Acute pain transmission in the mammalian nervous system begins with transduction of various noxious or injurious events in the periphery by primary sensory neurons referred to as nociceptors. In addition to functional and morphological distinctions from other sensory neurons, nociceptors have been distinguished by their expression of key signaling molecules involved in the transduction of injury to action potentials. Among these molecules is the recently clone vanilloid receptor, VR1, which is activated both by capsaicin (CAP), protons, and heat. Another distinctive feature is the very specific developmental and survival requirement f nociceptors for the neurotrophin, nerve growth (NGF) acting through its receptor, trkA. Recent Evidence suggests that both VR1 and trkA may be important in the induction of hyperalgesia states associated with chronic inflammation. The interactions between these two receptor systems that may be involved in such phenomena are the focus of the present proposal. Preliminary data indicates an acute sensitization of VR1 by NGF in cultured rat DRG neurons. Furthermore, we have now been able to reconstitute this phenomenon in a mammalian cell line by heterologous co-expression of both VR1 and trkA (CHO-VR1/trkA cells), thus facilitating study of the signal transduction process linking the two at the biochemical and molecular levels. Using patch clamp electrophysiology, we will characterize the capsaicin responsiveness of individual cells in the presence and absence of NGF. Phosphorylation/dephosphorylation reactions involved in the sensitization of NGF will be explored using pharmacological probes and site-directed mutations of trkA designed to inhibit or facilitate well characterized signaling pathways activated by NGF. Phosphorylation/dephosphorylation reactions involved in the sensitization by NGF will be explored using pharmacological probes and site-directed mutation of trkA designed to inhibit or facilitate well characterized signaling pathways activated by NGF. The amino acid residues of VR1 critical or facilitate well characterized signaling pathways activated by NGF. The amino acid residues of VR1 critical to its increased responsiveness after NGF will be assessed by site-directed mutations of key phosphorylation sites in VR1. We will determine the patterns of expression of neurotrophin receptors and VR1 at the protein and mRNA levels, in single physiologically characterized DRG neurons using immunocytochemistry and single pathways signaling activated by NGF. The amino acid residues of VR1 critical to its increased responsiveness after NGF will be assessed by site-directed mutations of key phosphorylation sites in VR1. We will determine the patterns of expression of neurotrophin receptors and VR1 at the protein and mRNA levels, in single physiologically characterized DRG neurons using immunocytochemistry and single cell RT-PCR to correlate their responsiveness to neurotrophins with particular complements of these key signaling molecules. Finally, using both DRG neurons and CHO- VR1/trkA cells we will determine whether NGF can sensitize cells to other activators of VR1, protons and heat. These studies will reveal mechanisms by which NGF can acutely sensitize nociceptors to inflammatory signals and thus yield insight into the mechanisms of induction of hyperalgesia as well as clues to possible therapeutic interventions.
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