Obstruction of the urethra results in numerous alterations in the urinary bladder that impair both its storage and emptying properties. The detailed molecular mechanisms responsible for these changes in function associated with obstruction-induced remodeling that have not been clearly delineated. This research project is based upon the hypothesis that the depressed function of the obstructed bladder is the result of alterations in both excitation-contraction coupling (Ca2+-availability) and the contractile apparatus (Ca2+-sensitivity and Ca2+-dependent activity) in detrusor muscle. To test this hypothesis, the following specific aims will be addressed using strips of detrusor muscle obtained from normal, decompensated, and compensated rabbit bladder: Aim 1: To correlate the force-velocity length relations of intact and skinned detrusor muscle associated with bladder obstruction and its reversal. This aim will determine the mechanism of altered mechanics at the tissue, cell and cross-bridge level. Chemically skinned muscle strips allow for the precise and direct control of the environment surrounding the contractile apparatus, bypassing the normal excitation-contraction pathway. Aim 2: To determine the relationship among agonist concentration, cytoplasmic [Ca2+] and the time course of contraction. Laser photolysis of caged-compounds will be used to initiate contraction while monitoring the intracellular [Ca2+] with Indo-1. This aim will determine if the altered contractility is due to altered calcium mobilization. Aim 3: To determine the time course of myosin light chain phosphorylation, [Ca2+], and isometric force in intact and myosin light chain phosphorylation, actin-activated myosin ATPase activity and isometric force in permeabilized tissues during various stimuli. This aim will test for alteration in the coupling of the Ca2+ signal to contractile activation. Aim 4: To determine the significance of other regulatory signaling pathways. The protein kinase C, mitogen-activated protein kinase, caldesmon phosphorylation cascade will be measured. This aim will determine if the loss of maintained contractile force in the decompensated bladder is due to alterations in thin filament regulation. The results of these studies, in conjunction with those in Project 1, will elucidate the specific steps of excitation-contraction coupling that are associated with bladder dysfunction. Moreover, the results of these studies will also determine which alterations in contractile or regulatory proteins, in collaboration with Project 2, are associated with bladder remodeling and will provide a more complete understanding of the cellular and molecular mechanisms responsible for the depressed bladder function associated with outlet obstruction.