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


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+)](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 microM) and pyridoxalphosphate-6-azophenyl-2'4'-disulphonate (PPADS; 20 microM), indicating that extracellular ATP is crucial for the [Ca(2+)](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 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+)](i) evoked by hypotonic stress. The concurrent activation of BK channels permits K(+) efflux that may contribute to regulatory volume decrease.
Hafting
T
T
Department of Molecular Biosciences, University of Oslo, Oslo, Norway.
Haug
T M
TM
Ellefsen
S
S
Sand
O
O
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D002522:Chlorocebus aethiops; D005470:Fluorometry; D016257:Fura-2; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology; D007668:Kidney / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism*; D058919:Purinergic P2 Receptor Antagonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017385:Sequence Homology; D013498:Suramin / Q000494:pharmacology; D014709:Vero Cells
D007038:Hypotonic Solutions; C546382:P2RY1 protein, human; D024681:Potassium Channels, Calcium-Activated; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D011732:Pyridoxal Phosphate; D013498:Suramin; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2