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(Journal of Leukocyte Biology. 2002;71:807-812.)
© 2002 by Society for Leukocyte Biology

Nitric oxide activates ATP-dependent K+ channels in human eosinophils

Andreas Schwingshackl*, Redwan Moqbel{dagger} and Marek Duszyk*

Departments of
* Physiology and
{dagger} Medicine, Pulmonary Research Group, University of Alberta, Edmonton, Canada

Correspondence: Dr. Marek Duszyk, Department of Physiology, University of Alberta, 7-46 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada. E-mail: marek.duszyk{at}ualberta.ca


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ABSTRACT
 
Nitric oxide (NO) affects the function of ion channels in many cell types, but its role in the regulation of eosinophil ion channels is unknown. In this study, we used the perforated patch-clamp method to investigate the effect of endogenous and exogenous NO on eosinophil ion channels. Using the NO synthase inhibitor, N-nitro-L-arginine methyl ester, we showed that endogenous NO did not affect the whole-cell current in eosinophil. However, two NO donors, S-nitroso-glutathione and S-nitroso-N-acetyl penicillamine, activated whole-cell currents via a NO/cGMP-dependent pathway. Ion substitution and pharmacological studies showed that NO-activated currents were carried by K+ ions, likely through ATP-dependent K+ channels (KATP). Although RT-PCR studies showed the expression of several classes of K+ channels in human eosinophils, NO donors affected only KATP channel function. We conclude that NO, at concentrations likely to be encountered in vivo, could prevent eosinophil activation by opening KATP channels.

Key Words: cGMP • diazoxide • KATP • RT-PCR


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INTRODUCTION
 
Plasma membrane ion channels are involved in stimulus-response coupling in many cell types [1 ]. However, relatively little is known about their role in eosinophil activation. Ca2+-activated K+ currents (KCa) were shown to affect eosinophil superoxide (O2-) production [2 ]. Furthermore, quinidine-sensitive K+ channels were implicated in promotion of eosinophil shrinkage during apoptosis [3 ]. Other studies have shown that eosinophils express inwardly rectifying K+ currents (Kir2.1) [4 ], but their functional role is unknown. The involvement of other ion channels, in particular Cl- channels, in eosinophil activation and O2- production has been demonstrated recently [5 ].

Nitric oxide (NO) is known to regulate ion channels in many cell types. NO activates KCa and adenosine 5'-triphosphate (ATP)-sensitive K+ channels (KATP) in smooth muscle [6 ], neuronal [7 ], endothelial [8 ], and colonic epithelial cells [9 ] via cGMP-dependent and -independent mechanisms [10 , 11 ]. In addition, NO activates cystic fibrosis transmembrane conductance regulator (CFTR) [12 ] and non-CFTR Cl- channels in lung epithelial cells [13 ]. In human airways, several cell types produce NO, including neutrophils [14 ], macrophages [15 ], epithelial cells [16 ], endothelial cells [17 ], and to a lesser extent, eosinophils [18 ]. An increased amount of NO was found in the exhaled air of asthmatic patients and was shown to correlate with lung eosinophilia [19 ]. NO may also be protective to airways reducing neutrophil recruitment [20 ] and O2- production [21 ]. However, the intracellular mechanisms that enable NO to exert such a wide range of effects in airways are not understood completely.

The aim of the present study was to investigate the effect of NO on ion channel function in human peripheral blood eosinophils. We hypothesized that endogenous and exogenous NO affects ion channel function via cGMP-dependent and/or -independent mechanisms. We found that endogenous NO did not affect channel function, but NO donors activated whole-cell currents via a cGMP-dependent pathway. Ion replacement studies indicated that the NO-activated current was carried by K+ ions. It is interesting that NO affected only KATP channel function, suggesting that these channels may represent a novel target to modulate eosinophil activation in asthma and related allergic diseases.


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MATERIALS AND METHODS
 
Chemicals and reagents
Amphotericin B, S-nitroso-glutathione (GSNO), 1-ethyl-2-benzimidazoline (1-EBIO), H89, diazoxide, glibenclamide, clotrimazole, and 4-aminopyridine (4-AP) were purchased from Sigma-Aldrich (Ontario, Canada). S-nitroso-N-acetyl penicillamine (SNAP), N-nitro-L-arginine methyl ester (L-NAME), and 1,2,4-oxadiazole-4,3-quinoxalin-1 (ODQ) were purchased from Alexis (San Diego, CA). 8-Br-cGMP was purchased from Calbiochem, (San Diego, CA).

Patch-clamp experiments in human peripheral blood eosinophils
Peripheral blood eosinophils were purified to homogeneity (<98%) from atopic asthmatic volunteers who had given their informed consent, as described previously [22 , 23 ]. Whole-cell recordings were obtained using the amphotericin B-perforated patch-clamp technique. In preliminary experiments, baseline whole-cell currents were recorded for up to 45 min. These experiments showed that the current stabilized after approximately 10 min, and all recordings were obtained under stable baseline conditions. Patch pipettes were pulled from borosilicate glass (A-M Systems, Carlsbourg, WA) using a Narishige puller (Tokyo, Japan).

Experiments were performed in the following bath solution (mM): 137 NaCl, 6.4 KCl, 4.3 Na2HPO4, 1.4 KH2PO4, 1.2 MgCl2, 0.5 CaCl2, 5 glucose. The pipette tip was dipped into pipette solution (mM): 137 KCl, 6.4 NaCl, 4.3 NaHPO2, 1.4 KH2PO2, 1.2 MgCl2, 0.5 CaCl2, 1 ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid, 4 glucose. The pipette was then back-filled with the same solution containing amphotericin B (240 µg/mL, Sigma-Aldrich, St. Louis, MO). When the contribution of K+ channels to the whole-cell current was measured, 137 mM Cs+ replaced equimolar K+ in the pipette solution. Similarly, to study the contribution of Cl- channels, 134 mM gluconate replaced equimolar Cl- in the bath and pipette solutions. All buffers were supplemented with 0.1% bovine serum albumin, and the pH was adjusted to 7.4. Experiments were performed at room temperature.

Liquid junction potentials (LJPs) develop during a patch-clamp recording whenever the pipette-filling solution is different from the bath solution [24 ]. Because LJPs could be misinterpreted as cellular effects, data were corrected for LJPs when they exceeded ±1 mV. An LJP was defined as the potential of the bath solution with respect to the pipette solution, and the membrane potential VM was calculated from the reading provided by the patch-clamp amplifier V, as VM = -V + LJP. LJPs were calculated using the generalized Henderson equation for N polyvalent ions:

where

u, C, and z represent the mobility, concentration, and valency of each ion species (i), respectively; and R, T, and F are the gas constant, temperature, and Faraday constant, respectively. Superscripts b and p denote bath and pipette solutions, respectively.

Pipette resistances were between 3 and 8 M{Omega}, and recordings were obtained using a patch-clamp amplifier (EPC-7; List Medical, Germany) in the voltage-clamp mode. The holding potential was -60 mV, and 20 mV steps, ranging from -80 to +80 mV, were applied every 200 ms. The cells were kept in the bath solution for 1 h before starting the experiments. After baseline currents stabilized, stimuli or blockers were added to the bath solution, and currents were recorded for up to 40 min. Data were analyzed using custom-written patch-clamp software (kindly provided by Dr. A. S. French, Dalhousie University, Canada). Statistical analysis of whole-cell currents was performed at +80 mV.

Reverse-transcriptase polymerase chain reaction (RT-PCR)
Total RNA was isolated from 2 x 106 eosinophils using the Qiagen RNeasy Mini kit (Qiagen, Chatsworth, CA). The average amount of RNA obtained from 2 x 106 eosinophils was 300 ng. One-third of the RNA was reverse-transcribed using superscript II RT (Gibco-BRL, Grand Island, NY) and random hexamers (50 A260 units; Boehringer Mannheim, Mannheim, Germany) as primers. Thereafter, PCR was performed in 20 µL reactions. The expression of K+ channels was studied using the primer pairs described in Table 1 . As a positive control for K+ channels, IK, BK, and Kv, mRNA from the human airway epithelial cell line A549 was used, and for TWIK-1 and TASK-2 expression, mRNA from Calu-3 cells was used. One-tenth of the cDNA was used in PCR experiments. DNA amplification was obtained by annealing for 45 s at 64°C for IK and BK, 56°C for Kv, 60°C for TWIK-1, and 51°C for TASK-2. This was followed by an elongation step at 72°C for 1 min. DNA sequences were amplified during 30 cycles. The sizes of the expected amplified products are shown in Table 1 .


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  		Table 1. List of the K+ Channels Tested, the Positions of the Primers Relative to the Published Data Sequences, the Expected Size of the RT-PCR Products, and the Base Sequence of the Primers in the 5' -> 3' Direction

Statistical analysis
Data are presented as means ± SE; n refers to the number of experiments. In bar diagrams, 100% represents the amplitude of baseline, whole-cell currents at +80 mV. Data are expressed as percentage of baseline currents. The unpaired, two-sided Student’s t-test was used to compare the means of two groups. Values of P < 0.05 were considered statistically significant.


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RESULTS
 
NO increases the whole-cell current in human eosinophils
Figure 1 shows the effects of two chemically different NO donors, GSNO and SNAP, on the whole-cell current in human eosinophils. In all recordings, the current-voltage (I-V) relationship was obtained in 20 mV steps from -80 mV to +80 mV. Statistical analysis of whole-cell currents was performed at +80 mV. In resting eosinophils, the baseline whole-cell current was 53 ± 13 pA (n=7). GSNO increased the current by 47 ± 7 pA (88%; n=4), whereas SNAP increased the current by 59 ± 18 pA (111%; n=4; P<0.05 in both sets of experiments). Furthermore, GSNO caused a shift in the reversal potential from -20 ± 2 to -29 ± 3 mV (n=4; P<0.05) and SNAP, from -20 ± 2 to -32 ± 0.5 mV (n=4; P<0.05).



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  		Figure 1. (A) A representative recording showing activation of the whole-cell current by GSNO (100 µM). (B) I-V relationship of the recording shown in panel A. GSNO shifted the reversal potential from -21 mV to -30 mV. (C) A representative recording showing activation of the whole-cell current by SNAP (100 µM). (D) I-V relationship of the recording shown in panel C. SNAP shifted the reversal potential from -18 mV to -31 mV.

To evaluate the role of endogenous and exogenous NO in the whole-cell current activation, we suppressed endogenous NO production with L-NAME, an inhibitor of NO synthases (NOS). L-NAME had no effect on the baseline current, indicating that endogenous NO did not affect channel function in resting eosinophils (n=4). Subsequent addition of SNAP (100 µM) increased the whole-cell current by 114 ± 10% (n=4; P<0.01), suggesting that exogenous NO mediated this increase.

NO stimulates the whole-cell current via a cGMP-dependent pathway
To investigate whether NO was acting on eosinophil ion channels via a cGMP-dependent or -independent mechanism, cells were incubated with 8-Br-cGMP, the soluble guanylyl cyclase inhibitor ODQ, or a protein kinase G inhibitor H89 before stimulation with SNAP (Fig. 2 ). ODQ (10 µM) and H89 (1 µM) had no effect on the baseline current and prevented subsequent activation of the current by SNAP (n=5 for both drugs). In other experiments, addition of 8-Br-cGMP increased the whole-cell current significantly, indicating that cGMP is involved in the activation of the observed current. The addition of 100 µM and 500 µM 8-Br-cGMP increased whole-cell currents by 27 ± 8% (n=4) and 124 ± 25% (n=4), respectively. The increase in whole-cell current caused by 500 µM 8-Br-cGMP was similar to that induced by SNAP (139±12%). All of these results suggest that exogenous NO activates the whole-cell current via a cGMP-dependent pathway.



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  		Figure 2. The NO/cGMP pathway is involved in the whole-cell current activation by SNAP. Baseline currents are expressed as 100%. ODQ (10 µM) had no effect on the baseline current (P>0.05; n=5) but inhibited the SNAP-induced increase (n=5). Similarly, H89 (1 µM) did not affect the baseline current (P>0.05; n=5) but inhibited stimulation of the whole-cell current by SNAP (n=5). 8-Br-cGMP (0.1 mM and 0.5 mM) increased the whole-cell current in a dose-dependent manner by 27 ± 8% and 124 ± 25% (n=4; P<0.05 in each series), respectively. (*P<0.05).

SNAP activates K+ channels
To investigate which ions participate in whole-cell currents evoked by NO, we replaced K+ or Cl- ions in the bath solution with Cs+ or gluconate, respectively (Fig. 3 ). In a gluconate-containing solution, SNAP increased the whole-cell current by 77 ± 25% (n=4; P<0.05) and shifted the reversal potential from -13 ± 1.5 to -26 ± 2 mV (n=4; P<0.05), indicating that Cl- currents do not contribute to the NO-induced current. In contrast, in a Cs+-containing solution, SNAP neither affected the amplitude of the whole-cell current (98±10%; n=7; P>0.05) nor the reversal potential (-14±2 mV and -15±2 mV in the absence and presence of SNAP, respectively; n=7; P>0.05). This suggests that currents activated by SNAP were carried by K+ ions.



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  		Figure 3. SNAP activates K+ but not Cl- channels. (A) A representative trace of a whole-cell current using Cs+-containing pipette and bath solution, showing that the addition of SNAP had no effect on the whole-cell current. (B) I-V relationship of the recording shown in panel A. (C) A representative trace of a whole-cell current in the presence of gluconate in the pipette and bath solution, showing that SNAP increased the whole-cell current. (D) I-V relationship of the recording shown in panel C.

The presence of K+ channels in eosinophils was investigated using openers of intermediate conductance KCa channels (IK) and KATP channels, 1-EBIO and diazoxide, respectively (Fig. 4 ). 1-EBIO increased the current by 64 ± 20% (500 µM; n=5), whereas diazoxide increased by 73 ± 19% (100 µM; n=10), demonstrating the presence of IK and KATP channels in eosinophils. In the presence of diazoxide, SNAP activated the whole-cell current further by 29 ± 10% (100 µM; n=5).



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  		Figure 4. Summary of the effects of K+ channel openers and blockers on the eosinophil whole-cell current. Baseline currents are expressed as 100%. Diazoxide alone increased the current by 73 ± 19% (100 µM; n=10) and in combination with SNAP by 102 ± 10% (n=5). Glibenclamide (5 µM) had no effect on the whole-cell current but inhibited subsequent current activation by SNAP (n=6 and n=4, respectively). 1-EBIO increased the current by 64 ± 20% (500 µM; n=5), whereas clotrimazole had no effect on the baseline current (10 µM; n=6) and did not affect subsequent current activation by SNAP (n=4). 4-AP inhibited the baseline current by 41 ± 5% (2 mM; n=7) and blocked subsequent current activation by SNAP (n=4). *, P < 0.05.

In other experiments, we studied activation of the whole-cell current by SNAP in the presence of different K+ channel blockers (Fig. 4) . An inhibitor of KATP channels, glibenclamide (5 µM and 100 µM) had no effect on the baseline current but prevented current activation by SNAP (n=6 and n=4, respectively). A blocker of IK channels, clotrimazole also had no effect on the baseline current (n=6), but it did not prevent current activation by SNAP (n=4; P<0.05), indicating that NO had no effect on IK channels. A chemical frequently used to block Kv channels, 4-AP (2 mM) inhibited the baseline current by 41 ± 5% (n=7; P<0.01) and also prevented its activation by SNAP (n=4).

Gene expression of K+ channels in eosinophils
Because KATP channels have been described in human eosinophils previously [4 , 25 ], we investigated the expression of IK and large conductance (BK) KCa channels, Kv, and two members of the K2P family, TWIK-1 and TASK-2 (Table 1) . Figure 5 shows representative RT-PCR experiments. Eosinophils express mRNA for IK, Kv, and TWIK-1 but not for BK and TASK-2 channels (n=6). The identity of the PCR products was confirmed by sequencing and comparison with the corresponding Genbank sequences.



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  		Figure 5. Expression of K+ channels in human eosinophils using RT-PCR. Eosinophils express mRNA for IK, Kv, and TWIK-1 but not BK and TASK-2. For IK, BK, and Kv, A549 cells were used as a positive control. For TWIK-1 and TASK-2, Calu-3 cells were used as a positive control. Data are representative of six different experiments. M, DNA standard (a 100-bp ladder).


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DISCUSSION
 
The central observation of this study is that an increased level of NO activates KATP channels in human peripheral blood eosinophils via a cGMP-dependent pathway. Eosinophils produce NO in a proinflammatory environment [26 ], and our aim was to investigate the effects of endogenous and exogenous NO on eosinophil ion channel function. An inhibitor of NOS, L-NAME had no effect on the whole-cell current, indicating that endogenous NO did not affect ion channel function. However, NO donors increased the whole-cell current significantly and shifted the reversal potential toward more negative values, consistent with the activation of K+ channels.

Activation of soluble guanylyl cyclase (sGC) and generation of cGMP are responsible for many biological effects of NO [13 , 27 ]. The results of this study show that the NO/cGMP-dependent pathway is also involved in the regulation of the whole-cell current in eosinophils because the NO effects could be eliminated by pretreatment of cells with a selective inhibitor of sGC, ODQ; activation of the whole-cell current by NO was abolished in the presence of a PKG inhibitor, H89; and application of membrane-permeable 8-Br-cGMP produced an effect similar to that of NO. Although these results are consistent with the regulation of eosinophil whole-cell current via the NO/cGMP-dependent pathway, they do not exclude the involvement of a cGMP-independent pathway in this process.

The baseline and NO-stimulated whole-cell currents showed a nearly linear I-V relationship and were neither time- nor voltage-dependent (Fig. 1) . This suggests that under our experimental conditions, a contribution of H+ channels to the whole-cell current was insignificant, because these channels show time-dependent activation at depolarizing voltages [28 ]. Our data indicate that Cl- and K+ channels dominate the whole-cell current in eosinophils. This conclusion is based on ion substitution studies and the use of ion channel blockers. Although Cl- channels account for ~50% of the whole-cell current in unstimulated eosinophils [5 ], substitution of Cl- with gluconate did not affect current activation by NO, indicating that NO donors activated K+ but not Cl- channels. It is important to notice that human peripheral blood eosinophils represent a heterogeneous population of cells, often with different sizes, or even different activation status. This heterogeneity contributes to the variability in baseline whole-cell currents observed in the present and previous studies [5 ].

RT-PCR studies showed that eosinophils express mRNA for the ß subunit of Kv channels, Ca2+-dependent K+ channels (IK), and a member of the K2P family, TWIK-1. The functional role of Kv channels in eosinophils remains unclear, because the nearly linear I-V relationship suggests that these channels do not make a significant contribution to the baseline or SNAP-induced current. Similarly, the TWIK-1 channels do not contribute to baseline or NO-stimulated currents, because these channels conduct an inwardly rectifying current. Studies from other laboratories suggested expression of KATP channels in human eosinophils [4 , 25 ].

The KATP channel is a heteromultimeric complex of a K+-selective pore and a sulfonylurea receptor (SUR), which are structurally unrelated to each other. It is composed of four inwardly rectifying K+ channel subunits, Kir6.1 or Kir6.2, and four SUR subunits, which belong to the family of ATP-binding cassette (ABC) transporters. Different combinations of the Kir6.2 or Kir6.1 subunits and the SUR1 and SUR2 account, in part, for the molecular and functional diversity of KATP channels. Depending on the combination of Kir6.x and SURx subunits, the single-channel conductance of KATP channels has been estimated between 10 and 80 pS in the presence of 140 mM K+ on both sides of the membrane [29 30 31 ].

Glibenclamide is an agent that binds with high specificity to sulfonylurea receptors, which belong to the ABC-transporter family (subfamily ABCC). This family also includes the CFTR Cl- channels. Although glibenclamide at the concentrations used in our experiments (5–100 µM) is known to affect CFTR function, previous studies have shown that eosinophils do not express CFTR mRNA [5 ]. Therefore, the effect of glibenclamide on the whole-cell current cannot be attributed to its effects on CFTR.

An opener of KATP channels, diazoxide, and NO donors activated a whole-cell current of ~50 pA, corresponding to a whole-cell conductance of 625 pS at +80 mV. The number of ion channels (N) activated by NO could be evaluated from the following relationship: G = {gamma} N P, where G- is the whole-cell conductance, {gamma}- is the single-channel conductance, and P- is the channel open probability. If we assume that the KATP channel conductance is 50 pS and that its open probability is P = 0.5, then the number of KATP channels activated by NO is equal to 25.

After activation of KATP channels by diazoxide, the subsequent addition of SNAP increased the whole-cell current further. A similar effect has been shown for mitochondrial KATP channels, where NO has been shown to activate KATP channels and potentiate the effect of diazoxide [32 ]. It is possible that diazoxide opens KATP channels partially, and NO is further increasing the channel open probability. Alternatively, eosinophils may possess different subtypes of KATP channels, and NO might open a different subpopulation than diazoxide.

In summary, eosinophils possess several types of K+ channels, but NO affects only KATP channel function. NO induces an outflow of K+ ions in eosinophils and thus causes membrane hyperpolarization. This reduces the cytoplasmic Ca2+ concentration; as a result, the eosinophils could be less excitable, and stronger stimuli are needed for depolarization. Thus, NO at concentrations likely to be encountered in vivo could perform a protective role, preventing eosinophil activation.


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ACKNOWLEDGEMENTS
 
This study was supported by grants from the Canadian Institute for Health Research. A. S. is an Alberta Heritage Foundation for Medical Research Fellow. M. D. and R. M. are Alberta Heritage Senior Medical Scholars.

Received October 22, 2001; revised January 22, 2002; accepted January 23, 2002.


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