In the lung, airway diameter is dramatically influenced by complex molecular interactions between peptides related from afferent sensory nerve endings and inflammatory mediators released from surrounding cells. A prominent class of inflammatory mediators are the leukotrienes C4 and D4 (LTC4 and LTD4) which when applied to gut, trachea, and pure cultures of sensory neurons elicit release of the tachykinins substance P (SP) and substance K (SK). Conversely, SP applied to isolated trachea elicits release of leukotriene E4 - like material. Release of TKs by leukotrienes (LTs) and release of LT by a TK supports the model of a """"""""relay mediator"""""""" role for LTs in which they trigger antidromic action potentials or directly produce Ca+ mediated TK release; TKs, in turn, cause further LT release and the cascading spread of inflammation is perpetuated. LTD4 produces release of SP and SK through separate mechanisms; LTD4 induced SP release is inhibited by tetrodotoxin (TTX) a blocker of Na+ action potentials, while LTD4 induced SK release is unaffected by TTX. We will further explore the phenomenon of differential SP and SK release by LTD4, determining 1) if SK is always colocalized with SP and 2) if different Ca+ channels or different gating mechanisms of the same type Ca+ channel mediate TTX sensitive and insensitive TK release. We will characterize TK evoked LT release further by 1) authenticating LTE4 release by SP, determining 2) if LTE is the initial LT released by SP, 3) whether an unknown released also by SP is a fourth LT, and 4) if SK, like SP, elicits LT release. A crucial, but largely unstudied feature of SP processing occurs ar the last step of biosynthesis, amidation of the final precursor form, SP-glycine (SP-G), to mature SP. Dietheyldithiocarbamate (DDC) is an inhibitor of this amidation reaction, and has been shown to produce differential increases in precursor, SP-G, in predominantly axonal versus terminal rich areas of the lung, suggesting site specificity of the amidation reaction, and/or differential transport of the two molecular forms. Using normal and DDC treated tissue, we will determine 1) whether there is conversion of SP-precursor to SP in vagal axons and/or terminals and 2) whether SP-precursor transport is retrograde as well as orthograde. Understanding of TK synthesis, transport, and release, and LT-tachykinin relay interactions is crucial to developing strategies to antagonize these important inflammatory components.
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