Variations in the intracellular concentration of calcium ions (Ca) play an important role in a wide range of cellular reactions and processes. In most cells, brief increases in Ca can result either from calcium entering the cell or calcium being released from a specialized compartment inside the cell. Calcium can be released from an intracellular compartment when an agonist (hormone or neurotransmitter) binds to a distinct receptor on the cell membrane. According to this model, binding of the agonist (first messenger) to the membrane receptor leads to the intracellular release of an agent (second messenger). The second messenger, in turn, releases calcium from the intracellular compartment. The proposed study will focus on characterizing the action of a novel second messenger, called inositol trisphophate (IP3), on nerve cells. The experiments will be carried out in giant molluscan neurons. Molluscan neurons constitute a particularly favorable prepartion in which to study calcium function and regulation. Some nerve cells display a prominent calcium component in the ionic basis of their excitability. Their cell bodies are quite large (0.1-0.5mm in diameter, about 10 times larger that mammalian neurons), and are easily identifiable for isolation. For these reasons, experimental manipulations can be made in these cells which would be impossible in the mammalian nervous system. Preliminary studies suggest that intracellular injections of inositol trisphosphate cause an increase in intracellular calcium stores. The proposed experiments will: 1. Look for an agonist that causes Ca to increase by puffing pharmacological agents on the cell membrane and measuring the level of Ca with calcium-selective probes made in the laboratory. 2. Compare the Ca increases induced by the agonist or IP3 by either puffing the agonist or injecting IP3 and measuring Ca. 3. Determine the localization of the intracellular Ca store by injecting IP3 at different depths fro the inner membrane of a nerve cell and measuring the free Ca change at each depth. 4. Determine the effects of IP3 injections on the electrical characteristics of the cells and correlate them with the intracellular free calcium level. The studies will suggest whether the action of some neurotransmitters on nerve cells is mediated by an increase in calcium released by inositol trisphosphate. The combination of these electrophysiological, pharmacological and biochemical techniques should prove useful to gather new and important information about nerve cell function and perhaps about disorders of the human central nervous system.
