Effect of inositol 1,3,4,5-tetrakisphosphate on inositol trisphosphate-activated Ca2+ signaling in mouse lacrimal acinar cells.

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)IP3), stimulated both intracellular Ca2+ mobilization and Ca2+ entry. Microinjection of inositol 1,3,4,5-tetrakisphosphate ((1,3,4,5)IP4), the inositol 1,4,5-trisphosphate-3-kinase product, was ineffective at mobilizing intracellular Ca2+ or activating Ca2+ entry. In lacrimal cells previously microinjected with submaximal levels of (2,4,5)IP3, the subsequent microinjection of low to moderate concentrations of (1,3,4,5)IP4 did not result in additional release of intracellular Ca2+, nor did it potentiate the Ca2+ entry phase attributable to (2,4,5)IP3. 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)IP3 induced further mobilization of intracellular Ca2+ and increased the elevated and sustained Ca2+ entry phase. Introduction of high concentrations of (1,3,4,5)IP4 appeared to inhibit or block the (2,4,5)IP3-induced Ca2+ entry phase. These results were consistent with the observed effect of (1,3,4,5)IP4 in permeabilized lacrimal cells, where (1,3,4,5)IP4 did not release cellular 45Ca2+ but at high concentrations inhibited the ability of submaximal concentrations of (2,4,5)IP3 to release 45Ca2+. Likewise, injection of a high concentration of (1,3,4,5)IP4 prior to injection of (2,4,5)IP3 blocked both release and influx of Ca2+. The inhibitory action of (1,3,4,5)IP4 on Ca2+ signaling observed in intact cells occurred at concentrations that might be obtained in agonist-stimulated cells. However, in permeabilized cells, (1,3,4,5)IP4 inhibited Ca2+ mobilization at concentrations exceeding those likely to occur in agonist-stimulated cells. These results suggest that physiologically relevant levels of (1,3,4,5)IP4 in the cell cytoplasm do not release Ca2+, nor do they potentiate inositol trisphosphate-induced Ca2+ entry across the plasma membrane. Rather, the possibility is raised that (1,3,4,5)IP4 or one of its metabolites could function as a negative feedback on Ca2+ mobilization by inhibiting inositol 1,4,5-trisphosphate-induced Ca2+ release.

In many cell types, surface receptor activation results in a complex, biphasic Ca 2ϩ response composed of an initial mobilization of internally stored Ca 2ϩ , followed by entry of extra-cellular Ca 2ϩ . An early event following activation of the muscarinic receptor is the breakdown of phosphatidylinositol 4,5bisphosphate 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 2ϩ 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 2ϩ mobilization from intracellular pools (1). However, the mechanism underlying the second phase of Ca 2ϩ 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).

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 Ͼ95% 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, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be 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. 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 M 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 2ϩ 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 ϫ (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 2ϩ ] 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 2ϩ ] i levels could be monitored prior to and during the microinjection procedure.
Preparation of Permeabilized Cells for 45 Ca 2ϩ 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 2ϩ was added so that the free Ca 2ϩ , 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 g/ml) and antimycin (10 M) when required. Cell permeabilization was achieved by incubation with 50 g/ml saponin and was complete (Ͼ95%) by 10 min as determined by trypan blue exclusion. The permeabilized cells were centrifuged once and resuspended in the intracellular solution lacking saponin for 45 Ca 2ϩ uptake.

45
Ca 2ϩ Uptake-The uptake and release of 45 Ca 2ϩ by permeabilized lacrimal cells were measured as described previously for guinea pig hepatocytes (15). Permeabilized cells were incubated with 1 Ci/ml of 45 Ca 2ϩ at a density of 0.75-1.0 mg/ml of protein, and uptake of 45 Ca 2ϩ was initiated by the addition of 3 mM-MgATP. Cell content of 45 Ca 2ϩ was determined by rapidly diluting 200-l samples of the cell suspension in 5 ml of ice-cold iso-osmotic sucrose (310 mM) containing EGTA (4 mM), and 0.1 Ci of [ 3 H]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 isoosmotic 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 2ϩ -free medium. Under these conditions, microinjection of a (1,3,4,5)IP 4 (10 mM pipette concentration, final cellular concentration, 100 -200 M (11)), did not mobilize intracellular Ca 2ϩ ([Ca 2ϩ ] i ), nor did it promote Ca 2ϩ entry into the cell on restoring the extracellular Ca 2ϩ (Fig. 1). Further, the injection of (1,3,4,5)IP 4 did not prevent the ability of thapsigargin to activate Ca 2ϩ 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 M), resulted in a submaximal Ca 2ϩ release and a submaximal level of Ca 2ϩ entry (Fig. 2a). Subsequent microinjection of additional (2,4,5)IP 3 released additional Ca 2ϩ and further increased the level of Ca 2ϩ entry (Fig. 2a). Control injections not containing an inositol phosphate did not modify the second Ca 2ϩ entry phase as compared with the first (Fig. 2b).
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 2ϩ release and Ca 2ϩ 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 2ϩ signaling in intact lacrimal cells. In Fig. 1, c and Fig. 1, lacrimal cells were incubated in a nominally Ca 2ϩ -free medium during the microinjections (indicated with arrows), while the horizontal bars indicate when extracellular Ca 2ϩ 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 2ϩ . The subsequent injection of submaximal (2,4,5)IP 3 (second arrow) caused a second, additional release of intracellular Ca 2ϩ , and the cumulative effects of these injections resulted in an increased level of Ca 2ϩ entry. b, as a control for any possible effect that the injection procedure itself may have on the Ca 2ϩ 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 M (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 2ϩ mobilization, nor did it potentiate Ca 2ϩ entry. However, with high concentrations of (1,3,4,5)IP 4 , the subsequent Ca 2ϩ entry phase was reduced, and in some cases it was blocked. Each experiment is representative of three to seven observations. 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 2ϩ -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 M to 10 mM) neither released additional intracellular Ca 2ϩ nor potentiated the Ca 2ϩ entry phase seen with the (2,4,5)IP 3 alone (n ϭ 15/15). Rather, high concentrations of microinjected (1,3,4,5)IP 4 appeared to reduce the subsequent Ca 2ϩ 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 M, n ϭ 6/6). Note that although the Ca 2ϩ entry phase appears blocked, Ca 2ϩ entry can still be activated by treatment with thapsigargin ( Fig. 1). Fig. 3 summarizes 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.
We also confirmed that (1,3,4,5)IP 4 was capable of inhibiting the Ca 2ϩ -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 2ϩ release and Ca 2ϩ entry phases of the response to (2,4,5) Fig. 2 are summarized. In each case, a single cell was initially injected with (2,4,5)IP 3 (1 mM in the pipette), and the Ca 2ϩ entry level was established. Subsequently, the same cell was injected with additional inositol phosphates, and the Ca 2ϩ entry level reexamined. Thus, 100% would indicate that there was no change in the level of the second sustained Ca 2ϩ entry phase when compared with the sustained Ca 2ϩ -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 2ϩ 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 2ϩ entry phase (to 11% with 10 mM (1,3,4,5)IP 4 ). Each data point represents three to five experiments. intracellular Ca 2ϩ pool by (1,4,5)IP 3 proportionally activates Ca 2ϩ 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 thapsigarginactivated calcium entry that does not involve interaction of (1,4,5)IP 3 with its receptor (11) (but see Ref. 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 3induced Ca 2ϩ entry in intact lacrimal cells most likely is a result of the inhibition of Ca 2ϩ 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 2ϩ 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 2ϩ entry in intact cells appear to be considerably less than those that inhibited Ca 2ϩ 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 2ϩ and entry of Ca 2ϩ across the plasma membrane. This action of IP 3 is presumably due to its ability to release Ca 2ϩ 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 2ϩ 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 2ϩ 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 2ϩ channel.