Spinal ephrinB/EphB signalling contributed to remifentanil-induced hyperalgesia via NMDA receptor.

BACKGROUND: One of the major unresolved issues in treating pain is the paradoxical hyperalgesia produced by opiates, and accumulating evidence implicate that EphBs receptors and ephrinBs ligands are involved in mediation of spinal nociceptive information and central sensitization, but the manner in which ephrinB/EphB signalling acts on spinal nociceptive information networks to produce hyperalgesia remains enigmatic. The objective of this research was to investigate the role of ephrinB/EphB signalling in remifentanil-induced hyperalgesia (RIH) and its downstream effector. METHODS: We characterized the remifentanil-induced pain behaviours by evaluating thermal hyperalgesia and mechanical allodynia in a rat hind paw incisional model. Protein expression of EphB1 receptor and ephrinB1 ligand in spinal dorsal horn cord was determined by Western blotting, and Fos was determined by immunohistochemistry assay, respectively. To figure out the manner in which ephrinB/EphB signalling acts with N-methyl-d-aspartic acid (NMDA) receptor, we used MK-801, an antagonist of NMDA receptor, trying to suppressed the hyperalgesia induced by ephrinB1-Fc, an agonist of ephrinB/EphB. RESULTS: Continuing infusion of remifentanil produced a thermal hyperalgesia and mechanical allodynia, which was accompanied with increased protein expression of spinal-level EphB1 receptor, ephrinB1 ligand and Fos; what appeared above was suppressed by pretreatment with EphB1-Fc, an antagonist of ephrinB/EphB or MK-801, and increased pain behaviours induced by intrathecal injection of ephrinB1-Fc, an agonist of ephrinB/EphB, were suppressed by MK-801. CONCLUSIONS: Our findings indicated that ephrinB/EphB signalling is involved in RIH. EphrinB/EphB signalling might be the upstream of NMDA receptor.

Interaction between Ephrins and mGlu5 metabotropic glutamate receptors in the induction of long-term synaptic depression in the hippocampus.

We applied the group-I metabotropic glutamate (mGlu) receptor agonist, 3,5-dihydroxyphenylglycine (DHPG), to neonatal or adult rat hippocampal slices at concentrations (10 microM) that induced a short-term depression (STD) of excitatory synaptic transmission at the Schaffer collateral/CA1 synapses. DHPG-induced STD was entirely mediated by the activation of mGlu5 receptors because it was abrogated by the mGlu5 receptor antagonist, MPEP [2-methyl-6-(phenylethynyl)pyridine], but not by the mGlu1 receptor antagonist, CPCCOEt [7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester]. Knowing that ephrin-Bs functionally interact with group-I mGlu receptors (Calò et al., 2005), we examined whether pharmacological activation of ephrin-Bs could affect DHPG-induced STD. We activated ephrin-Bs using their cognate receptor, EphB1, under the form of a preclustered EphB1/Fc chimera. Addition of clustered EphB1/Fc alone to the slices induced a small but nondecremental depression of excitatory synaptic transmission, which differed from the depression induced by 10 microM DHPG. Surprisingly, EphB1/Fc-induced synaptic depression was abolished by MPEP (but not by CPCCOEt) suggesting that it required the endogenous activation of mGlu5 receptors. In addition, coapplication of DHPG and EphB1/Fc, resulted in a large and nondecremental long-term depression. The effect of clustered EphB1/Fc was specific because it was not mimicked by unclustered EphB1/Fc or clustered EphA1/Fc. These findings raise the intriguing possibility that changes in synaptic efficacy mediated by mGlu5 receptors are under the control of the ephrin/Eph receptor system, and that the neuronal actions of ephrins can be targeted by drugs that attenuate mGlu5 receptor signaling.

Proangiogenic role of ephrinB1/EphB1 in basic fibroblast growth factor-induced corneal angiogenesis.

Corneal neovascularization is a vision-threatening condition caused by various ocular pathological conditions. The aim of this study was to evaluate the function of the ephrin ligands and Eph receptors in vitro and in vivo in corneal angiogenesis in a mouse model. The Eph tyrosine kinase receptors and their ligands, ephrins, are expressed on the cell surface. The functions of Eph and ephrins have been shown to regulate axonal guidance, segmentation, cell migration, and angiogenesis. Understanding the roles of Eph and ephrin in corneal angiogenesis may provide a therapeutic intervention for the treatment of angiogenesis-related disorders. Immunohistochemical studies demonstrated that ephrinB1 and EphB1 were expressed in basic fibroblast growth factor (bFGF)-induced vascularized corneas. EphB1 was specifically colocalized with vascular endothelial marker CD31 surrounded by type IV collagen. EphrinB1 was expressed in corneal-resident keratocytes and neutrophils. Recombinant ephrinB1-Fc, which induces EphB receptor activation, enhanced bFGF-induced tube formation in an in vitro aortic ring assay and promoted bFGF-induced corneal angiogenesis in vivo in a corneal pocket assay. Synergistically enhanced and sustained activation of extracellular signal-regulated kinase was noted in vascular endothelial cell lines after stimulation with ephrin B1 and bFGF combinations. These results suggest that ephrinB1 plays a synergistic role in corneal neovascularization.

Crystal structure of the ephrin-B1 ectodomain: implications for receptor recognition and signaling.

Eph receptors and their ephrin ligands are involved in various aspects of cell-cell communication during development, including axonal pathfinding in the nervous system and cell-cell interactions of the vascular endothelial cells. Recent structural studies revealed unique molecular features, not previously seen in any other receptor-ligand families, and explained many of the biochemical and signaling properties of Ephs and ephrins. However, unresolved questions remain regarding the potential oligomerization and clustering of these important signaling molecules. In this study, the biophysical properties and receptor-binding preferences of the extracellular domain of ephrin-B1 were investigated and its crystal structure was determined at 2.65 A resolution. Ephrin-B1 is a monomer both in solution and in the crystals, while it was previously shown that the closely related ephrin-B2 forms homodimers. The main structural difference between ephrin-B1 and ephrin-B2 is the conformation of the receptor-binding G-H loop and the partially disordered N-terminal tetramerization region of ephrin-B1. The G-H loop is structurally rigid in ephrin-B2 and adopts the same conformation in both the receptor-bound and unbound ligand, where it mediates receptor-independent homodimerization. In the ephrin-B1 structure, on the other hand, the G-H loop is not involved in any homotypic interactions and adopts a new, distinct conformation. The implications of the ephrin-B1 structure, in context of available ephrin-B1 mutagenesis data, for the mechanism of Eph-ephrin recognition and signaling initiation are discussed.