The cytokine interleukin-1 (IL-1) has a variety of effects in brain, including induction of fever, alteration of slow wave sleep, and alteration of neuroendocrine activity. To examine the potential sites of action of IL-1 in brain, we used iodine-125-labeled recombinant human interleukin-1 [( 125I]IL-1) to identify and characterize IL-1 receptors in crude membrane preparations of mouse (C57BL/6) hippocampus and to study the distribution of IL-1-binding sites in brain using autoradiography. In preliminary homogenate binding and autoradiographic studies, [125I]IL-1 alpha showed significantly higher specific binding than [125I]IL-1 beta. Thus, [125I]IL-1 alpha was used in all subsequent assays. The binding of [125I]IL-1 alpha was linear over a broad range of membrane protein concentrations, saturable, reversible, and of high affinity, with an equilibrium dissociation constant value of 114 +/- 35 pM and a maximum number of binding sites of 2.5 +/- 0.4 fmol/mg protein. In competition studies, recombinant human IL-1 alpha, recombinant human IL-1 beta, and a weak IL-1 beta analog. IL-1 beta +, inhibited [125I]IL-1 alpha binding to mouse hippocampus in parallel with their relative bioactivities in the T-cell comitogenesis assay, with inhibitory binding affinity constants of 55 +/- 18, 76 +/- 20, and 2940 +/- 742 pM, respectively; rat/human CRF and human tumor necrosis factor showed no effect on [125I]IL-1 alpha binding. Autoradiographic localization studies revealed very low densities of [125I]IL-1 alpha-binding sites throughout the brain, with highest densities present in the molecular and granular layers of the dentate gyrus of the hippocampus and in the choroid plexus. Quinolinic acid lesion studies demonstrated that the [125I]IL-1 alpha-binding sites in the hippocampus were localized to intrinsic neurons. After hypophysectomy, homogenate binding and autoradiographic studies showed that there was no apparent change in the relative density of IL-1 receptors in the hippocampus. The identification of IL-1 receptors in brain with characteristics similar to IL-1 receptors in immune and neuroendocrine tissues provides further support for a physiological role for IL-1 to regulate central nervous system activity.

RTI-55 (3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid methyl ester), one of the most potent inhibitors of dopamine uptake reported to date, was radioiodinated and tested as a probe for the cocaine receptor in Sprague-Dawley rat brain. Saturation and kinetic studies in the striatum revealed that [125I]RTI-55 bound to both a high- and low-affinity site. The Kd for the high-affinity site was 0.2 nM, while the Kd for the low-affinity site was 5.8 nM. The corresponding number of binding sites in the striatum was 37 and 415 pmol/g protein. The pharmacological profile of specific [125I]RTI-55 binding in the striatum was consistent with that of the dopamine transporter. Additionally, [125I]RTI-55 was found to bind with high affinity to the cerebral cortex. Scatchard analysis revealed a single high-affinity component of 0.2 nM with a density of 2.5 pmol/g protein. The pharmacological profile demonstrated by [125I]RTI-55 in the cerebral cortex matched that of the serotonin transporter. Autoradiographic analysis of sagittal brain sections with [125I]RTI-55 binding was consistent with these findings. Specific binding of [125I]RTI-55 was blocked by dopamine uptake inhibitors in areas rich in dopaminergic nerve terminals. Conversely, serotonin uptake inhibitors blocked the binding of [125I]RTI-55 in brain areas rich in serotonergic neurons. These results demonstrate that [125I]RTI-55 may be a very useful ligand for the dopamine and serotonin transporters.

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