©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Threshold for Inositol 1,4,5-Trisphosphate Action (*)

(Received for publication, January 12, 1996; and in revised form, March 15, 1996)

Ludwig Missiaen (§) Humbert De Smedt Jan B. Parys (¶) Ilse Sienaert (**) Sara Vanlingen Rik Casteels

From the Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We developed a unidirectional Ca efflux technique in which 60 cumulative doses of inositol 1,4,5-trisphosphate (InsP(3)), each lasting 6 s, were subsequently added to permeabilized A7r5 cells. This technique allowed an accurate determination of the threshold for InsP(3) action, which was around 32 nM InsP(3) under control conditions. The InsP(3)-induced Ca release was characterized by an initial rapid phase, after which the normalized rate progressively decreased. The slowing of the release was associated with a shift of the threshold to higher InsP(3) concentrations. Stimulatory concentrations of thimerosal (10 µM) shifted the threshold to 4.5 nM InsP(3) and increased both the cooperativity and the maximal normalized rate of Ca release. This low threshold was maintained when the thimerosal concentration was increased to inhibitory levels (100 µM) but then the effects on the cooperativity and on the normalized rate of Ca release disappeared. Oxidized glutathione (5 mM) was much less effective in stimulating the release and did not have an effect on the threshold or on the cooperativity. ATP (5 mM) stimulated the release despite a shift in threshold toward higher InsP(3) concentrations. Luminal Ca did not affect the threshold for InsP(3) action but stimulated the normalized release at each InsP(3) concentration. The inhibitory effect of 10 µM free cytosolic Ca was associated with a shift in threshold to higher InsP(3) concentrations and a decreased cooperativity of the release process. We conclude that this novel technique of accurately measuring the threshold for InsP(3) action under various experimental conditions has allowed us to refine the analysis of the kinetic parameters involved in the regulation of the InsP(3) receptor.


INTRODUCTION

Many hormones, neurotransmitters, and growth factors induce the hydrolysis of phosphatidylinositol 4,5-bisphosphate and thereby produce inositol 1,4,5-trisphosphate (InsP(3)) (^1)as an intracellular messenger(1) . Once threshold concentrations of InsP(3) are reached and conditions for the regenerative release of Ca are created(2) , InsP(3) mobilizes Ca from the nonmitochondrial stores through interaction with the InsP(3)R. The precise kinetics of the [InsP(3)] rise in an intact cell are unknown, but it is possible that there is a gradual increase in the [InsP(3)] before the first Ca response(3, 4) .

It is not clear how the Ca stores in cells respond to such progressively increasing [InsP(3)]. On the one hand, there is the technical difficulty in accurately measuring the Ca release induced by very low doses of InsP(3) above the background Ca leak. On the other hand, it is not clear whether classical dose-response relationships, obtained by acutely introducing one concentration of InsP(3), can predict the response when the [InsP(3)] is slowly increasing, especially if inactivation of the InsP(3)R (5) would occur. We have now developed a Ca efflux technique in which cumulative doses of InsP(3) are subsequently added. This technique, which mimics the gradual accumulation of InsP(3) at the onset of a Ca response, allows an accurate measurement of the threshold [InsP(3)] for InsP(3)-induced Ca release. With this technique, we have investigated how the InsP(3) thresholds are affected by thimerosal, GSSG, ATP, luminal Ca, and cytosolic Ca and how a repeated application of InsP(3) acts on the threshold value. This novel technique of measuring thresholds for InsP(3) action allowed a refined analysis of the kinetic parameters controlling the mechanisms of action of several regulators of the InsP(3)R.


MATERIALS AND METHODS

A7r5 cells, an established cell line derived from embryonic rat aorta, were used between the 7th and the 17th passage after receipt from the American Type Culture Collection (Bethesda, MD) and subcultured weekly by trypsinization. The cells were cultured at 37 °C in a 9% CO(2) incubator in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 3.8 mML-glutamine, 0.9% (v/v) nonessential amino acids, 85 IU ml penicillin, and 85 µg ml streptomycin. The cells were seeded in 12-well dishes (4 cm^2, Costar, MA) at a density of approximately 10^4 cells cm.

Ca fluxes on permeabilized cells were done on a thermostated plate at 25 °C. The culture medium was aspirated and replaced by 1 ml of permeabilization medium containing 120 mM KCl, 30 mM imidazole HCl (pH 6.8), 2 mM MgCl(2), 1 mM ATP, 1 mM EGTA, and 20 µg ml saponin. The saponin-containing solution was removed after 10 min, and the cells were washed once with a similar saponin-free solution. Ca uptake into the nonmitochondrial Ca stores was accomplished by incubation for 60 min in 2 ml of loading medium containing 120 mM KCl, 30 mM imidazole HCl (pH 6.8), 5 mM MgCl(2), 5 mM ATP, 0.44 mM EGTA, 10 mM NaN(3), and 100 nM free Ca. After this phase of Ca accumulation, the monolayers were incubated in 1 ml of efflux medium containing 120 mM KCl, 30 mM imidazole HCl (pH 6.8), 2 mM MgCl(2), 1 mM ATP, 1 mM EGTA, 100 nM free Ca, and 2 µM thapsigargin. The inclusion of thapsigargin was necessary to inhibit any further Ca-ATPase activity during the efflux. The main difference with our previous work(6, 7, 8, 9, 10, 11, 12, 13) is that the technique was modified to allow a sampling resolution of 6 s during the efflux. This was performed by automatically transferring the efflux medium from the cells into scintillation vials using a high speed P-50 Pump (Pharmacia Biotech, Inc.) connected to a Pharmacia LKB 158 SuperFrac fraction collector. The pumping rate was 1 ml/6 s. The advantage of this modification is that the rate of Ca release can be measured, and not only the extent of Ca release as in our previous work. The first 5 min of efflux were not monitored. InsP(3) was included in the efflux solutions during the time periods and at the concentrations indicated in the figures. At the end of the experiment the Ca remaining in the stores was released by incubation with 1 ml of a 2% sodium dodecyl sulfate solution for 30 min.


RESULTS AND DISCUSSION

Kinetics of InsP(3)-induced Ca Release

Permeabilized A7r5 cells slowly lost their accumulated Ca during incubation in an efflux medium containing 2 µM thapsigargin. The decrease in store Ca content was accelerated in the presence of 0.5 µM InsP(3) (Fig. 1A). The kinetics of the InsP(3)-induced Ca release are better resolved when the data are replotted as fractional loss or normalized Ca release as a function of time (Fig. 1B). The fractional loss or normalized Ca release is defined here as the amount of Ca leaving the stores in 6 s (i.e. the rate of Ca release) divided by the total store Ca content at that time. It is therefore independent of the specific activity of the label. Plotting the data in this way is particularly useful in conditions where the Ca content of the stores decreases, as in our experimental conditions with blocked Ca pumps. The fractional loss or normalized Ca release remained in control conditions with blocked Ca pumps fairly constant as a function of time. The release induced by InsP(3) was characterized by an initial rapid release, after which the fractional loss progressively decreased as the time of incubation in InsP(3) was prolonged. The normalized rate of Ca release immediately returned to basal level when InsP(3) was removed (Fig. 1B). Readdition of 0.5 µM InsP(3) under unidirectional Ca efflux conditions with inhibited Ca pumps again increased the fractional loss, although the maximal normalized rate of release during the second stimulation never exceeded that occurring at the end of the first InsP(3) challenge (Fig. 1B). A similar finding was observed when 0.5 µM InsP(3) was shortly removed at the maximum of the InsP(3) response (inset). If intrinsic inactivation of the InsP(3)R (5) would have caused the slowing of the release, a more pronounced release would have been expected upon restimulation with InsP(3). These findings confirm earlier work (6, 12, 14) that intrinsic desensitization of the InsP(3)R (5) can be excluded as the cause of the decreasing rate of Ca release. The normalized rates of spontaneous release before the first and after the second InsP(3) stimulation were almost identical, despite the 4-fold difference in store Ca content. This finding validates our approach to monitor the release kinetics using the fractional loss or normalized rate of Ca release as a parameter that is independent of the store Ca content.


Figure 1: Kinetics of the InsP(3)-induced Ca release from permeabilized A7r5 cells. The nonmitochondrial stores were loaded for 60 min at 100 nM free Ca and then incubated in an efflux medium containing 2 µM thapsigargin. The first 5 min of efflux were not monitored. A shows, on a logarithmic scale, the decrease of the Ca content of the stores during further incubation in efflux medium and its modification by 2 additions of 0.5 µM InsP(3), added as indicated by the bars above the trace. B shows, on a linear scale, the fractional loss (the amount of Ca leaving the stores in 6 s, divided by the total store Ca content at that time) as a function of time. The inset shows the effect of shortly removing 0.5 µM InsP(3) at the maximum of the InsP(3) response. Typical for four experiments.



The release was rather slow, since it was still significant at the end of the first stimulation period, i.e. after 3.5 min (Fig. 1B). This observation contrasts with the much faster kinetics observed in some other experimental systems (e.g. rat brain synaptosomes(15) ). These differences are unlikely to be due to a slow diffusion of Ca out of our permeabilized cell preparation, since the release dropped to control values only upon removing the InsP(3) and again increased upon restimulation (Fig. 1B). The long time period that is needed to empty the stores in A7r5 cells might be related to the larger amount of Ca ions that have to pass through the channels: more than 95% of the accumulated Ca is releasable by a maximal [InsP(3)] in A7r5 cells(7) , while the figure for rat brain synaptosomes is only 6%(15) .

The effect of the [InsP(3)] on the kinetics of the release is shown in Fig. 2. Increasing the [InsP(3)] from 0.2 to 1 µM increased the extent of Ca release, as deduced from the lower Ca content at the end of the efflux (Fig. 2A), confirming the ``quantal'' nature of the release in A7r5 cells(6) . Fig. 2B illustrates that the normalized maximal rate of Ca release was higher at 1 than at 0.2 µM InsP(3). The normalized rates of release at both concentrations of InsP(3) then gradually decreased and eventually became identical, i.e. they became independent of the [InsP(3)]. At these later time points, the efflux rates were still significantly higher than the basal efflux rate in the absence of InsP(3). These data obtained with the technique of Ca fluxes are in agreement with similar findings made with protocols in which the release was monitored with fluorescent Ca indicators in the cytosol (16, 17, 18) or in the store(19) . The arrows in Fig. 2B indicate the time at which the fractional loss has decreased to half of its maximal value. This phenomenon occurred earlier at 1 µM than at 0.2 µM InsP(3). This progressively decreasing normalized rate of Ca release also occurred in the presence of 0.1 µM InsP(3), although it took a much longer time for the release to reach half of its maximal rate (data not shown). A progressive decrease in the normalized rate of Ca release therefore occurred at all InsP(3) concentrations. The rate constant of this decline was, however, dependent on the [InsP(3)]. In contrast, Sugiyama and Goldman (19) concluded from very similar experiments that low and high concentrations of InsP(3) induced different patterns of Ca release, 0.1 µM InsP(3) only induced a monoexponential decline of the store Ca content, while higher concentrations induced a biphasic release. In contrast, we could only observe one pattern of Ca release with a slowly decreasing rate constant.


Figure 2: Effect of the [InsP(3)] on the kinetics of the InsP(3)-induced Ca release. A shows, on a logarithmic scale, the decrease of the Ca content of the stores during incubation in efflux medium and its modification by 0.2 (bullet) and 1 µM (circle) InsP(3), added as indicated by the bar above the traces. B shows, on a linear scale, the fractional loss for the two experimental conditions as a function of time. The arrows indicate the time at which the fractional loss has decreased to half of its maximal value. Typical for four experiments.



There is evidence that the InsP(3)R might be stimulated by Ca inside the store(6, 7, 8, 11, 13, 19, 20, 21, 22, 23, 24, 25) . Part of these effects could be exerted at the cytosolic side of the receptor(26, 27) , but other authors (8, 11, 24) have brought forward arguments against the latter hypothesis. Since the release process is associated with a decline in the luminal [Ca], we have compared the kinetics of the release induced by 1 µM InsP(3) in stores that were allowed to deplete by blocking their pumps with 2 µM thapsigargin either for 6 min (Fig. 3, A and B, closed circles) or for 21 min (Fig. 3, A and B, open circles) before the InsP(3) challenge. The maximal normalized rate of Ca release as well as the fractional loss at the later time points appreciably decreased when the InsP(3) challenge was given to the 4 times less filled stores (Fig. 3B). The effect of luminal Ca was also apparent when the increase in fractional loss was normalized to that in the presence of 10 µM A23187 given at the same time in parallel experiments (data not shown). These findings therefore confirm that the decreasing rate of Ca release in the continuous presence of InsP(3) (Fig. 1) is, at least partly, explained by the decreasing Ca content of the store. This interpretation of the data can explain why the normalized release rate decreased faster at high InsP(3) concentrations (Fig. 2B), because high concentrations induce a more rapid decrease in the store Ca content.


Figure 3: Effect of the level of store loading on the kinetics of InsP(3)-induced Ca release. The nonmitochondrial stores were loaded for 60 min at 100 nM free Ca and then incubated in efflux medium containing 2 µM thapsigargin. The efflux was allowed to proceed either for 6 min (more filled stores, bullet) or for 21 min (less filled stores, circle) before the InsP(3) challenge. A shows, on a logarithmic scale, the decline of the Ca content of the stores during the incubation in the efflux medium and its modification by the addition of 1 µM InsP(3) as indicated by the bar above the trace. B shows, on a linear scale, the corresponding fractional loss as a function of time. Typical for four experiments.



Threshold for InsP(3)-induced Ca Release

Fig. 4A illustrates the change of the Ca content of the stores by gradually increasing the [InsP(3)] from 10 nM to 10 µM over a period of 6 min, i.e. in 60 steps each lasting 6 s. The inset shows the extent of Ca release, i.e. the difference between the Ca content in the absence and presence of InsP(3), as a function of the cumulative [InsP(3)]. It was not possible to accurately fit the entire curve with the Hill equation. This might be due to the presence of both InsP(3)R-I and -III in A7r5 cells(11, 28) , by the presence of regulatory factors affecting the dose-response relationship (e.g. the decrease in luminal [Ca]), or by the fact that the rate of Ca release decreased at a rate constant that depended on the [InsP(3)] (Fig. 2B). Schrenzel et al.(29) therefore only used the data points in the lower range of InsP(3) concentrations to assess the cooperativity of the release process. We could obtain a perfect fit with a Hill coefficient of 2.0 if only the data obtained between 10 and 320 nM InsP(3) were used for the curve fitting. The fractional loss as a function of time is shown in Fig. 4B. This way of plotting the data allowed an accurate determination of the threshold for InsP(3) action. The time point, from where the fractional loss started to increase above base line, was determined and the corresponding [InsP(3)] at that time was then taken as threshold. This threshold in standard efflux medium was always around 32 nM InsP(3).


Figure 4: Effect of gradually increasing the [InsP(3)] on the Ca release. A shows, on a logarithmic scale, the decrease of the store Ca content. B shows, on a linear scale, the corresponding fractional loss during incubation in efflux medium and its modification by a gradual increase of the [InsP(3)] from 10 nM to 10 µM in 60 individual steps each lasting 6 s. The [InsP(3)] was increased in a logarithmic way, as shown by the full line in B. The inset in A shows, on a linear scale, the extent of Ca release, i.e. the difference between the Ca content in the absence and presence of InsP(3), as a function of the cumulative [InsP(3)]. The release at 10 µM InsP(3) was taken as 100%. Typical for four experiments.



Effect of Sulfhydryl Reagents

Thimerosal stimulates the InsP(3)R(10, 20, 21, 30, 31, 32, 33, 34, 35) . The stimulatory effect of 10 µM thimerosal was further characterized in Fig. 5, A and B. This concentration of thimerosal shifted the threshold for Ca release from 32 to 4.5 nM InsP(3). Also the maximal normalized rate of Ca release increased. The dose-response relationship for InsP(3)-induced Ca release became much steeper in the presence of 10 µM thimerosal (Fig. 5B). Thimerosal increased the calculated Hill coefficient from 2.0 to 4.5, at least if only the data points in the lower range of InsP(3) concentrations were fitted(29) .


Figure 5: Effect of 10 µM thimerosal on the InsP(3)-induced Ca release. A shows, on a logarithmic scale, the decline of the Ca content of the stores during incubation in efflux medium containing 10 µM thimerosal in 0.1% Me(2)SO (bullet) or containing 0.1% Me(2)SO only (circle) and its modification by a gradual logarithmic increase of the [InsP(3)] from 3.2 nM to 3.2 µM in 60 individual steps each lasting 6 s. B shows, on a linear scale, the fractional loss for the above-mentioned experimental conditions as a function of time. Typical for four experiments.



Higher concentrations of thimerosal become inhibitory(10, 32, 34) . In agreement with these findings, we observed that 316 µM thimerosal completely prevented any effect of InsP(3) (data not shown). The nature of this inhibition was further characterized using a lower, but still inhibitory, thimerosal concentration (100 µM, Fig. 6, A and B). This concentration of thimerosal still allowed InsP(3) to act at a lower threshold. However, the increased cooperativity was lost and the maximal normalized rate of Ca release became smaller (Fig. 6B). The impaired InsP(3)-induced Ca release in the presence of high concentrations of thimerosal was not the consequence of an increased passive Ca leak and, therefore, the lower store Ca content at the time of InsP(3) addition(10) .


Figure 6: Effect of 100 µM thimerosal on the InsP(3)-induced Ca release. A shows, on a logarithmic scale, the decline of the Ca content of the stores during incubation in efflux medium containing 100 µM thimerosal in 0.1% Me(2)SO (bullet) or containing 0.1% Me(2)SO only (circle) and its modification by a gradual logarithmic increase of the [InsP(3)] from 3.2 nM to 3.2 µM in 60 individual steps each lasting 6 s. B shows, on a linear scale, the fractional loss as a function of time. Typical for three experiments.



From these findings, we propose that thimerosal might act on at least two different sites on the InsP(3)R or, alternatively, on some proteins associated with the InsP(3)R. Interaction with one site results in the increased sensitivity for InsP(3), the increase in cooperativity and of the maximal normalized rate of Ca release. This interaction may also explain the decreased K(d) for InsP(3) binding(32, 34, 35, 36) . The other interaction site for thimerosal could be responsible for the inhibition by high thimerosal concentrations.

Oxidized glutathione (GSSG) can also stimulate the InsP(3)R, but this stimulation has so far only been reported in hepatocytes(20, 21, 35, 36, 37) . The effect of 5 mM GSSG in A7r5 cells was further characterized. A very modest stimulation of the release could be observed, but its overall effect was much smaller than that of 10 µM thimerosal (data not shown). GSSG had no effect on the threshold for InsP(3)-induced Ca release nor on the cooperativity. In contrast, the normalized rate of Ca release at each [InsP(3)] increased by about 20% (data not shown).

These functional data indicate that the two sulfhydryl reagents that we have tested (thimerosal and GSSG) exerted different effects on the InsP(3)R. Our findings are in agreement with InsP(3)-binding studies in permeabilized rat hepatocytes indicating that GSSG only increased the number of binding sites for InsP(3) without changing the K(d), whereas thimerosal both increased the number of binding sites and decreased the K(d)(36) . The effect of GSSG on Ca release in A7r5 cells was much smaller than reported in rat hepatocytes (20, 21, 36, 37) . This finding reinforces previous conclusions that InsP(3)Rs of different sources can be differently affected by sulfhydryl reagents(33) .

Effect of ATP

Fig. 7A compares the effect of progressively increasing the [InsP(3)] in the absence and presence of 5 mM ATP in a medium without Mg. ATP shifted the threshold for InsP(3) action to higher concentrations of InsP(3) (Fig. 7B). This phenomenon is explained by the known inhibitory effect of ATP on the InsP(3)-binding site(38) . Higher concentrations of InsP(3) overcame this inhibitory effect, at which stage the release became more pronounced. This stimulatory effect probably occurred via interaction with a regulatory ATP-binding site and not by interaction with the InsP(3)-binding site (39, 40, 41) . The overall effect was an increased Ca release at the higher InsP(3) concentrations in the presence of 5 mM ATP, as indicated by the lower Ca content of the stores at the end of the efflux (Fig. 7A).


Figure 7: Effect of 5 mM ATP on the InsP(3)-induced Ca release. A shows, on a logarithmic scale, the decline of the Ca content of the stores during incubation in efflux medium containing 5 mM ATP (bullet) or in control medium without ATP (circle) and its modification by a gradual logarithmic increase of the [InsP(3)] from 3.2 nM to 3.2 µM in 60 individual steps each lasting 6 s. The efflux medium did not contain MgCl(2). B shows, on a linear scale, the fractional loss as a function of time. Typical for four experiments.



Effect of Luminal Ca

Fig. 8A illustrates the effect of a gradual increase of the [InsP(3)] after 6 min of efflux and after 21 min of efflux, respectively. After 21 min of efflux, the stores contained 2 times less Ca than after 6 min in this particular experiment. Fig. 8B compares the normalized rates of Ca release in these two conditions. The level of store loading did not exert an effect on the threshold for InsP(3)-induced Ca release but potentiated the release at each [InsP(3)]. It should be stressed that the present technique did not allow us to study stores at a larger degree of depletion, in which the effects of the decreased luminal [Ca] are more pronounced(8, 11) , since not enough radioactivity is then left to sample every 6 s.


Figure 8: Effect of the level of store loading on the InsP(3)-induced Ca release. The nonmitochondrial stores were loaded for 60 min at 100 nM free Ca and then incubated in efflux medium containing 2 µM thapsigargin. The efflux was allowed to proceed for 6 min (bullet) or 21 min (circle) before the InsP(3) challenge. A shows, on a logarithmic scale, the change of the Ca content of the stores elicited by a gradual logarithmic increase of the [InsP(3)] from 3.2 nM to 3.2 µM in 60 individual steps each lasting 6 s. B shows, on a linear scale, the fractional loss as a function of time. Typical for four experiments.



Effect of Inhibitory Concentrations of Cytosolic Ca

High Ca concentrations inhibit the InsP(3)-induced Ca release(8, 13, 15, 42, 43, 44) . In hepatocytes, high Ca may reduce the size of the InsP(3)-sensitive store over the whole range of InsP(3) concentrations, including the very high ones(45, 46) . In A7r5 cells, this inhibition only occurred at the lower InsP(3) concentrations and vanished at higher InsP(3) concentrations(13) . The inhibition in the presence of 10 µM free Ca was further characterized in Fig. 9A. The more rapid decrease in the Ca content of the stores at 10 µM Ca in the absence of InsP(3) was due to Ca-Ca exchange and therefore did not represent an increased basal leak(7) . As can be observed in Fig. 9B, Ca shifted the threshold for Ca release to higher InsP(3) concentrations. It should be noted that the [InsP(3)] was increased up to 32 µM in the presence of 10 µM Ca, while the maximal [InsP(3)] was 3.2 µM in the control trace at 100 nM free Ca. The dose-response relationship for InsP(3)-induced Ca release was less steep in the presence of 10 µM Ca: the Hill coefficient decreased from 2.0 to 1.0 if, according to Schrenzel et al.(29) , only the data points in the lower range of InsP(3) concentrations were fitted.


Figure 9: Effect of 10 µM free Ca on the InsP(3)-induced Ca release. A shows, on a logarithmic scale, the Ca content of the stores during incubation in efflux medium buffered at 10 µM (bullet) or 100 nM (circle) free Ca. In the experiment at 10 µM Ca, the [InsP(3)] was gradually increased from 3.2 nM to 32 µM in 80 individual steps each lasting 6 s and in that at 100 nM Ca from 3.2 nM to 3.2 µM in 60 individual steps each lasting 6 s. B shows, on a linear scale, the fractional loss as a function of time. Typical for four experiments.



Lower concentrations of cytosolic Ca stimulate the InsP(3)-induced Ca release(8, 15, 42, 43, 44) . However, these effects were small in permeabilized A7r5 cells with relatively filled stores(8) . An analysis with the present Ca efflux technique in strongly depleted stores was technically impossible, since not enough radioactivity is then left to sample every 6 s.

Threshold of a Second InsP(3) Challenge

The normalized rate of InsP(3)-induced Ca release progressively decreased (Fig. 1B). It has been suggested that this phenomenon represents the transition of the InsP(3)R into a high affinity low permeability state(16, 17, 19) . This conclusion was reached by fitting the curve of the Ca release by two exponentials and subsequently calculating the rate constants and the amplitudes of the proposed two components of the Ca release. We have now directly measured the thresholds for Ca release during a first and a second InsP(3) stimulation under conditions with inhibited Ca pumps (Fig. 10). If the decreasing rate of Ca release would represent the transition from a low affinity to a high affinity state of the InsP(3)R, we would expect that the second InsP(3) challenge should require less InsP(3) to initiate Ca release. Fig. 10B failed to confirm this prediction, since the threshold for the second addition of InsP(3) was shifted toward higher InsP(3) concentrations. These results in A7r5 cells do not provide experimental evidence for the suggestion that the slowing of the release was caused by the formation of a high affinity conformation of the InsP(3)R. Hirose and Iino (14) also preincubated stores with InsP(3) and measured the InsP(3) sensitivity of the remaining stores. They did not find a change in EC for InsP(3)-induced Ca release. This discrepancy between their and our results might be explained by the dose of InsP(3) used for the first InsP(3) challenge. This [InsP(3)] was near threshold (30 nM) in the work of Hirose and Iino(14) , whereas the maximal concentration in Fig. 10(500 nM) was well above the EC for Ca release.


Figure 10: InsP(3)-induced Ca release during two consecutive stimulations. A shows, on a logarithmic scale, the change of the Ca content of the stores during incubation in efflux medium and its modification by two consecutive stimulation periods in which the [InsP(3)] was gradually increased on a logarithmic scale from 3.2 nM to 500 nM in 40 individual steps each lasting 6 s and then kept at 500 nM for another 2 min. The two periods of the InsP(3) challenge were interrupted by a 2.5-min incubation in efflux medium without InsP(3). B shows, on a linear scale, the superimposed curves of the fractional loss as a function of time for the first (bullet) and the second (circle) stimulation. The pattern of the InsP(3) addition was the same for both traces. Typical for five experiments.



Concluding Remarks on the Kinetics of InsP(3)-induced Ca Release

The response to a long-lasting stimulation with InsP(3) is characterized by an initial fast release, which then progressively slows down. We have observed that the maximal normalized rate of Ca release as well as the fractional loss at the later time points appreciably decreased when the InsP(3) challenge was given to stores with a lower Ca content (Fig. 3B). These findings support our proposal that the decreasing rate of Ca release in the continuous presence of InsP(3) is, at least partly, explained by the decreasing Ca content of the store. Although the store Ca content per se seems to control in a direct or indirect way the release properties of the stores and possibly the conformational state of the InsP(3)R, it may not be the only parameter that contributes to the quantal Ca release behavior. The fractional loss at each InsP(3) concentration decreased both after depleting the stores by preincubating them in efflux medium (Fig. 8B) and in efflux medium containing InsP(3) (Fig. 10B). However, there was one important difference between both procedures. The threshold for Ca release was not affected by preincubation in efflux medium, whereas it was shifted to higher InsP(3) concentrations following preincubation in InsP(3)-containing efflux medium. This finding indicates that the application of InsP(3) in unidirectional Ca efflux conditions with blocked Ca pumps is inducing effects different from those caused by a reduction of the store Ca content. Intrinsic desensitization of the InsP(3)R did not cause the decreasing rate of Ca release (Fig. 1B). Ca-dependent inactivation of the channel due to the proposed local build-up of Ca near the channel pore is still controversial(14, 26, 47) . We previously presented evidence that heterogeneity of the stores with respect to the expression of InsP(3)Rs also contributed to the quantal Ca release in A7r5 cells(11) . Such heterogeneity allows the more sensitive stores to be depleted faster than the less sensitive stores in the presence of low InsP(3) concentrations. This view can explain why the threshold for the second InsP(3) stimulation was shifted to higher InsP(3) concentrations. Tortorici et al.(48) recently also provided experimental evidence that heterogeneity in the affinity of InsP(3)Rs present in compartmentalized Ca pools contributes to the nonexponential nature of the InsP(3)-induced Ca release. In conclusion, both regulation of the InsP(3)R by luminal Ca and InsP(3)R heterogeneity can explain the complex kinetics of the InsP(3)-induced Ca release in A7r5 cells.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 32-16-345720; Fax: 32-16-345991; Ludwig.Missiaen{at}med.kuleuven.ac.be.

Senior Research Assistant of the Belgian National Foundation for Scientific Research (National Fonds voor Wetenschappelijk Onderzoek).

**
Research Assistant of the Belgian National Foundation for Scientific Research (National Fonds voor Wetenschappelijk Onderzoek).

(^1)
The abbreviations used are: InsP(3), inositol 1,4,5-trisphosphate; InsP(3)R, InsP(3) receptor; GSSG, oxidized glutathione; Me(2)SO, dimethyl sulfoxide.


ACKNOWLEDGEMENTS

We thank Drs. Colin W. Taylor and Karl-Heinz Krause for sending us their manuscripts prior to publication.


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