1,4-bisphosphate; IP3, inositol 1,4,5-trisphosphate; I-1-P, inositol. 1-phosphate; I-4-P, inositol 4-phosphate; 1-1,3,4-P3, inositol 1,3,4- trisphosphate; PI ...
Plant Physiol. (1993) 103: 637-647
Changes in Phosphatidylinositol Metabolism in Response to Hyperosmotic Stress in Daucus carota 1. Cells Grown in Suspension Culture' Myeon H. Cho, Stephen B. Shears, and Wendy F. BOSS*
Department of Botany, North Carolina State University, Raleigh, North Carolina 27695-761 2 (M.H.C., W.F.B.); and Laboratory of Cellular and Molecular Pharmacology, National lnstitute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 (S.B.S)
Carrot (Daucus carota L.) cells plasmolyzed within 30 s after adding sorbitol to increase the osmotic strength of the medium from 0.2 to 0.4 or 0.6 osmolal. However, there was no significant change in the polyphosphorylated inositol phospholipids or inositol phosphates or in inositol phospholipid metabolism within 30 s of imposing the hyperosmotic stress. Maximum changes in phosphatidylinositol 4-monophosphate (PIP) metabolism were detected at 5 min, at which time the cells appeared to adjust to the change in osmoticum. There was a 30% decrease in [3H]inositol-labeledPIP. The specific activity of enzymes involved in the metabolism of the inositol phospholipids also changed. The plasma membrane phosphatidylinositol (Pl) kinase decreased 50% and PIP-phospholipase C (PIP-PLC) increased 60% compared with the control values after 5 min of hyperosmotic stress. The PIP-PLC activity recovered to control levels by 10 min; however, the PI kinase activity remained below the control value, suggesting that the cells had reached a new steady state with regard to PIP biosynthesis. If cells were pretreated with okadaic acid, the protein phosphatase 1 and 2A inhibitor, the differences in enzyme activity resulting from the hyperosmotic stress were no longer evident, suggesting that an okadaic acid-sensitive phosphatase was activated in response to hyperosmotic stress. Our work suggests that, in this system, PIP is not involved in the initial response to hyperosmotic stress but may be involved in the recovery phase.
The signal transduction mechanisms involving inositol phospholipid-derived second messengers, IP3and diacylglycerol, are well characterized in many animal cells (Majerus et al., 1986; Berridge and Irvine, 1989; Michell, 1992). In these systems the response includes a rapid and transient decrease in PIPz and at least a 3-fold increase in Ir3.The increased IP3 releases calcium from internal stores, specifically the ER (Streb et al., 1983), thus activating calcium-dependent enzymes and altering cell physiology. Most components of the inositol phospholipid-mediated signal transduction pathway have been shown to be present in higher plants (for reviews, see Einspahr and Thompson, 1990; RincÓn and Boss, 1990; Chen et al., 1991; Gross and Boss, 1992; Hetherington and Drcibak, 1992). However, only
' This research was supported by grant No. DCB-8812580 from the National Science Foundation and in part by the North Carolina Agricultura1Research Service. * Corresponding author; fax 1-919-515-3436.
in the Samanea saman pulvini is there evidence for stimulusmediated turnover of polyphosphorylated inositol phospholipids and inositol phosphates within the rapid time scale seen in animal cells (Morse et al., 1987). Although results from in vitro studies and from IP3 microinjection in vivo have shown that Ir3can release Ca2+from internal stores such as vacuoles and tonoplast vesicles (Drcibak and Ferguson, 1985; Schumaker and Sze, 1987; Ranjeva et al., 1988; Gilroy et al., 1990), the complete pathway from a stimulus to Ca2+release has yet to be demonstrated in higher plants. When studying the functions of the inositol phospholipids, one needs to realize the multifaceted effects of the inositol phospholipids on cell physiology. For example, in addition to being the sources of second messengers, PIP and PIPz also can directly affect cell physiology by regulating P-type ATPases (Varsanyi et al., 1983; Schafer et al., 1987; Memon et al., 1989a; Chen and Boss, 1991), protein kinase C (Chauhan and Brockerhoff, 1988; Chauhan et al., 1989), and actin polymerization and nucleation sites (Lassing and Lindberg, 1985; Forscher, 1989). It has been shown that both PI and PIP kinase are present in the F-actin fractions from both plant (Tan and Boss, 1992) and animal cells (A431 mouse fibroblast cells) (Payrastre et al., 1991) and that the specific activities of the lipid kinases in the F-actin fraction change in response to externa1 stimuli. These data suggest that there is a change in intracellular enzyme distribution or covalent modification of the enzymes. In addition, we recently (Yang et al., 1993) have found a protein that not only regulates PI kinase activity but also binds and bundles actin and has translational elongation factor-la activity. Thus, changes in the metabolism of these very negatively charged phospholipids could be a part of a complex intracellular communication system among the plasma membrane, cytoskeleton, and protein synthesis machinery . Changes in inositol phospholipids had been shown to occur in Dunaliella salina as a result of hyperosmotic stress (EinAbbreviations: I, inositol; IP, inositol monophosphate; Ir2,inositol 1,4-bisphosphate; IP3, inositol 1,4,5-trisphosphate; I-1-P, inositol 1-phosphate; I-4-P, inositol 4-phosphate; 1-1,3,4-P3,inositol 1,3,4trisphosphate; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-monophosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; MeOH, methanol; OSM, osmolal.
Cho et al.
638
spahr et al., 1988), and changes in inositol phosphates have been reported for beet roots exposed to different osmoticum (Srivastava et al., 1989). Because cells grown in suspension culture provide a relatively uniform system for biochemical studies, we decided to study the effects of hyperosmotic stress on inositol lipid metabolism in this system in more detail. For these experiments we kept the cells in their normal medium and applied stress by adding sorbitol to the medium rather than placing the cells in sorbitol alone. Plasmolysis has been used over the years by cell biologists to study the properties of the plasma membrane of plant cells (Pfeffer, 1877; Lee-Stadelmann and Stadelmann, 1976). Plasmolysis of cells in response to hyperosmotic conditions is rapid (within seconds) and readily visible with the light microscope. Rapid membrane depolarization occurs as the cells plasmolyze (Slayman, 1982); however, the mechanism of signal transduction has not been elucidated. Tolerance to osmotic stress is important agriculturally. Recent advances in this field show promising results in altering cell physiology to withstand hyperosmotic stress (Tarczynski et al., 1993) and emphasize the importance of understanding the mechanisms by which cells respond and adjust to hyperosmotic conditions. The question we have addressed in this work is what role do the inositol phospholipids play in cellular responses to hyperosmotic stress? Our data indicate that the inositol phospholipids are involved in the recovery phase as the cells adjust to hyperosmotic conditions and that this response may be mediated by an okadaic acid-sensitive phosphatase. MATERIALS AND METHODS Plant Material
Wild carrot (Daucus carota L.) cells grown in suspension culture were transferred weekly and used 4 d after transfer as previously described (Chen and Boss, 1990). Chemicals
my0-[2-~H]Inositol(24.40 Ci mmol-') and PI-4-P (inositol2-3H[N]) (9.90 Ci mmol-') were purchased from New England Nuclear, and [y3'P]ATP was purchased from ICN Radiochemicals. 3H-Labeled inositol phosphate standards (I- 1-P, I-4-P, Ir2,I- 1,3,4-P3, Ir3,I- 1,3,4,5-tetrakisphosphate, I-hexaphosphate) were obtained from New England Nuclear. [14C]I(1,3,4)P3 was prepared from ['4C]inositol-labeled parotid acinar cells (Hughes et al., 1989). Dowex-1 (X8),chloride form (drymesh, 200-400), and okadaic acid were purchased ) prepared from Sigma, and okadaic acid stock (155 p ~ was in 10% DMSO according to manufacturer's guide. A11 other chemicals were analytical grade. Hyperosmotic Treatment and Okadaic Acid Pretreatment
The cells from severa1 125-mL culture flasks were combined into a 500-mL flask and redistributed by pipetting 22.5 mL of medium and cells into 125-mL flasks. The flasks were placed on a shaker for 30 min at 120 rpm and 25OC to preequilibrate the cells. The 30-min equilibration period is essential to stabilize the cells and minimize the effects of
Plant Physiol. Vol. 103, 1993
transfer. The conditioned culture medium had an osmolality of 0.2 OSM, determined by freezing point depression using an Osmette Precision Osmometer and a pH of 4.2. Hyperosinotic conditions were produced by adding 2.5 mL of 2 or 4 OSM sorbitol in conditioned medium containing 2 mM Mes baffer (pH 4.2) to conditioned medium (22.5 mL, pH 4.2). This gave a final osmoticum equivalent to 0.4 and 0.6 OSM, respectkely. An equal volume of conditioned medium containing :! mM Mes buffer (pH 4.2) was added for the control. At 30 s before the termination of the treatments or at time zero for the 30-s time points, the cells were quickly poured into conical tubes and centrifuged at 2000g for 30 s. Supernatant was pciured off, and cells were immediately placed on ice and homogenized or extracted for inositol phospholipid or inositol phosphate analysis. Cells were pretreated with okadaic acid for 1 h following the 30-min preequilibration. Okadaic acid was added iising a stock solution of 155 p~ okadaic acid in 10% (v/v) DlMSO to give a final concentration of 0.15 ~ L Mokadaic acid and 0.01% DMSO. The equivalent volume of 10% DMSO was added as a control. Observations of Plasmolysis
For the microscopy studies, the carrot suspension culture cells (0.1 g in 4.5 mL of conditioned medium, pH 4.2) were placed in a small Erlenmeyer flask (25 mL) and treated hyperosmotically by adding 0.5 mL of 2 or 4 OSM sorbitol in conditioned medium containing 2 IT~MMes (pH 4.2), to give a final osmolality of 0.4 and 0.6 OSM, respectively. A drop of cells was placed on the glass slide, and plasmolysis was observed over time using a Zeiss IM 35 microscope with a X63 neofluor lens. The first observations were made 30 to 40 s after the addition of osmoticum. [3Hllnositol Labeling
Cells (approximately 0.25 g fresh wt 25 mL-' of med:ium) were used 3 d after transfer. myo-[2-3H]Inositol(0.2pCi rnL-' for lipid analysis and 0.4 pCi mL-' for inositol phosphate analysis) was added to the cells for 18 h. The cells were harvested by brief centrifugation (2000g for 30 s). Extraction and Separation of lnositol Phosphates in HPLC
The centrifuged cells were quenched with a final concentration of 4% (v/v) perchloric acid (Kirk et al., 1990) for inositol phosphate analysis. Perchlorate-quenched cell extracts were neutralized with freon/octylamine (Kirk et al., 1990), and the inositol phosphates therein were resolvetl on an Adsorbosphere 5 p-SAX HPLC column (Alltech Associates, Deerfield, IL) using a slight modification of the salt gradient previously described (Menniti et al., 1990)generated from water and buffer (1 M ammonium dihydrogen phosphate, pH 3.35 with phosphoric acid): O to 10 min, 0% buffer; 10 to 85 min the buffer increased linearly from O to 35%; 85 to 130 min, the buffer increased linearly from 35 to 80%. Half-minute fractions were collected for 80 min, and 1-min fractions were collected thereafter. Scintillant was added to each of the fractions, and the radioactivity was assessed.
Phosphoinositide Metabolism and Hyperosmotic Stress
639
Lipid Extraction and Separation
PLC Assay
For lipid analysis 1.5 mL of ice-cold CHCl3:MeOH (1:2, v/v) was added to the centrifuged cells, and the lipids were extracted and separated by TLC as described by Cho et al. (1991). Briefly, the LK5D (Whatman) silica gel plates were presoaked in 1% potassium oxalate for 80 s, dried in a microwave for 10 min, and developed in CHC^MeOH: NH4OH:H2O (86:76:6:16, v/v/v/v). The distribution of the 3 H- and 32P-labeled lipids was quantitated with a Bioscan System 500 imaging scanner. The efficiency was 1% for 3H and 10% for 32P. 32P-Labeled phospholipids were visualized by autoradiography.
PLC activity was assayed according to the method of Melin et al. (1992). The standard reaction mixture contained 50 mM Tris-HCl (pH 6.0), 20 MM Ca2+ (a Ca 2+ /EGTA mixture was used as described by Owen [1976]), 0.2 mM PIP, and 4 to 6 Mg of membrane protein in a final volume of 50 ^L- The reaction was started by the addition of a PIP stock solution containing 3H-labeled PIP (1100 dpm nmol"1) in 0.1% deoxycholate, which was prepared by drying the lipid under a stream of nitrogen followed by sonication in 0.1% deoxycholate for 10 min. The reaction was stopped after 5 min at 25°C by addition of 1 mL of chloroform:methanol (2:1, v/v). After addition of 250 fiL of 1 N HC1, vortexing, and brief centrifugation in a table-top centrifuge, the radioactive reaction products were recovered in the upper phase, analyzed by ionexchange chromatography on a Dowex AG 1-X8 column (Berridge et al., 1983), and quantitated by liquid scintillation counting.
Plasma Membrane Isolation
Plasma membranes were isolated by aqueous two-phase partitioning as described by Chen and Boss (1990). The final plasma membrane pellet was resuspended in 30 mM Tris/ Mes buffer (pH 6.5) containing 15 mM MgQ2 and kept on ice until used for the enzyme assays. For the PIP-PLC assay, plasma membranes were resuspended in 50 mM Tris-HCl, pH 6.0. Protein was determined by the method of Lowry et al. (1951) with BSA as a standard. Analysis of PI Kinase
PI kinase activity was measured in the absence and presence of exogenous substrate. Equal amounts of plasma membrane protein were used for the control and treated cells within each experiment. The protein amount ranged from 15 to 40 fig for the experiments reported. The plasma membranes were resuspended in 40 nL of 30 mM Tris-Mes buffer (pH 6.5) containing 15 mM MgCl2. Phosphorylation was started by adding 10 pL of ATP stock solution, which contained 10 nCi of [7-32P]ATP (7000 Ci mmol~'), 6 mM ATP, 0.05% (v/v) Triton X-100, and 5 mM Na 2 MoO 4 in 30 mM Tris/Mes containing 15 mM MgCl2 (pH 6.5), to give a final concentration of 1.2 mM ATP, 1 mM Na2MoO4, and 0.01% Triton X100. When exogenous substrate (PI, 25 ^g/tube) was added, the final concentration of Triton X-100 was 0.2% and the pH was 8.0. The PI was presolubilized in 2% Triton X-100. The tubes were shaken by hand vigorously every 1 min at room temperature, and after 10 min, the reaction was stopped by adding 1.5 mL of ice-cold CHCl3:MeOH (1:2, v/v). The lipids were kept on ice until they were extracted.
RESULTS
Hyperosmotic Stress Induces Rapid Plasmolysis
The osmolality of the medium in which the carrot cells were growing was determined to be approximately 0.2 OSM. Cells in conditioned medium were turgid and contained large starch granules (Fig. 1A). When sorbitol was added to the medium to give the equivalent of a 0.4 OSM solution, the outer cells in a cluster could be seen to plasmolyze slightly (Fig. IB). At a final concentration of 0.6 OSM, plasmolysis was
ATPase Assay
Vanadate-sensitive ATPase was assayed by adding 50 nL of the plasma membrane-rich fraction (30-40 ^g of protein) to 350 /uL of reaction mixture to give a final concentration of 0.01% Triton X-100, 3 mM MgSO4, 30 mM Tris/Mes, 50 mM KC1, 1 mM NaN 3 in the presence or absence of 500 MM vanadate as described by Gallagher and Leonard (1982). The reaction was started by adding 100 juL of a 15 mM ATP stock solution and continued for 30 min at room temperature. The reaction was stopped by the addition of 250 pL of 20% icecold TCA. The Pi released was determined according to the method of Taussky and Shorr (1953).
Figure 1. Plasmolysis induced by hyperosmotic treatments using 0.4 and 0.6 OSM sorbitol in conditioned medium. Carrot cells were treated hyperosmotically by adding 0.4 and 0.6 OSM sorbitol to the conditioned medium. Small aliquots of cells were placed on a glass slide, and plasmolysis was observed with a Zeiss IM 35 microscope at X630 as described in "Materials and Methods." A, The control cells in conditioned medium; B, cells at 0.4 OSM after 1 min; C, cells at 0.6 OSM after 45 s; D, cells at 0.6 OSM after 5 min (in recovery phase). Arrowheads denote starch granules. Arrows denote the position of the plasma membrane and cell wall.
Cho et al.
640
clearly evident and complete in a11 cells within 30 s. The photograph in Figure 1C was taken after 45 s. The plasmolyzed cells could be seen to regain volume slightly over a 5min period (Fig. 1D). After 5 to 10 min the volume stabilized. The average decrease in diameter of the cells in 0.6 OSM sorbitol was 15 & 4.8% ( n = 6). The Distribution of [3H]lnositol-Labeled Lipids Changes after Hyperosmotic Stress
In the [3H]inositol-labeled carrot cells the predominant [3H]inositol-labeled lipid was PI (82-85% of the total, Table I). [3H]PIPand [3H]PIP2were relatively minor constituents (approximately 8 and 0.5%, respectively). When carrot cells were treated hyperosmotically with sorbitol in conditioned medium to give an osmoticum of 0.4 or 0.6 OSM, the total amount of 3H-labeled lipid did not change significantly at either 30 s or 5 min (Table I). Furthermore, within the time frame that plasmolysis was complete (30 s), there were no significant changes in any of the [3H]inositol-labeledlipids based either on percentage of total 3H-lipid or on cpm (Table I). After the 5-min treatment at either 0.4 or 0.6 OSM sorbitol, the proportion of [3H]PIP in the 3H-labeled lipids decreased by 25 and 32%, respectively, relative to the controls (Table I, P < 0.05). This effect on PIP, which appears to be a secondary response to osmotic stress, was not associated with any significant change in the levels of [3H]PIP2.Because we only detected changes in [3H]PIP, we focused our studies on PIP metabolism. The changes in [3H]PIPin response to osmotic stress could have resulted from stress-induced changes in the kinasel phosphatase cycle that interconverts PI and PIP. Osmotic stress also might have caused an increased rate of hydrolysis
Plant Physiol. Vol. 103, 1993
of PIP by PLC. One potential mechanism by which such putative regulation could be achieved is by covalent modification of the enzymes or of regulatory proteins. Therefore, we isolated the plasma membranes of carrot cells and assayed PI kinase activity and PIP hydrolysis by PLC as well as the plasma membrane vanadate-sensitive ATPase activity, specifically to search for any stable changes in the inherent activities of these enzymes. Hyperosmotic Stress Decreases the Plasma Membrane Vanadate-Sensitive ATPase Activity
Initially as a means of characterizing the plasma membrane fractions, we studied the vanadate-sensitive ATPase activity in plasma membranes isolated from stressed and nonstressed cells. Greater than 85% of the ATPase activity in the plasma membrane fraction from the carrot cells was inhibited by vanadate (data not shown). The specific activity of the vanadate-sensitive ATPase decreased when cells were treated hyperosmotically at 0.4 OSM by adding sorbitol to the conditioned medium (Fig. 2). A reduction in the ATPase-specific activity was detected within 1 min. The maximum decrease was observed after 5 min of hyperosmotic stress. Hyperosmotic Stress lncreases the Activity of PIP-PLC
Plasma membranes prepared from the stressed cells exhibited a transient increase in PIP hydrolysis by PLC. No ecfect was observed after 2 min of stress. A 60 & 6.4% stimulation in activity was observed after 5 min, and by 10 min the rate of hydrolysis of PIP had returned to the control leve1 (Fig. 3). Analysis of the water-soluble products by Dowex-1 anionexchange chromatography showed that more than 90% were
Table 1. Changes in the distribution ~f[~H]inosito/ lipids in response to 0.4 and 0.6 hyperosmotic stress for 30 s and 5 min
OSM
Carrot cells were labeled with [3H]inositol(1.25 pCi 0.1-' g fresh weight) for 18 h and treated hyperosmotically at 0.4 and 0.6 OSM in conditioned medium plus sorbitol for 30 s and 5 min. For the control, conditioned medium (0.2 OSM) was used. The experimental procedures are described in "Materials and Methods." [3Hllnositol-Labeled Lipids Treatment
PI
Lvso-PI
PIP
Lvso-PIP
PIP2
30 s
Control
5
a
0.4
OSM
0.6
OSM
min Control 0.4
OSM
0.6
OSM
83.0 f 2.7" (7496 f 54)b 83.1 f 2.8 (7464 f 89) 82.3 f 2.7 (7489 f 41)
2.5 f 0.7 (1 73 f 6) 2.7 f 0.7 (204 f 40) 3.2 f 1.1 (201 f 24)
8.1 f 0.9 (673 f 45) 7.5 f 1.2 (676 f 74) 7.6 1- 0.8 (660 f 19)
5.3 f 2.0 (324 f 23) 5.2 f 2.1 (302 f 18) 5.3 f 2.1 (316 +- 18)
0.6 f 0.1 (43 f 4) 0.5 f 0.1 (45 f 5) 0.6 f 0.2 (51 f 2)
83.1 f 1.6 (7511 f 53) 84.2 f 1.5 (7360 f 27) 85.3 f 1.5 (7557 f 106)
2.6 f 0.6 (214 f 44) 2.3 f 0.6 (259 f 86) 2.3 f 0.5 (179 f 24)
8.2 f 0.8 (738 f 9) 6.3 f 0.7 (549 f 15) 5.5 f 0.3 (505 f 8)
5.1 1.8 (345 f 17) 4.6 f 1.8 (318 f 14) 4.1 f 0.9 (327 f 52)
0.5 f 0.2 (38 f 10) 0.6 2 0.2 (38 f 10) 0.7 f 0.1 (50 f 9)
The numbers are the percentages of total [3H]inositol-labeledlipids and expressed as the means
The numbers in parentheses are the f SD of five or six values from three different experiments. cpm of each inositol lipid and are the average of two values from one representative experiment.
64 1
Phosphoinositide Metabolism and Hyperosmotic Stress 110 I
these experiments the same fresh weight of cells was used for each treatment, and the total radioactivity recovered from each treatment was almost identical. There were no significant increases in [3H]IP, [3H]IP2,or [3H]IP3after subjecting cells to osmotic stress (0.4 or 0.6 OSM) for either 5 min or 30 s (data not shown). Thus, we conclude that in the carrot cells stress-dependent increases in PLC-mediated hydrolysis of PIP are insufficient to alter the steady-state changes in levels of inositol phosphates to a substantial extent.
I
= O
L
. I -
c O o
y.
O
s
v
>
..->
. I -
. I -
o
a
40
-
al
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-
Hyperosmotic Stress Decreases PI Kinase Activity in lsolated Plasma Membranes
v)
a
a
20
a
10
I-
-
-
O 0
2
4
6
8
1
0
1
2
Time (min) Figure 2. Time course of changes in plasma membrane vanadatesensitive ATPase activity in response to 0.4 OSM hyperosmotic stress. Celis were treated hyperosmotically (0.4 OSM) as described in ”Materials and Methods.” Plasma membranes were isolated by aqueous two-phase partitioning, and the vanadate-sensitive ATPase was assayed. The results are means & SD of four values from two experiments.
IP2(data not shown). Thus, the decrease in [3H]PIPin stressed cells could have been partly caused by increased hydrolysis by PLC.
To determine whether or not there was a difference in PI kinase activity, plasma membranes were isolated from control and hyperosmotically treated cells. In vitro phosphorylation in the presence of [-p3’P]ATP indicated that plasma membranes from hyperosmotically stressed cells formed 40% less [32P]PIPthan membranes from the control cells. This decrease in PIP formation was not due to limited substrate availability because a similar decrease in PIP formation was observed in the presence of exogenous PI (25 &each assay) (Table 11). When changes in PI kinase activity were monitored over time, slight but significant changes were observed consistently in plasma membranes from control cells (Fig. 5). The effect was transient and may be caused in part by a “touch”induced response upon addition of the conditioned medium. More importantly at a11 times, the effect of osmotic stress was observed over and above that of the conditioned medium. After 2 min of hyperosmotic stress, there was a slight decrease in the amount of [32P]PIPformed in the in vitro phosphoryl-
h
.-c
20
E
T
[3H]lnositol Phosphates in lntact Cells Did Not Change after Hyperosmotic Stress
The analysis of [3H]inositol phosphates in [3H]inositollabeled plant cells is complicated by the large array of additional water-soluble metabolites (Loewus and Loewus, 1982; RincÓn et al., 1989; Coté et al., 1990). It is very difficult to specifically assay [3H]inositol phosphates against this high background of 3H-labeled compounds. In our experiments we have adapted an anion-exchange HPLC procedure that has been used successfully to resolve a number of inositol phosphates in extracts of animal cells (Balla et al., 1989; Menniti et al., 1990). The column was initially characterized with 3H-labeled standards of I-I-P, I-4-P, Ir2, I-1,3,4-P3, Ir3, 1-1,3,4,5-tetrakisphosphate,and I-hexaphosphate. Each sample also contained an interna1 standard of [‘4C]I-1,3,4-P3to account for run-to-run variability. The extract from control cells described in Figure 4 contained small 3H-labeledpeaks that coincided with the elution positions of I-I-P and I-4-P (combined total = 8089 dpm) plus IP2 (1,464 dpm). Ir3was not consistently detected. Two unidentified 3H peaks that migrated between IPz and IP3 and an unidentified 3H peak between IP and IP2 (a11denoted with ?) were observed to increase in the cells treated at 0.6 OSM. In
“ d o u
-
o
2
4
6
8
10
12
Time (min) Figure 3. Time-course study of the effects of hyperosmotic stress on PIP-PLC activity of plasma membranes isolated from hyperosmotically treated and nontreated carrot suspension-culture cells. Cells were treated hyperosmotically at 0.4 OSM in conditioned medium plus sorbitol for 2, 5, and 10 min. Conditioned medium was used for the control. Plasma membranes were isolated by aqueous two-phase partitioning as described in “Materials and Methods.” PLC activity was assayed using [3H]PIP as a substrate according to the method of Melin et al. (1992). Open circles, control; closed circles, hyperosmotic stress. The results are the means & SD of six values from three experiments.
Cho et al.
642 5000
c
.-O o 2
of the large amount of diacylglycerol in the plasma m.embranes. Lysophosphatidic acid, PIP, and an as yet unidentified phosphorylation product were also formed. PIP2 was barely detectable, which is consistent with previous studies of plasma membranes from higher plants (Sandelius and Sommarin, 1986; Sommarin and Sandelius, 1988; Chen and Boss, 1990; Memon and Boss, 1990; Gross and Boss, 1992).
4000
3000
) I
2
a
-
n
2000
Pretreatment of Cells with Okadaic Acid Eliminates the Effect of Hyperosmotic Stress on PI Kinase and PIP-PLC Activity
I ‘ - 1000 O
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5000
-
4000
-
3000
-
2000
-
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a
r ,“
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+ l(4)P
1(1*4,5)P
1000
O 5000
2
.-
4000
CI
o
2
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3000
2
a n
3 Y
Plant Physiol. Vol. 103, 1993
2000
1000
O
O
20
40
60
80
The effect of hyperosmotic stress on PI kinase and I’IPPLC activity persisted in isolated plasma membranes. Because protein phosphorylation/dephosphorylation is a rapid and effective means of altering enzyme activity and because there was evidence for phosphatases decreasing PI kinase activity (Chen and Boss, 1990; Memon and Boss, 1990), we as,ked whether inhibiting dephosphorylation would affect the response of the membrane enzymes to hyperosmotic stress. For these experiments we used okadaic acid, a polyether fatty acid, that inhibits protein phosphatases 1 and 2A (Bialojan and Takai, 1988). Okadaic acid is hydrophobic and readily enters cells. The effect of okadaic acid on the change in the PIP-PLC and PI kinase activity was studied both in vivo and in viitro. For the in vivo experiments cells were pretreated with okadaic acid (0.15 PM, final concentration) in DMSO or DMSO alone for 1 h. Okadaic acid pretreatment did not affect the plasmolysis (data not shown). As with the non-okadaic acidpretreated cells, plasmolysis was complete within 30 s. In okadaic acid-pretreated cells, hyperosmotic stress no loriger resulted in an increased PIP-PLC activity (Table 111). I t is interesting that okadaic acid pretreatment decreased the specific activity of PIP-PLC in both the control and hyperosmotic stressed cells to the same basal value (approximately 6 nimol min-’ mg-’ of protein). Okadaic acid pretreatment also eliminated the difference
Elution Time (min) Table II. The PI kinase activity of plasma membranes isolated from
Figure 4. HPLC elution profile of inositol phosphates from control and stressed cells. Cells were labeled with [3H]inositol(2.5 pCi 0.1 g-’ fresh weight) for 18 h and treated hyperosmotically at 0.4 and 0.6 OSM in conditioned medium plus sorbitol or in conditioned medium alone for 5 min (A, control; B, 0.4 OSM; C, 0.6 OSM). Total [’Hlinositol phosphates were extracted and separated in HPLC as described in “Materials and Methods.” The inositol phosphates were identified by comparison to the elution times of standards. The elution time for [’4C]1(1,3,4)P3, an interna1 standard, is indicated by the large arrow in each profile.
hyperosmotically treated and nontreated carrot suspension-culture cells Carrot suspension-culture cells were treated hyperosmotically for 5 min at 0.4 OSM in conditioned medium plus sorbitol and conditioned medium alone was used for the control. The plasma membranes were isolated by aqueous two-phase partitioning. The PI kinase activity was assayed in the absence and presence of exogenous substrate (Pl) as described in ”Materialsand Methods.” PI Kinase Activity [32P]PIPformed
Percentage of Control
pmol mg-’ min-’
ation assay in the presence of exogenous substrate. As with the PIP-PLC activity, the maximum decrease was found after 5 min of stress (Fig. 5). In contrast to the PIP-PLC activity (Fig. 3), the activity of PI kinase did not recover by 10 min but remained at a low leve1 (Fig. 5 ) . In the in vitro phosphorylation assay in which only endogenous substrate was used, i.e. isolated plasma membranes alone, the major phosphorylation product was phosphatidic acid. This is indicative
Using endogenous substrate
Control Hyperosmotic Using exogenous substrate Control Hyperosmotic a The data are the means different experiments.
265.9 f 24.0” 150.4 +- 40.4 1395.8 k 13.4
650.4 +- 236.8 4 SD of
1 O0 56.7 f 21.4 1O0
46.6 +- 24.0
at least six values from three
64 3
Phosphoinositide Metabolism and Hyperosmotic Stress
To determine whether or not the stress-regulated okadaic acid-sensitive phosphatase could be recovered with the plasma membrane, we isolated membranes from stressed and nonstressed cells and added okadaic acid (0.01-1 p ~ to) the phosphorylation reaction mixture. At a concentration of 0.01 p ~ okadaic , acid had no effect on the PI kinase activity of the membranes isolated from hyperosmotically stressed cells and decreased (by 15%) the [32P]PIPformed by membranes from control cells (Fig. 6). At higher concentrations (0.1 and 1 p ~ ) okadaic , acid decreased the [32P]PIPformed by membranes from hyperosmotically stressed cells. Importantly, in the in vitro experiments at all concentrations of okadaic acid added, the effect of osmotic stress on PI kinase activity was still evident (Fig. 6). The effect of okadaic acid on the plasma membrane PIPPLC also was studied. Adding okadaic acid to the isolated plasma membranes decreased the specific activity of PIPPLC by 40 and 20% for the control and hyperosmotic cells, respectively. It is interesting that adding okadaic acid to the plasma membranes isolated from hyperosmotically stressed cells decreased the specific activity of the PIP-PLC to approximately that of untreated membranes from control cells. Although the specific activity of the PIP-PLC decreased, the specific activity of PIP-PLC from hyperosmotically stressed cells remained higher than the okadaic acid-treated controls, i.e. adding okadaic acid to isolated plasma membranes did not overcome the effect of hyperosmotic stress.
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Time (min) Figure 5. Time course of changes in PI kinase activity in response to 0.4 OSM hyperosmotic stress. Cells were treated hyperosmotically (0.4 OSM) for various times, and for the control the same amount of buffer in conditioned medium was added. Plasma membranes were isolated from cells in each treatment by aqueous two-phase partitioning. PI kinase activity was assayed in the presence of exogenous substrate. Open circles, control; closed circles, hyperosmotic stress. The results are the means f SD of four values from two different experiments.
in PI kinase activity resulting from hyperosmotic stress. The PI kinase activity of plasma membrane from the okadaic acid-pretreated cells did not decrease when the cells were hyperosmotically stressed (Table IV). DMSO alone (the minus okadaic acid control) had no effect on the response to osmotic stress. The data suggested that an okadaic acid-sensitive phosphatase(s) was activated in response to hyperosmotic stress and that the phosphatase(s) was involved in the stressinduced increase in PIP-PLC activity and decrease in PI kinase activity.
DISCUSSION
We have shown that the metabolism of PIP changes as a result of hyperosmotic stress. After 5 min of stress there was a decrease in [3H]inositol-labeled PIP from whole cells. This was associated with a concomitant increase in PIP-PLC and a decrease in PI kinase-specific activity assayed in vitro. The observed changes in PIP metabolism did not correlate with the initial plasmolysis, which was complete within 30 s. The fact that the effects of hyperosmotic stress on the
Table 111. Effect of okadaic acid in vivo and in vitro on PIP-PLC activity
Cells were pretreated with 0.15 PM okadaic acid for 1 h. A n equivalent volume of DMSO was used for the control. After pretreatment,cells were treated hyperosmoticallyat 0.4 OSM in conditioned medium plus sorbitol. Plasma membranes were isolated by aqueous two-phase partitioning. For the in vitro treatment, okadaic acid (1 PM, final concentration)was added to reaction mixture. PIP-PLC was assaved as described in "Materiais and Methods." Pretreatment
Assay Condition
Treatment
PLC Activity
Percentage of Control
nmol min-' mg-'
Okadaic acid -
+ +
Control Hyperosmotic
f 0.82a
12.83 7.96 17.01 13.10
f 1.64 f 0.86
f 0.08 f 0.48 f 0.51
1O 0 78.0 f 1.1 141.7 f 6.4 74.9 f 6.8
Okadaic acid
Control
-
Hyperosmotic "The numbers are the averages
7.52 5.87 10.66 5.63
zk SD
+ -
+
f 1.16b f 0.92
1O 0 62.0 f 6.7 132.6 zk 9.1 102.1 f 7.2
of four values from two different experiments.
numbers are averages f SD of three values from two experiments.
bThe
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Plant Physiol. Vol. 103, 1993
Table IV. Effect of okadaic acid in vivo and in vitro on PI kina5e activity Cells were pretreated with 0.15 p~ okadaic acid for 1 h. An equivalent volume of DMSO was used for the control. After pretreatment, cells were treated hyperosmotically at 0.4 OSM in conditioned
medium plus sorbitol. Plasma membranes were isolated by aqueous two-phase partitioning. For the in vitro treatment, okadaic acid (1 p ~ final , concentration) was added to reaction mixture. PI kinase was assayed as described in "Materials and Methods." Pretreatment
Assay Condition
Treatment
PI Kinase Activity
Percentage of Control
pmol min-' mg-'
Okadaic acid
+ -
+
Control Hyperosmotic
-
+ -
Hyperosmotic
i-
The numbers are the averages &
1O0 82.8 rt 9.2 65.8 f 12.9 89.3 f 12.6
293 & 21.7 2 5 0 & 6.6 210 & 11.7 166 f 36.7
1o0 85.3 & 3.2 71.7& 5.6 56.7 f 12.5
Okadaic acid
Control
a
261 f 36.4" 216 f 17.0 172 2 23.8 233 f 23.2
SD of
four values from two different experiments.
plasma membrane enzymes persisted in the isolated membranes indicated that the enzymes or some regulator of enzyme activity was altered. Although there is no evidence for covalent modification of PI kinase per se (Carpenter and Cantley, 1990), our data suggest that the changes in PIP metabolism observed as a result of hyperosmotic stress are mediated by an okadaic acid-sensitive phosphatase or by other okadaic acid-sensitive processes such as cytoskeleton reorganization (Fernandez et al., 1990) or intracellular transport (Lucocq et al., 1991). Although PIP-PLC activity recovered to control values by 10 min, the PI kinase did not. These data suggest that, in these cells, PI kinase may be a more critica1 and sustaining factor for controlling steady-state PIP levels. Indeed, the PI kinase activity may reflect the physiological state of the cell. The decrease in the percentage of [3H]PIPand the tendency for the percentage of [3H]PI to increase after 5 min of hyperosmotic stress would be consistent with activation of a PIP phosphatase (monoesterase) as well as a decrease in PI kinase activity; however, we could not detect PIP phosphatase activity in isolated plasma membranes assayed with endogenous [32P]PIPor exogenous [3H]inositol PIP as substrates (data not shown). We started each experiment with the same fresh weight of cells. For each treatment, the inositol lipids and inositol phosphates were monitored as changes in cpm recovered as well as relative changes in the distribution of each lipid or inositol phosphate as a percentage of the total 3H recovered. [3H]PIPdecreased both in cpm and as percentage of total 3Hlipid. Because the [3H]PIPwas 10-fold higher than the [3H]PIPl and because the [3H]IP3was so low, it seems unlikely that an increase in the flux of PIP to PIP2 and I r 3 makes a significant contribution to the decrease in [3H]PIP. The profile of water-soluble 3H compounds indicates the complexity of inositol metabolism in plants, as others have shown (for review, see Coté et al., 1990; Loewus et al., 1990). We did not detect a net increase in [3H]IP or [3H]IP2 after osmotic stress. However, we could not exclude the possibility
that the transient increase in PLC activity in vitro caused a limited increase in the rate of release in [3H]IP2or that osmotic stress enhanced the hydrolysis of Ir2.Severa1 laboratories have shown that the inositol phosphates are metabolized rapidly by plant homogenates and plant membranes anel that the plant inositol phosphate phosphatases are not very sensitive to lithium (Loewus and Loewus, 1982; Drdbak et al., 1988; Joseph et al., 1989; Memon et al., 1989b). Thus, newly released IP2 may have been quickly degraded to inositol. In any event, within the limits of our measurements, inositol phospholipids do not appear to be involved ir1 the primary signaling in response to hyperosmotic stress in carrot cells. It is possible that selective pools of PIP and PIP2 are involved in the early signaling event; however, the magnitude of change certainly is not comparable to that of highly responsive animal cells such as the blowfly salivary gland (Bemdge, 1983) or iris muscle (Akhtar and Abdel-Latif, 1980). The change in PIP metabolism in the carrot cells appears to be secondary to the initial stimulus, perhaps prolonging or facilitating in this instance a change in cell shape and ultimately reflecting a change in the metabolic state of the cell. Einspahr et al. (1988) reported that increased [32P]PIPand [32P]PIP2occurred as a result of short-tem treatment of the halotolerant algae, D. salina, with NaCl. Based on these experiments we anticipated seeing an increase in [3H]inosit~llabeled PIP and PIP2 after hyperosmotic stress, but we found decreased recovery of [3H]PIP. The difference in the data might be due to differences in systems, halotolerant algae versus carrot cells, labeling positions, and, therefore, potential pools of labeled lipids, [32P]Piversus [3H]inositol,and stressinducing substances, NaCl versus sorbitol, respectively. Also, this discrepancy between algae and higher plants ma,y be explained by the difference in regulation of PLC. For example, there is no evidence for effects of GTP or GTP-7-5 on the PLC-mediated breakdown of polyphosphoinositides in higher plants (Melin et al., 1987; McMurray and Irvine, 1988; Tate et al., 1989; Pica1 et al., 1992), but the activity of PLC
Phosphoinositide Metabolism and Hyperosmotic Stress 400
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Okadaic Acid (1 0-6M) Figure 6. The effect of okadaic acid in vitro on PI kinase activity in plasma membranes isolated from control and stressed cells. Cells were treated hyperosmotically (0.4 OSM) for 5 min as described in “Material5 and Methods.” Okadaic acid (155 P M in 10% DMSO) was added to the lipid phosphorylation reaction mixture to give the final concentrations indicated. The PI kinase activity was assayed in the absence of exogenous substrate. Open circles, control; closed circles, hyperosmotic stress. The results are the means & SD of four values from two different experiments.
the PLC-mediated breakdown of polyphosphoinositides in higher plants (Melin et al., 1987; McMurray and Irvine, 1988; Tate et al., 1989; Pical et al., 1992), but the activity of PLC from D. salina has been reported to be stimulated by the nonhydrolyzable GTP analog, GTP-7-S (Einspahr et al., 1989). Srivastava et al. (1989) studied the effects of mannitol on IP3 production in red beet slices. IP3 was measured as [3H]IP3 displaced from the IP3-binding protein. The amount of putative IP3 decreased 10 min after beet root slices were placed in 0.4 M mannitol and increased after 10 min in 0.2 M mannitol. If the red beet slices were plasmolyzed in 0.4 M mannitol, the decrease in IP3 would be consistent with the down regulation of the inositol lipid metabolism observed in the plasmolyzed carrot cells. Salt stress resulted in reduced plasma membrane ATPase activity of tomato roots (Gronwald et al., 1990), suggesting that salt stress may impair the catalytic efficiency of the ATPase either by affecting the synthesis of positive or negative effectors or by affecting the lipid composition of the membrane. The carrot cell plasma membrane vanadate-sensitive ATPase activity also was reduced by hyperosmotic stress. The change in plasma membrane vanadate-sensitive ATPase activity previously has been shown to closely correlate with the PIP kinase activity in response to the cell-walldegrading enzyme, Driselase (Chen and Boss, 1990). Results of the time-course study of ATPase and PI kinase activity in
645
the osmotically stressed cells suggested that an initial decrease in ATPase activity may be unrelated to the level of PIP in plasma membranes; however, the later decrease in ATPase after 5 min may reflect the lower levels of PIP as the cells reach a new steady state. Negatively charged inositol lipids can directly affect the activity of P-type ATPases (Varsanyi et al., 1983; Schafer et al., 1987; Memon et al., 1989a; Chen and Boss, 1991). Within the context of signal transduction mechanisms the changes in inositol metabolism that we observed are slow and are probably down stream from the initial response. Slayman (1982) has shown that the plasma membrane of yeast cells depolarizes rapidly, within 30 s, in response to hyperosmotic stress, followed by a slower recovery period that lasts severa1 minutes. Such a depolarization could indicate a change in ion flux or membrane structure. Stretchactivated ion channels have been shown to be present in tobacco cells grown in suspension culture and guard cells (Edwards and Pickard, 1987; Schroeder and Hedrich, 1989). These channels are activated by stretching the plasma membranes, and therefore, their open probability may be changed rapidly in response to mechanical stimuli or as a result of volume or turgor changes. Physiological responses within the cells could be mediated by a resultant increase in cytosolic calcium or by some other factors, such as a change in H+, K+, or C1- ions. Calcium has been shown to activate PIP-PLC (Melin et al., 1987, 1992; Tate et al., 1989; Pical et al., 1992) and inhibit PI kinase activity (Kamada and Muto, 1992) in higher plants. One consequence of decreased PIP is a potential loss of gelsolin or profilin binding and a resultant increase in actin severing and a decrease in actin nucleation sites (Janmey and Stossel, 1987; Janmey et al., 1987; Lind et al., 1987). In vitro studies indicate that profilin can bind to PIP and PIPl and protect them from hydrolysis by PLC (Forscher, 1989; Goldschmidt-Clermont et al., 1990; Machesky et al., 1990). It is not yet clear, however, whether the increase in cytosolic calcium and the profilin release precede or follow PIP hydrolysis in vivo. The data suggest that profilin could link cell signaling at the membrane level to reorganization of the cytoskeleton. Because a considerable portion of the PI and PIP kinase activity is associated with the actin filament fraction in isolated plasma membranes (Tan and Boss, 1992), if there is a change in the cytoskeletal structure ( e g a loss of actin filaments and actin-binding proteins associated with the plasma membrane), this could contribute to the decrease in plasma membrane-associated PI kinase. Based on these results we propose the following working model for carrot cells hyperosmotically stressed with sorbitol: Hyperosmotic stress induces membrane depolarization and plasmolysis within 30 s. An okadaic acid-sensitive phosphatase that is not associated with the plasma membrane is activated. After about 5 min the cells are in a recovery phase in which the cells begin to adjust to the new osmoticum. During the recovery phase, PIP-PLC is transiently activated while there is a sustained decrease in PI kinase activity. The result is a decrease in PIP. The net decrease in PIP would contribute to the decrease plasma membrane ATPase and affect cytoskeleton reorganization. As a result of these and
Cho et al.
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other intracellular events, the physiological state of cells changes, and cells reach a new steady state. Received April 19, 1993; accepted June 19, 1993. Copyright Clearance Center: 0032-0889/93/103/0637/11.
\
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