The aim of this application is to establish by which mechanisms Cx-proteins improve cellular survival following injury. We have in preliminary observations established that Cx-expression antagonizes cell death indicating that the connexin proteins have death-inhibitory or anti-apoptotic activity. In specific we will ask: Does an adaptive remodeling and reorganization of Cx43 contribute to the high resistance to injury of Cx43 expressing cells? A Cx43-eGFP fusion protein has been stably expressed in C6 cells and time-lapse analysis has revealed that Cx43-eGFP undergo major structural reorganization after injury. We will here test the preposition that Cx43 reorganization represents an adaptive response that improves survival and that the lower resistance of Cx-deficient cells results from their limited ability to initiate the same process after injury on a single cell level. The analysis will be extended to include primary astrocytes transfected with an adenoviral vector encoding Cx43-GFP. By which mechanisms do connexin proteins increase cellular resistance to injury? Is formation of functional gap junction channels a prerequisite for their anti-apoptotic action? Alternatively, do mutant Cx's with deficient channel function also provide injury-resistance indicating that yet undefined actions of Cx.proteins are responsible for the increased survival? We have established several cell lines with stable expression of either wildtype Cx43, or Cx-mutants with deficiencies in either channel function or membrane localization. These clones represent a powerful tool to establish at which level the Cx-proteins antagonize cell death. Is the increase in cellular resistance associated with Cx-expression dictated by the phenotypic transformation? Does loss of cytoskeletal organization increase cellular sensitivity to injury? We will in these studies test the preposition that the Cx-induced phenotypic transformation observed, in part, may contribute to the increased cellular resistance. Does the death-inhibitory activity of Cx require ATP secretion? Do purinergic receptor blockers antagonize the Cx-induced increase in cellular resistance? It is here postulated that ATP secreted from Cx-expressing cells acts as an autocoid differentiation factor that increases the cellular resistance to injury. The effects on cellular resistance of long-term treatment with purinergic-receptor agonist and antagonists will here be analyzed. Defining the pathways, by which connexin proteins increase cellular resistance to injury, may provide a potential new therapeutic target for preventing cell death in acute pathologies as stroke and head trauma.
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