Isolation and Characterization of the Inositol Cyclic Phosphate Products of Polyphosphoinositide Cleavage by Phospholipase C

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The American Society of Biological Chemists, Inc. Vol. 260, No. 25, Issue of November 5, pp Printed in i! S. A. Isolation and Characterization of the Inositol Cyclic Phosphate Products of Polyphosphoinositide Cleavage by Phospholipase C PHYSIOLOGICAL EFFECTS IN PERMEABILIZED PLATELETS AND LIMULUS PHOTORECEPTOR CELLS* (Received for publication, May 28, 1985) David B. Wilson, Thomas M. Connolly, Teresa E. Bross, Philip W. MajerusS, William R. Sherman, Andrew N. Tyler, Leona J. Rubin, and Joel E. Brown From the Departments of Medicine, Biochemistry, Psychiatry, and Ophthulmology, Washington University School of Medicine, St. Louis, Missouri Cleavage of the polyphosphoinositides, catalyzed by phospholipase C purified from ram seminal vesicles, produces phosphorylated inositols containing cyclic phosphate esters (Wilson, D. B., Bross, T. E., Sherman, W. R., Berger, R. A., and Majerus, P. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, ). In the present study we describe the isolation and characterization of inositol l:%-cyclic 4-bisphosphate and inositol l:2-cyclic 4,5-trisphosphate, the two cyclic phosphate products of phospholipase C catalyzed cleavage of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, respectively. We established the structures of these two cyclic compounds through '*O labeling of phosphate moieties, phosphomonoesterase digestion, and fast atom bombardment-mass spectrometry. We examined the physiological effects of these compounds in two systems: saponin-permeabil- ized platelets loaded with 4SCa2+ and intact Limulus photoreceptors. Both inositol l:2-cyclic 4,5-trisphosphate and the noncyclic inositol 1,4,5-trisphosphate, but not inositol l:2-cyclic 4-bisphosphate, release 45Ca2+ from permeabilized platelets in a concentrationdependent manner. Injection of inositol l:2-cyclic 4,5- trisphosphate into Limulus ventral photoreceptor cells induces both a change in membrane conductance and a transient increase in intracellular calcium ion concentration similar to those induced by light. We injected inositol 1,4,5-trisphosphate and inositol l:2-cyclic 4,5- trisphosphate into the same photoreceptor cell and found that the cyclic compound is approximately five times more potent than the noncyclic compound in stimulating a conductance change. We speculate/ that inositol l:2-cyclic 4,5-trisphosphate may functiod as a / second messenger in stimulated cells. Stimulation of a variety of cells with the appropriate agonists results in the phospholipase C mediated cleavage of *This research was supported by National Institutes of Health Grants HLBI (Specialized Center for Research in Thrombosis), HL16634, and T32-HL (to P. W. M.); NS (to W. R. S.); EY and EY (to J. E. B.). The Mass Spectrometry Facility (RR00954) at Washington University is a Core Facility of the Diabetes Research and Training Center (AM 20579). 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 reprint requests should be addressed at: Division of Hematology-Oncology, Washington University School of Medicine, 660 South Euclid, St. Louis, MO phosphatidylinositol (PtdInsl) and the polyphosphoinositides, phosphatidylinositol 4-phosphate (PtdIns-4-P) and phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-Pz)(for reviews, see Refs. 1-4). Agonist-induced phosphoinositide metabolism generates a number of cellular messenger molecules. Phosphoinositide-derived diglyceride can serve to activate protein kinase C (5) or can be further metabolized by diglyceride and monoglyceride lipase activities to yield free arachidonic acid for icosanoid synthesis (6, 7). Another product of PtdIns-4,5-Pz hydrolysis by phospholipase C, inositol 1,4,5- trisphosphate (IP3), is a Ca'+-mobilizing agent in a number of experimental systems (4; 8-16). Dawson et al. (17) have shown that cleavage of PtdIns by phospholipase C produces a mixture of two water-soluble products: inositol 1-phosphate (IP1) and inositol l:2-cyclic phosphate (cip,). We recently showed that all three phosphoinositides are hydrolyzed by the same phospholipase C (18). Therefore, by analogy to the PtdIns reaction, cyclic 1:2- phosphate esters might be anticipated among the phospholipase C cleavage products of PtdIns-4-P and PtdIns-4,5-Pz. Recently, we employed "0 incorporation into the phospholi- pase C reaction products to demonstrate that cyclic phosphate esters are present among the water-soluble products of polyphosphoinositide cleavage by phospholipase C (19). We concluded that cleavage of PtdIns-4-P produces inositol 1,4- bisphosphate (IPz), and the cyclic product inositol l:2-cyclic 4-phosphate (cipz), while cleavage of PtdIns-4,5-Pz produces both inositol 1,4,5-trisphosphate (IP3) and the cyclic product inositol k2-cyclic 4,5-triphosphate (cip3). These two cyclic inositol polyphosphates are candidates for second messengers in cells. In the present study we have isolated cipz and cip3 and examined the physiological effects of these compounds in two systems: saponin-permeabilized platelets and intact Limulus photoreceptor cells. EXPERIMENTAL PROCEDURES Materials-Phospholipids, ATP, creatinine phosphate, and creatine phosphokinase were purchased from Sigma. Inositol l:2-cyclic phosphate (cyclohexylamine salt) was provided by Merck Sharp and The abbreviations used are: PtdIns, phosphatidylinositol; PtdIns- 4-P, phosphatidylinositol 4-phosphate; PtdIns-4,5-P2, phosphatidylinositol 4,5-bisphosphate; IP1, myo-inositol l-phosphate; IP2, myo- inositol l,4-bisphosphate; IP3, myo-inositol1,4,5-trisphosphate; cip1, myo-inositol 1:2-cyclic phosphate; cip2, myo-inositol 1:e-cyclic phosphate 4-monophosphate; cip3, myo-inositol l:2-cyclic phosphate 4 5 bisphosphate; GroPIns-4,5-P2, glycerophosphorylinositol 4,5-bisphosphate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, ethylene glycol bis(8-aminoethy1ether)-n,n,n',n'-tetraacetic acid; FAB, fast atom bombardment; HPLC, high performance liquid chromatography.

2 Isolation and Characterization of the Inositol Cyclic Phosphate Products Dohme, Rahway, NJ. H;80 (>95% enriched) was obtained from p~ stock solutions of I&, IP2, cip3, or cip, followed by incubation Monsanto/Mound Laboratories, Miamisburg, OH. Aequorin was ob- for 3 min at room temperature. The reactions were stopped by dilution tained from Dr. J. R. Blinks, Mayo Foundation, Rochester, MN. ["PI with 4 ml of 120 mm KC1,4 mm MgC12,0.5 mm EGTA, 20 mm Hepes, Orthophosphoric acid, my~-[~h]inositol, and %a'+ were obtained ph 7.1, and immediate filtration on 0.45-pm HAWP Millipore filters from New England Nuclear. [32P]Inositol 1,4-bisphosphate and ["PI previously soaked in 1% bovine serum albumin. The filters were inositol 1,4,5-trisphosphate were prepared from labeled erythrocyte washed twice with 4-ml portions of 120 mm KCl, 4 mm MgCl2, 0.5 ghosts using the method of Downes et al. (20). A sample of ox brain mm EGTA, 20mM Hepes, ph 7.1, and then counted in 10 ml of IP3 for use in the Limulus photoreceptor studies was a gift of Dr. R. Scintiverse I scintillation mixture to determine the &Ca" remaining F. Irvine, ARC Institute, Babraham, Cambridge, United Kingdom. in the cells. The difference in 45Ca2+ retained in control platelets [32P]PtdIns-4-P and [32P]PtdIns-4,5-Pz were isolated from labeled versus those treated with 1 ~ L M A23187 was considered to be 100% human erythrocytes (21) by column chromatography on neomycin- release. The &Ca released by inositol phosphates is expressed as a glass beads (22,23). [3H]PtdIns, [3H]PtdIns-4-P, and [3H]PtdIns-4,5- percentage of the *Ca'+ released by A Pz were isolated from [3H]inositol-labeled LM cells (24) by the same Intracellular Injection into Limulus Photoreceptors-Ventral rudimethods (22, 23). Phospholipase C enzymes were purified from ram mentary eyes (29) were dissected from Limulus polyphemus and seminal vesicles as described previously (18,24). Human platelet IP3- prepared for electrophysiology (30). Single photoreceptors were im- 5-phosphomonoesterase was purified by the method of Connolly et paled with double-barreled (0 glass) micropipettes that were used both al. (25). Small unilamellar phospholipid vesicles containing labeled to measure membrane voltage and to pressure inject intracellularly phosphoinositide and phosphatidylethanolamine were prepared as solutions out of both barrels. By this technique, the effects of injection described (26). of two soutions could be made at the same locus in a cell; thus spatial Phospholipase C Reaction-A typical reaction mixture contained differences in effects of injection (31, 32) did not confound the vesicles of phosphoinositide:phosphatidylethanolamine (1:0.4, mol/ findings. The relative sizes of injections out of both barrels were mol; 300 p~ [32P]phosphoinositide( cpm/nmol by Cerenkov compared optically (at 940 nm) by the technique of Corson and Fein counting)), 10 mm Tris acetate, ph 5.3, 37 mm NaCl, 1 mm CaCl2, (33). The volumes of individual injections were estimated to vary and 2 pg of phospholipase C in a total volume of 1 ml. The mixture from about 10"l to less than 10"' liters; the volume of a typical was incubated 15 min at 37 "C, and the reaction was then terminated Limulus ventral photoreceptor is about 5 X 10-l' liters. Values are by the addition of 5 ml of chloroform/methanol (l:l, v/v) and 1.5 ml given for the concentrations of substances in the electrode-filling of HzO. The upper phase of this reaction mixture was removed and solutions. In some experiments the cell was impaled with a second dried in a shaker evaporator after the addition of 10 pl of 1 M NaHC03. micropipette filled with 2 M KC1; this pipette was used to pass current The dried material was dissolved in 1 ml of water and the reaction in order to voltage-clamp a cell (34, 35). Changes in calcium ion products were separated by HPLC as described below. concentration within Limulus photoreceptors were monitored by ae- HPLC of Inositol Phosph~tes-~~P-labeled phosphorylated inositols quorin (36). The aequorin luminescence was detected by a photomulor the tritiated compounds were resolved on a Whatman Partisil 10 tiplier tube (R105UH, Hamamatsu) operated in the photon-counting SAX column (Cobert Associates, St. Louis, MO). The elution scheme mode at dry ice temperature. consisted of isocratic elution with 50 mm ammonium formate, ph Other Methods-The concentration of inositol phosphate solutions 6.25, for 10 min, followed by a linear gradient from 50 mm to 2.7 M was determined by phosphate analysis (37). In some experiments cip3 ammonium formate, ph 6.25, over the next 20 min, and then isocratic and IP3 were isolated from the same phospholipase C reaction mixture elution at 2.7 M ammonium formate, ph 6.25, for 20 min. The flow and used for injection into photoreceptors thereby allowing for estirate was 1 ml/min and 1-ml fractions were collected and assayed for mation of the concentrations of the two compounds by measurement radioactivity by Cerenkov counting. Under these conditions, the of radioactivity. Protein was measured by a Coomassie Blue protein following retention times were obtained inositol, 4 min; cipl, 8 min; assay (Bio-Rad). IPl, 23 min; Pi, 25 min; cip2, min; IP2, 30 min; cip3, 36 min; IP3, min. RESULTS Desalting-HPLC fractions containing cyclic or noncyclic inositol In a previous study (19) we demonstrated two water-soluble phosphates were desalted on a 1 X 90-cm Sephadex G-10 (Sigma) column equilibrated with 7% 1-propanol in water. The flow rate was products of phospholipase C cleavage of PtdIns-4-P, cip2, and 1.8 ml/min and 2-ml fractions were collected. Fractions containing IP2. The cyclic and noncyclic products were not fully resolved inositol phosphates were pooled, lyophilized, and dissolved in either by anion exchange HPLC at ph 4.3. Because cyclic inositol water or 100 mm Hepes, ph 7.4,200 mm KC1 at concentrations phosphates are unstable at low ph, we developed an HPLC ranging from 0.2 to 10 mm. method that operates at ph This method resolves the Fast Atom Bombardment (FAB)-Mass Spectrometry-FAB-mass cyclic and noncyclic inositol phosphate products of phosphospectra (27) were obtained using a VG ZAB 3F mass spectrometer equipped with a FAB source and operated in the double focusing lipase C cleavage of all three polyphoinositides. A chromatomode. Instrument conditions were as follows: primary beam of Xenon gram of the water-soluble products of phospholipase C-medi- 8 kev energy, 1 ma emission current. The ion source was operated ated cleavage of phospholipid vesicles containing [32P]PtdInsat 8 kev energy and instrument mass resolution was about P and [32P]PtdIns-4,5-Pz is shown in Fig. 1A. Four radio- Samples were dissolved in 2-3 p1 of water and applied to the FAB active components are resolved on this chromatogram. Two probe tip onto which 2-3 pl of glycerol had previously been deposited. of the components co-migrate with ["P]IP2 and [32P]IP3 Spectra were obtained by scanning at a rate of 100 s/decade from m/ z 800 and recorded on UV oscillographic paper. isolated from labeled erythrocyte ghosts (Fig. 1C). The other 45Ca2+ Release from Permeabilized Platelets-The 45Ca2+ release two represent [32P]cIPz and [32P]cIP3 based on criteria deassay was developed by Dr. Lawrence Brass, University of Pennsyl- scribed below. A chromatogram similar to that in Fig. 1A was vania. Human platelets were washed as described elsewhere (28) and obtained when [3H]inositol-labeled PtdIns-4-P and PtdInsresuspended just prior to use at a final concentration of 1.4 X IO9 4,5-P2 were substituted for the 32P-labeled lipids in the phoscells/ml in 137 mm NaC1,2.7 mm KCl, 1 mm MgC12, 3.3 mm NaH2P04, pholipase C reaction; this result suggests that all four peaks 5.6 mm glucose, 20 mm Hepes, ph 7.4. A 0.8-ml portion of the platelet contain inositol. When the individual products were desalted suspension was added to 2.4 ml of a solution containing 160 mm KCl, 5.3 mm MgC1, 3.3 mm ATP, 26.7 mm Hepes, ph 7.1,0.67 mm EGTA, and re-chromatographed, they eluted asingle peaks with the 6.7 mm creatine phosphate, and 13 units/ml creatine phosphokinase. same retention times. The platelets were permeabilized by the addition of 56 pl of a 1 mg/ In the presence of acid, inositol cyclic phosphate esters are ml saponin solution (final saponin concentration 5 pg/108 platelets) rapidly hydrolyzed to phosphomonoesters. Treatment of the followed by incubation at room temperature for 2 min. The permea- p,roducts of the phospholipase C reaction with acid prior to bilized platelets were loaded with 45CaZ+ by the addition of 3.2 p1 of a HPLC converted the [32P]cIPz and [32P]cIP3 products into 0.15 M solution of %ac12 (80 pci/pmol) followed by incubation at room temperature for 20 min. The platelet suspension was then their noncyclic counterparts (Fig. 1B). When acid hydrolysis diluted with 9.6 ml of 120 mm KCl, 4 mm MgCl,, 0.5 mm EGTA, and of cyclic phosphate esters is carried out in Hz180, the resultant 20 mm Hepes, ph 7.1. Ca'+ release was determined by adding 0.5 ml phosphomonoesters contain a single atom of "0. The HPLC of the diluted suspension to tubes containing varying amounts of 30 fractions corresponding to [32P]cIPz, [32P]IPz, [32P]cIP3, and

3 13498 Isolation and Characterization of the Inositol Cyclic Phosphate Products TABLE I "0 incorporation into the phosphate groups of cyclic and noncyclic inositol phosphates HPLC fractions corresponding to [32P]cIP2, [32P]IP2, [32P]cIP3, and [32P]IP3 were desalted, resuspended in 50 mm Hepes, ph 7.5,200 mm KCl, and tested for their ability to incorporate "0 into phosphate groups in response to treatment with acidified H2"O. Samples of Pi and chemically synthesized cipl were also treated in this fashion. The samples (approximately 3 nmol each) were exposed to acidified HZ1'O, lyophilized, and treated with alkaline phosphatase as described (19). The resultant inorganic phosphate was converted to the tertbutyldimethylsilyl derivative (32) for gas chromatography/mass spectroscopy using OV-17 resin (19)."0 enrichment of phosphate groups was measured using selected ion monitoring where the ratio of the m/z 385 peak to the m/z 383 peak was compared. The values shown represent the average of duplicate determinations. Compound mlz 385:383 Pi CIPl cip IPZ C1P.q IPS Samples of [32P]cIP3 and IP, were further characterized by FAB-mass spectrometry, a technique useful for the determination of the molecular weights of non-volatile compounds. The negative ion mass spectra for IP3 and cip, are shown in Fig. 2. The positive ion spectra were of inferior quality and are not presented. The sample of IP, analyzed was prepared from erythrocyte ghosts and was the free acid. The mass spectrum of IPS contained a peak at m/z 419, which corresponds to the mass of a compound whose molecular weight is FRACTION NUMBER 420 minus a proton. There was also a small peak at m/z 441 FIG. 1. Partisill0 SAX HPLC of the water-soluble products which corresponds to the monosodium salt of that compound of ["P]PtdIns-4-P and [s2p]ptdins-4,5-pa cleavage by phos- minus a proton. These molecular weights agree with the actual pholipase C. Small unilamellar vesicles containing [32P]PtdIns-4,5- molecular weight of IPS and it salt. The sample of [32P]cIP3 Pz (100 cpm/nmol), [32P]PtdIns-4-P(150 cpm/nmol), and phosphaanalyzed was prepared by HPLC chromatography followed by tidylethanolamine in a molar ratio of (1:0.25:0.4) were prepared and treated with phospholipase C as described under "Experimental Pro- gel filtration on Sephadex G-10 to remove excess salt. The cedures." The water-soluble products of the reaction were divided into two portions. One portion was subjected to HPLC (panel A). The otherportionwastreatedwith 1 N HC1 for 1min,frozen, lyophilized, resuspended in HzO, and then subjected to HPLC (panel B). Panel C shows the chromatogram for a mixture of erythrocyte [32P]IP2(eluting at fraction 30) and [32P]IP3(eluting at fraction 38). The ordinate is cpm of Cerenkov radiation. [32P]IP3 were desalted and tested for their ability to incorporate "0 into phosphate groups in acidified H2"0. The "0 content of the phosphomonoesters was measured after alkaline phosphatase treatment and conversion of the resultant inorganic phosphate to a volatile tert-butyldimethylsilyl derivative for gas chromatography/mass spectroscopy (38)."0 enrichment was determined by selected ion monitoring of the m/z 385 and 383 peaks of the Tris-tert-butyldimethylsilyl/ phosphate spectrum (19). In the absence of enrichment, the ratio of the m/z 385 peak to the m/z 383 peak is about (19). Ratios greater than are a measure of "0 enrich- co-migrated with [32P]IP2 and,'pi. Treatment of [32P]cIP3 produced compounds that co-migrated with [32P]cIP2 and 32Pi. ment. The m/z 385:383 ratios obtained for [32P]IP2, [32P]cP12, These results confirm that the HPLC peaks identified as cip2 [32P]IP3, and [32P]cIP3, are shown in Table I along with the and cip3 differ by a phosphate moietyin the 5-position. ratios for unenriched Pi and synthetic cip1. The [32P]cIP2 and Moreover, these findings suggest that cip3 may be metabo- [32P]cIP3 samples showed incorporation of "0 into phosphate, lized in uiuo in a fashion analogous to IP3, which appears to while IP2 and IP3 samples did not. The m/z 385:383 ratio was be degraded by a 5-phosphomonoesterase (9, 20, 25, 40-42). found to decrease in the order cipl > cip2 > cip3. This reflects The metabolism of cip3 by cell extracts is detailed in a dilution of the "0-enriched 1-position phosphate by the unenriched 4- and 5-position phosphates that are also released by alkaline phosphatase treatment. These data confirm the ex- istence of an acid labile cyclic phosphate ester in the products identified as cip2 and cip3. negative ion mass spectrum of cip, contained a peak at 401, which corresponds to the mass of a compound whose molecular weight is 402 minus a proton. Peaks at 423 and 445 represent mono- and disodium salts of this compound. The cip3 spectrum has a higher background, indicative of a lower sample concentration. The molecularweights obtained by FAB-mass spectrometry agree with the actual molecular weights of cip3 and its salts. The m/z 419 peak found in the IP, spectrum was not present in the cip3; this finding indicates that the cip3 sample was not contaminated with IP3. Samples of [32P]IP3 and [32P]cIP3(derived from the phospholipase C cleavage of the same preparation of [32P]PtdIns- 4,fi-p~) were treated with IP3-5-phosphomonoesterasepurified from human platelets, and the products were characterized as described in a separate report (39). Treatment of [32P]IP3 with the 5-phosphomonoesterase produced compounds that sep&ate report (39). In seven experiments, cipz comprised an average of 43% (range 28-58%) of the total water-soluble products of phospholipase C-mediated cleavage of labeled PtdIns-4-P, based on chromatograms of the reaction mixtures. In nine experi-

4 FIG. 2. Negative ion FAB-maw spectrum of [3zP]cIP3 (9 nmol) (right) and IP3 (20 nmol) (left) dissolved in glycerol. [5G-H]- pentamer of glycerol. Markings under upper trace indicate mass range Lower trace, XI; upper trace, X Isolation and Characterization of the Inositol Cyclic Phosphate Products MOBILIZATION OF CALCIUM IN PLATELETS IPS - cip,--- 'P* x CIpZ 0 I FIG. 3. Release of ",aa+ from saponin-permeabilized platelets in response to IP3 and cip3. The IP3 used in these experiments was from erythrocyte ghosts. The &Ca2+ release assays were performed as described under "Experimental Procedures." Data is graphed as a percentage of the &Ca2+ release elicited by 1 p~ calcium ionophore A Each point represents the mean & S.D. of 7-14 determinations using platelets from multiple donors, The permeabilized platelets accumulated an average of 3600 cpm of &(=a2+ prior to stimulation. In the absence of ATP the permeabilized cells accumulated only about 300 cpm over this same interval. The 45Ca2+ released by A23187 averaged 1700 cpm. ments, cip, comprised an average of 36% (range 19-55%) of the water-soluble products of PtdIns-4,5-P2 cleavage. Physiological Actions of Inositol Cyclic Phosphates-We also examined the physiological effects of CIPZ and cip3 in two systems. A number of studies have demonstrated that addition of IP3 to permeabilized cells results in mobilization of Caz+ from intracellular stores (for review, see Ref. 4). Consistent with observations in other permeabilized cell systems, saponin-treated platelets accumulate 45Ca2+ into intracellular storage pools in an ATP-dependent fashion. As shown in Fig. 3, addition of IP, to these 45Ca2+-loaded cells resulted in a concentration-dependent release of 45Caz+ from intracellular storage pools as shown in Fig. 3. At low concentrations ( PM) IP, mobilized Ca2+ more efficiently than cip3. Higher concentrations of the two compounds induced release of similar amounts of Caz+. The Caz+ release induced by cip3 does not require conversion to IP, prior to Ca" release. When [32P]cIP3(400 PM) was incubated with saponin-treated platelets, there was no conversion to [32P]IP3. Addition of IP2 or cipz at levels up to 4 PM did not cause 45Ca2+ release from permeabilized platelets (Fig. 3). 4.0" [MtNa-ZHI- [5G-H]- ["HIm/Z 401 m/z L Intracellular injection of IP, into intact Limulus ventral photoreceptors has been shown to induce electrical responses generated across the membrane mimicking those induced by light (31, 32). Also, injection of IP, has been shown to induce an increase in cytoplasmic Ca2+ as assessed by aequorin luminescence (16). As shown in Fig. 4A, injection of cip3 also induces an inward current similar to that induced by light. Brief pressure injection of cip3 elicited a depolarization that resembles the response of cells to illumination (Fig. 4, upper tracings) and an increase in intracellular Ca2+ as measured by aequorin luminescence (Fig. 4, middle tracing). The change in membrane voltage induced by microinjection of solutions containing equal concentrations of cip, and IP3 is shown in Fig. 4C. The depolarizations in response to cip3 were consistently larger than those induced by IP3 injections of approximately the same volume. Fig. 40 shows the tracing for a voltage clamp experiment in which a 0.2 mm solution of cip3 was compared to a 1.0 mm solution of IP3 using a doublebarrel micropipette. Nearly equal inward currents were induced by equal size injections of these two solutions, suggest- ing that cip3 is about five times as potent as IP3 in inducing a response in these cells. This difference in potency was consistently observed in several experiments in which various amounts of IP, and cip3 were injected. Intracellular injection of 1 mm c1p2 or 1 mm cipl into Limulus photoreceptor cells induced no consistent changes in membrane voltage or current (data not shown). \ DISCUSSION In this study we have isolated cipz and cip,, the cyclic phosphate products of polyphosphoinositide cleavage by phospholipase C, and confirmed the structure of these compounds through "0 labeling, phosphomonoesterase digestion, and FAB-mass spectrometry. Additionally, we have shown that one of these compounds, cip3, elicits physiological responses in two systems. In a previous report (19) we used "0 labeling of phosphate to show that phosphorylated inositols containing cyclic phosphate esters were present among the water-soluble products of phospholipase C mediated cleavage of all three polyphosphoinositides. Based on "0 labeling we concluded that the ratio of cyclic to noncyclic product in the phospholipase C reaction differed for the three substrates. About 70% of the product of PtdIns cleavage was cyclic, whereas with PtdIns- 4-P and PtdIns-4,5-P2 these percentages were 35% and 15%, respectively. In the present study we find using HPLC that 43% of the PtdIns-4-P cleavage product is cyclic and that 36% of the product of the PtdIns-4,5-Pz cleavage product is cyclic. We are uncertain why the "0 method used previously failed to detect the higher cip3 levels that wenow obtain consistently by direct isolation of the compound.

5 13500 Isolation and Characterization of the Inositol Cyclic Phosphate Products 40 r SM L n n I I I I 4 a 4 L 0 & Q FIG. 4. Electrophysiological effects of intracellular injections of cips and IPS. Samples of IPS and cip3 were dissolved in 50 mm Hepes, ph 7.5, 200 mm KCl. The bottom tracing of each set is a stimulus monitor. L denotes illumination, i denotes voltage-clamped current, V indicates membrane voltage, SM denotes stimulus monitor, LUM indicates aequorin luminescence monitor, filled arrows indicate cip3 injections, and empty urrows indicate IP3 injections. A, membrane current recorded by a voltage clamp technique in response to an injection of a 0.2 mm solution of cip3. B, change in membrane voltage and aequorin luminescence elicited by intracellular injection of 0.2 mm cip3. C, change in membrane voltage induced by injection of cip, and IP3 using a doublebarreled electrode containing 0.4 mm cip3 in one barrel and 0.4 mm IPS in the other. D, membrane current recorded by voltage clamp technique. The injections were made out of a double-barreled pipette containing 0.2 mm cip3 in one barrel and 1.0 mm IP, in the other. We find that cip3 causes release of Ca2+ from permeabilized platelets, but that this compound is less effective than IP,. cipz does not cause Ca2' release from these cells. These results are consistent with a study by Irvine et al. (12) who investigated the ability of various phosphorylated inositols to elicit Ca2+ release in permeabilized cells. These workers found the order of potency of the phosphorylated inositols studied to be Ins-1,4,5-P3 > Ins-2,4,5-P3 2 GroPIns-4,5-Pz > Ins-4,5-P2 >> Ins-l,4-P~. They concluded that phosphate groups at the 4- and 5-positions of inositol are required for Ca2' release, and that in addition there is a requirement for a phosphate moiety on the opposite end of the molecule, with a preference for the 1-position. cip, has a pair of phosphate groups on the 4- and 5-positions of inositol and another phosphate &esterified to the 1- and 2-positions, so it is not surprising that this molecule elicits Ca2+ release in permeabilized cells. Previous studies have shown that microinjection of IP, in Limulus photoreceptor cells induces changes in membrane conductance similar to those produced by light (31, 32). Ins- 4,5-P2 and GroPIns-4,5-P2 also elicit changes in membrane conductance in these cells. Ins-1,4-P2 induces weaker responses than IPS; IP, and phytic acid do not elicit responses. In this study we have shown that intracellular injection of cip, also induces changes in membrane conductance similar to those induced by light. Based on direct comparisons in the same cell, cip3 appears to be about five times as potent as IP3 in eliciting these responses. The finding that cip3 is less potent than IPS in releasing Ca2+ from permeabilized platelets may not be inconsistent with cip3 being more potent in eliciting changes in membrane conductance in the Limulus system. In Limulus photoreceptor cells, light-induced changes in membrane conductance do not require an increase in cytoplasmic Ca" (43). However, increases in cytoplasmic Ca2+ function in adaptation to light (36,43,44). cip, may prove to be less potent than IP, in inducing increases in cytoplasmic Ca" in Limulus photoreceptor cells. Therefore, we speculate that IPS and cip, may be distinct signal transducing molecules that have different functions. Several unresolved questions remain: 1) what levels of inositol cyclic phosphates are produced in stimulated cells? Previous studies measuring levels of IPS would have overlooked cip3 because of its acid lability. 2) Are there factors which alter the proportion of IPS uersus cip3 produced and could this in turn yield varied cellular responses? 3) What are the functions of IP2 and cip2 in signal transduction? In a separate paper (39) we present evidence that cip3 is degraded in cell extracts by 5-phosphomonoesterase cleavage, the same mechanism by which IP, is degraded in cells. Acknowledgments-We thank Monita Peacock for technical advice, and Dr. Michael Grayson and the McDonnell Douglas Corp. for permitting us to use the FAB-mass spectrometer. We also thank Dr. Lawrence Brass for providing us with his unpublished protqcol for 45Ca2+ release from platelets. REFERENCES 1. Majerus, P. W., Wilson, D. B., Connolly, T. M., Bross, T. E., and Neufeld, E. J. (1985) Trends Biochem. Sci. 10, Berridge, M. J. (1984) Biochem. J. 220, Majerus, P. W., Neufeld, E. J., and Wilson, D. B. (1984) Cell 37, Berridge, M. L.,and Irvine, R. F. (1984) Nature 312, Nishizuka, Y. (1984) Nature 308, Bell, R. L., Kennerly, D.A., Stanford, N., and Majerus, P. W. (1979) Proc. Natl. Acud. Sci. U. S. A. 76,

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