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Evidence That Outwardly Rectifying Cl− Channels Underlie Volume-Regulated Cl− Currents in Heart
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Swelling-induced Cl− current (ICl.swell) is present in most cardiac tissues, but the unitary channel underlying ICl.swell is unknown. We used the cell-attached patch-clamp technique to assess the properties of single channels underlying ICl.swell and the basally active Cl− current (ICl.b) in rabbit atrial myocytes. Under isotonic conditions, single outwardly rectifying Cl− channels (ORCCs) with a slope conductance of 28±1 pS at the reversal potential were observed in 21 (5.7%) of 367 patches. Unconditional kinetic analysis revealed at least three open and four closed-channel states. Hypotonic superfusion-induced swelling resulted in the appearance of active channels in 41 (15.5%) of 265 patches without channel activity under isotonic conditions and caused a second active channel to appear in 3 of 14 patches showing a single channel under isotonic conditions. Overall, channels were seen in 54 of 336 patches under hypotonic conditions (16.1%, P<.001 versus isotonic conditions). The current-voltage relations, reversal potential–[Cl−]o relations, open probability, and kinetics of swelling-induced channels were indistinguishable from those of ORCCs under isotonic conditions. Unitary ORCCs, ICl.b, and ICl.swell were strongly and similarly inhibited by tamoxifen. Swelling-induced increases in macroscopic Cl− current were attributable to an increase in the number of active ORCCs with no significant effects on single-channel amplitude or open probability. Estimated macroscopic currents based on cell surface area, patch dimensions, single-channel ORCC current amplitude, open probability, and density were consistent with measured values of ICl.b and ICl.swell. We conclude that ORCCs underlie volume-regulated basal and swelling-induced Cl− currents in isolated rabbit atrial myocytes.
Chloride channels have been found to be present in the plasma membranes of most cells and play potentially important roles in the control of cell volume, pHi, and membrane potential.1 2 Over the past few years, evidence has accumulated to show that the heart may express several types of Cl− channels.3 ICl.swell has been reported to exist in various cardiac cell types, such as canine atrial and ventricular cells,4 5 rabbit atrial cells,6 guinea pig atrial and ventricular cells,7 human atrial cells,8 9 10 and cultured chick heart cells.11 12 Although a 400-pS Cl− channel has been reported to be activated by exposure to hypotonic solution in neonatal rat cardiac cells,13 this channel has not been observed in adult cardiac cells. The nature of the unitary channel(s) underlying cardiac ICl.swell is thus unclear.
In rabbit atrial myocytes, we have previously observed ICl.b14 15 and more recently have found that ICl.b may be regulated by cell volume and α1-adrenoceptor–coupled, pertussis toxin–sensitive, G protein–mediated activation of PKC, a mechanism similar to that regulating ICl.swell in the same preparation.6 Although ORCCs of ≈60 pS were identified in inside-out patches excised from rabbit atrial myocytes with an I-V form similar to that of macroscopic ICl.b,15 a definitive identification between ORCC and ICl.b is still lacking. Furthermore, the nature of unitary channels underlying ICl.swell remains unknown. In the present study, we used the patch-clamp technique to identify unitary Cl− channels in the cell-attached mode in rabbit atrial myocytes during superfusion with isotonic and hypotonic solutions and to study their properties. Macroscopic ICl.b and ICl.swell were also studied with the use of the whole-cell voltage-clamp technique to compare them with single-channel currents under similar conditions. Preliminary data from these studies have been presented in abstract form.16 17
Materials and Methods
Preparation of Single Cells
Single atrial cells were obtained from rabbit hearts using a previously described dissociation technique.6 14 15 Briefly, rabbits (1.5 to 2.0 kg) were killed by a blow on the neck, and the hearts were quickly removed and perfused in the Langendorff mode, first with a modified HEPES-buffered Tyrode's solution at 37°C, then with a nominally Ca2+-free Tyrode's solution until the heart ceased to beat, and finally with the same solution containing 0.04% collagenase (CLSII, Worthington Biochemical) and 1.0% bovine serum albumin (Sigma Chemical Co) for 10 minutes. The left atrium was removed and further dissected into small pieces, and cell dissociation was achieved by gentle mechanical agitation. All cells studied were rod-shaped, exhibited clear cross striations, and lacked any visible blebs under isotonic conditions. Cell dimensions were determined with a calibrated graticule in the microscope, and cell volumes were estimated with assumed right cylindrical geometry according to the following equation: V=πL(W/2)2, where V, L, and W are cell volume, length, and width, respectively.
Electrophysiological Recording
The cell-attached configuration of the patch-clamp technique18 was used to record single Cl− channel currents. The tight-seal, whole-cell, voltage-clamp configuration of the patch-clamp technique was used to record the macroscopic ICl.b and ICl.swell and compare their I-V and other properties with those of single-channel currents under similar conditions. Recording pipettes were prepared from borosilicate glass electrodes (outer diameter, 1.5 mm) with tip resistances of 2 to 5 MΩ when filled with pipette solution. A bridge (3 mol/L KCl in agar salt) between the bath and a Ag/AgCl reference electrode immersed in pipette solution was used to minimize changes in liquid junction potential, and junction potentials were zeroed before establishing a membrane seal. A tight seal between the cell membrane and the pipette tip (seal resistance, >10 GΩ) was achieved by applying light suction. Recordings were made using an Axopatch 200A amplifier (Axon Instruments). Commercial software from Axon Instruments (pCLAMP 6; Clampex, Clampfit, Fetchex, Fetchan, and Pstat routines) was used for control of voltage-clamp protocols, data acquisition, and data analysis. Voltage-clamp pulses were generated by a 12-bit digital-to-analog converter. Single-channel currents were recorded at a gain of 500 mV/pA and low pass–filtered with an eight-pole Bessel filter at 5 kHz and stored on videotape or at 1 to 2 kHz and simultaneously digitized (Digidata 1200, Axon Instruments Inc) at a sampling rate of 2 to 5 kHz and stored on the hard disk of an IBM PC/AT compatible computer.
To obtain single-channel I-V relations, the membrane was clamped from a holding potential of 0 mV (relative to the RP) to a series of test potentials for 4 s at a time. The voltage of all cell-attached single-channel voltage clamps in the present study will be expressed as RP+V, where RP is the cell membrane resting potential and V is the transpatch voltage step applied by the amplifier, as would be measured at the intracellular side of the patch membrane. Hyperpolarizing and depolarizing pulses were imposed at 0.1 Hz in +10-mV increments between RP−40 and RP+140 mV. Ensemble averages of single-channel current were obtained by analyzing data from patches with a single channel and averaging the records of 60 two-second pulses. All command voltages and single-channel currents are displayed as they would be measured at the intracellular side of the membrane; ie, the values given are the negative of the values measured by the pipette at the extracellular surface.
The cell-attached patch configuration was checked at the end of each experiment by rupturing the patch to confirm a passage from the cell-attached to the whole-cell configuration. The average intracellular potential measured immediately after membrane rupture was 63.6±1.6 mV (n=53). All experiments were performed at 30±1°C. Inward cation currents such as Na+, Ca2+, and nonselective cation currents were prevented in single-channel studies by using the large impermeant cation NMDG as the only cation in the pipette solution. CdCl2 (200 μmol/L), BaCl2 (2 mmol/L), 4-aminopyridine (2 mmol/L), and TEA-Cl (10 mmol/L) were present continuously in the bath solution to block Ca2+ and K+ currents (inward rectifier, transient outward, and delayed rectifier K+ currents, respectively).
Solutions and Drugs
The modified Tyrode's solution for cell isolation contained (mmol/L) NaCl 126, KCl 5.4, CaCl2 2.0, MgCl2 1.0, NaH2PO4 0.33, glucose 10, and HEPES 10; pH was adjusted to 7.4 with NaOH. The high-K+ cell storage solution contained (mmol/L) KCl 20, KH2PO4 10, glucose 10, l-glutamic acid 70, β-hydroxybutyric acid 10, taurine 10, and EGTA 10, along with 1% albumin, pH 7.4 (KOH). Osmolarity was adjusted to 290 to 300 mOsm/kg H2O by adding mannitol.
The standard pipette (external) solution for cell-attached patch-clamp recording contained (mmol/L) NMDG-Cl 108, HEPES 5, and glucose 5.5 (total [Cl−]p=108 mmol/L). The pH was adjusted to 7.40 with NMDG-OH, and osmolarity was adjusted to 285 to 295 mOsm/kg H2O by adding mannitol. To evaluate the ionic selectivity of the channels studied, the concentration of the ion of interest was reduced by equimolar replacement of NMDG with Tris or of Cl− with aspartate in the pipette solution. When high [Cl−]p was needed, the concentration of NMDG-Cl in the pipette solution was increased. The pipette (internal) solution for whole-cell patch-clamp recordings contained (mmol/L) NMDG aspartate 100, NMDG-Cl 24, Mg2+-ATP 5, and HEPES 10 (total [Cl]i=24 mmol/L).
The standard hypotonic bath (external) solutions for both cell-attached and whole-cell recordings contained (mmol/L) NaCl 85, KCl 5.0, BaCl2 2, TEA-Cl 10, CdCl2 0.2, 4-aminopyridine 5, MgCl2 0.8, CaCl2 1.0, NaH2PO4 0.33, HEPES 10, and glucose 5.5; pH was adjusted to 7.4 with NaOH (220 mOsm/kg H2O); total [Cl]o was 108 mmol/L. When experiments were performed with decreased [Cl−], aspartate was used to replace Cl− at equimolar concentrations. The standard isotonic bath solution was the same as the standard hypotonic solution, but the osmolarity was adjusted to 290 to 310 (302±4) mOsm/kg H2O by adding mannitol. Solution osmolarities were measured by freezing-point depression (Osmomette A, Precision Systems Inc).
Data Analysis
Single-channel current amplitudes were measured relative to the 0 current level. For the analysis of Po and open- and closed-state kinetics, patches were held at the desired potential for at least 150 s (>100 000 transitions). Po was obtained for patches with only one open peak and one closed peak in the amplitude histogram from the ratio of the area under the curve representing open events (fitted with a gaussian equation by a curve-fitting program in Pstat) divided by the sum of the areas under the open- and closed-event histograms. The kinetics of open and closed events were analyzed for patches containing only one active channel (determined by all-points amplitude histogram) with a half-amplitude algorithm incorporated in Fetchan, and events were reviewed manually. Open and closed dwell-time analyses were performed using the unconditional distributions of these interval durations.19 The interval durations were logarithmically binned, and the number of events was transformed as a square root of the ordinate in order to keep the errors approximately constant throughout the plot of the dwell-time histogram.20 Log-binned open or closed dwell-time data were fitted by the maximum likelihood estimate or Marquardt least-squares method (program incorporated in Pstat) using 1, 2, 3, and 4 exponential terms. To test whether or not different models produce a statistically better fit, for each dwell-time distribution histogram we compared the sum of squared errors for each model (the F value) and the ratio of the natural logarithm of the maximum likelihood estimate (log likelihood ratio) for different models. The F statistic was evaluated at α levels of 90% and 95%. A log likelihood ratio of >2 was taken to indicate statistical significance.
All results are expressed as mean±SEM. Statistical comparisons were performed either by ANOVA with Scheffé contrasts for group data or by Student's t test when only two groups were compared. The χ2 test was applied to compare the prevalence of active channels under isotonic versus hypotonic conditions. A two-tailed probability of <5% was taken to indicate statistical significance.
Properties of Unitary Cl− Channel Current in Cell-Attached Patches
Under isotonic conditions (290 to 310 mOsm/kg H2O), most patches failed to show single-channel activity. However, in a minority of patches (21 [5.7%] of 367), single-channel activity of the type shown in Fig 1A⇓ was clearly visible. When present, channel activity was evident at either hyperpolarizing or depolarizing voltages immediately after the formation of cell-attached configuration and generally remained stable for >15 minutes. Single-channel currents showed strong outward rectification and reversed at 19±1 mV (n=6) positive to RP at [Cl−]p of 108 mmol/L. The average slope conductance of the channel for outward current was 49±1 pS (n=6) on the basis of linear regression of current versus voltage over the range RP+20 to RP+140 mV (slope conductance, 28±1 pS at reversal potential) when [Cl−]p was 108 mmol/L (Fig 1B⇓).
Three examples of single-channel currents elicited from a typical patch by 2-second hyperpolarizing (Fig 1C⇑, left) and depolarizing (Fig 1C⇑, right) pulses from the RP are shown at the bottom of Fig 1⇑. The respective voltage steps shown are ≈60 mV negative (Fig 1C⇑, left) and 60 mV positive (Fig 1C⇑, right) to the current reversal potential at 108 mmol/L [Cl−]p, which averaged RP+19 mV. Ensemble-averaged currents from 60 pulses are shown at the bottom of each panel and indicate that the current carried by this channel is time independent and outwardly rectifying.
To evaluate the ionic selectivity of the channel, we altered [Cl−]p by equimolar substitution with aspartate, producing the type of recordings shown in Fig 2A⇓-b, which had smaller outward currents and a more positive reversal potential than recordings at a physiological [Cl−]o of 108 mmol/L (Fig 2A⇓-a). Increasing the concentration of NMDG-Cl (to increase [Cl−]p) had opposite effects, resulting in the type of recordings shown in Fig 2A⇓-c. Mean (±SEM; where error bars are not visible, they fall within symbol for mean) I-V relations for single-channel current under each condition are shown in Fig 2B⇓. A decrease in [Cl−]p to 22 mmol/L decreased single-channel conductance in the outward direction (to 23±1 pS, over the range from RP+60 mV to RP+140 mV; slope conductance at reversal potential, 20±2 pS; n=4) and shifted the reversal potential to more positive values (53±3 mV). Opposite effects were seen with an increase in [Cl−]p to 208 mmol/L, which increased conductance in the outward direction (to 62±1 pS, over the range from RP+10 mV to RP+140 mV; slope conductance at reversal potential, 33±1 pS; n=3) and shifted the reversal potential to more negative voltages (RP−6±1 mV). When mean reversal potentials (expressed as voltages relative to RP) were plotted as a function of log ([Cl−]p), the points fell on a line (r=.987) with a slope of −57.7 mV per decade (Fig 2C⇓), indicating substantial Cl− selectivity of the channel. NMDG was the only cation in the pipette solution. Since cation channels are relatively impermeable to NMDG, inward currents across single channels studied with NMDG-Cl in the pipette are very unlikely to be due to cation entry into the cell. To address this possibility further, we eliminated NMDG from the pipette solution by substituting the even larger cation Tris for NMDG in the pipette. Fig 2D⇓ shows mean I-V relations obtained with either 108 or 0 mmol/L NMDG in the pipette. The mean reversal potential of single-channel currents obtained with Tris substitution was RP+17±2 mV (n=4), not significantly different from the value obtained with 108 mmol/L NMDG in the pipette (RP+19±1 mV). Single-channel conductance over the range between RP+20 mV and RP+140 mV averaged 51±2 pS (n=4) with 0 mmol/L NMDG in the pipette, not significantly different from the value of 49±1 pS obtained with the use of 108 mmol/L NMDG.
Channel activity at all voltages showed long-duration openings with either brief or relatively longer-lasting closings, as shown in Figs 1 through 3⇑⇑⇓. Amplitude histograms of unitary single-channel currents were consistent with the presence of only one channel with a single dominant closed and open level for each of the seven patches studied to evaluate Po. Po averaged 0.68±0.02 at RP+120 mV (n=6), 0.66±0.04 at RP−40 mV (n=7), 0.63±0.03 at RP+60 mV (n=6), 0.62±0.03 at RP+80 mV (n=5), 0.65±0.03 at RP+100 mV (n=6), and 0.70±0.03 at RP+140 mV (n=5) (P=NS for voltage dependence, ANOVA).
Channel kinetics were studied in seven patches in which single-channel activity was observed for at least 150 seconds. In each patch, single open and closed levels were determined from the all-points amplitude histogram. Results from a representative patch are shown in Fig 3⇑. During hyperpolarization (to 40 mV negative to RP), channel opening elicited an inward current. The open and closed analysis using the unconditional interval distribution and maximum likelihood fitting indicated that the channels must have a minimum of three open kinetic states (P<.01 for a model with three open states versus model with two, P>.05 for a model with four open states versus a model with three) with time constants of τ1=2.1 ms, τ2=15.9 ms, and τ3=120.8 ms and a minimum of four closed states (P<.05 for a model with four closed states versus a model with three, P<.01 for a model with four closed states versus a model with two, and P<.01 for a model with three closed states versus a model with two) with time constants of τ1=1.0 ms, τ2=6.1 ms, τ3=31.4 ms, and τ4=246.7 ms. Mean kinetic data from seven patches under isotonic conditions are shown in the Table⇓.
Effects of Hypotonic Cell Swelling on Single-Channel Activity
In the first series of experiments, patches lacking single-channel activity in the presence of isotonic superfusate were monitored during the induction of cell swelling. Superfusion with hypotonic bath solution (210 to 220 mOsm/kg H2O) for >15 minutes increased cell volume from 7895±524 μm3 (length, 102±3 μm; width, 9.8±0.3 μm) to 14778±1080 μm3 (length, 102±3 μm; width, 13.3±0.4 μm; n=54), representing an 88±6% increase in volume compared with control conditions (P<.001). Fig 4⇓ shows an example of a patch without single-channel activity under isotonic conditions that developed currents typical of ORCCs in the presence of hypotonic swelling. Of 220 patches lacking channel activity under isotonic conditions with [Cl−]p of 108 mmol/L that were followed for >15 minutes after the onset of hypotonic superfusion, 36 (16.4%) showed the type of response illustrated in Fig 4⇓. The onset of channel opening was observed an average of 15±2 minutes after changing to hypotonic superfusate.
Another set of patches (n=14) that demonstrated channel activity under isotonic conditions was observed during the induction of hypotonic swelling. In some of these (n=3), cell swelling revealed the presence of a second channel in the patch, with unitary current properties resembling those of the channels under basal conditions. Fig 5⇓ shows a representative recording of single-channel currents from these patches. Under isotonic conditions, the amplitude distribution histogram (right panel of Fig 5A⇓) showed only a single dominant open and closed level and was best fitted with a two-order gaussian equation (r=.94, mean current amplitude, −0.43 pA; Po, 0.65). Exposure of the same cell to hypotonic superfusate for 10 minutes caused the appearance of a second active channel in the same patch. The amplitude histogram (right panel of Fig 5B⇓) showed two open levels and one closed level and was best fit with a three-order gaussian equation (r=.96). The mean current amplitude at each open level was the same (−0.43 pA), and the amplitude distributions were consistent with two channels with Po of ≈0.6. These data suggest that the second channel activated by cell swelling is identical to the first channel in the same patch recorded under isotonic conditions.
Finally, in one channel studied in the presence of 22 mmol/L [Cl−]p, the induction of hypotonic swelling caused the reappearance of the same channel activity that had disappeared spontaneously after a period of activity under isotonic conditions.
Comparison of Properties of ORCCs Under Basal Conditions and in the Presence of Hypotonic Cell Swelling
Fig 6A⇓ shows mean (±SEM, where error bars are not visible they fall within the symbol for mean) I-V relations of single-channel activity recorded in the presence of isotonic and hypotonic superfusate with [Cl−]p of 108 mmol/L. The data are superimposed, indicating identical conductance properties. The reversal potential of single-channel current in the presence of hypotonic superfusate was RP+18±1 mV, not significantly different from that of the ORCC under basal isotonic conditions (RP+19±1 mV, P=NS). The slope conductance at the reversal potential of single-channel current carried by channels recorded in the presence of cell swelling was 27±1 pS (n=14), similar to that of channels studied under isotonic conditions (28±1 pS, P=NS). Fig 6B⇓ shows the effects of reduced [Cl−]p (22 mmol/L) on single-channel activity in the presence of hypotonic swelling and compares unitary currents at this [Cl−]p under hypotonic and isotonic conditions. At lower [Cl−]p, channels under hypotonic conditions showed a reduced conductance (slope conductance at the reversal potential averaged 20±1 pS) and more positive reversal potential (RP+54±3 mV, n=5) compared with results at 108 mmol/L [Cl−]p. When values of the reversal potential of single-channel activity under hypotonic conditions (open squares in Fig 6C⇓) at various [Cl−]p levels are plotted along with those of channels observed under isotonic conditions (open circles in Fig 6C⇓), they are virtually superimposed. The reversal potentials for single-channel currents recorded in the presence of hypotonic swelling fall close to the regression line obtained under isotonic conditions (dashed line), indicating high Cl− selectivity (57.7 mV per decade, r=.99). Furthermore, a similar selectivity for Cl− was observed for macroscopic ICl.b (56.3 mV per decade, r=.99, n=5) and ICl.swell (56.9 mV per decade, r=.999, n=5; data not shown).
The prevalence of channel activity was substantially greater in the presence of hypotonic swelling. Overall, single-channel activity was seen in 54 (16.1%) of 336 patches studied under hypotonic conditions with [Cl−]p of 108 or 22 mmol/L. Of these, 41 patches had failed to show activity under isotonic conditions and became active during exposure of the cell to hypotonic conditions. The remaining 13 patches showing channel activity were obtained by initially forming a patch under hypotonic conditions and constituted 18.3% of the 71 patches studied in this fashion. The overall prevalence of channel activity under hypotonic conditions was thus much greater than under isotonic conditions (Fig 6D⇑). On the other hand, the Po of channels under hypotonic conditions was similar to that of channels recorded under isotonic conditions (Table⇑).
Fig 7⇓ illustrates the kinetic properties of channels recorded under hypotonic conditions. As was the case under isotonic conditions, the open-time distributions were best fit with a three-exponential relation, and the closed-time distributions were best fit with a four-exponential relation. The mean time constants for both open and closed dwell times under hypotonic conditions (from 10 different patches) were of the same order as those observed under isotonic conditions (P>.05 for each time constant under hypotonic conditions compared with the corresponding time constant under isotonic conditions, Table). These data suggest that cell swelling activates the same set of Cl− channels (ORCCs) as recorded under isotonic conditions and increases macroscopic current by recruiting additional channels without altering the properties of those already activated.
Inhibition of ORCC, ICl.b, and ICl.swell by Tamoxifen
Recent studies have shown that tamoxifen (an antiestrogen) is a selective and potent inhibitor of ICl.swell in both cardiac myocytes7 and noncardiac cells.21 Vandenberg et al7 reported that tamoxifen inhibits whole-cell ICl.swell in guinea pig atrial and ventricular myocytes with a slow onset of action, suggesting that this agent may act from the intracellular surface of the channel. Therefore, we studied the effects of tamoxifen on ORCCs in cell-attached patches and on macroscopic ICl.b and ICl.swell.
As shown in Fig 8⇓, tamoxifen (10 μmol/L, 10 minutes) almost completely inhibited ORCCs activated by cell swelling. The same effect was observed in all four cell-attached patches studied. Tamoxifen also inhibited ICl.b and ICl.swell (Fig 9⇓). Fig 9A⇓ shows the effect of tamoxifen on ICl.b. Although the current showed no change over 20 minutes of recording before drug infusion, tamoxifen (10 μmol/L) caused time-dependent inhibition (44%, 71%, and 88% reduction at +40 mV and 29%, 52%, and 82% at −100 mV after 2, 4, and 7 minutes, respectively). The currents recorded after exposure to tamoxifen for 7 minutes are shown in Fig 9A⇓-c. The tamoxifen-sensitive current (Fig 9A⇓-d) reversed at −41.5 mV (estimated Cl− equilibrium potential, −39.3 mV). Fig 9B⇓ shows the effect of tamoxifen (10 μmol/L) on swelling-induced currents. Tamoxifen strongly inhibited the current (Fig 9B⇓-c) within 10 minutes. Fig 9B⇓-d shows average I-V curves from four cells: tamoxifen reduced total current by 90±2% and 76±4% at +40 mV and −100 mV, respectively. The pharmacological data shown in Figs 8 and 9⇓⇓ support the role of ORCCs in underlying both ICl.b and ICl.swell.
In the present study, we have demonstrated that unitary channel currents carried by ORCCs can be recorded in the cell-attached mode in isolated rabbit atrial myocytes under isotonic conditions. These channels carry inward current in the absence of permeable cations in the pipette (extracellular) solution, and changes in pipette Cl−concentration cause shifts in channel conductance and reversal potential consistent with a Cl−-selective channel. Several lines of evidence suggest that hypotonic cell swelling increases Cl− conductance by activating the same channel. These include (1) similar conductance, open probability, and kinetic properties of cell swelling–induced single-channel currents to ORCCs under hypotonic conditions, (2) a higher prevalence of active ORCCs in the presence of hypotonic superfusate–induced swelling and the swelling-induced appearance of active channels in patches that lack such channels under basal conditions, and (3) a similar response to tamoxifen of ORCC, ICl.b, and ICl.swell. The correspondence between the pharmacological response of ORCC, ICl.b, and ICl.swell, their Cl− dependence, and directly measured ICl.b and ICl.swell suggest that the latter are carried by ORCCs in rabbit atrial myocytes.
Relation Between Single-Channel and Macroscopic Cl− Currents in the Present Experiments
As indicated in "Results", the Cl− dependence of ORCC was indistinguishable from that of ICl.b and ICl.swell, as was the response to tamoxifen. To evaluate further the relation between macroscopic currents and ORCCs, we compared the estimated macroscopic current expected on the basis of the properties of ORCCs with directly measured whole-cell currents. The number of active channels per cell under isotonic conditions was estimated on the basis of the following formula: (CSA/PA)·(%prev), where CSA is cell surface area estimated from cell dimensions assuming right cylindrical geometry (π×length×width), PA is patch area estimated with an assumed circular electrode tip and a microscopically measured diameter of 2 μm, and %prev is the proportion of patches showing channel activity. This provides an estimate of 67 active channels per cell. Mean unitary current at RP+120 mV, which corresponds to a transmembrane potential of +56 mV based on the mean measured resting potential, was 4.62 pA. Multiplying this value by the measured Po of 0.68 and by the estimated number of channels per cell provides an expected macroscopic current of 210 pA at +56 mV. This compares with directly measured mean values of macroscopic ICl.b of 196 and 237 pA under isotonic conditions at +50 and +60 mV. The corresponding estimate under hypotonic conditions was obtained by multiplying the number of active channels per cell under isotonic conditions by ACThypo/ACTiso, where ACThypo and ACTiso are the mean prevalence of active channels per patch under hypotonic and isotonic conditions, respectively. This provides an estimate of 189 channels per cell and a predicted macroscopic current at +56 mV (RP+120 mV) of 593 pA, similar to the measured mean values of total macroscopic Cl− current of 568 and 678 pA at +50 and +60 mV, respectively, under hypotonic conditions. These results indicate that the ORCCs recorded in cell-attached patches can account for all of the ICl.b and ICl.swell measured by whole-cell voltage clamp. In combination with the similar kinetics and conductance of ORCCs observed under isotonic and hypotonic conditions, they suggest that cell swelling elicits ICl.swell by increasing the number of active ORCCs without altering the intrinsic behavior of active channels.
Comparison With Previous Studies of Unitary Cardiac Cl− Channels
The first cardiac Cl− current to have a unitary channel identified was the ICl.cAMP,22 whose biophysical properties resemble those of CFTR.23 24 25 The nature of single channels underlying other cardiac Cl− currents remains largely unknown. We previously reported the presence of ORCCs, with properties similar to sarcolemmal Cl− channels reported by Coronado and Latorre,26 in excised patches from rabbit atrial myocytes.15 In the present study, we report the observation of ORCCs during cell-attached patch recording on intact rabbit atrial myocytes and, for the first time, provide strong direct evidence for a role of ORCCs in underlying ICl.swell.
Very recent studies from Collier et al27 28 investigated the single-channel properties of ICl.PKC27 and ICl.Ca.28 In guinea pig ventricular cells, PKC-activated unitary Cl− channels have properties similar to those of cardiac CFTR Cl− channels.27 In canine ventricular myocytes, Ca2+ applied to the cytosolic surface of inside-out membrane patches activated small-conductance (1.0 to 1.3 pS) Cl− channels.28 The properties of cardiac ORCCs, including their conductance, kinetics, rectification, and Ca2+-sensitivity, are strikingly different from those of unitary cardiac Cl− channels of ICl.cAMP,22 ICl.PKC,27 and ICl.Ca.28
Relation of Cardiac ORCCs to Noncardiac ORCCs
ORCCs are found in a wide variety of mammalian tissues29 30 31 32 and may play a role in volume regulation, signal transduction, and transepithelial transport.33 34 35 36 The properties of cardiac ORCCs are in general quite similar to those of ORCCs in noncardiac cells in terms of conductance, outward rectification under symmetric Cl− gradients, and pharmacological properties. Although ORCCs have been observed often in cell-free patches from noncardiac tissues, they have been more difficult to record in the cell-attached mode. Moreover, the macroscopic equivalent of ORCCs in these tissues is also still uncertain, leading to uncertainty about their physiological function.36 37
Solc and Wine35 recorded single-channel activity during cell swelling in epithelial cells and observed an outwardly rectifying channel with properties somewhat different from ORCC, including a slightly greater conductance, greater stability of the open state, voltage-dependent inactivation, and a strong tendency for time-dependent rundown. They concluded that the two types of channel may be distinct proteins or different functional states of the same channel. In the present study, the properties of ORCCs recorded in the cell-attached mode under isotonic conditions and during cell swelling were identical and were similar to those of cardiac ORCCs in excised inside-out patches15 in terms of conductance, rectification, and voltage dependence.
Potential Significance of the Present Findings
Despite the broad distribution of ICl.swells that have been described in both cardiac4 5 6 7 8 9 10 11 12 13 and noncardiac tissues,38 39 40 41 42 the single-channel properties underlying the macroscopic behaviors of these currents are still unclear. The present study provides strong and direct evidence that ORCCs underlie volume-regulated Cl− currents in rabbit atrial cells. Further work is necessary in order to establish the role of ORCCs in volume-sensitive Cl− currents of other cardiac cell types and other species.
Our previous studies pointed toward a common ionic mechanism for ICl.swell and ICl.b in rabbit atrial myocytes.6 Both currents are outwardly rectifying, volume sensitive, inhibited by disulfonic stilbenes, and suppressed by α1-adrenergic stimulation through a PKC-mediated mechanism.6 The present work further supports the notion of a common mechanism for these currents by providing direct evidence that they are mediated by the same unitary channels. ICl.swell is conventionally considered to be activated by cell swelling and to be inactive under normal physiological conditions. It is possible, however, that such channels are active over a range of volume states, which includes basal conditions, and therefore may play a role even in the absence of pathological swelling. On the other hand, the cell isolation procedure and experimental manipulations may have allowed ORCCs to be recorded under isotonic conditions, despite a lack of activity under physiological conditions in vivo.
Stationary noise analysis studies of noncardiac ICl.swell have suggested a channel conductance in the range of 1 to 2 pS.38 39 40 41 Recently, Jackson and Strange42 showed that stationary noise analysis suggested a single-channel conductance of volume-sensitive currents of ≈1 pS at 0 mV. They then applied nonstationary noise analysis, which does not assume a constant number of functional channels, and obtained a single-channel conductance of 50.6±1 pS at +120 mV, in the range that we obtained for cardiac ORCCs at positive voltages. They suggested that activation of volume-sensitive anion channels may involve an abrupt switching of channels from an "off" state, where Po is zero, to an "on" state, where Po is substantial. Our results provide direct evidence supporting the theoretical basis of Jackson and Strange's analysis, in that hypotonic swelling appeared to increase current by increasing the number of channels in an active mode, without altering the Po of active channels. It has been reported that there may be a cytoplasmic inhibitor of ORCCs that restricts activity in cell-attached mode.43 44 Our results are consistent with these reports in that, under isotonic conditions, the prevalence of active channels and the Po of ORCCs in cell-attached patches are significantly lower than those of ORCCs in inside-out patches.15 Our findings are also compatible with preliminary observations reported by Zhang et al12 in 1992, who observed outwardly rectifying Cl− channels with a chord conductance of 31 pS during hypotonic swelling in cultured chick heart cells.
Cardiac ORCCs share many similarities with ORCCs in other tissues, including basic biophysical (eg, the characteristic outwardly rectifying I-V relation under symmetrical Cl−concentrations) and pharmacological (eg, inhibition by stilbene derivatives) properties. Like cardiac ORCCs,6 ORCCs from human airway epithelial cells are also inhibited by PKC45 and pertussis toxin–sensitive G proteins (Gαi-2).46 A recently cloned member of the CLC family, CLC-3, is also inhibited by PKC.47 CLC-3 is the most distantly related member of the CLC family (the 760–amino acid protein encoded by CLC-3 is only 24% homologous to previously reported CLC channels but has a similar hydropathy profile) and may represent a new branch of this gene family.48 Unlike other members of the CLC family but similar to cardiac ICl.b and ICl.swell, the current carried by CLC-3 shows strong outward rectification under symmetric Cl− and is inhibited by PKC and stilbene derivatives.6 15 47 The single-channel properties of CLC-3 also show similarities to those of cardiac ORCCs, including (1) strong outward rectification under symmetrical Cl− conditions, (2) 40-pS conductance (when intracellular Ca2+ is 200 nmol/L), (3) similar open- and closed-state kinetics, and (4) sensitivity to DIDS. Although CLC-3 appears not to be expressed in the heart,47 two new members of the CLC family, CLCN449 and CLCN5,50 which are strikingly similar to CLC-3, are expressed in the heart,49 50 and a deficiency in CLCN4 has been associated with cardiac abnormalities.49 Our finding that ORCC underlies cardiac volume-sensitive Cl− currents may help in the delineation of the cellular mechanisms by which these genes contribute to cardiac function.
We have obtained, for the first time, direct evidence that ORCCs underlie both ICl.b and ICl.swell in rabbit atrium and may therefore play a potentially important physiological role in cardiac electrophysiology. These findings provide new insights into subcellular mechanisms controlling Cl− movement across cardiac cell membranes and open up new avenues for the exploration of their molecular control.
Selected Abbreviations and Acronyms
|pipette Cl− concentration
|cystic fibrosis transmembrane conductance regulator
|cloned Cl− channel family
|basal macroscopic Cl− current
|Ca2+-dependent Cl− current
|cAMP-dependent Cl− current
|PKC-dependent Cl− current
|cell swelling–induced Cl− current
|outwardly rectifying Cl− channel
|protein kinase C
|open-channel probability
|resting potential
This study was supported by the Medical Research Council of Canada, the Quebec Heart Foundation, the Fonds de Recherche de l'Institut de Cardiologie de Montréal, and National Institutes of Health grant HL-52803 (Dr Hume). Dr Duan was supported by a graduate studentship and subsequently by a postdoctoral fellowship from the Medical Research Council of Canada. We thank Dr Lingyu Ye for excellent technical support, Dr J.L. Kenyon for suggestions regarding single-channel analysis, and Luce Bégin and Carolyn Gillis for secretarial assistance.
Previously published in abstract form (Biophys J. 1995;68:A110; Circulation. 1995;92[suppl I]:I-638).
- Received September 19, 1996.
- Accepted October 15, 1996.
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Hoffmann EK, Simonsen LO. Membrane mechanisms in volume and pH regulation in vertebrate cells. Physiol Rev. 1989;69:315-382.
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Sorota S. Swelling-induced chloride-sensitive current in canine atrial cells revealed by whole-cell patch-clamp method. Circ Res. 1992;70:679-687.
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Tseng G-N. Cell swelling increases membrane conductance of canine cardiac cells: evidence for a volume-sensitive Cl channel. Am J Physiol. 1992;262:C1056-C1068.
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Duan D, Fermini B, Nattel S. α-Adrenergic control of volume-regulated Cl− currents in rabbit atrial myocytes: characterization of a novel ionic regulatory mechanism. Circ Res. 1995;77:379-393.
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Vandenberg JI, Yoshida A, Kirk K, Powell T. Swelling-activated and isoprenaline-activated chloride currents in guinea pig cardiac myocytes have distinct electrophysiology and pharmacology. J Gen Physiol. 1994;104:997-1017.
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Oz MC, Sorota S. Forskolin stimulates swelling-induced chloride current, not cardiac cystic fibrosis transmembrane-conductance regulator current, in human cardiac myocytes. Circ Res. 1995;76:1063-1070.
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Li G-R, Feng J, Wang Z, Nattel S. Transmembrane chloride currents in human atrial myocytes. Am J Physiol. 1996;270:C500-C507.
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Zhang J-P, Rasmusson RL, Hall SK, Lieberman M. Hypo-osmotically activated Cl−conductance in cultured chick heart cells. J Gen Physiol. 1992;100:31a. Abstract.
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Duan D, Fermini B, Nattel S. Sustained outward current observed after Ito1 inactivation in rabbit atrial myocytes is a novel Cl− current. Am J Physiol. 1992;263:H1967-H1971.
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Duan D, Nattel S. Properties of single outwardly rectifying Cl− channels in heart. Circ Res. 1994;75:789-795.
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Duan D, Nattel S. Single outwardly-rectifying chloride channels in cell-attached patches from rabbit atrial myocytes. Biophys J. 1995;68:A110. Abstract.
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Duan D, Nattel S. A possible single-channel mechanism for volume-sensitive Cl− currents in rabbit atrial myocytes. Circulation. 1995;92(suppl I):I-638. Abstract.
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Hamill OP, Marty AJ, Neher E, Sakmann B, Sigworth FT. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1992;422:143-150.
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Hart P, Warth J, Levesque PC, Collier ML, Geary Y, Horowitz B, Hume JR. Cystic fibrosis gene encodes a cAMP-dependent chloride channel in heart. Proc Nat Acad Sci U S A. 1996;93:6343-6348.
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Collier ML, Hume JR. Unitary chloride channels activated by protein kinase C in guinea-pig ventricular myocytes. Circ Res. 1995;76:317-324.
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Collier ML, Levesque PC, Kenyon JL, Hume JR. Unitary Cl− channels activated by cytoplasmic Ca2+ in canine ventricular myocytes. Circ Res. 1996;78:936-944.
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Barres BA, Chun LL, Corey DP. Ion channels expression by white matter. Glia. 1988;21:10-30.
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Welsh MJ. Electrolyte transport by airway epithelia. Physiol Rev. 1987;67:1143-1184.
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Lukacs GL, Moczydlowski E. A chloride channel from lobster walking leg nerves: characterization of single-channel properties in planar bilayers. J Gen Physiol. 1990;96:707-733.
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Solc CK, Wine JJ. Swelling-induced and depolarization-induced Cl− channels in normal and cystic fibrosis epithelial cells. Am J Physiol. 1991;261:C658-C674.
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Gray MA, Harris A, Coleman L, Greenwell JR, Argent BE. Two types of chloride channel on duct cells cultured from human fetal pancreas. Am J Physiol. 1989;257:C240-C251.
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Stoddard JS, Steinbach JH, Simchowitz L. Whole cell Cl− currents in human neutrophils induced by cell swelling. Am J Physiol. 1993;265:C156-C165.
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Lewis RS, Ross PE, Cahalan MD. Chloride channels activated by osmotic stress in T lymphocytes. J Gen Physiol. 1993;101:801-826.
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Nilius B, Oike M, Zahradnik I, Droogmans G. Activation of a Cl− current by hypotonic volume increase in human endothelial cells. J Gen Physiol. 1994;103:787-805.
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Jackson PS, Strange K. Current activation occurs by abrupt switching of closed channels to an open state. J Gen Physiol. 1995;105:643-660.
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Li M, McCann JD, Anderson MP, Clancy JP, Liedtke CM, Nairn AC, Greengard P, Welsh MJ. Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway cells. Science. 1989;244:1353-1356.
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Schwiebert EM, Gruenert DC, Guggino WB, Stanton BA. G-protein Gα1-2 inhibits outwardly rectifying chloride channels in human airway epithelial cells. Am J Physiol. 1995;269:C451-C456.
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Slegtenhorst MA, Bassi MT, Borsani G, Wapenaar MC, Ferrero GB, Concillis L, Rugarli EI, Grillo A, Franco B, Zoghbi HY, Ballabio A. A gene from the Xp22.3 region shares homology with voltage-gated chloride channels. Hum Mol Genet. 1994;3:547-552.
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- Evidence That Outwardly Rectifying Cl− Channels Underlie Volume-Regulated Cl− Currents in HeartDayue Duan, Joseph R. Hume and Stanley NattelCirculation Research. 1997;80:103-113, originally published January 1, 1997http://dx.doi.org/10.1161/01.RES.80.1.103
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- Evidence That Outwardly Rectifying Cl− Channels Underlie Volume-Regulated Cl− Currents in HeartDayue Duan, Joseph R. Hume and Stanley NattelCirculation Research. 1997;80:103-113, originally published January 1, 1997http://dx.doi.org/10.1161/01.RES.80.1.103
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Welcome to Librarium Online!
Join our community of 80,000+ members and take part in the number one resource for Warhammer and Warhammer 40K discussion!
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I am getting back in Warhammer 40k after a couple of years. I used to play Dark Eldar and Deathguard and decided to jump back in with Chaos again. Having found out that were now at 5th edition I got the new rulebook and new Chaos Codex and after a quick read through the Codex I began to get really irritated. What happened to the Great Unclean One? What happened to Plaguebearers? What happened to the seperate "books of chaos" to field a Deathguard, World Eaters, etc. army? I feel like there is no room for originality with the new Chaos Codex. Is there any way that I can still field a legitimate Deathguard army because I am very disapointed with the new Codex.
P.S. I am actually pretty happy with the new 5th Edition rules though.
This is a very common reaction to the new chaos marine codex. Not surprising since it went from a forces and legions of chaos style to a renegade warband style.
Still, there are some things you can do. You have the option of using plaguebearers as the generic lesser daemons and the GUO as the generic Greater Daemon. I know, it just doesn't feel the same. But that's about the only way you're going to get the daemons back in the army short of apoc with a Chaos Daemons army in a 2nd org chart detachment. (But hey, it's a chance to start a second nurgle army!)
I used to use a lot of daemons personally. Since the 'dex came out they've been enjoying the hospitality of the foam on my shelves at home. Probably not what you wanted to hear. However, if you can focus more on the marine side of things with your deathguard, you can still make a very capable army.
The massive page of armory goodies is gone along with some of my old favorites, but what we've got now covers most of the goodies I used to angle for anyway. Not all, but most. And they did add a few new goodies that are quite nice as well (no more possibility to insta-kill or even force-weapon kill a daemon prince).
As deathguard, you'll have a very solid core army. Plague Marines are rock-hard, which is fantastic in 5th edition where they've also got scoring status. And you can now take raptors and give them an icon of nurgle if you want. Model up some plague-winged guys or whatever. Which is good because you can't fill that hole with Possessed anymore because they got gutted into virtual uselessness.
Frankly, the transition can be a bit rough. But the new 'dex can stand on it's own and still provide some nice variety. Just takes some getting used to.
Do you think it would be possible to field a Chaos Marine army with the GUO, Plaguebearers, and Nurglings from the Chaos Daemon Codex or would that be bending the rules a bit too much? To be honest, the GUO and plaguebearers were my main reasons for picking chaos (although I love plague marines too, just not as much). Ah, well. I guess I am hust going to have to get used to the new codex. Thanks for the reply Xardian.
If you liked the daemons more then the marines, why not pick the Chaos Daemons codex and see about building an all-daemon nurgle army?
If you like Daemons more than CSM then the Chaos Daemon Codex is for you. You will find a lot of nasties in it, especially if you're already dedicated to a particular God.
On the other Hand, the new Chaos Codex is a lot more balanced now and you should find a way to make a great Nurgle army. Personnally I had a pure Death Guard army with the 3rd edition, now I just have a lot of Plague Marines with feel no pain and I love it. I still use them in squad of 7 but I also like to field them in squad of 10... having a more flexible list is really nice.
On the other side I am now more inclined to use other type of dedicated troopers and I'm learning to love my Khorne Berserkers and my Thousand Sons more and more every game...
I cried too when I lost my Alpha Legion cultists. But I've learned to work with the new codex. It made my army list alot easier to read, and made it possible to field marines while drunk! So while it took away from ALOT of the fluff (I miss the Dark Chaplains too, repainted all my Word Bearers when we lost them), the new codex still gives you options for very yummy armies!
Yes, I did deep-strike your sister...
Necrons : W-8 D-3 L-7 (Sleeping in their TombFoam, waiting for a new codex)
Chaos Forces (CSM/CD): W-42 D-19 L-28
Apocalypse Games : W-4 D-0 L-1 (4 Titans, Brass Scorp, 3 Baneblades, 2 Bombers, 3 Fighters, and a Partrage in a Pear Tree)

subset: commoncrawl


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OTTAWA - The cost of medical marijuana in Canada is about to hit a new high as the government seeks to get out of the business.
Proposed regulations posted by Health Canada say medical marijuana would be treated more like any other medicine by creating a licensing scheme for production and distribution.
But the changes would nearly double the price of a gram of the drug, costing consumers an additional $166 million a year for the next 10 years.
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CBS-FM got a little face … er, uh … voice lift this week as it welcomed a new member to the Deep Voice Team. Joining Ziggy will be Earl Mann, a name that might not ring a bell, but whose voice will set off sirens once it you hear it.
Remember those epic NFL Films VHS tapes (YES, we said VHS) that highlighted a teams season in an hour with slow motion montages of game highlights while that perfect voice offered insightful commentary? That's Earl Mann. If you're from the New York/New Jersey area, you probably have the tapes covering the New York Giants Championship seasons from 1986 and 1990 (Phil Simms going 22-25 for 3 TDs in '86 and "WIDE RIGHT" from '90).
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Home » Fiction - YA » James Roy » Problem Child
Problem Child
Max Quigley doesn't think he's a bully. He's an "observer". He observes the habits, interests and peculiarities of other people, which he then points out. A lot. Triffin Nordstrom isn't really all that clever. He's a nerd. He reads too many fat books, makes medieval seige machines out of Lego, and probably speaks fluent Elvish. Plus he lives way out in the bush with his mum, who he calls Ulrika. Max and Triffin. It's a match made in hell, and it's going to get uglier than a second-hand cheesecake. In this hilarious look at playground politics, James Roy has created the ultimate odd couple, two boys who don't see eye to eye, but have to, somehow.


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A very easy to use website. Friendly, professional people that you deal with. Unfortunately I had to cancel my trip two days before arrival due to a medical emergency and they were nothing but courteous.


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like all khadi products i like this product for being natural as i don'tprefer chemical products as they are harmful in the long run.this isan excellent oil for hair and skin. i use it on my hair before washing them.it makes my hair stronger,more shiny,and healthy. its totally worth the price as very little needs to be applied
5 all in one...
almond oil is the natural moisturiser,and therefore sutaible for all skin type.it is also one of the most popular carrier oil,almond oil is rich with various vitamins nad as result is great for the skin.almond oil will not blok pore ,due to its fine texture.almond oil's property even extend to having benefits for hairs.also help make my hair stroger thicker and add shine as well as nourishing and smoothing hairs.almond oil is very good...or we can say great for digestion.almond oil can often provide relief from muscular pain.almond oil is greta for immune system.its correct sleep disorderd.almond oil help to improving memory.sweet almond oil on the other hand has all these wonderfull applications and has become more and more popular in the beauty products.almond oil improve comlpexion and glow,relieving dry and itching skin,cures chapped lips and body rashes,lighting dark circle and under eye,reducing fine line and wrinkles.and specially to hairs...this is very good product....i regular use almond oil with coconut oil and olive oil.i was suffering from hairfall problem,but i use coconut oil,olive oil with almond oil and aroma magic stimulating oil,right now i get rid from hair fall problem...i recommend almond oil to oil personly.this is very good product
5 All in One oil for everybody
Khadi Sweet Almond oil is a all around multi purpose product that can be used for hair and skin for all members of the family. I use it for hair and my mother uses it for her dry skin. The Khadi brand is an assurance of quality. It is value for money compared to some other brands in market. According to the lable it is good for brain/ memory and nervous system....but I have not consumed it internally. It is also a good massage oil even for babies. As I said an oil for the whole family...!
5 For Best Hair
One can use it for better and Healthy hair....... I have used it and i have got a best result for my hair. This hair is for all type hair. So i am recommding this product. This is non sticky hair oil for all season type. Almond contains vitamins and is rich in protien so that it isvery useful for hair as wll as skin.
4 Good one
Khadi products are very natural and this oil is no different. I love the original almond extracts in this one which is very beneficial to the hair. It is non sticky and leaves a pleasant fragrance in the hair making it healthy. It is a natural one and I think it will also be great for a body massage.


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Effect of Benzylic Oxygen on the Cytotoxic Activity for Colon 26 Cell Line of Phenolic Lignans
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The cytotoxic activity for colon 26 cell line of matairesinol, oxidized matairesinol, 9,9′-epoxylignan and oxidized 9,9′-epoxylignan were examined. (−)-Matairesinol (Mat 1) showed greatest cytotoxic activity (LC50=9 μg/ml) of the lactone-type lignans. 7,7′-Oxomatairesinol having same steric configuration as that of (−)-matairesinol showed greater activity (LC50=25 μg/ml) than hydroxy or mono-oxomatairesinol. The activities of 9,9′-epoxylignan and 7,7′-oxo-9,9′-epoxylignan having same steric configurations as (−)-matairesinol were weaker than that of corresponding matairesinols. Different activity levels were observed between enantiomers.
- Agricultural and Biological Chemistry
Agricultural and Biological Chemistry 70(12), 2942-2947, 2006-12-23
Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Forget for a moment the crisis and economic difficulties. This text speaks of astronomical amounts of money. Ah, let`s talk about Paris SG. Is that the Parisian emblem, according to some news will have 123 million to open its doors to Cristiano Ronaldo.
Yes, you read right. 123 million euros to try hiring the Portuguese captain at Real Madrid, which seems to be `sad` in the Spanish capital, according to recent news from the neighboring country.
According to the website http://Goal.com, the owners of Paris SG hope to convince Ronaldo to exchange the meringues by Parisian club in the summer market. The idea is to make the Portuguese playmaker highest paid player in the world (495 thousand euros per semana/25 million annually).
Thus, Ronaldo would win more than 5 million Samuel Eto`o, receiving 20 million in Russia.
what is your opinion about these rumors? Is that really true?
picture is not mine.
Source link: http://esporte.uol.com.br/futebol/campeonatos/espanhol/ultimas-noticias/2012/09/03/cristiano-ronaldo-diz-nao-querer-mais-falar-s


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Tlemcen Fruit Baskets
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What our Customers Say about our Service
"This was the first time we worked with the company and the experience was great. We are in South America and we sent gifts to several countries. We were afraid that we would not be able to control the delivery and/or the situation, so we repeatedly sent inquiries. We received immediate response to all our questions and doubts, we were informed of every package delivered or processed, and the corporate specialist that we worked with was very accurate, kind, helpful and professional, I highly recommend them. It is a serious and responsible company."
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"I had a last minute idea to send my friend in Belize a birthday gift basket. As a first time customer, I am delighted with the service. It was delivered right on time and my friend loved it. I especially loved the handwritten note with my message that was included because it gave the basket a personalized touch, as if I'd written it myself. I will definitely be using this company again."
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Source: The Wensleydale Heifer
The dishes are "hard to fault" at The Wensleydale Heifer in the Yorkshire Dales, says The Darlington & Stockton Times, which describes the food as "just brilliant". You and a friend can choose between a lobster or steak meal accompanied by a glass of bubbly each, for £39.
12-2:15pm Mon-Sat; 6-6:30pm Sun-Fri until 28 Feb, 2017
Source: Ripon Museums
Ripon Museums includes three unique historic buildings -- the Courthouse Museum, the Prison & Police Museum, and the Workhouse Museum & Garden. For £7.50, you can now get entry to all three museums, and an annual pass, so you can visit as many times as you like for a year.
Visit before 27 Nov: annual pass valid 12 months
Source: The Feversham Arms Hotel & Verbena Spa
Verbena Spa at the Feversham Arms Hotel, which is set in the "chocolate-box village" of Helmsley, is "pampered, gourmet heaven", according to the Daily Mirror. You can now visit the spa and get a facial, bubbly and use of the facilities for 38% less than usual.
10am-5pm Sun-Fri from 1 Nov, 2016-31 Mar, 2017
Source: Best Western Burn Hall Hotel
Explore the "rural beauty and rich history" (The Independent) of North Yorkshire with a countryside getaway at Best Western Burn Hall Hotel. A 2-night stay for two with daily breakfast and extra goodies, including afternoon tea, is now £89 -- that's over £90 off.
Until 30 Dec
Source: The Yorkshire Bar and Grill at the Best Western Monkbar
You can get afternoon tea and prosecco for two at The Yorkshire Bar and Grill in the Best Western Monkbar hotel in central York for £19 -- that's less than £10 per head.
2-5pm Mon-Sat until 28 Feb, 2017
Source: Aldwark Manor Golf & Spa Hotel
You can save up to 60% on a spa day at Aldwark Manor Golf & Spa Hotel, a 19th-century mansion near York. For £40, you'll get two treatments of your choice, all-day access to the facilities, and refreshments on arrival.
10am-6pm Mon-Fri until Mar 31, 2017
Source: The Grange Hotel York
Built in 1829, The Grange Hotel York is a "city-centre hotel with classic Georgian interiors", according to The Daily Telegraph. An overnight stay with a 3-course set-menu dinner in the hotel's 2-AA-Rosette restaurant is now £129, saving up to 46% on the usual price.
Sun-Thu 1 Nov-29 Dec, 2016; daily 1-31 Jan, 2017
Source: Best Western Dower House Hotel & Spa
For half the regular price, you and a friend can treat yourselves to a spa package for two at Imagine Spa at the Best Western Dower House & Spa. For £35, you'll both get half-day spa access and afternoon tea.
9am-8pm Mon-Fri 1 Oct-30 Dec afternoon tea is served from 12-3pm; +£10 Sat-Sun
Source: Best Western Crown Hotel
Mini breaks to the Best Western Crown Hotel in the North Yorkshire market town of Boroughbridge -- 30 minutes' drive from York -- are now £169. Two-night stays save 32% on the usual price and include dinner and a bottle of prosecco.
1 Nov, 2016-28 Feb, 2017
Source: Imagine Spa Hazlewood Castle
You can now get a pamper package for two at the Imagine Spa in Hazlewood Castle, including a 45-minute mud-chamber session and cream tea, for 56% less than usual.
9am-6pm Mon-Fri until 30 Dec; +£5 Sat-Sun
Source: Nidd Hall Hotel
Stay three nights, with meals and activities included, at a Grade II-listed mansion near the spa town of Harrogate with this exclusive offer. Mini breaks at Nidd Hall Hotel -- a Warner Leisure Hotels property -- are now £239 per person (based on two people sharing).
14 Oct; 4 Nov; 2, 9, 16 Dec arrivals
Source: North Yorkshire Moors Railway
You can hop aboard the award-winning North Yorkshire Moors Railway, which has featured in numerous films and television shows, including "Heartbeat" and "Harry Potter", for 41% less than usual.
Daily from 5 Sep-30 Oct
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