Hypotonic stress activates BK channels in clonal kidney cells via purinergic receptors, presumably of the P2Y1 subtype

Aim: Membrane stretch due to cell swelling may cause a minute leakage of adenosine triphosphate (ATP) that stimulates endogenous purinergic receptors. The following elevation of the cytosolic-free Ca 2+ concentration ([Ca 2+ ]i) may then participate in cell volume regulation. The aim of the present study was to test if purinergic receptors and large conductance Ca 2+ activated K + (BK) channels are activated in response to hypotonic stress in clonal kidney cells (Vero cells). Methods: The methods used are fura-2 microfluorometry, cell-attached patch clamp and reverse-transcriptase polymerase chain reaction (RT-PCR). Results: Subjecting cells to hypotonic stress for 10 s by exposure to a solution with 45% reduced osmolality induced a transient rise in [Ca 2+ ]i. This response persisted in virtually Ca 2+ -free extracellular solution, demonstrating that Ca 2+ was mainly released from intracellular stores. The hypotonically induced elevation of [Ca 2+ ]i was completely inhibited by the P2 receptor antagonists suramine (100 lm) and pyridoxalphosphate-6-azophenyl-2¢4¢-disulphonate (PPADS; 20 lm), indicating that extracellular ATP is crucial for the [Ca 2+ ]i increase. RT-PCR revealed the expression of mRNA for P2Y1 receptors in Vero cells. The putatively selective P2Y1 antagonist PPADS did completely block Ca 2+ responses to both ATP and hypotonic stress, suggesting that P2Y1 receptors are mediating the response. Furthermore, patch clamp recordings in cell-attached configuration revealed that BK channels are activated in response to hypotonic stress. Conclusion: Vero cells express functional purinergic receptors, presumably of the P2Y1 subtype. These receptors are responsible for the elevation of [Ca 2+ ]i evoked by hypotonic stress. The concurrent activation of BK channels