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PHYSIOLOGICAL ROLE OF APICAL POTASSIUM ION CHANNELS IN FROG SKIN BY WILLY VAN ... PDF

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J. Phy8"l. (1984), 356,pp. 79-95 79 With3text-ftgure PrintedinGreatBritain PHYSIOLOGICAL ROLE OF APICAL POTASSIUM ION CHANNELS IN FROG SKIN BY WILLY VAN DRIESSCHE From the Laboratorium voor Fy8iologie, Campus Gasthuisberg, B-3000 Leuven, Belgium (Received 13 March 1984) SUMMARY 1. TheK+permeability oftheapical membraneoffrogskin (Ranatemporaria) was analysed by recording the short-circuit current and its fluctuations in the presence ofa mucosa-to-serosa-oriented K+ concentration gradient. 2. Loading of the animals with KCl resulted in an augmentation of the Ba2+- blockable component of the short-circuit current and the plateau value of the K+-dependent relaxation noise. 3. Poisoning ofactive transport and exposing both sides ofthe epithelium to KCl Ringer solution caused an increase ofthe K+ current and its fluctuations recorded after restoring the inward-oriented K+ gradient. 4. Serosal quinidine (5x 10-4 M), which is thought to increase intracellular Ca2+ activity, depressed the K+ current and the relaxation noise. This effect was completely reversible. 5. Removal of Na+ from the serosal solution, which is known to result in an elevation of intracellular Ca2+ by abolishing the driving force for the Na+/Ca2+ exchanger, alsoreducedtheK+currentandtheLorentzianplateau. Bothparameters returnedtotheircontrolvaluesafterrestoringtheNa+gradientacrossthebasolateral membranes. 6. It is concluded from these experiments that the apical K+ permeability is controlled by factors which depend on the intracellular K+ and Ca2+ concentration and that the apical K+ channels may constitute a pathway for K+ secretion. INTRODUCTION Previous investigations showed that the apical membrane of frog skin (Rana temporaria) has a significant K+ permeability (Zeiske & Van Driessche, 1979). This was demonstrated by recording the transepithelial short-circuit current and by the analysis ofits fluctuations: in the presence of-a K+ gradient oriented from mucosa to serosa, an inward K+ current was recorded and the spectrum ofthe fluctuations in current contained a Lorentzian component. The latter observation indicates that the K+ channels open and close randomly (Van Driessche & Zeiske, 1980a). The K+ currents are blockable by mucosal Cs+ and Ba2+ and additional fluctuations in current were recorded in the presence of the latter cation. The analysis of these blocker-inducedfluctuationsallowedtheestimationofthesingle-channel currentand 80 W. VAN DRIESSCHE channel density (Van Driessche &Zeiske, 1980b). The K+ channels permeable for are different 'K+-like' cations (Rb+, NH + and Tl+), and the affinity ofthe K+-channel blocker, Ba2+, strongly depends on the nature of the permeating species (Zeiske & Van Driessche, 1983). With all these observations we were able to establish a picture ofthefactorswhich determine the molecular characteristics ofthe apical K+ channel. However, untilnowwewereignorantabout possiblephysiological functionforthese a K+ channels. Itwaspreviously observed by ourselves (Zeiske & Van Driessche, 1979) and others (Nagel & Hirschmann, 1980) that not all frogs had a skin with a measurable K+ permeability. SomebatchescontainedveryfewfrogswithaK+-permeable skin, while in others the majority displayed a significant K+ permeability. The cause for this difference among frogs could not be resolved until now. In this paper experiments whichweredesigned toresolvetheseproblems described. Thehypothesis that are was the apical K+ channels participate in the K+ homoeostasis ofthe frog by promoting K+ secretion when the animals experience large K+ uptake, after eating lot a e.g. a of insects. The results show that treatments of the preparation such that the intracellular K+ and/or Ca2+ concentrations can be expected to be altered, give rise to modifications ofthe K+ permeability. METHODS Frogsofthe speciesRanatemporariawereobtainedfrom Stein (Lauingen-Donau, F.R.G.). They werekeptinplastic containers at an ambient temperature of17 0C. The containers hadareservoir of101 tapwater, whichwasrefreshedeveryday. Theanimalswerefedmealwormsonadrysurface ofthe containers. Three days before the experiments, some ofthe frogs which were used for the K+-adaptationstudiesweretransferredfromthiscontainerintoasmallertank.Thistankcontained distilledwaterinwhich 15mM-KCl wasdissolved. In this container the animals were continuously in contactwith the solution. During this period they were not fed. On the day of the experiment the animals were double pithed and the abdominal skins were mounted in modified 'Ussing-type' Lucite chambers as described before (Van Driessche & Erlij, 1983). Theareaofskinincontactwiththesolutionwas0-4cm2. Thevoltageandcurrentelectrodes consisted of Ag/AgC1/1M-NaCl electrodes connected to the solutions by1 M-NaCl-filled agar bridges. Both chamber compartments were continuously perfused. In order to avoid microphonic noise, the perfusion was interrupted during the periods where the noise data were collected. The transepithelial current and voltage were recorded continuously on an X-T recorder. The transepithelial conductance was measured by recording the current changes caused by the application ofvoltage pulses of1-5 sduration. Theelectronic equipment used to record the transepithelial currents, the fluctuation in current andthemembrane conductance wasthe same asthatdescribedpreviously (Van Driessche & Erlij, 1983). However, the field-effect transistors (2N6451, National Semiconductors) which we used previously in the input stage ofthe low-noise voltage clamp (Van Driessche & Lindemann, 1978) were replaced by a newer type (SK146, Toshiba Corp., Tokyo, Japan). The reason for this replacementwastheimprovednoisecharacteristicsofthelattertype.Especially, theamplifiernoise atlowerfrequencies was much smaller. The fluctuations in current were recorded and analysed with an SBC 86/12A and SBC 80/30 (INTEL,SantaClara,U.S.A.)multiprocessorcomputersystem.Theaveragedandfinalspectrawere transmitted toa PDP11/34 computer andstored on aharddisk. The fluctuations in current were analysed in one ofthe following frequency ranges: (1) frequency range 1 was identical to the one usedin apreviouspaper (Van Driessche & Erlij, 1983), and covered the range0-2-670 Hz; (2) the fundamental and maximal frequency were1 and 900Hz, respectively, forfrequency range 2. The finalspectraanalysedwithfrequencyrange1 consistedofthreemergedspectrawhichwererecorded simultaneously,whilethoseanalysedwithfrequencyrange2werethesuperposition oftwospectra. APICAL K+ CHANNELS INFROGSKIN 81 It has been demonstrated for different membranes and several channels that the spectrum of the fluctuations of the current through channels which open and close randomly contains a Lorentziancomponent. ThespectraldensityofsuchaLorentziancomponentreachesaplateau(SO) inthelowerfrequencylimitanddecreasesby6dB/octaveatthehighestfrequencies.Thefrequency where the spectral density is 3dB below SO is called the corner frequency (fe). The frequency dependence ofthe powerdensity, SL(f), fulfils the followingequation: SLWf) = '+(f/f )2' (1) wherefrepresents the frequency. The presence ofsuch a Lorentzian component in the spectra recorded with KCl and NaCl Ringer solution on the mucosal and serosal sides, respectively, was usedasacriterionfortheexistenceofameasurableamountoffluctuatingK+channelsintheapical membrane. Themainadvantage ofthiscriterionisthatinoursystem, thefluctuatingK+currents are selectively recorded in the power spectrum: they give rise to a Lorentzian component with a fcof80Hz,whilefluctuationsofbackgroundcurrentsproducemostlylow-frequencynoise(G6gelein & Van Driessche, 1981). Themaindisadvantage ofthiscriterion isthattheplateauvaluedepends notonlyonthechanneldensity(M)butalsoonthesingle-channelconductance,theelectrochemical gradient across the apical membrane and on the rate constants ofthe open-close reaction (Van Driessche & Zeiske, 1980a). So, using the presence ofa Lorentzian in the spectrum as a criterion forasignificant K+conductance impliesthatweassumethattheelectrochemicalgradientandthe rateconstantsdoindeedremain constantduringthedifferentphysiological alterationsusedinthe experiments.Therefore,wealsorecordedtheK+current(IK)throughtheepithelium.Therecording of these currents is hampered by the presence of shunt currents which might, under certain circumstances become larger than IK. Therefore, we defined the IK as the amount of current blockable by2 mM-mucosal Ba2+. Frompreviousexperiments (Van Driessche & Zeiske, 1980b) we know that 60,uM-mucosal Ba2+ blocks 50% of IK. Thus, assuming simple Michaelis-Menten kinetics, 97% ofthe K+ channels should be blocked at2mM-mucosal Ba2+. Nevertheless, the K+ currents also depend on the above-mentioned parameters as SO does, although in a different way (see eqns. (3) and (4) in the Discussion). So the IK data have to be considered as information complementary to the noise data. As described in previous papers (Van Driessche & Zeiske, 1980a, b), most spectra contained, besides the Lorentzian noise component, a low-frequency noise component (SB) which was dominant in the low-frequency range. Its dependence on the frequency fulfils the following equation: SB(f) A/f', (2) = whereArepresentsthepowerdensityatafrequencyoft Hzandatheslopeinadouble-logarithmic representation. On the other hand, in the high-frequency range the amplifier noise surpassed the fluctuations of the K+ current. Due to the capacitance of the membrane, the amplifier noise increasesbecauseofthedecreaseofthe membraneimpedance (VanDriessche& Gullentops, 1982). AnestimationoftheLorentzianparameters(fcandSO)wasobtainedfromanon-linearregression (Van Driessche & Zeiske, 1980b) ofthe following equation to the data points: ST = SL+SB. The datapointsinthehighestfrequencyrangeofthespectrum(wheretheamplifiernoisewasdominant) were omitted in these calculations. Solution-. Thetissueswere bathed with Ringersolutionswithdifferentionic compositions. NaCl Ringer solution contained (in mM): NaCl, 115; KHCO3, 2-5; CaCl2, 1. KCl and choline chloride Ringersolutionscontained 115 mM-KCland 115 mM-cholinechloriderespectively, insteadofNaCl. The choline solutions were alwaysfreshly prepared on the day ofthe experiment. To measure the IK, Ba2+ (2 mM) was added as chloride to the KCI Ringer solution. Quinidine was obtained from Sigma (St. Louis, U.S.A.) and dissolved to give a concentration of5x 10-4 M in the NaCl Ringer solution used asserosal medium. Ouabain waspurchasedfrom Sigma (St. Louis, U.S.A.) and used ataconcentration of2x 10-5 Min the serosal solutions. All studiesweredoneatroomtemperature with air-saturated solutions at a pH of8-0. 82 W. VAN DRIESSCHE RESULTS Four types ofexperiments were done to modify the K+ permeability offrog skin. Wetriedtocorrelatethesechangeswithalterationsofintracellularionconcentrations. In the first two parts of the Results section, experiments are described in which changesinintracellular K+ concentration couldbe considered asprimarytriggersfor alterations in the apical K+ permeability. On the other hand, in the two following typesofexperimentintracellularCa2+ concentrationsweremanipulatedandfollowed by alterations in the K+ permeability, as judged from the alterations in relaxation noise amplitudes and the Ba2+-blockable K+ currents. (A) Exposure ofthefrogs to KCI-containing solutions It has been shown for other K+-transporting epithelia (distal portions of the nephron and large intestine) that K+ secretion is augmented after chronic dietary potassium loading (Hayslett & Binder, 1982). It might be that the large variability in K+ permeability among the frogs is also related to a difference in K+ load on the animals. Therefore, in analogy to studies byothers inseveral otherepithelia (Fisher, Binder & Hayslett, 1976; Silva, Brown & Epstein, 1977; Hayslett & Binder, 1982), we attempted to augment the K+ permeability by exposing the animals to high- K+-containing solutions and expected a larger K+ intake. Two groups of frogs composed ofa random selection ofanimals from the same batch from the supplier were subjected to two different treatments. (1) The first group (ten frogs) was kept under normal conditions and had access to normal tap water till the day of the experiment (see Methods). (2) Theexperimental group ofanimals (sixteen frogs) was transferred into separate containers three days before the experiments. In these containers the animals were continuously in contact with 15 mM-KCl solution. The skinsofbothgroupsweremountedinthechambersandcontinuouslyshort-circuited. Both sides ofthe epithelium were exposed to NaCl Ringer solution for45 min. After this equilibration period, all mucosal Na+ was isotonically replaced by K+. In the presence of this mucosa-to-serosa-oriented K+ concentration gradient, the K+ permeabilitycanbemonitoredbyrecordingthenoisespectrumandtheBa2+-blockable component of the short-circuit current (see Methods). From the ten frogs in the control group, only one skin showed a measurable K+ conductance, i.e. only for this skin could we fit a Lorentzian curve to the data points. For this experiment, the Lorentzian parameters were: SO = 1-08 x 10-21A2s/cm2 andf, = 76-2 Hz. Moreover, this was the only skin ofthe control group in which a significant K+ current could berecorded: IK = 2-8#sA/cm2. InthenineotherskinsaLorentziancomponentmight have been present in the spectrum, but the plateau would have been so small that the low-frequency noise and amplifier noise dominated over the entire frequency range. Also, the Ba2+-blockable K+ currents were small. The mean value calculated from these nine skins was: IK = 1 200+±69#uA/cm2. The experiments with the animals ofthe experimental group showed that skin offourteen out ofthe sixteen frogs had ameasurable K+permeability. The mean valuesforIK and the Lorentzian parameters calculatedfrom these fourteenexperimentswere: IK = 7-3+1F1 ,A/cm2, SO = 3-9+14x 10-21 A2s/cm2 andf, = 76-7+3-8 Hz. These results suggest that the in vivo load of the frogs with KCl elicits an APICAL K+ CHANNELS INFROG SKIN 83 augmentationofthe K+ permeability oftheapical membrane. Whetherthis K+ load causesanincreaseoftheintracellularK+concentrationisstillunknown.Consequently, the final mechanism responsible for the increase ofapical K+ permeability could be anincreaseoftheintracellularK+ concentration, themodification oftheintracellular concentrationofotherionslikeCa2+,alterationoftheintracellularATPconcentration, or the modification ofsome other metabolic or hormonal factor. (B) In vitro ouabain-KCI treatment Previously, we reported (Van Driessche & Zeiske, 1980a) that the apical K+ permeability was depressed after abolishing active transport with ouabain. In the present series ofexperiments the same observation was made in many experiments, although in others an augmentation of the Lorentzian plateau and IK could be observed. The short-circuit current recorded during this treatment is shown in Fig. 1A. After an equilibration period of45 min, during which the skin was exposed to NaCl Ringer solution on both sides, the K+ permeability was tested by recording thenoisespectraandIKinthepresenceofamucosa-to-serosa-oriented K+gradient. In theexperiment shown in Fig. 1 a Lorentzian component was present inthe spectrum (So= 0-75 x 10-21 A2s/cm2), and the Ba2+-blockable current was only 0-4#A/cm2 when the skin was exposed to KCl and NaCl Ringer solution on the mucosal and serosal sides, respectively. In the presence ofthese solutions, 2 x 10-5 M-ouabain was added to the serosal medium. After the addition of ouabain, the transepithelial current increased slowly and reached a peak after about 30-40 min and decreased again. Concomitantly, the transepithelial conductance increased considerably. After 50 min exposure to ouabain, the K+ permeability was tested by recording theIK and noise. In five experiments out often, the IK as well as the Lorentzian plateau were considerably augmented (see Fig. 1). In the five other experiments the Lorentzian component remained below background noise levels and theIK did not increase (not shown). After ouabain treatment we should expect a decrease ofIK and So, ifthe K+ permeabilityremainedunchanged. Becauseoftheelimination oftheelectrogenic Na+ transport across the basolateral border, the electrical driving force for K+ across the apical border should be reduced (Helman, Nagel & Fisher, 1981). On the other hand, the intracellular K+ concentration could be smaller after ouabain treatment, due to K+ loss in exchange for Na+. In the present experiments the mucosal compartment wasNa+free, sotheNa+entryshouldoccurthroughthebasolateral membranewhich has only a small Na+ permeability. Consequently the loss ofintracellular K+ should be hampered by the limited Na+ entry. On the other hand, intracellular K+ should be kept high because ofK+ entry through the apical border. So, it is likely that the increase inIK and So is not only a consequence of an increased driving force but is at least partly due to an augmented K+ permeability ofthe apical membrane. After 60 min of ouabain treatment, all serosal Na+ was replaced by K+ for 30 min. The purpose of this exposure of both sides of the skin to high K+ was to increase the intracellular K+ concentration. After switching back to NaCl Ringer solution on the serosal side we always recorded largerIK and So values than before basolateral K+ depolarization (see Fig. 1). Also, in the five skins in which ouabain alone did not increase theIK and the relaxation noise, the exposure to serosal KCl raised IK to 4-1+ 1-7,sA/cm2 and.So to 2-1+ 1-3 x 10-21 A2S/cm2. The result ofan experiment in 84 W. VANDRIESSCHE ._ A 9 Ba2+ K+ IBa2+, v X -IT Na+ Ba2+ -6 0 40 80 120 Time (min) B 10o-16- 10-17 10-18 ~~~4 'V E i' 2 0~ 10-21 *. lo-22 100 101 102 Frequency (Hz) Fig. 1A andB. Forlegend see opposite. APICAL K+ CHANNELS INFROG SKIN 85 C 10-18 10-21 ;l7o-211~-~ 4~ ~ ~ v\ E~~~~~~~~ 1001°0 102 103 Frequency (Hz) Fig. 1. Response offrog skin to KCl-ouabain treatment. Under control conditions, the skinwasexposedtoNaCiRingersolution (serosalside)andKClRingersolutionmucosall side). The current deflexions in A are caused by transepithelial voltage pulses of 1-5s duration and 3mV amplitude. A (current recording) and B (noise spectra) are data obtained from the same experiment. The spectra were recorded with frequency range 1 (see Methods). In this experiment the skin had a small, though measurable, K+ permeability: asmall Ba2+-blockable current (A) and a Lorentzian component (B) were recorded. AftertestingtheK+permeability (spectrum 1),ouabain (2x 10-6M)wasadded to the serosal solution. The transepithelial current generally reached a maximum after about 50min. The irregularities in the recording of the current before and after the maximumaregenerallypresent.Intheexperimentshown,theBa2+-blockablecurrentwas largerafter 1 hofouabainpoisoning thanundercontrol conditions. Also, the Lorentzian plateau was higher after ouabain (spectrum 2). After exposing the serosal side to KCl Ringer solution the K+ permeability was assessed again (spectrum 4). With KCl Ringer solutiononbothsides,thecurrentfluctuationsareveryclosetotheinstrumentationnoise (spectrum 3). Cshows the spectra recorded in another experiment before and after the same ouabain-KCl treatment. For clarity, the intermediately recorded spectra are omitted.InthiscasenosignificantK+permeabilitywasrecordedundercontrolconditions. Aftertheouabain-KCItreatmentaLorentziannoisecomponentaswellasaBa2+-blockable current of8-5 sA/cm2 wasrecorded. which no Lorentzian component was recorded under control conditions is displayed in Fig. 1C. After the ouabain-KCl treatment the relaxation noise clearly emerged above the background noise. As pointed out above, it is not clear how the intracellular K+ will change after inhibition of active transport under the conditions used. Nevertheless, it can be expected that the intracellular K+ concentration will increase when the skin is exposedtoKClRingersolutiononbothsides. Asthistreatmentcauses (afterouabain poisoning) an increase of the relaxation noise amplitude, we suggest that in these experiments, the intracellular K+ concentration acts as the primary trigger for the increase ofapical K+ permeability. Nevertheless, it cannot be excluded that other 86 W. VANDRIESSCHE intracellularionsalsomodulatetheapicalK+permeability.Ithasbeenshowninother preparationsthatsomeK+-channeltypesareactivatedbyCa2+.However,asweknow fromprevious studies (Zeiske & Van Driessche, 1983), the apical K+ channels infrog skin do not resemble the Ca2+-activated K+ channels, e.g. they are not blockable by tetraethylammonium (TEA) (Van Driessche & Zeiske, 1980a). In spite of this negativeargument,weperformedtheexperiments, describedinthenexttwosections, in which intracellular Ca2+ was altered. (C) The effect ofquinidine Quinidine has beenusedextensively to study iontransport indifferent tissues. Its effect on muscle contraction has been known for many years (Isaacson & Sandow, 1967), this effect having been attributed to an increase of intracellular Ca2+ concentration. Quinidine is thought to inhibit mitochondrial Ca2+ uptake, thus causinganincreaseofcytosolicCa2+activity(Carvalho, 1968;Batra, 1976). Recently, it has been shown that the addition ofquinidine to the serosal medium (4x 10-4 M) oftheisolatedurinarybladderimpairsNa+transportthroughthisepithelium (Taylor &Windhager, 1979; Windhager& Taylor, 1983;Arruda, 1983).ThereductionofNa+ transport is associated with an increase ofCa2+ release (Arruda & Sabatini, 1980). It was therefore suggested that quinidine causes a release ofCa2+ from intracellular organelles, thus increasing cytosolic Ca2+ concentration which is responsible for the reduction ofNa+ transport. We did six experiments in which quinidine (5x 10-4 M) was added to the serosal bathingsolutionwhich consistedofNaCl Ringersolution, whilethe mucosalsidewas bathed with KCl Ringer solution. The results ofa typical experiment are shown in Fig. 2. In the experiment shown, the Ba2+-blockable short-circuit current was 3 ,uA/cm2 under control conditions and the spectrum of the current fluctuations contained a Lorentzian component. After addition ofquinidine, the Ba2+-blockable currentdecreasedwithinabout 16 mintolessthan30% ofitscontrolvalue (Fig. 2A), and the plateau ofthe Lorentzian componentwasdepressedto the backgroundnoise level (Fig. 2B). After washing out quinidine from the serosal medium, the Ba2+- blockable current increased slowly and reached the control level after 60 min. Also, the Lorentzian noise increased after the removal of quinidine and the plateau increased to the control value in about 48 min. As can be seen from the current deflexionscausedbythevoltagepulses,thetransepithelialconductanceisirreversibly increased by quinidine, an observation which remains inexplicable. From these experiments we conclude that quinidine impairs the apical K+ permeabilityoftheskin.WhetherquinidinedirectlyaffectstheapicalK+conductance or acts via the augmentation ofintracellular Ca2+ concentration cannot be decided fromtheseexperiments.Therefore,weattemptedtomanipulatetheintracellularCa2+ concentration in a different way, by removal ofNa+ from the serosal solution. Fig.2.EffectofserosalquinidineontheapicalK+permeability.A,transepithelialcurrent recordedbefore, duringandafterwashingwithquinidine. B, spectrarecordedbeforeand after addition of quinidine. C, spectra (frequency range 2) recorded after wash-out of quinidine. Mucosal solution, KCl Ringersolution; serosalsolution, NaCI Ringersolution. The transepithelial conductance was measured with voltage pulses of 3 mV. The K+ current was recorded by adding 2 mm-Ba2+ to the mucosal solution. APICAL K+ CHANNELS INFROG SKIN 87 A ±5x101 M-quinidine *+2mM-Ba21 m m mr-_ 5. E U 2 2-5- :1 0 25 50 75 100 Time(min) B 10-20 E ce 10-21 Frequency (Hz) 10-19 C ,min 10-20 A E min C a coo 0min 10-21 I I A.I.A1 .A .a A aA IA . . A . . A.tA..... 100 101 U02 103 Frequency (Hz) Fig. 2. Forlegend see opposite. 88 W. VANDRIESSCHE A Na+ choline Choline Na+ Ba2+ - - 8 1- I 1 E Q- 4, _S 0 0 1 I I 0 15 30 45 60 Time(min) B a a a a g;- io-'9 E Control c 10-20 After During 4 4+44+-+ 10-21 N. N040:4+1, ... l11i00 ..a..Ia1II0*.i. a a I a1..1-.8.102 I.-;-;- ..sAA103 Frequency (Hz) Fig. 3. Effect ofreplacing all serosal Na+ by choline. A, transepithelial current. B, noise spectra (frequency range 1) recorded before, during and after replacing Na+ by choline. Mucosalsolution, KCl Ringersolutiontowhich2mM-Ba2+wasaddedtomeasuretheK+ current. The transepithelial conductance was measured with voltage pulsesof3 mV.

Description:
The K+ permeability of the apical membrane of frog skin (Rana temporaria) was activity, depressed the K+ current and the relaxation noise. to serosa, an inward K+ current was recorded and the spectrum of the fluctuations.
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