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+)](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.

Inhibition of BK channels contributes to the second phase of the response to TRH in clonal rat anterior pituitary cells.

AIM: Thyrotropin-releasing hormone (TRH) induces biphasic changes in the electrical activity, the cytosolic free Ca2+ concentration ([Ca2+]i), and prolactin secretion from both GH cells and native lactotrophs. It is well established that inhibition of erg channels contributes to the second phase of the TRH response. We have investigated if BK channels are also involved. RESULTS: The BK channels may be active at the resting membrane potential (open probability, Po=0.01) in clonal rat anterior pituitary cells (GH4), which makes it possible that inhibition of these channels may contribute to the reduced K+ conductance during the TRH response. The specific BK channel blocker iberiotoxin (IbTx, 100 nm) had no effect on the resting conductance at holding potentials negative to -40 mV, but significantly reduced the conductance at shallower membrane potentials. This corresponds to the voltage dependency of the sustained [Ca2+]i. Furthermore, IbTx increased the action potential frequency by 36% in spontaneously firing cells. During the second phase of the TRH response, the action potential frequency increased by 34%, concomitantly with 61% reduction of the Po of single BK channels. The protein kinase C (PKC)-activating phorbol ester TPA had no significant effect on BK channel Po within the normal range of the resting potential. CONCLUSION: The BK channels may contribute to the resting membrane conductance, and they are partially inhibited by TRH during the second phase. This modulation seems not to depend on PKC. We propose that inhibition of erg and BK channels acts in concert to enhance the cell excitability during the second phase of the response to TRH.

Purinergic activation of BK channels in clonal kidney cells (Vero cells).

We have studied the activation of a high-conductance channel in clonal kidney cells from African green monkey (Vero cells) using patch-clamp recordings and microfluorometric (fura-2) measurements of cytosolic Ca2+. The single-channel conductance in excised patches is 170 pS in symmetrical 140 mM KCl. The channel is highly selective for K+ and activated by membrane depolarization and application of Ca2+ to the cytoplasmatic side of the patch. The channel is, thus, a large-conductance Ca2+-activated K+ channel (BK channel). Cell-attached recordings revealed that the channel is inactive in unstimulated cells. Extracellular application of less than 0.1 microM ATP transiently increased the cytosolic Ca2+ concentration ([Ca2+]i) to about 550 nM, and induced membrane hyperpolarization caused by Ca2+-activated K+ currents. ATP stimulation also activated BK channels in cell-attached patches at both the normal-resting potential and during membrane hyperpolarization. The increase in [Ca2+]i was owing to Ca2+ release from internal stores, suggesting that Vero cells express G-protein-coupled purinergic receptors (P2Y) mediating IP3-induced release of Ca2+. The P2Y receptors were sensitive to both uracil triphosphate (UTP) and adenosine diphosphate (ADP), and the rank of agonist potency was ATP >> UTP >/= ADP. This result indicates the presence of both P2Y1 and P2Y2 receptors or a receptor subtype with untypical agonist sensitivity. It has previously been shown that hypotonic challenge activates BK channels in both normal and clonal kidney cells. The subsequent loss of KCl may be an important factor in cellular volume regulation. Our results support the idea of an autocrine role of ATP in this process. A minute release of ATP induced by hypotonically evoked membrane stretch may activate the P2Y receptors, subsequently increasing [Ca2+]i and thus causing K+ efflux through BK channels.