Our long term goal is to understand how signal transduction and gene expression in neurons are involved in synaptic plasticity, in particular, to understand the molecular mechanisms that permit a neuron to integrate coincident signals and to relay them to other cells. Nitric oxide (NO), a recently discovered novel type of second messenger, is a freely diffusible short-lived compound capable of acting within a limited volume in the vicinity of its original source.We have found recently that NO can act as an amplifier of calcium signals in neuronal cells. In this proposal we wish to test the hypothesis that NO and other gaseous messengers are involved in changes in synaptic plasticity by reprogramming the patterns of gene activity in neurons, alone or in concert with other second messenger molecules. We plan to identify and characterize the steps in the NO signaling cascade which synergize with the calcium signaling, to search for targets of NO and NO/Ca2+ action, to search for the other types of NO-Ca2+ interactions, to search for the other types of neuronal response to the coincident versus noncoincident action of NO accompanied by other stimuli and to investigate the cooperation or antagonism of NO with other signaling systems in the brain. We want to investigate the possibility that other gaseous messenger molecules exist and act in the brain and are relevant for gene expression.The unusual physical state of these compounds can help to explain many phenomena where activity in a limited volume or action without apparent system for secretion and/or reception are implicated. Our goal here is to design a sensitive general system suitable for the detection of weak or short-lasting effects on gene activity and to use it for a search of the CO (and other potential gaseous messengers) involvement in gene expression in neurons. Protein kinases are the crucial elements of most of the known signaling cascades in the cell. The ability to manipulate their activity provides both the instruments to dissect signaling networks and to introduce the desirable changes in the cell metabolism. We have prepared a family of highly specific and selective recombinant inhibitors of protein kinases, whose inhibitory action is based on the pseudosubstrate mechanism. Here we propose to increase potency of recombinant inhibitors, to prepare inhibitors with targeted and regulatable localization and activity and to use them as analytical tools to study complex signaling cascades in cells.
