We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'-[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743 -7481. To investigate whether this stimulation was due to a soluble c1 subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein By dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that By subunits, purified from bovine retinal transducin (By,), markedly stimulated the hydrolysis of phosphatidylinositol4,Sbisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by by, was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin a subunit and was additive to activation of phospholipase C by guanosine 5'-[3-0-thio]triphosphate. byt also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL-60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to By,, but was also observed with highly purified subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in granulocytes. Only one of these forms was sensitive to stimulation by by,, demonstrating that stimulation of phospholipase C by by subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein By subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins.The hydrolysis by phospholipase C of phosphatidylinositol 4,5-bisphosphate (PtdInsPJ to inositol 1,4,5-trisphosphate (InsP,) and diacylglycerol is a key mechanism by which many extracellular signalling molecules (hormones, growth factors and neurotransmitters) regulate the functions of their target cells [l, 21. There is ample evidence to suggest that many of the receptors interacting with these mediators stimulate phospholipase C via a guanine-nucleotide-binding protein (G protein) [3-51. In certain cell types, e.g.

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor (GPCR) superfamily and mediate several physiological responses, such as blood pressure, food intake, sedation and memory retention. To understand the interactions between the NPY Y1 receptor subtype and its ligands, computer modeling was applied to the natural peptide agonist, NPY and a small molecule antagonist, BIBP3226. An agonist and antagonist binding domain was elucidated using mutagenesis data for the Y1 receptor as well as for other GPCR families. The agonist and antagonist ligands which were investigated appear to share common residues for their interaction within the transmembrane regions of the Y1 receptor structure, including Gln120, Asn283 and His306. This is in contrast to findings with tachykinin receptors where the binding domains of the non-peptide antagonists have very little in common with the binding domains of the agonist, substance-P. In addition, a hydrogen bond between the hydroxyl group of Tyr36 of NPY and the side chain of Gln219, an interaction that is absent in the model complex between Y1 and the antagonist BIBP3226, is proposed as one of the potential interactions necessary for receptor activation.

To investigate the role of phospholipase C (PLC) in inflammatory processes, we tested 1-(6-((17␤-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), a widely used PLC inhibitor, in several in vitro and in vivo assays. We first examined the effects of U73122 on human phospholipase C-␤ (PLC-␤) isozymes and found that U73122 significantly inhibited recombinant human PLC-␤2, with an IC 50 of ϳ6 M. U73122 had little effect on PLC-␤1, PLC-␤3, or PLC-␤4. Consistent with its ability to inhibit PLC-␤2 enzymatic activity, U73122 reduced interleukin-8 and leukotriene B 4 -induced Ca 2ϩ flux and chemotaxis in human neutrophils in a concentration-dependent manner. In vivo, U73122 blocked carrageenan-induced hind paw edema in rats, carrageenan-induced macrophage and lymphocyte accumulation into subcutaneous chambers in dogs, lipopolysaccharide-induced macrophage, lymphocyte infiltration and prostaglandin E 2 production in a mouse peritonitis model, and 12-O-tetradecanoylphorbol-13-acetate-induced ear edema in mice. These results implicate PLC-dependent signaling pathways in the development of acute and chronic inflammatory responses in vivo.

A series of substituted dipiperidine compounds have been synthesized and identified as selective CCR2 antagonists. Combining the most favorable substituents led to the discovery of remarkably potent CCR2 antagonists displaying IC50 values in the nanomolar range. Compound 7a had outstanding selectivity over CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, and CCR8 and showed excellent efficacy in adjuvant-induced arthritis model, collagen-induced arthritis model, and allergic asthma model.

Myeloid differentiated human leukaemia (HL-60) cells contain a soluble phospholipase C that hydrolysed phosphatidylinositol 4.5-bisphosphate and was markedly stimulated by the metabolically stable GTP analogue guanosine 5′-[gamma-thio]triphosphate (GTP[S]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTP[S] occurred at 1.5 microM and 30 microM respectively. Other nucleotides (GTP, GDP, GMP, guanosine 5′-[beta-thio]diphosphate. ATP, adenosine 5′-[gamma-thio]triphosphate, UTP) did not affect phospholipase C activity, GTP[S] stimulation of inositol phosphate accumulation was inhibited by excess GDP, but not by ADP. The effect of GTP[S] on inositol phosphate formation was absolutely dependent on and markedly stimulated by free Ca2+ (median effective concn. approximately 100 nM). Analysis of inositol phosphates by anion-exchange chromatography revealed InsP3 as the major product of GTP[S]-stimulated phospholipase C activity. In the absence of GTP[S], specific phospholipase C activity was markedly decreased when tested at high protein concentrations, whereas GTP[S] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTP[S]-stimulated inositol phosphate formation were linear with time whether studied at low or high protein concentration, these results suggest that (a) phospholipase C is under an inhibitory constraint and (b) GTP[S] relieves this inhibition, most likely by activating a soluble GTP-binding protein.