Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells. Calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R) and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway. Activation of cGKI decreases IP3-stimulated elevations in intracellular calcium, induces smooth muscle relaxation and contributes to the antiproliferative and pro-apoptotic effects of NO/cGMP. Here we show that, in microsomal smooth muscle membranes, cGKIbeta phosphorylated the IP3R and cGKIbeta, and a protein of relative molecular mass 125,000 which we now identify as the IP3R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIbeta, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIbeta in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.

Signal transduction via NO/cGMP/cGKI 1 is involved in a variety of cellular mechanisms including smooth muscle contractility and platelet aggregation (1-4). cGKI affects smooth muscle tone by either decreasing the release of calcium from InsP 3 -sensitive stores (5-9) or by reducing calcium sensitivity of the contractile elements (10, 11). During the last years, several mechanisms were proposed for the action of cGKI mediating these effects. A decrease of the cytosolic calcium concentration by cGKI might involve reduced InsP 3 synthesis (12-15), enhanced calcium re-uptake by intracellular stores via CaATPase (16), or inhibition of calcium release via the InsP 3 R (17). The molecular mechanisms for these different possible intracellular calcium regulation pathways were only partly resolved up to now.Recently, we identified a 125-kDa cGKI substrate protein which was designated as inositol 1,4,5-trisphophate receptorassociated cGMP kinase substrate (IRAG). IRAG which is phosphorylated by cGKI is associated in a macromolecular complex with cGKI␤ and InsP 3 RI in smooth muscle (9). The observed perinuclear localization of heterologously expressed IRAG suggested the potential role of IRAG as a modulator of calcium release from intracellular stores. Indeed, functional studies revealed that IRAG inhibits InsP 3 -induced calcium release after activation of cGKI␤ with 8-pCPT-cGMP in COS-7 cells (9). However, the precise mechanism by which IRAG influences calcium release is still unknown.In the present study we investigated the molecular determinants for the interaction of IRAG and cGKI. It is shown that IRAG interacts specifically with the amino-terminal region containing the leucine zipper of cGKI␤. Phosphorylation of Ser 696 of IRAG is essential for the inhibition of InsP 3 -induced calcium release. EXPERIMENTAL PROCEDURESMaterials-The yeast strain EGY48 (MAT␣, his3, trp1, ura3, lex-A opx6 Leu2) and the yeast expression plasmids pEG202, pJG4-5, and pSH18-34 were used for the two-hybrid screen. Yeast media and dropout media lacking the appropriate amino acids were obtained from CLONTECH (Heidelberg, Germany) and Difco (Hamburg, Germany), respectively. The full-length rat cDNAs of the neuronal InsP 3 RI (S1Ϫ/ S2ϩ) (18) and the peripheral InsP 3 RI (S1Ϫ/S2Ϫ) (19) were a gift from Dr. Ilya Bezprozvanny (Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX).Antibodies-A polyclonal antibody specific for IRAG, raised in rabbits against recombinant IRAG 53-499 expressed in bacteria, was used for Western blot analysis at a dilution of 1:1000. Further antibodies were directed against cGKI (8) and InsP 3 RI (ABR Biochemicals).Plasmid Construction of Baits and Preys-The bovine IRAG cDNA was the template of all IRAG constructs (baits) which were synthesized by PCR. The generated IRAG amplicons were purified and inserted into the BamHI/EcoRI-digested expression plasmid pEG202 (20) in-frame with the DNA-binding domain of LexA, yielding pEG202-LexA/IRAG. The full-length and truncated PCR amplicons of bo...

4SC-101 is a novel immunosuppressive drug with excellent potential for the treatment of intestinal inflammation.

Immunosuppressive treatments of systemic lupus (SLE) remain associated with significant toxicities; hence, compounds with better toxicity profiles are needed. Dihydroorotate dehydrogenase (DHODH) inhibition with leflunomide has proven to be effective in autoimmune diseases including SLE, but leflunomide can cause a variety of side effects. We hypothesized that 4SC-101, a novel DHODH inhibitor with a more favorable toxicity profile, would be as effective as high-dose cyclophosphamide (CYC) in controlling experimental SLE of female MRL(Fas)lpr mice. Daily oral gavage of 30, 100, and 300 mg/kg 4SC-101 from 12 to 22 weeks of age was compared with either vehicle or CYC treatment (30 mg/kg/ week, i.p.) in terms of efficacy and toxicity. Three hundred milligrams per kilogram 4SC-101 was as effective as CYC in depleting spleen autoreactive T cells, B cells, and plasma cells as well as the respective DNA and RNA serum autoantibodies. This was associated with a comparable amelioration of the renal , dermal , and pulmonary SLE manifestations of MRL(Fas)lpr mice. However , even the highest dose of 4SC-101 had no effect on bone marrow neutrophil counts , which were significantly reduced in CYC-treated mice. Together , the novel DHODH inhibitor 4SC-101 is as effective as high dose CYC in controlling SLE without causing myelosuppression. Hence , DHODH inhibition with 4SC-101 might be suitable to treat active SLE with fewer side effects than CYC. (Am J Pathol

This trial provides first evidence of clinical efficacy of vidofludimus in IBD. Although the safety and tolerability profile seems favorable, long-term controlled studies are needed to further investigate its potential as novel IBD therapy.