INTRODUCTION

Monitoring directly the [Ca²⁺] inside the main Ca²⁺ store of the cell, the endoplasmic reticulum, has been difficult to achieve in intact cells. We have reported recently (1) that using HeLa cells expressing ER¹-targeted, low Ca²⁺ affinity aequorin reconstituted with coelenterazine n allows measuring reliably [Ca²⁺]_er in intact cells. In that study, however, the high Ca²⁺ levels reached at steady state in the ER (500-600 µM) led to a fast consumption of aequorin, allowing measurement of [Ca²⁺]_er for only a few minutes. We have now observed that aequorin consumption is 1 order of magnitude slower at 22 °C, and this allows performing long term comparative studies of the dynamics of [Ca²⁺]_er and [Ca²⁺]_c under different conditions, e.g. in the presence of extracellular InsP₃-producing agonists, Ca²⁺-ATPase inhibitors, or during incubation in Ca²⁺-free medium. Our results reveal several unexpected characteristics of the Ca²⁺ release phenomenon, e.g. that the magnitude of the changes of [Ca²⁺]_er after agonist action does not always correlate with the magnitude of the corresponding changes in [Ca²⁺]_c. A sharp agonist-induced cytosolic Ca²⁺ peak can be obtained with only little Ca²⁺ release from the stores, and further release is feedback inhibited by the increase in [Ca²⁺]_c, probably through the generation of microdomains of high [Ca²⁺].

EXPERIMENTAL PROCEDURES

Cell lysates of the EM26 cell clone (2) stably expressing the recombinant photoprotein were reconstituted overnight at 4 °C with 5 µM coelenterazine n, and incubated with solutions containing 110 mM KCl, 10 mM NaCl, 1 mM free Mg²⁺, 40 mM HEPES, pH 7.0, at 22 °C, and known concentrations of Ca²⁺ prepared using buffers with 5 mM HEDTA when necessary. The fractional rate of aequorin consumption (luminescence/total luminescence remaining, L/L_max) at steady state was calculated in every case after consuming all aequorin luminescence with 10 mM Ca²⁺. The procedures for fitting the curve to the experimental data and other details have been described previously (3).

The HeLa cell clone EM26 producing ER-targeted low Ca²⁺ affinity mutated aequorin (2) was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 0.2 mg/ml G418. Cell clones were plated onto 13-mm round coverslips. Before reconstituting aequorin, [Ca²⁺]_er was reduced by incubating the cells for 5 min at 37 °C with the SERCA inhibitor BHQ (10 µM) in KRB (Krebs-Ringer modified buffer: 125 mM NaCl, 5 mM KCl, 1 mM Na₃PO₄, 1 mM MgCl₂, 5.5 mM glucose, 20 mM HEPES, pH 7.4), supplemented with 3 mM EGTA. Cells were then incubated for 1 h at room temperature in KRB containing 0.5 mM EGTA, 10 µM BHQ, and either 0.5 µM coelenterazine n (for measuring [Ca²⁺]_er) or 4 µM fura-2-AM (for measuring [Ca²⁺]_c). The coverslip was then washed for 5 min in KRB containing 0.5 mM EGTA, 5% bovine serum albumin, and 10 µM BHQ, and finally placed (for aequorin measurements) in the perfusion chamber of a purpose-built thermostatized luminometer. [Ca²⁺]_er values were calculated from the luminescence records using a computer algorithm (3) which follows the curve shown in Fig. 1A. [Ca²⁺]_c measurement on cell populations was performed using a Cairn spectrophotometer equipped with a six-filter rotating wheel as described previously (4). Cells used for [Ca²⁺]_c measurements were always depleted of Ca²⁺ in the same way as those used for [Ca²⁺]_er measurements, to allow comparison of both types of data in the same conditions. Coelenterazine n, fura-2-AM, and BAPTA-AM were obtained from Molecular Probes. Other reagents were from Sigma (Madrid, Spain) or Merck (Darmstadt, Germany).

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RESULTS

Fig. 1A shows the calibration curve obtained at 22 °C of low Ca²⁺ affinity mutated aequorin reconstituted with coelenterazine n, a prosthetic group conferring still lower Ca²⁺ affinity (5). The curve obtained at 37 °C, reported previously (1), is also shown for comparison. We can observe that decreasing the temperature reduces the rate of aequorin consumption by nearly 1 order of magnitude at every [Ca²⁺]. This effect allows measuring [Ca²⁺] in the millimolar range for quite a long time (>15 min) without problems of aequorin consumption. Fig. 1B shows the effect of the addition of 1 mM Ca²⁺ to Ca²⁺-depleted HeLa cells. The luminescence record shows a Ca²⁺-triggered luminescence peak that contains most of the aequorin luminescence. Later addition of digitonin produces only a minor release of residual luminescence. The calibrated signal shows that addition of extracellular Ca²⁺ produces an increase in [Ca²⁺]_er which reaches a steady-state level around 600 µM (590 ± 90 µM, mean ± S.D., n = 44) within 8-10 min of Ca²⁺ addition. The rate of increase of [Ca²⁺]_er after extracellular Ca²⁺ addition was 1.8 ± 0.5 µM/s (n = 13), nearly 4 times slower than that observed at 37 °C (7 ± 1 µM/s, n = 15, calculated from the data reported in Ref. 1). However, the final steady-state [Ca²⁺]_er level was the same (compare with 550 ± 70 µM obtained at 37 °C, see Ref. 1). This suggests that the rate of Ca²⁺ leak from the ER may have a similar temperature dependence, because the steady-state [Ca²⁺]_er depends only on the balance between Ca²⁺ pumping and Ca²⁺ leak. In fact, the rate of Ca²⁺ leak from the ER, measured as the rate of [Ca²⁺]_er decrease after inhibition of the SERCA with BHQ or after incubation in Ca²⁺-free medium, increased 2-3-fold within that temperature interval (see below).

In Fig. 2, we compare the effect of adding increasing concentrations of histamine on both [Ca²⁺]_c and [Ca²⁺]_er. In the upper panel, we show the effect of the addition of 2.5 µM, 10 µM and 100 µM histamine on the [Ca²⁺]_c of HeLa cells, measured using fura-2. In every case, the agonist induces a sharp initial [Ca²⁺]_c peak, which is followed by a decreasing plateau lasting for several minutes. In the middle panel, the effect of the same histamine concentrations on [Ca²⁺]_er is shown. The higher concentration, 100 µM histamine, produced a fast [Ca²⁺]_er decrease of about 100 µM, coincident with the sharp cytosolic peak. Then, surprisingly, [Ca²⁺]_er stabilized at that level for several minutes still in the presence of histamine, turning up again when histamine was washed. Therefore, the decreasing plateau of [Ca²⁺]_c coincides with a steady-state [Ca²⁺]_er in which the stores keep at 80-90% of maximum loading. When the histamine concentration was only 10 µM, the initial phase of [Ca²⁺]_er decrease was smaller (around 50 µM), and [Ca²⁺]_er remained afterward at approximately the same levels as before, even though the [Ca²⁺]_c peak was not much different from that obtained with 100 µM histamine. With 2.5 µM histamine, the effect on [Ca²⁺]_er was negligible, and in many experiments it could not even be detected over the background noise. Instead, the [Ca²⁺]_c peak was smaller, but its amplitude was still near half the maximum obtained with 100 µM histamine. The effect of histamine on [Ca²⁺]_er was always potentiated when the increase in [Ca²⁺]_c was blocked by loading the cells with the Ca²⁺-chelator BAPTA. The lower panel shows the effect of the same three histamine concentrations on [Ca²⁺]_er in BAPTA-loaded cells. Histamine induced a much prolonged decrease in [Ca²⁺]_er in the three cases, and the rate was proportional to the histamine concentration.

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The potentiating effect of BAPTA loading could be attributed either to the inhibition of Ca²⁺ pumping or to a direct effect activating InsP₃-gated Ca²⁺ release. In our previous paper (1), we showed that simultaneous addition of 100 µM histamine and the SERCA inhibitor BHQ produced only additive effects, suggesting that the main effect of BAPTA was protecting the InsP₃ receptors from inhibition by the increase in [Ca²⁺]_c. We have made now a more complete study on this point, which provides further evidence in favor of this hypothesis. Fig. 3 shows the effect on [Ca²⁺]_er of the addition of 10 µM BHQ either alone or together with 2.5 µM, 10 µM, or 100 µM histamine, at 22 °C. In control cells (upper panel), there was no difference in the rate of emptying induced by BHQ or BHQ+2.5 µM histamine. After the addition of BHQ + 10 µM histamine, there was an initial drop in [Ca²⁺]_er, but the curve then followed the same slope as with BHQ alone. Finally, when we added BHQ + 100 µM histamine, we obtained a greater initial drop in [Ca²⁺]_er, but the emptying continued afterward at a rate similar to that obtained in the absence of histamine. The effect of histamine was very different in cells loaded with BAPTA. The lower panel of Fig. 3 shows that increasing concentrations of histamine induced Ca²⁺ release at an increasing rate, and following a near monoexponential decay. These experiments show clearly that the effect of BAPTA loading is not due to a decreased Ca²⁺ pumping. Similar data could be obtained at 37 °C, although at this temperature histamine was found to be more potent inducing Ca²⁺ release in control cells at every concentration. Fig. 4 (upper panel) shows that addition of increasing concentrations of histamine induced also here a biphasic decrease in [Ca²⁺]_er: a fast initial decrease, proportional to the histamine concentration, followed by a much slower phase of release. This second phase, however, was still somewhat faster here at 37 °C than that obtained in the absence of histamine. In any case, loading with BAPTA transformed again the curves to a near monoexponential decay at rates proportional to the histamine concentration (Fig. 4, lower panel).

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Careful analysis of the decay curves of [Ca²⁺]_er obtained after simultaneous addition of histamine and BHQ to cells loaded with BAPTA allowed us to study the possible regulation of the InsP₃-gated channels by [Ca²⁺]_er. Theoretically, if the ER would behave as a single compartment emptying at constant rate through the InsP₃ receptors, the decay of [Ca²⁺]_er in the absence of pumping should follow a single exponential. Fig. 5 compares the rate of emptying obtained experimentally after addition of BHQ and either 2.5 µM or 100 µM histamine, at 22 °C, with a monoexponential decay calculated to fit the initial part of the experimental curve. We can see that the experimental data follow the exponential most of the time, but clearly deviate from it when the [Ca²⁺]_er decreases below 150 µM, no matter what is the histamine concentration used. This finding, which has been reported previously using the same technique but with Sr²⁺ as a Ca²⁺ surrogate (6), provides evidence in favor that InsP₃ receptors require a certain level of [Ca²⁺]_er for maximal activity.

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We have studied finally the effect of histamine on [Ca²⁺]_er in the absence of extracellular Ca²⁺, as well as the behavior of [Ca²⁺]_er during incubation of the cells in Ca²⁺-free medium. Fig. 6 (upper panel) shows that when histamine is added to HeLa cells incubated in Ca²⁺-free medium, the initial [Ca²⁺]_c peak is similar to that obtained in Ca²⁺-containing medium, but the plateau phase is largely (though not completely) removed. If the cells are incubated for 10 or 20 min in Ca²⁺-free medium, the height of the peak was only reduced to 73 ± 5% (n = 4; 78% in the figure) and 69 ± 12% (n = 4; 81% in the figure) of the control value, respectively. In all the cases, it is apparent that the return of [Ca²⁺]_c to the base line is biphasic, with a final slow phase lasting for 1-2 min, which cannot be attributed to Ca²⁺ entry and must be due to Ca²⁺ release. The counterpart in the ER is shown in the lower panel. Addition of histamine in EGTA-containing medium produces a decrease in [Ca²⁺]_er, which has three consecutive phases from a kinetic point of view: a first rapid drop of about 60 µM, which must be responsible for the peak of [Ca²⁺]_c; a second phase of slower decrease, lasting for 1-2 min and getting 80-90 µM lower (coincident in time with the last part of the [Ca²⁺]_c peak); and a third phase, in which the rate of decrease in [Ca²⁺]_er approaches that obtained just by incubation in Ca²⁺-free medium. The same kinetics was observed when histamine was added after 10 or 20 min of incubation in Ca²⁺-free medium. It was surprising, however, to find that there was not a strict correlation between the height of the [Ca²⁺]_c peak and the [Ca²⁺]_er at the moment of histamine addition. The [Ca²⁺]_er level decreased to 59 ± 6% (n = 4; 60% in the figure) of the initial value after 10 min (when the [Ca²⁺]_c peak was 73 ± 5% of maximum) and to 29 ± 8% (n = 5; 30% in the figure) after 20 min (when the [Ca²⁺]_c peak was still 69 ± 12% of maximum). In these experiments, the half-time for emptying of the stores in Ca²⁺-free medium was 12 ± 3 min (n = 10).

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Similar effects were observed when the experiments were carried out at 37 °C, but histamine was in this case more potent, inducing Ca²⁺ release. Fig. 7 shows that addition of histamine in EGTA-containing medium produced a sharp [Ca²⁺]_c peak, followed by a short (30-40 s) phase in which the decrease in [Ca²⁺]_c was slower. If cells with full Ca²⁺ stores were incubated in Ca²⁺-free medium for 3 or 5 min, the [Ca²⁺]_c peak suffered only a minor decrease to 92 ± 6% (n = 4; 99% in the figure) and 82 ± 17% (n = 4; 91% in the figure) of the control, respectively. The lower panel shows the effect of histamine on [Ca²⁺]_er. Due to the fast aequorin consumption at 37 °C, histamine had to be added during the rising phase of [Ca²⁺]_er after addition of extracellular Ca²⁺. We can observe that histamine induces a decrease of [Ca²⁺]_er in two phases: a first rapid decrease from 500 µM to about 350 µM, followed by a slower decrease, which continues down to about 100 µM [Ca²⁺]_er. This second phase is still much faster than the rate of decrease in [Ca²⁺] induced only by EGTA (see the traces on the right). The same kinetics were observed if histamine was added after 3 or 5 min of incubation in Ca²⁺-free medium. As in the experiments at 22 °C, the initial drop was responsible for the sharp cytosolic peak and the second slower phase was coincident with the last part of the [Ca²⁺]_c peak. Again, the height of the [Ca²⁺]_c peak did not correlate with the [Ca²⁺]_er level, which decreased to 46 ± 10% (n = 4; 48% in the figure) after 3 min in Ca²⁺-free medium (the [Ca²⁺]_c peak decreased only to 92 ± 6%) and to 35 ± 10% (n = 5; 34% in the figure) after 5 min (the [Ca²⁺]_c peak decreased only to 82 ± 17%). The half-time for emptying of the stores in Ca²⁺-free medium in these experiments was about 3 min (170 ± 50 s, n = 8).

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DISCUSSION

We have measured directly the dynamics of [Ca²⁺] in the endoplasmic reticulum of intact HeLa cells. Using ER-targeted, low Ca²⁺ affinity mutated aequorin reconstituted with coelenterazine n, and because of the decrease in the rate of aequorin consumption at room temperature, we have been able to perform long-lasting experiments comparing the effects both in [Ca²⁺]_er and [Ca²⁺]_c of the addition of extracellular agonists and/or SERCA inhibitors, both in Ca²⁺-containing or Ca²⁺-free medium. The results obtained, some of them quite unexpected, provide a new view on the role of the intracellular Ca²⁺ stores in Ca²⁺ homeostasis.

When using aequorin, the possibility of artifacts due to heterogeneity in [Ca²⁺] or behavior of the pools containing the indicator should be always considered. Native aequorin had a extremely nonlinear sensitivity to Ca²⁺, and the signal could be therefore much affected by the presence of small compartments of high [Ca²⁺]. In the mutated aequorin we have used in this paper, the near abolition of one of the Ca²⁺-binding sites reduced considerably the slope of the calibration curve, which became near linear with [Ca²⁺] (see Fig. 1 and Ref. 2). Using mutated aequorin, therefore, the possibility of having artifacts due to small compartments with high [Ca²⁺] is much lower. On the other hand, because of consumption, aequorin is very well suited to detect heterogeneity in [Ca²⁺]. We have shown recently using theoretical models (6) that, in experiments such as that of Fig. 1B, we should obtain a peak after Ca²⁺ addition in the calibrated [Ca²⁺] signal if there were a compartment refilling to a much higher [Ca²⁺] than the rest. The reason for this artifact is that the signal would be dominated initially by the high [Ca²⁺] compartment until aequorin consumption in that compartment was near complete. Afterward, the [Ca²⁺] signal would return down to end monitoring the low [Ca²⁺] compartment isolated. Our experimental results show instead a smooth increase in [Ca²⁺], leading to a steady state that keeps flat until near 90% of aequorin has been consumed (see Fig. 1B). Therefore, we can conclude that there are no gross differences in [Ca²⁺] throughout the bulk of the ER.

Increasing the temperature from 22 °C to 37 °C accelerated nearly 4-fold the rate of increase in [Ca²⁺]_er after addition of extracellular Ca²⁺. On the other hand, the rate of Ca²⁺ leak from the stores, measured from the rate of decrease in [Ca²⁺]_er after addition of BHQ in 1 mM Ca²⁺-containing medium, also increased with the temperature from 2 ± 0.1 (n = 5) µM/s at 22 °C to 4.4 ± 0.8 (n = 4) µM/s at 37 °C. Similarly, emptying of the stores after incubation in Ca²⁺-free medium was 4-fold slower at 22 °C (half-time 12 min) than at 37 °C (half-time 3 min). Given that the steady-state [Ca²⁺]_er depends on the balance between these Ca²⁺-pumping and leak, it is not surprising to find that the steady-state [Ca²⁺]_er does not change with the temperature (590 ± 90 µM at 22 °C, compare with 550 ± 70 µM (Ref. 1), at 37 °C).

The experiments shown in Fig. 2 demonstrate that a relatively small and fast decrease in [Ca²⁺]_er is enough to produce a maximal [Ca²⁺]_c peak. A similar conclusion was reached recently observing the decrease in [Sr²⁺]_er and the [Sr²⁺]_c peak induced by caffeine in skeletal muscle myotubes (7). Therefore, the height of the [Ca²⁺]_c peak appears to be related much more with the rate of Ca²⁺ release than with the actual amount of Ca²⁺ released. In fact, 2.5 µM histamine produced an almost undetectable decrease in [Ca²⁺]_er and a near half-maximum peak in [Ca²⁺]_c. Additionally, it is also surprising to see from these experiments that the plateau of elevated [Ca²⁺]_c following the main peak coincides with a constant [Ca²⁺]_er level, still in the presence of histamine. This phenomenon has two main implications that deserve further discussion. First, the constant [Ca²⁺]_er level must be a consequence of the balance between increased Ca²⁺-pumping (following the increase in [Ca²⁺]_c) and reduced InsP₃-activated Ca²⁺ release. Second, the plateau of [Ca²⁺]_c must be due to an increased Ca²⁺ entry from the extracellular medium due to activation of the store-operated Ca²⁺ channels under these conditions.

Regarding the first conclusion, clearly activation of Ca²⁺ release through the InsP₃-gated channels at maximal rate could never be balanced by the activity of the SERCA, given that ion flux through channels is several orders of magnitude faster than the maximal pumping activity of Ca²⁺ ATPases. Partial emptying is also not due to heterogeneous distribution of the InsP₃ receptors, because in BAPTA-loaded cells histamine induces an almost complete emptying (see also Ref. 1). The only consistent explanation of these results is therefore that the InsP₃-gated channels become strongly inhibited after the initial histamine-induced Ca²⁺ release. The experiments shown in Figs. 3 and 4, where histamine-induced Ca²⁺ release is observed in the absence of Ca²⁺ pumping, indicate that the inhibition of the InsP₃ receptors is quite strong at 37 °C and near 100% at 22 °C. The stronger inhibition at low temperature is probably due to the reduced production of InsP₃ (8). As has been discussed already in Ref. 1, the mechanism for the inhibition most probably depends on the generation of microdomains of relatively high [Ca²⁺] in the vicinity of the InsP₃ receptors, which would inhibit Ca²⁺ release according to the known bell-shaped dependence of these channels with [Ca²⁺]_c (9-12). This mechanism seems to be perfectly designed from a physiological point of view to produce maximal [Ca²⁺]_c peaks with minimum Ca²⁺ release from the stores. In this way, the energetic cost is considerably reduced and, even more important, the cell is able to respond consecutively to several different stimuli because the Ca²⁺ store is maintained nearly full most of the time.

Regarding the second point, stimulation of Ca²⁺ entry by histamine in HeLa cells is believed to occur through the capacitative pathway (SOCC, store-operated calcium channels; see Refs. 13 and 14), a Ca²⁺ pathway that is activated through an unknown mechanism following the emptying of the intracellular Ca²⁺ stores. The activation of this pathway has been shown in several cell types to be proportional to the degree of emptying of the stores (15, 16). However, if the activation of Ca²⁺ entry by histamine was just a consequence of the emptying of the stores we observe, it is difficult to imagine a mechanism with such an exquisite sensitivity to detect the minute changes in the bulk [Ca²⁺]_er (<10%) induced by the different histamine concentrations. Alternative explanations could be: (i) selective emptying by the agonist of a store with specific functions in the activation of SOCC, although confocal microscopy studies have shown no specific sites for histamine-induced Ca²⁺ release in HeLa cells (17); (ii) direct activation of SOCC by the agonist through a mechanism either independent or cooperative with the emptying of the stores (18-20). Further study will be necessary to determine the correlation between the activation of SOCC and the actual level of [Ca²⁺]_er.

The results shown in Fig. 5 suggest also that Ca²⁺ release through the InsP₃-gated channels may be regulated by [Ca²⁺]_er. Ca²⁺ release follows a single exponential while [Ca²⁺]_er is above 150 µM, but slows down progressively when [Ca²⁺]_er gets below that. An alternative interpretation for this phenomenon could only be found in terms of heterogeneity of the rate of Ca²⁺ release through the InsP₃ receptors between different pools of the ER, assuming that there was a small portion of the ER having a rate of Ca²⁺ release several times slower than the rest. We have previously shown the same phenomenon using Sr²⁺ as a Ca²⁺ surrogate (6), although in that case the [Sr²⁺]_er required to activate maximally Sr²⁺ release was higher, around 500 µM. Regulation by [Ca²⁺]_er of the sensitivity of the InsP₃ receptors has been reported previously (21, 22), but some other groups failed to detect this effect (23-26) or found the regulation to be significant only at very low lumenal [Ca²⁺] (<10 µM, Ref. 27). Our data suggest that regulation of Ca²⁺ release by [Ca²⁺]_er takes place in intact cells, slowing Ca²⁺ release when [Ca²⁺]_er gets below 150 µM. This threshold was independent of the histamine concentration and therefore of the rate of Ca²⁺ release, so excluding alternative explanations such as the build-up of a diffusion membrane potential across the ER membrane. Our results cannot be interpreted either in terms of an effect of luminal Ca²⁺ acting at the cytosolic inhibitory site (28), because the same effect was observed (6) using cells loaded with Sr²⁺, which does not bind to the inhibitory site (1, 29, 30). Regarding the physiological significance of this mechanism, it is important to note that the stimulatory effect of Ca²⁺ saturates at [Ca²⁺]_er values around 150 µM, that is 30% of its steady-state value. This means that inhibition of Ca²⁺ release by this mechanism will probably take place only when cells are strongly stimulated. In those conditions, this mechanism may be designed as a negative feedback able to stop cell activation, avoiding complete depletion of Ca²⁺ of the ER.

Finally, another initially unexpected result from this study was the relative lack of correlation between the height of the histamine-induced [Ca²⁺]_c peaks and the [Ca²⁺]_er level during incubations in Ca²⁺-free medium. The height and the shape of the [Ca²⁺]_c peak were only slightly modified even when the [Ca²⁺]_er had been reduced to about 30% of the initial value (see Figs. 6 and 7). This result, however, could be expected from the data shown in Fig. 2. Given that a decrease in [Ca²⁺]_er of 50-100 µM is enough to produce a maximum [Ca²⁺]_c peak and that the affinity for Ca²⁺ of the most abundant Ca²⁺-binding proteins in the ER is in the millimolar range (31), we can predict that a similar amount of Ca²⁺ should be released after a fast [Ca²⁺]_er decrease from 500 µM to 450 µM or from 200 µM to 150 µM. In fact, the inhibition of the InsP₃ receptors by [Ca²⁺]_c limits the total amount of Ca²⁺ released after histamine action, and therefore the effect of the agonist on [Ca²⁺]_c becomes nearly independent of the [Ca²⁺]_er, within a certain range of levels (below 150 µM, Ca²⁺ release starts to be inhibited, see above). This may also be important from a physiological point of view, because this mechanism allows cell responses to InsP₃-producing extracellular agonists to be nearly independent of the Ca²⁺ content of the stores. On the other hand, these results suggest that the content of the Ca²⁺ stores cannot be adequately estimated from the height of the peaks of [Ca²⁺]_c induced by an agonist. Direct measurement of [Ca²⁺]_er appears now essential and will surely throw new light in the next few years on many long-known phenomena related to Ca²⁺ homeostasis.