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Knowledge regarding human bladder smooth muscle cell (SMC) physiology is very limited. Only a few specific medical therapies for bladder disorders have therefore been established. The objective of this study was to develop a model for videomicroscopy of bladder SMC contractions. Cells were isolated from human cystoprostatectomy specimens and cultured in a modified EMEM medium. These cells were identified as SMCs by means of immunohistochemistry. For videomicroscopy, the culture flasks were coated with a viscous agent to allow cell contraction. Contractions were visualized by means of a cell culture microscope with a time-lapse videosystem. For cholinergic stimulation of the cells, acetylcholine, in concentrations ranging from 100 microM to 10 mM, was applied. The percentage of contracting cells within the observation field was evaluated for quantitative analysis. In control experiments without contractile stimulant 6% of the cells were observed to contract. Stimulation with acetylcholine induced a significant dose-dependent increase to 47% in contracting cells. These results demonstrated that videomicroscopy is an appropriate tool to investigate the contraction mechanisms of bladder SMCs. This model offers the possibility of studying drug effects on the human detrusor in vitro.

SUMMARYCell suspensions of rainbow trout Oncorhynchus mykisspseudobranch, prepared by Ca2+ depletion and mechanical maceration,contained a distinct population of cells that always kept their relatively cuboidal shape and did not round up in suspension or proliferate after adhering to the surface of cell culture dishes. Phasecontrast microscopy revealed an extensive system of basal membrane invaginations, and Na+-K+-ATPase- and anionexchanger-like immunoreactivity could be localized in cell membranes. The cells were characterized by a high mitochondrial density. Using specific antibodies, V-ATPase subunit B was localized in the plasma membrane. Using a cytosensor microphysiometer, the rate of acid secretion of these cells was measured and compared with the activity of a gill cell preparation. Incubation of pseudobranch cells with bafilomycin A1 (10-6 moll-1), a specific inhibitor of V-ATPase, reduced the rate of acid secretion by about 10% under control conditions, while no effect of bafilomycin on the rate of acid secretion of gill cells was observed. Application of amiloride (5×10-5moll-1) reduced the rate of acid secretion in cells of both organs,pseudobranch and gills. Incubation of pseudobranch cells with DIDS(10-3 moll-1) resulted in a minor increase in the rate of proton secretion, but in cells prepared from the gills of rainbow trout acid secretion was reduced by about 30-40%. It is concluded that pseudobranch cells are equipped with various pathways to secrete protons, and that the anion exchange activity especially of pseudobranch cells appears to be different from that in gills.