Cellular swelling triggers the activation of Cl؊ channels (volume-sensitive outwardly rectifying (VSOR) Cl ؊ channels) in many cell types. Ensuing regulatory volume decrease has been considered the primary function of these channels. However, Cl ؊ channels, which share functional properties with volume-sensitive Cl ؊ channels, have been shown to be involved in other physiological processes, including cell proliferation and apoptosis, raising the question of their physiological roles and the signal transduction pathways involved in their activation. Here we report that exogenously applied H 2 O 2 elicited VSOR Cl ؊ channel activation. Furthermore, activation of these channels was found to be coupled to NAD(P)H oxidase activity. Also, epidermal growth factor, known to increase H 2 O 2 production, activated Cl ؊ channels with properties identical to swelling-sensitive Cl ؊ channels. It is concluded that NAD(P)H oxidasederived H 2 O 2 is the common signal transducing molecule that mediates the activation of these ubiquitously expressed anion channels under a variety of physiological conditions.

The fluorescence intensity of the dye 1,1'-dipropylox-adicarbocyanine (DiOC3-(5] has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (Vm) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na+-free K-/choline-media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155 mM. Calibration by the valinomycin "null point" procedure described by Laris et al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976, Biochim, Biophys. Acta 436:475-488) is shown to be valid only in the presence of the Cl- -channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN- as an indirect estimation of the membrane potential is found not to be applicable for the fast changes in Vm reported in this paper. Incubation with DiOC3-(5) for 5 min is demonstrated to reduce the Cl permeability by 26 +/- 5% and the NO3- permeability by 15 +/- 2%, while no significant effect of the probe could be demonstrated on the K+ permeability. Values for Vm, corrected for the inhibitory effect of the dye on the anion conductance, are estimated at -61 +/- 1 mV in isotonic standard NaCl medium, -78 +/- 3 mV in isotonic Na+-free choline medium and -46 +/- 1 mV in isotonic NaNO3 medium. The cell membrane is depolarized by addition of the K+ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na+-free choline medium, indicating that Vm is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO3- for all cellular and extracellular Cl- leads to a depolarization of the membrane, demonstrating that Vm is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K+ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 microS/cm2 for K+, 3.0 microS/cm2 for Na+, 0.6 microS/cm2 for Cl- and 8.7 microS/cm2 for NO3-. Addition of the Ca2+-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K+ permeability exceeds the Cl- permeability also after the addition of A23187. The K+ and Cl- conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 microS/cm2, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

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Abstract-This study aimed at identifying the type and functional significance of potassium channels and voltagedependent calcium channels (Ca v ) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents between Ϫ60 and Ϫ10 mV. Blockade of K ϩ channels with TEA inhibited 83% of the current at ϩ105 mV. Inhibition of K V channels with 4-AP inhibited 21% of the current. Blockade of calcium-sensitive voltage-gated K ϩ channels (BK Ca ) with charybdotoxin or iberiotoxin inhibited 89% and 82% of the current, respectively. Double immunofluorescence confirmed the presence of BK Ca and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74% with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BK Ca was confirmed in single-sampled JG cells by RT-PCR. The resting membrane potential of JG cells was Ϫ32 mV and activation of BK Ca with cAMP hyperpolarized cells on average 16 mV, and inhibition with TEA depolarized cells by 17 mV. The cells displayed typical high-voltage activated calcium currents sensitive to the L-type Ca v blocker calciseptine. RT-PCR analysis and double-immunofluorescence labeling showed coexpression of renin and L-type Ca v 1.2. The cAMP-mediated increase in exocytosis (measured as membrane capacitance) was inhibited by depolarization to ϩ10 mV, and this inhibitory effect was blocked with calciseptine, whereas K ϩ -blockers had no effect.

The K+ and Cl− currents activated by hypotonic cell swelling were studied in Ehrlich ascites tumour cells using the whole‐cell recording mode of the patch‐clamp technique. Currents were measured in the absence of added intracellular Ca2+ and with strong buffering of Ca2+. K+ current activated by cell swelling was measured as outward current at the Cl− equilibrium potential (ECl) under quasi‐physiological gradients. It could be abolished by replacing extracellular Na+ with K+, thereby cancelling the driving force. Replacement with other cations suggested a selectivity sequence of K+ > Rb+ > NH4≈ Na+≈ Li+; Cs+ appeared to be inhibitory. The current‐voltage relationship of the volume‐sensitive K+ current was well fitted with the Goldman‐Hodgkin‐Katz current equation between ‐130 and +20 mV with a permeability coefficient of around 10−6 cm s−1 with both physiological and high‐K+ extracellular solutions. The class III antiarrhythmic drug clofilium blocked the volume‐sensitive K+ current in a voltage‐independent manner with an IC50 of 32 μM. Clofilium was also found to be a strong inhibitor of the regulatory volume decrease response of Ehrlich cells. Cell swelling‐activated K+ currents of Ehrlich cells are voltage and calcium insensitive and are resistant to a range of K+ channel inhibitors. These characteristics are similar to those of the so‐called background K+ channels. Noise analysis of whole‐cell current was consistent with a unitary conductance of 5.5 pS for the single channels underlying the K+ current evoked by cell swelling, measured at 0 mV under a quasi‐physiological K+ gradient.