The filling of the urinary bladder is accomplished by coordinated contraction and relaxation of smooth muscle cells in different regions of the bladder. Emptying of the bladder is caused by prolonged maintenance of tension associated with low energy utilization, as in """"""""latch"""""""" which is characterized by slowly- or non-cycling cross-bridges. Bladder hypertrophy in partial obstruction is associated with alterations of the structure, energetics, and contractility of smooth muscle in the bladder wall. Experiments proposed in this application are designed to understand the intracellular mechanisms that regulate the bladder function in health and disease, specifically (a) the expression of myosin isoforms (SM1 and SM2) and (b) the actin-myosin interaction and actin-activated ATP hydrolysis, the in vitro correlate of muscle contraction, in hypertrophy following urethral obstruction in rabbit. Polymorphism of myosin in bladder hypertrophy and its effects on the intracellular structure and function of smooth muscle cells in the bladder wall will be investigated at mRNA, protein, and tissue levels. Immunofluorescence microscopy, utilizing antibodies specific to SM1 and SM2 isoforms, and S1 nuclease mapping of smooth muscle myosin heavy chain mRNA will be performed. Effects of alteration of myosin isoforms in the assembly of myosin filaments in smooth muscle cells from different regions of the bladder will be determined. Furthermore, we will determine if the difference in filament assembly, due to a change in myosin isoforms, has any effect on the actomyosin ATPase. These data will be helpful to see if a change in myosin isoforms affects the intracellular contractile machinery and the Ca2+ regulation of bladder contraction. Data on the myosin gene expression and the regulation of bladder function at cellular and genetic level during development and reversal of hypertrophy will help to develop better treatments for this lower urinary tract dysfunction.
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