CONTRACTIONS IN HUMAN DETRUSOR SMOOTH MUSCLE INDUCED BY HYPO-OSMOLAR SOLUTIONS

Abstract

Purpose The aim of this study was to investigate stretch activated channels in human detrusor using hypo-osmolar solutions to produce cell deformation. Stretch activated channels could provide another mechanism by which detrusor myocytes may be coupled. Materials And Methods Human detrusor removed at surgery was dissected into strips and also enzymatically digested and cultured. Strips (5 x 1 x 1 mm.) were mounted in an organ bath and perfused with gassed Tyrode's. Hypo-osmolar solutions were made by removal of NaCl. Gadolinium (Gd 3+), a blocker of stretch activated channels (SACs), and diltiazem, an L-type Ca ( 2+) channel antagonist were used at 10 micro M concentrations. Mean data +/- S.E.M. are expressed as a percentage of maximal tension produced by 1 micro M carbachol for each patient. Enzymatically disaggregated, human detrusor was cultured in flasks, passaged and placed on glass coverslips. Once confluent the cells were incubated with the Ca 2+ sensitive fluorochrome Fura-2AM. Coverslips were placed in a bath on the stage of EPI-fluorescence microscope and solutions were perfused through the bath (5 ml. per minute, 35C, pH 7.4). Changes in fluorescence emission ratio (proportional to changes in cytosolic Ca 2+) were measured. Results Hypo-osmolar solutions produced a tension increase in the strips and a Ca 2+ influx in the cells. In the strips in paired experiments Gd 3+ and diltiazem significantly reduced the response to hypo-osmolar solution (87% +/- 16% v 51% +/- 12.5%, p = 0.003, n = 10 for Gd ( 3+)), and (69% +/- 11% v 37% +/- 9%, p = 0.001, n = 9 for diltiazem). In Ca 2+ free solution responses were significantly reduced (65% +/- 10% v 21% +/- 8%, p = 0.001, n = 9). In the cells in paired experiments, 10 micro M Gd 3+ significantly reduced the elevation of cytosolic Ca 2+ in response to hypo-osmolar solutions (median 0 v 0.38 (62 cells, n = 7 bladders)), as did Ca 2+ free hypo-osmolar solution (median 0 v 0.44 (46 cells, n = 7)). 10 micro M diltiazem (L-type Ca 2+ channel antagonist) did not influence the response to hypo-osmolar solution (p = 0.14, median 0.5 v 0.54 (31 cells, n = 4)). Conclusions Hypo-osmolar solutions produced a tension increase in human detrusor that appears to be dependent on upon influx of Ca 2+ through stretch activated channels (SACs), influx of Ca 2+ through L-type Ca 2+ channels and also on release of intracellular Ca 2+.