Neuronal activity fluctuates in response to changes in activation of particular pathways. Target cells, in turn adapt to these altered activation patterns by modifying their sensitivity to neuronal stimuli (adaptive sensitivity). The postsynaptic cell may become more sensitive (supersensitive) or less sensitive to external stimuli subsequent to changes in neural activity or innervation. This neuronally-regulated plasticity creates normal adaptations to altered conditions. Similar mechanisms may underlie responses to pathological conditions and contribute to dysfunction in conditions ranging from diabetic neuropathy to neuronal degeneration, or trauma. Denervation supersensitivity of the rat parotid gland acinar cell provides an excellent model of neuronally regulated plasticity because of the simplicity of the innervation pattern and the accessibility of the target cells for detailed physiological and molecular studies. Previous studies have documented the role of innervation in modulating important salivary gland functional responses when diet or physiological status changes. Parotid mechanisms can be examined in vitro, where parasympathetic denervation supersensitivity is observed by analysis of cytosolic calcium responses to neurotransmitter receptor activation in individual cells. The studies proposed in this grant will focus on the mechanism of parasympathetic denervation supersensitivity of the cytosolic calcium response in rat parotid acinar cells. Supersensitivity of the heterologous type develops for receptors that regulate cytosolic calcium by different pathways, either by intracellular calcium release (mediate by inositol trisphosphate) or by ATP-mediated calcium influx. Imaging of individual acinar cells using Ca- sensitive fluorescent dyes suggests that responsiveness is regulated both by receptor-specific and by nonspecific processes of Ca modulation. Further studies will pharmacologically analyze transport and release mechanisms and mechanisms of phosphorylation that appear to be involved in muscarinic and substance P pathways as well as in the ATP (P2- purinoceptor)-activated ion influx. In addition, biochemical, physiological and molecular methods will evaluate the role of Cai handling mechanisms and release in normal an supersensitive cells. Similar strategies and whole cell patch clamp methods will be used to study purinoceptor activation, with the goal of determining whether changes in receptor number or modulation of the activity of the associated ion channel are factors in increased responsiveness. Characterization of the ATP receptor and it modulation has wide spread implications for salivary gland function as well as for many other cells in the body. Understanding of the properties of the P2z ATP receptor may lead to therapeutic opportunities as with other types of ATP receptors on secretory epithelia, which have been recognized as possible targets for treatment of the transport defect of cystic fibrosis. The long term goal is to characterize the P2 receptors and the molecular components that set sensitivity to Cai- modulating receptors in response to changes in neuronal input and to identify the neuronal signals that are involved.
