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Originally published In Press as doi:10.1074/jbc.M301181200 on August 17, 2003

J. Biol. Chem., Vol. 278, Issue 43, 41685-41690, October 24, 2003
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Vasopressin Type 1A Receptor Up-regulation by Cyclosporin A in Vascular Smooth Muscle Cells Is Mediated by Superoxide*

Alexandra Krauskopf{ddagger}**, Philippe Lhote{ddagger}, Manfred Mutter§, Jean-François Dufour¶, Urs T. Ruegg{ddagger}||, and Timo M. Buetler{ddagger}

From the {ddagger}Pharmacology Group, School of Pharmacy, University of Lausanne, 1015 Lausanne, Switzerland, §Institute of Molecular and Biological Chemistry, Institute of Molecular and Biological Chemistry-Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland, and Department of Clinical Pharmacology, University of Bern, Murtenstrasse 35, 3010 Bern, Switzerland

Received for publication, February 4, 2003 , and in revised form, August 15, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Based on our previous results, we investigated whether cyclosporin A (CsA)-induced vasopressin type 1A receptor up-regulation was mediated by free radicals. We report that CsA analogues with different affinities for cyclophilin and calcineurin were able to up-regulate vasopressin type 1A receptor and to generate free radicals in smooth muscle cells independently of calcineurin. Further, we demonstrate that the antioxidant N-acetyl-L-cysteine blocked the increase in vasopressin type 1A receptor mRNA and protein levels induced by CsA and that low concentrations of prooxidants were able to directly increase vasopressin type 1A receptor mRNA and protein levels. In addition, short exposure to CsA or pro-oxidants was sufficient to significantly increase vasopressin type 1A receptor mRNA and protein levels. Using cell-permeable forms of superoxide dismutase and catalase, we finally show that superoxide mediates the CsA-induced effects on vasopressin type 1A receptor. These results provide strong evidence that CsA-induced superoxide generation is causally involved in vasopressin type 1A receptor expression and demonstrate for the first time that low physiological concentrations of radicals, most probably superoxide, are able to directly affect cellular signaling to increase vasopressin type 1A receptor expression in rat aortic smooth muscle cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cyclic undecapeptide cyclosporin A (CsA)1 is the most widely used immunosuppressive drug to prevent transplant rejection and in the therapy of autoimmune diseases (1). CsA acts by binding to cyclophilin to inhibit calcineurin phosphatase activity, NF-AT dephosphorylation, and interleukin-2 expression, thus preventing T-lymphocyte proliferation (1–3).

The use of CsA is accompanied by mild to severe side effects, and the clinically most important are nephrotoxicity and hypertension (4–6). Both are likely caused by CsA-induced local vasoconstriction (7, 8). We have shown previously (9, 10) that CsA caused an elevation in cytosolic free calcium concentrations in rat and human aortic smooth muscle cells and enhanced vasoconstriction of rat aortic smooth muscle cells (RASMC) when these were stimulated with vasoconstrictor hormones such as endothelin-1, serotonin, angiotensin II, and vasopressin. At least for vasopressin and angiotensin II, the expression of their respective cell surface receptors was shown to be increased by CsA in rat and human aortic smooth muscle cells (10–12). Recently, we have shown that CsA up-regulated the vasopressin type 1A (V1A) receptor via an increase in the corresponding mRNA levels in RASMC (13). Up-regulation of vasoconstrictor hormone receptors may be responsible for the enhanced vasoconstriction under in vivo conditions, thus leading, via an increase in peripheral resistance, to hypertension and to a decrease in glomerular filtration (4–6). However, the exact mechanisms by which CsA enhances vasoconstriction have not yet been clarified.

Several lines of evidence point to a possible role of reactive oxygen species (ROS) as mediators leading to the side effects of CsA (14–17). Some studies have shown that CsA is able to produce ROS in vascular endothelial and mesangial cells (16, 18, 19). In addition, we have shown that CsA generates ROS in RASMC that were inhibited by antioxidants (20). ROS have been assigned a role of biological mediators in cellular signaling (21–23). For example, it has been shown that platelet-derived growth factor-induced cell proliferation was dependent on the cellular production of H2O2 (24). In addition, it has been demonstrated that ROS are able to activate several transcription factors, such as nuclear factor-{kappa}B and activated protein-1 (25–28). Recent studies have illustrated that not only H2O2 but also superoxide is involved in cellular signaling (22, 29–32). Thus, ROS seem to play a role as activators of signaling pathways leading to altered gene expression.

Using CsA analogues with different capacities to bind cyclophilin and calcineurin we demonstrate that CsA generates ROS and up-regulates V1A receptor in RASMC independently of cyclophilin and calcineurin. Further, we report that CsA generates superoxide and that it is likely the superoxide anion radical that is able to directly increase V1A receptor expression at the mRNA and protein levels.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Buffers—CsA, cyclosporin H (CsH), and PSC 833 were gifts from Novartis Pharma (Basel, Switzerland), and CsA analogues (cyclosporin C (CsC), [EtLeu4]CsA, [MeVal4]CsA, [EtVal4]CsA, [MeIle4]CsA, and [MeAla3EtVal4]CsA) were synthesized in the laboratory of M. Mutter. Fetal calf serum, oligo(dT) 12–18 primer, Moloney murine leukemia virus reverse transcriptase, dithiothreitol, and first strand buffer were from Invitrogen. Ciproxin was purchased from Bayer Pharma AG (Zürich, Switzerland), and 2',7'-dichlorofluorescin diacetate (2',7'-DCFH) was from Molecular Probes. Dulbecco's modified Eagle's medium (DMEM), polyethylene glycol-coupled bovine erythrocyte superoxide dismutase (PEG-SOD), polyethylene glycol-coupled bovine liver catalase (PEG-CAT), hydrogen peroxide, hypoxanthine, xanthine oxidase, and N-acetyl-L-cysteine were from Sigma, and tert-butylhydroperoxide was from Fluka (Buchs, Switzerland). Dimethylnaphthoquinone was purchased from Alexis Corporation (Läufelfingen, Switzerland), and {alpha}-tocopherol was from Calbiochem. [3H][Arg8]vasopressin ([3H]AVP) was obtained from PerkinElmer Life Sciences, and [Arg8]vasopressin (AVP) was from Bachem (Bubendorf, Switzerland). RNeasy Mini Kit, Qiashredder columns, and Taq DNA polymerase were from Qiagen. RQ1 RNase-free DNase and dNTPs were purchased from Promega. TaqMan probes and 2xTaqMan Universal PCR Master Mix were from PerkinElmer Life Sciences. Primers were custom synthesized by Qiagen. Stock solutions of chemicals were prepared in ethanol, buffer, or Me2SO. CsA stock solutions were prepared in ethanol at a concentration of 10 mM and diluted to either 1 or 10 µM for experiments, thus resulting in maximal final EtOH concentration of 0.1%. Fresh CsA stock solutions were prepared every week. In all experiments, 0.1% EtOH or Me2SO served as control and were set at 100%. The physiological salt solution contained 145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.2 mM CaCl2, 5mM Hepes, and 10 mM glucose, adjusted to pH 7.6.

Cultures of Smooth Muscle Cells—RASMC were prepared from aortae of male Wistar Kyoto rats (200–300 g) as described (9). RASMC were cultured in DMEM supplemented with essential and non-essential amino acids, vitamins, 10 µg/ml ciproxin, and 10% fetal calf serum and kept at 37 °C in a humidified atmosphere of 5% CO2 in air. For studies with CsA, cells were used at confluence (after 7 to 9 days of culture) between passages 6 and 11. Twenty-four h before experiments, culture media were replaced with fresh DMEM without fetal calf serum. Treatment of RASMC with CsA and other agents was always performed in serum-free DMEM.

[3H]AVP Binding—Binding experiments were carried out as described (13). Results are expressed in percent of control to normalize for inter-experimental variations. Control values ranged from 0.5 to 4.2 fmol/well in different experiments, and the intra-experimental variability was less than 5%.

Measurements of ROS by 2', 7'-DCFH—ROS measurements were performed as outlined in Ref. 20. Results are expressed in percent of control to normalize for inter-experimental variations. Because of the variability between experiments, the gain applied to the photomultiplier tube was adjusted before each experiment.

Isolation of Total RNA, DNase Treatment, and Reverse Transcription—Total RNA from RASMC cultured in 6-well plates was isolated using a RNeasy Mini Kit according to the manufacturer's instructions. All RNAs were quantified by spectrophotometrical determination of the absorption at 260 nm. The RNA integrity was assessed by electrophoresis in non-denaturing 1.2% agarose gels stained with ethidium bromide. DNase treatment and reverse transcription were executed as described (13).

Real-time Quantitative PCR Analysis—Real-time quantitative PCR analysis was performed with a PerkinElmer Life Sciences 7700 Sequence Detector (PerkinElmer Life Sciences) as illustrated (13). Results were normalized for GAPDH expression for each sample. Results are expressed in percent of control to normalize for inter-experimental variations. V1A receptor/GAPDH ratios of controls varied from 0.51 to 1.04 in different experiments, and the intra-experimental variability was less than 5%.

Data Analysis—Results are presented as the means of at least three independent experiments with bars indicating S.E. Statistical evaluation was performed by means of one-way analysis of variance followed by Newman Keuls or Dunnett post-tests using the software lnPlotPrism (GraphPad Software, San Diego, CA). Differences with a value of p < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CsA and Analogues Up-regulate the V1A Receptor—To investigate whether CsA depended on its binding to cyclophilin and/or calcineurin to up-regulate V1A receptor, we tested whether CsA analogues with differential affinities for cyclophilin and/or calcineurin affected V1A receptor expression. Like CsA, the analogues CsC and [EtLeu4]CsA bind both to cyclophilin and calcineurin. [MeVal4]CsA, [EtVal4]CsA, [MeIle4]-CsA, and [MeAla3EtVal4]CsA do not bind to calcineurin but have a high binding affinity for cyclophilin. The analogues CsH and PSC 833 bind to neither cyclophilin nor calcineurin. Fig. 1 illustrates that all analogues (tested at 1 µM) were able to significantly increase V1A receptor expression in RASMC after a 20-h pretreatment at 37 °C. This response was of similar magnitude as for CsA (about 3-fold), thus suggesting that CsA-induced V1A receptor up-regulation occurred independently of cyclophilin and/or calcineurin inhibition.



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  		FIG. 1.
Effect of CsA analogues on [3H]AVP binding. [3H]AVP binding was measured after a 20-h treatment at 37 °C of RASMC with CsA or analogues (1 µM). Dark gray bars represent CsA analogues that bind to both cyclophilin and calcineurin, light gray bars represent CsA analogues that only bind to cyclophilin but not to calcineurin, and white bars represent CsA analogues that bind neither to cyclophilin nor calcineurin. Data are means ± S.E. of at least five independent experiments done in triplicate. Asterisks indicate values significantly different from control at p < 0.001 (***).

 

ROS Formation by CsA Analogues in RASMC—As V1A receptor up-regulation appears to occur independently of the calcineurin/NF-AT pathway, we investigated whether ROS could be involved. To assess whether CsA requires cyclophilin and/or calcineurin for the generation of ROS, we tested whether analogues of CsA would be able to generate ROS. Because the oxidation of 2', 7'-DCFH has been used in many laboratories to detect cellular radical formation (33–36), we used this approach to measure ROS in RASMC. Cells were treated with CsA or the CsA analogues tested for V1A receptor up-regulation and 2', 7'-dichlorofluorescein (DCF) fluorescence was measured after 1 h. Fig. 2 demonstrates that all analogues were able to produce ROS. From these results, we conclude that CsA and analogues generate ROS independently of their ability to bind to cyclophilin and/or calcineurin. In experiments that are not shown, we verified that ROS formation by CsA analogues were blocked by antioxidants.



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  		FIG. 2.
ROS formation by CsA and analogues. RASMC were treated with CsA or analogues (10 µM) for 1 h at 37 °C, and DCF-fluorescence was measured. Dark gray bars represent CsA analogues that bind to both cyclophilin and calcineurin, light gray bars represent CsA analogues that only bind to cyclophilin but not to calcineurin, and white bars represent CsA analogues that bind neither to cyclophilin nor calcineurin. Values represent means ± S.E. of at least five independent experiments done in triplicate or quadruplicate. Asterisks indicate values significantly different from control at p < 0.01 (**) or at p < 0.001 (***).

 

Effect of Antioxidants on CsA-induced Increase in V1A Receptor mRNA and Protein Levels in RASMC—As CsA and all analogues tested generated ROS and increased V1A receptor expression, we tested whether the CsA-induced increase in V1A receptor expression could be blocked by antioxidants. Because it is known that several transcription factors are regulated by changes in the redox balance, the two antioxidants N-acetyl-L-cysteine (NAC) and {alpha}-tocopherol were tested on CsA-induced increase in V1A receptor mRNA and protein levels. Fig. 3 reveals that NAC was not only able to block the increase in V1A receptor mRNA levels induced by CsA after a 6-h treatment (A), but this antioxidant also decreased CsA-induced V1A receptor protein up-regulation after a 20-h treatment (B). The apparently higher levels of [3H]AVP binding in cells treated with CsA and NAC were statistically not different from control. Co-treatment of RASMC with CsA and {alpha}-tocopherol was equally effective in blocking CsA-induced V1A receptor up-regulation (data not shown).



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  		FIG. 3.
Effect of antioxidants on CsA-mediated increase in V1A receptor mRNA (A) and protein (B) levels. A, RASMC were exposed to CsA (1 or 10 µM) alone or in the presence of NAC during 6 h at 37 °C. V1A receptor mRNA levels were measured by real-time quantitative PCR and were normalized to GAPDH mRNA levels. B, RASMC were treated with CsA (1 or 10 µM) in the presence or absence of NAC for 20 h at 37 °C, and [3H]AVP binding was measured at the end. Results are expressed as the means ± S.E. of at least three independent experiments performed in triplicate or quadruplicate. Asterisks indicate values significantly different from CsA alone (1 or 10 µM) at p < 0.01 (**) or at p < 0.001 (***).

 

Effect of Pro-oxidants on V1A Receptor mRNA and Protein Levels—The fact that antioxidants were able to block CsA-mediated increase in V1A receptor mRNA and protein levels suggests that CsA-induced ROS generation may be responsible for this effect. To address this question, RASMC were treated with the peroxides hydrogen peroxide (H2O2) or tert-butylhydroperoxide (BHP) or the superoxide-generating systems dimethylnaphthoquinone (DMNQ) or hypoxanthine (HX)/xanthine oxidase (XO). V1A receptor mRNA expression was determined 6 h after treatment, and V1A receptor protein expression was determined 20 h after treatment. As shown in Fig. 4, all pro-oxidants were able to significantly increase V1A receptor mRNA (A) and V1A receptor protein (B) levels in RASMC.



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  		FIG. 4.
Effect of pro-oxidants on V1A receptor mRNA (A) and protein (B) levels. A, RASMC were treated with CsA (3 µM) or the pro-oxidants H2O2 (30 µM), BHP (3 µM), DMNQ (10 nM), or HX (10 µM)/XO (1 milliunit/ml) during 6 h at 37 °C. V1A receptor mRNA levels were determined by quantitative real-time PCR and were normalized to GAPDH mRNA levels. B, analysis of [3H]AVP binding after 20 h of treatment was performed with CsA (1 µM) or the same pro-oxidants as under A was measured. Results are means ± S.E. of at least three independent experiments done in triplicate or quadruplicate. Asterisks indicate values significantly different from control at p < 0.01 (**) or at p < 0.001 (***).

 

Effect of Incubation Time on CsA- or Pro-oxidants-induced V1A Receptor Expression—The number of V1A receptor was earlier found to be maximal after 20 h of treatment even though cell surface receptor expression was already significantly elevated after 8–12 h (13). In addition, we have shown that the effect of CsA on receptor protein expression was preceded by an increase in V1A receptor mRNA levels starting at 2 h and peaking at 6–10 h after CsA addition (13). These data suggest that shorter times of CsA exposure may be sufficient to induce V1A receptor up-regulation. If ROS are the critical mediators of this response, it is questionable whether ROS are produced for the full 20 h. Our data have shown that CsA-induced ROS generation was increased within the first hour, peaking at 45 min, followed by a steady decline up to 2 h (20). Additional investigations have shown that after 20 h CsA does no longer generate any detectable levels of ROS when incubated with 2', 7'-DCFH for the last hour (data not shown). Also, the pro-oxidants used in Fig. 4 very likely produced ROS only for a limited time before either the substrate was exhausted (HX), or the chemical was inactivated (H2O2, BHP, DMNQ). Therefore, we investigated whether short exposure of RASMC to CsA or pro-oxidants was sufficient to trigger V1A receptor up-regulation detected after 20 h. For this, cells were incubated with either CsA or DMNQ or HX/XO for 30 min or 1 h. Then the compounds were removed, and the cells were incubated in medium without compounds for a total time of 20 h before [3H]AVP binding was measured. Fig. 5 shows that exposure of RASMC to CsA or pro-oxidants for as little as 30 min was sufficient to significantly up-regulate V1A receptors after 20 h.



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  		FIG. 5.
Effect of CsA and pro-oxidant exposure time on V1A receptor expression. RASMC were exposed at 37 °C to CsA (10 µM)or the pro-oxidants DMNQ (10 nM) or HX (10 µM)/XO (1 milliunit/ml) for 30 min, 1 h, or 20 h. [3H]AVP binding was measured after 20 h (for details see text). Data are means ± S.E. of at least three independent experiments performed in triplicate. Asterisks indicate values significantly different from control at p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).

 

The Effect of PEG-SOD or PEG-CAT on CsA-induced ROS Formation and V1A Receptor Up-regulation—To test what kind of radical was generated by CsA and served as mediator of CsA-induced V1A receptor expression, we examined the effects of PEG-SOD that dismutates superoxide to hydrogen peroxide and PEG-CAT that reduces hydrogen peroxide to water (37, 38). RASMC were pretreated for 6 h with PEG-SOD (100 units/ml) or PEG-CAT (50 units/ml), and DCF-fluorescence and [3H]AVP binding were determined in the presence or absence of CsA as described under "Experimental Procedures." Fig. 6A shows that PEG-SOD increased the CsA-induced DCF-signal whereas PEG-CAT decreased this signal. On the other hand, PEG-SOD decreased CsA-mediated V1A receptor up-regulation whereas PEG-CAT had no effect (Fig. 6B). These results imply that CsA generated and that , but not H2O2, acted as mediator in V1A receptor up-regulation. Neither PEG-SOD nor PEG-CAT alone had a significant effect on basal ROS formation or [3H]AVP binding (data not shown).



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  		FIG. 6.
Effect of PEG-SOD and PEG-CAT on CsA-mediated increase in ROS generation (A) and [3H]AVP binding (B). RASMC were pretreated for 6 h at 37 °C with PEG-SOD (100 units/ml) or PEG-CAT (50 units/ml) in culture medium without serum. A, the medium was changed with medium containing CsA (1 µM) but no PEG-SOD or PEG-CAT, and DCF-fluorescence was determined 1 h later. B, the medium was changed with medium containing CsA (1 µM) but no PEG-SOD or PEG-CAT, and [3H]AVP binding was measured 24 h later. Data are means ± S.E. of at least four independent experiments performed in triplicate. Asterisks indicate values significantly different from CsA at p < 0.01 (**) or at p < 0.001 (***).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we provide evidence that superoxide serves as mediator in CsA-induced increase in V1A receptor mRNA and protein expression whereas the contribution of the calcineurin/NF-AT pathway in this effect is negligible. In antigen-triggered receptor signaling, calcium release will activate calcineurin to dephosphorylate NF-AT, which will translocate into the nucleus and induce transcription of genes such as interleukin-2. To exert its immunosuppressive activity, CsA has to bind to cyclophilin, and this complex will inhibit the protein phosphatase activity of calcineurin and consequently prevent the dephosphorylation of NF-AT and interleukin-2 expression (1).

We have shown earlier that 1 µM CsA was able to increase the number of V1A cell surface receptors (12) via an increase in the corresponding mRNA levels (13). This effect could be mediated by transcriptional up-regulation of the V1A receptor promoter involving the calcineurin/NF-AT pathway. Calcineurin has also been shown to play an important role in the regulation of other transcription factors, including NF-{kappa}B (39), Jun (40, 41), cAMP-response element-binding protein (42), Elk-1 (43), and others. Therefore, we studied whether CsA-induced V1A receptor up-regulation depended on calcineurin using CsA analogues with different affinities for cyclophilin and calcineurin. Our results demonstrate that all CsA analogues tested were able to increase [3H]AVP binding independently of their ability to bind to cyclophilin and/or calcineurin. In addition, as calcineurin is activated by calcium, sequestering of cytosolic calcium with the cell-permeable calcium chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester (BAPTA/AM) would inhibit calcineurin-dependent signaling. In independent experiments, we saw that treatment of RASMC with 10 µM BAPTA/AM was not able to block CsA-induced [3H]AVP binding (data not shown). Together, these results suggest that a role of calcineurin in CsA-induced V1A receptor up-regulation is highly unlikely.

In previous work we have shown that 1 µM CsA (representing peak plasma CsA concentrations encountered in CsA-treated patients (44)) produced significant amounts of ROS in RASMC that could be blocked by antioxidants (20). Another study has also found that 1 µM CsA generated ROS in cardiomyocytes (45). Small amounts of ROS are indispensable for many biochemical processes (23, 46). In recent years, considerable evidence has accumulated supporting a role for ROS as activators of signaling pathways and transcription factors (22, 28, 46). When the balance between pro- and antioxidants is disturbed in favor of the former, a situation of oxidative stress ensues (23). It is now well established that oxidative stress is involved in the pathogenesis of numerous diseases (22).

Because CsA-induced V1A receptor up-regulation occurred independently of calcineurin, our next goal was to elucidate whether the generation of ROS by CsA is implicated in the mechanisms leading to CsA-induced vasoconstriction, because oxidative stress has been proposed as a causative factor in the toxic side effects of CsA (14–16, 47). Enhanced superoxide () generation could significantly contribute to CsA-induced hypertension, because inactivates the important vasodilator nitric oxide. Diederich et al. (18) have shown that CsA treatment impairs nitric oxide-mediated relaxation in mesenteric resistance arteries of rats suggesting that increased formation could be responsible for the decrease in nitric oxide (48).

Our focus was to first investigate whether CsA-induced ROS formation could lead to V1A receptor up-regulation. Because all CsA analogues increased [3H]AVP binding, we tested whether they were able to produce ROS. Our results show that all CsA analogues tested were indeed able to generate significant amounts of ROS, independently of cyclophilin and/or calcineurin binding. Even if the relative fluorescence intensity varied between the analogues, ROS formation by CsA appears to be independent from cyclophilin or calcineurin. The small differences in fluorescence intensity could be related to the chemical structure of the CsA analogues. Because ROS can interfere directly (49) or indirectly via calmodulin kinase II (50) with calcineurin signaling it may be possible that ROS generated by CsA analogues could still act via calcineurin signaling. However, these two studies used considerably higher concentrations of pro-oxidants compared with this study. In fact, in additional experiments using DMNQ to inhibit calcineurin in an interleukin-2 reporter gene assay (51) concentrations in excess of 1 µM were needed to inhibit calcineurin signaling (IC50 = 6 µM) as compared with 10 nM DMNQ to increase V1A receptor expression (data not shown). These results suggest that ROS could serve as mediators in CsA-induced V1A receptor up-regulation but act independently of calcineurin.

To further investigate whether ROS are involved in CsA-induced vasoconstriction, the effect of antioxidants was assessed on CsA-mediated increase in V1A receptor at the mRNA and protein levels. Our findings demonstrate that NAC blocked the increase in V1A receptor mRNA and protein levels induced by CsA. Similar data were obtained with {alpha}-tocopherol (data not shown). Taken together, these data further support a causal role of ROS in CsA-mediated increase in V1A receptor mRNA and protein levels. It is interesting to note that both hydro- and lipophilic antioxidants were able to block the CsA-induced V1A receptor up-regulation suggesting a possible membrane component in CsA-induced ROS signaling. It should also be noted that it is unlikely that CsA can react to form radicals directly.

Our results of the effect of ROS on V1A receptor mRNA and protein levels suggest that donors were more potent than the peroxides. To address the question whether superoxide could be the mediator of CsA-induced V1A receptor expression, we used PEG coupled forms of SOD and CAT that penetrate the cells. First, we investigated the effect of PEG-SOD and PEG-CAT on CsA-induced ROS generation. Because DCFH reacts only with H2O2 in a cellular system but not with O2., superoxide generation can only be detected indirectly with this agent (36). If CsA was able to generate it would be expected that PEG-SOD would increase whereas PEG-CAT would decrease the CsA-induced DCF-signal that was indeed observed (Fig. 6A). The apparent increase in DCF fluorescence with PEG-SOD treatment suggests that the dismutation reaction was the rate-limiting step. It is interesting that this occurs despite the presence of large amounts of endogenous SOD in the cells and rapid spontaneous superoxide dismutation rates. Alternatively, it may be considered that excess SOD diverts superoxide from other redox reactions (for instance with nitrogen monoxide to form peroxynitrite), but we suggest that nitrogen monoxide and peroxynitrite do not play a role in CsA-induced DCFH oxidation.2

Second, if superoxide mediated the CsA-induced V1A receptor up-regulation, it would be expected that PEG-SOD would decrease whereas PEG-CAT would not affect the CsA effect. Our results confirm this notion and suggest that superoxide but not H2O2 was responsible for the CsA effect on V1A receptor up-regulation. In line with these results are additional data using the superoxide scavengers 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (1 mM) and coelenterazine (10 µM), which both decreased CsA-induced [3H]AVP binding in RASMC (data not shown). Consistently, additional data showed that the SOD inhibitor diethyldithiocarbamate enhanced both basal and CsA-induced [3H]AVP binding in RASMC but blocked CsA-induced DCF fluorescence (data not shown). These results suggest that (i) there is enough formed, perhaps in a restricted local environment, to directly induce or to interfere with downstream events, (ii) that the dismutation reaction appears to be the rate-limiting step in this process, and (iii) that the signaling function of must be very rapid to outcompete the dismutation reaction. Together, these data demonstrate that superoxide, but not H2O2, is the most likely ROS generated by CsA to up-regulate V1A receptor expression.

The effective concentrations produced in this study are low. To serve as signaling molecule, levels must be kept low, and in biological systems, SODs are present for this purpose. Superoxide alone is not very reactive and in biological systems is rapidly dismutated to H2O2 by the superoxide dismutases with a rate constant of 2 x 109 mol–1·s–1 (52). This ensures that a signal is transient and of short duration. Under normal conditions, H2O2 is removed by at least three enzyme systems, catalase, glutathione peroxidase, and peroxiredoxin (53, 54). In the presence of metal ions such as Fe2+ or Cu+, H2O2 can be transformed into hydroxyl radicals via the Fenton reaction (55). The highly reactive hydroxyl radical reacts with any biomolecules in its vicinity to cause cellular damage. However, hydroxyl radicals can also react with H2O2 to give rise to , and traces of Fe3+ can also react with H2O2 to reform (56).

Of the two radical species H2O2 and , mostly H2O2 has been shown to have biological effects. For example, H2O2 has been reported to activate several subtypes of the mitogen-activated protein kinases in aortic smooth muscle cells (57–59). Further, it has been shown that H2O2 stimulates c-jun and c-fos mRNA expression in RASMC (60, 61) and activates NF-{kappa}B in a number of cell types (26, 27). In comparison, the effects of on cellular signaling have been less investigated. A recent study demonstrated that DMNQ was able to induce phosphorylation of extracellular signal-related protein kinase, suggesting a direct effect of (32).

is dismutated to H2O2, but as discussed above, under certain conditions can also be formed from H2O2. Our data suggest that H2O2 is not able to mediate the CsA-induced up-regulation of the V1A receptor, because PEG-CAT was unable to influence the CsA effect (Fig. 6B). Because our results suggest that seems to be the mediator of the CsA-effect, it is likely that in our system, H2O2. is transformed into to up-regulate the V1A receptor. However, it may also be possible that both superoxide and peroxides could mediate this end point under different experimental conditions. More research is needed to investigate the possibility that also mediates the effect of H2O2. In addition, it should be noted that the concentration of H2O2 used in our study (30 µM) is in the lower range of concentrations (10 to 1000 µM) used by others (22, 23). It is unlikely that a cell will generate micromolar or even millimolar concentrations of H2O2 as second messenger, but it appears more practical to use the primary radical () and not a derivative thereof as a second messenger. Unfortunately, most studies using H2O2 have not investigated whether the observed effect could have been mediated by . Based on these considerations, in many cases the role of H2O2 as a direct mediator in cellular signaling may have to be reconsidered, and we suggest that in the future pure generators, together with specific inhibitors (PEG-SOD and PEG-CAT), should be tested in parallel with H2O2 to clarify their respective contribution to cellular signaling.

In summary, our results demonstrate that CsA generates superoxide and up-regulates V1A receptor independently of binding to cyclophilin and of its immunosuppressive activity. Further, we show for the first time that is most probably directly mediating the increase in V1A receptor mRNA and protein levels in RASMC.


   FOOTNOTES
 
* This work was supported by grants from the Swiss National Science Foundation (31-68315.02 and 3100A0-100513), the Roche Research Foundation, and the Fondation Herbette of the University of Lausanne. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** Current address: Department of Biomedical Sciences, University of Padova, 1-35121 Padova, Italy. Back

|| To whom correspondence should be addressed. Tel.: 4121-692-4531; Fax: 4121-692-4515; E-mail: urs.ruegg{at}dpharm.unil.ch.

1 The abbreviations used are: CsA, cyclosporin A; DCF, 2', 7'-dichlorofluorescein; ROS, reactive oxygen species; RASMC, rat aortic smooth muscle cells, DMEM, Dulbecco's modified Eagle's medium; 2', 7'-DCFH, 2', 7'-dichlorofluorescin diacetate; NAC, N-acetyl-L-cysteine; BHP, tert-butylhydroperoxide; DMNQ, dimethylnaphthoquinone; HX, hypoxanthine; XO, xanthine oxidase; PEG-SOD, polyethylene glycol-coupled superoxide dismutase; PEG-CAT, polyethylene glycol-coupled catalase; V1A, vasopressin type 1A; BAPTA/AM, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester; AVP, [Arg8]vasopressin; NF-AT, nuclear factor of activated T cells. Back

2 A. Krauskopf, P. Lhote, U. T. Ruegg, and T. M. Buetler, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Michael Lüthi (University of Bern, Bern, Switzerland) for help with quantitative PCR. We also thank Drs. Francis Hubler and Jean François Guichou (Swiss Federal Institute of Technology, Lausanne, Switzerland) for the synthesis of the CsA analogues and Drs. Angelo Azzi and Stéphanie Wagner for critically reading this manuscript.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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