Regulation of anion secretion by nitric oxide in human airway epithelial cells

Marek Duszyk

Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> and a Cl- channel, mediating the apical exit of either anion. Recent evidence indicates that forskolin stimulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, whereas 1-ethyl-2-benzimidazolinone (1-EBIO) increases Cl- 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
I<SUB>sc</SUB> = <IT>I</IT><SUB>control</SUB> + <FR><NU><IT>I</IT><SUB>GSNO</SUB> − <IT>I</IT><SUB>control</SUB></NU><DE>1 + (K&cjs0823;  C)<SUP><IT>n</IT></SUP></DE></FR>
where Isc is in µA/cm2, IGSNO is a peak current in the presence of GSNO, Icontrol is a basal current, and C is GSNO concentration. The two fitted parameters were K, the drug concentration that gave half-maximal Isc activation, and n, the Hill coefficient. Curve fitting was performed using Origin (Microcal Software, Northampton, MA). All data are presented as means ± SE, where n is the number of experiments. Student's t-test was used to determine the significant difference between two group means. A value of P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 · min-1 · 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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Fig. 1.   A: effect of NG-nitro-L-arginine methyl ester (L-NAME) and S-nitrosoglutathione (GSNO) on short-circuit current (Isc) in Calu-3 monolayers. L-NAME (1 mM both sides) transiently reduced Isc, and subsequent application of GSNO (100 µM both sides) significantly increased Isc. Current pulses represent responses to voltage pulses of 0.5 mV applied to monitor tissue conductance. B: activation of Isc by S-nitroso-N-acetylpenicillamine (SNAP; 100 µM both sides). The current was significantly reduced by application of diphenylamine-2-carboxylate (DPC; 1 mM apical).

Application of another NO donor, SNAP, which is biochemically different from GSNO, had a similar effect on Isc, suggesting a common mechanism of action via release of NO (n = 3, Fig. 1B). The fact that 100 µM GSH (carrier of NO in GSNO) had no effect on Isc further suggests that GSNO was acting via release of NO (n = 4). An average GSNO concentration-response curve is shown in Fig. 2. The maximum Isc response was seen at 100 µM GSNO, with a half-maximal effective concentration of 1.2 µM and a Hill coefficient of 1.6. 


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Fig. 2.   Average concentration-response curve for Calu-3 monolayers stimulated by GSNO. Solid line shows best fit of Michaelis-Menten function to the data (Km = 1.2 µM, n = 1.6) The number of measurements is shown at each data point.

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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Fig. 3.   Histogram showing effects of channel blockers and subsequent addition of GSNO (100 µM) on Isc. The dotted line represents control level (100%). DPC (1 mM, n = 5) decreased Isc by 76 ± 12%, and subsequent addition of GSNO had no significant effect on Isc. Amiloride (Amil; 50 µM, n = 9) and diltiazem (Dilt; 100 µM, n = 4) both had no effect on Isc and did not prevent its activation by GSNO. Charybdotoxin (CTX; 50 nM, n = 4) and clotrimazole (Clotr; 30 µM, n = 4) both decreased basal Isc, and in their presence GSNO had no significant effect on Isc. Niflumic acid (NA; 100 µM, n = 6) significantly reduced basal Isc but did not block the effect of GSNO because significant current activation was observed when GSNO was applied in the presence of NA. *P < 0.05. **P < 0.01.

To further resolve the conductance pathways activated by NO, nystatin was used to permeabilize either the apical or basolateral membrane and the appropriate transepithelial ion gradients were established to measure K+ and Cl- currents, respectively. Figure 4A shows that GSNO (100 µM) activated K+ current in apically permeabilized Calu-3 cells; Fig. 4B shows activation of Cl- current by GSNO in basolaterally permeabilized cells. These results indicate that GSNO activates both apical Cl- channels and basolateral K+ channels.


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Fig. 4.   A: effect of GSNO on basolateral membrane K+ currents after establishment of an apical-to-basolateral K+ gradient and permeabilization of the apical membrane with nystatin. The effect of GSNO was inhibited by CTX. B: effect of GSNO on apical membrane Cl- currents after establishment of an apical-to-basolateral Cl- gradient and permeabilization of the basolateral membrane. The effect of GSNO was inhibited by 100 µM DPC.

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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Fig. 5.   A: histogram showing cytoplasmic levels of cGMP and cAMP in Calu-3 cells after treatment with GSNO (0-1,000 µM) for 1 min in the absence and presence of a specific inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 20 µM). *P < 0.05 compared with control, n = 6. B: effect of 8-bromo-cGMP (8-BrcGMP; 100 µM) on Isc in Calu-3 cells. The data are representative of 5 different experiments.

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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Fig. 6.   A: effect of forskolin (10 µM) and GSNO (100 µM) on Isc in Calu-3 cells. B: statistical analysis of the effects of forskolin (10 µM) and GSNO (100 µM) (n = 12), 1-ethyl-2-benzimidazolinone (1-EBIO; 300 µM) and GSNO (n = 5), and thapsigargin (300 nM) and GSNO (n = 4) on Isc. *P < 0.05.



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Fig. 7.   Effect of GSNO (100 µM) on intracellular Ca2+ concentration indicated by the 340- to 380-nm ratio in fura 2-loaded cells. Data are presented as an averaged response from 6 preparations. The ratio increased from 1.32 ± 0.03 to 1.41 ± 0.04 after GSNO treatment (P < 0.01, Student's paired t-test; n = 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 · min-1 · 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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