Hypotonic stress activates BK channels in clonal kidney cells via purinergic receptors, presumably of the P2Y(1) 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 Ca2+ concentration ([Ca2+](i)) may then participate in cell volume regulation. The aim of the present study was to test if purinergic receptors and large conductance Ca2+ 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 [Ca2+](i). This response persisted in virtually Ca2+-free extracellular solution, demonstrating that Ca2+ was mainly released from intracellular stores. The hypotonically induced elevation of [Ca2+](i) was completely inhibited by the P2 receptor antagonists suramine (100 mu M) and pyridoxalphosphate-6-azophenyl-2'4'-disulphonate (PPADS; 20 mu M), indicating that extracellular ATP is crucial for the [Ca2+](i) increase. RT-PCR revealed the expression of mRNA for P2Y(1) receptors in Vero cells. The putatively selective P2Y(1) antagonist PPADS did completely block Ca2+ responses to both ATP and hypotonic stress, suggesting that P2Y(1) 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 P2Y(1) subtype. These receptors are responsible for the elevation of [Ca2+](i) evoked by hypotonic stress. The concurrent activation of BK channels permits K+ efflux that may contribute to regulatory volume decrease.