Zeta (ξ), a growth-related opioid receptor in developing rat cerebellum: identification and characterization

Endogenous opioids and opioid receptors (i.e. endogenous opioid systems) are expressed during neural ontogeny, and play a role in the development of the nervous system. Using [³H][Met⁵]-enkephalin, a potent ligand involved in neural growth, particularly cell proliferation, specific and saturable binding was detected in homogenates of 6-day-old rat cerebellum; the data were consistent with a single binding site. Scatchard analysis yielded a binding affinity (K_d) of 2.2 nM and a binding capacity (B_max) of 22.3 fmol/mg protein. Binding was linear with protein concentration, dependent on time, temperature, and pH, and was sensitive to Na⁺, Mg²⁺, and guanyl nucleotides. Optimal binding required protease inhibitors, and pretreatment of the homogenates with trypsin markedly reduced binding, suggesting that the binding site was proteinaceous in character. The [Met⁵]-enkephalin binding site was an integral membrane protein located in the nuclear fraction. Competition experiments indicated that [Met⁵]-enkephalin was the most potent displacer of [³H][Met⁵]-enkephalin, and that binding was stereospecific. In the adult rat cerebellum, non-opioid receptor binding of [³H][Met⁵]-enkephalin was recorded. μ and κ receptors were also found in the developing rat cerebellum, while μ, δ, and κ receptors were recorded in adult cerebellar tissue. The function, pharmacological and biochemical characteristics, subcellular distribution, and temporal expression of the [Met⁵]-enkephalin binding site suggest the presence of a unique opioid receptor, termed zeta (ξ), in the developing nervous system.