Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models.

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.

Evidence that the proposed novel human "neurokinin-4" receptor is pharmacologically similar to the human neurokinin-3 receptor but is not of human origin.

There have been proposals that the tachykinin receptor classification should be extended to include a novel receptor, the "neurokinin-4" receptor (NK-4R), which has a close homology with the human NK-3 receptor (hNK-3R). We compared the pharmacological and molecular biological characteristics of the hNK-3R and NK-4R. Binding experiments, with (125)I-[MePhe(7)]-NKB binding to HEK 293 cell membranes transiently expressing the hNK-3R (HEK 293-hNK-3R) or NK-4R (HEK 293-NK-4R), and functional studies (Ca(2+) mobilization in the same cells) revealed a similar profile of sensitivity to tachykinin agonists and antagonists for both receptors; i.e., in binding studies with the hNK-3R, MePhe(7)-NKB > NKB > senktide >> NKA = Substance P; with the NK-4R, MePhe(7)-NKB > NKB = senktide >> Substance P = NKA; and with antagonists, SB 223412 = SR 142801 > SB 222200 >> SR 48968 >> CP 99994 for both hNK-3R and NK-4R. Thus, the pharmacology of the two receptors was nearly identical. However, attempts to isolate or identify the NK-4R gene by using various molecular biological techniques were unsuccessful. Procedures, including nested polymerase chain reaction studies, that used products with restriction endonuclease sites specific for either hNK-3R or NK-4R, failed to demonstrate the presence of NK-4R in genomic DNA from human, monkey, mouse, rat, hamster, or guinea pig, and in cDNA libraries from human lung, brain, or heart, whereas the hNK-3R was detectable in the latter libraries. In view of the failure to demonstrate the presence of the putative NK-4R it is thought to be premature to extend the current tachykinin receptor classification.

Identification and molecular characterization of rat CXCR3: receptor expression and interferon-inducible protein-10 binding are increased in focal stroke.

We describe here the cloning and characterization of a rat homolog of the chemokine receptor CXCR3. The predicted amino acid sequence of rat CXCR3 contains 367 amino acid residues, sharing 96 and 87% amino acid sequence identity to the murine and human CXCR3, respectively. Among a large panel of chemokines tested, only interferon-inducible protein-10 (IP-10), interferon-gamma-induced monokine, and interferon-inducible T cell alpha-chemoattractant demonstrated specific abilities to induce an intracellular calcium mobilization response in human embryonic kidney 293 cells transfected with rat CXCR3 expression vector. (125)I-IP-10 competition binding studies to the CXCR3-transfected human embryonic kidney 293 cells demonstrated that human IP-10 and interferon-inducible T cell alpha-chemoattractant are more potent ligands than human interferon-gamma-induced monokine. Following our previous observation for the induced expression of IP-10 in focal stroke, we demonstrate here the time-dependent up-regulation of CXCR3 mRNA in the rat ischemic cortex after permanent occlusion of the middle cerebral artery. A significant increase in (125)I-IP-10-specific binding to ischemic cerebral cortical samples was obtained and paralleled the increase in CXCR3 mRNA expression. The changes in receptor expression and ligand binding correlate highly with known changes in leukocyte accumulation, and gliosis occurred after focal stroke. These data suggest that CXCR3/IP-10 may be a potential novel therapeutic target in focal stroke. In addition, the cloning of rat CXCR3 provides an important tool for the investigation of the pathophysiological role of CXCR3 in other rodent disease models.

Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14.

Urotensin-II (U-II) is a vasoactive 'somatostatin-like' cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.

Melanin-concentrating hormone is the cognate ligand for the orphan G-protein-coupled receptor SLC-1.

The underlying causes of obesity are poorly understood but probably involve complex interactions between many neurotransmitter and neuropeptide systems involved in the regulation of food intake and energy balance. Three pieces of evidence indicate that the neuropeptide melanin-concentrating hormone (MCH) is an important component of this system. First, MCH stimulates feeding when injected directly into rat brains; second, the messenger RNA for the MCH precursor is upregulated in the hypothalamus of genetically obese mice and in fasted animals; and third, mice lacking MCH eat less and are lean. MCH antagonists might, therefore, provide a treatment for obesity. However, the development of such molecules has been hampered because the identity of the MCH receptor has been unknown until now. Here we show that the 353-amino-acid human orphan G-protein-coupled receptor SLC-1 expressed in HEK293 cells binds MCH with sub-nanomolar affinity, and is stimulated by MCH to mobilize intracellular Ca2+ and reduce forskolin-elevated cyclic AMP levels. We also show that SLC-1 messenger RNA and protein is expressed in the ventromedial and dorsomedial nuclei of the hypothalamus, consistent with a role for SLC-1 in mediating the effects of MCH on feeding.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.

Evidence that the receptor for C4a is distinct from the C3a receptor.

The cDNAs encoding the human (hC3aR) and mouse C3a receptors (mC3aR) were functionally expressed in RBL-2H3 cells. A calcium mobilization assay was utilized to assess the biologic activity of human anaphylatoxins, and C3a synthetic peptide agonists on hC3aR and mC3aR cells and this activity was compared to the activity of the anaphylatoxins on human neutrophils. Both hC3aR and mC3aR cells responded in a concentration-dependent manner with a robust calcium mobilization response to C3a with 50% effective concentrations (EC50s) of 0.24 nM and 1.3 nM, respectively. The response obtained with hC3aR cells was similar to the response elicited by C3a on human neutrophils (EC50 0.77 nM). The potency of a C3a analogue synthetic peptide (WWGKKYRASKLGLAR), derived from the fifteen carboxy-terminal residues (63-77) of C3a, relative to C3a, in stimulating calcium mobilization differed on cells expressing the human vs. mouse receptors. While the peptide was approximately 10 fold less active than C3a in stimulating calcium mobilization on cells expressing the hC3aR (EC50 2.0 nM), the peptide was essentially equipotent to the native ligand when tested on cells expressing the mC3aR. Data obtained with C4a, purified from activated serum, were difficult to interpret due to possible trace contamination of the C4a with C5a. Subsequently, an alternative C4a isolation scheme was utilized, via cleavage in vitro of purified C4. Concentrations of this latter C4a preparation, of up to 3.3 microM, had no effect on calcium mobilization in human neutrophils or in cells stably expressing the cloned C3a receptors, an indication that C4a does not interact with the C3a receptor.