Effect of extracellular ATP on contraction, cytosolic calcium activity, membrane voltage and ion currents of rat mesangial cells in primary culture

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Abstract
1 The effects of extracellular ATP on contraction, membrane voltage (Vm), ion currents and intracellular calcium activity [Ca2+]i were studied in rat mesangial cells (MC) in primary culture. 2 Addition of extracellular ATP (10−5 and 10−4 m) to MC led to a cell contraction which was independent of extracellular calcium. 3 Membrane voltage (Vm) and ion currents were measured with the nystatin patch clamp technique. ATP induced a concentration-dependent transient depolarization of Vm (ED50: 2 × 10−6 m). During the transient depolarization ion currents were monitored simultaneously and showed an increase of the inward- and outward current. 4 In a buffer with a reduced extracellular chloride concentration (from 145 to 30 mm) ATP induced a depolarization augmented to −4 ± 4 mV. 5 ATP-γ-S and 2-methylthio-ATP depolarized Vm to the same extent as ATP, whereas α,β-methylene-ATP (all 10−5 m) had no effect on Vm. 6 The Ca2+ ionophore, A23187, depolarized Vm transiently from −51 ± 2 to −28 ± 4 mV and caused an increase of the inward current. 7 The intracellular calcium activity [Ca2+]i was measured with the fura-2 technique. ATP stimulated a concentration-dependent increase of [Ca2+]i (ED50: 5 × 10−6 m). The increase of [Ca2+]i was biphasic with an initial peak followed by a sustained plateau. 8 The [Ca2+]i peak was still present in an extracellular Ca2+-free buffer, whereas the plateau was abolished. Verapamil (10−4 m) did not inhibit the [Ca2+]i increase induced by ATP. 9 The data indicate that extracellular ATP contracts MC and is able to increase [Ca2+]i by the release of Ca2+ from intracellular stores and recruitment from the extracellular space. In addition ATP depolarizes Vm of MC by activating a Cl− conductance. The ATP-induced depolarization is mediated by a P2y receptor.