motilin receptors. In addition, macrolides have been shown to penetrate into the cell interior (3). One of the EM derivatives, FK506, clinically used as an immunosuppressant, has been shown to inhibit cytokine production, including IL-2 and IL-8, at the gene transcription level in activated T lymphocytes (19). This anti-inflammatory action may not be detected within an order of minutes. Taken together, in the present investigation, we have isolated a cholinomimetic action as a novel acute extracellular-phase behavior out of a broad spectrum of actions of macrolide antibiotics.
The airway submucosal gland has been demonstrated to exhibit a marked hypertrophy and hyperplasia in chronic inflammatory lung diseases (22). Furthermore, an upregulated secretory response specific to the cholinergic muscarinic pathway has also been reported in an experimental bronchitic airway (15). The anticholinergic effect of EM and its derivatives may exhibit a pronounced effectiveness in such pathological airways.
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ACKNOWLEDGEMENTS |
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We thank Brent Bell for reading the manuscript.
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FOOTNOTES |
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This work was supported by Grant-in-Aid no. 07670650 for Scientific Research from The Ministry of Education, Science, Sports and Culture, Japan (to T. Sasaki).
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. Shirato, First Dept. of Internal Medicine, Tohoku Univ. School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.
Received 20 August 1998; accepted in final form 26 February 1999.
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