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

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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>.