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

用胶原酶分离新生（0 ~ 14d）大鼠髓内集管，37℃低温培养3 ~ 24hr。使用膜片钳技术，我发现了两种不同类型的离子通道，150 pS的钙活化钾通道和30 pS的非选择性阳离子通道，在静息条件下，这两种通道的开放概率都很低（<0.001），但在应用0.1%血清后，这两种通道的开放概率都有所增加。这些通道可能对细胞生长和调节尿液中的离子含量起重要作用。其次，将细胞置于含3muM氟3- am的标准溶液中，在24℃下孵育40 min，用共聚焦激光扫描显微镜测量细胞内和细胞核钙含量。核质和核周荧光强度分别为19和18。因此，核强度与核周强度之比为1.02。将ATP （2-100muM）应用于浴液中，在有或没有钙的浴液中迅速增加核和核周强度。N/P比值分别为1.71 （1mM Ca）和1.53（游离Ca），表明核钙的增加与核周钙的增加是同步的，但与核周钙的增加无关。虽然UTP同样增加了细胞内钙，但AMP和ADP没有。这表明p_2 -嘌呤能受体的一个亚型为P_<2U>。

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>.