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NERVOUS SYSTEM CELL BIOLOGY
1Department of Physiology and Functional Genomics and McKnight Brain Institute, University of Florida, Gainesville, Florida; 2Department of Medicinal Chemistry, Welsh School of Pharmacy, Cardiff University, Cardiff, United Kingdom; 3Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Sakyo, Kyoto, Japan; and 4Department of Anesthesiology, University of Florida, Gainesville, Florida
Submitted 11 December 2006 ; accepted in final form 12 March 2007
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ABSTRACT |
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thiol-protein oxidoreductase; patch clamp; neuronal activity
Considering the above-described neuronal actions of MIF via its intrinsic TPOR activity, we became interested in whether another TPOR-containing protein, specifically Trx, could also serve as a modulator of neuronal activity and ANG II-induced responses. Trx, the canonical member for which the superfamily is named, is a 12-kDa protein that exhibits intrinsic TPOR activity via the active sequence -Cys-Gly-Pro-Cys- between residues 32 and 35 (2) and mainly operates as a disulfide reductase to maintain proteins in their reduced state (21). Trx itself is maintained in its reduced form by Trx reductase and reduced nicotinamide adenine dinucleotide phosphate (NADPH). Trx, along with the peroxiredoxin (Prx) family of enzymes, plays a critical intracellular role in the scavenging of reactive oxygen species (ROS) and in the redox regulation of protein function and signaling (21). In addition, Trx has many other functions such as redox regulation of transcription factors, inhibition of apoptosis, and immunomodulation (2, 22).
In the present study, our goal was to provide the first insight into the actions of Trx on neuronal activity by studying the effects of this protein on basal outward K+ currents, changes of which contribute to alterations in neuronal firing (29, 33). Furthermore, we have examined whether ANG II can modulate neuronal Trx expression and whether Trx can influence the actions of ANG II on outward K+ currents. Collectively, the data presented here support the idea that the TPOR activity of small proteins (including Trx) may serve as a regulator of both basal and stimulated neuronal activity.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Materials. Dulbecco's modified Eagle's medium (DMEM) was obtained from Invitrogen (Grand Island, NY). Rabbit anti-Trx1 polyclonal antibody was purchased from Chemicon International (Temecula, CA). Plasma-derived horse serum (PDHS), 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron), ANG II, PD-123319, goat anti-rabbit IgG, NADPH, insulin, 5,5'-dithiobis(2-nitrobenzoic acid) (DNTB), and all other chemicals were obtained from Sigma (St. Louis, MO). Mouse Trx and rat Trx reductase were purchased from American Diagnostica (Stamford, CT). PMX 464 [formerly AW464; 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-dienone] was obtained from Pharminox, where it was synthesized as detailed previously (35). Recombinant human (rh)Trx and C32S/C35S mutant rhTrx (rhC32S/C35S-Trx) were kindly provided by the Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital (15). Cells-to-cDNA II kits were purchased from Ambion (Austin, TX). Primers for Trx and 18S for real-time RT-PCR were obtained from Applied Biosystems (Foster City, CA).
Preparation of neuronal cultures. Neuronal cocultures were prepared from the brain stem and a hypothalamic block of newborn SD rats as described previously (25). Cultures were grown in DMEM containing 10% PDHS for a further 12–15 days before use.
Electrophysiological recordings. Electrophysiological recordings of K+ currents were carried out in cultured neurons as detailed by us recently with the whole cell configuration of the patch-clamp technique (17). Total K+ current was recorded by stepping from a holding potential of –80 mV to +10 mV for 100 ms every minute. IKv was measured directly by stepping from –40 mV to +10 mV for 100 ms. To inactivate IA, we applied depolarizing prepulses to –40 mV from a holding potential of –70 mV for 50 ms (37). In most cases, this 50-ms prepulse to –40 mV was able to inactivate this fast K+ current, although in some neurons a remnant component of outward inactivating current was present. For total K+ current and IKv, current amplitudes were measured at 50 ms from the onset of the test pulse. Current density was derived by dividing current amplitude (pA) by membrane capacitance (pF), which was measured with the Membrane test of pCLAMP 8.0. The average cell capacitance for neurons used in this study was 33.7 ± 1.3 pF (n = 137), ranging from 12 to 74 pF. IA was elicited by depolarization of the membrane potential to +42.5 mV from a holding potential of –110 mV for 100 ms every minute (17, 34). IA amplitude was measured as the peak current during the depolarizing pulse.
Analysis of Trx mRNA. cDNA was produced from control or ANG II-treated neuronal cultures with the Cells-to-cDNA II kit. Levels of Trx mRNA were quantified by real-time RT-PCR, essentially as described previously for MIF (28). Data were normalized to 18S RNA.
Analysis of Trx protein.
Proteins were isolated from control or ANG II-treated neuronal cultures, and the expression of Trx was assessed via Western immunoblots. This was achieved with rabbit anti-Trx1 (1:2,500; primary antibody), peroxidase-conjugated goat anti-rabbit IgG (1:10,000), and a chemiluminescence method using ECL Western Blotting reagents (Amersham Biosciences, Piscataway, NJ). All procedures were as detailed by us previously for the analysis of MIF (4). Trx protein levels were quantified by densitometry using a calibrated imaging densitometer (model GS710, Bio-Rad Laboratories, Hercules, CA) and were expressed as a ratio of Trx to 
-actin levels in the same samples. Quantity One software (Bio-Rad Laboratories) was used to validate the linearity of the obtained signals.
Drug solutions and intracellular application. Trx (mouse and human) and rhC32S/C35S-Trx were dissolved directly in the pipette solution. PMX 464 was dissolved in DMSO, followed by dilution in pipette solution to the required concentration (800 nM). The concentration of DMSO in the final solution was 0.005%. Trx, rhC32S/C35S-Trx, and PMX 464 were injected intracellularly via the patch pipette as detailed by us previously (28, 37). The concentrations of proteins and drugs given in RESULTS refer to the amounts injected at the pipette tip, and so are likely higher than the amounts that reach the site of action.
Insulin reduction assay. The insulin reduction assay, with modifications described by Kunkel et al. (12) and Pallis et al. (20), was used to assess the TPOR activity of native Trx (mouse and human), boiled Trx, and mutant Trx (rhC32S/C35S-Trx) and the ability of PMX 464 to inhibit TPOR activity. In brief, mouse Trx (0.8 µM) was incubated for 30 min at 37°C in 0.1 M Tris-Cl (pH 7.5)-2 mM EDTA buffer containing 1 mM NADPH, 0.16 U/ml Trx reductase, and 2.5 mg/ml insulin. Parallel incubations were performed with boiled mouse Trx or mouse Trx in the presence of PMX 464 (80 µM). Further incubations were performed with rhTrx or rhC32S/C35S-Trx. Reactions were stopped by the addition of buffer consisting of 6 M guanidine-HCl, 50 mM Tris (pH 8), and 10 mM DTNB. The reduction of the latter compound, by transfer of reducing equivalents from NADPH, was measured as an increase in absorbance at 405 nm.
Data analyses. Results are expressed as means ± SE. Statistical significance was evaluated with ANOVA1 and paired Student's t-test. Differences were considered significant at P < 0.05; n refers to the number of cells examined or the number of experiments.
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RESULTS |
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80–90% of the neurons tested, Trx (8 nM), applied via intracellular perfusion, produced a significant increase in IKv, measured at +10 mV. This effect became apparent by 10 min, increased slowly, and reached a maximum at 15–20 min after the start of Trx administration (Fig. 1). The current-voltage (I-V) relationships of IKv, recorded before and after treatment of neurons with the same concentration of Trx, are shown in Fig. 2. Trx increased the amplitude and density (pA/pF) of IKv at all tested potentials without a significant change in the threshold of IKv activation. The effect of Trx on IKv was concentration dependent, with the increase in current density (pA/pF) from 31.70 ± 0.56 (mean ± SE) in controls to 43.71 ± 2.89 at 8 nM of Trx (P < 0.01) (Fig. 3). The effect of Trx was statistically significant at concentrations of 0.8, 8, and 80 nM, but there was no effect of this protein at a concentration of 0.08 nM. Trx (8 nM) that had been denatured by boiling for 1 h did not alter IKv (Fig. 3). It should be noted that boiling reduced the TPOR activity to 5.90% of the unboiled (native) Trx, as determined via insulin reduction assays. In contrast to its stimulatory effect on IKv, Trx (8 nM) did not produce significant changes in the peak values of IA, recorded for 20 min after intracellular administration of Trx. (Fig. 4, A and B). The same concentration of Trx substantially increased total K+ current, activated by a depolarizing pulse to +10 mV from the holding membrane potential of –80 mV (Fig. 4C, top). Subtraction of the control recording from the Trx data revealed that the Trx-sensitive K+ current is a slow-activating current that does not exhibit any obvious inactivation during a 100-ms depolarizing pulse (Fig. 4C, bottom). The total K+ current density, measured at 50 ms from the onset of the test pulse and therefore reflecting predominantly IKv amplitude, revealed that Trx (8 nM) produced a significant increase in IKv (Fig. 4D).
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-carbon atoms (3, 19, 20, 35), on Trx-stimulated neuronal IKv. First we tested the effects of 800 nM PMX 464, injected intracellularly as described in MATERIALS AND METHODS, on basal neuronal IKv. As shown in Fig. 5A, PMX 464 alone at this concentration elicited a minor decrease in basal neuronal IKv, while its solvent (0.005% DMSO) had no effect on basal IKv (31.42 ± 1.26 to 31.41 ± 3.30 pA/pF; n = 7, P = 0.9959). Since 800 nM PMX 464 decreased the insulin-reducing ability of Trx (an indicator of TPOR activity) by 80.73%, these data suggest that inhibition of the TPOR activity of endogenous Trx by PMX 464 can elicit a reduction in neuronal IKv. This supports the idea that the effects of Trx on neuronal IKv are mediated via its TPOR activity. Next we tested the effects of 800 nM PMX 464 on the increase in neuronal IKv produced by 8 nM Trx. Trx and PMX 464 were premixed in pipette solution, in order to decrease the TPOR activity of Trx. Intracellular application of this mixture did not alter neuronal IKv, in contrast to the stimulatory action of Trx alone in a separate group of neurons (Fig. 5B). Inclusion of the PMX 464 solvent (0.005% DMSO) within the pipette solution did not alter the effects of Trx on neuronal IKv (30.95 ± 1.30 to 38.59 ± 1.72 pA/pF; n = 6, P < 0.01). Thus the data shown in Fig. 5B provide further support for the idea that the effects of Trx on IKv are mediated via its TPOR activity.
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Having demonstrated that the stimulatory action of Trx on neuronal IKv appears to involve a TPOR mechanism, we next tested whether this action of Trx involves scavenging of ROS, analogous to the actions of MIF on this K+ current (17). The strategy used here was to test the effect of Tiron, a cell-permeant low-molecular-weight phenolic compound and scavenger of intracellular superoxide anions (11, 36), on the Trx-induced increase in neuronal IKv. Neuronal cultures were pretreated with Tiron (1 mM) for 30 min, conditions that have been used previously to scavenge superoxides in neurons in culture (23). The Tiron pretreatment was followed by recordings of IKv before and after intracellular application of Trx (8 nM). The data presented in Fig. 5D demonstrate that under these experimental conditions Trx failed to produce any changes in IKv in the Tiron-pretreated neurons. Thus the data demonstrate that a superoxide scavenger can prevent the stimulatory action of Trx on neuronal IKv.
ANG II increases expression of Trx in neuronal cultures. ANG II elicits an increase in the expression of MIF mRNA and protein in hypothalamus-brain stem neuronal cultures from SD rats (4, 28). Using similar neuronal cultures, we examined whether ANG II (1 µM) increases the expression of Trx protein and mRNA. As demonstrated in Fig. 6A, ANG II increased Trx mRNA expression within 0.5 h. In addition, ANG II produced a time-dependent increase in Trx protein expression, with a maximal effect at 5 h after incubation (Fig. 6B).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The rationales for focusing on Trx in the present study were as follows. First, Trx exhibits both TPOR and antioxidant activity similar to MIF, and may exhibit the same effect on neuronal IKv as MIF. Second, both Trx and Trx reductase are located in many tissues including the brain, and in situ hybridization studies have revealed that Trx mRNA is highly expressed in certain regions that are involved in neuroendocrine and/or cardiovascular control, such as the PVN of the hypothalamus and the nucleus of the solitary tract (14). Thus if Trx serves as a negative regulator of ANG II, it may play a pivotal role in controlling sympathetic outflow and cardiovascular control. Third, in general, intracellular concentrations of Trx in several tissues are 100- to 1,000-fold less than those of glutathione (GSH), another common antioxidant defense (24). However, it is likely that the antioxidant role of Trx is more significant in neurons because the level of GSH is relatively low in these cells (21).
The mechanism of Trx action in modulating basal neuronal IKv appears to involve the intrinsic TPOR activity of the Trx molecule, which is mediated via its -Cys-X-X-Cys- motif at residues 32–35. We conclude this because the effect of Trx on basal neuronal IKv was abolished by simultaneous application of PMX 464, which attaches to the aforementioned cysteine residues and specifically inhibits the TPOR activity of Trx (3, 19, 20). The effect of Trx on IKv was also abolished by pretreatment with Tiron, which scavenges superoxide anions, indicating that scavenging or sensing of ROS may be critical to this Trx action. In addition, the rhC32S/C35S-Trx TPOR-negative mutant did not alter neuronal IKv. The data from the insulin reduction assay support the results of the patch-clamp experiments, strongly suggesting that Trx is stimulating IKv via its intrinsic TPOR activity.
On the basis of these data we speculate that Trx scavenges ROS via its TPOR activity and that this may be a significant mechanism for increasing IKv, since ROS can modulate the activity of membrane ion channels. For example, it has been demonstrated that the fast activation of certain IKv is mediated by oxidative processes (1), and also that ROS can alter K+ channel activity (10). Interestingly, this proposed mechanism of action of Trx is similar to that described for the stimulatory action of MIF on neuronal IKv (17). Despite the above-described findings, other possible mechanisms of action of Trx should also be considered. For example, Trx may reduce another protein disulfide, and that protein may directly modulate K+ channels and thus influence K+ current. A further possibility is that Prxs may serve as an effector of Trx to scavenge ROS instead of Trx itself. The expression of Prxs II-V has been observed in the cytoplasm of mouse neurons (7). If rat neurons also contain Prxs, there is a possibility that intracellular application of Trx may reduce the intracellular Prxs, and that the reduced Prxs in turn may scavenge ROS. Future electrophysiological recordings will assess the role of Prxs in the actions of Trx on basal neuronal IKv.
The mechanisms by which Trx at low concentrations inhibits ANG II-induced increases in neuronal IKv are not known but may also involve the above-described proposed TPOR/ROS mechanisms. We are proposing that this is the case because low levels of MIF, which do not alter basal neuronal firing, abolish the neuronal chronotropic actions of ANG II via a TPOR/ROS-scavenging mechanism (28). Thus the same may be true for Trx; at the low concentrations used it does not alter basal IKv but still may retain a sufficient level of TPOR activity and ROS-scavenging capability to inhibit the actions of ANG II, which depend on ROS generation (29).
Even though it has been shown that ANG II increases the expression of Trx protein in peripheral blood mononuclear cells from Wistar-Kyoto rats (31), the present studies are the first to demonstrate that ANG II increases Trx expression in neurons. The promoter region of the Trx gene contains many possible regulatory binding motifs compatible with inducible expression, including AP-1, AP-2, and NF-
B, etc. (8), and also has an antioxidant response element (16, 30). Activation of neuronal ANG II receptors results in the modulation of multiple intracellular signaling pathways (27), including the induction of AP-1 (6). Thus AP-1 will be a primary target in our future studies on the mechanisms of ANG II-induced Trx production.
In this study, 0.08–80 nM of Trx had concentration-dependent stimulatory effects on neuronal IKv. These levels of Trx may, at first glance, appear insignificant since the intracellular concentration of endogenous Trx is roughly in the range of 1–15 µM (24) and the intracellular content of another major antioxidant, GSH, is in the millimolar range (1–11 mM) in many other tissues (24). It is imperative, however, to recall that physiological redox status is a very delicate balance between the formation and the elimination of ROS. Many enzyme systems, including NAD(P)H oxidase, xanthine oxidase, and myeloperoxidase, produce ROS under normal conditions. On the other hand, many enzyme systems, including superoxide dismutase, the GSH system, the Trx system, and MIF scavenge ROS. The plethora of recent literature demonstrating that low amounts of various ROS have physiologically relevant functions, rather than pathological consequences, in cellular signaling indicates that even a small perturbation of the redox balance in the intracellular environment can be meaningful. Therefore, we speculate that, even at this small concentration, Trx may serve to produce enough increase in the total amount of ROS scavenging systems to tip the redox balance of the local intracellular environment.
To summarize, the present data provide in vitro evidence that Trx acts intracellularly to produce a specific modulatory action on one of the K+ currents that is the basis of the neuronal action potential and thus neuronal firing. Furthermore, this action of Trx likely involves a TPOR/ROS mechanism. However, this work raises some broader issues. The first is the issue of whether such actions of Trx on basal and ANG II-modulated K+ current occur in vivo in rat brain and contribute to regulation of the physiological actions of ANG II, similar to MIF (13). The second issue is whether TPOR may be a general mechanism for regulating neuronal activity and the actions of ANG II. The third issue is whether Trx and MIF, acting via similar TPOR mechanisms, are members of a novel class of intracellular inhibitors of ANG II action in neurons.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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