Hypotonic stress activates BK channels in clonal kidney cells via purinergic receptors, presumably of the P2Y 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+)) 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).

METHODS

Subjecting cells to hypotonic stress for 10 s by exposure to a solution with 45% reduced osmolality induced a transient rise in Ca(2+). 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+) was completely inhibited by the P2 receptor antagonists suramine (100 microM) and pyridoxalphosphate-6-azophenyl-2'4'-disulphonate (PPADS; 20 microM), indicating that extracellular ATP is crucial for the Ca(2+) 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 Ca(2+) 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 Ca(2+) evoked by hypotonic stress. The concurrent activation of BK channels permits K(+) efflux that may contribute to regulatory volume decrease.