Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
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ABSTRACT |
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Nitric oxide (NO) is continuously produced and released in human airways, but the biological significance of this process is unknown. In this study, we have used Calu-3 cells to investigate the effects of NO on transepithelial anion secretion. An inhibitor of NO synthase, NG-nitro-L-arginine methyl ester, reduced short- circuit current (Isc), whereas an NO donor, S-nitrosoglutathione (GSNO), increased Isc, with an EC50 ~1.2 µM. The NO-activated current was inhibited by diphenylamine-2-carboxylate, clotrimazole, and charybdotoxin. Selective permeabilization of cell membranes indicated that NO activated both apical anion channels and basolateral potassium channels. An inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, prevented activation of Isc by NO but not by 8-bromo-cGMP, suggesting that NO acts via a cGMP-dependent pathway. Sequential treatment of cells with forskolin and GSNO or 1-ethyl-2-benzimidazolinone and GSNO showed additive effects of these chemicals on Isc. Interestingly, GSNO elevated intracellular Ca2+ concentration ([Ca2+]i) but had no effect on Isc activated by thapsigargin. These results show that NO activates transepithelial anion secretion via a cGMP-dependent pathway that involves cross talk between NO and [Ca2+]i.
Calu-3 cells; guanosine 3'5'-cyclic monophosphate; chloride channels; potassium channels
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INTRODUCTION |
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NITRIC OXIDE (NO) produced in human airways is involved in physiological and pathophysiological events such as vasodilatation of pulmonary vessels, bronchodilation, smooth muscle relaxation, neurotransmission, and bacteriostasis (for review see Ref. 24). NO is synthesized via the oxidation of L-arginine to L-citrulline by the action of NO synthase (NOS), which has three isoforms. The endothelial and neuronal isoforms are Ca2+ dependent and generate small quantities of NO that participate in physiological functions via activation of the soluble guanylyl cyclase. The inducible isoform is Ca2+ independent and generates large, sustained amounts of NO that may be beneficial or harmful to the cells that produce it and those in the vicinity. All of these isoforms of NOS have been identified in the human respiratory tract, and all three are thought to contribute to NO production (43).
There is increasing evidence that constitutively produced NO plays an important role in the regulation of epithelial ion channels in the human respiratory tract. Both cGMP-dependent (10, 20) and -independent (14) pathways have been implicated in this process. In lung alveolar type II cells, NO has been shown to inhibit the activity of epithelial Na+ channels (7, 14, 19). Although the physiological role of this effect is unknown, it could be expected that, under inflammatory conditions, NO would promote lung edema formation by reducing the rate of alveolar fluid absorption. Interestingly, other studies have shown that inhaled NO prevents interleukin-1-induced edema formation in rat lung (13), and NO has been found beneficial in high-altitude pulmonary edema by improving arterial oxygenation (36). Similarly, it has been suggested that NO released from alveolar macrophages protects type II cells from undergoing apoptosis (9).
Endogenously produced NO has been shown before to stimulate
glycoconjugate secretion from human airway submucosal glands
(30), but the effect of NO on electrolyte secretion has
not been investigated. Submucosal gland serous cells express high
levels of cystic fibrosis (CF) transmembrane conductance
regulator (CFTR) Cl channels, and they make a significant
contribution to the quantity and composition of gland secretions.
They also represent a potential target in CF gene therapy, and for this
reason, it is important to understand their mechanisms of fluid and
electrolyte transport.
Most of our knowledge about ion movements in human airway
serous cells comes from studies performed on the Calu-3 cell line, which is derived from a lung adenocarcinoma (39).
Secretion of anions by Calu-3 cells results from the coupling of ion
transport processes in the apical and basolateral membranes
(6). CFTR in the apical membrane serves as both a
HCO channel, mediating the
apical exit of either anion. Recent evidence indicates that forskolin
stimulates HCO
secretion. These secretagogues must activate different signal transduction pathways because sequential treatment of Calu-3 cells with
forskolin and 1-EBIO reveals additivity between forskolin and 1-EBIO
effects (6).
The present study concerns the role of NO in transepithelial anion secretion in Calu-3 cells. Specifically, we have investigated whether NO, at concentrations likely to be encountered in vivo, can modulate anion secretion in airway submucosal glands. Inhaled NO is now frequently administered to patients with inflammatory diseases, and it is important to understand the effects of altered NO levels on transepithelial anion secretion. Our results indicate that constitutively produced NO modulates anion secretion and that NO donors can further increase the secretory effects produced by forskolin and 1-EBIO.
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METHODS |
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Cell culture.
Calu-3 cells were obtained from the American Type Culture Collection
(Manassas, VA). The cells were grown in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum, 60 µg/ml gentamicin
sulfate, 60 µg/ml penicillin G and 100 µg/ml streptomycin and
maintained in T75 tissue culture flasks (Costar, Cambridge, MA) at 37°C in a humidified atmosphere of 5% CO2 in air.
Confluent cell layers were incubated with 0.05% trypsin and 0.02%
EDTA in saline for 45 min to avoid selecting for a subpopulation. For transepithelial measurements, cells were seeded at 106
cells/cm2 onto Costar Transwell inserts (0.45- µm pore
size, 1-cm2 surface area) coated with human placental
collagen (Sigma, St. Louis, MO). For the first 6 days, cells were grown
submerged in culture medium that was changed every 2-3 days.
Subsequently, air interface culturing was used, in which the medium was
added only to the basolateral side of the inserts. Transepithelial
measurements were performed with whole inserts mounted into an Ussing
chamber (World Precision Instruments, Sarasota, FL) 10-30 days
after plating.
Transepithelial measurements. Standard techniques were used in Ussing chamber studies. Apical and basolateral solutions were maintained in water-jacketed glass chambers kept at 37°C. Chemicals were added from concentrated stock solutions, and both chambers were continuously and separately perfused to ensure proper oxygenation and stirring of the solutions. The transepithelial potential difference was clamped to zero by use of a DVC 1000 voltage/current amplifier (World Precision Instruments), and the resulting short- circuit current (Isc) was recorded through Ag-AgCl electrodes and 3 M NaCl-agar bridges. Initially, all cell monolayers were equilibrated with 10 ml of Krebs-Henseleit solution, which contained (in mM) 116 NaCl, 5 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 25 NaHCO3, and 10 glucose (pH was 7.4 when bubbled with 95% O2-5% CO2 at 37°C). The Isc was allowed to stabilize for 10-15 min before application of NO donors or other tested chemicals. Positive currents were defined as anion secretion or movement from serosal to mucosal side. The transepithelial conductance was continuously monitored and calculated with the use of Ohm's law by measuring current changes in response to 0.5-mV pulses. All Isc measurements were recorded on an IBM-compatible computer through an analog-to-digital board (DT2128 Data Translation, Marlboro, MA).
Apical membrane Cl current.
The effects of NO on apical membrane Cl
current
(ICl) were assessed after permeabilization of
the basolateral membrane with 360 µg/ml nystatin and establishment of
an apical-to-basolateral Cl
concentration gradient.
Basolateral NaCl was equimolarly exchanged for sodium gluconate, and
CaCl2 concentration was increased to 4 mM to compensate for
the Ca2+-buffering capacity of the gluconate. Under these
conditions, the contribution of basolateral ion cotransporters and
Na+-K+-ATPase to the Isc
are eliminated, and Isc represents
ICl as these ions move down their concentration
gradient through apical Cl
channels.
Basolateral membrane
K+ current.
The effects of NO on basolateral membrane K+ channels were
assessed after permeabilization of the apical membrane with 360 µg/ml
nystatin and establishment of an apical-to-basolateral K+
concentration gradient. Apical NaCl was replaced by equimolar potassium
gluconate, whereas basolateral NaCl was substituted with sodium
gluconate, and CaCl2 concentration was increased to 4 mM.
Under these conditions, the contribution of apical Cl
channels to Isc is eliminated, and measured
Isc represents K+ current as these
ions move down their concentration gradient through basolateral
K+ channels.
Measurement of cGMP and cAMP. Cells were seeded in 24-well plates (Becton Dickinson Labware) at a density of ~65,000 cells/well and grown to confluence as described in Cell culture. Monolayers were washed three times with bath solution and then placed into a fresh bath solution containing 3-isobutyl-1-methylxanthine (100 µM) or an equal volume of DMSO as a control and left for 5 min at room temperature. Cells were then treated with S-nitrosoglutathione (GSNO) for 60 s, after which the supernatant was replaced with acetic acid (300 µl, 5 mM) and frozen on dry ice for 10 min. The cells were lysed by boiling for 10 min in acetic acid, and the supernatant was kept for radioimmunoassay (RIA) detection of cGMP and cAMP by use of acetylated samples (16). Antibodies for the RIAs of cGMP and cAMP were gifts from Dr. Anthony Ho (Dept. of Physiology, University of Alberta, Edmonton, Canada). The measurements represent cGMP or cAMP that was generated and released into the medium during the period of exposure to drugs. Protein was measured with the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) with the use of bovine serum albumin as a standard.
Intracellular Ca2+ measurements. Monolayers of Calu-3 cells were grown on glass coverslips coated with collagen. Cells were loaded with 3 µM fura 2-AM for 60 min in the dark at 37°C. Afterward, they were washed three times and mounted into a cuvette filled with 2.5 ml of Krebs-Henseleit solution. Fluorescence was measured using a PTI spectrofluorometer (Photon Technology International) at 340 and 380 nm with irradiation at 510 nm, and the ratio of fluorescence intensity at 340 nm to that at 380 nm (340/380 ratio) was calculated. No attempt was made to calibrate the results for intracellular Ca2+ concentration ([Ca2+]i).
L-Citrulline assay. The activity of NOS was assayed by measuring the rate of conversion of L-[14C]arginine to L-[14C]citrulline (33). Briefly, the samples were incubated at 37°C with L-[14C]arginine (Amersham) in assay buffer containing 50 mM KH2PO4, 1 mM MgCl2, 0.2 mM CaCl2, 50 mM L-valine, 1 mM L-citrulline, 20 µM L-arginine, 0.1 mM NADPH, 10 µM tetrahydrobiopterin, and 1.5 mM dithiothreitol in the presence or absence of 1.5 mM NG-monomethyl-L-arginine (L-NMMA). EGTA (2 mM) was used to differentiate between Ca2+-dependent and -independent NOS. After a 20-min incubation, the reaction was terminated by dilution and removal of nonreacted L-arginine by means of AG-50W-X8 resin (Bio-Rad); the remaining radioactivity was counted using a liquid scintillation counter.
Chemicals. NG-nitro-L-arginine methyl ester (L-NAME) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) were purchased from Alexis Biochemicals (San Diego, CA), L-[U-14C]arginine from Amersham Life Science, and 1H-[1,2,4]oxadiazolol-[4,3-a]quinoxalin-1-one (ODQ) from Tocris Cookson (St. Louis, MO). Thapsigargin, forskolin, clotrimazole, 1-EBIO, amiloride, L-cis-diltiazem, charybdotoxin (CTX), diphenylamine-2-carboxylate (DPC), 8-bromo-cGMP (8-BrcGMP), and GSNO were purchased from Sigma. DPC, ODQ, and thapsigargin were prepared as 1,000-fold stock solutions in DMSO. SNAP, clotrimazole, forskolin, and 1-EBIO were made as 1,000-fold stock solutions in ethanol. Nystatin was prepared as a 180 mg/ml stock solution in DMSO and sonicated for 30 s just before use. All other compounds were made as stock solutions in distilled, deionized water.
Data analysis.
GSNO dose response was fitted by the Michaelis-Menten equation of the
form
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RESULTS |
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NOS activities in Calu-3 cells.
The activities of Ca2+-dependent and
Ca2+-independent (assayed in the presence of 1 mM EGTA) NO
synthases in Calu-3 cells were measured using a
L-[14C]arginine to
L-[14C]citrulline conversion assay
(33). In nonfractionated cytosols of Calu-3 cells, the
activities of Ca2+-dependent and
Ca2+-independent NO synthases were 1.89 ± 0.36 (pmol · min1 · mg
1 of
protein, n = 3) and 0.12 ± 0.04 (pmol · min
1 · mg
1 of
protein, n = 3), respectively. These results are in
agreement with similar studies performed with primary cultures of human airway epithelial cells (2) and suggest that Calu-3 cells
can be used as a model system to investigate the role of NO in the regulation of serous cell anion secretion.
Effects of endogenous and exogenous NO on anion
secretion.
We have studied 98 monolayers with standard Krebs-Henseleit solution on
the apical and basolateral membrane surfaces. The basal
Isc under these conditions averaged 15.2 ± 9.1 µA/cm2 (range 2.5-51.0 µA/cm2).
Application of L-NAME, an analog of L-arginine
that inhibits generation of NO, caused ~9% reduction of
Isc (n = 9; P = 0.002, two-tailed paired t-test; Fig.
1A). The average duration of
L-NAME effect was 3.8 ± 0.6 min (n = 7), but in two recordings, the effect lasted >10 min, and the current
returned to the baseline only after L-NAME had been washed
out from the bath solution. Application of the NO donor GSNO (100 µM)
increased Isc by 112.4 ± 16.7%
(n = 41). The response of Isc to
GSNO was also transient, characterized by a large initial peak,
followed by a second, smaller peak before reaching steady state (Fig.
1A). The maximal Isc response was used in all calculations.
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Pharmacological characterization of GSNO-activated
Isc.
It is generally accepted that apically located CFTR channels serve as
the conductive pathway for anion secretion in Calu-3 cells. To
determine the role of these and other ion channels in GSNO-mediated
Isc activation, a number of pharmacological
agents were applied to the bath solution prior to the addition of GSNO. A summary of these experiments is shown in Fig.
3. DPC, a blocker of CFTR, reduced basal
Isc by 76 ± 12% (n = 5).
In the presence of DPC, GSNO had no significant effect on
Isc. It has been shown previously that GSNO and
other NO donors inhibited amiloride-sensitive Na+ channels
in alveolar type II cells (7, 14, 19). In Calu-3 cells,
amiloride (50 µM) had no effect on Isc
(P > 0.05, n = 9), and subsequent
application of GSNO (100 µM) still significantly increased
Isc (P < 0.01, n = 9), indicating that amiloride-sensitive channels
did not contribute to Isc in Calu-3 cells. The
contribution of cyclic nucleotide-gated cation channels to the
Isc in rat tracheal epithelia has been shown
before (38). The role of these channels in Calu-3 cells
was investigated by using L-cis-diltiazem (100 µM, n = 4). Diltiazem had no effect on the basal
Isc and did not block the effect of GSNO,
indicating that cyclic nucleotide-gated channels did not contribute
significantly to Isc in Calu-3 cells and were
not targeted by GSNO. CTX (50 nM), a blocker of basolateral K+ channels, reduced the basal Isc
by ~25%, indicating that CTX-sensitive K+ channels
contribute to the basal current (n = 4). The fact that subsequent addition of GSNO had no significant effect on
Isc indicated that CTX-sensitive K+
channels were a target of GSNO-mediated activation of
Isc (Fig. 3). Similar effects were observed with
another blocker of Ca2+-dependent K+ channels,
clotrimazole (30 µM, n = 4). A blocker of
Cl channels, niflumic acid (NA; 100 µM), significantly
reduced basal Isc compared with control levels
(P < 0.05, n = 6). However, NA did not
block the effect of GSNO because significant current activation was
still observed when GSNO was applied in the presence of NA. In summary,
this study suggested that both apical Cl
channels and
basolateral K+ channels were likely targets for NO action
in Calu-3 cells.
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NO acts via a cGMP-dependent pathway
in Calu-3 cells.
The majority of biological effects of NO have been attributed to its
interaction with the heme component of soluble guanylyl cyclase (sGC)
and stimulation of enzymatic conversion of GTP to cGMP. To determine
whether the NO/cGMP-dependent pathway was involved in
Isc activation, we first measured cGMP levels in
Calu-3 cells after GSNO treatment. Incubation of cells with GSNO
(0-1,000 µM) for 1 min significantly increased the cytoplasmic
levels of cGMP (P < 0.05, n = 6) but
had no effect on intracellular cAMP (Fig. 5A). Pretreatment of cells
with a selective inhibitor of sGC, ODQ (10 µM), abolished the
increase in cGMP generation, indicating that NO effects are mediated
through activation of sGC. To directly demonstrate the role of cGMP in
the NO-induced activation of Isc, ODQ (10 µM)
was added to both apical and basolateral sides before application of
GSNO (Fig. 5B). Although ODQ had no effect alone on
Isc, it prevented activation of
Isc by GSNO but not by a membrane-permeable analog of cGMP, 8-BrcGMP (1 mM, n = 4). These results
indicate that cGMP plays a crucial role in NO-mediated current
activation in Calu-3 cells.
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Effects of forskolin, 1-EBIO, and
GSNO on Isc.
Sequential treatment of Calu-3 cells with forskolin and thapsigargin
(29) or forskolin and 1-EBIO (6) leads to
additive activation of Isc. The goal of this
study was to examine possible interactions between signal transduction
pathways activated by these agents and GSNO. Forskolin (10 µM) alone
caused a significant increase in Isc. Subsequent
application of GSNO (100 µM) further increased forskolin-activated
Isc, indicating independent actions of the cAMP-
and GSNO-stimulated pathways (n = 12, Fig.
6A). Similar potentiation of
the Isc response has been observed after
sequential treatment of Calu-3 cells with 1-EBIO (300 µM) and GSNO
(n = 5). Interestingly, GSNO had no effect on
Isc activated by an inhibitor of endoplasmic
reticulum Ca2+-ATPase, thapsigargin (300 nM,
n = 4; Fig. 6B). This suggests the
involvement of intracellular Ca2+ stores in
Isc activation by NO and that NO could be a
physiological regulator of [Ca2+]i in Calu-3
cells. Direct evidence for such interactions was sought by measuring
the ratio of the intensity of fluorescence emission at 340 and 380 nm
of fura 2-loaded cells irradiated at 510 nm (340/380 ratio, Fig.
7). GSNO (100 µM) increased the 340/380 ratio from 1.32 ± 0.03 to 1.41 ± 0.04 (P < 0.01, Student's paired t-test; n = 6).
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DISCUSSION |
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The present study shows that endogenously produced NO affects basal Isc in Calu-3 cells and that NO donors further increase the stimulatory effects of forskolin and 1-EBIO on Isc. NO exerts its effects via a cGMP-dependent pathway, and the most likely targets of its action are apical anion channels and basolateral K+ channels. These results are similar to earlier observations showing that NO donors stimulated, whereas NO synthase inhibitors (L-NAME or L-NMMA) inhibited, glycoconjugate secretion from isolated human airway submucosal glands (30).
NO is known to affect the function of epithelial ion channels,
including Na+- (7, 14, 19), Cl-
(10, 20), K+- (23), and
cGMP-gated channels (3). cGMP-dependent and -independent mechanisms have both been implicated in this regulation (14, 21). The amiloride-sensitive Na+ channels in
alveolar type II cells are known to be inhibited by NO donors (7,
14, 19). However, because contribution of these channels to
basal Isc in Calu-3 cells is not significant (40), it is not surprising that amiloride did not affect
activation of Isc by GSNO.
The expression of cyclic nucleotide-gated channels is both tissue and species specific, and NO has been shown to be a major physiological regulator of their function (3). The cGMP-gated channels contribute to electrolyte secretion in rat tracheal epithelium (38) and are potential targets for the regulation of ion movement by NO. Interestingly, the results of this study show that cyclic nucleotide-gated channels do not play a significant role in NO-dependent electrolyte secretion in Calu-3 cells.
Studies with intact and permeabilized Calu-3 monolayers revealed that NO activated both apical membrane anion channels and basolateral membrane K+ channels. At least three biophysically and pharmacologically distinct types of K+ channels are thought to contribute to the basolateral membrane K+ conductance: large-conductance Ca2+-activated K+ (BK) channels, intermediate conductance Ca2+-activated K+ (IK) channels, and cAMP-dependent K+ channels (37). Calu-3 cells have been shown before to express K+ channels with biophysical and pharmacological properties similar to the IK channel family (6). Although IK channels are directly activated by 1-EBIO via a Ca2+-dependent mechanism (32), their regulation by NO has not been studied. Another possible target for NO action are BK channels, which are regulated by the NO/cGMP-dependent pathway (11) and are present in the basolateral membrane of human airway epithelial cells (37).
Activation of sGC and generation of cGMP are responsible for many of the biological effects of NO (28). The results of this study show that the NO/cGMP-dependent pathway is also involved in the activation of Isc in Calu-3 cells because 1) application of 8-BrcGMP produced a similar effect to that of NO, 2) a correlation was observed between the activation of Isc and cGMP levels after NO treatment, and 3) the NO effects could be eliminated by pretreatment of cells with a selective inhibitor of sGC, ODQ. Although these results are consistent with the regulation of Isc via the NO/cGMP-dependent pathway, they do not exclude the involvement of a cGMP-independent pathway in this process. The role of the cGMP-independent pathway could be especially important under inflammatory conditions when large amounts of NO are generated, and NO groups could be introduced into some thiol and transition metals containing proteins, altering their properties and functions (12).
The concentration of NO donors used in our study is likely to yield NO
concentrations similar to those encountered in native tissues. Although
NO concentration in the airway surface liquid (ASL) has not been
measured directly, it has been shown that alveolar macrophages produce
0.1 nM · min1 · 106
cells
1 of NO (18), which may generate
micromolar concentrations in the ASL. Similarly, previous measurements
have shown that ~4 µM concentrations of nitrosothiols were reported
in distal airway fluid of patients with pneumonia (12),
2-4 µM concentrations of NO in brain during cerebral ichemia
(25), and ~0.3 µM concentrations of NO in mesenteric
resistance arteries (41). In addition, it has been shown
that 100 µM SNAP generates a stable NO concentration of 0.1 µM at
25°C (17). Therefore, it is reasonable to assume that NO
amounts used in our study are similar to those found in airways.
Forskolin and 1-EBIO have additive, and independent of the order of
addition, effects on Isc in Calu-3 cells
(6). The results of this study show that GSNO, when added
to either forskolin- or 1-EBIO-pretreated monolayers, further increases
Isc. Although NO has been suggested to activate
CFTR either directly (8) or through cGMP-dependent protein
kinase (42), this effect is not likely to play a
significant role in the presence of 10 µM forskolin, which is known
to maximally stimulate CFTR in Calu-3 cells (1). However,
in addition to CFTR, these cells also possess cAMP-independent,
DIDS-sensitive, outwardly rectifying Cl channels
(4). Channels with similar biophysical
characteristics were shown to be activated by NO in our earlier studies
(20), and their contribution to Isc
could explain the additive effects of forskolin and GSNO. Another
hypothesis involves the effects of NO on the epithelial barrier, in
particular, the function of tight junctions (44). The
modulation of tight junction permeability by NO could have a
significant effect because both transcellular and paracellular
pathways contribute to Isc. ClC-2 chloride
channels were recently found at the tight-junction complexes between
adjacent epithelial cells (15), but the effect of NO on
their activity is unknown.
The fact that application of GSNO to thapsigargin-pretreated cells had no further effect on Isc suggests the involvement of intracellular Ca2+ stores in current activation. A cross talk between NO and [Ca2+]i has been shown in several studies (for review see Ref. 5). Intracellular Ca2+ controls production of NO by Ca2+-dependent NO synthases, whereas NO regulates Ca2+ release from intracellular stores. The results of this study show that these interactions could play a significant role in the activation of anion secretion in human airways.
NO has increasingly been recognized as having an important signaling
role in the regulation of a variety of physiological functions in the
airways. NO has bacteriostatic effects at concentrations found in the
nose (26), controls electrolyte (10) and
mucus secretion (30), and increases cilia beat frequency
(35). Therefore, decreased NO production, as it is
observed in CF (22, 27), would be expected to contribute
to the pathology of the disease by altering the volume and composition
of ASL. Interestingly, recent studies with nasal epithelia from both
non-CF and CF patients suggested that NO had neither inhibitory effects
on amiloride-sensitive Na+ channels nor stimulatory effects
on Cl secretion (by CFTR or any other Cl
channel) in either tissue (34). However, NO significantly
elevated the intracellular Ca2+ concentration in both
tissues. These results are different from the effects of NO on
Cl
secretion reported in this study. Although the reason
for these discrepancies is not clear, it might be due to a difference
in tissue type because it is known that the regulation of epithelial ion channels differs among salt transporting tissues.
Inhaled NO is now administered to patients with inflammatory diseases to lower blood pressure and improve ventilation-perfusion matching (31). It is also reasonable to believe that drugs that affect endogenous NO synthesis will be used clinically in the future. Therefore, it is important to understand the effects of altered NO levels on transepithelial anion secretion. The results of the present study show the involvement of intracellular Ca2+ stores in activation of anion secretion in airway submucosal glands by NO. This suggests that pharmacological regulation of the NO/cGMP-dependent pathway of electrolyte and mucus secretion could represent a novel approach to controlling airway secretions and mucociliary clearance.
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ACKNOWLEDGEMENTS |
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I thank Dr. Anthony Ho for help with cAMP and cGMP assays, Drs. M. Radomski and S. F. P. Man for helpful discussions, and J. Sawicka for excellent technical help.
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FOOTNOTES |
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This work was supported by grants from the Canadian Cystic Fibrosis Foundation and the Canadian Institutes of Health Research.
Address for reprint requests and other correspondence: M. Duszyk, Dept. of Physiology, Univ. of Alberta, 7-46 Medical Sciences Bldg., Edmonton, Alberta T6G 2H7, Canada (E-mail: marek.duszyk{at}ualberta.ca).
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. Section 1734 solely to indicate this fact.
Received 19 June 2000; accepted in final form 20 March 2001.
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