Linda Werling - US grants

The objectives of this application are to investigate the roles of phencyclidine (PCP) and sigma receptors in various physiological processes, including regulation of catecholamine release, activation or inhibition of second messenger systems, and relationship to excitatory amino acid neurotransmitter systems. Although once thought to be a single receptor type due to the non-selectivity of the original drugs used to characterize sigma and PCP receptor-mediated effects, it is now clear that the two receptors are separate entities, based upon several lines of evidence including differential localization. PCP remains a major drug of abuse, although often the sensations it produces are extremely unpleasant. The unique sequelae elicited by PCP and benzomorphans, which interact with PCP and sigma receptors are markedly similar to symptoms of schizophrenia. The development of antagonists for these two receptor types could provide the basis for treatment of PCP abuse and schizophrenia. A clinically used compound thought to exert its antipsychotic properties at least partially through its antagonist action at the sigma receptor is haloperidol, originally identified as a dopamine antagonist. The association of sigma receptors with dopaminergic systems in some brain areas may factor into the "dopamine hypothesis of schizophrenia". There are at present no known antagonists for the PCP receptor, but a major advance in understanding the physiological function of this site has been its localization within the cation channel activated selectively by the excitatory amino acid NMDA. Whether this is the only location of the PCP receptor remains to be determined. The widespread localization of the two receptor types within the brain is suggestive of major significance in neurological processes, but relatively few of these have been characterized, especially those affected by sigma receptor activation. This project will concentrate on exploring the actions of the two receptor types in tissues where they have been identified and there is evidence of their activity. The P.I. has preliminary evidence suggesting that both sigma and PCP agonists inhibit the NMDA-stimulated release of radiolabeled dopamine from guinea pig striatum. Attempts will be made to separate actions mediated through the two receptor types by use of selective antagonists for sigma receptors. Results will be compared to those found for NMDA-stimulated release of radiolabeled norepinephrine from hippocampus. Radioligand binding will be used to investigate potential relationship of sigma to NMDA receptors compared to that described for PCP and NMDA receptors. Cerebellar granule or other cell cultures will be used to explore sigma and PCP receptor effects on several second messenger systems in the absence of potential complication by an intact neural network. Finally, an attempt to localize sigma and PCP receptors on interneurons or catecholaminergic terminals will be made utilizing tetrodotoxin and selective interneuron transmitter antagonists.

DESCRIPTION: Physiological effects produced by ingestion of the drug of abuse phencyclidine (PCP) are mediated by PCP acting through several receptors, ion channels, and neurotransmitter reuptake transporters. Among the receptors activated by PCP is the sigma receptor, located in motor and limbic areas of rodent, non-human primate, and human brains. In rodent striatum, nucleus accumbens, prefrontal cortex, hippocampus and cerebellum, we have shown that one role of the sigma receptors is regulation of catecholamine release. The catecholamine dopamine is critical to reinforcement, movement, and psychosis; the catecholamine norepinephrine is involved in mood stability, attention, and cognition. Interference with these neurotransmitter systems by PCP acting through the sigma receptor might be expected to contribute to the psychosis and dysphoria resulting from PCP abuse. Several second messengers involved in the stimulation of catecholamine release from brain tissue and cells have been identified, but nothing is known about the signal transduction pathways mediating sigma receptor inhibition of catecholamine release. In this project, the relationships among sigma receptor agonist binding, activation or inhibition of second messengers, and release of catecholamines will be established in cell models, including the human neuroblastoma cells SK-N-SH and SH-SY5Y for studies on regulation of norepinephrine release, and in rat pheochromocytoma (PC12) cells for regulation of dopamine release. These cell lines have proved valuable in understanding catecholamine release mechanisms in the past, and many second messenger systems have been studied in them. In some experiments not involving catecholamine release, we will use rat neonatal cerebellar granule cells in culture. We have used this cell type extensively to study sigma-1 receptor inhibition of arachidonic acid release. Sigma receptors of the sigma-1 subtype are thought to be coupled to their transducers via a G protein. By using antisense oligonucleotides to the alpha subunits of various inhibitory G proteins, we have begun to identify G protein(s) involved in sigma-1-mediated responses. The physiological relevance of the identified second messengers coupling sigma receptors to catecholamine release in cell models will be determined in brain slices using our established superfusion methodologies. Our findings should be helpful in designing appropriate therapies for abuse of PCP, and related psychotic syndromes.