The thrombin receptor (protease activated receptor-1 or PAR1) is a G-protein coupled receptor that is best known for its role in coagulation and hemostasis. PAR1 is also expressed in the central nervous system (CNS) and mediates a variety of important effects. Work performed during the previous funding period demonstrated unambiguous PAR1-mediated potentiation of N-methyl-D-aspartate (NMDA) receptors and assessed the role of this effect in neuronal injury during blood brain barrier breakdown, which allows PAR1 activators direct access to brain parenchyma. We obtained strong evidence showing that PAR1 activation can exacerbate neuronal damage in vivo following 4 different CNS insults --transient ischemia, transient hypoxia, traumatic brain injury, and intra-striatal injection of NMDA. In addition, we completed cellular experiments that lead to the intriguing and unexpected working hypothesis that activation of PAR1 in astrocytes releases glutamate, which depolarizes neurons resulting in a relief of Mg2+ block of NMDA receptors. This competitive renewal proposes cellular experiments that examine whether activation of PAR1 and other astrocytic Gaq-coupled receptors triggers release of an excitatory amino acid that acts as a glial-neuronal signal. We will use mixed co-cultures of PAR1 -/- neurons on wild type glial feeder layers (and the converse) to explore the mechanism of PAR1-mediated glial-neuronal signaling. Our preliminary data suggests that astrocytic Ca2+-activated C1 channels may control release of glutamate, and we thus will explore the hypothesis that glutamate release reflects permeation through open Ca2+-dependent C1- channels. Single channel recordings will be made to examine in detail properties and permeation characteristics of Ca2+-activated C1- channels. We will address the following five experimental questions, which will expand our understanding of signaling by PAR1 and other Gaq/11-1inked receptors in the CNS as well as help explain how PAR1 could be involved in a wide range of CNS injuries. 1. Does activation of astrocytic PAR1 depolarize neurons by stimulating glutamate release? 2. Does activation of astrocytic PAR1 potentiate NMDA-Rs through depolarization-induced relief of distal Mg 2+block? 3. Does PAR1-stimulated glutamate release from astrocytes involve permeation through Ca2+ -activated C1 channels? 4. Are the effects of PAR1 activation in cultured astrocytes shared by astrocytes in brain slices? 5. Is astrocytic release of glutamate a generalized response to activation of Gaq/11-coupled receptors?
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