Masters J G, Neal D E, Gillespie J I
Department of Surgical Sciences, The Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.
J Urol. 1999 Aug;162(2):581-9.
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.
Human detrusor removed at surgery was dissected into strips and also enzymatically digested and cultured. Strips (5x1x1 mm.) were mounted in an organ bath and perfused with gassed Tyrode's. Hypo-osmolar solutions were made by removal of NaCl. Gadolinium (Gd3+), a blocker of stretch activated channels (SACs), and diltiazem, an L-type Ca2+ channel antagonist were used at 10 microM concentrations. Mean data +/- S.E.M. are expressed as a percentage of maximal tension produced by 1 microM 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 Ca2+ 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 Ca2+) were measured.
Hypo-osmolar solutions produced a tension increase in the strips and a Ca2+ influx in the cells. In the strips in paired experiments Gd3+ and diltiazem significantly reduced the response to hypo-osmolar solution (87%+/-16% v. 51%+/-12.5%, p = 0.003, n = 10 for Gd3+), and (69%+/-11% v. 37%+/-9%, p = 0.001, n = 9 for diltiazem). In Ca2+ free solution responses were significantly reduced (65%+/-10% v. 21%+/-8%, p = 0.001, n = 9). In the cells in paired experiments, 10 microM Gd3+ significantly reduced the elevation of cytosolic Ca2+ in response to hypo-osmolar solutions (median 0 v. 0.38 (62 cells, n = 7 bladders)), as did Ca2+ free hypo-osmolar solution (median 0 v. 0.44 (46 cells, n = 7)). 10 microM diltiazem (L-type Ca2+ channel antagonist) did not influence the response to hypo-osmolar solution (p = 0.14, median 0.5 v. 0.54 (31 cells, n = 4)).
Hypo-osmolar solutions produced a tension increase in human detrusor that appears to be dependent on upon influx of Ca2+ through stretch activated channels (SACs), influx of Ca2+ through L-type Ca2+ channels and also on release of intracellular Ca2+.
本研究旨在利用低渗溶液使细胞变形，从而研究人逼尿肌中的牵张激活通道。牵张激活通道可能为逼尿肌细胞的耦联提供另一种机制。
将手术中切除的人逼尿肌切成条带，并进行酶消化和培养。将条带（5×1×1毫米）安装在器官浴槽中，并用通有气体的台氏液灌注。通过去除氯化钠来制备低渗溶液。使用浓度为10微摩尔的钆（Gd3+），一种牵张激活通道（SACs）的阻滞剂，以及地尔硫卓，一种L型钙通道拮抗剂。每个患者的平均数据±标准误表示为1微摩尔卡巴胆碱产生的最大张力的百分比。将酶解后的人逼尿肌在培养瓶中培养，传代并置于玻璃盖玻片上。细胞汇合后，用钙敏感荧光染料Fura-2AM孵育。将盖玻片置于落射荧光显微镜载物台上的浴槽中，溶液通过浴槽灌注（每分钟5毫升，35℃，pH 7.4）。测量荧光发射比率的变化（与胞质钙的变化成比例）。
低渗溶液使条带张力增加，细胞内钙流入。在配对实验中，条带中Gd3+和地尔硫卓显著降低了对低渗溶液的反应（Gd3+：87%±16%对51%±12.5%，p = 0.003，n = 10），（地尔硫卓：69%±11%对37%±9%，p = 0.001，n = 9）。在无钙溶液中，反应显著降低（65%±10%对21%±8%，p = 0.001，n = 9）。在配对实验的细胞中，10微摩尔Gd3+显著降低了对低渗溶液的胞质钙升高反应（中位数0对0.38（62个细胞，n = 7个膀胱）），无钙低渗溶液也有此效果（中位数0对0.44（46个细胞，n = 7））。10微摩尔地尔硫卓（L型钙通道拮抗剂）对低渗溶液的反应无影响（p = 0.14，中位数0.5对0.54（31个细胞，n = 4））。
低渗溶液使人类逼尿肌张力增加，这似乎依赖于通过牵张激活通道（SACs）的钙内流、通过L型钙通道的钙内流以及细胞内钙的释放。
