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• W1976527906 abstract "The nuclear factor of activated T-cells (NFAT), originally identified in T-cells, has since been shown to play a role in mediating Ca2+-dependent gene transcription in diverse cell types outside of the immune system. We have previously shown that nuclear accumulation of NFATc3 is induced in ileal smooth muscle by platelet-derived growth factor in a manner that depends on Ca2+ influx through L-type, voltage-dependent Ca2+ channels. Here we show that NFATc3 is also the predominant NFAT isoform expressed in cerebral artery smooth muscle and is induced to accumulate in the nucleus by UTP and other Gq/11-coupled receptor agonists. This induction is mediated by calcineurin and is dependent on sarcoplasmic reticulum Ca2+ release through inositol 1,4,5-trisphosphate receptors and extracellular Ca2+ influx through L-type, voltage-dependent Ca2+ channels. Consistent with results obtained in ileal smooth muscle, depolarization-induced Ca2+ influx fails to induce NFAT nuclear accumulation in cerebral arteries. We also provide evidence that Ca2+release by ryanodine receptors in the form of Ca2+ sparks may exert an inhibitory influence on UTP-induced NFATc3 nuclear accumulation and further suggest that UTP may act, in part, by inhibiting Ca2+ sparks. These results are consistent with a multifactorial regulation of NFAT nuclear accumulation in smooth muscle that is likely to involve several intracellular signaling pathways, including local effects of sarcoplasmic reticulum Ca2+release and effects attributable to global elevations in intracellular Ca2+. The nuclear factor of activated T-cells (NFAT), originally identified in T-cells, has since been shown to play a role in mediating Ca2+-dependent gene transcription in diverse cell types outside of the immune system. We have previously shown that nuclear accumulation of NFATc3 is induced in ileal smooth muscle by platelet-derived growth factor in a manner that depends on Ca2+ influx through L-type, voltage-dependent Ca2+ channels. Here we show that NFATc3 is also the predominant NFAT isoform expressed in cerebral artery smooth muscle and is induced to accumulate in the nucleus by UTP and other Gq/11-coupled receptor agonists. This induction is mediated by calcineurin and is dependent on sarcoplasmic reticulum Ca2+ release through inositol 1,4,5-trisphosphate receptors and extracellular Ca2+ influx through L-type, voltage-dependent Ca2+ channels. Consistent with results obtained in ileal smooth muscle, depolarization-induced Ca2+ influx fails to induce NFAT nuclear accumulation in cerebral arteries. We also provide evidence that Ca2+release by ryanodine receptors in the form of Ca2+ sparks may exert an inhibitory influence on UTP-induced NFATc3 nuclear accumulation and further suggest that UTP may act, in part, by inhibiting Ca2+ sparks. These results are consistent with a multifactorial regulation of NFAT nuclear accumulation in smooth muscle that is likely to involve several intracellular signaling pathways, including local effects of sarcoplasmic reticulum Ca2+release and effects attributable to global elevations in intracellular Ca2+. nuclear factor of activated T-cells voltage-dependent Ca2+ channel(s) platelet-derived growth factor epidermal growth factor sarcoplasmic reticulum ryanodine receptor inositol 1,4,5-trisphosphate IP3R, inositol 1,4,5-trisphosphate receptors large conductance, Ca2+-activated K+ reverse transcription Tris-buffered saline 2-aminoethoxydiphenyl borate Nuclear factor of activated T-cells (NFAT)1 was originally identified as the transcription factor responsible for mediating the Ca2+-dependent transcription of genes involved in T-cell activation (1Emmel E.A. Verweij C.L. Durand D.B. Higgins K.M. Lacy E. Crabtree G.R. Science. 1989; 246: 1617-1620Crossref PubMed Scopus (573) Google Scholar, 2Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar) but has since been shown to play a role in mediating Ca2+-dependent gene transcription in diverse cell types outside of the immune system, including neurons (3Graef I.A. Mermelstein P.G. Stankunas K. Neilson J.R. Deisseroth K. Tsien R.W. Crabtree G.R. Nature. 1999; 401: 703-708Crossref PubMed Scopus (456) Google Scholar), endothelial cells (4Ranger A.M. Grusby M.J. Hodge M.R. Gravallese E.M. de la Brousse F.C. Hoey T. Mickanin C. Baldwin H.S. Glimcher L.H. Nature. 1998; 392: 186-190Crossref PubMed Scopus (513) Google Scholar, 5Boss V. Wang X. Koppelman L.F., Xu, K. Murphy T.J. Mol. Pharmacol. 1998; 54: 264-272Crossref PubMed Scopus (49) Google Scholar), cardiac muscle (6Molkentin J.D., Lu, J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2219) Google Scholar), skeletal muscle (7Semsarian C., Wu, M.J., Ju, Y.K. Marciniec T. Yeoh T. Allen D.G. Harvey R.P. Graham R.M. Nature. 1999; 400: 576-581Crossref PubMed Scopus (377) Google Scholar, 8Musaro A. McCullagh K.J. Naya F.J. Olson E.N. Rosenthal N. Nature. 1999; 400: 581-585Crossref PubMed Scopus (557) Google Scholar), and smooth muscle (9Boss V. Abbott K.L. Wang X.F. Pavlath G.K. Murphy T.J. J. Biol. Chem. 1998; 273: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 10Stevenson A.S. Gomez M.F. Hill-Eubanks D.C. Nelson M.T. J. Biol. Chem. 2001; 276: 15018-15024Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The potential physiological roles for this transcription factor are also diverse and include the developmental regulation of slow twitch/fast twitch skeletal muscle fiber types (11Chin E.R. Olson E.N. Richardson J.A. Yang Q. Humphries C. Shelton J.M., Wu, H. Zhu W. Bassel-Duby R. Williams R.S. Genes Dev. 1998; 12: 2499-2509Crossref PubMed Scopus (839) Google Scholar) and smooth muscle cell precursor migration and vascular development during embryogenesis (12Graef I.A. Chen F. Chen L. Kuo A. Crabtree G.R. Cell. 2001; 105: 863-875Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). NFAT has also been implicated in the pathogenesis of cardiac (6Molkentin J.D., Lu, J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2219) Google Scholar) and skeletal (7Semsarian C., Wu, M.J., Ju, Y.K. Marciniec T. Yeoh T. Allen D.G. Harvey R.P. Graham R.M. Nature. 1999; 400: 576-581Crossref PubMed Scopus (377) Google Scholar, 8Musaro A. McCullagh K.J. Naya F.J. Olson E.N. Rosenthal N. Nature. 1999; 400: 581-585Crossref PubMed Scopus (557) Google Scholar) muscle hypertrophy and might be predicted to play a similar role in smooth muscle hypertrophy associated with, for example, atherosclerosis and bladder dysfunction. NFAT represents a family of transcription factors composed of four well characterized members, designated NFATc1 (NFAT2/c), NFATc2 (NFAT1/p), NFATc3 (NFAT4/x), and NFATc4 (NFAT3). A fifth putative member of the family (NFAT5) is a calcineurin-insensitive, constitutively nuclear phosphoprotein that has limited sequence similarity to other members of the NFAT family (13Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (319) Google Scholar). NFAT activation is regulated primarily through control of its subcellular localization (2Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar). Elevation of global Ca2+produced by a variety of mechanisms activates the Ca2+/calmodulin-dependent protein phosphatase, calcineurin (14Rusnak F. Mertz P. Physiol. Rev. 2000; 80: 1483-1521Crossref PubMed Scopus (1117) Google Scholar). Subsequent calcineurin-mediated dephosphorylation of specific NFAT serine residues induces a conformational change in the NFAT molecule that exposes nuclear localization signals, allowing import of NFAT into the nucleus (15Okamura H. Aramburu J. Garcia-Rodriguez C. Viola J.P. Raghavan A. Tahiliani M. Zhang X. Qin J. Hogan P.G. Rao A. Mol. Cell. 2000; 6: 539-550Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 16Shibasaki F. Price E.R. Milan D. McKeon F. Nature. 1996; 382: 370-373Crossref PubMed Scopus (434) Google Scholar, 17Zhu J. Shibasaki F. Price R. Guillemot J.C. Yano T. Dotsch V. Wagner G. Ferrara P. McKeon F. Cell. 1998; 93: 851-861Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 18Liu J. Koyano-Nakagawa N. Amasaki Y. Saito-Ohara F. Ikeuchi T. Imai S. Takano T. Arai N. Yokota T. Arai K. Mol. Biol. Cell. 1997; 8: 157-170Crossref PubMed Scopus (28) Google Scholar, 19Beals C.R. Clipstone N.A., Ho, S.N. Crabtree G.R. Genes Dev. 1997; 11: 824-834Crossref PubMed Scopus (342) Google Scholar, 20Masuda E.S. Liu J. Imamura R. Imai S.I. Arai K.I. Arai N. Mol. Cell. Biol. 1997; 17: 2066-2075Crossref PubMed Scopus (48) Google Scholar). The distribution of NFAT between nuclear and cytoplasmic compartments is dynamically regulated by the activity of nuclear kinases, which oppose the action of calcineurin (3Graef I.A. Mermelstein P.G. Stankunas K. Neilson J.R. Deisseroth K. Tsien R.W. Crabtree G.R. Nature. 1999; 401: 703-708Crossref PubMed Scopus (456) Google Scholar, 28Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Science. 1997; 275: 1930-1934Crossref PubMed Scopus (637) Google Scholar, 29Gomez del Arco P. Martinez-Martinez S. Maldonado J.L. Ortega-Perez I. Redondo J.M. J. Biol. Chem. 2000; 275: 13872-13878Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 30Porter C.M. Havens M.A. Clipstone N.A. J. Biol. Chem. 2000; 275: 3543-3551Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 31Chow C.W. Rincon M. Cavanagh J. Dickens M. Davis R.J. Science. 1997; 278: 1638-1641Crossref PubMed Scopus (300) Google Scholar). Additional mechanisms, which modulate the ability of calcineurin to associate with and/or dephosphorylate NFAT in the cytosol, provide regulation at the level of nuclear import (17Zhu J. Shibasaki F. Price R. Guillemot J.C. Yano T. Dotsch V. Wagner G. Ferrara P. McKeon F. Cell. 1998; 93: 851-861Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar,32Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 33Chow C.W. Dong C. Flavell R.A. Davis R.J. Mol. Cell. Biol. 2000; 20: 5227-5234Crossref PubMed Scopus (120) Google Scholar, 34Chow C.W. Davis R.J. Mol. Cell. Biol. 2000; 20: 702-712Crossref PubMed Scopus (98) Google Scholar). In the nucleus, NFAT associates with a transcriptional co-activator, an interaction that is required for significant NFAT-mediated transcriptional activity. NFAT family members have been shown to cooperatively bind to DNA with variety of cofactors, including AP-1 (24Jain J. McCaffrey P.G. Valge-Archer V.E. Rao A. Nature. 1992; 356: 801-804Crossref PubMed Scopus (429) Google Scholar, 25Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2917-2923Abstract Full Text PDF PubMed Google Scholar, 26Rooney J.W. Hoey T. Glimcher L.H. Immunity. 1995; 2: 473-483Abstract Full Text PDF PubMed Scopus (238) Google Scholar), GATA (6Molkentin J.D., Lu, J.R. Antos C.L. Markham B. Richardson J. Robbins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2219) Google Scholar, 8Musaro A. McCullagh K.J. Naya F.J. Olson E.N. Rosenthal N. Nature. 1999; 400: 581-585Crossref PubMed Scopus (557) Google Scholar, 27Wada H. Hasegawa K. Morimoto T. Kakita T. Yanazume T. Abe M. Sasayama S. J. Cell Biol. 2002; 156: 983-991Crossref PubMed Scopus (89) Google Scholar), and MEF2 (11Chin E.R. Olson E.N. Richardson J.A. Yang Q. Humphries C. Shelton J.M., Wu, H. Zhu W. Bassel-Duby R. Williams R.S. Genes Dev. 1998; 12: 2499-2509Crossref PubMed Scopus (839) Google Scholar), and in this way integrate Ca2+/calcineurin signaling with other signaling pathways, such as Ras, Rac, and protein kinase C. A sustained, global increase in intracellular Ca2+ has generally been considered a defining feature of NFAT-activating stimuli (35Timmerman L.A. Clipstone N.A., Ho, S.N. Northrop J.P. Crabtree G.R. Nature. 1996; 383: 837-840Crossref PubMed Scopus (472) Google Scholar, 36Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1564) Google Scholar, 37Crabtree G.R. Cell. 1999; 96: 611-614Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar, 38Crabtree G.R. Clipstone N.A. Annu. Rev. Biochem. 1994; 63: 1045-1083Crossref PubMed Scopus (627) Google Scholar). More recent evidence has shown that temporal modulation of Ca2+ signals in the form of Ca2+ oscillations or waves increases the coupling efficiency of Ca2+ signals to NFAT activation in nonexcitable cells (39Li W. Llopis J. Whitney M. Zlokarnik G. Tsien R.Y. Nature. 1998; 392: 936-941Crossref PubMed Scopus (777) Google Scholar, 40Dolmetsch R.E., Xu, K. Lewis R.S. Nature. 1998; 392: 933-936Crossref PubMed Scopus (1683) Google Scholar). Surprisingly, transient increases in intracellular Ca2+ induced by a depolarizing stimulus and mediated by flux through L-type voltage-dependent Ca2+ channels (VDCC), have also been shown to effectively stimulate a sustained increase in NFAT activity in hippocampal neurons (3Graef I.A. Mermelstein P.G. Stankunas K. Neilson J.R. Deisseroth K. Tsien R.W. Crabtree G.R. Nature. 1999; 401: 703-708Crossref PubMed Scopus (456) Google Scholar). In contrast, and counter to expectations, sustained increases in intracellular Ca2+induced by depolarization fail to induce nuclear accumulation of the NFATc3 isoform in ileal smooth muscle, although PDGF, which activates multiple intracellular pathways, is a potent stimulus for NFATc3 nuclear translocation in this tissue (10Stevenson A.S. Gomez M.F. Hill-Eubanks D.C. Nelson M.T. J. Biol. Chem. 2001; 276: 15018-15024Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Smooth muscle exhibits a diverse array of Ca2+ signals, including Ca2+ waves that traverse the length of the cell and display distinctive frequency and amplitude properties (41Jaggar J.H. Nelson M.T. Am. J. Physiol. 2000; 279: C1528-C1539Crossref PubMed Google Scholar, 42Iino M. Mol. Cell Biochem. 1999; 190: 185-190Crossref PubMed Google Scholar, 43Iino M. Kasai H. Yamazawa T. EMBO J. 1994; 13: 5026-5031Crossref PubMed Scopus (161) Google Scholar, 44Mayer E.A. Kodner A. Sun X.P. Wilkes J. Scott D. Sachs G. J. Membr. Biol. 1992; 125: 107-118Crossref PubMed Scopus (28) Google Scholar), and localized transient releases of Ca2+through sarcoplasmic reticulum (SR) ryanodine receptors (RyRs) in the form of Ca2+ sparks (45Nelson M.T. Cheng H. Rubart M. Santana L.F. Bonev A.D. Knot H.J. Lederer W.J. Science. 1995; 270: 633-637Crossref PubMed Scopus (1208) Google Scholar). The role that various Ca2+ signals and Ca2+-dependent transcription factors may play in the physiological or pathological regulation of gene expression in this phenotypically plastic tissue is largely unknown. Here we show that, in native cerebral artery smooth muscle cells, UTP and other Gq/11-coupled receptor agonists induce a calcineurin-mediated nuclear accumulation of NFATc3. This induction is dependent on SR Ca2+ release through IP3 receptors (IP3R) and further depends on extracellular Ca2+ influx through L-type VDCC. We also show that UTP may act to induce NFATc3 nuclear accumulation, at least in part, by suppressing Ca2+ sparks, suggesting a novel inhibitory role for Ca2+ sparks in the regulation of Ca2+-sensitive transcription factors. Epidermal growth factor (EGF) and platelet-derived growth factor BB (PDGF-BB) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), pinacidil was from RBI (Research Biochemical Inc.), fluo-4 and pluronic acid were from Molecular Probes, Inc. (Eugene, OR), ryanodine was from LC Laboratories, xestospongin C was from Calbiochem, and 2-aminoethoxydiphenyl borate (2-APB) was from Tocris. FK506 was kindly provided by Fujisawa. All other drugs and chemical reagents were from Sigma. Adult female CD-1 mice (20–25 g; Charles River Laboratories) were euthanized by peritoneal injection of pentobarbital solution (200 mg/kg). Cerebral arteries were dissected from the brain in ice-cold physiological saline solution (containing 135 mmol/liter NaCl, 5.9 mmol/liter KCl, 1.2 mmol/liter MgCl2, 11.6 mmol/liter Hepes, 11.5 mmol/liter glucose, pH 7.4) and cleaned of connective tissue. Endothelium-denuded vessels were prepared by passing an air bubble through the lumen of the artery. Total RNA was prepared from cerebral vessels using the Trizol-LS Reagent (Invitrogen), followed by DNase treatment and reverse transcription using oligo(dT) primers and the Sensiscript RT Kit (Qiagen), as described by the manufacturers. PCRs were performed using the AmpliTaq Gold (PerkinElmer Life Sciences) with the following sets of primer pairs: for NFATc2, F (5′-ACATCCGCGTGCCCGTGAAAGT-3′) and R (5′-CTCGGGGCAGTCTGTTGTTGGATG-3′); for NFATc1, F (5′-CATGCGCCCTCTGTGGCCCTCAAA-3′) and R (5′-GGAGCCTTCTCCACGAAAATG-3′); for NFATc4, F (5′-GAAGCTACCCTCCGGTACAGAG-3′) and R (5′-GCTTCATAGCTGGCTGTAGCC-3′); for NFATc3, F (5′-CTACTGGTGGCCATCCTGTTGT-3′) and R (AGCTCGTGGGCAGAGCGCTGAGAGCACTC-3′); and glyceraldehyde-3-phosphate dehydrogenase (CLONTECH). Amplification conditions were 94 °C for 10 min, 30–45 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min, and extension for 10 min at 72 °C. Amplified PCR products were separated by agarose gel electrophoresis and detected by ethidium bromide staining. Cerebral arteries (midcerebral, posterior, cerebellar, and basilar) from 2–4 mice were pooled and homogenized in sample preparation buffer (50 mm Tris-Cl, pH 6.8, 2% SDS, 100 mm dithiothreitol, 10% glycerol, and 0.1% bromphenol blue). Aliquots of cerebral artery and control tissue extracts, prepared in a similar manner, were separated by SDS-PAGE on 8% gels using the Laemmli buffering system. Proteins were transferred to immunoblot polyvinylidene difluoride membranes (Bio-Rad) and blocked by rocking for 1 h at room temperature in blocking buffer (Tris-buffered saline with 0.1% Tween 20 (TBST) and 5% nonfat dry milk). Blots were exposed to primary antibodies for 1 h, multiply washed with TBST, and treated with horseradish peroxidase-conjugated secondary antibody for 45 min, followed by a final series of washes with TBST. Primary (rabbit polyclonal anti-NFATc3; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and secondary (donkey anti-rabbit IgG; Santa Cruz Biotechnology) antibodies were prepared in blocking buffer, and all steps were performed at room temperature. Blots were developed using an enhanced chemiluminescence substrate (SuperSignal West Dura; Pierce) according to the manufacturer's instructions. Arteries were treated at room temperature as specified and then mounted onto glass slides. After air-drying for 5 min, the arteries were fixed with 4% formaldehyde in phosphate-buffered saline (pH 7.4) for 15 min, permeabilized with 0.2% Triton-X-100 in phosphate-buffered saline for 10 min, and blocked for 2 h with 2% bovine serum albumin in phosphate-buffered saline. Primary antibody (rabbit anti-NFATc3 (Santa Cruz Biotechnology) diluted 1:250 in 2% bovine serum albumin/phosphate-buffered saline) was applied overnight at 4 °C. Secondary antibody (Cy5 anti-rabbit IgG (Jackson Immuno Research Laboratories), 1:500 dilution) was applied for 1 h at 25 °C. Nuclei were identified using the fluorescent nucleic acid dye YOYO-1 (1:30,000 dilution). After washing, the vessels were mounted (Aqua Polymount mounting medium; Polysciences) and examined at ×40 magnification using a Bio-Rad 1000 laser-scanning confocal microscope. NFATc3 was detected by monitoring Cy5 fluorescence using an excitation wavelength of 650 nm and an emission wavelength of 670 nm. Specificity of immune staining was confirmed by the absence of fluorescence in arteries incubated with primary or secondary antibodies alone. For scoring of NFATc3-positive nuclei, multiple fields for each vessel were imaged and counted by two independent observers under double-blind conditions. For quantification, a cell was considered positive if co-localization (yellow) was observed in the nucleus, whereas a cell was considered negative if no co-localization (green only) was visualized. All imaging experiments were performed at room temperature. Arteries were loaded with 10 μm fluo-4-AM in physiological saline solution and 0.05% pluronic acid for 60 min and then kept in physiological saline solution for 30 min to allow fluo-4 de-esterification. The vessel ends were anchored beneath two stainless steel hooks to maintain the artery at the bottom of the chamber and provide a fixed imaging area. Arteries were illuminated with a krypton/argon laser at 488 nm and imaged using a Noran Oz laser-scanning confocal microscope and a ×60 water immersion lens (numerical aperture = 1.2). For Ca2+sparks detection, images of 58.1 × 54.5-μm (256 × 240 pixels) sections of the vessel wall were recorded every 16.7 ms (60 images/s). Under each condition, at least two different representative areas of the same artery were scanned for 10 s. Ca2+sparks were analyzed using custom software written in our laboratory by Dr. Adrian Bonev (using IDL 5.2; Research Systems Inc., Boulder, CO), which allows for off-line quantification of fluorescence changes in selected regions of a sample corresponding to boxes of defined dimensions positioned by eye within the sample. Ca2+ spark amplitude (F/F 0) was obtained by determining the fluorescence intensity within a 2.37-μm2(1.54 μm (7 pixels) × 1.54 μm (7 pixels)) area corresponding to a detected spark event (F), and dividing by a base line (F 0) that was determined by averaging 30 images without Ca2+ spark activity. Ca2+ spark frequency under a given condition was calculated by measuring the number of sparks that occurred in a 58.1 × 54.5-μm area (∼20 cells) scanned for 10 s. For detection of Ca2+ waves, images of the vessel wall (116.2 × 108.0 μm, or 512 × 480 pixels) were recorded every 531.9 ms (1.88 images/s). Under each condition, at least two different representative areas of the same artery were scanned for 213 s. Ca2+ waves were determined by analyzing recurrent changes in fluorescence intensity occurring in 2.2 × 2.2-μm (10 × 10 pixels) regions of individual myocytes and defined as a change inF/F 0 > 1.3 that remained elevated for >200 ms. Changes in global F/F 0were calculated by measuring the mean pixel value of images acquired at 1.88 images/s, before and after application of each drug. The same area was not scanned more than once to avoid introducing Ca2+signaling artifacts due to laser-induced cell damage. Results are expressed as means ± S.E., where applicable. All statistical analysis was performed using GraphPad software (Prism 3.0). Statistical significance was determined using one-way analysis of variance analysis followed by Bonferroni or Tukey-Kramer tests (for comparisons between up to five groups or at least six groups, respectively). We have used RT-PCR analysis and immunoblotting to identify NFAT isoforms expressed in native cerebral artery smooth muscle. Previous results from experiments employing the rat A7r5 aortic smooth muscle cell line suggested that the NFATc2 and NFATc1 isoforms are expressed in smooth muscle (9Boss V. Abbott K.L. Wang X.F. Pavlath G.K. Murphy T.J. J. Biol. Chem. 1998; 273: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), although expression of additional isoforms could not be ruled out in this study. In an RT-PCR analysis of total RNA isolated from cerebral arteries, we find no evidence for NFATc2 expression using primer pairs that efficiently amplify NFATc2 from spleen (Fig.1 A). Instead, we find that NFATc3 and, to a lesser extent, NFATc4, are expressed in cerebral arteries (Fig. 1 B). NFATc1 expression is generally very low to undetectable in unstimulated mouse cerebral arteries (Fig.1 B). The presence of NFATc3 protein in cerebral arteries was confirmed by Western analysis, which showed the presence of major bands corresponding to those identified in thymus (Fig. 1 C), a tissue that expresses predominantly the NFATc3 isoform (46Masuda E.S. Naito Y. Tokumitsu H. Campbell D. Saito F. Hannum C. Arai K. Arai N. Mol. Cell. Biol. 1995; 15: 2697-2706Crossref PubMed Scopus (198) Google Scholar). The predominant expression of NFATc3 in smooth muscle has led us to focus on this isoform, although it is likely that other NFAT isoforms may play important roles in smooth muscle, as suggested by others (9Boss V. Abbott K.L. Wang X.F. Pavlath G.K. Murphy T.J. J. Biol. Chem. 1998; 273: 19664-19671Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 12Graef I.A. Chen F. Chen L. Kuo A. Crabtree G.R. Cell. 2001; 105: 863-875Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar,47Abbott K.L. Loss 2nd, J.R. Robida A.M. Murphy T.J. Mol. Pharmacol. 2000; 58: 946-953Crossref PubMed Scopus (43) Google Scholar). UTP is an important vasoactive substance in the cerebral vasculature and has been previously shown to activate the NFATc1 isoform in cultured smooth muscle cells (47Abbott K.L. Loss 2nd, J.R. Robida A.M. Murphy T.J. Mol. Pharmacol. 2000; 58: 946-953Crossref PubMed Scopus (43) Google Scholar). In intact cerebral arteries, treatment with UTP induces nuclear translocation of NFATc3 as evidenced by colocalization of NFATc3 with the fluorescent nucleic acid dye, YOYO-1 (Fig.2 A). These results are summarized in Fig. 2 B, which shows that the number of NFATc3-positive nuclei in intact cerebral arteries is increased from 7.9% in untreated vessels to 43.2% in UTP-treated arteries. Similar results for control (5.8%) and UTP-treated conditions (50.7%) were obtained for endothelium-denuded arteries, indicating that this action of UTP on NFATc3 subcellular distribution is a direct effect on smooth muscle. Other Gq/11-coupled vasoconstrictors and peptide ligands for certain tyrosine kinase-linked growth factor receptors are capable of inducing NFATc3 translocation in cerebral artery smooth muscle, although the robustness of the response varies with the agonist used. Endothelin-1 is as effective as UTP in inducing NFATc3 nuclear accumulation (Fig. 2 C), whereas angiotensin II and the peptide ligand EGF are much less effective. Prostaglandin F2α induces a small, but significant, increase in NFAT nuclear accumulation that is comparable in magnitude with that induced by angiotensin II and EGF. PDGF, which is a smooth muscle mitogen and potent activator of NFATc3 nuclear accumulation in ileal smooth muscle (10Stevenson A.S. Gomez M.F. Hill-Eubanks D.C. Nelson M.T. J. Biol. Chem. 2001; 276: 15018-15024Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), is ineffective in cerebral artery smooth muscle. Calcineurin activity is sensitive to inhibition by the chemically unrelated immunosuppressive agents FK506 and cyclosporin A, which inhibit calcineurin by distinct mechanisms (48Ho S. Clipstone N. Timmermann L. Northrop J. Graef I. Fiorentino D. Nourse J. Crabtree G.R. Clin. Immunol. Immunopathol. 1996; 80: S40-S45Crossref PubMed Scopus (626) Google Scholar). To determine whether UTP-induced NFATc3 nuclear accumulation is calcineurin-dependent, we treated intact cerebral arteries with each of these agents prior to and/or concurrent with UTP treatment. Inhibition of calcineurin activity with either of these compounds completely abrogates UTP-induced NFATc3 nuclear accumulation (Fig. 3), indicating that this process is calcineurin-dependent. Calcineurin activity is strictly dependent on Ca2+/calmodulin (49Klee C.B. Ren H. Wang X. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar). In intact cerebral arteries, UTP induces an increase in global intracellular Ca2+characterized by an initial Ca2+ spike followed by a sustained elevated plateau phase (Fig.4 A; see also Ref. 41Jaggar J.H. Nelson M.T. Am. J. Physiol. 2000; 279: C1528-C1539Crossref PubMed Google Scholar). Although the magnitude of each phase is somewhat variable between and within vessels, this biphasic response is a consistent property of UTP-induced Ca2+ elevation. To determine whether SR-mediated Ca2+ release is involved in UTP-induced NFATc3 nuclear accumulation, we pretreated cerebral arteries with the SR Ca2+-ATPase inhibitor, thapsigargin, to deplete SR luminal Ca2+. This treatment prevents the increase in global Ca2+ induced by UTP (Fig. 4 B), indicating that intracellular calcium stores are required for this effect. To determine whether this calcium release from the SR induced by UTP contributes to UTP-induced NFATc3 nuclear accumulation, NFATc3 subcellular distribution was monitored immunohistochemically in cerebral arteries pretreated with thapsigargin. In these experiments, we employed cerebral arteries that had first been denuded of endothelium, as described under “Experimental Procedures.” In endothelium-denuded cerebral arteries, prior depletion of Ca2+ stores completely prevents UTP-induced NFATc3 nuclear accumulation (Fig.4 C). Under these conditions, thapsigargin alone has no effect on NFATc3 subcellular distribution. These data indicate that UTP mediates its effects on NFATc3 subcellular distribution, at least in part, through release of SR Ca2+. In nonexcitable cells, the sustained increase in intracellular Ca2+ required to maintain NFAT in the nucleus is provided by a capacitative mechanism by which depletion of intracellular Ca2+ stores is coupled to extracellular Ca2+ influx (50Serafini A.T. Lewis R.S. Clipstone N.A. Bram R.J. Fanger C. Fiering S. Herzenberg L.A. Crabtree G.R. Immunity. 1995; 3: 239-250Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 51Fanger C.M. Hoth M. Crabtree G.R. Lewis R.S. J. Cell Biol. 1" @default.
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• W1976527906 cites W1509578353 @default.
• W1976527906 cites W1601157315 @default.
• W1976527906 cites W1633206935 @default.
• W1976527906 cites W1650401181 @default.
• W1976527906 cites W1657298386 @default.
• W1976527906 cites W1666469474 @default.
• W1976527906 cites W1671239168 @default.
• W1976527906 cites W1753089048 @default.
• W1976527906 cites W18180971 @default.
• W1976527906 cites W1827812119 @default.
• W1976527906 cites W1965021019 @default.
• W1976527906 cites W1980321436 @default.
• W1976527906 cites W1982815320 @default.
• W1976527906 cites W1984947099 @default.
• W1976527906 cites W1987784311 @default.
• W1976527906 cites W1991163286 @default.
• W1976527906 cites W1993569708 @default.
• W1976527906 cites W2007774889 @default.
• W1976527906 cites W2011952922 @default.
• W1976527906 cites W2012218395 @default.
• W1976527906 cites W2013056080 @default.
• W1976527906 cites W2014577426 @default.
• W1976527906 cites W2032770343 @default.
• W1976527906 cites W2039842713 @default.
• W1976527906 cites W2043736479 @default.
• W1976527906 cites W2044406741 @default.
• W1976527906 cites W2047198875 @default.
• W1976527906 cites W2049982941 @default.
• W1976527906 cites W2053010478 @default.
• W1976527906 cites W2054180166 @default.
• W1976527906 cites W2061851471 @default.
• W1976527906 cites W2062469526 @default.
• W1976527906 cites W2074475161 @default.
• W1976527906 cites W2074803910 @default.
• W1976527906 cites W2077635738 @default.
• W1976527906 cites W2082502466 @default.
• W1976527906 cites W2087700080 @default.
• W1976527906 cites W2088264073 @default.
• W1976527906 cites W2088919308 @default.
• W1976527906 cites W2090536285 @default.
• W1976527906 cites W2092109656 @default.
• W1976527906 cites W2104987037 @default.
• W1976527906 cites W2105075836 @default.
• W1976527906 cites W2106522615 @default.
• W1976527906 cites W2118870612 @default.
• W1976527906 cites W2124047072 @default.
• W1976527906 cites W2125760322 @default.
• W1976527906 cites W2132325104 @default.
• W1976527906 cites W2135281145 @default.
• W1976527906 cites W2136341846 @default.
• W1976527906 cites W2137407985 @default.
• W1976527906 cites W2140707292 @default.
• W1976527906 cites W2145303230 @default.
• W1976527906 cites W2156433522 @default.
• W1976527906 cites W2156600279 @default.
• W1976527906 cites W2157614703 @default.
• W1976527906 cites W2160634299 @default.
• W1976527906 cites W2164497329 @default.
• W1976527906 cites W2165948633 @default.
• W1976527906 cites W2168393307 @default.
• W1976527906 cites W2172196619 @default.
• W1976527906 cites W2233204418 @default.
• W1976527906 cites W4245806460 @default.
• W1976527906 cites W4249759827 @default.
• W1976527906 cites W4313343508 @default.
• W1976527906 cites W4376453153 @default.
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