Inositol cyclic trisphosphate [inositol 1,2-(cyclic)-4,5-trisphosphate] is ...

Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6397-6401, September 1986 Biochemistry

Inositol cyclic trisphosphate [inositol 1,2-(cyclic)-4,5-trisphosphate] is formed upon thrombin stimulation of human platelets (phosphatidylinositol/inositol phosphates)

HIDEMI ISHII, THOMAS M. CONNOLLY, TERESA E. BROSS, AND PHILIP W. MAJERUS* Division of Hematology-Oncology, Departments of Internal Medicine and Biological Chemistry, Washington University School of Medicine, St. Louis, MO 63110

Contributed by Philip W. Majerus, May 27, 1986

Cleavage of polyphosphoinositides in vitro by ABSTRACT phospholipase C results in formation of both cyclic and noncyclic inositol phosphates. We have now isolated the cyclic product of phosphatidylinositol 4,5-bisphosphate cleavage, inositol 1,2-(cydic)-4,5-trisphosphate [cIns(1:2,4,5)P3], from thrombin-treated platelets. We found 0.2-0.4 nmol of cIns(1:2,4,5)P3 per 10' platelets at 10 sec after thrombin; none was found in unstimulated platelets or in platelets 10 min after thrombin addition. We conclude that clns(1:2,4,5)P3 is a major product of polyphosphoinositide metabolism in thrombinstimulated platelets. Enormous interest in phosphoinositide metabolism has followed the discoveries that these lipids serve as storage forms for a variety of messenger molecules released in response to specific extracellular signals (for reviews, see refs. 1-4). One of these messenger molecules, inositol 1,4,5-trisphosphate [Ins(1,4,5)P31, is able to mediate calcium ion mobilization (5). Inositol phosphates are released from their phospholipid precursors by the action ofphospholipase C. A single enzyme from ram seminal vesicles utilizes all three phosphoinositides (6), producing a mixture of inositol noncyclic and 1,2-cyclic phosphates (7-9). Inositol 1,2-(cyclic)-4,5-trisphosphate [cIns(1:2,4,5)P3], produced from phosphatidylinositol 4,5bisphosphate [PtdIns(4,5)P2], stimulates calcium mobilization (9) and serotonin secretion (10) in platelets with a potency similar to that of Ins(1,4,5)P3. clns(1:2,4,5)P3 was found to be =5 times more potent than Ins(1,4,5)P3 in producing a change in membrane conductance similar to that evoked by light when injected into Limulus photoreceptor cells (9). These findings suggested that cIns(1:2,4,5)P3 might serve a messenger function in stimulated cells. We now describe the isolation and identification of clns(1:2,4,5)P3 in thrombin-stimulated human platelets.

MATERIALS AND METHODS Materials. [32P]phosphoric acid was from New England Nuclear. [3H]Ins(1,4,5)P3 and [3H]clns(1:2,4,5)P3 were prepared as described (9). Matrex gel PBA-60 (phenyl boronate agarose) and Centricon 10 microconcentrators were from Amicon. The Partisil SAX HPLC column, silicic acid, and Partisil SAX pellicular guard resins were from Whatman. Dowex formate (AG 1 x 8, 200-400 mesh) was from Bio-Rad. All other materials were from Sigma or Fisher. Platelet Labeling. Human platelets were obtained from normal donors as described (11). The platelets were washed, suspended at 1 x 109 cells per ml in 15.4 mM Tris-HCl, pH 7.4/140 mM NaCl/5.6 mM glucose/bovine serum albumin (1 mg/ml), and incubated with [32P]orthophosphoric acid (100

,uCi/ml; 1 Ci = 37 GBq) for 90 min. They were then washed and suspended at 1 x 109 cells per ml in the same buffer. Isolation of Inositol Phosphates After Thrombin Stimulation. Platelet suspensions were incubated 5 min at 370C, and then stimulated by the addition of thrombin (5 units/ml). After 10 sec (or at other times where indicated), 24 ml of thrombin-treated and control platelet suspensions were transferred to glass tubes containing 6 ml of 2 M KC1/0.1 M EDTA and 84 ml of chloroform/methanol (2:5). At this time, an internal standard of 16,000 cpm of [3H]Ins(1,4,5)P3 (200 cpm/nmol) and 20,000 cpm of [3H]cIns(1:2,4,5)P3 (200 cpm/nmol) was added to the sample of unstimulated and thrombin-stimulated platelets, respectively. The mixtures were left at room temperature for 1 hr followed by 24 ml of chloroform and 12 ml of 2 M KC1/O.1 M EDTA. After extraction, the upper aqueous phase was diluted with H20 to the same conductivity as 50 mM NH4COOH (1.5 liters), and then applied at 85 ml/hr to a 1.5-ml Dowex formate column, which was equilibrated with 50 mM NH4COOH (pH 6.25). The column was washed successively with 20 ml of 50 mM NH4COOH, 50 ml of 300 mM NH4COOH, and 150 ml of 400 mM NH4COOH (pH 6.25); then the inositol trisphosphates were eluted with 30 ml of 1 M NH4COOH (pH 6.25). [Recovery of clns(1:2,4,5)P3 was --75% in this step.] The eluted fraction was desalted by lyophilization and then dissolved in 2 ml of 0.2 M triethylammonium bicarbonate (pH 8.8) (TEAB) containing 15 mM MgCl2. The dissolved sample was applied to a 1-ml column of phenyl boronate agarose, which was equilibrated in the same buffer. The flow-through fraction from the phenyl boronate column was collected and desalted by lyophilization. The lyophilized samples were dissolved in 3 ml of H20 and applied to 0.2-ml Dowex formate columns equilibrated with 50 mM NH4COOH (pH 6.25). The columns were washed with 16 ml of 400 mM NH4COOH (pH 6.25) and then the inositol trisphosphates were eluted with 700 mM NH4COOH (pH 6.25). A small amount of material was eluted later in 1 M NH4COOH (pH 6.25). The eluted fractions were desalted by lyophilization. HPLC of Inositol Trisphosphates. We used a precolumn of silicic acid (4 x 250 mm) (mounted before the injector loop) and a Partisil SAX guard column (4 x 50 mm) before a Partisil SAX HPLC separating column (4.6 x 250 mm) (9, 12). The flow rate was 1 ml/min and 1-ml fractions were collected and assayed for radioactivity in a Beckman liquid scintillation counter either by measuring Cerenkov radiation to determine 32p content or with Scintiverse scintillation fluid to determine the tritiated internal standard. Abbreviations: Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; Ins(1,3,4)P3, inositol 1,3,4-trisphosphate; cIns(1:2,4,5)P3, inositol 1,2-(cyclic)4,5-trisphosphate; Ptdlns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; Ins(1,4)P2, inositol 1,4-bisphosphate; Ins(1,3,4,5)P4, inositol

1,3,4,5-tetraphosphate.

*To whom reprint requests should be addressed at: Division of Hematology-Oncology, Washington University School of Medicine, 660 South Euclid, St. Louis, MO 63110.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6397

6398

Proc. Natl. Acad. Sci. USA 83 (1986)

Biochemistry: Ishii et al.

Acidification of Inositol Trisphosphates. A portion of lyophilized sample from the 700 mM NH4COOH eluate fraction from thrombin-treated platelets was mixed with an equal volume of 2 M HCl. After 2 min at room temperature, the sample was frozen in liquid nitrogen, lyophilized to remove acid, and then dissolved in H20. Ins(1,4,5)P3 5-Phosphomonoesterase Treatment of cIns(1:2,4,5)P3 Fraction. A sample of a 700 mM NH4COOH eluate fraction from thrombin-treated platelets was applied to HPLC. The cIns(1:2,4,5)P3-containing fractions were collected and lyophilized. The lyophilized sample containing 600 cpm of internal standard [3H]Ins(1:2,4,5)P3 and 2000 cpm of 3 P radioactivity was incubated at 370C with 50 mM 2-(Nmorpholino)ethanesulfonic acid, pH 6.5/3 mM MgCl2/60 FkM each unlabeled Ins(1,4,5)P3 and cIns(1:2,4,5)P3, [3H]Ins(1,4,5)P3 (2000 cpm), and 5.5 units of Ins(1,4,5)P3 5-phosphomonoesterase [1 unit = 1 nmol of Ins(1,4,5)P3 hydrolyzed per min] (13) in 0.37 ml for 100 min. The reaction was stopped by dilution with an equal volume of ice-cold H20 followed by filtration on a Centricon 10 microconcentrator at 4°C for 1 hr to remove protein.

RESULTS In previous studies of phosphoinositide breakdown in cells in response to agonists, reactions have been stopped by acidification prior to separation of the lipid substrates from the water-soluble inositol phosphate products. Since cIns(1:2,4,5)P3 is acid labile (8), this compound was not detected in prior studies. In preliminary experiments, we found that inositol phosphates are difficult to separate from precipitated protein upon neutral pH solvent extraction. However, reasonable yields (50-75%) of added radiolabeled cIns(1:2,4,5)P3 could be obtained in the aqueous phase of a chloroform/ methanol/water extract of platelets when high concentrations of KCl and EDTA were present. A further difficulty in detecting cIns(1:2,4,5)P3 in human platelets is that [3H]inositol labeling cannot be used satisfactorily because of the relative impermeability of platelets to inositol. Therefore, we used platelets labeled with 32P04, which required more extensive methods for separation of cIns(1:2,4,5)P3 from nucleotides and other phosphate esters. Separation of Nucleotides and Inorganic Phosphate from Inositol Phosphates. We used phenyl boronate agarose chromatography (14) to remove 32P-labeled nucleotides from our samples as shown in Fig. 1. Under the conditions used, nucleotides are retained, while inositol phosphates and inorganic phosphate are in the flow-through fraction. The flowthrough fraction was then applied to a second Dowex formate column, as described, to remove additional inorganic phosphate. After this step, the recovery of internal standard ranged from 25% to 30% with =80% in the 700 mM NH4COOH eluate and the remainder in the 1 M eluate. Characterization of [32P]clns(1:2,4,5)P3 from ThrombinStimulated Platelets. An HPLC chromatogram of 12% of the 700 mM NH4COOH eluate from thrombin-treated and control platelets is shown in Fig. 2. The thrombin-treated sample contains greater 32P radioactivity eluting with standard cIns(1:2,4,5)P3 and Ins(1,4,5)P3 (1012 cpm in this experiment). The identity of this radioactivity as cIns(1:2,4,5)P3 in samples from thrombin-treated platelets was established further as described below. There was also 32P radioactivity eluting later in a position suggesting a more anionic compound. In several experiments, we found variable amounts of 32P eluting in fractions 43-45 in the position of inositol trisphosphate, but it was never metabolized by the Ins(1,4,5)P3 5-phosphomonoesterase. Thus, it is likely to be inositol 1,3,4-trisphosphate [Ins(1,3,4)P3]. A standard of Ins(1,3,4)P3 prepared by treatment of [3H]inositol 1,3,4,5-tetraphosphate [Ins(1,3,4,5)P4]

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FIG. 1. Phenyl boronate chromatography. Samples were prepared from [32P]phosphate-labeled platelets. Samples from untreated (control) and thrombin-treated (THR) (5 units/ml) platelets were applied to separate 1-ml phenyl boronate agarose columns and eluted as described; l-ml fractions were collected.

(kindly provided by Robin Irvine) with Ins(1,4,5)P3 5phosphomonoesterase comigrates with Ins(1,4,5)P3 in our HPLC system. The HPLC chromatogram in Fig. 3 compares samples with and without acid treatment from thrombin-treated platelets. The 32P radioactivity in fractions 38-42 diminishes after acid treatment, shifting to fractions 42-46, where Ins(1,4,5)P3

elutes. The [3H]inositol-containing clns(1:2,4,5)P3 internal standard shows the same behavior with a shift in the position of elution from that of cIns(1:2,4,5)P3 to that of Ins(1,4,5)P3. We counted one-eighth of the sample in these runs shown in Fig. 3. In the pooled fractions 38-42, radioactivity decreased by 976 cpm after acid treatment, indicating loss of cIns(1:2,4,5)P3 with a corresponding increase in radioactivity of 650 cpm in fractions 43-46 corresponding to Ins(1,4,5)P3. The identity of clns(1:2,4,5)P3 was further established in another experiment in which we chromatographed a sample of the 700 mM NH4COOH eluate fraction from thrombintreated platelets (10 sec) on a Partisil SAX HPLC column and collected the cIns(1:2,4,5)P3 fractions. These fractions were pooled, lyophilized, and then incubated with equal concentrations (60 ,uM) of unlabeled Ins(1,4,5)P3 and cIns(1:2,4,5)P3 plus [3H]Ins(1,4,5)P3 and Ins(1,4,5)P3 5-phosphomonoesterase. Nearly all of the added [3H]Ins(1,4,5)P3 was converted to [3H]-inositol 1,4-bisphosphate [Ins(1,4)P2] as shown in Fig. 4. The internal standard [3H]cIns(1:2,4,5)P3 and the corresponding 32P-labeled compound from platelets were converted to cIns(1:2,4)P2 and inorganic phosphate to equal extents (28%). The incomplete hydrolysis of cIns(1:2,4,5)P3 is expected since the 5-phosphomonoesterase utilizes this substrate poorly compared to Ins(1,4,5)P3 (12). This result indicates that nearly all of the 32P radioactivity in Fig. 4 was cIns(1:2,4,5)P3. No inositol trisphosphate of either isomer was present in the fractions used in this experiment. Any Ins(1,4,5)P3 present would have been converted to [32p]Ins(1,4)P2, while any Ins(1,3,4)P3 would have remained as 32p radioactivity eluting in the position of the inositol trisphosphate isomers (fractions 47 and 48). In this experiment, 40% of the sample was counted, and the total recoveries in the various peaks were as follows: [3H]cIns(1:2,4)P2, 134 cpm; [3H]cIns(1:2,4,5)P3, 345 cpm; [32P]clns(1:2,4)P2 plus 32P04, 437 cpm; [32P]cIns(1:2,4,5)P3, 1180 cpm.


Proc. Natl. Acad. Sci. USA 83 (1986) I

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FRACTION NUMBER FIG. 2. HPLC of inositol trisphosphates from control and thrombin-treated platelets. Platelets were treated with thrombin and the inositol trisphosphate fraction was isolated as described. Equal 32P radioactivity was used for each chromatogram. o-o, 32P thrombin; -o, 32P control; o-o, [3H]clns(1:2,4,5,)P3 internal standard; Ae, [3H]Ins(1,4,5)P3 internal standard. Elution positions of standards are noted. Pi, inorganic phosphate; IP2, Ins(1,4)P2; cIP3, cIns(1:2,4,5)P3; IP3, inositol trisphosphate.

We found cIns(1:2,4,5)P3 in thrombin-treated platelets (10 sec) in each of three experiments. There was no clns(1:2,4,5)P3 found in unstimulated platelets or in platelets incubated for 10 min with thrombin. We used the internal [3H]clns(1:2,4,5)P3 standard to determine the amount of [32P]cIns(1:2,4,5)P3 formed in the platelets. ([32P]cIns(1:2,4,5)P3 was estimated

by determining the acid-labile radioactivity in the cIns(1:2,4,5)P3 fractions.) We estimated the specific activity of cIns(1:2,4,5)P3 by measuring the specific activity of its lipid precursor PtdIns(4,5)P2 in platelets incubated with 32P04 under the same conditions in a separate experiment. Based on these determinations, we calculate that 10 sec after 1P2 cIP3 IP3

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6400


Proc. Natl. Acad. Sci. USA 83 (1986)

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FIG. 4. HPLC after Ins(1,4,5)P3 5-phosphomonoesterase treatment of cIns(1:2,4,5)P3 fraction. A sample containing the pooled fractions corresponding to the clns(1:2,4,5)P3 region on HPLC chromatography was treated with the Ins(1,4,5)P3 5-phosphomonoesterase as described. a0-, 32p; 3H. Elution positions of standards are noted as in Fig. 2. The HPLC column used in this experiment was a different one from that used in Figs. 2 and 3, which explains the slightly different elution of standard compounds. See text for details. a-a,

thrombin addition, the levels of cIns(1:2,4,5)P3 were 0.22, 0.35, and 0.43 nmol per 109 platelets in three experiments. These values are similar to the decrease in mass of Ptdlns(4,5)P2 observed after thrombin stimulation of platelets (15, 16) and are also similar to the direct measurement of Ins(1,4,5)P3 mass in thrombin-stimulated platelets made by Rittenhouse (0.17 nmol per 109 platelets) (17). DISCUSSION Several recent reports indicate the presence of inositol cyclic phosphates in stimulated cells. Inositol 1,2-(cyclic)phosphate has been isolated from stimulated mouse pancreatic cells (18), kidney cells (19), platelets (20), and simian virus 40-transformed mouse cells (21). Numerous other studies of phosphoinositide metabolism have failed to detect cIns(1:2,4,5)P3 or other inositol cyclic phosphates because the conditions used for their isolation destroy these labile compounds. Irvine et al. (22) have recently shown that rat brain contains a kinase that converts Ins(1,4,5)P3 to Ins(1,3,4,5)P4. The Ins(1,3,4,5)P4 thus formed is converted to Ins(1,3,4)P3, presumably by a 5-phosphomonoesterase (23). These workers reported that cIns(1:2,4,5)P3 is a good substrate for the kinase enzyme, which would thereby make cIns(1:2,3,4,5)P4 (22). We found that Ins(1,4,5)P3 5-phosphomonoesterase converts the 32P radioactivity that migrates in fractions 50-55 (see Figs. 2 and 3) to a compound that elutes from HPLC in the position of the inositol trisphosphate isomers (data not shown). This result is consistent with the conversion of Ins(1,3,4,5)P4 to Ins(1,3,4)P3. Whether these compounds contain any cyclic esters-i.e., cIns(1:2,3,4,5)P4 and cIns(1:2,3,4)P3-is currently unknown since we do not know if our HPLC system will resolve these cyclic from the corresponding noncyclic compounds. Why do we find only cIns(1:2,4,5)P3 in the extract from platelets, whereas in enzymatic reactions with purified phospholipase C we observe both cyclic and noncyclic products? It is possible that Ins(1,4,5)P3 is also found in platelets and that we did not detect it. The present experiments cannot be used to determine the quantitative produc-

tion of Ins(1,4,5)P3 in stimulated platelets. Because we did not add an internal standard of Ins(1,4,5)P3 to the thrombintreated samples in our experiments, we cannot be certain that our failure to detect Ins(1,4,5)P3 did not result from its loss or degradation. Similarly, we cannot exclude the possibility

that Ins(1,4,5)P3 is formed in thrombin-stimulated platelets but is rapidly converted to Ins(1,4)P2 or Ins(1,3,4,5)P4 prior to our earliest (10 sec) time point. In either case, in stimulated platelets a major product of polyphosphoinositide metabolism is the cIns(1:2,4,5)P3 product. If cells form both cIns(1:2,4,5)P3 and Ins(1,4,5)P3, it may be that differences in the subsequent metabolism of these compounds determines their physiological functions. For example, the Ins(1,4,5)P3 5-phosphomonoesterase degrades Ins(1,4,5)P3 in preference to cIns(1:2,4,5)P3 by at least a factor of 10 (see Fig. 4). The relative physiological importance of the cyclic vs. noncyclic phosphate esters of inositol in vivo cannot be assessed with the limited information currently available. This research was supported by Grants HLBI 14147 (Specialized Center for Research in Thrombosis), HL 16634, and Training Grant T32 HLBI 07088 from the National Institutes of Health. 1. Majerus, P. W., Wilson, D. B., Connolly, T. M., Bross, T. E. & Neufeld, E. J. (1985) Trends Biochem. Sci. 10, 168-171. 2. Majerus, P. W., Neufeld, E. J. & Wilson, D. B. (1984) Cell 37,

701-703. 3. Berridge, M. L. & Irvine, R. F. (1984) Nature (London) 312, 315-321. 4. Nishizuka, Y. (1984) Nature (London) 308, 693-697. 5. Berridge, M. J. (1984) Biochem. J. 220, 345-360. 6. Wilson, D. B., Bross, T. E., Hofmann, S. L. & Majerus, P. W. (1984) J. Biol. Chem. 259, 11718-11724. 7. Dawson, R. M. C., Freinkel, N., Jungalwala, F. B. & Clarke, N. (1971) Biochem. J. 122, 605-607. 8. Wilson, D. B., Bross, T. E., Sherman, W. R., Berger, R. A. & Majerus, P. W. (1985) Proc. Nati. Acad. Sci. USA 82, 4013-4017. 9. Wilson, D. B., Connolly, T. M., Bross, T. E., Majerus, P. W., Sherman, W. R., Tyler, A. N., Rubin, L. J. & Brown, J. E. (1985) J. Biol. Chem. 260, 13496-13501. 10. Wilson, D. B., Connolly, T. M., Ross, T. S., Ishii, H., Bross,


11. 12. 13. 14.

15.

16.

T. E., Deckmyh, H., Brass, L. F. & Majerus, P. W. (1986) Adv. Prostaglandin Thromboxane Leukotrine Res., in press. Baenziger, N. L. & Majerus, P. W. (1974) Methods Enzymol. 31, 149-155. Connolly, T. M., Wilson, D. B., Bross, T. E. & Majerus, P. W. (1986) J. Biol. Chem. 261, 122-126. Connolly, T. M., Bross, T. E. & Majerus, P. W. (1985) J. Biol. Chem. 260, 7868-7874. Walseth, T. F., Gander, J. E., Eide, S. J., Krick, T. P. & Goldberg, N. D. (1983) J. Biol. Chem. 258, 1544-1558. Wilson, D. B., Neufeld, E. J. & Majerus, P. W. (1985) J. Biol. Chem. 260, 1046-1051. Broekman, M. J. (1984) Biochem. Biophys. Res. Commun. 120, 226-231.

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17. Rittenhouse, S. E. & Sasson, J. P. (1985) J. Biol. Chem. 260, 8657-8661.

18. Dixon, J. F. & Hokin, L. E. (1985) J. Biol. Chem. 260, 16068-16071. 19. Shayman, J. A., Auchus, R. J. & Morrison, A. R. (1985) Clin. Res. 33, 498A (abstr.). 20. Binder, H., Weber, P. C. & Siess, W. (1985) Anal. Biochem. 148, 220-227. 21. Koch, M. A. & Diringer, H. (1974) Biochem. Biophys. Res. Commun. 58, 361-367. 22. Irvine, R. F., Letcher, A. S., Heslop, J. P. & Berridge, M. J. (1986) Nature (London) 320, 631-634. 23. Batty, I. R., Nahorski, S. R. & Irvine, R. F. (1985) Biochem.

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