Gerard D. Gagné

Nitric oxide synthases (NOS Types I-III) generate nitric oxide (NO), which in turn activates soluble guanylyl cyclase (GC-S). The distribution of this NO-mediated (nitrinergic) signal transduction pathway in the body is unclear. A polyclonal monospecific antibody to rat cerebellum NOS-I and a monoclonal antibody to rat lung GC-S were employed to localize the protein components of this pathway in different rat organs and tissues. We confirmed the localization of NOS-I in neurons of the central and peripheral nervous system, where NO may regulate cerebral blood flow and mediate long-term potentiation. GC-S was located in NOS-negative neurons, indicating that NO acts as an intercellular signal molecule or neurotransmitter. However, NOS-I was not confined to neurons but was widely distributed over several non-neural cell types and tissues. These included glia cells, macula densa of kidney, epithelial cells of lung, uterus, and stomach, and islets of Langerhans. Our findings suggest that NOS-I is the most widely distributed isoform of NOS and, in addition to its neural functions, regulates secretion and non-vascular smooth muscle function. With the exception of bone tissue, NADPH-diaphorase (NADPH-d) activity was generally co-localized with NOS-I immunoreactivity in both neural and non-neural cells, and is a suitable histochemical marker for NOS-I but not a selective neuronal marker.

NFAT (nuclearfactor of activated T cell) proteins are expressed in most immune system cells and regulate the transcription of cytokine genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the Ca2+/calmodulin-dependent protein phosphatase 2B/calcineurin (CaN). Dephosphorylation of NFAT by CaN is required for NFAT nuclear localization. Current immunosuppressive drugs such as cyclosporin A and FK506 block CaN activity thus inhibiting nuclear translocation of NFAT and consequent cytokine gene transcription. The inhibition of CaN in cells outside of the immune system may contribute to the toxicities associated with cyclosporin A therapy. In a search for safer immunosuppressive drugs, we identified a series of 3,5-bistrifluoromethyl pyrazole (BTP) derivatives that block Th1 and Th2 cytokine gene transcription. The BTP compounds block the activation-dependent nuclear localization of NFAT as determined by electrophoretic mobility shift assays. Confocal microscopy of cells expressing fluorescent-tagged NFAT confirmed that the BTP compounds block calcium-induced movement of NFAT from the cytosol to the nucleus. Inhibition of NFAT was selective because the BTP compounds did not affect the activation of NF-κB and AP-1 transcription factors. Treatment of intact T cells with the BTP compounds prior to calcium ionophore-induced activation of CaN caused NFAT to remain in a highly phosphorylated state. However, the BTP compounds did not directly inhibit the dephosphorylation of NFAT by CaNin vitro, nor did the drugs block the dephosphorylation of other CaN substrates including the type II regulatory subunit of protein kinase A and the transcription factor Elk-1. The data suggest that the BTP compounds cause NFAT to be maintained in the cytosol in a phosphorylated state and block the nuclear import of NFAT and, hence, NFAT-dependent cytokine gene transcription by a mechanism other than direct inhibition of CaN phosphatase activity. The novel inhibitors described herein will be useful in better defining the cellular regulation of NFAT activation and may lead to identification of new therapeutic targets for the treatment of autoimmune disease and transplant rejection. NFAT (nuclearfactor of activated T cell) proteins are expressed in most immune system cells and regulate the transcription of cytokine genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the Ca2+/calmodulin-dependent protein phosphatase 2B/calcineurin (CaN). Dephosphorylation of NFAT by CaN is required for NFAT nuclear localization. Current immunosuppressive drugs such as cyclosporin A and FK506 block CaN activity thus inhibiting nuclear translocation of NFAT and consequent cytokine gene transcription. The inhibition of CaN in cells outside of the immune system may contribute to the toxicities associated with cyclosporin A therapy. In a search for safer immunosuppressive drugs, we identified a series of 3,5-bistrifluoromethyl pyrazole (BTP) derivatives that block Th1 and Th2 cytokine gene transcription. The BTP compounds block the activation-dependent nuclear localization of NFAT as determined by electrophoretic mobility shift assays. Confocal microscopy of cells expressing fluorescent-tagged NFAT confirmed that the BTP compounds block calcium-induced movement of NFAT from the cytosol to the nucleus. Inhibition of NFAT was selective because the BTP compounds did not affect the activation of NF-κB and AP-1 transcription factors. Treatment of intact T cells with the BTP compounds prior to calcium ionophore-induced activation of CaN caused NFAT to remain in a highly phosphorylated state. However, the BTP compounds did not directly inhibit the dephosphorylation of NFAT by CaNin vitro, nor did the drugs block the dephosphorylation of other CaN substrates including the type II regulatory subunit of protein kinase A and the transcription factor Elk-1. The data suggest that the BTP compounds cause NFAT to be maintained in the cytosol in a phosphorylated state and block the nuclear import of NFAT and, hence, NFAT-dependent cytokine gene transcription by a mechanism other than direct inhibition of CaN phosphatase activity. The novel inhibitors described herein will be useful in better defining the cellular regulation of NFAT activation and may lead to identification of new therapeutic targets for the treatment of autoimmune disease and transplant rejection. T cell antigen receptor bistrifluoromethyl pyrazole cyclosporin A nuclear factor of activated T cells NFAT homology calcineurin Chinese hamster ovary green fluorescent protein interleukin peripheral mononuclear cells nuclear factor enzyme-linked immunosorbent assay phorbol 12-mystrate 13-acetate FK506-binding protein Rous sarcoma virus N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine phosphate-buffered saline interferon glutathioneS-transferase Engagement of the T cell antigen receptor (TcR)1 with the antigen-major histocompatibility complex on antigen-presenting cells triggers a complex TcR signaling cascade that leads to T cell activation and cytokine secretion (1Chan A.C. Desai D.M. Weiss A. Annu. Rev. Immunol. 1994; 12: 555-592Crossref PubMed Scopus (500) Google Scholar). During this process, T cells express the autocrine growth factor interleukin 2 (IL-2), which promotes T cell proliferation by interacting with the IL-2 receptor, which is also up-regulated on activated T cells. The transcriptional regulation of the IL-2 gene has been extensively analyzed at the IL-2 promoter, a 275-bp region located upstream of the transcriptional start site of the gene (2Rothenberg E.V. Ward S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9358-9365Crossref PubMed Scopus (169) Google Scholar, 3Jain J. Loh C. Rao A. Curr. Opin. Immunol. 1995; 7: 333-342Crossref PubMed Scopus (501) Google Scholar). Several transcription factors have been identified to bind elements within this regulatory region, including AP-1, NF-κB, and the nuclear factor of activated T cells (NFAT) (2Rothenberg E.V. Ward S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9358-9365Crossref PubMed Scopus (169) Google Scholar). The transcription factor NFAT plays an essential role in IL-2 expression. Binding sites for NFATs have also been found within the promoter regions of several other cytokine genes, including IL-3, IL-4, IL-5, IL-8, IL-13, tumor necrosis factor α, granulocyte-macrophage colony-stimulating factor, and γ-IFN (4Rao A. Immunol. Today. 1994; 15: 274-281Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 5Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar). NFAT is a complex composed of a cytoplasmic subunit and an inducible nuclear component comprised of AP-1 (Fos/Jun) family members. At least four structurally related NFAT cytoplasmic subunit members, NFATp/NFAT1, NFATc/NFAT2, NFAT3, and NFATX/NFATc3/NFAT4, have been identified (5Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar). NFAT proteins share a conserved domain located toward the C terminus (6Jain J. Burgeon E. Badalian T.M. Hogan P.G. Rao A. J. Biol. Chem. 1995; 270: 4138-4145Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) that binds DNA and also participates in cooperative protein-protein interactions with AP-1 transcription factors (7Jain J. McCaffrey P. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (681) Google Scholar, 8Molkentin 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 (2218) Google Scholar). Immediately N-terminal to the DNA-binding domain is a second conserved module of ∼300 residues known as the NFAT homology (NFAT-h) region. The N terminus of NFAT, including the NFAT-h region, regulates nuclear/cytoplasm trafficking in response to changes in intracellular Ca2+ concentrations. In resting T cells, the protein is retained in the cytoplasm and its NFAT-h domain is heavily phosphorylated. Engagement of the TcR or treatment of cells with the Ca2+ ionophore activates the Ca2+/calmodulin-dependent Ser/Thr phosphatase, calcineurin. CaN dephosphorylates the NFAT-h domain, resulting in translocation of NFAT to the nucleus (9Liu J. Farmer J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3633) Google Scholar). The clinically important immunosuppressive drugs, cyclosporin A and FK506, act by binding to their respective immunophilins, cyclophilin and FKBP12 (9Liu J. Farmer J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3633) Google Scholar). The immunophilin-drug complex binds to CaN and inhibits CaN phosphatase activity, thus preventing the dephosphorylation and translocation of NFAT to the nucleus (10Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (773) Google Scholar, 11Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Gastinel L.N. Habuka N. Chen X. Maldonado F. Barker J. Nature. 1995; 378: 641-644Crossref PubMed Scopus (699) Google Scholar, 12Hemenway C.S. Heitman J. Cell. Biochem. Biophys. 1999; 30: 115-151Crossref PubMed Scopus (195) Google Scholar). As a substrate of CaN, NFAT is a secondary target of the action of these immunosuppressive drugs. NFAT inhibition is believed to account, at least in part, for the transcriptional inhibitory activity of these immunosuppressants. Both CsA and FK-506 have been shown to be effective in preventing organ graft rejection in the clinic (13Morris R.E. Busuttil R.W. Klintmalm G.B. Transplantation of the Liver; New Immunosuppressive Drugs. W. B. Saunders Co., Philadelphia1995: 760-786Google Scholar). In addition, CsA has been shown to be beneficial in reducing joint and disease in F. S. A. 1997; Google Scholar, J. J. K. 1993; PubMed Scopus Google Scholar). However, with the of of these and have their K. S. A. J. J. Proc. 1991; Google Scholar). are to be caused by the these binding to and inhibiting CaN in cells outside the immune system N. J. I. J. Immunol. 1996; PubMed Scopus Google Scholar, M.J. S.L. C.S. T. J. PubMed Scopus Google Scholar, F. P. M.J. S.L. B. J. 1991; PubMed Scopus Google Scholar). The identification of NFAT as a target in T cell activation a to the of immunosuppressive with the for and of the toward the identification of novel is to that target the NFAT transcription factor inhibiting the phosphatase, In this we the identification and of a novel series of NFAT that their a mechanism that not inhibition of the phosphatase, The IL-2 promoter the DNA from to J. Biol. Chem. Full Text PDF PubMed Google Scholar) was by from DNA and DNA was from expressing the DNA and the IL-2 promoter was as a and to the promoter of with the IL-2 promoter to The gene an from by and I. was I. The by with and with The was to the to cell cells with as described J.P. J. PubMed Scopus Google Scholar). for to and a cell was cells in and to cells at in and with and 2 and in in a of The cells for at in a and by The was and the cells by the of of cell 2 2 and at for of was to the cell and was with a peripheral mononuclear cells by of peripheral was from on the of The was with an of phosphate-buffered and peripheral mononuclear cells by at to at with was to and the cell for and IL-2 determined by as described of IL-2 in phosphate-buffered saline was to and at four with phosphate-buffered saline and with phosphate-buffered saline and for at or at and IL-2 was and in at was at in for 2 at and four with of IL-2 was and for at was by four and the of of of the four and of or at in was was to at for the of of 2 at was determined a IL-2 on the IL-2 of peripheral was from four on the of The was with an of phosphate-buffered and by at cells from in at for The with C for at and in in of with and and in in a of to of or of cells and of cells at for of was to the of a was by direct a compounds to on in of peripheral mononuclear cells to in cell to of A in was to a of for at with with on a on a direct cells for nuclear The cells and for at with or The cells for at with and The cells in of at The cell was in of A 2 and in of A The was in of C and of was and for in the The was at for of the was to a new and an of was and on for The nuclear was at for and in of C. The nuclear proteins by a determined by The binding with of nuclear in a of and 2 of The protein at for with of in these IL-2 promoter sites for NFAT to AP-1 to and NF-κB, The on The and on a fluorescent protein to the N-terminal regulatory domain of was and expressed in cells. of in cells was by microscopy a and a The cells, in a and maintained at the was the of a and a The was to at of to the of compounds to inhibit translocation of the nucleus. The cells with for prior to was and for also the drugs nuclear translocation of was and cells 2 for to nuclear import of or was and for to the of nuclear of was the and the was to cells. and with or for at The cells for at with the cells with and in 2 of 2 and The cell was and the four in by and was by with cells with phosphate-buffered saline and by in A and with The cell by at for at and the for in dephosphorylation assays. In phosphatase phosphorylated as a was by with and in kinase and for at in the of was with and in was with the of cell in a of of A phosphatase inhibitors as The to for at and by and by was by CaN activity was determined by the dephosphorylation of a to a in the subunit of kinase In the FK506 binding protein was in the of with was as described M.J. J. Scholar) with the subunit of protein from and with CaN in the of and FKBP12 in assay of and for at in a of The was by the of of an of at the was of was by and by as described Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In a search to novel of inhibiting IL-2 we a gene which of the T cell with the gene the transcriptional of a IL-2 promoter The of a identified BTP compounds and which inhibit IL-2 transcription with was identified in a search of the inhibit IL-2 gene activity. T cells with the gene the of the IL-2 promoter to gene activity was with and as described in the of of and are The of the BTP compounds to inhibit IL-2 gene transcription was determined in T cells to IL-2 by activation with and J.A. T. J. Immunol. Google Scholar). The BTP compounds IL-2 secretion in a with in the of The FK506, was as a in the assay and was in IL-2 secretion with an to IL-2 is a cytokine for T cell proliferation in response to or T. T. J. Immunol. Google Scholar). The BTP compounds IL-2 secretion proliferation and with to The was also in T cell proliferation in the with an to inhibit and T cell peripheral mononuclear cells and with A or by as described in the of of or proliferation was as described are The 3,5-bistrifluoromethyl pyrazole compounds identified on their to block IL-2 gene transcription in T cells in response to and IL-2 gene transcription is the activation of several transcription including NF-κB, AP-1 and NFAT (2Rothenberg E.V. Ward S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9358-9365Crossref PubMed Scopus (169) Google Scholar, 3Jain J. Loh C. Rao A. Curr. Opin. Immunol. 1995; 7: 333-342Crossref PubMed Scopus (501) Google Scholar). The activation of these transcription factors is the of and signaling regulated by the TcR and other such as The and to these T. T. J. Immunol. Google Scholar). better the BTP compounds these signaling we determined the of the BTP compounds on IL-2 transcription factors in cells. The was to transcription by the and the from the nucleus to the cytosol to the mechanism of BTP The BTP compounds the activation-dependent nuclear localization of NFAT as determined by electrophoretic mobility shift The NFAT inhibitory of was to the inhibition with CsA or FK506 The BTP compounds did not affect the of NFAT to bind directly to its IL-2 directly to the binding in not The BTP compounds selective inhibition of did not affect the nuclear or binding of NF-κB or AP-1 to their respective IL-2 elements Binding sites for NFATs have also been found within the promoter regions of several other cytokine genes, including IL-4, IL-5, and γ-IFN (4Rao A. Immunol. Today. 1994; 15: 274-281Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 5Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar). with their to inhibit NFAT the BTP compounds and secretion from activated cells and γ-IFN from peripheral T cells with of S. W. and J. in The of to block the nuclear localization of NFAT was in cells with green fluorescent protein with the N-terminal regulatory domain of As shown in cells cytoplasmic and the of nuclear of with the fluorescent to the nucleus However, in the of the movement of from the cytoplasm to the nucleus is A inhibition of nuclear localization is with CsA The nuclear localization of NFAT the activation of As Ca2+ or CaN is with the immunosuppressive drugs FK506 or NFAT is and from the nucleus L.A. J.P. Nature. 1996; PubMed Scopus Google Scholar). In cells with to cause nuclear of of BTP compounds a of from the nucleus not with cells. The movement of NFAT from the cytosol to the nucleus is the dephosphorylation of the N-terminal regulatory domain of NFAT by and as NFAT activation is by and by the immunosuppressive drugs CsA and FK506 S. F. K. E. S. PubMed Scopus Google Scholar, J. C. A. X. J. Hogan P.G. Rao A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). we determined the block in NFAT nuclear translocation was by inhibition of NFAT cells, expressing the N-terminal regulatory domain of for with was in cell by from cells with from cells the in the state of in response to J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). of cells with and the dephosphorylation of as determined by of the of The of on the NFAT state was to that with a known of the NFAT phosphatase, CaN The of to block NFAT dephosphorylation in cells caused to are of directly inhibiting the activity of CaN activity was determined by the dephosphorylation of protein kinase subunit by CaN, as described assay the was in the assay to inhibition of CaN by As shown in FK506 the dephosphorylation of in a with an of and to inhibit CaN activity in the assay at as as the for inhibition of cellular NFAT activation and IL-2 secretion and data the that not directly inhibit CaN activity. CsA and FK506 inhibit CaN activity with their respective (9Liu J. Farmer J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3633) Google Scholar). the of directly inhibiting CaN activity we the of the to inhibit CaN activity in cell to cellular factors inhibition of these an residues was as The transcription factor is a component of complex factor and regulates gene in response to a of of the domain of is by and protein has been to be a substrate of CaN, which the activation of T. S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). As shown in phosphorylated is by a and an in electrophoretic with cell The dephosphorylation of is in a by the of CsA or to the cell A and that is a CaN substrate T. S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). cell from cells for with CsA and inhibit the dephosphorylation of as T. S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). However, from cells for with or with direct of or to inhibit also to inhibit CaN activity in was as substrate dephosphorylation of NFAT by CaN is by the of these proteins CaN sites in A conserved located at the N terminus of the NFAT regulatory domain to CaN binding to NFAT, NFAT dephosphorylation J. F. A. Rao A. Hogan P.G. Cell. 1998; Full Text Full Text PDF Scopus Google Scholar). The of to block NFAT dephosphorylation that the not their by in that the BTP compounds block NFAT activation by a mechanism other than direct inhibition of CaN activity and thus novel immunosuppressive dephosphorylation by CaN in cell is not by cells with and the expressed was by was to of cell in of and at for in the or of CsA or at the with of and reducing and of was to was by as described The highly phosphorylated of NFAT have mobility in the and are with an as of NFAT have mobility and are by an as The 3,5-bistrifluoromethyl pyrazole compounds identified on their to block IL-2 gene transcription. IL-2 gene transcription is the activation of several transcription including NF-κB, AP-1 complex of and that the BTP compounds block the activation-dependent nuclear localization of The activity of NFAT proteins is tightly regulated by the phosphatase, A conserved located at the N terminus of the NFAT regulatory domain to CaN binding to NFAT, NFAT dephosphorylation J. F. A. Rao A. Hogan P.G. Cell. 1998; Full Text Full Text PDF Scopus Google Scholar). Dephosphorylation of NFAT by CaN is required for NFAT activation and nuclear localization. In this treatment of intact T cells with the BTP by CaN caused NFAT to remain in a highly phosphorylated state to the However, the BTP compounds did not directly inhibit the dephosphorylation of NFAT in nor did the drugs block the dephosphorylation of other CaN substrates including the type II regulatory subunit of protein kinase A and the transcription factor, Elk-1. the are not CaN inhibitors or inhibitors of The data suggest that the BTP compounds NFAT or inhibit NFAT dephosphorylation by a mechanism other than direct inhibition of CaN phosphatase activity. Dephosphorylation of NFAT is for import the nucleus. In resting cells, is phosphorylated on at least residues in the N-terminal regulatory domain J. C. A. X. J. Hogan P.G. Rao A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of these are activation with the calcium resulting in the of a nuclear localization within the regulatory domain and nuclear nuclear localization of NFAT the activation of As Ca2+ or CaN is with immunosuppressive drugs, CsA or NFAT is L.A. J.P. Nature. 1996; PubMed Scopus Google Scholar, C. J. C. Rao A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The of NFAT promotes the of the nuclear localization and the of a nuclear J. C. A. X. J. Hogan P.G. Rao A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In this the of BTP compounds to cells caused a of from the nucleus with cells not The the of NFAT in the cytosol by the activity the of an NFAT A of have been in the nuclear of NFATs including kinase kinase kinase kinase kinase N-terminal kinase and protein kinase C.R. P. 1997; PubMed Scopus Google Scholar, J. F. T. P. F. Cell. 1998; 93: Full Text Full Text PDF PubMed Scopus Google Scholar, M.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. S. Maldonado J.L. I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have to an of on the activity of and not However, in cells, these are regulated by kinase and to be affect the activation for these or other in NFAT The BTP compounds be from CsA by their to inhibit CaN activity in However, in cells, the BTP compounds and CsA inhibitory on NFAT activation and cytokine gene transcription. of intracellular calcium with and CsA caused to to the cytosol in a state and a of cytokine gene these is that inhibit CaN by intracellular Ca2+ The intracellular Ca2+ by be to the cellular signaling protein kinase and activation of calcium required for calcium to cellular CaN activation R.E. K. Lewis Nature. 1998; PubMed Scopus Google Scholar). affect the of CaN to NFAT in cells. has been that the with the phosphorylated of NFAT W. Lu PubMed Scopus Google Scholar). The domain of and the regulatory domain of NFAT the The binding of to NFAT inhibits the dephosphorylation of NFAT by CaN in vitro, and of in T cells activation of W. Lu PubMed Scopus Google Scholar). act by binding and thus block dephosphorylation of are to this NFAT dephosphorylation at residues for nuclear import and transcriptional activation J. C. A. X. J. Hogan P.G. Rao A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). that other in to CaN may in the regulation of NFAT the BTP compounds affect the NFAT state by a and the sites of regulatory domain in the of CsA and will be useful in this have the and of the bistrifluoromethyl pyrazole series of cytokine inhibitors with to and to inhibit IL-2 in and A. J.R. Chem. PubMed Scopus Google Scholar). these by that the BTP compounds at the of NFAT this is the of of The mechanism of action of the to be However, the data in this a mechanism of action in NFAT nuclear localization and NFAT-dependent cytokine gene expression. mechanism the from immunosuppressive drugs, CsA and may have as immunosuppressive drugs with and with CsA and FK506 of the of these compounds is to be related to inhibition of CaN in cells outside the immune system N. J. I. J. Immunol. 1996; PubMed Scopus Google Scholar, M.J. S.L. C.S. T. J. PubMed Scopus Google Scholar, F. P. M.J. S.L. B. J. 1991; PubMed Scopus Google Scholar). the novel inhibitors described herein will be useful in better defining the cellular regulation of NFAT activation and may lead to identification of new therapeutic targets for the treatment of autoimmune disease and transplant rejection.

We investigated the mechanism of increases in cyclic GMP levels in bovine superior cervical ganglion (SCG) in response to muscarinic receptor stimulation. Acetylcholine increased cyclic GMP levels in SCG. This increase was inhibited by NG-methyl-L-arginine (NMA), and the inhibition was reversed by L-arginine. Soluble nitric oxide (NO) synthase was partially purified from bovine SCG using 2',5'-ADP Sepharose affinity chromatography. The resulting enzyme activity was Ca2+/calmodulin dependent and required NADPH and tetrahydrobiopterin as cofactors. Superoxide dismutase protected and oxyhemoglobin blocked the effect of NO formed by the enzyme. NMA inhibited the activity of the NO synthase. In western blots, an antibody generated against rat brain NO synthase specifically recognized the NO synthase from SCG as a 155-kDa protein band. Immunohistochemistry using the same antibody demonstrated that NO synthase was localized in postganglionic neuronal cell bodies of the SCG. Immunofluorescent labeling showed that some of the cells staining positive for dopamine-beta-hydroxylase also contained NO synthase. Thus, NO is synthesized in specific cells within bovine SCG, including sympathetic neurons, and mediates the acetylcholine-induced stimulation of soluble guanylyl cyclase.