This project seeks to examine specific excitation-contraction coupling mechanisms in isolated bladder smooth muscle cells, and to determine if these processes are associated with the contractile dysfunction associated with chronic obstruction of the urinary bladder. Two important coupling process that have been implicated in this dysfunction will be examined: non-selective cation channels and calcium-induced calcium release (CICR). Cation channels that are activated by neurotransmitters and may be calcium permeant will be identified using simultaneous single-cell, patch-clamp methods and fura 2 calcium measurements. ATP acting on purinergic receptors plays an important role in urinary bladder function. The ligand- gated cation channels mediating purinergic excitatory currents in bladder smooth muscle cells will be identified and correlated with the properties of recently cloned P2X receptor/channels. The calcium permeation, biophysical and pharmacological properties of these channels in rabbit detrusor will be determined. CICR appears to be an important component of excitation/contraction coupling in urinary bladder myocytes. The ability of calcium currents and cation currents to release calcium from sarcoplasmic reticulum will be determine using simultaneous measurements of intracellular calcium and current in voltage-clamped myocytes. The expression of P2X genes will also be determined. These processes will be compared in cells dissociated from normal, decompensated, and reversed urinary bladders to determine the role of non-selective cation channels and CICR in post obstructive urinary bladder dysfunction. The extent to which CICR, non-selective cation channels or downstream calcium release/uptake processes are altered in decompensated bladders will shed light on the processes underlying bladders dysfunction associated with urinary outflow obstruction.
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