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Electrophysiological and optical methods for ATP-receptor-operated calcium channels in kidney tubule cells.

肾小管细胞中 ATP 受体操纵的钙通道的电生理学和光学方法。

基本信息

批准号：
05670038
负责人：
KAWAHARA Katsumasa
金额：
$ 1.34万
依托单位：
Chiba Univ.
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670038/
关键词：
neonatal rat Kidney inner medullary collecting duct P_2 receptor patch-clamp confocal laser scanning inicroscope nuclear envelope K^+ channel Ca^<2+> channel 細胞外ATP 細胞内Ca IP_3 腎集合管抗利尿ホルモンV2受容体 ATP受容体 Caチャネル K選択性チャネル

项目摘要

Neonatal (0-14d) rat inner medullary collecting ducts were isolated by collagenase and were shortly cultured (3-24hr) at 37ﾟC.Using the patch-clamp technique, I have found two different types of ion channels, Ca-activated potassium channel of 150 pS and non-selective cation channel of 30 pS.Open probabily of both channels was low (<0.001) at the resting condition, but it increased after application of 0.1% serum. These channels may play an important role for cell grawing and regulating ionic contents in the urine. Second, the cells were incubated in a standard solution containing 3muM fluo3-AM for 40 min at 24ﾟC,their intracellular and nuclear calcium was measured by the confocal laser scanning microscope. The level of fluoresent intensity of nucleoplasm and perinucleoplasm was 19 and 18, respectively. Therefore, a ratio between nuclear and perinuclear intensity was 1.02. Application of ATP (2-100muM) to the bath quickly increased both nuclear and perinuclear intensity with or without calcium in a bathing solution. The N/P ratio also increased to 1.71 (1mM Ca) and 1.53 (Ca free) , suggesting that nuclear calcium increase was synchronized with, but independent from the perinuclear calcium. Although UTP similarly increased the intracellular calcium, AMP and ADP did not. This indicates that a subtype of the P_2-purinergic receptor was P_<2U>.

用胶原酶分离新生（0- 14天）大鼠内髓集合管，在37 ℃培养3- 24小时，用膜片钳技术发现两种不同类型的离子通道，即150 pS的钙激活钾通道和30 pS的非选择性阳离子通道，两种通道开放的概率都很低（<0.001），而0.1%血清使其增加。这些通道可能在细胞生长和调节尿中离子含量方面发挥重要作用。第二，将细胞在含有3 μ M fluo 3-AM的标准溶液中于24 ℃孵育40 min，通过共聚焦激光扫描显微镜测量其细胞内和核内钙。核质和核周质的荧光强度水平分别为19和18。因此，核和核周强度之间的比率为1.02。ATP（2- 100 μ M）的应用浴迅速增加核和核周的强度与或不含钙的浴溶液。N/P比值也增加到1.71（1 mM Ca）和1.53（无Ca），表明核钙增加与核周钙同步，但不依赖于核周钙。虽然UTP同样增加细胞内钙，AMP和ADP没有。提示P_2嘌呤能受体的一种亚型为P_1<2U>。