Our major hypothesis for the last funded period was that reversal of EAA transporters and activation of Volume-Regulated Anion Channels (YRACs) are major sources of EAAs in rat cerebral ischemia and that inhibition of these routes of release would be neuroprotective. In support of the first part of this hypothesis, we found that elevated extracellular [K+] induced EAA release due to both reversal of the EAA transporter and activation of VRACs in primary asfrocyte cultures. In vivo, microdialysis studies in a rat temporary global ischemia model established that application via a microdialysis probe of dihydrokainaxe, an inhibitor of the astrocyte-specific EAA transporter GLT-1, or DNDS an anion channel inhibitor, led to potent suppression of EAA levels during the ischemic episode. If applied together these compounds reduced EAA levels in ischemia by over 80 percent. To check whether inhibition of VRACs is neuroprotective, we chose, on the basis of its high blood-brain barrier permeability, the estrogen receptor antagonist/agonist tamoxifen (TAM) that is also an efficient inhibitor of VRACs in vitro. In the rat middle cerebral artery occlusion model (rMCAO), 5 mg/kg TAM reduced infarction volume by up to 80 percent if applied just before the 2 hour ischemic episode or 3 h after initiation of ischemia. We propose to continue these studies along two lines. One will be devoted to molecular identification of VRACs and intracellular signalling events involved in the volume-dependent release of EAAs in primary astrocyte cultures. Our hypotheses for this part of the project are that more than one VRAC is involved in volume-dependent amino acid release, one or more of these channels are incorporated in calveolae signaling complexes and calmodulin and tyrosine kinases are involved in their volume-dependent activation. The second line of the study will be to explore the molecular mechanisms of TAM neuroprotection and evaluation of its therapeutic window with different dosages and with different durations of reversible middle cerebral artery occlusion. Our hypothesis here is that TAM is highly neuroprotective in rMCAo because it has multiple protective effects. These include inhibition of VRACs, suppression of Ca24 about/calmodulin-dependent nitric oxide production, and/or antioxidant action. We also cannot exclude that some portion of the protection may also be mediated by brain estrogen receptors. This second part of the project will test all these possibilities in animal studies. Both aspects of the project will add new basic knowledge on VRACs, with the potential for understanding their functions in the brain. The second half of the project that deals with neuroprotection has direct potential clinical implications, as TAM is known to be well tolerated in humans being widely used for breast cancer treatment.
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