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MS 11206 Received 5 June 2000; accepted after revision 18 July 2000.
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Although endothelium-derived relaxing factor is widely believed to be nitric oxide (for review, see Moncada et al. 1991), doubt still exists about its exact chemical identity (Feelisch et al. 1994). This is in part because nitric oxide can exist in different redox forms (Stamler et al. 1992b): nitrosonium cation (NO+), nitric oxide (NO·) and nitroxyl anion (NO-). Also, the lifespan of pure NO· is 
10 s in biological systems, mainly due to its fast (6·7 × 109 M-1 s-1) reaction with the superoxide anion to yield peroxynitrite (Koppenol et al. 1992). NO· forms stable complexes with haem and non-haem transition metal ions of metalloproteins, as well described for the direct activation of guanylyl cyclase (for review, see Ignarro, 1990; Moncada et al. 1991; Snyder, 1992). However, several cGMP-independent effects of NO· have been reported, which support the idea that NO·-related species like NO+ play important roles. Nitrosation and transnitrosation, reactions of NO+ and NO+-related species with nucleophiles, lead to the formation of nitroso compounds under neutral physiological conditions (Stamler et al. 1992a; Gow et al. 1998), consistent with protein function being modulated in a complex manner by NO· or NO·-related species. Indeed, nitrosation has been proposed recently as a new way of allosteric regulation of proteins (Stamler et al. 1997), and in particular, S-nitrosylation appears to be implicated in the modulation of several types of ion channels, including NMDA receptors (Lei et al. 1992), large conductance Ca2+-activated K+ channels (Bolotina et al. 1994), cyclic nucleotide-gated channels (Broillet & Firestein, 1997), L-type Ca2+ channels (Campbell et al. 1996), ryanodine receptors (Xu et al. 1998), and voltage-dependent Na+ channels (Li et al. 1998). In addition, S-nitrosylation of the small G-protein p21ras by S-nitroso-N-acetylpenicillamine (SNAP) triggers guanine nucleotide exchange and downstream signalling (Lander et al. 1997) subsequent to NMDA receptor activation (Yun et al. 1998). As any NO·-associated carrier will affect the redox state of NO·, hence its stability and the effectiveness of biological NO· transfer reactions, this suggests potentially significant roles for S-nitrosothiols (which have been detected in rat brain; Kluge et al. 1997) and dinitrosyl iron complexes (DNICs) which stabilize NO+.
Low molecular mass DNICs have been found in cells expressing high levels of the inducible NO synthase (NOS II). In these conditions, it is thought that the reaction of NO· with iron-sulfur centres of intracellular proteins, including mitochondrial aconitase involved in electron transport (Kennedy et al. 1997), results in the formation of high molecular mass DNICs (Henry et al. 1993). Such protein-bound dinitrosyl iron-dithiolate complexes are characterized by EPR spectra with g = 2·04 and g|| = 2·015 (Vithaythil et al. 1965; Henry et al. 1993). As well, NO· can react with free cellular iron, leading to the formation of low molecular mass DNICs, with distinct EPR spectra at room temperature, having cysteine or glutathione as ligands (Vanin, 1967). It has also been shown that an exchange of the dinitrosyl iron moiety between high and low molecular mass ligands is possible (Mülsch et al. 1991).
Proposed roles for such DNICs include storage and transport of forms of NO· (Mülsch et al. 1991, 1993; Muller et al. 1996). Since high molecular mass DNICs have been implicated in the disruption of mitochondrial electron transport (Kennedy et al. 1997), it has been concluded that these compounds have an intracellular site of action. However, low molecular mass DNICs are known to be released from cells expressing elevated levels of NOS II (Lancaster & Hibbs, 1990). It may be that the dinitrosyl iron moiety of low molecular mass DNICs is transferred to critical ligands of membrane proteins, and that such an action could modulate ion channel activity.
Here, we show in PC12 cells that transient external application of dinitrosyl iron-thiosulfate, a model low molecular mass DNIC, causes irreversible activation of a depolarizing inward current (IDNIC) blocked by the metal chelator DETC and by antagonists of receptor-operated calcium entry, gadolinium (25 µM) and SK&F 96365 (25 µM). This effect is not mimicked by NO· nor by compounds causing S-nitrosylation and does not involve the cGMP pathway. We propose that the formation of DNICs on the external surface of ion channels represents yet another pathway for ion channel regulation by NO·-related species.
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METHODS |
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Cell culture
PC12 cells (American Type Culture Collection) were grown at 37°C (95 % air, 5 % CO2) in RPMI 1640 with L-glutamine supplemented with 10 % horse serum, 5 % fetal calf serum, 100 u ml-1 penicillin and 100 u ml-1 streptomycin. Cells were plated on collagen-coated 35 mm diameter dishes (Falcon) for electrophysiology. Undifferentiated cells were used between 10 and 20 passages.
Electrophysiological recordings
Conventional whole-cell and outside-out patch-clamp recordings were made with an EPC 7 amplifier (List) at room temperature. Data were filtered (4-pole Bessel) at 0·1 or 1 kHz (-3 dB) and acquired directly at 0·5 or 5 kHz via a Cambridge Electronic Design (CED) 1401 interface. For whole-cell recording, pipettes of 2-4 M
resistance were made from thin-walled borosilicate glass (Poly Labo) using a vertical puller (Kopf). Normally (condition A, Table 1), the bath external solution was (mM): NaCl 140, KCl 5, CaCl2 2, MgCl2 2, glucose 11, Hepes 10, pH 7·3; and the pipette internal solution was (mM): KCl 140, CaCl2 1, MgCl2 2, EGTA 11, MgATP 5, Hepes 20, pH 7·3. For ion substitution experiments, extra- and intracellular solutions were modified as indicated in Table 1. Differences in junction potentials were minimized during solution changes using a 3 M KCl salt bridge. To calculate the relative permeabilities Po/Pi, where subscripts o and i denote external and internal ion species, respectively, the voltage form of the Goldman-Hodgkin-Katz equation was used:
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where R, T and F have their usual meaning. For PNa/PK eqn (1) was used and for PNa/PCl and PCl/PK the following equation:
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Differences between means were analysed using Wilcoxon's matched pairs test.
For single-channel recording, pipettes were made from thick-walled borosilicate glass (Hilgenberg), coated with beeswax to reduce associated capacitance and had resistances of 20-30 M. The external solution was as above, and the pipette internal solution was (mM): KMeSO3 140, CaCl2 1, MgCl2 2, EGTA 11, MgATP 5, Hepes 20, pH 7·3. Channel analysis was done with CED software. The mean open probability was determined by dividing the total measured channel open time by a fixed time (usually 1 min), just before and after the application of DETC and DTT. Individual channel open times were measured using an amplitude cursor set at a threshold value of 50 % of the mean amplitude. Each channel opening detected was visually 'inspected' before being accepted for final analysis. Differences between mean open probability were analysed using Wilcoxon's matched pairs test.
Preparation of NO·-related species
NO gas was synthesized by the reaction of 20 % FeSO4 with 40 % NaNO2 in 0·1 M HCl, and was purified first by passage through 10 % NaOH, then by low-temperature fractional sublimation in a glass high vacuum system and stored in a glass balloon under 300-500 mmHg pressure (Boese et al. 1995). Solutions of authentic NO were prepared by treating degassed (10 min) Millipore water (2 ml; 20°C) with pure NO for 10 min; NO gas from the headspace was then evacuated during 2-3 s. The NO concentration in solution was calculated as PNO (mmHg)/380, assuming that the saturating concentration of NO in water (760 mmHg; 20°C) is about 2 mM. Solutions containing NO were used immediately after preparation.
Paramagnetic low molecular mass dinitrosyl iron-thiosulfate was synthesized in a Thumberg vessel (Boese et al. 1995). A solution of thiosulfate (40 mM) in 15 mM Hepes buffer was placed in the vessel bottom, and 2 mM FeSO4 in the top. The aqueous nitrosyl complexes formed after addition of NO gas were mixed with the thiosulfate solution and then shaken under the NO atmosphere for 5-10 min, forming DNICs having a characteristic dark green colour. Dinitrosyl iron-thiosulfate was obtained as a 2 mM solution (Fe2+:ligand ratio, 1:20), characterized by EPR, and stored in liquid N2.
A solution of cysteine (50 mM) containing 7 mM Hepes buffer was used for synthesis of S-nitrosocysteine (CysNO) (Vanin et al. 1997). The solution was degassed, mixed in a Thumberg flask with NO gas (300-500 mmHg) for 2 min, and then air was allowed to enter the flask for 1 s, resulting in the oxidation of NO to NO2. Subsequent shaking of the solution for 5 min resulted in the appearance of an intense rose colour confirming the formation of S-nitrosothiols. S-Nitrosocysteine was obtained in quantitative yield as assayed by Griess' reaction after addition of mercuric chloride (Boese et al. 1995) and stored in liquid N2 until use.
Drugs were locally applied using pressurized (10 kPa) puffer pipettes (1-2 µm diameter) 200-300 µm from the cell. The pH of solutions was checked before each application. For CysNO, bolus applications were used, to avoid rapid degradation; pH was buffered with Hepes (20 mM).
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RESULTS |
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DNIC application activates depolarizing inward current in PC12 cells via an iron-, cGMP-, NO·- and S-nitrosylation-independent mechanism
A 3 min application of 50 µM dinitrosyl iron-thiosulfate (Fe2+:ligand ratio, 1:20) resulted in a slowly developing, maintained depolarization (Fig. 1A) and correspondingly, a slow activation of an inward current, which we term IDNIC (Fig. 1B). These effects approached stable levels 10-15 min after washout of dinitrosyl iron-thiosulfate and both were partially inhibited by subsequent application of 1 mM diethyldithiocarbamate (DETC), a metal chelator. The inhibitory effect of DETC supports the idea that dinitrosyl iron forms a complex with critical ligands (e.g. cysteine or histidine residues) of membrane proteins, in our case thereby leading to channel opening.
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A, a 3 min application of 50 µM dinitrosyl iron-thiosulfate provoked slowly developing depolarization (under current clamp, from -60 to -22 ± 7 mV, n = 3), which was partially inhibited by 1 mM DETC (58 ± 6 % of the initial depolarization, n = 3). B, corresponding activation of inward current (mean maximal amplitude 104 ± 28 pA, n = 10) under voltage clamp (V h, -60 mV; dotted line shows zero current level) in another cell by dinitrosyl iron-thiosulfate was also partially reversed by DETC (to 70 ± 5 % of the steady-state current, n = 6). | ||
One of the major targets of free radicals is polyunsaturated fatty acids of cell membranes, with their degradation causing alteration of membrane structure and function (Richter, 1987). Dinitrosyl iron-thiosulfate contains iron that may undergo the Haber-Weiss reaction, resulting in the production of hydroxyl radicals. To check if iron-dependent lipid peroxidation is responsible for the inward current, PC12 cells were challenged with 50 µM iron-thiosulfate (Fe2+:ligand ratio, 1:20) for 3 min. No inward current was activated by this treatment (Fig. 2A; n = 5).
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No slowly developing, stable inward current was elicited by a 3 min application of 50 µM iron-thiosulfate (A) (Fe2+:ligand ratio, 1:20), 100 µM NO· (B) or 200 µM CysNO (E). The DNIC-induced inward current was not prevented by a 20 min pretreatment with 1 µM ODQ (C) and was unaffected by a 3 min application of 5 mM DTT (D), in contrast to the inhibitory effect of 1 mM DETC. Vh, -60 mV throughout; dotted lines show zero current levels. | ||
Two major mechanisms for NO·-related modulation of channel activity are known, one involving nitrosylation and/or oxidation (Lei et al. 1992), and the other via the cGMP/protein kinase G pathway (Ignarro, 1990; Moncada et al. 1991; Snyder, 1992). To evaluate if NO· was able to directly activate IDNIC by nitrosylation or nitrosylation/oxidation, a degassed external solution containing 100 µM NO· was applied. No inward current was elicited (Fig. 2B; n = 5), but K+ currents were transiently activated by NO· (Fig. 3D and E; n = 3). Since NO·-associated increases in cGMP lead to modulation of cyclic nucleotide-gated channel activity via direct fixation of cGMP (Broillet & Firestein, 1997) or for other channel types, via protein kinase G-dependent phosphorylation (Koh et al. 1995; Clementi et al. 1996; Minowa et al. 1997), we tested if an increase in cGMP is necessary for the activation of IDNIC. When PC12 cells were pretreated (20 min) with the irreversible guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 1 µM), activation of IDNIC was unaffected (Fig. 2C; n = 4).
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Instantaneous current-voltage relationships in PC12 cells were obtained using voltage ramps (-60 to 80 mV; 1 s duration every 15 s; whole-cell recording). A, dinitrosyl iron-thiosulfate causes transient potentiation of outward K+ currents before activation of IDNIC. B, I-V plot for the currents at the times indicated in A: 1, control (before DNIC); 2-1, during DNIC, after subtraction of the control; 3-1, after DNIC (when IDNIC was maximal) after subtraction of the control. C, average I-V data from 11 cells, before and after DNIC. D, transient potentiation of K+ currents during application of 100 µM NO·. E, corresponding I-V relations at the indicated times (after subtraction of the control current). | ||
Dinitrosyl iron is an electrophilic nitrosating agent, due to the stabilization of the nitrosonium moiety. However, 5 mM dithiothreitol (DTT), a thiol reducing/denitrosylating agent, had no inhibitory effect on IDNIC (Fig. 2D; n = 6), indicating that S-nitrosothiol formation does not play a major role in the mechanism responsible for the development of IDNIC. CysNO, a more potent nitrosylating compound than NO·, was used to confirm the nitrosylation-independent activation of IDNIC. Application of 200 µM CysNO triggered the immediate activation of a transient inward current which was qualitatively quite different from IDNIC (Fig. 2E; n = 5). Moreover, exposure to 3-morpholinosydnonimine (SIN-1, 3 min, 400 µM), a peroxynitrite-producing compound and a potent nitrosylating and nitrating agent, resulted in no inward current activation (not shown; n = 3). In a similar manner, a 3 min challenge with S-nitroso-N-acetylpenicillamine (100 µM; n = 2), a more stable S-nitrosothiol, and sodium nitroprusside (100 µM; n = 3), which contains a mononitrosyl moiety, was without effect (not shown). Taken together, these results indicate that among various NO·-related compounds tested in PC12 cells, dinitrosyl iron-thiosulfate alone possesses the capacity to activate a slowly developing, depolarizing inward current in a maintained fashion.
NO· donor nature of dinitrosyl iron-thiosulfate
Because K+ currents are potentiated by NO· (Bolotina et al. 1994; Koh et al. 1995), we checked whether dinitrosyl iron-thiosulfate shares this effect. We observed a transient potentiation of K+ currents elicited by voltage ramp commands during dinitrosyl iron-thiosulfate application (Fig. 3A and B), which occurred well before the onset of significant amounts of IDNIC. Averaged I-V data before and after DNIC are given in Fig. 3C, showing clearly that when IDNIC is maximally activated, K+ currents are not potentiated. A similar transient potentiation of K+ currents was observed when cells were challenged with 100 µM NO· (Fig. 3D and E; n = 3), with no inward current being subsequently seen. These data suggest that even if dinitrosyl iron-thiosulfate has a NO· donor-like capacity, a potential modulatory effect of NO· is unlikely to account for the activation of the slowly developing, maintained IDNIC.
Extracellular localization of the site of action of dinitrosyl iron
Since the activation of IDNIC is NO· independent, we tried to elucidate the localization of the dinitrosyl iron target responsible for this inward current by including 100 µM dinitrosyl iron-thiosulfate in the pipette internal solution. Under these conditions, a rapid potentiation of K+ current which reached stable levels within 5 min was observed, but no inward current developed subsequently (Fig. 4A and B; n = 4). The same experiment was carried out with lower concentrations of EGTA and ATP, which are potential chelators of dinitrosyl iron. Again, K+ currents were potentiated, but to a greater extent and in a continual manner, while inward IDNIC-like current was absent (Fig 4C and D; n = 3). These results support the idea that dinitrosyl iron-thiosulfate-induced IDNIC arises from an extracellular target and that putative protein-bound DNIC may well be located on the external membrane surface when formed in vivo.
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Whole-cell recordings with ramp commands, as in Fig. 3. A, with 100 µM dinitrosyl iron-thiosulfate in the pipette internal solution (containing inter alia 11 mM EGTA/1 mM CaCl2 and 5 mM MgATP), no inward current was triggered, while outward K+ currents were increased, as seen in the I-V plots in B. C, effect of including 100 µM dinitrosyl iron-thiosulfate in the pipette internal solution (containing inter alia 100 µM EGTA/0 CaCl2 and 500 µM MgATP). Outward K+ currents were increased, and the voltage dependence of activation was shifted leftwards at later times (D). | ||
The DETC-sensitive component of IDNIC is a non-selective cationic current
In order to identify the ion species contributing to IDNIC, we examined the effects of ion replacement on IDNIC reversal potentials determined using voltage ramp commands. All mean reversal potential data given hereafter were obtained at least 10 min after DNIC application. Since replacement of intracellular K+ by Cs+ (condition B, Table 1) resulted in a linear I-V relationship for IDNIC (not shown), with a reversal potential of -1 ± 11 mV (n = 6), in the presence of intracellular K+, reversal potentials were determined after extrapolation of the inward part of the DNIC-activated current. When permeant extracellular ions were mainly Na+ and Cl- and permeant intracellular ions were K+ and Cl- (condition A, Table 1), the IDNIC reversal potential was -5 ± 11 mV (Fig. 3C and Table 1; n = 11), suggesting the activation of a membrane conductance poorly selective for cations and/or chloride. Relative permeability ratios were calculated using the Goldman-Hodgkin-Katz equation from reversal potentials determined under bi-ionic conditions (Table 1): PNa/PK 
1·1 (Erev = -1·5 ± 1·6 mV; n = 5; condition C); PCl/PK 1·8 (Erev = -10 ± 4 mV; n = 10; condition D); and PNa/PCl 0·73 (Erev = 12 ± 3 mV; n = 6; condition E). Taken together, these data suggest that Cl- and cations (K+, Na+) contribute 60 and 40 %, respectively, to IDNIC.
Table 1. Ionic selectivity of IDNIC, determined from quasi steady-state I-V relationships under different ionic conditions
| Ionic concentrations (o/i; mM) | Erev (mV) | Relative permeability |
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| Na+ | K+ | Cl- | NMDG+ | MeSO3- | Cs+ | |||
| A | 144/0 | 5/140 | 153/146 | - | - | - | -5 ± 11 | - |
| B | 144/0 | 5/0 | 153/146 | - | - | 0/140 | 1 ± 11 | - |
| C | 145/0 | 0/170 | 8/6 | - | 140/140 | - | -1·5 ± 1·6 | Na+/K+1·1 |
| D | - | 14/150 | 148/146 | 140/0 | - | - | -10 ± 4 | Cl-/K+1·8 |
| E | 149/8 | - | 153/146 | 0/140 | - | - | 12 ± 3* | Na+/Cl-0·7 |
| F | 149/8 | - | 153/146 | 0/140 | - | - | 5 ± 6* | Na+/Cl-0·3* |
Nevertheless, the above results cannot alone distinguish between a heterogeneous population of channels consisting of subsets of cation- and anion-selective channels, and a single population of channels permeable to both cations and anions. The inhibition of IDNIC by 1 mM DETC triggered a significant shift in the reversal potential of IDNIC (12 ± 3 to 5 ± 6 mV; n = 6; Wilcoxon's matched pairs test) when the anion Erev was -1 mV and the cation Erev was +75 mV (Table 1, condition F). This might suggest that IDNIC is due to a heterogeneous population of channels consisting of subsets of cation- and anion-selective channels having Pcation/Panion 1·5 and Pcation/Panion 0·3, respectively. The shift of the IDNIC reversal potential towards the anion Erev after DETC suggests that DETC acts preferentially on cationic currents, in agreement with the idea that the DETC-sensitive part of IDNIC is due to a non-selective cationic current. In any case, in physiological conditions, ECl is close to the resting membrane potential, and thus IDNIC would be largely due to Na+ entry rather than Cl- exit.
Because several types of non-selective cationic channels have been described in PC12 cells, we tried to identify the channel type responsible for IDNIC. The nicotinic channel blockers hexamethonium (100 µM, n = 4), d-tubocurarine (10 µM, n = 3), and 
-bungarotoxin (10 nM, n = 4) were tested and none were found to have an inhibitory action on IDNIC activation (data not shown), consistent with nicotinic receptor-channels not being responsible for IDNIC. The purinoreceptor blocker suramin (500 µM, n = 3) also had no inhibitory action on IDNIC activation, suggesting that DNIC does not directly activate such ATP-sensitive channels.
As activation of non-selective cationic channels contributing to receptor-mediated Ca2+ influx depolarize PC12 cells (Fasolato et al. 1990), Gd3+ and SK&F 96365, known antagonists of receptor-operated Ca2+ entry (Merritt et al. 1990), were tried. Both SK&F 96365 (25 µM) and Gd3+ (25 µM) caused about 50 % inhibition of IDNIC (Fig. 5), while on the other hand, 10 µM Cd2+, an inhibitor of voltage-dependent Ca2+ channels, was without effect (data not shown, n = 3). These results are consistent with a significant fraction of IDNIC being carried by non-selective cationic channels.
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A, DNIC-dependent inward current was inhibited (by 45 ± 4 %; n = 6) by application of 25 µM Gd3+. B, IIDN was inhibited (by 48 ± 4 %; n = 7) by application of 25 µM SK&F 96365. Vh, -60 mV. Dotted lines correspond to zero current levels. | ||
Single-channel analysis of dinitrosyl iron-thiosulfate-activated currents
Single-channel recordings were made from outside-out patches in the absence of Cl- in the pipette internal solution (so that inwardly directed currents were carried by cations). In these conditions, in all patches tested, dinitrosyl iron- thiosulfate application resulted in the maintained activation of inward currents at negative holding potentials having a predominant elementary conductance of 50 pS (Fig. 6). Such currents were inhibited by DETC, with the mean open probability after DNIC (0·081 ± 0·030) decreasing significantly to 0·012 ± 0·005 after DETC application (Fig. 6A-D; n = 5; Wilcoxon's matched pairs test), without a change in the elementary conductance. The reversal potential of these currents was -1 mV (Fig. 6E), in agreement with IDNIC being carried by non-selective cationic channels. Many of the openings were of short duration (<10 ms), although occasional bursts lasting >100 ms occurred. As found in whole-cell recordings, 5 mM DTT was ineffective (mean open probability was 0·041 ± 0·013 after DNIC and 0·05 ± 0·03 after DTT; n = 5; data not shown).
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A, representative recording showing activation of single-channel currents after dinitrosyl iron-thiosulfate (50 µM, 3 min application) in an outside-out patch from PC12 cells, and subsequent inhibition by DETC (1 mM). Breaks correspond to 5 min; Vh, -50 mV. B-C, channel activity at a higher time resolution before (B) and after (C) application of DETC. Dotted lines show the closed (c) and open (o) levels. The all-points amplitude histograms to the right of the current traces were made from 1 min segments before (B) and after (C) application of DETC. D, mean open channel probability calculated for successive 1 min intervals for the recording shown in A. E, I-V relationship of DNIC-activated currents. The regression line yields a single-channel conductance of 50 pS with a reversal potential of -1 mV. Each point represents mean values from amplitude histograms from 3-6 patches (the error bars are smaller than the symbols). Data were acquired at 5 kHz with a 1 kHz filter. The pipette internal solution was Cl- free. | ||
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DISCUSSION |
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We have shown that dinitrosyl iron-thiosulfate application causes irreversible activation of a depolarizing inward current in PC12 cells. This activation involves neither a direct effect of NO· through nitrosylation nor the cGMP pathway, but is inhibited by the metal chelator DETC. This may well represent a novel modulatory mechanism whereby the formation of a low molecular mass DNIC in vivo leads to activation of ion channels via direct extracellular complexing of the dinitrosyl iron moiety on such membrane proteins.
Nitric oxide-independent activation of IDNIC
NO·, being liposoluble, can be considered as a diffusive signal and a potential neuronal messenger (Snyder, 1992). Transduction of the NO· signal in target cells mainly occurs through stimulation of soluble guanylyl cyclase (sGC) and subsequent cGMP-dependent phosphorylation. Several ionic channels are modulated by this pathway: activation of ryanodine receptors (Clementi et al. 1996), activation of K+ channels (Koh et al. 1995) and inhibition of non-selective cationic channels (Minowa et al. 1997). Since low molecular mass DNICs are potential NO· donors, activation of sGC may occur. The rapidly developing, short-lived increase of K+ currents observed here after dinitrosyl iron-thiosulfate challenge was similar to that induced by NO· (Fig. 3), in agreement with the NO· donor character of low molecular mass DNICs. Nevertheless, several lines of evidence indicate that activation of IDNIC was NO· and cGMP independent. Firstly, exogenous NO· elicited no inward current. The development of IDNIC was not prevented by the sGC inhibitor ODQ, nor by the absence of GTP in the pipette internal solution, and dinitrosyl iron-thiosulfate-evoked inward single-channel currents were observed in outside-out patches. Furthermore, the differential effects of intra-/extracellular dinitrosyl iron-thiosulfate application on K+ currents (respectively stimulation and inhibition; Fig. 4), distinguishes between purely NO·-dependent events and NO+-Fe+-dependent events. Because IDNIC was only seen upon extracellular application of dinitrosyl iron-thiosulfate, this suggests an extracellular site of action, consistent with the reaction mechanism being both cGMP and NO· independent, as NO· is membrane permeant.
In contrast to the activation of IDNIC described here, non-selective cationic channel activity was decreased by NO·-releasing agents applied to the intracellular surface of inside-out patches from brown adipocytes with DTT reversing this effect. These actions were taken as evidence for a redox-based modulatory mechanism involving S-nitrosylation of sulfhydryl groups (Koivisto & Nedergaard, 1995).
Nitrosylation versus dinitrosyl iron-protein complex formation
Other ionic channels are sensitive to S-nitrosylation/oxidation of their cysteine residues caused by NO·, peroxynitrite or S-nitrosothiols (Bolotina et al. 1994; Xu et al. 1998). Dinitrosyl iron-dithiolate complexes are also S-nitrosylating agents, with for example, iron-catalysed S-nitrosylation followed by transnitrosation of a histidine leading to inhibition of glutathione reductase (Boese et al. 1997). However, in addition, low molecular mass DNICs readily form high molecular mass dinitrosyl iron-protein complexes (e.g. with serum albumin) without subsequent nitrosation (Boese et al. 1995). Potential ligands for dinitrosyl iron include cysteine, histidine, aspartate and glutamine residues.
Here, in PC12 cells, we suggest that IDNIC activation results from the formation of protein-bound dinitrosyl iron rather than S-nitrosylation. In agreement, IDNIC was evoked in an irreversible manner, neither NO· nor potent nitrosylating agents produced the slowly developing, maintained inward current that we term IDNIC, and DTT, a denitrosylating/reducing agent (Xu et al. 1998), was without effect (Fig. 2). Our data are consistent with the notion that the specificity of action of NO·-related species depends on the NO· carrier (Feelisch et al. 1994). Finally, IDNIC was inhibited by the metal chelator DETC, probably not due to slow denitrosylation (Arnelle et al. 1997), but rather the faster dinitrosyl iron chelation reaction. In any case, while we cannot completely reject the possibility that dinitrosyl iron-thiosulfate-associated nitrosation (nitrosylation) of DTT-inaccessible residues (cysteines) occurs, it should be noted that the molecular sizes of DTT and DETC are similar.
We propose that DNICs may have two, three or even four protein ligands, thereby permitting conformational transitions towards the most stable configuration. The ligands for DNIC formation on modulated channels may be colocalized on a single subunit, or alternatively, co-ordinated dinitrosyl iron binding between adjacent subunits may occur, as has been suggested for the modulatory actions of Ni2+ on cyclic nucleotide-gated channels (Gordon & Zagotta, 1995). Note that the co-ordinating sphere for dinitrosyl iron is not necessarily provided only by protein residues, as the ligands in the dinitrosyl iron-bovine serum albumin complex are free L-cysteine and the single reduced cysteinyl-thiol on the protein (Boese et al. 1995).
Irreversible activation of IDNIC: pathophysiological implications
As mentioned above, activation of IDNIC and the accompanying depolarizing current are irreversible. This suggests that these effects may be implicated in the long-term actions of NO·, including, for example, NO· modulation of synaptic transmission, and in pathological processes involving high levels of NO· production like excitotoxicity (Dawson et al. 1996), Parkinson's (Hantraye et al. 1996) and Alzheimer's (Meda et al. 1995) diseases. Also, Alzheimer's disease is characterized by extracellular accumulation of iron (Good et al. 1992), which may favour the extracellular formation of low molecular mass DNICs. Interrelationships between iron and NO· metabolism are well documented (Richardson & Ponka, 1997), with the formation of DNICs being associated with the loss of intracellular iron and cytotoxicity (Henry et al. 1993). Evidence for physiological roles of protein-bound dinitrosyl iron in vivo is less obvious, because of the detection limits of EPR measurements. EPR signals for such DNICs are best characterized in systems expressing NOS II where NO· production is elevated (Lancaster & Hibbs, 1990; Henry et al. 1993), and are considered as indicators of NO·-associated cytotoxicity. This supports the idea that DNIC formation and subsequent modulation of ion channel activity would occur mainly in pathophysiological processes. Nevertheless, the formation of DNICs in physiological conditions may be possible, as the formation of low molecular mass DNICs and high molecular mass dinitrosyl iron-protein complexes might also occur under conditions of constitutive NO· production (Mülsch et al. 1993). It may be that the generation of DNICs participates in the complex effects of NO· in neuroprotection or neurodegeneration (Lipton et al. 1993), perhaps via activation of IDNIC. The resultant stable depolarization might have dramatic effects on cellular survival. For example, apoptosis in mouse neocortical neurons is associated with an increase in delayed rectifier K+ current and loss of intracellular K+ (Yu et al. 1997).
The modulation of non-selective cationic channels by NO· probably has pathophysiological/physiological implications, since Ca2+ signalling is often involved. For example, NO·-dependent inhibition of NMDA receptors may represent a negative feedback for NO production, as Ca2+ entry is decreased (Lei et al. 1992). Direct activation of the 
subunit of cyclic nucleotide-gated channels by S-nitrosylation triggers an increase in Ca2+ permeability in several neuronal cells (Broillet & Firestein, 1997). As well, second-messenger-operated channels and store-operated Ca2+ channels responsible for Ca2+ influx are also modulated by NO· (Clementi & Meldolesi, 1997).
Here, we show that a DETC-sensitive component of IDNIC probably results from the activation of non-selective cationic channels. The inhibition of IDNIC by both Gd3+ and SK&F 96365 suggests that the cationic component of IDNIC might include some Ca2+ permeability, since these compounds have been described as inhibitors of receptor-operated Ca2+ entry. The potential implications of IDNIC in contributing to an irreversible calcium influx may have important consequences in pathophysiological processes where overproduction of NO· is involved.
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REFERENCES |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| Arnelle, D. R., Day, B. J. & Stamler, J. S. (1997). Diethyl dithiocarbamate-induced decomposition of S-nitrosothiols. Nitric Oxide 1, 56-64. | |
| Boese, M., Keese, M. A., Becker, K., Busse, R. & Mulsch, A. (1997). Inhibition of glutathione reductase by dinitrosyl-iron-dithiolate complex. Journal of Biological Chemistry 272, 21767-21773 | [Abstract/Full Text] |
| Boese, M., Mordvintcev, P. I., Vanin, A. F., Busse, R. & Mulsch, A. (1995). S-Nitrosation of serum albumin by dinitrosyl-iron complex. Journal of Biological Chemistry 270, 29244-29249 | [Abstract/Full Text] |
| Bolotina, V. M., Najibi, S., Palacino, J. J., Agano, P. J. & Cohen, R. A. (1994). Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368, 850-853 | [Medline] |
| Broillet, M. C. & Firestein, S. (1997). -Subunits of the olfactory cyclic nucleotide-gated channel form a nitric oxide activated Ca2+ channel. Neuron 18, 951-958 |
[Medline] |
| Campbell, D. L., Stamler, J. S. & Strauss, H. C. (1996). Redox modulation of L-type calcium channels in ferret ventricular myocytes. Dual mechanism regulation by nitric oxide and S-nitrosothiols. Journal of General Physiology 108, 277-293 | [Abstract] |
| Clementi, E. & Meldolesi, J. (1997). The cross-talk between nitric oxide and Ca2+: a story with a complex past and a promising future. Trends in Pharmacological Sciences 18, 266-269 | [Medline] |
| Clementi, E., Riccio, M., Sciorati, C., Nistico, G. & Meldolesi, J. (1996). The type 2 ryanodine receptor of neurosecretory PC12 cells is activated by cyclic ADP-ribose. Role of the nitric oxide/cGMP pathway. Journal of Biological Chemistry 271, 17739-17745 | [Abstract/Full Text] |
| Dawson, V. L., Kizushi, V. M., Huang, P. L., Snyder, S. H. & Dawson, T. M. (1996). Resistance to neurotoxicity in cortical cultures from neuronal nitric oxide synthase-deficient mice. Journal of Neuroscience 16, 2479-2487 | [Abstract] |
| Fasolato, C., Pizzo, P. & Pozzan, T. (1990). Receptor-mediated calcium influx in PC12 cells. ATP and bradykinin activate two independent pathways. Journal of Biological Chemistry 265, 20351-20355 | [Abstract] |
| Feelisch, M., te Poel, M., Zamora, R., Deussen, A. & Moncada, S. (1994). Understanding the controversy over the identity of EDRF. Nature 368, 62-65 | [Medline] |
| Good, P. F., Perl, D. P., Bierer, L. M. & Schmeidler, J. (1992). Selective accumulation of aluminum and iron in the neurofibrillary tangles of Alzheimer's disease: a laser microprobe (LAMMA) study. Annals of Neurology 31, 286-292 | [Medline] |
| Gordon, S. E. & Zagotta, W. N. (1995). Subunit interactions in coordination of Ni2+ in cyclic nucleotide-gated channels. Proceedings of the National Academy of Sciences of the USA 92, 10222-10226 | [Abstract] |
| Gow, A. J. & Stamler, J. S. (1998). Reactions between nitric oxide and haemoglobin under physiological conditions. Nature 391, 169-173 | [Medline] |
| Hantraye, P., Brouillet, E., Ferrante, R., Palfi, S., Dolan, R., Matthews, R. T. & Beal, M. F. (1996). Inhibition of neuronal nitric oxide synthase prevents MPTP-induced Parkinsonism in baboons. Nature Medicine 2, 1017-1021 | [Medline] |
| Henry, Y., Lepoivre, M., Drapier, J. C., Ducrocq, C., Boucher, J. L. & Guissani, A. (1993). EPR characterization of molecular targets for NO in mammalian cells and organelles. FASEB Journal 7, 1124-1134 | [Abstract] |
| Ignarro, L. J. (1990). Biosynthesis and metabolism of endothelium-derived nitric oxide. Annual Review of Pharmacology and Toxicology 30, 535-560 | [Medline] |
| Kennedy, M. C., Antholine, W. E. & Beinert, H. (1997). An EPR investigation of the products of the reaction of cytosolic and mitochondrial aconitases with nitric oxide. Journal of Biological Chemistry 272, 20340-20347 | [Abstract/Full Text] |
| Kluge, I., Gutteck-Amsler, U., Zollinger, M. & Do, K.Q. (1997). S-nitrosoglutathione in rat cerebellum: identification and quantification by liquid chromatography-mass spectrometry. Journal of Neurochemistry 69, 2599-2607 | [Abstract] |
| Koh, S. D., Campbell, J. D., Carl, A. & Sanders, K. M. (1995). Nitric oxide activates multiple potassium channels in canine colonic smooth muscle. The Journal of Physiology 489, 735-743 | [Medline] |
| Koivisto, A. & Nedergaard, J. (1995). Modulation of calcium-activated non-selective cation channel activity by nitric oxide in rat brown adipose tissue. The Journal of Physiology 486, 59-65 | [Medline] |
| Koppenol, W. H., Moreno, J. J., Pryor, W. A., Ischiropoulos, H. & Beckman, J. S. (1992). Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chemical Research in Toxicology 5, 834-842. | [Medline] |
| Lancaster, J. R. & Hibbs, J. R. (1990). EPR demonstration of iron-nitrosyl complex formation by cytotoxic activated macrophages. Proceedings of the National Academy of Sciences of the USA 87, 1223-1227 | [Abstract] |
| Lander, H. M., Hajjar, D. P., Hempstead, B. L., Mirza, U. A., Chait, B. T., Campbell, S. & Quilliam, L. A. (1997). A molecular redox switch on p21ras. Structural basis for the nitric oxide-p21ras interaction. Journal of Biological Chemistry 272, 4323-4236 | [Abstract/Full Text] |
| Lei, S. Z., Pan, Z. H., Aggarwal, S. K., Chen, H. S., Hartman, J., Sucher, N. J. & Lipton, S. A. (1992). Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex. Neuron 8, 1087-1099 | [Medline] |
| Li, Z., Chapleau, M. W., Bates, J. N., Bielefeldt, K., Lee, H. C. & Abboud, F. M. (1998). Nitric oxide as an autocrine regulator of sodium currents in baroreceptor neurons. Neuron 20, 1039-1049 | [Medline] |
| Lipton, S. A., Choi, Y. B., Pan, Z. H., Lei, S. Z., Chen, H. S., Sucher, N. J., Loscalzo, J., Singel, D. J. & Stamler, J. S. (1993). A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 364, 626-632 | [Medline] |
Meda, L., Cassatella, M. A., Szendrei, G. I., Otvos, L. Jr, Baron, P., Villalba, M., Ferrari, D. & Rossi, F. (1995). Activation of microglial cells by -amyloid protein and interferon- . Nature 374, 647-650 |
[Medline] |
| Merritt, J. E., Armstrong, W. P., Benham, C. D., Hallam, T. J., Jacob, R., Jaxa-Chamiec, A., Leigh, B. K., McCarthy, S. A., Moores, K. E. & Rink, T. J. (1990). SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochemical Journal 271, 515-522 | [Medline] |
| Minowa, T., Miwa, S., Kobayashi, S., Enoki, T., Zhang, X. F., Komuro, T., Iwamuro, Y. & Masaki, T. (1997). Inhibitory effect of nitrovasodilators and cyclic GMP on ET-1-activated Ca2+-permeable nonselective cation channel in rat aortic smooth muscle cells. British Journal of Pharmacology 120, 1536-1544 | [Medline] |
| Moncada, S., Palmer, R. M. & Higgs, E. A. (1991). Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacological Reviews 43, 109-142 | [Medline] |
| Muller, B., Kleschyov, A. L. & Stoclet, J. C. (1996). Evidence for n-acetylcysteine-sensitive nitric oxide storage as dinitrosyl-iron complexes in lipopolysaccharide-treated rat aorta. British Journal of Pharmacology 119, 1281-1285 | [Medline] |
| Mülsch, A., Mordvintcev, P. I., Vanin, A. F. & Busse, R. (1991). The potent vasodilating and guanylyl cyclase activating dinitrosyl-iron(II) complex is stored in a protein-bound form in vascular tissue and is released by thiols. FEBS Letters 294, 252-256 | [Medline] |
| Mülsch, A., Mordvintcev, P. I., Vanin, A. F. & Busse, R. (1993). Formation and release of dinitrosyl iron complexes by endothelial cells. Biochemical and Biophysical Research Communications 196, 1303-1308 | [Medline] |
| Richardson, D. R. & Ponka, P. (1997). The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochimicia et Biophysica Acta 1331, 1-40. | |
| Richter, C. (1987). Biophysical consequences of lipid peroxidation in membranes. Chemistry and Physics of Lipids 44, 175-189 | [Medline] |
| Snyder, S. H. (1992). Nitric oxide: first in a new class of neurotransmitters. Science 257, 494-496 | [Medline] |
| Stamler, J. S., Simon, D. I., Osborne, J. A., Mullins, M. E., Jaraki, O., Michel, T., Singel, D. J. & Loscalzo, J. (1992a). S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proceedings of the National Academy of Sciences of the USA 89, 444-448 | [Abstract] |
| Stamler, J. S., Singel, D. J. & Loscalzo, J. (1992b). Biochemistry of nitric oxide and its redox-activated forms. Science 258, 1898-1902 | [Medline] |
| Stamler, J. S., Toone, E. J., Lipton, S. A. & Sucher, N. J. (1997). (S)NO signals: translocation, regulation, and a consensus motif. Neuron 18, 691-696 | [Medline] |
| Vanin, A. F. (1967). Identification of divalent iron complexes with cysteine in biological systems by EPR method. Biokhimia 32, 228-232 (in Russian). | |
| Vanin, A. F., Malenkova, I. V. & Serezhenkov, V. A. (1997). Iron catalyzes both decomposition and synthesis of S-nitrosothiols: optical and electron paramagnetic resonance studies. Nitric Oxide 1, 191-203. | [Medline] |
| Vithayathil, A. J., Ternberg, J. L. & Commoner, B. (1965). Changes in electron spin resonance signals of rat liver during chemical carcinogenesis. Nature 207, 1246-1249 | [Medline] |
| Xu, L., Eu, J. P., Meissner, G. & Stamler, J. S. (1998). Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation. Science 279, 234-237 | [Abstract/Full Text] |
| Yu, S. P., Yeh, C. H., Sensi, S. L., Gwag, B. J., Canzoniero, L. M., Farhangrazi, Z. S., Ying, H. S., Tian, M., Dugan, L. L. & Choi, D. W. (1997). Mediation of neuronal apoptosis by enhancement of outward potassium current. Science 278, 114-117 | [Abstract/Full Text] |
| Yun, H. Y., Gonzalez-Zulueta, M., Dawson, V. L. & Dawson, T. M. (1998). Nitric oxide mediates N-methyl-D-aspartate receptor-induced activation of p21ras. Proceedings of the National Academy of Sciences of the USA 95, 5773-5778 | [Abstract] |
This work was in part supported by grants from the European Union (Biomed II PL950979), the Fondation de France, the Fondation pour la Recherche Médicale, the Ministère de l'Education Nationale, de la Recherche et de la Technologie (ACC-SV 9511021) and the CNRS. We thank C. Untereiner and D. Wagner for expert technical assistance, and Professor J.-C. Stoclet for discussion and critical reading of the manuscript.
Corresponding author
K. Takeda: Université Louis Pasteur de Strasbourg, CNRS UMR 7034, Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, B.P. 24, 67401 Illkirch, France.
Email: kt{at}aspirine.u-strasbg.fr
Author's present address
A. L. Kleyschov: University Hospital Hamburg-Eppendorf, Department of Internal Medicine, Division of Cardiology, Room 133, Martini Strasse 52, 20246 Hamburg, Germany.
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