Sigma receptors are saturable, high affinity binding sites for several important classes of psychotropic drugs including typical antipsychotic, antidepressant, anticonvulsant, and psychotomimetic compounds. They are likely to contribute to the beneficial and/or side-effect profile of these compounds. Sigma sites are present in high density in a number of tumor cell lines, and thus may play some role in carcinogenesis. We have investigated signal transduction mechanisms possibly utilized by sigma receptors, characterized a novel sigma-like binding site, and developed several new high affinity ligands for sigma sites which will be useful as agonists, antagonists, and imaging agents. 1) Effects of sigma ligands on intracellular calcium levels : Changes in intracellular free Ca++ ([Ca++]i) were measured in single human SK- N-SH neuroblastoma cells and rat cerebellar granule cells by the indo fluorescence ratio method. In SK-N-SH cells, sigma ligands produced immediate, dose-dependent increases in [Ca++]i. This effect was blocked by thapsigargin-pretreatment but not by removal of extracellular Ca++, indicating release from intracellular stores. In cerebellar granule cells, the effect of sigma ligands on depolarization-induced Ca++ entry was examined. KCl (55 mM) depolarized cells, producing a large """"""""spike"""""""" of [Ca++]i. Sigma ligands, when added to cells prior to addition of KCl, dose- dependently blocked the KCl-induced rise in [Ca++]i. For both effects, ligands lacking sigma affinity or ligands selective for sigma-1 receptors had little or no effect. Sigma-2 receptors may therefore be capable of signalling intracellular Ca++ release and of modulating voltage-gated Ca++ channels. Calcium may thus play a role in the cellular effects of sigma compounds. 2) Development of a Tc-99m-based sigma ligand suitable for tumor imaging: The high density of sigma receptors on many tumor cell lines and in biopsy samples of human breast tumors suggest that sigma receptors may be useful targets for the development of tumor imaging agents. We have developed a potential Tc-99m imaging agent by incorporating the N-methyl-2-piperidinyl ethylamine sigma receptor pharmacophore into a bisaminothiol chelate, which is known to form highly stable, neutral, and lipophilic complexes with Tc-99m. The Tc- 99m complex bound with high affinity to sites in T47D breast carcinoma cells with the pharmacological profile of sigma receptors. Furthermore, the Tc-99g derivative displaced [3H]DTG from guinea pig brain membranes with a Ki = 42.7 +/- 8.57 nM, further supporting labeling of sigma sites by the Tc-99m radioligand in tumor cells. Further characterization of this probe is underway in order to asses its utility in the diagnosis of cancer.
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