Unitechnologies (Switzerland)

Interleukin (IL)-6 is a pro-inflammatory cytokine now widely recognized to contribute to the molecular events that follow CNS injury. Little is known, however, about its action on axonal sprouting and regeneration in the brain. We addressed this issue using the model of transection of Schaffer collaterals in mice organotypic hippocampal slice cultures. Transection of slice cultures was associated with a marked release of IL-6 that could be neutralized by an IL-6 blocking antibody. We monitored functional recovery across the lesion by recording synaptic responses using a multi-electrode array. We found that application of IL-6 antibodies to the cultures after lesioning significantly reduced functional recovery across the lesion. Furthermore, the level of expression of the 43-kDa growth-associated protein (GAP-43) was lower in slices treated with the IL-6 neutralizing antibody than in those treated with a control IgG. Conversely, addition of exogenous IL-6 to the culture medium resulted in a dose-dependent enhancement of functional recovery across the lesion and a higher level of expression of GAP-43. Co-culture of CA3 hemi-slices from thy1-YFP mice with CA1 hemi-slices from wild-type animals confirmed that IL-6-treated co-cultures exhibited an increased number of growing fluorescent fibres across the lesion site. Taken together these data indicate that IL-6 plays an important role in CNS repair mechanisms by promoting regrowth and axon regeneration.

We have investigated the interaction of fluorescent peptide ligands with the G protein-coupled receptor NK2 using novel spectrofluorometric approaches. Several heptapeptide antagonists of structure PhCO-Xaa-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 were labelled on position 1 (Xaa) with the environment-sensitive nitrobenzoxadiazole (NBD) probe, differing only in the length of the spacer between the NBD group and the peptide. Upon binding of the labelled antagonist to NK2 receptors stably expressed in Chinese hamster ovary (CHO) cells, an increase in NBD fluorescence was observed when the spacer length was less than 10 A. Collisional quenching experiments using iodide and Co2+ ions were performed to define the accessibility of the NBD group on bound ligands to the solvent. By comparing ligands with spacer arms of varying lengths, we found that the binding pocket is buried at a depth of 5-10 A. In contrast, N-terminally NBD-labelled agonists, decapeptide neurokinin A (NKA) or heptapeptide Nle10-NKA[4-10], bound to the NK2 receptor were accessible to the solvent. Binding of fluorescent ligands to the NK2 receptor was accompanied by an enhancement in the fluorescence anisotropy. The changes in fluorescence properties were used to determine the kinetic parameters of antagonist binding and dissociation. These results indicate that the binding site on the NK2 receptor for the amino-terminal end of the heptapeptide antagonists is buried in the hydrophobic pocket of the receptor protein and clearly distinct from the binding site for the amino-terminal end of agonists, which is accessible to the solvent.(ABSTRACT TRUNCATED AT 250 WORDS)