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• W1978791874 abstract "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. 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. The cyclic undecapeptide cyclosporin A (CsA) 1The 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.1The 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. is the most widely used immunosuppressive drug to prevent transplant rejection and in the therapy of autoimmune diseases (1Borel J.F. Baumann G. Chapman I. Donatsch P. Fahr A. Mueller E.A. Vigouret J.M. Adv. Pharmacol. 1996; 35: 115-246Crossref PubMed Scopus (137) Google Scholar). CsA acts by binding to cyclophilin to inhibit calcineurin phosphatase activity, NF-AT dephosphorylation, and interleukin-2 expression, thus preventing T-lymphocyte proliferation (1Borel J.F. Baumann G. Chapman I. Donatsch P. Fahr A. Mueller E.A. Vigouret J.M. Adv. Pharmacol. 1996; 35: 115-246Crossref PubMed Scopus (137) Google Scholar, 2Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2203) Google Scholar, 3Aramburu J. Rao A. Klee C.B. Curr. Top. Cell Regul. 2000; 36: 237-295Crossref PubMed Scopus (274) Google Scholar).The use of CsA is accompanied by mild to severe side effects, and the clinically most important are nephrotoxicity and hypertension (4Kahan B.D. New Engl. J. Med. 1989; 321: 1725-1738Crossref PubMed Scopus (1626) Google Scholar, 5Mason J. Pharmacol. Rev. 1990; 41: 423-434PubMed Google Scholar, 6Textor S.C. Canzanello V.J. Taler S.J. Wilson D.J. Schwartz L.L. Augustine J.E. Raymer J.M. Romero J.C. Wiesner R.H. Krom R.A. Mayo Clin. Proc. 1994; 69: 1182-1193Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Both are likely caused by CsA-induced local vasoconstriction (7Lamb F.S. Webb R.C. Life Sci. 1987; 40: 2571-2578Crossref PubMed Scopus (69) Google Scholar, 8Rego A. Vargas R. Suarez K.R. Foegh M.L. Ramwell P.W. J. Pharmacol. Exp. Ther. 1990; 254: 799-808PubMed Google Scholar). We have shown previously (9Lo Russo A. Passaquin A.C. André P. Skutella M. Ruegg U.T. Br. J. Pharmacol. 1996; 118: 885-892Crossref PubMed Scopus (72) Google Scholar, 10Avdonin P.V. Cottet-Maire F. Afanasjeva G.V. Loktionova S.A. Lhote P. Ruegg U.T. Kidney Int. 1999; 55: 2407-2414Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) 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 (10Avdonin P.V. Cottet-Maire F. Afanasjeva G.V. Loktionova S.A. Lhote P. Ruegg U.T. Kidney Int. 1999; 55: 2407-2414Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 11Lo Russo A. Passaquin A.-C. Cox C. Ruegg U.T. J. Recept. Signal Transduct. Res. 1997; 17: 149-161Crossref PubMed Scopus (26) Google Scholar, 12Lo Russo A. Passaquin A.C. Ruegg U.T. Br. J. Pharmacol. 1997; 121: 248-252Crossref PubMed Scopus (18) Google Scholar). Recently, we have shown that CsA up-regulated the vasopressin type 1A (V1A) receptor via an increase in the corresponding mRNA levels in RASMC (13Cottet-Maire F. Avdonin P.V. Roulet E. Buetler T.M. Mermod N. Ruegg U.T. Br. J. Pharmacol. 2001; 132: 909-917Crossref PubMed Scopus (29) Google Scholar). 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 (4Kahan B.D. New Engl. J. Med. 1989; 321: 1725-1738Crossref PubMed Scopus (1626) Google Scholar, 5Mason J. Pharmacol. Rev. 1990; 41: 423-434PubMed Google Scholar, 6Textor S.C. Canzanello V.J. Taler S.J. Wilson D.J. Schwartz L.L. Augustine J.E. Raymer J.M. Romero J.C. Wiesner R.H. Krom R.A. Mayo Clin. Proc. 1994; 69: 1182-1193Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). 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 (14Ahmed S.S. Strobel H.W. Napoli K.L. Grevel J. J. Pharmacol. Exp. Ther. 1993; 265: 1047-1054PubMed Google Scholar, 15Wang C. Salahudeen A.K. Kidney Int. 1995; 47: 927-934Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 16Perez de Lema G. Arribas-Gomez I. Ruiz-Gines J.A. de Arriba G. Prieto A. Rodriguez-Puyol D. Rodriguez-Puyol M. Transplant. Proc. 1997; 29: 1241-1243Crossref PubMed Scopus (26) Google Scholar, 17Lopez-Ongil S. Hernandez-Perera O. Navarro-Antolin J. Perez de Lema G. Rodriguez-Puyol M. Lamas S. Rodriguez-Puyol D. Br. J. Pharmacol. 1998; 124: 447-454Crossref PubMed Scopus (115) Google Scholar). Some studies have shown that CsA is able to produce ROS in vascular endothelial and mesangial cells (16Perez de Lema G. Arribas-Gomez I. Ruiz-Gines J.A. de Arriba G. Prieto A. Rodriguez-Puyol D. Rodriguez-Puyol M. Transplant. Proc. 1997; 29: 1241-1243Crossref PubMed Scopus (26) Google Scholar, 18Diederich D. Skopec J. Diederich A. Dai F.X. Hypertension. 1994; 23: 957-961Crossref PubMed Scopus (111) Google Scholar, 19Galle J. Lehmann-Bodem C. Hubner U. Heinloth A. Wanner C. Nephrol. Dial. Transplant. 2000; 15: 339-346Crossref PubMed Scopus (66) Google Scholar). In addition, we have shown that CsA generates ROS in RASMC that were inhibited by antioxidants (20Krauskopf A. Buetler T.M. Nguyen N.S.D. Mace K. Ruegg U.T. Br. J. Pharmacol. 2002; 135: 977-986Crossref PubMed Scopus (25) Google Scholar). ROS have been assigned a role of biological mediators in cellular signaling (21Lander H.M. FASEB J. 1997; 11: 118-124Crossref PubMed Scopus (819) Google Scholar, 22Wolin M.S. Gupte S.A. Oeckler R.A. J. Vasc. Res. 2002; 39: 191-207Crossref PubMed Scopus (101) Google Scholar, 23Dröge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7355) Google Scholar). For example, it has been shown that platelet-derived growth factor-induced cell proliferation was dependent on the cellular production of H2O2 (24Sundaresan M. Yu Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2300) Google Scholar). In addition, it has been demonstrated that ROS are able to activate several transcription factors, such as nuclear factor-κB and activated protein-1 (25Schreck R. Bauerle A. Trends Cell Biol. 1991; 1: 39-43Abstract Full Text PDF PubMed Scopus (443) Google Scholar, 26Meyer M. Schreck R. Baeuerle P.A. EMBO J. 1993; 12: 2005-2015Crossref PubMed Scopus (1266) Google Scholar, 27Sen C.K. Packer L. FASEB J. 1996; 10: 709-720Crossref PubMed Scopus (1771) Google Scholar, 28Dalton T.D. Shertzer H.G. Puga A. Ann. Rev. Pharmacol. Toxicol. 1999; 39: 67-101Crossref PubMed Scopus (949) Google Scholar). Recent studies have illustrated that not only H2O2 but also superoxide is involved in cellular signaling (22Wolin M.S. Gupte S.A. Oeckler R.A. J. Vasc. Res. 2002; 39: 191-207Crossref PubMed Scopus (101) Google Scholar, 29Yang J.Q. Li S. Domann F.E. Buettner G.R. Oberley L.W. Mol. Carcinog. 1999; 26: 180-188Crossref PubMed Scopus (87) Google Scholar, 30Yang J.Q. Buettner G.R. Domann F.E. Li Q. Engelhardt J.F. Weydert C.D. Oberley L.W. Mol. Carcinog. 2002; 33: 206-218Crossref PubMed Scopus (42) Google Scholar, 31Zimmerman M.C. Lazartigues E. Lang J.A. Sinnayah P. Ahmad I.M. Spitz D.R. Davisson R.L. Circ. Res. 2002; 91: 1038-1045Crossref PubMed Scopus (332) Google Scholar, 32Huang W.-C. Chio C.-C. Chi K.-H. Wu H.-M. Lin W.-W. Exp. Cell Res. 2002; 277: 192-200Crossref PubMed Scopus (50) Google Scholar). 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 PROCEDURESChemicals 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 α-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 (9Lo Russo A. Passaquin A.C. André P. Skutella M. Ruegg U.T. Br. J. Pharmacol. 1996; 118: 885-892Crossref PubMed Scopus (72) Google Scholar). 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 (13Cottet-Maire F. Avdonin P.V. Roulet E. Buetler T.M. Mermod N. Ruegg U.T. Br. J. Pharmacol. 2001; 132: 909-917Crossref PubMed Scopus (29) Google Scholar). 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. 20Krauskopf A. Buetler T.M. Nguyen N.S.D. Mace K. Ruegg U.T. Br. J. Pharmacol. 2002; 135: 977-986Crossref PubMed Scopus (25) Google Scholar. 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 (13Cottet-Maire F. Avdonin P.V. Roulet E. Buetler T.M. Mermod N. Ruegg U.T. Br. J. Pharmacol. 2001; 132: 909-917Crossref PubMed Scopus (29) Google Scholar).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 (13Cottet-Maire F. Avdonin P.V. Roulet E. Buetler T.M. Mermod N. Ruegg U.T. Br. J. Pharmacol. 2001; 132: 909-917Crossref PubMed Scopus (29) Google Scholar). 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.RESULTSCsA 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.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 (33Rosenkranz A.R. Schmaldienst S. Stuhlmeier K.M. Chen W. Knapp W. Zlabinger G.J. J. Immunol. Methods. 1992; 156: 39-45Crossref PubMed Scopus (473) Google Scholar, 34Wang H. Joseph J.A. Free Radic. Biol. Med. 1999; 27: 612-616Crossref PubMed Scopus (1956) Google Scholar, 35Hempel S.L. Buettner G.R. O'Malley Y.Q. Wessels D.A. Flaherty D.M. Free Radic. Biol. Med. 1999; 27: 146-159Crossref PubMed Scopus (640) Google Scholar, 36Ischiropoulos H. Gow A. Thom S.R. Kooy N.W. Royall J.A. Crow J.P. Packer L. Methods in Enzymology: Oxygen Radicals in Biological Systems, Part D. Vol. 301. Academic Press, San Diego1999: 367-373Google Scholar), 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.Fig. 2ROS 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 (***).View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 α-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 α-tocopherol was equally effective in blocking CsA-induced V1A receptor up-regulation (data not shown).Fig. 3Effect 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 (***).View Large Image Figure ViewerDownload Hi-res image Download (PPT)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.Fig. 4Effect 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 (***).View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 (13Cottet-Maire F. Avdonin P.V. Roulet E. Buetler T.M. Mermod N. Ruegg U.T. Br. J. Pharmacol. 2001; 132: 909-917Crossref PubMed Scopus (29) Google Scholar). 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 (13Cottet-Maire F. Avdonin P.V. Roulet E. Buetler T.M. Mermod N. Ruegg U.T. Br. J. Pharmacol. 2001; 132: 909-917Crossref PubMed Scopus (29) Google Scholar). 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 (20Krauskopf A. Buetler T.M. Nguyen N.S.D. Mace K. Ruegg U.T. Br. J. Pharmacol. 2002; 135: 977-986Crossref PubMed Scopus (25) Google Scholar). 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.Fig. 5Effect 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 (***).View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 (37Beckman J. Minor Jr., R. White C. Repine J. Rosen G. Freeman B. J. Biol. Chem. 1988; 263: 6884-6892Abstract Full Text PDF PubMed Google Scholar, 38Cai H. Li Z. Dikalov S. Holland S.M. Hwang J. Jo H. Dudley Jr., S.C. Harrison D.G. J. Biol. Chem. 2002; 277: 48311-48317Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). 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 O2·¯ and that O2·¯ , 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).Fig. 6Effect 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 (***).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONIn 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." @default.
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• W1978791874 title "Vasopressin Type 1A Receptor Up-regulation by Cyclosporin A in Vascular Smooth Muscle Cells Is Mediated by Superoxide" @default.
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