This proposal is based on the unusual finding in animal models (rat, mouse) that nicotinic receptor agonists significantly enhance the inhibitory action of muscarinic receptor antagonists (atropine, 4-DAMP) on neurally evoked rat bladder contractions, presumably by impairing the non-adrenergic non-cholinergic (NANC component of the contraction. This interaction can be evoked by mixed nicotinic-muscarinic agonists like carbachol, or the cholinergic transmitter acetylcholine, indicating that this finding may have pathophysiological significance, particularly if modulated following bladder injury or insult. For example, the nicotinic-purinergic interaction appears to be constitutively active following spinal cord injury as rat bladders exhibit a higher sensitivity to muscarinic antagonists and, thus, become more 'cholinergic"""""""" as compared to normal bladders. In addition, after nicotinic-purinergic interaction the normal rat bladder smooth muscle exhibits much smaller contractions to the purinergic agonist, alpha-beta-methylene ATP suggesting that P2X1 receptor mechanisms may be impacted. We propose to investigate the role of purinergic P2X1 receptors in the nicotinic-purinergic interaction by: 1) utilizing pharmacological methods on the neurally and chemically evoked bladder contractions and 2) measuring changes in intracellular Ca2+ levels by calcium imaging. In addition, we will explore mechanisms underlying changes in nicotinic-purinergic interaction that occur in bladders after spinal cord injury or after bladder outlet obstruction. We hypothesize that the nicotinic binding site is plastic and is already activated in neurogenic bladders, thus explaining why atropine is more effective in inhibiting the neurally evoked contractions. We will utilize RT-PCR and immunohistochemical techniques to identify nicotinic receptor subtypes in normal bladders and to examine whether expression of these specific receptors is altered with SCI or obstruction. Depending on the nicotinic subunits identified with RT-PCR, specific ligands to measure nicotinic receptor binding affinity will be used to more fully characterize the nature of this unusual interaction. Finally, we will utilize readily available nicotinic receptor knockout mice to functionally confirm the modulatory role of specific nicotinic receptors on mouse bladder contractions.
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