Metabolism of Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate by the Oocytes of Xenopus laevis*

Christopher E. Sims and Nancy L. AllbrittonDagger

From the Department of Physiology and Biophysics, University of California, Irvine, California 92697-4560

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The pathway and kinetics of inositol 1,4,5-trisphosphate (IP3) metabolism were measured in Xenopus laevis oocytes and cytoplasmic extracts of oocytes. Degradation of microinjected IP3 in intact oocytes was similar to that in the extracts containing comparable concentrations of IP3 ([IP3]). The rate and route of metabolism of IP3 depended on the [IP3] and the intracellular free Ca2+ concentration ([Ca2+]). At low [IP3] (100 nM) and high [Ca2+] (>= 1 µM), IP3 was metabolized predominantly by inositol 1,4,5-trisphosphate 3-kinase (3-kinase) with a half-life of 60 s. As the [IP3] was increased, inositol polyphosphate 5-phosphatase (5-phosphatase) degraded progressively more IP3. At a [IP3] of 8 µM or greater, the dephosphorylation of IP3 was the dominant mode of IP3 removal irrespective of the [Ca2+]. At low [IP3] and low [Ca2+] (both <= 400 nM), the activities of the 5-phosphatase and 3-kinase were comparable. The calculated range of action of IP3 in the oocyte was ~300 µm suggesting that IP3 acts as a global messenger in oocytes. In contrast to IP3, inositol 1,3,4,5-tetrakisphosphate (IP4) was metabolized very slowly. The half-life of IP4 (100 nM) was 30 min and independent of the [Ca2+]. IP4 may act to sustain Ca2+ signals initiated by IP3. The half-life of both IP3 and IP4 in Xenopus oocytes was an order of magnitude or greater than that in small mammalian cells.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Ca2+ signals regulate a diverse array of cellular functions including secretion, cytoskeletal rearrangement, and gene transcription (1, 2). Modulations in [Ca2+]1 are used to transduce signals in nearly all cells including those of plants and animals (1-3). Generation and degradation of the second messenger IP3, which opens the IP3 receptor/Ca2+ channel on the endoplasmic reticulum, regulates the formation and termination of Ca2+ signals. Knowing the rates and pathways of IP3 degradation is fundamental to understanding Ca2+ wave formation. The metabolism of IP3 and its products by phosphatases and kinases is unfolding as an increasingly complex process (4-6). Two primary degradative pathways exist for IP3, but they differ in their relative importance among cell types and between species. Several isoforms of the 5-phosphatase dephosphorylate IP3 yielding inositol 1,4-bisphosphate (4-8). IP3 is also a substrate of the 3-kinase which phosphorylates IP3 to form IP4 (9-13). The binding of Ca2+/calmodulin to the 3-kinase enhances its activity (14-16). The 5-phosphatase also metabolizes IP4 to inositol 1,3,4-trisphosphate (I-1,3,4-P3). Current evidence suggests that the major function of the 5-phosphatase in the phosphoinositide cycle is to decrease the [IP3] and the concentration of IP4 ([IP4]). In contrast, the role of the 3-kinase is to generate another second messenger, IP4, as well as to decrease the [IP3]. An increasing amount of evidence suggests that IP4 is an important regulatory molecule in cells (6). Like IP3, IP4 may be involved in the regulation of the [Ca2+] (17-19). IP4 binds to the IP3 receptor/Ca2+ channel and releases Ca2+ from the endoplasmic reticulum although with a 10-fold lower potency than IP3. More intriguing, IP4 binds with high affinity to several intracellular proteins, synaptotagmin I and II, Gap1, Btk, and centaurin-alpha (20-24). The Ras GTPase-activity of Gap1 is stimulated by IP4, and IP4 may interact with synaptotagmin to inhibit synaptic transmission (20, 25). The pathway selected to metabolize IP3 may not only influence its rate of removal but also alter subsequent signal transduction within the cell.

Much of our mechanistic knowledge of Ca2+ wave propagation is derived from studies of Ca2+ waves in oocytes and eggs of Xenopus laevis. Formation of the Ca2+ wave, which follows the fertilization of an egg and which follows the activation of plasma-membrane receptors in oocytes, requires IP3 (26, 27). Work to date on IP3 metabolism in Xenopus oocytes has yielded conflicting results. Microinjection of concentrated [3H]IP3 into single Xenopus oocytes followed by separation of the metabolites by ion-exchange chromatography has suggested that the 5-phosphatase pathway prevailed (28). However, the addition of [3H]IP3 to a homogenate of oocytes or microinjection into ovarian follicles followed also by high pressure liquid chromatography (HPLC) separation have provided strong evidence for the 3-kinase as the primary route of IP3 degradation (29, 30). These discrepancies remain unresolved, despite the central role that the Xenopus oocyte has played in understanding Ca2+ wave propagation. All three of the studies discussed above found a surprisingly prolonged degradation time for IP3, 20 min or longer. This contrasts with smaller cells that degrade IP3 in a few seconds (31, 32). Establishing both the rate and pathway of IP3 metabolism in oocytes is required to understand the role of the phosphoinositide pathway in the generation and termination of Ca2+ signals and in interactions with other signal transduction cascades. The purpose of this investigation was to determine how IP3 and IP4 were degraded in X. laevis oocytes. Moreover, the goal was to quantitate the kinetic properties of these metabolic pathways in the cytoplasmic milieu.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Tritiated inositol and inositol phosphates were purchased from NEN Life Science Products. Nonradioactive IP3 was purchased from Calbiochem and Alexis (Woburn, MA), and nonradioactive IP4 and calpain inhibitor I (N-Ac-Leu-Leu-norleucinal) were purchased from Calbiochem. Phorbol 12-myristate 13-acetate was supplied by Alexis (Woburn, MA). EGTA "puriss" grade, was obtained from Fluka (Ronkonkoma, NY). Rhod-2, BAPTA, and calcium green-5N were supplied by Molecular Probes (Eugene, OR). All other reagents were purchased from Fisher.

EGTA-buffered Ca2+ Solutions-- An equimolar solution of EGTA and Ca2+ was prepared by the method of Tsien and Pozzan (33). Cytoplasmic extracts with varying [Ca2+] were made by addition of 10 mM EGTA and 10 mM EGTA with 10 mM Ca2+. The [Ca2+] in the EGTA-buffered extracts was estimated from the [Ca2+] in buffer A (135 mM KCl, 5 mM NaCl, 1 mM MgCl2, 10 mM HEPES, pH 7.4) which approximated the intracellular ionic environment and contained the same mixture of 10 mM EGTA and 10 mM EGTA with 10 mM Ca2+ as the EGTA-buffered extract. The [Ca2+] in the buffer solution was measured using the fluorescent Ca2+ indicators, rhod-2 and calcium green-5N as described by Allbritton and colleagues (34) and Haugland (35). The [Ca2+] in buffer A containing 10 mM EGTA and 10 mM Ca2+ was 10 µM.

Preparation of Oocytes and Cytoplasmic Extracts-- Female X. laevis frogs were purchased from Nasco (Modesto, CA). Oocytes were surgically obtained and prepared as described previously (34, 36). Cytoplasmic extracts were also made as described previously with the following exceptions (34, 37). To minimize proteolysis the oocytes and extract were maintained at 4 °C throughout the preparation. Calpain inhibitor I (10 µg/ml) was added to the extract to block the Ca2+-activated protease calpain. After isolation, the cytoplasmic extract was used immediately in experiments. Prolonged delays prior to use diminished the ability of the extract to metabolize inositol phosphates. This cytoplasmic preparation is ~90% pure cytoplasm (90 mg/ml protein) (34).

Measurement of IP3 Degradation in Intact Oocytes-- Oocytes were microinjected with 5-30 nl of [3H]IP3 (2 µM) contained in buffer A with or without BAPTA (100 mM). The volume of injectate was determined from the total number of counts contained in the oocyte. The oocytes were incubated at room temperature for the indicated times. Just prior (~10 s) to the end of the incubation period, the buffer solution surrounding the oocyte was removed to determine how much of the tritium label had leaked from the oocyte. Cells that leaked greater than 10% of the radioactive label were excluded from experiments. The intracellular reactions of the oocyte were terminated by rapid freezing with powdered dry ice. The frozen oocyte was then homogenized in chloroform:methanol (50 µl of 1:2) and buffer B (50 µl of 25 mM tetrabutylammonium hydrogen sulfate, 20 mM KH2PO4, pH 3.5) with 80 µg of hydrolyzed phytic acid added as a carrier to prevent nonspecific loss of inositol phosphates. Hydrolyzed phytic acid was prepared as described by Irvine and colleagues (38). After addition of chloroform (50 µl), the mixture was centrifuged at 15,000 × g, and the aqueous phase was separated by reverse phase HPLC.

Measurement of Inositol Phosphate Degradation in Cytoplasmic Extracts-- [Ca2+] in the cytosol was buffered at the indicated concentration by addition of 10 mM EGTA and 10 mM EGTA with 10 mM Ca2+. In some instances, phorbol 12-myristate 13-acetate (50 ng/ml) was included in the cytoplasmic mixture. [3H]IP3 or [3H]IP4 was then added to the oocyte cytosol. After the addition of each reagent to the cytosolic extract, it was gently mixed over a 5-s period by pipetting the mixture up and down. The cytoplasmic extract was incubated for the indicated time at room temperature (~20 °C). The reaction was stopped with a 16-fold excess volume of trifluoroacetic acid (10%) containing hydrolyzed phytic acid (0.8 mg/ml) and centrifuged at 15,000 × g. The supernatant was dried, resuspended in buffer B, and separated by reverse phase HPLC.

Reverse Phase HPLC-- Inositol phosphates were separated on an analytical C18 column (Alltech, San Jose, CA) maintained at 45 °C with an elution protocol modified from that of Shayman and Bement (39) and Sulpice et al. (40). The flow rate was 1 ml/min. During the first 22 min, fractions were collected every 0.25 min and thereafter every 1.25 min. To monitor the separation efficiency of the column, a set of standards previously added to and then extracted from cytosol was chromatographed each day. [3H]Inositol coeluted with inositol 1-phosphate as did the other isomers of inositol phosphate. The isomers of inositol bisphosphate were not distinguishable.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

To qualitatively determine the rate of IP3 metabolism in vivo, stage V/VI oocytes were microinjected with [3H]IP3 (2 µM) (n = 11). After incubation for 30 s, 1 min, or 5 min, the oocytes were rapidly frozen to terminate intracellular reactions. The inositol phosphates were extracted from the cytoplasm and separated by HPLC. Representative standard and experimental traces are shown in Fig. 1, A-C. Oocytes microinjected with small volumes of [3H]IP3 metabolized half of the [3H]IP3 in approximately 1 min (Fig. 1B). Longer times were required to degrade half of the [3H]IP3 when larger volumes were microinjected (Fig. 1C). In all of the experiments, the metabolic fraction that contained the most tritium was that of IP4 or IP3. In vivo and at an [IP3] of 2 µM and lower, IP3 was metabolized predominantly by the 3-kinase.


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Fig. 1.   Metabolism of IP3 by intact oocytes. An oocyte was microinjected with [3H]IP3 (7 nl (B) or 25 nl (C)) or [3H]IP3 and BAPTA (14 nl (D) or 20 nl (E)). After 1 (B), 5 (C and D), or 15 (E) min, the oocyte was frozen rapidly, and the inositol phosphates were extracted from the oocyte and chromatographed. Representative HPLC traces are shown in B-E. An HPLC trace of tritiated standards (~50 nCi each) which were added to and then extracted from cytoplasm is shown in A. The standards were inositol 1-phosphate (I-1-P1) (fraction 11), inositol 1,4-bisphosphate (I-1,4-P2) (fraction 19), I-1,3,4-P3 (fraction 68), IP3 (fraction 74), IP4 (fraction 92), and IP6 (fraction 105). B-E, the migration times of the accompanying set of standards are marked by arrows. The migration times of the standards in A-C differ from those in D and E since the samples were chromatographed on different HPLC systems. The standards eluted in the same order in all experiments. Since IP6 was not included in the standard mix for the experiments in D and E, the migration time of IP6 was not marked in these panels.

In other species the activity of the 3-kinase is increased by the binding of calmodulin and Ca2+ (11, 14-16). To determine whether [Ca2+] regulated the metabolism of IP3 in Xenopus oocytes, [3H]IP3 (2 µM) was coinjected with BAPTA (100 mM) (n = 10) to diminish the IP3-mediated increase in [Ca2+]. After a 1-, 5-, 15-, or 30-min incubation time, the inositol phosphates were extracted and separated. Remarkably, very little of the [3H]IP3 was metabolized by 5 min (Fig. 1D). The half-life of the microinjected IP3 was ~13 min. In contrast to the oocytes without BAPTA, substantial amounts of tritium did not accumulate in the IP4 fraction (Fig. 1, D and E). The activity of the 3-kinase was dramatically decreased in the oocytes containing BAPTA presumably due to a decrease in [Ca2+]. In these in vivo experiments, both diffusion and degradation altered [IP3] during the time course of the measurements. One minute after microinjection in the absence of degradation, the [IP3] at the microinjection site would still be 10 times greater than the final equilibrium concentration (41). Since [IP3] was also altered by diffusion, these experiments could not be used to measure quantitatively the rates and pathways of IP3 metabolism.

To provide a quantitative description of the metabolism of IP3, subsequent experiments were performed in a cytosolic extract. In past experiments, cytosolic preparations were greatly diluted during preparation, and the compartmentalization of proteins and membranes was abolished by homogenization (29, 30). To eliminate these disadvantages, we used a cytosolic extract that is ~90% of the concentration of undiluted cytoplasm (34). The cytosol contains nearly all of the intracellular organelles, and they retain many of their normal functions including the ability to transit through the cell cycle (34, 37, 42). These observations suggested that this extract could be used to define how [IP3] and [Ca2+] regulate the removal of IP3 in an environment similar to the cytoplasmic milieu. [3H]IP3 (100 nM) was added to a cytoplasmic extract that contained 10 mM EGTA with 10 mM Ca2+ ([Ca2+] ~10 µM). At varying times an aliquot of this cytosolic mixture was removed, and the inositol phosphates were extracted and chromatographed. The half-life of the [3H]IP3 was 60 s (Fig. 2B). Under these conditions most of the [3H]IP3 was converted to IP4. The half-life of IP4 was considerably longer than that of IP3. For incubation times of 5 min or less, the metabolism of IP4 was negligible (see also Fig. 4). By 10 min much of the tritium (~30-40%) initially added to the cytosol was no longer soluble after acid extraction; the [3H]inositol was recycled into a cellular component other than inositol or an inositol phosphate. These results are consistent with those obtained in the intact oocytes microinjected with [3H]IP3 suggesting that the cytosolic extract is a very good model for the intact oocyte.


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Fig. 2.   Metabolism of IP3 by cytoplasmic extracts of oocytes. A, the schematic of IP3 metabolism was valid for incubation times of 5 min and less when [Ca2+] in the extract was elevated (>5 µM) and for 10 min and less when [Ca2+] was very low (<100 nM). B and C, [3H]IP3 (100 nM) was added to a cytoplasmic extract with [Ca2+] buffered to 10 µ M (B) or to less than 100 nM (C). After varying incubation times, the inositol phosphates were isolated and separated by HPLC. [IPx] represents the concentration of IP3 (bullet ), IP4 (open circle ), or IPless or equal 2 (×). IPless or equal 2 represents the sum of the concentrations of inositol, inositol phosphate, and inositol bisphosphate. The traces are the best fits to the data points obtained with Origin (Microcal, Northampton, MA). B, the IP4 and IP3 data points were fit to an exponential function, whereas the IPless or equal 2 data points in B and all points in C were fit to a straight line. D, [3H]IP3 (100 nM) was added to a cytoplasmic extract with varying [Ca2+] and incubated for 1 min. The concentrations of the inositol phosphates were quantitated and graphed as in B and C. The traces are hand drawn.

For experimental time points of 5 min or less, the cytosolic reactions of IP3 could be segregated into two distinct pathways without common metabolites (Fig. 2A). In the 3-kinase pathway, IP4 accumulated with very little conversion of IP4 to the lower inositol phosphates. Consequently, [IP4] was used as an estimate of the activity of the 3-kinase for time points of 5 min or less. Inositol bisphosphate (IP2) was then formed almost exclusively by dephosphorylation of IP3 rather than by dephosphorylation of I-1,3,4-P3. Inositol and inositol monophosphate (IP1) were formed by subsequent dephosphorylation of that IP2. The products of the 5-phosphatase pathway were inositol, IP1, and IP2, and the sum of the concentrations of these three inositols ([IPless or equal 2]) was used to estimate the activity of the 5-phosphatase toward IP3. For times less than 5 min, neither inositol, IP1, nor IP2 accrued to a substantial degree (Fig. 2B). Neither of these inositol fractions exceeded 10% of the total tritium in the cytosol. The metabolite of IP4, I-1,3,4-P3, did not accumulate either. The chromatographic peak representing this isomer never contained more than 6% of the added tritium. Although both the 3-kinase and 5-phosphatase were active in the extract, the conversion of IP3 to IP4 dominated at these low [IP3] and for incubation times less than 5 min. Since the activity of the 5-phosphatase was much lower than that of the 3-kinase, the kinetic properties of the 3-kinase were estimated from the rate of formation of IP4. The kinetics of this reaction appeared first order; the rate of formation of IP4 was proportional to [IP3] (43). The first order rate constant (k) obtained by fitting the [IP4] versus time trace to an exponential was 10-2 s-1.

Since the prior in vivo experiments suggested that [Ca2+] regulated the metabolism of IP3 in oocytes, [3H]IP3 (100 nM) was added to a cytoplasmic extract which contained 10 mM EGTA decreasing [Ca2+] to less than 100 nM (Fig. 2C). Since the half-life of IP4 remained much greater than 5 min (see also Fig. 4), the data were displayed identically to that in Fig. 2B. At low [Ca2+] the half-life of IP3 was increased markedly to 10 min. In agreement with the in vivo results, the longer half-life was due to a decrease in the activity of the 3-kinase. Less than 7 and 25% of the IP3 was converted to IP4 in 1 and 10 min, respectively. As before, I-1,3,4-P3 did not accumulate. The rate of accrual of IP4 and IPless or equal 2 was similar and linear over time (0.04 nM/s) and nearly identical to the rate of accumulation of IPless or equal 2 in the presence of high [Ca2+] (Fig. 2, B and C). Formation of IPless or equal 2 was independent of [Ca2+] suggesting that the 5-phosphatase of Xenopus oocytes was not regulated by [Ca2+]. The unchanged production of [IPless or equal 2], despite a markedly decreased [IP4], also suggests that the lower inositol phosphates resulted predominantly from sequential dephosphorylation of IP3 rather than IP4.

To determine the range of [Ca2+] that potentiated the 3-kinase's activity, we varied the [Ca2+] in the extract by altering the ratio of 10 mM EGTA to 10 mM EGTA with 10 mM Ca2+. After addition of [3H]IP3 (100 nM) to the cytosolic preparation for 1 min, the inositol phosphates were extracted and chromatographed. As the [Ca2+] was increased from <100 nM to >2 µM, the amount of IP3 degraded increased from 7 to 40 nM. Half-maximal activation of the 3-kinase occurred at a [Ca2+] of 390 nM and maximal activation appeared by 1 µM (Fig. 2D). Several previous reports indicated that the 5-phosphatase was activated after phosphorylation by protein kinase C, whereas other experiments suggested that the 5-phosphatase activity was inactivated by protein kinase C (44-48). Addition of phorbol 12-myristate 13-acetate (50 ng/ml), a potent activator of protein kinase C, to cytosolic extracts with varying [Ca2+] did not alter the rate or pathway of IP3 metabolism (data not shown). Under these conditions, activation of protein kinase C did not alter the dephosphorylation of IP3 by the 5-phosphatase or phosphorylation of IP3 by the 3-kinase.

The Michaelis constant (Km) of the 5-phosphatase for IP3 is greater than 10 times that of the 3-kinase in many different tissues (4, 5). Therefore, the metabolic pathway of IP3 may depend on [IP3]. To determine how the route of degradation in Xenopus oocytes changed with [IP3], we varied the starting [IP3] in the cytosolic preparation from 100 nM to 30 µM. [3H]IP3 was incubated in the extract for 5 min followed by extraction and separation of the inositol phosphates. In the first series of experiments, [Ca2+] was maintained at 10 µM by addition of 10 mM EGTA with 10 mM Ca2+. For a starting [IP3] of less than 8 µM, the major metabolic pathway was conversion to IP4 by the 3-kinase (Fig. 3A). At an initial [IP3] of approximately 2 µM and greater, the rate of formation of IP4 was independent of [IP3] consistent with [IP3] being much greater than the Km of the 3-kinase. The rate of generation of IP4 at this high [IP3] was used to estimate the maximal velocity (Vmax) of the 3-kinase for IP3 (4 nM/s). For a starting [IP3] greater than 8 µM, [IPless or equal 2] continued to increase despite a plateau in the generation of IP4 (Fig. 3A). The additional IPless or equal 2 must originate from dephosphorylation of IP3 by the 5-phosphatase. At high [IP3], most of the IPless or equal 2 was formed by dephosphorylation of IP3 rather than IP4; IP3 was degraded chiefly by the 5-phosphatase.


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Fig. 3.   The effect of [IP3] on the pathway of IP3 metabolism. Varying concentrations of [3H]IP3 were added to a cytoplasmic extract with [Ca2+] buffered to 10 µM (A) or to less than 100 nM (B). After 5 min the cytosolic reactions were stopped, and the tritiated inositol phosphates were isolated and quantitated. [IP4] (open circle ) and [IPless or equal 2] (×) are plotted against the initial [IP3] in the cytoplasmic extract. The traces through the data points are hand drawn.

To determine how a decrease in [Ca2+] altered IP3 metabolism at different [IP3], the initial [IP3] was varied from 100 nM to 30 µM in a cytosolic preparation containing 10 mM EGTA (Fig. 3B). The [3H]IP3 was incubated in the extract for 5 min followed by acid extraction and chromatographic separation. For all starting [IP3], the rate of formation of IP4 was greatly decreased compared with that in the presence of micromolar [Ca2+]. In contrast, the rate of accrual of [IPless or equal 2] was nearly unchanged from that in the presence of high [Ca2+]. As before the 5-phosphatase was independent of [Ca2+]. Moreover, [IPless or equal 2] did not depend on [IP4] indicating that these lower inositol phosphates resulted mainly from the action of the 5-phosphatase on IP3. The rate formation of IP4 became independent of [IP3] when the initial [IP3] was ~10 µM or greater. This rate was used to estimate the Vmax of the 3-kinase at low [Ca2+] (1 nM/s). For most initial [IP3], IP3 was degraded mainly by dephosphorylation, and the rate of formation of [IPless or equal 2] was linearly related to the initial [IP3] (Fig. 3B). For an initial [IP3] of 1 µM or greater, [IP3] decreased negligibly during the course of the reaction (<= 20%). Since [IP3] was nearly constant during the reaction time and [IPless or equal 2] was linearly related to the starting [IP3], the reaction of the 5-phosphatase with IP3 was modeled as a first order reaction for [IP3] greater than 1 µM. The k of the 5-phosphatase for IP3 (6 × 10-4 s-1) was then estimated from the rate of formation of [IPless or equal 2]. When [Ca2+] was in the micromolar range, the metabolic pathway of IP3 depended on [IP3]. At low initial [IP3], IP3 was metabolized predominantly by the 3-kinase, whereas at high initial [IP3], the 5-phosphatase dominated. The crossover point from the 3-kinase to the 5-phosphatase occurred at an [IP3] of approximately 8 µM.

Since IP4 may be an important signaling molecule, the rate and pathway of IP4 degradation were determined. [3H]IP4 (100 nM or 10 µM) was added to a cytosolic preparation containing 10 mM EGTA with 10 mM Ca2+. At the indicated times the inositol phosphates were extracted from an aliquot of the reaction mixture and separated. For both initial [IP4], 50% of the [3H]IP4 was degraded by dephosphorylation in 30 min (Fig. 4). After a 5-min reaction time, only 5% of the IP4 was metabolized. For reaction times of 5 min or less, destruction of IP4 was negligible, and the reaction scheme depicted in Fig. 2A for IP3 was valid. Varying the [Ca2+] in the extract from <100 nM to 10 µM did not alter the rate of degradation of IP4. As in prior experiments, I-1,3,4-P3 did not accumulate to a substantial degree although [IPless or equal 2] increased with time. Conversion of IP4 to IP3 was not observed. Metabolism of IP4 and formation of IPless or equal 2 followed first order kinetics for initial IP4 concentrations of 100 nM and 10 µM. The k obtained by fitting the [IP4] or [IPless or equal 2] curves to an exponential was 4 × 10-4 s-1.


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Fig. 4.   Metabolism of IP4 by cytoplasmic extracts of oocytes. [3H]IP4 (10 µM) was added to a cytoplasmic extract which was then incubated for varying times. The inositol phosphates were isolated and quantitated. The concentrations of IPless or equal 2 (black-triangle, triangle ), IP4 (bullet , open circle ), and I-1,3,4-P3 (black-square, square ) are plotted against the incubation time. [Ca2+] in the extract was buffered to 10 µM (triangle , open circle , square ) or to less than 100 nM (black-triangle, bullet , black-square). The lines through the IP4 and IPless or equal 2 data points are fits to an exponential function, and the line through the I-1,3,4-P3 data points is hand drawn.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Intact oocytes and a cytoplasmic extract of oocytes were used to determine the pathways and estimate the rates of IP3 and IP4 metabolism in Xenopus oocytes. The pathway of IP3 degradation depended on both [IP3] and [Ca2+]. The 3-kinase was regulated by [Ca2+]; the measured Vmax and k of the 3-kinase decreased as [Ca2+] decreased. Lowering [Ca2+] from 1 µM to less than 100 nM diminished the k and the Vmax by a factor of 25 and 4, respectively. The k of the 3-kinase for IP3 in the presence of 10 mM EGTA ([Ca2+] < 100 nM) was estimated from the slope of the IP4 trace in Fig. 2C and the initial slope of the IP4 trace in Fig. 3B (k = 4 × 10-4 s-1). The Km calculated from the Vmax and k increased from 400 nM to 2.5 µM as [Ca2+] declined from greater than 1 µM to less than 100 nM. [Ca2+] regulated the 3-kinase by altering both the Vmax and Km of the enzyme for IP3. By releasing Ca2+, IP3 enhances its own degradation. The Km values of the 3-kinase and its regulation by Ca2+ in Xenopus oocytes are close to that reported in other species (4, 5, 10-12). These similar enzymatic features suggest that the Xenopus oocyte 3-kinase is regulated by calmodulin as is the case in other species.

The k value of the 5-phosphatase for IP3 was approximately twice that of the 3-kinase when [Ca2+] was low (~100 nM). However, when [Ca2+] was greater than 1 µM, the k value of the 3-kinase exceeded that of the 5-phosphatase by almost 20-fold. Although the Vmax of the 5-phosphatase for IP3 could not be determined, the Vmax must be greater than 16 nM s-1, the fastest rate measured for the 5-phosphatase (Fig. 3). The Km of the 5-phosphatase for IP3 must then be greater than 27 µM. The first order behavior of the 5-phosphatase at IP3 concentrations up to 30 µM also suggests that the Km is greater than 30 µM. The lower limit of the Km value and the absence of regulation by [Ca2+] are consistent with the properties of the 5-phosphatase in other species (4-8, 48-50). For all [Ca2+], the Km and Vmax values of the 5-phosphatase for IP3 were much greater than those of the 3-kinase for IP3. The relative values of the Km and Vmax of the 5-phosphatase and 3-kinase divided IP3 metabolism into three regions. The regions were defined by their [Ca2+] and [IP3] and were characterized by their different metabolic routes for IP3. At high [Ca2+] (>= 400 nM) but low [IP3] (<= 8 µM), IP3 was predominantly metabolized by the 3-kinase to IP4. The 5-phosphatase and the 3-kinase degraded roughly similar amounts of IP3 when both [Ca2+] and [IP3] were low (<400 nM and <1 µM, respectively). However, the 5-phosphatase was the dominant metabolic enzyme when [IP3] was greater than 8 µM irrespective of [Ca2+]. The regions that occur physiologically are not known since the intracellular range of [IP3] has yet to be defined.

IP4 was metabolized very slowly with a half-life of approximately 30 min. The k value of the 5-phosphatase for IP4 was similar to that for IP3. The reported Km of the 5-phosphatase for IP4 in other species is approximately 1 µM (7, 51). When the initial [IP4] was 10 µM, the metabolism of IP4 followed first order kinetics. Therefore, it is unlikely that the Km of the 5-phosphatase for IP4 in Xenopus oocytes was as low as 1 µM. When [Ca2+] was elevated, phosphorylation of IP3 to IP4 was much faster than degradation of IP4. Consequently, repeated or persistent stimulation of IP3 production in the presence of high nanomolar to micromolar [Ca2+], could result in IP4 concentrations that are far greater than that of IP3. Although opening of the IP3 receptor/Ca2+ channel requires 10-fold more IP4 than IP3, the accumulation of IP4 may result in physiologically important IP4-mediated Ca2+ release. IP4 may play a necessary role in sustained signal transduction.

The k and Vmax values of the oocyte's 3-kinase and 5-phosphatase were markedly lower than the values reported in other species (4-12, 48-50). Since IP3 microinjected into intact oocytes was also metabolized slowly, the long IP3 degradation times were not an artifact of the cytosolic preparation. Additionally, IP4 microinjected into intact oocytes has a half-life similar to that reported here in the cytosolic extract (29, 30). The lower k and Vmax values of the Xenopus oocyte compared with those of mammalian cells may be a reflection of the different size, temperature, and physiologic role of these cells. Slowing the degradation of IP3 may be important for signal transduction from the cell's surface to its interior. The 500-µm radius of the oocyte makes the transmission of information from the plasma membrane to the central core formidable. Slowing the metabolism of IP3 increases its range of action, the distance over which a spatially localized impulse of IP3 can diffuse (34). The range of action (R) of IP3 can be estimated from its diffusion coefficient (D) and half-life (t1/2) in cytoplasm: r = (2*D*t1/2)1/2. D for IP3 is 280 µm2 s-1, and the t1/2 for IP3 in the oocyte ranges from 1 to greater than 5 min. R for IP3 is ~300 µm suggesting that IP3 acts as a long-ranged messenger in the oocyte. In nearly all cell types, IP3 is a global messenger (Table I). Whereas the rate of degradation of IP3 and the size of a cell vary over several orders of magnitude, the range of action of IP3 is nearly the same as the cellular dimensions. IP3 transmits signals received at the plasma membrane throughout the cell, coordinating cell-wide activities.

                              
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Table I
The range of action of IP3

    ACKNOWLEDGEMENTS

We gratefully acknowledge Tobias Meyer and Lubert Stryer for stimulating discussions and H. N. Dao for technical assistance.

    FOOTNOTES

* This work was supported by the Arnold and Mabel Beckman Foundation, the Searle Scholars Program, and National Institute of Mental Health Grant MH45324.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 714-824-6493; Fax: 714-824-8540.

1 The abbreviations used are: [Ca2+], free Ca2+ concentration; I-1-P1, inositol 1-phosphate; IP1, all isomers of inositol monophosphate; I-1,4-P2, inositol 1,4-bisphosphate; IP2, all isomers of inositol bisphosphate; IP3, inositol 1,4,5-trisphosphate; I-1,3,4-P3, inositol 1,3,4-trisphosphate; IP4, inositol 1,3,4,5-tetrakisphosphate; [IP3], concentration of IP3; [IP4], concentration of IP4; IPless or equal 2, inositol, IP1, and IP2; [IPless or equal 2], the sum of the concentration of inositol, IP1 and IP2; 5-phosphatase, inositol polyphosphate 5-phosphatase; 3-kinase, inositol 1,4,5-trisphosphate 3-kinase; HPLC, high pressure liquid chromatography; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
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