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1Department of Biological Sciences, National University of Singapore, Singapore; 2Neuroscience Research Institute, Peking University and Department of Neurobiology, Peking University Health Science Center, Beijing, China; and 3The University Scholars Programme, National University of Singapore, Singapore
Submitted 17 November 2004; accepted in final form 12 January 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Ko and Kelly 1999; Stamler et al. 1997). NO is produced by nitric oxide synthase (NOS), the neuronal form (nNOS), which is activated by Ca2+ through calmodulin. nNOS-containing neurons are present in many areas of the CNS. Although nNOS neurons represent only roughly 1% of cell bodies in the cerebral cortex, virtually every neuron in the cortex is exposed to nNOS nerve terminals (Bredt et al. 1990; Salter et al. 1991). NO has been proposed as a retrograde messenger that coordinates the enhancement of both pre- and postsynaptic mechanisms involved in synaptic plasticity (Prast and Phillipu 2001). The actions of NO are a consequence of its influence on a variety of protein functions. It is well established that NO can exert its biological effects through the activation of guanylyl cyclase and the production of cGMP or through cGMP-independent mechanisms known as "redox signaling" (Stamler 1994). However, relatively few targets of the action of NO in neurons that could explain its role in CNS function have been identified.
K+ channels are widely expressed throughout the brain. They play a key role in membrane potential maintenance, neuronal excitability, and synaptic transmission. The properties of many K+ channels can be modulated through second-messenger pathways activated by neurotransmitters and other stimuli (Hille 1992; Levitan 1988). It has been shown that NO regulates several types of K+ channels, including ATP-dependent K+ channels and Ca2+-activated K+ channels, in both peripheral tissues and the CNS (Prast and Phillipu 2001). However, the biological mechanisms by which NO acts on the delayed-rectifier K+ channels, especially in the CNS, are not fully understood. Delayed-rectifier K+ channels (IK) have been found to contribute to the repolarization of membrane potentials following action potentials, and the modulation of spike discharge in many neurons. Different NO donors or caged NO exert diverse reactions on K+ channels, probably due to the dosages of NO or different regions of the CNS (Lang and Watson 1998; Prast and Phillipu 2001). Furthermore, both cGMP-dependent and -independent signaling pathways have been reported to involve in the modulation of Ca2+-activated K+ channel by NO (Bolotina et al. 1994; Klyachko et al. 2001; Zhou et al. 1996). The controversy of regulatory mechanism for NO on K+ channel activity may be resolved if sensitive, quantitative detection methods are developed to measure and distinguish NO derived from various NO donors (sources) in different in situ experimental systems. In the present study, we have developed an electrochemical NO sensor that can accurately evaluate the concentration of NO released from NO donors. This study aims to address effects of NO and its effective concentrations in regulating delayed-rectifier K+ channels, using a combination of patch-clamp electrophysiology and electrochemical NO sensor. We report here that different concentrations of NO either augment or block the delayed-rectifier K+ channel via a dual cGMP- and redox-dependent mechanism in mouse cerebral neocortical neurons.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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All experiments were performed using primary culture of mouse neocortical neurons. Cortices were obtained from 1-day-old postnatal Tac:Icr:Ha(ICR)fBR mice as described previously (Baughman et al. 1991; Goslin and Banker 1991). In brief, cortices were dissected from the brains under sterile conditions and were digested in 0.25% trypsin in Hank's balanced salt solution (HBSS) for 20 min at 37°C. Trypsin digestion was terminated by adding 10% fetal bovine serum (FBS) and then the tissue was triturated with a plastic pipette. After centrifugation for 5 min at 200 g, cell pellets were mechanically dissociated by triturating in culture medium (Eagle's minimal essential medium supplemented with 10% fetal calf serum, and 2 mM glutamine) until no cell clumps could be seen. After filtering through 70 µM nylon mesh (Millipore), the cells were seeded at a density of 2.5 x 105 cells/cm2 in 35-mm petri dishes containing circular 10-mm diameter poly-D-lysine (12.5 µg/ml)-coated coverslips. Cultures were maintained in a humidified CO2 incubator (5% CO2, 37°C). After 16–18 h, culture medium was changed with fresh culture medium. One day later, half of the medium was replaced with fresh medium, and 10 µM cytosine arabinoside was added to inhibit proliferation of nonneuronal cells. The cells were used for experiments for 
7 days. All recordings were made from cells cultured between days 4 and 7.
All animal experiments were approved by the Research Committee of the University Animal Holding Unit, National University of Singapore. All efforts were made to minimize animal suffering and to reduce the number of animal used.
Electrophysiological recording
All electrophysiological experiments were carried out at room temperature (22–24°C). Voltage-clamp recordings were made from cultured neocortical neurons using standard patch-clamp methods in the whole cell and cell-attached configurations. Currents were recorded using an EPC-9 amplifier (Heka Elektronik). Data acquisition was controlled by PULSE 8.63 (Heka Elektronik). Currents were filtered at 1–2 kHz and digitalized directly to a Macintosh Power Mac G4. Data were analyzed using PULSE-Fit (Heka Elektronik) and Igor-Pro (WaveMetrics, Lake Oswego, OR).
Patch pipettes were made from 1.5-mm borosilicate capillary glass (World Precision Instruments, Sarasota, FL) using a Sutter P-97 puller (Sutter Instrument, Novato, CA). The pipettes were fire-polished and tip resistance was 2–6 M
for whole cell and 12–17 M for single-channel recordings when filled with pipette solution (for contents, see following text).
For the whole cell recordings, the bath solution contained (in mM) 140 NaCl, 10 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 2 CdCl2, 10 glucose, and 0.001 tetrodotoxin (TTX) at pH 7.4. The pipette solution was composed of (in mM) 10 NaCl, 140 K-gluconate, 0.1 CaCl2, 4 MgCl2, 10 HEPES, and 1 EGTA at pH 7.4. At the beginning of each experiment, junction potential between pipette solution and bath solution was electronically adjusted to zero. No leakage subtraction was performed to the original recordings, and all cells with visible changes in leakage currents during the course of experiment were excluded from further analysis. Test pulses were made at 10-mV increments from –60 to +80 mV with the holding potential at –50 mV unless stated otherwise. Cells were continuously perfused with the bath solution containing test chemicals. All solutions were adjusted with osmolarity of 300 mosM/l and pH 7.4. Serial resistance (Rs) and capacity transients were electronically compensated. The mean whole-patch capacitance was 15.6 ± 7.8 pF (n = 68) and the mean Rs was 1.18 ± 0.12 M. Rs compensation of 80–90% was used, with a lag of 10 µs, and was periodically checked during the experiment.
For the cell-attached recordings, both pipette and bath solution containing 140 mM KCl and 5 mM NaCl. Rs compensation was not used. Patches (seal resistance: 5–15 G) were obtained on the soma of neurons. Data were analyzed using software TAC 4.02 (Bruxton, Seattle, WA). The channel open probability (Po) was calculated from the ratio between the open time and the total time. NPo was determined from data samples of a 60-s duration and defined as: NPo = 
(t1 + 2t2 + 3t3 +...+ ntn), where n is channel number, and t1, t2, and tn are the ratios of open time to total time for each channel at each current levels. The unit amplitude of channels and channel open duration were determined from all point histograms with a Gaussian curve. A current level higher than 50% of the unit channel current was considered to reflect a channel opening.
Measurement of NO release by SNAP using electrochemical sensor
Preparation of a saturated NO solution involves the meticulous exclusion of O2 as NO can be oxidized rapidly by O2 when its concentration is higher than a certain threshold. To produce a saturated NO solution free of O2, phosphate buffer solution (PBS) at pH 7.4 was first purged with pure nitrogen gas (99.9%, Soxal) for 30 min. The O2-free PBS buffer was then purged with pure NO gas (99.9%, Matheson) for 30 min and kept under an NO atmosphere until use. The saturated aqueous NO solution contained 
2.0 mM NO at 25°C at PNO = 1 atm (Lantoine et al. 1995). The diluted NO standards were made freshly for each experiment using O2-free, NO-saturated PBS that was kept in a glass flask with an air-tight rubber septum.
To measure NO release from the dissolved S-nitro-N-acetylpenicillamine (SNAP) in bath solution, we used a gold (Au) electrode of 2.0-mm diam (EG&G) prepared by polishing with aqueous slurries of fine alumina power (0.06 µm) on a polishing microcloth (Bioanalytical Systems) and was sonicated for 10 min in water. The electrode was then cleaned electrochemically in 0.02 M H2SO4 by potential cycling (CHI660A, CH Instruments, Austin, TX) across a range of –0.1 to +1.5 V at 10 V/s for 30 cycles. The pretreated Au electrode was dipped into 0.5% Nafion/ethanol solution for 5 s and then dried in air. This dipping was repeated three times. The resulting electrode is denoted as Nafion@Au.
All electrochemical measurements were performed on a BAS 100 B/W workstation (Bioanalytical Systems) controlled by the BAS 100 B/W software from a Gateway 2000 personal computer. The three-electrode system consisted of a Nafion@Au as the working electrode, an Ag/AgCl reference electrode, and a platinum wire auxiliary electrode. All potentials were quoted with respect to the reference. Prior to each measurement, the working electrode (Nafion@Au) was inserted in a solution containing 5.0 ml PBS buffer, and its potential was cycled between 0 and +800 mV at a rate of 50 mV/s until a steady response was obtained. The detection of NO was conducted in chronoamperometric mode and the solution was stirred mechanically during the experiment. The currents from the electrocatalytic oxidation of NO were recorded at the applied potential of +800 mV for monitoring the release of NO from SNAP (Bedioui et al. 1994; Do and Wu 2001; Miao et al. 2000).
Materials
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), except tetraethylammonium chloride (TEA), SNAP, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), and methylene blue (MB) from Calbiochem (La Jolla, CA). NO gas was purchased from Matheson Tri-Gas (Newark, CA). All cell culture reagents were purchased from GIBCO-Invitrogen (Grand Island, NY).
Data analysis
Results were expressed as means ± SE from at least three independent experiments. Statistical significance was determined by one-way ANOVA and the Student-Newman-Keuls post hoc test (SPSS for Windows, SPS, Chicago, IL) for unpaired data, with P < 0.05 representing significance. The normalized conductances were fitted to a least squares criterion procedure with the Boltzmann function
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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It has been previously shown that voltage-dependent K+ currents can be separated by their current kinetics and inactivation properties (Surmeier et al. 1994). In neocortical neurons, depolarization from –120 mV activates a rapidly inactivating A current (IA) and a delayed rectifier current (IK); depolarization from a more positive holding potential (–50 mV) activates only the IK because the IA was almost completely inactivated at this potential (Hamill et al. 1991; Storm 1990).
Figure 1, A and B, shows typical currents recorded under voltage clamp in a whole cell patch from a neocortical neuron in control bath solution. The bath solution contained 0.001 mM TTX to block voltage-gated Na+ channels and 2 mM Cd2+ to block voltage-gated Ca2+ channels and Ca2+-dependent K+ currents. The Cl– was replaced by gluconate– to eliminate the Cl– current. Two types of outward currents were observed. Following a range of depolarizing steps from –60 to +50 mV, the evoked currents showed the delay rectifier type of K+ currents (IK; Fig. 1A). This was further confirmed pharmacologically by adding of 10 mM TEA to the bath solution that blocked 75.6 ± 9.5% of the currents (Fig. 1C). When a 600-ms, –120-mV conditioning pulse was applied, a rapidly inactivating A current (IA) occurred (Fig. 1B). However, the IA current was not prominent, and only 7 in 30 cells measured showed IA current under the voltage protocols as shown in Fig. 1. Most cells, 21 in 30 cells measured, displayed delayed rectifier type of K+ current even the –120-mV conditioning pulse was applied. Addition of 10 mM TEA to the bath solution reduced slowly decaying current, leaving the fast component more prominent (Fig. 1D). The IA current could be isolated by subtracting the current evoked by a step depolarization at a holding potential of –50 mV from those evoked from –120 mV (Fig. 1E).
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Low concentration of NO donor SNAP and pure NO increase the whole cell IK current
The application of freshly prepared 20 µM SNAP to cells, held at –50 mV, resulted in a reversible increase in the whole cell IK current (Fig. 2A). The effects of SNAP were very rapid, starting 30 s after the application of the reagent, and reaching maximal values in 5 min. At +50 mV, the current increased 50.4 ± 6.0% (Fig. 2C), which was significantly higher than the control neuron with no SNAP treatment. However, when a –120-mV conditioning pulse was applied, the peak current remained unchanged while the final currents increased (Fig. 2B). At +50 mV, the final currents increased 29.2 ± 2.3% compared with the control, whereas the peak current increased only 3.1 ± 1.8%, which was not significantly different from the control (Fig. 2D). This suggests that 20 µM SNAP had little effect on the IA currents.
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High concentration of SNAP and pure NO decrease the whole cell K+ current
When the concentration of SNAP was increased to 100 µM, a rapid reduction of whole cell K+ current within 1 min was observed. The application of freshly prepared 100 µM SNAP to cells, held at –50 mV, resulted in fast reduction of the whole cell IK current (Fig. 3A). At +50 mV, the current decreased 65.1 ± 1.2% (Fig. 3C), significantly lower than the control. When a –120-mV conditioning pulse was applied, both the peak current and the final currents decreased dramatically (Fig. 3B). At +50 mV, the final currents decreased 62.9 ± 5.6%, whereas the peak current decreased 59.8 ± 2.6% compared with the control (P < 0.05; Fig. 3D). This suggested that 100 µM SNAP decreased both IK and IA currents. The reductions of the current were irreversible and persisted after washing for 5 min.
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Calibration and quantitative measurement of NO release from SNAP
Because SNAP releases NO continuously, it is important to know the NO concentration in the bath solution for the electrophysiological experiments. A chemically modified NO sensor was used to calibrate the real-time NO oxidation current at successive steps after injection of 0.5 µM dissolved NO at each step to the bath solution. Figure 4A shows a typical calibration of the NO current-concentration curve obtained with a Nafion@Au working electrode at +800 mV (vs. Ag/AgCl) for the successive addition of 0.5 µM NO. With the addition of NO, the current generated from the oxidation of NO increased correspondingly. The inset shows that the correlation between the current response and the concentration of NO was linear, expressed as I = 75.37 x [NO] –0.3143 (correlation coefficient r = 0.9998, P < 0.05). Figure 4B illustrates an amperogram for the detection of NO released from SNAP with the same Nafion@Au working electrode at +800 mV (vs. Ag/AgCl). Each step corresponded to an addition of 20 µM of SNAP. The inset shows that the current response was linear to the square root of SNAP concentration, instead of SNAP concentration, expressed as I = 2.135 x [SNAP]1/2 –0.1974 (r = 0.9990, P < 0.05). The following equation summarizes the mathematical relation between the [NO] and the [SNAP]1/2 when both reagents produced an equal amount of current detected at the same electrode
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10 mM stock solution of SNAP was made on the day of experiments and was stored at –20°C to minimize the degradation of SNAP. Effect of SNAP and pure NO on K+ single-channel activity
The whole cell voltage-clamp experiments described in the preceding text suggest that low concentration of SNAP (20 µM) and pure NO (0.1 µM) increased the IK currents amplitude but not the IA current. However, higher concentrations of SNAP (100 µM) and NO solution (0.5 µM) induced a blockade of both IK and IA K+ currents. We further confirmed this effect of NO on single K+ channel activity in cell-attached patches. The observation that IK and IA currents were differentially modified by different concentrations of NO suggests that NO may have kinetically distinct actions on the two major channels responsible for the K+ current in this preparation. To clarify this issue, we focused on the IK channel in the following single-channel recordings of this study.
The single-channel activities were recorded when a cell-attached patch was depolarized from 0 to 80 mV. The pipette solution contained 140 mM KCl, similar to the expected intracellular [K+], and thus the reversal potential for K+ was close to 0 mV. Patch membrane potentials were calculated from the equation: Vmem = RMP –Vcmd, where Vcmd was the patch depolarization step, and RMP was the resting membrane potential. Figure 5, A and B, shows an analysis of the single-channel conductance. Currents recorded from one patch on depolarization to various voltages are shown in Fig. 5A. Figure 5B displays averaged single-channel current amplitude in four cells each. Currents were linearly related to membrane potentials, and the slope conductance corresponding to the regression line shown in figure was 45.8 pS at pipette [K+] of 140 mM. The currents were sensitive to blockage of 10 mM TEA and 250 µM 4-aminopyridine (4-AP; Fig. 5, C and D). The ensemble averaged currents had a delayed rectifier property (Fig. 6B), suggesting that it was a single IK channel.
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; Broillet et al. 1996; Xu et al. 1998), we determined whether the reducing agent dithiothreitol (DTT) could recover the channel activity. DTT has been widely used to investigate S-nitrosylation and redox mechanisms of various proteins, including Ik channel in guinea pig cardiomyocytes (Bai et al. 2004), Ca2+-activated K+ channels (Abderrahmane et al. 1998) and ryanodine receptors (Menshikova et al. 2000). DTT itself has no effect on the macroscopic whole cell and single-channel Ik currents (Fig. 7, A and B), consistent with the results from colonic smooth muscle (Prasad and Goyal 2004). Figure 6, A and B, shows that after adding 5 mM DTT into the bath solution containing 100 µM SNAP, the K+ channel activities were restored and its NP0 increased to 0.03 ± 0.01 (n = 6). Similarly, our whole cell results show that after treatment with 5 mM DTT, the current was recovered to 65% of the control, which is comparable to single-channel data (Fig. 7C). The similarity in DTT effect (along with similar responses to 4-AP and TEA) on the whole cell currents and single-channel currents suggest that both currents arise from the activity of the same type of channel.
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Effect of PKG and cGMP inhibitor on K+ channel
Many of the cellular effects of NO are mediated by soluble guaylate cyclase (sGC) and cGMP. It has been shown that increases in cGMP levels mediate a large number of physiological actions of NO. Either nitregic nerve stimulation or administration of NO donors increases intracellular cGMP concentrations (Bredt and Snyder 1989; Torphy et al. 1986). To further explore cGMP signaling in the facilitation of IK channel activity, we used the inhibitors for sGC and cGMP dependent protein kinase G (PKG) to inhibit their actions. Figure 9C shows that under 40 µM SNAP treatment, the averaged NP0 is 0.48 ± 0.15 (n = 6). However, in the presence of 10 µM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a highly selective inhibitor of sGC, the averaged NP0 values in the presence of 40 µM SNAP were significantly decreased to 0.02 ± 0.01 (n = 3; Fig. 9, A and C). The reduction of channel activity could be reversed by addition of a membrane-permeable cGMP analogue, 8-Br-cGMP. Moreover, 100 µM 8-Br-cGMP has similar effect as 40 µM SNAP, it significantly increased NP0 from 0.04 ± 0.02 to 0.38 ± 0.14 (Fig. 9, B and C). Another sGC inhibitor, methylene blue (MB) 300 µM (C. H. Chen et al. 1998; Kang et al. 2005; Pineda et al. 1996) also significantly reduced NP0 in the presence of 40 µM SNAP to 0.19 ± 0.06 (n = 5; Fig. 9C). These results indicate that NO stimulates sGC and elevates intracellular cGMP, which leads to channel activation.
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; C. H. Chen et al. 1998; Pineda et al. 1996), inhibited the enhanced channel activity by 40 µM SNAP. The averaged value of NP0 in the presence of 40 µM SNAP was reduced to 0.11 ± 0.02 (n = 6). That the effect of H-8 was induced by inhibiting PKG was further confirmed by using Rp-8-Br-PET-cGMP, a more specific inhibitor of PKG than H-8. Figure 9C shows that addition of 100 µM Rp-8-Br-PET-cGMP completely abolished the effect of 40 µM SNAP. In addition, in the presence of 100 µM Rp-8-Br-PET-cGMP, the cGMP analogue 8-Br-cGMP 100 µM failed to reactivate the channel, suggesting that PKG-dependent signal transduction pathway was involved in the facilitation of K+ channel by a low dose of SNAP (40 µM) or pure NO (0.1 µM). Effect of superoxide on K+ channel activity
After establishing the optimum concentration of SNAP that could maximally increase the channel open probability, we investigated whether superoxide anion was involved in the process of the SNAP regulation of K+ channel. Several lines of evidence suggested that superoxide itself may affect K+ channel activity directly or through oxidizing K+ channel associated proteins (Armstead 2001; Brzezinska et al. 2005; Van der Vlies et al. 2002). The results in Figs. 6–8 show that reducing agent DTT was able to recover the channel open probability from a high dose of SNAP or NO solution, indicating that regulation process by high levels of NO maybe due to an oxidation mechanism. To test this hypothesis, we then test whether superoxide itself affects channel activity by applying a superoxide-generating agent, KO2, into the bath solution. KO2 spontaneously releases O
2– in the solution phase. Figure 10A illustrates an inhibitory effect of superoxide on the channel activity. The channel openings were recorded 3 min after adding KO2. Figure 10A shows that 20 µM KO2 dramatically decreased the NP0 from 0.04 ± 0.01 to 0.009 ± 0.0004 (n = 8). When the concentration of KO2 was further increased to 40 µM, the channel openings were entirely abolished. The open probability decreased as a function of the concentration of KO2 increased from 0 to 40 µM. The reduction of channel activity could be reversed by 800 Unit of superoxide dismutase (SOD; Fig. 10A).
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However, when we added 800 U of superoxide dismutase (SOD) to the typical control sample and observed the channel activity, there was no significant difference of NP0 values between the control and the SOD-treated group (Fig. 10B). When 800 U of SOD and 40 µM SNAP were co-introduced, a significant increase in the NP0 (0.4232 ± 0.1173) was observed (Fig. 10B). When 100 µM SNAP was added with 800 U of SOD, the channels almost closed (NP0 = 0.003 ± 0.003; Fig. 10B). Interestingly, when the reducing agent DTT was applied to the silent channel in the presence of high-dose SNAP, we found recovering of NP0 of the channel (NP0 = 0.028 ± 0.012; Fig. 10B). A comparison of the data in Fig. 10B with those in the absence of SOD (Fig. 8A) showed that there was no significant difference in the effect of SNAP with or without SOD on K+ channel. These results suggested that superoxide anion (O2–) did not exert any detectable effect on the SNAP regulation of K+ channel under our experimental conditions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Mode of NO action in modulating K+ channel can be specific to the channel type and differentially corresponding to NO concentration
Since the discovery of NO as a modulator of neuronal function in the brain (Garthwaite et al. 1988), many studies have shown that the role of NO in CNS is highly diverse. For example, NO inhibits outwardly rectifying K+ current in both the whole cell and the cell-attached patches in type I hair cells from rat semicircular canals (Chen and Eatock 2000). However, Browning et al. (1998) found that in cultured sympathetic ganglionic neurons, NO exerts dual opposing effects on neuronal potassium conductances, namely an inward current shift mediated through an inhibition of calcium-dependent potassium currents and induction of an outward current mediated by activation of the potassium delayed rectifier channel. The controversial effect of NO on K+ channel may be due to the different brain regions by which NO acts on or that the exact effective concentrations of NO at any particular instant are varied. Therefore we proceed to measure such concentrations using electrochemical NO sensor in this investigation. Based on Eq. 1, 20 µM SNAP equivalent to 0.13 µM of calibrated NO would lead to an increase in K+ channels opening. In contrast, 100 µM SNAP equivalent to 0.30 µM of calibrated NO would tend to close these channels. In recent years, the relationship between NO concentrations and its cellular physiology has caught sufficient attention. Development of sensitive and selective analytical methods for the measurement of NO in biological systems has received great interest. NO is difficult to measure in vivo because of its short half-life. In addition, the diffusion rates of NO in different tissues, redox environment and compartmentalization of NO targets are important factors in determining NO actions. Concentrations of NO in different tissues can vary from a few nanomolar to micromolar. In rat cerebellar slices, endogenous NO production ranges from 8 to 58 nM (Shibuki 1990). On stimulation of NMDA receptor, NO concentration increased from a low nanomolar range up to 200 nM in rat hippocampal slices (Ledo et al. 2002). In peripheral macrophages and endothelial cells, concentrations of NO could reach up to 1 µM (B. Chen et al. 1998; Malinski et al. 1993).
The modulation of Ik channels by NO has been investigated in vascular smooth muscle (Li et al. 1997; Waldron and Cole 1999). It showed that the open probability of a 49.1 pS Ik channel increased 10- to 25-fold by NO donor NONOate in a concentration-dependent manner (Li et al. 1997). The Ik channels in our study showed a conductance of 45.8 pS, in agreement with those reported elsewhere, such as those of bovine arterial smooth muscle (49.1 pS) (Li et al. 1997), rabbit portal vein smooth muscle (42 pS) (Ogata et al. 1997), canine atrial myocytes (35.5 pS) (Yue et al. 1996), and rat dorsal ganglion neurons (55 pS) (Safronov et al. 1996).
Voltage-dependent K+ channel is widely distributed throughout the brain. It is known that changes in functions of K+ channels will directly affect the neuronal activity because K+ channels function to set a cell's resting potential, repolarize the cell after an action potential, and control the shape and the threshold of action potential (Nestler et al. 2001). Therefore the modification of neuronal K+ currents by NO may affect neuronal membrane potential and thereby cause changes in the neuronal activity. Several lines of evidence have shown that delayed-rectifier K+ channels were involved in the pathological process of neuronal death mediated by NO (Bossy-Wetzel et al. 2004; Yu et al. 1997, 1998). The transient, A-type K+ channel was proposed to correlate with the induction of long-term potentiation (Johnston et al. 2003). The voltage-dependent K+ channels are expressed in both pre- and postsynaptic areas although the distribution patterns in cell body, dendrite, axon and axon terminal are different. As a possible retrograde messenger, NO can regulate both pre- and postsynaptic K+ channels, hence modulate neuronal synaptic plasticity.
Involvement of cGMP in NO signaling pathway
It is well known that NO donors activate the guanylate cyclase and cGMP-dependent pathways in most cells (Fischmeister and Mery 1996; White 1999). There is ample evidence showing NO-cGMP signaling cascade in the CNS and peripheral tissues. In fact, we are probably the first to demonstrate that a cGMP-dependent pathway in cerebral cortical neurons could be involved in facilitating the effect of low NO levels on the IK channel. The application of sGC inhibitors, ODQ and MB, and specific PKG inhibitors, H-8 and Rp-8-Br-PET-cGMP, abolished the facilitating effect of low NO concentration on IK channel. The addition of a cell-membrane-permeable cGMP analogue, 8-Br-cGMP, reversed the inhibitory effect of ODQ but not the effect of PKG inhibitor Rp-8-Br-PET-cGMP. Thus it can be concluded that low NO levels stimulated channel activity through the activation of sGC, leading to subsequent elevation of intracellular cGMP and activation of the PKG-mediated signaling pathway. Our results are consistent with findings in apical K+ channel of rat kidney, ATP-sensitive K+ channel from ventricular cells of guinea-pig hearts, and Ca2+-activated K+ channel in posterior pituitary nerve terminal (Klyachko et al. 2001; Lu and Wang 1996; Lu et al. 1998; Shinbo and Iijima 1997). Lu et al. (1998) found that NO enhanced K+ channel activity in cell-attached patches and the enhancement was abolished by ODQ.
Moreover, the intracellular concentration of cGMP dramatically increased after SNAP application. It was reported that intracellular cGMP concentrations increased 10 folds 2–3 min just after administration of NO donors (Bredt and Snyder 1989; Lu et al. 1998). We observed that the facilitating effect of low concentrations of NO or SNAP was very fast, starting in <5 s of pure NO application. We used whole cell and cell-attached configurations in our study because these two configurations maintained cells in their physiological environment. Although we did not include ATP and GTP in out patch pipette solution in whole cell experiments, the fast effect of low concentrations of SNAP or NO is unlikely due to S-nitrosylation mechanisms (i.e., the process of transferring a NO-group to a protein) because the half-reaction time of S-nitrosylation is much longer (Ahern et al. 2002). However, we cannot exclude the possibility that other cGMP-independent pathway may also involve in the facilitating effect of low level NO on IK channels.
Redox mechanism induced by NO
In recent years, discovery of endogenous sources of oxidizing and reducing agents have prompted the recognition of redox modulation of protein function and direct effect of NO on S-nitrosylation as an important mechanism for many cell types, thus affecting a variety of proteins (Jaffrey et al. 2001; Stamler et al. 1992, 2001). A regulatory mechanism of ion channel activity involving sulfhydryl to disulfide conversion has been described previously and "regulatory thiols" have been demonstrated to be related to activities of K+ channel (George and Shibata 1995; Trapp et al. 1998), N-methyl-D-aspartate-receptor channels (Choi et al. 2000), P2X receptors (Ennion and Evans 2002), and ryanodine receptor calcium release channels (Xia et al. 2000).
Being a reactive-free radical, the chemical reactions of NO are largely dictated by its redox state. Increasing evidence suggests that the various redox states of NO exist endogenously in biological tissues. It is established that NO can react with thiol-containing biomolecules (RSH) such as cysteine and glutathione (GSH) to form S-nitrosothiols (RSNOs), which then release nitrogen-containing compounds, including NO (Miao et al. 2000; Moran et al. 2001; Sheu et al. 2000). Further oxidation of critical thiols can possibly form disulfide bonds. This implies that high concentrations of NO derived from a high dose of SNAP (100 µM) or the NO solution (0.5 µM) could modify K+ channel by oxidation. We have found that decreases in channel activity elicited by NO could not be readily reversed by simply washing with perfusing solution. If this seemingly irreversible property was a consequence of NO oxidation or formation of adducts with free SH groups, such as cysteine residues on the K+ channel protein (or a closely associated regulatory protein), leading to disulfide-bound formation, it should be possible to reverse this effect with a reducing agent (sulfhydryl-regenerating agent) such as DTT (Miao et al. 2000). The data presented earlier in Figs. 6–8 showed that 5 mM DTT was able to recover the channel open probability from a high dose of SNAP or NO solution. Moreover, a similar effect of DTT was observed for the superoxide producing agent KO2 (Fig. 10). These data suggests that part of NO regulation process, particularly when the concentration of NO reached a critically high level, was due to an oxidation mechanism and could be reversed by reducing agents.
Our preliminary results showed that superoxide radical inhibits the activity of the IK channel. It is known that in the absence of the NOS substrate, arginine, NOS does not generate any NO (Pou et al. 1992). It is further noted that at low concentrations of arginine, a new catalytic function of NOS is to generate superoxide radicals (Xia et al. 1996). Because the reaction of superoxide dismutase with superoxide anion is less efficient than the combination of superoxide with trace levels of NO to form peroxynitrite (Beckman and Koppenol 1996). The inhibition of K+ channels by superoxide may explain the toxicity of superoxide related to NO and NOS in the CNS. However, the effect of NO in the presence of superoxide on the K+ channels is largely unknown and awaits further investigation.
In summary, the present study demonstrates observation of a dual effect of NO on IK channel in mouse neocortical neurons. Both the NO-cGMP signaling and the NO-triggered redox mechanism are involved in the modulation of IK channel activity. These findings have a fundamental relevance to neuronal synaptic activity because K+ channels play an important role in controlling the overall neuron excitability.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Address for reprint requests and other correspondence: F.-S. Sheu, Dept. of Biological Sciences, National University of Singapore, 14 Science Dr. 4, Singapore 117543, Singapore (E-mail: dbssfs{at}nus.edu.sg)
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) and the IA isolated in E (
, each injection of 0.5 µM NO. Inset: dose-response curve for NO. The correlation between the current size and NO concentration is displayed above the traces. B: amperometric curves of different concentrations of SNAP. 
