©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Effect of Inositol 1,3,4,5-Tetrakisphosphate on Inositol Trisphosphate-activated Ca Signaling in Mouse Lacrimal Acinar Cells (*)

(Received for publication, August 15, 1995; and in revised form, January 4, 1996)

G. St. J. Bird (§) J. W. Putney Jr.

From the Calcium Regulation Section, Laboratory of Cellular and Molecular Pharmacology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In mouse lacrimal acinar cells, microinjection of the metabolically stable analog of inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate ((2,4,5)IP(3)), stimulated both intracellular Ca mobilization and Ca entry. Microinjection of inositol 1,3,4,5-tetrakisphosphate ((1,3,4,5)IP(4)), the inositol 1,4,5-trisphosphate-3-kinase product, was ineffective at mobilizing intracellular Ca or activating Ca entry. In lacrimal cells previously microinjected with submaximal levels of(2, 4, 5) IP(3), the subsequent microinjection of low to moderate concentrations of (1, 3, 4, 5) IP(4) did not result in additional release of intracellular Ca, nor did it potentiate the Ca entry phase attributable to(2, 4, 5) IP(3). However, as previously demonstrated (Bird, G. S. J., Rossier, M. F., Hughes, A. R., Shears, S. B., Armstrong, D. L., and Putney, J. W., Jr. (1991) Nature 352, 162-165), additional injections of (2, 4, 5) IP(3) induced further mobilization of intracellular Ca and increased the elevated and sustained Ca entry phase. Introduction of high concentrations of (1, 3, 4, 5) IP(4) appeared to inhibit or block the (2, 4, 5) IP(3)-induced Ca entry phase. These results were consistent with the observed effect of (1, 3, 4, 5) IP(4) in permeabilized lacrimal cells, where (1, 3, 4, 5) IP(4) did not release cellular Ca but at high concentrations inhibited the ability of submaximal concentrations of(2, 4, 5) IP(3) to release Ca. Likewise, injection of a high concentration of(1, 3, 4, 5) IP(4) prior to injection of (2, 4, 5) IP(3) blocked both release and influx of Ca. The inhibitory action of(1, 3, 4, 5) IP(4) on Ca signaling observed in intact cells occurred at concentrations that might be obtained in agonist-stimulated cells. However, in permeabilized cells,(1, 3, 4, 5) IP(4) inhibited Ca mobilization at concentrations exceeding those likely to occur in agonist-stimulated cells. These results suggest that physiologically relevant levels of(1, 3, 4, 5) IP(4) in the cell cytoplasm do not release Ca, nor do they potentiate inositol trisphosphate-induced Ca entry across the plasma membrane. Rather, the possibility is raised that (1, 3, 4, 5) IP(4) or one of its metabolites could function as a negative feedback on Ca mobilization by inhibiting inositol 1,4,5-trisphosphate-induced Ca release.


INTRODUCTION

In many cell types, surface receptor activation results in a complex, biphasic Ca response composed of an initial mobilization of internally stored Ca, followed by entry of extracellular Ca. An early event following activation of the muscarinic receptor is the breakdown of phosphatidylinositol 4,5-bisphosphate generating the putative second messenger, inositol 1,4,5-trisphosphate ((1,4,5)IP(3))(^1)(1) , which can subsequently undergo complex metabolic processing(2) . The role played by the inositol phosphates in Ca homeostasis has been the subject of much study, and it is widely believed that (1, 4, 5) IP(3) is responsible for the first phase of Ca mobilization from intracellular pools(1) . However, the mechanism underlying the second phase of Ca entry is poorly understood(3) , and controversy particularly surrounds the role of the(1, 4, 5) IP(3) metabolite,(1, 3, 4, 5) IP(4), in this process(3, 4, 5, 6) .

In previous studies, it was reported that intracellular application of (1, 4, 5) IP(3) into lacrimal acinar cells, by means of perfusion of patch clamp pipettes in the whole cell configuration, resulted in transient activation of a Ca-activated K conductance. Sustained activation could only be achieved when(1, 3, 4, 5) IP(4) was applied together with (1, 4, 5) IP(3)(7, 8, 9, 10) . These studies suggested that both (1, 4, 5) IP(3) and(1, 3, 4, 5) IP(4) were necessary for the activation of Ca entry in these cells. Subsequently, we measured [Ca] changes in lacrimal cells with the Ca-sensitive fluorescent dye, fura-2, and introduced(1, 4, 5) IP(3) or its poorly metabolized analog, (2, 4, 5) IP(3) into the cells by microinjection(11) . These studies demonstrated that(1, 4, 5) IP(3) alone is both a necessary and sufficient signal for intracellular Ca ([Ca]) mobilization as well as Ca entry across the plasma membrane. However, as pointed out by Irvine(12) , possible augmenting actions of (1, 3, 4, 5) IP(4) on calcium signaling were not directly examined. In the present study, we have used fura-2-loaded mouse lacrimal acinar cells to examine effects of(1, 3, 4, 5) IP(4) on Ca entry induced by(2, 4, 5) IP(3). Our results indicate that physiological concentrations of(1, 3, 4, 5) IP(4) clearly do not augment but rather may inhibit (1, 4, 5) IP(3)-induced Ca signaling.


MATERIALS AND METHODS

Cell Isolation

Mouse lacrimal acinar cells were prepared essentially as described by Parod et al.(13) . Briefly, the excised glands from five mice (male CD-1; 30-40 g) were finely minced and treated for 1 min with 0.25 mg/10 ml trypsin (Sigma). The trypsin was then removed by centrifugation, followed by a 5-min incubation of the tissue fragments with 2 mg/10 ml soybean trypsin inhibitor (Sigma), in the presence of 2.5 mM EGTA. Finally, the acinar cells were isolated after treating the tissue with 4 mg/10 ml collagenase (Boehringer Mannheim) for 10 min. Viability of the isolated cells was >95% based on trypan blue exclusion. Throughout, all enzyme solutions were prepared in Dulbecco's modified Eagle's medium containing 0.5% (w/v) bovine serum albumin. Following isolation, the acinar cells were washed and resuspended in sterile Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 5 mM glutamine, 50 units/ml penicillin, and 50 units/ml streptomycin. The cells were then allowed to attach to glass coverslips coated with Matrigel (Collaborative Biomedical Products, Bedford, MA). Acinar cells were incubated on the glass coverslips for at least 3 h before use.

Fura-2 Loading

The attached cells were mounted in a Teflon chamber (Bionique) and incubated with 0.5 µM fura-2/AM (Molecular Probes) for 30 min at room temperature. The cells were then washed and bathed in a HEPES-buffered physiological saline solution (HPSS; 120.0 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO(4), 1.8 mM CaCl(2), 11.0 mM glucose, 20.0 mM HEPES, pH 7.4, 0.2% (w/v) bovine serum albumin) at room temperature for at least 30 min before Ca measurements were made.

Fluorescence Measurements

The fluorescence of the fura-2-loaded cells was monitored with a photomultiplier-based system, mounted on a Nikon Diaphot microscope equipped with a Nikon 40 times (1.3 N.A.) Neofluor objective. The fluorescence light source was provided by a Deltascan D101 (Photon Technology International Ltd.), equipped with a light path chopper and dual excitation monochromators. The light path chopper enabled rapid interchange between two excitation wavelengths (340 and 380 nm), and a photomultiplier tube monitored the emission fluorescence at 510 nm, selected by a barrier filter (Omega). All experiments were carried out at 24 °C. Calibration and calculation of [Ca](i) were carried out as described previously(11) .

Lacrimal Cell Microinjection

Mouse lacrimal cells were microinjected essentially as described before(11) . A solution consisting of 27 mM K(2)HPO(4), 8 mM Na(2)HPO(4), 26 mM KH(2)PO(4), pH 7.2, and 2 mM fura-2 (acid) was pressure-injected into cells via a glass micropipette attached to a WPI PV830 Picopump (World Precision Instruments, New Haven, CT). Prior to microinjection, lacrimal cells were loaded with fura-2 by incubation with fura-2/AM so that [Ca](i) levels could be monitored prior to and during the microinjection procedure.

Preparation of Permeabilized Cells for Ca Uptake Studies

Isolated cells were suspended in a medium resembling the intracellular milieu, which had the following composition (in mM): 20.0 NaCl, 100.0 KCl, 2.0 MgSO(4), 20.0 HEPES (pH 7.2), 1.0 EGTA. Total Ca was added so that the free Ca, calculated as described by Fabiato(14) , was 150 nM. The medium also contained an ATP-regenerating system (10 mM phosphocreatine, 10 units/ml creatine kinase) and the mitochondrial inhibitors oligomycin (10 µg/ml) and antimycin (10 µM) when required. Cell permeabilization was achieved by incubation with 50 µg/ml saponin and was complete (>95%) by 10 min as determined by trypan blue exclusion. The permeabilized cells were centrifuged once and resuspended in the intracellular solution lacking saponin for Ca uptake.

Ca Uptake

The uptake and release of Ca by permeabilized lacrimal cells were measured as described previously for guinea pig hepatocytes (15) . Permeabilized cells were incubated with 1 µCi/ml of Ca at a density of 0.75-1.0 mg/ml of protein, and uptake of Ca was initiated by the addition of 3 mM-MgATP. Cell content of Ca was determined by rapidly diluting 200-µl samples of the cell suspension in 5 ml of ice-cold iso-osmotic sucrose (310 mM) containing EGTA (4 mM), and 0.1 µCi of [^3H]mannose/ml for determination of trapped volume. The samples were subsequently filtered rapidly through GF/C (Whatman) glass fiber filters and washed with 5 ml of ice-cold iso-osmotic sucrose. The radioactive content of the filters was then determined by liquid scintillation spectroscopy.


RESULTS

During the microinjection of inositol phosphates, the mouse lacrimal cells were maintained in nominally Ca-free medium. Under these conditions, microinjection of a (1, 3, 4, 5) IP(4) (10 mM pipette concentration, final cellular concentration, 100-200 µM(11) ), did not mobilize intracellular Ca ([Ca](i)), nor did it promote Ca entry into the cell on restoring the extracellular Ca (Fig. 1). Further, the injection of (1, 3, 4, 5) IP(4) did not prevent the ability of thapsigargin to activate Ca entry in these cells (Fig. 1). As shown previously in lacrimal cells(11) , microinjection of submaximal concentrations of the metabolically stable(1, 4, 5) IP(3) analog,(2, 4, 5) IP(3) (1 mM pipette concentration, final cellular concentration 10-20 µM), resulted in a submaximal Ca release and a submaximal level of Ca entry (Fig. 2a). Subsequent microinjection of additional(2, 4, 5) IP released additional Ca and further increased the level of Ca entry (Fig. 2a). Control injections not containing an inositol phosphate did not modify the second Ca entry phase as compared with the first (Fig. 2b).


Figure 1: The effect of microinjected (1, 3, 4, 5) IP(4) on Ca signaling in a single mouse lacrimal acinar cell. Lacrimal cells were incubated in a nominally Ca-free medium, microinjected with 10 mM(1, 3, 4, 5) IP(4) (indicated by the arrow on the left), and the horizontal bar indicates when extracellular Ca was restored to 1.8 mM. This was followed, where indicated, by 2 µM thapsigargin.




Figure 2: The effect of inositol polyphosphate microinjection on Ca mobilization and entry in a mouse lacrimal acinar cell previously microinjected with a submaximal concentration of(2, 4, 5) IP^3. As in Fig. 1, lacrimal cells were incubated in a nominally Ca-free medium during the microinjections (indicated with arrows), while the horizontal bars indicate when extracellular Ca was restored to 1.8 mM. a, (2, 4, 5) IP(3) was first injected as indicated by the first arrow. The pipette solution contained 1 mM(2, 4, 5) IP(3), which gave sufficient intracellular (2, 4, 5) IP(3) to induce a submaximal release of Ca. The subsequent injection of submaximal (2, 4, 5) IP(3) (second arrow) caused a second, additional release of intracellular Ca, and the cumulative effects of these injections resulted in an increased level of Ca entry. b, as a control for any possible effect that the injection procedure itself may have on the Ca entry phase itself, a second injection was made in the absence of any inositol phosphate. In c and d, the protocol was similar to that described for a, except that the second injection was either 100 µM(1, 3, 4, 5) IP(4) (c) or 10 mM(1, 3, 4, 5) IP(4) (d). In all cases, the injection did not induce intracellular Ca mobilization, nor did it potentiate Ca entry. However, with high concentrations of (1, 3, 4, 5) IP(4), the subsequent Ca entry phase was reduced, and in some cases it was blocked. Each experiment is representative of three to seven observations.



Although it is apparent from these results and our earlier report (11) that(1, 4, 5) IP(3) provides both a necessary and sufficient signal for both intracellular Ca release and Ca entry, our previous study did not directly address the possibility that(1, 3, 4, 5) IP(4) might modulate or augment IP(3)-induced Ca signaling in intact lacrimal cells. In Fig. 1, c and d, the effect of (1, 3, 4, 5) IP(4) on cells previously microinjected with submaximal concentrations of(2, 4, 5) IP(3) was examined to determine if(1, 3, 4, 5) IP(4) could either mobilize additional intracellular Ca or potentiate the Ca entry phase induced by(2, 4, 5) IP(3) alone.

After establishing the response of a single lacrimal cell to a submaximal concentration of(2, 4, 5) IP(3), the cells were returned to a nominally Ca-free medium and microinjected a second time with different concentrations of(1, 3, 4, 5) IP(4) (Fig. 2, c and d). In all cases, (1, 3, 4, 5) IP(4) (pipette concentrations from 100 µM to 10 mM) neither released additional intracellular Ca nor potentiated the Ca entry phase seen with the(2, 4, 5) IP(3) alone (n = 15/15). Rather, high concentrations of microinjected (1, 3, 4, 5) IP(4) appeared to reduce the subsequent Ca entry phase, and the highest concentrations used almost completely blocked the entry phase (Fig. 2d, 10 mM(1, 3, 4, 5) IP(4) in the pipette, cellular concentration 100-200 µM, n = 6/6). Note that although the Ca entry phase appears blocked, Ca entry can still be activated by treatment with thapsigargin (Fig. 1). Fig. 3summarizes results of experiments showing that the inhibition of calcium entry by injected (1, 3, 4, 5) IP(4) was dependent on the concentration of (1, 3, 4, 5) IP(4) in the injection pipette.


Figure 3: Concentration effect curve for microinjected (1, 3, 4, 5) IP(4) on the Ca influx response to 10 µM(2, 4, 5) IP(3) in single lacrimal cells. The effects of(1, 3, 4, 5) IP(4) injection on the (2, 4, 5) IP(3)-induced Ca entry described in Fig. 2are summarized. In each case, a single cell was initially injected with(2, 4, 5) IP(3) (1 mM in the pipette), and the Ca entry level was established. Subsequently, the same cell was injected with additional inositol phosphates, and the Ca entry level reexamined. Thus, 100% would indicate that there was no change in the level of the second sustained Ca entry phase when compared with the sustained Ca-entry phase induced by the initial injection of (2, 4, 5) IP(3). As can be seen, a second injection of 1 mM(2, 4, 5) IP(3) results in an approximate doubling of the Ca entry; no such effect is seen when the second injection is carried out in the absence of inositol phosphates in the injection solution (control). In contrast, increasing the concentration of injected(1, 3, 4, 5) IP(4) decreases the second Ca entry phase (to 11% with 10 mM(1, 3, 4, 5) IP(4)). Each data point represents three to five experiments.



We next considered that the inhibitory effect of (1, 3, 4, 5) IP(4) might result from an inhibition of the action of(2, 4, 5) IP(3) at the IP(3) receptor. Thus, we examined the effect of(1, 3, 4, 5) IP(4) on Ca release from saponin-permeabilized mouse lacrimal cells, particularly to see if(1, 3, 4, 5) IP(4) could modulate the effect of a submaximal concentration of (2, 4, 5) IP(3) on Ca release (Fig. 4). In permeabilized lacrimal cells, (2, 4, 5) IP(3) released Ca in a dose-dependent fashion (EC = 6.5 ± 0.8 µM; maximal release was 46.2 ± 4.8% of the ATP-dependent Ca pool(16) ). As shown in Fig. 4, a submaximal concentration (10 µM) of (2, 4, 5) IP(3) released 31.8 ± 5.4% (n = 4) of the ATP-dependent Ca-pool (total ATP-dependent Ca pool = 6.40 ± 0.98 nmol of Ca/mg of protein; n = 4). Under these conditions,(1, 3, 4, 5) IP(4) (500 µM) did not cause significant Ca release (4.0 ± 4.9%; n = 4). When 10 µM(2, 4, 5) IP(3) and various concentrations of (1, 3, 4, 5) IP(4) were added together to the permeabilized cells,(1, 3, 4, 5) IP(4) did not augment Ca release due to(2, 4, 5) IP(3). Rather, consistent with the observations in single cells, concentrations of (1, 3, 4, 5) IP(4) greater than 10 µM apparently inhibited the ability of 10 µM(2, 4, 5) IP(3) to release Ca.


Figure 4: Concentration effect curve for the effect of (1, 3, 4, 5) IP(4) on the ability of 10 µM(2, 4, 5) IP(3) to induce Ca release from permeable lacrimal cells. Release of Ca from permeable lacrimal cells was determined as described under ``Materials and Methods.'' The data are expressed as the percentage released of the ATP-dependent Ca pool (6.40 ± 0.98 nmol of Ca/mg of protein). 10 µM(2, 4, 5) IP(3) maximally released 31.8 ± 5.4% of the pool, whereas(1, 3, 4, 5) IP(4) alone was ineffective up to 500 µM (4.0 ± 4.9%). The effect of increasing concentrations of(1, 3, 4, 5) IP(4) on the ability of 10 µM(2, 4, 5) IP(3) to release Ca are shown. At the highest concentration of(1, 3, 4, 5) IP(4) tested, the effect of 10 µM(2, 4, 5) IP(3) was reduced to 11.7 ± 2.1%. Results are means ± S.E. of four independent experiments.



We also confirmed that(1, 3, 4, 5) IP(4) was capable of inhibiting the Ca-mobilizing action of (2, 4, 5) IP(3) in intact cells. In experiments shown in Fig. 5, 10 mM(1, 3, 4, 5) IP(4) was injected into a single lacrimal cells prior to injection of 1 mM(2, 4, 5) IP(3). This resulted in a complete blockade of both the Ca release and Ca entry phases of the response to(2, 4, 5) IP(3).


Figure 5: Prior injection of(1, 3, 4, 5) IP(4) blocks both Ca release and Ca entry due to (2, 4, 5) IP(3) injection. Top, immediately prior to data collection, a single lacrimal acinar cell was injected with (1, 3, 4, 5) IP(4) (pipette concentration 10 mM); this blocked both Ca release and Ca entry due to(2, 4, 5) IP(3) injection (pipette concentration 1 mM). The response to thapsigargin was unaffected. Bottom, In a control experiment, a cell was mock-injected (injected with fura-2-containing diluent only);(2, 4, 5) IP(3) injection induced both release of intracellular Ca as well as Ca entry. The results illustrate findings from three independent experiments.




DISCUSSION

(1,3,4,5)IP(4) did not release intracellular Ca in intact or permeabilized cells, nor did it induce or facilitate Ca entry in intact cells. Rather, and surprisingly,(1, 3, 4, 5) IP(4) appeared to block the Ca entry phase induced by(2, 4, 5) IP(3) microinjection in intact cells. Results from experiments in permeabilized and intact cells would suggest that the inhibitory effect on the Ca entry phase may be due to (1, 3, 4, 5) IP(4) interfering with the ability of (2, 4, 5) IP(3) to maintain depletion of the intracellular Ca pool. It is now well established that depletion of the intracellular Ca pool by(1, 4, 5) IP(3) proportionally activates Ca entry(17) . Heparin, an antagonist of the(1, 4, 5) IP(3) receptor, blocks agonist-activated calcium entry presumably by virtue of its ability to prevent(1, 4, 5) IP(3)-induced depletion of intracellular stores(11) ; heparin does not block thapsigargin-activated calcium entry that does not involve interaction of(1, 4, 5) IP(3) with its receptor (11) (but see (18) ).(1, 3, 4, 5) IP(4) similarly blocked calcium entry as well as calcium release by (2, 4, 5) IP(3) but not responses to thapsigargin. Thus the antagonistic effect of high concentrations of(1, 3, 4, 5) IP(4) on(2, 4, 5) IP(3)-induced Ca entry in intact lacrimal cells most likely is a result of the inhibition of Ca release by(2, 4, 5) IP(3).

(1,3,4,5)IP(4) is metabolized in the cytoplasm of cells by a 5-phosphatase, and thus the question arises as to whether the injected material would persist long enough to exert any physiological action. The half-time for(1, 3, 4, 5) IP(4) in exocrine gland cells has been estimated to be on the order of 45 s to a minute(19) . Thus, since Ca entry was examined within 1 min after injection, and with a wide variety of concentrations, one would expect to have detected some effect of the injected(1, 3, 4, 5) IP(4) if in fact such could occur. Indeed, an inhibitory effect was seen at the higher concentrations of(1, 3, 4, 5) IP(4), an effect also seen with the addition of(1, 3, 4, 5) IP(4) to permeabilized acinar cells. Because(1, 3, 4, 5) IP(4) is metabolized to (1, 3, 4) IP(3) and subsequent metabolites, we cannot at present determine if this is a direct effect of(1, 3, 4, 5) IP(4) or an effect of a metabolic product. In fact, we note that the concentrations of(1, 3, 4, 5) IP(4) that inhibited Ca entry in intact cells appear to be considerably less than those that inhibited Ca mobilization in permeable cells, based on the estimate (11) that the injected material is diluted 50-100-fold. Such a discrepancy might result if a metabolite of(1, 3, 4, 5) IP(4) inhibits the(1, 4, 5) IP(3) response since the concentration of such a metabolite would be considerably diluted in the permeable cell experiments. Alternatively, if the effect is due to a more direct action of(1, 3, 4, 5) IP(4), then the discrepancy may reflect factors that influence IP(3) receptor sensitivity and accessibility in intact cells that are absent or changed in permeabilized cells.

In conclusion, these findings confirm and extend our previous conclusion (11) that IP(3) provides a necessary and sufficient signal for both the intracellular release of Ca and entry of Ca across the plasma membrane. This action of IP(3) is presumably due to its ability to release Ca from intracellular stores, resulting in the transmission of an as yet unknown message to the plasma membrane(3, 20, 21) . In the current studies, (1, 3, 4, 5) IP(4) neither mobilized Ca nor potentiated the effects of(2, 4, 5) IP(3). However, (1, 3, 4, 5) IP(4) was able to antagonize the effects of (2, 4, 5) IP(3) and reduce Ca entry, perhaps by interfering with the ability of(2, 4, 5) IP(3) to bind to its receptor, although this was not directly determined in the present study. Interestingly, Wilcox et al.(22) have reported that in neuronal cells,(1, 3, 4, 5) IP(4) can act as an agonist at the(1, 4, 5) IP(3) receptor. The reason for the different effects in these two different preparations is not known, but a possibility is that the two cell types express distinct forms of the (1, 4, 5) IP(3) receptor, both of which can bind (1, 3, 4, 5) IP(4) but with different effects on activation of the Ca channel.

These data raise the possibility that(1, 3, 4, 5) IP(4) could inhibit the effects of(1, 4, 5) IP(3) under physiological stimulation. Co-injection of 100 µM(1, 3, 4, 5) IP(4) together with 1 mM(2, 4, 5) IP(3) resulted in significant inhibition. Since (1, 4, 5) IP(3) is roughly 10-fold more potent than (2, 4, 5) IP(3), this would indicate that significant inhibition would occur when(1, 4, 5) IP(3) and(1, 3, 4, 5) IP(4) are present in approximately equal concentrations, a situation that occurs in most cases of sustained stimulation. On the other hand, from the data in permeable cells an approximately 30-100-fold excess of (1, 3, 4, 5) IP(4) over(1, 4, 5) IP(3) would be required for inhibition. The levels of(1, 3, 4, 5) IP(4) in maximally stimulated lacrimal acinar cells range from approximately equal to, to 2-3 times the level of(1, 4, 5) IP(3)(23) and do not to our knowledge reach the 30-100-fold excess level in any cell type. However, as argued above, the sensitivity of the(1, 4, 5) IP(3) receptor to(1, 4, 5) IP(3) and/or to(1, 3, 4, 5) IP(4) in intact cells may be markedly different than in permeable cells. Errors in estimates of cellular dilution of inositol phosphates are not an issue, because these would apply equally to the injected (2, 4, 5) IP(3) and the injected(1, 3, 4, 5) IP(4). Thus, although additional work is clearly needed, we suggest that inhibition by(1, 3, 4, 5) IP(4), or possibly one of its metabolites, of the (1, 4, 5) IP(3) response may occur under conditions of physiological stimulation, and this could represent yet another important negative feedback on Ca signaling, at least in this cell type.


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: NIEHS, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-3298.

(^1)
The inositol phosphates are abbreviated according to the ``Chilton Convention''(24) , as, for example, (1, 4, 5) IP(3) for D-myo-inositol 1,4,5-trisphosphate.


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