Intracellular Na+, K+ and Cl− activities in Ehrlich ascites tumor cells

Ehrlich ascites tumor cell membrane potential (V_m) and intracellular Na⁺, K⁺ and Cl⁻ activities were measured under steady-state conditions in normal saline medium (Na⁺ = 154, K⁺ = 6, Cl⁻ = 150 mequiv./l). Membrane potential was estimated to be −23.3 ± 0.8 mV using glass microelectrodes. Intracellular ion activities were estimated with similar glass electrodes rendered ion-selective by incorporation of ion-specific ionophores. Measurements of V_m and ion-activity differences were made in the same populations of cells. Under these conditions the intracellular Na⁺, K⁺ and Cl⁻ activities are 4.6 ± 0.5; 68.3 ± 8.0; and 43.6 ± 2.1 mequiv./l, respectively. The apparent activity coefficients for Na⁺ and K⁺ are 0.18 ± 0.02 and 0.41 ± 0.05 respectively. These are significantly lower than the activity coefficients expected for the ions in physiological salt solutions (0.71 and 0.73, respectively). The activity coefficient for intracellular Cl⁻ (0.67 ± 0.03), however, is close to that of the medium (0.73), and the transmembrane electrochemical potential difference for Cl⁻ is not different from zero. The results establish that the energy available from the Na⁺ electrochemical gradient is much greater than previously estimated from chemical measurements.     

Volume-induced increase of K+ and Cl− permeabilities in Ehrlich ascites tumor cells. Role of internal Ca2+

Summary Ehrlich ascites tumor cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but subsequently recover their volume within 5 to 10 min with an associated KCl loss. 1. The regulatory volume decrease was unaffected when nitrate was substituted for Cl^–, and was insensitive to bumetanide and DIDS. 2. Quinine, an inhibitor of the Ca²⁺-activated K⁺ pathway, blocked the volume recovery. 3. The hypotonic response was augmented by addition of the Ca²⁺ ionophore A23187 in the presence of external Ca²⁺, and also by a sudden increase in external Ca²⁺. The volume response was accelerated at alkaline pH. 4. The anti-calmodulin drugs trifluoperazine, pimozide, flupentixol, and chlorpromazine blocked the volume response. 5. Depletion of intracellular Ca²⁺ stores inhibited the regulatory volume decrease. 6. Consistent with the low conductive Cl^– permeability of the cell membrane there was no change in cell volume or Cl^– content when the K⁺ permeability was increased with valinomycin in isotonic medium. In contrast, addition of the Ca²⁺ ionophore A23187 in isotonic medium promoted Cl^– loss and cell shrinkage. During regulatory volume decrease valinomycin accelerated the net loss of KCl, indicating that the conductive Cl^– permeability was increased in parallel with and even more than the K⁺ permeability. It is proposed that separate conductive K⁺ and Cl^– channels are activated during regulatory volume decrease by release of Ca²⁺ from internal stores, and that the effect is mediated by calmodulin.     

A Ca2+-activated Cl− current in sheep parotid secretory cells

Previous studies have shown that the whole-cell current-voltage (I-V) relation of unstimulated sheep parotid cells is dominated by two K⁺ conductances, one outwardly and the other inwardly rectifying. We now show that once these K⁺ conductances are blocked by replacement of pipette K⁺ with Na⁺ and by the addition of 5 mmol/liter CsCl to the bath, there remains an outwardly rectifying conductance with a reversal potential of 0 mV. Replacement of 120 mmol/liter NaCl in the pipette solution with an equimolar amount of Na-glutamate shifted the reversal potential of this residual current to -55 mV, indicating that the conductance was Cl^? selective. The Cl^? current was activated by increasing the free Ca²⁺ in the pipette solution from 10 to 100 nmol/liter. When the Ca²⁺ concentration in the pipette solution was 10 nmol/liter, the relaxations observed in response to membrane depolarization could be fitted with a single exponential, whose time constant increased from 81 to 183 ms as the pipette potential was increased from -30 to +60 mV. Relaxation analysis showed that the current was activated by membrane depolarization. Reversal potential measurements in experiments in which external Cl^? was replaced with various anions, gave the following relative permeabilities: SCN^- (1.80) > I^- (1.09) > CI^- (1) > NO ₃ ^- (0.92) > Br^- (0.75). The relative conductances were: SCN^- (2.18) > I^- (1.07) > Cl^? (1.00) > Br^- (0.91) > NO ₃ ^- (0.50). The Cl^? current was blocked by NPPB (ID₅₀ ≈ 10 μm), DIDS (10 or 30 μmol/liter) and furosemide (100 μmol/liter).     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

Identification and regulation of whole-cell Cl− and Ca2+-activated K+ currents in cultured medullary thick ascending limb cells

The whole-cell patch-clamp technique has been used to study membrane currents in cultured rabbit medullary thick ascending limb (MTAL) epithelial cells. A Ca²⁺-activated K⁺ current was characterized by its voltage-dependent and Ca²⁺-dependent properties. When the extracellular K⁺ ion concentration was increased from 2 to 140 mm, the rereversal potential (E_k) was shifted from –85 to 0 mV with a slope of 46 mV per e-fold change. The Ca²⁺-activated K⁺ current is blocked by charybdotoxin (CTX) in a manner similar to the apical membrane Ca²⁺-activated K⁺ channel studied with the single channel patch-clamp technique. The results suggest that the Ca²⁺-activated K⁺ current is the predominant, large conductance and Ca²⁺-dependent K⁺ pathway in the cultured MTAL cell apical membrane. The biophysical properties and physiological regulation of a Cl^– current were also investigated. This current was activated by stimulation of intracellular cAMP using forskolin and isobutyl-1-methylxanthine (IBMX). The current-voltage (I–V) relationship of the Cl^– current showed an outward-rectifying pattern in symmetrical Cl^– solution. The Cl^– selectivity of the whole-cell current was confirmed by tail current analysis in different Cl^– concentration bath solutions. Several Cl^– channel blockers were found to be effective in blocking the outward-rectifying Cl^– current in MTAL cells. The cAMP-dependent Cl^– transport in MTAL cells was further confirmed by measuring changes in the intensity of Cl^– sensitive dye using fluorescence microscopy. These results suggest that the Cl^– channel in the apical or basolateral membrane of MTAL cells may be regulated by cAMP-dependent protein-kinase-induced phosphorylation.This study was supported by the National Institutes of Health grants GM46834 to L.L. and DK32753 to W.B.G., and by a Grant-in-Aid from the American Heart Association of Ohio to L.L.     

Phosphate transport and its relationship to cation movements in Ehrlich Lettré ascites tumor cells.

In view of the importance of P_i in the control of cell metabolism, it was of interest to study the mechanism and regulation of P_i uptake by ascites tumor cells. For this purpose, the incorporation of ³²P_i into Ehrlich Lettré cells was compared when competitive anions and inhibitors which alter cation movements were present. Anions such as sulfanilate (35 mm) and succinate (30 mm) decrease ³²P_i uptake by ca. 35%, suggesting that transport is mediated by a protein similar to the 100,000 M_r anion carrier isolated from erythrocyte membranes. Furosemide, a diuretic which bears a structural analogy to sulfanilate inhibitors of anion transport, also decreases ³²P_i incorporation at concentrations as low as 2 × 10^?5m. This inhibitor blocks cation exchange in ascites tumor cells, and from the present data, it is suggested that a possible function of the furosemidesensitive cation exchange protein is to facilitate anion transport. Ouabain, known to inhibit (Na⁺ + K⁺)-ATPase and its dephosphorylation, stimulates the rate of incorporation of ³²P_i into cells and also raises the net inorganic phosphate level. The stimulation of ³²P_i incorporation is decreased by sulfanilate or succinate. In contrast to the effects of ouabain, addition of 10 mm K⁺, which is known to stimulate (Na⁺ + K⁺)-ATPase and its dephosphorylation, decreases ³²P_i incorporation. These observations suggest that anion transport and energy-dependent Na⁺ and K⁺ movements may be closely coupled to the intact cell.     

Intracellular compartmentation of Na+, K+ and Cl− in the Ehrlich ascites tumor cell: Correlation with the membrane potential

Summary The intracellular distribution of Na⁺, K⁺, Cl^– and water has been studied in the Ehrlich ascites tumor cell. Comparison of the ion and water contents of whole cells with those of cells exposed to La³⁺ and mechanical stress indicated that La³⁺ treatment results in selective damage to the cell membrane and permits evaluation of cytoplasmic and nuclear ion concentrations. The results show that Na⁺ is sequestered within the nucleus, while K⁺ and Cl^– are more highly concentrated in the cell cytoplasm. Reduction of the [Na⁺] of the incubation medium by replacement with K⁺ results in reduced cytoplasmic [Na⁺], increased [Cl^–] and no change in [K⁺]. Nuclear concentrations of these ions are virtually insensitive to the cation composition of the medium. Concomitant measurements of the membrane potential were made. The potential in control cells was –13.7 mV. Reduction of [Na⁺] in the medium caused significant depolarization. The measured potential is describable by the Cl^– equilibrium potential and can be accounted for in terms of cation distributions and permeabilities. The energetic implications of the intracellular compartmentation of ions are discussed.     

Calcium-ion transport by intact Ehrlich ascites tumor cells at 0°C

At 0°C, where Ca²⁺ efflux is not observed, the uptake of Ca²⁺ by Ehrlich ascites tumor cells consists of four components: 1) An energy-dependent mitochondrial component, which is inhibited by uncouplers, respiratory inhibitors, and mitochondrial ATP-ase inhibitors. 2) Binding to the cell surface, which can be displaced by an EGTA wash. 3) An electrochemical gradient-dependent component, which is inhibited by agents which dissipate these gradients, such as proton ionophores, metabolic uncouplers, and valinomycin. The valinomycin inhibition of this transport component is dependent on K⁺ concentration. 4) Passive diffusion, which is dependent on Ca²⁺ concentration and is observed in the presence of inhibitors of the other components. The uptake of Ca²⁺ at 0°C is sensitive to ruthenium red presumably due to its competition with Ca²⁺ for cell binding sites.     

Cell swelling activates K+ and Cl− channels as well as nonselective,stretch-activated cation channels in ehrlich ascites tumor cells

Summary Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba²⁺ and hence presumably to Ca²⁺. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca²⁺ permeability, as part of the volume-activated signal system. In isolated insideout patches a Ca²⁺-dependent, inwardly rectifying K⁺ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mm K⁺ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K⁺ channel takes place after an increase in Ca²⁺ from 10^–7 to 10^–6 m which is in the physiological range. Patch-clamp studies in cellattached mode show K⁺ channels with spontaneous activity and with characteristics similar to those of the K⁺ channel seen in excised patches. The single-channel conductance for outward current at 5 mm external K⁺ is estimated at about 7 pS. A K⁺ channel with similar properties can be activated in the cellattached mode by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K⁺ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K⁺ channels following addition of Ca²⁺ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K⁺ channels in the presence of 3 mm Ca²⁺ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca²⁺ and could not be activated by membrane stretch (suction). In cell-attached mode singlechannel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by hypotonic exposure with a single-channel conductance at 7 pS (or less) and with a time delay at about 1 min. The number of open channels during RVD is estimated at 80 per cell. Two other types of Cl^– channels were regularly recorded in excised inside-out patches: a voltage-activated 400-pS channel and a 34-pS Cl^– channel which show properties similar to the Cl^– channel in the apical membrane in human airway epithelial cells. There is no evidence for a role in RVD for either of these two channels.     

Na+, Cl− cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase)

Ehrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. The volume recovery was inhibited when NO-3 was substituted for Cl-, and when Na+ was replaced by K+, or by choline (at 5 mM external K+). The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. The net uptake of Cl- was much larger than the value predicted from the conductive Cl- permeability. The undirectional 36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. During volume recovery the Cl- flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl-, or with an uptake via a K+,Na+,2Cl- cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl- in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+,Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+,Cl- cotransport system.     

Ca2+-dependent Cl− efflux in tonoplast-free cells ofNitellopsis obtusa

Summary In order to demonstrate the presence of a Ca²⁺-activated Cl^–-channel in theNitellopsis plasmalemma, tonoplast-free cells were prepared and their intracellular Ca²⁺ concentration was modified by internal perfusion. An increase in the Ca²⁺ concentration caused a large Cl^– efflux with a concomitant depolarization of the membrane potential. These changes were for the most part reversible. The critical Ca²⁺ concentration was about 4.0 m. Neither the Cl^– efflux nor the membrane depolarization showed a time-dependent inactivation. A Cl^–-channel blocker, A-9-C (9-anthracenecarboxylic acid) reduced both the Cl^– efflux and the magnitude of the membrane potential depolarization. A small increase in the intracellular Ca²⁺ concentration, which is caused by membrane excitation of tonoplast-free cells is not sufficient to activate this Ca²⁺-dependent Cl^–-channel.     

Regulatory volume increase in Ehrlich ascites tumor cells is mediated by the 1Na ∶ 1K ∶ 2Cl cotransport system

Summary After swelling in hyposmotic solution, Ehrlich ascites tumor cells shrink towards their original volume. Upon restoration of isosmolality (300 mOsm) the cells initially shrink but subsequently recover volume. This regulatory volume increase (RVI) is completely blocked when [Na⁺] _o or [Cl^–] _o is reduced by 50% in the presence of normal [K⁺] _o . With normal [NaCl] _o but less than 2 mm [K⁺] _o , not only is volume recovery blocked but the cells lose KCl and shrink. When [K⁺] _o is increased to 5 mm there is a rapid net uptake of K⁺ and Cl^– which results in volume recovery. This suggests that the reswelling phase requires the simultaneous presence of Na⁺, K⁺, and Cl^–. Although ouabain has no effect on volume recovery, bumetanide completely blocks RVI by inhibiting a cotransport pathway that mediates the net uptake of Na⁺, K⁺ and Cl^– in the ratio of 1Na1K2Cl. Na⁺ that accumulates is then replaced by K⁺ via the Na/K pump.I wish to thank my colleague, Dr. Thomas C. Smith for advice and helpful comments during the course of these studies. The excellent technical assistance provided by Rebecca Corcoran-Merrill is gratefully acknowledged.This investigation was supported by Grant CA 32927 from the National Cancer Institute, U.S. Public Health Service.     

Hydrochlorothiazide action on the apical Cl−, Ca2+ and K+ conductances in rabbit gallbladder epithelium. Presence of an apamin-sensitive,Ca2+-activated K+ conductance

In the rabbit gallbladder epithelium, hydrochlorothiazide (HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na⁺-Cl^– symport, to depolarize the apical membrane potential and to enhance the cell-to-lumen Cl^– backflux (radiochemically measured), this increase being SITS-sensitive. To better investigate the causes of the depolarization and the Cl^– backflux increase, cells were punctured with conventional microelectrodes on the luminal side (incubation in bicarbonate-free saline at 27°C) and the apical membrane potential (V _m) was studied either with prolonged single impalements or with a set of short multiple impalements. The maximal depolarization was of 3–4 mV and was reached with 2.5 × 10^–4 m HCTZ. It was significantly enhanced by reducing luminal Cl^– concentration to 30 mm; it was abolished by SCN^–, furosemide, SITS; it was insensitive to DPC. SITS converted the depolarization into a hyperpolarization of about 4 mV; this latter was apamin, nifedipine and verapamil sensitive. It was concluded that HCTZ concomitantly opens apical Cl^– and (probably) Ca²⁺ conductances and, indirectly, a Ca²⁺-sensitive, apamin inhibitable K⁺ conductance: since the intracellular Cl^– activity is maintained above the value predicted at the electrochemical equilibrium, the opening of the apical Cl^– conductance depolarizes V _mand enhances Cl^– backflux. In the presence of apamin or verapamil, to avoid the hyperpolarizing effects due to HCTZ, the depolarization elicited by this drug was fully developed (7–10 mV) and proved to be Ca²⁺ insensitive. On this basis and measuring the transepithelial resistance and the apical/basolateral resistance ratio, the Cl^– conductance opened by HCTZ has been estimated and the Cl^– backflux increase calculated: it proved to be in the order of that observed radiochemically. The importance of this Cl^– leak to the lumen in the overall inhibition of the transepithelial NaCl transport by HCTZ has been evaluated.This research was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Rome, Italy. We are very grateful to prof. G. Meyer and dr. G. Bottà for helpful discussion and criticism.     

Differential Effect of High Extracellular Ca2+ on K+ and Cl− Conductances in Murine Osteoclasts

Effects of the extracellular Ca²⁺ concentration ([Ca²⁺] _o ) on whole cell membrane currents were examined in mouse osteoclastic cells generated from bone marrow/stromal cell coculture. The major resting conductance in the presence of 1 mm Ca²⁺ was mediated by a Ba²⁺-sensitive, inwardly rectifying K⁺ (IR_K) current. A rise in [Ca²⁺] _o (5–40 mm) inhibited the IR_K current and activated an 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS)-sensitive, outwardly rectifying Cl⁻ (OR_Cl) current. The activation of the OR_Cl current developed slowly and needed higher [Ca²⁺] _o than that required to inhibit the IR_K current. The inhibition of the IR_K current consisted of two components, initial and subsequent late phases. The initial inhibition was not affected by intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or guanosine 5′-O-(2-thiodiphosphate) (GDPβS). The late inhibition, however, was enhanced by GTPγS and attenuated by GDPβS, suggesting that GTP-binding proteins mediate this inhibition. The activation of the OR_Cl current was suppressed by pretreatment with pertussis toxin, but not potentiated by GTPγS. An increase in intracellular Ca²⁺ level neither reduced the IR_K current nor activated the OR_Cl current. Staurosporine, an inhibitor for protein kinase C, did not modulate the [Ca²⁺] _o -induced changes in the IR_K and OR_Cl conductances. These results suggest that high [Ca²⁺] _o had a dual action on the membrane conductance of osteoclasts, an inhibition of an IR_K conductance and an activation of an OR_Cl conductance. The two conductances modulated by [Ca²⁺] _o may be involved in different phases of bone resorption because they differed in Ca²⁺ sensitivity, temporal patterns of changes and regulatory mechanisms. Received: 28 May 1996/Revised: 28 January 1997     

Extracellular ATP activates both Ca2+- and cAMP-dependent Cl− conductances in rat epididymal cells

Activation of Ca²⁺ and cAMP-dependent Cl^– conductances by extracellular ATP was studied using the whole-cell patch clamp technique. Immediately after addition of extracellular ATP (10 m), activation of wholecell Cl^– current exhibiting delayed inactivation and activation kinetics at hyperpolarizing and depolarizing voltages, respectively, was observed. After prolonged activation, the kinetic characteristics of the ATP-induced Cl^– current became time- and voltage-independent. When applied to the later phase of the ATP-activated whole-cell current, the disulfonic acid stilbene DIDS (200 m) could only inhibit 64% of the current while diphenylamine-dicarboxylic acid (DPC, 1 mm) completely inhibited it. Inclusion of a peptide inhibitor for protein kinase A (PKI, 10 nm) in the pipette solution blocked ATP-induced time- and voltage-independent current activation but did not affect the delayed activating and inactivating current activation but did not affect the delayed activating and inactivating current which could be totally blocked by DIDS. Anion selectivity sequence was determined in the presence of either PKI or DIDS and found to be significantly different. Increased pipette EGTA (10 mm) or treatment of the cells with trifluoperazine (40 m), an inhibitor of calmodulin, suppressed both types of ATP-induced Cl^– currents. No current activation by ATP was observed when cells were dialyzed with the IP₃ receptor blocker, heparin (10 ng/ml). These results suggest that extracellular ATP activates IP₃-linked Ca²⁺-dependent regulatory pathway, which in turn activates cAMP-dependent pathway, leading to activation of both Ca²⁺ and cAMP-dependent Cl^– conductances in epididymal cells.The authors wish to thank Mr. W.O. Fu for technical assistance. This work was supported by the Croucher Foundation, the University and Polytechnic Grants Committee.     

Niflumic Acid Affects Store-Operated Ca2+-Permeable (SOC) and Ca2+-Dependent K+ and Cl− Ion Channels and Induces Apoptosis in K562 Cells

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells. However, the precise mechanisms by which NSAIDs facilitate apoptosis in tumor cells are not clear. In the present study, we show that niflumic acid (NA), a member of the fenamates group of NSAIDs and Cl^? and Ca²⁺-activated Cl^? (CAC) channels blocker, induced apoptosis (by ~8 %, 24 h treatment) and potentiated (by 8–10 %) apoptotic effect of endoplasmic reticulum Ca²⁺ mobilizer thapsigargin (Tg) in human erythroleukemic K562 cell line. The whole-cell patch clamp and Fluo-3 flow cytometric experiments confirmed an inhibitory effect of NA (100 and 300 µM) on store-operated (SOC) channels. We also found that NA-blocked CAC channels were activated by acute application of Tg (2 µM) in K562 cells. NA blockage of CAC channels was accompanied by activation of Ca²⁺-activated K⁺ (SK4) channels. The observed effects of NA were not connected with COX-2 inhibition since 100-nM NA (IC₅₀ for COX-2 inhibition) did not induce either apoptosis or affect the channels activity. We conclude that inhibition of SOC channels plays a major role in NA-induced apoptosis. Increased apoptotic levels in Tg-treated K562 cells in the presence of NA may be due to the blockage of CAC and stimulation of SK4 channels in addition to SOC channels inhibition.     

The bestrophin- and TMEM16A-associated Ca2+-activated Cl– channels in vascular smooth muscles

The presence of Ca²⁺-activated Cl^– currents (I_Cl(Ca)) in vascular smooth muscle cells (VSMCs) is well established. I_Cl(Ca) are supposedly important for arterial contraction by linking changes in [Ca²⁺]_i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with I_Cl(Ca). Two distinct I_Cl(Ca) are characterized in VSMCs; the cGMP-dependent I_Cl(Ca) dependent upon bestrophin expression and the ‘classical’ Ca²⁺-activated Cl^– current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical I_Cl(Ca). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A’s role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl^– channels. It is suggested that TMEM16A expression modulates voltage-gated Ca²⁺ influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.     

Reduced growth of Ehrlich ascites tumor cells in creatine depleted mice fed β-guanidinopropionic acid

The effect of implantation of Ehrlich ascites tumor (EAT) cells of creatine distribution was investigated. It was also studied how depletion of creatine by feeding creatine-analogue β-guanidinopropionic acid (β-GPA) affects the growth of EAT cells in mice. Enhanced mobilization of creatine from host tissues to EAT cells against a greater concentration gradient was observed. The creatine (but not creatinine) level in blood plasma was lowered to 22% of the normal value by β-GPA feeding alone and assimilation of ¹⁴C-creatine into EAT cells was inhibited. The growth of EAT cells was significantly reduced and the duration of survival of mice after implantation of EAT cells was extended when the creatine concentration was decreased. A decrease in daily food consumption and the degree of muscle atrophy after implantation of EAT cells was less in β-GPA than control groups. In the creatine-depleted mice, the rate of increase in total EAT cell number and the volume of abdominal ascites were approximately half of the control values, and more dead EAT cells were observed. These results suggest that supplementation of β-GPA inhibits creatine transfer to EAT cells and reduces the growth of cancer cells.     

The bestrophin- and TMEM16A-associated Ca2+-activated Cl– channels in vascular smooth muscles

The presence of Ca²⁺-activated Cl^– currents (I_Cl(Ca)) in vascular smooth muscle cells (VSMCs) is well established. I_Cl(Ca) are supposedly important for arterial contraction by linking changes in [Ca²⁺]_i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with I_Cl(Ca). Two distinct I_Cl(Ca) are characterized in VSMCs; the cGMP-dependent I_Cl(Ca) dependent upon bestrophin expression and the ‘classical’ Ca²⁺-activated Cl^– current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical I_Cl(Ca). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A’s role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl^– channels. It is suggested that TMEM16A expression modulates voltage-gated Ca²⁺ influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.     

Coexpression and characterization of the human large-conductance Ca2+-activated K+ channel α + β1 subunits in HEK293 cells

Large-conductance Ca²⁺-activated K⁺ channel is formed by a tetramer of the pore-forming α-subunit and distinct accessory β-subunits (β1–β4) which contribute to BK_Ca channel molecular diversity. Accumulative evidences indicate that not only α-subunit alone but also the α + β subunit complex and/or β-subunit might play an important role in modulating various physiological functions in most mammalian cells. To evaluate the detailed pharmacological and biophysical properties of α + β1 subunit complex or β1-subunit in BK_Ca channel, we established an expression system that reliably coexpress hSloα + β1 subunit complex in HEK293 cells. The coexpression of hSloα + β1 subunit complex was evaluated by western blotting and immunolocalization, and then the single-channel kinetics and pharmacological properties of expressed hSloα + β1 subunit complex were investigated by cell-attached and outside-out patches, respectively. The results in this study showed that the expressed hSloα + β1 subunit complex demonstrated to be fully functional for its typical single-channel traces, Ca²⁺-sensitivity, voltage-dependency, high conductance (151 ± 7 pS), and its pharmacological activation and inhibition.