Synthesis and in vitro evaluation of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)-pteridines as dual immunosuppressive and anti-inflammatory agents.

Screening of a pteridine-based compound library led to the identification of compounds exhibiting immunosuppressive as well as anti-inflammatory activity. Optimization afforded a series of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)pteridine analogues. The most potent congeners in this series displayed low nM IC(50) values in the Mixed Lymphocyte Reaction (MLR) assay.

Immunosuppressive activity of a new pteridine derivative (4AZA1378) alleviates severity of TNBS-induced colitis in mice.

Besides TNF, activated T cells play a central role in the pathogenesis of inflammatory bowel diseases such as Crohn's disease. New therapies are still awaited to cure these often debilitating diseases. Natural occurring pteridines such as tetrahydrobiopterin (BH4) and neopterin have been reported to have immune modulating activities.

Anti-inflammatory activity of a pteridine derivative (4AZA2096) alleviates TNBS-induced colitis in mice.

Elevated production of tumor necrosis factor (TNF) plays a central role in the pathogenesis of many inflammatory diseases, such as rheumatoid arthritis and Crohn's disease. Naturally occurring pteridine analogs have been reported to have potent immunomodulatory activity, especially on TNF production. The aim of this study is to identify small molecule TNF inhibitiors derived from pteridine and to prove their in vivo efficacy in an inflammatory model.

A novel Ca2+-induced Ca2+ release mechanism in A7r5 cells regulated by calmodulin-like proteins.

Intracellular Ca2+ release is involved in setting up Ca2+ signals in all eukaryotic cells. Here we report that an increase in free Ca2+ concentration triggered the release of up to 41 +/- 3% of the intracellular Ca2+ stores in permeabilized A7r5 (embryonic rat aorta) cells with an EC50 of 700 nm. This type of Ca2+-induced Ca2+ release (CICR) was neither mediated by inositol 1,4,5-trisphosphate receptors nor by ryanodine receptors, because it was not blocked by heparin, 2-aminoethoxydiphenyl borate, xestospongin C, ruthenium red, or ryanodine.

Pharmacology of inositol trisphosphate receptors.

In almost all cells, cytosolic Ca(2+) is a crucial intracellular messenger, regulating many cellular processes. In non-excitable as well as in some excitable cells, Ca(2+) release from the intracellular stores into the cytoplasm is primarily initiated by the second messenger inositol 1,4,5-trisphosphate (IP(3)), which interacts with the IP(3) receptor (IP(3)R), a tetrameric intracellular Ca(2+)-release channel. This review focuses on the pharmacological modulation of the various functionally important sub-domains of the IP(3)R, including the IP(3)-binding domain, calmodulin-binding sites, adenine nucleotide-binding sites and the sites for interaction for FK506-binding proteins and other regulators.

The role of calmodulin for inositol 1,4,5-trisphosphate receptor function.

Intracellular calcium release is a fundamental signaling mechanism in all eukaryotic cells. The ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP(3)R) are intracellular calcium release channels. Both channels can be regulated by calcium and calmodulin (CaM).

Inhibition of the inositol trisphosphate receptor of mouse eggs and A7r5 cells by KN-93 via a mechanism unrelated to Ca2+/calmodulin-dependent protein kinase II antagonism.

KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, concentration-dependently and reversibly inhibited inositol 1,4,5-trisphosphate receptor (IP(3)R)-mediated [Ca(2+)](i) signaling in mouse eggs and permeabilized A7r5 smooth muscle cells, two cell types predominantly expressing type-1 IP(3)R (IP(3)R-1). KN-92, an inactive analog, was ineffective. The inhibitory action of KN-93 on Ca(2+) signaling depended neither on effects on IP(3) metabolism nor on the filling grade of Ca(2+) stores, suggesting a direct action on the IP(3)R.

Localization and function of a calmodulin-apocalmodulin-binding domain in the N-terminal part of the type 1 inositol 1,4,5-trisphosphate receptor.

Calmodulin (CaM) is a ubiquitous protein that plays a critical role in regulating cellular functions by altering the activity of a large number of proteins, including the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). CaM inhibits IP3 binding in both the presence and absence of Ca2+ and IP3-induced Ca2+ release in the presence of Ca2+. We have now mapped and characterized a Ca2+-independent CaM-binding site in the N-terminal part of the type 1 IP3R (IP3R1).

Mapping of the ATP-binding sites on inositol 1,4,5-trisphosphate receptor type 1 and type 3 homotetramers by controlled proteolysis and photoaffinity labeling.

Submillimolar ATP concentrations strongly enhance the inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, by binding specifically to ATP-binding sites on the IP(3) receptor (IP(3)R). To locate those ATP-binding sites on IP(3)R1 and IP(3)R3, both proteins were expressed in Sf9 insect cells and covalently labeled with 8-azido-[alpha-(32)P]ATP. IP(3)R1 and IP(3)R3 were then purified and subjected to a controlled proteolysis, and the labeled proteolytic fragments were identified by site-specific antibodies.

Basic properties of an inositol 1,4,5-trisphosphate-gated channel in carp olfactory cilia.

In addition to the activation of cAMP-dependent pathways, odorant binding to its receptor can lead to inositol 1,4,5-trisphosphate (InsP3) production that may induce the opening of plasma membrane channels. We therefore investigated the presence and nature of such channels in carp olfactory cilia. Functional analysis was performed by reconstitution of the olfactory cilia in planar lipid bilayers (tip-dip method).

Ca2+-calmodulin inhibits Ca2+ release mediated by type-1, -2 and -3 inositol trisphosphate receptors.

InsP(3) binding to type-1, but not type-3, InsP(3) receptors is inhibited by calmodulin in a Ca(2+)-independent fashion [Cardy and Taylor (1998) Biochem. J. 334, 447-455], and Ca(2+) mobilization by type-1 InsP(3) receptors of cerebellum is inhibited by calmodulin [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc.

Adenine-nucleotide binding sites on the inositol 1,4,5-trisphosphate receptor bind caffeine, but not adenophostin A or cyclic ADP-ribose.

Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP3R) results in a more pronounced Ca2+ release in the presence of inositol 1,4,5-trisphosphate (IP3). We have expressed the cDNAs encoding two putative adenine-nucleotide binding sites of the neuronal form of IP3R-1 as glutathione S-transferase (GST)-fusion proteins in bacteria. Specific [alpha-32P]ATP binding was observed for the two GST-fusion proteins, representing aa 1710-1850 and aa 1944-2040 of IP3R-1.

Modulation of inositol 1,4,5-trisphosphate binding to the recombinant ligand-binding site of the type-1 inositol 1,4, 5-trisphosphate receptor by Ca2+ and calmodulin.

A recombinant protein (Lbs-1) containing the N-terminal 581 amino acids of the mouse type 1 inositol 1,4,5-trisphosphate receptor (IP3R-1), including the complete IP3-binding site, was expressed in the soluble fraction of E. coli. The characteristics of IP3 binding to this protein were similar as observed previously for the intact IP3R-1.

ATP-induced Ca2+ signals in bronchial epithelial cells.

Ca2+-dependent Cl- secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators are released. The mechanism of intracellular Ca2+ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o-. Experiments on both intact and permeabilized cells revealed that only inositol 1,4,5-trisphosphate (InsP3) receptors and not ryanodine receptors are involved in intracellular Ca2+ release.

Functional properties of the type-3 InsP3 receptor in 16HBE14o- bronchial mucosal cells.

The type-3 inositol 1,4,5-trisphosphate (InsP3) receptor is the major isoform expressed in 16HBE14o- cells from bronchial mucosa, representing 93% at the mRNA level as determined by ratio reverse transcription-polymerase chain reaction and about 81% at the protein level as determined with isoform-specific antibodies (Sienaert, I., Huyghe, S., Parys, J.

Inhibition of inositol trisphosphate-induced calcium release by cyclic ADP-ribose in A7r5 smooth-muscle cells and in 16HBE14o- bronchial mucosal cells.

Ca2+ release from intracellular stores occurs via two families of intracellular channels, each with their own specific agonist: Ins(1, 4,5)P3 for the Ins(1,4,5)P3 receptor and cyclic ADP-ribose (cADPR) for the ryanodine receptor. We now report that cADPR inhibited Ins(1, 4,5)P3-induced Ca2+ release in permeabilized A7r5 cells with an IC50 of 20 microM, and in permeabilized 16HBE14o- bronchial mucosal cells with an IC50 of 35 microM. This inhibition was accompanied by an increase in specific [3H]Ins(1,4,5)P3 binding.

Molecular and functional evidence for multiple Ca2+-binding domains in the type 1 inositol 1,4,5-trisphosphate receptor.

Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) by Ca2+. To define the structural determinants for Ca2+ binding, cDNAs encoding GST fusion proteins that covered the complete linear cytosolic sequence of the InsP3R-1 were expressed in bacteria. The fusion proteins were screened for Ca2+ and ruthenium red binding through the use of 45Ca2+ and ruthenium red overlay procedures.

Synergism between hypotonically induced calcium release and fatty acyl-CoA esters induced calcium release from intracellular stores.

The non-mitochondrial Ca2+ stores in permeabilized A7r5 cells responded to a decrease in Mg-ATP concentration with a pronounced Ca2+ release if 20 microM CoA was present. This release was rather specific for the preincubation or removal of ATP. ATP gamma S was much less effective and AMP-PNP, GTP, ITP, CTP, UTP, ADP, AMP, adenosine and adenine had no effect.

Effect of adenine nucleotides on myo-inositol-1,4,5-trisphosphate-induced calcium release.

The effects of a whole series of adenine nucleotides on Ins(1,4,5)P3-induced Ca2+ release were characterized in permeabilized A7r5 smooth-muscle cells. Several adenine nucleotides activated the Ins(1, 4,5)P3 receptor. It was observed that 3'-phosphoadenosine 5'-phosphoulphate, CoA, di(adenosine-5')tetraphosphate (Ap4A) and di(adenosine-5')pentaphosphate (Ap5A) were more effective than ATP.

Slow kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release: is the release 'quantal' or 'non-quantal'?

Inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from intracellular stores is generally assumed to be a 'quantal' process because low InsP3 concentrations mobilize less Ca2+ than high concentrations and a submaximal concentration does not release all the InsP3-mobilizable Ca2+. However, some recent reports questioned the generally accepted view that a low dose of InsP3 is unable to empty the whole store. We have now challenged the stores of permeabilized A7r5 cells in InsP3 for much longer periods than previously reported, to assess directly whether the slow phase of the release would empty the whole store (a non-quantal response) or only a fraction of it (a quantal response).

Isoform diversity of the inositol trisphosphate receptor in cell types of mouse origin.

Previous reports suggested the expression of four or five different Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] isoforms in mouse cells [Ross, Danoff, Schell, Snyder and Ullrich (1992) Proc. Natl. Acad.

Characterization of a cytosolic and a luminal Ca2+ binding site in the type I inositol 1,4,5-trisphosphate receptor.

To study the Ca2+ regulation of the inositol 1,4,5-trisphosphate receptor (InsP3R) at the molecular level, we expressed various cytosolic and luminal regions of the mouse type I InsP3R as glutathione S-transferase fusion proteins. 45Ca2+ and ruthenium red overlay studies and Stains-all spectra and staining revealed both a cytosolic and a luminal Ca2+ binding site. The luminal Ca2+ binding site was mapped to the nonconserved acidic subregion of the luminal loop between amino acids 2463 and 2528.

Mechanisms responsible for quantal Ca2+ release from inositol trisphosphate-sensitive calcium stores.

Activation of cells by hormones, growth factors or neurotransmitters leads to an increased production of inositol trisphosphate (InsP3) and, after activation of the InsP3 receptor (InsP3R), to Ca2+ release from intracellular Ca2+ stores. The release of intracellular Ca2+ is characterised by a graded response when submaximal doses of agonists are used. The basic phenomenon, called "quantal Ca2+ release", is that even the maintained presence of a submaximal dose of agonist or of InsP3 for long time periods (up to 20 min) provokes only a partial release of Ca2+.

Threshold for inositol 1,4,5-trisphosphate action.

We developed a unidirectional 45Ca2+ efflux technique in which 60 cumulative doses of inositol 1,4,5-trisphosphate (InsP3), each lasting 6 s, were subsequently added to permeabilized A7r5 cells. This technique allowed an accurate determination of the threshold for InsP3 action, which was around 32 nM InsP3 under control conditions. The InsP3-induced Ca2+ release was characterized by an initial rapid phase, after which the normalized rate progressively decreased.

Hypotonically induced calcium release from intracellular calcium stores.

Osmotic cell swelling induced by hypotonic stress is associated with a rise in intracellular Ca2+ concentration, which is at least partly due to a release of Ca2+ from internal stores. Since osmotic influx of water dilutes the cytoplasmic milieu, we have investigated how nonmitochondrial Ca2+ stores in permeabilized A7r5 cells respond to a reduction in cytoplasmic tonicity. We now present experimental evidence for a direct Ca2+ release from the stores when exposed to a hypotonic medium.