release induced by inositol 1,4,5-trisphosphate (InsP3) in saponin-treated ... properties of the receptor to InsP3 and of its associated Ca2+ channel. They have ...
Vol. 264, No. 30, Issue of October 25, pp. 17665-17673, 1989 Printed in U.S.A.
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society fo1 Biochemistry and Molecular Biology, Inc.
Fast Kinetics of Calcium Release Induced bymyo-Inositol Trisphosphate in Permeabilized Rat Hepatocytes* (Received for publication, April 4, 1989)
Philippe ChampeilS, Laurent Combettesg,Brigitte Berthon§, Edith Doucetg, Stephane Orlowski$, and Michel. Claret8 From the §Unite de Recherche Institut National de la Sante etde la Recherche Medicale, U274, Universite Paris Sud, bGt.443, 91405 Orsay, France and the SDepartement de Biologie, Service de Biophysique et Unite de Recherches Associee a Centre National de IC! Recherche Scientifique 219, Centre d'Etudes Nucleaires de Saclay, 91191 Gif-sur-Yvette Cedex, France
ible delay of 100-300 ms (Sage and Rink, 1987; Merritt and Rink, 1987); in patch-clamped liver cells, flash photolysis of an inactive precursor (caged InsP,) delivered from the patch clamppipette produces the Ca2+response within 250 ms, again after a minimal delay of 100 ms (Capiod et al., 1988). The ability of internal pools to release Ca2+rapidly has also been suggested by studies showing that hormonal stimulation of single hepatocytes loaded with aequorin induces repeated transientchangesratherthan a slow regularrisein the internal Ca2+concentration (e.g. see Woods et al., 1986). The time course of the InsP,-induced Ca2+efflux cannot therefore be accurately determinedby conventional spectrofluorimetry, since with this method the totalmixing and response time is of the order of seconds, leading to underestimation of the speed of InsP3 action (see, for instance, Joseph et at., 1984; Meyer et al., 1988 Combetteset al., 1989). T o investigate the fast kineticsof the Ca2+ fluxesactivated by InsP3, we have developed a stopped-flow method capable of measuring, on a 10-ms time scale, the Ca2+ fluxesresulting from rapid mixing of saponin-treated cells with the InsP3 messenger. The results presented hereshow that InsP, opens Ca2+channels with almost no detectable delay (less than 20 ms), indicating that the sequence of biochemical steps between InsP3 binding andCa2' release is not the rate-limiting Several authors have suggested that myo-inositol trisphos- step in the action of InsP3-releasing hormones. Our kinetic phate (InsP,),' the water-soluble messenger derived from the measurements have allowed us to deduce several molecular of its associated Ca2+ hydrolysis of phosphatidylinositol bisphosphate, opens Ca2+ properties of the receptor to InsP3 and channels in the internal stores of a variety of cells (Berridge, channel. They have also shown that in suspensions of per1984; Downes and Miclhell, 1985; Ehrlich and Watras, 1988; meabilized hepatocytes, part of the response to InsP, comes Suarez-Isla et al., 1988). The InsP3-stimulatedefflux of Ca2+ from a vesicular fraction which is released from the cells into along its gradient depends little on temperature and does not their supernatant in thepresence of saponin. requirethepresence of ATPorotherphosphoryldonors EXPERIMENTALPROCEDURES (Prentki et al., 1984; S.mith et al., 1985; Taylor and Putney, Cell Perrneabilization-Hepatocytes were prepared fromfemale 1985; Joseph and Williamson, 1986; Meyer et al., 1988). The whole process is fast. For instance, in permeabilized basophilic Wistarrat livers andincubatedin Eagle'smedium a t a n initial leukemia cells, the minimal time required for the binding of concentration of 2 X lo6 cells/ml as described previously (Combettes et al., 1988). A volume of 20-80 ml of cells was centrifuged and washed InsP, and opening of the Ca2+ channels has been estimated twice (50 X g for 1 min) with a n Eagle's medium lacking Ca2+, then to be lower than a few #seconds(Meyer et al., 1988); in intact resuspended a t 37 "C for 5 min in a cytosol-like medium containing human platelets and parotid cells, stopped-flow experiments 100 mM KC], 20 m M NaCI, 5 mM MgC12,0.96 mM NaH2P0,, 25 m M have shown that internal Ca2+ isreleased within 600 ms to 1 Hepes buffer (pH 7.25 a t room temperature), 1.5 m M Na2ATP, 5 m M creatine phosphate, 5 pg/ml creatine kinase, and 10 p~ fluorescent s of the addition of In~l?~-releasing agonists, with an irreduc-
We used a stopped-flow method for determining the kinetic properties (between 10 ms and 10 s) of the Ca2+ release induced by inositol 1,4,5-trisphosphate (InsP3) in saponin-treated rat hepatocytes. Preliminary experiments ensured that the indicator was able to monitor rapid changes in free Ca2+ reliably. At 20 "C, a maximally efficient concentration of 10 pM InsP3 released Ca2+with a half-time of 150-300 ms, the initial rate being about 1-2 nmol of Ca2+/mgof cellprotein/s. The delay between the a.ddition of 10 p~ InsP3 and the onset of Ca2+release was shorter than 20 ms, suggesting that the opening process of Ca2+ channels after binding of InsP3 to receptors is completed within a few milliseconds. Half-maximal initial rates for Ca2+ release occurred at about 1 p~ InsP, (Hill index was 1.6). The resultingCa2+efflux had a moderate temperature dependence. It could not be fitted to a single exponential. After low speed centrifugation of saponin-treated cells (1000 X g for 1 min), part of the InsP3-sensitive Caz+ pool was recovlered in the cell-free supernatant fraction, suggesting that the response to InsP3 arises from a vesicular fraction which may diffuse from the saponin-treated cells into the medium.
Ca2+buffer quin2, in the presence of a few pM contaminating CaZ+ plus endogenous Ca2+. Saponinwas added a t a final concentration of the payment of page charges. This article must therefore be hereby 50 pg/ml; this allowed permeabilization of the plasma membrane but which thus retained theirability for active marked "advertisement" in accordance with 18 U.S.C. Section 1734 not of internal Ca2+ stores accumulation of Ca'+. After this 5-minperiod, the permeabilized cells solely to indicate this fact. The abbreviations used are: InsPs, inositol 1,4,5-trisphosphate; were transferred on ice and used within a few hours. More than 98% quin2, 2-[(2-amino-5-methylphenoxy)methyl]-6-methoxy-8-amino- of the cells were freely permeable to trypan blue. Before the experiquinoline-N,N,N',N'-tetraaceticacid EGTA, [ethylenebis(oxyethy- ment, 10 ml of cells were rewarmed to the chosen temperaturefor 51enenitrilo))tetraaceticacid CI2E8,octaethylene glycol dodecyl mon- 20 min (which allowed them to accumulate significant amounts of oether; Hepes, 4-(2-hydro:cyethyl)-l-piperazineethanesulfonic acid. Ca") and then introduced into the large syringe of the stopped-flow
* The costs of publication of this article were defrayed in part by
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system. At that time, thecell protein concentration ranged from 2 to 2.8 mg/ml. Cell Supernatant-Hepatocytes were permeabilized with saponin as indicated above, except that the cell suspension was slightly more concentrated (2-4 X lo6 cells/ml). The permeabilizedcells were centrifuged a t 1000 X g for 1 min. The supernatant was stored on ice before being usedfor stopped-flow experiments andfor parallel measurements in a conventional spectrofluorimeter of the total amounts of Caz+released by InsPB orionomycin. The supernatant was totally devoid of cells as indicated by photon microscopy. Optical Measurements-Ca2+ movements were measured from the observed changes in the fluorescence of the Ca2+-sensitivedye quin2 (excitation wavelength, 335 nm; bandwidth, 4 nm). Observations on a time scale ranging from5 s to several minutes were performed with a conventional fluorimeter, either Jobin & Yvon or SLM 4000 (emission wavelength, 510 nm; bandwidth, 8 or 16 nm). Measurements with subsecond time resolution were made withaDionexD-110 stopped-flow apparatus, in which nitrogen pressure forces the contents of two syringes through a mixing chamber into an observation cell designed for fluorescence monitoring after theflow has stopped. The dead timeof the apparatuswas 3-6 ms (see below). We used two syringes with different diameters, so that the mixing volume ratio was 1:16. A relatively wide band filter, centered a t 531 nm (MTO 531c), was used for fluorescence detection in order to maximize the collection of emitted light. The fluorescence signal was digitalized as describedelsewhere (Guillainet al., 1980; Champeilet al.,1983). During the period of time chosen, 178 data points were stored and, afterwards, 35 more were collected on alonger time scale. In all experiments reported here, the fluorescencenoise was reduced by averaging 4-10 shots. Rate of Interaction of Quin.2 with Calcium-Preliminary experiments to test the ability of quin2 to equilibrate rapidly with a new free Ca2+concentration were carried out at 20 "C in the cytosol-like medium from which ATP and the ATP regenerating system were absent. In these experiments, as indicated Fig. in 1, the smallsyringe in the stopped-flow apparatus contained a concentrated solution of quin2 which was either Caz+-deprived(Fig. 1A) or Caz+-saturated (Fig. 1B). The large syringe contained a Ca*+-EGTA buffer, so that the final free Ca" concentration after mixing could be set atvarious levels. When Ca2+-saturatedquin2 was mixed with EGTA only (Fig. 1B, lower trace), the fluorescence of quin2 dropped in a monophasic way; half-time was 16 ms a t 20 "C, which corresponded to 43 s-', in agreement with previous results (Quast et al., 1984; Martin et al., 1985; Rosenfeld et al., 1985). For higher final concentrations of free Ca2+,the amplitudeof the fluorescence drop was smaller, and its halftime was shorter (see Fig. 1B). When quin2was initially deprived of Ca2+ and then mixed with a medium with a given concentration of free Ca", the stopped-flow recording allowed measurement of the rate at which fluorescencerose(Fig.1A).However, for highCa2+ concentrations, equilibration of the quin2 fluorescence to its final value was so fast that part or all of the signal amplitude was lost during the dead time of the instrument, which is 3-6 ms (see upper traces in Fig. 1A). A plot of the final fluorescence level, measured as a function of the calculated final free Ca2+ concentration (Fig. lC), gave the standard titration curve of quin2 fluorescence (which can also be obtained with any conventional fluorimeter). The dissociation constant Kd was about 100 nM, taking 170 nM astheapparent dissociation constant for the Ca2+-EGTAcomplex in the presence of 3.5 mM free magnesium at pH 7.25 (Champeil et al., 1985). In Fig. lD, the rate constants observed for the fluorescence changes were plotted as a function of the final free Ca2+ concentration. This type of plot allows derivation both of the rate of CaZ+dissociation from the quin2.Ca2+complex (from the intercept, k- = 40 s-l) and of the bimolecular rate of Ca2+ bindingto its ligand (from the slope, k+ 4 x lo8 M-' s-l), since koba= k- + k + .[Ca"]. Both rates are compatible with the Kd actually measured, since Kd = k - / k + . These rates insured reliable monitoring by quin2 of the changes in the free Ca2+ concentration occurring in subsequent experiments. Preliminary Measurementswith Sarcoplasmic ReticulumVesiclesA further test of the stopped-flow mixing and detection device was performed using sarcoplasmic reticulum vesicles as a control system. When sarcoplasmic reticulum vesicles (0.1 mg of protein/ml), prepared as previously described (Champeil et al., 1985), were suspended in the cytosol-like medium at 20 "C with no ATP, addition of ATP (1.5 mM) initiated Ca2+uptake by the sarcoplasmic reticulum vesicles, and subsequent addition of ionophore (ionomycin) or detergent (for example C12Es)reversed the large ATP-induced accumulation of Ca2+ in these vesicles; this could be observed using a conventional fluori-
ouin2-EGTA
I
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,
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FIG. 1. Stopped-flow resolution of quina fluorescence changes for different final free Ca2' concentrations.A, 1 volume of concentrated Ca2+-deprived quin2 (160 p M quin2, 1.6 mM EGTA) was mixedin theDionex stopped-flow device with 16 volumes of Ca2'-EGTA buffer containing 2 mM EGTA plus various amounts of Ca2+. B, same experiment, except that quin2 was initially Ca2+saturated; the smallsyringe contained 160p M quin2 and320 pM Ca2+. C, plot of the final fluorescence levels observed in the above experiments, as a function of the final free Ca2+ concentration. Triangles and circles refer to experiments depicted in A and B, respectively. D, plot of the observed rate constantsfor fluorescence equilibration. The experiments were performed in a cytosol-like medium lacking ATP and the ATP-regenerating system and containing 100 mM KCl, 20 mM NaCI, 25 mM Hepes, 1 mM NaHzPOa, and 3.5 mM MgC12 (pH 7.25, 20 "C). The apparent dissociation constant of the Ca2'-EGTA complex under these conditionswas taken as 170 nM. meter.On the other hand, sarcoplasmicreticulum vesicles whose active pumping of Ca2+in a quin2-containing medium had reached a steady state were introduced into the large syringe of the stoppedflow device, and detergent (C12E8)was added from the small syringe; we found that thestopped-flow equipment did allow satisfactory time resolution of the resulting rise in fluorescence (not shown). Similar experiments in which only water was added from the small syringe resulted in no fluorescence changes, as expected. Chemicals-The InsPS used here was fromSigma(Ref. I-4009), quin2 and ionomycin were fromCalbiochem, and C12E, was from Nikko Chemicals Co., Tokyo. RESULTS
Time Course of InsPs-induced Calcium Release from Internal Stores in Permeabilized Rat Hepatocytes-As previously 1986; observed(Burgess et al.,1984; Joseph et al.,1984, Combettes et al., 1988, 1989), saponin-treated hepatocytes displayed a substantial release of Ca2+in response to Imp3. The inset to Fig. 2 shows a typical experiment designed to test InsPs-induced release of Ca2+in permeabilized cells, and performed at 20 "C with a conventional fluorimeter, in which calibration with EGTA and Ca2+is easier than in a stopped-
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Fast Kinetics of InsPs-induced Ca2+Release time I
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cells,quin2 Ins Pa EGTAT
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time , s FIG. 2. InsPs-inducedl release of Caa+ from permeabilized hepatocytes. The inset shows a typical experiment performed in a conventional fluorimeter. Permeabilized cells (2 X 10g/ml, in the presence of 0.05 mg/ml saponin, kept on ice) were warmed to 20 "C in their permeabilization medium, which contained 100 mMKC1,20 mM NaCl, 25 mM Hepes, 1 mM NaHZPO,, 5 mM MgCl,, 1.5 m M Na2ATP, 5 mM creatine phosphate, 5 units/ml creatine kinase, and 10 pM quin2 (pH 7.25). Fluorescence (left scale)stabilized at a certain level, corresponding here to 106 nM free Caz+ (rightscale). Two additions of 3 pM InsP3 each raised the Ca2+concentration in the medium to 180 nM. This was followed by two additions of 2 pg/ml ionomycin each, 0.5 mM EGTA, and 0.5 mM Ca2'. Following the addition of InsP3, the total Ca2+titrated by 10 p~ quin2, estimated from the relation (Ca2+]fi.e. = Kd.[CaZ'],/([quin2], - [Ca*'],), rose from 5 to 6.4 p ~ indicating , that 1.4 p~ Ca2+was released by the InsPs messenger. Main frame, stopped-flow experiments. A similar suspension of Ca2+-loadedpermeabilized cells was introduced into the large syringe of the stopped-flow device and mixed at a 16:l (v/v) ratio with InsP3 (final concentration 3 p ~ upper , trace) or water (lower trace). Control experiments showed that the turbidity of the cell suspension, recorded at an excitation wavelength of 540 nm, did not vary as a result of InsP, addition (not shown). Traces are means of five sequential shots. They have been shifted arbitrarily along the y-axis. The dashed line is the fit to a single exponential of the data points collected between rl.01 and 1 s. It is clearly not sufficient to describe adequately the experimental data.
flow apparatus. In this experiment, the cells initially accumulated Ca", resulting in a certain free Caz+ concentration in the medium. Addition of InsP3 at thispoint significantly enhanced fluorescence; this enhancement was not due to InsPB contamination byCa", because a second addition of InsPs had no effect. InsP3 was used here at a concentration (3 p ~ which ) maximally depleted the sensitive intracellular Ca2+pool (see dose-response curves for InsP3 action in Fig. 4); however, subsequent addition of ionomycin revealed that the Ca" stores in the cells could be further emptied. Last, addition of EGTA and thenof excess Ca2+allowed calibration of the fluorescence signal and its conversion into free Ca2+ (right scale in the inset). In this experiment, InsP3 raised the free Ca2+ concentration from 106 to 180 nM in the presence of 10 p M quin2, thus implying that 1.4 p M Ca2+was released
17667
from the internal store upon addition of the InsP3 messenger (see legend to Fig. 2). When estimated from several experiments ( n = 11), InsP3 and ionomycin released 2.1 f 0.14 and 3.2 +- 0.16 p~ Ca2+in the medium, respectively. For a cell protein content of 2-2.8 mg/ml, the InsP3-mediated rise in the Ca2+concentration thus corresponds to therelease of 0.51 nmol of Ca2+/mg of cell protein, values which are in the range of those previously reported in liver cells (Burgess et al., 1984; Joseph et al., 1984; Combettes et al., 1988, 1989). It has to be noted that in our experiments permeabilized cells were stored on ice before being rewarmed to 20 "C and challenged with InsP3, a protocol which was required for parallel analysis of Ca2+movements in stopped-flow experiments. To monitor the true rate of the InsP3-induced rise in the free Ca2+concentration, Caz+-loadedpermeabilized cells were transferred into thelarge syringe of our stopped-flow system. Inspa, diluted in water, was introduced into thesmall syringe, so that its final concentration after mixing was 3 pM. The upper trace in Fig. 2 illustrates the time course of the fluorescence rise typically observed after mixing. Despite its relatively small amplitude (8-10% of the initial fluorescence, as expected from the fluorimeter recording shown in the inset), the rise in fluorescence was easily detected by the stoppedflow device. The individual traces recorded in the presence of InsP3 were highly reproducible but contained a significant level of noise, which was reduced by averaging 4-10 shots. When water was substituted for InsP3 in the small syringe, the fluorescence trace was flat (lower trace, Fig. 2; in this figure, as in Figs. 4A and 5B below, the InsP3-dependent traces have been arbitrarilyshifted along the y-axis with respect to the control trace: both of course start from the same initial fluorescence level). The results showed the following: (i) Ca2+release proceeded with almost no detectable delay on the time scale of our experiments. Closer examination of the traces recorded in several different experimentsas well as of an average of all of them to reduce the noise level (not shown) allowed us to conclude that there might in fact be a very small delay before the onset of fluorescence rise but that, under the conditions of these experiments(20 "C,3 p~ InsP3), it was restricted to the first 10-20 ms (see also Figs. 4 and 5) and could probably be accounted for by the noninstantaneous response of quin2 to the changes in calcium concentration (80-100 s-l around pCa 7, see Fig. 1D). (ii) Ca2+release in the presence of InsP3 was fairly rapid. The half-time derived from experiments performed a t 20 "C in the presence of 3 p~ InsP3 ranged between 200 and 300 ms (see Figs. 2 and 5 ) . On the basis of the calibration performed in parallel experiments with a conventional fluorimeter on the same batch of cells incubated under identical conditions (as for the inset to Fig. 2), the initial rate of Ca2+release ( u ) was calculated from the initial slope of the rise in fluorescence: for instance, in Fig. 2, u = 0.5 nmol of Ca2+/mgof protein/0.6 S, or about 1nmol of Ca2+/mgof protein/s. Again, for different experiments, this initial rate ranged between 1 and 2 nmol/ mgls. (iii) Due to our data acquisition system, we were also able to collect data points at relatively long intervals (see dots in Fig. 2). Fig. 2 shows that the fluorescence rise displayed a "trailing tail" between 1 and 3 s. The data points collected between 0.01 and 1 s were fitted to a single exponential, as shown by the dashed line in Fig. 2. The discrepancy between experimental points and the predicted curve, clearly seen for long time periods, indicates that thetime course of the InsP3induced Ca2+release is not adequately described by a single exponential (more data below).
Fast Kinetics of InsP3-induced Ca" Release
17668
Temperature Dependence of the Kinetics of Calcium Release-After the above data had been obtained at 20 "C, the rate of InsP3-induced Ca2+was monitored at several temperatures between 10 and 37 "C, taking into account the temperature dependence of the quin2 fluorescence level (a high temperature reduces the fluorescence quantum yield of fluorophores). This is illustrated in Fig. 3, in which the intensity (volts) of quin2 fluorescence emission has been directly plotted. Fig. 3 shows that between 17 and 31 "C, the apparent half-time approximately doubled every 10 "C. On the other hand, the initial rate of InsPs-induced Ca2+release a t different temperatures could be evaluated from the ratio of the initial rate of fluorescence change to the initial fluorescence level, provided the cells had imposed similar free Ca2+concentrations at all temperatures; this was the case between 17 and 31 "C for the experiments illustrated in Fig. 3, as evaluated from parallel experiments performed on the same batch of cells with a conventional fluorimeter (not shown). Again, the initial rate of InsP3-induced Ca2+release at different temperatures, estimated in this way, approximately doubled every 10 "C (inset toFig. 3). At more extreme temperatures (10 and 37 "C), differences in Ca2+load hampered accurate determination of the effect of temperature on Ca2+release. Thus, at 10 "C, Ca2+accumulation was much slower, and the preequilibration period was probably too short toload internal stores maximally. On the other hand,at 37 "C, we obtained evidence
in these parallel fluorimeter experiments that a slow timedependent reduction occurred in both the InsP3- and ionomycin-sensitive Ca2+ pools during the 5-15-min period required to load the stopped-flow syringes and acquire data points. This accounted for the apparently less efficient release observed at 37 "Cthan at31 "C (data notshown). In another series of experiments in which we optimized the acquisition conditions to reveal any initial delay, it was found that the very short delay previously detected at 20 "C was also probably slightly temperature-dependent. Despite these limitations,we estimated that theQlo for the Ca2+release rate was close to 2 (E, = 12 kcal/mol or 50 kJ/mol), a value slightly higher than the one expected from passive diffusion through ion channels (1.4).
Imp3 Dose-response Curve-In subsequent experiments, different concentrations of InsP3 were added to Ca2+-loaded permeabilized cells. Fig. 4A shows a typical series of recordings for experiments performed at 20 "C. Here, again, individual curves were arbitrarily shifted in relation to each other along the y-axis: all of them start from the same initial fluorescence level. The results showed the following: (i) Theamplitude of the total rise in fluorescence (after 10
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FIG. 3. Temperature dependence of Inspa-induced CaZ+release. Experiments identical to the one described in Fig. 2 were performed at different temperatures (the cells internal stores were also equilibrated with Ca" at the indicated temperature). The fluorescence signal actually measured was directly plotted as a function of time. Points were collected for up to 10 s (dots). Inset, estimates of the half-time (squares) and of the ratio of the initial rate of the increase in fluorescence to the initial level of fluorescence (circles) were plotted as a function of temperature on a logarithmic scale (see text). a.u., arbitrary units.
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FIG. 4. InsPBdependence of Ca" release. A, the experimental protocol was identical to that described in Fig. 2, except that InsP3 was added at different final concentrations, as indicated. Typical traces of Ca2+release initiated by various concentrations of InsP3. B, initial rates (circles) and amplitudes (squares) of the resulting fluorescence changes, as afunction of the InsPa concentration (log scale). The data (arbitrary units (Q.u.))were derived from a set of experiments different from the one shown in A, in which more concentrations were tested. C, direct plot of the initial rate of Ca2+ release versus the InsPs concentration (same experiment as in B ) .
Fast Kinetics of InsP3-induced Ca2+Release
17669
s) resulting from thea.ddition of InsP3 varied with the InsP3 concentration, showing an apparent ECso of about 0.2 p M (squares in Fig. 4B), consistent withprevious estimates with S T conventionalspectrofluorimeters or in &Ca2+ experiments (Burgess et ul., 1984; Joseph et al., 1984; Combettes et al., CE 1989). (ii) The traces shown in Fig. 4A did not reveal any apparent delay between InsP3 addition and the initiation of Ca2+ release. However, when the InsP3-inducedfluorescence rise was recorded under conditions designed to optimize detection of few any initial lag (i.e.storing 178 data points during the first Io hundreds of milliseconds and the residual 35 up to 10 s), a small delay between the addition of InsPs and the onset of fluorescence rise couldl be detected; as mentioned above, this '5 delay was very short at high InsP3 concentrations, but it Io increased to 30-50 m.s when the InsP3 concentration was reduced to low values, e.g. 0.4-1 PM InsP3 (not shown). This IP3 lrnin may result from rate-limiting binding of the agonist to its U receptor, as would be the case if, for instance, k+ were 1-2 X lo7 ~ - s-1. 1 (iii) The rateof the InsP3-inducedCa2+ release was strongly dependent on the InsPs concentration, since it was slow a t I I I 1 I I I low concentrations ( e g 0.3 p M ) and reached a maximal value .. ,......I' for high concentrations (e.g. 10 pM InsP3). This asymptotic ._... . behavior is consistent .with the view that only a finite number *..' of InsP,-activated Ca"+ channels are available in the memsupernatant ..,-".. brane of the internal store. As this point, the InsP3 dependence of the kinetics of Ca2+ release was further explored by focusing on the initial rateof Ca2+release, which reflects the properties and number of the openchannels available. Thisinitialrate,measuredin a separate series of experiments, was plotted in Fig. 4B, as a function of the InsP3Concentration (open circles).Half-maximal rate of Ca2+ rekase occurred at 1 p~ InsP,. Thus the initial rate of the release process had an apparent affinityfor ) was poorer than the apparent affinity InsP3 (1 p ~ which deduced from measuring the final extent of Ca2+ release or 1 I I I 1 IlIlllllLYI thetotal fluorescence rise (0.2 PM, see above). This poor 0 0.4 1.0 3.0 apparent affinity of 3. pM, deduced fromkineticmeasuretime , s ments, reflects the binding properties of the InsPs receptor FIG. 5 . Saponin-induced extraction of InsPs-sensitive and more reliably than the higher affinity of 0.2 p~ does, since -insensitive Ca2+pools. A, saponin-treated cells, 2 X lo6 cells/ml the latteraverages true binding propertiesof the receptor and (T), were centrifuged at 1000 x g for 1 min and the supernatant( S ) time-dependent emptyingof the Ca2+pool. and pellet ( P )were tested for retention of InsPs-sensitive Ca" pools We wondered whether cooperative opening of channels by and total Caz+pools by successive additions of 3 PM InsPs (ZPa),5 InsP3 could be determined from our measurements. In fact,a pg/ml ionomycin (Zo), 30 p~ Ca2+ (C), and 3 mM EGTA (E).Left direct plot of the Ca2+ release initial rates provided evidence truce, perrneabilized cells; central truce,supernatant; right trace,pellet (the pellet was resuspended in the same volume of quin2-containing for a slight cooperativity (Fig. 4C) A Hill plot (not shown) cytosolic medium). B, stopped-flow recordings of InsP3-induced Ca2+ allowed to determine a cooperativity index of nH = 1.6 f 0.2 release, either with permeabilized cells (lower truce) or with the 1000 (three separate experiments). The significance of this Hill x g supernatant of a more concentrated cell suspension of 4 X IO6 cells/ml (upper truce) (conditions as in Fig. 2). For clarity, the upper coefficient will be considered below (see "Discussion"). Last, it is noteworthy that the individual rises in fluores- truce was arbitrarily shifted along the y-axis. cence were not monophasic, since a prolonged slow Ca2+efflux was observed at all the InsP3 concentrations tested, but more ionomycin. When this cell suspension was centrifuged at a especially at thelow concentrations, asshown in Fig. 4A (see speed and for a period of time (1000 X g for 1 min) sufficient data points collected up to 10 s; quantitative analysis in Fig. to pelletmore than 99.9% of suspended permeabilized cells as 6 below). judged fromphoton microscopy, the cell-free supernatant was Partial Extraction of the InsP3-sensitiue Calcium Storesensitive to both InsP3 and ionomycin (see central truce in During these experiments, we incidentally found that saponin Fig. 5A). This supernatant contained about 50% of the pronot only interacted with the hepatocyte external membrane, teins originally present in thecell, presumably themajor part thus allowing InsP, freeaccess to the cytoplasm, but also of the soluble proteins asindicated by the fact that more than released into the cell supernatant a significant proportion of 90% of the cell lactate dehydrogenase activity was recovered the Ca2+-storing compartments, including a fraction of the in this supernatant (data not shown). Although the aim of InsPs-sensitive pool. A typical experiment is illustrated in the present study was not to undertake a complete study of Fig. 5A. The left truce shows that permeabilized hepatocytes this Ca2+-containing fraction, preliminary results performed in this experiment displayed the usual release of Ca2+upon on 10 separate experimentsrevealed that 30-40% of the InsP3successive additions of maximal concentrations of Imp3 and and ionomycin-sensitive Ca2+pools were recovered in the cell-
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Fast Kinetics of InsP3-induced Ca2+Release
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- @
ophasic behavior of the permeabilizationprocess. Tentative BiexponentialAnalysis of Fluorescence Rises-We mentioned above thattheInsP3-induced fluorescencerise could not be fitted to a single exponential. We present here the results of an attempt to fit the fluorescence rises to a linear combination of two exponentials with different rates and amplitudes. The fit was done as previously described (Champeil et al., 1983). This analysis matched the experimental points very well (the theoretical lines were not drawn in our figures becausethey would have masked the experimental points). The resultof this analysis is shown in Fig. 6; the rate constants of the fast and slow phases of InsP3-induced Ca2+ release differed by a factor of 5-10, which made them easily detectable. The rates for the two components variedwith temperature (Fig. 6A). The relative contribution of the slow phase to the totalCa2+ release remained approximately constant when the temperature was varied (Fig. 6B). The concentration of InsP3 influenced the rate constants of both the fast and slow phases (Fig. 6C). At low Inspa concentrations, the amplitude of the slow phase dominated the total signal amplitude (Fig. 6D); the latter resultwas obtained both a t 20 and 37 "C, with permeabilized cells and with the acellular supernatant (not shown). DISCUSSION
Rate of InsP3-induced Release of Calcium-Phosphatidylinositol bisphosphate breakdown is a key step that mediates various Ca2+-controlledprocesses in several cell functions, 0' through Inspa-induced openingof Ca2+ channels in the membrane of the cell internal Ca'+ store (Downes and Michell, J l l l I 1985; Berridge, 1984). The aim of the present work was to 0.1 1 10 0.1 1 10 estimate the rate of channel opening triggered by InsP3 and [ I n s P31 , p M ( log scale) the rate of the resulting Ca2+ release by using a stopped-flow method able to detect Ca2+movements within 10 ms of InsP3 FIG.6. Tentative biexponential analysis of the InsPa-in- addition. Our results show that maximal concentrations of duced Ca2+release curves (data from Figs. 3 and 4, B and C): temperature dependence ( A and B ) and InsPs dependence (C InsP3 mobilize Ca2+ without any significant delay and with and D ) of the rate constants ( A and C)and amplitudes ( B and half-times of 150-300 ms at 20 "C or evenless at higher D ) observed for both the fast component (closedsymbols) and temperature. These half-times are much shorter than what the slow component (open symbols). In B, the fastand slow was previously derived from conventional fluorimetry experamplitudes have not been plotted independently, since part of their iments, emphasizing that the latter technique canonly protemperature dependence arises from the temperature dependence of vide minimum estimates of the initial rates of Ca2+ release the fluorophore quantum yield; rather, the relative weight of the slow (Joseph and Williamson, 1986; Combettes et at., 1989). The component, i.e. the ratio (slow amplitude)/(slow amplitude + fast amplitude) has been plotted (open squares). The concentration of upper limitof 20 ms for the delay between InsP3 addition and InsP3 was 3 PM in A and B; temperature was 20 "C in C and D. a.u., the onsetof Ca2+ release clearlyindicates that channel openarbitrary units. ing is an extremely fast process. Consequently, the delay of 100-300 ms between the addition of stimulating agonists and the [Ca2+Iiriseobserved with intact cells in other studies free supernatant. The righttrace in Fig. 5A shows that a significant fraction of the Ca2+ responsewas also retained in (Sage and Rink,1987; Merritt and Rink,1987),or therecently the cell pellet. Note that thecell pellet probably took Ca2+up reported 100-ms delay between the flashing of caged InsP3 and the Ca2+-dependent opening of potassium channels in the again during resuspension in the new cytosol-like medium. These results indicate that saponin treatmentof rat hepato- plasma membrane (Capiod et al., 1988), are not due to slow cytes releases into the mediuma closed vesicular system InsP3-induced channel opening. In addition, the half-times (150-300 ms)arecertainly containing a Ca2" store mobilizable by the InsP3 messenger forCa2+releaseobservedhere sufficient to account for the repetitive spikes of cytosolic Ca2+ and theionophore. described elsewhere in a single hepatocyte exposed to horIn view of these findings, it was important to establish whether the InsP3-sensitive vesicular Ca2+ store detected in monal stimulation (Woods et al., 1986; Jacob et al., 1988 for references in other nonexcitable cells). thesupernatant displayed thesamekineticpropertiesas permeabilized cells. Experiments performed with the stopped- Since the quin2 fluorescence level reflects the amount of flow device indicated that this was the case. Thus, as shown Ca2+ titratedby the dye, the initial rate of the InsP3-induced in Fig. 5B after adequate normalization, the kinetic features rise in fluorescence in the stopped-flow experiment can be of the InsP3-induced Ca2+release from the supernatant were converted into a rate of CaZ+release from the internal store. similar to thoseobserved with permeabilizedcells. In common This conversion required parallel calibration of the fluoreswith cells, the Ca2+ release from the vesicular compartment cence signal; as explained under "Results," this allowed us to estimate an initial Ca2+ release rate of about 1-2 nmol of proceeded without significantdelay (t20 ms)and witha similar half-time. In addition, also it displayeda second phase, Ca2+/mgof protein/s. Since the volume of isolated rat liver especially at low InsP3 (not shown),confirming the nonmon- cells is about 2.4 pl/mg protein, such a rate would allow, in I
I
I
I
I
Fast Kinetics of InsP3-induced Ca2' Release
17671
an intact cell, a rate fcor the rise in the totalcytoplasmic Ca2+ occurs with the acetylcholine receptor (see Changeux et al., concentration of (1-2 )/2.4 nmol. pl" .s-l, i.e. 4-8 p M every 10 1984, for review). Such a cooperativity has also been assigned cytosolic ms. Because of the buffering capacity of the cell cytoplasm, a key role for generating the repetitive spikes in the actual rise in the free Ca2+ concentration will, however, calcium levels observed upon hormonal stimulation of many be much slower. A similar rate in muscle cells would be fast cells (Meyer and Stryer, 1988; see, however, Wakui et at., enough to allow the Imp3route tobe a possible candidate for 1989). However, we want to point out that cooperativity of the Ca2+ release curves (which is demonstrated in both our theexcitation-contraction coupling mechanisminsmooth muscle, but probably not in fast skeletal muscle, a system in experiments and in those of Meyer et al., 1988),does not which the role of Inr;P3 is controversial (Suarez-Isla et al., necessarily imply cooperativity of the InsPs-induced opening 1988; Walker et al., 1987; Vilven and Coronado, 1988; see also of thechannels. An apparent cooperativitymight be the Caswell and Brandt, 1989). Consistent with this view, the artifactual resultof refilling of Ca" stores by the ATP-driven presence of InsP3 rec,eptors in fast muscle has not yet been pump, which would reduce the net InsP3-inducedCa2+efflux to a greater extent for low concentrations of InsPs (i.e. for demonstrated. Intrinsic Properties of the InsP3 Receptor-Assuming that low unidirectional efflux rates) than forhigher concentraCa2+reaccumulation by the intera hepatocyte is50-200 fmol/ tions. The assumption that the amountof InsP3 receptors in mg cell protein (see Slpat et al., 1986; Guillemette etal., 1987; nal cell stores partially compensated the Ca2+ leak when low Mauger et al., 1989), tlhe initial rate of the InsPs-inducedCa2+ InsP3concentrations were used mightexplain why a low release (1-2 nmol of Ca2+/mgof protein/s) can also be con- concentration of InsP3 (e.g. 0.2 PM) didnot totally empty the verted into an amount of Ca" ions that flow through one wholeCa2+pool (see, however, below). T o conclude, it is channel per second this amounts 1-2 to nmol of Ca2+/50-200 therefore of critical importance to evaluateprecisely the relfmol of receptor/s, i.e. to lo4 ions per channel per second. ative rates of InsP3-induced Ca2+ releaseand pump-mediated When evaluating this figure, it should be remembered that reuptake of Ca2+, agoal which we are actively pursuing. The virtual absenceof delay between the addition of satutheconcentration of freeCa2+ stored inside theinternal M or less; rating concentrations of InsPs and the onsetof Ca2+release compartment is probably of theorder of therefore, if such a channel were bathed between two com- might have a structural implication, because it suggests that partments, one of them containing 100 mM divalent cations there isa close structural connectionbetween the receptorfor as occurs in many el.ectrophysiologica1 studies, this would InsPs and the Ca2+ channel itself. This possibility is in line result in lo6 ions flowing through the channel per second, with similar previousproposals concerning smooth muscle of cells (Smith et al., 1985), basophilic leukemia cells (Meyer et rather than lo4, assuming an almost proportional increase al., 1988), and, again, similar to what is known about the the unidirectional efflux through the channel. This correor acetylcholine receptor, for instance. sponds to2 X IO6electrical charges per channel per second a unitarycurrent of 2 lo6 X 1.6. Coulomb/s, i.e. 0.3 PA. We would now like to analyze furthertheInsP3 doseThis currentwould be even larger if, as mightbe the case, the responsecurve we obtained (Fig. 4). Our observation of a of InsP3-induced Ca2+ channels fluctuate between open and closed conformations saturation limitfor theinitialrate even in thepresence of a saturating amount of InsP3. Sucha release of high InsPs concentrationssuggests that under these channel mightwell be detectable by electrophysiological tech- conditions (for instance when 3-10 p~ InsPsisaddedto niques; the calculated value of its unitary current is in fact of permeabilized cells), the diffusion-limited encounter of InsP3 of channel opening: the same orderof magnitude as the unitary current observed and its receptordoes not restrict the rate for the channel opened by InsP3 in smooth muscle sarco- InsP3 binding is faster than the subsequent conformational or biochemical steps underlying the opening process; under plasmic reticulum (Ehrlich and Watras,1988). Our data also reveal a moderate temperature dependence these conditions, the absence of a detectable delay between of the rate of Ca2+efflux through the channels opened by InsPs addition and Ca2+ release in turn implies that channel InsP3. Qlo is close to 2, i.e. higher than 1.4, the value expected opening rapidly follows InsPa binding, within less than 20 ms for a purely passive efflux through an open channel (see for a t 20 "C (see "Results"). For thelower concentrations of InsP3 instance Hille, 1984; Smith et al., 1985; Joseph and William- tested, we could resolve a short delay between the addition of son, 1986; Meyer et o:l., 1988). A Qlo higher than 1.4 may InsPs and the onset of Ca2+release, which then can probably reflect the temperaturedependence of the number of channels be attributed to rate-limiting InsPs binding,since channel opened by a saturatingamount of InsPa rather than the opening itself is fast. Nevertheless, this delay was very short permeability characteristics of the opened channel itself, since compared to the time course of Ca2+ release in the presence the binding of an agonist does not necessarily increase to 1.0 of low concentrations of 1 n s P ~(Fig. U ) ,so that binding of the probability of the opening of ion channels (seeHille, 1984; InsP, rapidly reaches equilibrium. We conclude that the"iniHeidmann etal., 1983 for examples). In fact, an InsP, channeltial rates" of Ca2+release, plotted in Fig. 4, B and C, which in opening efficiency of less than one could be physiologically fact are themaximal rates measured in the first 100 ms or so, relevant if the effect o:f the agonist were modulated by other do reflect the properties of the form of receptor available for cytoplasmic regulators. InsP3 binding, which thus has a relatively poor dissociation The dose-response curve for the initial rateof Ca2+release constant of about 1p M . It is clear that the InsPs concentration shows evidence for a cooperative effectof InsP3 (see the points required to produce a half-maximal final extentof Ca2+ release at low concentrations in the direct plot of the release rate, will be lower (we found 0.2 p ~ ) since , a small number of open Fig. 4C). Cooperativity, however, was low (Hill coefficient 1.6 channels may succeed in emptying a Ca2+ pool as efficiently k 0.2; see also Williamson et al., 1985). A higher apparent as a large number of open channels, although less rapidly. A cooperativity has been previously described under different similar differencein apparent affinities between rates and experimental conditions in basophilic leukemia cells (Meyer amplitudes of Ca2+ releasehas been previously observed (Wilet al., 1988). Obviously this is an important issue. True co- liamson et al., 1985; Meyer et d . , 1988). Therefore, for the operative activation of the ca" channel would imply that at description of the Inspa receptor, the poor apparent affinity least two InsP3 molecules must bind to thereceptor in order deduced from the kinetic measurementsof release rates (Fig. t o open the channel, a situation which would resemble what 4) is more significant than the affinity deduced from meas-
17672
Fast Kinetics of InsP3-induced ea2+Release
urements of the final Ca2+ contents. Detailed Analysis of the Calcium Release Curves-We wish to discuss some of the possible reasons for the nonmonophasic nature of our Ca2+release curves. Their nonmonophasic character was not due to thenonlinear dependence of the fluorescence level of quin2 on the Ca2+ concentration, since such nonlinearity would tend to make fluorescence curves flatter than the supposedly exponential Ca2+release curve, whereas we have to account for lasting rises in fluorescence that persisted after fast movements. Deviation from monophasic behavior was probably also not due to metabolically induced disappearance of InsP3, since the biphasic pattern was observed at all temperatures (Figs. 3 and 6B), whereas the enzymes which metabolize InsP3 are markedly inhibited at low temperature. In addition, the time required for InsP3 breakdown in permeabilized liver cells, where the ratio of cell volume to medium volume is about 1%,amounts to several minutes (Joseph et al., 1984; Leslie et al., 1988). Last, the possibility that a productof InsP3metabolization or a contaminant, and not InsP3 itself, was responsible for the slow phase can also probably be excluded because of the relative predominance of this phase at low InsP3 concentrations. Another possible reason for nonmonophasic Ca2+release curves might be geometrical heterogeneity among membranous stores carrying a single type of Ca2+channel, e.g. the coexistence of vesicles of different sizes, or of similar sizes but with either a high or a low surface density of channels (Bernhardtand Neumann, 1982). Inthis case, the small vesicles would empty first, because of a favorable surface to volume ratio. Nonexponential curves might also be generated because of the presence of Ca2+-bufferingsubstances such as calsequestrin-like proteins (Feher and Briggs, 1982) inside the Caz+ storage compartments of liver (Volpe et al., 1988; Damiani et al., 1988; Hashimoto et al., 1988). Such behavior might also occur if the permeability through the opened Ca2+ channels were modulated by the rise in concentration of the cytoplasmic Ca2+or the drop in concentration of the Ca2+ inside the stores. However, in all these cases, it wouldbe difficult to understand why the amplitude of the fast phase would specifically vanish at low InsP3 concentrations (the component associated with slow efflux seems to have a higher apparent affinity for InsP3 than theone associated with fast efflux (see Figs. 4 and 6D). The existence of two different pools, both sensitive to InsP3 and containing different types of receptors, might also be involved to account for the nonmonophasic nature of our Ca2+ release curves. For instance, the tentative hypothesis might be put forward that the slower phase of Ca2+ efflux, which displays high affinity for InsP3, originates from stores colocalized with the plasma membrane (Dunlop and Larkins, 1988; Guillemette et al., 1988),whereas the faster phase,which displays poorer affinity for InsP3, originates from organelles more evenly distributed throughout the cell interior (OSullivan et al., 1989). However, the fact that inboth permeabilized cells and 1000 X g supernatants, theCa2+release curves were comparable in their biphasicity (see Fig. 5 C ) , would then imply that thetwo putative types of Ca2+ storewere released by the saponin treatment to similar extents; this would be a surprising coincidence. Last, an attractive hypothesis is that thetwo phases of Ca2+ release reflect the existence of two different states of a single receptor to InsP3. Thus, this nonmonophasic behavior could be the consequence of spontaneous inactivation of the InsPa receptor, shortly after ligand binding and activation, and its conversion into a species with poor permeability and high affinity for the agonist, as has been documented for the
acetylcholine receptor and other channels (see Heidmann et al., 1983; Changeux et al., 1984; Hille, 1984). Consistent with this view, studies of InsP3 binding in liver (Spat et al., 1986; Guillemette et al., 1988; Mauger et al., 1989) have reported the presence of one or several binding sites displaying much higher affinities (1-200 nM) than thepoor (micromolar) affinity found in the present work. However, as opposed to the acetylcholine receptor for which total desensitization is observed, the receptor to InsP3 would only experience partial desensitization. The maintenance of a low permeability through the partially inactivated associated channel would then account for the slow phase of Ca2+release we observed. Furthermore, this proposal may also explain that no pharmacological desensitization of the InsP3 receptor over minutes or hours has been observed yet (Prentki et al., 1985; Leslie et al., 1988; Vilven and Coronado, 1988). Such a partial desensitization would also provide a mechanistic explanation of the “quantal” propertiesof hormone-evoked Ca2+release recently described by Muallem et al. (1989). In their system, these authors excluded the assumption that the inability of submaximal InsP3 concentrations to release all the InsP3-mobilizable Ca2+could be the result of pump-leak turnover across the pool membrane. Cellular Localization of InsP3-sensitive Pools-Our results have also shown that a fraction of the InsP3-sensitive Ca2+ store was released from the cells intothe medium upon saponin treatment. The experimental conditions used here (2-4 x lo6 cells/ml were incubated for 5 min with 50 pgof saponin/ml) result in minimal alteration of the rat hepatocytes, as established previously (Burgess et al., 1983; Fiskum, 1985; Wassler et al., 1987; Combettes et al., 1988). Electron microscopy revealed the expected disruption of the plasma membrane continuity and thedepletion of cytoplasmic background staining material, and the plasma membrane was freely permeable to trypan blue, lactate dehydrogenase, and other soluble proteins. Although this was not the major aim of this work, this detergent-induced release of a closed vesicular system sensitive toInsP3has been characterized in preliminary experiments. First, a significant although variable fraction of the InsP3-sensitive pool was always found in a 1000 X g supernatant of the permeabilized cells. Second, the InsP3-sensitiveCa2+ releasing stores were relatively heavy structures, since centrifugation at 9000 X gfor 5-20 min progressively spinned them down (not shown). Finally, the kinetic properties of the InsP3-sensitive vesicular system, as analyzed in our stopped-flow experiments, were similar to those of the original permeabilized cells in terms of half-time, apparent absence of delay, InsP3 dependence, and nonmonophasic behavior (Fig. 5B). These data are consistent with the view that saponin has released into the medium a fraction of a pool of preexisting organelles, such as theCa2+-sequesteringcalciosomes recently described in liver and other cells by using antibodies raised against calsequestrin and Ca2+-M$+-ATPase (Volpe et al., 1988; Hashimoto et al., 1988). In fractionation studies, these organelles dissociate from the usual enzyme markers of the other intracellular organelles including the endoplasmic reticulum. Although they distribute evenly throughout the cytoplasm, some appear to be adjacent to the plasma membrane (Volpe et al., 1988). Furthermore, it has been observed that Inspa binds to a significant extent to isolated fractions enriched in plasma membranes (Guillemette et al., 1987, 19881, and release Ca2+from a store colocalized with the cell plasma membrane (Dunlop and Larkins, 1988), indicating the existence of perimembrane structures sensitive to InsP3, as originally hypothesized by Putney (1986). In that case, the obser-
Fast Kineticsof Imp3-induced ea2+Release vation of a loss of the Ca2+ storesupon addition of saponin may suggest that a fraction of the subplasmalemmal Ca2+ pumping organelles could be displaced because of disruption of the cell membrane continuity by the detergent. However, another hypothesis cannot be rejected; the vesicular Ca2+store found i.n the cell supernatant could well be an anomaly, artifactually generated by the saponin treatment itself (“saposomes?”). The detergent could promote a vesiculation and a loss of selected parts of the intracellular membranes. Work is in progress in our laboratory to elucidate the mechanism underlying saponin actionin cells. In thiscontext, it should be mentioned that saponin-induced partial extraction of the InsP3-sensitive Ca2+pool was not observed when other cells, neuroblastoma cells, were used rather than hepatocytes. Irrespective o:f its significance, the observation that supernatants of saponin-treated hepatocytes contain a significant fraction of the InsP3-sensitive Caz+-storing compartment might at least provide a starting point for future attempts tocharacterize furtherthe InsPs receptor in thesecells and reconstitute it in lblack lipid membranes for electrophysiology studies. Acknowledgments-We wish to thank J. P. Mauger, Th. Capiod, D. Ogden, Th. Heidmann, F. Guillain, H. Goudeau, and M. Garrigos for fruitful discussions, and P. Belle and M. Dreyfus for their help in typing and editing the manuscript. REFERENCES Bernhardt, J., and Neumann, E.(1982) Biophys. Chem. 15,327-341 Berridge, M. J. (1984) Biochem. J. 2 2 0 , 345-360 Burgess, G. M., McKinney, J. S., Fabiato, A., Leslie, B.A., and Putney, J. W., Jr. (1983) J. Biol. Chem. 2 5 8 , 15336-15345 Burgess, G.M., Godfrey, P. P.,McKinney, J. S., Berridge, M. J., Irvine, R. F., and Putney, J. W. (1984) Nature 309, 63-66 Capiod, T., Ogden, D. C., Trentham, D. R., and Walker, J. W. (1988) J. Physiol. (Lond.) 406, 124P Caswell, A. H., and Brandt, N. R. (1989) Trends Biochem. Sci. 14, 161-165 Champeil, P., Gingold, MY. P., Guillain, F., and Inesi, G. (1983) J. Biol. Chem. 258,4453-4458 Champeil, P., Guillain, F., VBnien,C., and Gingold, M. P. (1985) Biochemistry 24,69-81 Changeux, J. P., Devillers-ThiBry, A., and Chemouilli, P. (1984) Science 225, 1335-1345 Combettes, L., Dumont, Mr.,Berthon, B., Erlinger, S., and Claret, M. (1988) J. Biol. Chem. 2 6 3 , 2299-2303 Combettes, L., Berthon, E., Doucet, E., Erlinger, S., and Claret, M. (1989) J. Biol. Chem. 2 6 4 , 157-167 Damiani, E., Spamer, C., Heilmann, C., Salvatori, S., and Margreth, A. (1988) J. Biol. Chem. 263, 340-343 Downes, C . P., and Michell, R. H. (1985) in Molecular Mechanisms of Transmembrane Signtrlling (Cohen, P., and Houslay, M. D., eds) pp. 3-56, Elsevier Science Publishers B. V., Amsterdam Dunlop, M. E., and Larkins, R. G . (1988) Biochem. J. 247,407-415 Ehrlich, B. E., and Watras, J . (1988) Nature 3 3 6 , 583-586 Feher, J. J., and Briggs, F. N. (1982) J. Biol. Chem. 2 5 7 , 1019110199 Fiskum, G . (1985) Cell Calcium 6,25-37 Guillain, F., Gingold, M. P., Biischlen, S., and Champeil, P. (1980) J . Biol. Chem. 2 5 5 , 2072-2076 Guillemette, G., Balla, T., Baukal, A. J., Spat, A., and Catt, K. J.
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