Extracellular ATP has been shown to modulate a variety of important biological processes, such as, secretion, cellular growth, and vasodilation, among others. ATP has also been shown to induce Cl- secretion in airway epithelial cells that is non-CFTR (cystic ficrosis transmembrane regulatory protein)-dependent. This observation has raised the possibility of the use of nucleotides for the treatment of cystic fibrosis (CF). Characterization of the fundamental molecular mechanisms that control P2 purinoceptors signal transduction will be valuable for the potential therapeutic development of nucleotides in the treatment of CF. CF treatment with nucleotides may produce conditions of prolonged exposure to the nucleotide agonists that could induce desensitization. However, the molecular mechanisms of desensitization of the P2U purinoceptor are currently unclear. We propose to rigorously investigate the long-term desensitization of the P2U purinoceptor system in the human epidermoid A431 cells and in 1321N1 astrocytomas transfected with a cloned P2U purinoceptor. The A43l cells respond to extracellular ATP in a variety of ways by means of purinoceptors. Untransfected 1321N1 system represent an ideal negative control.
A specific aim of this proposal is to determine if the function of P2U purinoceptors is controlled by modulation of the number of receptors on the plasma membrane and to examine if down- regulation of purinoceptors occurs during long-term desensitization. We will examine the down regulation of the purinoceptor by monitoring a variety of receptor function markers: mobilization of intracellular stores of calcium, formation of IP3 and DAG, and stimulation of calcium fluxes. The pharmacological data will be supported also by binding experiments in which we will examine the ATP binding parameters (using radioactively labeled ligands) before and after desensitization. In addition, a photoreactive analog of ATP, (3-O-(4-benzoyl)benzoyl) ATP, and polyclonal antibodies (Western analysis) will be used to measure the amount of P2 purinoceptors in the plasma membrane of A431 cells and transfected 1321N1 cells. The amount of receptors during the desensitization process may also be controlled by regulation of transcription of the P2U purinoceptor gene. Therefore, we will also use molecular techniques to examine the level of expression of the purinoceptor. Using these various approaches (pharmacological, biochemical and molecular) we will also examine the resensitization of the cells after chronic exposure to agonist, and the involvement of protein synthesis in the recovery. The characterization of the long- term desensitization of the P2 purinoceptor will be important for the future development of nucleotides as therapeutic agents against CF. Future goals of our research will include the elucidation of the molecular regulatory mechanisms that modulate P2 purinoceptor signaling and desensitization. Minority students (undergraduate and graduate) will directly participate in this study and will learn modem techniques used in biotechnology, biochemistry and molecular biology. Students will become competent in the application of biotechnology to a basic science-biomedical problem.
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