Detection, characterization and biological activities of [bisphospho-thr3,9]ODN, an endogenous molecular form of ODN released by astrocytes.

Astrocytes synthesize and release endozepines, a family of regulatory neuropeptides, including diazepam-binding inhibitor (DBI) and its processing fragments such as the octadecaneuropeptide (ODN). At the molecular level, ODN interacts with two types of receptors, i.e. it acts as an inverse agonist of the central-type benzodiazepine receptor (CBR), and as an agonist of a G protein-coupled receptor (GPCR). ODN exerts a wide range of biological effects mediated through these two receptors and, in particular, it regulates astrocyte activity through an autocrine/paracrine mechanism involving the metabotropic receptor. More recently, it has been shown that Müller glial cells secrete phosphorylated DBI and that bisphosphorylated ODN ([bisphospho-Thr(3,9)]ODN, bpODN) has a stronger affinity for CBR than ODN. The aim of the present study was thus to investigate whether bpODN is released by mouse cortical astrocytes and to compare its potency to ODN. Using a radioimmunoassay and mass spectrometry analysis we have shown that bpODN as well as ODN were released in cultured astrocyte supernatants. Both bpODN and ODN increased astrocyte calcium event frequency but in a very different range of concentration. Indeed, ODN stimulatory effect decreased at concentrations over 10(-10)M whereas bpODN increased the calcium event frequency at similar doses. In vivo effects of bpODN and ODN were analyzed in two behavioral paradigms involving either the metabotropic receptor (anorexia) or the CBR (anxiety). As previously described, ODN (100ng, icv) induced a significant reduction of food intake. Similar effect was achieved with bpODN but at a 10 times higher dose (1000 ng, icv). Similarly, and contrasting with our hypothesis, bpODN was also 10 times less potent than ODN to induce anxiety-related behavior in the elevated zero maze test. Thus, the present data do not support that phosphorylation of ODN is involved in receptor selectivity but indicate that it rather weakens ODN activity.