The overall aim of the experiments described in this proposal is to investigate factors involved in the regulation of serotonin receptors at a cellular and molecular level. Their are several subtypes of serotonin receptors that can be distinguished both pharmacologically and in terms of their biochemical effector mechanisms. Serotonin, acting at either 5-HT1a or 5-HT1b receptors, appears to regulate the activity of adenylate cyclase, whereas 5-HT1c and 5-HT2 receptors function through the stimulation of phosphoinositide hydrolysis. The subtypes of serotonin receptors are independently distributed in the central nervous system and appear to be involved in several major neuropsychiatric disorders, including depression and anxiety. Progress in understanding at the molecular level the regulation of serotonin receptors has been hindered by the need to use homogenates of heterogeneous tissues such as the cerebral cortex. The present experiments will focus on the use of clonal lines of cultured cells that express subtypes of serotonin receptors. We have recently developed a clonal line of cells derived from a rat anterior pituitary tumor that appears to express 5-HT2 receptors based on preliminary results of radioligand binding assays. Epitheliomas of the choroid plexus found in transgenic mice carrying the SV40 T-antigen encoding gene have been used to develop a second line of cells that appear to express 5-HT1c receptors. The properties of the receptors on these cell lines will be determined using radioligand binding assays. The ability of agonists at serotonin receptors to stimulate hydrolysis of phosphoinositides and mobilize intracellular calcium will be investigated, and the coupling of serotonin receptors to these effector systems will be explored. The regulation of serotonin receptors in response to pretreatment with agonists and antagonists will be examined. Chronic administration of antidepressants that are antagonists at 5-HT2 receptors results in a decrease in the density and a diminution of the function of these receptors. The apparent down-regulation of receptors induced by antagonists will be examined at a cellular level to elucidate of mechanism for this paradoxical response. Studies of desensitization and the sequestration of receptors in response to treatment with agonists or nonspecific activators will be carried out in each of the cell lines. The effects of regulation of receptors on the rate at which 5-HT1c and HT2 receptors are synthesized and degraded will be determined. A precise understanding of these phenomenon is central to an understanding of the functioning of serotonin receptors at synapses in the central nervous system and should have implications for the understanding and treatment of neuropsychiatric disorders such as depression and anxiety.
